Molecular identification of StAR and 3βHSD1 and characterization in response to GnIH stimulation in protogynous hermaphroditic grouper (Epinephelus coioides).

PubMed

Wang, Qingqing; Qi, Xin; Tang, Haipei; Guo, Yin; Li, Shuisheng; Li, Gaofei; Yang, Xiaoli; Zhang, Haifa; Liu, Xiaochun; Lin, Haoran

2017-04-01

Gonadal steroids are critical factors in reproduction and sex reverse process. StAR (steroidogenic acute regulatory protein), transferring the cholesterol from the outer mitochondrial membrane to the inner membrane, is the rate-limiting factor of steroidogenesis. 3βHSD (3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase), converting Δ5-steroids into Δ4-steroids, is an important oxidoreductase in steroidogenesis. In the present study, StAR and 3βHSD1 were cloned and characterized from protogynous orange-spotted grouper. StAR cDNA contains an 861bp open reading frame (ORF), encoding a predicted protein of 286 amino acids, and the ORF of 3βHSD1 was 1125bp, encoding a predicted protein of 374 amino acids. The transcript of StAR was mainly expressed in gonad, while 3βHSD1 mRNA was predominantly detected in brain and gonad. In the previous study, we found the expression of GnIH mRNA level in male, as well as in 17 alpha-methyltestosterone (MT)-induced male fish was significantly higher than in female fish, this indicating that GnIH/GnIHR signaling might be involved in the regulation of sex reversal and male maintenance. In order to figure out the function of GnIH in steroidogenesis, the expression of StAR and 3βHSD1 regulated by GnIH was examined. In vitro study showed that treatment of cultured ovary fragments with gGnIH peptides significantly stimulated the expression of StAR and 3βHSD1. In addition, the mRNA levels of StAR and 3βHSD1 were significantly increased after intraperitoneal injection (i.p.) with gGnIH peptides. Moreover, during MT-induced sex change from female to male, the levels of StAR mRNA significantly increased by 5.2, 24.8 and 353.5 folds, and that of 3βHSD1 mRNA by 3.5, 32.5 and 55.4 folds at the 2nd, 4th and 6th week after MT implantation, respectively. Collectively, our results indicate that GnIH may be involved in the regulation of sex reversal or male maintenance by stimulating the expression of StAR and 3βHSD1 in protogynous grouper. Copyright © 2017 Elsevier Inc. All rights reserved.

Artemisinin disrupts androgen responsiveness of human prostate cancer cells by stimulating the 26S proteasome-mediated degradation of the androgen receptor protein.

PubMed

Steely, Andrea M; Willoughby, Jamin A; Sundar, Shyam N; Aivaliotis, Vasiliki I; Firestone, Gary L

2017-10-01

Androgen receptor (AR) expression and activity is highly linked to the development and progression of prostate cancer and is a target of therapeutic strategies for this disease. We investigated whether the antimalarial drug artemisinin, which is a sesquiterpene lactone isolated from the sweet wormwood plant Artemisia annua, could alter AR expression and responsiveness in cultured human prostate cancer cell lines. Artemisinin treatment induced the 26S proteasome-mediated degradation of the receptor protein, without altering AR transcript levels, in androgen-responsive LNCaP prostate cancer cells or PC-3 prostate cancer cells expressing exogenous wild-type AR. Furthermore, artemisinin stimulated AR ubiquitination and AR receptor interactions with the E3 ubiquitin ligase MDM2 in LNCaP cells. The artemisinin-induced loss of AR protein prevented androgen-responsive cell proliferation and ablated total AR transcriptional activity. The serine/threonine protein kinase AKT-1 was shown to be highly associated with artemisinin-induced proteasome-mediated degradation of AR protein. Artemisinin treatment activated AKT-1 enzymatic activity, enhanced receptor association with AKT-1, and induced AR serine phosphorylation. Treatment of LNCaP cells with the PI3-kinase inhibitor LY294002, which inhibits the PI3-kinase-dependent activation of AKT-1, prevented the artemisinin-induced AR degradation. Furthermore, in transfected receptor-negative PC-3 cells, artemisinin failed to stimulate the degradation of an altered receptor protein (S215A/S792A) with mutations in its two consensus AKT-1 serine phosphorylation sites. Taken together, our results indicate that artemisinin induces the degradation of AR protein and disrupts androgen responsiveness of human prostate cancer cells, suggesting that this natural compound represents a new potential therapeutic molecule that selectively targets AR levels.

Distinct patterns of dysregulated expression of enzymes involved in androgen synthesis and metabolism in metastatic prostate cancer tumors

PubMed Central

Mitsiades, Nicholas; Sung, Clifford C.; Schultz, Nikolaus; Danila, Daniel C.; He, Bin; Eedunuri, Vijay Kumar; Fleisher, Martin; Sander, Chris; Sawyers, Charles L.; Scher, Howard I.

2012-01-01

Androgen receptor (AR) signaling persists in castration-resistant prostate carcinomas (CRPCs), due to several mechanisms that include increased AR expression and intratumoral androgen metabolism. We investigated the mechanisms underlying aberrant expression of transcripts involved in androgen metabolism in CRPC. We compared gene expression profiles and DNA copy number alteration (CNA) data from 29 normal prostate tissue samples, 127 primary prostate carcinomas (PCas) and 19 metastatic PCas. Steroidogenic enzyme transcripts were evaluated by qRT-PCR in PCa cell lines and circulating tumor cells (CTCs) from CRPC patients. Metastatic PCas expressed higher transcript levels for AR and several steroidogenic enzymes, including SRD5A1, SRD5A3, and AKR1C3, while expression of SRD5A2, CYP3A4, CYP3A5 and CYP3A7 was decreased. This aberrant expression was rarely associated with CNAs. Instead, our data suggest distinct patterns of coordinated aberrant enzyme expression. Inhibition of AR activity by itself stimulated AKR1C3 expression. The aberrant expression of the steroidogenic enzyme transcripts were detected in CTCs from CRPC patients. In conclusion, our findings identify substantial interpatient heterogeneity and distinct patterns of dysregulated expression of enzymes involved in intratumoral androgen metabolism in PCa. These steroidogenic enzymes represent targets for complete suppression of systemic and intratumoral androgen levels, an objective that is supported by the clinical efficacy of the CYP17 inhibitor abiraterone. A comprehensive AR axis targeting approach via simultaneous, frontline enzymatic blockade and/or transcriptional repression of several steroidogenic enzymes, in combination with GnRH analogs and potent anti-androgens, would represent a powerful future strategy for PCa management. PMID:22971343

PecS regulates the urate-responsive expression of type 1 fimbriae in Klebsiella pneumoniae CG43.

PubMed

Wang, Zhe-Chong; Liu, Chia-Jui; Huang, Ying-Jung; Wang, Yu-Seng; Peng, Hwei-Ling

2015-12-01

In the Klebsiella pneumoniae CG43 genome, the divergently transcribed genes coding for PecS, the MarR-type transcription factor, and PecM, the drug metabolite transporter, are located between the type 1 and type 3 fimbrial gene clusters. The intergenic sequence pecO between pecS and pecM contains three putative PecS binding sites and a CpxR box. Electrophoretic mobility shift assay revealed that the recombinant PecS and CpxR could specifically bind to the pecO sequence, and the specific interaction of PecS and pecO could be attenuated by urate. The expression of pecS and pecM was negatively regulated by CpxAR and PecS, and was inducible by exogenous urate in the absence of cpxAR. Compared with CG43S3ΔcpxAR, the derived mutants CG43S3ΔcpxARΔpecS and CG43S3ΔcpxARΔpecSΔpecM exerted similar levels of sensitivity to H2O2 or paraquat, but higher levels of mannose-sensitive yeast agglutination activity and FimA production. The promoter activity and transcript levels of fimA in CG43S3ΔcpxAR were also increased by deleting pecS. However, no binding activity between PecS and the fimA promoter could be observed. Nevertheless, PecS deletion could reduce the expression of the global regulator HNS and release the negative effect of HNS on FimA expression. In CG43S3ΔcpxAR, the expression of FimA as well as PecS was inducible by urate, whilst urate-induced FimA expression was inhibited by the deletion of pecS. Taken together, we propose that K. pneumoniae PecS indirectly and negatively regulates the expression of type 1 fimbriae, and the regulation is urate-inducible in the absence of CpxAR.

[Effect of epidermal growth factor and testosterone on androgen receptor activation in urethral plate fibroblasts in hypospadias].

PubMed

Lin, Junshan; Xie, Cheng; Chen, Ruiqing; Li, Dumiao

2016-05-01

To investigate androgen receptor (AR) expression and the effect of epidermal growth factor (EGF) and testosterone on AR expression level.
 EGF or different concentrations of testosterone were incubated with the primary urethral plate fibroblasts from patients with hypospadias. The levels of AR expression in the fibroblasts were detected by immunocytochemical assays and graphical analysis.
 There was no significant difference in AR activation under physiological concentrations (3×10(-8) mol/L) of testosterone between the control and the distal hypospadias group (P>0.05). However, there was a significant decrease in AR activation in the proximal hypospadias group compared to that in the control group (P<0.001). Under the concentration of 3×10(-6) mol/L, the effects of testosterone on AR activation were dramatically different in the three groups (control group>distal hypospadias group>proximal hypospadias group, P<0.001). AR activation level in the group of proximal hypospadias was improved most obviously when EGF and physiological concentration of testosterone were employed in the urethral plate fibroblasts from hypospadias patients (P<0.001), and it was improved more in the distal hypospadias group than that in the control group (P=0.02).
 AR expression and activation in the urethral plate fibroblasts from hypospadias patients are abnormal. EGF can be used to improve AR activation in fibroblasts from different types of hypospadias, especially in the proximal type.

Androgen receptor (AR) promotes clear cell renal cell carcinoma (ccRCC) migration and invasion via altering the circHIAT1/miR-195-5p/29a-3p/29c-3p/CDC42 signals.

PubMed

Wang, Kefeng; Sun, Yin; Tao, Wei; Fei, Xiang; Chang, Chawnshang

2017-05-28

Increasing evidence has demonstrated that the androgen receptor (AR) plays important roles to promote the metastasis of clear cell renal cell carcinoma (ccRCC). The detailed mechanisms, especially how AR functions via altering the circular RNAs (circRNAs) remain unclear. Here we identified a new circRNA (named as circHIAT1) whose expression was lower in ccRCCs than adjacent normal tissues. Targeting AR could suppress ccRCC cell progression via increasing circHIAT1 expression. ChIP assay and luciferase assay demonstrated that AR suppressed circHIAT1 expression via regulating its host gene, Hippocampus Abundant Transcript 1 (HIAT1) expression at the transcriptional level. The consequences of AR-suppressed circHIAT1 resulted in deregulating miR-195-5p/29a-3p/29c-3p expressions, which increased CDC42 expression to enhance ccRCC cell migration and invasion. Increasing this newly identified signal via circHIAT1 suppressed AR-enhanced ccRCC cell migration and invasion. Together, these results suggested that circHIAT1 functioned as a metastatic inhibitor to suppress AR-enhanced ccRCC cell migration and invasion. Targeting this newly identified AR-circHIAT1-mediated miR-195-5p/29a-3p/29c-3p/CDC42 signals may help us develop potential new therapies to better suppress ccRCC metastasis. Copyright © 2017 Elsevier B.V. All rights reserved.

The A3 adenosine receptor agonist CF502 inhibits the PI3K, PKB/Akt and NF-kappaB signaling pathway in synoviocytes from rheumatoid arthritis patients and in adjuvant-induced arthritis rats.

PubMed

Ochaion, A; Bar-Yehuda, S; Cohen, S; Amital, H; Jacobson, K A; Joshi, B V; Gao, Z G; Barer, F; Patoka, R; Del Valle, L; Perez-Liz, G; Fishman, P

2008-08-15

The A(3) adenosine receptor (A(3)AR) is over-expressed in inflammatory cells and was defined as a target to combat inflammation. Synthetic agonists to this receptor, such as IB-MECA and Cl-IB-MECA, exert an anti-inflammatory effect in experimental animal models of adjuvant- and collagen-induced arthritis. In this study we present a novel A(3)AR agonist, CF502, with high affinity and selectivity at the human A(3)AR. CF502 induced a dose dependent inhibitory effect on the proliferation of fibroblast-like synoviocytes (FLS) via de-regulation of the nuclear factor-kappa B (NF-kappaB) signaling pathway. Furthermore, CF502 markedly suppressed the clinical and pathological manifestations of adjuvant-induced arthritis (AIA) in a rat experimental model when given orally at a low dose (100 microg/kg). As is typical of other G-protein coupled receptors, the A(3)AR expression level was down-regulated shortly after treatment with agonist CF502 in paw and in peripheral blood mononuclear cells (PBMCs) derived from treated AIA animals. Subsequently, a decrease in the expression levels of protein kinase B/Akt (PKB/Akt), IkappaB kinase (IKK), I kappa B (IkappaB), NF-kappaB and tumor necrosis factor-alpha (TNF-alpha) took place. In addition, the expression levels of glycogen synthase kinase-3 beta (GSK-3beta), beta-catenin, and poly(ADP-ribose)polymerase (PARP), known to control the level and activity of NF-kappaB, were down-regulated upon treatment with CF502. Taken together, CF502 inhibits FLS growth and the inflammatory manifestations of arthritis, supporting the development of A(3)AR agonists for the treatment of rheumatoid arthritis.

Skp2 regulates androgen receptor through ubiquitin-mediated degradation independent of Akt/mTOR pathways in prostate cancer.

PubMed

Li, Bo; Lu, Wenfu; Yang, Qing; Yu, Xiuping; Matusik, Robert J; Chen, Zhenbang

2014-04-01

The intervention of advanced prostate cancer (PCa) in patients has been commonly depending on androgen deprivation therapy. Despite of tremendous research efforts, however, molecular mechanisms on AR regulation remain poorly understood, particularly for castration resistant prostate cancer (CRPC). Targeting AR and associated factors is considered an effective strategy in PCa treatment. Human prostate cancer cells were used in this study. Manipulations of Skp2 expression were achieved by Skp2 shRNA/siRNA or overexpression of plasmids. Dual luciferase reporter assay was applied for AR activity assessment. Western blot, ubiquitination assay, immunoprecipitation, and immunofluorescence were applied to detect the proteins. Our results demonstrated that Skp2 directly involves the regulation of AR expression through ubiquitination-mediated degradation. Skp2 interacted with AR protein in PCa cells, and enforced expression of Skp2 resulted in a decreased level and activity of AR. By contrast, Skp2 knockdown increased the protein accumulation and activity of AR. Importantly, changes of AR contributed by Skp2 led to subsequent alterations of PSA level in PCa cells. AR ubiquitination was significantly increased upon Skp2 overexpression but greatly reduced upon Skp2 knockdown. AR mutant at K847R abrogated Skp2-mediated ubiquitination of AR. NVP-BEZ235, a dual PI3K/mTOR inhibitor, remarkably inhibited Skp2 level with a striking elevation of AR. The results indicate that Skp2 is an E3 ligase for proteasome-dependent AR degradation, and K847 on AR is the recognition site for Skp2-mediated ubiquitination. Our findings reveal an essential role of Skp2 in AR signaling. © 2013 Wiley Periodicals, Inc.

Transcriptional up-regulation of the human androgen receptor by androgen in bone cells.

PubMed

Wiren, K M; Zhang, X; Chang, C; Keenan, E; Orwoll, E S

1997-06-01

Androgen regulation of androgen receptor (AR) expression has been observed in a variety of tissues, generally as inhibition, and is thought to attenuate cellular responses to androgen. AR is expressed in osteoblasts, the bone-forming cell, suggesting direct actions of androgens on bone. Here we characterized the effect of androgen exposure on AR gene expression in human osteoblastic SaOS-2 and U-2 OS cells. Treatment of osteoblastic cells with the nonaromatizable androgen 5alpha-dihydrotestosterone increased AR steady state messenger RNA levels in a time- and dose-dependent fashion. Reporter assays with 2.3 kilobases of the proximal 5'-flanking region of the human AR promoter linked to the chloramphenicol acetyltransferase gene in transfected cultures showed that up-regulation of AR promoter activity by androgen was time and dose dependent. Treatment with other steroid hormones, including progesterone, 17beta-estradiol, and dexamethasone, was without effect. The antiandrogen hydroxyflutamide completely antagonized androgen up-regulation. Thus, in contrast to many other androgen target tissues, androgen exposure increases steady state AR messenger RNA levels in osteoblasts. This regulation occurs at least partially at the level of transcription, is mediated by the 5'-promoter region of the AR gene, and is dependent on functional AR. These results suggest that physiological concentrations of androgens have significant effects on AR expression in skeletal tissue.

NRIP/DCAF6 stabilizes the androgen receptor protein by displacing DDB2 from the CUL4A-DDB1 E3 ligase complex in prostate cancer.

PubMed

Chen, Hsin-Hsiung; Fan, Ping; Chang, Szu-Wei; Tsao, Yeou-Ping; Huang, Hsiang-Po; Chen, Show-Li

2017-03-28

Both nuclear receptor interaction protein (NRIP) and DNA damage binding protein 2 (DDB2) belong to the Cullin 4 (CUL4)-DDB1 binding protein family and are androgen receptor (AR)-interacting proteins. Here, we investigated the expression patterns of the NRIP, DDB2 and AR proteins in human prostate cancer tissues and found that the expression levels of NRIP and AR were higher, but the DDB2 level was lower, in prostate cancer tissues than in non-neoplastic controls, suggesting NRIP as a candidate tumor promoter and DDB2 as a tumor suppressor in prostate cancer. Furthermore, both NRIP and DDB2 shared the same AR binding domain; they were competitors for the AR, but not for DDB1 binding, in the AR-DDB2-DDB1-CUL4A complex. Conclusively, NRIP stabilizes the AR protein by displacing DDB2 from the AR-DDB2 complex. Consistent with our hypothesis, a specific expression pattern with high levels of NRIP and AR, together with a low level of DDB2, was found more frequently in the human prostate cancer tissues with a cribriform pattern than in non-cribriform tumors, suggesting that disruption of the balance between NRIP and DDB2 may change AR protein homeostasis and contribute to pathogenesis in certain aggressive types of prostate cancer.

NRIP/DCAF6 stabilizes the androgen receptor protein by displacing DDB2 from the CUL4A-DDB1 E3 ligase complex in prostate cancer

PubMed Central

Tsao, Yeou-Ping; Huang, Hsiang-Po; Chen, Show-Li

2017-01-01

Both nuclear receptor interaction protein (NRIP) and DNA damage binding protein 2 (DDB2) belong to the Cullin 4 (CUL4)-DDB1 binding protein family and are androgen receptor (AR)-interacting proteins. Here, we investigated the expression patterns of the NRIP, DDB2 and AR proteins in human prostate cancer tissues and found that the expression levels of NRIP and AR were higher, but the DDB2 level was lower, in prostate cancer tissues than in non-neoplastic controls, suggesting NRIP as a candidate tumor promoter and DDB2 as a tumor suppressor in prostate cancer. Furthermore, both NRIP and DDB2 shared the same AR binding domain; they were competitors for the AR, but not for DDB1 binding, in the AR-DDB2-DDB1-CUL4A complex. Conclusively, NRIP stabilizes the AR protein by displacing DDB2 from the AR-DDB2 complex. Consistent with our hypothesis, a specific expression pattern with high levels of NRIP and AR, together with a low level of DDB2, was found more frequently in the human prostate cancer tissues with a cribriform pattern than in non-cribriform tumors, suggesting that disruption of the balance between NRIP and DDB2 may change AR protein homeostasis and contribute to pathogenesis in certain aggressive types of prostate cancer. PMID:28212551

KDM4B and KDM4A promote endometrial cancer progression by regulating androgen receptor, c-myc, and p27kip1

PubMed Central

Kwan, Suet-Ying; Chen, Limo; Chen, Jin-Hong; Ying, Zuo-Lin; Zhou, Ye; Gu, Wei; Wang, Li-Hua; Cheng, Wei-Wei; Zeng, Jianfang; Wan, Xiao-Ping; Mok, Samuel C.; Wong, Kwong-Kwok; Bao, Wei

2015-01-01

Epidemiological evidence suggests that elevated androgen levels and genetic variation related to the androgen receptor (AR) increase the risk of endometrial cancer (EC). However, the role of AR in EC is poorly understood. We report that two members of the histone demethylase KDM4 family act as major regulators of AR transcriptional activityin EC. In the MFE-296 cell line, KDM4B and AR upregulate c-myc expression, while in AN3CA cells KDM4A and AR downregulate p27kip1. Additionally, KDM4B expression is positively correlated with AR expression in EC cell lines with high baseline AR expression, while KDM4A and AR expression are positively correlated in low-AR cell lines. In clinical specimens, both KDM4B and KDM4A expression are significantly higher in EC tissues than that in normal endometrium. Finally, patients with alterations in AR, KDM4B, KDM4A, and c-myc have poor overall and disease-free survival rates. Together, these findings demonstrate that KDM4B and KDM4A promote EC progression by regulating AR activity. PMID:26397136

Insights into arsenic multi-operons expression and resistance mechanisms in Rhodopseudomonas palustris CGA009

PubMed Central

Zhao, Chungui; Zhang, Yi; Chan, Zhuhua; Chen, Shicheng; Yang, Suping

2015-01-01

Arsenic (As) is widespread in the environment and causes numerous health problems. Rhodopseudomonas palustris has been regarded as a good model organism for studying arsenic detoxification since it was first demonstrated to methylate environmental arsenic by conversion to soluble or gaseous methylated species. However, the detailed arsenic resistance mechanisms remain unknown though there are at least three arsenic-resistance operons (ars1, ars2, and ars3) in R. palustris. In this study, we investigated how arsenic multi-operons contributed to arsenic detoxification in R. palustris. The expression of ars2 or ars3 operons increased with increasing environmental arsenite (As(III)) concentrations (up to 1.0 mM) while transcript of ars1 operon was not detected in the middle log-phase (55 h). ars2 operon was actively expressed even at the low concentration of As(III) (0.01 μM), whereas the ars3 operon was expressed at 1.0 μM of As(III), indicating that there was a differential regulation mechanism for the three arsenic operons. Furthermore, ars2 and ars3 operons were maximally transcribed in the early log-phase where ars2 operon was 5.4-fold higher than that of ars3 operon. A low level of ars1 transcript was only detected at 43 h (early log-phase). Arsenic speciation analysis demonstrated that R. palustris could reduce As(V) to As(III). Collectively, strain CGA009 detoxified arsenic by using arsenic reduction and methylating arsenic mechanism, while the latter might occur with the presence of higher concentrations of arsenic. PMID:26441915

StAR protein and steroidogenic enzyme expressions in the rat Harderian gland.

PubMed

Falvo, Sara; Chieffi Baccaria, Gabriella; Spaziano, Giuseppe; Rosati, Luigi; Venditti, Massimo; Di Fiore, Maria Maddalena; Santillo, Alessandra

2018-03-01

The Harderian gland (HG) of the rat (Rattus norvegicus) secretes copious amounts of lipids, such as cholesterol. Here we report a study of the expressions of the StAR protein and key steroidogenic enzymes in the HG of male and female rats. The objective of the present investigation was to ascertain (a) whether the rat HG is involved in steroid production starting with cholesterol, and (b) whether the pattern of gene and protein expressions together with the enzymatic activities display sexual dimorphism. The results demonstrate, for the first time, the expression of StAR gene and protein, and Cyp11a1, Hsd3b1, Hsd17b3, Srd5a1, Srd5a2 and Cyp19a1 genes in the rat HG. StAR mRNA and protein expressions were much greater in males than in females. Immunohistochemical analysis demonstrated a non-homogeneous StAR distribution among glandular cells. Hsd17b3 and Cyp19a1 mRNA levels were higher in males than in females, whereas Srd5a1 mRNA levels were higher in females than in males. No significant differences were observed in mRNA levels of Cyp11a1, Hsd3b1 and Srd5a2 between sexes. Furthermore, the in vitro experiments demonstrated a higher 5α-reductase activity in the female as compared to the male HG vice versa a higher P450 aro activity in males as compared to females. These results suggest that the Harderian gland can be classified as a steroidogenic tissue because it synthesizes cholesterol, expresses StAR and steroidogenic enzymes involved in both androgen and estrogen synthesis. The dimorphic expression and activity of the steroidogenic enzymes may suggest sex-specific hormonal effects into the HG physiology. Copyright © 2018 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.

Complement C3a binding to its receptor as a negative modulator of Th2 response in liver injury in trichloroethylene-sensitized mice.

PubMed

Wang, Feng; Zha, Wan-sheng; Zhang, Jia-xiang; Li, Shu-long; Wang, Hui; Ye, Liang-ping; Shen, Tong; Wu, Chang-hao; Zhu, Qi-xing

2014-08-17

Trichloroethylene (TCE) is a major occupational health hazard and causes occupational medicamentosa-like dermatitis (OMLDT) and liver damage. Recent evidence suggests immune response as a distinct mode of action for TCE-induced liver damage. This study aimed to explore the role of the key complement activation product C3a and its receptor C3aR in TCE-induced immune liver injury. A mouse model of skin sensitization was induced by TCE in the presence and absence of the C3aR antagonist SB 290157. Liver function was evaluated by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in conjunction with histopathological characterizations. C3a and C3aR were detected by immunohistochemistry and C5b-9 was assessed by immunofluorescence. IFN-γ and IL4 expressions were determined by flow cytometry and ELISA. The total sensitization rate was 44.1%. TCE sensitization caused liver cell necrosis and inflammatory infiltration, elevated serum ALT and AST, expression of C3a and C3aR, and deposition of C5b-9 in the liver. IFN-γ and IL-4 expressions were up-regulated in spleen mononuclear cells and their serum levels were also increased. Pretreatment with SB 290157 resulted in more inflammatory infiltration in the liver, higher levels of AST, reduced C3aR expression on Kupffer cells, and decreased IL-4 levels while IFN-γ remained unchanged. These data demonstrate that blocking of C3a binding to C3aR reduces IL4, shifts IFN-γ and IL-4 balance, and aggravates TCE-sensitization induced liver damage. These findings reveal a novel mechanism whereby modulation of Th2 response by C3a binding to C3a receptor contributes to immune-mediated liver damage by TCE exposure. Copyright © 2014. Published by Elsevier Ireland Ltd.

Androgen receptor activity modulates responses to cisplatin treatment in bladder cancer.

PubMed

Kashiwagi, Eiji; Ide, Hiroki; Inoue, Satoshi; Kawahara, Takashi; Zheng, Yichun; Reis, Leonardo O; Baras, Alexander S; Miyamoto, Hiroshi

2016-08-02

Cisplatin (CDDP)-based combination chemotherapy remains the mainstream treatment for advanced bladder cancer. However, its efficacy is often limited due to the development of resistance for which underlying mechanisms are poorly understood. Meanwhile, emerging evidence has indicated the involvement of androgen-mediated androgen receptor (AR) signals in bladder cancer progression. In this study, we aimed to investigate whether AR signals have an impact on sensitivity to CDDP in bladder cancer cells. UMUC3-control-short hairpin RNA (shRNA) cells with endogenous AR and AR-negative 647V/5637 cells stably expressing AR were significantly more resistant to CDDP treatment at its pharmacological concentrations, compared with UMUC3-AR-shRNA and 647V-vector/5637-vector control cells, respectively. A synthetic androgen R1881 significantly reduced CDDP sensitivity in UMUC3, 647V-AR, or 5637-AR cells, and the addition of an anti-androgen hydroxyflutamide inhibited the effect of R1881. In these AR-positive cells, R1881 treatment also induced the expression levels of NF-κB, which is known to involve CDDP resistance, and its phosphorylated form, as well as nuclear translocation of NF-κB. In CDDP-resistant bladder cancer sublines established following long-term culture with CDDP, the expression levels of AR as well as NF-κB and phospho-NF-κB were considerably elevated, compared with respective control sublines. In bladder cancer specimens, there was a strong trend to correlate between AR positivity and chemoresistance. These results suggest that AR activation correlates with CDDP resistance presumably via modulating NF-κB activity in bladder cancer cells. Targeting AR during chemotherapy may thus be a useful strategy to overcome CDDP resistance in patients with AR-positive bladder cancer.

Docosahexaenoic acid inhibits IL-6 expression via PPARγ-mediated expression of catalase in cerulein-stimulated pancreatic acinar cells.

PubMed

Song, Eun Ah; Lim, Joo Weon; Kim, Hyeyoung

2017-07-01

Cerulein pancreatitis mirrors human acute pancreatitis. In pancreatic acinar cells exposed to cerulein, reactive oxygen species (ROS) mediate inflammatory signaling by Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3, and cytokine induction. Docosahexaenoic acid (DHA) acts as an agonist of peroxisome proliferator activated receptor γ (PPARγ), which mediates the expression of some antioxidant enzymes. We hypothesized that DHA may induce PPARγ-target catalase expression and reduce ROS levels, leading to the inhibition of JAK2/STAT3 activation and IL-6 expression in cerulein-stimulated acinar cells. Pancreatic acinar AR42J cells were treated with DHA in the presence or absence of the PPARγ antagonist GW9662, or treated with the PPARγ agonist troglitazone, and then stimulated with cerulein. Expression of IL-6 and catalase, ROS levels, JAK2/STAT3 activation, and nuclear translocation of PPARγ were assessed. DHA suppressed the increase in ROS, JAK2/STAT3 activation, and IL-6 expression induced nuclear translocation of PPARγ and catalase expression in cerulein-stimulated AR42J cells. Troglitazone inhibited the cerulein-induced increase in ROS and IL-6 expression, but induced catalase expression similar to DHA in AR42J cells. GW9662 abolished the inhibitory effect of DHA on cerulein-induced increase in ROS and IL-6 expression in AR42J cells. DHA-induced expression of catalase was suppressed by GW9662 in cerulein-stimulated AR42J cells. Thus, DHA induces PPARγ activation and catalase expression, which inhibits ROS-mediated activation of JAK2/STAT3 and IL-6 expression in cerulein-stimulated pancreatic acinar cells. Copyright © 2017. Published by Elsevier Ltd.

14-3-3η Amplifies Androgen Receptor Actions in Prostate Cancer

PubMed Central

Titus, Mark A.; Tan, Jiann-an; Gregory, Christopher W.; Ford, O. Harris; Subramanian, Romesh R.; Fu, Haian; Wilson, Elizabeth M.; Mohler, James L.; French, Frank S.

2009-01-01

Purpose Androgen receptor (AR) abundance and AR-regulated gene expression in castration-recurrent prostate cancer (CaP) are indicative of AR activation in the absence of testicular androgen. AR transactivation of target genes in castration-recurrent CaP occurs in part through mitogen signaling that amplifies the actions of AR and its coregulators. Herein we report on the role of 14-3-3η in AR action. Experimental Design and Results AR and 14-3-3η co-localized in COS cell nuclei with and without androgen and 14-3-3η promoted AR nuclear localization in the absence of androgen. 14-3-3η interacted with AR in cell-free binding and coimmunoprecipitation assays. In the recurrent human CaP cell line, CWR-R1, native endogenous AR transcriptional activation was stimulated by 14-3-3η at low DHT concentrations and was increased by EGF. Moreover, the DHT and EGF dependent increase in AR transactivation was inhibited by a dominant negative 14-3-3η. In the CWR22 CaP xenograft model, 14-3-3η expression was increased by androgen, suggesting a feed-forward mechanism that potentiates both 14-3-3η and AR actions. 14-3-3η mRNA and protein decreased following castration of tumor bearing mice and increased in tumors of castrate mice after treatment with testosterone. CWR22 tumors that recurred 5 months after castration contained 14-3-3η levels similar to the androgen-stimulated tumors removed before castration. In a human prostate tissue microarray of clinical specimens, 14-3-3η localized with AR in nuclei and the similar amounts expressed in castration-recurrent CaP, androgen-stimulated CaP and benign prostatic hyperplasia were consistent with AR activation in recurrent CaP. Conclusion 14-3-3η enhances androgen and mitogen induced AR transcriptional activity in castration-recurrent CaP. PMID:19996220

Exendin-4 impairs the autophagic flux to induce apoptosis in pancreatic acinar AR42J cells by down-regulating LAMP-2.

PubMed

Li, Zhiqiang; Zhu, Shaihong; Huang, Lihua; Shang, Mingming; Yu, Can; Zhu, Hongwei; Han, Duo; Huang, Hui; Yu, Xiao; Li, Xia

2018-02-05

This study aimed to explore the mechanism of impaired autophagy flux induced by exendin-4 and its role on cell apoptosis in pancreatic AR42J cells. The AR42J cells were treated with various concentration of exendin-4 for several time points to assess its cytotoxicity by MTT assay. Then the AR42J cells were treated by 10pM exendin-4 for 72 h, the cell death was analyzed by flow cytometry and caspase-3 level was examined by Western blot with or without the pretreatment of z-VAD-fmk to testify whether exendin-4 induces the cell apoptosis. The protein levels of LC3B, p62 and LAMP-2 were assessed by Western blot, the mRNA level of LAMP-2 was quantified by quantitative PCR in the absence or presence of LAMP-2 over-expression plasmid and the expression and activity of CatB and CatL were tested by ELISA or activity assay methods in AR42J cells treated by exendin-4. The normal rats and the diabetes-model rats by high-fat and high-sugar diet for two month then with streptozotocin intraperitoneally were subcutaneously injected with exendin-4 for 10 weeks to test the expression of LAMP-2 mRNA and protein in the pancreas. Cells pretreated with Bafilomycin A1 were detected for LC3B and p62 expressions by Western blot. Cells pretreated by 3-MA were used to assess whether 3-MA can protect from exendin-4 cytotoxicity. We found that exendin-4 can decrease the AR42J cell viability as well as increase the cell death and cleaved caspase-3 level, which all can be inhibited by z-VAD-fmk. Exendin-4 can downregulate the expression of LAMP-2 and then impair the autophagy flux to induce the accumulation of LC3B-II and p62, but cannot change the expression and activity of CatB and CatL. Bafilomycin A1 almostly have no impact on the change of LC3B and p62 protein levels induced by exendin-4. Both 3-MA and overexpressed LAMP-2 can reduce the cytotoxicity of exendin-4. Therefore, we considered the down-regulation of LAMP-2 which can impair the autophagy flux by inhibiting the fusion of autophagosomes with lysosomes to induce the AR42J cell apoptosis as the potential mechanism of chronic pancreatitis induced by exendin-4. Copyright © 2018 Elsevier Inc. All rights reserved.

An experimental study on amelioration of dyslipidemia-induced atherosclesis by Clematichinenoside through regulating Peroxisome proliferator-activated receptor-α mediated apolipoprotein A-I, A-II and C-III.

PubMed

Liu, Chao; Guo, Qianqian; Lu, Mengchen; Li, Yunman

2015-08-15

Prevention or amelioration the prevalence of atherosclerosis has been an effective strategy in the management of cardiovascular diseases. The aim of the study was to scrutinize the effect of Clematichinenoside (AR) on dyslipidemia-induced atherosclerosis and explore its capability on expression of Peroxisome proliferator-activated receptor-α (PPAR-alpha), apolipoprotein A-I (APOA1) and A-II (APOA2), and suppression of apolipoprotein C-III (APOC3) genes and proteins. In the present study, we investigated atherosclerosis effect of AR using a combination of high-fat diet and balloon injury model in rabbits. The levels of biochemical indicators were evaluated in plasma, liver and HepG2 cells using immunoassay technology. In order to expose the underlying mechanism, we evaluated the regulation of PPAR-alpha, APOA1, APOA2 and APOC3 expressions by AR, and we further evaluated the interactions between them after transfection with shRNA (shPPAR-alpha) and, the action of PPAR-alpha in HepG2 cells. We could find that AR markedly promoted the PPAR-alpha transfer from cytoplasm to nucleus which resulted in the alteration of APOA1, APOA2 and APOC3 expressions in HepG2 cells. Moreover, AR significantly reduced total cholesterol, triglycerides and low-density lipoprotein cholesterol (LDL-C) levels, and elevated high-density lipoprotein cholesterol (HDL-C) level, which play an important role in dyslipidemia-induced atherosclerosis. In conclusion, AR ameliorated atherosclerosis via the regulation of hepatic lipid metabolism, and AR also contributed to the activation of PPAR-alpha, APOA1, APOA2 and APOC3. Therefore, AR could be a potential therapeutic agent in the treatment of atherosclerosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Expression profiles and functional associations of endogenous androgen receptor and caveolin-1 in prostate cancer cell lines.

PubMed

Bennett, Nigel C; Hooper, John D; Johnson, David W; Gobe, Glenda C

2014-05-01

In prostate cancer (PCa) patients, the protein target for androgen deprivation and blockade therapies is androgen receptor (AR). AR interacts with many proteins that function to either co-activate or co-repress its activity. Caveolin-1 (Cav-1) is not found in normal prostatic epithelium, but is found in PCa, and may be an AR co-regulator protein. We investigated cell line-specific signatures and associations of endogenous AR and Cav-1 in six PCa cell lines of known androgen sensitivity: LNCaP (androgen sensitive); 22Rv1 (androgen responsive); PC3, DU145, and ALVA41 (androgen non-reliant); and RWPE1 (non-malignant). Protein and mRNA expression profiles were compared and electron microscopy used to identify cells with caveolar structures. For cell lines expressing both AR and Cav-1, knockdown techniques using small interfering RNA against AR or Cav-1 were used to test whether diminished expression of one affected the other. Co-sedimentation of AR and Cav-1 was used to test their association. A reporter assay for AR genomic activity was utilized following Cav-1 knockdown. AR-expressing LNCaP and 22Rv1 cells had low endogenous Cav-1 mRNA and protein. Cell lines that expressed little or no AR (DU145, PC3, ALVA41, and RWPE1) expressed high endogenous levels of Cav-1. AR knockdown in LNCaP cells had little effect on Cav-1, but Cav-1 knockdown inhibited AR expression and genomic activity. These data show endogenous AR and Cav-1 mRNA and protein expression is inversely related in PCa cells, with Cav-1 acting on the androgen/AR signaling axis possibly as an AR co-activator, demonstrated by diminished AR genomic activity following Cav-1 knockdown. © 2013 Wiley Periodicals, Inc.

β3-Adrenoceptor activation upregulates apolipoprotein A-I expression in HepG2 cells, which might further promote cholesterol efflux from macrophage foam cells.

PubMed

Gao, Xia-Qing; Li, Yan-Fang; Jiang, Zhi-Li

2017-01-01

The aim of this study was to explore the effects of β 3 -adrenoceptor (β 3 -AR) activation on HepG2 cells and its influence on cholesterol efflux from macrophage foam cells. HepG2 cells were cultured and treated with the β 3 -AR agonist, BRL37344, and antagonist, SR52390A, and the expression of apolipoprotein (Apo) A-I, ApoA-II, ApoB, and β 3 -AR in the supernatants and cells was determined. The expression of peroxisome proliferator-activated receptor (PPAR) γ and PPARα in the HepG2 cells was also assessed. Next, using the RAW264.7 macrophage foam cell model, we also assessed the influence of the HepG2 cell supernatants on lipid efflux. The cholesterol content of the foam cells was also measured, and the cholesterol efflux from the macrophages was examined by determining 3 H-labeled cholesterol levels. Expression of ATP-binding cassette transporter (ABC) A1 and ABCG1 of the macrophage foam cells was also assessed. β 3 -AR activation increased ApoA-I expression in both the HepG2 cells and the supernatants; PPARγ expression was upregulated, but PPARα expression was not. Treatment with GW9662 abolished the increased expression of ApoA-I induced by the β 3 -AR agonist. The HepG2 cell supernatants decreased the lipid accumulation and increased the cholesterol efflux from the macrophage foam cells. ABCA1 expression, but not ABCG1 expression, increased in the macrophage foam cells treated with BRL37344-treated HepG2 cell supernatants. Activation of β 3 -AR in HepG2 cells upregulates ApoA-I expression, which might further promote cholesterol efflux from macrophage foam cells. PPARγ might be required for the induction of ApoA-I expression.

β1- and β2-adrenergic receptor stimulation differ in their effects on PGC-1α and atrogin-1/MAFbx gene expression in chick skeletal muscle.

PubMed

Shimamoto, Saki; Ijiri, Daichi; Kawaguchi, Mana; Nakashima, Kazuki; Tada, Osamu; Inoue, Hiroki; Ohtsuka, Akira

2017-09-01

Adrenaline changes expression of the genes encoding peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α), which is known as a regulator of muscle size, and atrogin-1/muscle atrophy F-box (MAFbx), which is a muscle-specific ubiquitin ligase. However, the subtype of β-adrenergic receptor (β-AR) involved in regulating these genes in skeletal muscle is not yet well defined. In this study, the effects of intraperitoneal injection of adrenaline and three β 1-3 -AR selective agonists on chick skeletal muscle metabolism were examined, to evaluate the functions of β-AR subtypes. Adrenaline decreased atrogin-1/MAFbx mRNA levels accompanied by an increase in PGC-1α mRNA and protein levels. However, among the three selective agonists, only the β 1 -AR agonist, dobutamine, increased PGC-1α mRNA and protein levels, while the β 2 -AR agonist, clenbuterol, suppressed atrogin-1/MAFbx mRNA levels. In addition, preinjection of the β 1 -AR antagonist, acebutolol, and the β 2 -AR antagonist, butoxamine, inhibited the adrenaline-induced increase in PGC-1α mRNA levels and the decrease in atrogin-1/MAFbx mRNA levels, respectively. Compared with adrenaline administration, the β 3 -AR agonist, BRL37344, decreased PGC-1α mRNA levels and increased atrogin-1/MAFbx mRNA levels. These results suggest that, in chick skeletal muscle, PGC-1α is induced via the β 1 -AR, while atrogin-1/MAFbx is suppressed via the β 2 -AR. Copyright © 2017. Published by Elsevier Inc.

Protein arginine methyltransferase 5 functions as an epigenetic activator of the androgen receptor to promote prostate cancer cell growth.

PubMed

Deng, X; Shao, G; Zhang, H-T; Li, C; Zhang, D; Cheng, L; Elzey, B D; Pili, R; Ratliff, T L; Huang, J; Hu, C-D

2017-03-02

Protein arginine methyltransferase 5 (PRMT5) is an emerging epigenetic enzyme that mainly represses transcription of target genes via symmetric dimethylation of arginine residues on histones H4R3, H3R8 and H2AR3. Accumulating evidence suggests that PRMT5 may function as an oncogene to drive cancer cell growth by epigenetic inactivation of several tumor suppressors. Here, we provide evidence that PRMT5 promotes prostate cancer cell growth by epigenetically activating transcription of the androgen receptor (AR) in prostate cancer cells. Knockdown of PRMT5 or inhibition of PRMT5 by a specific inhibitor reduces the expression of AR and suppresses the growth of multiple AR-positive, but not AR-negative, prostate cancer cells. Significantly, knockdown of PRMT5 in AR-positive LNCaP cells completely suppresses the growth of xenograft tumors in mice. Molecular analysis reveals that PRMT5 binds to the proximal promoter region of the AR gene and contributes mainly to the enriched symmetric dimethylation of H4R3 in the same region. Mechanistically, PRMT5 is recruited to the AR promoter by its interaction with Sp1, the major transcription factor responsible for AR transcription, and forms a complex with Brg1, an ATP-dependent chromatin remodeler, on the proximal promoter region of the AR gene. Furthermore, PRMT5 expression in prostate cancer tissues is significantly higher than that in benign prostatic hyperplasia tissues, and PRMT5 expression correlates positively with AR expression at both the protein and mRNA levels. Taken together, our results identify PRMT5 as a novel epigenetic activator of AR in prostate cancer. Given that inhibiting AR transcriptional activity or androgen synthesis remains the major mechanism of action for most existing anti-androgen agents, our findings also raise an interesting possibility that targeting PRMT5 may represent a novel approach for prostate cancer treatment by eliminating AR expression.

An AKT activity threshold regulates androgen-dependent and androgen-independent PSA expression in prostate cancer cell lines.

PubMed

Paliouras, Miltiadis; Diamandis, Eleftherios P

2008-06-01

The androgen receptor (AR) plays an important role in early prostate cancer by activating transcription of a number of genes participating in cell proliferation and growth and cancer progression. However, as the cancer progresses, prostate cancer cells transform from an androgen-dependent to an androgen-independent state. Androgen-independent prostate cancer can manifest itself in several forms, including a percentage of cancers that show reduced levels of prostate-specific antigen (PSA) and can progress without the need for the ligand or active receptor. Therefore, our goal was to examine the role of intracellular signaling pathways in an androgen-independent prostate cancer in vitro model. Using the cell line PC3(AR)(2), we stimulated cells with 5-alpha-dihydrotestosterone (DHT) and epidermal growth factor (EGF) and then analyzed PSA expression. We observed lower PSA expression when cells were jointly stimulated with DHT and EGF, and this was associated with an increase in AKT activity. We examined the role of AKT in AR activity and PSA expression by creating stable PC3(AR)(2) cell lines transfected with a PI3K-Ras-effector loop mutant. These cell lines showed lower DHT-stimulated PSA expression that correlated to changes in the phosphorylated state of AR. Therefore, we propose an in vitro androgen-independent model in which a PI3K/AKT activity threshold and subsequent AR transactivation regulate PSA expression.

Evaluation of C3a receptor expression on human leucocytes by the use of novel monoclonal antibodies

PubMed Central

ZWIRNER, J; GÖTZE, O; BEGEMANN, G; KAPP, A; KIRCHHOFF, K; WERFEL, T

1999-01-01

Varying results have been published in the past regarding the reactivity of different leucocyte subpopulations, including neutrophils, monocytes and B lymphocytes, to the anaphylatoxin C3a and its degradation product C3a(desArg). To better characterize the cellular distribution of C3a receptor (C3aR) expression, monoclonal antibodies against two different epitopes on the third extracellular domain of the human C3aR were generated. Quantification of C3aR as compared with C5aR densities was performed on peripheral blood leucocytes by quantitative indirect immunofluorescence. Eosinophils and basophils expressed similar numbers of C3aR and C5aR molecules/cell. On eosinophils 10 700±4500 (mean±SD) C3aR and 14 700±4100 C5aR were found, whereas basophils carried 8100±2100 C3aR and 13 500±3800 C5aR. Monocytes expressed approximately six times more C5aR than C3aR molecules on their surface (6000±2500 C3aR versus 34 100±9300 C5aR molecules) whereas on neutrophils, the expression of C5aR was more than 20 times higher than the expression of C3aR (3100±1000 C3aR versus 63 500±12 200 C5aR). No C3aR expression was detectable on peripheral blood-derived B lymphocytes and on tonsillar B cells before and after stimulation with interleukin-2/Staphylococcus aureus Cowan strain I. Our findings correspond well with the paucity of data on C3a-induced functional activities in monocytes and neutrophils and suggest that eosinophilic and basophilic granulocytes represent the primary effector cells in the peripheral blood which can be stimulated by C3a. PMID:10447728

Pig StAR: mRNA expression and alternative splicing in testis and Leydig cells, and association analyses with testicular morphology traits.

PubMed

Zhang, Yanghai; Cui, Yang; Zhang, Xuelian; Wang, Yimin; Gao, Jiayang; Yu, Ting; Lv, Xiaoyan; Pan, Chuanying

2018-05-31

Steroidogenic acute regulatory protein (StAR), primarily expressed in Leydig cells (LCs) in the mammalian testes, is essential for testosterone biosynthesis and male fertility. However, no previous reports have explored the expression profiles, alternative splicing and genetic variations of StAR gene in pig. The aim of current study was to explore the expression profiles in different tissues and different types of testicular cells (LCs; spermatogonial stem cells, SSCs; Sertoli cells, SCs), to identify different splice variants and their expression levels, as well as to detect the indel polymorphism in pig StAR gene. Expression analysis results revealed that StAR was widely expressed in all tested tissues and the expression level in testis was significantly higher than that in other tissues (P < 0.01); among different types of testicular cells, the StAR mRNA expression level was significantly higher in LCs than others (P < 0.05). Furthermore, three splice variants, StAR-a, StAR-b and StAR-c, were first found in pig. Further study showed StAR-a was highly expressed in both testis and LCs when compared with other variants (P < 0.01), suggesting StAR-a was the primary variant at StAR gene post-transcription and may facilitate the combination and transportation of cholesterol with StAR. In addition, a 5-bp duplicated deletion (NC_010457.5:g.5524-5528 delACTTG) was verified in the porcine StAR gene, which was closely related to male testicular morphology traits (P < 0.05), and we speculated that the allele "D" of StAR gene might be a positive allele. Briefly, the current findings suggest that StAR and StAR-a play imperative roles in male fertility and the 5-bp indel can be a potential DNA marker for the marker-assisted selection in boar. Copyright © 2018 Elsevier Inc. All rights reserved.

Growth differentiation factor 9 reverses activin A suppression of steroidogenic acute regulatory protein expression and progesterone production in human granulosa-lutein cells.

PubMed

Shi, Feng-Tao; Cheung, Anthony P; Klausen, Christian; Huang, He-Feng; Leung, Peter C K

2010-10-01

We have reported that growth differentiation factor 9 (GDF9) can enhance activin A (β(A)β(A))-induced inhibin B (αβ(B)) secretion in human granulosa-lutein (hGL) cells, but its effects on steroidogenic acute regulatory protein (StAR), ovarian steroidogenic enzymes, and progesterone production are unknown. We undertook this study to further evaluate GDF9 in this regard. hGL cells from women undergoing in vitro fertilization treatment were cultured with and without small interfering RNA (siRNA) transfection targeted at inhibin α-subunit or GDF9 before treatment with GDF9, activin A, FSH, or combinations. We compared StAR, P450 side-chain cleavage enzyme, and 3β-hydroxysteroid dehydrogenase expression in hGL cells and progesterone levels in culture media after these treatments. mRNA, protein, and hormone levels were assessed with real-time RT-PCR, immunoblotting, and ELISA, respectively. Data were analyzed by ANOVA followed by Tukey's test. Activin A alone reduced basal and FSH-induced progesterone production by decreasing the expression of StAR protein, which regulates the rate-limiting step in steroidogenesis but not P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase. GDF9 attenuated these activin A effects on StAR and progesterone. After transfection of α-subunit siRNA, activin A level increased (P < 0.001), whereas basal and activin A-induced inhibin B levels (with and without GDF9) decreased. Furthermore, the effects of GDF9 in reversing activin A suppression of progesterone production were attenuated (P < 0.001). Transfection of GDF9 siRNA decreased GDF9 as expected and led to lower StAR expression and progesterone secretion than those observed with activin A treatment alone. GDF9 attenuates the suppressive effects of activin A on StAR expression and progesterone production by increasing the expression of inhibin B, which acts as an activin A competitor.

Histone demethylase JMJD1A promotes alternative splicing of AR variant 7 (AR-V7) in prostate cancer cells.

PubMed

Fan, Lingling; Zhang, Fengbo; Xu, Songhui; Cui, Xiaolu; Hussain, Arif; Fazli, Ladan; Gleave, Martin; Dong, Xuesen; Qi, Jianfei

2018-05-15

Formation of the androgen receptor splicing variant 7 (AR-V7) is one of the major mechanisms by which resistance of prostate cancer to androgen deprivation therapy occurs. The histone demethylase JMJD1A (Jumonji domain containing 1A) functions as a key coactivator for AR by epigenetic regulation of H3K9 methylation marks. Here, we describe a role for JMJD1A in AR-V7 expression. While JMJD1A knockdown had no effect on full-length AR (AR-FL), it reduced AR-V7 levels in prostate cancer cells. Reexpression of AR-V7 in the JMJD1A-knockdown cells elevated expression of select AR targets and partially rescued prostate cancer cell growth in vitro and in vivo. The AR-V7 protein level correlated positively with JMJD1A in a subset of human prostate cancer specimens. Mechanistically, we found that JMJD1A promoted alternative splicing of AR-V7 through heterogeneous nuclear ribonucleoprotein F (HNRNPF), a splicing factor known to regulate exon inclusion. Knockdown of JMJD1A or HNRNPF inhibited splicing of AR-V7, but not AR-FL, in a minigene reporter assay. JMJD1A was found to interact with and promote the recruitment of HNRNPF to a cryptic exon 3b on AR pre-mRNA for the generation of AR-V7. Taken together, the role of JMJD1A in AR-FL coactivation and AR-V7 alternative splicing highlights JMJD1A as a potentially promising target for prostate cancer therapy.

Methylation of the PMEPA1 gene, a negative regulator of the androgen receptor in prostate cancer.

PubMed

Sharad, Shashwat; Ravindranath, Lakshmi; Haffner, Michael C; Li, Hua; Yan, Wusheng; Sesterhenn, Isabell A; Chen, Yongmei; Ali, Amina; Srinivasan, Alagarsamy; McLeod, David G; Yegnasubramanian, Srinivasan; Srivastava, Shiv; Dobi, Albert; Petrovics, Gyorgy

2014-06-01

The prostate transmembrane protein androgen induced 1 (PMEPA1) gene is highly expressed in prostate epithelial cells and is a direct transcriptional target for the androgen receptor (AR). AR protein levels are controlled by the AR-PMEPA1 negative feedback loop through NEDD4-E3 ligase. Reduced expression of PMEPA1 observed in prostate tumors, suggests that loss of PMEPA1 may play critical roles in prostate tumorigenesis. This study focuses on epigenetic mechanisms of reduced PMEPA1 expression in the cancer of the prostate (CaP). Benign (n = 77) and matched malignant (n = 77) prostate epithelial cells were laser capture micro-dissected from optimum cutting temperature embedded frozen prostate sections from 42 Caucasian American (CA) and 35 African American (AA) cases. Purified DNA specimens were analyzed for CpG methylation of the PMEPA1 gene. PMEPA1 mRNA expression levels were evaluated by qRT-PCR. Analysis of PMEPA1 methylation and mRNA expression in the same tumor cell populations indicated a significant inverse correlation between mRNA expression and methylation in CaP (P = 0.0115). We noted higher frequency of CpG methylation within the evaluated first intronic region of the PMEPA1 gene in prostate tumors of CA men as compared with AA. In CaP cell lines, PMEPA1 expression was induced and AR protein levels were diminished in response to treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (decitabine). Cell culture-based studies demonstrated that decitabine restores PMEPA1 expression in AR-positive CaP cell lines. This report reveals the potential role of PMEPA1 gene methylation in the regulation of AR stability. Thus, downregulation of PMEPA1 may result in increased AR protein levels and function in CaP cells, contributing to prostate tumorigenesis.

Methylation of the PMEPA1 gene, a negative regulator of the androgen receptor in prostate cancer

PubMed Central

Sharad, Shashwat; Ravindranath, Lakshmi; Haffner, Michael C; Li, Hua; Yan, Wusheng; Sesterhenn, Isabell A; Chen, Yongmei; Ali, Amina; Srinivasan, Alagarsamy; McLeod, David G; Yegnasubramanian, Srinivasan; Srivastava, Shiv; Dobi, Albert; Petrovics, Gyorgy

2014-01-01

The prostate transmembrane protein androgen induced 1 (PMEPA1) gene is highly expressed in prostate epithelial cells and is a direct transcriptional target for the androgen receptor (AR). AR protein levels are controlled by the AR-PMEPA1 negative feedback loop through NEDD4-E3 ligase. Reduced expression of PMEPA1 observed in prostate tumors, suggests that loss of PMEPA1 may play critical roles in prostate tumorigenesis. This study focuses on epigenetic mechanisms of reduced PMEPA1 expression in the cancer of the prostate (CaP). Benign (n = 77) and matched malignant (n = 77) prostate epithelial cells were laser capture micro-dissected from optimum cutting temperature embedded frozen prostate sections from 42 Caucasian American (CA) and 35 African American (AA) cases. Purified DNA specimens were analyzed for CpG methylation of the PMEPA1 gene. PMEPA1 mRNA expression levels were evaluated by qRT-PCR. Analysis of PMEPA1 methylation and mRNA expression in the same tumor cell populations indicated a significant inverse correlation between mRNA expression and methylation in CaP (P = 0.0115). We noted higher frequency of CpG methylation within the evaluated first intronic region of the PMEPA1 gene in prostate tumors of CA men as compared with AA. In CaP cell lines, PMEPA1 expression was induced and AR protein levels were diminished in response to treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (decitabine). Cell culture-based studies demonstrated that decitabine restores PMEPA1 expression in AR-positive CaP cell lines. This report reveals the potential role of PMEPA1 gene methylation in the regulation of AR stability. Thus, downregulation of PMEPA1 may result in increased AR protein levels and function in CaP cells, contributing to prostate tumorigenesis. PMID:24694733

Amphiregulin mediates hCG-induced StAR expression and progesterone production in human granulosa cells.

PubMed

Fang, Lanlan; Yu, Yiping; Zhang, Ruizhe; He, Jingyan; Sun, Ying-Pu

2016-04-26

Progesterone plays critical roles in maintaining a successful pregnancy at the early embryonic stage. Human chorionic gonadotropin (hCG) rapidly induces amphiregulin (AREG) expression. However, it remains unknown whether AREG mediates hCG-induced progesterone production. Thus, the objective of this study was to investigate the role of AREG in hCG-induced progesterone production and the underlying molecular mechanism in human granulosa cells; primary cells were used as the experimental model. We demonstrated that the inhibition of EGFR and the knockdown of AREG abolished hCG-induced steroidogenic acute regulatory protein (StAR) expression and progesterone production. Importantly, follicular fluid AREG levels were positively correlated with progesterone levels in the follicular fluid and serum. Treatment with AREG increased StAR expression and progesterone production, and these stimulatory effects were abolished by EGFR inhibition. Moreover, activation of ERK1/2, but not PI3K/Akt, signaling was required for the AREG-induced up-regulation of StAR expression and progesterone production. Our results demonstrate that AREG mediates hCG-induced StAR expression and progesterone production in human granulosa cells, providing novel evidence for the role of AREG in the regulation of steroidogenesis.

Prognostic relevance of proliferation markers (Ki-67, PHH3) within the cross-relation of ERG translocation and androgen receptor expression in prostate cancer.

PubMed

Goltz, Diane; Montani, Matteo; Braun, Martin; Perner, Sven; Wernert, Nicolas; Jung, Klaus; Dietel, Manfred; Stephan, Carsten; Kristiansen, Glen

2015-12-01

We evaluated the prognostic value of the mitosis-associated marker phosphorylated histone H3 (PHH3) and Ki-67 in prostate cancer with respect to ERG status and androgen receptor (AR) expression.PHH3 and Ki-67 expression was immunohistochemically detected and digitally quantitated in a radical prostatectomy cohort (n = 640). The results were correlated to clinicopathological parameters including biochemical recurrence times. Prognostic values of PHH3 and Ki-67 were analysed by Cox regression and Kaplan-Meier statistics.In prostate cancer, mean Ki-67 and PHH3 rates were 3.40% (95%CI 3.16-3.63%) and 0.0152% (95%CI 0.0112-0.0191%), respectively.Ki-67 showed a significant correlation with Gleason scores, pT status, margin status, and AR expression, while PHH3 showed a significant correlation with Gleason scores and pT status. Univariate analyses for biochemical recurrence times demonstrated a significant prognostic value for median Ki-67 rate and for the PHH3 rate of the 90th percentile. Of importance, in patient subgroups stratified according to AR expression and ERG translocation, the prognostic power of proliferation markers PHH3 and Ki-67 was markedly enhanced in ERG translocation negative and high-level AR expressing ERG translocation positive prostate cancers.As expected, the proliferation markers PHH3 and Ki-67 predict adverse outcome of prostate cancer and have a particularly pronounced prognostic value in specific molecular subsets of prostate cancer (ERG- or AR+).

ING3 promotes prostate cancer growth by activating the androgen receptor.

PubMed

Nabbi, Arash; McClurg, Urszula L; Thalappilly, Subhash; Almami, Amal; Mobahat, Mahsa; Bismar, Tarek A; Binda, Olivier; Riabowol, Karl T

2017-05-16

The androgen receptor (AR) is a major driver of prostate cancer, and increased AR levels and co-activators of the receptor promote the development of prostate cancer. INhibitor of Growth (ING) proteins target lysine acetyltransferase or lysine deacetylase complexes to the histone H3K4Me3 mark of active transcription, to affect chromatin structure and gene expression. ING3 is a stoichiometric member of the TIP60 lysine acetyltransferase complex implicated in prostate cancer development. Biopsies of 265 patients with prostate cancer were stained for ING3, pan-cytokeratin, and DNA. LNCaP and C4-2 androgen-responsive cells were used for in vitro assays including immunoprecipitation, western blotting, Luciferase reporter assay and quantitative polymerase chain reaction. Cell viability and migration assays were performed in prostate cancer cell lines using scrambled siRNA or siRNA targeting ING3. We find that ING3 levels and AR activity positively correlate in prostate cancer. ING3 potentiates androgen effects, increasing expression of androgen-regulated genes and androgen response element-driven reporters to promote growth and anchorage-independent growth. Conversely, ING3 knockdown inhibits prostate cancer cell growth and invasion. ING3 activates the AR by serving as a scaffold to increase interaction between TIP60 and the AR in the cytoplasm, enhancing receptor acetylation and translocation to the nucleus. Activation is independent of ING3's ability to target the TIP60 complex to H3K4Me3, identifying a previously unknown chromatin-independent cytoplasmic activity for ING3. In agreement with in vitro observations, analysis of The Cancer Genome Atlas (TCGA) data (n = 498) and a prostate cancer tissue microarray (n = 256) show that ING3 levels are higher in aggressive prostate cancers, with high levels of ING3 predicting shorter patient survival in a low AR subgroup. Including ING3 levels with currently used indicators such as the Gleason score provides more accurate prognosis in primary prostate cancer. In contrast to the majority of previous reports suggesting tumor suppressive functions in other cancers, our observations identify a clear oncogenic role for ING3, which acts as a co-activator of AR in prostate cancer. Data from TCGA and our previous and current tissue microarrays suggest that ING3 levels correlate with AR levels and that in patients with low levels of the receptor, ING3 level could serve as a useful prognostic biomarker.

[Influence of carbon monoxide on the expression of inducible nitric oxide synthase mRNA in guinea pigs with allergic rhinitis.].

PubMed

Yu, Shao-Qing; Zhang, Ru-Xin; Chen, Ying-Jian; Yan, Zhi-Qiang; Wu, Ge-Ping; Wang, Yan-Sheng; Chen, Jian-Qiu; Zhu, Chun-Sheng; Li, Gen-Hong

2009-12-01

To study the impact of carbon monoxide (CO) on expression levels of inducible nitric oxide synthase (iNOS) mRNA in guinea pigs with allergic rhinitis (AR). Twenty four guinea pigs were divided randomly into four study groups with 6 guinea pigs in each. The guinea pigs in the first group were treated with saline only (Group 1, the healthy controls). The remaing guinea pigs were sensitized by ovalbumin and thus establishing the AR models. After sensitization, the animals in the second group remained untreated (Group 2, AR control group). The third group was treated with Hemin as the induction group, and the fourth group was treated with Zinc protoporphyrin (ZnPP) as the suppression group. The plasma concentration of carboxyhemoglobin (COHb) was measured, which represents the concentration of CO. The expression levels of Heme oxygenase-1 (HO-1) and NOS mRNAs in nasal mucosa were determined by fluorescent quantitative RT-PCR. AR models were established successfully in all study guinea pigs. The concentrations of COHb (x(-) +/- s) in plasma of the second group (2.27% +/- 1.13%) were significantly (q = 4.10, P < 0.01) higher than those of healthy controls (1.08% +/- 0.24%). The plasma concentration of COHb in the third group (3.17% +/- 0.68%) were also significantly higher (q = 3.12, P < 0.05) than those in the second group. The expression levels of HO-1 and iNOS in nasal mucosa of the second group [(7.80 +/- 1.60) x 10(-3) and (5.81 +/- 0.05) x 10(-3), respectively] were also significantly (q equals 5.52 and 7.21, respectively, P < 0.01) higher than those of controls [(1.96 +/- 0.71) x 10(-3) and (0.97 +/- 0.05) x 10(-3), respectively]. The expression levels of HO-1 and iNOS in the nasal mucosa of the third group [(11.89 +/- 4.78) x 10(-3) and (7.42 +/- 0.70) x 10(-3), respectively] were significantly (q equals 3.86 and 2.22, P < 0.05) higher than those of the second group. The expression levels of HO-1 and iNOS in nasal mucosa of the fourth group [(3.82 +/- 0.98) x 10(-3) and (2.34 +/- 0.04) x 10(-3), respectively] were significantly (q equals 3.76 and 5.18, P < 0.05) lower than those in the second group. Endogenous carbon monoxide influenced the expression levels of iNOS in nasal mocusa in guinea pigs with AR.

Nicotine Induced CpG methylation of Pax6 binding motif in StAR promoter reduces the gene expression and cortisol production

PubMed Central

Wang, Tingting; Chen, Man; Liu, Lian; Cheng, Huaiyan; Yan, You-E; Feng, Ying-Hong; Wang, Hui

2011-01-01

Steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in the synthesis of steroid hormones, essential to fetal development. We have reported that the StAR expression in fetal adrenal is inhibited in a rat model of nicotine-induced intrauterine growth retardation (IUGR). Here using primary human fetal adrenal cortex (pHFAC) cells and a human fetal adrenal cell line NCI-H295A, we show that nicotine inhibits StAR expression and cortisol production in a dose- and time-dependent manner, and prolongs the inhibitory effect on cells proliferating over 5 passages after termination of nicotine treatment. Methylation detection within the StAR promoter region uncovers a single site CpG methylation at nt −377 that is sensitive to nicotine treatment. Nicotine-induced alterations in frequency of this point methylation correlates well with the levels of StAR expression, suggesting an important role of the single site in regulating StAR expression. Further studies using bioinformatics analysis and siRNA approach reveal that the single CpG site is part of the Pax6 binding motif (CGCCTGA) in the StAR promoter. The luciferase activity assays validate that Pax6 increases StAR gene expression by binding to the glucagon G3-like motif (CGCCTGA) and methylation of this site blocks Pax6 binding and thus suppresses StAR expression. These data identify a nicotine-sensitive CpG site at the Pax6 binding motif in the StAR promoter that may play a central role in regulating StAR expression. The results suggest an epigenetic mechanism that may explain how nicotine contributes to onset of adult diseases or disorders such as metabolic syndrome via fetal programming. PMID:21971485

β3-adrenergic receptor activation induces TGFβ1 expression in cardiomyocytes via the PKG/JNK/c-Jun pathway.

PubMed

Xu, Zhongcheng; Wu, Jimin; Xin, Junzhou; Feng, Yenan; Hu, Guomin; Shen, Jing; Li, Mingzhe; Zhang, Youyi; Xiao, Han; Wang, Li

2018-06-05

In heart failure, the expression of cardiac β 3 -adrenergic receptors (β 3 -ARs) increases. However, the precise role of β 3 -AR signaling within cardiomyocytes remains unclear. Transforming growth factor β1 (TGFβ1) is a crucial cytokine mediating the cardiac remodeling that plays a causal role in the progression of heart failure. Here, we set out to determine the effect of β 3 -AR activation on TGFβ1 expression in rat cardiomyocytes and examine the underlying mechanism. The selective β 3 -AR agonist BRL37344 induced an increase in TGFβ1 expression and the phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun in β 3 -AR-overexpressing cardiomyocytes. Those effects of BRL37344 were suppressed by a β 3 -AR antagonist. Moreover, the inhibition of JNK and c-Jun activity by a JNK inhibitor and c-Jun siRNA blocked the increase in TGFβ1 expression upon β 3 -AR activation. A protein kinase G (PKG) inhibitor also attenuated β 3 -AR-agonist-induced TGFβ1 expression and the phosphorylation of JNK and c-Jun. In conclusion, the β 3 -AR activation in cardiomyocytes increases the expression of TGFβ1 via the PKG/JNK/c-Jun pathway. These results help us further understand the role of β 3 -AR signaling in heart failure. Copyright © 2018 Elsevier Inc. All rights reserved.

Analytical Validation and Clinical Qualification of a New Immunohistochemical Assay for Androgen Receptor Splice Variant-7 Protein Expression in Metastatic Castration-resistant Prostate Cancer.

PubMed

Welti, Jonathan; Rodrigues, Daniel Nava; Sharp, Adam; Sun, Shihua; Lorente, David; Riisnaes, Ruth; Figueiredo, Ines; Zafeiriou, Zafeiris; Rescigno, Pasquale; de Bono, Johann S; Plymate, Stephen R

2016-10-01

The androgen receptor splice variant-7 (AR-V7) has been implicated in the development of castration-resistant prostate cancer (CRPC) and resistance to abiraterone and enzalutamide. To develop a validated assay for detection of AR-V7 protein in tumour tissue and determine its expression and clinical significance as patients progress from hormone-sensitive prostate cancer (HSPC) to CRPC. Following monoclonal antibody generation and validation, we retrospectively identified patients who had HSPC and CRPC tissue available for AR-V7 immunohistochemical (IHC) analysis. Nuclear AR-V7 expression was determined using IHC H score (HS) data. The change in nuclear AR-V7 expression from HSPC to CRPC and the association between nuclear AR-V7 expression and overall survival (OS) was determined. Nuclear AR-V7 expression was significantly lower in HSPC (median HS 50, interquartile range [IQR] 17.5-90) compared to CRPC (HS 135, IQR 80-157.5; p<0.0001), and in biopsy tissue taken before (HS 80, IQR 30-136.3) compared to after (HS 140, IQR 105-167.5; p=0.007) abiraterone or enzalutamide treatment. Lower nuclear AR-V7 expression at CRPC biopsy was associated with longer OS (hazard ratio 1.012, 95% confidence interval 1.004-1.020; p=0.003). While this monoclonal antibody primarily binds to AR-V7 in PC biopsy tissue, it may also bind to other proteins. We provide the first evidence that nuclear AR-V7 expression increases with emerging CRPC and is prognostic for OS, unlike antibody staining for the AR N-terminal domain. These data indicate that AR-V7 is important in CRPC disease biology; agents targeting AR splice variants are needed to test this hypothesis and further improve patient outcome from CRPC. In this study we found that levels of the protein AR-V7 were higher in patients with advanced prostate cancer. A higher level of AR-V7 identifies a group of patients who respond less well to certain prostate cancer treatments and live for a shorter period of time. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Expression of androgen receptor and estrogen receptor-alpha in the developing pituitary gland of male sheep lamb.

PubMed

Huang, Li-Bo; Yuan, Xue-Jun

2011-09-01

To explore the expression of androgen receptor (AR) and estrogen receptor alpha (ERα) in the developing pituitary of male lamb, we detected AR and ERα expression in the anterior pituitary of lambs aged 2-7 months old by immunohistochemistry. The results showed that both AR immunoreactivity (AR-ir) and ERα immunoreactivity (ERα-ir) were localized in the nuclei of anterior pituitary cell. The percentage of the anterior pituitary cells expressing ERα fluctuated from 8.79±0.02% to 11.80±0.04% during the examined stages, but fell significantly to the lowest level at 6 months. While the proportion of AR-ir showed significant changes, it was in 11.52±1.26% at 2 months, it firstly increased to 19.86±1.03% at 3 months, and then significantly decreased to 8.18±1.17% at 6 months (P<0.05). The expression of both AR-ir and ERα-ir were the lowest level at 6 months old. By staining for PCNA, we observed that the changes in expression of AR and ERα at different lamb ages did not result from cell proliferation of anterior pituitary cells. These results indicate that both AR and ERα are important in regulation of secretary function of anterior pituitary in sheep lamb, although the related mechanism needs to be elucidated further. Copyright © 2011 Elsevier B.V. All rights reserved.

PCA3 noncoding RNA is involved in the control of prostate-cancer cell survival and modulates androgen receptor signaling

PubMed Central

2012-01-01

Background PCA3 is a non-coding RNA (ncRNA) that is highly expressed in prostate cancer (PCa) cells, but its functional role is unknown. To investigate its putative function in PCa biology, we used gene expression knockdown by small interference RNA, and also analyzed its involvement in androgen receptor (AR) signaling. Methods LNCaP and PC3 cells were used as in vitro models for these functional assays, and three different siRNA sequences were specifically designed to target PCA3 exon 4. Transfected cells were analyzed by real-time qRT-PCR and cell growth, viability, and apoptosis assays. Associations between PCA3 and the androgen-receptor (AR) signaling pathway were investigated by treating LNCaP cells with 100 nM dihydrotestosterone (DHT) and with its antagonist (flutamide), and analyzing the expression of some AR-modulated genes (TMPRSS2, NDRG1, GREB1, PSA, AR, FGF8, CdK1, CdK2 and PMEPA1). PCA3 expression levels were investigated in different cell compartments by using differential centrifugation and qRT-PCR. Results LNCaP siPCA3-transfected cells significantly inhibited cell growth and viability, and increased the proportion of cells in the sub G0/G1 phase of the cell cycle and the percentage of pyknotic nuclei, compared to those transfected with scramble siRNA (siSCr)-transfected cells. DHT-treated LNCaP cells induced a significant upregulation of PCA3 expression, which was reversed by flutamide. In siPCA3/LNCaP-transfected cells, the expression of AR target genes was downregulated compared to siSCr-transfected cells. The siPCA3 transfection also counteracted DHT stimulatory effects on the AR signaling cascade, significantly downregulating expression of the AR target gene. Analysis of PCA3 expression in different cell compartments provided evidence that the main functional roles of PCA3 occur in the nuclei and microsomal cell fractions. Conclusions Our findings suggest that the ncRNA PCA3 is involved in the control of PCa cell survival, in part through modulating AR signaling, which may raise new possibilities of using PCA3 knockdown as an additional therapeutic strategy for PCa control. PMID:23130941

Androgen receptor signals regulate UDP-glucuronosyltransferases in the urinary bladder: a potential mechanism of androgen-induced bladder carcinogenesis.

PubMed

Izumi, Koji; Zheng, Yichun; Hsu, Jong-Wei; Chang, Chawnshang; Miyamoto, Hiroshi

2013-02-01

UDP-glucuronosyltransferases (UGTs), major phase II drug metabolism enzymes, play an important role in urinary bladder cancer initiation by detoxifying carcinogens. We aimed to determine if androgens regulate UGT expression via the androgen receptor (AR) pathway in the bladder. Real-time reverse transcription-polymerase chain reaction and Western blot analyses were used to assess UGT1A levels in the normal urothelium SVHUC cell line stably expressed with AR and in bladder tissues from AR knockout (ARKO) and castrated male mice. Immunohistochemistry was also performed in radical cystectomy specimens. Dihydrotestosterone (DHT) treatment in SVHUC-AR reduced mRNA expression of all the UGT1A subtypes (19-75% decrease), and hydroxyflutamide antagonized the DHT effects. In contrast, DHT showed only marginal effects on UGT1A expression in SVHUC-Vector. Of note were higher expression levels of UGT1As in SVHUC-Vector than in SVHUC-AR. In ARKO mice, all the Ugt1a subtypes were up-regulated, compared to wild-type littermates. In wild-type male mice, castration increased the expression of Ugt1a8, Ugt1a9, and Ugt1a10. Additionally, wild-type female mice had higher levels of Ugt1a than wild-type males. Immunohistochemical studies showed strong (3+) UGT1A staining in 11/24 (46%) cancer tissues, which was significantly lower than in corresponding benign tissues [17/18 (94%) cases (P = 0.0009)]. These results suggest that androgen-mediated AR signals promote bladder carcinogenesis by down-regulating the expression of UGTs in the bladder. Copyright © 2011 Wiley Periodicals, Inc.

Seasonal changes and sexual dimorphism in gene expression of StAR protein, steroidogenic enzymes and sex hormone receptors in the frog brain.

PubMed

Santillo, Alessandra; Falvo, Sara; Di Fiore, Maria Maddalena; Chieffi Baccari, Gabriella

2017-05-15

The brain of amphibians contains all the key enzymes of steroidogenesis and has a high steroidogenic activity. In seasonally-breeding amphibian species brain steroid levels fluctuate synchronously with the reproductive cycle. Here we report a study of gene expression of StAR protein, key steroidogenic enzymes and sex hormone receptors in the telencephalon (T) and diencephalon-mesencephalon (D-M) of male and female reproductive and post-reproductive Pelophylax esculentus, a seasonally breeding anuran amphibian. Significant differences in gene expression were observed between (a) the reproductive and post-reproductive phase, (b) the two brain regions and (c) male and female frogs. During the reproductive phase, star gene expression increased in the male (both T and D-M) but not in the female brain. Seasonal fluctuations in expression levels of hsd3b1, hsd17b1, srd5a1 and cyp19a1 genes for neurosteroidogenic enzymes occurred in D-M region of both sexes, with the higher levels in reproductive period. Moreover, the D-M region generally showed higher levels of gene expression than the T region in both sexes. Gene expression was higher in females than males for most genes, suggesting higher neurosteroid production in female brain. Seasonal and sex-linked changes were also observed in gene expression for androgen (ar) and estrogen (esr1, esr2) receptors, with the males showing the highest ar levels in reproductive phase and the highest esr1 and esr2 levels in post-reproductive phase; in contrast, females showed the maximum expression for all three genes in reproductive phase. The results are the first evidence for seasonal changes and sexual dimorphism of gene expression of the neurosteroidogenic pathway in amphibians. Copyright © 2016 Elsevier Inc. All rights reserved.

β3 adrenergic receptor in the kidney may be a new player in sympathetic regulation of renal function.

PubMed

Procino, Giuseppe; Carmosino, Monica; Milano, Serena; Dal Monte, Massimo; Schena, Giorgia; Mastrodonato, Maria; Gerbino, Andrea; Bagnoli, Paola; Svelto, Maria

2016-09-01

To date, the study of the sympathetic regulation of renal function has been restricted to the important contribution of β1- and β2-adrenergic receptors (ARs). Here we investigate the expression and the possible physiologic role of β3-adrenergic receptor (β3-AR) in mouse kidney. The β3-AR is expressed in most of the nephron segments that also express the type 2 vasopressin receptor (AVPR2), including the thick ascending limb and the cortical and outer medullary collecting duct. Ex vivo experiments in mouse kidney tubules showed that β3-AR stimulation with the selective agonist BRL37344 increased intracellular cAMP levels and promoted 2 key processes in the urine concentrating mechanism. These are accumulation of the water channel aquaporin 2 at the apical plasma membrane in the collecting duct and activation of the Na-K-2Cl symporter in the thick ascending limb. Both effects were prevented by the β3-AR antagonist L748,337 or by the protein kinase A inhibitor H89. Interestingly, genetic inactivation of β3-AR in mice was associated with significantly increased urine excretion of water, sodium, potassium, and chloride. Stimulation of β3-AR significantly reduced urine excretion of water and the same electrolytes. Moreover, BRL37344 promoted a potent antidiuretic effect in AVPR2-null mice. Thus, our findings are of potential physiologic importance as they uncover the antidiuretic effect of β3-AR stimulation in the kidney. Hence, β3-AR agonism might be useful to bypass AVPR2-inactivating mutations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The β3 Adrenergic Receptor Agonist BRL37344 Exacerbates Atrial Structural Remodeling Through iNOS Uncoupling in Canine Models of Atrial Fibrillation.

PubMed

Wang, Xiaobing; Wang, Ruifeng; Liu, Guangzhong; Dong, Jingmei; Zhao, Guanqi; Tian, Jingpu; Sun, Jiayu; Jia, Xiuyue; Wei, Lin; Wang, Yuping; Li, Weimin

2016-01-01

The role of the β3-adrenergic receptor (β3-AR) agonist BRL37344 in atrial fibrillation (AF) structural remodeling and the underlying mechanisms as a therapeutic target were investigated. Four groups of dogs were evaluated: sham, pacing, β3-AR agonist BRL37344 (β3-AGO), and β3-AR antagonist L748337 (β3-ANT) groups. Dogs in the pacing, β3-AGO and β3-ANT groups were subjected to rapid atrial pacing for four weeks. Atrial structure and function, AF inducibility and duration, atrial myocyte apoptosis and interstitial fibrosis were assessed. Atrial superoxide anions were evaluated by fluorescence microscopy and colorimetric assays. Cardiac nitrate+nitrite levels were used to assess nitric oxide (NO) production. Protein and mRNA expression of β3-AR, neuronal NO synthase (nNOS), inducible NO synthase (iNOS), endothelial NO synthase (eNOS) and guanosine triphosphate cyclohydrolase-1 (GCH-1) as well as tetrahydrobiopterin (BH4) levels were measured. β3-AR was up-regulated in AF. Stimulation of β3-AR significantly increased atrial myocyte apoptosis, fibrosis and atrial dilatation, resulting in increased AF induction and prolonged duration. These effects were attenuated by β3-ANT. Moreover, β3-AGO reduced BH4 and NO production and increased superoxide production, which was inhibited by the specific iNOS inhibitor, 1400w β3-AGO also increased iNOS but decreased eNOS and had no effect on nNOS expression in AF. β3-AR stimulation resulted in atrial structural remodeling by increasing iNOS uncoupling and related oxidative stress. β3-AR up-regulation and iNOS uncoupling might be underlying AF therapeutic targets. © 2016 The Author(s) Published by S. Karger AG, Basel.

[Effects of 18β-glycyrrhetinic Acid on the Expression of CCL11, AQP1 and EOS in Nasal Mucosa of Allergic Rhinitis Rats].

PubMed

Li, Juan-li; Xi, Ke-hu; Hou, Yun; Jiang, Ying; Gui, Yan; Wang, You-hu; Zhang, Fu-hong; Zhang, Xiao-bing

2015-05-01

To investigate the effects of 18β-glycyrrhetinic acid (GA) on the expression of eotaxin 1 (CCL11), aquaporin protein 1 (AQP1) and eosinophil (EOS) in nasal mucosa of allergic rhinitis (AR) rats. Seventy six Wistar rats were randomly divided into 4 groups, normal control (NC) group, AR model (AR) group, loratadine (LOA) group and 18β-GA group. All the mice in AR, LOA and 18β-GA groups were sensitized intraperitoneally with OVA and AL(OH), from day 1-14, then induced by intranasal administration with OVA from day 14-21, while the mice in NC group were sensitized with saline. The mice in both LOA and 18β-GA group were given LOA and 18β-GA once a day respectively from the 21 d, while the mice in AR and NC groups were administrated with saline. At the end of 1 week, 2 weeks and 3 weeks, the behavioral changes of mice were observed and recorded, the level of CCL11 mRNA was measured by RT-QPCR, and AQP1 expression was investiaged by SP staing. EOS in nasal mucosa was studied with the methods of HE staining. Compared with NC group, AR group showed typical AR symptoms. With the treatments, AR symptom scores and the expression levels of CCL11, AQP1 and EOS in nasal mucosa were improved significantly (P<0. 05). When compared with AR group, the above statistics in LOA group were down-regulated evidently at different points in time (P<. 05). At the end of 1 week, the above detection results in 18β-GA group were lower than those in AR group (P<0. 05). At the end of 2 weeks, those parameters approached to the levels of LOA and NC group significantly. 18β-GA administration could down-regulate the expression levels of CCL11, AQP1 and EOS in nasal mucosa of allergic rhinitis rats and cast effects on inhibiting the progress of AR.

AR-V7 in Peripheral Whole Blood of Patients with Castration-resistant Prostate Cancer: Association with Treatment-specific Outcome Under Abiraterone and Enzalutamide.

PubMed

Seitz, Anna Katharina; Thoene, Silvia; Bietenbeck, Andreas; Nawroth, Roman; Tauber, Robert; Thalgott, Mark; Schmid, Sebastian; Secci, Ramona; Retz, Margitta; Gschwend, Jürgen E; Ruland, Jürgen; Winter, Christof; Heck, Matthias M

2017-11-01

It has been demonstrated that androgen receptor splice variant 7 (AR-V7) expression in circulating tumor cells (CTCs) predicts poor treatment response in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone or enzalutamide. To develop a practical and robust liquid profiling approach for direct quantification of AR-V7 in peripheral whole blood without the need for CTC capture and to determine its potential for predicting treatment response in mCRPC patients. Whole blood samples from a prospective biorepository of 85 mCRPC patients before treatment initiation with abiraterone (n=56) or enzalutamide (n=29) were analyzed via droplet digital polymerase chain reaction. The association of AR-V7 status with prostate-specific antigen (PSA) response defined by PSA decline ≥50% and with PSA-progression-free survival (PSA-PFS), clinical PFS, and overall survival (OS) was assessed. High AR-V7 expression levels in whole blood were detectable in 18% (15/85) of patients. No patient with high AR-V7 expression achieved a PSA response, and AR-V7 status was an independent predictor of PSA response in multivariable logistic regression analysis (p=0.03). High AR-V7 expression was associated with shorter PSA-PFS (median 2.4 vs 3.7 mo; p<0.001), shorter clinical PFS (median 2.7 vs 5.5 mo; p<0.001), and shorter OS (median 4.0 vs. 13.9 mo; p<0.001). On multivariable Cox regression analysis, high AR-V7 expression remained an independent predictor of shorter PSA-PFS (hazard ratio [HR] 7.0, 95% confidence interval [CI] 2.3-20.7; p<0.001), shorter clinical PFS (HR 2.3, 95% CI 1.1-4.9; p=0.02), and shorter OS (HR 3.0, 95% CI 1.4-6.3; p=0.005). Testing of AR-V7 mRNA levels in whole blood is a simple and promising approach to predict poor treatment outcome in mCRPC patients receiving abiraterone or enzalutamide. We established a method for determining AR-V7 status in whole blood. This test predicted treatment resistance in patients with metastatic castration-resistant prostate cancer undergoing treatment with abiraterone or enzalutamide. Prospective validation is needed before application to clinical practice. Copyright © 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.

3-bromopyruvate and buthionine sulfoximine effectively kill anoikis-resistant hepatocellular carcinoma cells

PubMed Central

Lee, Minjong; Jo, Ara; Lee, Seulki; Kim, Jong Bin; Chang, Young; Nam, Joon Yeul; Cho, Hyeki; Cho, Young Youn; Cho, Eun Ju; Lee, Jeong-Hoon; Yu, Su Jong; Yoon, Jung-Hwan

2017-01-01

Background & aims Acquisition of anoikis resistance is a prerequisite for metastasis in hepatocellular carcinoma (HCC). However, little is known about how energy metabolism and antioxidant systems are altered in anoikis-resistant (AR) HCC cells. We evaluated anti-tumor effects of a combination treatment of 3-bromopyruvate (3-BP) and buthionine sulfoximine (BSO) in AR HCC cells. Methods We compared glycolysis, reactive oxygen species (ROS) production, and chemoresistance among Huh-BAT, HepG2 HCC cells, and the corresponding AR cells. Expression of hexokinase II, gamma-glutamylcysteine synthetase (rGCS), and epithelial–mesenchymal transition (EMT) markers in AR cells was assessed. Anti-tumor effects of a combination treatment of 3-BP and BSO were evaluated in AR cells and an HCC xenograft mouse model. Results AR HCC cells showed significantly higher chemoresistance, glycolysis and lower ROS production than attached cells. Expression of hexokinase II, rGCS, and EMT markers was higher in AR HCC cells than attached cells. A combination treatment of 3-BP/BSO effectively suppressed proliferation of AR HCC cells through apoptosis by blocking glycolysis and enhancing ROS levels. In xenograft mouse models, tumor growth derived from AR HCC cells was significantly suppressed in the group treated with 3-BP/BSO compared to the group treated with 3-BP or sorafenib. Conclusions These results demonstrated that a combination treatment of 3-BP/BSO had a synergistic anti-tumor effect in an AR HCC model. This strategy may be an effective adjuvant therapy for patients with sorafenib-resistant HCC. PMID:28362858

3-bromopyruvate and buthionine sulfoximine effectively kill anoikis-resistant hepatocellular carcinoma cells.

PubMed

Lee, Minjong; Jo, Ara; Lee, Seulki; Kim, Jong Bin; Chang, Young; Nam, Joon Yeul; Cho, Hyeki; Cho, Young Youn; Cho, Eun Ju; Lee, Jeong-Hoon; Yu, Su Jong; Yoon, Jung-Hwan; Kim, Yoon Jun

2017-01-01

Acquisition of anoikis resistance is a prerequisite for metastasis in hepatocellular carcinoma (HCC). However, little is known about how energy metabolism and antioxidant systems are altered in anoikis-resistant (AR) HCC cells. We evaluated anti-tumor effects of a combination treatment of 3-bromopyruvate (3-BP) and buthionine sulfoximine (BSO) in AR HCC cells. We compared glycolysis, reactive oxygen species (ROS) production, and chemoresistance among Huh-BAT, HepG2 HCC cells, and the corresponding AR cells. Expression of hexokinase II, gamma-glutamylcysteine synthetase (rGCS), and epithelial-mesenchymal transition (EMT) markers in AR cells was assessed. Anti-tumor effects of a combination treatment of 3-BP and BSO were evaluated in AR cells and an HCC xenograft mouse model. AR HCC cells showed significantly higher chemoresistance, glycolysis and lower ROS production than attached cells. Expression of hexokinase II, rGCS, and EMT markers was higher in AR HCC cells than attached cells. A combination treatment of 3-BP/BSO effectively suppressed proliferation of AR HCC cells through apoptosis by blocking glycolysis and enhancing ROS levels. In xenograft mouse models, tumor growth derived from AR HCC cells was significantly suppressed in the group treated with 3-BP/BSO compared to the group treated with 3-BP or sorafenib. These results demonstrated that a combination treatment of 3-BP/BSO had a synergistic anti-tumor effect in an AR HCC model. This strategy may be an effective adjuvant therapy for patients with sorafenib-resistant HCC.

Origin, timing, and gene expression profile of adventitious rooting in Arabidopsis hypocotyls and stems.

PubMed

Welander, Margareta; Geier, Thomas; Smolka, Anders; Ahlman, Annelie; Fan, Jing; Zhu, Li-Hua

2014-02-01

Adventitious root (AR) formation is indispensable for vegetative propagation, but difficult to achieve in many crops. Understanding its molecular mechanisms is thus important for such species. Here we aimed at developing a rooting protocol for direct AR formation in stems, locating cellular AR origins in stems and exploring molecular differences underlying adventitious rooting in hypocotyls and stems. In-vitro-grown hypocotyls or stems of wild-type and transgenic ecotype Columbia (Col-0) of Arabidopsis thaliana were rooted on rooting media. Anatomy of AR formation, qRT-PCR of some rooting-related genes and in situ GUS expression were carried out during rooting from hypocotyls and stems. We developed a rooting protocol for AR formation in stems and traced back root origins in stems by anatomical and in situ expression studies. Unlike rooting in hypocotyls, rooting in stems was slower, and AR origins were mainly from lateral parenchyma of vascular bundles and neighboring starch sheath cells as well as, to a lesser extent, from phloem cap and xylem parenchyma. Transcript levels of GH3-3, LBD16, LBD29, and LRP1 in hypocotyls and stems were similar, but transcript accumulation was delayed in stems. In situ expression signals of DR5::GUS, LBD16::GUS, LBD29::GUS, and rolB::GUS reporters in stems mainly occurred at the root initiation sites, suggesting their involvement in AR formation. We have developed an efficient rooting protocol using half-strength Lepoivre medium for studying AR formation in stems, traced back the cellular AR origins in stems, and correlated expression of rooting-related genes with root initiation sites.

Attenuation of alpha2A-adrenergic receptor expression in neonatal rat brain by RNA interference or antisense oligonucleotide reduced anxiety in adulthood.

PubMed

Shishkina, G T; Kalinina, T S; Dygalo, N N

2004-01-01

Brain alpha2-adrenergic receptors (alpha2-ARs) have been implicated in the regulation of anxiety, which is associated with stress. Environmental treatments during neonatal development could modulate the level of brain alpha2-AR expression and alter anxiety in adults, suggesting possible involvement of these receptors in early-life programming of anxiety state. The present study was undertaken to determine whether the reduction of the expression of A subtype of these receptors most abundant in the neonatal brain affects anxiety-related behavior in adulthood. We attenuated the expression of alpha2A-ARs during neonatal life by two different sequence specific approaches, antisense technology and RNA interference. Treatment of rats with the antisense oligodeoxynucleotide or short interfering RNA (siRNA) against alpha2A-ARs on the days 2-4 of their life, produced a marked acute decrease in the levels of both alpha2A-AR mRNA and [3H]RX821002 binding sites in the brainstem into which drugs were injected. The decrease of alpha2A-AR expression in the neonatal brainstem influenced the development of this receptor system in the brain regions as evidenced by the increased number of [3H]RX821002 binding sites in the hypothalamus of adult animals with both neonatal alpha2A-AR knockdown treatments; also in the frontal cortex of antisense-treated, and in the hippocampus of siRNA-treated adult rats. These adult animals also demonstrated a decreased anxiety in the elevated plus-maze as evidenced by an increased number of the open arm entries, greater proportion of time spent in the open arms, and more than a two-fold increase in the number of exploratory head dips. The results provide the first evidence that the reduction in the brain expression of a gene encoding for alpha2A-AR during neonatal life led to the long-term neurochemical and behavioral alterations. The data suggests that alterations in the expression of the receptor-specific gene during critical periods of brain development may be involved in early-life programming of anxiety-related behavior.

Characterization of children with FLT3-ITD acute myeloid leukemia: a report from the AIEOP AML-2002 study group.

PubMed

Manara, E; Basso, G; Zampini, M; Buldini, B; Tregnago, C; Rondelli, R; Masetti, R; Bisio, V; Frison, M; Polato, K; Cazzaniga, G; Menna, G; Fagioli, F; Merli, P; Biondi, A; Pession, A; Locatelli, F; Pigazzi, M

2017-01-01

Recurrent molecular markers have been routinely used in acute myeloid leukemia (AML) for risk assessment at diagnosis, whereas their post-induction monitoring still represents a debated issue. We evaluated the prognostic value and biological impact of minimal residual disease (MRD) and of the allelic ratio (AR) of FLT3-internal-tandem duplication (ITD) in childhood AML. We retrospectively screened 494 children with de novo AML for FLT3-ITD mutation, identifying 54 harboring the mutation; 51% of them presented high ITD-AR at diagnosis and had worse event-free survival (EFS, 19.2 versus 63.5% for low ITD-AR, <0.05). Forty-one percent of children with high levels of MRD after the 1st induction course, measured by a patient-specific real-time-PCR, had worse EFS (22.2 versus 59.4% in low-MRD patients, P<0.05). Next, we correlated these parameters with gene expression, showing that patients with high ITD-AR or persistent MRD had characteristic expression profiles with deregulated genes involved in methylation and acetylation. Moreover, patients with high CyclinA1 expression presented an unfavorable EFS (20.3 versus 51.2% in low CyclinA1 group, P<0.01). Our results suggest that ITD-AR levels and molecular MRD should be considered in planning clinical management of FLT3-ITD patients. Different transcriptional activation of epigenetic and oncogenic profiles may explain variability in outcome among these patients, for whom novel therapeutic approaches are desirable.

Aurora Kinase A Promotes AR Degradation via the E3 Ligase CHIP.

PubMed

Sarkar, Sukumar; Brautigan, David L; Larner, James M

2017-08-01

Reducing the levels of the androgen receptor (AR) is one of the most viable approaches to combat castration-resistant prostate cancer. Previously, we observed that proteasomal-dependent degradation of AR in response to 2-methoxyestradiol (2-ME) depends primarily on the E3 ligase C-terminus of HSP70-interacting protein (STUB1/CHIP). Here, 2-ME stimulation activates CHIP by phosphorylation via Aurora kinase A (AURKA). Aurora A kinase inhibitors and RNAi knockdown of Aurora A transcript selectively blocked CHIP phosphorylation and AR degradation. Aurora A kinase is activated by 2-ME in the S-phase as well as during mitosis, and phosphorylates CHIP at S273. Prostate cancer cells expressing an S273A mutant of CHIP have attenuated AR degradation upon 2-ME treatment compared with cells expressing wild-type CHIP, supporting the idea that CHIP phosphorylation by Aurora A activates its E3 ligase activity for the AR. These results reveal a novel 2-ME→Aurora A→CHIP→AR pathway that promotes AR degradation via the proteasome that may offer novel therapeutic opportunities for prostate cancer. Mol Cancer Res; 15(8); 1063-72. ©2017 AACR . ©2017 American Association for Cancer Research.

Transcript Levels of Androgen Receptor Variant 7 and Ubiquitin-Conjugating Enzyme 2C in Hormone Sensitive Prostate Cancer and Castration-Resistant Prostate Cancer.

PubMed

Lee, Chan Ho; Ku, Ja Yoon; Ha, Jung Min; Bae, Sun Sik; Lee, Jeong Zoo; Kim, Choung-Soo; Ha, Hong Koo

2017-01-01

This study is designed to identify the androgen receptor variant 7 (AR-V7) status, clinical significance of AR-V7 in hormone sensitive prostate cancer (HSPC). Then, we evaluated AR-V7 and changes of its target gene, ubiquitin-conjugating enzyme E2C (UBE2C) which is an anaphase-promoting complex/cyclosome (APC/C)-specific ubiquitin-conjugating enzyme, in castration-resistant prostate cancer (CRPC) in serial tumor biopsies from patients receiving androgen deprivation therapy. We used RT-PCR and Q-PCR assay to evaluate AR-V7, androgen receptor full length (AR-FL), and UBE2C in tumor biopsies from patients with HSPC and CRPC. We examined associations between mRNA expression of AR-V7 and clinicopathologic factors. Furthermore, to identify other potential genes involved in the development of CRPC, RNA sequencing was conducted, using paired prostate cancer (PCa) tissues obtained immediately prior to treatment and at the time of therapeutic resistance. A total of 13 HSPC patients and three CRPC patients were enrolled. Neither a high Gleason score (score of 8 and 9) nor a high risk of PCa (a high risk of locally advanced PCa according to NCCN guidelines) was correlated with mRNA expression of AR-V7 in HSPC (P = 0.153 and P = 0.215). The mRNA expression of AR-FL, but not AR-V7, was significantly associated with the mRNA expression of UBE2C level in HSPC (P = 0.007). However, increased expression of AR-V7, not AR-FL, paralleled increased expression of UBE2C in the CRPC specimens (P = 0.03). AR-V7 expression status before ADT was likely related to shorter CRPC development in patients treating ADT. The result of the RNA-sequencing analysis using serial samples from the same patient before and after castration demonstrated an increased level of the PI3K regulatory subunit 1 (P = 0.018). Our study revealed the role of UBE2C as a marker of the androgen signaling pathway in PCa. Differential gene expression analysis using serial samples from the same patient before and after castration revealed potential genes and pathways involved in development of CRPC. Further studies are needed to determine whether these genes and pathways are potential therapeutic target for CRPC. Prostate 77:60-71, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Nicotine induced CpG methylation of Pax6 binding motif in StAR promoter reduces the gene expression and cortisol production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Tingting; Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland; Chen, Man

Steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in the synthesis of steroid hormones, essential to fetal development. We have reported that the StAR expression in fetal adrenal is inhibited in a rat model of nicotine-induced intrauterine growth retardation (IUGR). Here using primary human fetal adrenal cortex (pHFAC) cells and a human fetal adrenal cell line NCI-H295A, we show that nicotine inhibits StAR expression and cortisol production in a dose- and time-dependent manner, and prolongs the inhibitory effect on cells proliferating over 5 passages after termination of nicotine treatment. Methylation detection within the StAR promoter region uncovers a singlemore » site CpG methylation at nt -377 that is sensitive to nicotine treatment. Nicotine-induced alterations in frequency of this point methylation correlates well with the levels of StAR expression, suggesting an important role of the single site in regulating StAR expression. Further studies using bioinformatics analysis and siRNA approach reveal that the single CpG site is part of the Pax6 binding motif (CGCCTGA) in the StAR promoter. The luciferase activity assays validate that Pax6 increases StAR gene expression by binding to the glucagon G3-like motif (CGCCTGA) and methylation of this site blocks Pax6 binding and thus suppresses StAR expression. These data identify a nicotine-sensitive CpG site at the Pax6 binding motif in the StAR promoter that may play a central role in regulating StAR expression. The results suggest an epigenetic mechanism that may explain how nicotine contributes to onset of adult diseases or disorders such as metabolic syndrome via fetal programming. -- Highlights: Black-Right-Pointing-Pointer Nicotine-induced StAR inhibition in two human adrenal cell models. Black-Right-Pointing-Pointer Nicotine-induced single CpG site methylation in StAR promoter. Black-Right-Pointing-Pointer Persistent StAR inhibition and single CpG methylation after nicotine termination. Black-Right-Pointing-Pointer Single CpG methylation located at Pax6 binding motif regulates StAR expression.« less

ERG Expression Levels in Prostate Tumors Reflect Functional Status of the Androgen Receptor (AR) as a Consequence of Fusion of ERG with AR Regulated Gene Promoters

DTIC Science & Technology

2010-01-01

mathematically defined in the following formulas. ARGI = Σ (I PMEPA1 + IPSA +INKX3.1+IODC1+IAMD1) ARFI=CI=ARGI+IERG Detection of TMPRSS2-ERG Fusion Junctions...ERG expression into the Androgen Receptor Function Index, ARFI. ARGI = Σ (I PMEPA1 + IPSA +INKX3.1+IODC1+IAMD1) ARFI=CI=ARGI+IERG In the high ERG

Diminished androgen and estrogen receptors and aromatase levels in hypogonadal diabetic men: reversal with testosterone.

PubMed

Ghanim, Husam; Dhindsa, Sandeep; Abuaysheh, Sanaa; Batra, Manav; Kuhadiya, Nitesh D; Makdissi, Antoine; Chaudhuri, Ajay; Dandona, Paresh

2018-03-01

One-third of males with type 2 diabetes (T2DM) have hypogonadism, characterized by low total and free testosterone concentrations. We hypothesized that this condition is associated with a compensatory increase in the expression of androgen receptors (AR) and that testosterone replacement reverses these changes. We also measured estrogen receptor and aromatase expression. This is a randomized double-blind placebo-controlled trial. Thirty-two hypogonadal and 32 eugonadal men with T2DM were recruited. Hypogonadal men were randomized to receive intramuscular testosterone or saline every 2 weeks for 22 weeks. We measured AR, ERα and aromatase expression in peripheral blood mononuclear cells (MNC), adipose tissue and skeletal muscle in hypogonadal and eugonadal males with T2DM at baseline and after 22 weeks of treatment in those with hypogonadism. The mRNA expression of AR, ERα (ESR1) and aromatase in adipose tissue from hypogonadal men was significantly lower as compared to eugonadal men, and it increased significantly to levels comparable to those in eugonadal patients with T2DM following testosterone treatment. AR mRNA expression was also significantly lower in MNC from hypogonadal patients compared to eugonadal T2DM patients. Testosterone administration in hypogonadal patients also restored AR mRNA and nuclear extract protein levels from MNC to that in eugonadal patients. In the skeletal muscle, AR mRNA and protein expression are lower in men with hypogonadism. Testosterone treatment restored AR expression levels to that comparable to levels in eugonadal men. We conclude that, contrary to our hypothesis, the expression of AR, ERα and aromatase is significantly diminished in hypogonadal men as compared to eugonadal men with type 2 diabetes. Following testosterone replacement, there is a reversal of these deficits. © 2018 European Society of Endocrinology.

Deletion of RhoA in Progesterone Receptor-Expressing Cells Leads to Luteal Insufficiency and Infertility in Female Mice.

PubMed

El Zowalaty, Ahmed E; Li, Rong; Zheng, Yi; Lydon, John P; DeMayo, Francesco J; Ye, Xiaoqin

2017-07-01

Ras homolog gene family, member A (RhoA) is widely expressed throughout the female reproductive system. To assess its role in progesterone receptor-expressing cells, we generated RhoA conditional knockout mice RhoAd/d (RhoAf/f-Pgr-Cre+/-). RhoAd/d female mice had comparable mating activity, serum luteinizing hormone, prolactin, and estradiol levels and ovulation with control but were infertile with progesterone insufficiency, indicating impaired steroidogenesis in RhoAd/d corpus luteum (CL). RhoA was highly expressed in wild-type luteal cells and conditionally deleted in RhoAd/d CL. Gestation day 3.5 (D3.5) RhoAd/d ovaries had reduced numbers of CL, less defined corpus luteal cord formation, and disorganized CL collagen IV staining. RhoAd/d CL had lipid droplet and free cholesterol accumulation, indicating the availability of cholesterol for steroidogenesis, but disorganized β-actin and vimentin staining, indicating disrupted cytoskeleton integrity. Cytoskeleton is important for cytoplasmic cholesterol movement to mitochondria and for regulating mitochondria. Dramatically reduced expression of mitochondrial markers heat shock protein 60 (HSP60), voltage-dependent anion channel, and StAR was detected in RhoAd/d CL. StAR carries out the rate-limiting step of steroidogenesis. StAR messenger RNA expression was reduced in RU486-treated D3.5 wild-type CL and tended to be induced in progesterone-treated D3.5 RhoAd/d CL, with parallel changes of HSP60 expression. These data demonstrated the in vivo function of RhoA in CL luteal cell cytoskeleton integrity, cholesterol transport, StAR expression, and progesterone synthesis, and a positive feedback on StAR expression in CL by progesterone signaling. These findings provide insights into mechanisms of progesterone insufficiency.

Differential regulation of insulin-like growth factor-I receptor gene expression by wild type and mutant androgen receptor in prostate cancer cells.

PubMed

Schayek, Hagit; Seti, Hila; Greenberg, Norman M; Sun, Shihua; Werner, Haim; Plymate, Stephen R

2010-07-29

The progression of prostate cancer from an organ-confined, androgen-sensitive disease to a metastatic one is associated with dysregulation of androgen receptor (AR)-regulated target genes and with a decrease in insulin-like growth factor-I receptor (IGF-IR) expression. To investigate the differential effects of wild type (wt) and mutant AR on IGF-IR levels we employed a series of isogenic prostate-derived cell lines and human xenografts. We show that basal and phosphorylated IGF-IR levels progressively decreased as prostate cancer cells became more tumorigenic and metastatic. In addition, we show that wt, but not mutant, AR along with dihydrotestosterone treatment increased IGF-IR promoter activity and endogenous IGF-IR levels. ChIP analysis show enhanced AR binding to the IGF-IR promoter in AR-overexpressing cells. Finally, wt AR-overexpressing cells display an enhanced proliferation rate. In summary, we provide evidence that activated wt AR enhances IGF-IR transcription in prostate cancer cells via a mechanism that involves AR binding to the IGF-IR promoter. AR mutations alter the ability of the mutated protein to regulate IGF-IR expression. Our results suggest that prostate cancer progression is associated with a decrease in IGF-IR expression that could be the result of impaired ability of AR to stimulate IGF-IR gene expression. 2010 Elsevier Ireland Ltd. All rights reserved.

Differential regulation of insulin-like growth factor-I receptor gene expression by wild type and mutant androgen receptor in prostate cancer cells

PubMed Central

Schayek, Hagit; Seti, Hila; Greenberg, Norman M.; Sun, Shihua; Werner, Haim; Plymate, Stephen R.

2010-01-01

The progression of prostate cancer from an organ-confined, androgen-sensitive disease to a metastatic one is associated with dysregulation of androgen receptor (AR)-regulated target genes and with a decrease in insulin-like growth factor-I receptor (IGF-IR) expression. To investigate the differential effects of wild type (wt) and mutant AR on IGF-IR levels we employed a series of isogenic prostate-derived cell lines and human xenografts. We show that basal and phosphorylated IGF-IR levels progressively decreased as prostate cancer cells became more tumorigenic and metastatic. In addition, we show that wt, but not mutant, AR along with dihydrotestosterone treatment increased IGF-IR promoter activity and endogenous IGF-IR levels. ChIP analysis show enhanced AR binding to the IGF-IR promoter in AR-overexpressing cells. Finally, wt AR-overexpressing cells display an enhanced proliferation rate. In summary, we provide evidence that activated wt AR enhances IGF-IR transcription in prostate cancer cells via a mechanism that involves AR binding to the IGF-IR promoter. AR mutations alter the ability of the mutated protein to regulate IGF-IR expression. Our results suggest that prostate cancer progression is associated with a decrease in IGF-IR expression that could be the result of impaired ability of AR to stimulate IGF-IR gene expression. PMID:20417685

Carbidopa abrogates L-dopa decarboxylase coactivation of the androgen receptor and delays prostate tumor progression.

PubMed

Wafa, Latif A; Cheng, Helen; Plaa, Nathan; Ghaidi, Fariba; Fukumoto, Takahiro; Fazli, Ladan; Gleave, Martin E; Cox, Michael E; Rennie, Paul S

2012-06-15

The androgen receptor (AR) plays a central role in prostate cancer progression to the castration-resistant (CR) lethal state. L-Dopa decarboxylase (DDC) is an AR coactivator that increases in expression with disease progression and is coexpressed with the receptor in prostate adenocarcinoma cells, where it may enhance AR activity. Here, we hypothesize that the DDC enzymatic inhibitor, carbidopa, can suppress DDC-coactivation of AR and retard prostate tumor growth. Treating LNCaP prostate cancer cells with carbidopa in transcriptional assays suppressed the enhanced AR transactivation seen with DDC overexpression and decreased prostate-specific antigen (PSA) mRNA levels. Carbidopa dose-dependently inhibited cell growth and decreased survival in LNCaP cell proliferation and apoptosis assays. The inhibitory effect of carbidopa on DDC-coactivation of AR and cell growth/survival was also observed in PC3 prostate cancer cells (stably expressing AR). In vivo studies demonstrated that serum PSA velocity and tumor growth rates elevated ∼2-fold in LNCaP xenografts, inducibly overexpressing DDC, were reverted to control levels with carbidopa administration. In castrated mice, treating LNCaP tumors, expressing endogenous DDC, with carbidopa delayed progression to the CR state from 6 to 10 weeks, while serum PSA and tumor growth decreased 4.3-fold and 5.4-fold, respectively. Our study is a first time demonstration that carbidopa can abrogate DDC-coactivation of AR in prostate cancer cells and tumors, decrease serum PSA, reduce tumor growth and delay CR progression. Since carbidopa is clinically approved, it may be readily used as a novel therapeutic strategy to suppress aberrant AR activity and delay prostate cancer progression. Copyright © 2011 UICC.

SMILE upregulated by metformin inhibits the function of androgen receptor in prostate cancer cells.

PubMed

Lee, Seung-Yon; Song, Chin-Hee; Xie, Yuan-Bin; Jung, Chaeyong; Choi, Hueng-Sik; Lee, Keesook

2014-11-28

Metformin, a diabetes drug, has been reported to inhibit the growth of prostate cancer cells. In this study, we investigated the effect and action mechanism of metformin on the function of androgen receptor (AR), a key molecule in the proliferation of prostate cancer cells. Metformin was found to reduce androgen-dependent cell growth and the expression of AR target genes by inhibiting AR function in prostate cancer cells such as LNCaP and C4-2 cells. Interestingly, metformin upregulated the protein level of small heterodimer partner-interacting leucine zipper (SMILE), a coregulator of nuclear receptors, and knockdown of SMILE expression with shRNA abolished the inhibitory effect of metformin on AR function. Further studies revealed that SMILE protein itself suppressed the transactivation of AR, and its ectopic expression resulted in the repressed expression of endogenous AR target genes, PSA and NKX3.1, in LNCaP cells. In addition, SMILE protein physically interacted with AR and competed with the AR coactivator SRC-1 to modulate AR transactivation. As expected, SMILE repressed androgen-dependent growth of LNCaP and C4-2 cells. Taken together, these results suggest that SMILE, which is induced by metformin, functions as a novel AR corepressor and may mediate the inhibitory effect of metformin on androgen-dependent growth of prostate cancer cells. Copyright © 2014. Published by Elsevier Ireland Ltd.

Immunohistochemical panel to characterize canine prostate carcinomas according to aberrant p63 expression.

PubMed

Fonseca-Alves, Carlos Eduardo; Kobayashi, Priscila Emiko; Rivera Calderón, Luis Gabriel; Felisbino, Sérgio Luis; Rinaldi, Jaqueline de Carvalho; Drigo, Sandra Aparecida; Rogatto, Silvia Regina; Laufer-Amorim, Renée

2018-01-01

An unusual variant of prostate adenocarcinoma (PC) expressing nuclear p63 in secretory cells instead of the typical basal expression has been reported in men. Nevertheless, the biological behavior and clinical significance of this phenomenon is unknown. In dogs, this unusual PC subtype has not been described. In this study, p63 immunoexpression was investigated in 90 canine PCs and 20 normal prostate tissues (NT). The p63 expression pattern in luminal or basal cells was confirmed in a selected group of 26 PCs and 20 NT by immunohistochemistry and/or Western blotting assays. Eleven canine PC samples aberrantly expressing p63 (p63+) in secretory cells were compared with 15 p63 negative (p63-) cases in the context of several molecular markers (high molecular weight cytokeratin-HMWC, CK8/18, CK5, AR, PSA, chromogranin, NKX3.1, PTEN, AKT and C-MYC). P63+ samples were positive for CK5, HMWC and CK8/18 and negative for PSA, NKX3.1, PTEN and chromogranin. Five p63+ PCs were negative for AR, and the remaining six samples had low AR expression. In contrast, p63- PC showed AR and PSA positive expression in all 15 samples. Only five p63- PCs were positive for CK5. Both p63+ and p63- PC samples showed higher cytoplasmic AKT expression and nuclear C-MYC staining in comparison with normal tissues. Metastatic (N = 12) and non-metastatic (N = 14) PCs showed similar immunoexpression for all markers tested. In contrast to human PC, canine PC aberrantly expressing p63 showed higher expression levels of HMWC and CK5 and lower levels of NKX3.1. Canine p63+ PC is a very rare PC group showing a distinct phenotype compared to typical canine PC, including AR and PSA negative expression. Although in a limited number of cases, p63 expression was not associated with metastasis in canine PC, and cytoplasmic p63 expression was observed in animals with shorter survival time, similar to human PC cases.

Effects of dietary omega-3/omega-6 fatty acid ratios on reproduction in the young breeder rooster.

PubMed

Feng, Yun; Ding, Yu; Liu, Juan; Tian, Ye; Yang, Yanzhou; Guan, Shuluan; Zhang, Cheng

2015-03-21

Polyunsaturated fatty acids (PUFAs) are necessary for the body's metabolism, growth and development. Although PUFAs play an important role in the regulation of reproduction, their role in testis development in the rooster is unknown. The present study was conducted to investigate the effects of omega-3/omega-6 (n-3/n-6, PUFAs) ratios on reproductive performance in young breeder roosters. Plasma levels of reproductive hormones, testis development, and reproductive hormone receptor and StAR mRNA expression were also assessed. Although PUFAs (n-3/n-6: 1/4.15) had no significant effect on the testis index (P > 0.05), the spermatogonial development and germ cell layers were increased. Moreover, serum levels of hormones (GnRH, FSH, LH and T) on day 35 were also significantly increased by PUFAs (n-3/n-6: 1/4.15). To investigate whether PUFAs regulate the expression of hormone receptors and StAR, real time-PCR was used to measure GnRHR, FSHR, LHR and StAR mRNA levels. PUFAs significantly increased the mRNA levels of all of these genes. These results indicate that PUFAs enhance the reproductive performance of young roosters by increasing hormone secretion and function, the latter by up-regulating receptor expression. These findings provide a sound basis for a balanced n-3/n-6 PUFA ratio being beneficial to young rooster reproduction.

C3aR and C5aR1 act as key regulators of human and mouse β-cell function.

PubMed

Atanes, Patricio; Ruz-Maldonado, Inmaculada; Pingitore, Attilio; Hawkes, Ross; Liu, Bo; Zhao, Min; Huang, Guo Cai; Persaud, Shanta J; Amisten, Stefan

2018-02-01

Complement components 3 and 5 (C3 and C5) play essential roles in the complement system, generating C3a and C5a peptides that are best known as chemotactic and inflammatory factors. In this study we characterised islet expression of C3 and C5 complement components, and the impact of C3aR and C5aR1 activation on islet function and viability. Human and mouse islet mRNAs encoding key elements of the complement system were quantified by qPCR and distribution of C3 and C5 proteins was determined by immunohistochemistry. Activation of C3aR and C5aR1 was determined using DiscoverX beta-arrestin assays. Insulin secretion from human and mouse islets was measured by radioimmunoassay, and intracellular calcium ([Ca 2+ ]i), ATP generation and apoptosis were assessed by standard techniques. C3 and C5 proteins and C3aR and C5aR1 were expressed by human and mouse islets, and C3 and C5 were mainly localised to β- and α-cells. Conditioned media from islets exposed for 1 h to 5.5 and 20 mM glucose stimulated C3aR and C5aR1-driven beta-arrestin recruitment. Activation of C3aR and C5aR1 potentiated glucose-induced insulin secretion from human and mouse islets, increased [Ca 2+ ]i and ATP generation, and protected islets against apoptosis induced by a pro-apoptotic cytokine cocktail or palmitate. Our observations demonstrate a functional link between activation of components of the innate immune system and improved β-cell function, suggesting that low-level chronic inflammation may improve glucose homeostasis through direct effects on β-cells.

Synergistic activation of the androgen receptor by bombesin and low-dose androgen.

PubMed

Dai, Jie; Shen, Ruoqian; Sumitomo, Makoto; Stahl, Rosalyn; Navarro, Daniel; Gershengorn, Marvin C; Nanus, David M

2002-07-01

Neuropeptide growth factors such as bombesinare implicated in progression to androgen-independent prostate cancer (PC). We examined the impact of bombesin on androgen receptor (AR)-mediated gene expression. The AR together with the AR-responsive probasin ARR(3)tk-luc or PSA-pPUE-ELB-luc promoter was cotransfected into Swiss 3T3 and PC-3 cells, both of which express high-affinity bombesin receptors; the cells were incubated with bombesin (0-50 nM) and dihydrotestosterone (DHT; 0-10 nM), and luciferase activities were measured. DHT increased transcription approximately 40-fold at doses of 1 and 10 nM but had no effect at 10 pM. Bombesin alone, or with 1 or 10 nM DHT, did not further increase transcription. However, 5 nM bombesin and 10 pM DHT, doses that by themselves had no effect, resulted in a approximately 20 fold increase in transcription (P < 0.005). This synergistic effect was blocked by bombesin receptor antagonists and recombinant neutral endopeptidase, which hydrolyzes bombesin. Bombesin and DHT together also increased binding of nuclear extracts from PC-3 cells transfected with AR to a consensus androgen response element in mobility shift assays and increased the level of secreted prostate-specific antigen in LNCaP cell supernatant compared with DHT or bombesin alone. Immunoprecipitation of AR from (32)P-labeled LNCaP cells revealed that 5 nM bombesin + 10 pM DHT induced AR phosphorylation comparable with 1 nM DHT, whereas bombesin or 10 pM DHT alone did not. These data indicate that bombesin can synergize with low (castrate) levels of DHT to induce AR-mediated transcription and suggest that neuropeptides promote AR-mediated signaling in androgen-independent prostate cancer.

Androgen receptor (AR) inhibitor ErbB3-binding protein-1 (Ebp1) is not targeted by the newly identified AR controlling signaling axis heat-shock protein HSP27 and microRNA miR-1 in prostate cancer cells.

PubMed

Stope, Matthias B; Peters, Stefanie; Großebrummel, Hannah; Zimmermann, Uwe; Walther, Reinhard; Burchardt, Martin

2015-03-01

Androgen receptor (AR) networks are predominantly involved in prostate cancer (PCa) progression; consequently, factors of AR regulation represent promising targets for PCa therapy. The ErbB3-binding protein 1 (Ebp1) is linked to AR suppression and chemoresistance by so far unknown mechanisms. In this study, an assumed regulation of Ebp1 by the newly identified AR controlling signaling axis heat-shock protein 27 (HSP27)-microRNA-1 (miR-1) was examined. Transfection experiments were carried out overexpressing and knockdown HSP27 and miR-1, respectively, in LNCaP and PC-3 cells. Afterward, HSP27- and miR-1-triggered Ebp1 protein expression was monitored by Western blotting. AR-positive LNCaP cells and AR-negative PC-3 cells possessed diverse basal expression levels of Ebp1. However, subsequent studies revealed no differences in cellular Ebp1 concentrations after modulation of HSP27 and miR-1. Furthermore, docetaxel incubation experiments exhibited no effects on Ebp1 protein synthesis. In PCa, Ebp1 has been described as a regulator of AR functionality and as an effector of PCa therapy resistance. Our data suggest that Ebp1 functionality is independent from heat-shock-protein-regulated progression networks in PCa.

The steroidogenic response and corpus luteum expression of the steroidogenic acute regulatory protein after human chorionic gonadotropin administration at different times in the human luteal phase.

PubMed

Kohen, Paulina; Castro, Olga; Palomino, Alberto; Muñoz, Alex; Christenson, Lane K; Sierralta, Walter; Carvallo, Pilar; Strauss, Jerome F; Devoto, Luigi

2003-07-01

This study was designed 1) to assess corpus luteum (CL) steroidogenesis in response to exogenous human chorionic gonadotropin (hCG) at different times during the luteal phase, 2) to examine the effect of hCG on steroidogenic acute regulatory protein (StAR) expression within the CL, 3) to correlate StAR expression and luteal steroidogenic responses to hCG, and 4) to determine whether endogenous LH regulates ovarian steroidogenesis in the early luteal phase. Blood was collected before and after hCG treatment for steroid and hCGbeta determinations. CL were obtained at the time of surgery to assess StAR gene and protein expression. During the early luteal phase various women received the GnRH antagonist for 24-48 h; some of them also received hCG 24 h after the GnRH antagonist. A slight steroidogenic response to hCG was observed in early luteal phase; 17alpha-hydroxyprogesterone, but not progesterone (P4), levels were significantly increased 8 h post-hCG, indicating a differential response by the granulosa and theca-lutein cells. The 1.6- and 4.4-kb StAR transcripts and the 37-kDa preprotein and 30-kDa mature StAR protein did not change post-hCG administration in early luteal phase CL. In contrast, the StAR 4.4- and 1.6-kb transcripts diminished significantly (P < 0.05) after the antagonist treatment. Immunohistochemical staining for StAR protein was weak, particularly in granulosa-lutein cells. Treatment with hCG restored StAR mRNA and protein and plasma P4 levels within 24 h in antagonist-treated women. hCG stimulated the highest plasma concentrations of P4 and estradiol in the midluteal phase, indicating its greatest steroidogenic capacity. Midluteal tissue StAR gene and protein expression increased by 1.6- and 1.4-fold after 24 h of hCG treatment, respectively. Administration of hCG resulted in the greatest increment in plasma P4 (4-fold) and 17alpha-hydroxyprogesterone (3-fold) levels over baseline in the late luteal phase. This was associated with an increase in StAR mRNA (3.5-fold) and protein (1.8-fold). Collectively, these data indicate that 1) the hCG-stimulated steroidogenic response is dependent on the age of the CL; 2) the early luteal phase CL is relatively insensitive to exogenous hCG in the presence of normal pituitary gonadotropin support, but becomes responsive when the latter is withdrawn; 3) the hCG-stimulated steroidogenic response in the mid- and late luteal phase is correlated with increased StAR mRNA and protein abundance; and 4) there are differential responses of small and large luteal cells to hCG stimulation that depend upon the age of the CL.

Regulation of Androgen Receptor Expression Alters AMPK Phosphorylation in the Endometrium: In Vivo and In Vitro Studies in Women with Polycystic Ovary Syndrome

PubMed Central

Li, Xin; Pishdari, Bano; Cui, Peng; Hu, Min; Yang, Hong-Ping; Guo, Yan-Rong; Jiang, Hong-Yuan; Feng, Yi; Billig, Håkan; Shao, Ruijin

2015-01-01

The failure of reproductive success in polycystic ovary syndrome (PCOS) patients could be in part due to endometrial dysfunction. However, no studies have investigated any causality between androgen, androgen receptor (AR) expression, and adenosine monophosphate activated protein kinase (AMPK) activation in the endometrium under physiological and pathological conditions. In the present study, we show that 1) endometrial AR expression levels fluctuate in non-PCOS and PCOS patients during the menstrual cycle; 2) the menstrual phase-dependent alteration of p-AMPKα expression occurs in non-PCOS patients but not in PCOS patients; 3) AR expression is higher in PCOS patients than non-PCOS patients during hyperplasia while AMPKα activation (indicated by the ratio of p-AMPKα to AMPKα); and 4) co-localization of AR and Ki-67 in epithelial cell nuclei is observed in endometrial hyperplasia. Importantly, using in vitro human tissue culture and an in vivo 5α-dihydrotestosterone-treated rat model, we show that the action of androgen on AMPKα activation is likely mediated through nuclear AR, especially in epithelial cells. Collectively, we present evidence that AR expression and AMPKα activation depend on menstrual cycle phase and the presence of PCOS, and the data suggest that AR-mediated regulation of AMPKα activation might play a role in the development of endometrial hyperplasia. PMID:26681917

ACK1/TNK2 Regulates Histone H4 Tyr88-phosphorylation and AR Gene Expression in Castration-Resistant Prostate Cancer.

PubMed

Mahajan, Kiran; Malla, Pavani; Lawrence, Harshani R; Chen, Zhihua; Kumar-Sinha, Chandan; Malik, Rohit; Shukla, Sudhanshu; Kim, Jongphil; Coppola, Domenico; Lawrence, Nicholas J; Mahajan, Nupam P

2017-06-12

The androgen receptor (AR) is critical for the progression of prostate cancer to a castration-resistant (CRPC) state. AR antagonists are ineffective due to their inability to repress the expression of AR or its splice variant, AR-V7. Here, we report that the tyrosine kinase ACK1 (TNK2) phosphorylates histone H4 at tyrosine 88 upstream of the AR transcription start site. The WDR5/MLL2 complex reads the H4-Y88-phosphorylation marks and deposits the transcriptionally activating H3K4-trimethyl marks promoting AR transcription. Reversal of the pY88-H4 epigenetic marks by the ACK1 inhibitor (R)-9bMS-sensitized naive and enzalutamide-resistant prostate cancer cells and reduced AR and AR-V7 levels to mitigate CRPC tumor growth. Thus, a feedforward ACK1/pY88-H4/WDR5/MLL2/AR epigenetic circuit drives CRPC and is necessary for maintenance of the malignant state. Copyright © 2017 Elsevier Inc. All rights reserved.

Increased aryl hydrocarbon receptor expression in patients with allergic rhinitis.

PubMed

Wei, P; Hu, G-H; Kang, H-Y; Yao, H-B; Kou, W; Liu, H; Hong, S-L

2014-02-01

A predominant Th17 population is a marker of allergic rhinitis (AR). As a ligand-activated transcription factor, the aryl hydrocarbon receptor (AhR) plays a vital role in promoting or inhibiting the development of specific Th cells. However, its role in AR remains undefined. To analyze the potential role of AhR in the pathogenesis of AR. In total, 30 AR patients and 13 healthy controls were recruited for this study and AR patients had clinical features, as demonstrated by rhinoconjunctivitis quality of life questionnaires, total symptom scores and visual analog scale scores. The expression of AhR, IL-17 and IL-22 and the presence of Th17 cells in peripheral blood mononuclear cells were measured before and after treatment with the nontoxic AhR ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Pretreatment ITE studies revealed that all AR patients had a significant increase in AhR expression compared with controls and AhR expression positively correlated with clinical parameters. After ITE intervention, a severe reduction in the differentiation of Th17 cells and the production of IL-17 and IL-22 was noted in both AR patients and normal subjects. Simultaneously, a dramatic enhancement of AhR expression was also observed in all healthy controls, but not in AR patients. The results suggested that the AhR may be one of the mechanisms underlying the Th17 response during the pathogenesis of AR and AhR levels were closely related to clinical severity in all AR patients. Additionally, ITE may represent a new drug candidate in the treatment of AR.

MicroRNAs Are Mediators of Androgen Action in Prostate and Muscle

PubMed Central

Narayanan, Ramesh; Jiang, Jinmai; Gusev, Yuriy; Jones, Amanda; Kearbey, Jeffrey D.; Miller, Duane D.; Schmittgen, Thomas D.; Dalton, James T.

2010-01-01

Androgen receptor (AR) function is critical for the development of male reproductive organs, muscle, bone and other tissues. Functionally impaired AR results in androgen insensitivity syndrome (AIS). The interaction between AR and microRNA (miR) signaling pathways was examined to understand the role of miRs in AR function. Reduction of androgen levels in Sprague-Dawley rats by castration inhibited the expression of a large set of miRs in prostate and muscle, which was reversed by treatment of castrated rats with 3 mg/day dihydrotestosterone (DHT) or selective androgen receptor modulators. Knockout of the miR processing enzyme, DICER, in LNCaP prostate cancer cells or tissue specifically in mice inhibited AR function leading to AIS. Since the only function of miRs is to bind to 3′ UTR and inhibit translation of target genes, androgens might induce miRs to inhibit repressors of AR function. In concordance, knock-down of DICER in LNCaP cells and in tissues in mice induced the expression of corepressors, NCoR and SMRT. These studies demonstrate a feedback loop between miRs, corepressors and AR and the imperative role of miRs in AR function in non-cancerous androgen-responsive tissues. PMID:21048966

Molecular cloning, characterization, and expression profiles of androgen receptors in spotted scat (Scatophagus argus).

PubMed

Chen, H P; Deng, S P; Dai, M L; Zhu, C H; Li, G L

2016-04-07

Androgen plays critical roles in vertebrate reproductive systems via androgen receptors (ARs). In the present study, the full-length spotted scat (Scatophagus argus) androgen receptor (sAR) cDNA sequence was cloned from testis. The sAR cDNA measured 2448 bp in length with an open-reading frame of 2289 bp, encoding 763 amino acids. Amino acid alignment analyses showed that the sARs exhibited highly evolutionary conserved functional domains. Phylogenetically, the sARs clustered within the ARβ common vertebrate group. Real-time polymerase chain reaction (RT-PCR) revealed that sAR expression varied in level and distribution throughout the tissues of both females and males. sAR expression was detected during testicular development by quantitative RT-PCR. The results showed that the highest transcription of sARs was observed in the mid-testicular stage, and remained at a high expression level until the late-testicular stage. In addition, the effects of 17α-methyltestosterone (MT) and estrogen (E2) on the expression of sARs in ovaries were determined using quantitative RT-PCR. sAR expression increased at 12 and 24 h post-MT treatment and decreased with E2 treatment. The present study provides preliminary evidence indicating gonadal plasticity of spotted scat under exogenous steroidal hormone treatments. It also provides a theoretical basis for sex reversal and production of artificial pseudo-males for female monosex breeding.

Effects of fluoride and aluminum on expressions of StAR and P450scc of related steroidogenesis in guinea pigs' testis.

PubMed

Dong, Chunguang; Cao, Jinling; Cao, Chunfang; Han, Yichao; Wu, Shouyan; Wang, Shaolin; Wang, Jundong

2016-03-01

A lot of studies have shown that fluoride and aluminum have toxic effect on male reproductive system, but the mechanism of which and the interaction between fluoride and aluminum is still unknown. This study investigated the effects of fluoride (NaF) or/and aluminum (AlCl3) on serum testosterone level, gene and protein expression levels of Steroidogenic Acute Regulatory Protein (StAR) and Cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) in the testes of guinea pigs. Fifty-two guinea pigs were divided randomly into four groups (Control, HiF, HiAl and HiF + HiAl). Fluoride (150 mg NaF/L) or/and aluminum (300 mg AlCl3/L) were orally administrated to male guinea pigs for 13 weeks. The results showed that F and Al reduced number and elevated abnormal ratio of sperm. Meanwhile, the concentrations of serum testosterone in all experimental groups were decreased. P450scc protein expression was significantly reduced in all treatment groups, and StAR expression was decreased remarkably in HiF group and HiF + HiAl group. The levels of StAR mRNA in three groups were reduced by 53.9%, 21.4% and 33.4%, respectively, while the expressions of P450scc mRNA were reduced by 67.8%, 17.0% and 47.8%. Therefore, we concluded that F induced the reduction in testosterone and sperm amount, and thus in lower fertility, which might occur as a consequence of depressed StAR and P450scc mRNA expression. There were no synergistic effects between F and Al, instead, Al weakened the toxicity of F to some extents. The results indicated that Al had antagonism effects on F. Copyright © 2015 Elsevier Ltd. All rights reserved.

ABA-stimulated SoDOG1 expression is after-ripening inhibited during early imbibition of germinating Sisymbrium officinale seeds.

PubMed

Carrillo-Barral, Néstor; Matilla, Angel J; García-Ramas, Cristina; Rodríguez-Gacio, María Del Carmen

2015-12-01

DELAY OF GERMINATION 1 (AtDOG1) was the first gene identified as dormancy-associated, but its physiological role in germination is far from being understood. Here, an orthologue of AtDOG1 in Sisymbrium officinale (SoDOG1; KM009050) is being reported. Phylogenetically, the SoDOG1 gene is included into the dicotyledonous group together with DOG1 from Arabidopsis thaliana (EF028470), Brassica rapa (AC189537), Lepidium papillosum (JX512183, JX512185) and Lepidium sativum (GQ411192). The SoDOG1 expression peaked at the onset of the silique maturation stage and there was presence of SoDOG1-mRNA in the freshly collected viable dry seed (i.e. AR0). The SoDOG1 transcripts were also found in other organs, such as open and closed flowers and to a lesser degree in roots and stems. We have previously reported in S. officinale seeds in which sensu stricto germination is positively affected by nitrate and both testa and micropylar endosperm ruptures are temporally separated. In dry viable seeds, the SoDOG1-mRNA level in three different after-ripening (AR) status was AR0 ≈ AR7 (optimal AR) < AR27 (optimal AR was almost lost). The presence of nitrate in the AR0 seed imbibition medium markedly decreased the SoDOG1 expression during sensu stricto germination. However, the nitrate stimulated the SoDOG1 expression during imbibition of AR7 compared to AR0. At the early AR0 seed imbibition (3-6 h), exogenous ABA provoked a very strong stimulation of the SoDOG1 expression. AR inhibits ABA-induced SoDOG1 expression during early germination and gibberellins (GA) can partially mimic this AR effect. A view on the integration of all found results in the sensu stricto germination of S. officinale was conducted. © 2015 Scandinavian Plant Physiology Society.

Study on predictive role of AR and EGFR family genes with response to neoadjuvant chemotherapy in locally advanced breast cancer in Indian women.

PubMed

Singh, L C; Chakraborty, Anurupa; Mishra, Ashwani K; Devi, Thoudam Regina; Sugandhi, Nidhi; Chintamani, Chintamani; Bhatnagar, Dinesh; Kapur, Sujala; Saxena, Sunita

2012-06-01

Locally advanced breast cancer (LABC) remains a clinical challenge as the majority of patients with this diagnosis develop distant metastases despite appropriate therapy. We analyzed expression of steroid and growth hormone receptor genes as well as gene associated with metabolism of chemotherapeutic drugs in locally advanced breast cancer before and after neoadjuvant chemotherapy (NACT) to study whether there is a change in gene expression induced by chemotherapy and whether such changes are associated with tumor response or non-response. Fifty patients were included with locally advanced breast cancer treated with cyclophosphamide, adriamycin, 5-fluorouracil (CAF)-based neoadjuvant chemotherapy before surgery. Total RNA was extracted from 50 match samples of pre- and post-NACT tumor tissues. RNA expression levels of epidermal growth factor receptor family genes including EGFR, ERBB2, ERBB3, androgen receptor (AR), and multidrug-resistance gene 1 (MDR1) were determined by quantitative real-time reverse transcriptase-polymerase chain reaction. Responders show significantly high levels of pre-NACT AR gene expression (P = 0.016), which reduces following NACT (P = 0.008), and hence can serve as a useful tool for the prediction of the success of neoadjuvant chemotherapy in individual cancer patients with locally advanced breast carcinoma. Moreover, a significant post-therapeutic increase in the expression levels of EGFR and MDR1 gene in responders (P = 0.026 and P < 0.001) as well as in non-responders (P = 0.055, P = 0.001) suggests that expression of these genes changes during therapy but they do not have any impact on tumor response, whereas a post-therapeutic reduction was observed in AR in responders. This indicates an independent predictive role of AR with response to NACT.

[MicroRNA in neurodegenerative disorders].

PubMed

Sobue, Gen

2013-01-01

MicroRNAs (miRNAs) bind to the 3'-untranslated region of mRNA, and thereby suppress the gene expression. Recent studies suggest that miRNAs modify the pathogenesis of cancer and neurodegeneration. Our study demonstrated that the expression levels of miR-196a is increased in a mouse model of spinal and bulbar muscular atrophy (SBMA), a neurodegenerative disease caused by the expansion of polyglutamine in androgen receptor (AR). In cultured neuronal cells, miR-196a decayed the mutant AR mRNA via silencing CUG triplet repeat RNA binding protein 2, a potent miR-196a targeting mRNA, which contributed to stabilize the mutant AR mRNA. Adeno-associated virus vector-mediated delivery of this miRNA attenuates the expression of the mutant AR, resulting in the mitigation of motor neuron degeneration in the SBMA mice. Introduction of miRNA appears to be a novel therapeutic strategy for devastating neurodegenerative diseases.

Drug membrane transporters and CYP3A4 are affected by hypericin, hyperforin or aristoforin in colon adenocarcinoma cells.

PubMed

Šemeláková, M; Jendželovský, R; Fedoročko, P

2016-07-01

Our previous results have shown that the combination of hypericin-mediated photodynamic therapy (HY-PDT) at sub-optimal dose with hyperforin (HP) (compounds of Hypericum sp.), or its stable derivative aristoforin (AR) stimulates generation of reactive oxygen species (ROS) leading to antitumour activity. This enhanced oxidative stress evoked the need for an explanation for HY accumulation in colon cancer cells pretreated with HP or AR. Generally, the therapeutic efficacy of chemotherapeutics is limited by drug resistance related to the overexpression of drug efflux transporters in tumour cells. Therefore, the impact of non-activated hypericin (HY), HY-PDT, HP and AR on cell membrane transporter systems (Multidrug resistance-associated protein 1-MRP1/ABCC1, Multidrug resistance-associated protein 2-MRP2/ABCC2, Breast cancer resistance protein - BCRP/ABCG2, P-glycoprotein-P-gp/ABCC1) and cytochrome P450 3A4 (CYP3A4) was evaluated. The different effects of the three compounds on their expression, protein level and activity was determined under specific PDT light (T0+, T6+) or dark conditions (T0- T6-). We found that HP or AR treatment affected the protein levels of MRP2 and P-gp, whereas HP decreased MRP2 and P-gp expression mostly in the T0+ and T6+ conditions, while AR decreased MRP2 in T0- and T6+. Moreover, HY-PDT treatment induced the expression of MRP1. Our data demonstrate that HP or AR treatment in light or dark PDT conditions had an inhibitory effect on the activity of individual membrane transport proteins and significantly decreased CYP3A4 activity in HT-29 cells. We found that HP or AR significantly affected intracellular accumulation of HY in HT-29 colon adenocarcinoma cells. These results suggest that HY, HP and AR might affect the efficiency of anti-cancer drugs, through interaction with membrane transporters and CYP3A4. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

The Development of Adultoid Reproductives and Brachypterous Neotenic Reproductives From the Last Instar Nymphs in Reticulitermes labralis (Isoptera: Rhinotermitidae): A Comparative Study

PubMed Central

Su, Xiao Hong; Xue, Wei; Liu, He; Chen, Jiao Ling; Zhang, Xiao Jing; Xing, Lian Xi; Liu, Ming Hua

2015-01-01

Secondary reproductives develop primarily from nymphs. However, they have been rarely studied; in particular, the development of adultoid reproductives (AR) with floppy wings is still unclear. In this study, the change in juvenile hormone (JH) levels, vitellogenin gene expression, and oogenesis during the development of AR and brachypterous neotenic reproductives (BN) from the last instar nymphs of Reticulitermes labralis are investigated and compared. The results showed that the AR derived from the last instar nymphs by molting, and they were more similar to neotenic reproductives in morphology. In addition, the paired AR were not able to survive in the absence of workers. In R. labralis, the process of the last instar nymphs developing into AR and BN took an increase in JH level as a starting point. The JH level of the last instar nymphs molting into BN was approximately 1.5-fold higher than that of the AR. Additionally, The JHIII level of BN peaked on day 5, and that of AR peaked on day 10, which induced the onset of vitellogenesis in BN and AR, respectively. After molting, the vitellogenin gene expression levels of both BN and AR initially increased and then declined, and the expression levels in the BN were significantly higher than those in the AR. In addition, the oocytes of BN matured earlier than those of the AR, and the number of eggs laid by the BN was higher than the number laid by the AR. Our results demonstrate that, in R. labralis, the last instar nymphs can develop into AR, which are significantly different from BN in their development. PMID:26494776

Thymic stromal lymphopoietin expression is increased in nasal epithelial cells of patients with mugwort pollen sensitive-seasonal allergic rhinitis.

PubMed

Zhu, Dong-dong; Zhu, Xue-wei; Jiang, Xiao-dan; Dong, Zhen

2009-10-05

Excessive expression of thymic stromal lymphopoietin (TSLP) has been demonstrated in asthmatic airway epithelia and in nasal epithelia from animal models of allergic rhinitis (AR), but the evidence of expression of TSLP in nasal epithelial cells (NECs) of patients with AR is lacking. We aimed to investigate the expression of TSLP in NECs of patients with mugwort sensitive-seasonal AR and determine whether it is associated with severity of symptoms and the number of infiltrated eosinophils in nasal mucosa. NECs specimens were obtained by scraping with plastic curettes from the nasal inferior turbinates of patients with mugwort pollen sensitive-seasonal AR (n = 22) and nonallergic controls (n = 11) during last peak mugwort pollen season. The severity of nasal symptom was assessed using a Visual Analog Scale (VAS). In addition, serum mugwort pollen IgE levels were tested from each patient. In situ hybridization (ISH) was performed to test the messenger RNA (mRNA) of TSLP in the NECs. Furthermore, immunohistochemical staining (IHC) was scored to evaluate the expression of TSLP and eosinophil cell count was made by May-Grünwald/Giemsa staining. The correlation between expression of TSLP and all other parameters was analyzed in this study. The mRNA level of TSLP was significantly increased in NECs of patients with AR compared with the nonallergic control group (P < 0.05). In addition, IHC results showed that expression of TSLP in NECs from patients with AR was up-regulated which was correlated with VAS score (r = 0.598; P < 0.05) and nasal eosinophils count (r = 0.702; P < 0.05), but it was unrelated with mugwort pollen specific IgE level. These preliminary findings indicate a potential relationship between TSLP expression, severity of symptoms and nasal eosinophils count in pathogenesis of AR, but TSLP expression did not correlate with mugwort pollen specific IgE level. The elevated expression of TSLP might play a critical role in local atopical responses of AR. In the future, the TSLP has the potential to be one of the most important molecular markers for AR diagnoses and assessment.

Androgen receptor mitigates postoperative disease progression of hepatocellular carcinoma by suppressing CD90+ populations and cell migration and by promoting anoikis in circulating tumor cells

PubMed Central

Lai, Hsueh-Chou; Yeh, Chun-Chieh; Jeng, Long-Bin; Huang, Shang-Fen; Liao, Pei-Ying; Lei, Fu-Ju; Cheng, Wei-Chun; Hsu, Cheng-Lung; Cai, Xiujun; Chang, Chawnshang; Ma, Wen-Lung

2016-01-01

Purpose Although hepatectomy and liver transplantation surgery for hepatocellular carcinoma (HCC) are effective treatment modalities, the risk of recurrence remains high, particularly in patients with a high number of circulating tumor cells (CTCs) expressing cancer stem/progenitor cell markers. Androgen receptor (AR) signaling has been shown to suppress HCC metastasis in rodent models of HCC. In this study, we investigated whether AR is associated with postoperative HCC recurrence. Experimental Design CTCs were obtained from patients with HCC who had undergone hepatectomy to investigate whether they are associated with disease outcome. AR knockout was introduced in two mouse models of spontaneous HCC (carcinogen- and hepatitis B virus-related HCC) to delineate the role that AR plays in HCC recurrence. Biological systems analysis was used to investigate the cellular and molecular mechanisms. Results We found that the expression of AR in CTCs was negatively associated with HCC recurrence/progression after hepatectomy. Our results suggest that AR-mediated suppression of HCC recurrence/progression is governed by a three-pronged mechanism. First, AR suppresses the expression of CD90 in CTCs by upregulating Histone 3H2A. Second, AR suppresses cell migration at the transcriptome level. Third, AR promotes anoikis of CTCs via dysregulation of cytoskeletal adsorption. Conclusions The results indicate that AR expression may be the gatekeeper of postoperative HCC recurrence. Therefore, targeting AR in presurgical down-staging procedures may serve as a secondary prevention measure against HCC recurrence in the future. PMID:27340775

β2-Adrenergic Receptor Agonists Inhibit the Proliferation of 1321N1 Astrocytoma CellsS⃞

PubMed Central

Toll, L.; Jimenez, L.; Waleh, N.; Jozwiak, K.; Woo, A.Y.-H.; Xiao, R.-P.; Bernier, M.

2011-01-01

Astrocytomas and glioblastomas have been particularly difficult to treat and refractory to chemotherapy. However, significant evidence has been presented that demonstrates a decrease in astrocytoma cell proliferation subsequent to an increase in cAMP levels. The 1321N1 astrocytoma cell line, as well as other astrocytomas and glioblastomas, expresses β2-adrenergic receptors (β2-ARs) that are coupled to Gs activation and consequent cAMP production. Experiments were conducted to determine whether the β2-AR agonist (R,R′)-fenoterol and other β2-AR agonists could attenuate mitogenesis and, if so, by what mechanism. Receptor binding studies were conducted to characterize β2-AR found in 1321N1 and U118 cell membranes. In addition, cells were incubated with (R,R′)-fenoterol and analogs to determine their ability to stimulate intracellular cAMP accumulation and inhibit [3H]thymidine incorporation into the cells. 1321N1 cells contain significant levels of β2-AR as determined by receptor binding. (R,R′)-fenoterol and other β2-AR agonists, as well as forskolin, stimulated cAMP accumulation in a dose-dependent manner. Accumulation of cAMP induced a decrease in [3H]thymidine incorporation. There was a correlation between concentration required to stimulate cAMP accumulation and inhibit [3H]thymidine incorporation. U118 cells have a reduced number of β2-ARs and a concomitant reduction in the ability of β2-AR agonists to inhibit cell proliferation. These studies demonstrate the efficacy of β2-AR agonists for inhibition of growth of the astrocytoma cell lines. Because a significant portion of brain tumors contain β2-ARs to a greater extent than whole brain, (R,R′)-fenoterol, or some analog, may be useful in the treatment of brain tumors after biopsy to determine β2-AR expression. PMID:21071556

Baicalein suppresses the androgen receptor (AR)-mediated prostate cancer progression via inhibiting the AR N-C dimerization and AR-coactivators interaction.

PubMed

Xu, Defeng; Chen, Qiulu; Liu, Yalin; Wen, Xingqiao

2017-12-01

Androgen receptor (AR) plays a critical role in prostate cancer (PCa) development and progression. Androgen deprivation therapy with antiandrogens to reduce androgen biosynthesis or prevent androgens from binding to AR are widely used to suppress AR-mediated PCa growth. However, most of ADT may eventually fail with development of the castration resistance after 12-24 months. Here we found that a natural product baicalein can effectively suppress the PCa progression via targeting the androgen-induced AR transactivation with little effect to AR protein expression. PCa cells including LNCaP, CWR22Rv1, C4-2, PC-3, and DU145, were treated with baicalein and luciferase assay was used to evaluate their effect on the AR transactivation. Cell growth and IC 50 were determined by MTT assay after 48 hrs treatment. RT-PCR was used to evaluate the mRNA levels of AR target genes including PSA, TMPRSS2, and TMEPA1. Western blot was used to determine AR and PSA protein expression. The natural product of baicalein can selectively inhibit AR transactivation with little effect on the other nuclear receptors, including ERα, and GR. At a low concentration, 2.5 μM of baicalein effectively suppresses the growth of AR-positive PCa cells, and has little effect on AR-negative PCa cells. Mechanism dissection suggest that baicalein can suppress AR target genes (PSA, TMPRSS2, and TMEPA1) expression in both androgen responsive LNCaP cells and castration resistant CWR22Rv1 cells, that may involve the inhibiting the AR N/C dimerization and AR-coactivators interaction. Baicalein may be developed as an effective anti-AR therapy via its ability to inhibit AR transactivation and AR-mediated PCa cell growth.

An agonist to the A3 adenosine receptor inhibits colon carcinoma growth in mice via modulation of GSK-3 beta and NF-kappa B.

PubMed

Fishman, Pnina; Bar-Yehuda, Sara; Ohana, Gil; Barer, Faina; Ochaion, Avivit; Erlanger, Abigail; Madi, Lea

2004-04-01

A(3) adenosine receptor (A(3)AR) activation with the specific agonist CF101 has been shown to inhibit the development of colon carcinoma growth in syngeneic and xenograft murine models. In the present study, we looked into the effect of CF101 on the molecular mechanisms involved in the inhibition of HCT-116 colon carcinoma in mice. In tumor lesions derived from CF101-treated mice, a decrease in the expression level of protein kinase A (PKA) and an increase in glycogen synthase kinase-3 beta (GSK-3 beta) was observed. This gave rise to downregulation of beta-catenin and its transcriptional gene products cyclin D1 and c-Myc. Further mechanistic studies in vitro revealed that these responses were counteracted by the selective A(3)AR antagonist MRS 1523 and by the GSK-3 beta inhibitors lithium and SB216763, confirming that the observed effects were A(3)AR and GSK-3 beta mediated. CF101 downregulated PKB/Akt expression level, resulting in a decrease in the level and DNA-binding capacity of NF-kappa B, both in vivo and in vitro. Furthermore, the PKA and PKB/Akt inhibitors H89 and Worthmannin mimicked the effect of CF101, supporting their involvement in mediating the response to the agonist. This is the first demonstration that A(3)AR activation induces colon carcinoma growth inhibition via the modulation of the key proteins GSK-3 beta and NF-kappa B.

Pacing-induced regional differences in adenosine receptors mRNA expression in a swine model of dilated cardiomyopathy.

PubMed

Del Ry, Silvia; Cabiati, Manuela; Lionetti, Vincenzo; Aquaro, Giovanni D; Martino, Alessandro; Mattii, Letizia; Morales, Maria-Aurora

2012-01-01

The adenosinergic system is essential in the mediation of intrinsic protection and myocardial resistance to insult; it may be considered a cardioprotective molecule and adenosine receptors (ARs) represent potential therapeutic targets in the setting of heart failure (HF). The aim of the study was to test whether differences exist between mRNA expression of ARs in the anterior left ventricle (LV) wall (pacing site: PS) compared to the infero septal wall (opposite region: OS) in an experimental model of dilated cardiomyopathy. Cardiac tissue was collected from LV PS and OS of adult male minipigs with pacing-induced HF (n = 10) and from a control group (C, n = 4). ARs and TNF-α mRNA expression was measured by Real Time-PCR and the results were normalized with the three most stably expressed genes (GAPDH, HPRT1, TBP). Immunohistochemistry analysis was also performed. After 3 weeks of pacing higher levels of expression for each analyzed AR were observed in PS except for A(1)R (A(1)R: C = 0.6±0.2, PS = 0.1±0.04, OS = 0.04±0.01, p<0.0001 C vs. PS and OS respectively; A(2A)R: C = 1.04±0.59, PS = 2.62±0.79, OS = 2.99±0.79; A(2B)R: C = 1.2±0.1, PS = 5.59±2.3, OS = 1.59±0.46; A(3)R: C = 0.76±0.18, PS = 8.40±3.38, OS = 4.40±0.83). Significant contractile impairment and myocardial hypoperfusion were observed at PS after three weeks of pacing as compared to OS. TNF-α mRNA expression resulted similar in PS (6.3±2.4) and in OS (5.9±2.7) although higher than in control group (3.4±1.5). ARs expression was mainly detected in cardiomyocytes. This study provided new information on ARs local changes in the setting of LV dysfunction and on the role of these receptors in relation to pacing-induced abnormalities of myocardial perfusion and contraction. These results suggest a possible therapeutic role of adenosine in patients with HF and dyssynchronous LV contraction.

Androgen receptor agonism promotes an osteogenic gene program in preadipocytes

PubMed Central

Hartig, Sean M.; Feng, Qin; Ochsner, Scott A.; Xiao, Rui; McKenna, Neil J.; McGuire, Sean E.; He, Bin

2013-01-01

Androgens regulate body composition by interacting with the androgen receptor (AR) to control gene expression in a tissue-specific manner. To identify novel regulatory roles for AR in preadipocytes, we created a 3T3-L1 cell line stably expressing human AR. We found AR expression is required for androgen-mediated inhibition of 3T3-L1 adipogenesis. This inhibition is characterized by decreased lipid accumulation, reduced expression of adipogenic genes, and induction of genes associated with osteoblast differentiation. Collectively, our results suggest androgens promote an osteogenic gene program at the expense of adipocyte differentiation. PMID:23567971

Androgen receptor mediated epigenetic regulation of CRISP3 promoter in prostate cancer cells.

PubMed

Pathak, Bhakti R; Breed, Ananya A; Deshmukh, Priyanka; Mahale, Smita D

2018-07-01

Cysteine-rich secretory protein 3 (CRISP3) is one of the most upregulated genes in prostate cancer. Androgen receptor (AR) plays an important role not only in initial stages of prostate cancer development but also in the advanced stage of castration-resistant prostate cancer (CRPC). Role of AR in regulation of CRISP3 expression is not yet known. In order to understand the regulation of CRISP3 expression, various overlapping fragments of CRISP3 promoter were cloned in pGL3 luciferase reporter vector. All constructs were transiently and stably transfected in PC3 (CRISP3 negative) and LNCaP (CRISP3 positive) cell lines and promoter activity was measured by luciferase assay. Promoter activity of LNCaP stable clones was significantly higher than PC3 stable clones. Further in CRISP3 negative PC3 and RWPE-1 cells, CRISP3 promoter was shown to be silenced by histone deacetylation. Treatment of LNCaP cells with DHT resulted in increase in levels of CRISP3 transcript and protein. AR dependency of CRISP3 promoter was also evaluated in LNCaP stable clones by luciferase assay. To provide molecular evidence of epigenetic regulation of CRISP3 promoter and its response to DHT, ChIP PCR was performed in PC3 and LNCaP cells. Our results demonstrate that CRISP3 expression in prostate cancer cells is androgen dependent and in AR positive cells, CRISP3 promoter is epigenetically regulated by AR. Copyright © 2018 Elsevier Ltd. All rights reserved.

Molecular identification of an androgen receptor and its changes in mRNA levels during 17α-methyltestosterone-induced sex reversal in the orange-spotted grouper Epinephelus coioides.

PubMed

Shi, Yu; Liu, Xiaochun; Zhang, Haifa; Zhang, Yong; Lu, Danqi; Lin, Haoran

2012-09-01

Androgens play a crucial role in sex differentiation, sexual maturation, and spermatogenesis in vertebrates. The action of androgens is mediated via androgen receptors (ARs). The present study reports the cloning of the cDNA sequence of the ar in the orange-spotted grouper, with high expression in testis and relatively low in subdivision of brain areas. The cDNA sequence of ar was 2358 bp, encoding a protein of 759 amino acids (aa). Phylogenetic analysis showed that the ar cDNA sequence was closely related to that of threespot wrasse (Halichoeres trimaculatus) and medaka (Oryzias latipes) arβ. As deduced from the phylogenetic tree and the high amino acid identity with the ARβ subtype of other teleosts, grouper ar seems to be more closely related to the beta than the alpha subtype cloned to date. In the first week after 17α-methyltestosterone (MT) implantation, the transcript levels of ar in the hypothalamus declined significantly, and consistently stayed at low level expression to the second week, but increased back to the control levels in the third and fourth week. In the gonad, the mRNA expression of ar was not changed in the first week compared with the control, but increased significantly in the second week, consistently reached the highest level in the third week, dropped slightly but still higher than that of the control in the fourth week. The expression pattern of ar in hypothalamus and gonad during MT-induced sex reversal suggests the involvement of ar in regulating this process in the orange-spotted grouper. The present study provides the data of the changes in the mRNA levels of ar during MT-induced sex reversal in detail to help understand the complicated signals under sex reversal. Copyright © 2012 Elsevier Inc. All rights reserved.

Comprehensive Profiling of the Androgen Receptor in Liquid Biopsies from Castration-resistant Prostate Cancer Reveals Novel Intra-AR Structural Variation and Splice Variant Expression Patterns.

PubMed

De Laere, Bram; van Dam, Pieter-Jan; Whitington, Tom; Mayrhofer, Markus; Diaz, Emanuela Henao; Van den Eynden, Gert; Vandebroek, Jean; Del-Favero, Jurgen; Van Laere, Steven; Dirix, Luc; Grönberg, Henrik; Lindberg, Johan

2017-08-01

Expression of the androgen receptor splice variant 7 (AR-V7) is associated with poor response to second-line endocrine therapy in castration-resistant prostate cancer (CRPC). However, a large fraction of nonresponding patients are AR-V7-negative. To investigate if a comprehensive liquid biopsy-based AR profile may improve patient stratification in the context of second-line endocrine therapy. Peripheral blood was collected from patients with CRPC (n=30) before initiation of a new line of systemic therapy. We performed profiling of circulating tumour DNA via low-pass whole-genome sequencing and targeted sequencing of the entire AR gene, including introns. Targeted RNA sequencing was performed on enriched circulating tumour cell fractions to assess the expression levels of seven AR splice variants (ARVs). Somatic AR variations, including copy-number alterations, structural variations, and point mutations, were combined with ARV expression patterns and correlated to clinicopathologic parameters. Collectively, any AR perturbation, including ARV, was detected in 25/30 patients. Surprisingly, intra-AR structural variation was present in 15/30 patients, of whom 14 expressed ARVs. The majority of ARV-positive patients expressed multiple ARVs, with AR-V3 the most abundantly expressed. The presence of any ARV was associated with progression-free survival after second-line endocrine treatment (hazard ratio 4.53, 95% confidence interval 1.424-14.41; p=0.0105). Six out of 17 poor responders were AR-V7-negative, but four carried other AR perturbations. Comprehensive AR profiling, which is feasible using liquid biopsies, is necessary to increase our understanding of the mechanisms underpinning resistance to endocrine treatment. Alterations in the androgen receptor are associated with endocrine treatment outcomes. This study demonstrates that it is possible to identify different types of alterations via simple blood draws. Follow-up studies are needed to determine the effect of such alterations on hormonal therapy. Copyright © 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.

The Long Non-Coding RNA XIST Interacted with MiR-124 to Modulate Bladder Cancer Growth, Invasion and Migration by Targeting Androgen Receptor (AR).

PubMed

Xiong, Yaoyao; Wang, Long; Li, Yuan; Chen, Minfeng; He, Wei; Qi, Lin

2017-01-01

Long non-coding RNA (lncRNA) X-inactive specific transcript (XIST) is involved in the progression of several tumors. The interaction between lncRNA and miRNA or miRNA's target genes is reported to play crucial roles in malignancy. In addition, Androgen receptor (AR) is considered to be involved in bladder cancer progression. In this study, we investigated the role of XIST in human bladder cancer and its interaction with miR-124 and AR. XIST and AR expression was detected in bladder tumor samples and cell lines. Effects of XIST and AR on bladder cancer cells growth, invasion and migration were analyzed. Bioinformatic analysis and luciferase assays were used to identify the interaction among XIST, AR and miR-124. The correlations of miR-124 with XIST and AR in bladder cancer samples were statistically analyzed. XIST and AR were upregulated in bladder cancer tissues and positively correlated. Higher XIST and AR expression were related to poorer TNM stage of bladder cancer. XIST knockdown reduced bladder cancer cells' proliferation, invasion and migration. While this inhibitory effect could be partially restored by AR overexpression. XIST inhibited miR-124 expression by directly targeting. Moreover, miR-124 could bind to the 3'UTR of AR to regulate its expression. MiR-124 inhibition partially restored the XIST knockdown-induced reduction of AR, c-myc, p27, MMP13 and MMP9 expression. In bladder cancer tissues, miR-124 level was inversely correlated with the expression of XIST and AR, respectively. These findings indicated that XIST might be an oncogenic lncRNA that promoted the bladder cancer growth, invasion and migration via miR-124 dependent AR regulation. © 2017 The Author(s). Published by S. Karger AG, Basel.

Increased Expression of miR-146a in Children With Allergic Rhinitis After Allergen-Specific Immunotherapy.

PubMed

Luo, Xi; Hong, Haiyu; Tang, Jun; Wu, Xingmei; Lin, Zhibin; Ma, Renqiang; Fan, Yunping; Xu, Geng; Liu, Dabo; Li, Huabin

2016-03-01

MicroRNAs (miRs) were recently recognized to be important for immune cell differentiation and immune regulation. However, whether miRs were involved in allergen-specific immunotherapy (SIT) remains largely unknown. This study sought to examine changes in miR-146a and T regulatory cells in children with persistent allergic rhinitis (AR) after 3 months of subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT). Twenty-four HDM-sensitized children with persistent AR were enrolled and treated with SCIT (n=13) or SLIT (n=11) for 3 months. Relative miR-146a and Foxp3 mRNA expression, the TRAF6 protein level, and the ratio of post-treatment to baseline IL-10+CD4+ T cells between the SCIT and SLIT groups were examined in the peripheral blood mononuclear cells (PBMCs) of AR patients using quantitative reverse transcription polymerase chain reaction (qRT-PCR), flow cytometry, and Western blot analysis, respectively. Serum levels of IL-5 and IL-10 were determined using ELISA. After 3 months of SIT, both the TNSS and INSS scores were significantly decreased compared to the baseline value (P<0.01). The relative expression of miR-146a and Foxp3 mRNA was significantly increased after both SCIT and SLIT (P<0.01). The ratio of post-treatment to baseline IL-10⁺CD4⁺ T cells and the serum IL-10 level were significantly increased in both the SCIT and SLIT groups (P<0.01), whereas the TRAF6 protein level and serum IL-5 level were significantly decreased (P<0.01). No significant differences in these biomarkers were observed between the SCIT and SLIT groups. Our findings suggest that miR-146a and its related biomarkers may be comparably modulated after both SCIT and SLIT, highlighting miR-146a as a potential therapeutic target for the improved management of AR.

Docosahexaenoic acid inhibits the growth of hormone-dependent prostate cancer cells by promoting the degradation of the androgen receptor.

PubMed

Hu, Zhimei; Qi, Haixia; Zhang, Ruixue; Zhang, Kun; Shi, Zhemin; Chang, Yanan; Chen, Linfeng; Esmaeili, Mohsen; Baniahmad, Aria; Hong, Wei

2015-09-01

Epidemiological and preclinical data have demonstrated the preventative effects of ω-3 polyunsaturated fatty acids, including docosahexaenoic acid (DHA), on prostate cancer. However, there are inconsistencies in these previous studies and the underlying mechanisms remain to be elucidated. In the present study, the androgen receptor (AR), which is a transcription factor involved in cell proliferation and prostate carcinogenesis, was identified as a target of DHA. It was revealed that DHA inhibited hormone‑dependent growth of LNCaP prostate cancer cells. Reverse transcription-quantitative polymerase chain reaction analysis revealed that treatment with DHA caused no alteration in the transcribed mRNA expression levels of the AR gene. However, immunoblotting revealed that this treatment reduces the protein expression level of the AR. The androgen‑induced genes were subsequently repressed by treatment with DHA. It was demonstrated that DHA exhibits no effect on the translation process of the AR, however, it promotes the proteasome‑mediated degradation of the AR. Therefore, the present study provided a novel mechanism by which DHA exhibits an inhibitory effect on growth of prostate cancer cells.

Myofibroblast androgen receptor expression determines cell survival in co-cultures of myofibroblasts and prostate cancer cells in vitro.

PubMed

Palethorpe, Helen M; Leach, Damien A; Need, Eleanor F; Drew, Paul A; Smith, Eric

2018-04-10

Fibroblasts express androgen receptor (AR) in the normal prostate and during prostate cancer development. We have reported that loss of AR expression in prostate cancer-associated fibroblasts is a poor prognostic indicator. Here we report outcomes of direct and indirect co-cultures of immortalised AR-positive (PShTert-AR) or AR-negative (PShTert) myofibroblasts with prostate cancer cells. In the initial co-cultures the AR-negative PC3 cell line was used so AR expression and signalling were restricted to the myofibroblasts. In both direct and indirect co-culture with PShTert-AR myofibroblasts, paracrine signalling to the PC3 cells slowed proliferation and induced apoptosis. In contrast, PC3 cells proliferated with PShTert myofibroblasts irrespective of the co-culture method. In direct co-culture PC3 cells induced apoptosis in and destroyed PShTerts by direct signalling. Similar results were seen in direct co-cultures with AR-negative DU145 and AR-positive LNCaP and C4-2B prostate cancer cell lines. The AR ligand 5α-dihydrotestosterone (DHT) inhibited the proliferation of the PShTert-AR myofibroblasts, thereby reducing the extent of their inhibitory effect on cancer cell growth. These results suggest loss of stromal AR would favour prostate cancer cell growth in vivo , providing an explanation for the clinical observation that reduced stromal AR is associated with a poorer outcome.

Myofibroblast androgen receptor expression determines cell survival in co-cultures of myofibroblasts and prostate cancer cells in vitro

PubMed Central

Palethorpe, Helen M.; Leach, Damien A.; Need, Eleanor F.; Drew, Paul A.; Smith, Eric

2018-01-01

Fibroblasts express androgen receptor (AR) in the normal prostate and during prostate cancer development. We have reported that loss of AR expression in prostate cancer-associated fibroblasts is a poor prognostic indicator. Here we report outcomes of direct and indirect co-cultures of immortalised AR-positive (PShTert-AR) or AR-negative (PShTert) myofibroblasts with prostate cancer cells. In the initial co-cultures the AR-negative PC3 cell line was used so AR expression and signalling were restricted to the myofibroblasts. In both direct and indirect co-culture with PShTert-AR myofibroblasts, paracrine signalling to the PC3 cells slowed proliferation and induced apoptosis. In contrast, PC3 cells proliferated with PShTert myofibroblasts irrespective of the co-culture method. In direct co-culture PC3 cells induced apoptosis in and destroyed PShTerts by direct signalling. Similar results were seen in direct co-cultures with AR-negative DU145 and AR-positive LNCaP and C4-2B prostate cancer cell lines. The AR ligand 5α-dihydrotestosterone (DHT) inhibited the proliferation of the PShTert-AR myofibroblasts, thereby reducing the extent of their inhibitory effect on cancer cell growth. These results suggest loss of stromal AR would favour prostate cancer cell growth in vivo, providing an explanation for the clinical observation that reduced stromal AR is associated with a poorer outcome. PMID:29721186

CrMAPK3 regulates the expression of iron-deficiency-responsive genes in Chlamydomonas reinhardtii.

PubMed

Fei, Xiaowen; Yu, Junmei; Li, Yajun; Deng, Xiaodong

2017-05-16

Under iron-deficient conditions, Chlamydomonas exhibits high affinity for iron absorption. Nevertheless, the response, transmission, and regulation of downstream gene expression in algae cells have not to be investigated. Considering that the MAPK pathway is essential for abiotic stress responses, we determined whether this pathway is involved in iron deficiency signal transduction in Chlamydomonas. Arabidopsis MAPK gene sequences were used as entry data to search for homologous genes in Chlamydomonas reinhardtii genome database to investigate the functions of mitogen-activated protein kinase (MAPK) gene family in C. reinhardtii under iron-free conditions. Results revealed 16 C. reinhardtii MAPK genes labeled CrMAPK2-CrMAPK17 with TXY conserved domains and low homology to MAPK in yeast, Arabidopsis, and humans. The expression levels of these genes were then analyzed through qRT-PCR and exposure to high salt (150 mM NaCl), low nitrogen, or iron-free conditions. The expression levels of these genes were also subjected to adverse stress conditions. The mRNA levels of CrMAPK2, CrMAPK3, CrMAPK4, CrMAPK5, CrMAPK6, CrMAPK8, CrMAPK9, and CrMAPK11 were remarkably upregulated under iron-deficient stress. The increase in CrMAPK3 expression was 43-fold greater than that in the control. An RNA interference vector was constructed and transformed into C. reinhardtii 2A38, an algal strain with an exogenous FOX1:ARS chimeric gene, to silence CrMAPK3. After this gene was silenced, the mRNA levels and ARS activities of FOX1:ARS chimeric gene and endogenous CrFOX1 were decreased. The mRNA levels of iron-responsive genes, such as CrNRAMP2, CrATX1, CrFTR1, and CrFEA1, were also remarkably reduced. CrMAPK3 regulates the expression of iron-deficiency-responsive genes in C. reinhardtii.

Early postnatal maternal separation causes alterations in the expression of β3-adrenergic receptor in rat adipose tissue suggesting long-term influence on obesity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miki, Takanori, E-mail: mikit@med.kagawa-u.ac.jp; Liu, Jun-Qian; Ohta, Ken-ichi

Highlights: •High-fat diet intake following maternal separation did not cause body weight gain. •However, levels of metabolism-related molecules in adipose tissue were altered. •Increased levels of prohibitin mRNA in white fat were observed. •Attenuated levels of β3-adrenergic receptor mRNA were observed in brown fat. •Such alterations in adipose tissue may contribute to obesity later in life. -- Abstract: The effects of early postnatal maternal deprivation on the biological characteristics of the adipose tissue later in life were investigated in the present study. Sprague–Dawley rats were classified as either maternal deprivation (MD) or mother-reared control (MRC) groups. MD was achieved bymore » separating the rat pups from their mothers for 3 h each day during the 10–15 postnatal days. mRNA levels of mitochondrial uncoupling protein 1 (UCP-1), β3-adrenergic receptor (β3-AR), and prohibitin (PHB) in the brown and white adipose tissue were determined using real-time RT-PCR analysis. UCP-1, which is mediated through β3-AR, is closely involved in the energy metabolism and expenditure. PHB is highly expressed in the proliferating tissues/cells. At 10 weeks of age, the body weight of the MRC and MD rats was similar. However, the levels of the key molecules in the adipose tissue were substantially altered. There was a significant increase in the expression of PHB mRNA in the white adipose tissue, while the β3-AR mRNA expression decreased significantly, and the UCP-1 mRNA expression remained unchanged in the brown adipose tissue. Given that these molecules influence the mitochondrial metabolism, our study indicates that early postnatal maternal deprivation can influence the fate of adipose tissue proliferation, presumably leading to obesity later in life.« less

A single oral dose of flavan-3-ols enhances energy expenditure by sympathetic nerve stimulation in mice.

PubMed

Kamio, Naoya; Suzuki, Takuma; Watanabe, Yuto; Suhara, Yoshitomo; Osakabe, Naomi

2016-02-01

Numerous clinical studies have found that ingestion of chocolate reduces the risk of metabolic syndrome, however, the mechanisms were remain unclear. We have reported that a single dose of a flavan-3-ol fraction derived from cocoa (FL) enhanced energy expenditure (EE) and increased the mRNA expression levels of uncoupling proteins (UCPs) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), and the protein level of phosphorylated AMP-activated protein kinase (AMPK)α in tissues, along with plasma adrenaline level. In the present study, we examined whether the EE enhancing activity of FL is mediated by adrenergic effect using several adrenalin receptor (AR) blockers. In the first study, mice were butoxamine, as β2AR blocker, with vehicle or 10mg/kg FL orally. We found that pretreatment with butoxamine prevented the increases of EE, the mRNA expression of UCP-3, and phosphorylated AMPKα that were induced in the gastrocnemius muscle of mice by 10mg/kg FL. Secondly, mice were given SR52930, as β3AR blocker. Pretreatment with SR52930 prevented the increases of EE, the mRNA expression of UCP-3, and phosphorylated AMPKα that were induced in the gastrocnemius muscle of mice by 10mg/kg FL. Pretreatment with a combination of both blockers also reduced the increments in mRNA expression levels of UCPs and PGC-1α, however, phosphorylated AMPKα in skeletal muscle was rather increased. These results suggest that the ability of a single oral dose of FL to enhance metabolic activity is mediated by sympathetic nerve system (SNS). Copyright © 2015. Published by Elsevier Inc.

Specific glycogen synthase kinase-3 inhibition reduces neuroendocrine markers and suppresses neuroblastoma cell growth.

PubMed

Carter, Yvette M; Kunnimalaiyaan, Selvi; Chen, Herbert; Gamblin, T Clark; Kunnimalaiyaan, Muthusamy

2014-05-01

Neuroblastoma is a common neuroendocrine (NE) tumor that presents in early childhood, with a high incidence of malignancy and recurrence. The glycogen synthase kinase-3 (GSK-3) pathway is a potential therapeutic target, as this pathway has been shown to be crucial in the management of other NE tumors. However, it is not known which isoform is necessary for growth inhibition. In this study, we investigated the effect of the GSK-3 inhibitor AR-A014418 on the different GSK-3 isoforms in neuroblastoma. NGP and SH-5Y-SY cells were treated with 0-20 μM of AR-A014418 and cell viability was measured by MTT assay. Expression levels of NE markers CgA and ASCL1, GSK-3 isoforms, and apoptotic markers were analyzed by western blot. Neuroblastoma cells treated with AR-A014418 had a significant reduction in growth at all doses and time points (P<0.001). A reduction in growth was noted in cell lines on day 6, with 10 μM (NGP-53% vs. 0% and SH-5Y-SY-38% vs. 0%, P<0.001) treatment compared to control, corresponding with a noticeable reduction in tumor marker ASCL1 and CgA expression. Treatment of neuroblastoma cell lines with AR-A014418 reduced the level of GSK-3α phosphorylation at Tyr279 compared to GSK-3β phosphorylation at Tyr216, and attenuated growth via the maintenance of apoptosis. This study supports further investigation to elucidate the mechanism(s) by which GSK-3α inhibition downregulates the expression of NE tumor markers and growth of neuroblastoma.

AR-12 suppresses dengue virus replication by down-regulation of PI3K/AKT and GRP78.

PubMed

Chen, Hsin-Hsin; Chen, Chien-Chin; Lin, Yee-Shin; Chang, Po-Chun; Lu, Zi-Yi; Lin, Chiou-Feng; Chen, Chia-Ling; Chang, Chih-Peng

2017-06-01

Dengue virus (DENV) infection has become a public health issue of worldwide concern and is a serious health problem in Taiwan, yet there are no approved effective antiviral drugs to treat DENV. The replication of DENV requires both viral and cellular factors. Targeting host factors may provide a potential antiviral strategy. It has been known that up-regulation of PI3K/AKT signaling and GRP78 by DENV infection supports its replication. AR-12, a celecoxib derivative with no inhibiting activity on cyclooxygenase, shows potent inhibitory activities on both PI3K/AKT signaling and GRP78 expression levels, and recently has been found to block the replication of several hemorrhagic fever viruses. However the efficacy of AR-12 in treating DENV infection is still unclear. Here, we provide evidence to show that AR-12 is able to suppress DENV replication before or after virus infection in cell culture and mice. The antiviral activities of AR-12 are positive against infection of the four different DENV serotypes. AR-12 significantly down-regulates the PI3K/AKT activity and GRP78 expression in DENV infected cells whereas AKT and GRP78 rescue are able to attenuate anti-DENV effect of AR-12. Using a DENV-infected suckling mice model, we further demonstrate that treatment of AR-12 before or after DENV infection reduces virus replication and mice mortality. In conclusion, we uncover the potential efficacy of AR-12 as a novel drug for treating dengue. Copyright © 2017 Elsevier B.V. All rights reserved.

Roles of methyltrienolone (R1881) in AKTs and AR expression patterns of cultured granulosa-lutein cells.

PubMed

Nekoonam, Saeid; Naji, Mohammad; Mortezaee, Keywan; Amidi, Fardin

2018-05-11

AR-mediated androgen signaling plays a key role in female reproductive system. Granulosa-lutein cells (GCs) are the main sites for expression of androgen receptor (AR). There is also a close relation between AKT signaling and AR. Here, we assayed the role for a synthetic AR ligand methyltrienolone (R1881) in expressions of AKTs and AR. Controlled ovarian hyperstimulation (COH) was performed in 20 normal women. Mural GCs were isolated by filtration method, cultured, and passaged. Then, the cells were starved for 48 h with 10% charcoal stripped FBS. The cells were then treated with R1881, bicalutamide (AR blocker), LY294002 (PI3K/AKT pathway blocker), and combination of them for 48 h. Finally, GCs were evaluated for quantitative real-time PCR analysis of AKT1, AKT2, AKT3, and AR, and also Western blot assessment of total AKT and phosphorylated AKT (p-AKT) [Ser473 and Thr308]. Addition of R1881 to the GCs culture showed high expressions of AKT1, AKT2, and AKT3 (P ≤ 0.05 vs LY294002 group and bicalutamide group). Expressions of AKT1 and AKT2 were decreased in the GCs under exposure to bicalutamide or LY294002 (P ≤ 0.05 vs R1881). AKT1, AKT2, and AKT3 showed decreased rates of expressions in the LY294002 + bicalutamide group (P ≤ 0.05 vs R1881). AR, total AKT and p-AKT showed no significant differences between groups. Our findings indicate that 46 h exposure with R1881 could affect AKTs expressions in the GCs of pre-ovulatory phase, but it cannot promote AR expression and AKTs activation. © 2018 Wiley Periodicals, Inc.

Androgen Regulation of 5α-Reductase Isoenzymes in Prostate Cancer: Implications for Prostate Cancer Prevention

PubMed Central

Li, Jin; Ding, Zhiyong; Wang, Zhengxin; Lu, Jing-Fang; Maity, Sankar N.; Navone, Nora M.; Logothetis, Christopher J.; Mills, Gordon B.; Kim, Jeri

2011-01-01

The enzyme 5α-reductase, which converts testosterone to dihydrotestosterone (DHT), performs key functions in the androgen receptor (AR) signaling pathway. The three isoenzymes of 5α-reductase identified to date are encoded by different genes: SRD5A1, SRD5A2, and SRD5A3. In this study, we investigated mechanisms underlying androgen regulation of 5α-reductase isoenzyme expression in human prostate cells. We found that androgen regulates the mRNA level of 5α-reductase isoenzymes in a cell type–specific manner, that such regulation occurs at the transcriptional level, and that AR is necessary for this regulation. In addition, our results suggest that AR is recruited to a negative androgen response element (nARE) on the promoter of SRD5A3 in vivo and directly binds to the nARE in vitro. The different expression levels of 5α-reductase isoenzymes may confer response or resistance to 5α-reductase inhibitors and thus may have importance in prostate cancer prevention. PMID:22194926

Wnt/Beta-Catenin, Foxa2, and CXCR4 Axis Controls Prostate Cancer Progression

DTIC Science & Technology

2013-07-01

NT1 cells with or without Foxa2 were cultured in 5% strip medium in the presence or absence of androgen. Cell growth curve was measured by WTS...assays. Over-expression of Foxa2 increased NT1 cell growth even in the absence of androgen. Figure 3. knocking-down Foxa2 did not affect PC3 cell ...in NeoTag1 cells decreased AR level; however, AR 6 activity did not change. Foxa2 target genes were up-regulated in NT1 /Foxa2 over-expressing

X chromosome gain is related to increased androgen receptor expression in male breast cancer.

PubMed

Di Oto, Enrico; Biserni, Giovanni B; Varga, Zsuzsanna; Morandi, Luca; Cucchi, Maria C; Masetti, Riccardo; Foschini, Maria P

2018-05-25

X chromosome gain has been previously described in male breast cancer (MBC). Androgen receptor (AR) gene is located on X chromosome. The aim of this study was to investigate the role of the X chromosome gain in the development of MBC and its relation with AR gene copy number and expression.The X chromosome status was assessed in 66 cases of male invasive and in situ duct breast carcinoma, in 34 cases of gynecomastia associated with cancer, and in 11 cases of tumor-free gynecomastia. Cases were tested by fluorescence in situ hybridization (FISH) to assess the X chromosome status and AR amplification. AR expression was studied by immunohistochemistry (IHC). In addition, AR methylation status was assessed.X chromosome gain was observed in 74.7% of invasive duct carcinoma, in 20.6% of in situ duct carcinoma, and in 14.6% of gynecomastia when associated with cancer, while all cases of tumor-free gynecomastia showed wild X chromosome asset. AR gene copy number when increased paralleled the number of X chromosomes. AR IHC expression was observed in 100% of MBC tested. AR gene methylation status revealed low level or absence of methylation.These data suggest that X chromosome can play a role in the neoplastic transformation of male breast epithelium. X chromosome gain is paralleled by AR gene polysomy. Polysomic AR genes show low methylation levels and high AR protein expression on IHC. These data should be taken into consideration for MBC treatment planning.

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells.

PubMed

El-Heliebi, Amin; Hille, Claudia; Laxman, Navya; Svedlund, Jessica; Haudum, Christoph; Ercan, Erkan; Kroneis, Thomas; Chen, Shukun; Smolle, Maria; Rossmann, Christopher; Krzywkowski, Tomasz; Ahlford, Annika; Darai, Evangelia; von Amsberg, Gunhild; Alsdorf, Winfried; König, Frank; Löhr, Matthias; de Kruijff, Inge; Riethdorf, Sabine; Gorges, Tobias M; Pantel, Klaus; Bauernhofer, Thomas; Nilsson, Mats; Sedlmayr, Peter

2018-03-01

Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices. © 2017 American Association for Clinical Chemistry.

Transcriptional activation of LON Gene by a new form of mitochondrial stress: A role for the nuclear respiratory factor 2 in StAR overload response (SOR).

PubMed

Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Isaac, Sara; Eden, Amir; Lauria, Ines; Langer, Thomas; Orly, Joseph

2015-06-15

High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peri-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Aldose reductase mediates retinal microglia activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Kun-Che; Shieh, Biehuoy; Petrash, J. Mark, E-mail: mark.petrash@ucdenver.edu

Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1{sup GFP} mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migrationmore » in vivo. When tested on an AR{sup WT} background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy. - Highlights: • AR inhibition prevents retinal microglial activation. • Endotoxin-induced ocular cytokine production is reduced in AR null mice. • Overexpression of AR spontaneously induces retinal microglial activation.« less

Identification of Androgen Receptor-Specific Enhancer RNAs

DTIC Science & Technology

2017-08-01

determine the role of AR- eRNA-regulation of DHRS4- AS1 in castration resistance, we knocked out DHRS4-AS1 by CRISPR /Cas9 technology in 22Rv1...DHRS4-AS1 as a regulator for AR3 expression. A, Knockout of DHRS4-AS1 by CRISPR /Cas9. B, DHRS4-AS1 knockout suppresses AR3 mRNA level as determined by...DHRS4-AS1 KO by CRISPR /Cas9  DHRS4-AS1 KO and rescue assays suggest that DHRS4-AS1 can promote castration resistance Reportable Outcomes A manuscript

A Novel Role for the Receptor of the Complement Cleavage Fragment C5a, C5aR1, in CCR5-Mediated Entry of HIV into Macrophages.

PubMed

Moreno-Fernandez, Maria E; Aliberti, Julio; Groeneweg, Sander; Köhl, Jörg; Chougnet, Claire A

2016-04-01

The complement system is an ancient pattern recognition system that becomes activated during all stages of HIV infection. Previous studies have shown that C5a can enhance the infection of monocyte-derived macrophages and T cells indirectly through the production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α and the attraction of dendritic cells. C5a exerts its multiple biologic functions mainly through activation of C5a receptor 1 (C5aR1). Here, we assessed the role of C5aR1 as an enhancer of CCR5-mediated HIV infection. We determined CCR5 and C5aR1 heterodimer formation in myeloid cells and the impact of C5aR1 blockade on HIV entry and genomic integration. C5aR1/CCR5 heterodimer formation was identified by immunoprecipitation and western blotting. THP-1 cells and monocyte-derived macrophages (MDM) were infected by R5 laboratory strains or HIV pseudotyped for the vesicular stomatitis virus (VSV) envelope. Levels of integrated HIV were measured by quantitative PCR after targeting of C5aR1 by a C5aR antagonist, neutralizing C5aR1 monoclonal antibody (mAb) or hC5a. C5aR1 was also silenced by specific siRNA prior to viral entry. We found that C5aR1 forms heterodimers with the HIV coreceptor CCR5 in myeloid cells. Targeting C5aR1 significantly decreased integration by R5 viruses but not by VSV-pseudotyped viruses, suggesting that C5aR1 is critical for viral entry. The level of inhibition achieved with C5aR1-blocking reagents was comparable to that of CCR5 antagonists. Mechanistically, C5aR1 targeting decreased CCR5 expression. MDM from CCR5Δ32 homozygous subjects expressed levels of C5aR1 similar to CCR5 WT individuals, suggesting that mere C5aR1 expression is not sufficient for HIV infection. HIV appeared to preferentially enter THP-1 cells expressing high levels of both C5aR1 and CCR5. Targeted reduction of C5aR1 expression in such cells reduced HIV infection by ~50%. Our data thus suggest that C5aR1 acts as an enhancer of CCR5-mediated HIV entry into macrophages, the targeting of which may prove useful to reduce HIV infection by R5 strains.

Transcription factor Brn-3α mRNA in cancers, relationship with AR, ER receptors and AKT/m-TOR pathway components

NASA Astrophysics Data System (ADS)

Spirina, L. V.; Gorbunov, A. K.; Chigevskaya, S. Y.; Usynin, Y. A.; Kondakova, I. V.; Slonimskaya, E. M.; Usynin, E. A.; Choinzonov, E. L.; Zaitseva, O. S.

2017-09-01

Transcription factors POU4F1 (neurogenic factor Brn-3α) play a pivotal role in cancers development. The aim of the study was to reveal the Brn-3α expression, AR, ER expression in cancers development, association with AKT/mTOR pathway activation. 30 patients with locally advanced prostate cancer, 20 patients with papillary thyroid cancer, T2-3N0-1M0 stages and 40 patients with renal cell cancer T2-3N0M0-1 were involved into the study. The expressions of Brn-3α, AR, ERα, components of AKT/m-TOR signaling pathway genes were performed by real-time PCR. The dependence of Brn-3α expression on mRNA levels of steroid hormone receptors and components of AKT/m-TOR signaling pathway in studied cancers were shown. High levels of mRNA of nuclear factor, steroid hormone receptors were found followed by the activation of this signaling pathway in prostate cancer tissue. The reduction of transcription factor Brn-3α was accompanied with tumor invasive growth with increasing rates of AR, ER and 4E-BP1 mRNA. Thyroid cancer development happened in a case of a Brn-3α and steroid hormone receptors decrease. The activation of AKT/m-TOR signaling pathway was established in the metastatic renal cancers, accompanied with the increase of ER mRNA. But there was no correlation between the steroid receptor and Brn-3α. One-direction changes of Brn-3α were observed in the development of prostate and thyroid cancer due to its effect on the steroid hormone receptors and the activation of AKT/m-TOR signaling pathway components. The influence of this factor on the development of the kidney cancer was mediated through m-TOR activity modifications, the key enzyme of oncogenesis.

Calcium affecting protein expression in longan under simulated acid rain stress.

PubMed

Pan, Tengfei; Li, Yongyu; Ma, Cuilan; Qiu, Dongliang

2015-08-01

Longan (Dimocarpus longana Lour. cv. Wulongling) of uniform one-aged seedlings grown in pots were selected to study specific proteins expressed in leaves under simulated acid rain (SiAR) stress and exogenous Ca(2+) regulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that there was a protein band specifically expressed under SiAR of pH 2.5 stress for 15 days with its molecular weight of about 23 kD. A 17 kD protein band specifically expressed after SiAR stress 5 days. Compared with pH 2.5, the pH 3.5 of SiAR made a less influence to protein expression. Two-dimensional electrophoresis (2-DE) results showed that six new specific proteins including C4 (20.2 kD pI 6.0), F (24 kD pI 6.35), B3 (22.3 kD pI 6.35), B4 (23.5 kD pI 6.5), C5 (21.8 kD pI 5.6), and C6 (20.2 kD pI 5.6) specifically expressed. C4 always expressed during SiAR stress. F expressed under the stress of pH 2.5 for 15 days and expressed in all pH SiAR stress for 20 days. The expression of proteins including B3, C5, and C6 was related to pH value and stress intensity of SiAR. The expression of B4 resulted from synergistic effects of SiAR and Ca. The expression of G1 (Mr 19.3 kD, pI 4.5), G2 (Mr 17.8 kD, pI 4.65), G3 (Mr 16.6 kD, pI 4.6), and G4 (Mr 14.7 kD, pI 4.4) enhanced under the treatment of 5 mM ethylene glycol tetraacetic acid (EGTA) and 2 mM chlorpromazine (CPZ). These proteins showed antagonistic effects and might be relative to the Ca-calmodulin (Ca-CaM) system of longan in response to SiAR stress.

Adrenergic Receptors in Individual Ventricular Myocytes: The Beta-1 and Alpha-1B Are in All Cells, the Alpha-1A Is in a Subpopulation, and the Beta-2 and Beta-3 Are Mostly Absent.

PubMed

Myagmar, Bat-Erdene; Flynn, James M; Cowley, Patrick M; Swigart, Philip M; Montgomery, Megan D; Thai, Kevin; Nair, Divya; Gupta, Rumita; Deng, David X; Hosoda, Chihiro; Melov, Simon; Baker, Anthony J; Simpson, Paul C

2017-03-31

It is unknown whether every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), β1, β2, β3, α1A, and α1B. The β1 and β2 are thought to be the dominant myocyte ARs. Quantify the 5 cardiac ARs in individual ventricular myocytes. We studied ventricular myocytes from wild-type mice, mice with α1A and α1B knockin reporters, and β1 and β2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of extracellular signal-regulated kinase and phospholamban), and contraction. We found that the β1 and α1B were present in all myocytes. The α1A was present in 60%, with high levels in 20%. The β2 and β3 were detected in only ≈5% of myocytes, mostly in different cells. In intact heart, 30% of total β-ARs were β2 and 20% were β3, both mainly in nonmyocytes. The dominant ventricular myocyte ARs present in all cells are the β1 and α1B. The β2 and β3 are mostly absent in myocytes but are abundant in nonmyocytes. The α1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with β1 and α1B only; 60% that also have the α1A; and 5% each that also have the β2 or β3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms. © 2017 American Heart Association, Inc.

Aromatase Deficient Female Mice Demonstrate Altered Expression of Molecules Critical for Renal Calcium Reabsorption

NASA Astrophysics Data System (ADS)

Öz, Orhan K.; Hajibeigi, Asghar; Cummins, Carolyn; van Abel, Monique; Bindels, René J.; Kuro-o, Makoto; Pak, Charles Y. C.; Zerwekh, Joseph E.

2007-04-01

The incidence of kidney stones increases in women after the menopause, suggesting a role for estrogen deficiency. In order to determine if estrogen may be exerting an effect on renal calcium reabsorption, we measured urinary calcium excretion in the aromatase-deficient female mouse (ArKO) before and following estrogen therapy. ArKO mice had hypercalciuria that corrected during estrogen administration. To evaluate the mechanism by which estrogen deficiency leads to hypercalciuria, we examined the expression of several proteins involved in distal tubule renal calcium reabsorption, both at the message and protein levels. Messenger RNA levels of TRPV5, TRPV6, calbindin-D28K, the Na+/Ca++ exchanger (NCX1), and the plasma membrane calcium ATPase (PMCA1b) were significantly decreased in kidneys of ArKO mice. On the other hand, klotho mRNA levels were elevated in kidneys of ArKO mice. ArKO renal protein extracts had lower levels of calbindin-D28K but higher levels of the klotho protein. Immunochemistry demonstrated increased klotho expression in ArKO kidneys. Estradiol therapy normalized the expression of TRPV5, calbindin-D28K, PMCA1b and klotho. Taken together, these results demonstrate that estrogen deficiency produced by aromatase inactivation is sufficient to produce a renal leak of calcium and consequent hypercalciuria. This may represent one mechanism leading to the increased incidence of kidney stones following the menopause in women.

The expression of β3-adrenoceptors and their function in the human prostate.

PubMed

Suzuki, Takahisa; Otsuka, Atsushi; Matsumoto, Rikiya; Furuse, Hiroshi; Ozono, Seiichiro

2016-02-01

Little is known about β3-adrenoceptor (AR) expression and function in human prostate. We examined the expression and distribution of β-AR subtypes in normal prostate and benign prostatic hyperplasia (BPH) tissues, and investigated which selective β-AR subtype agonist was most involved in the relaxation of isolated human prostate strips. Messenger RNA (mRNA) expression for β1-, β2-, and β3 -ARs was investigated using reverse transcriptase-polymerase chain reactions (RT-PCR). Quantitative analysis of mRNA expression of β-AR subtypes between normal prostate and BPH tissues was performed using quantitative RT-PCR (qPCR). Distributions were examined by immunohistochemistry (IHC). Strips of human normal prostate or BPH were suspended in organ baths and exposed to isoproterenol, dobutamine, procaterol, and TRK-380 to investigate their relaxant effects on KCl-induced contractions, and their inhibitory effects on electrical field stimulation (EFS)-induced contractions. We confirmed the presence of mRNA for β1-, β2-, and β3-ARs both in normal prostate and in BPH tissues. For β3-AR, mRNA expression in BPH tissues was significantly higher than in normal prostate tissues, but there was no significant difference in β1- and β2-AR expression between normal and BPH tissues. IHC revealed differences in staining intensity between smooth muscle cells and glandular cells, with different proportions for different β-AR subtypes. Staining of β3-AR was particularly intense in smooth muscle cells as opposed to glandular cells. Isoproterenol and TRK-380 significantly decreased the tone of KCl-induced contractions of the normal prostate strips. The rank order of relaxant effects was isoproterenol > TRK-380 > procaterol > dobutamine. All selective β-AR agonists significantly decreased the amplitude of EFS-induced contractions of the normal prostate strips. The rank order of inhibitory effects was isoproterenol > dobutamine >TRK-380 > procaterol. In BPH strips, all selective β-AR agonists showed no significant relaxant or inhibitory effects on KCl- or EFS-induced contractions. β3 -AR is abundant in human prostate smooth muscle, whose relaxation is mediated by β1- and β3-AR stimulation. β3-AR agonists may have clinical use in the treatment of male non-BPH patients or neurogenic bladder patients with voiding dysfunction. © 2015 Wiley Periodicals, Inc.

Coagulation factor VII is regulated by androgen receptor in breast cancer.

PubMed

Naderi, Ali

2015-02-01

Androgen receptor (AR) is widely expressed in breast cancer; however, there is limited information on the key molecular functions and gene targets of AR in this disease. In this study, gene expression data from a cohort of 52 breast cancer cell lines was analyzed to identify a network of AR co-expressed genes. A total of 300 genes, which were significantly enriched for cell cycle and metabolic functions, showed absolute correlation coefficients (|CC|) of more than 0.5 with AR expression across the dataset. In this network, a subset of 35 "AR-signature" genes were highly co-expressed with AR (|CC|>0.6) that included transcriptional regulators PATZ1, NFATC4, and SPDEF. Furthermore, gene encoding coagulation factor VII (F7) demonstrated the closest expression pattern with AR (CC=0.716) in the dataset and factor VII protein expression was significantly associated to that of AR in a cohort of 209 breast tumors. Moreover, functional studies demonstrated that AR activation results in the induction of factor VII expression at both transcript and protein levels and AR directly binds to a proximal region of F7 promoter in breast cancer cells. Importantly, AR activation in breast cancer cells induced endogenous factor VII activity to convert factor X to Xa in conjunction with tissue factor. In summary, F7 is a novel AR target gene and AR activation regulates the ectopic expression and activity of factor VII in breast cancer cells. These findings have functional implications in the pathobiology of thromboembolic events and regulation of factor VII/tissue factor signaling in breast cancer. Copyright © 2014 Elsevier Inc. All rights reserved.

Steroidogenic acute regulatory protein (StAR) gene expression construct: Development, nanodelivery and effect on reproduction in air-breathing catfish, Clarias batrachus.

PubMed

Rathor, Pravesh Kumar; Bhat, Irfan Ahmad; Rather, Mohd Ashraf; Gireesh-Babu, Pathakota; Kumar, Kundan; Purayil, Suresh Babu Padinhate; Sharma, Rupam

2017-11-01

Steroidogenic acute regulatory protein (StAR) is responsible for the relocation of cholesterol across mitochondrial membrane in vertebrates and is, therefore, a key factor in regulating the rate and timing of steroidogenesis. In the present study, we developed chitosan nanoparticle (CNP) conjugated StAR gene construct (CNP-pcDNA4-StAR) in a eukaryotic expression vector, pcDNA4/HisMax A. CNPs of 135.4nm diameter, 26.7mV zeta potential and 0.381 polydispersity index were used for conjugation. The loading efficiency (LE) of pcDNA4-StAR construct with CNPs was found to be 86%. After the 24h of intramuscular injection, the CNP-pcDNA4-StAR plasmid could be detected from testis, brain, kidney and muscle tissues of Clarias batrachus. The transcript levels of important reproductive genes viz. cyp11a1, cyp17a1, 3β-hsd, 17β-hsd and cyp19a1 in CNP-pcDNA4-StAR treated group were initially low up to 24h, but significantly increased subsequently up to 120h. In naked pcDNA4-StAR treated group, the mRNA level of 3β-hsd, 17β-hsd and cyp19a1 increased initially up to 24h, while cyp11a1 and cyp17a1 increased up to 48h and then started declining. Similar results were obtained for 11-Ketotestosterone and 17β-estradiol. The results indicate relatively long lasting effects of nano-conjugated construct compared to the construct alone. Furthermore, the histopathology of gonads and liver authenticates its possible role in the gonadal development in fish without any adverse effect. Copyright © 2017 Elsevier B.V. All rights reserved.

Cotargeting of Androgen Synthesis and Androgen Receptor Expression as a Novel Treatment for Castration Resistant Prostate Cancer

DTIC Science & Technology

2017-08-01

expression of both AR and AR-V7 in CRPC cells, and that knockdown or inhibition of PRMT5 suppresses growth of CRPC cells. These results support our...correlates with the expression of AR [9]. Preliminary data strongly suggest that PRMT5 regulates prostate cancer cell growth through epigenetic...findings as follow. 3B-1. Inhibition of PRMT5 suppresses cell growth and the expression of AR and AR-V7 in CRPC cells. We established inducible

Aurora A regulates expression of AR-V7 in models of castrate resistant prostate cancer.

PubMed

Jones, Dominic; Noble, Martin; Wedge, Steve R; Robson, Craig N; Gaughan, Luke

2017-02-16

Androgen receptor variants (AR-Vs) provide a mechanism of therapy evasion in castrate-resistant prostate cancer (CRPC), yet mechanisms of regulation remain largely unknown. Here we investigate the role of Aurora A kinase on AR-Vs in models of CRPC and show depletion of Aurora A reduces AR-V target gene expression. Importantly, knockdown of Aurora A reconfigures splicing of AR pre-mRNA to discriminately down-regulate synthesis of AR-V transcripts, including AR-V7, without effecting full-length AR mRNA; and as a consequence, AR-V-driven proliferation and survival of CRPC cells is markedly reduced. Critically, these effects are reproduced by Aurora A inhibition. We show that Aurora A levels increase in advanced disease and AURKA is an AR-V target gene demonstrating a positive feedback mechanism of androgenic signalling in CRPC. In all, our data suggests that Aurora A plays a pivotal role in regulation of AR-V7 expression and represents a new therapeutic target in CRPC.

Aurora A regulates expression of AR-V7 in models of castrate resistant prostate cancer

PubMed Central

Jones, Dominic; Noble, Martin; Wedge, Steve R.; Robson, Craig N.; Gaughan, Luke

2017-01-01

Androgen receptor variants (AR-Vs) provide a mechanism of therapy evasion in castrate-resistant prostate cancer (CRPC), yet mechanisms of regulation remain largely unknown. Here we investigate the role of Aurora A kinase on AR-Vs in models of CRPC and show depletion of Aurora A reduces AR-V target gene expression. Importantly, knockdown of Aurora A reconfigures splicing of AR pre-mRNA to discriminately down-regulate synthesis of AR-V transcripts, including AR-V7, without effecting full-length AR mRNA; and as a consequence, AR-V-driven proliferation and survival of CRPC cells is markedly reduced. Critically, these effects are reproduced by Aurora A inhibition. We show that Aurora A levels increase in advanced disease and AURKA is an AR-V target gene demonstrating a positive feedback mechanism of androgenic signalling in CRPC. In all, our data suggests that Aurora A plays a pivotal role in regulation of AR-V7 expression and represents a new therapeutic target in CRPC. PMID:28205582

Hormonal regulation of β-myosin heavy chain expression in the mouse left ventricle.

PubMed

Patrizio, Mario; Musumeci, Marco; Piccone, Ambra; Raggi, Carla; Mattei, Elisabetta; Marano, Giuseppe

2013-03-01

We investigated the influence of sex hormones on the expression of α- and β-cardiac myosin heavy chain isoforms (α-MHC and β-MHC) in C57bl/6 mice of both sexes under physiological and pathological conditions. In the left ventricles (LVs) of fertile female mice, β-MHC expression was tenfold higher compared with the age-matched males, whereas no difference was found in α-MHC expression. These differences disappeared after ovariectomy or in immature mice. We also found a sex-related difference in expression of β-adrenoceptors (β1-AR), as mRNA levels of this gene were 40% lower in fertile females compared with males of the same age but did not differ in prepubertal or ovariectomized animals. Interestingly, the deletion of both β1- and β2-ARs abolished sex difference of β-MHC expression, as mRNA levels in the LVs of knockout males were increased and reached values comparable to those of knockout females. Moreover, the β1-AR antagonist metoprolol induced about a threefold increase in β-MHC expression in adult male mice. The capability of gender to regulate β-MHC expression was also evaluated in the presence of hemodynamic overload. Thoracic aortic coarctation (TAC) produced cardiac hypertrophy along with a 12-fold increase in β-MHC and a 50% decrease in β1-AR expression in males but not in females, thus abolishing the gender difference observed in sham animals for such genes. By contrast, TAC did not change β2-AR expression. In conclusion, our results show that the expression of β-MHC and β1-AR in the LVs undergo gender-related and correlated changes under both physiological and pathological conditions and suggest a role of β1-AR-mediated signaling.

The Early Effects of Rapid Androgen Deprivation on Human Prostate Cancer.

PubMed

Shaw, Greg L; Whitaker, Hayley; Corcoran, Marie; Dunning, Mark J; Luxton, Hayley; Kay, Jonathan; Massie, Charlie E; Miller, Jodi L; Lamb, Alastair D; Ross-Adams, Helen; Russell, Roslin; Nelson, Adam W; Eldridge, Matthew D; Lynch, Andrew G; Ramos-Montoya, Antonio; Mills, Ian G; Taylor, Angela E; Arlt, Wiebke; Shah, Nimish; Warren, Anne Y; Neal, David E

2016-08-01

The androgen receptor (AR) is the dominant growth factor in prostate cancer (PCa). Therefore, understanding how ARs regulate the human transcriptome is of paramount importance. The early effects of castration on human PCa have not previously been studied 27 patients medically castrated with degarelix 7 d before radical prostatectomy. We used mass spectrometry, immunohistochemistry, and gene expression array (validated by reverse transcription-polymerase chain reaction) to compare resected tumour with matched, controlled, untreated PCa tissue. All patients had levels of serum androgen, with reduced levels of intraprostatic androgen at prostatectomy. We observed differential expression of known androgen-regulated genes (TMPRSS2, KLK3, CAMKK2, FKBP5). We identified 749 genes downregulated and 908 genes upregulated following castration. AR regulation of α-methylacyl-CoA racemase expression and three other genes (FAM129A, RAB27A, and KIAA0101) was confirmed. Upregulation of oestrogen receptor 1 (ESR1) expression was observed in malignant epithelia and was associated with differential expression of ESR1-regulated genes and correlated with proliferation (Ki-67 expression). This first-in-man study defines the rapid gene expression changes taking place in prostate cancer (PCa) following castration. Expression levels of the genes that the androgen receptor regulates are predictive of treatment outcome. Upregulation of oestrogen receptor 1 is a mechanism by which PCa cells may survive despite castration. Copyright © 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Effects of targeted deletion of A1 adenosine receptors on postischemic cardiac function and expression of adenosine receptor subtypes.

PubMed

Morrison, R Ray; Teng, Bunyen; Oldenburg, Peter J; Katwa, Laxmansa C; Schnermann, Jurgen B; Mustafa, S Jamal

2006-10-01

To examine ischemic tolerance in the absence of A(1) adenosine receptors (A(1)ARs), isolated wild-type (WT) and A(1)AR knockout (A(1)KO) murine hearts underwent global ischemia-reperfusion, and injury was measured in terms of functional recovery and efflux of lactate dehydrogenase (LDH). Hearts were analyzed by real-time RT-PCR both at baseline and at intervals during ischemia-reperfusion to determine whether compensatory expression of other adenosine receptor subtypes occurs with either A(1)AR deletion and/or ischemia-reperfusion. A(1)KO hearts had higher baseline coronary flow (CF) and left ventricular developed pressure (LVDP) than WT hearts, whereas heart rate was unchanged by A(1)AR deletion. After 20 min of ischemia, CF was attenuated in A(1)KO compared with WT hearts, and this reduction persisted throughout reperfusion. Final recovery of LVDP was decreased in A(1)KO hearts (54.4 +/- 5.1 vs. WT 81.1 +/- 3.4% preischemic baseline) and correlated with higher diastolic pressure during reperfusion. Postischemic efflux of LDH was greater in A(1)KO compared with WT hearts. Real-time RT-PCR demonstrated the absence of A(1)AR transcript in A(1)KO hearts, and the message for A(2A), A(2B), and A(3) adenosine receptors was similar in uninstrumented A(1)KO and WT hearts. Ischemia-reperfusion increased A(2B) mRNA expression 2.5-fold in both WT and A(1)KO hearts without changing A(1) or A(3) expression. In WT hearts, ischemia transiently doubled A(2A) mRNA, which returned to preischemic level upon reperfusion, a pattern not observed in A(1)KO hearts. Together, these data affirm the cardioprotective role of A(1)ARs and suggest that induced expression of other adenosine receptor subtypes may participate in the response to ischemia-reperfusion in isolated murine hearts.

Androgen receptor polyglutamine repeat length affects receptor activity and C2C12 cell development.

PubMed

Sheppard, Ryan L; Spangenburg, Espen E; Chin, Eva R; Roth, Stephen M

2011-10-20

Testosterone (T) has an anabolic effect on skeletal muscle and is believed to exert its local effects via the androgen receptor (AR). The AR harbors a polymorphic stretch of glutamine repeats demonstrated to inversely affect receptor transcriptional activity in prostate and kidney cells. The effects of AR glutamine repeat length on skeletal muscle are unknown. In this study we examined the effect of AR CAG repeat length on AR function in C2C12 cells. AR expression vectors harboring 14, 24, and 33 CAG repeats were used to assess AR transcriptional activity. C2C12 cell proliferation, differentiation, gene expression, myotube formation, and myonuclear fusion index were assessed. Transcriptional activity increased with increasing repeat length and in response to testosterone (AR14 = 3.91 ± 0.26, AR24 = 25.21 ± 1.72, AR33 = 36.08 ± 3.22 relative light units; P < 0.001). Ligand activation was increased for AR33 (2.10 ± 0.04) compared with AR14 (1.54 ± 0.09) and AR24 (1.57 ± 0.05, P < 0.001). AR mRNA expression was elevated in each stably transfected line. AR33 cell proliferation (20,512.3 ± 1,024.0) was decreased vs. AR14 (27,604.17 ± 1,425.3; P < 0.001) after 72 h. Decreased CK activity in AR14 cells (54.9 ± 2.9 units/μg protein) in comparison to AR33 (70.8 ± 8.1) (P < 0.05) was noted. The myonuclear fusion index was lower for AR14 (15.21 ± 3.24%) and AR33 (9.97 ± 3.14%) in comparison to WT (35.07 ± 5.60%, P < 0.001). AR14 and AR33 cells also displayed atypical myotube morphology. RT-PCR revealed genotype differences in myostatin and myogenin expression. We conclude that AR polyglutamine repeat length is directly associated with transcriptional activity and alters the growth and development of C2C12 cells. This polymorphism may contribute to the heritability of muscle mass in humans.

Effect of Lipopolysaccharide on Progesterone Production during Luteinization of Granulosa and Theca cells In Vitro.

PubMed

Shimizu, Takashi; Echizenya, Riku; Miyamoto, Akio

2016-04-01

The aim of this study is to examine the effect of lipopolysaccharide (LPS) on progesterone production during luteinization of granulosa and theca cells isolated from bovine large follicles. Granulosa and theca cells isolated from large follicles of bovine ovaries were exposed to LPS under appropriate hormone conditions in vitro. Progesterone (P4) production in theca cells, but not granulosa cells, was decreased by long-term exposure of LPS. Long-term exposure of LPS suppressed the gene expression of luteinizing hormone receptor in theca cells. Although long-term exposure of LPS did not affect the expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxy-steroid dehydrogenase (3β-HSD) genes, it did inhibit the protein expression of StAR and 3β-HSD in theca cells. These findings suggest that theca cells, rather than granulosa cells, are susceptible to LPS during luteinization and that LPS inhibits P4 production by decreasing protein levels of StAR during luteinization of theca cells. © 2016 Wiley Periodicals, Inc.

Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas.

PubMed

Kleb, Brittany; Estécio, Marcos R H; Zhang, Jiexin; Tzelepi, Vassiliki; Chung, Woonbok; Jelinek, Jaroslav; Navone, Nora M; Tahir, Salahaldin; Marquez, Victor E; Issa, Jean-Pierre; Maity, Sankar; Aparicio, Ana

2016-03-03

Small cell prostate carcinoma (SCPC) morphology is rare at initial diagnosis but often emerges during prostate cancer progression and portends a dismal prognosis. It does not express androgen receptor (AR) or respond to hormonal therapies. Clinically applicable markers for its early detection and treatment with effective chemotherapy are needed. Our studies in patient tumor-derived xenografts (PDX) revealed that AR-negative SCPC (AR(-)SCPC) expresses neural development genes instead of the prostate luminal epithelial genes characteristic of AR-positive castration-resistant adenocarcinomas (AR(+)ADENO). We hypothesized that the differences in cellular lineage programs are reflected in distinct epigenetic profiles. To address this hypothesis, we compared the DNA methylation profiles of AR(-) and AR(+) PDX using methylated CpG island amplification and microarray (MCAM) analysis and identified a set of differentially methylated promoters, validated in PDX and corresponding donor patient samples. We used the Illumina 450K platform to examine additional regions of the genome and the correlation between the DNA methylation profiles of the PDX and their corresponding patient tumors. Struck by the low frequency of AR promoter methylation in the AR(-)SCPC, we investigated this region's specific histone modification patterns by chromatin immunoprecipitation. We found that the AR promoter was enriched in silencing histone modifications (H3K27me3 and H3K9me2) and that EZH2 inhibition with 3-deazaneplanocin A (DZNep) resulted in AR expression and growth inhibition in AR(-)SCPC cell lines. We conclude that the epigenome of AR(-) is distinct from that of AR(+) castration-resistant prostate carcinomas, and that the AR(-) phenotype can be reversed with epigenetic drugs.

Androgen receptor expression in the human vagina under different physiological and treatment conditions.

PubMed

Baldassarre, M; Perrone, A M; Giannone, F A; Armillotta, F; Battaglia, C; Costantino, A; Venturoli, S; Meriggiola, M C

2013-01-01

Recent data report an important role of testosterone (T) in modulating female sexual responses, but little is known about the expression and distribution of androgen receptor (AR) in the human vagina. Therefore, the aims of our study were to evaluate the expression of AR in the human vagina in premenopausal (PrM) and menopausal (M) women and in T-treated women. Vaginal biopsies were obtained from PrM and postmenopausal women and from women with gender identity disorder (female to male (FtM)) receiving exogenous T. AR gene and protein expression levels in vaginal tissues were determined by real-time PCR and western blot analysis, respectively, whereas the localization of AR in vaginal mucosa and stroma was performed by immunohistochemistry. ARs were detected by immunostaining both in the mucosa and stroma. In vaginal mucosa, AR density score decreases with age but does not change with T administration. In stromal tissue, AR density score does not change with age but significantly increases with T administration (P<0.01). AR protein expression was significantly increased in FtM subjects (P<0.001). The expression of AR messenger RNA (mRNA) evaluated by Real-time PCR showed a significantly higher mRNA expression in FtM versus M patients (P<0.01) and in PrM versus M subjects (P<0.05). In conclusion, we found AR protein and mRNA expression both in the epithelium and stroma of the human vagina in all groups of women. A negative correlation exists between age and AR expression in the vaginal mucosa. T administration increases AR expression in both the mucosa and stroma.

A Potential Role for Endoplasmic Reticulum Stress in Progesterone Deficiency in Obese Women.

PubMed

Takahashi, Nozomi; Harada, Miyuki; Hirota, Yasushi; Zhao, Lin; Azhary, Jerilee M K; Yoshino, Osamu; Izumi, Gentaro; Hirata, Tetsuya; Koga, Kaori; Wada-Hiraike, Osamu; Fujii, Tomoyuki; Osuga, Yutaka

2017-01-01

Obesity in reproductive-aged women is associated with a shorter luteal phase and lower progesterone levels. Lipid accumulation in follicles of obese women compromises endoplasmic reticulum (ER) function, activating ER stress in granulosa cells. We hypothesized that ER stress activation in granulosa-lutein cells (GLCs) would modulate progesterone production and contribute to obesity-associated progesterone deficiency. Pretreatment with an ER stress inducer, tunicamycin or thapsigargin, inhibited human chorionic gonadotropin (hCG)-stimulated progesterone production in cultured human GLCs. Pretreatment of human GLCs with tunicamycin inhibited hCG-stimulated expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) messenger RNAs (mRNAs) without affecting expression of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), as determined by real-time quantitative polymerase chain reaction. Pretreatment with tunicamycin also inhibited hCG-stimulated expression of StAR protein and 3β-HSD enzyme activity in cultured human GLCs, as determined by Western blot analysis and an enzyme immunoassay, respectively, but did not affect hCG-induced intracellular 3',5'-cyclic adenosine monophosphate accumulation. Furthermore, tunicamycin attenuated hCG-induced protein kinase A and extracellular signal-regulated kinase activation, as determined by Western blot analysis. In vivo administration of tunicamycin to pregnant mare serum gonadotropin-treated immature mice prior to hCG treatment inhibited the hCG-stimulated increase in serum progesterone levels and hCG-induced expression of StAR and 3β-HSD mRNA in the ovary without affecting serum estradiol levels or the number of corpora lutea. Our findings indicate that ER stress in the follicles of obese women contributes to progesterone deficiency by inhibiting hCG-induced progesterone production in granulosa cells. Copyright © 2017 by the Endocrine Society.

Blockade of adenosine A2A receptor enhances CD8+ T cells response and decreases regulatory T cells in head and neck squamous cell carcinoma.

PubMed

Ma, Si-Rui; Deng, Wei-Wei; Liu, Jian-Feng; Mao, Liang; Yu, Guang-Tao; Bu, Lin-Lin; Kulkarni, Ashok B; Zhang, Wen-Feng; Sun, Zhi-Jun

2017-06-07

Cancer immunotherapy offers a promising approach in cancer treatment. The adenosine A2A receptor (A2AR) could protect cancerous tissues from immune clearance via inhibiting T cells response. To date, the role of A2AR in head and neck squamous cell carcinoma (HNSCC) has not been investigated. Here, we sought to explore the expression and immunotherapeutic value of A2AR blockade in HNSCC. The expression of A2AR was evaluated by immunostaining in 43 normal mucosae, 48 dysplasia and 165 primary HNSCC tissues. The immunotherapeutic value of A2AR blockade was assessed in vivo in genetically defined immunocompetent HNSCC mouse model. Immunostaining of HNSCC tissue samples revealed that increased expression of A2AR on tumor infiltrating immune cells correlated with advanced pathological grade, larger tumor size and positive lymph node status. Elevated A2AR expression was also detected in recurrent HNSCC and HNSCC tissues with induction chemotherapy. The expression of A2AR was found to be significantly correlated with HIF-1α, CD73, CD8 and Foxp3. Furthermore, the increased population of CD4 + Foxp3 + regulatory T cells (Tregs), which partially expressed A2AR, was observed in an immunocompetent mouse model that spontaneously develops HNSCC. Pharmacological blockade of A2AR by SCH58261 delayed the tumor growth in the HNSCC mouse model. Meanwhile, A2AR blockade significantly reduced the population of CD4 + Foxp3 + Tregs and enhanced the anti-tumor response of CD8 + T cells. These results offer a preclinical proof for the administration of A2AR inhibitor on prophylactic experimental therapy of HNSCC and suggest that A2AR blockade can be a potential novel strategy for HNSCC immunotherapy.

Progress toward heterologous expression of active G-protein-coupled receptors in Saccharomyces cerevisiae: Linking cellular stress response with translocation and trafficking

PubMed Central

O'Malley, Michelle A; Mancini, J Dominic; Young, Carissa L; McCusker, Emily C; Raden, David; Robinson, Anne S

2009-01-01

High-level expression of mammalian G-protein-coupled receptors (GPCRs) is a necessary step toward biophysical characterization and high-resolution structure determination. Even though many heterologous expression systems have been used to express mammalian GPCRs at high levels, many receptors are improperly trafficked or are inactive in these systems. En route to engineering a robust microbial host for GPCR expression, we have investigated the expression of 12 GPCRs in the yeast Saccharomyces cerevisiae, where all receptors are expressed at the mg/L scale. However, only the human adenosine A2a (hA2aR) receptor is active for ligand-binding and located primarily at the plasma membrane, whereas other tested GPCRs are mainly retained within the cell. Selective receptors associate with BiP, an ER-resident chaperone, and activated the unfolded protein response (UPR) pathway, which suggests that a pool of receptors may be folded incorrectly. Leader sequence cleavage of the expressed receptors was complete for the hA2aR, as expected, and partially cleaved for hA2bR, hCCR5R, and hD2LR. Ligand-binding assays conducted on the adenosine family (hA1R, hA2aR, hA2bR, and hA3R) of receptors show that hA2aR and hA2bR, the only adenosine receptors that demonstrate leader sequence processing, display activity. Taken together, these studies point to translocation as a critical limiting step in the production of active mammalian GPCRs in S. cerevisiae. PMID:19760666

Prokaryotic arsenate reductase enhances arsenate resistance in Mammalian cells.

PubMed

Wu, Dan; Tao, Xuanyu; Wu, Gaofeng; Li, Xiangkai; Liu, Pu

2014-01-01

Arsenic is a well-known heavy metal toxicant in the environment. Bioremediation of heavy metals has been proposed as a low-cost and eco-friendly method. This article described some of recent patents on transgenic plants with enhanced heavy metal resistance. Further, to test whether genetic modification of mammalian cells could render higher arsenic resistance, a prokaryotic arsenic reductase gene arsC was transfected into human liver cancer cell HepG2. In the stably transfected cells, the expression level of arsC gene was determined by quantitative real-time PCR. Results showed that arsC was expressed in HepG2 cells and the expression was upregulated by 3 folds upon arsenate induction. To further test whether arsC has function in HepG2 cells, the viability of HepG2-pCI-ArsC cells exposed to arsenite or arsenate was compared to that of HepG2-pCI cells without arsC gene. The results indicated that arsC increased the viability of HepG2 cells by 25% in arsenate, but not in arsenite. And the test of reducing ability of stably transfected cells revealed that the concentration of accumulated trivalent arsenic increased by 25% in HepG2-pCI-ArsC cells. To determine the intracellular localization of ArsC, a fusion vector with fluorescent marker pEGFP-N1-ArsC was constructed and transfected into.HepG2. Laser confocal microscopy showed that EGFP-ArsC fusion protein was distributed throughout the cells. Taken together, these results demonstrated that prokaryotic arsenic resistant gene arsC integrated successfully into HepG2 genome and enhanced arsenate resistance of HepG2, which brought new insights of arsenic detoxification in mammalian cells.

Transcriptional and posttranscriptional regulation of Bacillus sp. CDB3 arsenic-resistance operon ars1

PubMed Central

Yu, Xuefei; Zheng, Wei; Bhat, Somanath; Aquilina, J. Andrew

2015-01-01

Bacillus sp. CDB3 possesses a novel eight-gene ars cluster (ars1, arsRYCDATorf7orf8) with some unusual features in regard to expression regulation. This study demonstrated that the cluster is a single operon but can also produce a short three-gene arsRYC transcript. A hairpin structure formed by internal inverted repeats between arsC and arsD was shown to diminish the expression of the full operon, thereby probably acting as a transcription attenuator. A degradation product of the arsRYC transcript was also identified. Electrophoretic mobility shift analysis demonstrated that ArsR interacts with the ars1 promoter forming a protein-DNA complex that could be impaired by arsenite. However, no interaction was detected between ArsD and the ars1 promoter, suggesting that the CDB3 ArsD protein may not play a regulatory role. Compared to other ars gene clusters, regulation of the Bacillus sp. CDB3 ars1 operon is more complex. It represents another example of specific mRNA degradation in the transporter gene region and possibly the first case of attenuator-mediated regulation of ars operons. PMID:26355338

Tumour-specific HMG-CoAR is an independent predictor of recurrence free survival in epithelial ovarian cancer.

PubMed

Brennan, Donal J; Brändstedt, Jenny; Rexhepaj, Elton; Foley, Michael; Pontén, Fredrik; Uhlén, Mathias; Gallagher, William M; O'Connor, Darran P; O'Herlihy, Colm; Jirstrom, Karin

2010-04-01

Our group previously reported that tumour-specific expression of the rate-limiting enzyme in the mevalonate pathway, 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMG-CoAR) is associated with more favourable tumour parameters and a good prognosis in breast cancer. In the present study, the prognostic value of HMG-CoAR expression was examined in tumours from a cohort of patients with primary epithelial ovarian cancer. HMG-CoAR expression was assessed using immunohistochemistry (IHC) on tissue microarrays (TMA) consisting of 76 ovarian cancer cases, analysed using automated algorithms to develop a quantitative scoring model. Kaplan Meier analysis and Cox proportional hazards modelling were used to estimate the risk of recurrence free survival (RFS). Seventy-two tumours were suitable for analysis. Cytoplasmic HMG-CoAR expression was present in 65% (n = 46) of tumours. No relationship was seen between HMG-CoAR and age, histological subtype, grade, disease stage, estrogen receptor or Ki-67 status. Patients with tumours expressing HMG-CoAR had a significantly prolonged RFS (p = 0.012). Multivariate Cox regression analysis revealed that HMG-CoAR expression was an independent predictor of improved RFS (RR = 0.49, 95% CI (0.25-0.93); p = 0.03) when adjusted for established prognostic factors such as residual disease, tumour stage and grade. HMG-CoAR expression is an independent predictor of prolonged RFS in primary ovarian cancer. As HMG-CoAR inhibitors, also known as statins, have demonstrated anti-neoplastic effects in vitro, further studies are required to evaluate HMG-CoAR expression as a surrogate marker of response to statin treatment, especially in conjunction with current chemotherapeutic regimens.

Tumour-specific HMG-CoAR is an independent predictor of recurrence free survival in epithelial ovarian cancer

PubMed Central

2010-01-01

Background Our group previously reported that tumour-specific expression of the rate-limiting enzyme in the mevalonate pathway, 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMG-CoAR) is associated with more favourable tumour parameters and a good prognosis in breast cancer. In the present study, the prognostic value of HMG-CoAR expression was examined in tumours from a cohort of patients with primary epithelial ovarian cancer. Methods HMG-CoAR expression was assessed using immunohistochemistry (IHC) on tissue microarrays (TMA) consisting of 76 ovarian cancer cases, analysed using automated algorithms to develop a quantitative scoring model. Kaplan Meier analysis and Cox proportional hazards modelling were used to estimate the risk of recurrence free survival (RFS). Results Seventy-two tumours were suitable for analysis. Cytoplasmic HMG-CoAR expression was present in 65% (n = 46) of tumours. No relationship was seen between HMG-CoAR and age, histological subtype, grade, disease stage, estrogen receptor or Ki-67 status. Patients with tumours expressing HMG-CoAR had a significantly prolonged RFS (p = 0.012). Multivariate Cox regression analysis revealed that HMG-CoAR expression was an independent predictor of improved RFS (RR = 0.49, 95% CI (0.25-0.93); p = 0.03) when adjusted for established prognostic factors such as residual disease, tumour stage and grade. Conclusion HMG-CoAR expression is an independent predictor of prolonged RFS in primary ovarian cancer. As HMG-CoAR inhibitors, also known as statins, have demonstrated anti-neoplastic effects in vitro, further studies are required to evaluate HMG-CoAR expression as a surrogate marker of response to statin treatment, especially in conjunction with current chemotherapeutic regimens. PMID:20359358

Vitamin D receptor expression and potential role of vitamin D on cell proliferation and steroidogenesis in goat ovarian granulosa cells.

PubMed

Yao, Xiaolei; Zhang, Guomin; Guo, Yixuan; Ei-Samahy, Mohamed; Wang, Shuting; Wan, Yongjie; Han, Le; Liu, Zifei; Wang, Feng; Zhang, Yanli

2017-10-15

This study aimed to investigate the expression of the vitamin D receptor (VDR) in goat follicles and to determine the effects of Vit D 3 supplementation on goat granulosa cells (GCs) function linked to follicular development. The results demonstrated that VDR was prominently localized in GCs, with expression increasing with follicle diameter. Addition of Vit D 3 (1α,25-(OH) 2 VD 3 ; 10 nM) to GCs caused an increase in VDR and in steroidogenic acute regulator (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA expression. Additionally, Vit D 3 increased the cyclic adenosine monophosphate (cAMP), estradiol (E 2 ), and progesterone (P 4 ) levels, while it decreased anti-müllerian hormone receptor (AMHR) and follicle-stimulating hormone receptor (FSHR) mRNA expression (P < 0.05). Addition of FSH remarkably increased E 2, P 4 , and cAMP levels (P < 0.05), and Vit D 3 further enhanced the E 2 and cAMP levels in the presence of FSH (P < 0.05). Vit D 3 significantly induced the mRNA expression of CDK4 and CyclinD1, and downregulated P21 gene expression (P < 0.05). In addition, Vit D 3 significantly decreased reactive oxygen species (ROS) production and increased the mRNA and protein expression of superoxide dismutase 2 (SOD2) and catalase (CAT) (P < 0.05). In conclusion, VDR is expressed in GCs of the goat ovaries and Vit D 3 might play an important role in GCs proliferation by regulating cellular oxidative stress and cell cycle-related genes. Meanwhile, Vit D 3 enhances the E 2 and P 4 output of GCs by regulating the expression of 3β-HSD and StAR and the level of cAMP, which regulate steroidogenesis, supporting a potential role for Vit D 3 in follicular development. Copyright © 2017 Elsevier Inc. All rights reserved.

Differential expression of androgen, estrogen, and progesterone receptors in benign prostatic hyperplasia

PubMed Central

Song, Lingmin; Shen, Wenhao; Zhang, Heng; Wang, Qiwu; Wang, Yongquan; Zhou, Zhansong

2016-01-01

This study aimed to identify the differential expression levels of androgen receptor (AR), estrogen receptors (ERα, ERβ), and progesterone receptor (PGR) between normal prostate and benign prostatic hyperplasia (BPH). The combination of immunohistochemistry, quantitative real-time reverse transcription polymerase chain reaction, and Western blotting assay was used to identify the distribution and differential expression of these receptors at the immunoactive biomarker, transcriptional, and protein levels between 5 normal human prostate tissues and 40 BPH tissues. The results were then validated in a rat model of BPH induced by testosterone propionate and estradiol benzoate. In both human and rat prostate tissues, AR was localized mainly to epithelial and stromal cell nuclei; ERα was distributed mainly to stromal cells, but not exclusively; ERβ was interspersed in the basal layer of epithelium, but sporadically in epithelial and stromal cells; PGR was expressed abundantly in cytoplasm of epithelial and stromal cells. There were decreased expression of ERα and increased expression of PGR, but no difference in the expression of ERβ in the BPH compared to the normal prostate of both human and rat. Increased expression of AR in the BPH compared to the normal prostate of human was observed, however, the expression of AR in the rat prostate tissue was decreased. This study identified the activation of AR and PGR and repression of ERα in BPH, which indicate a promoting role of AR and PGR and an inhibitory role of ERα in the pathogenesis of BPH. PMID:27294569

C5aR expression in a novel GFP reporter gene knock-in mouse: implications for the mechanism of action of C5aR signaling in T cell immunity1

PubMed Central

Dunkelberger, Jason; Zhou, Lin; Miwa, Takashi; Song, Wen-Chao

2012-01-01

C5aR is a G protein-coupled receptor for the anaphylatoxin C5a and mediates many pro-inflammatory reactions. C5aR signaling has also been shown to regulate T cell immunity, but its sites and mechanism of action in this process remains uncertain. Here, we created a green fluorescence protein (GFP) knock-in mouse and used GFP as a surrogate marker to examine C5aR expression. GFP was knocked into the 3′-untranslated region (3′-UTR) of C5aR by gene targeting. We show that GFP is expressed highly on Gr-1+CD11b+ cells in the blood, spleen and bone marrow (BM), and moderately on CD11b+F4/80+ circulating leukocytes and elicited peritoneal macrophages. No GFP is detected on resting or activated T lymphocytes, nor on splenic myeloid or plasmacytoid dendritic cells. In contrast, 5–20% cultured BM-derived dendritic cells expressed GFP. Interestingly, GFP knock-in prevented cell surface but not intracellular C5aR expression. We conclude that C5aR is unlikely to play an intrinsic role on murine T cells and primary DCs. Instead, its effect on T cell immunity in vivo may involve CD11b+F4/80+ or other C5aR-expressing leukocytes. Further, our data reveal a surprising role of the 3′UTR of C5aR mRNA in regulating C5aR protein targeting to the plasma membrane. PMID:22430734

The phytochemical 3,3'-diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells.

PubMed

Palomera-Sanchez, Zoraya; Watson, Gregory W; Wong, Carmen P; Beaver, Laura M; Williams, David E; Dashwood, Roderick H; Ho, Emily

2017-09-01

Androgen receptor (AR) is a transcription factor involved in normal prostate physiology and prostate cancer (PCa) development. 3,3'-Diindolylmethane (DIM) is a promising phytochemical agent against PCa that affects AR activity and epigenetic regulators in PCa cells. However, whether DIM suppresses PCa via epigenetic regulation of AR target genes is unknown. We assessed epigenetic regulation of AR target genes in LNCaP PCa cells and showed that DIM treatment led to epigenetic suppression of AR target genes involved in DNA repair (PARP1, MRE11, DNA-PK). Decreased expression of these genes was accompanied by an increase in repressive chromatin marks, loss of AR occupancy and EZH2 recruitment to their regulatory regions. Decreased DNA repair gene expression was associated with an increase in DNA damage (γH2Ax) and up-regulation of genomic repeat elements LINE1 and α-satellite. Our results suggest that DIM suppresses AR-dependent gene transcription through epigenetic modulation, leading to DNA damage and genome instability in PCa cells. Published by Elsevier Inc.

Expression of Hormone Receptors and HER-2 in Benign and Malignant Salivary Gland Tumors.

PubMed

Can, Nhu Thuy; Lingen, Mark W; Mashek, Heather; McElherne, James; Briese, Renee; Fitzpatrick, Carrie; van Zante, Annemieke; Cipriani, Nicole A

2018-03-01

With the advent of targeted therapies, expression of sex hormone receptors and HER-2 in salivary gland tumors (SGTs) is of clinical interest. Previous reports of estrogen (ER) and progesterone (PR) receptor expression have varied. Androgen receptor (AR) and HER-2 overexpression are frequently reported in salivary duct carcinoma (SDC), but have not been studied systematically in other SGTs. This study examines ER, PR, AR, and HER-2 expression in SGTs. Immunohistochemistry for ER, PR, AR, and HER-2 was performed on 254 SGTs (134 malignant). ER, PR, and AR expression was scored using Allred system. HER-2 expression was scored using Dako HercepTest guidelines. FISH for HER-2 amplification was performed on select cases with HER-2 overexpression (2-3+). No SGT demonstrated strong expression of ER or PR. Combined strong AR and HER-2 expression was seen in 22 carcinomas: 14/25 SDC, 3/16 poorly differentiated, two oncocytic, and one each carcinoma ex pleomorphic adenoma, squamous cell, and intraductal carcinoma. Eighteen additional high grade carcinomas had HER-2 overexpression with absent, weak, or moderate AR expression; eight high grade carcinomas had isolated strong AR expression with 0-1+ HER-2 staining. Of 15 tested cases, six demonstrated HER-2 amplification by FISH, all of which had 3+ immunoreactivity. Neither benign nor malignant SGTs had strong expression of ER or PR. None of the benign SGTs overexpressed AR or HER-2. Coexpression of AR and HER-2 should not define SDC, but immunostaining should be considered in high grade salivary carcinomas, as some show overexpression and may benefit from targeted therapy.

Multimodal actions of the phytochemical sulforaphane suppress both AR and AR-V7 in 22Rv1 cells: Advocating a potent pharmaceutical combination against castration-resistant prostate cancer.

PubMed

Khurana, Namrata; Kim, Hogyoung; Chandra, Partha K; Talwar, Sudha; Sharma, Pankaj; Abdel-Mageed, Asim B; Sikka, Suresh C; Mondal, Debasis

2017-11-01

Prostate cancer (PCa) cells expressing full-length androgen receptor (AR-FL) are susceptible to androgen deprivation therapy (ADT). However, outgrowth of castration-resistant prostate cancer (CRPC) can occur due to the expression of constitutively active (ligand-independent) AR splice variants, particularly AR-V7. We previously demonstrated that sulforaphane (SFN), an isothiocyanate phytochemical, can decrease AR-FL levels in the PCa cell lines, LNCaP and C4-2B. Here, we examined the efficacy of SFN in targeting both AR-FL and AR-V7 in the CRPC cell line, CWR22Rv1 (22Rv1). MTT cell viability, wound-heal assay, and colony forming unit (CFU) measurements revealed that 22Rv1 cells are resistant to the anti-androgen, enzalutamide (ENZ). However, co-exposure to SFN sensitized these cells to the potent anticancer effects of ENZ (P<0.05). Immunoblot analyses showed that SFN (5-20 µM) rapidly decreases both AR-FL and AR-V7 levels, and immunofluorescence microscopy (IFM) depicted decreased AR in both cytoplasm and nucleus with SFN treatment. SFN increased both ubiquitination and proteasomal activity in 22Rv1 cells. Studies using a protein synthesis inhibitor (cycloheximide) or a proteasomal inhibitor (MG132) indicated that SFN increases both ubiquitin-mediated aggregation and subsequent proteasomal-degradation of AR proteins. Previous studies reported that SFN inhibits the chaperone activity of heat-shock protein 90 (Hsp90) and induces the nuclear factor erythroid-2-like 2 (Nrf2) transcription factor. Therefore, we investigated whether the Hsp90 inhibitor, ganetespib (G) or the Nrf2 activator, bardoxolone methyl (BM) can similarly suppress AR levels in 22Rv1 cells. Low doses of G and BM, alone or in combination, decreased both AR-FL and AR-V7 levels, and combined exposure to G+BM sensitized 22Rv1 cells to ENZ. Therefore, adjunct treatment with the phytochemical SFN or a safe pharmaceutical combination of G+BM may be effective against CRPC cells, especially those expressing AR-V7.

Multimodal actions of the phytochemical sulforaphane suppress both AR and AR-V7 in 22Rv1 cells: Advocating a potent pharmaceutical combination against castration-resistant prostate cancer

PubMed Central

Khurana, Namrata; Kim, Hogyoung; Chandra, Partha K.; Talwar, Sudha; Sharma, Pankaj; Abdel-Mageed, Asim B.; Sikka, Suresh C.; Mondal, Debasis

2017-01-01

Prostate cancer (PCa) cells expressing full-length androgen receptor (AR-FL) are susceptible to androgen deprivation therapy (ADT). However, outgrowth of castration-resistant prostate cancer (CRPC) can occur due to the expression of constitutively active (ligand-independent) AR splice variants, particularly AR-V7. We previously demonstrated that sulforaphane (SFN), an isothiocyanate phytochemical, can decrease AR-FL levels in the PCa cell lines, LNCaP and C4-2B. Here, we examined the efficacy of SFN in targeting both AR-FL and AR-V7 in the CRPC cell line, CWR22Rv1 (22Rv1). MTT cell viability, wound-heal assay, and colony forming unit (CFU) measurements revealed that 22Rv1 cells are resistant to the anti-androgen, enzalutamide (ENZ). However, co-exposure to SFN sensitized these cells to the potent anticancer effects of ENZ (P<0.05). Immunoblot analyses showed that SFN (5–20 µM) rapidly decreases both AR-FL and AR-V7 levels, and immunofluorescence microscopy (IFM) depicted decreased AR in both cytoplasm and nucleus with SFN treatment. SFN increased both ubiquitination and proteasomal activity in 22Rv1 cells. Studies using a protein synthesis inhibitor (cycloheximide) or a proteasomal inhibitor (MG132) indicated that SFN increases both ubiquitin-mediated aggregation and subsequent proteasomal-degradation of AR proteins. Previous studies reported that SFN inhibits the chaperone activity of heat-shock protein 90 (Hsp90) and induces the nuclear factor erythroid-2-like 2 (Nrf2) transcription factor. Therefore, we investigated whether the Hsp90 inhibitor, ganetespib (G) or the Nrf2 activator, bardoxolone methyl (BM) can similarly suppress AR levels in 22Rv1 cells. Low doses of G and BM, alone or in combination, decreased both AR-FL and AR-V7 levels, and combined exposure to G+BM sensitized 22Rv1 cells to ENZ. Therefore, adjunct treatment with the phytochemical SFN or a safe pharmaceutical combination of G+BM may be effective against CRPC cells, especially those expressing AR-V7. PMID:28901514

FOXA1 promotes tumor cell proliferation through AR involving the Notch pathway in endometrial cancer

PubMed Central

2014-01-01

Background Increasing evidence suggests that forkhead box A1 (FOXA1) is frequently dysregulated in many types of human cancers. However, the exact function and mechanism of FOXA1 in human endometrial cancer (EC) remains unclear. Methods FOXA1 expression, androgen receptor (AR) expression, and the relationships of these two markers with clinicopathological factors were determined by immunohistochemistry analysis. FOXA1 and AR were up-regulated by transient transfection with plasmids, and were down-regulated by transfection with siRNA or short hairpin RNA (shRNA). The effects of FOXA1 depletion and FOXA1 overexpression on AR-mediated transcription as well as Notch pathway and their impact on EC cell proliferation were examined by qRT-PCR, western blotting, co-immunoprecipitation, ChIP-PCR, MTT, colony-formation, and xenograft tumor–formation assays. Results We found that the expression of FOXA1 and AR in ECs was significantly higher than that in a typical hyperplasia and normal tissues. FOXA1 expression was significantly correlated with AR expression in clinical tissues. High FOXA1 levels positively correlated with pathological grade and depth of myometrial invasion in EC. High AR levels also positively correlated with pathological grade in EC. Moreover, the expression of XBP1, MYC, ZBTB16, and UHRF1, which are downstream targets of AR, was promoted by FOXA1 up-regulation or inhibited by FOXA1 down-regulation. Co-immunoprecipitation showed that FOXA1 interacted with AR in EC cells. ChIP-PCR assays showed that FOXA1 and AR could directly bind to the promoter and enhancer regions upstream of MYC. Mechanistic investigation revealed that over-expression of Notch1 and Hes1 proteins by FOXA1 could be reversed by AR depletion. In addition, we showed that down-regulation of AR attenuated FOXA1-up-regulated cell proliferation. However, AR didn’t influence the promotion effect of FOXA1 on cell migration and invasion. In vivo xenograft model, FOXA1 knockdown reduced the rate of tumor growth. Conclusions These results suggest that FOXA1 promotes cell proliferation by AR and activates Notch pathway. It indicated that FOXA1 and AR may serve as potential gene therapy in EC. PMID:24512546

The NLR-related protein NWD1 is associated with prostate cancer and modulates androgen receptor signaling.

PubMed

Correa, Ricardo G; Krajewska, Maryla; Ware, Carl F; Gerlic, Motti; Reed, John C

2014-03-30

Prostate cancer (PCa) is among the leading causes of cancer-related death in men. Androgen receptor (AR) signaling plays a seminal role in prostate development and homeostasis, and dysregulation of this pathway is intimately linked to prostate cancer pathogenesis and progression. Here, we identify the cytosolic NLR-related protein NWD1 as a novel modulator of AR signaling. We determined that expression of NWD1 becomes elevated during prostate cancer progression, based on analysis of primary tumor specimens. Experiments with cultured cells showed that NWD1 expression is up-regulated by the sex-determining region Y (SRY) family proteins. Gene silencing procedures, in conjunction with transcriptional profiling, showed that NWD1 is required for expression of PDEF (prostate-derived Ets factor), which is known to bind and co-regulate AR. Of note, NWD1 modulates AR protein levels. Depleting NWD1 in PCa cell lines reduces AR levels and suppresses activity of androgen-driven reporter genes. NWD1 knockdown potently suppressed growth of androgen-dependent LNCaP prostate cancer cells, thus showing its functional importance in an AR-dependent tumor cell model. Proteomic analysis suggested that NWD1 associates with various molecular chaperones commonly related to AR complexes. Altogether, these data suggest a role for tumor-associated over-expression of NWD1 in dysregulation of AR signaling in PCa.

Elevated nitrogen metabolism and nitric oxide production are involved in Arabidopsis resistance to acid rain.

PubMed

Qiao, Fang; Zhang, Xi-Min; Liu, Xiang; Chen, Juan; Hu, Wen-Jun; Liu, Ting-Wu; Liu, Ji-Yun; Zhu, Chun-Quan; Ghoto, Kabir; Zhu, Xue-Yi; Zheng, Hai-Lei

2018-06-01

Acid rain (AR) can induce great damages to plants and could be classified into different types according to the different SO 4 2- /NO 3 - ratio. However, the mechanism of plants' responding to different types of AR has not been elucidated clearly. Here, we found that nitric-rich simulated AR (N-SiAR) induced less leaves injury as lower necrosis percentage, better physiological parameters and reduced oxidative damage in the leaves of N-SiAR treated Arabidopsis thaliana compared with sulfate and nitrate mixed (SN-SiAR) or sulfuric-rich (S-SiAR) simulated AR treated ones. Of these three types of SiAR, N-SiAR treated Arabidopsis maintained the highest of nitrogen (N) content, nitrate reductase (NR) and nitrite reductase (NiR) activity as well as N metabolism related genes expression level. Nitric oxide (NO) content showed that N-SiAR treated seedlings had a higher NO level compared to SN-SiAR or S-SiAR treated ones. A series of NO production and elimination related reagents and three NO production-related mutants were used to further confirm the role of NO in regulating acid rain resistance in N-SiAR treated Arabidopsis seedlings. Taken together, we concluded that an elevated N metabolism and enhanced NO production are involved in the tolerance to different types of AR in Arabidopsis. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Functional characterization of the beta-adrenergic receptor subtypes expressed by CA1 pyramidal cells in the rat hippocampus.

PubMed

Hillman, Kristin L; Doze, Van A; Porter, James E

2005-08-01

Recent studies have demonstrated that activation of the beta-adrenergic receptor (AR) using the selective beta-AR agonist isoproterenol (ISO) facilitates pyramidal cell long-term potentiation in the cornu ammonis 1 (CA1) region of the rat hippocampus. We have previously analyzed beta-AR genomic expression patterns of 17 CA1 pyramidal cells using single cell reverse transcription-polymerase chain reaction, demonstrating that all samples expressed the beta2-AR transcript, with four of the 17 cells additionally expressing mRNA for the beta1-AR subtype. However, it has not been determined which beta-AR subtypes are functionally expressed in CA1 for these same pyramidal neurons. Using cell-attached recordings, we tested the ability of ISO to increase pyramidal cell action potential (AP) frequency in the presence of subtype-selective beta-AR antagonists. ICI-118,551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol] and butoxamine [alpha-[1-(t-butylamino)ethyl]-2,5-dimethoxybenzyl alcohol) hydrochloride], agents that selectively block the beta2-AR, produced significant parallel rightward shifts in the concentration-response curves for ISO. From these curves, apparent equilibrium dissociation constant (K(b)) values of 0.3 nM for ICI-118,551 and 355 nM for butoxamine were calculated using Schild regression analysis. Conversely, effective concentrations of the selective beta1-AR antagonists CGP 20712A [(+/-)-2-hydroxy-5-[2-([2-hydroxy-3-(4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy)propyl]amino)ethoxy]-benzamide methanesulfonate] and atenolol [4-[2'-hydroxy-3'-(isopropyl-amino)propoxy]phenylacetamide] did not significantly affect the pyramidal cell response to ISO. However, at higher concentrations, atenolol significantly decreased the potency for ISO-mediated AP frequencies. From these curves, an apparent atenolol K(b) value of 3162 nM was calculated. This pharmacological profile for subtype-selective beta-AR antagonists indicates that beta2-AR activation is mediating the increased AP frequency. Knowledge of functional AR expression in CA1 pyramidal neurons will aid future long-term potentiation studies by allowing selective manipulation of specific beta-AR subtypes.

Peripheral α2-β1 adrenergic interactions mediate the ghrelin response to brain urocortin 1 in rats

PubMed Central

Yakabi, Koji; Harada, Yumi; Takayama, Kiyoshige; Ro, Shoki; Ochiai, Mitsuko; lizuka, Seiichi; Hattori, Tomohisa; Wang, Lixin; Taché, Yvette

2018-01-01

Summary The autonomic nervous system (ANS) conveys neuronal input from the brain to the stomach. We investigated mechanisms through which urocortin 1 (UCN1) injected intracerebroventricularly (ICV, 300 pmol/rat) inhibits circulating ghrelin in rats. This was achieved by assessing (1) the induction of c-fos gene expression as a marker of neuronal activation in specific hypothalamic and caudal brainstem regulating ANS; (2) the influence of vagotomy and pharmacological blockade of central and peripheral α- and β-adrenergic receptor (AR) on ICV UCN1 -induced reduction of plasma ghrelin levels (determined by ELISA); and (3) the relevance of this pathway in the feeding response to a fast in rats. UCN1 increased c-fos mRNA expression in key brain sites influencing sympathetic activity namely the hypothalamic paraventricular and ventromedial nuclei, locus coeruleus, nucleus of the solitary tract, and rostral ventrolateral medulla, by 16-, 29-, 6-, 37-, and 13-fold, respectively. In contrast, the dorsal motor nucleus of the vagus had little c-fos mRNA expression and ICV UCN1 induced a similar reduction in acylated ghrelin in the sham-operated (31%) and vagotomized (41%) rats. An intraperitoneal (IP) injection of either a non-selective α- or selective α2-AR antagonist reduced, while a selective α2-AR agonist enhanced ICV UCN1-induced suppression of plasma acylated ghrelin levels. In addition, IP injection of a non-selective β- or selective β1-AR agonist blocked, and selective β1-AR antagonist augmented, the ghrelin response to ICV UCN1. The IP injections of a selective α1- or non-selective β or β2-AR antagonists, or any of the pretreatments given ICV had no effect. ICV UCN1 reduced the 2-h food intake in response to a fast by 80%, and this effect was partially prevented by a selective α2-AR antagonist. These data suggest that ICV UCN1 reduces plasma ghrelin mainly through the brain sympathetic component of the ANS and peripheral AR specifically α2-AR activation and inactivation of β1-AR. The α2-AR pathway contributes to the associated reduction in food intake. PMID:25265283

Biomarker Associations with Efficacy of Abiraterone Acetate and Exemestane in Postmenopausal Patients with Estrogen Receptor-Positive Metastatic Breast Cancer.

PubMed

Li, Weimin; O'Shaughnessy, Joyce; Hayes, Daniel; Campone, Mario; Bondarenko, Igor; Zbarskaya, Irina; Brain, Etienne; Stenina, Marina; Ivanova, Olga; Graas, Marie-Pascale; Neven, Patrick; Ricci, Deborah; Griffin, Thomas; Kheoh, Thian; Yu, Margaret; Gormley, Michael; Martin, Jason; Schaffer, Michael; Zelinsky, Kathy; De Porre, Peter; Johnston, Stephen R D

2016-12-15

Abiraterone may suppress androgens that stimulate breast cancer growth. We conducted a biomarker analysis of circulating tumor cells (CTCs), formalin-fixed paraffin-embedded tissues (FFPETs), and serum samples from postmenopausal estrogen receptor (ER) + breast cancer patients to identify subgroups with differential abiraterone sensitivity. Patients (randomized 1:1:1) were treated with 1,000 mg/d abiraterone acetate + 5 mg/d prednisone (AA), AA + 25 mg/d exemestane (AAE), or exemestane. The biomarker population included treated patients (n = 293). The CTC population included patients with ≥3 baseline CTCs (n = 104). Biomarker [e.g., androgen receptor (AR), ER, Ki-67, CYP17] expression was evaluated. Cox regression stratified by prior therapies in the metastatic setting (0/1 vs. 2) and setting of letrozole/anastrozole (adjuvant vs. metastatic) was used to assess biomarker associations with progression-free survival (PFS). Serum testosterone and estrogen levels were lowered and progesterone increased with AA. Baseline AR or ER expression was not associated with PFS in CTCs or FFPETs for AAE versus exemestane, but dual positivity of AR and ER expression was associated with improved PFS [HR, 0.41; 95% confidence interval (CI), 0.16-1.07; P = 0.070]. For AR expression in FFPETs obtained <1 year prior to first dose (n = 67), a trend for improved PFS was noted for AAE versus exemestane (HR, 0.56; 95% CI, 0.24-1.33; P = 0.19). An AA pharmacodynamic effect was shown by decreased serum androgen and estrogen levels and increased progesterone. AR and ER dual expression in CTCs and newly obtained FFPETs may predict AA sensitivity. Clin Cancer Res; 22(24); 6002-9. ©2016 AACR. ©2016 American Association for Cancer Research.

Bone vs. fat: Embryonic origin of progenitors determines response to androgen in adipocytes and osteoblasts

PubMed Central

Wiren, Kristine M.; Hashimoto, Joel G.; Semirale, Anthony A.; Zhang, Xiao-Wei

2011-01-01

Although androgen is considered an anabolic hormone, the consequences of androgen receptor (AR) overexpression in skeletally-targeted AR-transgenic lines highlight the detrimental effect of enhanced androgen sensitivity on cortical bone quality. A compartment-specific anabolic response is observed only in male but not female AR3.6-transgenic (tg) mice, with increased periosteal bone formation and calvarial thickening. To identify anabolic signaling cascades that have the potential to increase bone formation, qPCR array analysis was employed to define expression differences between AR3.6-tg and wild-type (WT) periosteal tissue. Notably, categories that were significantly different between the two genotypes included axonal guidance, CNS development and negative regulation of Wnt signaling with a node centered on stem cell pathways. Further, fine mapping of AR3.6-tg calvaria revealed that anabolic thickening in vivo is not uniform across the calvaria, occurring only in frontal but not parietal bones. Multipotent fraction 1 progenitor populations from both genotypes were cultured separately as frontal bone neural crest stem-like cells (fNCSC) and parietal bone mesenchymal stem-like cells (pMSC). Both osteoblastic and adipogenic differentiation in these progenitor populations was influenced by embryonic lineage and by genotype. Adipogenesis was enhanced in WT fNCSC compared to pMSC, but transgenic cultures showed strong suppression of lipid accumulation only in fNCSC cells. Osteoblastogenesis was significantly increased in transgenic fNCSC cultures compared to WT, with elevated alkaline phosphatase (ALP) activity and induction of mineralization and nodule formation assessed by alizarin red and von Kossa staining. Osteocalcin (OC) and ALP mRNA levels were also increased in fNCSC cultures from AR3.6-tg vs. WT, but in pMSC cultures ALP mRNA levels, mineralization and nodule formation were decreased in AR3.6-tg cells. Expression differences identified by array in long bone periosteal tissue from AR3.6-tg vs. WT were recapitulated in the fNCSC samples while pMSCs profiles reflected cortical expression. These observations reveal the opposing effects of androgen signaling on lineage commitment and osteoblast differentiation that is enhanced in cells derived from a neural crest origin but inhibited in cells derived from a mesodermal origin, consistent with in vivo compartment-specific responses to androgen. Combined, these results highlight the complex action of androgen in the body that is dependent on the embryonic lineage and developmental origin of the cell. Further, these data these data suggest that the periosteum surrounding long bone is derived from neural crest. PMID:21704206

Localization of the androgen-synthesizing enzymes, androgen receptor, and sex steroids in the vagina: possible implications for the treatment of postmenopausal sexual dysfunction.

PubMed

Bertin, Jonathan; Dury, Alain Y; Ouellet, Johanne; Pelletier, Georges; Labrie, Fernand

2014-08-01

To better understand the mechanisms underlying the beneficial effects of the intravaginal administration of dehydroepiandrosterone (DHEA) observed in postmenopausal women on sexual dysfunction. To identify the distribution of the androgen-synthesizing enzymes as well as androgen receptor (AR) and measure steroid levels in the monkey vagina. The cynomolgus monkey (Macaca fascicularis), the closest model to the human, has been used to measure the expression levels of steroidogenic enzymes and androgen receptor by quantitative reverse transcription polymerase chain reaction (n=4), confirmed by immunohistochemistry, and immunofluorescence (n=3). DHEA and its androgenic metabolites were quantified by LC-MS/MS (n=4). The presence of SRD5A1, SRD5A2, HSD17B3, AR as well as nerve fibers (PGP 9.5) was investigated, and steroid levels were measured. AR is widely distributed within the vaginal epithelium and also in the lamina propria with a lower expression in the muscularis layer and blood vessel walls. Androgen-forming enzymes, on the other hand, are expressed in the vaginal stratified squamous epithelium at a relatively high level where they are uniformly distributed from the basal membrane up to the superficial keratinized cells. The enzymes are at a lower level in blood vessel walls and zona muscularis where nerve fibers are localized. DHEA and its androgen metabolites are present at biologically significant concentrations in the monkey vagina. The enzymes responsible for androgen formation as well as AR are at the highest level in the superficial layer of the stratified epithelium and muscularis layers of the vagina. These data provide a potential explanation for the described role of androgens in regulating vaginal lubrication, smooth muscle activity, blood flow, and the neuronal activity potentially involved in the correction of sexual dysfunction. © 2014 International Society for Sexual Medicine.

Small glutamine-rich tetratricopeptide repeat-containing protein alpha is present in human ovaries but may not be differentially expressed in relation to polycystic ovary syndrome.

PubMed

Butler, Miriam S; Yang, Xing; Ricciardelli, Carmela; Liang, Xiaoyan; Norman, Robert J; Tilley, Wayne D; Hickey, Theresa E

2013-06-01

To evaluate the expression and function of small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA), an androgen receptor (AR) molecular chaperone, in human ovarian tissues. Examine the effect of SGTA on AR subcellular localization in granulosa tumor cells (KGN) and SGTA expression in ovarian tissues. University-based research laboratory. Archived tissues from premenopausal women and granulosa cells from infertile women receiving assisted reproduction. None. AR subcellular localization and SGTA protein or mRNA levels. SGTA and AR proteins were expressed in the cytoplasm of KGN cells and exposure to androgen stimulated AR nuclear localization. SGTA protein knockdown increased AR nuclear localization at low (0-0.1 nmol/L) but not high (1-10 nmol/L) concentrations of androgen hormone. In ovarian tissues, SGTA was localized to the cytoplasm of granulosa cells at all stages of folliculogenesis and in thecal cells of antral follicles. SGTA protein levels were similar when comparing primordial and primary follicles within core biopsies (n = 40) from women with and without polycystic ovary syndrome (PCOS). Likewise, SGTA mRNA levels were not significantly different in granulosa cells from preovulatory follicles after hyperstimulation of women with and without PCOS. SGTA is present in human ovaries and has the potential to modulate AR signalling, but it may not be differentially expressed in PCOS. Copyright © 2013 American Society for Reproductive Medicine. All rights reserved.

Effects of dietary soybean isoflavones (SI) on reproduction in the young breeder rooster.

PubMed

Heng, Dai; Zhang, Tao; Tian, Ye; Yu, Shangyu; Liu, Wenbo; Xu, Kaili; Liu, Juan; Ding, Yu; Zhu, Baochang; Yang, Yanzhou; Zhang, Cheng

2017-02-01

Soybean isoflavones (SIs) are phytoestrogens that competitive with estrogens in body. Although SIs play an important role in reproduction, their role in testicular development in roosters is unknown. This study was conducted to investigate the effect of SIs on testicular development and serum reproductive hormone profiles in young breeder roosters (70-133days old). Gene expression of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD), which are related to testosterone synthesis, in rooster testis were also evaluated after treatment with different SI doses. Although SIs had no significant effect on body weight, 5mg/kg SIs significantly increased the testis index and serum levels of reproductive hormones (gonadotropin releasing hormone, follicle- stimulating hormone, luteinizing hormone, and testosterone).To further investigate whether SIs regulate hormone synthesis via StAR, p450scc, 3β-HSD, real time-PCR was performed to measure the mRNA levels of the corresponding genes. The results showed that 5mg/kg of SIs significantly increased StAR mRNA levels. However, there were no significant effects on p450scc or 3β-HSD mRNA levels. Moreover, the spermatogonial development and the number of germ cell layers were increased by treatment with 5mg/kg of SIs. These results suggest that SIs promote testicular growth by increasing reproductive hormone secretion, which is closely related to StAR expression, to positively regulate reproduction in young roosters. Copyright © 2016 Elsevier B.V. All rights reserved.

High-fat diet aggravates 2,2',4,4'-tetrabromodiphenyl ether-inhibited testosterone production via DAX-1 in Leydig cells in rats.

PubMed

Zhang, Zhan; Yu, Yongquan; Xu, Hengsen; Wang, Chao; Ji, Minghui; Gu, Jun; Yang, Lu; Zhu, Jiansheng; Dong, Huibin; Wang, Shou-Lin

2017-05-15

Growing evidence has revealed that a high-fat diet (HFD) could lead to disorders of glycolipid metabolism and insulin-resistant states, and HFDs have been associated with the inhibition of testicular steroidogenesis. Our previous study demonstrated that 2,2',4,4'-tetrabromodiphenyl ether (BDE47) could increase the risk of diabetes in humans and reduce testosterone production in rats. However, whether the HFD affects BDE47-inhibited testosterone production by elevating insulin levels and inducing related pathways remains unknown. In male rats treated with BDE47 by gavage for 12 weeks, the HFD significantly increased the BDE47 content of the liver and testis and increased the weight of the adipose tissue; increased macrovesicular steatosis in the liver and the levels of triglycerides, fasting glucose and insulin; further aggravated the disruption of the seminiferous epithelium; and lowered the level of testosterone, resulting in fewer sperm in the epididymis. Of note, the HFD enhanced BDE47-induced DAX-1 expression and decreased the expression levels of StAR and 3β-HSD in the testicular interstitial compartments in rats. In isolated primary Leydig cells from rats, BDE47 or insulin increased DAX-1 expression, decreased the expression of StAR and 3β-HSD, and reduced testosterone production, which was nearly reversed by knocking down DAX-1. These results indicated that the HFD aggravates BDE47-inhibited testosterone production through hyperinsulinemia, and the accumulation of testicular BDE47 that induces the up-regulation of DAX-1 and the subsequent down-regulation of steroidogenic proteins, i.e., StAR and 3β-HSD, in Leydig cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Green tea polyphenol EGCG blunts androgen receptor function in prostate cancer

PubMed Central

Siddiqui, Imtiaz A.; Asim, Mohammad; Hafeez, Bilal B.; Adhami, Vaqar M.; Tarapore, Rohinton S.; Mukhtar, Hasan

2011-01-01

Androgen deprivation therapy is the major treatment for advanced prostate cancer (PCa). However, it is a temporary remission, and the patients almost inevitably develop hormone refractory prostate cancer (HRPC). HRPC is almost incurable, although most HRPC cells still express androgen receptor (AR) and depend on the AR for growth, making AR a prime drug target. Here, we provide evidence that epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, is a direct antagonist of androgen action. In silico modeling and FRET-based competition assay showed that EGCG physically interacts with the ligand-binding domain of AR by replacing a high-affinity labeled ligand (IC50 0.4 μM). The functional consequence of this interaction was a decrease in AR-mediated transcriptional activation, which was due to EGCG mediated inhibition of interdomain N-C termini interaction of AR. Treatment with EGCG also repressed the transcriptional activation by a hotspot mutant AR (T877A) expressed ectopically as well as the endogenous AR mutant. As the physiological consequence of AR antagonism, EGCG repressed R1881-induced PCa cell growth. In a xenograft model, EGCG was found to inhibit AR nuclear translocation and protein expression. We also observed a significant down-regulation of androgen-regulated miRNA-21 and up-regulation of a tumor suppressor, miRNA-330, in tumors of mice treated with EGCG. Taken together, we provide evidence that EGCG functionally antagonizes androgen action at multiple levels, resulting in inhibition of PCa growth.—Siddiqui, I. A., Asim, M., Hafeez, B. B., Adhami, V. M., Tarapore, R. S., Mukhtar, H. Green tea polyphenol EGCG blunts androgen receptor function in prostate cancer. PMID:21177307

BMP15 suppresses progesterone production by down-regulating StAR via ALK3 in human granulosa cells.

PubMed

Chang, Hsun-Ming; Cheng, Jung-Chien; Klausen, Christian; Leung, Peter C K

2013-12-01

In addition to somatic cell-derived growth factors, oocyte-derived growth differentiation factor (GDF)9 and bone morphogenetic protein (BMP)15 play essential roles in female fertility. However, few studies have investigated their effects on human ovarian steroidogenesis, and fewer still have examined their differential effects or underlying molecular determinants. In the present study, we used immortalized human granulosa cells (SVOG) and human granulosa cell tumor cells (KGN) to compare the effects of GDF9 and BMP15 on steroidogenic enzyme expression and investigate potential mechanisms of action. In SVOG cells, neither GDF9 nor BMP15 affects the mRNA levels of P450 side-chain cleavage enzyme or 3β-hydroxysteroid dehydrogenase. However, treatment with BMP15, but not GDF9, significantly decreases steroidogenic acute regulatory protein (StAR) mRNA and protein levels as well as progesterone production. These suppressive effects, along with the induction of Sma and Mad-related protein (SMAD)1/5/8 phosphorylation, are attenuated by cotreatment with 2 different BMP type I receptor inhibitors (dorsomorphin and DMH-1). Furthermore, depletion of activin receptor-like kinase (ALK)3 using small interfering RNA reverses the effects of BMP15 on SMAD1/5/8 phosphorylation and StAR expression. Similarly, knockdown of ALK3 abolishes BMP15-induced SMAD1/5/8 phosphorylation in KGN cells. These results provide evidence that oocyte-derived BMP15 down-regulates StAR expression and decreases progesterone production in human granulosa cells, likely via ALK3-mediated SMAD1/5/8 signaling. Our findings suggest that oocyte may play a critical role in the regulation of progesterone to prevent premature luteinization during the late stage of follicle development.

StAR Enhances Transcription of Genes Encoding the Mitochondrial Proteases Involved in Its Own Degradation

PubMed Central

Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Lauria, Ines; Langer, Thomas

2014-01-01

Steroidogenic acute regulatory protein (StAR) is essential for steroid hormone synthesis in the adrenal cortex and the gonads. StAR activity facilitates the supply of cholesterol substrate into the inner mitochondrial membranes where conversion of the sterol to a steroid is catalyzed. Mitochondrial import terminates the cholesterol mobilization activity of StAR and leads to mounting accumulation of StAR in the mitochondrial matrix. Our studies suggest that to prevent mitochondrial impairment, StAR proteolysis is executed by at least 2 mitochondrial proteases, ie, the matrix LON protease and the inner membrane complexes of the metalloproteases AFG3L2 and AFG3L2:SPG7/paraplegin. Gonadotropin administration to prepubertal rats stimulated ovarian follicular development associated with increased expression of the mitochondrial protein quality control system. In addition, enrichment of LON and AFG3L2 is evident in StAR-expressing ovarian cells examined by confocal microscopy. Furthermore, reporter studies of the protease promoters examined in the heterologous cell model suggest that StAR expression stimulates up to a 3.5-fold increase in the protease gene transcription. Such effects are StAR-specific, are independent of StAR activity, and failed to occur upon expression of StAR mutants that do not enter the matrix. Taken together, the results of this study suggest the presence of a novel regulatory loop, whereby acute accumulation of an apparent nuisance protein in the matrix provokes a mitochondria to nucleus signaling that, in turn, activates selected transcription of genes encoding the enrichment of mitochondrial proteases relevant for enhanced clearance of StAR. PMID:24422629

Hypoxia reduces testosterone synthesis in mouse Leydig cells by inhibiting NRF1-activated StAR expression

PubMed Central

Zou, Zhiran; Wang, Dan; Lu, Yapeng; Dong, Zhangji; Zhu, Li

2017-01-01

Male fertility disorders play a key role in half of all infertility cases. Reduction in testosterone induced by hypoxia might cause diseases in reproductive system and other organs. Hypoxic exposure caused a significant decrease of NRF1. Software analysis reported that the promoter region of steroidogenic acute regulatory protein (StAR) contained NRF1 binding sites, indicating NRF1 promoted testicular steroidogenesis. The purpose of this study is to determine NRF1 is involved in testosterone synthesis; and under hypoxia, the decrease of testosterone synthesis is caused by lower expression of NRF1. We designed both in vivo and in vitro experiments. Under hypoxia, the expressions of NRF1 in Leydig cells and testosterone level were significantly decreased both in vivo and in vitro. Overexpression and interference NRF1 could induced StAR and testosterone increased and decreased respectively. ChIP results confirmed the binding of NRF1 to StAR promoter region. In conclusion, decline of NRF1 expression downregulated the level of StAR, which ultimately resulted in a reduction in testosterone synthesis. PMID:28146428

Java Web Start based software for automated quantitative nuclear analysis of prostate cancer and benign prostate hyperplasia.

PubMed

Singh, Swaroop S; Kim, Desok; Mohler, James L

2005-05-11

Androgen acts via androgen receptor (AR) and accurate measurement of the levels of AR protein expression is critical for prostate research. The expression of AR in paired specimens of benign prostate and prostate cancer from 20 African and 20 Caucasian Americans was compared to demonstrate an application of this system. A set of 200 immunopositive and 200 immunonegative nuclei were collected from the images using a macro developed in Image Pro Plus. Linear Discriminant and Logistic Regression analyses were performed on the data to generate classification coefficients. Classification coefficients render the automated image analysis software independent of the type of immunostaining or image acquisition system used. The image analysis software performs local segmentation and uses nuclear shape and size to detect prostatic epithelial nuclei. AR expression is described by (a) percentage of immunopositive nuclei; (b) percentage of immunopositive nuclear area; and (c) intensity of AR expression among immunopositive nuclei or areas. The percent positive nuclei and percent nuclear area were similar by race in both benign prostate hyperplasia and prostate cancer. In prostate cancer epithelial nuclei, African Americans exhibited 38% higher levels of AR immunostaining than Caucasian Americans (two sided Student's t-tests; P < 0.05). Intensity of AR immunostaining was similar between races in benign prostate. The differences measured in the intensity of AR expression in prostate cancer were consistent with previous studies. Classification coefficients are required due to non-standardized immunostaining and image collection methods across medical institutions and research laboratories and helps customize the software for the specimen under study. The availability of a free, automated system creates new opportunities for testing, evaluation and use of this image analysis system by many research groups who study nuclear protein expression.

Differential expression of oestrogen receptor isoforms and androgen receptor in the normal vulva and vagina compared with vulval lichen sclerosus and chronic vaginitis.

PubMed

Taylor, A H; Guzail, M; Al-Azzawi, F

2008-02-01

Although the expression of the oestrogen receptor (ER) alpha isoform and androgen receptor (AR) has been examined in vulval lichen sclerosus (VLS), the distribution pattern of ERalpha, ERbeta and AR has not been described in chronic atrophic vaginitis nor correlated with markers of proliferation (Ki-67) in either of these diseased tissues. To measure the levels and distribution of ERalpha, ERbeta and AR immunoreactivity in relation to Ki-67 in normal and diseased vulva and vagina. The expression of ERalpha, ERbeta and AR in relation to the proliferation marker Ki-67 in VLS, squamous hyperplasia of the vulva and chronic atrophic vaginitis was determined by immunohistomorphometric analysis and compared with that in normal vulva and vagina. VLS showed similar ERalpha and ERbeta expression in the 'epidermal' and 'dermal' tissue layers to that of normal vulvae, whereas AR expression appeared to be absent in most cases. ERbeta and Ki-67 expression was correlated with ERalpha expression but only in the 'fibrovascular' layer of the vulva. ERalpha expression was absent from the 'fibromuscular' layer of diseased vulvae, while ERbeta expression was absent in normal tissues but was highly expressed in diseased vulvae. ERalpha expression was significantly correlated with AR expression in the fibrovascular layer of the vagina and inversely correlated with Ki-67 staining in the parabasal cells of the epidermis in patients with chronic atrophic vaginitis. These data suggest that ER expression and levels may be implicated in the aetiopathology of VLS and chronic atrophic vaginitis.

CIP2A is a candidate therapeutic target in clinically challenging prostate cancer cell populations.

PubMed

Khanna, Anchit; Rane, Jayant K; Kivinummi, Kati K; Urbanucci, Alfonso; Helenius, Merja A; Tolonen, Teemu T; Saramäki, Outi R; Latonen, Leena; Manni, Visa; Pimanda, John E; Maitland, Norman J; Westermarck, Jukka; Visakorpi, Tapio

2015-08-14

Residual androgen receptor (AR)-signaling and presence of cancer stem-like cells (SCs) are the two emerging paradigms for clinically challenging castration-resistant prostate cancer (CRPC). Therefore, identification of AR-target proteins that are also overexpressed in the cancer SC population would be an attractive therapeutic approach.Our analysis of over three hundred clinical samples and patient-derived prostate epithelial cultures (PPECs), revealed Cancerous inhibitor of protein phosphatase 2A (CIP2A) as one such target. CIP2A is significantly overexpressed in both hormone-naïve prostate cancer (HN-PC) and CRPC patients . CIP2A is also overexpressed, by 3- and 30-fold, in HN-PC and CRPC SCs respectively. In vivo binding of the AR to the intronic region of CIP2A and its functionality in the AR-moderate and AR-high expressing LNCaP cell-model systems is also demonstrated. Further, we show that AR positively regulates CIP2A expression, both at the mRNA and protein level. Finally, CIP2A depletion reduced cell viability and colony forming efficiency of AR-independent PPECs as well as AR-responsive LNCaP cells, in which anchorage-independent growth is also impaired.These findings identify CIP2A as a common denominator for AR-signaling and cancer SC functionality, highlighting its potential therapeutic significance in the most clinically challenging prostate pathology: castration-resistant prostate cancer.

Regulation of Androgen Receptor-Mediated Transcription by RPB5 Binding Protein URI/RMP ▿

PubMed Central

Mita, Paolo; Savas, Jeffrey N.; Djouder, Nabil; Yates, John R.; Ha, Susan; Ruoff, Rachel; Schafler, Eric D.; Nwachukwu, Jerome C.; Tanese, Naoko; Cowan, Nicholas J.; Zavadil, Jiri; Garabedian, Michael J.; Logan, Susan K.

2011-01-01

Androgen receptor (AR)-mediated transcription is modulated by interaction with coregulatory proteins. We demonstrate that the unconventional prefoldin RPB5 interactor (URI) is a new regulator of AR transcription and is critical for antagonist (bicalutamide) action. URI is phosphorylated upon androgen treatment, suggesting communication between the URI and AR signaling pathways. Whereas depletion of URI enhances AR-mediated gene transcription, overexpression of URI suppresses AR transcriptional activation and anchorage-independent prostate cancer cell growth. Repression of AR-mediated transcription is achieved, in part, by URI binding and regulation of androgen receptor trapped clone 27 (Art-27), a previously characterized AR corepressor. Consistent with this idea, genome-wide expression profiling in prostate cancer cells upon depletion of URI or Art-27 reveals substantially overlapping patterns of gene expression. Further, depletion of URI increases the expression of the AR target gene NKX-3.1, decreases the recruitment of Art-27, and increases AR occupancy at the NKX-3.1 promoter. While Art-27 can bind AR directly, URI is bound to chromatin prior to hormone-dependent recruitment of AR, suggesting a role for URI in modulating AR recruitment to target genes. PMID:21730289

Catecholamine-Induced β2-adrenergic receptor activation mediates desensitization of gastric cancer cells to trastuzumab by upregulating MUC4 expression.

PubMed

Shi, Ming; Yang, Zhengyan; Hu, Meiru; Liu, Dan; Hu, Yabin; Qian, Lu; Zhang, Wei; Chen, Hongyu; Guo, Liang; Yu, Ming; Song, Lun; Ma, Yuanfang; Guo, Ning

2013-06-01

Trastuzumab is currently used for patients with Her2(+) advanced gastric cancer. However, the response rate to trastuzumab among the patients is low. The molecular mechanisms underlying trastuzumab resistance in gastric cancer are unknown. Our in vitro data show that activation of β2-adrenergic receptor (β2-AR) triggered by catecholamine caused "targeting failure" of trastuzumab in gastric cancer cells. The antitumor activities of trastuzumab were significantly impeded by chronic catecholamine stimulation in gastric cancer cells and in the mice bearing human gastric cancer xenografts. Mechanistically, catecholamine induced upregulation of the MUC4 expression at both transcription and protein levels via activating STAT3 and ERK. The effects of catecholamine could be effectively blocked by β2-AR antagonist ICI-118,551, indicating that β2-AR-mediated signaling pathway plays a key role in upregulation of MUC4, which was previously demonstrated to interfere with the recognition and physical binding of trastuzumab to Her2 molecules. Moreover, a significant elevation of the MUC4 level was observed in the xenograft tissues in nude mice chronically treated with isoproterenol. Knockdown of MUC4 restored the binding activities of trastuzumab to Her2-overexpressing gastric cancer cells. In addition, coexpression of β2-AR and MUC4 were observed in gastric cancer tissues. Our data indicated a novel trastuzumab resistance mechanism, by which catecholamine-induced β2-AR activation mediates desensitization of gastric cancer cells to trastuzumab through upregulating the MUC4 expression.

Osteopontin-c mediates the upregulation of androgen responsive genes in LNCaP cells through PI3K/Akt and androgen receptor signaling.

PubMed

Tilli, Tatiana Martins; Ferreira, Luciana Bueno; Gimba, Etel Rodrigues Pereira

2015-04-01

Androgen receptor (AR) signaling is a key pathway modulating prostate cancer (PCa) progression. Several steps in this pathway have been investigated in order to propose novel treatment strategies for advanced PCa. Total osteopontin (OPN) has been described as a biomarker for PCa, in addition to its role in activating the progression of this tumor. Based on the known effects of the OPNc splice variant on PCa progression, the present study investigated whether this isoform can also modulate AR signaling. In order to test this, an in vitro model was used in which LNCaP cells were cultured in the presence of conditioned medium (CM) secreted by PCa cells overexpressing OPNc (OPNc-CM). The activation of AR signaling was evaluated by measuring the expression levels of AR-responsive genes (ARGs) using quantitative polymerase chain reaction and specific oligonucleotides. The data demonstrated that all nine tested ARGs ( Fgf8 , TMPRSS2 , Greb1 , Cdk2 , Ndrg1 , Cdk1 , Pmepa1 , Psa and Ar ) are significantly upregulated in response to OPNc-CM compared with LNCaP cells cultured in CM secreted by control cells transfected with empty expression vector. The specific involvement of OPNc was demonstrated by depleting OPNc from OPNc-CM using an anti-OPNc neutralizing antibody. In addition, by using a phosphoinositide 3-kinase (PI3K)-specific inhibitor and AR antagonists, such as flutamide and bicalutamide, it was also observed that upregulation of ARGs in response to OPNc-CM involves PI3K signaling and depends on the AR. In conclusion, these data indicated that OPNc is able to activate AR signaling through the PI3K pathway and the AR. These data further corroborate our previous data, revealing the OPNc splice variant to be a key molecule that is able to modulate key signaling pathways involved in PCa progression.

Osteopontin-c mediates the upregulation of androgen responsive genes in LNCaP cells through PI3K/Akt and androgen receptor signaling

PubMed Central

TILLI, TATIANA MARTINS; FERREIRA, LUCIANA BUENO; GIMBA, ETEL RODRIGUES PEREIRA

2015-01-01

Androgen receptor (AR) signaling is a key pathway modulating prostate cancer (PCa) progression. Several steps in this pathway have been investigated in order to propose novel treatment strategies for advanced PCa. Total osteopontin (OPN) has been described as a biomarker for PCa, in addition to its role in activating the progression of this tumor. Based on the known effects of the OPNc splice variant on PCa progression, the present study investigated whether this isoform can also modulate AR signaling. In order to test this, an in vitro model was used in which LNCaP cells were cultured in the presence of conditioned medium (CM) secreted by PCa cells overexpressing OPNc (OPNc-CM). The activation of AR signaling was evaluated by measuring the expression levels of AR-responsive genes (ARGs) using quantitative polymerase chain reaction and specific oligonucleotides. The data demonstrated that all nine tested ARGs (Fgf8, TMPRSS2, Greb1, Cdk2, Ndrg1, Cdk1, Pmepa1, Psa and Ar) are significantly upregulated in response to OPNc-CM compared with LNCaP cells cultured in CM secreted by control cells transfected with empty expression vector. The specific involvement of OPNc was demonstrated by depleting OPNc from OPNc-CM using an anti-OPNc neutralizing antibody. In addition, by using a phosphoinositide 3-kinase (PI3K)-specific inhibitor and AR antagonists, such as flutamide and bicalutamide, it was also observed that upregulation of ARGs in response to OPNc-CM involves PI3K signaling and depends on the AR. In conclusion, these data indicated that OPNc is able to activate AR signaling through the PI3K pathway and the AR. These data further corroborate our previous data, revealing the OPNc splice variant to be a key molecule that is able to modulate key signaling pathways involved in PCa progression. PMID:25789054

Nuclear Factor-κB Promotes Urothelial Tumorigenesis and Cancer Progression via Cooperation with Androgen Receptor Signaling.

PubMed

Inoue, Satoshi; Ide, Hiroki; Mizushima, Taichi; Jiang, Guiyang; Netto, George J; Gotoh, Momokazu; Miyamoto, Hiroshi

2018-06-01

We investigated the role of NF-κB in the development and progression of urothelial cancer as well as cross-talk between NF-κB and androgen receptor (AR) signals in urothelial cells. Immunohistochemistry in surgical specimens showed that the expression levels of NF-κB/p65 ( P = 0.015)/phospho-NF-κB/p65 ( P < 0.001) were significantly elevated in bladder tumors, compared with those in nonneoplastic urothelial tissues. The rates of phospho-NF-κB/p65 positivity were also significantly higher in high-grade ( P = 0.015)/muscle-invasive ( P = 0.033) tumors than in lower grade/non-muscle-invasive tumors. Additionally, patients with phospho-NF-κB/p65-positive muscle-invasive bladder cancer had significantly higher risks of disease progression ( P < 0.001) and cancer-specific mortality ( P = 0.002). In immortalized human normal urothelial SVHUC cells stably expressing AR, NF-κB activators and inhibitors accelerated and prevented, respectively, their neoplastic transformation induced by a chemical carcinogen 3-methylcholanthrene. Bladder tumors were identified in 56% (mock), 89% (betulinic acid), and 22% (parthenolide) of N -butyl- N -(4-hydroxybutyl)nitrosamine-treated male C57BL/6 mice at 22 weeks of age. NF-κB activators and inhibitors also significantly induced and reduced, respectively, cell proliferation/migration/invasion of AR-positive bladder cancer lines, but not AR-knockdown or AR-negative lines, and their growth in xenograft-bearing mice. In both nonneoplastic and neoplastic urothelial cells, NF-κB activators/inhibitors upregulated/downregulated, respectively, AR expression, whereas AR overexpression was associated with increases in the expression levels of NF-κB/p65 and phospho-NF-κB/p65. Thus, NF-κB appeared to be activated in bladder cancer, which was associated with tumor progression. NF-κB activators/inhibitors were also found to modulate tumorigenesis and tumor outgrowth in AR-activated urothelial cells. Accordingly, NF-κB inhibition, together with AR inactivation, has the potential of being an effective chemopreventive and/or therapeutic approach for urothelial carcinoma. Mol Cancer Ther; 17(6); 1303-14. ©2018 AACR . ©2018 American Association for Cancer Research.

Effects of prenatal stress and monoaminergic perturbations on the expression of serotonin 5-HT₄ and adrenergic β₂ receptors in the embryonic mouse telencephalon.

PubMed

Chen, Angela; Kelley, Lauren D S; Janušonis, Skirmantas

2012-06-12

The serotonin 5-HT(4) receptor (5-HT(4)R) is coded by a complex gene that produces four mRNA splice variants in mice (5-HT(4(a))R, 5-HT(4(b))R, 5-HT(4(e))R, 5-HT(4(f))R). This receptor has highly dynamic expression in brain development and its splice variants differ in their developmental trajectories. Since 5-HT(4)Rs are important in forebrain function (including forebrain control of serotonergic activity in the brainstem), we investigated the susceptibility of 5-HT(4)R expression in the mouse embryonic telencephalon to prenatal maternal stress and altered serotonin (5-hydroxytryptamine, 5-HT) levels. Because the gene coding the adrenergic β(2) receptor (β(2)AR) is embedded in the 5-HT(4)R gene, we also investigated whether 5-HT(4)R mRNA levels were modulated by selective β(2)AR agents. Timed-pregnant C57BL/6 mice were treated beginning at embryonic day (E) 14 and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to assess the mRNA levels of all 5-HT(4)R splice variants and β(2)AR in the embryonic telencephalon at E17. Maternal prenatal stress and 5-HT depletion with pCPA, a tryptophan hydroxylase inhibitor, reduced the levels of the 5-HT(4(b))R splice variant. Terbutaline (a selective β(2)AR agonist) and ICI 118,551 (a selective β(2)AR antagonist) had no effect on β(2)AR and 5-HT(4)R mRNA levels. These results show that prenatal stress and reduced 5-HT levels can alter 5-HT(4)R expression in the developing forebrain and that some 5-HT(4)R splice variants may be more susceptible than others. Copyright © 2012 Elsevier B.V. All rights reserved.

Modulation of Androgen Receptor Signaling in Hormonal Therapy-Resistant Prostate Cancer Cell Lines

PubMed Central

Marques, Rute B.; Dits, Natasja F.; Erkens-Schulze, Sigrun; van IJcken, Wilfred F. J.; van Weerden, Wytske M.; Jenster, Guido

2011-01-01

Background Prostate epithelial cells depend on androgens for survival and function. In (early) prostate cancer (PCa) androgens also regulate tumor growth, which is exploited by hormonal therapies in metastatic disease. The aim of the present study was to characterize the androgen receptor (AR) response in hormonal therapy-resistant PC346 cells and identify potential disease markers. Methodology/Principal Findings Human 19K oligoarrays were used to establish the androgen-regulated expression profile of androgen-responsive PC346C cells and its derivative therapy-resistant sublines: PC346DCC (vestigial AR levels), PC346Flu1 (AR overexpression) and PC346Flu2 (T877A AR mutation). In total, 107 transcripts were differentially-expressed in PC346C and derivatives after R1881 or hydroxyflutamide stimulations. The AR-regulated expression profiles reflected the AR modifications of respective therapy-resistant sublines: AR overexpression resulted in stronger and broader transcriptional response to R1881 stimulation, AR down-regulation correlated with deficient response of AR-target genes and the T877A mutation resulted in transcriptional response to both R1881 and hydroxyflutamide. This AR-target signature was linked to multiple publicly available cell line and tumor derived PCa databases, revealing that distinct functional clusters were differentially modulated during PCa progression. Differentiation and secretory functions were up-regulated in primary PCa but repressed in metastasis, whereas proliferation, cytoskeletal remodeling and adhesion were overexpressed in metastasis. Finally, the androgen-regulated genes ENDOD1, MCCC2 and ACSL3 were selected as potential disease markers for RT-PCR quantification in a distinct set of human prostate specimens. ENDOD1 and ACSL3 showed down-regulation in high-grade and metastatic PCa, while MCCC2 was overexpressed in low-grade PCa. Conclusions/Significance AR modifications altered the transcriptional response to (anti)androgens in therapy-resistant cells. Furthermore, selective down-regulation of genes involved in differentiation and up-regulation of genes promoting proliferation and invasion suggest a disturbed balance between the growth and differentiation functions of the AR pathway during PCa progression. These findings may have implications in the current treatment and development of novel therapeutical approaches for metastatic PCa. PMID:21829708

The effects and possible mechanism of β2AR gene expression in cardiocytes of canines with heart failure.

PubMed

Gong, Haibin; San, Yu; Wang, Lei; Lv, Qian; Chen, Libin

2017-07-01

The objective of the present study was to observe the changes of β 2 -adrenergic receptor (β 2 AR) protein expression in a canine model of heart failure (HF), and the function of cardiocytes after transfection with Adv-β 2 AR. The canine model of chronic HF was induced by rapid right ventricular pacing and cardiocytes were isolated with collagenase II. Cardiocytes were transfected with Adv-β 2 AR to observe contractile function with a motion edge-detection system of single cells. Expression of β 2 AR protein in cardiocytes was measured by immunoblotting and the levels of intracellular cAMP were measured by ELISA. Compared with the control group (the sham group), the expression of β 2 AR protein in HF cardiocytes did not change, but the basal (1 mM Ca 2+ ) contraction amplitude percentage (1.809±0.922 vs. 1.120±0.432%, P<0.05), the maximum contraction amplitude percentage (14.855±2.377 vs. 10.784±2.675%, P<0.01) and the basal levels of intracellular cAMP (9.39±2.54 vs. 5.26±0.95 pmol/ml, n=6, P<0.05) of HF cardiocytes were significantly decreased. However, when HF cardiocytes were transfected with Adv-β 2 AR and cultured for 48 h, compared with the non-transfected group, the basal contraction amplitude percentage (0.851±0.324 vs. 1.629±0.522%, P<0.05), the maximum contraction amplitude percentage (9.260±2.208% vs. 12.205±1.437%, P<0.01) and the basal levels of intracellular cAMP (5.26±0.95 vs. 9.03±1.03 pmol/ml, n=6, P<0.05) of cardiocytes in the transfected group were significantly increased. In conclusion, the expression of β 2 AR protein in HF cardiocytes did not change, but contraction function was impaired. The moderate overexpression of β 2 AR gene in the HF cardiocytes increased the levels of intracellular cAMP and improved contraction function.

Do β3-adrenergic receptors play a role in guinea pig detrusor smooth muscle excitability and contractility?

PubMed Central

Afeli, Serge A. Y.; Hristov, Kiril L.

2012-01-01

In many species, β3-adrenergic receptors (β3-ARs) have been reported to play a primary role in pharmacologically induced detrusor smooth muscle (DSM) relaxation. However, their role in guinea pig DSM remains controversial. The aim of this study was to investigate whether β3-ARs are expressed in guinea pig DSM and to evaluate how BRL37344 and L-755,507, two selective β3-AR agonists, modulate guinea pig DSM excitability and contractility. We used a combined experimental approach including RT-PCR, patch-clamp electrophysiology, and isometric DSM tension recordings. β3-AR mRNA message was detected in freshly isolated guinea pig DSM single cells. BRL37344 but not L-755,507 caused a slight decrease in DSM spontaneous phasic contraction amplitude and frequency in a concentration-dependent manner. In the presence of atropine (1 μM), only the spontaneous phasic contractions frequency was inhibited by BRL37344 at higher concentrations. Both BRL37344 and L-755,507 significantly decreased DSM carbachol-induced phasic and tonic contractions in a concentration-dependent manner. However, only BRL37344 inhibitory effect was partially antagonized by SR59230A (10 μM), a β3-AR antagonist. In the presence of atropine, BRL37344 and L-755,507 had no inhibitory effect on electrical field stimulation-induced contractions. Patch-clamp experiments showed that BRL37344 (100 μM) did not affect the DSM cell resting membrane potential and K+ conductance. Although β3-ARs are expressed at the mRNA level, they play a minor to no role in guinea pig DSM spontaneous contractility without affecting cell excitability. However, BRL37344 and L-755,507 have pronounced inhibitory effects on guinea pig DSM carbachol-induced contractions. The study outlines important DSM β3-ARs species differences. PMID:21993887

Functional coupling of rat myometrial alpha 1-adrenergic receptors to Gh alpha/tissue transglutaminase 2 during pregnancy.

PubMed

Dupuis, Morgan; Lévy, Arlette; Mhaouty-Kodja, Sakina

2004-04-30

Gh alpha protein, which exhibits both transglutaminase and GTPase activities, represents a new class of GTP-binding proteins. In the present study, we characterized Gh alpha in rat uterine smooth muscle (myometrium) and followed its expression during pregnancy by reverse transcription-PCR and Western blot. We also measured transglutaminase and GTP binding functions and used a smooth muscle cell line to evaluate the role of Gh alpha in cell proliferation. The results show that pregnancy is associated with an up-regulation of Gh alpha expression at both the mRNA and protein level. Gh alpha induced during pregnancy is preferentially localized to the plasma membrane. This was found associated with an increased ability of plasma membrane preparations to catalyze Ca(2+)-dependent incorporation of [(3)H]putrescine into casein in vitro. In the cytosol, significant changes in the level of immunodetected Gh alpha and transglutaminase activity were seen only at term. Activation of alpha1-adrenergic receptors (alpha1-AR) enhanced photoaffinity labeling of plasma membrane Gh alpha. Moreover, the level of alpha1-AR-coupled Gh alpha increased progressively with pregnancy, which parallels the active period of myometrial cell proliferation. Overexpression of wild type Gh alpha in smooth muscle cell line DDT1-MF2 increased alpha1-AR-induced [(3)H]thymidine incorporation. A similar response was obtained in cells expressing the transglutaminase inactive mutant (C277S) of Gh alpha. Together, these findings underscore the role of Gh alpha as signal transducer of alpha1-AR-induced smooth muscle cell proliferation. In this context, pregnant rat myometrium provides an interesting physiological model to study the mechanisms underlying the regulation of the GTPase function of Gh alpha

Nicotine Suppressed Fetal Adrenal StAR Expression via YY1 Mediated-Histone Deacetylation Modification Mechanism.

PubMed

Liu, Lian; Wang, Jian-Fei; Fan, Jie; Rao, Yi-Song; Liu, Fang; Yan, You-E; Wang, Hui

2016-09-03

Steroidogenic acute regulatory (StAR) protein plays a pivotal role in steroidogenesis. Previously, we have demonstrated that prenatal nicotine exposure suppressed fetal adrenal steroidogenesis via steroidogenic factor 1 deacetylation. This study further explored the potential role of the transcriptional repressor Yin Yang 1 (YY1) in nicotine-mediated StAR inhibition. Nicotine was subcutaneously administered (1.0 mg/kg) to pregnant rats twice per day and NCI-H295A cells were treated with nicotine. StAR and YY1 expression were analyzed by real-time PCR, immunohistochemistry, and Western blotting. Histone modifications and the interactions between the YY1 and StAR promoter were assessed using chromatin immunoprecipitation (ChIP). Prenatal nicotine exposure increased YY1 expression and suppressed StAR expression. ChIP assay showed that there was a decreasing trend for histone acetylation at the StAR promoter in fetal adrenal glands, whereas H3 acetyl-K14 at the YY1 promoter presented an increasing trend following nicotine exposure. Furthermore, in nicotine-treated NCI-H295A cells, nicotine enhanced YY1 expression and inhibited StAR expression. ChIP assay showed that histone acetylation decreased at the StAR promoter in NCI-H295A cells and that the interaction between the YY1 and StAR promoter increased. These data indicated that YY1-medicated histone deacetylation modification in StAR promoters might play an important role in the inhibitory effect of nicotine on StAR expression.

Nicotine Suppressed Fetal Adrenal StAR Expression via YY1 Mediated-Histone Deacetylation Modification Mechanism

PubMed Central

Liu, Lian; Wang, Jian-Fei; Fan, Jie; Rao, Yi-Song; Liu, Fang; Yan, You-E; Wang, Hui

2016-01-01

Steroidogenic acute regulatory (StAR) protein plays a pivotal role in steroidogenesis. Previously, we have demonstrated that prenatal nicotine exposure suppressed fetal adrenal steroidogenesis via steroidogenic factor 1 deacetylation. This study further explored the potential role of the transcriptional repressor Yin Yang 1 (YY1) in nicotine-mediated StAR inhibition. Nicotine was subcutaneously administered (1.0 mg/kg) to pregnant rats twice per day and NCI-H295A cells were treated with nicotine. StAR and YY1 expression were analyzed by real-time PCR, immunohistochemistry, and Western blotting. Histone modifications and the interactions between the YY1 and StAR promoter were assessed using chromatin immunoprecipitation (ChIP). Prenatal nicotine exposure increased YY1 expression and suppressed StAR expression. ChIP assay showed that there was a decreasing trend for histone acetylation at the StAR promoter in fetal adrenal glands, whereas H3 acetyl-K14 at the YY1 promoter presented an increasing trend following nicotine exposure. Furthermore, in nicotine-treated NCI-H295A cells, nicotine enhanced YY1 expression and inhibited StAR expression. ChIP assay showed that histone acetylation decreased at the StAR promoter in NCI-H295A cells and that the interaction between the YY1 and StAR promoter increased. These data indicated that YY1-medicated histone deacetylation modification in StAR promoters might play an important role in the inhibitory effect of nicotine on StAR expression. PMID:27598153

Infarct-Induced Steroidogenic Acute Regulatory Protein: A Survival Role in Cardiac Fibroblasts

PubMed Central

Anuka, Eli; Yivgi-Ohana, Natalie; Eimerl, Sarah; Garfinkel, Benjamin; Melamed-Book, Naomi; Chepurkol, Elena; Aravot, Dan; Zinman, Tova; Shainberg, Asher; Hochhauser, Edith

2013-01-01

Steroidogenic acute regulatory protein (StAR) is indispensable for steroid hormone synthesis in the adrenal cortex and the gonadal tissues. This study reveals that StAR is also expressed at high levels in nonsteroidogenic cardiac fibroblasts confined to the left ventricle of mouse heart examined 3 days after permanent ligation of the left anterior descending coronary artery. Unlike StAR, CYP11A1 and 3β-hydroxysteroid dehydrogenase proteins were not observed in the postinfarction heart, suggesting an apparent lack of de novo cardiac steroidogenesis. Work with primary cultures of rat heart cells revealed that StAR is induced in fibroblasts responding to proapoptotic treatments with hydrogen peroxide or the kinase inhibitor staurosporine (STS). Such induction of StAR in culture was noted before spontaneous differentiation of the fibroblasts to myofibroblasts. STS induction of StAR in the cardiac fibroblasts conferred a marked resistance to apoptotic cell death. Consistent with that finding, down-regulation of StAR by RNA interference proportionally increased the number of STS-treated apoptotic cells. StAR down-regulation also resulted in a marked increase of BAX activation in the mitochondria, an event known to associate with the onset of apoptosis. Last, STS treatment of HeLa cells showed that apoptotic demise characterized by mitochondrial fission, cytochrome c release, and nuclear fragmentation is arrested in individual HeLa cells overexpressing StAR. Collectively, our in vivo and ex vivo evidence suggests that postinfarction expression of nonsteroidogenic StAR in cardiac fibroblasts has novel antiapoptotic activity, allowing myofibroblast precursor cells to survive the traumatized event, probably to differentiate and function in tissue repair at the infarction site. PMID:23831818

Testosterone enables growth and hypertrophy in fusion impaired myoblasts that display myotube atrophy: deciphering the role of androgen and IGF-I receptors.

PubMed

Hughes, David C; Stewart, Claire E; Sculthorpe, Nicholas; Dugdale, Hannah F; Yousefian, Farzad; Lewis, Mark P; Sharples, Adam P

2016-06-01

We have previously highlighted the ability of testosterone (T) to improve differentiation and myotube hypertrophy in fusion impaired myoblasts that display reduced myotube hypertrophy via multiple population doublings (PD) versus their parental controls (CON); an observation which is abrogated via PI3K/Akt inhibition (Deane et al. 2013). However, whether the most predominant molecular mechanism responsible for T induced hypertrophy occurs directly via androgen receptor or indirectly via IGF-IR/PI3K/Akt pathway is currently debated. PD and CON C2C12 muscle cells were exposed to low serum conditions in the presence or absence of T (100 nM) ± inhibitors of AR (flutamide/F, 40 μm) and IGF-IR (picropodophyllin/PPP, 150 nM) for 72 h and 7 days (early/late muscle differentiation respectively). T increased AR and Akt abundance, myogenin gene expression, and myotube hypertrophy, but not ERK1/2 activity in both CON and PD cell types. Akt activity was not increased significantly in either cell type with T. Testosterone was also unable to promote early differentiation in the presence of IGF-IR inhibitor (PPP) yet still able to promote appropriate later increases in myotube hypertrophy and AR abundance despite IGF-IR inhibition. The addition of the AR inhibitor powerfully attenuated all T induced increases in differentiation and myotube hypertrophy with corresponding reductions in AR abundance, phosphorylated Akt, ERK1/2 and gene expression of IGF-IR, myoD and myogenin with increases in myostatin mRNA in both cell types. Interestingly, despite basally reduced differentiation and myotube hypertrophy, PD cells showed larger T induced increases in AR abundance vs. CON cells, a response abrogated in the presence of AR but not IGF-IR inhibitors. Furthermore, T induced increases in Akt abundance were sustained despite the presence of IGF-IR inhibition in PD cells only. Importantly, flutamide alone reduced IGF-IR mRNA in both cell types across time points, with an observed reduction in activity of ERK and Akt, suggesting that IGF-IR was transcriptionally regulated by AR. However, where testosterone increased AR protein content there was no increases observed in IGF-IR gene expression. This suggested that sufficient AR was important to enable normal IGF-IR expression and downstream signalling, yet elevated levels of AR due to testosterone had no further effect on IGF-IR mRNA, despite testosterone increasing Akt abundance in the presence of IGF-IR inhibitor. In conclusion, testosterones ability to improve differentiation and myotube hypertrophy occurred predominately via increases in AR and Akt abundance in both CON and PD cells, with fusion impaired cells (PD) showing an increased responsiveness to T induced AR levels. Finally, T induced increases in myotube hypertrophy (but not early differentiation) occurred independently of upstream IGF-IR input, however it was apparent  that normal AR function in basal conditions was required for adequate IGF-IR gene expression and downstream ERK/Akt activity.

Exploring the Interaction Mechanism Between Cyclopeptide DC3 and Androgen Receptor Using Molecular Dynamics Simulations and Free Energy Calculations

PubMed Central

Zhang, Huimin; Song, Tianqing; Yang, Yizhao; Fu, Chenggong; Li, Jiazhong

2018-01-01

Androgen receptor (AR) is a key target in the discovery of anti-PCa (Prostate Cancer) drugs. Recently, a novel cyclopeptide Diffusa Cyclotide-3 (DC3), isolated from Hedyotisdiffusa, has been experimentally demonstrated to inhibit the survival and growth of LNCap cells, which typically express T877A-mutated AR, the most frequently detected point mutation of AR in castration-resistant prostate cancer (CRPC). But the interaction mechanism between DC3 and AR is not clear. Here in this study we aim to explore the possible binding mode of DC3 to T877A-mutated AR from molecular perspective. Firstly, homology modeling was employed to construct the three-dimensional structure of the cyclopeptide DC3 using 2kux.1.A as the template. Then molecular docking, molecular dynamics (MD) simulations, and molecular mechanics/generalized Born surface area (MM-GBSA) methods were performed to determine the bind site and explore the detailed interaction mechanism of DC3-AR complex. The obtained results suggested that the site formed by H11, loop888-893, and H12 (site 2) was the most possible position of DC3 binding to AR. Besides, hydrogen bonds, hydrophobic, and electrostatic interactions play dominant roles in the recognition and combination of DC3-AR complex. The essential residues dominant in each interaction were specifically revealed. This work facilitates our understanding of the interaction mechanism of DC3 binding to AR at the molecular level and contributes to the rational cyclopeptide drug design for prostate cancer. PMID:29755968

Exploring the Interaction Mechanism between Cyclopeptide DC3 and Androgen Receptor Using Molecular Dynamics Simulations and Free Energy Calculations

NASA Astrophysics Data System (ADS)

Zhang, Huimin; Song, Tianqing; Yang, Yizhao; Fu, Chenggong; Li, Jiazhong

2018-04-01

Androgen receptor (AR) is a key target in the discovery of anti-PCa (Prostate Cancer) drugs. Recently, a novel cyclopeptide Diffusa Cyclotide-3 (DC3), isolated from Hedyotisdiffusa, has been experimentally demonstrated to inhibit the survival and growth of LNCap cells, which typically express T877A-mutated AR, the most frequently detected point mutation of AR in castration-resistant prostate cancer (CRPC). But the interaction mechanism between DC3 and AR is not clear. Here in this study we aim to explore the possible binding mode of DC3 to T877A-mutated AR from molecular perspective. Firstly, homology modeling was employed to construct the three-dimensional structure of the cyclopeptide DC3 using 2kux.1.A as the template. Then molecular docking, molecular dynamics (MD) simulations and molecular mechanics/generalized Born surface area (MM-GBSA) methods were performed to determine the bind site and explore the detailed interaction mechanism of DC3-AR complex. The obtained results suggested that the site formed by H11, loop888-893 and H12 (site 2) was the most possible position of DC3 binding to AR. Besides, hydrogen bonds, hydrophobic and electrostatic interactions play dominant roles in the recognition and combination of DC3-AR complex. The essential residues dominant in each interaction were specifically revealed. This work facilitates our understanding of the interaction mechanism of DC3 binding to AR at the molecular level and contributes to the rational cyclopeptide drug design for prostate cancer.

Exploring the Interaction Mechanism Between Cyclopeptide DC3 and Androgen Receptor Using Molecular Dynamics Simulations and Free Energy Calculations.

PubMed

Zhang, Huimin; Song, Tianqing; Yang, Yizhao; Fu, Chenggong; Li, Jiazhong

2018-01-01

Androgen receptor (AR) is a key target in the discovery of anti-PCa (Prostate Cancer) drugs. Recently, a novel cyclopeptide Diffusa Cyclotide-3 (DC3), isolated from Hedyotisdiffusa , has been experimentally demonstrated to inhibit the survival and growth of LNCap cells, which typically express T877A-mutated AR, the most frequently detected point mutation of AR in castration-resistant prostate cancer (CRPC). But the interaction mechanism between DC3 and AR is not clear. Here in this study we aim to explore the possible binding mode of DC3 to T877A-mutated AR from molecular perspective. Firstly, homology modeling was employed to construct the three-dimensional structure of the cyclopeptide DC3 using 2kux.1.A as the template. Then molecular docking, molecular dynamics (MD) simulations, and molecular mechanics/generalized Born surface area (MM-GBSA) methods were performed to determine the bind site and explore the detailed interaction mechanism of DC3-AR complex. The obtained results suggested that the site formed by H11, loop888-893, and H12 (site 2) was the most possible position of DC3 binding to AR. Besides, hydrogen bonds, hydrophobic, and electrostatic interactions play dominant roles in the recognition and combination of DC3-AR complex. The essential residues dominant in each interaction were specifically revealed. This work facilitates our understanding of the interaction mechanism of DC3 binding to AR at the molecular level and contributes to the rational cyclopeptide drug design for prostate cancer.

Jasmonates act positively in adventitious root formation in petunia cuttings.

PubMed

Lischweski, Sandra; Muchow, Anne; Guthörl, Daniela; Hause, Bettina

2015-09-22

Petunia is a model to study the process of adventitious root (AR) formation on leafy cuttings. Excision of cuttings leads to a transient increase in jasmonates, which is regarded as an early, transient and critical event for rooting. Here, the role of jasmonates in AR formation on petunia cuttings has been studied by a reverse genetic approach. To reduce the endogenous levels of jasmonates, transgenic plants were generated expressing a Petunia hybrida ALLENE OXIDE CYCLASE (PhAOC)-RNAi construct. The transgenic plants exhibited strongly reduced PhAOC transcript and protein levels as well as diminished accumulation of cis-12-oxo-phytodienoic acid, jasmonic acid and jasmonoyl-isoleucine after wounding in comparison to wild type and empty vector expressing plants. Reduced levels of endogenous jasmonates resulted in formation of lower numbers of ARs. However, this effect was not accompanied by altered levels of auxin and aminocyclopropane carboxylate (ACC, precursor of ethylene) or by impaired auxin and ethylene-induced gene expression. Neither activity of cell-wall invertases nor accumulation of soluble sugars was altered by jasmonate deficiency. Diminished numbers of AR in JA-deficient cuttings suggest that jasmonates act as positive regulators of AR formation in petunia wild type. However, wound-induced rise in jasmonate levels in petunia wild type cuttings seems not to be causal for increased auxin and ethylene levels and for sink establishment.

Prostate Cancer Characteristics Associated with Response to Pre-Receptor Targeting of the Androgen Axis

PubMed Central

Mostaghel, Elahe A.; Morgan, Andrew; Zhang, Xiaotun; Marck, Brett T.; Xia, Jing; Hunter-Merrill, Rachel; Gulati, Roman; Plymate, Stephen; Vessella, Robert L.; Corey, Eva; Higano, Celestia S.; Matsumoto, Alvin M.; Montgomery, R. Bruce; Nelson, Peter S.

2014-01-01

Background Factors influencing differential responses of prostate tumors to androgen receptor (AR) axis-directed therapeutics are poorly understood, and predictors of treatment efficacy are needed. We hypothesized that the efficacy of inhibiting DHT ligand synthesis would associate with intra-tumoral androgen ratios indicative of relative dependence on DHT-mediated growth. Methods We characterized two androgen-sensitive prostate cancer xenograft models after androgen suppression by castration in combination with the SRD5A inhibitor, dutasteride, as well as a panel of castration resistant metastases obtained via rapid autopsy. Results In LuCaP35 tumors (intra-tumoral T:DHT ratio 2∶1) dutasteride suppressed DHT to 0.02 ng/gm and prolonged survival vs. castration alone (337 vs.152 days, HR 2.8, p = 0.0015). In LuCaP96 tumors (T:DHT 10∶1), survival was not improved despite similar DHT reduction (0.02 ng/gm). LuCaP35 demonstrated higher expression of steroid biosynthetic enzymes maintaining DHT levels (5-fold higher SRD5A1, 41 fold higher, 99-fold higher RL-HSD, p<0.0001 for both), reconstitution of intra-tumoral DHT (to ∼30% of untreated tumors), and ∼2 fold increased expression of full length AR. In contrast, LuCaP96 demonstrated higher levels of steroid catabolizing enzymes (6.9-fold higher AKR1C2, 3000-fold higher UGT2B15, p = 0.002 and p<0.0001 respectively), persistent suppression of intra-tumoral DHT, and 6–8 fold induction of full length AR and the ligand independent V7 AR splice variant. Human metastases demonstrated bio-active androgen levels and AR full length and AR splice-variant expression consistent with the range observed in xenografts. Conclusions Intrinsic differences in basal steroidogenesis, as well as variable expression of full length and splice-variant AR, associate with response and resistance to pre-receptor AR ligand suppression. Expression of steroidogenic enzymes and AR isoforms may serve as potential biomarkers of sensitivity to potent AR-axis inhibition and should be validated in additional models. PMID:25356728

Prostate cancer characteristics associated with response to pre-receptor targeting of the androgen axis.

PubMed

Mostaghel, Elahe A; Morgan, Andrew; Zhang, Xiaotun; Marck, Brett T; Xia, Jing; Hunter-Merrill, Rachel; Gulati, Roman; Plymate, Stephen; Vessella, Robert L; Corey, Eva; Higano, Celestia S; Matsumoto, Alvin M; Montgomery, R Bruce; Nelson, Peter S

2014-01-01

Factors influencing differential responses of prostate tumors to androgen receptor (AR) axis-directed therapeutics are poorly understood, and predictors of treatment efficacy are needed. We hypothesized that the efficacy of inhibiting DHT ligand synthesis would associate with intra-tumoral androgen ratios indicative of relative dependence on DHT-mediated growth. We characterized two androgen-sensitive prostate cancer xenograft models after androgen suppression by castration in combination with the SRD5A inhibitor, dutasteride, as well as a panel of castration resistant metastases obtained via rapid autopsy. In LuCaP35 tumors (intra-tumoral T:DHT ratio 2:1) dutasteride suppressed DHT to 0.02 ng/gm and prolonged survival vs. castration alone (337 vs.152 days, HR 2.8, p = 0.0015). In LuCaP96 tumors (T:DHT 10:1), survival was not improved despite similar DHT reduction (0.02 ng/gm). LuCaP35 demonstrated higher expression of steroid biosynthetic enzymes maintaining DHT levels (5-fold higher SRD5A1, 41 fold higher, 99-fold higher RL-HSD, p<0.0001 for both), reconstitution of intra-tumoral DHT (to ∼30% of untreated tumors), and ∼2 fold increased expression of full length AR. In contrast, LuCaP96 demonstrated higher levels of steroid catabolizing enzymes (6.9-fold higher AKR1C2, 3000-fold higher UGT2B15, p = 0.002 and p<0.0001 respectively), persistent suppression of intra-tumoral DHT, and 6-8 fold induction of full length AR and the ligand independent V7 AR splice variant. Human metastases demonstrated bio-active androgen levels and AR full length and AR splice-variant expression consistent with the range observed in xenografts. Intrinsic differences in basal steroidogenesis, as well as variable expression of full length and splice-variant AR, associate with response and resistance to pre-receptor AR ligand suppression. Expression of steroidogenic enzymes and AR isoforms may serve as potential biomarkers of sensitivity to potent AR-axis inhibition and should be validated in additional models.

Comparative Proteomic Analysis Reveals the Effects of Exogenous Calcium against Acid Rain Stress in Liquidambar formosana Hance Leaves.

PubMed

Hu, Wen-Jun; Wu, Qian; Liu, Xiang; Shen, Zhi-Jun; Chen, Juan; Liu, Ting-Wu; Chen, Juan; Zhu, Chun-Quan; Wu, Fei-Hua; Chen, Lin; Wei, Jia; Qiu, Xiao-Yun; Shen, Guo-Xin; Zheng, Hai-Lei

2016-01-04

Acid rain (AR) impacts forest health by leaching calcium (Ca) away from soils and plants. Ca is an essential element and participates in various plant physiological responses. In the present study, the protective role of exogenous Ca in alleviating AR stress in Liquidambar formosana Hance at the physiological and proteomic levels was examined. Our results showed that low Ca condition resulted in the chlorophyll content and photosynthesis decreasing significantly in L. formosana leaves; however, these effects could be reversed by high Ca supplementation. Further proteomic analyses successfully identified 81 differentially expressed proteins in AR-treated L. formosana under different Ca levels. In particular, some of the proteins are involved in primary metabolism, photosynthesis, energy production, antioxidant defense, transcription, and translation. Moreover, quantitative real time polymerase chain reaction (qRT-PCR) results indicated that low Ca significantly increased the expression level of the investigated Ca-related genes, which can be reversed by high Ca supplementation under AR stress. Further, Western blotting analysis revealed that exogenous Ca supply reduced AR damage by elevating the expression of proteins involved in the Calvin cycle, reactive oxygen species (ROS) scavenging system. These findings allowed us to better understand how woody plants respond to AR stress at various Ca levels and the protective role of exogenous Ca against AR stress in forest tree species.

PPARγ regulates exocrine pancreas lipase.

PubMed

Danino, Hila; Naor, Ronny Peri-; Fogel, Chen; Ben-Harosh, Yael; Kadir, Rotem; Salem, Hagit; Birk, Ruth

2016-12-01

Pancreatic lipase (triacylglycerol lipase EC 3.1.1.3) is an essential enzyme in hydrolysis of dietary fat. Dietary fat, especially polyunsaturated fatty acids (PUFA), regulate pancreatic lipase (PNLIP); however, the molecular mechanism underlying this regulation is mostly unknown. As PUFA are known to regulate expression of proliferator-activated receptor gamma (PPARγ), and as we identified in-silico putative PPARγ binding sites within the putative PNLIP promoter sequence, we hypothesized that PUFA regulation of PNLIP might be mediated by PPARγ. We used in silico bioinformatics tools, reporter luciferase assay, PPARγ agonists and antagonists, PPARγ overexpression in exocrine pancreas AR42J and primary cells to study PPARγ regulation of PNLIP. Using in silico bioinformatics tools we mapped PPARγ binding sites (PPRE) to the putative promoter region of PNLIP. Reporter luciferase assay in AR42J rat exocrine pancreas acinar cells transfected with various constructs of the putative PNLIP promoter showed that PNLIP transcription is significantly enhanced by PPARγ dose-dependently, reaching maximal levels with multi PPRE sites. This effect was significantly augmented in the presence of PPARγ agonists and reduced by PPARγ antagonists or mutagenesis abrogating PPRE sites. Over-expression of PPARγ significantly elevated PNLIP transcript and protein levels in AR42J cells and in primary pancreas cells. Moreover, PNLIP expression was up-regulated by PPARγ agonists (pioglitazone and 15dPGJ2) and significantly down-regulated by PPARγ antagonists in non-transfected rat exocrine pancreas AR42J cell line cells. PPARγ transcriptionally regulates PNLIP gene expression. This transcript regulation resolves part of the missing link between dietary PUFA direct regulation of PNLIP. Copyright © 2016 Elsevier B.V. All rights reserved.

Arabidopsis SHR and SCR transcription factors and AUX1 auxin influx carrier control the switch between adventitious rooting and xylogenesis in planta and in in vitro cultured thin cell layers.

PubMed

Della Rovere, F; Fattorini, L; D'Angeli, S; Veloccia, A; Del Duca, S; Cai, G; Falasca, G; Altamura, M M

2015-03-01

Adventitious roots (ARs) are essential for vegetative propagation. The Arabidopsis thaliana transcription factors SHORT ROOT (SHR) and SCARECROW (SCR) affect primary/lateral root development, but their involvement in AR formation is uncertain. LAX3 and AUX1 auxin influx carriers contribute to primary/lateral root development. LAX3 expression is regulated by SHR, and LAX3 contributes to AR tip auxin maximum. In contrast, AUX1 involvement in AR development is unknown. Xylogenesis is induced by auxin plus cytokinin as is AR formation, but the genes involved are largely unknown. Stem thin cell layers (TCLs) form ARs and undergo xylogenesis under the same auxin plus cytokinin input. The aim of this research was to investigate SHR, SCR, AUX1 and LAX3 involvement in AR formation and xylogenesis in intact hypocotyls and stem TCLs in arabidopsis. Hypocotyls of scr-1, shr-1, lax3, aux1-21 and lax3/aux1-21 Arabidopsis thaliana null mutant seedlings grown with or without auxin plus cytokinin were examined histologically, as were stem TCLs cultured with auxin plus cytokinin. SCR and AUX1 expression was monitored using pSCR::GFP and AUX1::GUS lines, and LAX3 expression and auxin localization during xylogenesis were monitored by using LAX3::GUS and DR5::GUS lines. AR formation was inhibited in all mutants, except lax3. SCR was expressed in pericycle anticlinally derived AR-forming cells of intact hypocotyls, and in cell clumps forming AR meristemoids of TCLs. The apex was anomalous in shr and scr ARs. In all mutant hypocotyls, the pericycle divided periclinally to produce xylogenesis. Xylary element maturation was favoured by auxin plus cytokinin in shr and aux1-21. Xylogenesis was enhanced in TCLs, and in aux1-21 and shr in particular. AUX1 was expressed before LAX3, i.e. in the early derivatives leading to either ARs or xylogenesis. AR formation and xylogenesis are developmental programmes that are inversely related, but they involve fine-tuning by the same proteins, namely SHR, SCR and AUX1. Pericycle activity is central for the equilibrium between xylary development and AR formation in the hypocotyl, with a role for AUX1 in switching between, and balancing of, the two developmental programmes. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company.

Ankyrin G expression is associated with androgen receptor stability, invasiveness, and lethal outcome in prostate cancer patients.

PubMed

Wang, Tingting; Abou-Ouf, Hatem; Hegazy, Samar A; Alshalalfa, Mohammed; Stoletov, Konstantin; Lewis, John; Donnelly, Bryan; Bismar, Tarek A

2016-12-01

Ankyrin G (ANK3) is a member of the Ankyrin family, which functions to provide cellular stability by anchoring the cytoskeleton to the plasma membrane. Deregulation of ANK3 expression has been observed in multiple human cancers but its mechanism remains unknown. ANK3 expression in relation to disease progression and patients' outcome was investigated in two cohorts of prostate cancer (PCA). Mechanistic studies were carried out in vitro and in vivo using several PCA cell lines and the avian embryo model. Silencing ANK3 resulted in significant reduction of cell proliferation through an AR-independent mechanism. Decreased ANK3 expression delayed S phase to G2/M cell cycle transition and reduced the expression of cyclins A and B. However, cells with knocked-down ANK3 exhibited significant increase in cell invasion through an AR-dependent mechanism. Furthermore, we found that ANK3 is a regulator of AR protein stability. ANK3 knockdown also promoted cancer cell invasion and extravasations in vivo using the avian embryo model (p < 0.01). In human samples, ANK3 expression was dramatically upregulated in high grade intraepithelial neoplasia (HGPIN) and localized PCA (p < 0.0001). However, it was downregulated castration resistant stage (p < 0.0001) and showed inverse relation to Gleason score (p < 0.0001). In addition, increased expression of ANK3 in cancer tissues was correlated with better cancer-specific survival of PCA patients (p = 0.012). Silencing ANK3 results in significant reduction of cell proliferation through an AR-independent mechanism. ANK3 knockdown results in significant increase in cell invasion through an AR-dependent mechanism. ANK3 is a regulator of AR protein stability. ANK3 knockdown also promotes cancer cell invasion and extravasation in vivo using the avian embryo model.

iTRAQ-Based Proteomic Analysis Reveals Potential Regulation Networks of IBA-Induced Adventitious Root Formation in Apple

PubMed Central

Lei, Chao; Fan, Sheng; Li, Ke; Meng, Yuan; Mao, Jiangping; Han, Mingyu; Zhao, Caiping; Bao, Lu; Zhang, Dong

2018-01-01

Adventitious root (AR) formation, which is controlled by endogenous and environmental factors, is indispensable for vegetative asexual propagation. However, comprehensive proteomic data on AR formation are still lacking. The aim of this work was to study indole-3-butyric acid (IBA)-induced AR formation in the dwarf apple rootstock ‘T337’. In this study, the effect of IBA on AR formation was analysed. Subsequent to treatment with IBA, both the rooting rate and root length of ‘T337’ increased significantly. An assessment of hormone levels in basal stem cuttings suggested that auxin, abscisic acid, and brassinolide were higher in basal stem cuttings that received the exogenous IBA application; while zeatin riboside, gibberellins, and jasmonic acid were lower than non-treated basal stem cuttings. To explore the underlying molecular mechanism, an isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic technique was employed to identify the expression profiles of proteins at a key period of adventitious root induction (three days after IBA treatment). In total, 3355 differentially expressed proteins (DEPs) were identified. Many DEPs were closely related to carbohydrate metabolism and energy production, protein homeostasis, reactive oxygen and nitric oxide signaling, and cell wall remodeling biological processes; as well as the phytohormone signaling, which was the most critical process in response to IBA treatment. Further, RT-qPCR analysis was used to evaluate the expression level of nine genes that are involved in phytohormone signaling and their transcriptional levels were mostly in accordance with the protein patterns. Finally, a putative work model was proposed. Our study establishes a foundation for further research and sheds light on IBA-mediated AR formation in apple as well as other fruit rootstock cuttings. PMID:29495482

Identification of an IL-1-induced gene expression pattern in AR+ PCa cells that mimics the molecular phenotype of AR- PCa cells.

PubMed

Thomas-Jardin, Shayna E; Kanchwala, Mohammed S; Jacob, Joan; Merchant, Sana; Meade, Rachel K; Gahnim, Nagham M; Nawas, Afshan F; Xing, Chao; Delk, Nikki A

2018-06-01

In immunosurveillance, bone-derived immune cells infiltrate the tumor and secrete inflammatory cytokines to destroy cancer cells. However, cancer cells have evolved mechanisms to usurp inflammatory cytokines to promote tumor progression. In particular, the inflammatory cytokine, interleukin-1 (IL-1), is elevated in prostate cancer (PCa) patient tissue and serum, and promotes PCa bone metastasis. IL-1 also represses androgen receptor (AR) accumulation and activity in PCa cells, yet the cells remain viable and tumorigenic; suggesting that IL-1 may also contribute to AR-targeted therapy resistance. Furthermore, IL-1 and AR protein levels negatively correlate in PCa tumor cells. Taken together, we hypothesize that IL-1 reprograms AR positive (AR + ) PCa cells into AR negative (AR - ) PCa cells that co-opt IL-1 signaling to ensure AR-independent survival and tumor progression in the inflammatory tumor microenvironment. LNCaP and PC3 PCa cells were treated with IL-1β or HS-5 bone marrow stromal cell (BMSC) conditioned medium and analyzed by RNA sequencing and RT-QPCR. To verify genes identified by RNA sequencing, LNCaP, MDA-PCa-2b, PC3, and DU145 PCa cell lines were treated with the IL-1 family members, IL-1α or IL-1β, or exposed to HS-5 BMSC in the presence or absence of Interleukin-1 Receptor Antagonist (IL-1RA). Treated cells were analyzed by western blot and/or RT-QPCR. Comparative analysis of sequencing data from the AR + LNCaP PCa cell line versus the AR - PC3 PCa cell line reveals an IL-1-conferred gene suite in LNCaP cells that is constitutive in PC3 cells. Bioinformatics analysis of the IL-1 regulated gene suite revealed that inflammatory and immune response pathways are primarily elicited; likely facilitating PCa cell survival and tumorigenicity in an inflammatory tumor microenvironment. Our data supports that IL-1 reprograms AR + PCa cells to mimic AR - PCa gene expression patterns that favor AR-targeted treatment resistance and cell survival. © 2018 Wiley Periodicals, Inc.

Silodosin inhibits prostate cancer cell growth via ELK1 inactivation and enhances the cytotoxic activity of gemcitabine.

PubMed

Kawahara, Takashi; Aljarah, Ali Kadhim; Shareef, Hasanain Khaleel; Inoue, Satoshi; Ide, Hiroki; Patterson, John D; Kashiwagi, Eiji; Han, Bin; Li, Yi; Zheng, Yichun; Miyamoto, Hiroshi

2016-06-01

Biological significance of ELK1, a transcriptional factor whose phosphorylation is necessary for c-fos proto-oncogene activation, in prostate cancer remains far from fully understood. In this study, we aim to investigate the role of ELK1 in tumor growth as well as the efficacy of a selective α1A-adrenergic blocker, silodosin, in ELK1 activity in prostate cancer cells. We first immunohistochemically determined the levels of phospho-ELK1 (p-ELK1) expression in radical prostatectomy specimens. We then assessed the effects of ELK1 knockdown via short hairpin RNA and silodosin on cell proliferation, migration, and invasion in prostate cancer lines. The levels of p-ELK1 expression were significantly higher in carcinoma than in benign (P < 0.001) or high-grade prostatic intraepithelial neoplasia (HGPIN) (P = 0.002) as well as in HGPIN than in benign (P < 0.001). Kaplan-Meier and log-rank tests revealed that moderate-strong positivity of p-ELK1 in carcinomas tended to correlate with biochemical recurrence after radical prostatectomy (P = 0.098). In PC3 and DU145 expressing ELK1 (mRNA/protein) but no androgen receptor (AR), ELK1 silencing resulted in considerable decreases in the expression of c-fos as well as in cell migration/invasion and matrix metalloproteinase-2 expression, but not in cell viability. Silodosin treatment reduced the expression/activity of ELK1 in these cells as well as the viability of AR-positive LNCaP and C4-2 cells and the migration of both AR-positive and AR-negative cells, but not the viability of AR-negative or ELK1-negative cells. Interestingly, silodosin significantly increased sensitivity to gemcitabine, but not to cisplatin or docetaxel, even in AR-negative cells. ELK1 is likely to be activated in prostate cancer cells and promote tumor progression. Furthermore, silodosin that inactivates ELK1 in prostate cancer cells not only inhibits their growth but also enhances the cytotoxic activity of gemcitabine. Thus, ELK1 inhibition has the potential of being a therapeutic approach for prostate cancer. © 2016 Wiley Periodicals, Inc.

Beta-Adrenergic Receptor Expression in Muscle Cells

NASA Technical Reports Server (NTRS)

Young, Ronald B.; Bridge, K.; Vaughn, J. R.

1999-01-01

beta-adrenergic receptor (bAR) agonists presumably exert their physiological action on skeletal muscle cells through the bAR. Since the signal generated by the bAR is cyclic AMP (cAMP), experiments were initiated in primary chicken muscle cell cultures to determine if artificial elevation of intracellular cAMP by treatment with forskolin would alter the population of bAR expressed on the surface of muscle cells. Chicken skeletal muscle cells after 7 days in culture were employed for the experiments because muscle cells have attained a steady state with respect to muscle protein metabolism at this stage. Cells were treated with 0-10 uM forskolin for a total of three days. At the end of the 1, 2, and 3 day treatment intervals, the concentration of cAMP and the bAR population were measured. Receptor population was measured in intact muscle cell cultures as the difference between total binding of [H-3]CGP-12177 and non-specific binding of [H-3]CGP-12177 in the presence of 1 uM propranolol. Intracellular cAMP concentration was measured by radioimmunoassay. The concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. Increasing concentrations of forskolin also led to an increase in (beta)AR population, with a maximum increase of approximately 50% at 10 uM. This increase in (beta)AR population was apparent after only 1 day of treatment, and the pattern of increase was maintained for all 3 days of the treatment period. Thus, increasing the intracellular concentration of cAMP leads to up-regulation of (beta)AR population. Clenbuterol and isoproterenol gave similar effects on bAR population. The effect of forskolin on the quantity and apparent synthesis rate of the heavy chain of myosin (mhc) were also investigated. A maximum increase of 50% in the quantity of mhc was observed at 0.2 UM forskolin, but higher concentrations of forskolin reduced the quantity of mhc back to control levels.

Alkylresorcinol synthases expressed in Sorghum bicolor root hairs play an essential role in the biosynthesis of the allelopathic benzoquinone sorgoleone.

PubMed

Cook, Daniel; Rimando, Agnes M; Clemente, Thomas E; Schröder, Joachim; Dayan, Franck E; Nanayakkara, N P Dhammika; Pan, Zhiqiang; Noonan, Brice P; Fishbein, Mark; Abe, Ikuro; Duke, Stephen O; Baerson, Scott R

2010-03-01

Sorghum bicolor is considered to be an allelopathic crop species, producing phytotoxins such as the lipid benzoquinone sorgoleone, which likely accounts for many of the allelopathic properties of Sorghum spp. Current evidence suggests that sorgoleone biosynthesis occurs exclusively in root hair cells and involves the production of an alkylresorcinolic intermediate (5-[(Z,Z)-8',11',14'-pentadecatrienyl]resorcinol) derived from an unusual 16:3Delta(9,12,15) fatty acyl-CoA starter unit. This led to the suggestion of the involvement of one or more alkylresorcinol synthases (ARSs), type III polyketide synthases (PKSs) that produce 5-alkylresorcinols using medium to long-chain fatty acyl-CoA starter units via iterative condensations with malonyl-CoA. In an effort to characterize the enzymes responsible for the biosynthesis of the pentadecyl resorcinol intermediate, a previously described expressed sequence tag database prepared from isolated S. bicolor (genotype BTx623) root hairs was first mined for all PKS-like sequences. Quantitative real-time RT-PCR analyses revealed that three of these sequences were preferentially expressed in root hairs, two of which (designated ARS1 and ARS2) were found to encode ARS enzymes capable of accepting a variety of fatty acyl-CoA starter units in recombinant enzyme studies. Furthermore, RNA interference experiments directed against ARS1 and ARS2 resulted in the generation of multiple independent transformant events exhibiting dramatically reduced sorgoleone levels. Thus, both ARS1 and ARS2 are likely to participate in the biosynthesis of sorgoleone in planta. The sequences of ARS1 and ARS2 were also used to identify several rice (Oryza sativa) genes encoding ARSs, which are likely involved in the production of defense-related alkylresorcinols.

Alkylresorcinol Synthases Expressed in Sorghum bicolor Root Hairs Play an Essential Role in the Biosynthesis of the Allelopathic Benzoquinone Sorgoleone[W][OA

PubMed Central

Cook, Daniel; Rimando, Agnes M.; Clemente, Thomas E.; Schröder, Joachim; Dayan, Franck E.; Nanayakkara, N.P. Dhammika; Pan, Zhiqiang; Noonan, Brice P.; Fishbein, Mark; Abe, Ikuro; Duke, Stephen O.; Baerson, Scott R.

2010-01-01

Sorghum bicolor is considered to be an allelopathic crop species, producing phytotoxins such as the lipid benzoquinone sorgoleone, which likely accounts for many of the allelopathic properties of Sorghum spp. Current evidence suggests that sorgoleone biosynthesis occurs exclusively in root hair cells and involves the production of an alkylresorcinolic intermediate (5-[(Z,Z)-8′,11′,14′-pentadecatrienyl]resorcinol) derived from an unusual 16:3Δ9,12,15 fatty acyl-CoA starter unit. This led to the suggestion of the involvement of one or more alkylresorcinol synthases (ARSs), type III polyketide synthases (PKSs) that produce 5-alkylresorcinols using medium to long-chain fatty acyl-CoA starter units via iterative condensations with malonyl-CoA. In an effort to characterize the enzymes responsible for the biosynthesis of the pentadecyl resorcinol intermediate, a previously described expressed sequence tag database prepared from isolated S. bicolor (genotype BTx623) root hairs was first mined for all PKS-like sequences. Quantitative real-time RT-PCR analyses revealed that three of these sequences were preferentially expressed in root hairs, two of which (designated ARS1 and ARS2) were found to encode ARS enzymes capable of accepting a variety of fatty acyl-CoA starter units in recombinant enzyme studies. Furthermore, RNA interference experiments directed against ARS1 and ARS2 resulted in the generation of multiple independent transformant events exhibiting dramatically reduced sorgoleone levels. Thus, both ARS1 and ARS2 are likely to participate in the biosynthesis of sorgoleone in planta. The sequences of ARS1 and ARS2 were also used to identify several rice (Oryza sativa) genes encoding ARSs, which are likely involved in the production of defense-related alkylresorcinols. PMID:20348430

A single cell level measurement of StAR expression and activity in adrenal cells.

PubMed

Lee, Jinwoo; Yamazaki, Takeshi; Dong, Hui; Jefcoate, Colin

2017-02-05

The Steroidogenic acute regulatory protein (StAR) directs mitochondrial cholesterol uptake through a C-terminal cholesterol binding domain (CBD) and a 62 amino acid N-terminal regulatory domain (NTD) that contains an import sequence and conserved sites for inner membrane metalloproteases. Deletion of the NTD prevents mitochondrial import while maintaining steroidogenesis but with compromised cholesterol homeostasis. The rapid StAR-mediated cholesterol transfer in adrenal cells depends on concerted mRNA translation, p37 StAR phosphorylation and controlled NTD cleavage. The NTD controls this process with two cAMP-inducible modulators of, respectively, transcription and translation SIK1 and TIS11b/Znf36l1. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA resolves slow RNA splicing at the gene loci in cAMP-induced Y-1 cells and transfer of individual 3.5 kB mRNA molecules to mitochondria. StAR transcription depends on the CREB coactivator CRTC2 and PKA inhibition of the highly inducible suppressor kinase SIK1 and a basal counterpart SIK2. PKA-inducible TIS11b/Znf36l1 binds specifically to highly conserved elements in exon 7 thereby suppressing formation of mRNA and subsequent translation. Co-expression of SIK1, Znf36l1 with 3.5 kB StAR mRNA may limit responses to pulsatile signaling by ACTH while regulating the transition to more prolonged stress. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A single cell level measurement of StAR expression and activity in adrenal cells

PubMed Central

Lee, Jinwoo; Yamazaki, Takeshi; Dong, Hui; Jefcoate, Colin

2018-01-01

The Steroidogenic acute regulatory protein (StAR) directs mitochondrial cholesterol uptake through a C-terminal cholesterol binding domain (CBD) and a 62 amino acid N-terminal regulatory domain (NTD) that contains an import sequence and conserved sites for inner membrane metalloproteases. Deletion of the NTD prevents mitochondrial import while maintaining steroidogenesis but with compromised cholesterol homeostasis. The rapid StAR-mediated cholesterol transfer in adrenal cells depends on concerted mRNA translation, p37 StAR phosphorylation and controlled NTD cleavage. The NTD controls this process with two cAMP-inducible modulators of, respectively, transcription and translation SIK1 and TIS11b/Znf36l1. High-resolution fluorescence in situ hybridization (HR-FISH) of StAR RNA resolves slow RNA splicing at the gene loci in cAMP-induced Y-1 cells and transfer of individual 3.5 kb mRNA molecules to mitochondria. StAR transcription depends on the CREB coactivator CRTC2 and PKA inhibition of the highly inducible suppressor kinase SIK1 and a basal counterpart SIK2. PKA-inducible TIS11b/Znf36l1 binds specifically to highly conserved elements in exon 7 thereby suppressing formation of mRNA and subsequent translation. Co-expression of SIK1, Znf36l1 with 3.5 kb StAR mRNA may limit responses to pulsatile signaling by ACTH while regulating the transition to more prolonged stress PMID:27521960

High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor

PubMed Central

O’Malley, Michelle A.; Lazarova, Tzvetana; Britton, Zachary T.; Robinson, Anne S.

2007-01-01

The G-protein coupled receptors (GPCRs) are a class of membrane proteins that trigger cellular responses to external stimuli, and are believed to be targets for nearly half of all pharmaceutical drugs on the market. However, little is known regarding their folding and cellular interactions, as well as what factors are crucial for their activity. Further structural characterization of GPCRs has largely been complicated by problems with expression, purification, and preservation of activity in vitro. Previously, we have demonstrated high-level expression (~4 mg/L of culture) of functional human adenosine A2a receptor fused to a green fluorescent protein (A2aR-GFP) from Saccharomyces cerevisiae. In this work we re-engineered A2aR with a purification tag, developed an adequate purification scheme, and performed biophysical characterization on purified receptors. Milligram amounts per liter of culture of A2aR and A2aR-GFP were functionally expressed in S. cerevisiae, with a C-terminal deca-histidine tag. Lysis procedures were developed for optimal membrane protein solubilization and recovery through monitoring fluorescence of A2aR-GFP-His10. One-step purification of the protein was achieved through immobilized metal affinity chromatography. After initial solubilization in n-dodecyl-β-D-maltoside (DDM), a combination of added cholesterol hemisuccinate (CHS) in 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS) was required to stabilize the functional state of the protein. Isolated A2aR under these conditions was found to be largely alpha-helical, and properly incorporated into a mixed-micelle environment. The A2a-His10 receptor was purified in quantities of 6 +/− 2 mg/L of culture, with ligand-binding yields of 1 mg/L, although all protein bound to xanthine affinity resin. This represents the highest purified total and functional yields for A2aR yet achieved from any heterologous expression system. PMID:17591446

Sex-specific hormone receptors in urothelial carcinomas of the human urinary bladder: a comparative analysis of clinicopathological features and survival outcomes according to receptor expression.

PubMed

Tuygun, Can; Kankaya, Duygu; Imamoglu, Abdurrahim; Sertcelik, Ayse; Zengin, Kursad; Oktay, Murat; Sertcelik, Nurettin

2011-01-01

To investigate the expression of sex-specific hormone receptors in normal bladder urothelium and urothelial carcinomas (UCs) of the bladder, and to analyze clinicopathological features and survival outcomes according to receptor expression. We evaluated the clinical data and tumor specimens of 139 patients with bladder cancer (BC). In addition, 72 samples of normal urothelium were included. Immunohistochemistry was performed using streptavidin-biotin peroxidase method, a monoclonal androgen receptor (AR), and an estrogen receptor-β (ERβ) antibody on paraffin-embedded tissue sections. Expression levels of each receptor were assessed by evaluating 500 tumor cells for each case and the percentage of positively-stained nuclei was recorded. None of the 58 male control cases showed any AR and ERβ expression. Five (35, 71%) of the 14 female control cases expressed ERβ. Of the 139 patients with UCs, 71 (51, 07%) expressed AR (62 male vs. 9 female; P = 0.413) and 44 (31, 65%) (39 male vs. 5 female; P = 0.402) showed ERβ expression (P < 0.001). No significant relationship was found between ERβ expression levels and tumor grades, and stages (P = 0.441; P = 0.247). AR expression was significantly lower in T2-tumors (21%) than in Ta-tumors (60%) and T1-tumors (60%) (P < 0.001). It was significantly higher in low-grade papillary UCs (64%) compared with high-grade papillary UCs (44%) and infiltrative high-grade UCs (17%) (P = 0.039; P < 0.001). Data of 79 patients with noninvasive BC were eligible to present, with a median 29 months follow-up. AR expression level did not influence recurrence-free survival (RFS) and progression-free survival (PFS) (P = 0.095; P = 0.110). No significant association was found between ERβ expression level and RFS (P = 0.293). PFS in patients with lower ERβ-expressing tumors was significantly better than that in patients with higher ERβ-expressing tumors (P = 0.035). Multivariate analysis confirmed this significant influence on PFS (P = 0.025). Although ERβ expression had no impact on histopathological tumor characteristics, decrease in its expression may be associated with better PFS rates in patients with noninvasive BC. Conversely, loss of AR expression was associated with higher grade UCs and invasive UCs, but had no prognostic effect on survival. Finally, sex-specific hormone receptors alone cannot be responsible for gender differences in BC rates because they were expressed in similar rates in both sexes. Copyright © 2011 Elsevier Inc. All rights reserved.

Steroidogenic acute regulatory protein (StAR) overexpression attenuates HFD-induced hepatic steatosis and insulin resistance.

PubMed

Qiu, Yanyan; Sui, Xianxian; Zhan, Yongkun; Xu, Chen; Li, Xiaobo; Ning, Yanxia; Zhi, Xiuling; Yin, Lianhua

2017-04-01

Non-alcoholic fatty liver disease (NAFLD) covers a wide spectrum of liver pathology. Intracellular lipid accumulation is the first step in the development and progression of NAFLD. Steroidogenic acute regulatory protein (StAR) plays an important role in the synthesis of bile acid and intracellular lipid homeostasis and cholesterol metabolism. We hypothesize that StAR is involved in non-alcoholic fatty liver disease (NAFLD) pathogenesis. The hypothesis was identified using free fatty acid (FFA)-overloaded NAFLD in vitro model and high-fat diet (HFD)-induced NAFLD mouse model transfected by recombinant adenovirus encoding StAR (StAR). StAR expression was also examined in pathology samples of patients with fatty liver by immunohistochemical staining. We found that the expression level of StAR was reduced in the livers obtained from fatty liver patients and NAFLD mice. Additionally, StAR overexpression decreased the levels of hepatic lipids and maintained the hepatic glucose homeostasis due to the activation of farnesoid x receptor (FXR). StAR overexpression attenuated the impairment of insulin signaling in fatty liver. This protective role of StAR was owing to a reduction of intracellular diacylglycerol levels and the phosphorylation of PKCε. Furthermore, FXR inactivation reversed the observed beneficial effects of StAR. The present study revealed that StAR overexpression can reduce hepatic lipid accumulation, regulate glucose metabolism and attenuate insulin resistance through a mechanism involving the activation of FXR. Our study suggests that StAR may be a potential therapeutic target for NAFLD. Copyright © 2017 Elsevier B.V. All rights reserved.

Targeting AR Variant-Coactivator Interactions to Exploit Prostate Cancer Vulnerabilities.

PubMed

Magani, Fiorella; Peacock, Stephanie O; Rice, Meghan A; Martinez, Maria J; Greene, Ann M; Magani, Pablo S; Lyles, Rolando; Weitz, Jonathan R; Burnstein, Kerry L

2017-11-01

Castration-resistant prostate cancer (CRPC) progresses rapidly and is incurable. Constitutively active androgen receptor splice variants (AR-Vs) represent a well-established mechanism of therapeutic resistance and disease progression. These variants lack the AR ligand-binding domain and, as such, are not inhibited by androgen deprivation therapy (ADT), which is the standard systemic approach for advanced prostate cancer. Signaling by AR-Vs, including the clinically relevant AR-V7, is augmented by Vav3, an established AR coactivator in CRPC. Using mutational and biochemical studies, we demonstrated that the Vav3 Diffuse B-cell lymphoma homology (DH) domain interacted with the N-terminal region of AR-V7 (and full length AR). Expression of the Vav3 DH domain disrupted Vav3 interaction with and enhancement of AR-V7 activity. The Vav3 DH domain also disrupted AR-V7 interaction with other AR coactivators: Src1 and Vav2, which are overexpressed in PC. This Vav3 domain was used in proof-of-concept studies to evaluate the effects of disrupting the interaction between AR-V7 and its coactivators on CRPC cells. This disruption decreased CRPC cell proliferation and anchorage-independent growth, caused increased apoptosis, decreased migration, and resulted in the acquisition of morphological changes associated with a less aggressive phenotype. While disrupting the interaction between FL-AR and its coactivators decreased N-C terminal interaction, disrupting the interaction of AR-V7 with its coactivators decreased AR-V7 nuclear levels. Implications: This study demonstrates the potential therapeutic utility of inhibiting constitutively active AR-V signaling by disrupting coactivator binding. Such an approach is significant, as AR-Vs are emerging as important drivers of CRPC that are particularly recalcitrant to current therapies. Mol Cancer Res; 15(11); 1469-80. ©2017 AACR . ©2017 American Association for Cancer Research.

Androgen resistance in squirrel monkeys (Saimiri spp.).

PubMed

Gross, Katherine L; Westberry, Jenne M; Hubler, Tina R; Sadosky, Patti W; Singh, Ravinder J; Taylor, Robert L; Scammell, Jonathan G

2008-08-01

The goal of this study was to understand the basis for high androgen levels in squirrel monkeys (Saimiri spp.). Mass spectrometry was used to analyze serum testosterone, androstenedione, and dihydrotestosterone of male squirrel monkeys during the nonbreeding (n = 7) and breeding (n = 10) seasons. All hormone levels were elevated compared with those of humans, even during the nonbreeding season; the highest levels occurred during the breeding season. The ratio of testosterone to dihydrotestosterone in squirrel monkeys is high during the breeding season compared to man. Squirrel monkeys may have high testosterone to compensate for inefficient metabolism to dihydrotestosterone. We also investigated whether squirrel monkeys have high androgens to compensate for low-activity androgen receptors (AR). The response to dihydrotestosterone in squirrel monkey cells transfected with AR and AR-responsive reporter plasmids was 4-fold, compared with 28-fold in human cells. This result was not due to overexpression of cellular FKBP51, which causes glucocorticoid and progestin resistance in squirrel monkeys, because overexpression of FKBP51 had no effect on dihydrotestosterone-stimulated reporter activity in a human fibroblast cell line. To test whether the inherently low levels of FKBP52 in squirrel monkeys contribute to androgen insensitivity, squirrel monkey cells were transfected with an AR expression plasmid, an AR-responsive reporter plasmid, and a plasmid expressing FKBP52. Expression of FKBP52 decreased the EC50 or increased the maximal response to dihydrotestosterone. Therefore, the high androgen levels in squirrel monkeys likely compensate for their relatively low 5 alpha-reductase activity during the breeding season and AR insensitivity resulting from low cellular levels of FKBP52.

FOXA1 promotes tumor progression in prostate cancer and represents a novel hallmark of castration-resistant prostate cancer.

PubMed

Gerhardt, Josefine; Montani, Matteo; Wild, Peter; Beer, Marc; Huber, Fabian; Hermanns, Thomas; Müntener, Michael; Kristiansen, Glen

2012-02-01

Forkhead box protein A1 (FOXA1) modulates the transactivation of steroid hormone receptors and thus may influence tumor growth and hormone responsiveness in prostate cancer. We therefore investigated the correlation of FOXA1 expression with clinical parameters, prostate-specific antigen (PSA) relapse-free survival, and hormone receptor expression in a large cohort of prostate cancer patients at different disease stages. FOXA1 expression did not differ significantly between benign glands from the peripheral zone and primary peripheral zone prostate carcinomas. However, FOXA1 was overexpressed in metastases and particularly in castration-resistant cases, but was expressed at lower levels in both normal and neoplastic transitional zone tissues. FOXA1 levels correlated with higher pT stages and Gleason scores, as well as with androgen (AR) and estrogen receptor expression. Moreover, FOXA1 overexpression was associated with faster biochemical disease progression, which was pronounced in patients with low AR levels. Finally, siRNA-based knockdown of FOXA1 induced decreased cell proliferation and migration. Moreover, in vitro tumorigenicity was inducible by ARs only in the presence of FOXA1, substantiating a functional cooperation between FOXA1 and AR. In conclusion, FOXA1 expression is associated with tumor progression, dedifferentiation of prostate cancer cells, and poorer prognosis, as well as with cellular proliferation and migration and with AR signaling. These findings suggest FOXA1 overexpression as a novel mechanism inducing castration resistance in prostate cancer. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Concentrations of the adrenocorticotropic hormone, corticosterone and sex steroid hormones and the expression of the androgen receptor in the pituitary and adrenal glands of male turkeys (Meleagris gallopavo) during growth and development.

PubMed

Kiezun, J; Kaminska, B; Jankowski, J; Dusza, L

2015-01-01

Androgens take part in the regulation of puberty and promote growth and development. They play their biological role by binding to a specific androgen receptor (AR). The aim of this study was to evaluate the expression of AR mRNA and protein in the pituitary and adrenal glands, to localize AR protein in luteinizing hormone (LH)-producing pituitary and adrenocortical cells, to determine plasma concentrations of adrenocorticotropic hormone (ACTH) and corticosterone and the concentrations of corticosterone, testosterone (T), androstenedione (A4) and oestradiol (E2) in the adrenal glands of male turkeys at the age of 4, 8, 12, 16, 20, 24 and 28weeks. The concentrations of hormones and the expression of AR varied during development. The expression of AR mRNA and protein in pituitary increased during the growth. The increase of AR mRNA levels in pituitary occurred earlier than increase of AR protein. The percentage of pituitary cells expressing ARs in the population of LH-secreting cells increased in week 20. It suggests that AR expression in LH-producing pituitary cells is determined by the phase of development. The drop in adrenal AR mRNA and protein expression was accompanied by an increase in the concentrations of adrenal androgens. Those results could point to the presence of a compensatory mechanism that enables turkeys to avoid the potentially detrimental effects of high androgen concentrations. Our results will expand our knowledge of the role of steroids in the development of the reproductive system of turkeys from the first month of age until maturity. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of an Enriched Extract of Paeoniflorin, a Monoterpene Glycoside used in Chinese Herbal Medicine, on Cholesterol Metabolism in a Hyperlipidemic Rat Model

PubMed Central

Hu, Huiming; Zhu, Qiaoqiao; Su, Jie; Wu, Yajun; Zhu, Yanchen; Wang, Yin; Fang, Hui; Pang, Minxia; Li, Bo; Chen, Suhong; Lv, Guiyuan

2017-01-01

Background Paeoniflorin is a monoterpene glycoside extracted from the roots of Paeonia lactiflora and is used in Chinese herbal medicine to treat hyperlipidemia. The aim of this study was to evaluate the effects of an enriched extract of paeoniflorin on cholesterol levels, hemodynamics, and oxidative stress in a hyperlipidemic rat model. Material/Methods Male Sprague-Dawley rats were fed high-cholesterol diets and treated with three different doses of paeoniflorin for 12 weeks. The effects of paeoniflorin treatment were assessed on cholesterol levels, cholesterol metabolism, red blood cell vascular flow using hemorheology, antioxidant enzymes, and expression of the rate-limiting enzyme in the mevalonate pathway, 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMG-CoAR). Rat liver histology and immunohistochemical analysis were performed to evaluate the expression of nuclear factor erythroid 2–related factor 2 (Nrf2), cytochrome P450 7A1 (CYP7A1), and peroxisome proliferator-activated receptors (PPAR)-α. Protein expression HMG-CoAR, low-density lipoprotein receptor (LDLR), PPAR-α and CYP7A1 was measured by Western blotting. Antioxidant activity in rat liver was determined by measuring superoxide dismutase (SOD) and malondialdehyde (MDA). Results Serum and hepatic cholesterol, hepatic steatosis and the products of cholesterol metabolism were reduced by paeoniflorin treatment, which also reduced the activity of HMG-CoAR and upregulated the expression of LDLR, PPAR-α, and CYP7A1 expression, increased SOD, decreased MDA, and upregulated Nrf2 expression. Conclusions The findings of this study in a rat model of hyperlipidemia have shown that paeoniflorin regulates hepatic cholesterol synthesis and metabolism and may also protect the liver from oxidative stress. PMID:28706181

Expression of alpha-AR subtypes in T lymphocytes and role of the alpha-ARs in mediating modulation of T cell function.

PubMed

Bao, Jing-Yin; Huang, Yan; Wang, Feng; Peng, Yu-Ping; Qiu, Yi-Hua

2007-01-01

Previous work in our laboratory has shown that alpha-adrenoreceptors (alpha-ARs) and beta-ARs exist on lymphocytes from functional profile, and that the receptors mediate the regulation of lymphocyte function by catecholamines. In the present study, we directly examined the expression of alpha-AR subtypes, alpha(1)-AR and alpha(2)-AR mRNAs, in T lymphocytes and explored the roles of the alpha-AR subtypes and intracellular signal transduction mechanisms linked to the receptors in mediating the modulation of T lymphocyte function. T lymphocytes from mesenteric lymph nodes of rats were purified by using a nylon wool column. Reverse transcription polymerase chain reaction was used to detect the expression of alpha(1)-AR and alpha(2)-AR mRNAs in the freshly isolated T cells and the mitogen concanavalin A (Con A)-activated lymphocytes. Colorimetric methylthiazoletetrazolium assay was employed to measure lymphocyte proliferation induced by Con A. Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) levels in the Con A-stimulated lymphocyte culture supernatants were examined by enzyme-linked immunosorbent assay. T cells expressed both alpha(1)-AR and alpha(2)-AR mRNAs. The expression of both alpha(1)-AR and alpha(2)-AR mRNAs was significantly higher in the Con A-activated lymphocytes than in the resting lymphocytes. Phenylephrine, a selective alpha(1)-AR agonist, had no evident effect on lymphocyte proliferation nor on IFN-gamma and IL-4 production induced by Con A. However, the selective alpha(2)-AR agonist clonidine attenuated Con A-induced lymphocyte proliferation as well as IFN-gamma and IL-4 production. The inhibited lymphocyte proliferation and IFN-gamma and IL-4 production by clonidine were blocked by yohimbine, an alpha(2)-AR antagonist. Either phospholipase C inhibitor U-73122 or protein kinase C inhibitor chelerythrine partially prevented the suppressive effect of clonidine on Con A-stimulated lymphocyte proliferation and IL-4 production. T lymphocytes express both alpha(1)-ARs and alpha(2)-ARs, but only the alpha(2)-ARs participate in the suppressive modulation of lymphocyte proliferation and cytokine production in vitro. The inhibitory effect of alpha(2)-AR stimulation on lymphocyte function is partially mediated via the phospholipase C-protein kinase C pathway. (c) 2008 S. Karger AG, Basel.

Probing Androgen Receptor Signaling in Circulating Tumor Cells in Prostate Cancer

DTIC Science & Technology

2014-07-01

androgen driven) and PSMA (androgen suppressed). Application of this CTC-based assay in a small cohort of men with prostate cancer revealed that the AR...activity in CTCs as measured by PSA and PSMA expression levels could be used to monitor the treatment of patients undergoing AR targeting therapies...transitioned the laboratory to the 3rd generation CTC iChip technology (4), and have adapted the PSA/ PSMA assay for use with this new CTC isolation

Apoptotic effect of a novel kefir product, PFT, on multidrug-resistant myeloid leukemia cells via a hole-piercing mechanism

PubMed Central

GHONEUM, MAMDOOH; GIMZEWSKI, JAMES

2014-01-01

We examined the apoptotic effect of a novel Probiotics Fermentation Technology (PFT) kefir grain product; PFT is a natural mixture composed primarily of Lactobacillus kefiri P-IF, a specific strain of L. kefiri with unique growth characteristics. The aim of this study was to examine the apoptotic effect of PFT on human multidrug-resistant (MDR) myeloid leukemia (HL60/AR) cells in vitro and explore the mechanistic approach underlying its effect. HL60/AR cells were cultured with PFT (0.6–5.0 mg/ml) for 3 days. The apoptotic effect of PFT was assessed through examination of percent apoptosis, caspase 3 activation, Bcl-2 expression levels and changes in mitochondrial membrane potential (MMP). PFT induced apoptosis in HL60/AR cells in a dose-dependent manner which was maximal at 67.5% for 5 mg/ml. Induction of apoptosis was associated with activation of caspase 3, decreased expression of Bcl-2 and decreased polarization of MMP. In addition, PFT showed a unique characteristic of piercing holes in HL60/AR cells, as indicated by AFM studies. This hole induction may be responsible for the apoptotic effect on cancer cells. These results suggest that PFT may act as a potential therapy for the treatment of MDR leukemia. PMID:24430613

Effects of velvet antler polypeptide on sexual behavior and testosterone synthesis in aging male mice

PubMed Central

Zang, Zhi-Jun; Tang, Hong-Feng; Tuo, Ying; Xing, Wei-Jie; Ji, Su-Yun; Gao, Yong; Deng, Chun-Hua

2016-01-01

Twenty-four-month-old male C57BL/6 mice with low serum testosterone levels were used as a late-onset hypogonadism (LOH) animal model for examining the effects of velvet antler polypeptide (VAP) on sexual function and testosterone synthesis. These mice received VAP for 5 consecutive weeks by daily gavage at doses of 100, 200, or 300 mg kg−1 body weight per day (n = 10 mice per dose). Control animals (n = 10) received the same weight-based volume of vehicle. Sexual behavior and testosterone levels in serum and interstitial tissue of testis were measured after the last administration of VAP. Furthermore, to investigate the mechanisms of how VAP affects sexual behavior and testosterone synthesis in vivo, the expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) in Leydig cells was also measured by immunofluorescence staining and quantitative real-time PCR. As a result, VAP produced a significant improvement in the sexual function of these aging male mice. Serum testosterone level and intratesticular testosterone (ITT) concentration also increased in the VAP-treated groups. The expression of StAR, P450scc, and 3β-HSD was also found to be enhanced in the VAP-treated groups compared with the control group. Our results suggested that VAP was effective in improving sexual function in aging male mice. The effect of velvet antler on sexual function was due to the increased expression of several rate-limiting enzymes of testosterone synthesis (StAR, P450scc, and 3β-HSD) and the following promotion of testosterone synthesis in vivo. PMID:26608944

Effects of velvet antler polypeptide on sexual behavior and testosterone synthesis in aging male mice.

PubMed

Zang, Zhi-Jun; Tang, Hong-Feng; Tuo, Ying; Xing, Wei-Jie; Ji, Su-Yun; Gao, Yong; Deng, Chun-Hua

2016-01-01

Twenty-four-month-old male C57BL/6 mice with low serum testosterone levels were used as a late-onset hypogonadism (LOH) animal model for examining the effects of velvet antler polypeptide (VAP) on sexual function and testosterone synthesis. These mice received VAP for 5 consecutive weeks by daily gavage at doses of 100, 200, or 300 mg kg-1 body weight per day (n = 10 mice per dose). Control animals (n = 10) received the same weight-based volume of vehicle. Sexual behavior and testosterone levels in serum and interstitial tissue of testis were measured after the last administration of VAP. Furthermore, to investigate the mechanisms of how VAP affects sexual behavior and testosterone synthesis in vivo, the expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) in Leydig cells was also measured by immunofluorescence staining and quantitative real-time PCR. As a result, VAP produced a significant improvement in the sexual function of these aging male mice. Serum testosterone level and intratesticular testosterone (ITT) concentration also increased in the VAP-treated groups. The expression of StAR, P450scc, and 3β-HSD was also found to be enhanced in the VAP-treated groups compared with the control group. Our results suggested that VAP was effective in improving sexual function in aging male mice. The effect of velvet antler on sexual function was due to the increased expression of several rate-limiting enzymes of testosterone synthesis (StAR, P450scc, and 3β-HSD) and the following promotion of testosterone synthesis in vivo.

Aerobic exercise protects against pressure overload-induced cardiac dysfunction and hypertrophy via β3-AR-nNOS-NO activation

PubMed Central

Li, Wenju; Li, Xiaoli; Zheng, Qiangsun; Niu, Xiaolin

2017-01-01

Aerobic exercise confers sustainable protection against cardiac hypertrophy and heart failure (HF). Nitric oxide synthase (NOS) and nitric oxide (NO) are known to play an important role in exercise-mediated cardioprotection, but the mechanism of NOS/NO stimulation during exercise remains unclear. The aim of this study is to determine the role of β3-adrenergic receptors (β3-ARs), NOS activation, and NO metabolites (nitrite and nitrosothiols) in the sustained cardioprotective effects of aerobic exercise. An HF model was constructed by transverse aortic constriction (TAC). Animals were treated with either moderate aerobic exercise by swimming for 9 weeks and/or the β3-AR-specific inhibitor SR59230A at 0.1 mg/kg/hour one day after TAC operation. Myocardial fibrosis, myocyte size, plasma catecholamine (CA) level, cardiac function and geometry were assessed using Masson’s trichrome staining, FITC-labeled wheat germ agglutinin staining, enzyme-linked immuno sorbent assay (ELISA) and echocardiography, respectively. Western blot analysis was performed to elucidate the expression of target proteins. The concentration of myocardial NO production was evaluated using the nitrate reductase method. Myocardial oxidative stress was assessed by detecting the concentration of myocardial super oxidative dismutase (SOD), malonyldialdehyde (MDA), and reactive oxygen species (ROS). Aerobic exercise training improved dilated left ventricular function and partially attenuated the degree of cardiac hypertrophy and fibrosis in TAC mice. Moreover, the increased expression of β3-AR, activation of neuronal NOS (nNOS), and production of NO were detected after aerobic exercise training in TAC mice. However, selective inhibition of β3-AR by SR59230A abolished the upregulation and activation of nNOS induced NO production. Furthermore, aerobic exercise training decreased the myocardial ROS and MDA contents and increased myocardial levels of SOD; both effects were partially attenuated by SR59230A. Our study suggested that aerobic exercise training could improve cardiac systolic function and alleviate LV chamber dilation, cardiac fibrosis and hypertrophy in HF mice. The mechanism responsible for the protective effects of aerobic exercise is associated with the activation of the β3-AR-nNOS-NO pathway. PMID:28622359

Aerobic exercise protects against pressure overload-induced cardiac dysfunction and hypertrophy via β3-AR-nNOS-NO activation.

PubMed

Wang, Bin; Xu, Ming; Li, Wenju; Li, Xiaoli; Zheng, Qiangsun; Niu, Xiaolin

2017-01-01

Aerobic exercise confers sustainable protection against cardiac hypertrophy and heart failure (HF). Nitric oxide synthase (NOS) and nitric oxide (NO) are known to play an important role in exercise-mediated cardioprotection, but the mechanism of NOS/NO stimulation during exercise remains unclear. The aim of this study is to determine the role of β3-adrenergic receptors (β3-ARs), NOS activation, and NO metabolites (nitrite and nitrosothiols) in the sustained cardioprotective effects of aerobic exercise. An HF model was constructed by transverse aortic constriction (TAC). Animals were treated with either moderate aerobic exercise by swimming for 9 weeks and/or the β3-AR-specific inhibitor SR59230A at 0.1 mg/kg/hour one day after TAC operation. Myocardial fibrosis, myocyte size, plasma catecholamine (CA) level, cardiac function and geometry were assessed using Masson's trichrome staining, FITC-labeled wheat germ agglutinin staining, enzyme-linked immuno sorbent assay (ELISA) and echocardiography, respectively. Western blot analysis was performed to elucidate the expression of target proteins. The concentration of myocardial NO production was evaluated using the nitrate reductase method. Myocardial oxidative stress was assessed by detecting the concentration of myocardial super oxidative dismutase (SOD), malonyldialdehyde (MDA), and reactive oxygen species (ROS). Aerobic exercise training improved dilated left ventricular function and partially attenuated the degree of cardiac hypertrophy and fibrosis in TAC mice. Moreover, the increased expression of β3-AR, activation of neuronal NOS (nNOS), and production of NO were detected after aerobic exercise training in TAC mice. However, selective inhibition of β3-AR by SR59230A abolished the upregulation and activation of nNOS induced NO production. Furthermore, aerobic exercise training decreased the myocardial ROS and MDA contents and increased myocardial levels of SOD; both effects were partially attenuated by SR59230A. Our study suggested that aerobic exercise training could improve cardiac systolic function and alleviate LV chamber dilation, cardiac fibrosis and hypertrophy in HF mice. The mechanism responsible for the protective effects of aerobic exercise is associated with the activation of the β3-AR-nNOS-NO pathway.

Differential regulation of metabolic pathways by androgen receptor (AR) and its constitutively active splice variant, AR-V7, in prostate cancer cells

PubMed Central

Shafi, Ayesha A.; Putluri, Vasanta; Arnold, James M.; Tsouko, Efrosini; Maity, Suman; Roberts, Justin M.; Coarfa, Cristian; Frigo, Daniel E.; Putluri, Nagireddy; Sreekumar, Arun; Weigel, Nancy L.

2015-01-01

Metastatic prostate cancer (PCa) is primarily an androgen-dependent disease, which is treated with androgen deprivation therapy (ADT). Tumors usually develop resistance (castration-resistant PCa [CRPC]), but remain androgen receptor (AR) dependent. Numerous mechanisms for AR-dependent resistance have been identified including expression of constitutively active AR splice variants lacking the hormone-binding domain. Recent clinical studies show that expression of the best-characterized AR variant, AR-V7, correlates with resistance to ADT and poor outcome. Whether AR-V7 is simply a constitutively active substitute for AR or has novel gene targets that cause unique downstream changes is unresolved. Several studies have shown that AR activation alters cell metabolism. Using LNCaP cells with inducible expression of AR-V7 as a model system, we found that AR-V7 stimulated growth, migration, and glycolysis measured by ECAR (extracellular acidification rate) similar to AR. However, further analyses using metabolomics and metabolic flux assays revealed several differences. Whereas AR increased citrate levels, AR-V7 reduced citrate mirroring metabolic shifts observed in CRPC patients. Flux analyses indicate that the low citrate is a result of enhanced utilization rather than a failure to synthesize citrate. Moreover, flux assays suggested that compared to AR, AR-V7 exhibits increased dependence on glutaminolysis and reductive carboxylation to produce some of the TCA (tricarboxylic acid cycle) metabolites. These findings suggest that these unique actions represent potential therapeutic targets. PMID:26378018

Antiandrogenic activities of diesel exhaust particle extracts in PC3/AR human prostate carcinoma cells.

PubMed

Kizu, Ryoichi; Okamura, Kazumasa; Toriba, Akira; Mizokami, Atsushi; Burnstein, Kerry L; Klinge, Carolyn M; Hayakawa, Kazuichi

2003-12-01

We collected diesel exhaust particles (DEPs) emitted from three diesel-engine vehicles--a car, a bus, and a truck--in daily use, and prepared DEP extracts (DEPEs), designated as EC, EB, or ET, respectively. The androgenic and antiandrogenic effects of the DEPE samples were examined by a luciferase reporter assay in human prostate carcinoma PC3/AR cells transiently transfected with a prostate specific antigen gene promoter-driven luciferase expression vector pGLPSA5.8. PC3/AR is a subline of human prostate carcinoma PC3 transformed to stably express wild-type human androgen receptor (AR). While DEPE samples did not exhibit any androgenic effect, they exerted antiandrogenic effect, inhibiting dihydrotestosterone (10 pM) -induced luciferase activity by 24 to 52% at an extract concentration of 10 microg/ml. The antiandrogenic effect was greater in the following order: ET > EB > EC. Co-treatment of PC3/AR cells with SKF-525A, a nonselective inhibitor of cytochrome P450 (CYP) enzymes, enhanced the antiandrogenic effect, indicating that the antiandrogenic effect is caused by intact species of DEPE constituents. The antiandrogenic effect of DEPE samples was reversed by alpha-naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist. The antiandrogenic activity of a DEPE sample correlated with its AhR agonist activity assayed in PC3/AR cells transiently transfected with CYP1A1 gene promoter-driven luciferase expression vector pLUC1A1. Equimolar mixtures of ten polycyclic aromatic hydrocarbons (PAHs) having four or more rings, structures found in the DEPEs, showed significant antiandrogenic effects and AhR agonist activity at concentrations equivalent to those found in DEPE samples. Further, DEPE samples elicited only antiandrogenic effects in recombinant yeast cells, which express beta-galactosidase in response to androgen. A competitive AR binding assay showed that AR-binding constituents exist in DEPE samples, indicating that greater part of AR-binding constituents in DEPEs are AR antagonists. All these findings show that DEPE samples exhibit significant antiandrogenic effect in cell-based transcription assay and that this effect is due in part to the constituents with AhR agonist activity including PAHs and to the constituents with AR antagonist activity.

The Correlation Between PARP1 and BRCA1 in AR Positive Triple-negative Breast Cancer.

PubMed

Luo, Jiayan; Jin, Juan; Yang, Fang; Sun, Zijia; Zhang, Wenwen; Shi, Yaqin; Xu, Jing; Guan, Xiaoxiang

2016-01-01

Triple-negative breast cancer (TNBC) lacks estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) expression and thus cannot benefit from conventional hormonal or anti-HER2 targeted therapies. Anti-androgen therapy has shown a certain effect on androgen receptor (AR) positive TNBC. The emerging researches have proved that poly (ADP-ribose) polymerase (PARP) inhibitor is effective in BRCA1-deficient breast cancers. We demonstrated that combination of AR antagonist (bicalutamide) and PARP inhibitor (ABT-888) could inhibit cell viability and induce cell apoptosis significantly whatever in vitro or in vivo setting in AR-positive TNBC. Previous studies have proved that both BRCA1 and PARP1 have close connections with AR in prostate cancer. We explored the correlation among AR, PARP1 and BRCA1 in TNBC for the first time. After BRCA1 overexpression, the expression of AR and PARP1 were decreased in mRNA and protein levels. Additionally, AR positively regulated PARP1 while PARP1 also up-regulated AR expression in vitro. We also confirmed BRCA1 expression was negatively correlated with AR and PARP1 in TNBC patients using a tissue microarray with TNBC patient samples. These results suggest that the combination of bicalutamide and PARP inhibitor may be a potential strategy for TNBC patients and merits further evaluation.

Neuroendocrine mediators up-regulate alpha1b- and alpha1d-adrenergic receptor subtypes in human monocytes.

PubMed

Rouppe van der Voort, C; Kavelaars, A; van de Pol, M; Heijnen, C J

1999-03-01

Beta2- and alpha2-adrenergic receptors (AR) are thought to be the main AR subtypes to exert the effects of catecholamines on the immune system. However, in the present study, we demonstrate that another subtype of AR can be induced in human monocytes. Expression of alpha1b- and alpha1d-AR mRNA can be obtained by culturing freshly isolated human peripheral blood monocytes with the neuroendocrine mediators dexamethasone or the beta2-AR agonist terbutaline. Using the human monocytic cell line THP-1, we demonstrate that increased levels of alpha1b- and alpha1d-mRNA are accompanied by increased levels of receptor protein as determined by Western blot analysis and radioligand binding assays. This study describes for the first time regulated expression of alpha1-AR subtypes in human monocytes.

Nitrate affects sensu-stricto germination of after-ripened Sisymbrium officinale seeds by modifying expression of SoNCED5, SoCYP707A2 and SoGA3ox2 genes.

PubMed

Carrillo-Barral, Néstor; Matilla, Angel J; Rodríguez-Gacio, María del Carmen; Iglesias-Fernández, Raquel

2014-03-01

The influence of nitrate upon the germination of Sisymbrium officinale seeds is not entirely controlled by after-ripening (AR), a process clearly influenced by nitrate. Recently, we have reported that nitrate affects sensu-stricto germination of non-AR (AR0) seeds by modifying the expression of crucial genes involved in the metabolism of GA and ABA. In this study, we demonstrate that nitrate affects also the germination of AR seeds because: (i) the AR negatively alters the ABA sensitivity being the seed more ABA-sensible as the AR is farthest from optimal (AR0 and AR20 versus AR7); in the presence of diniconazole (DZ), a competitive inhibitor of ABA 8'-hydroxylase, testa rupture is affected while the endosperm rupture is not. (ii) AR7 seed-coat rupture is not inhibited by paclobutrazol (PBZ) suggesting that nitrate can act by a mechanism GA-independent. (iii) The germination process is accelerated by nitrate, most probably by the increase in the expression of SoNCED5, SoCYP707A2 and SoGA3ox2 genes. Taken together, these and previous results demonstrate that nitrate promotes germination of AR and non-AR seeds through transcriptional changes of different genes involved in ABA and GA metabolism. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Expression of the cancer-testis antigen BORIS correlates with prostate cancer.

PubMed

Cheema, Zubair; Hari-Gupta, Yukti; Kita, Georgia-Xanthi; Farrar, Dawn; Seddon, Ian; Corr, John; Klenova, Elena

2014-02-01

BORIS, a paralogue of the transcription factor CTCF, is a member of the cancer-testis antigen (CT) family. BORIS is normally present at high levels in the testis; however it is aberrantly expressed in various tumors and cancer cell lines. The main objectives of this study were to investigate BORIS expression together with sub-cellular localization in both prostate cell lines and tumor tissues, and assess correlations between BORIS and clinical/pathological characteristics. We examined BORIS mRNA expression, protein levels and cellular localization in a panel of human prostate tissues, cancer and benign, together with a panel prostate cell lines. We also compared BORIS levels and localization with clinical/pathological characteristics in prostate tumors. BORIS was detected in all inspected prostate cancer cell lines and tumors, but was absent in benign prostatic hyperplasia. Increased levels of BORIS protein positively correlated with Gleason score, T-stage and androgen receptor (AR) protein levels in prostate tumors. The relationship between BORIS and AR was further highlighted in prostate cell lines by the ability of ectopically expressed BORIS to activate the endogenous AR mRNA and protein. BORIS localization in the nucleus plus cytoplasm was also associated with higher BORIS levels and Gleason score. Detection of BORIS in prostate tumors suggests potential applications of BORIS as a biomarker for prostate cancer diagnosis, as an immunotherapy target and, potentially, a prognostic marker of more aggressive prostate cancer. The ability of BORIS to activate the AR gene indicates BORIS involvement in the growth and development of prostate tumors. © 2013 Wiley Periodicals, Inc.

Monoterpene limonene induces brown fat-like phenotype in 3T3-L1 white adipocytes.

PubMed

Lone, Jameel; Yun, Jong Won

2016-05-15

Several dietary compounds that are able to induce the brown fat-like phenotype in white adipocytes have been considered for treatment of obesity due to their ability to increase energy expenditure. Here, we report that limonene induces the brown fat-like phenotype in 3T3-L1 adipocytes by increasing expression of brown adipocyte-specific genes and proteins. Limonene-induced browning in white adipocytes was investigated by determining expression levels of brown fat-specific genes and proteins by real-time RT-PCR, immunoblot analysis, and immunocytochemical staining. Limonene enhanced mitochondrial biogenesis, as evidenced by increased mitochondrial content and immunofluorescent intensity. Limonene also significantly elevated protein levels of HSL, PLIN, p-AMPK, p-ACC, ACO, COX4, CPT1, and CYT C, suggesting its possible role in enhancement of lipolysis and lipid catabolism. Increased expression of PRDM16, UCP1, C/EBPβ, and other brown fat-specific markers by limonene was possibly mediated by activation of β3-adnergenic receptor (β3-AR), as inhibition of β3-AR inhibited up-regulation of brown fat-specific markers. Similarly, limonene-mediated activation of ERK and up-regulation of key brown adipocyte specific markers were eliminated by treatment with ERK antagonist. Taken together, these results suggest that limonene induces browning of 3T3-L1 adipocytes via activation of β3-AR and the ERK signaling pathway. In conclusion, our findings suggest that limonene plays a dual modulatory role in induction of the brown adipocyte-like phenotype as well as promotion of lipid metabolism and thus may have potential therapeutic implications for treatment of obesity. Copyright © 2016 Elsevier Inc. All rights reserved.

Reconfiguring the AR-TIF2 Protein–Protein Interaction HCS Assay in Prostate Cancer Cells and Characterizing the Hits from a LOPAC Screen

PubMed Central

Fancher, Ashley T.; Hua, Yun; Camarco, Daniel P.; Close, David A.; Strock, Christopher J.

2016-01-01

Abstract The continued activation of androgen receptor (AR) transcription and elevated expression of AR and transcriptional intermediary factor 2 (TIF2) coactivator observed in prostate cancer (CaP) recurrence and the development of castration-resistant CaP (CRPC) support a screening strategy for small-molecule inhibitors of AR-TIF2 protein–protein interactions (PPIs) to find new drug candidates. Small molecules can elicit tissue selective effects, because the cells of distinct tissues express different levels and cohorts of coregulatory proteins. We reconfigured the AR-TIF2 PPI biosensor (PPIB) assay in the PC-3 CaP cell line to determine whether AR modulators and hits from an AR-TIF2 PPIB screen conducted in U-2 OS cells would behave differently in the CaP cell background. Although we did not observe any significant differences in the compound responses between the assay performed in osteosarcoma and CaP cells, the U-2 OS AR-TIF2 PPIB assay would be more amenable to screening, because both the virus and cell culture demands are lower. We implemented a testing paradigm of counter-screens and secondary hit characterization assays that allowed us to identify and deprioritize hits that inhibited/disrupted AR-TIF2 PPIs and AR transcriptional activation (AR-TA) through antagonism of AR ligand binding or by non-specifically blocking nuclear receptor trafficking. Since AR-TIF2 PPI inhibitor/disruptor molecules act distally to AR ligand binding, they have the potential to modulate AR-TA in a cell-specific manner that is distinct from existing anti-androgen drugs, and to overcome the development of resistance to AR antagonism. We anticipate that the application of this testing paradigm to characterize the hits from an AR-TIF2 PPI high-content screening campaign will enable us to prioritize the AR-TIF2 PPI inhibitor/disruptor leads that have potential to be developed into novel therapeutics for CaP and CRPC. PMID:27606620

Volatile arsenic species released from Escherichia coli expressing the AsIII S-adenosylmethionine methyltransferase gene.

PubMed

Yuan, Chungang; Lu, Xiufen; Qin, Jie; Rosen, Barry P; Le, X Chris

2008-05-01

Biological systems, ranging from bacteria and fungi to humans, can methylate arsenic. Recent studies have suggested that the AsIII S-adenosylmethionine methyltransferase (arsM) gene in bacteria was responsible for the removal of arsenic as the volatile arsines from the bacteria. However, there has been no direct measure of the arsines released from bacteria cultures. We describe here an integrated system incorporating the bacterial incubation and volatile arsenic species analysis, and we demonstrate its application to the identification of the volatile arsines produced in bacterial cultures. The headspace of the bacterial cultures was purged with helium, and the volatile arsenic species were trapped in a chromatographic column immersed in liquid nitrogen. The cryogenically trapped arsines [AsH3, (CH3)AsH2, (CH3)2AsH, and (CH3)3As] were separated by gas chromatography and were detected by inductively coupled plasma mass spectrometry. A hydride generation system was coupled to the bacterial culture system, allowing for spiking standards and for generating calibration arsines necessary for quantitative analysis. Both bacteria containing the arsM gene or its variant arsMC2 gene were able to produce 400-500 ng of trimethylarsine. No trimethylarsine was detectable in bacteria lacking the arsM gene (containing the vector plasmid as negative control). These results confirm that arsM is responsible for releasing arsenic as volatile species from the arsenic-resistant bacteria. Our results also show traces of AsH3, CH3AsH2, and (CH3)2AsH in cultures of bacteria expressing arsM. The method detection limits for AsH3, CH3AsH2, (CH3)2AsH, and (CH3)3As were 0.5, 0.5, 0.7, and 0.6 pg, respectively. The ability to quantify trace levels of these volatile arsenic species makes it possible to study the biotransformation and biochemical roles of the evolution of these volatile arsenic species by biological systems.

The Yeast Anaerobic Response Element AR1b Regulates Aerobic Antifungal Drug-dependent Sterol Gene Expression*

PubMed Central

Gallo-Ebert, Christina; Donigan, Melissa; Liu, Hsing-Yin; Pascual, Florencia; Manners, Melissa; Pandya, Devanshi; Swanson, Robert; Gallagher, Denise; Chen, WeiWei; Carman, George M.; Nickels, Joseph T.

2013-01-01

Saccharomyces cerevisiae ergosterol biosynthesis, like cholesterol biosynthesis in mammals, is regulated at the transcriptional level by a sterol feedback mechanism. Yeast studies defined a 7-bp consensus sterol-response element (SRE) common to genes involved in sterol biosynthesis and two transcription factors, Upc2 and Ecm22, which direct transcription of sterol biosynthetic genes. The 7-bp consensus SRE is identical to the anaerobic response element, AR1c. Data indicate that Upc2 and Ecm22 function through binding to this SRE site. We now show that it is two novel anaerobic AR1b elements in the UPC2 promoter that direct global ERG gene expression in response to a block in de novo ergosterol biosynthesis, brought about by antifungal drug treatment. The AR1b elements are absolutely required for auto-induction of UPC2 gene expression and protein and require Upc2 and Ecm22 for function. We further demonstrate the direct binding of recombinant expressed S. cerevisiae ScUpc2 and pathogenic Candida albicans CaUpc2 and Candida glabrata CgUpc2 to AR1b and SRE/AR1c elements. Recombinant endogenous promoter studies show that the UPC2 anaerobic AR1b elements act in trans to regulate ergosterol gene expression. Our results indicate that Upc2 must occupy UPC2 AR1b elements in order for ERG gene expression induction to take place. Thus, the two UPC2-AR1b elements drive expression of all ERG genes necessary for maintaining normal antifungal susceptibility, as wild type cells lacking these elements have increased susceptibility to azole antifungal drugs. Therefore, targeting these specific sites for antifungal therapy represents a novel approach to treat systemic fungal infections. PMID:24163365

Functional potentiation of leptin-signal transducer and activator of transcription 3 signaling by the androgen receptor.

PubMed

Fan, WuQiang; Yanase, Toshihiko; Nishi, Yoshihiro; Chiba, Seiichi; Okabe, Taijiro; Nomura, Masatoshi; Yoshimatsu, Hironobu; Kato, Shigeaki; Takayanagi, Ryoichi; Nawata, Hajime

2008-12-01

Hypogonadism is associated with increased fat mass and dysregulation of metabolic homeostasis in men. Our previous study revealed that androgen receptor (AR)-null male mice (ARL-/Y) develop late-onset obesity and are leptin-resistant. The present study evaluated how hypothalamic AR contributes to central leptin-signal transducer and activator of transcription 3 (STAT3) signaling. We evaluated leptin action in wild-type and ARL-/Y mice, the anatomic co-relationship between AR and leptin signaling in the hypothalamus, and the effects of AR on leptin-mediated STAT3 transactivation and nuclear translocation. AR deletion in male mice results in a weaker leptin-induced suppression of food intake and body weight drop even before the onset of overt obesity. In wild-type male but not female mice, AR was highly expressed in various hypothalamic nuclei that also expressed the long-form leptin receptor (OBRB) and co-resided with OBRB directly in the arcuate neurons. In vitro, AR significantly enhanced STAT3-mediated transcription of leptin target genes including POMC and SOCS3. This effect relied on the AR N-terminal activation function-1 (AF-1) domain and was specific to AR in that none of the other sex steroid hormone receptors tested showed similar effects. AR enhanced the low concentrations of leptin-induced STAT3 nuclear translocation in vitro, and ARL-/Y mice receiving leptin had impaired STAT3 nuclear localization in the arcuate neurons. These findings indicate that AR in the hypothalamus functions as a regulator of central leptin-OBRB-STAT3 signaling and has a physiological role in energy homeostasis and metabolic regulation in male mice.

Overexpression of androgen receptor and forkhead-box A1 protein in apocrine breast carcinoma.

PubMed

Sasahara, Manami; Matsui, Akira; Ichimura, Yoshiko; Hirakata, Yuuko; Murata, Yuuya; Marui, Eiji

2014-03-01

Apocrine breast carcinoma often lacks estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor type-2 (HER2) expression. Accordingly, development of a new treatment strategy is important for this type of cancer. The growth stimulus through the androgen receptor (AR) can be a candidate for targeted treatment. Therefore, we examined the factors related to AR transcription. We immunohistochemically evaluated 54 apocrine cancer lesions for ER, PgR, AR, HER2, Ki-67, forkhead-box protein A1 (FOXA1), and prostate-specific antigen (PSA) expression. ER, PgR, and HER2 were expressed at a low level, thus 44 out of 54 (81.4%) cases were of triple-negative breast cancer. AR, PSA and FOXA1 were expressed in 100% (54/54), 48% (26/54) and 93% (50/54) of cases, respectively. Most of apocrine breast carcinomas were immunohistochemically-positive for AR and FOXA1. Anti-androgenic therapies can potentially serve as a cancer-targeting therapy for apocrine breast carcinoma.

Therapeutic targeting of sunitinib-induced AR phosphorylation in renal cell carcinoma.

PubMed

Adelaiye-Ogala, Remi; Damayanti, Nur P; Orillion, Ashley R; Arisa, Sreevani; Chintala, Sreenivasulu; Titus, Mark A; Kao, Chinghai; Pili, Roberto

2018-03-23

Androgen receptor (AR) plays a crucial role in the development and progression of prostate cancer. AR expression has also been reported in other solid tumors, including renal cell carcinoma (RCC), but its biological role here remains unclear. Through integrative analysis of a reverse phase protein array (RPPA), we discovered increased expression of AR in an RCC patient-derived xenograft model of acquired resistance to the receptor tyrosine kinase inhibitor (RTKi) sunitinib. AR expression was increased in RCC cell lines with either acquired or intrinsic sunitinib resistance in vitro. An AR signaling gene array profiler indicated elevated levels of AR target genes in sunitinib-resistant cells. Sunitinib-induced AR transcriptional activity was associated with increased phosphorylation of serine 81 (pS81) on AR. Additionally, AR overexpression resulted in acquired sunitinib resistance, and the AR antagonist enzalutamide-induced AR degradation and attenuated AR downstream activity in sunitinib-resistant cells, also indicated by decreased secretion of human kallikrein 2 (KLK2). Enzalutamide-induced AR degradation was rescued by either proteasome inhibition or by knockdown of the AR ubiquitin ligase speckle-type POZ protein (SPOP). In vivo treatment with enzalutamide and sunitinib demonstrated that this combination efficiently induced tumor regression in an RCC model following acquired sunitinib resistance. Overall, our results suggest the potential role of AR as a target for therapeutic interventions, in combination with RTKi, to overcome drug resistance in RCC. Copyright ©2018, American Association for Cancer Research.

Cloning, cell-type specificity, and regulatory function of the mouse alpha(1B)-adrenergic receptor promoter.

PubMed

Zuscik, M J; Piascik, M T; Perez, D M

1999-12-01

The functionality of a 3422-base pair promoter fragment from the mouse alpha(1B)-adrenergic receptor (alpha(1B)AR) gene was examined. This fragment, cloned from a mouse genomic library, was found to have significant sequence homology to the known human and rat alpha(1B)AR promoters. However, the consensus motif of several key cis-acting elements is not conserved among the rat, human, and mouse genes, suggesting species specificity. Confirming fidelity of the murine promoter, robust in vitro expression of a chloramphenicol acetyltransferase (CAT) reporter was detected in known alpha(1B)AR-expressing BC(3)H1, NB41A3, and DDT(1)MF-2 cells transiently transfected with a promoter-CAT construct. Conversely, minimal CAT expression was detected in known alpha(1B)AR-negative RAT-1 and R3T3 cells. These findings were extended by transfecting the same promoter-CAT construct into various primary cell types. In support of the hypothesis that alpha(1)ARs are differentially expressed in the smooth muscle of the vasculature, primary cultures of superior mesenteric and renal artery vascular smooth muscle cells showed significantly stronger CAT expression than did vascular smooth muscle cells derived from pulmonary, femoral, and iliac arteries. Primary osteoblastic bone-forming cells, which are known to be alpha(1B)AR negative, showed minimal CAT expression. Indicating regulatory function through cis-acting elements, RAT-1, R3T3, NB41A3, BC(3)H1, and DDT(1)MF2 cells transfected with the promoter-CAT construct all showed increased CAT production when challenged with forskolin or hypoxic conditions. Additionally, tissue-specific regulation of the promoter was observed when cells were simultaneously challenged with both forskolin and hypoxia. These results collectively demonstrate that a 3.4-kb PvuII fragment of the murine alpha(1B)AR gene promoter can: 1) drive tissue-specific production of a CAT reporter in both clonal and primary cell lines; and 2) confer tissue-specific regulation of that CAT reporter when induced by challenge with forskolin and/or hypoxic conditions.

PCA3 Silencing Sensitizes Prostate Cancer Cells to Enzalutamide-mediated Androgen Receptor Blockade.

PubMed

Özgür, Emre; Celik, Ayca Iribas; Darendeliler, Emin; Gezer, Ugur

2017-07-01

Prostate cancer (PCa) is an androgen-dependent disease. Novel anti-androgens (i.e. enzalutamide) have recently been developed for the treatment of patients with metastatic castration-resistant prostate cancer (CRPC). Evidence is accumulating that prostate cancer antigen 3 (PCA3) is involved in androgen receptor (AR) signaling. Here, in combination with enzalutamide-mediated AR blockade, we investigated the effect of PCA3 targeting on the viability of PCa cells. In hormone-sensitive LNCaP cells, AR-overexpressing LNCaP-AR + cells and VCaP cells (representing CRPC), PCA3 was silenced using siRNA oligonucleotides. Gene expression and cell viability was assessed in PCA3-silenced and/or AR-blocked cells. PCA3 targeting reduced the expression of AR-related genes (i.e. prostate-specific antigen (PSA) and prostate-specific transcript 1 (non-protein coding) (PCGEM1)) and potentiated the effect of enzalutamide. Proliferation of PCa cells was suppressed upon PCA3 silencing with a greater effect in LNCaP-AR + cells. Furthermore, PCA3 silencing sensitized PCa cells to enzalutamide-induced loss of cell growth. PCA3, as a therapeutic target in PCa, might be used to potentiate AR antagonists. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Epigenetic modulation of AR gene expression in prostate cancer DU145 cells with the combination of sodium butyrate and 5'-Aza-2'-deoxycytidine.

PubMed

Fialova, Barbora; Luzna, Petra; Gursky, Jan; Langova, Katerina; Kolar, Zdenek; Trtkova, Katerina Smesny

2016-10-01

The androgen receptor (AR) plays an essential role in the development and progression of prostate cancer. Castration-resistant prostate cancer (CRPC) is a consequence of androgen deprivation therapy. Unchecked CRPC followed by metastasis is lethal. Some CRPCs show decreased AR gene expression due to epigenetic mechanisms such as DNA methylation and histone deacetylation. The aim of this study was to epigenetically modulate the methylated state of the AR gene leading to targeted demethylation and AR gene expression in androgen-independent human prostate cancer DU145 cell line, representing the CRPC model with very low or undetectable AR levels. The cell treatment was based on single and combined applications of two epigenetic inhibitors, sodium butyrate (NaB) as histone deacetylases inhibitor and 5'-Aza-2'-deoxycytidine (Aza-dC) as DNA methyltransferases inhibitor. We found that the Aza-dC in combination with NaB may help reduce the toxicity of higher NaB concentrations in cancer cells. In normal RWPE-1 cells and even stronger in cancer DU145 cells, the combined treatment induced both AR gene expression on the mRNA level and increased histone H4 acetylation in AR gene promoter. Also activation and maintenance of G2/M cell cycle arrest and better survival in normal RWPE-1 cells compared to cancer DU145 cells were observed after the treatments. These results imply the selective toxicity effect of both inhibitors used and their potentially more effective combined use in the epigenetic therapy of prostate cancer patients.

Effect of treadmill exercise on 5-HT, 5-HT1A receptor and brain derived neurophic factor in rats after permanent middle cerebral artery occlusion.

PubMed

Lan, Xiaofang; Zhang, Meng; Yang, Wan; Zheng, Zongju; Wu, Yuan; Zeng, Qian; Liu, Shudong; Liu, Ke; Li, Guangqin

2014-05-01

It has been well documented that exercise promotes neurological rehabilitation in patients with cerebral ischemia. However, the exact mechanisms have not been fully elucidated. This study aimed to discuss the effect of treadmill exercise on expression levels of 5-HT, 5-HT1A receptor (5-HT1AR) and brain derived neurophic factor (BDNF) in rat brains after permanent middle cerebral artery occlusion (pMCAO). A total of 55 rats were randomly divided into 3 groups: pMCAO group, pMCAO and treadmill exercise (pMCAO + Ex) group, and sham-operated group. Rats in pMCAO + Ex group underwent treadmill exercise for 16 days. Neurological function was evaluated by modified Neurological Severity Scores (mNSS). High-performance liquid chromatography-electrochemical detection system was used to determine the content of 5-HT in cortex tissues. The protein levels of 5-HT1AR, BDNF and synaptophysin were measured by Western blot. The mNSS in pMCAO + Ex group was lower than that in pMCAO group on day 19 post-MCAO (p < 0.001). The content of 5-HT dropped to 3.81 ± 1.86 ng/ml in pMCAO group (43.84 ± 2.05 ng/ml in sham-operated group), but increased in pMCAO + Ex group (10.06 ± 1.80 ng/ml). The protein expressions levels of synaptophysin, 5-HT1AR and BDNF were downregulated after cerebral ischemia (p < 0.05), and upregulated after treadmill exercise (p < 0.05). These results indicate that treadmill exercise improves neurologic function, enhances neuronal plasticity and upregulates the levels of 5-HT, 5-HT1AR and BDNF in rats with pMCAO.

β-Adrenergic Receptor-Mediated Cardiac Contractility is Inhibited via Vasopressin Type 1A-Receptor-Dependent Signaling

PubMed Central

Tilley, Douglas G.; Zhu, Weizhong; Myers, Valerie D.; Barr, Larry A.; Gao, Erhe; Li, Xue; Song, Jianliang; Carter, Rhonda L.; Makarewich, Catherine A.; Yu, Daohai; Troupes, Constantine D.; Grisanti, Laurel A.; Coleman, Ryan C.; Koch, Walter J.; Houser, Steven R.; Cheung, Joseph Y.; Feldman, Arthur M.

2014-01-01

Background Enhanced arginine vasopressin (AVP) levels are associated with increased mortality during end-stage human heart failure (HF), and cardiac AVP type 1A receptor (V1AR) expression becomes increased. Additionally, mice with cardiac-restricted V1AR overexpression develop cardiomyopathy and decreased β-adrenergic receptor (βAR) responsiveness. This led us to hypothesize that V1AR signaling regulated βAR responsiveness and in doing so contributes to HF development. Methods and Results Transaortic constriction resulted in decreased cardiac function and βAR density and increased cardiac V1AR expression, effects reversed by a V1AR-selective antagonist. Molecularly, V1AR stimulation led to decreased βAR ligand affinity, as well as βAR-induced Ca2+ mobilization and cAMP generation in isolated adult cardiomyocytes, effects recapitulated via ex vivo Langendorff analysis. V1AR-mediated regulation of βAR responsiveness was demonstrated to occur in a previously unrecognized Gq protein-independent/GRK-dependent manner. Conclusions This newly discovered relationship between cardiac V1AR and βAR may be informative for the treatment of patients with acute decompensated HF and elevated AVP. PMID:25205804

Molecular biology of castration-resistant prostate cancer: basis for the novel therapeutic targets.

PubMed

Mellado, Begoña; Marin Aguilera, Mercedes; Pereira, Maria Veronica

2013-06-01

Prostate cancer cells express the androgen receptor (AR) and need the presence of androgens to survive. Androgen suppression is the gold standard first-line therapy for metastatic disease. Almost all prostate cancer patients initially respond to hormonal therapy, but most of them gradually develop castration-resistant progression. Recent evidence has shown that progression at the castration resistant prostate cancer (CRPC) stage is often mediated by AR signalling. Importantly, subsequent AR androgen inhibition, by abiraterone acetate or enzalutamide, has shown to improve patients' survival. Several mechanisms that enhance AR signalling in an androgen-depleted environment have been elucidated:(1) AR mutations that allow activation by low androgen levels or by other endogenous steroids, (2) AR amplification and/or overexpression,(3)increased local intracrine synthesis of androgens, (4) changes in AR cofactors and (5) cross-talk with cytokines and growth factors. Today, there are under development a number of novel agents targeting the AR signaling pathway. This article reviews the postulated mechanisms of AR-driven resistance to androgen suppression that have contributed to the development of new hormonal therapeutic strategies in prostate cancer.

Ars insulator identified in sea urchin possesses an activity to ensure the transgene expression in mouse cells.

PubMed

Tajima, Shoji; Shinohara, Keiko; Fukumoto, Maiko; Zaitsu, Reiko; Miyagawa, Junichi; Hino, Shinjiro; Fan, Jun; Akasaka, Koji; Matsuoka, Masao

2006-04-01

Sea urchin arylsulfatase (Ars) gene locus has features of an insulator, i.e., blocking of enhancer and promoter interaction, and protection of a transgene against positional effects [Akasaka et al. (1999) Cell. Mol. Biol. 45, 555-565]. To examine the effect of Ars insulator on long-term expression of a transgene, the insulator was inserted into LTR of retrovirus vector harboring hrGFP gene as a reporter, and then introduced into mouse myoblast cells. The isolated clones transduced with the reporter gene with or without Ars insulator were cultured for more than 20 wk in the absence of a selection reagent, and the expression of hrGFP was periodically determined. Expression of hrGFP in four clones transduced with the reporter gene without Ars insulator was completely silenced after 20 wk of culture. On the other hand, hrGFP was expressed in all clones with Ars insulator inserted in one of the two different orientations. Histone H3 deacetylation and DNA methylation of the 5'LTR promoter region, signs for heterochromatin and silencing, were suppressed in the clones that were expressing hrGFP. Ars insulator is effective in maintaining a transgene in mouse cells in an orientation-dependent manner, and will be a useful tool to ensure stable expression of a transgene.

Pomegranate Polyphenols Downregulate Expression of Androgen Synthesizing Genes in Human Prostate Cancer Cells Overexpressing the Androgen Receptor

PubMed Central

Hong, Mee Young; Seeram, Navindra P.; Heber, David

2008-01-01

Prostate cancer is dependent on circulating testosterone in its early stages and is treatable with radiation and surgery. However, recurrent prostate tumors advance to an androgen-independent state where they progress in the absence of circulating testosterone leading to metastasis and death. During the development of androgen independence, prostate cancer cells are known to increase intracellular testosterone synthesis which maintains cancer cell growth in the absence of significant amounts of circulating testosterone. Overexpression of the androgen receptor (AR) occurs in androgen-independent prostate cancer and has been proposed as another mechanism promoting the development of androgen independence. The LNCaP-AR cell line is engineered to overexpress AR but is otherwise similar to the widely studied LNCaP cell line. We have previously shown that pomegranate extracts inhibit both androgen-dependent and androgen-independent prostate cancer cell growth. In the present study, we examined the effects of pomegranate polyphenols, ellagitannin-rich extract and whole juice extract on the expression of genes for key androgen synthesizing enzymes and the AR. We measured expression of the HSD3B2, AKR1C3 and SRD5A1 genes for the respective androgen synthesizing enzymes in LNCaP, LNCaP-AR, and DU-145 human prostate cancer cells. A two-fold suppression of gene expression was considered statistically significant. Pomegranate polyphenols inhibited gene expression and AR most consistently in the LNCaP-AR cell line (P =.05). Therefore, inhibition by pomegranate polyphenols of gene expression involved in androgen synthesis enzymes and the AR may be of particular importance in androgen-independent prostate cancer cells and the subset of human prostate cancers where AR is upregulated. PMID:18479901

Beta-3 adrenergic agonists reduce pulmonary vascular resistance and improve right ventricular performance in a porcine model of chronic pulmonary hypertension.

PubMed

García-Álvarez, Ana; Pereda, Daniel; García-Lunar, Inés; Sanz-Rosa, David; Fernández-Jiménez, Rodrigo; García-Prieto, Jaime; Nuño-Ayala, Mario; Sierra, Federico; Santiago, Evelyn; Sandoval, Elena; Campelos, Paula; Agüero, Jaume; Pizarro, Gonzalo; Peinado, Víctor I; Fernández-Friera, Leticia; García-Ruiz, José M; Barberá, Joan A; Castellá, Manuel; Sabaté, Manel; Fuster, Valentín; Ibañez, Borja

2016-07-01

Beta-3 adrenergic receptor (β3AR) agonists have been shown to produce vasodilation and prevention of ventricular remodeling in different conditions. Given that these biological functions are critical in pulmonary hypertension (PH), we aimed to demonstrate a beneficial effect of β3AR agonists in PH. An experimental study in pigs (n = 34) with chronic PH created by pulmonary vein banding was designed to evaluate the acute hemodynamic effect and the long-term effect of β3AR agonists on hemodynamics, vascular remodeling and RV performance in chronic PH. Ex vivo human experiments were performed to explore the expression of β3AR mRNA and the vasodilator response of β3AR agonists in pulmonary arteries. Single intravenous administration of the β3AR agonist BRL37344 produced a significant acute reduction in PVR, and two-weeks treatment with two different β3AR selective agonists, intravenous BRL37344 or oral mirabegron, resulted in a significant reduction in PVR (median of -2.0 Wood units/m(2) for BRL37344 vs. +1.5 for vehicle, p = 0.04; and -1.8 Wood units/m(2) for mirabegron vs. +1.6 for vehicle, p = 0.002) associated with a significant improvement in magnetic resonance-measured RV performance. Histological markers of pulmonary vascular proliferation (p27 and Ki67) were significantly attenuated in β3AR agonists-treated pigs. β3AR was expressed in human pulmonary arteries and β3AR agonists produced vasodilatation. β3AR agonists produced a significant reduction in PVR and improved RV performance in experimental PH, emerging as a potential novel approach for treating patients with chronic PH.

Enhanced free cholesterol, SREBP-2 and StAR expression in human NASH.

PubMed

Caballero, Francisco; Fernández, Anna; De Lacy, Antonio M; Fernández-Checa, Jose C; Caballería, Juan; García-Ruiz, Carmen

2009-04-01

Non-alcoholic fatty liver disease (NAFLD) pathogenesis remains unknown. Due to the emerging role of free cholesterol (FC) in NAFLD, our aim was to examine the correlation between FC accumulation in patients with NAFLD and the expression of enzymes that regulate cholesterol homeostasis. Filipin staining, indicative of FC accumulation, and real-time PCR analyses were performed in 31 NAFLD patients and in seven controls. All NASH patients (n=14) and 4 out of 17 patients with steatosis exhibited filipin staining compared to controls (0 out of 7 subjects with normal liver histology and BMI). Sterol regulatory element-binding protein-2 (SREBP-2) mRNA levels were 7- and 3-fold higher in NASH and steatosis patients, respectively, compared to controls. Since hydroxymethylglutaryl-CoA (HMG-CoA) reductase is the key enzyme in cholesterol synthesis and transcriptionally controlled by SREBP-2 we measured its mRNA levels, being 3- to 4-fold higher in NAFLD compared to controls, without any difference between NASH and steatosis patients. Fatty acid synthase (FAS) and SREBP-1c expression were not significantly induced in NAFLD, while ATP-binding cassette sub-family G member 1 (ABCG1), a transporter involved in cholesterol egress, and acyl-CoA-cholesterol acyltransferase mRNA levels were modestly increased (1.5- to 2.5-fold, p<0.05), regardless of fibrosis. Interestingly, mRNA levels of steroidogenic acute regulatory protein (StAR), a mitochondrial-cholesterol transporting polypeptide, increased 7- and 15-fold in steatosis and NASH patients, respectively, compared to controls. FC increases in NASH and correlates with SREBP-2 induction. Moreover, StAR overexpression in NASH suggests that mitochondrial FC may be a player in disease progression and a novel target for intervention.

Prohibitin promotes androgen receptor activation in ER-positive breast cancer

PubMed Central

Liu, Pengying; Xu, Yumei; Zhang, Wenwen; Li, Yan; Tang, Lin; Chen, Weiwei; Xu, Jing; Sun, Qian; Guan, Xiaoxiang

2017-01-01

ABSTRACT Prohibitin (PHB) is an evolutionarily conserved protein with multiple functions in both normal and cancer cells. Androgen receptor (AR) was reported to act as a different role in the ER-positive and ER-negative breast cancer. However, little is known about the role of PHB and whether PHB could regulate AR expression in the ER-positive breast cancer. Here, we determined the expression and clinical outcomes of PHB in breast cancer samples using 121 breast cancer tissues and published databases, and investigated the role of PHB in breast cancer cell growth, apoptosis and cell cycle arrest in the ER-positive breast cancer cells. We obtained the expression of PHB is significantly low in breast cancer samples, and low PHB expression positively correlated with poor prognosis of breast cancer. We detected that PHB could inhibit breast cancer cell proliferation, change cell cycle distribution and promote cell apoptosis in the ER-positive breast cancer cells. Moreover, we found PHB could significantly increase AR expression in both mRNA and protein levels in the ER-positive breast cancer cells. Additionally, a significant positive correlation between PHB and AR expression was identified in the 121 breast cancer tissues. PHB and AR expression are associated with prognosis in the ER-positive breast cancer patients. Our results indicate that PHB promotes AR activation in ER-positive breast cancer, making PHB and AR potential molecular targets for ER-positive breast cancer therapy. PMID:28272969

B-DIM impairs radiation-induced survival pathways independently of androgen receptor expression and augments radiation efficacy in prostate cancer.

PubMed

Singh-Gupta, Vinita; Banerjee, Sanjeev; Yunker, Christopher K; Rakowski, Joseph T; Joiner, Michael C; Konski, Andre A; Sarkar, Fazlul H; Hillman, Gilda G

2012-05-01

Increased consumption of cruciferous vegetables is associated with decreased risk in prostate cancer (PCa). The active compound in cruciferous vegetables appears to be the self dimerized product [3,3'-diindolylmethane (DIM)] of indole-3-carbinol (I3C). Nutritional grade B-DIM (absorption-enhanced) has proven safe in a Phase I trial in PCa. We investigated the anti-cancer activity of B-DIM as a new biological approach to improve the effects of radiotherapy for hormone refractory prostate cancer cells, which were either positive or negative for androgen receptor (AR) expression. B-DIM inhibited cell growth in a dose-dependent manner in both PC-3 (AR-) and C4-2B (AR+) cell lines. B-DIM was effective at increasing radiation-induced cell killing in both cell lines, independently of AR expression. B-DIM inhibited NF-κB and HIF-1α DNA activities and blocked radiation-induced activation of these transcription factors in both PC-3 and C4-2B cells. In C4-2B (AR+) cells, AR expression and nuclear localization were significantly increased by radiation. However, B-DIM abrogated the radiation-induced AR increased expression and trafficking to the nucleus, which was consistent with decreased PSA secretion. In vivo, treatment of PC-3 prostate tumors in nude mice with B-DIM and radiation resulted in significant primary tumor growth inhibition and control of metastasis to para-aortic lymph nodes. These studies demonstrate that B-DIM augments radiation-induced cell killing and tumor growth inhibition. B-DIM impairs critical survival signaling pathways activated by radiation, leading to enhanced cell killing. These novel observations suggest that B-DIM could be used as a safe compound to enhance the efficacy of radiotherapy for castrate-resistant PCa. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Near-IR Resonance Raman Spectroscopy of Archaerhodopsin 3: Effects of Transmembrane Potential

PubMed Central

Saint Clair, Erica C.; Ogren, John I.; Mamaev, Sergey; Russano, Daniel; Kralj, Joel M.; Rothschild, Kenneth J.

2013-01-01

Archaerhodopsin 3 (AR3) is a light driven proton pump from Halorubrum sodomense that has been used as a genetically targetable neuronal silencer and an effective fluorescent sensor of transmembrane potential. Unlike the more extensively studied bacteriorhodopsin (BR) from Halobacterium salinarum, AR3 readily incorporates into the plasma membrane of both E. coli and mammalian cells. Here, we used near-IR resonance Raman confocal microscopy to study the effects of pH and membrane potential on the AR3 retinal chromophore structure. Measurements were performed both on AR3 reconstituted into E. coli polar lipids and in vivo in E. coli expressing AR3 in the absence and presence of a negative transmembrane potential. The retinal chromophore structure of AR3 is in an all-trans configuration almost identical to BR over the entire pH range from 3–11. Small changes are detected in the retinal ethylenic stretching frequency and Schiff Base (SB) hydrogen bonding strength relative to BR which may be related to a different water structure near the SB. In the case of the AR3 mutant D95N, at neutral pH an all-trans retinal O-like species (Oall-trans) is found. At higher pH a second 13-cis retinal N-like species (N13-cis) is detected which is attributed to a slowly decaying intermediate in the red-light photocycle of D95N. However, the amount of N13-cis detected is less in E. coli cells but is restored upon addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or sonication, both of which dissipate the normal negative membrane potential. We postulate that these changes are due to the effect of membrane potential on the N13-cis to M13-cis levels accumulated in the D95N red-light photocycle and on a molecular level by the effects of the electric field on the protonation/deprotonation of the cytoplasmic accessible SB. This mechanism also provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane potential. PMID:23189985

Mechanisms involved in 3',5'-cyclic adenosine monophosphate-mediated inhibition of the ubiquitin-proteasome system in skeletal muscle.

PubMed

Gonçalves, Dawit A P; Lira, Eduardo C; Baviera, Amanda M; Cao, Peirang; Zanon, Neusa M; Arany, Zoltan; Bedard, Nathalie; Tanksale, Preeti; Wing, Simon S; Lecker, Stewart H; Kettelhut, Isis C; Navegantes, Luiz C C

2009-12-01

Although it is well known that catecholamines inhibit skeletal muscle protein degradation, the molecular underlying mechanism remains unclear. This study was undertaken to investigate the role of beta(2)-adrenoceptors (AR) and cAMP in regulating the ubiquitin-proteasome system (UPS) in skeletal muscle. We report that increased levels of cAMP in isolated muscles, promoted by the cAMP phosphodiesterase inhibitor isobutylmethylxanthine was accompanied by decreased activity of the UPS, levels of ubiquitin-protein conjugates, and expression of atrogin-1, a key ubiquitin-protein ligase involved in muscle atrophy. In cultured myotubes, atrogin-1 induction after dexamethasone treatment was completely prevented by isobutylmethylxanthine. Furthermore, administration of clenbuterol, a selective beta(2)-agonist, to mice increased muscle cAMP levels and suppressed the fasting-induced expression of atrogin-1 and MuRF-1, atrogin-1 mRNA being much more responsive to clenbuterol. Moreover, clenbuterol increased the phosphorylation of muscle Akt and Foxo3a in fasted rats. Similar responses were observed in muscles exposed to dibutyryl-cAMP. The stimulatory effect of clenbuterol on cAMP and Akt was abolished in muscles from beta(2)-AR knockout mice. The suppressive effect of beta(2)-agonist on atrogin-1 was not mediated by PGC-1alpha (peroxisome proliferator-activated receptor-gamma coactivator 1alpha known to be induced by beta(2)-agonists and previously shown to inhibit atrogin-1 expression), because food-deprived PGC-1alpha knockout mice were still sensitive to clenbuterol. These findings suggest that the cAMP increase induced by stimulation of beta(2)-AR in skeletal muscles from fasted mice is possibly the mechanism by which catecholamines suppress atrogin-1 and the UPS, this effect being mediated via phosphorylation of Akt and thus inactivation of Foxo3.

β2 Adrenoceptors are underexpressed in peripheral blood mononuclear cells and associated with a better metabolic profile in central obesity.

PubMed

Leite, Fernanda; Lima, Margarida; Marino, Franca; Cosentino, Marco; Ribeiro, Laura

2017-01-01

Background: Central obesity (CO) is an inflammatory disease. Because immune cells and adipocytes are catecholamines(CA)-producing cells, we studied the expression of adrenoceptors (AR) in peripheral blood mononuclear cells (PBMCs) hypothesizing a distinct adrenergic pattern in inflammatory obesity. Methods: AR expression was assessed in blood donors categorized by waist circumference (WC) (CO: WC≥0.80 m in women and ≥0.94 m in men). Following a pilot study for all AR subtypes, we measured β 2 AR expression in fifty-seven individuals and correlated this result with anthropometric, metabolic and inflammatory parameters. A ratio (R) between AR mRNA of CO and non-CO<0.5 was considered under and >2.0 over expression. Results: The pilot study revealed no differences between groups, except for β 2 AR mRNA. CO individuals showed underexpression of β 2 AR relatively to those without CO (R=0.08; p=0.009). β 2 AR expression inversely correlated with triacylglycerol (r=-0.271; p=0.041), very low-density lipoprotein-cholesterol (r=-0.313; p=0.018) and leptin (r=-0.392; p=0.012) and positively with high-density lipoprotein-cholesterol (r=0.310: p=0.045) plasma levels. Multiple logistic regression analysis showed a protective effect of β 2 AR expression (≥2x10-6) [odds ratio (OR) 0.177 with respective confidence interval of 95% (95% CI) (0.040- 0.796)] for the occurrence of CO. A higher association was found for women as compared to men (Ξ9:1) [OR 8.972 (95% CI) (1.679-47.949)]. Conclusion: PBMCs β 2 AR, underexpressed in centrally obese, are associated with a better metabolic profile and showed a protective role for the development of CO. The discovery of β 2 AR as a new molecular marker of obesity subphenotypes in PBMCs might contribute to clarify the adrenergic immunomodulation of inflammatory obesity.

β2 Adrenoceptors are underexpressed in peripheral blood mononuclear cells and associated with a better metabolic profile in central obesity

PubMed Central

Leite, Fernanda; Lima, Margarida; Marino, Franca; Cosentino, Marco; Ribeiro, Laura

2017-01-01

Background: Central obesity (CO) is an inflammatory disease. Because immune cells and adipocytes are catecholamines(CA)-producing cells, we studied the expression of adrenoceptors (AR) in peripheral blood mononuclear cells (PBMCs) hypothesizing a distinct adrenergic pattern in inflammatory obesity. Methods: AR expression was assessed in blood donors categorized by waist circumference (WC) (CO: WC≥0.80 m in women and ≥0.94 m in men). Following a pilot study for all AR subtypes, we measured β2AR expression in fifty-seven individuals and correlated this result with anthropometric, metabolic and inflammatory parameters. A ratio (R) between AR mRNA of CO and non-CO<0.5 was considered under and >2.0 over expression. Results: The pilot study revealed no differences between groups, except for β2AR mRNA. CO individuals showed underexpression of β2AR relatively to those without CO (R=0.08; p=0.009). β2AR expression inversely correlated with triacylglycerol (r=-0.271; p=0.041), very low-density lipoprotein-cholesterol (r=-0.313; p=0.018) and leptin (r=-0.392; p=0.012) and positively with high-density lipoprotein-cholesterol (r=0.310: p=0.045) plasma levels. Multiple logistic regression analysis showed a protective effect of β2AR expression (≥2x10-6) [odds ratio (OR) 0.177 with respective confidence interval of 95% (95% CI) (0.040- 0.796)] for the occurrence of CO. A higher association was found for women as compared to men (Ξ9:1) [OR 8.972 (95% CI) (1.679-47.949)]. Conclusion: PBMCs β2AR, underexpressed in centrally obese, are associated with a better metabolic profile and showed a protective role for the development of CO. The discovery of β2AR as a new molecular marker of obesity subphenotypes in PBMCs might contribute to clarify the adrenergic immunomodulation of inflammatory obesity. PMID:28824322

Transcription Profiling of Bacillus subtilis Cells Infected with AR9, a Giant Phage Encoding Two Multisubunit RNA Polymerases.

PubMed

Lavysh, Daria; Sokolova, Maria; Slashcheva, Marina; Förstner, Konrad U; Severinov, Konstantin

2017-02-14

Bacteriophage AR9 is a recently sequenced jumbo phage that encodes two multisubunit RNA polymerases. Here we investigated the AR9 transcription strategy and the effect of AR9 infection on the transcription of its host, Bacillus subtilis Analysis of whole-genome transcription revealed early, late, and continuously expressed AR9 genes. Alignment of sequences upstream of the 5' ends of AR9 transcripts revealed consensus sequences that define early and late phage promoters. Continuously expressed AR9 genes have both early and late promoters in front of them. Early AR9 transcription is independent of protein synthesis and must be determined by virion RNA polymerase injected together with viral DNA. During infection, the overall amount of host mRNAs is significantly decreased. Analysis of relative amounts of host transcripts revealed notable differences in the levels of some mRNAs. The physiological significance of up- or downregulation of host genes for AR9 phage infection remains to be established. AR9 infection is significantly affected by rifampin, an inhibitor of host RNA polymerase transcription. The effect is likely caused by the antibiotic-induced killing of host cells, while phage genome transcription is solely performed by viral RNA polymerases. IMPORTANCE Phages regulate the timing of the expression of their own genes to coordinate processes in the infected cell and maximize the release of viral progeny. Phages also alter the levels of host transcripts. Here we present the results of a temporal analysis of the host and viral transcriptomes of Bacillus subtilis infected with a giant phage, AR9. We identify viral promoters recognized by two virus-encoded RNA polymerases that are a unique feature of the phiKZ-related group of phages to which AR9 belongs. Our results set the stage for future analyses of highly unusual RNA polymerases encoded by AR9 and other phiKZ-related phages. Copyright © 2017 Lavysh et al.

Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

NASA Astrophysics Data System (ADS)

Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

2014-01-01

We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

HSD3B2, HSD17B1, HSD17B2, ESR1, ESR2 and AR expression in infertile women with endometriosis.

PubMed

Osiński, Maciej; Wirstlein, Przemysław; Wender-Ożegowska, Ewa; Mikołajczyk, Mateusz; Jagodziński, Paweł Piotr; Szczepańska, Małgorzata

2018-01-01

The development of endometriosis is associated with changes in the expression of genes encoding the 3β-hydroxysteroid dehydrogenase type II (HSD3B2) and 17β-hydroxysteroid dehydrogenase type II (HSD17B2), estrogen receptors 1 (ESR1) and 2 (ESR2) and the androgen receptor (AR). However, little is known about the expression of HSD3B2, HSD17B1, HSD17B2, ESR1 ESR2 and AR during the endometrial phases in eutopic endometrium from infertile women with endometriosis. Using RT-qPCR analysis, we assessed the expression of the studied genes in the follicular and luteal phases in eutopic endometrium from fertile women (n = 17) and infertile women (n = 35) with endometriosis. In the mid-follicular eutopic endometrium, we observed a significant increase in HSD3B2 transcript levels in all infertile women with endometriosis (p = 0.003), in infertile women with stage I/II endometriosis (p = 0.008) and in infertile women with stage III/IV endometriosis (p = 0.009) compared to all fertile women. There was a significant increase in ESR1 tran-scripts in all infertile women with endometriosis (p = 0.008) and in infertile women with stage I/II endometriosis (p = 0.019) and in infertile women with stage III/IV endometriosis (p = 0.023) compared to all fertile women. In the mid-luteal eutopic endometrium, we did not observe significant differences in HSD3B2, HSD17B1, HSD17B2, ESR1, ESR2 and AR transcripts between infertile women with endometriosis and fertile women. Observed significant increase in HSD3B2 and ESR1 transcripts in follicular eutopic endometrium from infer-tile women with endometriosis may be related to abnormal biological effect of E2 in endometrium, further affecting the development of human embryos.

Chronic stress enhances progression of periodontitis via α1-adrenergic signaling: a potential target for periodontal disease therapy.

PubMed

Lu, Huaixiu; Xu, Minguang; Wang, Feng; Liu, Shisen; Gu, Jing; Lin, Songshan

2014-10-17

This study assessed the roles of chronic stress (CS) in the stimulation of the sympathetic nervous system and explored the underlying mechanisms of periodontitis. Using an animal model of periodontitis and CS, the expression of tyrosine hydroxylase (TH) and the protein levels of the α1-adrenergic receptor (α1-AR) and β2-adrenergic receptor (β2-AR) were assessed. Furthermore, human periodontal ligament fibroblasts (HPDLFs) were stimulated with lipopolysaccharide (LPS) to mimic the process of inflammation. The proliferation of the HPDLFs and the expression of α1-AR and β2-AR were assessed. The inflammatory-related cytokines interleukin (IL)-1β, IL-6 and IL-8 were detected after pretreatment with the α1/β2-AR blockers phentolamine/propranolol, both in vitro and in vivo. Results show that periodontitis under CS conditions enhanced the expression of TH, α1-AR and β2-AR. Phentolamine significantly reduced the inflammatory cytokine levels. Furthermore, we observed a marked decrease in HPDLF proliferation and the increased expression of α1-ARfollowing LPS pretreatment. Pretreatment with phentolamine dramatically ameliorated LPS-inhibited cell proliferation. In addition, the blocking of α1-ARsignaling also hindered the upregulation of the inflammatory-related cytokines IL-1β, IL-6 and IL-8. These results suggest that CS can significantly enhance the pathological progression of periodontitis by an α1-adrenergic signaling-mediated inflammatory response. We have identified a potential therapeutic target for the treatment of periodontal disease, particularly in those patients suffering from concurrent CS.

High levels of the AR-V7 Splice Variant and Co-Amplification of the Golgi Protein Coding YIPF6 in AR Amplified Prostate Cancer Bone Metastases.

PubMed

Djusberg, Erik; Jernberg, Emma; Thysell, Elin; Golovleva, Irina; Lundberg, Pia; Crnalic, Sead; Widmark, Anders; Bergh, Anders; Brattsand, Maria; Wikström, Pernilla

2017-05-01

The relation between androgen receptor (AR) gene amplification and other mechanisms behind castration-resistant prostate cancer (CRPC), such as expression of constitutively active AR variants and steroid-converting enzymes has been poorly examined. Specific aim was to examine AR amplification in PC bone metastases and to explore molecular and functional consequences of this, with the long-term goal of identifying novel molecular targets for treatment. Gene amplification was assessed by fluorescence in situ hybridization in cryo-sections of clinical PC bone metastases (n = 40) and by PCR-based copy number variation analysis. Whole genome mRNA expression was analyzed using H12 Illumina Beadchip arrays and specific transcript levels were quantified by qRT-PCR. Protein localization was analyzed using immunohistochemistry and confocal microscopy. The YIPF6 mRNA expression was transiently knocked down and stably overexpressed in the 22Rv1 cell line as representative for CRPC, and effects on cell proliferation, colony formation, migration, and invasion were determined in vitro. Extracellular vesicles (EVs) were isolated from cell cultures using size-exclusion chromatography and enumerated by nanoparticle tracking analysis. Protein content was identified by LC-MS/MS analysis. Blood coagulation was measured as activated partial thromboplastin time (APTT). Functional enrichment analysis was performed using the MetaCore software. AR amplification was detected in 16 (53%) of the bone metastases examined from CRPC patients (n = 30), and in none from the untreated patients (n = 10). Metastases with AR amplification showed high AR and AR-V7 mRNA levels, increased nuclear AR immunostaining, and co-amplification of genes such as YIPF6 in the AR proximity at Xq12. The YIPF6 protein was localized to the Golgi apparatus. YIPF6 overexpression in 22Rv1 cells resulted in reduced cell proliferation and colony formation, and in enhanced EV secretion. EVs from YIPF6 overproducing 22Rv1 cells were enriched for proteins involved in blood coagulation and, accordingly, decreased the APTT in a dose-dependent fashion. AR amplified CRPC bone metastases show high AR-V7 expression that probably gives resistance to AR-targeting drugs. Co-amplification of the Golgi protein coding YIPF6 gene with the AR may enhance the secretion of pro-coagulative EVs from cancer cells and thereby stimulate tumor progression and increase the coagulopathy risk in CRPC patients. Prostate 77: 625-638, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Enhanced tubuloglomerular feedback in mice with vascular overexpression of A1 adenosine receptors

PubMed Central

Oppermann, Mona; Qin, Yan; Lai, En Yin; Eisner, Christoph; Li, Lingli; Huang, Yuning; Mizel, Diane; Fryc, Justyna; Wilcox, Christopher S.; Briggs, Josephine; Schnermann, Jurgen

2009-01-01

Adenosine 1 receptors (A1AR) in the kidney are expressed in the vasculature and the tubular system. Pharmacological inhibition or global genetic deletion of A1AR causes marked reductions or abolishment of tubuloglomerular feedback (TGF) responses. To assess the function of vascular A1AR in TGF, we generated transgenic mouse lines in which A1AR expression in smooth muscle was augmented by placing A1AR under the control of a 5.38-kb fragment of the rat smooth muscle α-actin promoter and first intron (12). Two founder lines with highest expression in the kidney [353 ± 42 and 575 ± 43% compared with the wild type (WT)] were used in the experiments. Enhanced expression of A1AR at the expected site in these lines was confirmed by augmented constrictor responses of isolated afferent arterioles to administration of the A1AR agonist N6-cyclohexyladenosine. Maximum TGF responses (0–30 nl/min flow step) were increased from 8.4 ± 0.9 mmHg in WT (n = 21) to 14.2 ± 0.7 mmHg in A1AR-transgene (tg) 4 (n = 22; P < 0.0001), and to 12.6 ± 1.2 mmHg in A1AR-tg7 (n = 12; P < 0.02). Stepwise changes in perfusion flow caused greater numerical TGF responses in A1AR-tg than WT in all flow ranges with differences reaching levels of significance in the intermediate flow ranges of 7.5–10 and 10–15 nl/min. Proximal-distal single-nephron glomerular filtration rate (SNGFR) differences (free-flow micropuncture) were also increased in A1AR-tg, averaging 6.25 ± 1.5 nl/min compared with 2.6 ± 0.51 nl/min in WT (P = 0.034). Basal plasma renin concentrations as well as the suppression of renin secretion after volume expansion were similar in A1AR-tg and WT mice, suggesting lack of transgene expression in juxtaglomerular cells. These data indicate that A1AR expression in vascular smooth muscle cells is a critical component for TGF signaling and that changes in renal vascular A1AR expression may determine the magnitude of TGF responses. PMID:19741017

Androgen-androgen receptor system improves chronic inflammatory conditions by suppressing monocyte chemoattractant protein-1 gene expression in adipocytes via transcriptional regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morooka, Nobukatsu, E-mail: amorooka@gunma-u.ac.jp; Ueguri, Kei; Yee, Karen Kar Lye

Age-related decreases in sex hormones are closely related to chronic inflammation in obesity and metabolic diseases. Particularly, the molecular basis of androgen activity in regulating inflammation and controlling metabolism remains largely unknown. Obese adipocytes secrete monocyte chemoattractant protein-1 (MCP-1), a key chemokine that promotes the infiltration of monocytes/macrophages into adipose tissue, thereby leading to metabolic disorders. Here, we studied the role of androgen-androgen receptor (AR) action in regulating MCP-1 expression in adipose tissue. We observed the induction of Mcp-1 expression in 3T3-L1 adipocytes co-cultured with RAW264.7 macrophages. Additionally, Mcp-1 expression was upregulated by culturing in conditioned medium derived from inflammatorymore » macrophages (M1-Mφ) containing tumor necrosis factor-alpha (TNF-α). We found that sex hormones downregulated TNF-α-induced Mcp-1 and interleukin (Il)-6 expression in 3T3-L1 adipocytes. Furthermore, luciferase-reporter analysis indicated that MCP-1 promoter activity was predominantly suppressed by dihydrotestosterone (DHT)-AR interactions through functional canonical nuclear factor-kappa B (NF-κB) sites, whereas non-canonical NF-κB site containing important flanking sequences exhibited minor contributions to DHT-AR transcriptional repression. These findings suggested that androgen-AR suppressed obesity-induced chronic inflammation in adipose tissue. - Highlights: • DHT, non-aromatizable androgen suppresses Mcp-1 expression in adipocytes. • Mcp-1 transcription was negatively regulated by DHT-AR action. • DHT-AR selectively regulates Mcp-1 transcription through distinct NF-κB sites.« less

Benzo[a]pyrene Reduces Testosterone Production in Rat Leydig Cells via a Direct Disturbance of Testicular Steroidogenic Machinery

PubMed Central

Chung, Jin-Yong; Lee, Seung Gee; Park, Ji-Eun; Yoon, Yong-Dal; Yoo, Ki Soo; Yoo, Young Hyun

2011-01-01

Background: Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH), is a ubiquitous environmental pollutant that is currently suspected of being an endocrine disruptor. The testis is an important target for PAHs, yet insufficient attention has been paid to their effects on steroidogenesis in Leydig cells. Objective: We hypothesized that long-term exposure to low concentrations of B[a]P might disrupt testosterone production in Leydig cells via an alteration of steroidogenic proteins. Results: Oral exposure to B[a]P reduced serum and intratesticular fluid testosterone levels in rats. However, we did not observe serious testicular atrophy or azoospermia, although spermatogonial apoptosis was significantly increased. Compared with control cells, Leydig cells primed with B[a]P in vivo produced less testosterone in response to human chorionic gonadotropin (hCG) or dibutyl cyclic adenosine monophosphate in vitro. Of note, the reduction of testosterone levels was accompanied by decreased expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD), as well as increased levels of cytochrome P450 side chain cleavage (P450scc), in Leydig cells. The up-regulation of P450scc expression after exposure to B[a]P appears to be associated with a compensatory mechanism for producing the maximum amount of pregnenolone with the minimum amount of transported cholesterol by StAR; the down-regulation of 3β-HSD may occur because B[a]P can negatively target 3β-HSD, which is required for testosterone production. Conclusions: B[a]P exposure can decrease epididymal sperm quality, possibly by disturbing testosterone levels, and StAR may be a major steroidogenic protein that is targeted by B[a]P or other PAHs. PMID:21737371

Gut immune deficits in LEW.1AR1-iddm rats partially overcome by feeding a diabetes-protective diet.

PubMed

Crookshank, Jennifer A; Patrick, Christopher; Wang, Gen-Sheng; Noel, J Ariana; Scott, Fraser W

2015-07-01

The gut immune system and its modification by diet have been implicated in the pathogenesis of type 1 diabetes (T1D). Therefore, we investigated gut immune status in non-diabetes-prone LEW.1AR1 and diabetes-prone LEW.1AR1-iddm rats and evaluated the effect of a low antigen, hydrolysed casein (HC)-based diet on gut immunity and T1D. Rats were weaned onto a cereal-based or HC-based diet and monitored for T1D. Strain and dietary effects on immune homeostasis were assessed in non-diabetic rats (50-60 days old) and rats with recent-onset diabetes using flow cytometry and immunohistochemistry. Immune gene expression was analysed in mesenteric lymph nodes (MLN) and jejunum using quantitative RT-PCR and PCR arrays. T1D was prevented in LEW.1AR1-iddm rats by feeding an HC diet. Diabetic LEW.1AR1-iddm rats had fewer lymphoid tissue T cells compared with LEW.1AR1 rats. The percentage of CD4(+)  Foxp3(+) regulatory T (Treg) cells was decreased in pancreatic lymph nodes (PLN) of diabetic rats. The jejunum of 50-day LEW.1AR1-iddm rats contained fewer CD3(+) T cells, CD163(+) M2 macrophages and Foxp3(+) Treg cells. Ifng expression was increased in MLN and Foxp3 expression was decreased in the jejunum of LEW.1AR1-iddm rats; Ifng/Il4 was decreased in jejunum of LEW.1AR1-iddm rats fed HC. PCR arrays revealed decreased expression of M2-associated macrophage factors in 50-day LEW.1AR1-iddm rats. Wheat peptides stimulated T-cell proliferation and activation in MLN and PLN cells from diabetic LEW.1AR1-iddm rats. LEW.1AR1-iddm rats displayed gut immune cell deficits and decreased immunoregulatory capacity, which were partially corrected in animals fed a low antigen, protective HC diet consistent with other models of T1D. © 2015 John Wiley & Sons Ltd.

A screen for transcription factor targets of glycogen synthase kinase-3 highlights an inverse correlation of NFκB and androgen receptor signaling in prostate cancer.

PubMed

Campa, Victor M; Baltziskueta, Eder; Bengoa-Vergniory, Nora; Gorroño-Etxebarria, Irantzu; Wesołowski, Radosław; Waxman, Jonathan; Kypta, Robert M

2014-09-30

Expression of Glycogen Synthase Kinase-3 (GSK-3) is elevated in prostate cancer and its inhibition reduces prostate cancer cell proliferation, in part by reducing androgen receptor (AR) signaling. However, GSK-3 inhibition can also activate signals that promote cell proliferation and survival, which may preclude the use of GSK-3 inhibitors in the clinic. To identify such signals in prostate cancer, we screened for changes in transcription factor target DNA binding activity in GSK-3-silenced cells. Among the alterations was a reduction in AR DNA target binding, as predicted from previous studies, and an increase in NFκB DNA target binding. Consistent with the latter, gene silencing of GSK-3 or inhibition using the GSK-3 inhibitor CHIR99021 increased basal NFκB transcriptional activity. Activation of NFκB was accompanied by an increase in the level of the NFκB family member RelB. Conversely, silencing RelB reduced activation of NFκB by CHIR99021. Furthermore, the reduction of prostate cancer cell proliferation by CHIR99021 was potentiated by inhibition of NFκB signaling using the IKK inhibitor PS1145. Finally, stratification of human prostate tumor gene expression data for GSK3 revealed an inverse correlation between NFκB-dependent and androgen-dependent gene expression, consistent with the results from the transcription factor target DNA binding screen. In addition, there was a correlation between expression of androgen-repressed NFκB target genes and reduced survival of patients with metastatic prostate cancer. These findings highlight an association between GSK-3/AR and NFκB signaling and its potential clinical importance in metastatic prostate cancer.

A screen for transcription factor targets of Glycogen Synthase Kinase-3 highlights an inverse correlation of NFκB and Androgen Receptor Signaling in Prostate Cancer

PubMed Central

Campa, Victor M.; Baltziskueta, Eder; Bengoa-Vergniory, Nora; Gorroño-Etxebarria, Irantzu; Wesołowski, Radosław; Waxman, Jonathan; Kypta, Robert M.

2014-01-01

Expression of Glycogen Synthase Kinase-3 (GSK-3) is elevated in prostate cancer and its inhibition reduces prostate cancer cell proliferation, in part by reducing androgen receptor (AR) signaling. However, GSK-3 inhibition can also activate signals that promote cell proliferation and survival, which may preclude the use of GSK-3 inhibitors in the clinic. To identify such signals in prostate cancer, we screened for changes in transcription factor target DNA binding activity in GSK-3-silenced cells. Among the alterations was a reduction in AR DNA target binding, as predicted from previous studies, and an increase in NFκB DNA target binding. Consistent with the latter, gene silencing of GSK-3 or inhibition using the GSK-3 inhibitor CHIR99021 increased basal NFκB transcriptional activity. Activation of NFκB was accompanied by an increase in the level of the NFκB family member RelB. Conversely, silencing RelB reduced activation of NFκB by CHIR99021. Furthermore, the reduction of prostate cancer cell proliferation by CHIR99021 was potentiated by inhibition of NFκB signaling using the IKK inhibitor PS1145. Finally, stratification of human prostate tumor gene expression data for GSK3 revealed an inverse correlation between NFκB-dependent and androgen-dependent gene expression, consistent with the results from the transcription factor target DNA binding screen. In addition, there was a correlation between expression of androgen-repressed NFκB target genes and reduced survival of patients with metastatic prostate cancer. These findings highlight an association between GSK-3/AR and NFκB signaling and its potential clinical importance in metastatic prostate cancer. PMID:25327559

Tadalafil modulates aromatase activity and androgen receptor expression in a human osteoblastic cell in vitro model.

PubMed

Aversa, A; Fittipaldi, S; Bimonte, V M; Wannenes, F; Papa, V; Francomano, D; Greco, E A; Lenzi, A; Migliaccio, S

2016-02-01

Phosphodiesterase type-5 inhibitor (PDE5i) tadalafil administration in men with erectile dysfunction is associated with increased testosterone/estradiol ratio, leading to hypothesize a potential increased effect of androgen action on target tissues. We aimed to characterize, in a cellular model system in vitro, the potential modulation of aromatase and sex steroid hormone receptors upon exposure to tadalafil (TAD). Human osteoblast-like cells SAOS-2 were chosen as an in vitro model system since osteoblasts are target of steroid hormones. Cells were tested for viability upon TAD exposure, which increased cell proliferation. Then, cells were treated with/without TAD for several times to evaluate potential modulation in PDE5, aromatase (ARO), androgen (AR) and estrogen (ER) receptor expression. Osteoblasts express significant levels of both PDE5 mRNA and protein. Exposure of cells to increasing concentrations of TAD (10(-8)-10(-7) M) decreased PDE5 mRNA and protein expression. Also, TAD inhibited ARO mRNA and protein expression leading to an increase in testosterone levels in the supernatants. Interestingly, TAD increased total AR mRNA and protein expression and decreased ERα, with an increased ratio of AR/ER, suggesting preferential androgenic vs estrogenic pathway activation. Our results demonstrate for the first time that TAD decreases ARO expression and increases AR protein expression in human SAOS-2, strongly suggesting a new control of steroid hormones pathway by PDE5i. These findings might represent the first evidence of translational actions of PDE5i on AR, which leads to hypothesize a growing relevance of this molecule in men with prostate cancer long-term treated with TAD for sexual rehabilitation.

Genome-Wide Analysis of Androgen Receptor Targets Reveals COUP-TF1 as a Novel Player in Human Prostate Cancer

PubMed Central

Perets, Ruth; Kaplan, Tommy; Stein, Ilan; Hidas, Guy; Tayeb, Shay; Avraham, Eti; Ben-Neriah, Yinon; Simon, Itamar; Pikarsky, Eli

2012-01-01

Androgen activity plays a key role in prostate cancer progression. Androgen receptor (AR) is the main mediator of androgen activity in the prostate, through its ability to act as a transcription mediator. Here we performed a genome-wide analysis of human AR binding to promoters in the presence of an agonist or antagonist in an androgen dependent prostate cancer cell line. Many of the AR bound promoters are bound in all examined conditions while others are bound only in the presence of an agonist or antagonist. Several motifs are enriched in AR bound promoters, including the AR Response Element (ARE) half-site and recognition elements for the transcription factors OCT1 and SOX9. This suggests that these 3 factors could define a module of co-operating transcription factors in the prostate. Interestingly, AR bound promoters are preferentially located in AT rich genomic regions. Analysis of mRNA expression identified chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) as a direct AR target gene that is downregulated upon binding by the agonist liganded AR. COUP-TF1 immunostaining revealed nucleolar localization of COUP-TF1 in epithelium of human androgen dependent prostate cancer, but not in adjacent benign prostate epithelium. Stromal cells both in human and mouse prostate show nuclear COUP-TF1 staining. We further show that there is an inverse correlation between COUP-TF1 expression in prostate stromal cells and the rising levels of androgen with advancing puberty. This study extends the pool of recognized putative AR targets and identifies a negatively regulated target of AR – COUP-TF1 – which could possibly play a role in human prostate cancer. PMID:23056316

Genome-wide analysis of androgen receptor targets reveals COUP-TF1 as a novel player in human prostate cancer.

PubMed

Perets, Ruth; Kaplan, Tommy; Stein, Ilan; Hidas, Guy; Tayeb, Shay; Avraham, Eti; Ben-Neriah, Yinon; Simon, Itamar; Pikarsky, Eli

2012-01-01

Androgen activity plays a key role in prostate cancer progression. Androgen receptor (AR) is the main mediator of androgen activity in the prostate, through its ability to act as a transcription mediator. Here we performed a genome-wide analysis of human AR binding to promoters in the presence of an agonist or antagonist in an androgen dependent prostate cancer cell line. Many of the AR bound promoters are bound in all examined conditions while others are bound only in the presence of an agonist or antagonist. Several motifs are enriched in AR bound promoters, including the AR Response Element (ARE) half-site and recognition elements for the transcription factors OCT1 and SOX9. This suggests that these 3 factors could define a module of co-operating transcription factors in the prostate. Interestingly, AR bound promoters are preferentially located in AT rich genomic regions. Analysis of mRNA expression identified chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) as a direct AR target gene that is downregulated upon binding by the agonist liganded AR. COUP-TF1 immunostaining revealed nucleolar localization of COUP-TF1 in epithelium of human androgen dependent prostate cancer, but not in adjacent benign prostate epithelium. Stromal cells both in human and mouse prostate show nuclear COUP-TF1 staining. We further show that there is an inverse correlation between COUP-TF1 expression in prostate stromal cells and the rising levels of androgen with advancing puberty. This study extends the pool of recognized putative AR targets and identifies a negatively regulated target of AR - COUP-TF1 - which could possibly play a role in human prostate cancer.

Transcriptional role of androgen receptor in the expression of long non-coding RNA Sox2OT in neurogenesis

PubMed Central

Tosetti, Valentina; Sassone, Jenny; Ferri, Anna L. M.; Taiana, Michela; Bedini, Gloria; Nava, Sara; Brenna, Greta; Di Resta, Chiara; Pareyson, Davide; Di Giulio, Anna Maria; Carelli, Stephana

2017-01-01

The complex architecture of adult brain derives from tightly regulated migration and differentiation of precursor cells generated during embryonic neurogenesis. Changes at transcriptional level of genes that regulate migration and differentiation may lead to neurodevelopmental disorders. Androgen receptor (AR) is a transcription factor that is already expressed during early embryonic days. However, AR role in the regulation of gene expression at early embryonic stage is yet to be determinate. Long non-coding RNA (lncRNA) Sox2 overlapping transcript (Sox2OT) plays a crucial role in gene expression control during development but its transcriptional regulation is still to be clearly defined. Here, using Bicalutamide in order to pharmacologically inactivated AR, we investigated whether AR participates in the regulation of the transcription of the lncRNASox2OTat early embryonic stage. We identified a new DNA binding region upstream of Sox2 locus containing three androgen response elements (ARE), and found that AR binds such a sequence in embryonic neural stem cells and in mouse embryonic brain. Our data suggest that through this binding, AR can promote the RNA polymerase II dependent transcription of Sox2OT. Our findings also suggest that AR participates in embryonic neurogenesis through transcriptional control of the long non-coding RNA Sox2OT. PMID:28704421

Transcriptional role of androgen receptor in the expression of long non-coding RNA Sox2OT in neurogenesis.

PubMed

Tosetti, Valentina; Sassone, Jenny; Ferri, Anna L M; Taiana, Michela; Bedini, Gloria; Nava, Sara; Brenna, Greta; Di Resta, Chiara; Pareyson, Davide; Di Giulio, Anna Maria; Carelli, Stephana; Parati, Eugenio A; Gorio, Alfredo

2017-01-01

The complex architecture of adult brain derives from tightly regulated migration and differentiation of precursor cells generated during embryonic neurogenesis. Changes at transcriptional level of genes that regulate migration and differentiation may lead to neurodevelopmental disorders. Androgen receptor (AR) is a transcription factor that is already expressed during early embryonic days. However, AR role in the regulation of gene expression at early embryonic stage is yet to be determinate. Long non-coding RNA (lncRNA) Sox2 overlapping transcript (Sox2OT) plays a crucial role in gene expression control during development but its transcriptional regulation is still to be clearly defined. Here, using Bicalutamide in order to pharmacologically inactivated AR, we investigated whether AR participates in the regulation of the transcription of the lncRNASox2OTat early embryonic stage. We identified a new DNA binding region upstream of Sox2 locus containing three androgen response elements (ARE), and found that AR binds such a sequence in embryonic neural stem cells and in mouse embryonic brain. Our data suggest that through this binding, AR can promote the RNA polymerase II dependent transcription of Sox2OT. Our findings also suggest that AR participates in embryonic neurogenesis through transcriptional control of the long non-coding RNA Sox2OT.

Effects of maternal dietary selenium (Se-enriched yeast) on testis development, testosterone level and testicular steroidogenesis-related gene expression of their male kids in Taihang Black Goats.

PubMed

Shi, Lei; Song, Ruigao; Yao, Xiaolei; Duan, Yunli; Ren, Youshe; Zhang, Chunxiang; Yue, Wenbin; Lei, Fulin

2018-07-01

To investigate the effects of maternal dietary selenium (Se-enriched yeast) on testis development, testosterone level and steroidogenesis-related gene expression in testis of their male kids, selected pregnant Taihang Black Goats were randomly allotted to four treatment groups. They were fed the basal gestation and lactation diets supplemented with 0 (control), 0.5, 2.0 and 4.0 mg of Se/kg DM. Thirty days after weaning, testes were collected from the kids. After the morphological development status of testis was examined, tissue samples were collected for analyzing testosterone concentration and histological parameters. Testosterone synthesis-related genes were detected using real-time PCR. Localization and quantification of androgen receptor (AR) in testis of goats were determined by immunohistochemical and western blot analysis. The results show that Se supplementation in the diet of dams led to higher (p < 0.05) testicular weight, volume, length, width, transverse and vertical grith of their male kids. Excessive Se (4.0 mg/kg) can inhibit the development of testis by decreasing testicular weight and volume. The density of spermatogenic cells and Leydig cells in the Se treatment groups was significantly (p < 0.05) higher than that in the control. Maternal dietary Se did not affect the thickness of testes, thickness of germinal epithelium and diameter of seminiferous tubule. Se supplemented in the diet of dams improved the testosterone level in testis tissue and serum, and promote the expression of testosterone-related genes. The mRNA expression of StAR, 3β-HSD and CYP11A1 was decreased with the increasing dietary Se levels of dams. Maternal dietary Se can improve the AR protein abundance in testis of their offspring. AR immunopositive product was detected in Leydig cells, peritubular myoid cells, perivascular smooth muscle cells, primary spermatocytes and spermatids. The expression of AR in spermatogenetic cells is stage specific. This study suggests that maternal dietary Se can influence the testis development and spermatogenesis of their male kids by modulating testosterone synthesis in goats. More attention should be given to the potential role of maternal nutrition in improving reproductive performance of their offspring. Copyright © 2018 Elsevier Inc. All rights reserved.

CD147 modulates androgen receptor activity through the Akt/Gsk-3β/β-catenin/AR pathway in prostate cancer cells.

PubMed

Fang, Fang; Qin, Yingxin; Hao, Feng; Li, Qiang; Zhang, Wei; Zhao, Chen; Chen, Shuang; Zhao, Liangzhong; Wang, Liguo; Cai, Jianhui

2016-08-01

The androgen signaling pathway serves an important role in the development of prostate cancer. β-Catenin is an androgen receptor (AR) cofactor and augments AR signaling. Glycogen synthase kinase-3β (GSK-3β), a target of phosphorylated serine/threonine protein kinase B (p-Akt), regulates β-catenin stability. In addition, β-catenin, a coregulator of AR, physically interacts with AR and enhances AR-mediated target gene transcription. The multifunctional glycoprotein cluster of differentiation (CD) 147 is highly expressed on the cell surface of the majority of cancer cells, and it promotes tumor invasion, metastasis and growth. In the present study, the molecular effects of CD147 on the Akt/GSK-3β/β-catenin/AR signaling network were investigated in LNCaP cells. Using short hairpin-mediated RNA knockdown of CD147 in LNCaP cells, it was demonstrated that downregulation of CD147 resulted in inhibitory phosphorylation of GSK-3β, and then promoted degeneration of β-catenin and reduced nuclear accumulation of β-catenin. In addition, immunoprecipitation studies demonstrated that CD147 downregulation decreased the formation of a complex between β-catenin and AR. It was shown that CD147 knockdown suppressed the expression of the AR target gene prostate-specific antigen and the growth of AR-positive LNCaP cells. Furthermore, inhibition of PI3K/Akt with LY294002 augmented CD147-mediated function. The present study indicates that the PI3K/Akt pathway may facilitate CD147-mediated activation of the AR pathway.

The Suppressor of AAC2 Lethality SAL1 Modulates Sensitivity of Heterologously Expressed Artemia ADP/ATP Carrier to Bongkrekate in Yeast

PubMed Central

Wysocka-Kapcinska, Monika; Torocsik, Beata; Turiak, Lilla; Tsaprailis, George; David, Cynthia L.; Hunt, Andrea M.; Vekey, Karoly; Adam-Vizi, Vera; Kucharczyk, Roza; Chinopoulos, Christos

2013-01-01

The ADP/ATP carrier protein (AAC) expressed in Artemia franciscana is refractory to bongkrekate. We generated two strains of Saccharomyces cerevisiae where AAC1 and AAC3 were inactivated and the AAC2 isoform was replaced with Artemia AAC containing a hemagglutinin tag (ArAAC-HA). In one of the strains the suppressor of ΔAAC2 lethality, SAL1, was also inactivated but a plasmid coding for yeast AAC2 was included, because the ArAACΔsal1Δ strain was lethal. In both strains ArAAC-HA was expressed and correctly localized to the mitochondria. Peptide sequencing of ArAAC expressed in Artemia and that expressed in the modified yeasts revealed identical amino acid sequences. The isolated mitochondria from both modified strains developed 85% of the membrane potential attained by mitochondria of control strains, and addition of ADP yielded bongkrekate-sensitive depolarizations implying acquired sensitivity of ArAAC-mediated adenine nucleotide exchange to this poison, independent from SAL1. However, growth of ArAAC-expressing yeasts in glycerol-containing media was arrested by bongkrekate only in the presence of SAL1. We conclude that the mitochondrial environment of yeasts relying on respiratory growth conferred sensitivity of ArAAC to bongkrekate in a SAL1-dependent manner. PMID:24073201

Lack of α2C-Adrenoceptor Results in Contrasting Phenotypes of Long Bones and Vertebra and Prevents the Thyrotoxicosis-Induced Osteopenia

PubMed Central

Cruz Grecco Teixeira, Marilia Bianca; Martins, Gisele Miyamura; Miranda-Rodrigues, Manuela; De Araújo, Iasmin Ferreira; Oliveira, Ricardo; Brum, Patrícia Chakur; Azevedo Gouveia, Cecilia Helena

2016-01-01

A series of studies have demonstrated that activation of the sympathetic nervous system (SNS) causes osteopenia via β2-adrenoceptor (β2-AR) signaling. However, in a recent study, we found an unexpected and generalized phenotype of high bone mass in female mice with chronic sympathetic hyperactivity, due to double gene inactivation of adrenoceptors that negatively regulate norepinephrine release, α2A-and α2C-AR (α2A/2C-AR-/-). These findings suggest that β2-AR is not the single adrenoceptor involved in bone turnover regulation and show that α2-AR signaling may also mediate the SNS actions in the skeleton. In addition, we found that α2A/2C-AR-/- animals are resistant to the thyrotoxicosis-induced osteopenia, suggesting that thyroid hormone (TH), when in supraphysiological levels, interacts with the SNS to control bone mass and structure, and that this interaction may also involve α2-AR signaling. In the present study, to further investigate these hypotheses and to discriminate the roles of α2-AR subtypes, we have evaluated the bone phenotype of mice with the single gene inactivation of α2C-AR subtype, which mRNA expression was previously shown to be down regulated by triiodothyronine (T3). A cohort of 30 day-old female α2CAR-/- mice and their wild-type (WT) controls were treated with a supraphysiological dose of T3 for 30 or 90 days, which induced a thyrotoxic state in both mouse lineages. The micro-computed tomographic (μCT) analysis showed that α2C-AR-/- mice present lower trabecular bone volume (BV/TV) and number (Tb.N), and increased trabecular separation (Tb.Sp) in the femur compared with WT mice; which was accompanied by decreased bone strength (determined by the three-point bending test) in the femur and tibia. The opposite was observed in the vertebra, where α2C-AR-/- mice show increased BV/TV, Tb.N and trabecular thickness (Tb.Th), and decreased Tb.Sp, compared with WT animals. In spite of the contrasting bone phenotypes of the femur and L5, thyrotoxicosis negatively regulated most of the micro architectural features of the trabecular bone in both skeletal sites of WT, but not of α2C-AR-/- mice. T3 treatment also decreased biomechanical properties (maximum load and ultimate load) in the femur and tibia of WT, but not of knockout mice. The mRNA expression of osteocalcin, a marker of mature osteoblasts, and tartrate-resistant acid phosphatase, which is expressed by osteoclasts and is involved in collagen degradation, was increased by T3 treatment only in WT, and not in α2C-AR-/- mice. Altogether, these findings suggest that α2C-AR subtype mediates the effects of the SNS in the bone in a skeletal site-dependent manner, and that thyrotoxicosis depends on α2C-AR signaling to promote bone loss, which sustains the hypothesis of a TH-SNS interaction to modulate bone remodeling and structure. PMID:26815679

Lack of α2C-Adrenoceptor Results in Contrasting Phenotypes of Long Bones and Vertebra and Prevents the Thyrotoxicosis-Induced Osteopenia.

PubMed

Cruz Grecco Teixeira, Marilia Bianca; Martins, Gisele Miyamura; Miranda-Rodrigues, Manuela; De Araújo, Iasmin Ferreira; Oliveira, Ricardo; Brum, Patrícia Chakur; Azevedo Gouveia, Cecilia Helena

2016-01-01

A series of studies have demonstrated that activation of the sympathetic nervous system (SNS) causes osteopenia via β2-adrenoceptor (β2-AR) signaling. However, in a recent study, we found an unexpected and generalized phenotype of high bone mass in female mice with chronic sympathetic hyperactivity, due to double gene inactivation of adrenoceptors that negatively regulate norepinephrine release, α2A-and α2C-AR (α2A/2C-AR-/-). These findings suggest that β2-AR is not the single adrenoceptor involved in bone turnover regulation and show that α2-AR signaling may also mediate the SNS actions in the skeleton. In addition, we found that α2A/2C-AR-/- animals are resistant to the thyrotoxicosis-induced osteopenia, suggesting that thyroid hormone (TH), when in supraphysiological levels, interacts with the SNS to control bone mass and structure, and that this interaction may also involve α2-AR signaling. In the present study, to further investigate these hypotheses and to discriminate the roles of α2-AR subtypes, we have evaluated the bone phenotype of mice with the single gene inactivation of α2C-AR subtype, which mRNA expression was previously shown to be down regulated by triiodothyronine (T3). A cohort of 30 day-old female α2CAR-/- mice and their wild-type (WT) controls were treated with a supraphysiological dose of T3 for 30 or 90 days, which induced a thyrotoxic state in both mouse lineages. The micro-computed tomographic (μCT) analysis showed that α2C-AR-/- mice present lower trabecular bone volume (BV/TV) and number (Tb.N), and increased trabecular separation (Tb.Sp) in the femur compared with WT mice; which was accompanied by decreased bone strength (determined by the three-point bending test) in the femur and tibia. The opposite was observed in the vertebra, where α2C-AR-/- mice show increased BV/TV, Tb.N and trabecular thickness (Tb.Th), and decreased Tb.Sp, compared with WT animals. In spite of the contrasting bone phenotypes of the femur and L5, thyrotoxicosis negatively regulated most of the micro architectural features of the trabecular bone in both skeletal sites of WT, but not of α2C-AR-/- mice. T3 treatment also decreased biomechanical properties (maximum load and ultimate load) in the femur and tibia of WT, but not of knockout mice. The mRNA expression of osteocalcin, a marker of mature osteoblasts, and tartrate-resistant acid phosphatase, which is expressed by osteoclasts and is involved in collagen degradation, was increased by T3 treatment only in WT, and not in α2C-AR-/- mice. Altogether, these findings suggest that α2C-AR subtype mediates the effects of the SNS in the bone in a skeletal site-dependent manner, and that thyrotoxicosis depends on α2C-AR signaling to promote bone loss, which sustains the hypothesis of a TH-SNS interaction to modulate bone remodeling and structure.

The Role of Retinal Determination Gene Network (RDGN) in Hormone Signaling Transduction and Prostate Tumorigenesis

DTIC Science & Technology

2012-10-01

support with our hypothesis, expressions of AR co-repressors (48-50), HDAC1, HDAC3 or SirT1 inhibit the ligand-induced AR activation at different...signaling and androgen-dependent growth. We hypothesis that DACH1/Six1/Eya pathway is an endogenous regulator of AR trans- activation and contributes to...mechanism. Inhibitory function of Eya1 on AR transactivation required a phosphates activity and could be enhanced by ectopic expression of co-repressors

Nanoscale organization of {beta}{sub 2}-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vobornik, Dusan; Rouleau, Yanouchka; Haley, Jennifer

2009-04-24

Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize {beta}{sub 2}-adrenergic receptors ({beta}{sub 2}AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the {beta}{sub 2}AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline,more » the size of domains containing the {beta}{sub 2}AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of {beta}{sub 2}AR are observed in the membrane of the HEK293 cells that stably overexpress {beta}{sub 2}AR-GFP and {beta}{sub 2}AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use {beta}{sub 2}AR-Venus fusion proteins as models for {beta}{sub 2}AR function. These observations are critical for designing future cell models and assays based on {beta}{sub 2}AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.« less

Specific beta1-adrenergic receptor silencing with small interfering RNA lowers high blood pressure and improves cardiac function in myocardial ischemia.

PubMed

Arnold, Anne-Sophie; Tang, Yao Liang; Qian, Keping; Shen, Leping; Valencia, Valery; Phillips, Michael Ian; Zhang, Yuan Clare

2007-01-01

Beta-blockers are widely used and effective for treating hypertension, acute myocardial infarction (MI) and heart failure, but they present side-effects mainly due to antagonism of beta2-adrenergic receptor (AR). Currently available beta-blockers are at best selective but not specific for beta1 or beta2-AR. To specifically inhibit the expression of the beta1-AR, we developed a small interfering RNA (siRNA) targeted to beta1-AR. Three different sequences of beta1 siRNA were delivered into C6-2B cells with 90% efficiency. One of the three sequences reduced the level of beta1-AR mRNA by 70%. The siRNA was highly specific for beta1-AR inhibition with no overlap with beta2-AR. To test this in vivo, systemic injection of beta1 siRNA complexed with liposomes resulted in efficient delivery into the heart, lung, kidney and liver, and effectively reduced beta1-AR expression in the heart without altering beta2-AR. beta1 siRNA significantly lowered blood pressure of spontaneously hypertensive rats (SHR) for at least 12 days and reduced cardiac hypertrophy following a single injection. Pretreatment with beta1 siRNA 3 days before induction of MI in Wistar rats significantly improved cardiac function, as demonstrated by dP/dt and electrocardiogram following the MI. The protective mechanism involved reduction of cardiomyocyte apoptosis in the beta1 siRNA-treated hearts. The present study demonstrates the possibility of using siRNA for treating cardiovascular diseases and may represent a novel beta-blocker specific for beta1-AR.

Androgen Receptor Variant AR-V9 Is Coexpressed with AR-V7 in Prostate Cancer Metastases and Predicts Abiraterone Resistance.

PubMed

Kohli, Manish; Ho, Yeung; Hillman, David W; Van Etten, Jamie L; Henzler, Christine; Yang, Rendong; Sperger, Jamie M; Li, Yingming; Tseng, Elizabeth; Hon, Ting; Clark, Tyson; Tan, Winston; Carlson, Rachel E; Wang, Liguo; Sicotte, Hugues; Thai, Ho; Jimenez, Rafael; Huang, Haojie; Vedell, Peter T; Eckloff, Bruce W; Quevedo, Jorge F; Pitot, Henry C; Costello, Brian A; Jen, Jin; Wieben, Eric D; Silverstein, Kevin A T; Lang, Joshua M; Wang, Liewei; Dehm, Scott M

2017-08-15

Purpose: Androgen receptor (AR) variant AR-V7 is a ligand-independent transcription factor that promotes prostate cancer resistance to AR-targeted therapies. Accordingly, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The purpose of this study was to understand whether other AR variants may be coexpressed with AR-V7 and promote resistance to AR-targeted therapies. Experimental Design: We utilized complementary short- and long-read sequencing of intact AR mRNA isoforms to characterize AR expression in CRPC models. Coexpression of AR-V7 and AR-V9 mRNA in CRPC metastases and circulating tumor cells was assessed by RNA-seq and RT-PCR, respectively. Expression of AR-V9 protein in CRPC models was evaluated with polyclonal antisera. Multivariate analysis was performed to test whether AR variant mRNA expression in metastatic tissues was associated with a 12-week progression-free survival endpoint in a prospective clinical trial of 78 CRPC-stage patients initiating therapy with the androgen synthesis inhibitor, abiraterone acetate. Results: AR-V9 was frequently coexpressed with AR-V7. Both AR variant species were found to share a common 3' terminal cryptic exon, which rendered AR-V9 susceptible to experimental manipulations that were previously thought to target AR-V7 uniquely. AR-V9 promoted ligand-independent growth of prostate cancer cells. High AR-V9 mRNA expression in CRPC metastases was predictive of primary resistance to abiraterone acetate (HR = 4.0; 95% confidence interval, 1.31-12.2; P = 0.02). Conclusions: AR-V9 may be an important component of therapeutic resistance in CRPC. Clin Cancer Res; 23(16); 4704-15. ©2017 AACR . ©2017 American Association for Cancer Research.

Adenosine A3 receptor elicits chemoresistance mediated by multiple resistance-associated protein-1 in human glioblastoma stem-like cells.

PubMed

Torres, Angelo; Vargas, Yosselyn; Uribe, Daniel; Jaramillo, Catherine; Gleisner, Alejandra; Salazar-Onfray, Flavio; López, Mercedes N; Melo, Rómulo; Oyarzún, Carlos; San Martín, Rody; Quezada, Claudia

2016-10-11

MRP1 transporter correlates positively with glioma malignancy and the Multiple Drug Resistance (MDR) phenotype in Glioblastoma Multiforme (GBM). Evidence shows that the MRP1 transporter is controlled by the adenosine signalling axis. The aim of this study was to identify the role of adenosine on the MDR phenotype in Glioblastoma Stem-like Cells (GSCs), the cell population responsible for the tumorigenic and chemoresistance capabilities of this tumour. We found that GSCs have increased intrinsic capacity to generate extracellular adenosine, thus controlling MRP1 transporter expression and activity via activation of the adenosine A3 receptor (A3AR). We showed PI3K/Akt and MEK/ERK1/2 signaling pathways downstream A3AR to control MRP1 in GSCs. In vitro pharmacological blockade of A3AR had a chemosensitizing effect, enhancing the actions of antitumour drugs and decreasing cell viability and proliferation of GSCs. In addition, we produced an in vivo xenograft model by subcutaneous inoculation of human GSCs in NOD/SCID-IL2Rg null mice. Pharmacological blockade of A3AR generated a chemosensitizing effect, enhancing the effectiveness of the MRP1 transporter substrate, vincristine, reducing tumour size and the levels of CD44 and Nestin stem cell markers as well as the Ki-67 proliferation indicator. In conclusion, we demonstrated the chemosensitizing effect of A3AR blockade on GSCs.

Bisphenol A down-regulates rate-limiting Cyp11a1 to acutely inhibit steroidogenesis in cultured mouse antral follicles.

PubMed

Peretz, Jackye; Flaws, Jodi A

2013-09-01

Bisphenol A (BPA) is the backbone of polycarbonate plastic products and the epoxy resin lining of aluminum cans. Previous studies have shown that exposure to BPA decreases sex steroid hormone production in mouse antral follicles. The current study tests the hypothesis that BPA first decreases the expression levels of the steroidogenic enzyme cytochrome P450 side-chain cleavage (Cyp11a1) and steroidogenic acute regulatory protein (StAR) in mouse antral follicles, leading to a decrease in sex steroid hormone production in vitro. Further, the current study tests the hypothesis that these effects are acute and reversible after removal of BPA. Exposure to BPA (10μg/mL and 100μg/mL) significantly decreased expression of Cyp11a1 and StAR beginning at 18h and 72h, respectively, compared to controls. Exposure to BPA (10μg/mL and 100μg/mL) significantly decreased progesterone levels beginning at 24h and decreased androstenedione, testosterone, and estradiol levels at 72h and 96h compared to controls. Further, after removing BPA from the culture media at 20h, expression of Cyp11a1 and progesterone levels were restored to control levels by 48h and 72h, respectively. Additionally, expression of StAR and levels of androstenedione, testosterone, and estradiol never decreased compared to controls. These data suggest that BPA acutely decreases expression of Cyp11a1 as early as 18h and this reduction in Cyp11a1 may lead to a decrease in progesterone production by 24h, followed by a decrease in androstenedione, testosterone, and estradiol production and expression of StAR at 72h. Therefore, BPA exposure likely targets Cyp11a1 and steroidogenesis, but these effects are reversible with removal of BPA exposure. Copyright © 2013 Elsevier Inc. All rights reserved.

Inhibitor of p52 NF-κB subunit and androgen receptor (AR) interaction reduces growth of human prostate cancer cells by abrogating nuclear translocation of p52 and phosphorylated ARser81

PubMed Central

Mehraein-Ghomi, Farideh; Church, Dawn R.; Schreiber, Cynthia L.; Weichmann, Ashley M.; Basu, Hirak S.; Wilding, George

2015-01-01

Accumulating evidence shows that androgen receptor (AR) activation and signaling plays a key role in growth and progression in all stages of prostate cancer, even under low androgen levels or in the absence of androgen in the castration-resistant prostate cancer. Sustained activation of AR under androgen-deprived conditions may be due to its interaction with co-activators, such as p52 NF-κB subunit, and/or an increase in its stability by phosphorylation that delays its degradation. Here we identified a specific inhibitor of AR/p52 interaction, AR/p52-02, via a high throughput screen based on the reconstitution of Gaussia Luciferase. We found that AR/p52-02 markedly inhibited growth of both castration-resistant C4-2 (IC50 ∼6 μM) and parental androgen-dependent LNCaP (IC50 ∼4 μM) human prostate cancer cells under low androgen conditions. Growth inhibition was associated with significantly reduced nuclear p52 levels and DNA binding activity, as well as decreased phosphorylation of AR at serine 81, increased AR ubiquitination, and decreased AR transcriptional activity as indicated by decreased prostate-specific antigen (PSA) mRNA levels in both cell lines. AR/p52-02 also caused a reduction in levels of p21WAF/CIP1, which is a direct AR targeted gene in that its expression correlates with androgen stimulation and mitogenic proliferation in prostate cancer under physiologic levels of androgen, likely by disrupting the AR signaling axis. The reduced level of cyclinD1 reported previously for this compound may be due to the reduction in nuclear presence and activity of p52, which directly regulates cyclinD1 expression, as well as the reduction in p21WAF/CIP1, since p21WAF/CIP1 is reported to stabilize nuclear cyclinD1 in prostate cancer. Overall, the data suggest that specifically inhibiting the interaction of AR with p52 and blocking activity of p52 and pARser81 may be an effective means of reducing castration-resistant prostate cancer cell growth. PMID:26622945

Downregulation of aldose reductase is responsible for developmental abnormalities of the silkworm purple quail-like mutant (q-lp).

PubMed

Wang, Pingyang; Bi, Simin; Wei, Weiyang; Qiu, Zhiyong; Xia, Dingguo; Shen, Xingjia; Zhao, Qiaoling

2018-05-03

Aldose reductase (AR) is a rate-limiting enzyme in the polyol pathway and is also the key enzyme involved in diabetic complications. The silkworm purple quail-like mutant (q-l p ) exhibits pigmented dots on its epidermis. The q-l p mutant also shows developmental abnormalities and decreased vitality. In this study, fat bodies from the q-l p mutant and the wildtype 932VR strain were subjected to two-dimensional gel electrophoresis (2-DE) analysis, and the Bombyx mori AR (BmAR) protein was found to be significantly downregulated in the q-l p mutant. The expression of BmAR at the mRNA level was also significantly downregulated, as verified through quantitative reverse transcription PCR (qRT-PCR). Knockdown of the expression of BmAR via RNAi resulted in a reduction of silkworm weight. The sorbitol level in q-l p was significantly lower than in the wildtype. These results suggested that the BmAR gene is closely related to the development of the q-l p mutant. Investigation of the cause of BmAR downregulation in the q-l p mutant could contribute to revealing the function of AR in insects and offers a new method of identifying AR inhibitors for the treatment of diabetic complications. Copyright © 2017. Published by Elsevier B.V.

COMPARISON OF THE EFFECTS OF TWO AR ANTAGONISTS ON ANDROGEN DEPENDENT TISSUES WEIGHTS AND HORMONE LEVELS IN MALE RATS AND ON EXPRESSION OF THREE ANDROGEN DEPENDENT GENES IN THE VENTRAL PROSTATE

EPA Science Inventory

Comparison of the effects of two AR antagonists on tissue weights and hormone levels in male rats and on expression of three androgen dependent genes in the ventral prostate
VS Wilson, CR Wood, GA Held, CS Lambright, JS Ostby, JR Furr, LE Gray Jr. US EPA, ORD, NHEERL, RTD, ...

CCAAT/enhancer-binding protein β is involved in the breed-dependent transcriptional regulation of 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4)-isomerase in adrenal gland of preweaning piglets.

PubMed

Li, Xian; Li, Runsheng; Jia, Yimin; Sun, Zhiyuan; Yang, Xiaojing; Sun, Qinwei; Zhao, Ruqian

2013-11-01

The enzyme 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4)-isomerase (3β-HSD) catalyzes the biosynthesis of all steroid hormones. The molecular mechanisms regulating porcine adrenal 3β-HSD expression in different breeds are still poorly understood. In this study, we aimed to compare the expression of 3β-HSD between preweaning purebred Large White (LW) and Erhualian (EHL) piglets and to explore the potential factors regulating 3β-HSD transcription. EHL had significantly higher serum levels of cortisol (P<0.01) and testosterone (P<0.01), which were associated with significantly higher expression of 3β-HSD mRNA (P<0.01) and protein (P<0.05) in the adrenal gland, compared with LW piglets. The 5' flanking region of the porcine 3β-HSD gene showed significant sequence variations between breeds, and the sequence of EHL demonstrated an elevated promoter activity (P<0.05) in luciferase reporter gene assay. Higher adrenal expression of 3β-HSD in EHL was accompanied with higher CCAAT/enhancer binding protein β (C/EBPβ) expression (P<0.05), enriched histone H3 acetylation (P<0.05) and C/EBPβ binding to 3β-HSD promoter (P<0.05). In addition, higher androgen receptor (AR) (P=0.06) and lower glucocorticoid receptor (GR) (P<0.05) were detected in EHL. Co-immunoprecipitation analysis revealed interactions of C/EBPβ with both AR and GR. These results indicate that the C/EBPβ binding to 3β-HSD promoter is responsible, at least in part, for the breed-dependent 3β-HSD expression in adrenal gland of piglets. The sequence variations of 3β-HSD promoter and the interactions of AR and/or GR with C/EBPβ may also participate in the regulation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effects of a Proteasome Inhibitor on Cardiomyocytes in a Pressure-Overload Hypertrophy Rat Model: An Animal Study.

PubMed

Kim, In-Sub; Jo, Won-Min

2017-06-01

The ubiquitin-proteasome system (UPS) is an important pathway of proteolysis in pathologic hypertrophic cardiomyocytes. We hypothesize that MG132, a proteasome inhibitor, might prevent hypertrophic cardiomyopathy (CMP) by blocking the UPS. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and androgen receptor (AR) have been reported to be mediators of CMP and heart failure. This study drew upon pathophysiologic studies and the analysis of NF-κB and AR to assess the cardioprotective effects of MG132 in a left ventricular hypertrophy (LVH) rat model. We constructed a transverse aortic constriction (TAC)-induced LVH rat model with 3 groups: sham (TAC-sham, n=10), control (TAC-cont, n=10), and MG132 administration (TAC-MG132, n=10). MG-132 (0.1 mg/kg) was injected for 4 weeks in the TAC-MG132 group. Pathophysiologic evaluations were performed and the expression of AR and NF-κB was measured in the left ventricle. Fibrosis was prevalent in the pathologic examination of the TAC-cont model, and it was reduced in the TAC-MG132 group, although not significantly. Less expression of AR, but not NF-κB, was found in the TAC-MG132 group than in the TAC-cont group (p<0.05). MG-132 was found to suppress AR in the TAC-CMP model by blocking the UPS, which reduced fibrosis. However, NF-κB expression levels were not related to UPS function.

Differential expression of miR-34b and androgen receptor pathway regulate prostate cancer aggressiveness between African-Americans and Caucasians

PubMed Central

Kato, Taku; Yamamura, Soichiro; Tanaka, Yuichiro; Majid, Shahana; Saini, Sharanjot; Varahram, Shahryari; Kulkarni, Priyanka; Dasgupta, Pritha; Mitsui, Yozo; Sumida, Mitsuho; Tabatabai, Laura; Deng, Guoren; Kumar, Deepak; Dahiya, Rajvir

2017-01-01

African-Americans are diagnosed with more aggressive prostate cancers and have worse survival than Caucasians, however a comprehensive understanding of this health disparity remains unclear. To clarify the mechanisms leading to this disparity, we analyzed the potential involvement of miR-34b expression in African-Americans and Caucasians. miR-34b functions as a tumor suppressor and has a multi-functional role, through regulation of cell proliferation, cell cycle and apoptosis. We found that miR-34b expression is lower in human prostate cancer tissues from African-Americans compared to Caucasians. DNA hypermethylation of the miR-34b-3p promoter region showed significantly higher methylation in prostate cancer compared to normal samples. We then sequenced the promoter region of miR-34b-3p and found a chromosomal deletion in miR-34b in African-American prostate cancer cell line (MDA-PCA-2b) and not in Caucasian cell line (DU-145). We found that AR and ETV1 genes are differentially expressed in MDA-PCa-2b and DU-145 cells after overexpression of miR-34b. Direct interaction of miR-34b with the 3’ untranslated region of AR and ETV1 was validated by luciferase reporter assay. We found that miR-34b downregulation in African-Americans is inversely correlated with high AR levels that lead to increased cell proliferation. Overexpression of miR-34b in cell lines showed higher inhibition of cell proliferation, apoptosis and G1 arrest in the African-American cells (MDA-PCa-2b) compared to Caucasian cell line (DU-145). Taken together, our results show that differential expression of miR-34b and AR are associated with prostate cancer aggressiveness in African-Americans. PMID:28039468

Effects of dietary arachidonic acid on cortisol production and gene expression in stress response in Senegalese sole (Solea senegalensis) post-larvae.

PubMed

Martins, Dulce Alves; Rocha, Filipa; Castanheira, Filipa; Mendes, Ana; Pousão-Ferreira, Pedro; Bandarra, Narcisa; Coutinho, Joana; Morais, Sofia; Yúfera, Manuel; Conceição, Luís E C; Martínez-Rodríguez, Gonzalo

2013-10-01

Dietary fatty acids, particularly arachidonic acid (ARA), affect cortisol and may influence the expression of genes involved in stress response in fish. The involvement of ARA on stress, lipid, and eicosanoid metabolism genes, in Senegalese sole, was tested. Post-larvae were fed Artemia presenting graded ARA levels (0.1, 0.4, 0.8, 1.7, and 2.3%, dry matter basis), from 22 to 35 days after hatch. Whole-body cortisol levels were determined, before and 3 h after a 2 min air exposure, as well as the expression of phospholipase A2 (PLA 2 ), cyclooxygenase-2 (COX-2), steroidogenic acute regulatory protein (StAR), glucocorticoid receptors (GRs), phosphoenolpyruvate carboxykinase (PEPCK), and peroxisome proliferator-activated receptor alpha (PPARα). Relative growth rate (6.0-7.8% day(-1)) and survival at the end of the experiment (91-96%) and after stress (100%) were unaffected. Fish reflected dietary ARA content and post-stress cortisol increased with ARA supply up to 1.7%, whereas 2.3% ARA seemed to enhance basal cortisol slightly and alter the response to stress. Results suggested that elevating StAR transcription might not be necessary for a short-term response to acute stress. Basal cortisol and PLA 2 expression were strongly correlated, indicating a potential role for this enzyme in steroidogenesis. Under basal conditions, larval ARA was associated with GR1 expression, whereas the glucocorticoid responsive gene PEPCK was strongly related with cortisol but not GR1 mRNA levels, suggesting the latter might not reflect the amount of GR1 protein in sole. Furthermore, a possible role for PPARα in the expression of PEPCK following acute stress is proposed.

AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon-induced allelic variation and meiosis-associated expression.

PubMed

Klimyuk, V I; Jones, J D

1997-01-01

Based on homologies between the yeast DMC1 and the lily LIM15 meiosis-specific genes, degenerate PCR primers were designed that amplified the Arabidopsis DMC1 gene (AtDMC1). AtDMC1 genomic DNA (8 kb) was sequenced, and the transcript was characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) and by 5' and 3' RACE (rapid amplification of cDNA ends). The AtDMC1 gene contains 15 exons and 14 introns. RNA in situ hybridization analysis showed that expression of the AtDMC1 is restricted to pollen mother cells in anthers and to megaspore mother cells in ovules. The AtDMC1 promoter was fused to the GUS reporter gene, and conferred meiosis-associated expression in both male and female floral lineages. Comparison of AtDMC1 isolated from Landsberg erecta ecotype to its Columbia allele ArLIM15, revealed the presence of a 1874 bp transposon-like element within the promoter region of ArLIM15. RT-PCR analysis showed that the expression levels of AtDMC1 and ArLIM15 are similar. Possible uses for the AtDMC1 promoter are discussed.

Platelets Express Activated P2Y12 Receptor in Patients With Diabetes Mellitus.

PubMed

Hu, Liang; Chang, Lin; Zhang, Yan; Zhai, Lili; Zhang, Shenghui; Qi, Zhiyong; Yan, Hongmei; Yan, Yan; Luo, Xinping; Zhang, Si; Wang, Yiping; Kunapuli, Satya P; Ye, Hongying; Ding, Zhongren

2017-08-29

Platelets from patients with diabetes mellitus are hyperactive. Hyperactivated platelets may contribute to cardiovascular complications and inadequate responses to antiplatelet agents in the setting of diabetes mellitus. However, the underlying mechanism of hyperactivated platelets is not completely understood. We measured P2Y 12 expression on platelets from patients with type 2 diabetes mellitus and on platelets from rats with diabetes mellitus. We also assayed platelet P2Y 12 activation by measuring cAMP and VASP phosphorylation. The antiplatelet and antithrombotic effects of AR-C78511 and cangrelor were compared in rats. Finally, we explored the role of the nuclear factor-κB pathway in regulating P2Y 12 receptor expression in megakaryocytes. Platelet P2Y 12 levels are 4-fold higher in patients with type 2 diabetes mellitus compared with healthy subjects. P2Y 12 expression correlates with ADP-induced platelet aggregation (r=0.89, P <0.01). P2Y 12 in platelets from patients with diabetes mellitus is constitutively activated. Although both AR-C78511, a potent P2Y 12 inverse agonist, and cangrelor have similar antiplatelet efficacy on platelets from healthy subjects, AR-C78511 exhibits more powerful antiplatelet effects on diabetic platelets than cangrelor (aggregation ratio 36±3% versus 49±5%, respectively, P <0.05). Using a FeCl 3 -injury mesenteric arteriole thrombosis model in rats and an arteriovenous shunt thrombosis model in rats, we found that the inverse agonist AR-C78511 has greater antithrombotic effects on GK rats with diabetes mellitus than cangrelor (thrombus weight 4.9±0.3 mg versus 8.3±0.4 mg, respectively, P <0.01). We also found that a pathway involving high glucose-reactive oxygen species-nuclear factor-κB increases platelet P2Y 12 receptor expression in diabetes mellitus. Platelet P2Y 12 receptor expression is significantly increased and the receptor is constitutively activated in patients with type 2 diabetes mellitus, which contributes to platelet hyperactivity and limits antiplatelet drug efficacy in type 2 diabetes mellitus. © 2017 American Heart Association, Inc.

Volatile Arsenic Species Released from Escherichia coli Expressing the AsIII S-adenosylmethionine Methyltransferase Gene

PubMed Central

YUAN, CHUNGANG; LU, XIUFEN; QIN, JIE; ROSEN, BARRY P.; LE, X. CHRIS

2015-01-01

Biological systems, ranging from bacteria and fungi to humans, can methylate arsenic. Recent studies have suggested that the AsIII S-adenosylmethionine methyltransferase (arsM) gene in bacteria was responsible for the removal of arsenic as the volatile arsines from the bacteria. However, there has been no direct measure of the arsines released from bacteria cultures. We describe here an integrated system incorporating the bacterial incubation and volatile arsenic species analysis, and we demonstrate its application to the identification of the volatile arsines produced in bacterial cultures. The headspace of the bacterial cultures was purged with helium, and the volatile arsenic species were trapped in a chromatographic column immersed in liquid nitrogen. The cryogenically trapped arsines [AsH3, (CH3)AsH2, (CH3)2AsH, and (CH3)3As] were separated by gas chromatography and were detected by inductively coupled plasma mass spectrometry. A hydride generation system was coupled to the bacterial culture system, allowing for spiking standards and for generating calibration arsines necessary for quantitative analysis. Both bacteria containing the arsM gene or its variant arsMC2 gene were able to produce 400–500 ng of trimethylarsine. No trimethylarsine was detectable in bacteria lacking the arsM gene (containing the vector plasmid as negative control). These results confirm that arsM is responsible for releasing arsenic as volatile species from the arsenic-resistant bacteria. Our results also show traces of AsH3, CH3AsH2, and (CH3)2AsH in cultures of bacteria expressing arsM. The method detection limits for AsH3, CH3AsH2, (CH3)2AsH, and (CH3)3As were 0.5, 0.5, 0.7, and 0.6 pg, respectively. The ability to quantify trace levels of these volatile arsenic species makes it possible to study the biotransformation and biochemical roles of the evolution of these volatile arsenic species by biological systems. PMID:18522094

High throughput microscopy identifies bisphenol AP, a bisphenol A analog, as a novel AR down-regulator.

PubMed

Stossi, Fabio; Dandekar, Radhika D; Bolt, Michael J; Newberg, Justin Y; Mancini, Maureen G; Kaushik, Akash K; Putluri, Vasanta; Sreekumar, Arun; Mancini, Michael A

2016-03-29

Prostate cancer remains a deadly disease especially when patients become resistant to drugs that target the Androgen Receptor (AR) ligand binding domain. At this stage, patients develop recurring castrate-resistant prostate cancers (CRPCs). Interestingly, CRPC tumors maintain dependency on AR for growth; moreover, in CRPCs, constitutively active AR splice variants (e.g., AR-V7) begin to be expressed at higher levels. These splice variants lack the ligand binding domain and are rendered insensitive to current endocrine therapies. Thus, it is of paramount importance to understand what regulates the expression of AR and its splice variants to identify new therapeutic strategies in CRPCs. Here, we used high throughput microscopy and quantitative image analysis to evaluate effects of selected endocrine disruptors on AR levels in multiple breast and prostate cancer cell lines. Bisphenol AP (BPAP), which is used in chemical and medical industries, was identified as a down-regulator of both full length AR and the AR-V7 splice variant. We validated its activity by performing time-course, dose-response, Western blot and qPCR analyses. BPAP also reduced the percent of cells in S phase, which was accompanied by a ~60% loss in cell numbers and colony formation in anchorage-independent growth assays. Moreover, it affected mitochondria size and cell metabolism. In conclusion, our high content analysis-based screening platform was used to classify the effect of compounds on endogenous ARs, and identified BPAP as being capable of causing AR (both full-length and variants) down-regulation, cell cycle arrest and metabolic alterations in CRPC cell lines.

Direct Regulation of Androgen Receptor Activity by Potent CYP17 Inhibitors in Prostate Cancer Cells*

PubMed Central

Soifer, Harris S.; Souleimanian, Naira; Wu, Sijian; Voskresenskiy, Anatoliy M.; Kisaayak Collak, Filiz; Cinar, Bekir; Stein, Cy A.

2012-01-01

TOK-001 and abiraterone are potent 17-heteroarylsteroid (17-HAS) inhibitors of Cyp17, one of the rate-limiting enzymes in the biosynthesis of testosterone from cholesterol in prostate cancer cells. Nevertheless, the molecular mechanism underlying the prevention of prostate cell growth by 17-HASs still remains elusive. Here, we assess the effects of 17-HASs on androgen receptor (AR) activity in LNCaP and LAPC-4 cells. We demonstrate that both TOK-001 and abiraterone reduced AR protein and mRNA expression, and antagonized AR-dependent promoter activation induced by androgen. TOK-001, but not abiraterone, is an effective apparent competitor of the radioligand [3H]R1881 for binding to the wild type and various mutant AR (W741C, W741L) proteins. In agreement with these data, TOK-001 is a consistently superior inhibitor than abiraterone of R1881-induced transcriptional activity of both wild type and mutant AR. However, neither agent was able to trans-activate the AR in the absence of R1881. Our data demonstrate that phospho-4EBP1 levels are significantly reduced by TOK-001 and to a lesser extent by abiraterone alcohol, and suggest a mechanism by which cap-dependent translation is suppressed by blocking assembly of the eIF4F and eIF4G complex to the mRNA 5′ cap. Thus, the effects of these 17-HASs on AR signaling are complex, ranging from a decrease in testosterone production through the inhibition of Cyp17 as previously described, to directly reducing both AR protein expression and R1881-induced AR trans-activation. PMID:22174412

Differentially methylated genes and androgen receptor re-expression in small cell prostate carcinomas

PubMed Central

Kleb, Brittany; Estécio, Marcos R.H.; Zhang, Jiexin; Tzelepi, Vassiliki; Chung, Woonbok; Jelinek, Jaroslav; Navone, Nora M.; Tahir, Salahaldin; Marquez, Victor E.; Issa, Jean-Pierre; Maity, Sankar; Aparicio, Ana

2016-01-01

ABSTRACT Small cell prostate carcinoma (SCPC) morphology is rare at initial diagnosis but often emerges during prostate cancer progression and portends a dismal prognosis. It does not express androgen receptor (AR) or respond to hormonal therapies. Clinically applicable markers for its early detection and treatment with effective chemotherapy are needed. Our studies in patient tumor–derived xenografts (PDX) revealed that AR–negative SCPC (AR−SCPC) expresses neural development genes instead of the prostate luminal epithelial genes characteristic of AR–positive castration-resistant adenocarcinomas (AR+ADENO). We hypothesized that the differences in cellular lineage programs are reflected in distinct epigenetic profiles. To address this hypothesis, we compared the DNA methylation profiles of AR− and AR+ PDX using methylated CpG island amplification and microarray (MCAM) analysis and identified a set of differentially methylated promoters, validated in PDX and corresponding donor patient samples. We used the Illumina 450K platform to examine additional regions of the genome and the correlation between the DNA methylation profiles of the PDX and their corresponding patient tumors. Struck by the low frequency of AR promoter methylation in the AR−SCPC, we investigated this region's specific histone modification patterns by chromatin immunoprecipitation. We found that the AR promoter was enriched in silencing histone modifications (H3K27me3 and H3K9me2) and that EZH2 inhibition with 3-deazaneplanocin A (DZNep) resulted in AR expression and growth inhibition in AR−SCPC cell lines. We conclude that the epigenome of AR− is distinct from that of AR+ castration-resistant prostate carcinomas, and that the AR− phenotype can be reversed with epigenetic drugs. PMID:26890396

Bone stroma-derived cells change coregulators recruitment to androgen receptor and decrease cell proliferation in androgen-sensitive and castration-resistant prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Villagran, Marcelo A.; Gutierrez-Castro, Francisco A.; Pantoja, Diego F.

Prostate cancer (CaP) bone metastasis is an early event that remains inactive until later-stage progression. Reduced levels of circulating androgens, due to andropause or androgen deprivation therapies, alter androgen receptor (AR) coactivator expression. Coactivators shift the balance towards enhanced AR-mediated gene transcription that promotes progression to androgen-resistance. Disruptions in coregulators may represent a molecular switch that reactivates latent bone metastasis. Changes in AR-mediated transcription in androgen-sensitive LNCaP and androgen-resistant C4-2 cells were analyzed for AR coregulator recruitment in co-culture with Saos-2 and THP-1. The Saos-2 cell line derived from human osteosarcoma and THP-1 cell line representing human monocytes were usedmore » to display osteoblast and osteoclast activity. Increased AR activity in androgen-resistant C4-2 was due to increased AR expression and SRC1/TIF2 recruitment and decreased SMRT/NCoR expression. AR activity in both cell types was decreased over 90% when co-cultured with Saos-2 or THP-1 due to dissociation of AR from the SRC1/TIF2 and SMRT/NCoR coregulators complex, in a ligand-dependent and cell-type specific manner. In the absence of androgens, Saos-2 decreased while THP-1 increased proliferation of LNCaP cells. In contrast, both Saos-2 and THP-1 decreased proliferation of C4-2 in absence and presence of androgens. Global changes in gene expression from both CaP cell lines identified potential cell cycle and androgen regulated genes as mechanisms for changes in cell proliferation and AR-mediated transactivation in the context of bone marrow stroma cells. - Highlights: • Decreased corepressor expression change AR in androgen-resistance prostate cancer. • Bone stroma-derived cells change AR coregulator recruitment in prostate cancer. • Bone stroma cells change cell proliferation in androgen-resistant cancer cells. • Global gene expression in CaP cells is modified by bone stroma cells in co-cultures. • Potential new multi-subunit coactivator complexes for AR in CaP bone metastasis.« less

MEIS1 functions as a potential AR negative regulator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cui, Liang; Department of Urology, Civil Aviation General Hospital/Civil Aviation Medical College of Peking University, Beijing 100123; Li, Mingyang

2014-10-15

The androgen receptor (AR) plays critical roles in human prostate carcinoma progression and transformation. However, the activation of AR is regulated by co-regulators. MEIS1 protein, the homeodomain transcription factor, exhibited a decreased level in poor-prognosis prostate tumors. In this study, we investigated a potential interaction between MEIS1 and AR. We found that overexpression of MEIS1 inhibited the AR transcriptional activity and reduced the expression of AR target gene. A potential protein–protein interaction between AR and MEIS1 was identified by the immunoprecipitation and GST pull-down assays. Furthermore, MEIS1 modulated AR cytoplasm/nucleus translocation and the recruitment to androgen response element in prostatemore » specific antigen (PSA) gene promoter sequences. In addition, MEIS1 promoted the recruitment of NCoR and SMRT in the presence of R1881. Finally, MEIS1 inhibited the proliferation and anchor-independent growth of LNCaP cells. Taken together, our data suggests that MEIS1 functions as a novel AR co-repressor. - Highlights: • A potential interaction was identified between MEIS1 and AR signaling. • Overexpression of MEIS1 reduced the expression of AR target gene. • MEIS1 modulated AR cytoplasm/nucleus translocation. • MEIS1 inhibited the proliferation and anchor-independent growth of LNCaP cells.« less

MUC1-C ONCOPROTEIN CONFERS ANDROGEN-INDEPENDENT GROWTH OF HUMAN PROSTATE CANCER CELLS

PubMed Central

Rajabi, Hasan; Ahmad, Rehan; Jin, Caining; Joshi, Maya Datt; Guha, Minakshi; Alam, Maroof; Kharbanda, Surender; Kufe, Donald

2012-01-01

Background The mucin 1 (MUC1) heterodimeric oncoprotein is overexpressed in human prostate cancers with aggressive pathologic and clinical features. However, few insights are available regarding the functional role of MUC1 in prostate cancer. Methods Effects of MUC1-C on AR expression were determined by RT-PCR, immunoblotting and AR promoter activation. Coimmunoprecipitations, direct binding assays and chromatin immunoprecipitation (ChIP) studies were performed to assess the interaction between MUC1-C and AR. Cells were analyzed for invasion, growth in androgen-depleted medium and sensitivity to MUC1-C inhibitors. Results The present studies in androgen-dependent LNCaP and LAPC4 prostate cancer cells demonstrate that the oncogenic MUC1-C subunit suppresses AR expression. The results show that MUC1-C activates a posttranscriptional mechanism involving miR-135b-mediated downregulation of AR mRNA levels. The results further demonstrate that MUC1-C forms a complex with AR through a direct interaction between the MUC1-C cytoplasmic domain and the AR DNA-binding domain. In addition, MUC1-C associates with AR in a complex that occupies the PSA promoter. The interaction between MUC1-C and AR is associated with induction of the epithelial-mesenchymal transition (EMT) and increased invasion. MUC1-C also conferred growth in androgen-depleted medium and resistance to bicalutamide treatment. Moreover, expression of MUC1-C resulted in sensitivity to the MUC1-C inhibitor GO-203 with inhibition of growth in vitro. GO-203 treatment also inhibited growth of established tumor xenografts in nude mice. Conclusions These findings indicate that MUC1-C suppresses AR expression in prostate cancer cells and confers a more aggressive androgen-independent phenotype that is sensitive to MUC1-C inhibition. PMID:22473899

Effect of fetal exposure to bisphenol A on brain mediated by X-chromosome inactivation.

PubMed

Kumamoto, Takayuki; Oshio, Shigeru

2013-01-01

Recent studies have reported that bisphenol A (BPA) influences brain development in fetal exposure to mice. The X-chromosome codes many neurodevelopment-related genes leading to abnormal development, such as mental retardation and intellectual deficiency. For females, most of expressions of X-linked genes are regulated by X-chromosome inactivation (XCI), which occurs during fetal period, and this mechanism is regulated by Xist and its antisense, Tsix. To clarify the possibility of X-mediated effect as a mechanism of neurodevelopmental disorders by BPA, pregnant ICR mice were orally administered 0.02 or 50 mg/kg of BPA on gestational days 6 and 15. Postnatally at days 2, 4 and weeks 3 and 7, mRNA expression of XCI-regulating factors (Xist and Tsix), X-linked neurodevelopment-related genes (Fmr1, Gdi1, Nlgn3, Pak3 and Ophn1), and sexual differentiation-related genes (ERα, ERβ and AR) were examined in cerebrums of female pups. Anogenital distance (AGD) and serum estradiol were also examined. In the 50 mg/kg exposed-group, reduced Xist, Fmr1, Gdi1, Nlgn3, and Pak3 and increased Tsix were observed simultaneously. Moderately reduced Xist, Gdi1, Nlgn3 and Pak3 were observed at 0.02 mg/kg BPA. ERα, ERβ and AR expression changes, shortened AGDs and reduced estradiol levels were observed in each exposure group. Fetal exposure to BPA changed expression of XCI-regulating factors and may alter the expression levels of X-linked neurodevelopment-related genes disrupting the XCI mechanism and function. This X-mediated effect is considered one of the mechanisms of various BPA-induced neurodevelopmental disorders.

Adenosine A2A Receptor Activation and Macrophage-mediated Experimental Glomerulonephritis

PubMed Central

Garcia, Gabriela E.; Truong, Luan D.; Li, Ping; Zhang, Ping; Du, Jie; Chen, Jiang-Fan; Feng, Lili

2010-01-01

In immune-induced inflammation, leukocytes are key mediators of tissue damage. Since A2A adenosine receptors (A2AR) are endogenous suppressors of inflammation, we examined cellular and molecular mechanisms of kidney damage to determine whether selective activation of A2AR will suppress inflammation in a rat model of glomerulonephritis. Activation of A2AR reduced the degree of kidney injury in both the acute inflammatory phase and the progressive phase of glomerulonephritis. This protection against acute and chronic inflammation was associated with suppression of the glomerular expression of the MDC/CCL22 chemokine and down-regulation of MIP-1α/CCL3, RANTES/CCL5, MIP-1β/CCL4, and MCP-1/CCL2 chemokines. The expression of anti-inflammatory cytokines, IL-4 and IL-10, also increased. The mechanism for these anti-inflammatory responses to the A2AR agonist was suppression of macrophages function. A2AR expression was increased in macrophages, macrophage-derived chemokines were reduced in response to the A2AR agonist, and chemokines not expressed in macrophages did not respond to A2AR activation. Thus, activation of the A2AR on macrophages inhibits immune-associated inflammation. In glomerulonephritis, A2AR activation modulates inflammation and tissue damage even in the progressive phase of glomerulonephritis. Accordingly, pharmacological activation of A2AR could be developed into a novel treatment for glomerulonephritis and other macrophage-related inflammatory diseases. PMID:17898087

Assessment of different biomarkers provides valuable diagnostic standards in the evaluation of the risk of acute rejection.

PubMed

Zheng, Jin; Ding, Xiaoming; Tian, Xiaohui; Jin, Zhankui; Pan, Xiaoming; Yan, Hang; Feng, Xinshun; Hou, Jun; Xiang, Heli; Ren, Li; Tian, Puxun; Xue, Wujun

2012-09-01

Acute rejection (AR) is a strong risk factor for chronic rejection in renal transplant recipients. Accurate and timely diagnosis of AR episodes is very important for disease control and prognosis. Therefore, objectively evaluated the immune status of patients is essential in the field of post-transplantation treatment. This longitudinal study investigated the usefulness of five biomarkers, human leukocyte antigen (HLA)-G5 and sCD30 level in sera, intracellular adenosine triphosphate (iATP) release level of CD4(+) T cells, and granzyme B/perforin expression in peripheral blood mononuclear cells (PBMCs) and biopsies, to detect AR and the resolution of biomarkers in a total of 84 cases of renal transplantation. The data demonstrated that recipients with clinical or biopsy proven rejection significantly increased iATP release level of CD4(+) T cells, and elevated sCD30 but lowered HLA-G5 level in sera compared with individuals with stable graft function. Expression levels of granzyme B and perforin were also elevated in PBMCs and graft biopsies of AR patients. Taken together, we identified that upregulation of sCD30, iATP, granzyme B, perforin, and downregulation of HLA-G5 could provide valuable diagnostic standards to identify those recipients in the risk of AR. And iATP may be a better biomarker than others for predicting the graft rejection episode.

Bypass Mechanisms of the Androgen Receptor Pathway in Therapy-Resistant Prostate Cancer Cell Models

PubMed Central

Marques, Rute B.; Dits, Natasja F.; Erkens-Schulze, Sigrun; van Weerden, Wytske M.; Jenster, Guido

2010-01-01

Background Prostate cancer is initially dependent on androgens for survival and growth, making hormonal therapy the cornerstone treatment for late-stage tumors. However, despite initial remission, the cancer will inevitably recur. The present study was designed to investigate how androgen-dependent prostate cancer cells eventually survive and resume growth under androgen-deprived and antiandrogen supplemented conditions. As model system, we used the androgen-responsive PC346C cell line and its therapy-resistant sublines: PC346DCC, PC346Flu1 and PC346Flu2. Methodology/Principal Findings Microarray technology was used to analyze differences in gene expression between the androgen-responsive and therapy-resistant PC346 cell lines. Microarray analysis revealed 487 transcripts differentially-expressed between the androgen-responsive and the therapy-resistant cell lines. Most of these genes were common to all three therapy-resistant sublines and only a minority (∼5%) was androgen-regulated. Pathway analysis revealed enrichment in functions involving cellular movement, cell growth and cell death, as well as association with cancer and reproductive system disease. PC346DCC expressed residual levels of androgen receptor (AR) and showed significant down-regulation of androgen-regulated genes (p-value = 10−7). Up-regulation of VAV3 and TWIST1 oncogenes and repression of the DKK3 tumor-suppressor was observed in PC346DCC, suggesting a potential AR bypass mechanism. Subsequent validation of these three genes in patient samples confirmed that expression was deregulated during prostate cancer progression. Conclusions/Significance Therapy-resistant growth may result from adaptations in the AR pathway, but androgen-independence may also be achieved by alternative survival mechanisms. Here we identified TWIST1, VAV3 and DKK3 as potential players in the bypassing of the AR pathway, making them good candidates as biomarkers and novel therapeutical targets. PMID:20976069

3',5'-cyclic adenosine monophosphate response element binding protein up-regulated cytochrome P450 lanosterol 14alpha-demethylase expression involved in follicle-stimulating hormone-induced mouse oocyte maturation.

PubMed

Ning, Gang; Ouyang, Hong; Wang, Songbo; Chen, Xiufen; Xu, Baoshan; Yang, Jiange; Zhang, Hua; Zhang, Meijia; Xia, Guoliang

2008-07-01

Cytochrome P450 lanosterol 14alpha-demethylase (CYP51) is a key enzyme in sterols and steroids biosynthesis that can induce meiotic resumption in mouse oocytes. The present study investigated the expression mechanism and function of CYP51 during FSH-induced mouse cumulus oocyte complexes (COCs) meiotic resumption. FSH increased cAMP-dependent protein kinase (PKA) RIIbeta level and induced cAMP response element-binding protein (CREB) phosphorylation and CYP51 expression in cumulus cells before oocyte meiotic resumption. Moreover, CYP51 and epidermal growth factor (EGF)-like factor [amphiregulin (AR)] expression were blocked by (2)-naphthol-AS-Ephosphate (KG-501) (a drug interrupting the formation of CREB functional complex). KG-501 and RS21607 (a specific inhibitor of CYP51 activity) inhibited oocyte meiotic resumption, which can be partially rescued by progesterone. These two inhibitors also inhibited FSH-induced MAPK phosphorylation. EGF could rescue the suppression by KG-501 but not RS21607. Furthermore, type II PKA analog pairs, N(6)-monobutyryl-cAMP plus 8-bromo-cAMP, increased PKA RIIbeta level and mimicked the action of FSH, including CREB phosphorylation, AR and CYP51 expression, MAPK activation, and oocyte maturation. All these data suggest that CYP51 plays a critical role in FSH-induced meiotic resumption of mouse oocytes. CYP51 and AR gene expression in cumulus cells are triggered by FSH via a type II PKA/CREB-dependent signal pathway. Our study also implicates that CYP51 activity in cumulus cells participates in EGF receptor signaling-regulated oocyte meiotic resumption.

Antiarrhythmic Effects of Beta3-adrenergic Receptor Stimulation in a Canine Model of Ventricular Tachycardia

PubMed Central

Zhou, Shengmei; Tan, Alex Y.; Paz, Offir; Ogawa, Masahiro; Chou, Chung-Chuan; Hayashi, Hideki; Nihei, Motoki; Fishbein, Michael C.; Chen, Lan S.; Lin, Shien-Fong; Chen, Peng-Sheng

2009-01-01

Background Beta3-adrenergic receptor (beta3-AR) stimulation inhibits cardiac contractility. Objective To test the hypothesis that beta3-AR stimulation is antiarrhythmic. Methods We implanted a radiotransmitter for continuous ECG monitoring in 18 dogs with a tendency for high incidence of spontaneous ventricular tachycardia (VT). Ten of 18 had subcutaneous continuous BRL37344 (beta3-AR agonist) infusion (experimental group) for 1 month. The other dogs were controls. Western blotting studies were performed on tissues sampled from the noninfarcted left ventricular free wall of all dogs that survived the 60-day follow up period. Results Phase-2 VT appeared significantly later in the experimental group than in the control group (p<0.05). The number of VT episodes in the experimental group was significantly lower than control during both the first month (0.5 ± 0.95 episode/d vs. 2.6 ± 2.3 episode/d) and the second month (0.2 ± 0.2 episode/d vs. 1.2 ± 1.1 episode/d, p<0.05 for both). The experimental group had shorter QTc than control (p<0.002). The experimental group had decreased protein levels for sodium calcium exchanger and dihydropyridine receptor, increased beta3-AR expression, without changes in beta1-AR, beta2-AR. The average heart weight and the left ventricular free wall thickness in the experimental group (226 ± 17 g and 15.1 ± 1.2 mm, respectively) was significantly lower than control (265 ± 21 g and 17.4 ± 2.5 mm, respectively, p<0.05 for both). There was no difference in the incidences of sudden cardiac death (SCD) in these two groups of dogs. Conclusion Beta3-AR stimulation significantly reduces the occurrence of ventricular tachycardia. PMID:18242556

Cerebral Artery Alpha-1 AR Subtypes: High Altitude Long-Term Acclimatization Responses

PubMed Central

Goyal, Ravi; Goyal, Dipali; Chu, Nina; Van Wickle, Jonathan; Longo, Lawrence D.

2014-01-01

In response to hypoxia and other stress, the sympathetic (adrenergic) nervous system regulates arterial contractility and blood flow, partly through differential activities of the alpha1 (α1) - adrenergic receptor (AR) subtypes (α1A-, α1B-, and α1D-AR). Thus, we tested the hypothesis that with acclimatization to long-term hypoxia (LTH), contractility of middle cerebral arteries (MCA) is regulated by changes in expression and activation of the specific α1-AR subtypes. We conducted experiments in MCA from adult normoxic sheep maintained near sea level (300 m) and those exposed to LTH (110 days at 3801 m). Following acclimatization to LTH, ovine MCA showed a 20% reduction (n = 5; P<0.05) in the maximum tension achieved by 10−5 M phenylephrine (PHE). LTH-acclimatized cerebral arteries also demonstrated a statistically significant (P<0.05) inhibition of PHE-induced contractility in the presence of specific α1-AR subtype antagonists. Importantly, compared to normoxic vessels, there was significantly greater (P<0.05) α1B-AR subtype mRNA and protein levels in LTH acclimatized MCA. Also, our results demonstrate that extracellular regulated kinase 1 and 2 (ERK1/2)-mediated negative feedback regulation of PHE-induced contractility is modulated by α1B-AR subtype. Overall, in ovine MCA, LTH produces profound effects on α1-AR subtype expression and function. PMID:25393740

Cerebral artery alpha-1 AR subtypes: high altitude long-term acclimatization responses.

PubMed

Goyal, Ravi; Goyal, Dipali; Chu, Nina; Van Wickle, Jonathan; Longo, Lawrence D

2014-01-01

In response to hypoxia and other stress, the sympathetic (adrenergic) nervous system regulates arterial contractility and blood flow, partly through differential activities of the alpha1 (α1) - adrenergic receptor (AR) subtypes (α1A-, α1B-, and α1D-AR). Thus, we tested the hypothesis that with acclimatization to long-term hypoxia (LTH), contractility of middle cerebral arteries (MCA) is regulated by changes in expression and activation of the specific α1-AR subtypes. We conducted experiments in MCA from adult normoxic sheep maintained near sea level (300 m) and those exposed to LTH (110 days at 3801 m). Following acclimatization to LTH, ovine MCA showed a 20% reduction (n = 5; P<0.05) in the maximum tension achieved by 10-5 M phenylephrine (PHE). LTH-acclimatized cerebral arteries also demonstrated a statistically significant (P<0.05) inhibition of PHE-induced contractility in the presence of specific α1-AR subtype antagonists. Importantly, compared to normoxic vessels, there was significantly greater (P<0.05) α1B-AR subtype mRNA and protein levels in LTH acclimatized MCA. Also, our results demonstrate that extracellular regulated kinase 1 and 2 (ERK1/2)-mediated negative feedback regulation of PHE-induced contractility is modulated by α1B-AR subtype. Overall, in ovine MCA, LTH produces profound effects on α1-AR subtype expression and function.

BMP4 and BMP7 Suppress StAR and Progesterone Production via ALK3 and SMAD1/5/8-SMAD4 in Human Granulosa-Lutein Cells.

PubMed

Zhang, Han; Klausen, Christian; Zhu, Hua; Chang, Hsun-Ming; Leung, Peter C K

2015-11-01

Adequate production of progesterone by the corpus luteum is critical to the successful establishment of pregnancy. In animal models, bone morphogenetic protein (BMP) 4 and BMP7 have been shown to suppress either basal or gonadotropin-induced progesterone production, depending on the species examined. However, the effects of BMP4 and BMP7 on progesterone production in human granulosa cells are unknown. In the present study, we used immortalized (SVOG) and primary human granulosa-lutein cells to investigate the effects of BMP4 and BMP7 on steroidogenic acute regulatory protein (StAR) expression and progesterone production and to examine the underlying molecular mechanism. Treatment of primary and immortalized human granulosa cells with recombinant BMP4 or BMP7 decreased StAR expression and progesterone accumulation. In SVOG cells, the suppressive effects of BMP4 and BMP7 on StAR expression were blocked by pretreatment with inhibitors of activin receptor-like kinase (ALK)2/3/6 (dorsomorphin) or ALK2/3 (DMH1) but not ALK4/5/7 (SB-431542). Moreover, small interfering RNA-mediated depletion of ALK3, but not ALK2 or ALK6, reversed the effects of BMP4 and BMP7 on StAR expression. Likewise, BMP4- and BMP7-induced phosphorylation of SMAD 1/5/8 was reversed by treatment with DMH1 or small interfering RNA targeting ALK3. Knockdown of SMAD4, the essential common SMAD for BMP/TGF-β signaling, abolished the effects of BMP4 and BMP7 on StAR expression. Our results suggest that BMP4 and BMP7 down-regulate StAR and progesterone production via ALK3 and SMAD1/5/8-SMAD4 signaling in human granulosa-lutein cells.

Androgen receptor polyglutamine repeat length (AR-CAGn) modulates the effect of testosterone on androgen-associated somatic traits in Filipino young adult men.

PubMed

Ryan, Calen P; Georgiev, Alexander V; McDade, Thomas W; Gettler, Lee T; Eisenberg, Dan T A; Rzhetskaya, Margarita; Agustin, Sonny S; Hayes, M Geoffrey; Kuzawa, Christopher W

2017-06-01

The androgen receptor (AR) mediates expression of androgen-associated somatic traits such as muscle mass and strength. Within the human AR is a highly variable glutamine short-tandem repeat (AR-CAGn), and CAG repeat number has been inversely correlated to AR transcriptional activity in vitro. However, evidence for an attenuating effect of long AR-CAGn on androgen-associated somatic traits has been inconsistent in human populations. One possible explanation for this lack of consistency is that the effect of AR-CAGn on AR bioactivity in target tissues likely varies in relation to circulating androgen levels. We tested whether relationships between AR-CAGn and several androgen-associated somatic traits (waist circumference, lean mass, arm muscle area, and grip strength) were modified by salivary (waking and pre-bed) and circulating (total) testosterone (T) levels in young adult males living in metropolitan Cebu, Philippines (n = 675). When men's waking T was low, they had a reduction in three out of four androgen-associated somatic traits with lengthening AR-CAGn (p < .1), consistent with in vitro research. However, when waking T was high, we observed the opposite effect-lengthening AR-CAGn was associated with an increase in these same somatic traits. Our finding that longer AR-CAGn predicts greater androgen-associated trait expression among high-T men runs counter to in vitro work, but is generally consistent with the few prior studies to evaluate similar interactions in human populations. Collectively, these results raise questions about the applicability of findings derived from in vitro AR-CAGn studies to the receptor's role in maintaining androgen-associated somatic traits in human populations. © 2017 Wiley Periodicals, Inc.

Bisphenol A down-regulates rate-limiting Cyp11a1 to acutely inhibit steroidogenesis in cultured mouse antral follicles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peretz, Jackye, E-mail: peretz@illinois.edu; Flaws, Jodi A., E-mail: jflaws@illinois.edu

Bisphenol A (BPA) is the backbone of polycarbonate plastic products and the epoxy resin lining of aluminum cans. Previous studies have shown that exposure to BPA decreases sex steroid hormone production in mouse antral follicles. The current study tests the hypothesis that BPA first decreases the expression levels of the steroidogenic enzyme cytochrome P450 side-chain cleavage (Cyp11a1) and steroidogenic acute regulatory protein (StAR) in mouse antral follicles, leading to a decrease in sex steroid hormone production in vitro. Further, the current study tests the hypothesis that these effects are acute and reversible after removal of BPA. Exposure to BPA (10more » μg/mL and 100 μg/mL) significantly decreased expression of Cyp11a1 and StAR beginning at 18 h and 72 h, respectively, compared to controls. Exposure to BPA (10 μg/mL and 100 μg/mL) significantly decreased progesterone levels beginning at 24 h and decreased androstenedione, testosterone, and estradiol levels at 72 h and 96 h compared to controls. Further, after removing BPA from the culture media at 20 h, expression of Cyp11a1 and progesterone levels were restored to control levels by 48 h and 72 h, respectively. Additionally, expression of StAR and levels of androstenedione, testosterone, and estradiol never decreased compared to controls. These data suggest that BPA acutely decreases expression of Cyp11a1 as early as 18 h and this reduction in Cyp11a1 may lead to a decrease in progesterone production by 24 h, followed by a decrease in androstenedione, testosterone, and estradiol production and expression of StAR at 72 h. Therefore, BPA exposure likely targets Cyp11a1 and steroidogenesis, but these effects are reversible with removal of BPA exposure. - Highlights: • BPA may target Cyp11a1 to inhibit steroidogenesis in antral follicles. • BPA may decrease the expression of Cyp11a1 prior to inhibiting steroidogenesis. • The adverse effects of BPA on steroidogenesis in antral follicles are reversible.« less

The Ars insulator facilitates I-SceI meganuclease-mediated transgenesis in the sea urchin embryo.

PubMed

Ochiai, Hiroshi; Sakamoto, Naoaki; Suzuki, Kenichi; Akasaka, Koji; Yamamoto, Takashi

2008-09-01

For the efficient generation of transgenic sea urchins, we have adopted an I-SceI meganuclease-mediated transgenesis method. Several types of promoter-GFP gene constructs flanked by two I-SceI recognition sequences were co-injected with I-SceI into sea urchin fertilized eggs. Using cell-lineage-specific promoter constructs, the frequency of transgene expression was elevated, and their level of mozaicism was reduced. The addition of the Ars insulator sequence, which is known to block the enhancer activity and protect transgenes from position effects, led to a reduction in ectopic transgene expression and an elevation of transgene expression frequency in this I-SceI-mediated system. However, the magnitude of the effects of the Ars insulator was dependent upon the promoter constructs. QPCR analysis also showed that the Ars insulator increases the transgene copy number. These results suggest that the I-SceI-mediated method using the Ars insulator is advantageous for transgenesis in the sea urchin embryo.

Triterpene and Flavonoid Biosynthesis and Metabolic Profiling of Hairy Roots, Adventitious Roots, and Seedling Roots of Astragalus membranaceus.

PubMed

Park, Yun Ji; Thwe, Aye Aye; Li, Xiaohua; Kim, Yeon Jeong; Kim, Jae Kwang; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Park, Sang Un

2015-10-14

Astragalus membranaceus is an important traditional Chinese herb with various medical applications. Astragalosides (ASTs), calycosin, and calycosin-7-O-β-d-glucoside (CG) are the primary metabolic components in A. membranaceus roots. The dried roots of A. membranaceus have various medicinal properties. The present study aimed to investigate the expression levels of genes related to the biosynthetic pathways of ASTs, calycosin, and CG to investigate the differences between seedling roots (SRs), adventitious roots (ARs), and hairy roots (HRs) using quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR study revealed that the transcription level of genes involved in the AST biosynthetic pathway was lowest in ARs and showed similar patterns in HRs and SRs. Moreover, most genes involved in the synthesis of calycosin and CG exhibited the highest expression levels in SRs. High-performance liquid chromatography (HPLC) analysis indicated that the expression level of the genes correlated with the content of ASTs, calycosin, and CG in the three different types of roots. ASTs were the most abundant in SRs. CG accumulation was greater than calycosin accumulation in ARs and HRs, whereas the opposite was true in SRs. Additionally, 40 metabolites were identified using gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS). Principal component analysis (PCA) documented the differences among SRs, ARs, and HRs. PCA comparatively differentiated among the three samples. The results of PCA showed that HRs were distinct from ARs and SRs on the basis of the dominant amounts of sugars and clusters derived from closely similar biochemical pathways. Also, ARs had a higher concentration of phenylalanine, a precursor for the phenylpropanoid biosynthetic pathway, as well as CG. TCA cycle intermediates levels including succinic acid and citric acid indicated a higher amount in SRs than in the others.

Distinct Gene Expression Patterns between Nasal Mucosal Cells and Blood Collected from Allergic Rhinitis Sufferers.

PubMed

Watts, Annabelle M; West, Nicholas P; Cripps, Allan W; Smith, Pete K; Cox, Amanda J

2018-06-19

Investigations of gene expression in allergic rhinitis (AR) typically rely on invasive nasal biopsies (site of inflammation) or blood samples (systemic immunity) to obtain sufficient genetic material for analysis. New methodologies to circumvent the need for invasive sample collection offer promise to further the understanding of local immune mechanisms relevant in AR. A within-subject design was employed to compare immune gene expression profiles obtained from nasal washing/brushing and whole blood samples collected during peak pollen season. Twelve adults (age: 46.3 ± 12.3 years) with more than a 2-year history of AR and a confirmed grass pollen allergy participated in the study. Gene expression analysis was performed using a panel of 760 immune genes with the NanoString nCounter platform on nasal lavage/brushing cell lysates and compared to RNA extracted from blood. A total of 355 genes were significantly differentially expressed between sample types (9.87 to -9.71 log2 fold change). The top 3 genes significantly upregulated in nasal lysate samples were Mucin 1 (MUC1), Tight Junction Protein 1 (TJP1), and Lipocalin-2 (LCN2). The top 3 genes significantly upregulated in blood samples were cluster of differentiation 3e (CD3E), FYN Proto-Oncogene Src Family Tyrosine Kinase (FYN) and cluster of differentiation 3d (CD3D). Overall, the blood and nasal lavage samples showed vastly distinct gene expression profiles and functional gene pathways which reflect their anatomical and functional origins. Evaluating immune gene expression of the nasal mucosa in addition to blood samples may be beneficial in understanding AR pathophysiology and response to allergen challenge. © 2018 S. Karger AG, Basel.

Sex steroid receptors and apoptosis-related proteins are differentially expressed in polycystic ovaries of adult dogs.

PubMed

Chuffa, Luiz Gustavo de Almeida; Lupi Júnior, Luiz Antonio; da Maia Lima, Alfredo Feio

2016-02-01

In Polycystic Ovaries (PCOs), the dynamics of sex hormone receptors and follicle-related apoptotic signaling remain unknown. In this study, we investigated the expression of androgen receptors (AR), estrogen receptors (ERα and ERβ), and apoptosis-related molecules (BAX, active caspase-3, Bcl-2 and Survivin) on different follicular stages of PCOs in adult dogs. Clinical evidences of high estradiol and testosterone levels, persistent estrus and vaginal discharge were observed. Inhibin B immunolabeling was increased in primary and 2 to 5-mm follicles, and a marked epithelial hyperplasia was common in the ovarian surface. Ovarian epithelia and primary follicles showed low expression of AR, ERα, and ERβ, whereas a moderate immunoexpression of AR was found in theca cells of secondary follicles and cysts. In PCOs, growing follicles displayed ERα expression, and secondary follicles exhibited higher ERβ expression. In addition, while few ERα-positive cells were found in the cysts, ERβ was moderately expressed in growing follicles and cysts. BAX was upregulated in the ovarian epithelium, primary follicles, and in the wall of follicular cysts. Active caspase-3 was significantly downregulated in the epithelium, primary follicles, and follicular cysts, whereas growing follicles had a strong immunoexpression in the granulosa cells. Bcl-2 and survivin were increased in the epithelium and primary follicles, and only survivin was upregulated in secondary and growing follicles. While Bcl-2 had a diffuse immunexpression in the follicular cysts, survivin was overexpressed by these cells. We concluded that sex steroid receptors and apoptotic proteins are differentially expressed in the follicles of adult dogs with PCOs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Interrelation of androgen receptor and miR-30a and miR-30a function in ER-, PR-, AR+ MDA-MB-453 breast cancer cells.

PubMed

Lyu, Shuhua; Liu, Han; Liu, Xia; Liu, Shan; Wang, Yahong; Yu, Qi; Niu, Yun

2017-10-01

The association between androgen-induced androgen receptor (AR) activating signal and microRNA (miR)-30a was investigated, as well as the function of miR-30a in estrogen receptor-negative (ER - ), progesterone receptor-negative (PR - ), and AR-positive (AR + ) MDA-MB-453 breast cancer cells. Androgen-induced AR activating signal upregulated the expression of AR, and downregulated the expression of miR-30a, b and c. Bioinformatics analysis indicated a putative miR-30a, b and c binding site in the 3'-untranslated region of AR mRNA. It was confirmed that the AR gene is a direct target of miR-30a, whereas AR does not target the miR-30a promoter, and AR activating signal may indirectly downregulate miR-30a through other cell signaling pathways. In this positive feedback mechanism AR is then upregulated through miR-30a. Overexpression of miR-30a inhibited cell proliferation, whereas inhibition of miR-30a expression by specific antisense oligonucleotides, increased cell growth. Previously, androgen-induced AR activating signal was demonstrated to inhibit cell proliferation in ER - , PR - and AR + MDA-MB-453 breast cancer cells, but AR activating signal downregulated the expression of miR-30a, relieving the inhibition of MDA-MB-453 cell growth. Therefore, in MDA-MB-453 breast cancer cells, miR-30a has two different functions regarding cell growth: Inhibition of cell proliferation through a positive feedback signaling pathway; and the relative promotion of cell proliferation through downregulation of miR-30a. Thus, the association between AR activating signal and microRNAs is complex, and microRNAs may possess different functions due to different signaling pathways. Although the results of the present study were obtained in one cell line, they contribute to subsequent studies on ER - , PR - and AR + breast cancer.

Expression of androgen receptor through androgen-converting enzymes is associated with biological aggressiveness in prostate cancer.

PubMed

Wako, K; Kawasaki, T; Yamana, K; Suzuki, K; Jiang, S; Umezu, H; Nishiyama, T; Takahashi, K; Hamakubo, T; Kodama, T; Naito, M

2008-04-01

The association between the expression of androgen receptor (AR) or androgen-converting enzymes and malignant potential in prostate cancer (PCa) was examined. PCa specimens from 44 cases of stage II, 10 cases of stage III, four cases of stage IV and two recurrent cases were semi-quantitatively studied with immunohistochemistry for AR and androgen-converting enzymes. The expression scores for AR, 5alpha-reductase type 1 (SRD5A1), 5alpha-reductase type 2 (SRD5A2), and aldo-keto reductase family 1 member C3 (AKR1C3) in the metastatic lesion of stage IV or recurrent cancer (n = 6) were 284.2 (30.1), 300 (0.0), 279.2 (51) and 254.2 (74.9), respectively; these scores were significantly higher than the respective scores of 121.8 (82.1), 135.1 (59.7), 167.0 (66.4) and 150.5 (62.8) for stage II and III cancer (n = 54) (p<0.001, p<0.001, p = 0.002 and p = 0.018, respectively). The expression scores for AR and SRD5A1 in stage II and III cancer with Gleason score 7 (n = 19) were 128.7 (72.3) and 150.5 (52.9); these were significantly higher than the scores of 78.8 (67.2) and 100.0 (39.6), respectively, for cancers with a Gleason score of < or =6 (n = 20) (p = 0.032 and p = 0.002, respectively). The expression scores for AR, SRD5A1 and AKR1C3 in stage II and III cancer with primary Gleason pattern > or =4 (n = 21) were 158.1 (84.3), 158.3 (61.1) and 173.8 (64.8); these were significantly higher than the scores of 98.6 (72.8), 120.3 (54.7) and 135.6 (57.6), respectively, for cancers with primary Gleason pattern < or =3 (n = 33) (p = 0.011, p = 0.026 and p = 0.034, respectively). Within Gleason score 9 cancer, the expression scores for AR and SRD5A1 in the primary lesion of stage IV (n = 3) were 276.7 (5.8) and 283.3 (28.9); these scores were significantly higher than the scores of 182.1 (86.0) and 140.0 (56.6), respectively, for stage II and III cancer (n = 7) (p = 0.027 and p = 0.001, respectively). Both AR and androgen-converting enzymes were upregulated in high-grade or advanced PCa.

Androgen Receptor Functional Analyses by High Throughput Imaging: Determination of Ligand, Cell Cycle, and Mutation-Specific Effects

PubMed Central

Szafran, Adam T.; Szwarc, Maria; Marcelli, Marco; Mancini, Michael A.

2008-01-01

Background Understanding how androgen receptor (AR) function is modulated by exposure to steroids, growth factors or small molecules can have important mechanistic implications for AR-related disease therapies (e.g., prostate cancer, androgen insensitivity syndrome, AIS), and in the analysis of environmental endocrine disruptors. Methodology/Principal Findings We report the development of a high throughput (HT) image-based assay that quantifies AR subcellular and subnuclear distribution, and transcriptional reporter gene activity on a cell-by-cell basis. Furthermore, simultaneous analysis of DNA content allowed determination of cell cycle position and permitted the analysis of cell cycle dependent changes in AR function in unsynchronized cell populations. Assay quality for EC50 coefficients of variation were 5–24%, with Z' values reaching 0.91. This was achieved by the selective analysis of cells expressing physiological levels of AR, important because minor over-expression resulted in elevated nuclear speckling and decreased transcriptional reporter gene activity. A small screen of AR-binding ligands, including known agonists, antagonists, and endocrine disruptors, demonstrated that nuclear translocation and nuclear “speckling” were linked with transcriptional output, and specific ligands were noted to differentially affect measurements for wild type versus mutant AR, suggesting differing mechanisms of action. HT imaging of patient-derived AIS mutations demonstrated a proof-of-principle personalized medicine approach to rapidly identify ligands capable of restoring multiple AR functions. Conclusions/Significance HT imaging-based multiplex screening will provide a rapid, systems-level analysis of compounds/RNAi that may differentially affect wild type AR or clinically relevant AR mutations. PMID:18978937

Adenosine A3 receptor elicits chemoresistance mediated by multiple resistance-associated protein-1 in human glioblastoma stem-like cells

PubMed Central

Torres, Angelo; Vargas, Yosselyn; Uribe, Daniel; Jaramillo, Catherine; Gleisner, Alejandra; Salazar-Onfray, Flavio; López, Mercedes N.; Melo, Rómulo; Oyarzún, Carlos; Martín, Rody San; Quezada, Claudia

2016-01-01

MRP1 transporter correlates positively with glioma malignancy and the Multiple Drug Resistance (MDR) phenotype in Glioblastoma Multiforme (GBM). Evidence shows that the MRP1 transporter is controlled by the adenosine signalling axis. The aim of this study was to identify the role of adenosine on the MDR phenotype in Glioblastoma Stem-like Cells (GSCs), the cell population responsible for the tumorigenic and chemoresistance capabilities of this tumour. We found that GSCs have increased intrinsic capacity to generate extracellular adenosine, thus controlling MRP1 transporter expression and activity via activation of the adenosine A3 receptor (A3AR). We showed PI3K/Akt and MEK/ERK1/2 signaling pathways downstream A3AR to control MRP1 in GSCs. In vitro pharmacological blockade of A3AR had a chemosensitizing effect, enhancing the actions of antitumour drugs and decreasing cell viability and proliferation of GSCs. In addition, we produced an in vivo xenograft model by subcutaneous inoculation of human GSCs in NOD/SCID-IL2Rg null mice. Pharmacological blockade of A3AR generated a chemosensitizing effect, enhancing the effectiveness of the MRP1 transporter substrate, vincristine, reducing tumour size and the levels of CD44 and Nestin stem cell markers as well as the Ki-67 proliferation indicator. In conclusion, we demonstrated the chemosensitizing effect of A3AR blockade on GSCs. PMID:27634913

Androgen Receptor and its Splice Variant, AR-V7, Differentially Regulate FOXA1 Sensitive Genes in LNCaP Prostate Cancer Cells

PubMed Central

Krause, William C.; Shafi, Ayesha A.; Nakka, Manjula; Weigel, Nancy L.

2014-01-01

Prostate cancer (PCa) is an androgen-dependent disease, and tumors that are resistant to androgen ablation therapy often remain androgen receptor (AR) dependent. Among the contributors to castration-resistant PCa are AR splice variants that lack the ligand-binding domain (LBD). Instead, they have small amounts of unique sequence derived from cryptic exons or from out of frame translation. The AR-V7 (or AR3) variant is constitutively active and is expressed under conditions consistent with CRPC. AR-V7 is reported to regulate a transcriptional program that is similar but not identical to that of AR. However, it is unknown whether these differences are due to the unique sequence in AR-V7, or simply to loss of the LBD. To examine transcriptional regulation by AR-V7, we have used lentiviruses encoding AR-V7 (amino acids 1-627 of AR with the 16 amino acids unique to the variant) to prepare a derivative of the androgen-dependent LNCaP cells with inducible expression of AR-V7. An additional cell line was generated with regulated expression of AR-NTD (amino acids 1-660 of AR); this mutant lacks the LBD but does not have the AR-V7 specific sequence. We find that AR and AR-V7 have distinct activities on target genes that are co-regulated by FOXA1. Transcripts regulated by AR-V7 were similarly regulated by AR-NTD, indicating that loss of the LBD is sufficient for the observed differences. Differential regulation of target genes correlates with preferential recruitment of AR or AR-V7 to specific cis-regulatory DNA sequences providing an explanation for some of the observed differences in target gene regulation. PMID:25008967

Progesterone production requires activation of caspase-3 in preovulatory granulosa cells in a serum starvation model.

PubMed

An, Li-Sha; Yuan, Xiao-Hua; Hu, Ying; Shi, Zi-Yun; Liu, Xiao-Qin; Qin, Li; Wu, Gui-Qing; Han, Wei; Wang, Ya-Qin; Ma, Xu

2012-11-01

Granulosa cells proliferate, differentiate, and undergo apoptosis throughout follicular development. Previous studies have demonstrated that stimulation of progesterone production is accompanied by caspase-3 activation. Moreover, we previously reported that arsenic enhanced caspase-3 activity coupled with progesterone production. Inhibition of caspase-3 activity can significantly inhibit progesterone production induced by arsenic or follicle-stimulating hormone (FSH). Here, we report that serum starvation induces caspase-3 activation coupled with augmentation of progesterone production. Serum starvation also increased the levels of cytochrome P450 cholesterol side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein, both of which may contribute to progesterone synthesis in preovulatory granulosa cells. Inhibition of caspase-3 activity resulted in a decrease in progesterone production. Deactivation of caspase-3 activity by caspase-3 specific inhibitor also resulted in decreases in P450scc and StAR expression, which may partly contribute to the observed decrease in progesterone production. Our study demonstrates for the first time that progesterone production in preovulatory granulosa cells is required for caspase-3 activation in a serum starvation model. Inhibition of caspase-3 activity can result in decreased expression of the steroidogenic proteins P450scc and StAR. Our work provides further details on the relationship between caspase-3 activation and steroidogenesis and indicates that caspase-3 plays a critical role in progesterone production by granulosa cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Role of androgen receptor on cyclic mechanical stretch-regulated proliferation of C2C12 myoblasts and its upstream signals: IGF-1-mediated PI3K/Akt and MAPKs pathways.

PubMed

Ma, Yiming; Fu, Shaoting; Lu, Lin; Wang, Xiaohui

2017-07-15

To detect the effects of androgen receptor (AR) on cyclic mechanical stretch-modulated proliferation of C2C12 myoblasts and its pathways: roles of IGF-1, PI3K and MAPK. C2C12 were randomly divided into five groups: un-stretched control, six or 8 h of fifteen percent stretch, and six or 8 h of twenty percent stretch. Cyclic mechanical stretch of C2C12 were completed using a computer-controlled FlexCell Strain Unit. Cell proliferation and IGF-1 concentration in medium were detected by CCK8 and ELISA, respectively. Expressions of AR and IGF-1R, and expressions and activities of PI3K, p38 and ERK1/2 in stretched C2C12 cells were determined by Western blot. ①The proliferation of C2C12 cells, IGF-1 concentration in medium, expressions of AR and IGF-1R, and activities of PI3K, p38 and ERK1/2 were increased by 6 h of fifteen percent stretch, while decreased by twenty percent stretch for six or 8 h ②The fifteen percent stretch-increased proliferation of C2C12 cells was reversed by AR inhibitor, Flutamide. ③The increases of AR expression, activities of PI3K, p38 and ERK1/2 resulted from fifteen percent stretch were attenuated by IGF-1 neutralizing antibody, while twenty percent stretch-induced decreases of the above indicators were enhanced by recombinant IGF-1. ④Specific inhibitors of p38, ERK1/2 and PI3K all decreased the expression of AR in fifteen percent and twenty percent of stretched C2C12 cells. Cyclic mechanical stretch modulated the proliferation of C2C12 cells, which may be attributed to the alterations of AR via IGF-1-PI3K/Akt and IGF-1-MAPK (p38, ERK1/2) pathways in C2C12 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

High-fat diet aggravates 2,2′,4,4′-tetrabromodiphenyl ether-inhibited testosterone production via DAX-1 in Leydig cells in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Zhan; Yu, Yongquan

Growing evidence has revealed that a high-fat diet (HFD) could lead to disorders of glycolipid metabolism and insulin-resistant states, and HFDs have been associated with the inhibition of testicular steroidogenesis. Our previous study demonstrated that 2,2′,4,4′-tetrabromodiphenyl ether (BDE47) could increase the risk of diabetes in humans and reduce testosterone production in rats. However, whether the HFD affects BDE47-inhibited testosterone production by elevating insulin levels and inducing related pathways remains unknown. In male rats treated with BDE47 by gavage for 12 weeks, the HFD significantly increased the BDE47 content of the liver and testis and increased the weight of the adiposemore » tissue; increased macrovesicular steatosis in the liver and the levels of triglycerides, fasting glucose and insulin; further aggravated the disruption of the seminiferous epithelium; and lowered the level of testosterone, resulting in fewer sperm in the epididymis. Of note, the HFD enhanced BDE47-induced DAX-1 expression and decreased the expression levels of StAR and 3β-HSD in the testicular interstitial compartments in rats. In isolated primary Leydig cells from rats, BDE47 or insulin increased DAX-1 expression, decreased the expression of StAR and 3β-HSD, and reduced testosterone production, which was nearly reversed by knocking down DAX-1. These results indicated that the HFD aggravates BDE47-inhibited testosterone production through hyperinsulinemia, and the accumulation of testicular BDE47 that induces the up-regulation of DAX-1 and the subsequent down-regulation of steroidogenic proteins, i.e., StAR and 3β-HSD, in Leydig cells. - Highlights: • High-fat diet (HFD) aggravates the accumulation of BDE47 in liver and testis in rats. • HFD aggravates BDE47-inhibited testosterone production via DAX-1 in Leydig cells. • HFD enhances BDE47-induced the disorder of glycolipid metabolism and hyperinsulinemia. • Both hyperinsulinemia and accumulation of BDE47 inhibited testosterone via DAX-1.« less

Effects of resveratrol and other wine polyphenols on the proliferation, apoptosis and androgen receptor expression in LNCaP cells.

PubMed

Ferruelo, A; Romero, I; Cabrera, P M; Arance, I; Andrés, G; Angulo, J C

2014-01-01

To address the effect of resveratrol and other red wine polyphenols on cell proliferation, apoptosis and androgen receptor (AR) expression in human prostate cancer LNCaP cells. LNCaP cells (5 × 102) were cultured in microtiter plate modules and treated with gallic acid, tannic acid and quercetin (1, 5 and 10 μM), rutin and morin (25, 50 and 75 μM) and resveratrol (5, 10 and 25 μM). To address the extent of proliferation at 24, 48, 72 and 96 hours, a colorimetric immunoassay method was used. An activity caspase 3/7 detection assay was used to disclose apoptosis at 24, 48 and 72 hours. AR mARN levels were determined by real time RT-PCR. All polyphenols studied significantly inhibited (P<.05) cell proliferation compared to control. However, there were moderate differences between them. Resveratrol was the strongest inhibitor at different times and doses. Also, caspase-3 and caspase-7 activity was significantly higher (P<.05) than control in the presence of all the compounds, but the earlier response was achieved by resveratrol. Resveratrol, quercetin and morin were the only nutrients that significantly inhibited AR mRNA expression. Again resveratrol produced the highest inhibition (90-250 times less than control), followed by morin (67-100 times) and quercetin (55-91 times). All polyphenols studied showed important antiproliferative effects and induced apoptosis when added to LNCaP cells culture. We confirm that resveratrol, morin and quercetin may achieve such effect through reduced expression of AR. The synergistic effects of these compounds and their potential to prevent progression of hormone-dependent prostate cancer merit further study. Copyright © 2014 AEU. Published by Elsevier Espana. All rights reserved.

Expression of estrogen, estrogen related and androgen receptors in adrenal cortex of intact adult male and female rats.

PubMed

Trejter, Marcin; Jopek, Karol; Celichowski, Piotr; Tyczewska, Marianna; Malendowicz, Ludwik K; Rucinski, Marcin

2015-01-01

Adrenocortical activity in various species is sensitive to androgens and estrogens. They may affect adrenal cortex growth and functioning either via central pathways (CRH and ACTH) or directly, via specific receptors expressed in the cortex and/or by interfering with adrenocortical enzymes, among them those involved in steroidogenesis. Only limited data on expression of androgen and estrogen receptors in adrenal glands are available. Therefore the present study aimed to characterize, at the level of mRNA, expression of these receptors in specific components of adrenal cortex of intact adult male and female rats. Studies were performed on adult male and female (estrus) Wistar rats. Total RNA was isolated from adrenal zona glomerulosa (ZG) and fasciculate/reticularis (ZF/R). Expression of genes were evaluated by means of Affymetrix® Rat Gene 1.1 ST Array Strip and QPCR. By means of Affymetrix® Rat Gene 1.1 ST Array we examined adrenocortical sex differences in the expression of nearly 30,000 genes. All data were analyzed in relation to the adrenals of the male rats. 32 genes were differentially expressed in ZG, and 233 genes in ZF/R. In the ZG expression levels of 24 genes were lower and 8 higher in female rats. The more distinct sex differences were observed in the ZF/R, in which expression levels of 146 genes were lower and 87 genes higher in female rats. Performed analyses did not reveal sex differences in the expression levels of both androgen (AR) and estrogen (ER) receptor genes in the adrenal cortex of male and female rats. Therefore matrix data were validated by QPCR. QPCR revealed higher expression levels of AR gene both in ZG and ZF/R of male than female rats. On the other hand, QPCR did not reveal sex-related differences in the expression levels of ERα, ERβ and non-genomic GPR30 (GPER-1) receptor. Of those genes expression levels of ERα genes were the highest. In studied adrenal samples the relative expression of ERα mRNA was higher than ERβ mRNA. In adrenals of adult male and female rats expression levels of estrogen-related receptors ERRα and ERRβ were similar, and only in the ZF/R of female rats ERRγ expression levels were significantly higher than in males. We also analyzed expression profile of three isoforms of steroid 5α-reductase (Srd5a1, Srd5a2 and Srd5a3) and aromatase (Cyp19a1) and expression levels of all these genes were similar in ZG and ZF/R of male and female rats. In contrast to Affymetrix microarray data QPCR revealed higher expression levels of AR gene in adrenal glands of the male rats. In adrenals of both sexes expression levels of ERa, ERb, non-genomic GPR30 (GPER-1), ERR α and ERRβ receptors were comparable. The obtained results suggest that acute steroidogenic effect of estrogens on corticosteroid secretion may be mediated by non-genomic GPR30.

Potent antiandrogen and androgen receptor activities of an Angelica gigas-containing herbal formulation: identification of decursin as a novel and active compound with implications for prevention and treatment of prostate cancer.

PubMed

Jiang, Cheng; Lee, Hyo-Jeong; Li, Guang-xun; Guo, Junming; Malewicz, Barbara; Zhao, Yan; Lee, Eun-Ok; Lee, Hyo-Jung; Lee, Jae-Ho; Kim, Min-Seok; Kim, Sung-Hoon; Lu, Junxuan

2006-01-01

Androgen and androgen receptor (AR)-mediated signaling are crucial for the development of prostate cancer. Identification of novel and naturally occurring phytochemicals that target androgen and AR signaling from Oriental medicinal herbs holds exciting promises for the chemoprevention of this disease. In this article, we report the discovery of strong and long-lasting antiandrogen and AR activities of the ethanol extract of a herbal formula (termed KMKKT) containing Korean Angelica gigas Nakai (AGN) root and nine other Oriental herbs in the androgen-dependent LNCaP human prostate cancer cell model. The functional biomarkers evaluated included a suppression of the expression of prostate-specific antigen (PSA) mRNA and protein (IC50, approximately 7 microg/mL, 48-hour exposure) and an inhibition of androgen-induced cell proliferation through G1 arrest and of the ability of androgen to suppress neuroendocrine differentiation at exposure concentrations that did not cause apoptosis. Through activity-guided fractionation, we identified decursin from AGN as a novel antiandrogen and AR compound with an IC50 of approximately 0.4 microg/mL (1.3 micromol/L, 48-hour exposure) for suppressing PSA expression. Decursin also recapitulated the neuroendocrine differentiation induction and G1 arrest actions of the AGN and KMKKT extracts. Mechanistically, decursin in its neat form or as a component of AGN or KMKKT extracts inhibited androgen-stimulated AR translocation to the nucleus and down-regulated AR protein abundance without affecting the AR mRNA level. The novel antiandrogen and AR activities of decursin and decursin-containing herbal extracts have significant implications for the chemoprevention and treatment of prostate cancer and other androgen-dependent diseases.

Preparation of nanobubbles carrying androgen receptor siRNA and their inhibitory effects on androgen-independent prostate cancer when combined with ultrasonic irradiation.

PubMed

Wang, Luofu; Zhang, Miao; Tan, Kaibin; Guo, Yanli; Tong, Haipeng; Fan, Xiaozhou; Fang, Kejing; Li, Rui

2014-01-01

The objective of this study was to investigate nanobubbles carrying androgen receptor (AR) siRNA and their in vitro and in vivo anti-tumor effects, when combined with ultrasonic irradiation, on androgen-independent prostate cancer (AIPC). Nanobubbles carrying AR siRNA were prepared using poly-L-lysine and electrostatic adsorption methods. Using C4-2 cell activity as a testing index, the optimal irradiation parameters (including the nanobubble number/cell number ratio, mechanical index [MI], and irradiation time) were determined and used for transfection of three human prostate cancer cell lines (C4-2, LNCaP, and PC-3 cells). The AR expression levels were investigated with RT-PCR and Western blot analysis. Additionally, the effects of the nanobubbles and control microbubbles named SonoVue were assessed via imaging in a C4-2 xenograft model. Finally, the growth and AR expression of seven groups of tumor tissues were assessed using the C4-2 xenograft mouse model. The nanobubbles had an average diameter of 609.5±15.6 nm and could effectively bind to AR siRNA. Under the optimized conditions of a nanobubble number/cell number ratio of 100∶1, an MI of 1.2, and an irradiation time of 2 min, the highest transfection rates in C4-2, LNCaP, and PC-3 cells were 67.4%, 74.0%, and 63.96%, respectively. In the C4-2 and LNCaP cells, treatment with these binding nanobubbles plus ultrasonic irradiation significantly inhibited cell growth and resulted in the suppression of AR mRNA and protein expression. Additionally, contrast-enhanced ultrasound showed that the nanobubbles achieved stronger signals than the SonoVue control in the central hypovascular area of the tumors. Finally, the anti-tumor effect of these nanobubbles plus ultrasonic irradiation was most significant in the xenograft tumor model compared with the other groups. Nanobubbles carrying AR siRNA could be potentially used as gene vectors in combination with ultrasonic irradiation for the treatment of AIPC.

Preparation of Nanobubbles Carrying Androgen Receptor siRNA and Their Inhibitory Effects on Androgen-Independent Prostate Cancer when Combined with Ultrasonic Irradiation

PubMed Central

Tan, Kaibin; Guo, Yanli; Tong, Haipeng; Fan, Xiaozhou; Fang, Kejing; Li, Rui

2014-01-01

Objective The objective of this study was to investigate nanobubbles carrying androgen receptor (AR) siRNA and their in vitro and in vivo anti-tumor effects, when combined with ultrasonic irradiation, on androgen-independent prostate cancer (AIPC). Materials and Methods Nanobubbles carrying AR siRNA were prepared using poly-L-lysine and electrostatic adsorption methods. Using C4-2 cell activity as a testing index, the optimal irradiation parameters (including the nanobubble number/cell number ratio, mechanical index [MI], and irradiation time) were determined and used for transfection of three human prostate cancer cell lines (C4-2, LNCaP, and PC-3 cells). The AR expression levels were investigated with RT-PCR and Western blot analysis. Additionally, the effects of the nanobubbles and control microbubbles named SonoVue were assessed via imaging in a C4-2 xenograft model. Finally, the growth and AR expression of seven groups of tumor tissues were assessed using the C4-2 xenograft mouse model. Results The nanobubbles had an average diameter of 609.5±15.6 nm and could effectively bind to AR siRNA. Under the optimized conditions of a nanobubble number/cell number ratio of 100∶1, an MI of 1.2, and an irradiation time of 2 min, the highest transfection rates in C4-2, LNCaP, and PC-3 cells were 67.4%, 74.0%, and 63.96%, respectively. In the C4-2 and LNCaP cells, treatment with these binding nanobubbles plus ultrasonic irradiation significantly inhibited cell growth and resulted in the suppression of AR mRNA and protein expression. Additionally, contrast-enhanced ultrasound showed that the nanobubbles achieved stronger signals than the SonoVue control in the central hypovascular area of the tumors. Finally, the anti-tumor effect of these nanobubbles plus ultrasonic irradiation was most significant in the xenograft tumor model compared with the other groups. Conclusion Nanobubbles carrying AR siRNA could be potentially used as gene vectors in combination with ultrasonic irradiation for the treatment of AIPC. PMID:24798477

The expression status of TRX, AR, and cyclin D1 correlates with clinicopathological characteristics and ER status in breast cancer.

PubMed

Huang, Weisun; Nie, Weiwei; Zhang, Wenwen; Wang, Yanru; Zhu, Aiyu; Guan, Xiaoxiang

2016-01-01

The ER signaling pathway plays a critical role in breast cancer. ER signaling pathway-related proteins, such as TRX, AR, and cyclin D1, may have an important function in breast cancer. However, the ways that they influence breast cancer development and progression are still unclear. A total of 101 Chinese female patients diagnosed with invasive ductal breast adenocarcinoma were retrospectively enrolled in the study. The expression levels of TRX, AR, and cyclin D1 were detected by immunohistochemistry and analyzed via correlation with clinicopathological characteristics and the expression status of ER, PR, and HER2. The expression status of TRX, AR, and cyclin D1 was not associated with the patient's age, menopausal status, tumor size, or histological differentiation (P>0.05), but was positively correlated with ER and PR (P<0.001, respectively). Most (66/76, 86.8) TRX-positive patients were also HER2-positive (P=0.003). Of AR- or cyclin D1-positive patients, most had relatively earlier I-II tumor stage (P=0.005 and P=0.047, respectively) and no metastatic lymph node involvement (P=0.008 and P=0.005, respectively). TRX was found to be positively correlated with ER and PR expression, whereas it was negatively correlated with HER2 expression. In addition, we found that the positive expression of AR and cyclin D1 was correlated with lower TNM stage and fewer metastatic lymph nodes, and it was more common in ER-positive breast cancer than in the basal-like subtype. This may indicate that AR and cyclin D1 are good predictive and prognostic factors and closely interact with ER signaling pathway. Further studies will be necessary to investigate the response and clinical outcomes of treatment targeting TRX, AR, and cyclin D1.

Androgen receptor (AR) suppresses miRNA-145 to promote renal cell carcinoma (RCC) progression independent of VHL status

PubMed Central

Chen, Yuan; Sun, Yin; Rao, Qun; Xu, Hua; Li, Lei; Chang, Chawnshang

2015-01-01

Mutational inactivation of the VHL tumor suppressor plays key roles in the development of renal cell carcinoma (RCC), and mutated VHL-mediated VEGF induction has become the main target for the current RCC therapy. Here we identified a signal pathway of VEGF induction by androgen receptor (AR)/miRNA-145 as a new target to suppress RCC progression. Mechanism dissection revealed that AR might function through binding to the androgen receptor element (ARE) located on the promoter region of miRNA-145 to suppress p53's ability to induce expression of miRNA-145 that normally suppresses expression of HIF2α/VEGF/MMP9/CCND1. Suppressing AR with AR-shRNA or introducing exogenous miRNA-145 mimic can attenuate RCC progression independent of VHL status. MiR-145 mimic in preclinical RCC orthotopic xenograft mouse model revealed its efficacy in suppression of RCC progression. These results together identified signals by AR-suppressed miRNA-145 as a key player in the RCC progression via regulating HIF2α/VEGF/MMP9/CCND1 expression levels. Blockade of the newly identified signal by AR inhibition or miRNA-145 mimics has promising therapeutic benefit to suppress RCC progression. PMID:26304926

Role of androgen receptor and associated lysine-demethylase coregulators, LSD1 and JMJD2A, in localized and advanced human bladder cancer.

PubMed

Kauffman, Eric C; Robinson, Brian D; Downes, Martin J; Powell, Leagh G; Lee, Ming Ming; Scherr, Douglas S; Gudas, Lorraine J; Mongan, Nigel P

2011-12-01

Bladder cancer is approximately three times more common in men as compared to women. We and others have previously investigated the contribution of androgens and the androgen receptor (AR) to bladder cancer. JMJD2A and LSD1 are recently discovered AR coregulator proteins that mediate AR-dependent transcription via recently described histone lysine-demethylation (KDM) mechanisms. We used immunohistochemistry to examine JMJD2A, LSD1, and AR expression in 72 radical cystectomy specimens, resulting in evaluation of 129 tissue samples (59 urothelial carcinoma, 70 benign). We tested levels of these proteins for statistical association with clinicopathologic variables and patient survival. Expression of these markers was also assessed in human bladder cancer cell lines. The effects of pharmacological inhibition of LSD1 on the proliferation of these bladder cancer cells was determined. JMJD2A and AR levels were significantly lower in malignant versus benign urothelium, while increased LSD1 levels were observed in malignant urothelium relative to benign. A significant reduction in all three proteins occurred with cancer stage progression, including muscle invasion (JMJD2A/LSD1/AR), extravesical extension (JMJD2A/LSD1), and lymph node metastasis (JMJD2A/AR). Lower JMJD2A intensity correlated with additional poor prognostic features, including lymphovascular invasion, concomitant carcinoma in situ and tobacco usage, and predicted significantly worse overall survival. Pharmacological inhibition of LSD1 suppressed bladder cancer cell proliferation and androgen-induced transcription. Our results support a novel role for the AR-KDM complex in bladder cancer initiation and progression, identify JMJD2A as a promising prognostic biomarker, and demonstrate targeting of the KDM activity as an effective potential approach for bladder cancer growth inhibition. Copyright © 2011 Wiley Periodicals, Inc.

The Steroidogenic Acute Regulatory Protein (StAR) Is Regulated by the H19/let-7 Axis.

PubMed

Men, Yi; Fan, Yanhong; Shen, Yuanyuan; Lu, Lingeng; Kallen, Amanda N

2017-02-01

The steroidogenic acute regulatory protein (StAR) governs the rate-limiting step in steroidogenesis, and its expression varies depending on the needs of the specific tissue. Tight control of steroid production is essential for multiple processes involved in reproduction, including follicular development, ovulation, and endometrial synchronization. Recently, there has been a growing interest in the role of noncoding RNAs in the regulation of reproduction. Here we demonstrate that StAR is a novel target of the microRNA let-7, which itself is regulated by the long noncoding RNA (lncRNA) H19. Using human and murine cell lines, we show that overexpression of H19 stimulates StAR expression by antagonizing let-7, which inhibits StAR at the post-transcriptional level. Our results uncover a novel mechanism underlying the regulation of StAR expression and represent the first example of lncRNA-mediated control of the rate-limiting step of steroidogenesis. This work thus adds to the body of literature describing the multiple roles in oncogenesis, cellular growth, glucose metabolism, and now regulation of steroidogenesis, of this complex lncRNA. Copyright © 2017 by the Endocrine Society.

IL-18 cleavage triggers cardiac inflammation and fibrosis upon β-adrenergic insult.

PubMed

Xiao, Han; Li, Hao; Wang, Jing-Jing; Zhang, Jian-Shu; Shen, Jing; An, Xiang-Bo; Zhang, Cong-Cong; Wu, Ji-Min; Song, Yao; Wang, Xin-Yu; Yu, Hai-Yi; Deng, Xiang-Ning; Li, Zi-Jian; Xu, Ming; Lu, Zhi-Zhen; Du, Jie; Gao, Wei; Zhang, Ai-Hua; Feng, Yue; Zhang, You-Yi

2018-01-01

Rapid over-activation of β-adrenergic receptor (β-AR) upon stress leads to cardiac inflammation, a prevailing factor that underlies heart injury. However, mechanisms by which acute β-AR stimulation induce cardiac inflammation still remain unknown. Here, we set out to identify the crucial role of inflammasome/interleukin (IL)-18 in initiating and maintaining cardiac inflammatory cascades upon β-AR insult. Male C57BL/6 mice were injected with a single dose of β-AR agonist, isoproterenol (ISO, 5 mg/kg body weight) or saline subcutaneously. Cytokine array profiling demonstrated that chemokines dominated the initial cytokines upregulation specifically within the heart upon β-AR insult, which promoted early macrophage infiltration. Further investigation revealed that the rapid inflammasome-dependent activation of IL-18, but not IL-1β, was the critical up-stream regulator for elevated chemokine expression in the myocardium upon ISO induced β1-AR-ROS signalling. Indeed, a positive correlation was observed between the serum levels of norepinephrine and IL-18 in patients with chest pain. Genetic deletion of IL-18 or the up-stream inflammasome component NLRP3 significantly attenuated ISO-induced chemokine expression and macrophage infiltration. In addition, IL-18 neutralizing antibodies selectively abated ISO-induced chemokines, proinflammatory cytokines and adhesion molecules but not growth factors. Moreover, blocking IL-18 early after ISO treatment effectively attenuated cardiac inflammation and fibrosis. Inflammasome-dependent activation of IL-18 within the myocardium upon acute β-AR over-activation triggers cytokine cascades, macrophage infiltration and pathological cardiac remodelling. Blocking IL-18 at the early stage of β-AR insult can successfully prevent inflammatory responses and cardiac injuries. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.

Suppression of LIM and SH3 Domain Protein 1 (LASP1) Negatively Regulated by Androgen Receptor Delays Castration Resistant Prostate Cancer Progression.

PubMed

Dejima, Takashi; Imada, Kenjiro; Takeuchi, Ario; Shiota, Masaki; Leong, Jeffrey; Tombe, Tabitha; Tam, Kevin; Fazli, Ladan; Naito, Seiji; Gleave, Martin E; Ong, Christopher J

2017-02-01

LIM and SH3 domain protein 1 (LASP1) has been implicated in several human malignancies and has been shown to predict PSA recurrence in prostate cancer. However, the anti-tumor effect of LASP1 knockdown and the association between LASP1 and the androgen receptor (AR) remains unclear. The aim of this study is to clarify the significance of LASP1 as a target for prostate cancer, and to test the effect of silencing LASP1 in vivo using antisense oligonucleotides (ASO). A tissue microarray (TMA) was performed to characterize the differences in LASP1 expression in prostate cancer treated after hormone deprivation therapy. Flow cytometry was used to analyze cell cycle. We designed LASP1 ASO for knockdown of LASP1 in vivo studies. The expression of LASP1 in TMA was increased after androgen ablation and persisted in castration resistant prostate cancer (CRPC). Also in TMA, compared with LNCaP cell, LASP1 expression is elevated in CRPC cell lines (C4-2 and VehA cells). Interestingly, suppression of AR elevated LASP1 expression conversely, AR activation decreased LASP1 expression. Silencing of LASP1 reduced cell growth through G1 arrest which was accompanied by a decrease of cyclin D1. Forced overexpression of LASP1 promoted cell cycle and induced cell growth which was accompanied by an increase of cyclin D1. Systemic administration of LASP1 ASO with athymic mice significantly inhibited tumor growth in CRPC xenografts. These results indicate that LASP1 is negatively regulated by AR at the transcriptional level and promotes tumor growth through induction of cell cycle, ultimately suggesting that LASP1 may be a potential target in prostate cancer treatment. Prostate 77:309-320, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The RNA binding protein Ars2 supports hematopoiesis at multiple levels.

PubMed

Elahi, Seerat; Egan, Shawn M; Holling, G Aaron; Kandefer, Rachel L; Nemeth, Michael J; Olejniczak, Scott H

2018-05-15

Recent biochemical characterization of Arsenic resistance protein 2 (Ars2) has established it as central to determining the fate of nascent RNA polymerase II (RNAPII) transcripts. Through interactions with the nuclear 5'-7-methylguanosine (7mG) cap binding complex (CBC), Ars2 promotes co-transcriptional processing coupled with nuclear export or degradation of several classes of RNAPII transcripts, allowing for gene expression programs that facilitate rapid and sustained proliferation of immortalized cells in culture. However, rapidly dividing cells in culture do not represent the physiological condition of the vast majority of cells in an adult mammal. To examine functions of Ars2 in a physiological setting we generated inducible Ars2 knockout mice and found that deletion of Ars2 from adult mice resulted in defective hematopoiesis in bone marrow and thymus. Importantly, only some of this defect could be explained by the requirement of Ars2 for rapid proliferation, which we found to be cell-type specific in vivo. Rather Ars2 was required for survival of developing thymocytes and for limiting differentiation of bone marrow resident long-term hematopoietic stem cells (LT-HSCs). As a result, Ars2 knockout led to rapid thymic involution and loss of the ability of mice to regenerate peripheral blood following myeloablation. These in vivo data demonstrate that Ars2 expression is important at several steps of hematopoiesis, likely because Ars2 acts on gene expression programs underlying essential cell fate decisions such as the decision to die, to proliferate, or to differentiate. Copyright © 2018. Published by Elsevier Inc.

Estrogen induces androgen-repressed SOX4 expression to promote progression of prostate cancer cells.

PubMed

Yang, Muyi; Wang, Jing; Wang, Lin; Shen, Chengwu; Su, Bo; Qi, Mei; Hu, Jing; Gao, Wei; Tan, Weiwei; Han, Bo

2015-09-01

The sex determing region Y-box 4 (SOX4) gene is a critical developmental transcriptional factor that is overexpressed in prostate cancer (PCa). While we and others have investigated the role of SOX4 overexpression in PCa, the molecular mechanism underlying its aberrant expression remains unclear. Immunohistochemistry were utilized to detect SOX4 expression and the correlation between estrogen receptor β (ERβ), androgen receptor (AR) and SOX4 in a cohort of 94 clinical specimens. Real-time quantitative PCR and Western blotting were used to study the transcript and protein expression levels. Immunofluorescence staining and co-immunoprecipitation were performed to assess the interaction and subcellular location of ERβ and AR. Chromatin immunoprecipitation (ChIP) assays and Luciferase reporter assays were performed to explore the binding and transcriptional activities of ERβ and AR to the SOX4 promoter. Cellular function was evaluated by MTS, invasion and wound healing assays. SOX4 expression is up-regulated in Castration-Resistant Prostate Cancer (CRPC) tumors compared to hormone-dependent PCa (HDPC) cases. Increased expression was also observed in PCa cells after long-term androgen-deprivation treatment (ADT). In vitro data indicated that SOX4 is an AR transcriptional target and down-regulated by dihydrotestosterone (DHT) via AR. 17β-estradiol (E2) up-regulates SOX4 expression in the absence of androgen through the formation of a protein complex between ERβ and AR. Knockdown of AR or ERβ blocks the E2-induced SOX4 expression. ChIP assays confirmed that both ERβ and AR bind to the SOX4 promoter in response to E2. Functionally, silencing SOX4 significantly attenuates the proliferative effect, as well as the capacity of migration and invasion of E2 on PCa cells. Clinically, overexpression of SOX4 is significantly associated with ERβ expression in PCa. In addition, this association is still retained in CRPC patients with poor prognosis. These findings suggest that SOX4 is a novel DHT-repressed AR-target gene. E2 could promote proliferation of PCa cells through the up-regulation of SOX4 under androgen-depleted environment. Our data provides a possible molecular basis for the overexpression of SOX4 in CRPC and may facilitate the detection and prevention of the emergence of CRPC. © 2015 Wiley Periodicals, Inc.

17β-estradiol exerts anticancer effects in anoikis-resistant hepatocellular carcinoma cell lines by targeting IL-6/STAT3 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Seulki, E-mail: sl10f@naver.com; Lee, Minjong, E-mail: minjonglee2@naver.com; Kim, Jong Bin, E-mail: kkimjp@hanmail.net

17β-Estradiol (E2) has been proven to exert protective effects against HCC; however, its mechanism on HCC proliferation and suppression of invasion remains to be further explored. Because HCC up-regulates serum Interleukin-6 (IL-6) levels and Signal Transducer and Activator of Transcription 3 (STAT3), molecular agents that attenuate IL-6/STAT3 signaling can potentially suppress HCC development. In this study, we examined involvement of E2 in anoikis resistance that induces invasion capacities and chemo-resistance. Huh-BAT and HepG2 cells grown under anchorage-independent condition were selected. The anoikis-resistant (AR) cells showed stronger chemo-resistance against sorafenib, doxorubicin, 5-fluorouracil and cisplatin compared to adherent HCC cells. AR HCCmore » cells exhibited decreased expression of E-cadherin and increased expression of the N-cadherin and vimentin compared to adherent HCC cells. We then demonstrated that E2 suppressed cell proliferation in AR HCC cells. IL-6 treatment enhanced invasive characteristics, and E2 reversed it. Regarding mechanism of E2, it decreased in the phosphorylation of STAT3 that overexpressed on AR HCC cells. The inhibitory effect of E2 on cell growth was accompanied with cell cycle arrest at G2/M phase and caspase-3/9/PARP activation through c-Jun N-terminal Kinase (JNK) phosphorylation. Taken together, these findings suggested that E2 inhibited the proliferation of AR HCC cells through down-regulation of IL-6/STAT3 signaling. Thus, E2 can be a potential therapeutic drug for treatment of metastatic or chemo-resistant HCC. -- Highlights: •Anoikis-resistant HCC cells characterized chemo-resistant and metastatic potentials. •17β-Estradiol down-regulated IL-6/STAT3 signaling in anoikis-resistant HCC cells. •17β-Estradiol suppressed cell proliferation by inducing G2/M phase arrest and apoptosis though JNK phosphorylation.« less

The protective effect of Hif3a RNA interference and HIF-prolyl hydroxylase inhibition on cardiomyocytes under anoxia-reoxygenation.

PubMed

Drevytska, T; Gonchar, E; Okhai, I; Lynnyk, O; Mankovska, I; Klionsky, D; Dosenko, V

2018-06-01

The aim of this study was to investigate the molecular mechanisms underlying the protective effects of hypoxia-inducible factor (HIF) signaling pathway activation in cardiomyocytes under anoxia-reoxygenation (A/R) injury. In this study, rat neonatal cardiomyocytes were pretreated with anti-Hif3A/Hif-3α siRNA or HIF-prolyl hydroxylase inhibitor prior to A/R injury. Our results showed that both HIF3A silencing and HIF-prolyl hydroxylase inhibition effectively increased the cell viability during A/R, led to changes in mRNA expression of HIF1-target genes, and reduced the loss of mitochondrial membrane potential (Δψ m ). Furthermore, application of anti-Hif3a siRNA led to an increase in mRNA expression of Epo, Igf1, Slc2a1/Glut-1, and Slc2a4/Glut-4. Similar results were observed with HIF-prolyl hydroxylase inhibition, which additionally upregulated the mRNA expression of Epor, Tert, and Pdk1. Hif3a RNA-interference and application of HIF-prolyl hydroxylase inhibitor during A/R modelling led to an increase of Δψ m on 11.5 and 11.9 mV respectively, compared to the control groups. Thus, Hif3a RNA interference and HIF-prolyl hydroxylase inhibition protect cardiomyocytes against A/R injury via the HIF signaling pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

Antiandrogenic mechanisms of pesticides in human LNCaP prostate and H295R adrenocortical carcinoma cells.

PubMed

Robitaille, Christina N; Rivest, Patricia; Sanderson, J Thomas

2015-01-01

Several pesticides suspected or known to have endocrine disrupting effects were screened for pro- or antiandrogenic properties by determining their effects on proliferation, prostatic-specific antigen (PSA) secretion and androgen receptor (AR) expression, and AR phosphorylation in androgen-dependent LNCaP human prostate cancer cells, as well as on the expression and catalytic activity of the enzyme CYP17 in H295R human adrenocortical carcinoma cells, an in vitro model of steroidogenesis. Effects on SRD5A gene expression were determined in both cell lines. Benomyl, vinclozolin, and prochloraz, but not atrazine, concentration dependently (1-30 μM) decreased dihydrotestosterone (DHT)-stimulated proliferation of LNCaP cells. All pesticides except atrazine decreased DHT-stimulated PSA secretion, AR nuclear accumulation, and AR phosphorylation on serines 81 and 213 in LNCaP cells. Benomyl and prochloraz, but not vinclozolin or atrazine, decreased levels of CYP17 gene and protein expression, as well as catalytic activity in H295R cells. In the case of prochloraz, some of these effects corresponded with cytotoxicity. H295R cells expressed AR protein and SRD5A1, but not SRD5A2 transcripts. SRD5A1 gene expression in H295R cells was increased by 10 nM DHT, whereas in LNCaP cells significant induction was observed by 0.1 nM DHT. AR protein expression in H295R cells was not increased by DHT. Vinclozolin decreased DHT-induced SRD5A1 gene expression in LNCaP, but not H295R cells, indicating a functional difference of AR between the cell lines. In conclusion, pesticides may exert antiandrogenic effects through several mechanisms that are cell type-specific, including AR antagonism and down-regulation or catalytic inhibition of androgen biosynthetic enzymes, such as CYP17 and SRD5A1. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Autocrine Complement Inhibits IL10-Dependent T-Cell Mediated Antitumor Immunity to Promote Tumor Progression

PubMed Central

Wang, Yu; Sun, Sheng-Nan; Liu, Qing; Yu, Yang-Yang; Guo, Jian; Wang, Kun; Xing, Bao-Cai; Zheng, Qing-Feng; Campa, Michael J.; Patz, Edward F.; Li, Shi-You; He, You-Wen

2016-01-01

In contrast to its inhibitory effects on many cells, IL-10 activates CD8+ tumor infiltrating lymphocytes (TILs) and enhances their antitumor activity. However, CD8+ TILs do not routinely express IL-10 as autocrine complement C3 inhibits IL-10 production through complement receptors C3aR and C5aR. CD8+ TILs from C3-deficient mice, however, express IL-10 and exhibit enhanced effector function. C3-deficient mice are resistant to tumor development in a T cell- and IL-10-dependent manner; human TILs expanded with IL-2 plus IL-10 increase the killing of primary tumors in vitro compared to IL-2 treated TILs. Complement-mediated inhibition of antitumor immunity is independent of the PD-1/PD-L1 immune checkpoint pathway. Our findings suggest that complement receptors C3aR and C5aR expressed on CD8+ TILs represent a novel class of immune checkpoints that could be targeted for tumor immunotherapy. Moreover, incorporation of IL-10 in the expansion of TILs and in gene-engineered T cells for adoptive cell therapy enhances their antitumor efficacy. PMID:27297552

Indole-3-butyric acid promotes adventitious rooting in Arabidopsis thaliana thin cell layers by conversion into indole-3-acetic acid and stimulation of anthranilate synthase activity.

PubMed

Fattorini, L; Veloccia, A; Della Rovere, F; D'Angeli, S; Falasca, G; Altamura, M M

2017-07-11

Indole-3-acetic acid (IAA), and its precursor indole-3-butyric acid (IBA), control adventitious root (AR) formation in planta. Adventitious roots are also crucial for propagation via cuttings. However, IBA role(s) is/are still far to be elucidated. In Arabidopsis thaliana stem cuttings, 10 μM IBA is more AR-inductive than 10 μM IAA, and, in thin cell layers (TCLs), IBA induces ARs when combined with 0.1 μM kinetin (Kin). It is unknown whether arabidopsis TCLs produce ARs under IBA alone (10 μM) or IAA alone (10 μM), and whether they contain endogenous IAA/IBA at culture onset, possibly interfering with the exogenous IBA/IAA input. Moreover, it is unknown whether an IBA-to-IAA conversion is active in TCLs, and positively affects AR formation, possibly through the activity of the nitric oxide (NO) deriving from the conversion process. Revealed undetectable levels of both auxins at culture onset, showing that arabidopsis TCLs were optimal for investigating AR-formation under the total control of exogenous auxins. The AR-response of TCLs from various ecotypes, transgenic lines and knockout mutants was analyzed under different treatments. It was shown that ARs are better induced by IBA than IAA and IBA + Kin. IBA induced IAA-efflux (PIN1) and IAA-influx (AUX1/LAX3) genes, IAA-influx carriers activities, and expression of ANTHRANILATE SYNTHASE -alpha1 (ASA1), a gene involved in IAA-biosynthesis. ASA1 and ANTHRANILATE SYNTHASE -beta1 (ASB1), the other subunit of the same enzyme, positively affected AR-formation in the presence of exogenous IBA, because the AR-response in the TCLs of their mutant wei2wei7 was highly reduced. The AR-response of IBA-treated TCLs from ech2ibr10 mutant, blocked into IBA-to-IAA-conversion, was also strongly reduced. Nitric oxide, an IAA downstream signal and a by-product of IBA-to-IAA conversion, was early detected in IAA- and IBA-treated TCLs, but at higher levels in the latter explants. Altogether, results showed that IBA induced AR-formation by conversion into IAA involving NO activity, and by a positive action on IAA-transport and ASA1/ASB1-mediated IAA-biosynthesis. Results are important for applications aimed to overcome rooting recalcitrance in species of economic value, but mainly for helping to understand IBA involvement in the natural process of adventitious rooting.

ArI/ArII laser induced fluorescence system for measurement of neutral and ion dynamics in a large scale helicon plasma.

PubMed

Kelly, R F; Meaney, K D; Gilmore, M; Desjardins, T R; Zhang, Y

2016-11-01

In order to investigate the role of both neutral and ion dynamics in large-scale helicon discharges, a laser induced fluorescence (LIF) system capable of measuring both ArI and ArII fluorescence using a single tunable laser is being developed. The system is based on a >250 mW solid state laser. For ArI measurements, the laser pumps the metastable ( 2 P 0 3/2 )4s level to the ( 2 P 0 1/2 )4p level using 696.7352 nm light, and fluorescence radiation from decay to the ( 2 P 0 1/2 )4s level at 772.6333 nm is observed. For ArII, currently in development, the metastable ( 3 P)3d 4 F 7/2 level will be pumped to the ( 3 P)4p 4 D 0 7/2 level using 688.8511 nm, and decay fluorescence to the ( 3 P)4s 4 P 5/2 level at 434.9285 nm measured. Here all wavelengths are in a vacuum.

Zebrafish (Danio rerio) androgen receptor: sequence homology and up-regulation by the fungicide vinclozolin.

PubMed

Smolinsky, Amanda N; Doughman, Jennifer M; Kratzke, Liên-Thành C; Lassiter, Christopher S

2010-03-01

Steroid hormones regulate gene expression in organisms by binding to receptor proteins. These hormones include the androgens, which signal through androgen receptors (ARs). Endocrine disrupters (EDCs) are chemicals in the environment that adversely affect organisms by binding to nuclear receptors, including ARs. Vinclozolin, a fungicide used on fruit and vegetable crops, is a known anti-androgen, a type of EDC that blocks signals from testosterone and its derivatives. In order to better understand the effects of EDCs, further research on androgen receptors and other hormone signaling pathways is necessary. In this study, we demonstrate the evolutionary conservation between the genomic structure of the human and zebrafish ar genes and find that ar mRNA expression increases in zebrafish embryos exposed to vinclozolin, which may be evolutionarily conserved as well. At 48 and 72 h post-fertilization, vinclozolin-treated embryos express ar mRNA 8-fold higher than the control level. These findings suggest that zebrafish embryos attempt to compensate for the presence of an anti-androgen by increasing the number of androgen receptors available.

Ralstonia solanacearum novel E3 ubiquitin ligase (NEL) effectors RipAW and RipAR suppress pattern-triggered immunity in plants.

PubMed

Nakano, Masahito; Oda, Kenji; Mukaihara, Takafumi

2017-07-01

Ralstonia solanacearum is the causal agent of bacterial wilt in solanaceous crops. This pathogen injects more than 70 effector proteins into host plant cells via the Hrp type III secretion system to cause a successful infection. However, the function of these effectors in plant cells, especially in the suppression of plant immunity, remains largely unknown. In this study, we characterized two Ralstonia solanacearum effectors, RipAW and RipAR, which share homology with the IpaH family of effectors from animal and plant pathogenic bacteria, that have a novel E3 ubiquitin ligase (NEL) domain. Recombinant RipAW and RipAR show E3 ubiquitin ligase activity in vitro. RipAW and RipAR localized to the cytoplasm of plant cells and significantly suppressed pattern-triggered immunity (PTI) responses such as the production of reactive oxygen species and the expression of defence-related genes when expressed in leaves of Nicotiana benthamiana. Mutation in the conserved cysteine residue in the NEL domain of RipAW completely abolished the E3 ubiquitin ligase activity in vitro and the ability to suppress PTI responses in plant leaves. These results indicate that RipAW suppresses plant PTI responses through the E3 ubiquitin ligase activity. Unlike other members of the IpaH family of effectors, RipAW and RipAR had no leucine-rich repeat motifs in their amino acid sequences. A conserved C-terminal region of RipAW is indispensable for PTI suppression. Transgenic Arabidopsis plants expressing RipAW and RipAR showed increased disease susceptibility, suggesting that RipAW and RipAR contribute to bacterial virulence in plants.

A synthetic decursin analog with increased in vivo stability suppresses androgen receptor signaling in vitro and in vivo.

PubMed

Zhang, Yong; Shaik, Ahmad Ali; Xing, Chengguo; Chai, Yubo; Li, Li; Zhang, Jinhui; Zhang, Wei; Kim, Sung-Hoon; Lü, Junxuan; Jiang, Cheng

2012-10-01

Targeting androgen receptor (AR) signaling with agents distinct from current antagonist drugs remains a rational approach to the prevention and treatment of prostate cancer (PCa). Our previous studies have shown that decursin and isomer decursinol angelate (DA), isolated from the Korean medicinal herb Angelica gigas Nakai, interrupt AR signaling and possess anti-PCa activities in vitro. In the LNCaP PCa cell model, these pyranoccoumarin compounds exhibit properties distinct from currently used antagonists (e.g., Casodex). However, both are rapidly de-esterified to decursinol, a partial AR agonist. We report here that a synthetic decursin analog, decursinol phenylthiocarbamate (DPTC), has greater in vivo stability than the parent compounds. DPTC-decursinol conversion was undetectable in mice. Furthermore, in LNCaP cells, DPTC decreased prostate specific antigen (PSA) expression, down-regulated AR abundance and mRNA and inhibited AR nuclear translocation. The effect of DPTC on AR and PSA mRNA and protein abundance was also observed in VCaP cells expressing wild type AR. DPTC inhibited growth of both PCa cell lines through G(1) cell cycle arrest and apoptosis, as did decursin and DA. Furthermore, i.p. administration of DPTC for 3 weeks suppressed the expression of AR target genes probasin and Nkx3.1 in mouse prostate glands. Overall, our data suggest that DPTC represents a prototype lead compound for development of in vivo stable and active novel decursin analogs for the prevention or therapy of PCa.

Functional analysis of the sea urchin-derived arylsulfatase (Ars)-element in mammalian cells.

PubMed

Watanabe, Satoshi; Watanabe, Sachiko; Sakamoto, Naoaki; Sato, Masahiro; Akasaka, Koji

2006-09-01

An insulator is a DNA sequence that has both enhancer-blocking activity, through its ability to modify the influence of neighboring cis-acting elements, and a barrier function that protects a transgene from being silenced by surrounding chromatin. Previously, we isolated and characterized a 582-bp-long element from the sea urchin arylsulfatase gene (Ars). This Ars-element was effective in sea urchin and Drosophila embryos and in plant cells. To investigate Ars-element activity in mammalian cells, we placed the element between the cytomegalovirus enhancer and a luciferase (luc) expression cassette. In contrast to controls lacking the Ars-element, NIH3T3 and 293T cells transfected with the element-containing construct displayed reduced luciferase activities. The Ars-element therefore acts as an enhancer-blocking element in mammalian cells. We assessed the barrier activity of the Ars-element using vectors in which a luc expression cassette was placed between two elements. Transfection experiments demonstrated that luc activity in these vectors was approximately ten-fold higher than in vectors lacking elements. Luc activities were well maintained even after 12 weeks in culture. Our observations demonstrate that the Ars-element has also a barrier activity. These results indicated that the Ars-element act as an insulator in mammalian cells.

Partial agonist clonidine mediates alpha(2)-AR subtypes specific regulation of cAMP accumulation in adenylyl cyclase II transfected DDT1-MF2 cells.

PubMed

Limon-Boulez, I; Bouet-Alard, R; Gettys, T W; Lanier, S M; Maltier, J P; Legrand, C

2001-02-01

alpha2-Adrenergic receptor (alpha(2)-AR) activation in the pregnant rat myometrium at midterm potentiates beta(2)-AR stimulation of adenylyl cyclase (AC) via Gbetagamma regulation of the type II isoform of adenylyl cyclase. However, at term, alpha(2)-AR activation inhibits beta(2)-AR stimulation of AC. This phenomenon is associated with changes in alpha(2)-AR subtype expression (midterm alpha(2A/D)-AR > alpha(2B)-AR; term alpha(2B) >or =alpha(2A/D)-AR), without any change in ACII mRNA, suggesting that alpha(2A/D)- and alpha(2B)-AR differentially regulate beta(2)-cAMP production. To address this issue, we have stably expressed the same density of alpha(2A/D)- or alpha(2B)-AR with AC II in DDT1-MF2 cells. Clonidine (partial agonist) increased beta(2)-AR-stimulated cAMP production in alpha(2A/D)-AR-ACII transfectants but inhibited it in alpha(2B)-AR-ACII transfectants. In contrast, epinephrine (full agonist) enhanced beta(2)-stimulated ACII in both alpha(2A)- and alpha(2B)-ACII clonal cell lines. 4-Azidoanilido-[alpha-(32)P]GTP-labeling of activated G proteins indicated that, in alpha(2B)-AR transfectants, clonidine activated only Gi(2), whereas epinephrine, the full agonist, effectively coupled to Gi(2) and Gi(3). Thus, partial and full agonists selectively activate G proteins that lead to drug specific effects on effectors. Moreover, these data indicate that Gi(3) activation is required for potentiation of beta(2)-AR stimulation of AC by alpha(2A/D) and alpha(2B)-AR in DDT1-MF2 cells. This may reflect an issue of the amount of Gbetagamma released upon receptor activation and/or betagamma composition of Gi(3) versus Gi(2).

Exogenous galanin attenuates spatial memory impairment and decreases hippocampal β-amyloid levels in rat model of Alzheimer's disease.

PubMed

Li, Lei; Yu, Liling; Kong, Qingxia

2013-11-01

One of the major pathological characteristics of Alzheimer's disease (AD) is the presence of enhanced deposits of beta-amyloid peptide (Aβ). The neuropeptide galanin (GAL) and its receptors are overexpressed in degenerating brain regions in AD. The functional consequences of galaninergic systems plasticity in AD are unclear. The objective of the present study was to investigate whether exogenous galanin could attenuate spatial memory impairment and hippocampal Aβ aggregation in rat model of AD. The effects of Aβ, galanin, galanin receptor 1 agonist M617 and galanin receptor 2 agonist AR-M1896 on spatial memory were tested by Morris water maze. The effects of Aβ, galanin, M617 and AR-M1896 on hippocampal Aβ protein expression were evaluated by western blot assay. The expression of galanin, galanin receptors 1 and 2 in rats' hippocampus were detected by real time PCR and western blot assay. The results showed that (1) Galanin administration was effective in improving the spatial memory and decreasing hippocampal Aβ levels after intracerebroventricular injection of Aβ; (2) AR-M1896 rather than M617 could imitate these effects of galanin; (3) GAL and GALR2 mRNA and protein levels increased significantly in hippocampus after Aβ administration, while GALR1 mRNA and protein levels did not change; (4) GAL, AR-M1896 and M617 administration did not show significant effect on GAL, GalR1 and GalR2 mRNA and protein levels in hippocampus after Aβ administration. These results implied that galanin receptor 2, but not receptor 1 was involved in the protective effects against spatial memory impairment and hippocampal Aβ aggregation.

Splicing Factor Prp8 Interacts With NES(AR) and Regulates Androgen Receptor in Prostate Cancer Cells.

PubMed

Wang, Dan; Nguyen, Minh M; Masoodi, Khalid Z; Singh, Prabhpreet; Jing, Yifeng; O'Malley, Katherine; Dar, Javid A; Dhir, Rajiv; Wang, Zhou

2015-12-01

Androgen receptor (AR) plays a pivotal role in the development of primary as well as advanced castration-resistant prostate cancer. Previous work in our lab identified a novel nuclear export signal (NES) (NES(AR)) in AR ligand-binding domain essential for AR nucleocytoplasmic trafficking. By characterizing the localization of green fluorescence protein (GFP)-tagged NES(AR), we designed and executed a yeast mutagenesis screen and isolated 7 yeast mutants that failed to display the NES(AR) export function. One of those mutants was identified as the splicing factor pre-mRNA processing factor 8 (Prp8). We further showed that Prp8 could regulate NES(AR) function using short hairpin RNA knockdown of Prp8 coupled with a rapamycin export assay in mammalian cells and knockdown of Prp8 could induce nuclear accumulation of GFP-tagged AR in PC3 cells. Prp8 expression was decreased in castration-resistant LuCaP35 xenograft tumors as compared with androgen-sensitive xenografts. Laser capture microdissection and quantitative PCR showed Prp8 mRNA levels were decreased in human prostate cancer specimens with high Gleason scores. In prostate cancer cells, coimmunoprecipitation and deletion mutagenesis revealed a physical interaction between Prp8 and AR mainly mediated by NES(AR). Luciferase assay with prostate specific antigen promoter-driven reporter demonstrated that Prp8 regulated AR transcription activity in prostate cancer cells. Interestingly, Prp8 knockdown also increased polyubiquitination of endogenous AR. This may be 1 possible mechanism by which it modulates AR activity. These results show that Prp8 is a novel AR cofactor that interacts with NES(AR) and regulates AR function in prostate cancer cells.

Circadian rhythm genes mediate fenvalerate-induced inhibition of testosterone synthesis in mouse Leydig cells.

PubMed

Guo, Yichen; Shen, Ouxi; Han, Jingjing; Duan, Hongyu; Yang, Siyuan; Zhu, Zhenghong; Tong, Jian; Zhang, Jie

2017-01-01

Fenvalerate (Fen), a widely used pesticide, is known to impair male reproductive functions by mechanisms that remain to be elucidated. Recent studies indicated that circadian clock genes may play an important role in successful male reproduction. The aim of this study was to determine the effects of Fen on circadian clock genes involved in the biosynthesis of testosterone using TM3 cells derived from mouse Leydig cells. Data demonstrated that the circadian rhythm of testosterone synthesis in TM3 cells was disturbed following Fen treatment as evidenced by changes in the circadian rhythmicity of core clock genes (Bmal1, Rev-erbα, Rorα). Further, the observed altered rhythms were accompanied by increased intracellular Ca 2+ levels and modified steroidogenic acute regulatory (StAR) mRNA expression. Thus, data suggested that Fen inhibits testosterone synthesis via pathways involving intracellular Ca 2+ and clock genes (Bmal1, Rev-Erbα, Rorα) as well as StAR mRNA expression in TM3 cells.

A herbal medicine, saikokaryukotsuboreito, improves serum testosterone levels and affects sexual behavior in old male mice.

PubMed

Zang, Zhi Jun; Ji, Su Yun; Dong, Wang; Zhang, Ya Nan; Zhang, Er Hong; Bin, Zhang

2015-06-01

Late-onset hypogonadism (LOH) is a clinical syndrome characterized with aging and declined serum testosterone levels. Sexual symptoms are also essential for the diagnosis of LOH. Testosterone replacement therapy is used widely to treat LOH. However, the side effects of it should not be ignored, such as fluid retention, hypertension and spermatogenic suppression. Therefore, alternate treatment modalities have been pursued. Herbal medicines used widely in China have achieved satisfying results with little side effects. Nonetheless, there are few pharmacological researches on them. In this study, 24-month-old mice were used as LOH animal models to explore the pharmacological effects of a herbal medicine, saikokaryukotsuboreito (SKRBT), on serum testosterone levels and sexual functions. Furthermore, the expression of steroidogenic acute regulatory (StAR) protein, a kind of rate-limiting enzyme of testosterone synthesis, was also examined. As a result, SKRBT improved the serum testosterone levels of these mice at a dose of 300 and 450 mg/kg. Multiple measures of sexual behavior were enhanced. The expression of StAR was also increased. Therefore, this study suggested that SKRBT can improve the serum testosterone levels by activating the expression of StAR and might be a viable option to treat sexual symptoms caused by LOH.

Modulation of intracellular Ca(2+) via alpha(1B)-adrenoreceptor signaling molecules, G alpha(h) (transglutaminase II) and phospholipase C-delta 1.

PubMed

Kang, Sung Koo; Kim, Dae Kyong; Damron, Derek S; Baek, Kwang Jin; Im, Mie-Jae

2002-04-26

We characterized the alpha(1B)-adrenoreceptor (alpha(1B)-AR)-mediated intracellular Ca(2+) signaling involving G alpha(h) (transglutaminase II, TGII) and phospholipase C (PLC)-delta 1 using DDT1-MF2 cell. Expression of wild-type TGII and a TGII mutant lacking transglutaminase activity resulted in significant increases in a rapid peak and a sustained level of intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to activation of the alpha(1B)-AR. Expression of a TGII mutant lacking the interaction with the receptor or PLC-delta 1 substantially reduced both the peak and sustained levels of [Ca(2+)](i). Expression of TGII mutants lacking the interaction with PLC-delta 1 resulted in a reduced capacitative Ca(2+) entry. Reduced expression of PLC-delta 1 displayed a transient elevation of [Ca(2+)](i) and a reduction in capacitative Ca(2+) entry. Expression of the C2-domain of PLC-delta 1, which contains the TGII interaction site, resulted in reduction of the alpha(1B)-AR-evoked peak increase in [Ca(2+)](i), while the sustained elevation in [Ca(2+)](i) and capacitative Ca(2+) entry remained unchanged. These findings demonstrate that stimulation of PLC-delta 1 via coupling of the alpha(1B)-AR with TGII evokes both Ca(2+) release and capacitative Ca(2+) entry and that capacitative Ca(2+) entry is mediated by the interaction of TGII with PLC-delta 1.

Roles of the Adenosine Receptor and CD73 in the Regulatory Effect of γδ T Cells

PubMed Central

Liang, Dongchun; Zuo, Aijun; Shao, Hui; Chen, Mingjiazi; Kaplan, Henry J.; Sun, Deming

2014-01-01

The adenosine A2A receptor (A2AR), the main functional adenosine receptor on murine T cells, plays a unique role in the attenuation of inflammation and tissue damage in vivo. Here, we showed that, of the immune cell types tested, activated γδ T cells expressed the highest levels of A2AR mRNA and that A2AR ligation inhibited αβ T cell activation, but enhanced γδ T cell activation. We also showed that the inhibitory effect of an adenosine receptor agonist on autoreactive T cells was prevented by addition of a low percentage of activated γδ T cells. Furthermore, compared to resting cells, activated γδ T cells expressed significantly lower levels of CD73, an enzyme involved in the generation of extracellular adenosine. Exogenous AMP had a significant inhibitory effect on autoreactive T cell responses, but only in the presence of CD73+ γδ T cells, and this effect was abolished by a CD73 inhibitor. Our results show that expression of increased amounts of A2AR allows γδ T cells to bind adenosine and thereby attenuate its suppressive effect, while decreased expression of CD73 results in less generation of adenosine in the inflammatory site. Together, these events allow activated γδ T cells to acquire increased proinflammatory activity, leading to augmented autoimmune responses. PMID:25268760

[Effect of Warm Acupuncture on the Levels of Serum Immunoglobulin E, Interleukin-1 β and Tumor Necrosis Factor-α in Rats with Allergic Rhinitis].

PubMed

Zheng, Xian-Li; Tian, Yong-Ping; Luo, Hai-Yan; Zhao, Yao-Dong; Liu, Xiang-Yi; Jiang, Ying; Ma, Cheng-Xu; Wang, Ming-Juan; Liu, Min

2018-01-25

To observe the effect of warm acupuncture on behavior and contents of serum immunoglobulin E(IgE), interleukin-1 β(IL-1 β) and tumor necrosis factor-α(TNF-α) in allergic rhinitis(AR) rats, so as to explore its mechanism underlying improving AR. Forty Wistar rats were randomly divided into four groups: control group, model group, medication group and warm acupuncture group(10 rats/group). The AR model was established by intraperitoneal injection of sensitization and nasal drip. The rats in the medication group were given fluticasone propionate nasal spray, daily for 10 days. Warm acupuncture was applied to "Fengchi"(GB 20), "Yintang"(GV 29), "Yingxiang"(LI 20) for 60 seconds, once daily for 10 days. Behavioral scores were used to evaluate behavioral changes in rats. Enzyme linked immunosorbent assay (ELISA) was used to detect the expression levels of serum IgE, IL-1 β and TNF-α. Behavioral scores of the model group were significantly higher than those of the control group 0, 3, 7 and 10 days after modeling ( P <0.05). After treatment, the behavioral scores of medication group and the warm acupuncture group were lower than those of the model group ( P <0.05), and the score was more lower in the warm acupuncture group than in the medication group ( P <0.05). Compared with the control group, the levels of serum IgE, IL-1 β and TNF-α in the model group were all increased ( P <0.01), while the levels of serum IgE, IL-1 β and TNF-α were decreased in the medication and warm acupuncture groups after treatment in comparison with the model group ( P <0.05, P <0.01). Compared with the medication group, the levels of serum IgE, IL-1 β and TNF-α were significantly lower in the warm acupuncture group ( P <0.05, P <0.01). The expression levels of IgE, IL-1 β and TNF-α were elevated in serum after AR attack. Warm acupuncture can improve the symptoms of AR rats, which may be associated to its effect in inhibiting the expression of serum IgE, IL-1 β and TNF-α.

Role of leptin in delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx.

PubMed

Banerjee, A; Meenakumari, K J; Krishna, A

2010-08-01

An adiposity-associated rise in leptin occurs at the time of delayed embryonic development in Cynopterus sphinx. The aim of present study was to examine the mechanism by which leptin may inhibit progesterone, and therefore could be responsible for delayed development. The study showed a significant increase in circulating leptin level during the period of increased fat accumulation, which coincided with significant decrease in serum progesterone level and delayed embryonic development in C. sphinx. The study showed increased Ob-R expression in the corpus luteum and in the utero-embryonic unit during the period of delayed embryonic development. The in vitro study showed suppressive effect of leptin on progesterone synthesis. The effect of high dose of leptin on ovarian steroidogenesis was found to be mediated through decreased expression of StAR and LH-R proteins in the ovary. The treatment with leptin caused increased expression of STAT 3 and iNOS proteins in the ovary, which correlated with decreased expression of StAR protein in the ovary. The inhibitory effects of leptin on progesterone synthesis in the ovary are thus mediated through STAT 3 and iNOS-NO signaling pathways. This study further demonstrated low expression of PCNA coinciding with the increased concentration of the leptin receptor in the utero-embryonic unit and high circulating leptin level during November. In conclusion, adiposity associated increased leptin level during November-December might play role in suppressing progesterone synthesis in the corpus luteum as well as suppressing the rate of cell-proliferation in the utero-embryonic unit thereby causing delayed embryonic development in C. sphinx. Copyright 2010 Elsevier Inc. All rights reserved.

Expression of the androgen receptor in the testes and the concentrations of gonadotropins and sex steroid hormones in male turkeys (Meleagris gallopavo) during growth and development.

PubMed

Kiezun, J; Leska, A; Kaminska, B; Jankowski, J; Dusza, L

2015-04-01

Androgens, including testosterone (T) and androstenedione (A4), are essential for puberty, fertility and sexual functions. The biological activity of those hormones is mediated via the androgen receptor (AR). The regulation of androgen action in birds is poorly understood. Therefore, the present study analysed mRNA and protein expression of AR in the testes, plasma concentrations of the luteinizing hormone (LH), follicle-stimulating hormone (FSH), T, A4 and oestradiol (E2), as well as the levels of T, A4 and E2 in testicular homogenates of male turkeys (Meleagris gallopavo) at the age of 4, 8, 12, 16, 20, 24 and 28weeks. Plasma concentrations of LH and FSH, as well as plasma and testicular levels of T and A4 began to increase at 20weeks of age. The lowest plasma levels of E2 were noted at 20weeks relative to other growth stages. The 20th week of life seems to be the key phase in the development of the reproductive system of turkeys. The AR protein was found in the nuclei of testicular cells in all examined growth stages. Higher expression of AR protein in the testes beginning at 20weeks of age was accompanied by high plasma concentrations of LH and high plasma and testicular levels of androgens. This relationship seems to be necessary to regulate male sexual function. Copyright © 2014 Elsevier Inc. All rights reserved.

Activin A, B and AB decrease progesterone production by down-regulating StAR in human granulosa cells.

PubMed

Chang, Hsun-Ming; Cheng, Jung-Chien; Huang, He-Feng; Shi, Feng-Tao; Leung, Peter C K

2015-09-05

Activins are homo- or heterodimers of inhibin β subunits that play important roles in the reproductive system. Our previous work has shown that activins A (βAβA), B (βBβB) and AB (βAβB) induce aromatase/estradiol, but suppress StAR/progesterone production in human granulosa-lutein cells. However, the underlying molecular determinants of these effects have not been examined. In this continuing study, we used immortalized human granulosa cells (SVOG) to investigate the effects of activins in regulating StAR/progesterone and the potential mechanisms of action. In SVOG cells, activins A, B and AB produced comparable down-regulation of StAR expression and progesterone production. In addition, all three activin isoforms induced equivalent phosphorylation of both SMAD2 and SMAD3. Importantly, the activin-induced down-regulation of StAR, increase in SMAD2/3 phosphorylation, and decrease in progesterone were abolished by the TGF-β type I receptor inhibitor SB431542. Interestingly, the small interfering RNA-mediated knockdown of ALK4 but not ALK5 reversed the activin-induced suppression of StAR. Furthermore, the knockdown of SMAD4 or SMAD2 but not SMAD3 abolished the inhibitory effects of all three activin isoforms on StAR expression. These results provide evidence that activins A, B and AB down-regulate StAR expression and decrease progesterone production in human granulosa cells, likely via an ALK4-mediated SMAD2/SMAD4-dependent pathway. Our findings provide important insights into the molecular mechanisms underlying the regulatory effects of activins on human granulosa cell steroidogenesis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Reduction in expression of the benign AR transcriptome is a hallmark of localised prostate cancer progression.

PubMed

Stuchbery, Ryan; Macintyre, Geoff; Cmero, Marek; Harewood, Laurence M; Peters, Justin S; Costello, Anthony J; Hovens, Christopher M; Corcoran, Niall M

2016-05-24

Despite the importance of androgen receptor (AR) signalling to prostate cancer development, little is known about how this signalling pathway changes with increasing grade and stage of the disease. To explore changes in the normal AR transcriptome in localised prostate cancer, and its relation to adverse pathological features and disease recurrence. Publically accessible human prostate cancer expression arrays as well as RNA sequencing data from the prostate TCGA. Tumour associated PSA and PSAD were calculated for a large cohort of men (n=1108) undergoing prostatectomy. We performed a meta-analysis of the expression of an androgen-regulated gene set across datasets using Oncomine. Differential expression of selected genes in the prostate TCGA database was probed using the edgeR Bioconductor package. Changes in tumour PSA density with stage and grade were assessed by Student's t-test, and its association with biochemical recurrence explored by Kaplan-Meier curves and Cox regression. Meta-analysis revealed a systematic decline in the expression of a previously identified benign prostate androgen-regulated gene set with increasing tumour grade, reaching significance in nine of 25 genes tested despite increasing AR expression. These results were confirmed in a large independent dataset from the TCGA. At the protein level, when serum PSA was corrected for tumour volume, significantly lower levels were observed with increasing tumour grade and stage, and predicted disease recurrence. Lower PSA secretion-per-tumour-volume is associated with increasing grade and stage of prostate cancer, has prognostic relevance, and reflects a systematic perturbation of androgen signalling.

Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription.

PubMed

Lee, Jinwoo; Foong, Yee Hoon; Musaitif, Ibrahim; Tong, Tiegang; Jefcoate, Colin

2016-07-05

The steroidogenic acute regulatory protein (StAR) has been proposed to serve as the switch that can turn on/off steroidogenesis. We investigated the events that facilitate dynamic StAR transcription in response to cAMP stimulation in MA-10 Leydig cells, focusing on splicing anomalies at StAR gene loci. We used 3' reverse primers in a single reaction to respectively quantify StAR primary (p-RNA), spliced (sp-RNA/mRNA), and extended 3' untranslated region (UTR) transcripts, which were quantitatively imaged by high-resolution fluorescence in situ hybridization (FISH). This approach delivers spatio-temporal resolution of initiation and splicing at single StAR loci, and transfers individual mRNA molecules to cytoplasmic sites. Gene expression was biphasic, initially showing slow splicing, transitioning to concerted splicing. The alternative 3.5-kb mRNAs were distinguished through the use of extended 3'UTR probes, which exhibited distinctive mitochondrial distribution. Combining quantitative PCR and FISH enables imaging of localization of RNA expression and analysis of RNA processing rates. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Variation in neural V1aR predicts sexual fidelity and space use among male prairie voles in semi-natural settings

PubMed Central

Ophir, Alexander G.; Wolff, Jerry O.; Phelps, Steven M.

2008-01-01

Although prairie voles (Microtus ochrogaster) are socially monogamous, males vary in both sexual and spatial fidelity. Most males form pairbonds, cohabit with one female, and defend territories. Wandering males, in contrast, have expansive home ranges that overlap many males and females. In the laboratory, pairing is regulated by arginine vasopressin and its predominant CNS receptor, vasopressin 1a receptor (V1aR). We investigated individual differences in forebrain V1aR expression of male prairie voles in mixed-sex seminatural enclosures. Individual differences in V1aR were compared with space use measured by radio telemetry and paternity determined with microsatellite markers. Animals engaging in extra-pair fertilizations (EPFs) as either wanderers or paired residents overlapped significantly more in same- and opposite-sex home ranges. Surprisingly, neither social fidelity measured by space use nor sexual fidelity measured by paternity was associated with V1aR expression in the ventral pallidum (VPall) or lateral septum, areas causally related to pairbond formation. In contrast, V1aR expression in the posterior cingulate/retrosplenial cortex (PCing) and laterodorsal thalamus (LDThal), areas implicated in spatial memory, strongly covaried with space use and paternity. Animals engaging in EPFs either as wanderers or paired residents exhibited low levels of LDThal and PCing V1aR expression. Individual differences in brain and behavior parallel differences between prairie voles and promiscuous congeners. The concordance among space use, paternity, and V1aR in spatial circuits suggests a common link between the mechanisms of spatial behaviors and success at EPF. The combined data demonstrate how organismal biology can inform our understanding of individual and species differences in behavioral mechanisms. PMID:18212120

Growth Inhibition by Testosterone in an Androgen Receptor Splice Variant-Driven Prostate Cancer Model.

PubMed

Nakata, Daisuke; Nakayama, Kazuhide; Masaki, Tsuneo; Tanaka, Akira; Kusaka, Masami; Watanabe, Tatsuya

2016-12-01

Castration resistance creates a significant problem in the treatment of prostate cancer. Constitutively active splice variants of androgen receptor (AR) have emerged as drivers for resistance to androgen deprivation therapy, including the next-generation androgen-AR axis inhibitors abiraterone and enzalutamide. In this study, we describe the characteristics of a novel castration-resistant prostate cancer (CRPC) model, designated JDCaP-hr (hormone refractory). JDCaP-hr was established from an androgen-dependent JDCaP xenograft model after surgical castration. The expression of AR and its splice variants in JDCaP-hr was evaluated by immunoblotting and quantitative reverse transcription-polymerase chain reaction. The effects of AR antagonists and testosterone on JDCaP-hr were evaluated in vivo and in vitro. The roles of full-length AR (AR-FL) and AR-V7 in JDCaP-hr cell growth were evaluated using RNA interference. JDCaP-hr acquired a C-terminally truncated AR protein during progression from the parental JDCaP. The expression of AR-FL and AR-V7 mRNA was upregulated by 10-fold in JDCaP-hr compared with that in JDCaP, indicating that the JDCaP and JDCaP-hr models simulate castration resistance with some clinical features, such as overexpression of AR and its splice variants. The AR antagonist bicalutamide did not affect JDCaP-hr xenograft growth, and importantly, testosterone induced tumor regression. In vitro analysis demonstrated that androgen-independent prostate-specific antigen secretion and cell proliferation of JDCaP-hr were predominantly mediated by AR-V7. JDCaP-hr cell growth displayed a bell-shaped dependence on testosterone, and it was suppressed by physiological concentrations of testosterone. Testosterone induced rapid downregulation of both AR-FL and AR-V7 expression at physiological concentrations and suppressed expression of the AR target gene KLK3. Our findings support the clinical value of testosterone therapy, including bipolar androgen therapy, in the treatment of AR-overexpressed CRPC driven by AR splice variants that are not clinically actionable at present. Prostate 76:1536-1545, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Norepinephrine upregulates VEGF, IL-8, and IL-6 expression in human melanoma tumor cell lines: implications for stress-related enhancement of tumor progression

PubMed Central

Yang, Eric V.; Kim, Seung-jae; Donovan, Elise L.; Chen, Min; Gross, Amy C.; Webster Marketon, Jeanette I.; Barsky, Sanford H.; Glaser, Ronald

2009-01-01

Studies suggest that stress can be a co-factor for the initiation and progression of cancer. The catecholamine stress hormone, norepinephrine (NE), may influence tumor progression by modulating the expression of factors implicated in angiogenesis and metastasis. The goal of this study was to examine the influence of NE on the expression of VEGF, IL-8, and IL-6 by the human melanoma cell lines, C8161, 1174MEL, and Me18105. Cells were treated with NE and levels of VEGF, IL-8, and IL-6 were measured using ELISA and real-time PCR. The expression of β-adrenergic receptors (β-ARs) mRNA and protein were also assessed. Finally, immunohistochemitry was utilized to examine the presence of β1- and β2-AR in primary and metastatic human melanoma biopsies. We show that NE treatment upregulated production of VEGF, IL-8, and IL-6 in C8161 cells and to a lesser extent 1174MEL and Me18105 cells. The upregulation was associated with induced gene expression. The effect on C8161 cells was mediated by both β1- and β2-ARs. Furthermore, 18 of 20 melanoma biopsies examined expressed β2-AR while 14 of 20 melanoma biopsies expressed β1-AR. Our data support the hypothesis that NE can stimulate the aggressive potential of melanoma tumor cells, in part, by inducing the production VEGF, IL-8, and IL-6. This line of research further suggests that interventions targeting components of the activated sympathetic-adrenal medullary (SAM) axis, or the utilization of β-AR blocking agents, may represent new strategies for slowing down the progression of malignant disease and improving cancer patients’ quality of life. PMID:18996182

Elevated expression of steroidogenesis pathway genes; CYP17, GATA6 and StAR in prenatally androgenized rats.

PubMed

Jahromi, Marziyeh Salehi; Tehrani, Fahimeh Ramezani; Noroozzadeh, Mahsa; Zarkesh, Maryam; Ghasemi, Asghar; Zadeh-Vakili, Azita

2016-11-15

It is believed that excess androgen exposure of the fetus, via altered gene expression, causes hyperandrogenism a key feature of polycystic ovary syndrome (PCOS). The aim of this study was to evaluate expression of Cytochrome P450-17 (CYP17), GATA-binding protein (GAGT6) and Steroidogenic acute regulatory protein (StAR), genes of adult female rats prenatally exposed to androgen excess, closely reflect endocrine and ovarian disturbances of PCOS in women, by comparing them during different phases of estrus cycle with those of non-treated rats. Both the adult prenatally testosterone exposed and control rats (n=23, each) were divided into four groups based on their observed vaginal smear (proestrus, estrus, metestrus and diestrus) and the relative expression of CYP17, GATA6 and StAR genes was measured in ovarian theca cells using Cyber-green Real-Time PCR. Serum sex steroid hormones and gonadotropins levels were measured using the ELISA method; a comparison of these two groups showed that there was an overall increase in the studied genes (CYP17; 2.39 fold change, 95% CI: 1.23-3.55; P<0.05, GATA6; 2.08 fold change, 95% CI: 1.62-2.55; P<0.0001, and StAR; 1.4 fold change, 95% CI: 1.02-1.78; P<0.05), despite variations in different phases with maximum elevation for all genes in diestrus. The changes observed may impair the normal development of ovaries that mediate the programming of adult PCOS. Copyright © 2016 Elsevier B.V. All rights reserved.

ArI/ArII laser induced fluorescence system for measurement of neutral and ion dynamics in a large scale helicon plasma

NASA Astrophysics Data System (ADS)

Kelly, R. F.; Meaney, K. D.; Gilmore, M.; Desjardins, T. R.; Zhang, Y.

2016-11-01

In order to investigate the role of both neutral and ion dynamics in large-scale helicon discharges, a laser induced fluorescence (LIF) system capable of measuring both ArI and ArII fluorescence using a single tunable laser is being developed. The system is based on a >250 mW solid state laser. For ArI measurements, the laser pumps the metastable (2P03/2)4s level to the (2P01/2)4p level using 696.7352 nm light, and fluorescence radiation from decay to the (2P01/2)4s level at 772.6333 nm is observed. For ArII, currently in development, the metastable (3P)3d 4F7/2 level will be pumped to the (3P)4p 4D07/2 level using 688.8511 nm, and decay fluorescence to the (3P)4s 4P5/2 level at 434.9285 nm measured. Here all wavelengths are in a vacuum.

Reproductive Hormones and Their Receptors May Affect Lung Cancer.

PubMed

Dou, Mengmeng; Zhu, Keyan; Fan, Zhirui; Zhang, Yuxuan; Chen, Xiufang; Zhou, Xueliang; Ding, Xianfei; Li, Lifeng; Gu, Zhaosen; Guo, Maofeng; Yan, Ming; Deng, Xiaoming; Shen, Peihong; Wang, Shuling

2017-01-01

In contrast to men, women have experienced a rapid increase in lung cancer mortality. Numerous studies have found that the sex differences in lung cancer are due to reproductive hormones. Experiments in female mice with and without ovariectomy were performed to explore the possible mechanism by which sex hormones (and their receptors) influence lung cancer. Twenty-four female C57BL/6 mice aged 56-62 days were randomly divided into the ovariectomized group and the control group. In the ovariectomized group, the bilateral ovaries were removed via the dorsal approach, while the control group underwent a sham operation with bilateral ovarian fat resection at the same sites. After 3 weeks of recovery, Lewis lung cancer cells were transplanted into these mice by subcutaneous inoculation of a tumour cell suspension to establish the ovariectomized lung cancer model. Beginning on the 6th day after subcutaneous inoculation, mouse weight and transplanted tumour volume were measured every 3 days. After 3 weeks, all the mice were killed by cervical dislocation, and we measured the tumour weight. Mouse serum and tumour tissues were removed. Then, the serum levels of E2 (oestradiol) and T (testosterone) were detected by ELISA; the protein expression levels of AR (androgen receptor), ERα (oestrogen receptor α) and ERβ (oestrogen receptor β) were detected by Western Blot and IHC (immunohistochemistry); and the mRNA expression levels of AR, ERα and ERβ were detected by qRT-PCR (quantitative real-time polymerase chain reaction) in the ovariectomized and control groups. Compared with the control group, both mouse weight and transplanted tumour volume increased rapidly in the ovariectomized group, and the transplanted tumour weight was significantly heavier in the ovariectomized group (1.83±0.40 and 3.13±0.43, P<0.05). E2 and T serum levels decreased exponentially in the ovariectomized group, while the E2/T ratio increased compared with the control group (E2: 55.88±11.45 and 78.21±9.37; T: 0.82±0.14 and 1.46±0.16; ratio: 69.62±14.43±29.81 and 52.22±5.42; all P<0.05). The Western blot and IHC results indicated that AR, ERα and ERβ protein expression levels were obviously higher in transplanted tumour and lung tissues from the ovariectomized group, with particular increases in ERβ in transplanted tumour tissue and in ERα in lung tissue. The PCR results also showed markedly higher mRNA expression levels of AR, ERα and ERβ in the ovariectomized group, and in particular, ERβ in transplanted tumour tissue and ERα in lung tissue were significantly increased in the ovariectomized group. Ovariectomy decreased E2 and T serum levels and increased the E2/T ratio in mice, and this imbalance in the internal environment promoted the growth of transplanted tumours. Sex hormone disorder not only promoted transplanted tumour growth but also significantly reduced the protein and mRNA expression levels of sex hormone receptors. The metabolism of E2 and T may affect the growth, proliferation and metabolism of lung cancer cells, and the mechanism by which sex hormones and their receptors influence lung cancer is worthy of further research. © 2017 The Author(s). Published by S. Karger AG, Basel.

Altered prostate epithelial development and IGF-1 signal in mice lacking the androgen receptor in stromal smooth muscle cells.

PubMed

Yu, Shengqiang; Zhang, Caixia; Lin, Chiu-Chun; Niu, Yuanjie; Lai, Kuo-Pao; Chang, Hong-chiang; Yeh, Shauh-Der; Chang, Chawnshang; Yeh, Shuyuan

2011-04-01

Androgens and the androgen receptor (AR) play critical roles in the prostate development via mesenchymal-epithelial interactions. Smooth muscle cells (SMC), differentiated from mesenchyme, are one of the basic components of the prostate stroma. However, the roles of smooth muscle AR in prostate development are still obscure. We established the smooth muscle selective AR knockout (SM-ARKO) mouse model using the Cre-loxP system, and confirmed the ARKO efficiency at RNA, DNA and protein levels. Then, we observed the prostate morphology changes, and determined the epithelial proliferation, apoptosis, and differentiation. We also knocked down the AR in a prostate smooth muscle cell line (PS-1) to confirm the in vivo findings and to probe the mechanism. The AR was selectively and efficiently knocked out in the anterior prostates of SM-ARKO mouse. The SM-ARKO prostates have defects with loss of infolding structures, and decrease of epithelial proliferation, but with little change of apoptosis and differentiation. The mechanism studies showed that IGF-1 expression level decreased in the SM-ARKO prostates and AR-knockdown PS-1 cells. The decreased IGF-1 expression might contribute to the defective development of SM-ARKO prostates. The AR in SMCs plays important roles in the prostate development via the regulation of IGF-1 signal. Copyright © 2010 Wiley-Liss, Inc.

Converging evidence for the association of functional genetic variation in the serotonin receptor 2a gene with prefrontal function and olanzapine treatment.

PubMed

Blasi, Giuseppe; De Virgilio, Caterina; Papazacharias, Apostolos; Taurisano, Paolo; Gelao, Barbara; Fazio, Leonardo; Ursini, Gianluca; Sinibaldi, Lorenzo; Andriola, Ileana; Masellis, Rita; Romano, Raffaella; Rampino, Antonio; Di Giorgio, Annabella; Lo Bianco, Luciana; Caforio, Grazia; Piva, Francesco; Popolizio, Teresa; Bellantuono, Cesario; Todarello, Orlando; Kleinman, Joel E; Gadaleta, Gemma; Weinberger, Daniel R; Bertolino, Alessandro

2013-09-01

Serotonin (5-hydroxytryptamine) receptor 2a (5-HT2AR) signaling is important for modulation of corticostriatal pathways and prefrontal activity during cognition. Furthermore, newer antipsychotic drugs target 5-HT2AR. A single-nucleotide polymorphism in the 5-HT2AR gene (HTR2A rs6314, C>T; OMIM 182135) has been weakly associated with differential 5-HT2AR signaling and with physiologic as well as behavioral effects. To use a hierarchical approach to determine the functional effects of this single-nucleotide polymorphism on 5-HT2AR messenger RNA and protein expression, on prefrontal phenotypes linked with genetic risk for schizophrenia, and on treatment with olanzapine. In silico predictions, in vitro, and case-control investigations. Academic and clinical facilities. The postmortem study included 112 brains from healthy individuals; the in vivo investigation included a total sample of 371 healthy individuals and patients with schizophrenia. EXPOSURES Patients received olanzapine monotherapy for 8 weeks. In silico predictions, messenger RNA, and protein expression in postmortem human prefrontal cortex and HeLa cells, functional magnetic resonance imaging prefrontal activity and behavior during working memory and attention in healthy individuals, and response to an 8-week trial of olanzapine treatment in patients with schizophrenia. Bioinformatic analysis predicted that rs6314 alters patterns of splicing, with possible effects on HTR2A expression. Moreover, the T allele was associated with reduced prefrontal messenger RNA expression in postmortem prefrontal cortex, with reduced protein expression in vitro, inefficient prefrontal blood oxygen level-dependent functional magnetic resonance imaging response during working memory and attentional control processing, and impaired working memory and attention behavior, as well as with attenuated improvement in negative symptoms after olanzapine treatment. Our results suggest that HTR2A rs6314 affects 5-HT2AR expression and functionally contributes to genetic modulation of known endophenotypes of schizophrenia-like higher-level cognitive behaviors and related prefrontal activity, as well as response to treatment with olanzapine.

Androgen receptor polyglutamine expansion drives age-dependent quality control defects and muscle dysfunction.

PubMed

Nath, Samir R; Yu, Zhigang; Gipson, Theresa A; Marsh, Gregory B; Yoshidome, Eriko; Robins, Diane M; Todi, Sokol V; Housman, David E; Lieberman, Andrew P

2018-05-29

Skeletal muscle has emerged as a critical, disease-relevant target tissue in spinal and bulbar muscular atrophy, a degenerative disorder of the neuromuscular system caused by a CAG/polyglutamine (polyQ) expansion in the androgen receptor (AR) gene. Here, we used RNA-Seq to identify pathways that are disrupted in diseased muscle using AR113Q knock-in mice. This analysis unexpectedly identified significantly diminished expression of numerous ubiquitin-proteasome pathway genes in AR113Q muscle, encoding approximately 30% of proteasome subunits and 20% of E2 ubiquitin conjugases. These changes were age-, hormone- and glutamine length-dependent and arose due to a toxic gain-of-function conferred by the mutation. Moreover, altered gene expression was associated with decreased level of the proteasome transcription factor NRF1 and its activator DDI2 and resulted in diminished proteasome activity. Ubiquitinated ADRM1 was detected in AR113Q muscle, indicating the occurrence of stalled proteasomes in mutant mice. Finally, diminished expression of Drosophila orthologues of NRF1 or ADRM1 promoted the accumulation of polyQ AR protein and increased toxicity. Collectively, these data indicate that AR113Q muscle develops progressive proteasome dysfunction that leads to the impairment of quality control and the accumulation of polyQ AR protein, key features that contribute to the age-dependent onset and progression of this disorder.

Characterization of a C3a receptor in rainbow trout and Xenopus: the first identification of C3a receptors in nonmammalian species

USGS Publications Warehouse

Boshra, Hani; Wang, Tiehui; Hove-Madsen, Leif; Hansen, John D.; Li, Jun; Matlapudi, Anjun; Secombes, Christopher J.; Tort, Lluis; Sunyer, J. Oriol

2005-01-01

Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.

Urea Synthesis and Excretion in Aedes aegypti Mosquitoes Are Regulated by a Unique Cross-Talk Mechanism

PubMed Central

Isoe, Jun; Scaraffia, Patricia Y.

2013-01-01

Aedes aegypti mosquitoes do not have a typical functional urea cycle for ammonia disposal such as the one present in most terrestrial vertebrates. However, they can synthesize urea by two different pathways, argininolysis and uricolysis. We investigated how formation of urea by these two pathways is regulated in females of A. aegypti. The expression of arginase (AR) and urate oxidase (UO), either separately or simultaneously (ARUO) was silenced by RNAi. The amounts of several nitrogen compounds were quantified in excreta using mass spectrometry. Injection of mosquitoes with either dsRNA-AR or dsRNA-UO significantly decreased the expressions of AR or UO in the fat body (FB) and Malpighian tubules (MT). Surprisingly, the expression level of AR was increased when UO was silenced and vice versa, suggesting a cross-talk regulation between pathways. In agreement with these data, the amount of urea measured 48 h after blood feeding remained unchanged in those mosquitoes injected with dsRNA-AR or dsRNA-UO. However, allantoin significantly increased in the excreta of dsRNA-AR-injected females. The knockdown of ARUO mainly led to a decrease in urea and allantoin excretion, and an increase in arginine excretion. In addition, dsRNA-AR-injected mosquitoes treated with a specific nitric oxide synthase inhibitor showed an increase of UO expression in FB and MT and a significant increase in the excretion of nitrogen compounds. Interestingly, both a temporary delay in the digestion of a blood meal and a significant reduction in the expression of several genes involved in ammonia metabolism were observed in dsRNA-AR, UO or ARUO-injected females. These results reveal that urea synthesis and excretion in A. aegypti are tightly regulated by a unique cross-talk signaling mechanism. This process allows blood-fed mosquitoes to regulate the synthesis and/or excretion of nitrogen waste products, and avoid toxic effects that could result from a lethal concentration of ammonia in their tissues. PMID:23755226

Fabrication and evaluation of osteoblastic differentiation of human mesenchymal stem cells on novel CaO-SiO2-P2O5-B2O3 glass-ceramics.

PubMed

Lee, Jae Hyup; Seo, Jun-Hyuk; Lee, Kyung Mee; Ryu, Hyun-Seung; Baek, Hae-Ri

2013-07-01

Apatite-wollastonite glass-ceramics have high mechanical strength, and CaO-SiO2 -B2 O3 glass-ceramics showed excellent bioactivity and high biodegradability. A new type of CaO-SiO2 -P2 O5 -B2 O3 system of bioactive glass-ceramics (BGS-7) was fabricated, and the effect and usefulness was evaluated via bioactivity using simulated body fluid and human mesenchymal stem cells (hMSCs). The purpose of this study was to compare BGS-7 and hydroxyapatite (HA) using hMSCs in order to evaluate the bioactivity of BGS-7 and its possibility as a bone graft extender. Alkaline phosphatase (ALP) staining, ALP activity, cell proliferation 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, Alizarin Red-S (AR-S) staining, calcium levels, the mRNA expression of ALP, osteocalcin, osteopontin, and runt-related transcription factor 2 (runx-2) using reverse-transcription polymerase chain reaction (RT-PCR) and the protein expression of osteocalcin and runx-2 using Western blot were measured by transplanting hMSC onto a tissue culture plate, HA, and BGS-7. The ALP staining and AR-S staining of BGS-7 was greater than that of HA and control. The ALP value of BGS-7 was significantly higher than that of HA and control. The MTS results showed that BGS-7 had a higher value than the groups transplanted onto HA and control on day 15. The calcium level was higher than the control in both HA and BGS-7, and was especially high in BGS-7. There were more mineral products on BGS-7 than on the HA when analyzed by scanning electron microscopy. The mRNA expression of ALP, osteopontin, osteocalcin, and runx-2 were higher on BGS-7 than on HA and the control when analyzed by RT-PCR. The relative gene expression of osteopontin and runx-2 were found to be higher on BGS-7 than on HA and the control by Western blot. Accordingly, it is predicted that BGS-7 would have high biocompatibility and good osteoconductivity, and presents a possibility as a new bone graft extender. © 2013, Copyright the Authors. Artificial Organs © 2013, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

miR-22 and miR-29a Are Members of the Androgen Receptor Cistrome Modulating LAMC1 and Mcl-1 in Prostate Cancer.

PubMed

Pasqualini, Lorenza; Bu, Huajie; Puhr, Martin; Narisu, Narisu; Rainer, Johannes; Schlick, Bettina; Schäfer, Georg; Angelova, Mihaela; Trajanoski, Zlatko; Börno, Stefan T; Schweiger, Michal R; Fuchsberger, Christian; Klocker, Helmut

2015-07-01

The normal prostate as well as early stages and advanced prostate cancer (PCa) require a functional androgen receptor (AR) for growth and survival. The recent discovery of microRNAs (miRNAs) as novel effector molecules of AR disclosed the existence of an intricate network between AR, miRNAs and downstream target genes. In this study DUCaP cells, characterized by high content of wild-type AR and robust AR transcriptional activity, were chosen as the main experimental model. By integrative analysis of chromatin immunoprecipitation-sequencing (ChIP-seq) and microarray expression profiling data, miRNAs putatively bound and significantly regulated by AR were identified. A direct AR regulation of miR-22, miR-29a, and miR-17-92 cluster along with their host genes was confirmed. Interestingly, endogenous levels of miR-22 and miR-29a were found to be reduced in PCa cells expressing AR. In primary tumor samples, miR-22 and miR-29a were less abundant in the cancerous tissue compared with the benign counterpart. This specific expression pattern was associated with a differential DNA methylation of the genomic AR binding sites. The identification of laminin gamma 1 (LAMC1) and myeloid cell leukemia 1 (MCL1) as direct targets of miR-22 and miR-29a, respectively, suggested a tumor-suppressive role of these miRNAs. Indeed, transfection of miRNA mimics in PCa cells induced apoptosis and diminished cell migration and viability. Collectively, these data provide additional information regarding the complex regulatory machinery that guides miRNAs activity in PCa, highlighting an important contribution of miRNAs in the AR signaling.

Characterization of squamous esophageal cells resistant to bile acids at acidic pH: implication for Barrett's esophagus pathogenesis

PubMed Central

Goldman, Aaron; Chen, Hwu Dau Rw; Roesly, Heather B.; Hill, Kimberly A.; Tome, Margaret E.; Dvorak, Bohuslav; Bernstein, Harris

2011-01-01

Barrett's esophagus (BE) is a premalignant condition, where normal squamous epithelium is replaced by intestinal epithelium. BE is associated with an increased risk of developing esophageal adenocarcinoma (EAC). However, the BE cell of origin is not clear. We hypothesize that BE tissue originates from esophageal squamous cells, which can differentiate to columnar cells as a result of repeated exposure to gastric acid and bile acids, two components of refluxate implicated in BE pathology. To test this hypothesis, we repeatedly exposed squamous esophageal HET1A cells to 0.2 mM bile acid (BA) cocktail at pH 5.5 and developed an HET1AR-resistant cell line. These cells are able to survive and proliferate after repeated 2-h treatments with BA at pH 5.5. HET1AR cells are resistant to acidification and express markers of columnar differentiation, villin, CDX2, and cytokeratin 8/18. HET1AR cells have increased amounts of reactive oxygen species, concomitant with a decreased level and activity of manganese superoxide dismutase compared with parental cells. Furthermore, HET1AR cells express proteins and activate signaling pathways associated with inflammation, cell survival, and tumorigenesis that are thought to contribute to BE and EAC development. These include STAT3, NF-κB, epidermal growth factor receptor (EGFR), cyclooxygenase-2, interleukin-6, phosphorylated mammalian target of rapamycin (p-mTOR), and Mcl-1. The expression of prosurvival and inflammatory proteins and resistance to cell death could be partially modified by inhibition of STAT3 signaling. In summary, our study shows that long-term exposure of squamous cells to BA at acidic pH causes the cells to display the same characteristics and markers as BE. PMID:21127259

Acupuncture Prevents the Impairment of Hippocampal LTP Through β1-AR in Vascular Dementia Rats.

PubMed

Xiao, Ling-Yong; Wang, Xue-Rui; Yang, Jing-Wen; Ye, Yang; Zhu, Wen; Cao, Yan; Ma, Si-Ming; Liu, Cun-Zhi

2018-02-13

It is widely accepted that the synaptic dysfunction and synapse loss contribute to the cognitive deficits of vascular dementia (VD) patients. We have previously reported that acupuncture improved cognitive function in rats with VD. However, the mechanisms involved in acupuncture improving cognitive ability remain to be elucidated. The present study aims to investigate the pathways and molecules involved in the neuroprotective effect of acupuncture. We assessed the effects of acupuncture on hippocampal long-term potentiation (LTP), the most prominent cellular model of memory formation. Acupuncture enhanced LTP and norepinephrine (NE) levels in the hippocampus. Inhibition of the β-adrenergic receptor (AR), but not the α-AR, was able to block the effects of acupuncture on hippocampal LTP. Furthermore, inhibition of β1-AR, not β2-AR, abolished the enhanced LTP induced by acupuncture. The expression analysis revealed a significant upregulation of β1-AR and unchanged β2-AR with acupuncture, which supported the above findings. Specifically, increased β1-ARs in the dentate gyrus were expressed on neurons exclusively. Taken together, the present data supports a beneficial role of acupuncture in synaptic plasticity challenged with VD. A likely mechanism is the increase of NE and activation of β1-AR in the hippocampus.

Activation of Beta-Catenin Signaling in Androgen Receptor–Negative Prostate Cancer Cells

PubMed Central

Wan, Xinhai; Liu, Jie; Lu, Jing-Fang; Tzelepi, Vassiliki; Yang, Jun; Starbuck, Michael W.; Diao, Lixia; Wang, Jing; Efstathiou, Eleni; Vazquez, Elba S.; Troncoso, Patricia; Maity, Sankar N.; Navone, Nora M.

2012-01-01

Purpose To study Wnt/beta-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the beta-catenin–androgen receptor (AR) interaction. Experimental Design We performed beta-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of beta-catenin–mediated transcription), and sequenced the beta-catenin gene in MDA PCa 118a, MDA PCa 118b, MDA PCa 2b, and PC-3 prostate cancer (PCa) cells. We knocked down beta-catenin in AR-negative MDA PCa 118b cells and performed comparative gene-array analysis. We also immunohistochemically analyzed beta-catenin and AR in 27 bone metastases of human CRPCs. Results Beta-catenin nuclear accumulation and TOP-flash reporter activity were high in MDA PCa 118b but not in MDA PCa 2b or PC-3 cells. MDA PCa 118a and 118b cells carry a mutated beta-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA PCa 118b cells with downregulated beta-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant beta-catenin. Finally, we found nuclear localization of beta-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher’s exact test), suggesting that reduced AR expression enables Wnt/beta-catenin signaling. Conclusion We identified a previously unknown downstream target of beta-catenin, HAS2, in PCa, and found that high beta-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone-metastatic PCa. These findings may guide physicians in managing these patients. PMID:22298898

TOFA (5-tetradecyl-oxy-2-furoic acid) reduces fatty acid synthesis, inhibits expression of AR, neuropilin-1 and Mcl-1 and kills prostate cancer cells independent of p53 status.

PubMed

Guseva, Natalya V; Rokhlin, Oskar W; Glover, Rebecca A; Cohen, Michael B

2011-07-01

A key player in prostate cancer development and progression is the androgen receptor (AR). Tumor-associated lipogenesis can protect cancer cells from carcinogenic- and therapeutic-associated treatments. Increased synthesis of fatty acids and cholesterol is regulated by androgens through induction of several genes in androgen-responsive cancer cells. Acetyl-CoA-carboxylase-α (ACCA) is a key enzyme in the regulation of fatty acids synthesis. Here we show that AR binds in vivo to intron regions of human ACCA gene. We also show that the level of ACCA protein in LNCaP depends on AR expression and that DHT treatment increases ACCA expression and fatty acid synthesis. Inhibition of ACCA by TOFA (5-tetradecyl-oxy-2-furoic acid) decreases fatty acid synthesis and induces caspase activation and cell death in most PCa cell lines. Our data suggest that TOFA can kill cells via the mitochondrial pathway since we found cytochrome c release after TOFA treatment in androgen sensitive cell lines. The results also imply that the pro-apoptotic effect of TOFA may be mediated via a decrease of neuropilin-1(NRP1) and Mcl-1expression. We have previously reported that Mcl-1 is under AR regulation and plays an important role in resistance to drug-induced apoptosis in prostate cancer cells, and NRP1 is known to regulate Mcl-1 expression. Here, we show for the first time that NRP1 expression is under AR control. Taken together, our data suggest that TOFA is a potent cell death inducing agent in prostate cancer cells.

Behavioral Analysis of Genetically Modified Mice Indicates Essential Roles of Neurosteroidal Estrogen

PubMed Central

Honda, Shin-Ichiro; Wakatsuki, Toru; Harada, Nobuhiro

2011-01-01

Aromatase in the mouse brain is expressed only in the nerve cells of specific brain regions with a transient peak during the neonatal period when sexual behaviors become organized. The aromatase-knockout (ArKO) mouse, generated to shed light on the physiological functions of estrogen in the brain, exhibited various abnormal behaviors, concomitant with undetectable estrogen and increased androgen in the blood. To further elucidate the effects of neurosteroidal estrogens on behavioral phenotypes, we first prepared an brain-specific aromatase transgenic (bsArTG) mouse by introduction of a human aromatase transgene controlled under a −6.5 kb upstream region of the brain-specific promoter of the mouse aromatase gene into fertilized mouse eggs, because the −6.5 kb promoter region was previously shown to contain the minimal essential element responsible for brain-specific spatiotemporal expression. Then, an ArKO mouse expressing the human aromatase only in the brain was generated by crossing the bsArTG mouse with the ArKO mouse. The resulting mice (ArKO/bsArTG mice) nearly recovered from abnormal sexual, aggressive, and locomotive (exploratory) behaviors, in spite of having almost the same serum levels of estrogen and androgen as the adult ArKO mouse. These results suggest that estrogens locally synthesized in the specific neurons of the perinatal mouse brain directly act on the neurons and play crucial roles in the organization of neuronal networks participating in the control of sexual, aggressive, and locomotive (exploratory) behaviors. PMID:22654807

A Recurrent Germline Mutation in the 5'UTR of the Androgen Receptor Causes Complete Androgen Insensitivity by Activating Aberrant uORF Translation.

PubMed

Hornig, Nadine C; de Beaufort, Carine; Denzer, Friederike; Cools, Martine; Wabitsch, Martin; Ukat, Martin; Kulle, Alexandra E; Schweikert, Hans-Udo; Werner, Ralf; Hiort, Olaf; Audi, Laura; Siebert, Reiner; Ammerpohl, Ole; Holterhus, Paul-Martin

2016-01-01

A subset of patients with monogenic disorders lacks disease causing mutations in the protein coding region of the corresponding gene. Here we describe a recurrent germline mutation found in two unrelated patients with complete androgen insensitivity syndrome (CAIS) generating an upstream open reading frame (uORF) in the 5' untranslated region (5'-UTR) of the androgen receptor (AR) gene. We show in patient derived primary genital skin fibroblasts as well as in cell-based reporter assays that this mutation severely impacts AR function by reducing AR protein levels without affecting AR mRNA levels. Importantly, the newly generated uORF translates into a polypeptide and the expression level of this polypeptide inversely correlates with protein translation from the primary ORF of the AR thereby providing a model for AR-5'UTR mediated translational repression. Our findings not only add a hitherto unrecognized genetic cause to complete androgen insensitivity but also underline the importance of 5'UTR mutations affecting uORFs for the pathogenesis of monogenic disorders in general.

A Recurrent Germline Mutation in the 5’UTR of the Androgen Receptor Causes Complete Androgen Insensitivity by Activating Aberrant uORF Translation

PubMed Central

Hornig, Nadine C.; de Beaufort, Carine; Denzer, Friederike; Cools, Martine; Wabitsch, Martin; Ukat, Martin; Kulle, Alexandra E.; Schweikert, Hans-Udo; Werner, Ralf; Hiort, Olaf; Audi, Laura; Siebert, Reiner; Ammerpohl, Ole; Holterhus, Paul-Martin

2016-01-01

A subset of patients with monogenic disorders lacks disease causing mutations in the protein coding region of the corresponding gene. Here we describe a recurrent germline mutation found in two unrelated patients with complete androgen insensitivity syndrome (CAIS) generating an upstream open reading frame (uORF) in the 5’ untranslated region (5’-UTR) of the androgen receptor (AR) gene. We show in patient derived primary genital skin fibroblasts as well as in cell-based reporter assays that this mutation severely impacts AR function by reducing AR protein levels without affecting AR mRNA levels. Importantly, the newly generated uORF translates into a polypeptide and the expression level of this polypeptide inversely correlates with protein translation from the primary ORF of the AR thereby providing a model for AR-5′UTR mediated translational repression. Our findings not only add a hitherto unrecognized genetic cause to complete androgen insensitivity but also underline the importance of 5′UTR mutations affecting uORFs for the pathogenesis of monogenic disorders in general. PMID:27110943

The antiandrogenic effect of finasteride against a mutant androgen receptor

PubMed Central

Chhipa, Rishi Raj; Zhang, Haitao; Ip, Clement

2011-01-01

Finasteride is known to inhibit Type 2 5α-reductase and thus block the conversion of testosterone to dihydrotestosterone (DHT). The structural similarity of finasteride to DHT raises the possibility that finasteride may also interfere with the function of the androgen receptor (AR). Experiments were carried out to evaluate the antiandrogenic effect of finasteride in LNCaP, C4-2 and VCaP human prostate cancer cells. Finasteride decreased DHT binding to AR, and DHT-stimulated AR activity and cell growth in LNCaP and C4-2 cells, but not in VCaP cells. LNCaP and C4-2 (derived from castration-resistant LNCaP) cells express the T877A mutant AR, while VCaP cells express the wild-type AR. When PC-3 cells, which are AR-null, were transfected with either the wild-type or the T877A mutant AR, only the mutant AR-expressing cells were sensitive to finasteride inhibition of DHT binding. Peroxiredoxin-1 (Prx1) is a novel endogenous facilitator of AR binding to DHT. In Prx1-rich LNCaP cells, the combination of Prx1 knockdown and finasteride was found to produce a greater inhibitory effect on AR activity and cell growth than either treatment alone. The observation suggests that cells with a low expression of Prx1 are likely to be more responsive to the antiandrogenic effect of finasteride. Additional studies showed that the efficacy of finasteride was comparable to that of bicalutamide (a widely used non-steroidal antiandrogen). The implication of the above findings is discussed in the context of developing strategies to improve the outcome of androgen deprivation therapy. PMID:21386657

Transition from androgenic to neurosteroidal action of 5α-androstane-3α, 17β-diol through the type A γ-aminobutyric acid receptor in prostate cancer progression.

PubMed

Xia, Ding; Lai, Doan V; Wu, Weijuan; Webb, Zachary D; Yang, Qing; Zhao, Lichao; Yu, Zhongxin; Thorpe, Jessica E; Disch, Bryan C; Ihnat, Michael A; Jayaraman, Muralidharan; Dhanasekaran, Danny N; Stratton, Kelly L; Cookson, Michael S; Fung, Kar-Ming; Lin, Hsueh-Kung

2018-04-01

Androgen ablation is the standard of care prescribed to patients with advanced or metastatic prostate cancer (PCa) to slow down disease progression. Unfortunately, a majority of PCa patients under androgen ablation progress to castration-resistant prostate cancer (CRPC). Several mechanisms including alternative intra-prostatic androgen production and androgen-independent androgen receptor (AR) activation have been proposed for CRPC progression. Aldo-keto reductase family 1 member C3 (AKR1C3), a multi-functional steroid metabolizing enzyme, is specifically expressed in the cytoplasm of PCa cells; and positive immunoreactivity of the type A γ-aminobutyric acid receptor (GABA A R), an ionotropic receptor and ligand-gated ion channel, is detected on the membrane of PCa cells. We studied a total of 72 radical prostatectomy cases by immunohistochemistry, and identified that 21 cases exhibited positive immunoreactivities for both AKR1C3 and GABA A R. In the dual positive cancer cases, AKR1C3 and GABA A R subunit α 1 were either expressed in the same cells or in neighboring cells. Among several possible substrates, AKR1C3 reduces 5α-dihydrotesterone (DHT) to form 5α-androstane-3α, 17β-diol (3α-diol). 3α-diol is a neurosteroid that acts as a positive allosteric modulator of the GABA A R in the central nervous system (CNS). We examined the hypothesis that 3α-diol-regulated pathological effects in the prostate are GABA A R-dependent, but are independent of the AR. In GABA A R-positive, AR-negative human PCa PC-3 cells, 3α-diol significantly stimulated cell growth in culture and the in ovo chorioallantoic membrane (CAM) xenograft model. 3α-diol also up-regulated expression of the epidermal growth factor (EGF) family of growth factors and activation of EGF receptor (EGFR) and Src as measured by quantitative polymerase chain reaction and immunoblotting, respectively. Inclusion of GABA A R antagonists reversed 3α-diol-stimulated tumor cell growth, expression of EGF family members, and activation of EGFR and Src to the level observed in untreated cells. Results from the present study suggest that 3α-diol may act as an alternative intra-prostatic neurosteroid that activates AR-independent PCa progression. The involvement of AKR1C3-mediated steroid metabolisms in modulating GABA A R activation and promoting PCa progression requires continued studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Targeting Androgen Receptor to Suppress Macrophage-induced EMT and Benign Prostatic Hyperplasia (BPH) Development

PubMed Central

Lu, Tianjing; Lin, Wen-Jye; Izumi, Kouji; Wang, Xiaohai; Xu, Defeng; Fang, Lei-Ya; Li, Lei; Jiang, Qi

2012-01-01

Early studies suggested macrophages might play roles in inflammation-associated benign prostatic hyperplasia (BPH) development, yet the underlying mechanisms remain unclear. Here we first showed that CD68+ macrophages were identified in both epithelium and the stromal area of human BPH tissues. We then established an in vitro co-culture model with prostate epithelial and macrophage cell lines to study the potential impacts of infiltrating macrophages in the BPH development and found that co-culturing prostate epithelial cells with macrophages promoted migration of macrophages. In a three-dimensional culture system, the sphere diameter of BPH-1 prostate cells was significantly increased during coculture with THP-1 macrophage cells. Mechanism dissection suggested that expression levels of epithelial-mesenchymal transition (EMT) markers, such as N-cadherin, Snail, and TGF-β2, were increased, and administration of anti-TGF-β2 neutralizing antibody during co-culture suppressed the EMT and THP-1-mediated growth of BPH-1 cells, suggesting THP-1 might go through EMT to influence the BPH development and progression. Importantly, we found that modulation of androgen receptor (AR) in BPH-1 and mPrE cells significantly increased THP-1 and RAW264.7 cell migration, respectively, and enhanced expression levels of EMT markers, suggesting that AR in prostate epithelial cells might play a role in promoting macrophage-mediated EMT in prostate epithelial cells. Silencing AR function via an AR degradation enhancer, ASC-J9, decreased the macrophage migration to BPH-1 cells and suppressed EMT marker expression. Together, these results provide the first evidence to demonstrate that prostate epithelial AR function is important for macrophage-mediated EMT and proliferation of prostate epithelial cells, which represents a previously unrecognized role of AR in the cross-talk between macrophages and prostate epithelial cells. These results may provide new insights for a new therapeutic approach to battle BPH via targeting AR and AR-mediated inflammatory signaling pathways. PMID:22915828

Targeting androgen receptor to suppress macrophage-induced EMT and benign prostatic hyperplasia (BPH) development.

PubMed

Lu, Tianjing; Lin, Wen-Jye; Izumi, Kouji; Wang, Xiaohai; Xu, Defeng; Fang, Lei-Ya; Li, Lei; Jiang, Qi; Jin, Jie; Chang, Chawnshang

2012-10-01

Early studies suggested macrophages might play roles in inflammation-associated benign prostatic hyperplasia (BPH) development, yet the underlying mechanisms remain unclear. Here we first showed that CD68(+) macrophages were identified in both epithelium and the stromal area of human BPH tissues. We then established an in vitro co-culture model with prostate epithelial and macrophage cell lines to study the potential impacts of infiltrating macrophages in the BPH development and found that co-culturing prostate epithelial cells with macrophages promoted migration of macrophages. In a three-dimensional culture system, the sphere diameter of BPH-1 prostate cells was significantly increased during coculture with THP-1 macrophage cells. Mechanism dissection suggested that expression levels of epithelial-mesenchymal transition (EMT) markers, such as N-cadherin, Snail, and TGF-β2, were increased, and administration of anti-TGF-β2 neutralizing antibody during co-culture suppressed the EMT and THP-1-mediated growth of BPH-1 cells, suggesting THP-1 might go through EMT to influence the BPH development and progression. Importantly, we found that modulation of androgen receptor (AR) in BPH-1 and mPrE cells significantly increased THP-1 and RAW264.7 cell migration, respectively, and enhanced expression levels of EMT markers, suggesting that AR in prostate epithelial cells might play a role in promoting macrophage-mediated EMT in prostate epithelial cells. Silencing AR function via an AR degradation enhancer, ASC-J9, decreased the macrophage migration to BPH-1 cells and suppressed EMT marker expression. Together, these results provide the first evidence to demonstrate that prostate epithelial AR function is important for macrophage-mediated EMT and proliferation of prostate epithelial cells, which represents a previously unrecognized role of AR in the cross-talk between macrophages and prostate epithelial cells. These results may provide new insights for a new therapeutic approach to battle BPH via targeting AR and AR-mediated inflammatory signaling pathways.

Glycogen synthase kinase-3 inhibitor AR-A014418 suppresses pancreatic cancer cell growth via inhibition of GSK-3-mediated Notch1 expression.

PubMed

Kunnimalaiyaan, Selvi; Gamblin, T Clark; Kunnimalaiyaan, Muthusamy

2015-09-01

Glycogen synthase kinase-3 (GSK-3) can act as either a tumour promoter or suppressor by its inactivation depending on the cell type. There are conflicting reports on the roles of GSK-3 isoforms and their interaction with Notch1 in pancreatic cancer. It was hypothesized that GSK-3α stabilized Notch1 in pancreatic cancer cells thereby promoting cellular proliferation. The pancreatic cancer cell lines MiaPaCa2, PANC-1 and BxPC-3, were treated with 0-20 μM of AR-A014418 (AR), a known GSK-3 inhibitor. Cell viability was determined by the MTT assay and Live-Cell Imaging. The levels of Notch pathway members (Notch1, HES-1, survivin and cyclinD1), phosphorylated GSK-3 isoforms, and apoptotic markers were determined by Western blot. Immunoprecipitation was performed to identify the binding of GSK-3 specific isoform to Notch1. AR-A014418 treatment had a significant dose-dependent growth reduction (P < 0.001) in pancreatic cancer cells compared with the control and the cytotoxic effect is as a result of apoptosis. Importantly, a reduction in GSK-3 phosphorylation lead to a reduction in Notch pathway members. Overexpression of active Notch1 in AR-A014418-treated cells resulted in the negation of growth suppression. Immunoprecipitation analysis revealed that GSK-3α binds to Notch1. This study demonstrates for the first time that the growth suppressive effect of AR-A014418 on pancreatic cancer cells is mainly mediated by a reduction in phosphorylation of GSK-3α with concomitant Notch1 reduction. GSK-3α appears to stabilize Notch1 by binding and may represent a target for therapeutic development. Furthermore, downregulation of GSK-3 and Notch1 may be a viable strategy for possible chemosensitization of pancreatic cancer cells to standard therapeutics. © 2015 International Hepato-Pancreato-Biliary Association.

Advanced glycation end products alter steroidogenic gene expression by granulosa cells: an effect partially reversible by vitamin D.

PubMed

Merhi, Z; Buyuk, E; Cipolla, M J

2018-06-01

Does vitamin D attenuate the adverse effects of advanced glycation end products (AGEs) on steroidogenesis by human granulosa cells (GCs)? AGEs alter the expression of genes important in steroidogenesis while 1,25-dihydroxyvitamin D3 (vit D3) in vitro attenuates some of the actions of AGEs on steroidogenic gene expression, possibly by downregulating the expression of the pro-inflammatory cell membrane receptor for AGEs (RAGE). Vitamin D attenuates the pro-inflammatory effects of AGEs in non-ovarian tissues. Women who were undergoing IVF were enrolled. Follicular fluid samples (n = 71) were collected and cumulus GCs (n = 12) were treated in culture. Follicular fluid levels of the anti-inflammatory soluble RAGE (sRAGE), AGEs and 25-hydroxyvitamin D (25-OHD) were quantified for possible correlations. GCs of each participant were split equally and treated with either media alone (control) or with human glycated albumin (HGA as a precursor for AGEs) with or without vit D3 after which RT-PCR and immunofluorescence were performed and cell culture media estradiol (E2) levels were compared. In follicular fluid, sRAGE levels were positively correlated with 25-OHD levels. HGA treatment (i) increased CYP11A1 (by 48%), 3β-HSD (by 38%), StAR (by 42%), CYP17A1 (by 30%) and LHR (by 37%) mRNA expression levels (P < 0.05 for all) but did not alter CYP19A1 or FSHR mRNA expression levels; and (ii) increased E2 release in cell culture media (P = 0.02). Vit D3 treatment (i) downregulated RAGE mRNA expression by 33% and RAGE protein levels by 44% (P < 0.05); (ii) inhibited the HGA-induced increase in CYP11A1, StAR, CYP17A1 and LHR mRNA levels, but not the increase in 3β-HSD mRNA levels; and (iii) did not inhibit the HGA-induced E2 release in cell culture media. This study used luteinized GCs that were collected from women who received gonadotropins thus the results obtained may not fully extrapolate to non-luteinized GCs in vivo. This study suggests that there is a relationship between AGEs and their receptors (RAGE and sRAGE) with vitamin D. Understanding the interaction between AGEs and vitamin D in ovarian physiology could lead to a more targeted therapy for the treatment of ovarian dysfunction. Funding was received from NIH (R01 NS045940), American Society for Reproductive Medicine, Ferring Pharmaceuticals Inc., and University of Vermont College of Medicine Bridge Funds. All authors have nothing to disclose.

Concurrent hypothalamic gene expression under acute and chronic long days: Implications for initiation and maintenance of photoperiodic response in migratory songbirds.

PubMed

Mishra, Ila; Bhardwaj, Sanjay K; Malik, Shalie; Kumar, Vinod

2017-01-05

Hypothalamic expression of the thyroid hormone (TH) responsive gonadostimulatory (eya3, cga, tshβ, dio2, dio3, gnrh, gnih) and neurosteroid pathway genes (androgen receptor [ar], aromatase [cyp19], estrogen receptor [er] α and β) was examined in photosensitive redheaded buntings exposed to 2 (acute, experiment 1) or 12 (chronic, experiment 2) long days (16L:8D). Experiment 2 also included a photorefractory group. Acute long days caused a significant increase in eya3, cga, tshβ, dio2 and gnrh and decrease in dio3 mRNA levels. eya3, cga and tshβ expressions were unchanged after the chronic long days. We also found increased cyp19, erα and erβ mRNA levels after acute, and increased cyp19 and decreased erβ levels after the chronic long-day exposure. Photorefractory buntings showed expression patterns similar to that in the photosensitive state, except for high gnrh and gnih and low dio3 mRNA levels. Consistent with gene expression patterns, there were changes in fat deposition, body mass, testis size, and plasma levels of testosterone, tri-iodothyronine and thyroxine. These results show concurrent photostimulation of the TH-signalling and neurosteroid pathways, and extend the idea, based on differences in gene expression, that transitions in seasonal photoperiodic states are accomplished at the transcriptional levels in absolute photorefractory species. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Cardiac Alpha1-Adrenergic Receptors: Novel Aspects of Expression, Signaling Mechanisms, Physiologic Function, and Clinical Importance

PubMed Central

O’Connell, Timothy D.; Jensen, Brian C.; Baker, Anthony J.

2014-01-01

Adrenergic receptors (AR) are G-protein-coupled receptors (GPCRs) that have a crucial role in cardiac physiology in health and disease. Alpha1-ARs signal through Gαq, and signaling through Gq, for example, by endothelin and angiotensin receptors, is thought to be detrimental to the heart. In contrast, cardiac alpha1-ARs mediate important protective and adaptive functions in the heart, although alpha1-ARs are only a minor fraction of total cardiac ARs. Cardiac alpha1-ARs activate pleiotropic downstream signaling to prevent pathologic remodeling in heart failure. Mechanisms defined in animal and cell models include activation of adaptive hypertrophy, prevention of cardiac myocyte death, augmentation of contractility, and induction of ischemic preconditioning. Surprisingly, at the molecular level, alpha1-ARs localize to and signal at the nucleus in cardiac myocytes, and, unlike most GPCRs, activate “inside-out” signaling to cause cardioprotection. Contrary to past opinion, human cardiac alpha1-AR expression is similar to that in the mouse, where alpha1-AR effects are seen most convincingly in knockout models. Human clinical studies show that alpha1-blockade worsens heart failure in hypertension and does not improve outcomes in heart failure, implying a cardioprotective role for human alpha1-ARs. In summary, these findings identify novel functional and mechanistic aspects of cardiac alpha1-AR function and suggest that activation of cardiac alpha1-AR might be a viable therapeutic strategy in heart failure. PMID:24368739

Activation of p300 histone acetyltransferase activity and acetylation of the androgen receptor by bombesin in prostate cancer cells.

PubMed

Gong, J; Zhu, J; Goodman, O B; Pestell, R G; Schlegel, P N; Nanus, D M; Shen, R

2006-03-30

Androgen receptor signaling in prostate cancer cells is augmented by the androgen receptor (AR) coactivator p300, which transactivates and acetylates the AR in the presence of dihydrotestosterone (DHT). As prostate cancer (PC) cells progress to androgen independence, AR signaling remains intact, indicating that other factors stimulate AR activities in the absence of androgen. We previously reported that neuropeptide growth factors could transactivate the AR in the presence of very low concentrations of DHT. Here, we examine the involvement of p300 in neuropeptide activation of AR signaling. Transfection of increasing concentrations of p300 in the presence of bombesin into PC-3 cells resulted in a linear increase in AR transactivation, suggesting that p300 acts as a coactivator in neuropeptide-mediated AR transactivation. P300 is endowed with histone acetyltransferase (HAT) activity. Therefore, we examine the effect of bombesin on p300 HAT activity. At 4 h after the addition of bombesin, p300 HAT activity increased 2.0-fold (P<0.01). Incubation with neutral endopeptidase, which degrades bombesin, or bombesin receptor antagonists blocked bombesin-induced p300 HAT activity. To explore the potential signaling pathways involved in bombesin-induced p300 HAT activity, we examined Src and PKCdelta pathways that mediate bombesin signaling. Inhibitors of Src kinase activity or Src kinase siRNA blocked bombesin-induced p300 HAT activity, whereas PKCdelta inhibitors or PKCdelta siRNA significantly increased bombesin-induced p300 HAT activity suggesting that Src kinase and PKCdelta kinase are involved in the regulation of p300 HAT activity. As AR is acetylated in the presence of 100 nM DHT, we next examined whether bombesin-induced p300 HAT activity would result in enhanced AR acetylation. Bombesin-induced AR acetylation at the same motif KLKK observed in DHT-induced acetylation. Elimination of p300 using p300 siRNA reduced AR acetylation, demonstrating that AR acetylation was mediated by p300. AR acetylation results in AR transactivation and the expression of the AR-regulated gene prostate-specific antigen (PSA). Therefore, we examined bombesin-induced AR transactivation and PSA expression in the presence and absence of p300 siRNA and found inhibition of p300 expression reduced bombesin-induced AR transactivation and PSA expression. Together these results demonstrate that bombesin, via Src and PKCdelta signaling pathways, activates p300 HAT activity which leads to enhanced acetylation of AR resulting in increased expression of AR-regulated genes.

Axenfeld-Rieger syndrome: further clinical and array delineation of four unrelated patients with a 4q25 microdeletion.

PubMed

Titheradge, Hannah; Togneri, Fiona; McMullan, Dominic; Brueton, Louise; Lim, Derek; Williams, Denise

2014-07-01

Axenfeld-Rieger syndrome (ARS) is an autosomal dominant disorder with variable expressivity. It is characterized by dysgenesis of the anterior segment of the eye together with dental, cardiac, and umbilical anomalies. There is a high incidence of secondary high tension glaucoma. It is a genetically heterogeneous condition due to deletion or mutations of FOXC1 (6p25) or PITX2 (4q25). We report on four unrelated patients with overlapping microdeletions encompassing PITX2 at 4q25. We compare the genotypes and phenotypes of these newly described ARS patients and discuss the involvement of contiguous genes. Patients 1, 2, and 3 had mild learning difficulties, not typically seen in patients with ARS. We implicate the adjacent neuronally expressed genes; NEUROG2, UGT8, NDST3, and PRSS12 as potentially causal. Our findings support the use of microarray analysis in ARS patients for full prognostic information in infants presenting with ARS-like phenotypes. © 2014 Wiley Periodicals, Inc.

Probiotics as a Complementary Therapy in the Model of Cadmium Chloride Toxicity: Crosstalk of β-Catenin, BDNF, and StAR Signaling Pathways.

PubMed

Kadry, Mai O; Megeed, Rehab Abdel

2018-02-09

Cadmium chloride (CdCl 2 ) is a ubiquitous environmental toxicant that causes a variety of disturbances in biological systems, including brain dysfunction and testicular tissue degeneration. On the other hand, it is supposed that beneficial properties of probiotic bacteria (Lactobacillus and Acidobacillus) are related to their capacity to adhere or bind different targets, thus leading to improved intestinal microbial balance and other benefits to the host. Bearing aforementioned in mind, the present study was undertaken to investigate the protective effect of probiotic supplementation against cadmium chloride-induced brain and testis toxicity in mice model. Animals received Lactobacillus and Acidobacillus either alone or added to folic acid for 1 week before CdCl2 intoxication in a dose of 20 mg/kg BW followed by probiotics (5 × 10 9) and/or folic acid (12 mg/kg) treatment for 3 weeks. The levels of malondialdehyde (MDA), butyrl choline esterase (BCHE), reduced glutathione (GSH), and total superoxide dismutase (SOD) activities were investigated. Finally, cadmium neurotoxicity was determined by estimating the gene expression of β-catenin and brain-derived neurotrophic factor (BDNF), as well as estimating the alterations in testicular function by determining acid phosphatase level in addition to steroidogenic acute regulatory protein (StAR) and 17-hydroxy steroid dehydrogenase (17-β HSD) gene expression. Based on our results, we can conclude that exposure of mice to cadmium chloride resulted in a significant elevation in MDA, BCHE levels accompanied with a significant reduction in GSH and SOD activities compared to the control value. CdCl 2 also downregulated the gene expression of β-catenin and BDNF, as well as acid phosphatase level, in addition to StAR and 17-β HSD gene expression. These deviated parameters were significantly modulated in the co-treated animals with probiotics compared with the cadmium-treated group. In conclusion, Lactobacillus and folic acid in a mixture with cadmium acted beneficially to an organism, increasing the cadmium excretion in feces, and consequently increasing β-catenin and BDNF in brain tissue and StAR and 17-β HSD in testis and improving their functions. Histoarchitecture analysis confirmed these results.

High-yield soluble expression, purification and characterization of human steroidogenic acute regulatory protein (StAR) fused to a cleavable Maltose-Binding Protein (MBP).

PubMed

Sluchanko, Nikolai N; Tugaeva, Kristina V; Faletrov, Yaroslav V; Levitsky, Dmitrii I

2016-03-01

Steroidogenic acute regulatory protein (StAR) is responsible for the rapid delivery of cholesterol to mitochondria where the lipid serves as a source for steroid hormones biosynthesis in adrenals and gonads. Despite many successful investigations, current understanding of the mechanism of StAR action is far from being completely clear. StAR was mostly obtained using denaturation/renaturation or in minor quantities in a soluble form at decreased temperatures that, presumably, limited the possibilities for its consequent detailed exploration. In our hands, existing StAR expression constructs could be bacterially expressed almost exclusively as insoluble forms, even upon decreased expression temperatures and in specific strains of Escherichia coli, and isolated protein tended to aggregate and was difficult to handle. To maximize the yield of soluble protein, optimized StAR sequence encompassing functional domain STARD1 (residues 66-285) was fused to the C-terminus of His-tagged Maltose-Binding Protein (MBP) with the possibility to cleave off the whole tag by 3C protease. The developed protocol of expression and purification comprising of a combination of subtractive immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography allowed us to obtain up to 25 mg/1 L culture of completely soluble StAR protein, which was (i) homogenous according to SDS-PAGE, (ii) gave a single symmetrical peak on a gel-filtration, (iii) showed the characteristic CD spectrum and (iv) pH-dependent ability to bind a fluorescently-labeled cholesterol analogue. We conclude that our strategy provides fully soluble and native StAR protein which in future could be efficiently used for biotechnology and drug discovery aimed at modulation of steroids production. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of selenium on the proliferation, apoptosis and testosterone production of sheep Leydig cells in vitro.

PubMed

Shi, Lei; Song, Ruigao; Yao, Xiaolei; Ren, Youshe

2017-04-15

The objective of this study was to investigate the effects of selenium (Se) on in vitro proliferation, apoptosis and testosterone production of sheep Leydig cells and its underlying mechanism. Leydig cells were collected from 8-month-old sheep and divided into four treatment groups (0, 2.0, 4.0 and 8.0 μmol/L Se). After treatment with Se for 48 h, the MTT and flow cytometric assay were used to detect cell proliferation and apoptosis. Testosterone level in the culture medium was determined by ELISA. The mRNA expression and protein abundance of cell cycle, apoptosis and testosterone synthesis-related genes were detected using real-time PCR and western blot analysis. The results showed that the highest percentage of live and apoptotic cells was obtained in the 2.0 and 8.0 μmol/L group, respectively. In the Se treatment groups, the proliferation rate of Leydig cells and the expression of cell cycle-related genes were decreased with the increasing Se supplementation in the culture medium. The percentage of apoptotic cells was increased with the increasing Se level, which was consistent with the expression of pro-apoptosis genes. The highest GSH-Px activity and lowest ROS content were also observed in the 2.0 μmol/L group. Appropriate Se level (2.0 μmol/L) can significantly increase the expression of p-ERK1/2, StAR and 3β-HSD, and improve the testosterone synthesis. Compared with the control group, PD0325901 could significantly inhibit the production of testosterone and the protein abundance of p-ERK1/2, StAR and 3β-HSD. Se treatment can mitigate the inhibition effect of PD0325901 and the testosterone secretion between the 2.0 μmol/L and control group was not significantly different. These results demonstrate that Se can affect the proliferation and apoptosis of Leydig cells by regulating cellular oxidative stress and the expressions of cell cycle and apoptosis-related genes. Se can also enhance the testosterone production of Leydig cells by activating the ERK signaling pathway and the expression of its downstream genes (StAR and 3β-HSD), which could be closely related to the regulating roles of Se in male fertility and spermatogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

CX4945 suppresses the growth of castration-resistant prostate cancer cells by reducing AR-V7 expression.

PubMed

Deng, Chuangzhong; Chen, Jieping; Guo, Shengjie; Wang, Yanjun; Zhou, Qianghua; Li, Zaishang; Yang, Xingping; Yu, Xingsu; Zhang, Zhenfeng; Zhou, Fangjian; Han, Hui; Yao, Kai

2017-08-01

The aberrant expression of casein kinase 2 (CK2) has been reported to be involved in the tumorigenesis and progression of prostate cancer. The inhibition of CK2 activity represses androgen-dependent prostate cancer cells by attenuating the androgen receptor (AR) signaling pathway. In this study, we examined the effect of CK2 inhibition in castration-resistant prostate cancer (CRPC) cells, in which AR variants (ARVs) play a predominant role. A newly synthetic CK2 selective inhibitor CX4945 was utilized to study the effect of CK2 inhibition in CRPC cells by CCK8 assay and colony formation assay. Protein and mRNA levels of full-length AR (AR-FL) and AR-V7 were determined by qPCR and western blot, respectively. The nuclear translocation of p50 and p65 was assessed to reflect the activity of the NF-κB pathway. CX4945 reduced the proliferation of CRPC cells in a dose-dependent and time-dependent manner. AR-V7 rather than AR-FL was downregulated by CX4945 in both the mRNA and protein level. Furthermore, CX4945 could restore the sensitivity of CRPC cells to bicalutamide. The analysis of possible mechanisms demonstrated that the inhibition of CK2 diminished the phosphorylation of p65 at ser529 and thus attenuated the activity of the NF-κB pathway. The inhibition of CK2 by CX4945 can repress the viability of CRPC cells and restore their sensitivity to anti-androgen therapy by suppressing AR-V7. This finding presents a potential option for the treatment of prostate cancer, especially CRPC.

The RNA helicase DDX39B and its paralog DDX39A regulate androgen receptor splice variant AR-V7 generation.

PubMed

Nakata, Daisuke; Nakao, Shoichi; Nakayama, Kazuhide; Araki, Shinsuke; Nakayama, Yusuke; Aparicio, Samuel; Hara, Takahito; Nakanishi, Atsushi

2017-01-29

Mounting evidence suggests that constitutively active androgen receptor (AR) splice variants, typified by AR-V7, are associated with poor prognosis and resistance to androgen deprivation therapy in prostate cancer patients. However, mechanisms governing the generation of AR splice variants are not fully understood. In this study, we aimed to investigate the dynamics of AR splice variant generation using the JDCaP prostate cancer model that expresses AR splice variants under androgen depletion. Microarray analysis of JDCaP xenografts before and after expression of AR splice variants suggested that dysregulation of RNA processing pathways is likely involved in AR splice variant generation. To explore factors contributing to generation of AR-V7 mRNA, we conducted a focused RNA interference screen in AR-V7-positive JDCaP-hr cells using an shRNA library targeting spliceosome-related genes. This screen identified DDX39B as a regulator of AR-V7 mRNA expression. Simultaneous knockdown of DDX39B and its paralog DDX39A drastically and selectively downregulated AR-V7 mRNA expression in multiple AR-V7-positive prostate cancer cell lines. DDX39B was upregulated in relapsed JDCaP xenografts expressing AR splice variants, suggesting its role in expression of AR splice variants. Taken together, our findings offer insight into the mechanisms of AR splice variant generation and identify DDX39 as a potential drug target for the treatment of AR splice variant-positive prostate cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Das, Dibash K.; The Graduate Center Departments of Biology and Biochemistry, The City University of New York, New York, NY 10016; Department of Medicine, Weill Cornell Medicine, Cornell University, New York, NY 10065

Prostate cancer (PCa) is frequently diagnosed in men, and dysregulation of microRNAs is characteristic of many cancers. MicroRNA-1207-3p is encoded at the non-protein coding gene locus PVT1 on the 8q24 human chromosomal region, an established PCa susceptibility locus. However, the role of microRNA-1207-3p in PCa is unclear. We discovered that microRNA-1207-3p is significantly underexpressed in PCa cell lines in comparison to normal prostate epithelial cells. Increased expression of microRNA-1207-3p in PCa cells significantly inhibits proliferation, migration, and induces apoptosis via direct molecular targeting of FNDC1, a protein which contains a conserved protein domain of fibronectin (FN1). FNDC1, FN1, and themore » androgen receptor (AR) are significantly overexpressed in PCa cell lines and human PCa, and positively correlate with aggressive PCa. Prostate tumor FN1 expression in patients that experienced PCa-specific death is significantly higher than in patients that remained alive. Furthermore, FNDC1, FN1 and AR are concomitantly overexpressed in metastatic PCa. Consequently, these studies have revealed a novel microRNA-1207-3p/FNDC1/FN1/AR regulatory pathway in PCa. - Graphical abstract: miR-1207-3p/FNDC1/FN1/AR is a novel regulatory pathway in prostate cancer. - Highlights: • Expression of microRNA-1207-3p is significantly lost in prostate cancer (PCa) cells. • MicroRNA-1207-3p regulates proliferation, apoptosis, and migration via direct molecular targeting of the 3′UTR of FNDC1. • MicroRNA-1207-3p regulates proliferation, apoptosis, and migration via direct molecular targeting of the 3′UTR of FNDC1. • FNDC1, FN1, and AR are concurrently overexpressed in metastatic PCa.« less

Characteristics of Men Who Report Persistent Sexual Symptoms After Finasteride Use for Hair Loss.

PubMed

Basaria, Shehzad; Jasuja, Ravi; Huang, Grace; Wharton, Whitney; Pan, Hong; Pencina, Karol; Li, Zhuoying; Travison, Thomas G; Bhawan, Jag; Gonthier, Renaud; Labrie, Fernand; Dury, Alain Y; Serra, Carlo; Papazian, Allen; O'Leary, Michael; Amr, Sami; Storer, Thomas W; Stern, Emily; Bhasin, Shalender

2016-12-01

Some men who use finasteride for hair loss report persistent sexual and other symptoms after discontinuing finasteride therapy. To determine whether these persistent symptoms after discontinuation of finasteride use are due to androgen deficiency, decreased peripheral androgen action, or persistent inhibition of steroid 5α-reductase (SRD5A) enzymes. Finasteride users, who reported persistent sexual symptoms after discontinuing finasteride (group 1); age-matched finasteride users who did not report sexual symptoms (group 2); and healthy men who had never used finasteride (group 3). Sexual function, mood, affect, cognition, hormone levels, body composition, functional magnetic resonance imaging (fMRI) response to sexually and affectively valenced stimuli, nucleotide sequences of androgen receptor (AR), SRD5A1, and SRD5A2; expression levels of androgen-dependent genes in skin. Academic medical center. Symptomatic finasteride users were similar in body composition, strength, and nucleotide sequences of AR, SRD5A1, and SRD5A2 genes to asymptomatic finasteride users and nonusers. Symptomatic finasteride users had impaired sexual function, higher depression scores, a more negative affectivity balance, and more cognitive complaints than men in groups 2 and 3 but had normal objectively assessed cognitive function. Testosterone, dihydrotestosterone, 5α-androstane-3α,17β-diol-glucuronide, testosterone to dihydrotestosterone and androsterone glucuronide to etiocholanolone glucuronide ratios, and markers of peripheral androgen action and expression levels of AR-dependent genes in skin did not differ among groups. fMRI blood oxygen level-dependent responses to erotic and nonerotic stimuli revealed abnormal function in brain circuitry linked to sexual arousal and major depression. We found no evidence of androgen deficiency, decreased peripheral androgen action, or persistent peripheral inhibition of SRD5A in men with persistent sexual symptoms after finasteride use. Symptomatic finasteride users revealed depressed mood and fMRI findings consistent with those observed in depression.

Characteristics of Men Who Report Persistent Sexual Symptoms After Finasteride Use for Hair Loss

PubMed Central

Basaria, Shehzad; Jasuja, Ravi; Huang, Grace; Wharton, Whitney; Pan, Hong; Pencina, Karol; Li, Zhuoying; Travison, Thomas G.; Bhawan, Jag; Gonthier, Renaud; Labrie, Fernand; Dury, Alain Y.; Serra, Carlo; Papazian, Allen; O'Leary, Michael; Amr, Sami; Storer, Thomas W.; Stern, Emily

2016-01-01

Context: Some men who use finasteride for hair loss report persistent sexual and other symptoms after discontinuing finasteride therapy. Objective: To determine whether these persistent symptoms after discontinuation of finasteride use are due to androgen deficiency, decreased peripheral androgen action, or persistent inhibition of steroid 5α-reductase (SRD5A) enzymes. Participants: Finasteride users, who reported persistent sexual symptoms after discontinuing finasteride (group 1); age-matched finasteride users who did not report sexual symptoms (group 2); and healthy men who had never used finasteride (group 3). Outcomes: Sexual function, mood, affect, cognition, hormone levels, body composition, functional magnetic resonance imaging (fMRI) response to sexually and affectively valenced stimuli, nucleotide sequences of androgen receptor (AR), SRD5A1, and SRD5A2; expression levels of androgen-dependent genes in skin. Setting: Academic medical center. Results: Symptomatic finasteride users were similar in body composition, strength, and nucleotide sequences of AR, SRD5A1, and SRD5A2 genes to asymptomatic finasteride users and nonusers. Symptomatic finasteride users had impaired sexual function, higher depression scores, a more negative affectivity balance, and more cognitive complaints than men in groups 2 and 3 but had normal objectively assessed cognitive function. Testosterone, dihydrotestosterone, 5α-androstane-3α,17β-diol-glucuronide, testosterone to dihydrotestosterone and androsterone glucuronide to etiocholanolone glucuronide ratios, and markers of peripheral androgen action and expression levels of AR-dependent genes in skin did not differ among groups. fMRI blood oxygen level-dependent responses to erotic and nonerotic stimuli revealed abnormal function in brain circuitry linked to sexual arousal and major depression. Conclusions: We found no evidence of androgen deficiency, decreased peripheral androgen action, or persistent peripheral inhibition of SRD5A in men with persistent sexual symptoms after finasteride use. Symptomatic finasteride users revealed depressed mood and fMRI findings consistent with those observed in depression. PMID:27662439

Adenosine receptor expression in an experimental animal model of myocardial infarction with preserved left ventricular ejection fraction.

PubMed

Cabiati, Manuela; Martino, Alessandro; Mattii, Letizia; Caselli, Chiara; Prescimone, Tommaso; Lionetti, Vincenzo; Morales, Maria-Aurora; Del Ry, Silvia

2014-07-01

Adenosine, a purine nucleoside and a "retaliatory metabolite" in ischemia, is ubiquitous in the body and increases 100-fold during ischemia. Its biological actions are mediated by four adenosine receptors (ARs): A(1), A(2A), A(2B) and A(3). The aim of this study was to determine possible myocardial alterations in AR expression in an experimental animal model of myocardial infarction (MI) with a preserved left ventricular (LV) ejection fraction. LV tissue was collected from sexually mature male farm pigs with MI (n = 6) induced by permanent surgical ligation of the left anterior descending coronary artery and from five healthy pigs (C). mRNA expression of A(1)R, A(2A)R, A(2B)R, A(3)R and TNF-α was determined by real-time PCR in tissue collected from border (BZ) and remote zones (RZ) of the infarcted area and from LV of C. BZ, RZ and samples of C were stained immunohistochemically to investigate A(3)R immunoreaction. After 4 weeks a different regulation of ARs was observed. A(1)R mRNA expression was significantly lower in the infarct regions than in controls (C = 0.75 ± 0.2, BZ = 0.05 ± 0.2, RZ = 0.07 ± 0.02 p = 0.0025, p = 0.0016, C vs. BZ and RZ, respectively). Conversely A(3)R was higher in infarct areas (C = 0.94 ± 0.2, BZ = 2.85 ± 0.5, RZ = 3.48 ± 1.0, p = 0.048 C vs. RZ). No significant differences were observed for A(2A)R (C = 1.58 ± 0.6, BZ = 0.42 ± 0.1, RZ = 1.37 ± 0.6) and A(2B)R (C = 1.66 ± 0.2, BZ = 1.54 ± 0.5, RZ = 1.25 ± 0.4). A(3)R expression was confirmed by immunohistochemical analysis and was principally localized in cardiomyocytes. TNF-α mRNA results were: C 0.41 ± 0.25; BZ 1.60 ± 0.19; RZ 0.17 ± 0.04. The balance between A(1)R and A(3)R as well as between A(2A)R and A(2B)R was consistent with adaptative retaliatory anti-ischemic adenosinergic changes in the infarcted heart with preserved LV function.

Small Molecule-Induced Complement Factor D (Adipsin) Promotes Lipid Accumulation and Adipocyte Differentiation

PubMed Central

Jang, Byung-Hyun; Chang, Seo-Hyuk; Yun, Ui Jeong; Park, Ki-Moon; Waki, Hironori; Li, Dean Y.; Tontonoz, Peter; Park, Kye Won

2016-01-01

Adipocytes are differentiated by various transcriptional cascades integrated on the master regulator, Pparγ. To discover new genes involved in adipocyte differentiation, preadipocytes were treated with three newly identified pro-adipogenic small molecules and GW7845 (a Pparγ agonist) for 24 hours and transcriptional profiling was analyzed. Four genes, Peroxisome proliferator-activated receptor γ (Pparγ), human complement factor D homolog (Cfd), Chemokine (C-C motif) ligand 9 (Ccl9), and GIPC PDZ Domain Containing Family Member 2 (Gipc2) were induced by at least two different small molecules but not by GW7845. Cfd and Ccl9 expressions were specific to adipocytes and they were altered in obese mice. Small hairpin RNA (shRNA) mediated knockdown of Cfd in preadipocytes inhibited lipid accumulation and expression of adipocyte markers during adipocyte differentiation. Overexpression of Cfd promoted adipocyte differentiation, increased C3a production, and led to induction of C3a receptor (C3aR) target gene expression. Similarly, treatments with C3a or C3aR agonist (C4494) also promoted adipogenesis. C3aR knockdown suppressed adipogenesis and impaired the pro-adipogenic effects of Cfd, further suggesting the necessity for C3aR signaling in Cfd-mediated pro-adipogenic axis. Together, these data show the action of Cfd in adipogenesis and underscore the application of small molecules to identify genes in adipocytes. PMID:27611793

Effects of triclosan (TCS) on hormonal balance and genes of hypothalamus-pituitary- gonad axis of juvenile male Yellow River carp (Cyprinus carpio).

PubMed

Wang, Fan; Liu, Fei; Chen, Wanguang; Xu, Ruijie; Wang, Wei

2018-02-01

Triclosan (TCS) is a broad spectrum antimicrobial agent which has been widely dispersed and determinated in the aquatic environment. However, the effects of TCS on reproductive endocrine in male fish are poorly understood. In this study, male Yellow River carp (Cyprinus carpio) were exposed to 0, 1/5, 1/10 and 1/20 LC 50 (96 h LC 50 of TCS to carp) TCS under semi-static conditions for 42 d. Vitellogenin (Vtg), 17β-estradiol (E 2 ), testosterone(T), gonadotropin (GtH), and gonadotropin-releasing hormone (GnRH) levels were measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, we also examined the mRNA expressions of aromatase, GtHs-β, GnRH, estrogen receptor (Er), and androgen receptor (Ar) by quantitative Real-time Polymerase Chain Reaction (qRT-PCR). TCS induced Vtg levels of hepatopancreas, E 2 levels of serum, and inhibited Ar and Er mRNA levels, suggesting that the induction of Vtg production by TCS was indirectly caused by non-Er pathways. TCS-induced Vtg levels by interfering with the reproductive axis at plenty of latent loci of male carps: (a) TCS exposure increased the aromatase mRNA expression of hypothalamus and gonad aromatase, consequently increasing serum concentrations of E 2 to induce Vtg in hepatopancreas; (b) TCS treatment changed GtH-β and GnRH mRNA expression and secretion, causing the disturbance of reproductive endocrine; (c) TCS exposure decreased Ar mRNA levels, indicating potential Ar-mediated antiandrogen action. These mechanisms showed that TCS may induce Vtg production in male carp by non-Er-mediated pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

Characterization and Developmental Expression Profile of the Steroidogenic Acute Regulatory Protein (StAR) in the Gonad-Mesonephros Complex of Lithobates sylvaticus.

PubMed

Robertson, Courtney; Pauli, Bruce D; Trudeau, Vance L; Navarro-Martín, Laia

2016-01-01

The steroidogenic acute regulatory (StAR) protein is responsible for the movement of cholesterol across mitochondrial membranes and is therefore a key factor in regulating the timing and rate of steroidogenesis. In this study, we characterized the coding region of the star gene in the ranid Lithobates sylvaticus and studied star mRNA levels in steroidogenic tissues during development and under natural conditions. Our results support previous research showing that the StAR sequence is well conserved. We determined that star is expressed in both the interrenal and gonadal tissues of adults and in the tadpole gonad-mesonephros complex (GMC). The mRNA levels of star in the GMC were found to increase during tadpole development, reaching a maximum between Gosner stages (Gs) 32 and 38. We observed a significant drop in star mRNA levels at the end of prometamorphosis (Gs40-41), just before the start of the metamorphic climax. Significant differences in star levels between females and males, with males presenting higher levels than females, were detected at Gs36-38. To our knowledge, this is the first study that reports transitory star sex differences in tadpoles' developing GMC. Our results suggest an involvement of StAR in anuran late male GMC formation and development that requires further investigation. © 2016 S. Karger AG, Basel.

Inhibition of androgen receptor by decoy molecules delays progression to castration-recurrent prostate cancer.

PubMed

Myung, Jae-Kyung; Wang, Gang; Chiu, Helen H L; Wang, Jun; Mawji, Nasrin R; Sadar, Marianne D

2017-01-01

Androgen receptor (AR) is a member of the steroid receptor family and a therapeutic target for all stages of prostate cancer. AR is activated by ligand binding within its C-terminus ligand-binding domain (LBD). Here we show that overexpression of the AR NTD to generate decoy molecules inhibited both the growth and progression of prostate cancer in castrated hosts. Specifically, it was shown that lentivirus delivery of decoys delayed hormonal progression in castrated hosts as indicated by increased doubling time of tumor volume, prolonged time to achieve pre-castrate levels of serum prostate-specific antigen (PSA) and PSA nadir. These clinical parameters are indicative of delayed hormonal progression and improved therapeutic response and prognosis. Decoys reduced the expression of androgen-regulated genes that correlated with reduced in situ interaction of the AR with androgen response elements. Decoys did not reduce levels of AR protein or prevent nuclear localization of the AR. Nor did decoys interact directly with the AR. Thus decoys did not inhibit AR transactivation by a dominant negative mechanism. This work provides evidence that the AR NTD plays an important role in the hormonal progression of prostate cancer and supports the development of AR antagonists that target the AR NTD.

Inhibition of androgen receptor by decoy molecules delays progression to castration-recurrent prostate cancer

PubMed Central

Myung, Jae-Kyung; Wang, Gang; Chiu, Helen H. L.; Wang, Jun; Mawji, Nasrin R.; Sadar, Marianne D.

2017-01-01

Androgen receptor (AR) is a member of the steroid receptor family and a therapeutic target for all stages of prostate cancer. AR is activated by ligand binding within its C-terminus ligand-binding domain (LBD). Here we show that overexpression of the AR NTD to generate decoy molecules inhibited both the growth and progression of prostate cancer in castrated hosts. Specifically, it was shown that lentivirus delivery of decoys delayed hormonal progression in castrated hosts as indicated by increased doubling time of tumor volume, prolonged time to achieve pre-castrate levels of serum prostate-specific antigen (PSA) and PSA nadir. These clinical parameters are indicative of delayed hormonal progression and improved therapeutic response and prognosis. Decoys reduced the expression of androgen-regulated genes that correlated with reduced in situ interaction of the AR with androgen response elements. Decoys did not reduce levels of AR protein or prevent nuclear localization of the AR. Nor did decoys interact directly with the AR. Thus decoys did not inhibit AR transactivation by a dominant negative mechanism. This work provides evidence that the AR NTD plays an important role in the hormonal progression of prostate cancer and supports the development of AR antagonists that target the AR NTD. PMID:28306720

Expression of ARs in triple negative breast cancer tumors: a potential prognostic factor?

PubMed

Giannos, Aris; Filipits, Martin; Zagouri, Flora; Brandstetter, Anita; Tsigginou, Alexandra; Sotiropoulou, Maria; Papaspyrou, Irene; Sergentanis, Theodoros N; Psaltopoulou, Theodora; Rodolakis, Alexandros; Antsaklis, Aris; Dimopoulos, Meletios-Athanasios; Dimitrakakis, Constantine

2015-01-01

In light of the controversial published literature, this study aims to examine the potential prognostic role of AR immunohistochemical expression in triple negative breast cancer (TNBC). Ninety patients with TNBC were included in this study; the associations between AR expression (Allred score), clinicopathological variables (stage, grade, histological subtype, tumor size, nodal status, age at diagnosis, Ki67 expression, and p53 expression), and overall survival were evaluated. AR expression was not associated with stage, grade, histological subtype, tumor size, nodal status, age at diagnosis, Ki67 expression, and p53 expression. AR immunopositivity was not associated with overall survival either at the univariate or at the multivariate Cox regression analysis (multivariate hazard ratio =0.66, 95% confidence interval: 0.26-1.70, P=0.393). AR expression does not seem to play a prognostic role in TNBC.

Detection of AR-V7 mRNA in whole blood may not predict the effectiveness of novel endocrine drugs for castration-resistant prostate cancer.

PubMed

Takeuchi, Takumi; Okuno, Yumiko; Hattori-Kato, Mami; Zaitsu, Masayoshi; Mikami, Koji

2016-01-01

A splice variant of androgen receptor (AR), AR-V7, lacks in androgen-binding portion and leads to aggressive cancer characteristics. Reverse transcription-polymerase chain reactions (PCRs) and subsequent nested PCRs for the amplification of AR-V7 and prostate-specific antigen (PSA) transcripts were done for whole blood of patients with prostate cancer and male controls. With primary reverse transcription PCRs, AR-V7 and PSA were detected in 4.5% and 4.7% of prostate cancer, respectively. With nested PCRs, AR-V7 messenger RNA (mRNA) was positive in 43.8% of castration-sensitive prostate cancer and 48.1% of castration-resistant prostate cancer (CRPC), while PSA mRNA was positive in 6.3% of castration-sensitive prostate cancer and 18.5% of CRPC. Whole-blood samples of controls showed AR-V7 mRNA expression by nested PCR. Based on multivariate analysis, expression of AR-V7 mRNA in whole blood was not significantly correlated with clinical parameters and PSA mRNA in blood, while univariate analysis showed a correlation between AR-V7 mRNA and metastasis at initial diagnosis. Detection of AR-V7 mRNA did not predict the reduction of serum PSA in patients with CRPC following abiraterone and enzalutamide administration. In conclusion, AR-V7 mRNA expression in normal hematopoietic cells may have annihilated the manifestation of aggressiveness of prostate cancer and the prediction of the effectiveness of abiraterone and enzalutamide by the assessment of AR-V7 mRNA in blood.

Differential splicing of human androgen receptor pre-mRNA in X-linked reifenstein syndrome, because of a deletion involving a putative branch site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ris-Stalpers, C.; Verleun-Mooijman, M.C.T.; Blaeij, T.J.P. de

1994-04-01

The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of >6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-pointmore » sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR, as tested in cotransfection studies. It is concluded that the partial androgen-insensitivity syndrome of this patient is the consequence of the limited amount of wild-type AR protein expressed in androgen target cells, resulting from the deletion of the intron 2 putative BPS. 42 refs., 6 figs., 1 tab.« less

L-rhamnose induces browning in 3T3-L1 white adipocytes and activates HIB1B brown adipocytes.

PubMed

Choi, Minji; Mukherjee, Sulagna; Kang, Nam Hyeon; Barkat, Jameel Lone; Parray, Hilal Ahmad; Yun, Jong Won

2018-06-01

Induction of the brown adipocyte-like phenotype in white adipocytes (browning) is considered as a novel strategy to fight obesity due to the ability of brown adipocytes to increase energy expenditure. Here, we report that L-rhamnose induced browning by elevating expression levels of beige-specific marker genes, including Cd137, Cited1, Tbx1, Prdm16, Tmem26, and Ucp1, in 3T3-L1 adipocytes. Moreover, L-rhamnose markedly elevated expression levels of proteins involved in thermogenesis both in 3T3-L1 white and HIB1B brown adipocytes. L-rhamnose treatment in 3T3-L1 adipocytes also significantly elevated protein levels of p-HSL, p-AMPK, ACOX, and CPT1 as well as reduced levels of ACC, FAS, C/EBPα, and PPARγ, suggesting its possible role in enhancement of lipolysis and lipid catabolism as well as reduced adipogenesis and lipogenesis, respectively. The quick technique of efficient molecular docking provided insight into the strong binding of L-rhamnose to the fat-digesting glycine residue of β 3 -adrenergic receptor (AR), indicating strong involvement of L-rhamnose in fat metabolism. Further examination of the molecular mechanism of L-rhamnose revealed that it induced browning of 3T3-L1 adipocytes via coordination of multiple signaling pathways through β 3 -AR, SIRT1, PKA, and p-38. To the best of our knowledge, this is the first study to demonstrate that L-rhamnose plays multiple modulatory roles in the induction of white fat browning, activation of brown adipocytes, as well as promotion of lipid metabolism, thereby demonstrating its therapeutic potential for treatment of obesity. © 2018 IUBMB Life, 70(6):563-573, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

Production and Characterization of Bacillus thuringiensis Cry1Ac-Resistant Cotton Bollworm Helicoverpa zea (Boddie)▿

PubMed Central

Anilkumar, Konasale J.; Rodrigo-Simón, Ana; Ferré, Juan; Pusztai-Carey, Marianne; Sivasupramaniam, Sakuntala; Moar, William J.

2008-01-01

Laboratory-selected Bacillus thuringiensis-resistant colonies are important tools for elucidating B. thuringiensis resistance mechanisms. However, cotton bollworm, Helicoverpa zea, a target pest of transgenic corn and cotton expressing B. thuringiensis Cry1Ac (Bt corn and cotton), has proven difficult to select for stable resistance. Two populations of H. zea (AR and MR), resistant to the B. thuringiensis protein found in all commercial Bt cotton varieties (Cry1Ac), were established by selection with Cry1Ac activated toxin (AR) or MVP II (MR). Cry1Ac toxin reflects the form ingested by H. zea when feeding on Bt cotton, whereas MVP II is a Cry1Ac formulation used for resistance selection and monitoring. The resistance ratio (RR) for AR exceeded 100-fold after 11 generations and has been maintained at this level for nine generations. This is the first report of stable Cry1Ac resistance in H. zea. MR crashed after 11 generations, reaching only an RR of 12. AR was only partially cross-resistant to MVP II, suggesting that MVP II does not have the same Cry1Ac selection pressure as Cry1Ac toxin against H. zea and that proteases may be involved with resistance. AR was highly cross-resistant to Cry1Ab toxin but only slightly cross-resistant to Cry1Ab expressing corn leaf powder. AR was not cross-resistant to Cry2Aa2, Cry2Ab2-expressing corn leaf powder, Vip3A, and cypermethrin. Toxin-binding assays showed no significant differences, indicating that resistance was not linked to a reduction in binding. These results aid in understanding why this pest has not evolved B. thuringiensis resistance, and highlight the need to choose carefully the form of B. thuringiensis protein used in experiments. PMID:18024681

Fucci2a: a bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice.

PubMed

Mort, Richard Lester; Ford, Matthew Jonathan; Sakaue-Sawano, Asako; Lindstrom, Nils Olof; Casadio, Angela; Douglas, Adam Thomas; Keighren, Margaret Anne; Hohenstein, Peter; Miyawaki, Atsushi; Jackson, Ian James

2014-01-01

Markers of cell cycle stage allow estimation of cell cycle dynamics in cell culture and during embryonic development. The Fucci system incorporates genetically encoded probes that highlight G1 and S/G2/M phases of the cell cycle allowing live imaging. However the available mouse models that incorporate Fucci are beset by problems with transgene inactivation, varying expression level, lack of conditional potential and/or the need to maintain separate transgenes-there is no transgenic mouse model that solves all these problems. To address these shortfalls we re-engineered the Fucci system to create 2 bicistronic Fucci variants incorporating both probes fused using the Thosea asigna virus 2A (T2A) self cleaving peptide. We characterize these variants in stable 3T3 cell lines. One of the variants (termed Fucci2a) faithfully recapitulated the nuclear localization and cell cycle stage specific florescence of the original Fucci system. We go on to develop a conditional mouse allele (R26Fucci2aR) carefully designed for high, inducible, ubiquitous expression allowing investigation of cell cycle status in single cell lineages within the developing embryo. We demonstrate the utility of R26Fucci2aR for live imaging by using high resolution confocal microscopy of ex vivo lung, kidney and neural crest development. Using our 3T3 system we describe and validate a method to estimate cell cycle times from relatively short time-lapse sequences that we then apply to our neural crest data. The Fucci2a system and the R26Fucci2aR mouse model are compelling new tools for the investigation of cell cycle dynamics in cell culture and during mouse embryonic development.

Central vasopressin V1a receptor activation is independently necessary for both partner preference formation and expression in socially monogamous male prairie voles.

PubMed

Donaldson, Zoe R; Spiegel, Lauren; Young, Larry J

2010-02-01

The neuropeptide arginine vasopressin (AVP) modulates a variety of species-specific social behaviors. In socially monogamous male prairie voles, AVP acts centrally via vasopressin V1a receptor (V1aR) to facilitate mating induced partner preferences. The display of a partner preference requires at least 2 temporally distinct processes: social bond formation as well as its recall, or expression. Studies to date have not determined in which of these processes V1aR acts to promote partner preferences. Here, male prairie voles were administered intracerebroventricularly a V1aR antagonist (AVPA) at different time points to investigate the role of V1aR in social bond formation and expression. Animals receiving AVPA prior to cohabitation with mating or immediately prior to partner preference testing failed to display a partner preference, while animals receiving AVPA immediately after cohabitation with mating and control animals receiving vehicle at all 3 time points displayed partner preferences. These results suggest that V1aR signaling is necessary for both the formation and expression of partner preferences and that these processes are dissociable. (c) 2009 APA, all rights reserved.

Effects of long-term heat stress and dietary restriction on the expression of genes of steroidogenic pathway and small heat-shock proteins in rat testicular tissue.

PubMed

Bozkaya, F; Atli, M O; Guzeloglu, A; Kayis, S A; Yildirim, M E; Kurar, E; Yilmaz, R; Aydilek, N

2017-08-01

The aim was to investigate the effects of long-term heat stress and dietary restriction on the expression of certain genes involving in steroidogenic pathway and small heat-shock proteins (sHSPs) in rat testis. Sprague Dawley rats (n = 24) were equally divided into four groups. Group I and II were kept at an ambient temperature of 22°C, while Groups III and IV were reared at 38°C for 9 weeks. Feed was freely available for Group I and Group III, while Group II and Group IV were fed 60% of the diet consumed by their ad libitum counterparts. At the end of 9 weeks, testicles were collected under euthanasia. Total RNA was isolated from testis tissue samples. Expression profiles of the genes encoding androgen-binding protein, follicle-stimulating hormone receptor, androgen receptor, luteinising hormone receptor, steroidogenic acute regulatory protein (StAR), cyclooxygenase-2 and sHSP genes were assessed at mRNA levels using qPCR. Long-term heat stress decreased the expression of StAR and HspB10 genes while dietary restriction upregulated StAR gene expression. The results suggested that long-term heat stress negatively affected the expression of StAR and HspB10 genes and the dietary restriction was able to reverse negative effect of heat stress on the expression of StAR gene in rat testis. © 2016 Blackwell Verlag GmbH.

In vitro biocompatibility, inflammatory response, and osteogenic potential of 4 root canal sealers: Sealapex, Sankin apatite root sealer, MTA Fillapex, and iRoot SP root canal sealer.

PubMed

Chang, Seok-Woo; Lee, So-Youn; Kang, Soo-Kyung; Kum, Kee-Yeon; Kim, Eun-Cheol

2014-10-01

The objective of this study was to compare the cytotoxicity, inflammatory response, osteogenic effect, and the signaling mechanism of these biologic activities of 4 calcium compound-based root canal sealers (ie, Sealapex [Sybron Kerr, WA], apatite root sealer [ARS; Dentsply Sankin, Tokyo, Japan], MTA Fillapex [Angelus Indústria de Produtos Odontológicos S/A, Londrina, PR, Brazil], and iRoot SP [Innovative BioCreamix Inc, Vancouver, Canada]) in human periodontal ligament cells. Cytotoxicity was assessed using the 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide assay. Levels of inflammatory mediators were measured by enzyme-linked immunosorbent assay, reverse-transcription polymerase chain reaction, and Western blot analysis. Osteogenic potential was evaluated by alkaline phosphatase activity, alizarin red staining, and marker genes by reverse-transcription polymerase chain reaction. The signal transduction pathways were examined by Western blotting. None of the sealers were cytotoxic. ARS, MTA Fillapex, and iRoot SP induced a lower expression of proinflammatory mediators than Sealapex. All sealers increased ALP activity and the formation of mineralized nodules and up-regulated the expression of osteoblastic marker messenger RNA. ARS, MTA Fillapex, and iRoot SP showed superior osteogenic potential compared with Sealapex. The expression and/or activation of integrin receptors and downstream signaling molecules, including focal adhesion kinase, paxillin, Akt, mitogen-activated protein kinase, and nuclear factor κB, was induced by ARS, MTA Fillapex, and iRoot SP treatment but not by Sealapex treatment. We show for the first time that ARS, MTA Fillapex, and iRoot SP induce a lower expression of inflammatory mediators and enhance osteoblastic differentiation of PDLCs via the integrin-mediated signaling pathway compared with Sealapex. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Prolactin- and testosterone-induced carboxypeptidase-D correlates with increased nitrotyrosines and Ki67 in prostate cancer.

PubMed

Thomas, Lynn N; Merrimen, Jennifer; Bell, David G; Rendon, Ricardo; Too, Catherine K L

2015-11-01

Carboxypeptidase-D (CPD) cleaves C-terminal arginine for conversion to nitric oxide (NO) by nitric oxide synthase (NOS). Prolactin (PRL) and androgens stimulate CPD gene transcription and expression, which increases intracellular production of NO to promote viability of prostate cancer (PCa) cells in vitro. The current study evaluated whether hormonal upregulation of CPD and NO promote PCa cell viabilty in vivo, by correlating changes in expression of CPD and nitrotyrosine residues (products of NO action) with proliferation marker Ki67 and associated proteins during PCa development and progression. Fresh prostate tissues, obtained from 40 men with benign prostatic hyperplasia (BPH) or PCa, were flash-frozen at the time of surgery and used for RT-qPCR analysis of CPD, androgen receptor (AR), PRL receptor (PRLR), eNOS, and Ki67 levels. Archival paraffin-embedded tissues from 113 men with BPH or PCa were used for immunohistochemical (IHC) analysis of CPD, nitrotyrosines, phospho-Stat5 (for activated PRLR), AR, eNOS/iNOS, and Ki67. RT-qPCR and IHC analyses showed strong AR and PRLR expression in benign and malignant prostates. CPD mRNA levels increased ∼threefold in PCa compared to BPH, which corresponded to a twofold increase in Ki67 mRNA levels. IHC analysis showed a progressive increase in CPD from 11.4 ± 2.1% in benign to 21.8 ± 3.2% in low-grade (P = 0.007), 40.7 ± 4.0% in high-grade (P < 0.0001) and 50.0 ± 9.5% in castration-recurrent PCa (P < 0.0001). Immunostaining for nitrotyrosines and Ki67 mirrored these increases during PCa progression. CPD, nitrotyrosines, and Ki67 tended to co-localize, as did phospho-Stat5. CPD, nitrotyrosine, and Ki67 levels were higher in PCa than in benign and tended to co-localize, along with phospho-Stat5. The strong correlation in expression of these proteins in benign and malignant prostate tissues, combined with abundant AR and PRLR, supports in vitro evidence that the CPD-Arg-NO pathway is involved in the regulation of PCa cell proliferation. It further highlights a role for PRL in the development and progression of PCa. © 2015 Wiley Periodicals, Inc.

Synergistic Activation of Steroidogenic Acute Regulatory Protein Expression and Steroid Biosynthesis by Retinoids: Involvement of cAMP/PKA Signaling

PubMed Central

Manna, Pulak R.; Slominski, Andrzej T.; King, Steven R.; Stetson, Cloyce L.

2014-01-01

Both retinoic acid receptors (RARs) and retinoid X receptors (RXRs) mediate the action of retinoids that play important roles in reproductive development and function, as well as steroidogenesis. Regulation of steroid biosynthesis is principally mediated by the steroidogenic acute regulatory protein (StAR); however, the modes of action of retinoids in the regulation of steroidogenesis remain obscure. In this study we demonstrate that all-trans retinoic acid (atRA) enhances StAR expression, but not its phosphorylation (P-StAR), and progesterone production in MA-10 mouse Leydig cells. Activation of the protein kinase A (PKA) cascade, by dibutyrl-cAMP or type I/II PKA analogs, markedly increased retinoid-responsive StAR, P-StAR, and steroid levels. Targeted silencing of endogenous RARα and RXRα, with small interfering RNAs, resulted in decreases in 9-cis RA-stimulated StAR and progesterone levels. Truncation of and mutational alterations in the 5′-flanking region of the StAR gene demonstrated the importance of the −254/−1-bp region in retinoid responsiveness. An oligonucleotide probe encompassing an RXR/liver X receptor recognition motif, located within the −254/−1-bp region, specifically bound MA-10 nuclear proteins and in vitro transcribed/translated RXRα and RARα in EMSAs. Transcription of the StAR gene in response to atRA and dibutyrl-cAMP was influenced by several factors, its up-regulation being dependent on phosphorylation of cAMP response-element binding protein (CREB). Chromatin immunoprecipitation studies revealed the association of phosphorylation of CREB, CREB binding protein, RXRα, and RARα to the StAR promoter. Further studies elucidated that hormone-sensitive lipase plays an important role in atRA-mediated regulation of the steroidogenic response that involves liver X receptor signaling. These findings delineate the molecular events by which retinoids influence cAMP/PKA signaling and provide additional and novel insight into the regulation of StAR expression and steroidogenesis in mouse Leydig cells. PMID:24265455

The phosphorylated C-terminus of cAR1 plays a role in cell-type-specific gene expression and STATa tyrosine phosphorylation.

PubMed

Briscoe, C; Moniakis, J; Kim, J Y; Brown, J M; Hereld, D; Devreotes, P N; Firtel, R A

2001-05-01

cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on multicellular development when expressed in wild-type cells. These findings suggest that the phosphorylated C-terminus of cAR1 may be involved in regulating aspects of receptor-mediated processes, is not essential for GBF function, and may play a role in mediating subsequent development. Copyright 2001 Academic Press.

Semicomprehensive analysis of the postnatal age-related changes in the mRNA expression of sex steroidogenic enzymes and sex steroid receptors in the male rat hippocampus.

PubMed

Kimoto, Tetsuya; Ishii, Hirotaka; Higo, Shimpei; Hojo, Yasushi; Kawato, Suguru

2010-12-01

Although sex steroids play a crucial role in the postnatal brain development, the age-related changes in the hippocampal steroidogenesis remain largely unknown. We performed comprehensive investigations for the mRNA expressions of 26 sex steroidogenic enzymes/proteins and three sex steroid receptors in the male rat hippocampus, at the ages of postnatal day (PD) 1, PD4, PD7, PD10, PD14, 4 wk, and 12 wk (adult), by RT-PCR/Southern blotting analysis. The relative expression levels of these enzymes/receptors at PD1 were Srd5a1 > Star > Ar ∼ Hsd17b4 ∼ Hsd17b1 ∼ Hsd17b7 ∼ Esr1 ∼ Srd5a2 > Hsd17b3 > Esr2 > Cyp11a1 > Cyp17a1 > Cyp19a1 ∼ Hsd17b2 > 3β-hydroxysteroid dehydrogenase I. The mRNA levels of essential enzymes for progesterone/testosterone/estradiol metabolisms (Cyp17a1, Hsd17b7, and Cyp19a1) were approximately constant between PD1 and PD14 and then declined toward the adult levels. Cyp11a1 increased during PD4-PD14 and then considerably decreased toward the adult level (∼8% of PD1). Hsd17b1, Hsd17b2, and 3β-hydroxysteroid dehydrogenase I mRNA decreased approximately monotonously. Hsd17b3 increased to approximately 200% of PD1 during PD4-PD14 and was maintained at this high level. The 5α-reductase mRNA was maintained constant (Srd5a1) or decreased monotonically (Srd5a2) toward the adult level. The Esr1 level peaked at PD4 and decreased toward the adult level, whereas Ar greatly increased during PD1-PD14 and was maintained at this high level. The Star and Hsd17b4 levels were maintained constant from neonate to adult. These results suggest that the hippocampal sex steroidogenic properties are substantially altered during the postnatal development processes, which might contribute to brain sexual maturation.

The androgen receptor governs the execution, but not programming, of male sexual and territorial behaviors

PubMed Central

Juntti, Scott A; Tollkuhn, Jessica; Wu, Melody V; Fraser, Eleanor J; Soderborg, Taylor; Tan, Stella; Honda, Shin-Ichiro; Harada, Nobuhiro; Shah, Nirao M

2010-01-01

SUMMARY Testosterone and estrogen are essential for male behaviors in vertebrates. How these two signaling pathways interact to control masculinization of the brain and behavior remains to be established. Circulating testosterone activates the androgen receptor (AR) and also serves as the source of estrogen in the brain. We have used a genetic strategy to delete AR specifically in the mouse nervous system. This approach permits us to determine the function of AR in sexually dimorphic behaviors in males while maintaining circulating testosterone levels within the normal range. We find that AR mutant males exhibit masculine sexual and territorial displays, but they have striking deficits in specific components of these behaviors. Taken together with the surprisingly limited expression of AR in the developing brain, our findings indicate that testosterone acts as a precursor to estrogen to masculinize the brain and behavior, and signals via AR to control the levels of male behavioral displays. PMID:20435002

[Glucocorticoid pathway mediated the inhibition of testosterone in rats exposed to dibutyl phthalate].

PubMed

Zhang, Xiao-feng; Zheng, Jing; Li, Zi; Zhang, Yang

2009-08-01

To investigate the inhibitory mechanisms of testosterone (T) biosynthesis in rats exposed to dibutyl phthalate (DBP). Male Wistar rats were randomly divided into five groups by weight, including 0.25, 0.50, 1.00, 2.00 g/kg DBP groups and corn oil control group, with 16 rats in each group. DBP was administered by gavage once a day. After 30 days exposure, eight rats in each group were killed and the others were killed after 15 days without DBP administration. The levels of T and glucocorticoid (GC) in serum were determined by radioimmunoassay. The expression levels of 11 beta-dedroxysteriod dehydrogenase (11 beta-HSD) mRNA and steroidogenesis acute regulatory protein (StAR) mRNA were determined by RT-PCR. The protein expression level of glucocorticoid receptor (GR) was investigated by Western blotting. During exposure period, in 1.00 and 2.00 g/kg DBP groups, the levels of T were (0.260 +/- 0.218) ng/ml and (0.260 +/- 0.342) ng/ml, the levels of GC were (13.470 +/- 5.661) ng/ml and (13.740 +/- 3.977) ng/ml, the levels of T and GC in control group were (1.045 +/- 1.222) ng/ml and (9.224 +/- 3.496) ng/ml. There were statistic differences between 1.00 and 2.00 g/kg DBP groups and control group (t(T) values were -2.295 and -2.295, t(GC) values were 2.159 and 2.296, respectively, P < 0.05). The expression level of StAR mRNA was significantly down-regulated in 1.00 and 2.00 g/kg DBP groups, while StAR/beta-Actin values were 0.657 +/- 0.060 and 0.407 +/- 0.033, and compared to control group (0.871 +/- 0.081), there was statistic difference (t values were -3.707 and -8.037, P < 0.05). In 1.00 and 2.00 g/kg DBP groups, the expression of 11 beta-HSD mRNA and the expression of GR protein were increased in DBP dose-dependent manner, while 11 beta-HSD/beta-Actin values were 0.538 +/- 0.138 and 0.988 +/- 0.133, and GR/beta-Actin were 0.785 +/- 0.106 and 0.956 +/- 0.076, respectively. There were statistic difference, as compared to the controls (0.285 +/- 0.106 and 0.275 +/- 0.035) (t(11 beta-HSD/beta-Actin) values were 2.829 and 7.860, t(GR/beta-Actin) values were 8.064 and 10.77, respectively, P < 0.05).Linear correlation and regression revealed that there were positive correlation between DBP dose and the expression levels of 11 beta-HSD mRNA and GR protein, with r values of 0.766 and 0.790, respectively. In post-exposure period, there were no statistic differences of all above index among DBP groups and control group. DBP might inhibit T production in rats through GR mediation.

Minoxidil may suppress androgen receptor-related functions.

PubMed

Hsu, Cheng-Lung; Liu, Jai-Shin; Lin, An-Chi; Yang, Chih-Hsun; Chung, Wen-Hung; Wu, Wen-Guey

2014-04-30

Although minoxidil has been used for more than two decades to treat androgenetic alopecia (AGA), an androgen-androgen receptor (AR) pathway-dominant disease, its precise mechanism of action remains elusive. We hypothesized that minoxidil may influence the AR or its downstream signaling. These tests revealed that minoxidil suppressed AR-related functions, decreasing AR transcriptional activity in reporter assays, reducing expression of AR targets at the protein level, and suppressing AR-positive LNCaP cell growth. Dissecting the underlying mechanisms, we found that minoxidil interfered with AR-peptide, AR-coregulator, and AR N/C-terminal interactions, as well as AR protein stability. Furthermore, a crystallographic analysis using the AR ligand-binding domain (LBD) revealed direct binding of minoxidil to the AR in a minoxidil-AR-LBD co-crystal model, and surface plasmon resonance assays demonstrated that minoxidil directly bound the AR with a K(d) value of 2.6 µM. Minoxidil also suppressed AR-responsive reporter activity and decreased AR protein stability in human hair dermal papilla cells. The current findings provide evidence that minoxidil could be used to treat both cancer and age-related disease, and open a new avenue for applications of minoxidil in treating androgen-AR pathway-related diseases.

Minoxidil may suppress androgen receptor-related functions

PubMed Central

Hsu, Cheng-Lung; Liu, Jai-Shin; Lin, An-Chi; Yang, Chih-Hsun; Chung, Wen-Hung; Wu, Wen-Guey

2014-01-01

Although minoxidil has been used for more than two decades to treat androgenetic alopecia (AGA), an androgen-androgen receptor (AR) pathway-dominant disease, its precise mechanism of action remains elusive. We hypothesized that minoxidil may influence the AR or its downstream signaling. These tests revealed that minoxidil suppressed AR-related functions, decreasing AR transcriptional activity in reporter assays, reducing expression of AR targets at the protein level, and suppressing AR-positive LNCaP cell growth. Dissecting the underlying mechanisms, we found that minoxidil interfered with AR-peptide, AR-coregulator, and AR N/C-terminal interactions, as well as AR protein stability. Furthermore, a crystallographic analysis using the AR ligand-binding domain (LBD) revealed direct binding of minoxidil to the AR in a minoxidil-AR-LBD co-crystal model, and surface plasmon resonance assays demonstrated that minoxidil directly bound the AR with a Kd value of 2.6 μM. Minoxidil also suppressed AR-responsive reporter activity and decreased AR protein stability in human hair dermal papilla cells. The current findings provide evidence that minoxidil could be used to treat both cancer and age-related disease, and open a new avenue for applications of minoxidil in treating androgen-AR pathway-related diseases. PMID:24742982

Lgr4 promotes prostate tumorigenesis through the Jmjd2a/AR signaling pathway.

PubMed

Zhang, Jianwei; Li, Qi; Zhang, Shaojin; Xu, Quanquan; Wang, Tianen

2016-11-15

Lgr4 (leucine-rich repeat domain containing G protein-coupled receptor 4) is implicated in the transcriptional regulation of multiple histone demethylases in the progression of diverse cancers, but there are few reports concerning the molecular mechanism by which Lgr4 regulates histone demethylase activation in prostate cancer (PCa) progression. As Jmjd2a is a histone demethylase, in the current study, we investigated the relationship between interaction Lgr4 with Jmjd 2a and Jmjd2a/androgen receptor (AR) signaling pathway in PCa progression. Firstly, Lgr4 was overexpressed by transfecting pcDNA3.1(+)/Lgr4 plasmids into PCa (LNCaP and PC-3) cell lines. Next, we found that Lgr4 overexpression promoted Jmjd2a mRNA expression, reduced cell apoptosis and arrested cell cycle in the S phase, these effects were reversed by Jmjd2a silencing. Moreover, Lgr4 overexpression markedly elevated AR levels and its interaction with Jmjd2a, which was tested by co-immunoprecipitation and luciferase reporter assays. Furthermore, interaction AR with PSA promoter (containing an AR response element) was obviously improved by Lgr4 overexpression, and PSA silencing reduced Lgr4-induced cell apoptosis and cell cycle arrest in PCa cells. Taken together, Lgr4 may be a novel tumor marker providing new mechanistic insights into PCa progression. Lgr4 activates Jmjd2a/AR signaling pathway to promote interaction AR with PSA promoter, causing reduction of PCa apoptosis and cell cycle arrest. Copyright © 2016 Elsevier Inc. All rights reserved.

Tumor suppressor REIC/DKK-3 and co-chaperone SGTA: Their interaction and roles in the androgen sensitivity

PubMed Central

Kato, Yuiko; Ishiguro-Oonuma, Toshina; Udagawa, Chihiro; Rungsuriyawiboon, Oumaporn; Azakami, Daigo; Michishita, Masaki; Ariyoshi, Yuichi; Ueki, Hideo; Nasu, Yasutomo; Kumon, Hiromi; Watanabe, Masami; Omi, Toshinori

2016-01-01

REIC/DKK-3 is a tumor suppressor, however, its intracellular physiological functions and interacting molecules have not been fully clarified. Using yeast two-hybrid screening, we found that small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), known as a negative modulator of cytoplasmic androgen receptor (AR) signaling, is a novel interacting partner of REIC/DKK-3. Mammalian two-hybrid and pull-down assay results indicated that the SGTA-REIC/DKK-3 interaction involved the N-terminal regions of both REIC/DKK-3 and SGTA and that REIC/DKK-3 interfered with the dimerization of SGTA, which is a component of the AR complex and a suppressor of dynein motor-dependent AR transport and signaling. A reporter assay in human prostate cancer cells that displayed suppressed AR signaling by SGTA showed recovery of AR signaling by REIC/DKK-3 expression. Considering these results and our previous data that REIC/DKK-3 interacts with the dynein light chain TCTEX-1, we propose that the REIC/DKK-3 protein interferes with SGTA dimerization, promotes dynein-dependent AR transport and then upregulates AR signaling. PMID:26658102

Consumption of high ω-3 fatty acid diet suppressed prostate tumorigenesis in C3(1) Tag mice.

PubMed

Akinsete, Juliana A; Ion, Gabriela; Witte, Theodore R; Hardman, W Elaine

2012-01-01

Prostate cancer incidence and mortality are high in the Western world and high ω-6/ω-3 PUFA in the Western diet may be a contributing factor. We investigated whether changing from a diet that approximates ω-6 fat content of the Western diet to a high ω-3 fat diet at adulthood might reduce prostate cancer risk. Female SV 129 mice that had consumed a high ω-6 diet containing corn oil for 2 weeks were bred with homozygous C3(1)Tag transgenic male mice. All male offspring were weaned to the corn oil diet (CO) until postpuberty when half of the male offspring were transferred to a high ω-3 diet containing canola oil and fish oil concentrate (FS). High ω-3 diet increased ω-3 and decreased ω-6 fat content of mice tissues. Average weights of prostate and genitourinary bloc were significantly lower in mice consuming high ω-3 diet at adulthood (CO-FS) than mice fed a lifetime high ω-6 diet (CO-CO). There was slower progression of tumorigenesis in dorsalateral prostate of CO-FS than in CO-CO mice. CO-FS mice had slightly lower plasma testosterone level at 24 and 40 weeks, significantly lower estradiol level at 40 weeks and significantly less expressed androgen receptor (AR) in the dorsalateral prostate at 40 weeks than CO-CO mice. Consumption of high ω-3 diet lowered the expression of genes expected to increase proliferation and decrease apoptosis in dorsalateral prostate. Our results suggest that consumption of high ω-3 diet slows down prostate tumorigenesis by lowering estradiol, testosterone and AR levels, promoting apoptosis and suppressing cell proliferation in C3(1)Tag mice.

Consumption of high ω-3 fatty acid diet suppressed prostate tumorigenesis in C3(1) Tag mice

PubMed Central

Ion, Gabriela; Witte, Theodore R.; Hardman, W.Elaine

2012-01-01

Prostate cancer incidence and mortality are high in the Western world and high ω-6/ω-3 PUFA in the Western diet may be a contributing factor. We investigated whether changing from a diet that approximates ω-6 fat content of the Western diet to a high ω-3 fat diet at adulthood might reduce prostate cancer risk. Female SV 129 mice that had consumed a high ω-6 diet containing corn oil for 2 weeks were bred with homozygous C3(1)Tag transgenic male mice. All male offspring were weaned to the corn oil diet (CO) until postpuberty when half of the male offspring were transferred to a high ω-3 diet containing canola oil and fish oil concentrate (FS). High ω-3 diet increased ω-3 and decreased ω-6 fat content of mice tissues. Average weights of prostate and genitourinary bloc were significantly lower in mice consuming high ω-3 diet at adulthood (CO-FS) than mice fed a lifetime high ω-6 diet (CO–CO). There was slower progression of tumorigenesis in dorsalateral prostate of CO-FS than in CO–CO mice. CO-FS mice had slightly lower plasma testosterone level at 24 and 40 weeks, significantly lower estradiol level at 40 weeks and significantly less expressed androgen receptor (AR) in the dorsalateral prostate at 40 weeks than CO–CO mice. Consumption of high ω-3 diet lowered the expression of genes expected to increase proliferation and decrease apoptosis in dorsalateral prostate. Our results suggest that consumption of high ω-3 diet slows down prostate tumorigenesis by lowering estradiol, testosterone and AR levels, promoting apoptosis and suppressing cell proliferation in C3(1)Tag mice. PMID:22045025

Depot differences in steroid receptor expression in adipose tissue: possible role of the local steroid milieu.

PubMed

Rodriguez-Cuenca, S; Monjo, M; Proenza, A M; Roca, P

2005-01-01

Sex hormones play an important role in adipose tissue metabolism by activating specific receptors that alter several steps of the lipolytic and lipogenic signal cascade in depot- and sex-dependent manners. However, studies focusing on steroid receptor status in adipose tissue are scarce. In the present study, we analyzed steroid content [testosterone (T), 17beta-estradiol (17beta-E2), and progesterone (P4)] and steroid receptor mRNA levels in different rat adipose tissue depots. As expected, T levels were higher in males than in females (P = 0.031), whereas the reverse trend was observed for P4 (P < 0.001). It is noteworthy that 17beta-E2 adipose tissue levels were higher in inguinal than in the rest of adipose tissues for both sexes, where no sex differences in 17beta-E2 tissue levels were noted (P = 0.010 for retroperitoneal, P = 0.005 for gonadal, P = 0.018 for mesenteric). Regarding steroid receptor levels, androgen (AR) and estrogen receptor (ER)alpha and ERbeta densities were more clearly dependent on adipose depot location than on sex, with visceral depots showing overall higher mRNA densities than their subcutaneous counterparts. Besides, expression of ERalpha predominated over ERbeta expression, and progesterone receptor (PR-B form and PR-A+B form) mRNAs were identically expressed regardless of anatomic depot and sex. In vitro studies in 3T3-L1 cells showed that 17beta-E2 increased ERalpha (P = 0.001) and AR expression (P = 0.001), indicating that estrogen can alter estrogenic and androgenic signaling in adipose tissue. The results highlighted in this study demonstrate important depot-dependent differences in the sensitivity of adipose tissues to sex hormones between visceral and subcutaneous depots that could be related to metabolic situations observed in response to sex hormones.

Systemic and Gene Modified Mesenchymal Stem Cell Therapy for Metastatic Prostate Cancer

DTIC Science & Technology

2009-05-01

downregulate the expression of growth factors , receptor tyrosine kinases, proteases, and AR both in vitro and in vivo only when the cells express high... growth , and apoptosis as shown in Figure 1A. Downregulation of growth factors , FGF, Mdk, PDGF, Ptn, TGFb1 and TGFb3, tyrosine kinase receptors Ffgr3 and... growth factors and receptor tyrosine kinases mainly in andro- gen-sensitive and AR-overexpressing cells. The effect was more pronounced in Erk, Ras, and

Up regulation of the steroid hormone synthesis regulator HSD3B2 is linked to early PSA recurrence in prostate cancer.

PubMed

Neubauer, Emily; Latif, Morwari; Krause, Jenny; Heumann, Asmus; Armbrust, Moritz; Luehr, Clara; Fraune, Christoph; Hube-Magg, Claudia; Kluth, Martina; Möller-Koop, Christina; Sauter, Guido; Simon, Ronald; Beyer, Burkhard; Pompe, Raisa S; Thederan, Imke; Schlomm, Thorsten; Büscheck, Franziska

2018-05-24

HSD3B2 plays a crucial role in steroid hormone biosynthesis and is thus of particular interest in hormone dependent tumors such as prostate cancer. To clarify the clinical relevance of HSD3B2 expression in prostate cancer, we analyzed HSD3B2 protein expression by immunohistochemistry on our preexisting tissue microarray with 12.247 annotated cancers. Compared with normal tissue cytoplasmic HSD3B2 staining was stronger in prostate cancers. In 9371 interpretable cancers, HSD3B2 expression was found in 95.5% of cancers and was considered weak in 29.9%, moderate in 40.7% and strong in 24.9%. HSD3B2 up regulation was linked to advanced pathological tumor stage (pT), high Gleason grade, elevated preoperative PSA levels (p < 0.0001 each), lymph node metastasis (p = 0.0019), accelerated cell proliferation (p < 0.0001), androgen receptor (AR) expression (p < 0.0001), and early biochemical recurrence (p < 0.0001). HSD3B2 up regulation was only marginally more frequent in ERG positive (98%) than in ERG negative cancers (94%; p < 0.0001) and was strongly linked to deletions of 5q and 6q (p < 0.0001 each). Multivariate analyses showed that the prognostic impact of HSD3B2 expression was independent of established preoperative, but not of postoperative prognostic parameters. In summary, the results of our study demonstrate that HSD3B2 is strongly up regulated in a fraction of prostate cancers that are characterized by increased AR signaling, adverse tumor phenotype and early biochemical recurrence. Copyright © 2018 Elsevier Inc. All rights reserved.

Effects of 17alpha-methyltestosterone exposure on steroidogenesis and cyclin-B mRNA expression in previtellogenic oocytes of Atlantic cod (Gadus morhua).

PubMed

Kortner, Trond M; Arukwe, Augustine

2007-11-01

Steroid hormone (estrogens and androgens) synthesis and regulation involve a large number of enzymes and potential biochemical pathways. In the context of these biochemical pathways, it is believed that the true rate-limiting step in acute steroid production is the movement of cholesterol across the mitochondrial membrane by the steroidogenic acute regulatory (StAR) protein and the subsequent conversion to pregnenolone by cytochrome P450-mediated side-chain cleavage (P450scc) enzyme. Oocyte development is a complex process that is triggered by the maturation-promoting factor (MPF) involving cyclin-B as a regulatory factor. In the present study, we evaluated the endocrine effects of 17alpha-methyltestosterone (MT) on steroidogenic pathways of Atlantic cod (Gadus morhua), using an in vitro previtellogenic oocyte culture technique that is based on an agarose floating method. Tissue was cultured in a humidified incubator at 10 degrees C for 1, 5, 10 and 20 days with different concentrations of the synthetic androgen MT (0 (control), 1, 10, 100 and 1000 microM) dissolved in ethanol (0.3%). Gene expressions for StAR, P450scc, aromatase-alpha (P450aromA) and cyclin-B were detected using validated real-time PCR with specific primer pairs. Cellular localization of the StAR protein and P450scc were performed using the immunohistochemical technique with antisera prepared against synthetic peptide for both proteins. Steroid hormones (estradiol-17beta: E2 and testosterone: T) levels were estimated using enzyme immunoassay. Our data showed significant concentration-specific increase (at day 1 and 5) and decrease (at day 10 and 20) of the StAR mRNA expression after exposure to MT. P450scc expression showed a MT concentration-specific decrease during the exposure periods and cyclin-B mRNA expression was decreased in MT concentration-dependent manner at days 10 and 20 (reaching almost total inhibition after exposure to 1000 microM MT). MT exposure produced variable effects on the P450aromA mRNA expression that can be described as concentration-specific increase (day 1) and decrease (days 5 and 10). Cellular localization of the StAR protein and P450scc demonstrated their expression mainly in ovarian follicular cells. MT produced an apparent concentration-and time-dependent increase of E2 and T levels. Thus, the present study reveals some novel effects of pharmaceutical endocrine disruptor on the development of previtellogenic oocytes in cod. The impaired steroidogenesis and hormonal imbalance reported in the present study may have potential consequences for the vitellogenic process and overt fecundity in teleosts.

Comparative biomarker expression and RNA integrity in biospecimens derived from radical retropubic and robot-assisted laparoscopic prostatectomies.

PubMed

Ricciardelli, Carmela; Bianco-Miotto, Tina; Jindal, Shalini; Dodd, Thomas J; Cohen, Penelope A; Marshall, Villis R; Sutherland, Peter D; Samaratunga, Hemamali; Kench, James G; Dong, Ying; Wang, Hong; Clements, Judith A; Risbridger, Gail P; Sutherland, Robert L; Tilley, Wayne D; Horsfall, David J

2010-07-01

Knowledge of preanalytic conditions that biospecimens are subjected to is critically important because novel surgical procedures, tissue sampling, handling, and storage might affect biomarker expression or invalidate tissue samples as analytes for some technologies. We investigated differences in RNA quality, gene expression by quantitative real-time PCR, and immunoreactive protein expression of selected prostate cancer biomarkers between tissues from retropubic radical prostatectomy (RRP) and robot-assisted laparoscopic prostatectomy (RALP). Sections of tissue microarray of 23 RALP and 22 RRP samples were stained with antibodies to androgen receptor (AR) and prostate-specific antigen (PSA) as intersite controls, and 14 other candidate biomarkers of research interest to three laboratories within the Australian Prostate Cancer BioResource tissue banking network. Quantitative real-time PCR was done for AR, PSA (KLK3), KLK2, KLK4, and HIF1A on RNA extracted from five RALP and five RRP frozen tissue cores. No histologic differences were observed between RALP and RRP tissue. Biomarker staining grouped these samples into those with increased (PSA, CK8/18, CKHMW, KLK4), decreased (KLK2, KLK14), or no change in expression (AR, ghrelin, Ki67, PCNA, VEGF-C, PAR2, YB1, p63, versican, and chondroitin 0-sulfate) in RALP compared with RRP tissue. No difference in RNA quality or gene expression was detected between RALP and RRP tissue. Changes in biomarker expression between RALP and RRP tissue exist at the immunoreactive protein level, but the etiology is unclear. Future studies should account for changes in biomarker expression when using RALP tissues, and mixed cohorts of RALP and RRP tissue should be avoided.

Expression of AR, 5αR1 and 5αR2 in bladder urothelial carcinoma and relationship to clinicopathological factors.

PubMed

Hata, Shuko; Ise, Kazue; Azmahani, Abdullah; Konosu-Fukaya, Sachiko; McNamara, Keely May; Fujishima, Fumiyoshi; Shimada, Keiji; Mitsuzuka, Koji; Arai, Yoichi; Sasano, Hironobu; Nakamura, Yasuhiro

2017-12-01

Bladder urothelial carcinoma is increasing in incidence with age and its prognosis could become worse when accompanied with metastasis. Effective treatment of these advanced patients is required and it becomes important to understand its underlying biology of this neoplasm, especially with regard to its biological pathways. A potential proposed pathway is androgen receptor (AR)-mediated intracellular signaling but the details have remained relatively unexplored. The expression of AR, 5α-reductase type1 (5αR1) and 5α-reductase type2 (5αR2) were examined in the bladder cancer cell line T24 and surgical pathology specimens. We also evaluated the status of androgen related cell proliferation and migration using the potent, non-aromatizable androgen agonist 5α-dihydrotestosterone (DHT). DHT treatment significantly increased AR mRNA expression level, but not those of 5αR1 and 5αR2 in T24 cells. DHT also suppressed cellular migration with weaker and opposite effects on cell proliferation. A significant inverse correlation was detected between pT stage and AR, 5αR1 and 5αR2 immunoreactivity. Inverse correlations detected between tumor grade and AR/androgen metabolizing enzyme also suggested that the loss of AR and androgen-producing enzymes could be associated with tumor progression. Effects of DHT on cells also suggest that androgens may regulate cellular behavior. Copyright © 2017 Elsevier Inc. All rights reserved.

A Non-imaging High Throughput Approach to Chemical Library Screening at the Unmodified Adenosine-A3 Receptor in Living Cells.

PubMed

Arruda, Maria Augusta; Stoddart, Leigh A; Gherbi, Karolina; Briddon, Stephen J; Kellam, Barrie; Hill, Stephen J

2017-01-01

Recent advances in fluorescent ligand technology have enabled the study of G protein-coupled receptors in their native environment without the need for genetic modification such as addition of N-terminal fluorescent or bioluminescent tags. Here, we have used a non-imaging plate reader (PHERAstar FS) to monitor the binding of fluorescent ligands to the human adenosine-A 3 receptor (A 3 AR; CA200645 and AV039), stably expressed in CHO-K1 cells. To verify that this method was suitable for the study of other GPCRs, assays at the human adenosine-A 1 receptor, and β 1 and β 2 adrenoceptors (β 1 AR and β 2 AR; BODIPY-TMR-CGP-12177) were also carried out. Affinity values determined for the binding of the fluorescent ligands CA200645 and AV039 to A 3 AR for a range of classical adenosine receptor antagonists were consistent with A 3 AR pharmacology and correlated well ( R 2 = 0.94) with equivalent data obtained using a confocal imaging plate reader (ImageXpress Ultra). The binding of BODIPY-TMR-CGP-12177 to the β 1 AR was potently inhibited by low concentrations of the β 1 -selective antagonist CGP 20712A (pK i 9.68) but not by the β 2 -selective antagonist ICI 118551(pK i 7.40). Furthermore, in experiments conducted in CHO K1 cells expressing the β 2 AR this affinity order was reversed with ICI 118551 showing the highest affinity (pK i 8.73) and CGP20712A (pK i 5.68) the lowest affinity. To determine whether the faster data acquisition of the non-imaging plate reader (~3 min per 96-well plate) was suitable for high throughput screening (HTS), we screened the LOPAC library for inhibitors of the binding of CA200645 to the A 3 AR. From the initial 1,263 compounds evaluated, 67 hits (defined as those that inhibited the total binding of 25 nM CA200645 by ≥40%) were identified. All compounds within the library that had medium to high affinity for the A 3 AR (pK i ≥6) were successfully identified. We found three novel compounds in the library that displayed unexpected sub-micromolar affinity for the A 3 AR. These were K114 (pK i 6.43), retinoic acid p -hydroxyanilide (pK i 6.13) and SU 6556 (pK i 6.17). Molecular docking of these latter three LOPAC library members provided a plausible set of binding poses within the vicinity of the established orthosteric A 3 AR binding pocket. A plate reader based library screening using an untagged receptor is therefore possible using fluorescent ligand opening the possibility of its use in compound screening at natively expressed receptors.

Downregulation of androgen receptors by NaAsO2 via inhibition of AKT-NF-κB and HSP90 in castration resistant prostate cancer.

PubMed

Kim, Yunlim; Park, Sang Eun; Moon, Jeong-Weon; Kim, Bong-Min; Kim, Ha-Gyeong; Jeong, In Gab; Yoo, Sangjun; Ahn, Jae Beom; You, Dalsan; Pak, Jhang Ho; Kim, Sujong; Hwang, Jung Jin; Kim, Choung-Soo

2017-07-01

Androgen and androgen receptor (AR) play essential roles in the development and maintenance of prostate cancer. The recently identified AR splice variants (AR-Vs) have been considered as a plausible mechanism for the primary resistance against androgen deprivation therapy (ADT) in castration-resistant prostate cancer (CRPC). Sodium meta-arsenite (NaAsO 2 ; KML001; Kominox), a trivalent arsenical, is an orally bioavailable and water soluble, which is currently in phase I/II clinical trials for the treatment of prostate cancer. It has a potent anti-cancer effect on prostate cancer cells and xenografts. The aim of this study was to examine the effect of NaAsO 2 on AR signaling in LNCaP and 22Rv1 CRPC cells. We used hormone-sensitive LNCaP cells, hormone-insensitive 22Rv1 cells, and CRPC patient-derived primary cells. We analyzed anti-cancer effect of NaAsO 2 using real-time quantitative reverse transcription-PCR, Western blotting, immunofluorescence staining and CellTiter Glo® luminescent assay. Statistical evaluation of the results was performed by one-way ANOVA. NaAsO 2 significantly reduced the translocation of AR and AR-Vs to the nucleus as well as their level in LNCaP and 22Rv1 cells. Besides, the level of the prostate-specific antigen (PSA), downstream target gene of AR, was also decreased. This compound was also an effective modulator of AKT-dependent NF-κB activation which regulates AR. NaAsO 2 significantly inhibited phosphorylation of AKT and expression and nuclear translocation of NF-κB. We then investigated the effect of NaAsO 2 on AR stabilization. NaAsO 2 promoted HSP90 acetylation by down-regulating HDAC6, which reduces the stability of AR in prostate cancer cells. Here, we show that NaAsO 2 disrupts AR signaling at multiple levels by affecting AR expression, stability, and degradation in primary tumor cell cultures from prostate cancer patients as well as CRPC cell lines. These results suggest that NaAsO 2 could be a novel therapeutics for prostate cancer. © 2017 Wiley Periodicals, Inc.

A Low-Protein, High-Carbohydrate Diet Stimulates Thermogenesis in the Brown Adipose Tissue of Rats via ATF-2.

PubMed

de França, Suélem A; dos Santos, Maísa P; Przygodda, Franciele; Garófalo, Maria Antonieta R; Kettelhut, Isis C; Magalhães, Diego A; Bezerra, Kalinne S; Colodel, Edson M; Flouris, Andreas D; Andrade, Cláudia M B; Kawashita, Nair H

2016-03-01

The aim of this study was to evaluate thermogenesis in the interscapular brown adipose tissue (IBAT) of rats submitted to low-protein, high-carbohydrate (LPHC) diet and the involvement of adrenergic stimulation in this process. Male rats (~100 g) were submitted to LPHC (6%-protein; 74%-carbohydrate) or control (C; 17%-protein; 63%-carbohydrate) isocaloric diets for 15 days. The IBAT temperature was evaluated in the rats before and after the administration of noradrenaline (NA) (20 µg 100 g b w(-1) min(-1)). The expression levels of uncoupling protein 1 (UCP1) and other proteins involved in the regulation of UCP1 expression were determined by Western blot (Student's t test, P ≤ 0.05). The LPHC diet promoted a 1.1 °C increase in the basal temperature of IBAT when compared with the basal temperature in the IBAT of the C group. NA administration promoted a 0.3 °C increase in basal temperature in the IBAT of the C rats and a 0.5 °C increase in the IBAT of the LPHC group. The level of UCP1 increased 60% in the IBAT of LPHC-fed rats, and among the proteins involved in its expression, such as β3-AR and α1-AR, there was a 40% increase in the levels of p38-MAPK and a 30% decrease in CREB when compared to the C rats. The higher sympathetic flux to IBAT, which is a consequence of the administration of the LPHC diet to rats, activates thermogenesis and increases the expression of UCP1 in the tissue. Our results suggest that the increase in UCP1 content may occur via p38 MAPK and ATF2.

Resistance to docetaxel in prostate cancer is associated with androgen receptor activation and loss of KDM5D expression

PubMed Central

Komura, Kazumasa; Jeong, Seong Ho; Hinohara, Kunihiko; Qu, Fangfang; Wang, Xiaodong; Hiraki, Masayuki; Azuma, Haruhito; Lee, Gwo-Shu Mary; Kantoff, Philip W.; Sweeney, Christopher J.

2016-01-01

The androgen receptor (AR) plays an essential role in prostate cancer, and suppression of its signaling with androgen deprivation therapy (ADT) has been the mainstay of treatment for metastatic hormone-sensitive prostate cancer for more than 70 y. Chemotherapy has been reserved for metastatic castration-resistant prostate cancer (mCRPC). The Eastern Cooperative Oncology Group-led trial E3805: ChemoHormonal Therapy Versus Androgen Ablation Randomized Trial for Extensive Disease in Prostate Cancer (CHAARTED) showed that the addition of docetaxel to ADT prolonged overall survival compared with ADT alone in patients with metastatic hormone-sensitive prostate cancer. This finding suggests that there is an interaction between AR signaling activity and docetaxel sensitivity. Here we demonstrate that the prostate cancer cell lines LNCaP and LAPC4 display markedly different sensitivity to docetaxel with AR activation, and RNA-seq analysis of these cell lines identified KDM5D (lysine-specific demethylase 5D) encoded on the Y chromosome as a potential mediator of this sensitivity. Knocking down KDM5D expression in LNCaP leads to docetaxel resistance in the presence of dihydrotestosterone. KDM5D physically interacts with AR in the nucleus, and regulates its transcriptional activity by demethylating H3K4me3 active transcriptional marks. Attenuating KDM5D expression dysregulates AR signaling, resulting in docetaxel insensitivity. KDM5D deletion was also observed in the LNCaP-derived CRPC cell line 104R2, which displayed docetaxel insensitivity with AR activation, unlike parental LNCaP. Dataset analysis from the Oncomine database revealed significantly decreased KDM5D expression in CRPC and poorer prognosis with low KDM5D expression. Taking these data together, this work indicates that KDM5D modulates the AR axis and that this is associated with altered docetaxel sensitivity. PMID:27185910

Designed modulation of sex steroid signaling inhibits telomerase activity and proliferation of human prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Verma, Vikas; Sharma, Vikas; Singh, Vishal

The predominant estrogen-receptor (ER)-β signaling in normal prostate is countered by increased ER-α signaling in prostate cancer (CaP), which in association with androgen-receptor (AR) signaling results in pathogenesis of the disease. However CaP treatments mostly target AR signaling which is initially effective but eventually leads to androgen resistance, hence simultaneous targeting of ERs has been proposed. A novel series of molecules were designed with multiple sex-steroid receptor modulating capabilities by coalescing the pharmacophores of known anti-CaP molecules that act via modulation of ER(α/β) and/or AR, viz. 3,3′diindolylmethane (DIM), mifepristone, toremifene, tamoxifen and raloxifene. N,N-diethyl-4-((2-(4-methoxyphenyl)-1H-indol-3-yl)methyl) aniline (DIMA) was identified as themore » most promising structure of this new series. DIMA increased annexin-V labelling, cell-cycle arrest and caspase-3 activity, and decreased expression of AR and prostate specific antigen in LNCaP cells, in vitro. Concurrently, DIMA increased ER-β, p21 and p27 protein levels in LNCaP cells and exhibited ∼ 5 times more selective binding for ER-β than ER-α, in comparison to raloxifene. DIMA exhibited a dose-dependent ER-β agonism and ER-α antagonism in classical gene reporter assay and decreased hTERT (catalytic subunit of telomerase) transcript levels in LNCaP at 3.0 μM (P < 0.05). DIMA also dose-dependently decreased telomerase enzyme activity in prostate cancer cells. It is thus concluded that DIMA acts as a multi-steroid receptor modulator and effectively inhibits proliferation of prostate cancer cells through ER-β mediated telomerase inhibition, by countering actions of ER-α and AR. Its unique molecular design can serve as a lead structure for generation of potent agents against endocrine malignancies like the CaP.« less

Expression Plasmids for Use in Candida glabrata

PubMed Central

Zordan, Rebecca E.; Ren, Yuxia; Pan, Shih-Jung; Rotondo, Giuseppe; Peñas, Alejandro De Las; Iluore, Joseph; Cormack, Brendan P.

2013-01-01

We describe a series of CEN/ARS episomal plasmids containing different Candida glabrata promoters, allowing for a range of constitutive or regulated expression of proteins in C. glabrata. The set of promoters includes three constitutive promoters (EGD2pr, HHT2pr, PDC1pr), two macrophage/phagocytosis-induced promoters (ACO2pr, LYS21pr), and one nutritionally regulated promoter (MET3pr). Each promoter was cloned into two plasmid backbones that differ in their selectable marker, URA3, or the dominant-selectable NAT1 gene, which confers resistance to the drug nourseothricin. Expression from the 12 resulting plasmids was assessed using GFP as a reporter and flow cytometry or quantitative reverse-transcription polymerase chain reaction to assess expression levels. Together this set of plasmids expands the toolkit of expression vectors available for use with C. glabrata. PMID:23934995

The anti-oestrogen fulvestrant (ICI 182,780) reduces the androgen receptor expression, ERK1/2 phosphorylation and cell proliferation in the rat ventral prostate.

PubMed

Fernandes, S A F; Gomes, G R O; Siu, E R; Damas-Souza, D M; Bruni-Cardoso, A; Augusto, T M; Lazari, M F M; Carvalho, H F; Porto, C S

2011-10-01

This study proposed to investigate further the role of oestrogens during pubertal growth of rat ventral prostate, by analysing the effect of anti-oestrogen fulvestrant (ICI 182,780) on the expression of androgen (AR) and oestrogen receptors (ESR1 and ESR2), mitogen-activated protein kinase (ERK1/2) phosphorylation, and expression of Ki-67, a biomarker for cell proliferation. Ventral prostates were obtained from 90-day-old rats treated once a week for 2 months with vehicle (control) or ICI 182,780 (10 mg/rat, s.c.). Transcripts for AR, ESR1 and ESR2 were evaluated by quantitative real-time polymerase chain reaction. Expression of AR, ESR1, ESR2, total and phospho-ERK1/2 was analysed by Western blot or immunofluorescence. Ki-67-positive cells and myosin heavy chain were detected by immunohistochemistry. Cylindrical epithelial cells slightly taller, epithelial dysplasia and an increase in smooth muscle layer were observed in the ventral prostate from ICI 182,780-treated rats. ICI 182,780 did not change the mRNA, but decreased the protein levels for AR in the ventral prostate. The expression of ESR1 (mRNA and protein) was upregulated by ICI 182,780, but no changes were observed on ESR2 expression (mRNA and protein). ICI 182,780 decreased the phosphorylation state of ERK1/2, with no changes in total ERK1/2 levels. Ki-67-positive cells in the ventral prostate were also decreased by ICI 182,780. In conclusion, ICI 182,780 induces downregulation of AR expression and may block the translocation of ESR1 and ESR2 from the nucleus to the plasma membrane, decreasing ERK1/2 phosphorylation and prostatic epithelial cell proliferation. These findings provide a basis for physiological roles of oestrogen in the ventral prostate. Further studies with fulvestrant are necessary in benign prostate hyperplasia and prostatic cancer models. © 2010 The Authors. International Journal of Andrology © 2011 European Academy of Andrology.

The relationship between rumen acidosis resistance and expression of genes involved in regulation of intracellular pH and butyrate metabolism of ruminal epithelial cells in steers.

PubMed

Schlau, N; Guan, L L; Oba, M

2012-10-01

Past research has focused on the prevention and management of subacute rumen acidosis by manipulating the ration; however, the severity of acidosis varies even among animals fed a common high-grain diet. The objectives of this study were to compare the ruminal volatile fatty acid (VFA) profile and expression of genes involved in the metabolism of butyrate, the VFA most extensively metabolized by the ruminal epithelium, and intracellular pH regulation in ruminal epithelial cells between acidosis-resistant (AR) and acidosis-susceptible (AS) steers. Acidosis indexes (area per day under pH 5.8 divided by dry matter intake) were measured for 17 steers fed a common high-grain diet, and the 3 steers with the lowest (1.4 ± 1.2 pH∙min/kg) and the 3 with the highest values (23.9 ± 7.4 pH∙min/kg) were classified as AR and AS, respectively, and used in the subsequent study. The steers were force-fed a diet containing 85% grain at 60% of the expected daily intake (5.8 ± 0.8 and 5.6 ± 0.6 kg for AR and AS, respectively) within 30 min. Mean ruminal pH over the postprandial 6-h period was higher for AR compared with AS (6.02 vs. 5.55), and mean total VFA concentration was 74% for AR compared with AS (122 vs. 164 mM). Molar proportion of butyrate in the ruminal fluid was 139% higher for AR compared with AS (17.5 vs. 7.33 mol/100 mol of VFA). Expression of monocarboxylate cotransporter isoform 1, sodium hydrogen exchanger isoforms 1 and 2, and anion exchangers (downregulated in adenoma and putative anion exchanger, isoform 1) did not differ between AR and AS steers. However, expression of sodium hydrogen exchanger isoform 3, which imports Na(+) to the epithelial cell and exports H(+) to the rumen, was 176% higher in AR steers than in AS steers. Higher ruminal pH for AR might be partly due to a faster rate of VFA absorption, lower VFA production, or both. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Autocrine Complement Inhibits IL10-Dependent T-cell-Mediated Antitumor Immunity to Promote Tumor Progression.

PubMed

Wang, Yu; Sun, Sheng-Nan; Liu, Qing; Yu, Yang-Yang; Guo, Jian; Wang, Kun; Xing, Bao-Cai; Zheng, Qing-Feng; Campa, Michael J; Patz, Edward F; Li, Shi-You; He, You-Wen

2016-09-01

In contrast to its inhibitory effects on many cells, IL10 activates CD8(+) tumor-infiltrating lymphocytes (TIL) and enhances their antitumor activity. However, CD8(+) TILs do not routinely express IL10, as autocrine complement C3 inhibits IL10 production through complement receptors C3aR and C5aR. CD8(+) TILs from C3-deficient mice, however, express IL10 and exhibit enhanced effector function. C3-deficient mice are resistant to tumor development in a T-cell- and IL10-dependent manner; human TILs expanded with IL2 plus IL10 increase the killing of primary tumors in vitro compared with IL2-treated TILs. Complement-mediated inhibition of antitumor immunity is independent of the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) immune checkpoint pathway. Our findings suggest that complement receptors C3aR and C5aR expressed on CD8(+) TILs represent a novel class of immune checkpoints that could be targeted for tumor immunotherapy. Moreover, incorporation of IL10 in the expansion of TILs and in gene-engineered T cells for adoptive cell therapy enhances their antitumor efficacy. Our data suggest novel strategies to enhance immunotherapies: a combined blockade of complement signaling by antagonists to C3aR, C5aR, and anti-PD-1 to enhance anti-PD-1 efficacy; a targeted IL10 delivery to CD8(+) TILs using anti-PD-1-IL10 or anti-CTLA4-IL10 fusion proteins; and the addition of IL10 in TIL expansion for adoptive cellular therapy. Cancer Discov; 6(9); 1022-35. ©2016 AACR.See related commentary by Peng et al., p. 953This article is highlighted in the In This Issue feature, p. 932. ©2016 American Association for Cancer Research.

RNase L Suppresses Androgen Receptor Signaling, Cell Migration and Matrix Metalloproteinase Activity in Prostate Cancer Cells

PubMed Central

Dayal, Shubham; Zhou, Jun; Manivannan, Praveen; Siddiqui, Mohammad Adnan; Ahmad, Omaima Farid; Clark, Matthew; Awadia, Sahezeel; Garcia-Mata, Rafael; Shemshedini, Lirim; Malathi, Krishnamurthy

2017-01-01

The interferon antiviral pathways and prostate cancer genetics converge on a regulated endoribonuclease, RNase L. Positional cloning and linkage studies mapped Hereditary Prostate Cancer 1 (HPC1) to RNASEL. To date, there is no correlation of viral infections with prostate cancer, suggesting that RNase L may play additional roles in tumor suppression. Here, we demonstrate a role of RNase L as a suppressor of androgen receptor (AR) signaling, cell migration and matrix metalloproteinase activity. Using RNase L mutants, we show that its nucleolytic activity is dispensable for both AR signaling and migration. The most prevalent HPC1-associated mutations in RNase L, R462Q and E265X, enhance AR signaling and cell migration. RNase L negatively regulates cell migration and attachment on various extracellular matrices. We demonstrate that RNase L knockdown cells promote increased cell surface expression of integrin β1 which activates Focal Adhesion Kinase-Sarcoma (FAK-Src) pathway and Ras-related C3 botulinum toxin substrate 1-guanosine triphosphatase (Rac1-GTPase) activity to increase cell migration. Activity of matrix metalloproteinase (MMP)-2 and -9 is significantly increased in cells where RNase L levels are ablated. We show that mutations in RNase L found in HPC patients may promote prostate cancer by increasing expression of AR-responsive genes and cell motility and identify novel roles of RNase L as a prostate cancer susceptibility gene. PMID:28257035

The post-menopausal ovary displays a unique pattern of steroidogenic enzyme expression.

PubMed

Havelock, Jon C; Rainey, William E; Bradshaw, Karen D; Carr, Bruce R

2006-01-01

While menopause results in the loss of cyclic steroid production, evidence exists for persistent, albeit reduced, ovarian androgen production. In order to continue to synthesize ovarian androgens, the steroidogenic enzymes necessary for androgen biosynthesis must be present. Few studies have selectively analysed some of the steroidogenic enzymes present in the post-menopausal ovary (PMO), and a comprehensive study of this matter has never been undertaken. RNA and protein were obtained from PMO, pre-menopausal ovarian stroma, corpora lutea (CL), ovarian follicles, placenta, and myometrium. Oligonucleotide microarray analysis was performed to compare the gene expression profiles of PMO with pre-menopausal ovarian stroma. Real-time RT-PCR was performed for LH/HCG receptor (LHCGR), steroidogenic acute regulatory (StAR), cholesterol side-chain cleavage (CYP11A), 3beta-hydroxysteroid dehydrogenase type I (HSD3B1) and type II (HSD3B2, 3betaHSD), 17a-hydroxylase (CYP17), cytochrome b5 (CytB5), and aromatase (CYP19). Western blot analysis was performed for StAR, CYP11A, CYP17,and 3betaHSD. The PMO and pre-menopausal ovarian stroma had a similar pattern of steroidogenic enzyme expression. The PMO had persistent, but reduced, levels of LHCGR and most steroidogenic enzymes. CYP19 and HSD3B2 mRNA were greatly reduced in PMO in comparison with CL (50-fold and 2000-fold less respectively). HSD3B2 was not detectable in PMO by western analysis. This study supports the idea that the PMO retains some steroidogenic capacity. However, based on steroidogenic enzyme expression, the PMO has a unique pattern of steroidogenic enzyme expression that favors Delta5 steroid formation over Delta4 steroid formation.

Expression of a hyperactive androgen receptor leads to androgen-independent growth of prostate cancer cells.

PubMed

Hsieh, Chen-Lin; Cai, Changmeng; Giwa, Ahmed; Bivins, Aaronica; Chen, Shao-Yong; Sabry, Dina; Govardhan, Kumara; Shemshedini, Lirim

2008-07-01

Cellular changes that affect the androgen receptor (AR) can cause prostate cancer to transition from androgen dependent to androgen independent, which is usually lethal. One common change in prostate tumors is overexpression of the AR, which has been shown to lead to androgen-independent growth of prostate cancer cells. This led us to hypothesize that expression of a hyperactive AR would be sufficient for androgen-independent growth of prostate cancer cells. To test this hypothesis, stable lune cancer prostate (LNCaP) cell lines were generated, which express a virion phosphoprotein (VP)16-AR hybrid protein that contains full-length AR fused to the strong viral transcriptional activation domain VP16. This fusion protein elicited as much as a 20-fold stronger transcriptional activity than the natural AR. Stable expression of VP16-AR in LNCaP cells yielded androgen-independent cell proliferation, while under the same growth conditions the parental LNCaP cells exhibited only androgen-dependent growth. These results show that expression of a hyperactive AR is sufficient for androgen-independent growth of prostate cancer cells. To study the molecular basis of this enhanced growth, we measured the expression of soluble guanylyl cyclase-alpha1 (sGCalpha1), a subunit of the sGC, an androgen-regulated gene that has been shown to be involved in prostate cancer cell growth. Interestingly, the expression of sGCalpha1 is androgen independent in VP16-AR-expressing cells, in contrast to its androgen-induced expression in control LNCaP cells. RNA(I)-dependent inhibition of sGCalpha1 expression resulted in significantly reduced proliferation of VP16-AR cells, implicating an important role for sGCalpha1 in the androgen-independent growth of these cells.

Adenosine A1 Receptor Protects Against Cisplatin Ototoxicity by Suppressing the NOX3/STAT1 Inflammatory Pathway in the Cochlea

PubMed Central

Kaur, Tejbeer; Borse, Vikrant; Sheth, Sandeep; Sheehan, Kelly; Ghosh, Sumana; Tupal, Srinivasan; Jajoo, Sarvesh; Mukherjea, Debashree; Rybak, Leonard P.

2016-01-01

Cisplatin is a commonly used antineoplastic agent that produces ototoxicity that is mediated in part by increasing levels of reactive oxygen species (ROS) via the NOX3 NADPH oxidase pathway in the cochlea. Recent studies implicate ROS generation in mediating inflammatory and apoptotic processes and hearing loss by activating signal transducer and activator of transcription (STAT1). In this study, we show that the adenosine A1 receptor (A1AR) protects against cisplatin ototoxicity by suppressing an inflammatory response initiated by ROS generation via NOX3 NADPH oxidase, leading to inhibition of STAT1. Trans-tympanic administration of the A1AR agonist R-phenylisopropyladenosine (R-PIA) inhibited cisplatin-induced ototoxicity, as measured by auditory brainstem responses and scanning electron microscopy in male Wistar rats. This was associated with reduced NOX3 expression, STAT1 activation, tumor necrosis factor-α (TNF-α) levels, and apoptosis in the cochlea. In vitro studies in UB/OC-1 cells, an organ of Corti immortalized cell line, showed that R-PIA reduced cisplatin-induced phosphorylation of STAT1 Ser727 (but not Tyr701) and STAT1 luciferase activity by suppressing the ERK1/2, p38, and JNK mitogen-activated protein kinase (MAPK) pathways. R-PIA also decreased the expression of STAT1 target genes, such as TNF-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced cisplatin-mediated apoptosis. These data suggest that the A1AR provides otoprotection by suppressing NOX3 and inflammation in the cochlea and could serve as an ideal target for otoprotective drug therapy. SIGNIFICANCE STATEMENT Cisplatin is a widely used chemotherapeutic agent for the treatment of solid tumors. Its use results in significant and permanent hearing loss, for which no US Food and Drug Administration-approved treatment is currently available. In this study, we targeted the cochlear adenosine A1 receptor (A1AR) by trans-tympanic injections of the agonist R-phenylisopropyladenosine (R-PIA) and showed that it reduced cisplatin-induced inflammation and apoptosis in the rat cochlea and preserved hearing. The mechanism of protection involves suppression of the NOX3 NADPH oxidase enzyme, a major target of cisplatin-induced reactive oxygen species (ROS) generation in the cochlea. ROS initiates an inflammatory and apoptotic cascade in the cochlea by activating STAT1 transcription factor, which is attenuated by R-PIA. Therefore, trans-tympanic delivery of A1AR agonists could effectively treat cisplatin ototoxicity. PMID:27053204

Adenosine A1 Receptor Protects Against Cisplatin Ototoxicity by Suppressing the NOX3/STAT1 Inflammatory Pathway in the Cochlea.

PubMed

Kaur, Tejbeer; Borse, Vikrant; Sheth, Sandeep; Sheehan, Kelly; Ghosh, Sumana; Tupal, Srinivasan; Jajoo, Sarvesh; Mukherjea, Debashree; Rybak, Leonard P; Ramkumar, Vickram

2016-04-06

Cisplatin is a commonly used antineoplastic agent that produces ototoxicity that is mediated in part by increasing levels of reactive oxygen species (ROS) via the NOX3 NADPH oxidase pathway in the cochlea. Recent studies implicate ROS generation in mediating inflammatory and apoptotic processes and hearing loss by activating signal transducer and activator of transcription (STAT1). In this study, we show that the adenosine A1 receptor (A1AR) protects against cisplatin ototoxicity by suppressing an inflammatory response initiated by ROS generation via NOX3 NADPH oxidase, leading to inhibition of STAT1. Trans-tympanic administration of the A1AR agonist R-phenylisopropyladenosine (R-PIA) inhibited cisplatin-induced ototoxicity, as measured by auditory brainstem responses and scanning electron microscopy in male Wistar rats. This was associated with reduced NOX3 expression, STAT1 activation, tumor necrosis factor-α (TNF-α) levels, and apoptosis in the cochlea. In vitro studies in UB/OC-1 cells, an organ of Corti immortalized cell line, showed that R-PIA reduced cisplatin-induced phosphorylation of STAT1 Ser(727) (but not Tyr(701)) and STAT1 luciferase activity by suppressing the ERK1/2, p38, and JNK mitogen-activated protein kinase (MAPK) pathways.R-PIA also decreased the expression of STAT1 target genes, such as TNF-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced cisplatin-mediated apoptosis. These data suggest that the A1AR provides otoprotection by suppressing NOX3 and inflammation in the cochlea and could serve as an ideal target for otoprotective drug therapy. Cisplatin is a widely used chemotherapeutic agent for the treatment of solid tumors. Its use results in significant and permanent hearing loss, for which no US Food and Drug Administration-approved treatment is currently available. In this study, we targeted the cochlear adenosine A1 receptor (A1AR) by trans-tympanic injections of the agonist R-phenylisopropyladenosine (R-PIA) and showed that it reduced cisplatin-induced inflammation and apoptosis in the rat cochlea and preserved hearing. The mechanism of protection involves suppression of the NOX3 NADPH oxidase enzyme, a major target of cisplatin-induced reactive oxygen species (ROS) generation in the cochlea. ROS initiates an inflammatory and apoptotic cascade in the cochlea by activating STAT1 transcription factor, which is attenuated byR-PIA. Therefore, trans-tympanic delivery of A1AR agonists could effectively treat cisplatin ototoxicity. Copyright © 2016 the authors 0270-6474/16/363962-16$15.00/0.

Androgen Receptor-Mediated Growth Suppression of HPr-1AR and PC3-Lenti-AR Prostate Epithelial Cells

PubMed Central

Bolton, Eric C.

2015-01-01

The androgen receptor (AR) mediates the developmental, physiologic, and pathologic effects of androgens including 5α-dihydrotestosterone (DHT). However, the mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells are not well understood, though they are central to prostate development, homeostasis, and neoplasia. Here, we identify androgen-responsive genes that restrain cell cycle progression and proliferation of human prostate epithelial cell lines (HPr-1AR and PC3-Lenti-AR), and we investigate the mechanisms through which AR regulates their expression. DHT inhibited proliferation of HPr-1AR and PC3-Lenti-AR, and cell cycle analysis revealed a prolonged G1 interval. In the cell cycle, the G1/S-phase transition is initiated by the activity of cyclin D and cyclin-dependent kinase (CDK) complexes, which relieve growth suppression. In HPr-1AR, cyclin D1/2 and CDK4/6 mRNAs were androgen-repressed, whereas CDK inhibitor, CDKN1A, mRNA was androgen-induced. The regulation of these transcripts was AR-dependent, and involved multiple mechanisms. Similar AR-mediated down-regulation of CDK4/6 mRNAs and up-regulation of CDKN1A mRNA occurred in PC3-Lenti-AR. Further, CDK4/6 overexpression suppressed DHT-inhibited cell cycle progression and proliferation of HPr-1AR and PC3-Lenti-AR, whereas CDKN1A overexpression induced cell cycle arrest. We therefore propose that AR-mediated growth suppression of HPr-1AR involves cyclin D1 mRNA decay, transcriptional repression of cyclin D2 and CDK4/6, and transcriptional activation of CDKN1A, which serve to decrease CDK4/6 activity. AR-mediated inhibition of PC3-Lenti-AR proliferation occurs through a similar mechanism, albeit without down-regulation of cyclin D. Our findings provide insight into AR-mediated regulation of prostate epithelial cell proliferation. PMID:26372468

High-expression β(1) adrenergic receptor/cell membrane chromatography method based on a target receptor to screen active ingredients from traditional Chinese medicines.

PubMed

Yue, Yuan; Xue, Hui; Wang, Xin; Yang, Qian; Song, Yanhong; Li, Xiaoni

2014-02-01

β-Adrenergic receptors are important targets for drug discovery. We have developed a new β1 -adrenergic receptor cell membrane chromatography (β1 AR-CMC) with offline ultra-performance LC (UPLC) and MS method for screening active ingredients from traditional Chinese medicines. In this study, Chinese hamster ovary-S cells with high β1 AR expression levels were established and used to prepare a cell membrane stationary phase in a β1 AR-CMC model. The retention fractions were separated and identified by the UPLC-MS system. The screening results found that isoimperatorin from Rhizoma et Radix Notopterygii was the targeted component that could act on β1 AR in similar manner of metoprolol as a control drug. In addition, the biological effects of active component were also investigated in order to search for a new type of β1 AR antagonist. It will be a useful method for drug discovery as a leading compound resource. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

AVP and Glu systems interact to regulate levels of anxiety in BALB/cJ mice.

PubMed

An, Xiao-Lei; Tai, Fa-Dao

2014-07-01

Whilethe roles of glutamic acid (Glu), arginine vasopressin (AVP) and their respective receptors in anxiety have been thoroughly investigated, the effects of interactions among Glu, N-methyl-D-aspartic acid (NMDA) receptor, AVP and a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor on anxiety are still unclear. In the present study, the agonist and antagonist of the NMDA receptor and AMPA receptor, as well as the antagonist of AVP V1 receptor (V1aR) were introduced into BALB/cJ mice by intracerebroventricular microinjection, and the anxiety-like behaviors of the mice were evaluated by open field and elevated plus-maze tests. Compared with C57BL/6 mice, BALB/cJ mice displayed higher levels of anxiety-like behavior. Significant anxiolytic effects were found in the NMDA receptor antagonist (MK-801) and the AMPA receptor or V1aR antagonist (SSRI49415), as well as combinations of AVP/MK-801 and SSRI49415/DNQX. These results indicated that anxiety-like behaviors expressed in BALB/CJ mice may be due to a coordination disorder among glutamate, NMDA receptor, AMPA receptor, AVP and V1aR, resulting in the up-regulation of the NMDA receptor and V1aR and down-regulation of the AMPA receptor. However, because the AMPA receptor can execute its anxiolytic function by suppressing AVP and V1aR, we cannot exclude the possibility of the NMDA receptor being activated by AVP acting on V1aR.

Protein Arginine Methyltransferase 7 Regulates Cellular Response to DNA Damage by Methylating Promoter Histones H2A and H4 of the Polymerase δ Catalytic Subunit Gene, POLD1*

PubMed Central

Karkhanis, Vrajesh; Wang, Li; Tae, Sookil; Hu, Yu-Jie; Imbalzano, Anthony N.; Sif, Saïd

2012-01-01

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage. PMID:22761421

Protein arginine methyltransferase 7 regulates cellular response to DNA damage by methylating promoter histones H2A and H4 of the polymerase δ catalytic subunit gene, POLD1.

PubMed

Karkhanis, Vrajesh; Wang, Li; Tae, Sookil; Hu, Yu-Jie; Imbalzano, Anthony N; Sif, Saïd

2012-08-24

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage.

Human PIRH2 Enhances Androgen Receptor Signaling through Inhibition of Histone Deacetylase 1 and Is Overexpressed in Prostate Cancer

PubMed Central

Logan, Ian R.; Gaughan, Luke; McCracken, Stuart R. C.; Sapountzi, Vasileia; Leung, Hing Y.; Robson, Craig N.

2006-01-01

The androgen receptor (AR) is a hormone-dependent transcription factor critically involved in human prostate carcinogenesis. Optimal transcriptional control of androgen-responsive genes by AR may require complex interaction among multiple coregulatory proteins. We have previously shown that the AR coregulator TIP60 can interact with human PIRH2 (hPIRH2). In this study, we uncover important new functional role(s) for hPIRH2 in AR signaling: (i) hPIRH2 interacts with AR and enhances AR-mediated transcription with a dynamic pattern of recruitment to androgen response elements in the prostate-specific antigen (PSA) gene; (ii) hPIRH2 interacts with the AR corepressor HDAC1, leading to reduced HDAC1 protein levels and inhibition of transcriptional repression; (iii) hPIRH2 is required for optimal PSA expression; and (iv) hPIRH2 is involved in prostate cancer cell proliferation. In addition, overexpression of hPIRH2 protein was detected in 73 of 82 (89%) resected prostate cancers, with a strong correlation between increased hPIRH2 expression and aggressive disease, as signified by high Gleason sum scores and the presence of metastatic disease (P = <0.0001 and 0.0004, respectively). Collectively, our data establish hPIRH2 as a key modulator of AR function, opening a new direction for targeted therapy in aggressive human prostate cancer. PMID:16914734

Human PIRH2 enhances androgen receptor signaling through inhibition of histone deacetylase 1 and is overexpressed in prostate cancer.

PubMed

Logan, Ian R; Gaughan, Luke; McCracken, Stuart R C; Sapountzi, Vasileia; Leung, Hing Y; Robson, Craig N

2006-09-01

The androgen receptor (AR) is a hormone-dependent transcription factor critically involved in human prostate carcinogenesis. Optimal transcriptional control of androgen-responsive genes by AR may require complex interaction among multiple coregulatory proteins. We have previously shown that the AR coregulator TIP60 can interact with human PIRH2 (hPIRH2). In this study, we uncover important new functional role(s) for hPIRH2 in AR signaling: (i) hPIRH2 interacts with AR and enhances AR-mediated transcription with a dynamic pattern of recruitment to androgen response elements in the prostate-specific antigen (PSA) gene; (ii) hPIRH2 interacts with the AR corepressor HDAC1, leading to reduced HDAC1 protein levels and inhibition of transcriptional repression; (iii) hPIRH2 is required for optimal PSA expression; and (iv) hPIRH2 is involved in prostate cancer cell proliferation. In addition, overexpression of hPIRH2 protein was detected in 73 of 82 (89%) resected prostate cancers, with a strong correlation between increased hPIRH2 expression and aggressive disease, as signified by high Gleason sum scores and the presence of metastatic disease (P = <0.0001 and 0.0004, respectively). Collectively, our data establish hPIRH2 as a key modulator of AR function, opening a new direction for targeted therapy in aggressive human prostate cancer.

SKCa Channels Blockage Increases the Expression of Adenosine A2A Receptor in Jurkat Human T Cells

PubMed Central

Regaya, Imed; Aidi-Knani, Sabrine; By, Youlet; Condo, Jocelyne; Gerolami, Victoria; Berge-Lefranc, Jean-Louis; Ben Hamida, Jeannette; Sabatier, Jean-Marc; Fenouillet, Emmanuel; Guieu, Régis

2013-01-01

Abstract Adenosine is a nucleoside displaying various biological effects via stimulation of four G-protein–coupled receptors, A1, A2A, A2B, and A3. Adenosine also modulates voltage-gated (Kv) and small conductance calcium-activated (SKCa) potassium channels. The effect of these potassium channels on the expression of adenosine receptors is poorly understood. We evaluated the action of BgK (a natural Kv channel blocker) and Lei-Dab7 (a synthetic SKCa channel blocker) on the expression of adenosine A2A receptors (A2AR) in Jurkat human T cells. We found that Lei-Dab7, but not BgK, increased the maximal binding value of the tritiated ligand ZM241385 to A2AR in a dose-dependent manner (+45% at 5 nM; +70% at 50 nM as compared to control). These results were further confirmed by Western blotting using a specific monoclonal antibody to human A2AR. The ligand affinity-related dissociation constant and A2AR mRNA amount were not significantly modified by either drug. We suggest that modulation of SKCa channels can influence membrane expression of A2AR and thus has a therapeutic potential. PMID:23593569

Houttuynia cordata aqueous extract attenuated glycative and oxidative stress in heart and kidney of diabetic mice.

PubMed

Hsu, Cheng-Chin; Yang, Hui-Ting; Ho, Jing-Jing; Yin, Mei-Chin; Hsu, Jen-Ying

2016-03-01

The anti-glycative and anti-oxidative effects from Houttuynia cordata leaves aqueous extract (HCAE) in heart and kidney of diabetic mice were examined. HCAE, at 1 or 2 %, was supplied in drinking water for 8 weeks. Plasma glucose and blood urea nitrogen (BUN) levels and creatine phosphokinase (CPK) activity were measured. The production of oxidative and inflammatory factors was determined. Activity and protein expression of associated enzymes or regulators were analyzed. HCAE intake at both doses lowered plasma glucose and BUN levels, and CPK activity and also restored creatinine clearance rate in diabetic mice. HCAE intake, only at 2 %, retained plasma insulin levels (P < 0.05). HCAE reduced reactive oxygen species, protein carbonyl, interleukin-6, tumor necrosis factor-alpha, N (ε) -(carboxymethyl)-lysine, pentosidine and fructose levels, and reserved glutathione content in heart and kidney of diabetic mice (P < 0.05). Diabetes enhanced aldose reductase (AR) activity and protein expression in heart and kidney (P < 0.05). HCAE intake at both doses decreased renal AR activity and protein expression, but only at 2 % lowered cardiac AR activity and protein expression (P < 0.05). Diabetes increased protein expression of RAGE, p47(phox) and gp91(phox), nuclear factor kappa-B (NF-κB) p50, NF-κB p65 and mitogen-activated protein kinase in heart and kidney (P < 0.05). HCAE intake only at 2 % limited RAGE expression, but at 1 and 2 % downregulated p47(phox), NF-κB p65 and p-p38 expression in these organs (P < 0.05). These findings suggest that Houttuynia cordata leaves aqueous extract could ameliorate cardiac and renal injury under diabetic condition.

Ethylene and auxin interaction in the control of adventitious rooting in Arabidopsis thaliana.

PubMed

Veloccia, A; Fattorini, L; Della Rovere, F; Sofo, A; D'Angeli, S; Betti, C; Falasca, G; Altamura, M M

2016-12-01

Adventitious roots (ARs) are post-embryonic roots essential for plant survival and propagation. Indole-3-acetic acid (IAA) is the auxin that controls AR formation; however, its precursor indole-3-butyric acid (IBA) is known to enhance it. Ethylene affects many auxin-dependent processes by affecting IAA synthesis, transport and/or signaling, but its role in AR formation has not been elucidated. This research investigated the role of ethylene in AR formation in dark-grown Arabidopsis thaliana seedlings, and its interaction with IAA/IBA. A number of mutants/transgenic lines were exposed to various treatments, and mRNA in situ hybridizations were carried out and hormones were quantified In the wild-type, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) at 0.1 μM enhanced AR formation when combined with IBA (10 μM), but reduced it when applied alone; this effect did not occur in the ein3eil1 ethylene-insensitive mutant. ACC inhibited the expression of the IAA-biosynthetic genes WEI2, WEI7, and YUC6, but enhanced IBA-to-IAA conversion, as shown by the response of the ech2ibr10 mutant and an increase in the endogenous levels of IAA. The ethylene effect was independent of auxin-signaling by TIR1-AFB2 and IBA-efflux by ABCG carriers, but it was dependent on IAA-influx by AUX1/LAX3.Taken together, the results demonstrate that a crosstalk involving ethylene signaling, IAA-influx, and IBA-to-IAA conversion exists between ethylene and IAA in the control of AR formation. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Activation of β-catenin signaling in androgen receptor-negative prostate cancer cells.

PubMed

Wan, Xinhai; Liu, Jie; Lu, Jing-Fang; Tzelepi, Vassiliki; Yang, Jun; Starbuck, Michael W; Diao, Lixia; Wang, Jing; Efstathiou, Eleni; Vazquez, Elba S; Troncoso, Patricia; Maity, Sankar N; Navone, Nora M

2012-02-01

To study Wnt/β-catenin in castrate-resistant prostate cancer (CRPC) and understand its function independently of the β-catenin-androgen receptor (AR) interaction. We carried out β-catenin immunocytochemical analysis, evaluated TOP-flash reporter activity (a reporter of β-catenin-mediated transcription), and sequenced the β-catenin gene in MDA prostate cancer 118a, MDA prostate cancer 118b, MDA prostate cancer 2b, and PC-3 prostate cancer cells. We knocked down β-catenin in AR-negative MDA prostate cancer 118b cells and carried out comparative gene-array analysis. We also immunohistochemically analyzed β-catenin and AR in 27 bone metastases of human CRPCs. β-Catenin nuclear accumulation and TOP-flash reporter activity were high in MDA prostate cancer 118b but not in MDA prostate cancer 2b or PC-3 cells. MDA prostate cancer 118a and MDA prostate cancer 118b cells carry a mutated β-catenin at codon 32 (D32G). Ten genes were expressed differently (false discovery rate, 0.05) in MDA prostate cancer 118b cells with downregulated β-catenin. One such gene, hyaluronan synthase 2 (HAS2), synthesizes hyaluronan, a core component of the extracellular matrix. We confirmed HAS2 upregulation in PC-3 cells transfected with D32G-mutant β-catenin. Finally, we found nuclear localization of β-catenin in 10 of 27 human tissue specimens; this localization was inversely associated with AR expression (P = 0.056, Fisher's exact test), suggesting that reduced AR expression enables Wnt/β-catenin signaling. We identified a previously unknown downstream target of β-catenin, HAS2, in prostate cancer, and found that high β-catenin nuclear localization and low or no AR expression may define a subpopulation of men with bone metastatic prostate cancer. These findings may guide physicians in managing these patients.

Role of SRC-3delta4 in the Progression and Metastasis of Castration-Resistant Prostate Cancer

DTIC Science & Technology

2013-10-01

Expression of SRC-3∆4, GAPDH, and AR target genes including PSA, KLK2, IGFBP5, Cyclin A2, and UBE2C was determined by RT-qPCR analysis . Data are...Expression of AR (B), GAPDH, and TMPRSS2- ERG (C) was determined by RT-qPCR analysis . Data are presented using the comparative Ct method, in which GAPDH...input. An irrelevant region (1800 bp downstream of transcription start site) was served as a negative control. (E) and (F). ChIP analysis of SRC-3∆4’s

Modulation of steroidogenesis by vitamin D3 in granulosa cells of the mouse model of polycystic ovarian syndrome.

PubMed

Bakhshalizadeh, Shabnam; Amidi, Fardin; Alleyassin, Ashraf; Soleimani, Masoud; Shirazi, Reza; Shabani Nashtaei, Maryam

2017-06-01

Polycystic ovarian syndrome (PCOS) is the most common endocrine disorder of women of reproductive age characterized by polycystic ovarian morphology, anovulation or oligomenorrhea, and hyperandrogenism. It is shown that disruption in the steroidogenesis pathway caused by excess androgen in PCOS is a critical element of abnormal folliculogenesis and failure in dominant follicle selection. Vitamin D plays an important role in the regulation of ovulatory dysfunction and can influence genes involved in steroidogenesis in granulosa cells. In the present study, we investigated the effects of vitamin D3 on steroidogenic enzyme expression and activities in granulosa cell using a PCOS mouse model. In our study, the PCOS mouse model was developed by the injection of dehydroepiandrosterone (DHEA) for 20 days. The mRNA and protein expression levels of genes involved in steroidogenesis in granulosa cells were compared between polycystic and normal ovaries using real-time PCR and Western blotting assays. Granulosa cells of DHEA-induced PCOS mice were then cultured with and without vitamin D3 and mRNA and protein expression levels of steroidogenic enzymes and serum 17beta-estradiol and progesterone levels were investigated using qRT-PCR, western blot, and radioimmunoassay, respectively. Steroidogenic enzymes including Cyp11a1, StAR, Cyp19a1, and 3β-HSD were upregulated in granulosa cells of PCOS mice when compared to normal mice. Treatment with vitamin D3 decreased mRNA and protein expression levels of steroidogenic enzymes in cultured granulosa cells. Vitamin D3 also decreased aromatase and 3β-HSD activity that leads to decreased 17beta-estradiol and progesterone release. This study suggests that vitamin D3 could modulate the steroidogenesis pathway in granulosa cells of PCOS mice that may lead to improving follicular development and maturation. This is a step towards a possible conceivable treatment for PCOS. AMHR-II: anti-müllerian hormone receptor-II; 3β-HSD: 3β-hydroxysteroid dehydrogenase; Cyp11a1: Cytochrome P450 Family 11 Subfamily A Member 1; Cyp19a1: cytochrome P450 aromatase; DHEA: dehydroepiandrosterone; FSH: follicle stimulating hormone; FSHR: follicle stimulating hormone receptor; IVF: in vitro fertilization; 25OHD: 25-hydroxy vitamin D; OHSS: ovarian hyperstimulation syndrome; PCOS: polycystic ovarian syndrome; P450scc: P450 side-chain cleavage enzyme; StAR: steroidogenic acute regulatory protein; VDRs: vitamin D receptors.

Intratumoral conversion of adrenal androgen precursors drives androgen receptor-activated cell growth in prostate cancer more potently than de novo steroidogenesis.

PubMed

Kumagai, Jinpei; Hofland, Johannes; Erkens-Schulze, Sigrun; Dits, Natasja F J; Steenbergen, Jacobie; Jenster, Guido; Homma, Yukio; de Jong, Frank H; van Weerden, Wytske M

2013-11-01

Despite an initial response to hormonal therapy, patients with advanced prostate cancer (PC) almost always progress to castration-resistant disease (CRPC). Although serum testosterone (T) is reduced by androgen deprivation therapy, intratumoral T levels in CRPC are comparable to those in prostate tissue of eugonadal men. These levels could originate from intratumoral conversion of adrenal androgens and/or from de novo steroid synthesis. However, the relative contribution of de novo steroidogenesis to AR-driven cell growth is unknown. The relative contribution of androgen biosynthetic pathways to activate androgen receptor (AR)-regulated cell growth and expression of PSA, FKBP5, and TMPRSS2 was studied at physiologically relevant levels of adrenal androgen precursors and intermediates of de novo androgen biosynthesis in human prostate cancer cell lines, PC346C, VCaP, and LNCaP. In PC346C and VCaP, responses to pregnenolone and progesterone were absent or minimal, while large effects of adrenal androgen precursors were found. VCaP CRPC clones overexpressing CYP17A1 did not acquire an increased ability to use pregnenolone or progesterone to activate AR. In contrast, all precursors stimulated growth and gene expression in LNCaP cells, presumably resulting from the mutated AR in these cells. Our data indicate that at physiological levels of T precursors PC cells can generally convert adrenal androgens, while de novo steroidogenesis is not generally possible in PC cells and is not able to support AR transactivation and PC growth. © 2013 Wiley Periodicals, Inc.

Beta-Adrenergic Receptor Population is Up-Regulated in Chicken Skeletal Muscle Cells Treated with Forskolin

NASA Technical Reports Server (NTRS)

Bridge, K. Y.; Young, R. B.; Vaughn, J. R.

1998-01-01

Skeletal muscle hypertrophy is promoted by in vivo administration of beta-adrenergic receptor (betaAR) agonists. These compounds presumably exert their physiological action through the betaAR, and alterations in the population of betaAR could potentially change the ability of the cell to respond to the betaAR agonists. Since the intracellular chemical signal generated by the betaAR is cyclic AMP (cAMP), experiments were initiated in primary chicken muscle cell cultures to determine if artificial elevation of intracellular cAMP by treatment with forskolin would alter the population of functional betaAR expressed on the surface of muscle cells. Chicken skeletal muscle cells after 7 days in culture were employed for the experiments because muscle cells have attained a steady state with respect to muscle protein metabolism at this stage. Cells were treated with 0-10 microM forskolin for a total of three days. At the end of the 1, 2, and 3 day treatment intervals, the concentration of cAMP and the betaAR population were measured. Receptor population was measured in intact muscle cell cultures as the difference between total binding of [H-3]CGP-12177 and non-specific binding of [H-3]CGP-12177 in the presence of 1 microM propranolol. Intracellular cAMP concentration was measured by radioimmunoassay. The concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. Increasing concentrations of forskolin also led to an increase in betaAR population, with a maximum increase of approximately 50% at 10 microM. This increase in PAR population was apparent after only 1 day of treatment, and the pattern of increase was maintained for all 3 days of the treatment period. Thus, increasing the intracellular concentration of cAMP leads to up-regulation of betaAR population. The effect of forskolin on the quantity and apparent synthesis rate of the heavy chain of myosin (mhc) were also investigated. A maximum increase of 50% in the quantity of mhc was observed at 0.2 microM forskolin, but higher concentrations of forskolin reduced the quantity of mhc back to control levels.

Androgen Receptor Involvement in Rat Amelogenesis: An Additional Way for Endocrine-Disrupting Chemicals to Affect Enamel Synthesis.

PubMed

Jedeon, Katia; Loiodice, Sophia; Salhi, Khaled; Le Normand, Manon; Houari, Sophia; Chaloyard, Jessica; Berdal, Ariane; Babajko, Sylvie

2016-11-01

Endocrine-disrupting chemicals (EDCs) that interfere with the steroid axis can affect amelogenesis, leading to enamel hypomineralization similar to that of molar incisor hypomineralization, a recently described enamel disease. We investigated the sex steroid receptors that may mediate the effects of EDCs during rat amelogenesis. The expression of androgen receptor (AR), estrogen receptor (ER)-α, and progesterone receptor was dependent on the stage of ameloblast differentiation, whereas ERβ remained undetectable. AR was the only receptor selectively expressed in ameloblasts involved in final enamel mineralization. AR nuclear translocation and induction of androgen-responsive element-containing promoter activity upon T treatment, demonstrated ameloblast responsiveness to androgens. T regulated the expression of genes involved in enamel mineralization such as KLK4, amelotin, SLC26A4, and SLC5A8 but not the expression of genes encoding matrix proteins, which determine enamel thickness. Vinclozolin and to a lesser extent bisphenol A, two antiandrogenic EDCs that cause enamel defects, counteracted the actions of T. In conclusion, we show, for the first time, the following: 1) ameloblasts express AR; 2) the androgen signaling pathway is involved in the enamel mineralization process; and 3) EDCs with antiandrogenic effects inhibit AR activity and preferentially affect amelogenesis in male rats. Their action, through the AR pathway, may specifically and irreversibly affect enamel, potentially leading to the use of dental defects as a biomarker of exposure to environmental pollutants. These results are consistent with the steroid hormones affecting ameloblasts, raising the issue of the hormonal influence on amelogenesis and possible sexual dimorphism in enamel quality.

Variation of bacterial communities and expression of Toll-like receptor genes in the rumen of steers differing in susceptibility to subacute ruminal acidosis.

PubMed

Chen, Yanhong; Oba, Masahito; Guan, Le Luo

2012-10-12

In order to determine differences in the ruminal bacterial community and host Toll-like receptor (TLR) gene expression of beef cattle with different susceptibility to acidosis, rumen papillae and content were collected from acidosis-susceptible (AS, n=3) and acidosis-resistant (AR, n=3) steers. The ruminal bacterial community was characterized using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real time PCR (qRT-PCR) analysis. Global R analysis of bacterial profile similarity revealed that bacterial diversity was significantly different between AR and AS groups for both rumen content (P=0.001) and epithelial (P=0.002) communities. The copy number of total bacterial 16S rRNA genes in content of AS steers was 10-fold higher than that of AR steers, and the copy number of total 16S rRNA genes of epimural bacteria in AR steers was positively correlated with ruminal pH (r=0.59, P=0.04), and negatively correlated with total VFA concentration (r=-0.59, P=0.05). The expressions of host TLR2 and 4 genes were significantly higher in AR steers compared to those in AS steers. These findings enhance our understanding about the ruminal microbial ecology and host gene expression changes that may be useful in the prevention of ruminal acidosis. Copyright © 2012 Elsevier B.V. All rights reserved.

AR-V7 in circulating tumor cells cluster as a predictive biomarker of abiraterone acetate and enzalutamide treatment in castration-resistant prostate cancer patients.

PubMed

Okegawa, Takatsugu; Ninomiya, Naoki; Masuda, Kazuki; Nakamura, Yu; Tambo, Mitsuhiro; Nutahara, Kikuo

2018-06-01

We examined whether androgen receptor splice variant 7 (AR-V7) in circulating tumor cell(CTC)clusters can be used to predict survival in patients with bone metastatic castration resistant-prostate cancer (mCRPC) treated with abiraterone or enzalutamide. We retrospectively enrolled 98 patients with CRPC on abiraterone or enzalutamide, and investigated the prognostic value of CTC cluster detection (+ v -) and AR-V7 detection (+ v -) using a CTC cluster detection - based AR-V7 mRNA assay. We examined ≤50% prostate-specific antigen (PSA) responses, PSA progression-free survival (PSA-PFS), clinical and radiological progression-free survival (radiologic PSF), and overall survival (OS). We then assessed whether AR-V7 expression in CTC clusters identified after On-chip multi-imaging flow cytometry was related to disease progression and survival after first-line systemic therapy. All abiraterone-treated or enzalutamide-treated patients received prior docetaxel. The median follow-up was 20.7 (range: 3.0-37.0) months in the abiraterone and enzalutamide cohorts, respectively. Forty-nine of the 98 men (50.0%) were CTC cluster (-), 23 of the 98 men (23.5%) were CTC cluster(+)/AR-V7(-), and 26 of the 98 men (26.5%) were CTC cluster(+)/AR-V7(+). CTC cluster(+)/AR-V7(+) patients were more likely to have EOD ≥3 at diagnosis (P = 0.003), pain (P = 0.023), higher alkaline phosphatase levels (P < 0.001), and visceral metastases (P < 0.001). On multivariable analysis, pretherapy CTC cluster(+), CTC cluster(+)/AR-V7(-), and ALP >UNL were independently associated with a poor PSA-PFS, radiographic PFS, and OS in abiraterone-treated patients and enzalutamide-treated patients. The CTC clusters and AR-V7-positive CTC clusters detected were important for assessing the response to abiraterone or enzalutamide therapy and for predicting disease outcome. © 2018 Wiley Periodicals, Inc.

Targeting chromatin binding regulation of constitutively active AR variants to overcome prostate cancer resistance to endocrine-based therapies

PubMed Central

Chan, Siu Chiu; Selth, Luke A.; Li, Yingming; Nyquist, Michael D.; Miao, Lu; Bradner, James E.; Raj, Ganesh V.; Tilley, Wayne D.; Dehm, Scott M.

2015-01-01

Androgen receptor (AR) variants (AR-Vs) expressed in prostate cancer (PCa) lack the AR ligand binding domain (LBD) and function as constitutively active transcription factors. AR-V expression in patient tissues or circulating tumor cells is associated with resistance to AR-targeting endocrine therapies and poor outcomes. Here, we investigated the mechanisms governing chromatin binding of AR-Vs with the goal of identifying therapeutic vulnerabilities. By chromatin immunoprecipitation and sequencing (ChIP-seq) and complementary biochemical experiments, we show that AR-Vs display a binding preference for the same canonical high-affinity androgen response elements (AREs) that are preferentially engaged by AR, albeit with lower affinity. Dimerization was an absolute requirement for constitutive AR-V DNA binding and transcriptional activation. Treatment with the bromodomain and extraterminal (BET) inhibitor JQ1 resulted in inhibition of AR-V chromatin binding and impaired AR-V driven PCa cell growth in vitro and in vivo. Importantly, this was associated with a novel JQ1 action of down-regulating AR-V transcript and protein expression. Overall, this study demonstrates that AR-Vs broadly restore AR chromatin binding events that are otherwise suppressed during endocrine therapy, and provides pre-clinical rationale for BET inhibition as a strategy for inhibiting expression and chromatin binding of AR-Vs in PCa. PMID:25908785

Markers of epithelial-to-mesenchymal transition reflect tumor biology according to patient age and Gleason score in prostate cancer

PubMed Central

Jędroszka, Dorota; Hamouz, Raneem; Górniak, Karolina; Bednarek, Andrzej K.

2017-01-01

Introduction Prostate carcinoma (PRAD) is one of the most frequently diagnosed malignancies amongst men worldwide. It is well-known that androgen receptor (AR) plays a pivotal role in a vast majority of prostate tumors. However, recent evidence emerged stating that estrogen receptors (ERs) may also contribute to prostate tumor development. Moreover, progression and aggressiveness of prostate cancer may be associated with differential expression genes of epithelial-to-mesenchymal transition (EMT). Therefore we aimed to assess the significance of receptors status as well as EMT marker genes expression among PRAD patients in accordance to their age and Gleason score. Materials and methods We analyzed TCGA gene expression profiles of 497 prostate tumor samples according to 43 genes involved in EMT and 3 hormone receptor genes (AR, ESR1, ESR2) as well as clinical characteristic of cancer patients. Then patients were divided into four groups according to their age and 5 groups according to Gleason score. Next, we evaluated PRAD samples according to relationship between the set of variables in different combinations and compared differential expression in subsequent groups of patients. The analysis was applied using R packages: FactoMineR, gplots, RColorBrewer and NMF. Results MFA analysis resulted in distinct grouping of PRAD patients into four age categories according to expression level of AR, ESR1 and ESR2 with the most distinct group of age less than 50 years old. Further investigations indicated opposite expression profiles of EMT markers between different age groups as well as strong association of EMT gene expression with Gleason score. We found that depending on age of prostate cancer patients and Gleason score EMT genes with distinctly altered expression are: KRT18, KRT19, MUC1 and COL4A1, CTNNB1, SNAI2, ZEB1 and MMP3. Conclusions Our major observation is that prostate cancer from patients under 50 years old compared to older ones has entirely different EMT gene expression profiles showing potentially more aggressive invasive phenotype, despite Gleason score classification. PMID:29206234

Markers of epithelial-to-mesenchymal transition reflect tumor biology according to patient age and Gleason score in prostate cancer.

PubMed

Jędroszka, Dorota; Orzechowska, Magdalena; Hamouz, Raneem; Górniak, Karolina; Bednarek, Andrzej K

2017-01-01

Prostate carcinoma (PRAD) is one of the most frequently diagnosed malignancies amongst men worldwide. It is well-known that androgen receptor (AR) plays a pivotal role in a vast majority of prostate tumors. However, recent evidence emerged stating that estrogen receptors (ERs) may also contribute to prostate tumor development. Moreover, progression and aggressiveness of prostate cancer may be associated with differential expression genes of epithelial-to-mesenchymal transition (EMT). Therefore we aimed to assess the significance of receptors status as well as EMT marker genes expression among PRAD patients in accordance to their age and Gleason score. We analyzed TCGA gene expression profiles of 497 prostate tumor samples according to 43 genes involved in EMT and 3 hormone receptor genes (AR, ESR1, ESR2) as well as clinical characteristic of cancer patients. Then patients were divided into four groups according to their age and 5 groups according to Gleason score. Next, we evaluated PRAD samples according to relationship between the set of variables in different combinations and compared differential expression in subsequent groups of patients. The analysis was applied using R packages: FactoMineR, gplots, RColorBrewer and NMF. MFA analysis resulted in distinct grouping of PRAD patients into four age categories according to expression level of AR, ESR1 and ESR2 with the most distinct group of age less than 50 years old. Further investigations indicated opposite expression profiles of EMT markers between different age groups as well as strong association of EMT gene expression with Gleason score. We found that depending on age of prostate cancer patients and Gleason score EMT genes with distinctly altered expression are: KRT18, KRT19, MUC1 and COL4A1, CTNNB1, SNAI2, ZEB1 and MMP3. Our major observation is that prostate cancer from patients under 50 years old compared to older ones has entirely different EMT gene expression profiles showing potentially more aggressive invasive phenotype, despite Gleason score classification.

Steroid synthesis by primary human keratinocytes; implications for skin disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hannen, Rosalind F., E-mail: r.f.hannen@qmul.ac.uk; Michael, Anthony E.; Jaulim, Adil

2011-01-07

Research highlights: {yields} Primary keratinocytes express the steroid enzymes required for cortisol synthesis. {yields} Normal primary human keratinocytes can synthesise cortisol. {yields} Steroidogenic regulators, StAR and MLN64, are expressed in normal epidermis. {yields} StAR expression is down regulated in eczema and psoriatic epidermis. -- Abstract: Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary humanmore » keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3{beta}HSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7-{sup 3}H]-pregnenolone through each steroid intermediate to [7-{sup 3}H]-cortisol in cultured PHK. Trilostane (a 3{beta}HSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data show that PHK are capable of extra-adrenal cortisol synthesis, which could be a fundamental pathway in skin biology with implications in psoriasis and atopic dermatitis.« less

Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target

PubMed Central

Asim, Mohammad; Massie, Charles E.; Orafidiya, Folake; Pértega-Gomes, Nelma; Warren, Anne Y.; Esmaeili, Mohsen; Selth, Luke A.; Zecchini, Heather I.; Luko, Katarina; Qureshi, Arham; Baridi, Ajoeb; Menon, Suraj; Madhu, Basetti; Escriu, Carlos; Lyons, Scott; Vowler, Sarah L.; Zecchini, Vincent R.; Shaw, Greg; Hessenkemper, Wiebke; Russell, Roslin; Mohammed, Hisham; Stefanos, Niki; Lynch, Andy G.; Grigorenko, Elena; D’Santos, Clive; Taylor, Chris; Lamb, Alastair; Sriranjan, Rouchelle; Yang, Jiali; Stark, Rory; Dehm, Scott M.; Rennie, Paul S.; Carroll, Jason S.; Griffiths, John R.; Tavaré, Simon; Mills, Ian G.; McEwan, Iain J.; Baniahmad, Aria; Tilley, Wayne D.; Neal, David E.

2016-01-01

Background: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling. Methods: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ2 tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided. Results: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts. Conclusions: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa. PMID:26657335

Adaptive Activation of a Stress Response Pathway Improves Learning and Memory Through Gs and β-Arrestin-1-Regulated Lactate Metabolism.

PubMed

Dong, Jun-Hong; Wang, Yi-Jing; Cui, Min; Wang, Xiao-Jing; Zheng, Wen-Shuai; Ma, Ming-Liang; Yang, Fan; He, Dong-Fang; Hu, Qiao-Xia; Zhang, Dao-Lai; Ning, Shang-Lei; Liu, Chun-Hua; Wang, Chuan; Wang, Yue; Li, Xiang-Yao; Yi, Fan; Lin, Amy; Kahsai, Alem W; Cahill, Thomas Joseph; Chen, Zhe-Yu; Yu, Xiao; Sun, Jin-Peng

2017-04-15

Stress is a conserved physiological response in mammals. Whereas moderate stress strengthens memory to improve reactions to previously experienced difficult situations, too much stress is harmful. We used specific β-adrenergic agonists, as well as β 2 -adrenergic receptor (β2AR) and arrestin knockout models, to study the effects of adaptive β2AR activation on cognitive function using Morris water maze and object recognition experiments. We used molecular and cell biological approaches to elucidate the signaling subnetworks. We observed that the duration of the adaptive β2AR activation determines its consequences on learning and memory. Short-term formoterol treatment, for 3 to 5 days, improved cognitive function; however, prolonged β2AR activation, for more than 6 days, produced harmful effects. We identified the activation of several signaling networks downstream of β2AR, as well as an essential role for arrestin and lactate metabolism in promoting cognitive ability. Whereas Gs-protein kinase A-cyclic adenosine monophosphate response element binding protein signaling modulated monocarboxylate transporter 1 expression, β-arrestin-1 controlled expression levels of monocarboxylate transporter 4 and lactate dehydrogenase A through the formation of a β-arrestin-1/phospho-mitogen-activated protein kinase/hypoxia-inducible factor-1α ternary complex to upregulate lactate metabolism in astrocyte-derived U251 cells. Conversely, long-term treatment with formoterol led to the desensitization of β2ARs, which was responsible for its decreased beneficial effects. Our results not only revealed that β-arrestin-1 regulated lactate metabolism to contribute to β2AR functions in improved memory formation, but also indicated that the appropriate management of one specific stress pathway, such as through the clinical drug formoterol, may exert beneficial effects on cognitive abilities. Copyright © 2016 Society of Biological Psychiatry. All rights reserved.

Inhibition of androgen receptor and β-catenin activity in prostate cancer

PubMed Central

Lee, Eugine; Madar, Aviv; David, Gregory; Garabedian, Michael J.; DasGupta, Ramanuj; Logan, Susan K.

2013-01-01

Androgen receptor (AR) is the major therapeutic target in aggressive prostate cancer. However, targeting AR alone can result in drug resistance and disease recurrence. Therefore, simultaneous targeting of multiple pathways could in principle be an effective approach to treating prostate cancer. Here we provide proof-of-concept that a small-molecule inhibitor of nuclear β-catenin activity (called C3) can inhibit both the AR and β-catenin–signaling pathways that are often misregulated in prostate cancer. Treatment with C3 ablated prostate cancer cell growth by disruption of both β-catenin/T-cell factor and β-catenin/AR protein interaction, reflecting the fact that T-cell factor and AR have overlapping binding sites on β-catenin. Given that AR interacts with, and is transcriptionally regulated by β-catenin, C3 treatment also resulted in decreased occupancy of β-catenin on the AR promoter and diminished AR and AR/β-catenin target gene expression. Interestingly, C3 treatment resulted in decreased AR binding to target genes accompanied by decreased recruitment of an AR and β-catenin cofactor, coactivator-associated arginine methyltransferase 1 (CARM1), providing insight into the unrecognized function of β-catenin in prostate cancer. Importantly, C3 inhibited tumor growth in an in vivo xenograft model and blocked renewal of bicalutamide-resistant sphere-forming cells, indicating the therapeutic potential of this approach. PMID:24019458

The role of α1-adrenergic receptors in regulating metabolism: increased glucose tolerance, leptin secretion and lipid oxidation.

PubMed

Shi, Ting; Papay, Robert S; Perez, Dianne M

2017-04-01

The role of α 1 -adrenergic receptors (α 1 -ARs) and their subtypes in metabolism is not well known. Most previous studies were performed before the advent of transgenic mouse models and utilized transformed cell lines and poorly selective antagonists. We have now studied the metabolic regulation of the α 1A - and α 1B -AR subtypes in vivo using knock-out (KO) and transgenic mice that express a constitutively active mutant (CAM) form of the receptor, assessing subtype-selective functions. CAM mice increased glucose tolerance while KO mice display impaired glucose tolerance. CAM mice increased while KO decreased glucose uptake into white fat tissue and skeletal muscle with the CAM α 1A -AR showing selective glucose uptake into the heart. Using indirect calorimetry, both CAM mice demonstrated increased whole body fatty acid oxidation, while KO mice preferentially oxidized carbohydrate. CAM α 1A -AR mice displayed significantly decreased fasting plasma triglycerides and glucose levels while α 1A -AR KO displayed increased levels of triglycerides and glucose. Both CAM mice displayed increased plasma levels of leptin while KO mice decreased leptin levels. Most metabolic effects were more efficacious with the α 1A -AR subtype. Our results suggest that stimulation of α 1 -ARs results in a favorable metabolic profile of increased glucose tolerance, cardiac glucose uptake, leptin secretion and increased whole body lipid metabolism that may contribute to its previously recognized cardioprotective and neuroprotective benefits.

A spontaneously immortalized Schwann cell line from aldose reductase-deficient mice as a useful tool for studying polyol pathway and aldehyde metabolism.

PubMed

Niimi, Naoko; Yako, Hideji; Takaku, Shizuka; Kato, Hiroshi; Matsumoto, Takafumi; Nishito, Yasumasa; Watabe, Kazuhiko; Ogasawara, Saori; Mizukami, Hiroki; Yagihashi, Soroku; Chung, Sookja K; Sango, Kazunori

2018-03-01

The increased glucose flux into the polyol pathway via aldose reductase (AR) is recognized as a major contributing factor for the pathogenesis of diabetic neuropathy, whereas little is known about the functional significance of AR in the peripheral nervous system. Spontaneously immortalized Schwann cell lines established from long-term cultures of AR-deficient and normal C57BL/6 mouse dorsal root ganglia and peripheral nerves can be useful tools for studying the physiological and pathological roles of AR. These cell lines, designated as immortalized knockout AR Schwann cells 1 (IKARS1) and 1970C3, respectively, demonstrated distinctive Schwann cell phenotypes, such as spindle-shaped morphology and immunoreactivity to S100, p75 neurotrophin receptor, and vimentin, and extracellular release of neurotrophic factors. Conditioned media obtained from these cells promoted neuronal survival and neurite outgrowth of cultured adult mouse dorsal root ganglia neurons. Microarray and real-time RT-PCR analyses revealed significantly down-regulated mRNA expression of polyol pathway-related enzymes, sorbitol dehydrogenase and ketohexokinase, in IKARS1 cells compared with those in 1970C3 cells. In contrast, significantly up-regulated mRNA expression of aldo-keto reductases (AKR1B7 and AKR1B8) and aldehyde dehydrogenases (ALDH1L2, ALDH5A1, and ALDH7A1) was detected in IKARS1 cells compared with 1970C3 cells. Exposure to reactive aldehydes (3-deoxyglucosone, methylglyoxal, and 4-hydroxynonenal) significantly up-regulated the mRNA expression of AKR1B7 and AKR1B8 in IKARS1 cells, but not in 1970C3 cells. Because no significant differences in viability between these two cell lines after exposure to these aldehydes were observed, it can be assumed that the aldehyde detoxification is taken over by AKR1B7 and AKR1B8 in the absence of AR. © 2017 International Society for Neurochemistry.

Androgen Receptor Expression in Epithelial and Stromal Cells of Prostatic Carcinoma and Benign Prostatic Hyperplasia.

PubMed

Filipovski, Vanja; Kubelka-Sabit, Katerina; Jasar, Dzengis; Janevska, Vesna

2017-08-15

Prostatic carcinoma (PCa) derives from prostatic epithelial cells. However stromal microenvironment, associated with malignant epithelium, also plays a role in prostatic carcinogenesis. Alterations in prostatic stromal cells contribute to the loss of growth control in epithelial cells that lead to progression of PCa. To analyse the differences between Androgen Receptor (AR) expression in both epithelial and stromal cells in PCa and the surrounding benign prostatic hyperplasia (BPH) and to compare the results with tumour grade. Samples from 70 cases of radical prostatectomy specimens were used. The expression and intensity of the signal for AR was analysed in the epithelial and stromal cells of PCa and BPH, and the data was quantified using histological score (H-score). AR showed significantly lower expression in both epithelial and stromal cells of PCa compared to BPH. In PCa a significant positive correlation of AR expression was found between stromal and epithelial cells of PCa. AR expression showed a correlation between the stromal cells of PCa and tumour grade. AR expression is reduced in epithelial and stromal cells of PCa. Expression of AR in stromal cells of PCa significantly correlates with tumour grade.

Serotonin hyperinnervation and upregulated 5-HT2A receptor expression and motor-stimulating function in nigrostriatal dopamine-deficient Pitx3 mutant mice.

PubMed

Li, Li; Qiu, Guozhen; Ding, Shengyuan; Zhou, Fu-Ming

2013-01-23

The striatum receives serotonin (5-hydroxytryptamine, 5-HT) innervation and expresses 5-HT2A receptors (5-HT2ARs) and other 5-HT receptors, raising the possibility that the striatal 5-HT system may undergo adaptive changes after chronic severe dopamine (DA) loss and contribute to the function and dysfunction of the striatum. Here we show that in transcription factor Pitx3 gene mutant mice with a selective, severe DA loss in the dorsal striatum mimicking the DA denervation in late Parkinson's disease (PD), both the 5-HT innervation and the 5-HT2AR mRNA expression were increased in the dorsal striatum. Functionally, while having no detectable motor effect in wild type mice, the 5-HT2R agonist 2,5-dimethoxy-4-iodoamphetamine increased both the baseline and l-dopa-induced normal ambulatory and dyskinetic movements in Pitx3 mutant mice, whereas the selective 5-HT2AR blocker volinanserin had the opposite effects. These results demonstrate that Pitx3 mutant mice are a convenient and valid mouse model to study the compensatory 5-HT upregulation following the loss of the nigrostriatal DA projection and that the upregulated 5-HT2AR function in the DA deficient dorsal striatum may enhance both normal and dyskinetic movements. Copyright © 2012 Elsevier B.V. All rights reserved.

Expression of Steroid Receptors in Ameloblasts during Amelogenesis in Rat Incisors.

PubMed

Houari, Sophia; Loiodice, Sophia; Jedeon, Katia; Berdal, Ariane; Babajko, Sylvie

2016-01-01

Endocrine disrupting chemicals (EDCs) play a part in the modern burst of diseases and interfere with the steroid hormone axis. Bisphenol A (BPA), one of the most active and widely used EDCs, affects ameloblast functions, leading to an enamel hypomineralization pattern similar to that of Molar Incisor Hypomineralization (MIH). In order to explore the molecular pathways stimulated by BPA during amelogenesis, we thoroughly investigated the receptors known to directly or indirectly mediate the effects of BPA. The expression patterns of high affinity BPA receptors (ERRγ, GPR30), of ketosteroid receptors (ERs, AR, PGR, GR, MR), of the retinoid receptor RXRα, and PPARγ were established using RT-qPCR analysis of RNAs extracted from microdissected enamel organ of adult rats. Their expression was dependent on the stage of ameloblast differentiation, except that of ERβ and PPARγ which remained undetectable. An additional large scale microarray analysis revealed three main groups of receptors according to their level of expression in maturation-stage ameloblasts. The expression level of RXRα was the highest, similar to the vitamin D receptor (VDR), whereas the others were 13 to 612-fold lower, with AR and GR being intermediate. Immunofluorescent analysis of VDR, ERα and AR confirmed their presence mainly in maturation- stage ameloblasts. These data provide further evidence that ameloblasts express a specific combination of hormonal receptors depending on their developmental stage. This study represents the first step toward understanding dental endocrinology as well as some of the effects of EDCs on the pathophysiology of amelogenesis.

Expression of Steroid Receptors in Ameloblasts during Amelogenesis in Rat Incisors

PubMed Central

Houari, Sophia; Loiodice, Sophia; Jedeon, Katia; Berdal, Ariane; Babajko, Sylvie

2016-01-01

Endocrine disrupting chemicals (EDCs) play a part in the modern burst of diseases and interfere with the steroid hormone axis. Bisphenol A (BPA), one of the most active and widely used EDCs, affects ameloblast functions, leading to an enamel hypomineralization pattern similar to that of Molar Incisor Hypomineralization (MIH). In order to explore the molecular pathways stimulated by BPA during amelogenesis, we thoroughly investigated the receptors known to directly or indirectly mediate the effects of BPA. The expression patterns of high affinity BPA receptors (ERRγ, GPR30), of ketosteroid receptors (ERs, AR, PGR, GR, MR), of the retinoid receptor RXRα, and PPARγ were established using RT-qPCR analysis of RNAs extracted from microdissected enamel organ of adult rats. Their expression was dependent on the stage of ameloblast differentiation, except that of ERβ and PPARγ which remained undetectable. An additional large scale microarray analysis revealed three main groups of receptors according to their level of expression in maturation-stage ameloblasts. The expression level of RXRα was the highest, similar to the vitamin D receptor (VDR), whereas the others were 13 to 612-fold lower, with AR and GR being intermediate. Immunofluorescent analysis of VDR, ERα and AR confirmed their presence mainly in maturation- stage ameloblasts. These data provide further evidence that ameloblasts express a specific combination of hormonal receptors depending on their developmental stage. This study represents the first step toward understanding dental endocrinology as well as some of the effects of EDCs on the pathophysiology of amelogenesis. PMID:27853434

Linked Tumor-Selective Virus Replication and Transgene Expression from E3-Containing Oncolytic Adenoviruses†

PubMed Central

Zhu, Mingzhu; Bristol, J. Andrew; Xie, Yuefeng; Mina, Mervat; Ji, Hong; Forry-Schaudies, Suzanne; Ennist, David L.

2005-01-01

Historically, the adenoviral E3 region was found to be nonessential for viral replication in vitro. In addition, adenoviruses whose genome was more than approximately 105% the size of the native genome were inefficiently packaged. These profound observations were used experimentally to insert transgenes into the adenoviral backbone. More recently, however, the reintroduction of the E3 region into oncolytic adenoviruses has been found to positively influence antitumor efficacy in preclinical models and clinical trials. In the studies reported here, the granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA sequence has been substituted for the E3-gp19 gene in oncolytic adenoviruses that otherwise retained the E3 region. Five viruses that differed slightly in the method of transgene insertion were generated and compared to Ar6pAE2fGmF (E2F/GM/ΔE3), a previously described E3-deleted oncolytic adenovirus encoding GM-CSF. In all of the viruses, the human E2F-1 promoter regulated E1A expression and GM-CSF expression was under the control of the adenoviral E3 promoter and the packaging signal was relocated immediately upstream from the right terminal repeat. The E3-gp19-deleted viruses had similar cytolytic properties, as measured in vitro by cytotoxicity assays, but differed markedly in their capacity to express and secrete GM-CSF. Ar15pAE2fGmF (E2F/GM/E3b), the virus that produced the highest levels of GM-CSF and retained the native GM-CSF leader sequence, was selected for further analysis. The E2F/GM/E3b and E2F/GM/ΔE3 viruses exhibited similar cytotoxic activity and GM-CSF production in several tumor cell lines in vitro. However, when compared in vivo in nude mouse xenograft tumor models, E2F/GM/E3b spread through tumors to a greater extent, resulted in higher peak GM-CSF and total exposure levels in both tumor and serum, and was more efficacious than the E3-deleted virus. Using the matched WI-38 (parental) and WI-38-VA13 (simian virus 40 large T antigen transformed) cell pair, GM-CSF was shown to be selectively produced in cells expressing high levels of E2F, indicating that the tumor-selective E2F promoter controlled E1A and GM-CSF expression. PMID:15827160

Intraspecific variation in estrogen receptor alpha and the expression of male sociosexual behavior in two populations of prairie voles.

PubMed

Cushing, Bruce S; Razzoli, Maria; Murphy, Anne Z; Epperson, Pamela M; Le, Wei-Wei; Hoffman, Gloria E

2004-08-06

Estrogen (E) regulates a variety of male sociosexual behaviors. We hypothesize that there is a relationship between the distribution of estrogen receptor alpha (ERalpha) and the degree of male social behavior. To test this hypothesis, ERalpha immunoreactivity (IR) was compared in prairie voles (Microtus ochrogaster) from Illinois (IL), which are highly social, and Kansas (KN), which are less social. The expression of androgen receptors (AR) in males also was compared between populations. The expression of ERalpha and AR were compared in brains from KN and IL males and females using immunocytochemistry (ICC). There were significant intrapopulational differences, with males expressing less ERalpha-IR than females in the medial preoptic area, ventromedial nucleus, ventrolateral portion of the hypothalamus, and bed nucleus of the stria terminalis (BST). IL males also displayed less ERalpha-IR in the medial amygdala (MeA) than IL females. While IL males expressed significantly less ERalpha-IR in the BST and MeA than KN males, there was no difference in AR-IR. Differences in the pattern of ERalpha-IR between KN and IL males were behaviorally relevant, as low levels of testosterone (T) were more effective in restoring sexual activity in castrated KN males than IL males. The lack of difference in AR combined with lower expression of ERalpha-IR in IL males suggests that behavioral differences in response to T are associated with aromatization of T to E and that reduced sensitivity to E may facilitate prosocial behavior in males.

Temporal Alterations in Vascular Angiotensin Receptors and Vasomotor Response in Offspring of Protein-restricted Rat Dams

PubMed Central

SATHISHKUMAR, Kunju; BALAKRISHNAN, Meena; CHINNATHAMBI, Vijayakumar; GAO, Haijun; YALLAMPALLI, Chandra

2012-01-01

Objective Examine temporal alterations in vascular angiotensin II (ANG II) receptors (AT1R and AT2R) and determine vascular response to ANG II in growth-restricted offspring. Study design Offspring of pregnant rats fed low-protein (6%) and control (20%) diet were compared. Results Prenatal protein restriction reprogrammed AT1aR mRNA expression in males’ mesenteric arteries to cause 1.7- and 2.3-fold increases at 3 and 6 months of age associated with arterial pressure increases of 10 and 33 mmHg, respectively; however, in females, increased AT1aR expression (2-fold) and arterial pressure (15 mmHg) occurred only at 6 months. Prenatal protein restriction did not affect AT2R expression. Losartan abolished hypertension, suggesting that AT1aR plays a primary role in arterial pressure elevation. Vasoconstriction to ANG II was exaggerated in all protein-restricted offspring, with greater potency and efficacy in males. Conclusion Prenatal protein restriction increased vascular AT1R expression and vasoconstriction to ANG II, possibly contributing to programmed hypertension. PMID:22537420

On the possibility of developing quasi-CW high-power high-pressure laser on 4p-4s transition of ArI with electron beam—optical pumping: quenching of 4s (3P2) lower laser level

NASA Astrophysics Data System (ADS)

Ionin, A. A.; Kholin, I. V.; L'dov, A. Yu; Seleznev, L. V.; Ustinovskii, N. N.; Zayarnyi, D. A.

2017-12-01

A new electron beam-optical procedure is proposed for quasi-cw pumping of high-pressure large-volume He-Ar laser on the 4p[1/2]1-4s[3/2]20 argon atom transition at the wavelength of 912.5 nm. It consists of creation and maintenance of a necessary density of the 4s[3/2]20 metastable state in the gain medium by a fast electron beam and subsequent optical pumping of the upper laser level via the classical three-level scheme using a laser diode. Absorption probing is used to study collisional quenching of Ar* metastable in electron-beam-excited high-pressure He-Ar mixtures with a low content of argon. The rate constants for plasma-chemical reactions Ar*  +  He  +  Ar  >  Ar2*   +  He (3.6  ±  0.4)  ×  10-33 cm6 s-1, Ar*  +  2He  >  HeAr*  +  He (4.4  ±  0.9)  ×  10-36 cm6 s-1 and Ar*  +  He  >  Products  +  He (2.4  ±  0.3)  ×  10-15 cm3 s-1 were for the first time measured.

Androgen receptor is overexpressed in boys with severe hypospadias, and ZEB1 regulates androgen receptor expression in human foreskin cells

PubMed Central

Qiao, Liang; Tasian, Gregory E.; Zhang, Haiyang; Cao, Mei; Ferretti, Max; Cunha, Gerald R.; Baskin, Laurence S.

2012-01-01

INTRODUCTION ZEB1 is overexpressed in patients with severe hypospadias. We examined the interaction between ZeB1 and the androgen receptor (AR) in vitro and the expression of AR in boys with hypospadias. RESULTS ZEB1 and AR colocalize to the nucleus. Estrogen upregulated ZEB1 and AR expression. Chromatin immunoprecipitation (ChIP) demonstrated that ZEB1 binds to an E-box sequence in the AR gene promoter. AR expression is higher in subjects with severe hypospadias than those with mild hypospadias and control subjects (P < 0.05). ZEB1 physically interacts with AR in human foreskin cells. DISCUSSION AR is overexpressed in patients with severe hypospadias. Environmental estrogenic compounds may increase the risk of hypospadias by facilitating the interaction between ZEB1 and AR. METHODS Hs68 cells, a fibroblast cell line derived from neonatal human foreskin, were exposed to 0, 10, and 100 nmol/l of estrogen, after which the cellular localization of ZEB1 and AR was assessed using immunocytochemistry. To determine if ZEB1 interacted with the AR gene, ChIP was performed using ZEB1 antibody and polymerase chain reaction (PCR) for AR. Second, AR expression was quantified using real-time PcR and western blot in normal subjects (n = 32), and subjects with mild (n = 16) and severe hypospadia (n = 16). PMID:22391641

Regulation of Thyroid Hormone-, Oestrogen- and Androgen-Related Genes by Triiodothyronine in the Brain of Silurana tropicalis

PubMed Central

Duarte-Guterman, Paula; Trudeau, Vance L

2010-01-01

Amphibian metamorphosis is an excellent example of hormone-dependent control of development. Thyroid hormones (THs) regulate almost all aspects of metamorphosis, including brain development and larval neuroendocrine function. Sex steroids are also important for early brain function, although little is known about interactions between the two hormonal systems. In the present study, we established brain developmental profiles for thyroid hormone receptors (tralpha and trbeta), deiodinases (dio1, dio2 and dio3), aromatase (cyp19) mRNA and activity, oestrogen receptors (eralpha and erbeta), androgen receptor (ar) and 5α-reductases (srd5alpha1 and srd5alpha2) mRNA during Silurana (Xenopus) tropicalis metamorphosis. Real-time reverse transcriptase-polymerase chain reaction analyses revealed that all of the genes were expressed in the brain and for most of the genes expression increased during development, with the exception of dio2, srd5alpha1 and srd5alpha2. The ability of premetamorphic tadpoles to respond to exogenous THs was used to investigate the regulation of TH- and sex steroid-related genes in the brain during development. Exposure of premetamorphic tadpoles to triiodothyronine (T3; 0, 0.5, 5 and 50 nm) for 48 h resulted in concentration-dependent increases in trbeta, dio2, dio3, eralpha and erbeta. Expression of srd5alpha2 showed large increases (six- to 7.5-fold) for all three concentrations of T3. No changes were detected in dio1, ar and cyp19 transcript levels; however, cyp19 activity increased significantly at 50 nm T3. The results obtained suggest that expression of TH-related genes and er during development could be regulated by rising levels of THs, as previously documented in Lithobates (Rana) pipiens. The positive regulation of srd5alpha by T3 in the brain suggests that endogenous TH levels help maintain or control the rate at which srd5alpha mRNA levels decrease as metamorphosis progresses. Finally, we have identified sex steroid-related genes that are responsive to T3, providing additional evidence of crosstalk between THs and sex steroids in the tadpole brain. PMID:20626568

Fulvestrant (ICI 182,780) down-regulates androgen receptor expression and diminishes androgenic responses in LNCaP human prostate cancer cells.

PubMed

Bhattacharyya, Rumi S; Krishnan, Aruna V; Swami, Srilatha; Feldman, David

2006-06-01

The androgen receptor (AR) plays a key role in the development and progression of prostate cancer. Targeting the AR for down-regulation would be a useful strategy for treating prostate cancer, especially hormone-refractory or androgen-independent prostate cancer. In the present study, we showed that the antiestrogen fulvestrant [ICI 182,780 (ICI)] effectively suppressed AR expression in several human prostate cancer cells, including androgen-independent cells. In LNCaP cells, ICI (10 micromol/L) treatment decreased AR mRNA expression by 43% after 24 hours and AR protein expression by approximately 50% after 48 hours. We further examined the mechanism of AR down-regulation by ICI in LNCaP cells. ICI did not bind to the T877A-mutant AR present in the LNCaP cells nor did it promote proteasomal degradation of the AR. ICI did not affect AR mRNA or protein half-life. However, ICI decreased the activity of an AR promoter-luciferase reporter plasmid transfected into LNCaP cells, suggesting a direct repression of AR gene transcription. As a result of AR down-regulation by ICI, androgen induction of prostate-specific antigen mRNA and protein expression were substantially attenuated. Importantly, LNCaP cell proliferation was significantly inhibited by ICI treatment. Following 6 days of ICI treatment, a 70% growth inhibition was seen in androgen-stimulated LNCaP cells. These data show that the antiestrogen ICI is a potent AR down-regulator that causes significant inhibition of prostate cancer cell growth. Our study suggests that AR down-regulation by ICI would be an effective strategy for the treatment of all prostate cancer, especially AR-dependent androgen-independent prostate cancer.

Amalaki rasayana, a traditional Indian drug enhances cardiac mitochondrial and contractile functions and improves cardiac function in rats with hypertrophy.

PubMed

Kumar, Vikas; Aneesh, Kumar A; Kshemada, K; Ajith, Kumar G S; Binil, Raj S S; Deora, Neha; Sanjay, G; Jaleel, A; Muraleedharan, T S; Anandan, E M; Mony, R S; Valiathan, M S; Santhosh, Kumar T R; Kartha, C C

2017-08-17

We evaluated the cardioprotective effect of Amalaki Rasayana (AR), a rejuvenating Ayurvedic drug prepared from Phyllanthus emblica fruits in the reversal of remodeling changes in pressure overload left ventricular cardiac hypertrophy (LVH) and age-associated cardiac dysfunction in male Wistar rats. Six groups (aging groups) of 3 months old animals were given either AR or ghee and honey (GH) orally; seventh group was untreated. Ascending aorta was constricted using titanium clips in 3 months old rats (N = 24; AC groups) and after 6 months, AR or GH was given for further 12 months to two groups; one group was untreated. Histology, gene and protein expression analysis were done in heart tissues. Chemical composition of AR was analyzed by HPLC, HPTLC and LC-MS. AR intake improved (P < 0.05) cardiac function in aging rats and decreased LVH (P < 0.05) in AC rats as well as increased (P < 0.05) fatigue time in treadmill exercise in both groups. In heart tissues of AR administered rats of both the groups, SERCA2, CaM, Myh11, antioxidant, autophagy, oxidative phosphorylation and TCA cycle proteins were up regulated. ADRB1/2 and pCREB expression were increased; pAMPK, NF-kB were decreased. AR has thus a beneficial effect on myocardial energetics, muscle contractile function and exercise tolerance capacity.

Syringic Acid Extracted from Herba dendrobii Prevents Diabetic Cataract Pathogenesis by Inhibiting Aldose Reductase Activity

PubMed Central

Wei, Xiaoyong; Chen, Dan; Yi, Yanchun; Qi, Hui; Gao, Xinxin; Fang, Hua; Gu, Qiong; Wang, Ling; Gu, Lianquan

2012-01-01

Objective. Effects of Syringic acid (SA) extracted from dendrobii on diabetic cataract (DC) pathogenesis were explored. Methods. Both in vitro and in vivo DC lens models were established using D-gal, and proliferation of HLEC exposed to SA was determined by MMT assay. After 60-day treatment with SA, rat lens transparency was observed by anatomical microscopy using a slit lamp. SA protein targets were extracted and isolated using 2-DE and MALDI TOF/TOF. AR gene expression was investigated using qRT-PCR. Interaction sites and binding characteristics were determined by molecule-docking techniques and dynamic models. Results. Targeting AR, SA provided protection from D-gal-induced damage by consistently maintaining lens transparency and delaying lens turbidity development. Inhibition of AR gene expression by SA was confirmed by qRT-PCR. IC50 of SA for inhibition of AR activity was 213.17 μg/mL. AR-SA binding sites were Trp111, His110, Tyr48, Trp20, Trp79, Leu300, and Phe122. The main binding modes involved hydrophobic interactions and hydrogen bonding. The stoichiometric ratio of non-covalent bonding between SA and AR was 1.0 to 13.3. Conclusion. SA acts to prevent DC in rat lenses by inhibiting AR activity and gene expression, which has potential to be developed into a novel drug for therapeutic management of DC. PMID:23365598

Expression of β1- and β2-adrenoceptors in different subtypes of interneurons in the medial prefrontal cortex of mice.

PubMed

Liu, Y; Liang, X; Ren, W-W; Li, B-M

2014-01-17

Noradrenaline acting via β-adrenoceptors (β-ARs) in the CNS plays an important role in learning/memory and cognitive functions. β-ARs have been shown to be expressed in cortical pyramidal and subcortical principal cells. However, little is known about β-AR expression in different subtypes of GABAergic neurons. Here, we report that both β1- and β2-ARs are expressed in a majority of GABAergic interneurons in the medial prefrontal cortex of mice, including parvalbumin (PV)-, calretinin (CR)-, calbindin D-28k (CB)-, somatostatin (SST)- and Reelin-immunoreactive (ir) interneurons. Relative to PV-, CB-, SST- and Reelin-ir interneurons, CR-ir interneurons are less likely to express β1- and β2-ARs. SST-ir interneurons are more likely to express β2-AR compared with the other subtypes of interneurons. The present results are of significance for understanding the role of β-ARs in prefrontal cortical functions. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Correlation of A2bAR and KLF4/KLF15 with Obesity-Dyslipidemia Induced Inflammation in Uygur Population

PubMed Central

Wang, Cuizhe; Ha, Xiaodan; Li, Wei; Xu, Peng; Gu, Yajuan; Wang, Tingting; Wang, Yan; Xie, Jianxin; Zhang, Jun

2016-01-01

In this paper, the researchers collected visceral adipose tissue from the Uygur population, which were divided into two groups: the normal control group (NC, n = 50, 18.0 kg/m2 ≤ BMI ≤ 23.9 kg/m2) and the obese group (OB, n = 45, BMI ≥ 28 kg/m2), and then use real-time PCR to detect the mRNA expression level of key genes involved in inflammation signaling pathway. The findings suggest that, in obese status, the lower expression level of A2bAR, KLF4, and KLF15 of visceral adipose tissue may correlate with obese-dyslipidemia induced inflammation in Uygur population. PMID:27199507

32 CFR 571.3 - Waiver enlistment criteria.

Code of Federal Regulations, 2010 CFR

2010-07-01

... document their waiver requests, as prescribed by AR 601-210 or AR 40-501. (3) The approval authorities for various types of waiver requests are set forth in AR 601-210. Commanders at levels below the approval... substantiate a meritorious case. (4) Unless otherwise stated in AR 601-210, waivers are valid for 6 months. (b...

β-Adrenergic Regulation of Cardiac Progenitor Cell Death Versus Survival and Proliferation

PubMed Central

Khan, Mohsin; Mohsin, Sadia; Avitabile, Daniele; Siddiqi, Sailay; Nguyen, Jonathan; Wallach, Kathleen; Quijada, Pearl; McGregor, Michael; Gude, Natalie; Alvarez, Roberto; Tilley, Douglas G.; Koch, Walter J.; Sussman, Mark A.

2013-01-01

Rationale Short-term β-adrenergic stimulation promotes contractility in response to stress but is ultimately detrimental in the failing heart because of accrual of cardiomyocyte death. Endogenous cardiac progenitor cell (CPC) activation may partially offset cardiomyocyte losses, but consequences of long-term β-adrenergic drive on CPC survival and proliferation are unknown. Objective We sought to determine the relationship between β-adrenergic activity and regulation of CPC function. Methods and Results Mouse and human CPCs express only β2 adrenergic receptor (β2-AR) in conjunction with stem cell marker c-kit. Activation of β2-AR signaling promotes proliferation associated with increased AKT, extracellular signal-regulated kinase 1/2, and endothelial NO synthase phosphorylation, upregulation of cyclin D1, and decreased levels of G protein–coupled receptor kinase 2. Conversely, silencing of β2-AR expression or treatment with β2-antagonist ICI 118, 551 impairs CPC proliferation and survival. β1-AR expression in CPC is induced by differentiation stimuli, sensitizing CPC to isoproterenol-induced cell death that is abrogated by metoprolol. Efficacy of β1-AR blockade by metoprolol to increase CPC survival and proliferation was confirmed in vivo by adoptive transfer of CPC into failing mouse myocardium. Conclusions β-adrenergic stimulation promotes expansion and survival of CPCs through β2-AR, but acquisition of β1-AR on commitment to the myocyte lineage results in loss of CPCs and early myocyte precursors. PMID:23243208

SLCO2B1 and SLCO1B3 as New Targets for Enhancing Androgen Deprivation Therapy for Prostate Cancer

DTIC Science & Technology

2016-10-01

Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as...a significantly lower level of SLCO2B1 expression, compared to those carrying the minor (risk) allele. SLCO2B1 expression levels are directly...Special Reporting Requirements…………………………………… 18 9. Appendices…………………………………………………………… .19 1 1, Introduction The androgen receptor ( AR ) plays an essential

NF-κB and androgen receptor variant expression correlate with human BPH progression.

PubMed

Austin, David C; Strand, Douglas W; Love, Harold L; Franco, Omar E; Jang, Alex; Grabowska, Magdalena M; Miller, Nicole L; Hameed, Omar; Clark, Peter E; Fowke, Jay H; Matusik, Robert J; Jin, Ren J; Hayward, Simon W

2016-04-01

Benign prostatic hyperplasia (BPH) is a common, chronic progressive disease. Inflammation is associated with prostatic enlargement and resistance to 5α-reductase inhibitor (5ARI) therapy. Activation of the nuclear factor-kappa B (NF-κB) pathway is linked to both inflammation and ligand-independent prostate cancer progression. NF-κB activation and androgen receptor variant (AR-V) expression were quantified in transition zone tissue samples from patients with a wide range of AUASS from incidental BPH in patients treated for low grade, localized peripheral zone prostate cancer to advanced disease requiring surgical intervention. To further investigate these pathways, human prostatic stromal and epithelial cell lines were transduced with constitutively active or kinase dead forms of IKK2 to regulate canonical NF-κB activity. The effects on AR full length (AR-FL) and androgen-independent AR-V expression as well as cellular growth and differentiation were assessed. Canonical NF-κB signaling was found to be upregulated in late versus early stage BPH, and to be strongly associated with non-insulin dependent diabetes mellitus. Elevated expression of AR-variant 7 (AR-V7), but not other AR variants, was found in advanced BPH samples. Expression of AR-V7 significantly correlated with the patient AUASS and TRUS volume. Forced activation of canonical NF-κB in human prostatic epithelial and stromal cells resulted in elevated expression of both AR-FL and AR-V7, with concomitant ligand-independent activation of AR reporters. Activation of NF-κB and over expression of AR-V7 in human prostatic epithelial cells maintained cell viability in the face of 5ARI treatment. Activation of NF-κB and AR-V7 in the prostate is associated with increased disease severity. AR-V7 expression is inducible in human prostate cells by forced activation of NF-κB resulting in resistance to 5ARI treatment, suggesting a potential mechanism by which patients may become resistant to 5ARI therapy. © 2015 Wiley Periodicals, Inc.

Inverse Regulation of DHT Synthesis Enzymes 5α-Reductase Types 1 and 2 by the Androgen Receptor in Prostate Cancer.

PubMed

Audet-Walsh, Étienne; Yee, Tracey; Tam, Ingrid S; Giguère, Vincent

2017-04-01

5α-Reductase types 1 and 2, encoded by SRD5A1 and SRD5A2, are the two enzymes that can catalyze the conversion of testosterone to dihydrotestosterone, the most potent androgen receptor (AR) agonist in prostate cells. 5α-Reductase type 2 is the predominant isoform expressed in the normal prostate. However, its expression decreases during prostate cancer (PCa) progression, whereas SRD5A1 increases, and the mechanism underlying this transcriptional regulatory switch is still unknown. Interrogation of SRD5A messenger RNA expression in three publicly available data sets confirmed that SRD5A1 is increased in primary and metastatic PCa compared with nontumoral prostate tissues, whereas SRD5A2 is decreased. Activation of AR, a major oncogenic driver of PCa, induced the expression of SRD5A1 from twofold to fourfold in three androgen-responsive PCa cell lines. In contrast, AR repressed SRD5A2 expression in this context. Chromatin-immunoprecipitation studies established that AR is recruited to both SRD5A1 and SRD5A2 genes following androgen stimulation but initiates transcriptional activation only at SRD5A1 as monitored by recruitment of RNA polymerase II and the presence of the H3K27Ac histone mark. Furthermore, we showed that the antiandrogens bicalutamide and enzalutamide block the AR-mediated regulation of both SRD5A1 and SRD5A2, highlighting an additional mechanism explaining their beneficial effects in patients. In summary, we identified an AR-dependent transcriptional regulation that explains the differential expression of 5α-reductase types 1 and 2 during PCa progression. Our work thus defines a mechanism by which androgens control their own synthesis via differential regulatory control of the expression of SRD5A1 and SRD5A2. Copyright © 2017 Endocrine Society.

Biological Differences Between Prostate Cancer Cells that Metastasize to Bone Versus Soft Tissue Sites

DTIC Science & Technology

2004-12-01

regulations of the United States Government and the University of Michigan. If the descriptive paragraph indicates that a "Comprehensive Written" informed...tumor samples expressing >50% AR and 41.5% (100/265) expressing ᝺% AR. Overall expression of AR was down regulated with median AR expression of...A. et al. Cancer statistics, 2003. CA Cancer J Clin 53, 5-26 (2003). 2. Rubin, M.A. et al. Rapid ("warm") autopsy study for procurement of

Mitochondrial Fusion and ERK Activity Regulate Steroidogenic Acute Regulatory Protein Localization in Mitochondria

PubMed Central

Duarte, Alejandra; Castillo, Ana Fernanda; Podestá, Ernesto J.; Poderoso, Cecilia

2014-01-01

The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR. PMID:24945345

Energies and transition rates in Ge-like ions between In XVIII and Ce XXVII

NASA Astrophysics Data System (ADS)

Chen, Zhan-Bin; Wang, Kai

2017-03-01

The energy levels, wavelengths, oscillator strengths, and radiative electric dipole (E1), magnetic quadrupole (M2) transition probabilities for Ge-like ions (49 ≤ Z ≤ 58) among the lowest 88 fine-structure levels belonging to the ([Ar] 3d10)4s24p2, ([Ar] 3d10)4s24p4d, ([Ar] 3d10)4s4p3, ([Ar] 3d10)4s4p24d, ([Ar] 3d10)4s24d2, and ([Ar] 3d10)4p4 configurations are calculated using the fully relativistic multiconfiguration Dirac-Fock (MCDF) approach including the correlations within the n = 7 complex, Breit interaction (BI) and quantum electrodynamics (QED) effects. For comparison, an independent calculation using the many-body perturbation theory (MBPT) method is also carried out to confirm the present energy levels accuracy, taking Xe XXIII as an example. The present results are compared with available experimental and theoretical results and good agreement is obtained. These accurate theoretical data are useful for controlled thermonuclear fusion research, plasma physics, and astrophysical applications.

Spinal motor and sensory neurons are androgen targets in an acrobatic bird.

PubMed

Fuxjager, Matthew J; Schultz, J Douglas; Barske, Julia; Feng, Ni Y; Fusani, Leonida; Mirzatoni, Anahid; Day, Lainy B; Hau, Michaela; Schlinger, Barney A

2012-08-01

Sex steroids affect the motivation to court mates, but less is known about how they influence motor movements associated with courtship behavior. Steroidal control of motor function may be especially important for species in which courtship requires superior strength, stamina, and neuromuscular coordination. Here we use the golden-collared manakin (Manacus vitellinus) to examine whether the neuromuscular circuitry that controls motoric aspects of courtship activity is sensitive to androgens. Males of this tropical species attract mates by rapidly jumping among branches in a courtship arena and using their wings to produce loud wing snaps. Testosterone activates this display via the androgen receptor (AR), and past work reveals that manakins injected with radio-labeled T ((3)H-T) accumulate radioactivity in the spinal cord. Thus, we used quantitative PCR to measure AR, estrogen receptor-α (ER-α) subtype, and aromatase (AROM) mRNA in spinal cords of male and female manakins and zebra finches. Expression of AR, but not ER-α or aromatase, was higher throughout the manakin spinal cord compared with the zebra finch. Next, we tested whether AR-expressing skeletal muscles are innervated by motor and sensory neurons that also express AR. To do this, we backfilled spinal neurons by injecting fluorescent tracers into select AR-sensitive wing and leg muscles of wild caught male and female manakins. We then removed these spinal cords and measured AR expression with in situ hybridization. Both sexes showed abundant AR mRNA in the cervical and lumbosacral spinal enlargements as well as in dorsal root ganglia attached to these enlargements. Together our findings suggest that androgens act widely on peripheral motor and sensory circuits in golden-collared manakins to influence wing snapping displays.

Identification of Novel Transplantable GPCR Recycling Motif for Drug Discovery

PubMed Central

Nooh, Mohammed M.; Mancarella, Salvatore; Bahouth, Suleiman W.

2016-01-01

β1-adrenergic receptor (β1-AR) agonists and antagonists are widely used in the treatment of major cardiovascular diseases such as heart failure and hypertension. The β1-AR like other G protein-couple receptors (GPCR) is endocytosed in response to intense agonist activation. Recycling of the agonist-internalized β1-AR is dependent on its carboxy-terminal type-1 PSD-95/DLG/ZO1 (PDZ) and on phospho-serine312 in the third intracellular loop of the β1-AR. Progressive elongation of the β1-AR at its C-tail inactivated the PDZ-biding domain and inhibited the recycling of the β1-AR. However, fusing a twenty amino acid peptide derived from the multiple cloning region of the mammalian expression vector pCDNA3 to the C-tail of the β1-AR (β1-AR[+20]) produced a chimeric β1-AR that recycled rapidly and efficiently. The β1-AR[+20] recycled in a type-1 PDZ and phospho-Ser312-independent manner, indicating that this peptide provided a general GPCR recycling signal. Fusing the enhanced yellow fluorescent protein (EYFP) down-stream of β1-AR[+20] generated a β1-AR-EYFP chimera that was expressed on the membrane and recycled efficiently after agonist-induced internalization. This construct trafficked in a PDZ-SNX27/retromer-independent manner. We also fused EYFP to the N-terminus of the β1-AR to created EYFP-WT β1-AR. This construct recycled in PDZ and SNX27/retromer dependent manner. These β1-AR-EYFP constructs would be useful for high throughput screening (HTS) programs to identify new entities that would interfere with the recycling of agonist internalized GPCR that traffic in PDZ-dependent vs. PDZ-independent roadmaps. PMID:27645110

Genome-wide analysis of AR binding and comparison with transcript expression in primary human fetal prostate fibroblasts and cancer associated fibroblasts.

PubMed

Nash, Claire; Boufaied, Nadia; Mills, Ian G; Franco, Omar E; Hayward, Simon W; Thomson, Axel A

2017-05-05

The androgen receptor (AR) is a transcription factor, and key regulator of prostate development and cancer, which has discrete functions in stromal versus epithelial cells. AR expressed in mesenchyme is necessary and sufficient for prostate development while loss of stromal AR is predictive of prostate cancer progression. Many studies have characterized genome-wide binding of AR in prostate tumour cells but none have used primary mesenchyme or stroma. We applied ChIPseq to identify genomic AR binding sites in primary human fetal prostate fibroblasts and patient derived cancer associated fibroblasts, as well as the WPMY1 cell line overexpressing AR. We identified AR binding sites that were specific to fetal prostate fibroblasts (7534), cancer fibroblasts (629), WPMY1-AR (2561) as well as those common among all (783). Primary fibroblasts had a distinct AR binding profile versus prostate cancer cell lines and tissue, and showed a localisation to gene promoter binding sites 1 kb upstream of the transcriptional start site, as well as non-classical AR binding sequence motifs. We used RNAseq to define transcribed genes associated with AR binding sites and derived cistromes for embryonic and cancer fibroblasts as well as a cistrome common to both. These were compared to several in vivo ChIPseq and transcript expression datasets; which identified subsets of AR targets that were expressed in vivo and regulated by androgens. This analysis enabled us to deconvolute stromal AR targets active in stroma within tumour samples. Taken together, our data suggest that the AR shows significantly different genomic binding site locations in primary prostate fibroblasts compared to that observed in tumour cells. Validation of our AR binding site data with transcript expression in vitro and in vivo suggests that the AR target genes we have identified in primary fibroblasts may contribute to clinically significant and biologically important AR-regulated changes in prostate tissue. Copyright © 2017. Published by Elsevier B.V.

Heterodimerization with beta2-adrenergic receptors promotes surface expression and functional activity of alpha1D-adrenergic receptors.

PubMed

Uberti, Michelle A; Hague, Chris; Oller, Heide; Minneman, Kenneth P; Hall, Randy A

2005-04-01

The alpha1D-adrenergic receptor (alpha1D-AR) is a G protein-coupled receptor (GPCR) that is poorly trafficked to the cell surface and largely nonfunctional when heterologously expressed by itself in a variety of cell types. We screened a library of approximately 30 other group I GPCRs in a quantitative luminometer assay for the ability to promote alpha1D-AR cell surface expression. Strikingly, these screens revealed only two receptors capable of inducing robust increases in the amount of alpha1D-AR at the cell surface: alpha1B-AR and beta2-AR. Confocal imaging confirmed that coexpression with beta2-AR resulted in translocation of alpha1D-AR from intracellular sites to the plasma membrane. Additionally, coimmunoprecipitation studies demonstrated that alpha1D-AR and beta2-AR specifically interact to form heterodimers when coexpressed in HEK-293 cells. Ligand binding studies revealed an increase in total alpha1D-AR binding sites upon coexpression with beta2-AR, but no apparent effect on the pharmacological properties of the receptors. In functional studies, coexpression with beta2-AR significantly enhanced the coupling of alpha1D-AR to norepinephrine-stimulated Ca2+ mobilization. Heterodimerization of beta2-AR with alpha1D-AR also conferred the ability of alpha1D-AR to cointernalize upon beta2-AR agonist stimulation, revealing a novel mechanism by which these different adrenergic receptor subtypes may regulate each other's activity. These findings demonstrate that the selective association of alpha1D-AR with other receptors is crucial for receptor surface expression and function and also shed light on a novel mechanism of cross talk between alpha1- and beta2-ARs that is mediated through heterodimerization and cross-internalization.

Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking.

PubMed

Perkins, Lydia A; Yan, Qi; Schmidt, Brigitte F; Kolodieznyi, Dmytro; Saurabh, Saumya; Larsen, Mads Breum; Watkins, Simon C; Kremer, Laura; Bruchez, Marcel P

2018-02-06

Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged β2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

Identification of glycogen synthase kinase 3α as a therapeutic target in melanoma

PubMed Central

Madhunapantula, SubbaRao V.; Sharma, Arati; Gowda, Raghavendra; Robertson, Gavin P.

2014-01-01

Summary Deregulated expression or activity of kinases can lead to melanomas, but often the particular kinase isoform causing the effect is not well established, making identification and validation of different isoforms regulating disease development especially important. To accomplish this objective, an siRNA screen was undertaken that which identified glycogen synthase kinase 3α (GSK3α) as an important melanoma growth regulator. Melanocytes and melanoma cell lines representing various stages of melanoma tumor progression expressed both GSK3α and GSK3β, but analysis of tumors in patients with melanoma showed elevated expression of GSK3α in 72% of samples, which was not observed for GSK3β. Furthermore, 80% of tumors in patients with melanoma expressed elevated levels of catalytically active phosphorylated GSK3α (pGSK3αY279), but not phosphorylated GSK3β (pGSK3βY216). siRNA-mediated reduction in GSK3α protein levels reduced melanoma cell survival and proliferation, sensitized cells to apoptosis-inducing agents and decreased xenografted tumor development by up to 56%. Mechanistically, inhibiting GSK3α expression using siRNA or the pharmacological agent AR-A014418 arrested melanoma cells in the G0/G1 phase of the cell cycle and induced apoptotic death to retard tumorigenesis. Therefore, GSK3α is a key therapeutic target in melanoma. PMID:24034838

Differential research of inflammatory and related mediators in BPH, histological prostatitis and PCa.

PubMed

Huang, T R; Wang, G C; Zhang, H M; Peng, B

2018-02-14

Prostate cancer (PCa) is one of the most common male malignancies in the world. It was aimed to investigate differential expression of inflammatory and related factors in benign prostatic hyperplasia (BPH), prostate cancer (PCa), histological prostatitis (HP) and explore the role of Inducible nitric oxide synthase (iNOS), (VEGF) Vascular endothelial growth factor, androgen receptor (AR) and IL-2, IL-8 and TNF-α in the occurrence and development of prostate cancer. RT-PCR was used to detect the mRNA expression level of iNOS, VEGF, AR and IL-2, IL-8 and TNF-α in BPH, PCa and BPH+HP. Western blotting and immunohistochemical staining were used to detect the protein levels of various proteins in three diseases. The results showed the mRNA and protein levels of iNOS, VEGF and IL-2, IL-8 and TNF-α were significantly increased in PCa and BPH+HP groups compared with BPH group (p < .05), while the AR was significantly lower than those in PCa and BPH+HP groups (p < .05). There was no significant difference in the mRNA and protein levels of iNOS, VEGF, AR and IL-2, IL-8 and TNF-α between PCa and BPH+HP groups (p > .05). iNOS, VEGF, AR and IL-2, IL-8 and TNF-α are involved in the malignant transformation of prostate tissue and play an important role in the development and progression of Prostate cancer (PCa). © 2018 Blackwell Verlag GmbH.

Role of alpha- and beta-adrenergic receptors in cardiomyocyte differentiation from murine-induced pluripotent stem cells.

PubMed

Li, Xiao-Li; Zeng, Di; Chen, Yan; Ding, Lu; Li, Wen-Ju; Wei, Ting; Ou, Dong-Bo; Yan, Song; Wang, Bin; Zheng, Qiang-Sun

2017-02-01

Induced pluripotent stem cell (iPSC)-derived cardiomyocytes are a promising source of cells for regenerative heart disease therapies, but progress towards their use has been limited by their low differentiation efficiency and high cellular heterogeneity. Previous studies have demonstrated expression of adrenergic receptors (ARs) in stem cells after differentiation; however, roles of ARs in fate specification of stem cells, particularly in cardiomyocyte differentiation and development, have not been characterized. Murine-induced pluripotent stem cells (miPSCs) were cultured in hanging drops to form embryoid bodies, cells of which were then differentiated into cardiomyocytes. To determine whether ARs regulated miPSC differentiation into cardiac lineages, effects of the AR agonist, epinephrine (EPI), on miPSC differentiation and underlying signalling mechanisms, were evaluated. Treatment with EPI, robustly enhanced miPSC cardiac differentiation, as indicated by increased expression levels of cardiac-specific markers, GATA4, Nkx2.5 and Tnnt2. Although β-AR signalling is the foremost signalling pathway in cardiomyocytes, EPI-enhanced cardiac differentiation depended more on α-AR signalling than β-AR signalling. In addition, selective activation of α 1 -AR signalling with specific agonists induced vigorous cardiomyocyte differentiation, whereas selective activation of α 2 - or β-AR signalling induced no or less differentiation, respectively. EPI- and α 1 -AR-dependent cardiomyocyte differentiation from miPSCs occurred through specific promotion of CPC proliferation via the MEK-ERK1/2 pathway and regulation of miPS cell-cycle progression. These results demonstrate that activation of ARs, particularly of α 1 -ARs, promoted miPSC differentiation into cardiac lineages via MEK-ERK1/2 signalling. © 2016 John Wiley & Sons Ltd.

PDE4 and mAKAPβ are nodal organizers of β2-ARs nuclear PKA signaling in cardiac myocytes.

PubMed

Bedioune, Ibrahim; Lefebvre, Florence; Lechêne, Patrick; Varin, Audrey; Domergue, Valérie; Kapiloff, Michael S; Fischmeister, Rodolphe; Vandecasteele, Grégoire

2018-05-03

β1- and β2-adrenergic receptors (β-ARs) produce different acute contractile effects on the heart partly because they impact on different cytosolic pools of cAMP-dependent protein kinase (PKA). They also exert different effects on gene expression but the underlying mechanisms remain unknown. The aim of this study was to understand the mechanisms by which β1- and β2-ARs regulate nuclear PKA activity in cardiomyocytes. We used cytoplasmic and nuclear targeted biosensors to examine cAMP signals and PKA activity in adult rat ventricular myocytes upon selective β1- or β2-ARs stimulation. Both β1- and β2-AR stimulation increased cAMP and activated PKA in the cytoplasm. While the two receptors also increased cAMP in the nucleus, only β1-ARs increased nuclear PKA activity and up-regulated the PKA target gene and pro-apoptotic factor, inducible cAMP element repressor (ICER). Inhibition of PDE4, but not Gi, PDE3, GRK2 nor caveolae disruption disclosed nuclear PKA activation and ICER induction by β2-ARs. Both nuclear and cytoplasmic PKI prevented nuclear PKA activation and ICER induction by β1-ARs, indicating that PKA activation outside the nucleus is required for subsequent nuclear PKA activation and ICER mRNA expression. Cytoplasmic PKI also blocked ICER induction by β2-AR stimulation (with concomitant PDE4 inhibition). However, in this case nuclear PKI decreased ICER up-regulation by only 30%, indicating that other mechanisms are involved. Down-regulation of mAKAPβ partially inhibited nuclear PKA activation upon β1-AR stimulation, and drastically decreased nuclear PKA activation upon β2-AR stimulation in the presence of PDE4 inhibition. β1- and β2-ARs differentially regulate nuclear PKA activity and ICER expression in cardiomyocytes. PDE4 insulates a mAKAPβ-targeted PKA pool at the nuclear envelope that prevents nuclear PKA activation upon β2-AR stimulation.

[Triptolide reverses apatinib resistance in gastric cancer cell line MKN45 via inhibition of heat shock protein 70].

PubMed

Teng, F; Xu, Z Y; Lyu, H; Wang, Y P; Wang, L J; Huang, T; Sun, J C; Zhu, H T; Ni, Y X; Cheng, X D

2018-02-23

Objective: To investigate the effect of triptolide, a specific inhibitor of heat shock protein 70 (HSP70), on apatinib resistance in gastric cancer cells line MKN45. Methods: The apatinib-resistant cells (MKN45/AR) and MKN45 parental cells were treated with apatinib, triptolide and apatinib combined with triptolide, respectively. CCK-8 assay was performed to determine the half maximal inhibitory concentration (IC(50)) of MKN45/AR and MKN45 cells in the presence of different treatment. The mRNA expression of heat shock protein gene (HSPA1A and HSPA1B) was detected by RT-PCR, while the protein expression of heat shock protein 70 was analyzed using Western blot in MKN45/AR and MKN45 cells. Results: The IC(50) values of apatinib-sensitive and apatinib-resistant MKN45 cells were 10.411 μmol/L and 70.527 μmol/L, respectively, showing a significant difference ( P <0.05). The mRNA expression of HSPA1A and HSPA1B in MKN45/AR cells was significantly higher than that in MKN45 cells ( P <0.001). The protein expression of heat shock protein 70 was significantly decreased after 0.25 μmol/L triptolide treatment in MKN45/AR cells ( P <0.01). When heat shock protein 70 was inhibited by triptolide, the IC(50) value of apatinib in MKN45/AR cells was reduced to 11.679 μmol/L, which was significantly lower than cells treated with apatinib alone ( P <0.05). Conclusions: The apatinib-resistant MKN45 cells have high levels of heat shock protein 70. Low doses of triptolide can significantly inhibit heat shock protein 70, leading to reverse the resistance phenotype of MKN45/AR cells. Therefore, inhibition of heat shock protein 70 provides a new therapy strategy for patients with apatinib resistance.

CL316,243, a β3-adrenergic receptor agonist, induces muscle hypertrophy and increased strength.

PubMed

Puzzo, Daniela; Raiteri, Roberto; Castaldo, Clotilde; Capasso, Raffaele; Pagano, Ester; Tedesco, Mariateresa; Gulisano, Walter; Drozd, Lisaveta; Lippiello, Pellegrino; Palmeri, Agostino; Scotto, Pietro; Miniaci, Maria Concetta

2016-11-22

Studies in vitro have demonstrated that β3-adrenergic receptors (β3-ARs) regulate protein metabolism in skeletal muscle by promoting protein synthesis and inhibiting protein degradation. In this study, we evaluated whether activation of β3-ARs by the selective agonist CL316,243 modifies the functional and structural properties of skeletal muscles of healthy mice. Daily injections of CL316,243 for 15 days resulted in a significant improvement in muscle force production, assessed by grip strength and weight tests, and an increased myofiber cross-sectional area, indicative of muscle hypertrophy. In addition, atomic force microscopy revealed a significant effect of CL316,243 on the transversal stiffness of isolated muscle fibers. Interestingly, the expression level of mammalian target of rapamycin (mTOR) downstream targets and neuronal nitric oxide synthase (NOS) was also found to be enhanced in tibialis anterior and soleus muscles of CL316,243 treated mice, in accordance with previous data linking β3-ARs to mTOR and NOS signaling pathways. In conclusion, our data suggest that CL316,243 systemic administration might be a novel therapeutic strategy worthy of further investigations in conditions of muscle wasting and weakness associated with aging and muscular diseases.