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Sample records for inhibit ligand-dependent transactivation

  1. Reconstruction of ligand-dependent transactivation of Choristoneura fumiferana ecdysone receptor in yeast.

    PubMed

    Tran, H T; Askari, H B; Shaaban, S; Price, L; Palli, S R; Dhadialla, T S; Carlson, G R; Butt, T R

    2001-07-01

    Ecdysteroids play an important role in regulating development and reproduction in insects. Interaction of 20-hydroxyecdysone (20E) with ecdysone receptor (EcR) as a heterodimer with ultraspiracle (USP) protein triggers the activation of 20E-responsive genes. In this paper we describe a ligand-mediated transactivation system in yeast using the spruce budworm Choristoneura fumiferana ecdysone receptor (CfEcR). Coexpression of C. fumiferana USP (CfUSP) with CfEcR in yeast led to constitutive transcription of the reporter gene. However, deletion of the A/B domain of CfUSP abolished constitutive activity observed for the CfUSP:CfEcR complex. Replacement of USP with its mammalian homolog retinoid X receptors (RXRs) abolished the constitutive activity of the heterodimer but it did not restore EcR ligand-mediated transactivation. These data suggest that USP and its A/B domain play a role in the constitutive function of CfEcR:USP in yeast. A ligand-mediated transactivation was observed when GRIP1, a mouse coactivator gene, was added to EcR:RXR or EcR:DeltaA/BUSP complexes. Deletion of the A/B domain of EcR in the context of DeltaA/BEcR:RXR:GRIP1 or DeltaA/BEcR:DeltaA/BUSP:GRIP1 dramatically improved the ligand-dependent transactivation. This is the first example of highly efficient ligand-dependent transactivation of insect EcR in yeast. Analysis of transactivation activity of different ecdysteroidal compounds showed that the yeast system remarkably mimics the response observed in insect tissue culture cells and whole insect systems. The results open the way to develop assays that can be used to screen novel species-specific ecdysone agonist/antagonist insecticides.

  2. ATF3 inhibits PPARγ-stimulated transactivation in adipocyte cells

    SciTech Connect

    Jang, Min-Kyung; Jung, Myeong Ho

    2015-01-02

    Highlights: • ATF3 inhibits PPARγ-stimulated transcriptional activation. • ATF3 interacts with PPARγ. • ATF3 suppresses p300-mediated transcriptional coactivation. • ATF3 decreases the binding of PPARγ and recruitment of p300 to PPRE. - Abstract: Previously, we reported that activating transcription factor 3 (ATF3) downregulates peroxisome proliferator activated receptor (PPARγ) gene expression and inhibits adipocyte differentiation in 3T3-L1 cells. Here, we investigated another role of ATF3 on the regulation of PPARγ activity. ATF3 inhibited PPARγ-stimulated transactivation of PPARγ responsive element (PPRE)-containing reporter or GAL4/PPARγ chimeric reporter. Thus, ATF3 effectively repressed rosiglitazone-stimulated expression of adipocyte fatty acid binding protein (aP2), PPARγ target gene, in 3T3-L1 cells. Coimmunoprecipitation and GST pulldown assay demonstrated that ATF3 interacted with PPARγ. Accordingly, ATF3 prevented PPARγ from binding to PPRE on the aP2 promoter. Furthermore, ATF3 suppressed p300-mediated transcriptional coactivation of PPRE-containing reporter. Chromatin immunoprecipitation assay showed that overexpression of ATF3 blocked both binding of PPARγ and recruitment of p300 to PPRE on aP2 promoter induced by rosiglitazone treatment in 3T3-L1 cells. Taken together, these results suggest that ATF3 interacts with PPARγ and represses PPARγ-mediated transactivation through suppression of p300-stimulated coactivation in 3T3-L1 cells, which may play a role in inhibition of adipocyte differentiation.

  3. Rapid Glucocorticoid Feedback Inhibition of ACTH Secretion Involves Ligand-Dependent Membrane Association of Glucocorticoid Receptors

    PubMed Central

    Deng, Qiong; Riquelme, Denise; Trinh, Loc; Low, Malcolm J.; Tomić, Melanija; Stojilkovic, Stanko

    2015-01-01

    The hypothesis that rapid glucocorticoid inhibition of pituitary ACTH secretion mediates a feedforward/feedback mechanism responsible for the hourly glucocorticoid pulsatility was tested in cultured pituitary cells. Perifusion with 30 pM CRH caused sustained the elevation of ACTH secretion. Superimposed corticosterone pulses inhibited CRH-stimulated ACTH release, depending on prior glucocorticoid clearance. When CRH perifusion started after 2 hours of glucocorticoid-free medium, corticosterone levels in the stress range (1 μM) caused a delayed (25 min) and prolonged inhibition of CRH-stimulated ACTH secretion, up to 60 minutes after corticosterone withdrawal. In contrast, after 6 hours of glucocorticoid-free medium, basal corticosterone levels inhibited CRH-stimulated ACTH within 5 minutes, after rapid recovery 5 minutes after corticosterone withdrawal. The latter effect was insensitive to actinomycin D but was prevented by the glucocorticoid receptor antagonist, RU486, suggesting nongenomic effects of the classical glucocorticoid receptor. In hypothalamic-derived 4B cells, 10 nM corticosterone increased immunoreactive glucocorticoid receptor content in membrane fractions, with association and clearance rates paralleling the effects on ACTH secretion from corticotrophs. Corticosterone did not affect CRH-stimulated calcium influx, but in AtT-20 cells, it had biphasic effects on CRH-stimulated Src phosphorylation, with early inhibition and late stimulation, suggesting a role for Src phosphorylation on the rapid glucocorticoid feedback. The data suggest that the nongenomic/membrane effects of classical GR mediate rapid and reversible glucocorticoid feedback inhibition at the pituitary corticotrophs downstream of calcium influx. The sensitivity and kinetics of these effects is consistent with the hypothesis that pituitary glucocorticoid feedback is part of the mechanism for adrenocortical ultradian pulse generation. PMID:26121342

  4. MEF/ELF4 transactivation by E2F1 is inhibited by p53.

    PubMed

    Taura, Manabu; Suico, Mary Ann; Fukuda, Ryosuke; Koga, Tomoaki; Shuto, Tsuyoshi; Sato, Takashi; Morino-Koga, Saori; Okada, Seiji; Kai, Hirofumi

    2011-01-01

    Myeloid elf-1-like factor (MEF) or Elf4 is an E-twenty-six (ETS)-related transcription factor with strong transcriptional activity that influences cellular senescence by affecting tumor suppressor p53. MEF downregulates p53 expression and inhibits p53-mediated cellular senescence by transcriptionally activating MDM2. However, whether p53 reciprocally opposes MEF remains unexplored. Here, we show that MEF is modulated by p53 in human cells and mice tissues. MEF expression and promoter activity were suppressed by p53. While we found that MEF promoter does not contain p53 response elements, intriguingly, it contains E2F consensus sites. Subsequently, we determined that E2F1 specifically binds to MEF promoter and transactivates MEF. Nevertheless, E2F1 DNA binding and transactivation of MEF promoter was inhibited by p53 through the association between p53 and E2F1. Furthermore, we showed that activation of p53 in doxorubicin-induced senescent cells increased E2F1 and p53 interaction, diminished E2F1 recruitment to MEF promoter and reduced MEF expression. These observations suggest that p53 downregulates MEF by associating with and inhibiting the binding activity of E2F1, a novel transcriptional activator of MEF. Together with previous findings, our present results indicate that a negative regulatory mechanism exists between p53 and MEF.

  5. Groucho suppresses Pax2 transactivation by inhibition of JNK-mediated phosphorylation.

    PubMed

    Cai, Yi; Brophy, Patrick D; Levitan, Inna; Stifani, Stefano; Dressler, Gregory R

    2003-10-15

    Pax proteins are DNA-binding transcription factors that regulate embryonic development through the activation and repression of downstream target genes. The Pax2 gene is absolutely required for kidney development and for patterning specific regions of the nervous system such as the eye, ear and hindbrain. The Pax2/5/8 family of proteins contains both transcription activation and repression domains. The activation domain of Pax2 is phosphorylated by the c-Jun N-terminal kinase (JNK) to enhance Pax2-dependent transcription. In this report, we demonstrate that the Groucho/TLE family protein, Grg4, interacts with Pax2 to suppress transactivation. Grg4 is able to specifically inhibit phosphorylation of the Pax2 activation domain, even in the presence of activated JNK. Furthermore, the Grg4 interaction and suppression of phosphorylation depends on Pax2 binding to its target DNA sequence and is independent of histone deacetylation. These data suggest a new model for Groucho mediated suppression of transcription through the specific inhibition of modifications in the activation domain of a transactivator.

  6. Hydroxyurea inhibits the transactivation of the HIV-long-terminal repeat (LTR) promoter

    PubMed Central

    Calzado, M A; Macho, A; Lucena, C; Muñoz, E

    2000-01-01

    HIV-1 gene expression is regulated by the promoter/enhancer located within the U3 region of the proviral 5′ LTR that contains multiple potential cis-acting regulatory sites. Here we describe that the inhibitor of the cellular ribonucleoside reductase, hydroxyurea (HU), inhibited phorbol myristate acetate- or tumour necrosis factor-alpha-induced HIV-1-LTR transactivation in both lymphoid and non-lymphoid cells in a dose-dependent manner within the first 6 h of treatment, with a 50% inhibitory concentration of 0·5 mm. This inhibition was found to be specific for the HIV-1-LTR since transactivation of either an AP-1-dependent promoter or the CD69 gene promoter was not affected by the presence of HU. Moreover, gel-shift assays in 5.1 cells showed that HU prevented the binding of the NF-κB to the κB sites located in the HIV-1-LTR region, but it did not affect the binding of both the AP-1 and the Sp-1 transcription factors. By Western blots and cell cycle analyses we detected that HU induced a rapid dephosphorylation of the pRB, the product of the retinoblastoma tumour suppressor gene, and the cell cycle arrest was evident after 24 h of treatment. Thus, HU inhibits HIV-1 promoter activity by a novel pathway that implies an inhibition of the NF-κB binding to the LTR promoter. The present study suggests that HU may be useful as a potential therapeutic approach for inhibition of HIV-1 replication through different pathways. PMID:10792382

  7. CI-988 Inhibits EGFR Transactivation and Proliferation Caused by Addition of CCK/Gastrin to Lung Cancer Cells.

    PubMed

    Moody, Terry W; Nuche-Berenguer, Bernardo; Moreno, Paola; Jensen, Robert T

    2015-07-01

    Cholecystokinin (CCK) receptors are G-protein coupled receptors (GPCR) which are present on lung cancer cells. CCK-8 stimulates the proliferation of lung cancer cells, whereas the CCK2R receptor antagonist CI-988 inhibits proliferation. GPCR for some gastrointestinal hormones/neurotransmitters mediate lung cancer growth by causing epidermal growth factor receptor (EGFR) transactivation. Here, the role of CCK/gastrin and CI-988 on EGFR transactivation and lung cancer proliferation was investigated. Addition of CCK-8 or gastrin-17 (100 nM) to NCI-H727 human lung cancer cells increased EGFR Tyr(1068) phosphorylation after 2 min. The ability of CCK-8 to cause EGFR tyrosine phosphorylation was blocked by CI-988, gefitinib (EGFR tyrosine kinase inhibitor), PP2 (Src inhibitor), GM6001 (matrix metalloprotease inhibitor), and tiron (superoxide scavenger). CCK-8 nonsulfated and gastrin-17 caused EGFR transactivation and bound with high affinity to NCI-H727 cells, suggesting that the CCK2R is present. CI-988 inhibited the ability of CCK-8 to cause ERK phosphorylation and elevate cytosolic Ca(2+). CI-988 or gefitinib inhibited the basal growth of NCI-H727 cells or that stimulated by CCK-8. The results indicate that CCK/gastrin may increase lung cancer proliferation in an EGFR-dependent manner.

  8. XAP2, a novel hepatitis B virus X-associated protein that inhibits X transactivation.

    PubMed Central

    Kuzhandaivelu, N; Cong, Y S; Inouye, C; Yang, W M; Seto, E

    1996-01-01

    The hepatitis B virus X protein is a promiscuous transcriptional transactivator. Transactivation by the X protein is most likely mediated through binding to different cellular factors. Using the yeast two-hybrid method, we have isolated a clone that encodes a novel X-associated cellular protein: XAP2. X and XAP2 interactions also occur in vitro. Antiserum raised against XAP2 recognizes a cytoplasmic protein with an apparent molecular mass of 36 kDa. The interaction between X and XAP2 requires a small region on X containing amino acids 13-26. From Northern blot analyses, XAP2 is ubiquitously expressed in both liver-derived and non-liver-derived cell lines as well as in normal non-liver tissues. In contrast, XAP2 is expressed in very low level in the normal human liver. In transfection assays, overexpression of XAP2 abolishes transactivation by the X protein. Based on these results, we suggest that XAP2 is an important cellular negative regulator of the X protein, and that X-XAP2 interaction may play a role in HBV pathology. PMID:8972861

  9. Caffeic acid phenethyl ester downregulates phospholipase D1 via direct binding and inhibition of NFκB transactivation

    SciTech Connect

    Park, Mi Hee; Kang, Dong Woo; Jung, Yunjin; Choi, Kang-Yell; Min, Do Sik

    2013-12-06

    Highlights: •We found CAFÉ, a natural product that suppresses expression and activity of PLD1. •CAPE decreased PLD1 expression by inhibiting NFκB transactivation. •CAPE rapidly inhibited PLD activity via its binding to a Cys837 of PLD1. •PLD1 downregulation by CAPE inhibited invasion and proliferation of glioma cells. -- Abstract: Upregulation of phospholipase D (PLD) is functionally linked with oncogenic signals and tumorigenesis. Caffeic acid phenethyl ester (CAPE) is an active compound of propolis extract that exhibits anti-proliferative, anti-inflammatory, anti-oxidant, and antineoplastic properties. In this study, we demonstrated that CAPE suppressed the expression of PLD1 at the transcriptional level via inhibition of binding of NFκB to PLD1 promoter. Moreover, CAPE, but not its analogs, bound to a Cys837 residue of PLD1 and inhibited enzymatic activity of PLD. CAPE also decreased activation of matrix metalloproteinases-2 induced by phosphatidic acid, a product of PLD activity. Ultimately, CAPE-induced downregulation of PLD1 suppressed invasion and proliferation of glioma cells. Taken together, the results of this study indicate that CAPE might contribute to anti-neoplastic effect by targeting PLD1.

  10. Transcriptional Corepressor SMILE Recruits SIRT1 to Inhibit Nuclear Receptor Estrogen Receptor-related Receptor γ Transactivation*

    PubMed Central

    Xie, Yuan-Bin; Park, Jeong-Hoh; Kim, Don-Kyu; Hwang, Jung Hwan; Oh, Sangmi; Park, Seung Bum; Shong, Minho; Lee, In-Kyu; Choi, Hueng-Sik

    2009-01-01

    SMILE (small heterodimer partner interacting leucine zipper protein) has been identified as a corepressor of the glucocorticoid receptor, constitutive androstane receptor, and hepatocyte nuclear factor 4α. Here we show that SMILE also represses estrogen receptor-related receptor γ (ERRγ) transactivation. Knockdown of SMILE gene expression increases ERRγ activity. SMILE directly interacts with ERRγ in vitro and in vivo. Domain mapping analysis showed that SMILE binds to the AF2 domain of ERRγ. SMILE represses ERRγ transactivation partially through competition with coactivators PGC-1α, PGC-1β, and GRIP1. Interestingly, the repression of SMILE on ERRγ is released by SIRT1 inhibitors, a catalytically inactive SIRT1 mutant, and SIRT1 small interfering RNA but not by histone protein deacetylase inhibitor. In vivo glutathione S-transferase pulldown and coimmunoprecipitation assays validated that SMILE physically interacts with SIRT1. Furthermore, the ERRγ inverse agonist GSK5182 enhances the interaction of SMILE with ERRγ and SMILE-mediated repression. Knockdown of SMILE or SIRT1 blocks the repressive effect of GSK5182. Moreover, chromatin immunoprecipitation assays revealed that GSK5182 augments the association of SMILE and SIRT1 on the promoter of the ERRγ target PDK4. GSK5182 and adenoviral overexpression of SMILE cooperate to repress ERRγ-induced PDK4 gene expression, and this repression is released by overexpression of a catalytically defective SIRT1 mutant. Finally, we demonstrated that ERRγ regulates SMILE gene expression, which in turn inhibits ERRγ. Overall, these findings implicate SMILE as a novel corepressor of ERRγ and recruitment of SIRT1 as a novel repressive mechanism for SMILE and ERRγ inverse agonist. PMID:19690166

  11. Orphan nuclear receptor small heterodimer partner inhibits transforming growth factor-beta signaling by repressing SMAD3 transactivation.

    PubMed

    Suh, Ji Ho; Huang, Jiansheng; Park, Yun-Yong; Seong, Hyun-A; Kim, Dongwook; Shong, Minho; Ha, Hyunjung; Lee, In-Kyu; Lee, Keesook; Wang, Li; Choi, Hueng-Sik

    2006-12-22

    Orphan nuclear receptor small heterodimer partner (SHP) is an atypical member of the nuclear receptor superfamily; SHP regulates the nuclear receptor-mediated transcription of target genes but lacks a conventional DNA binding domain. In this study, we demonstrate that SHP represses transforming growth factor-beta (TGF-beta)-induced gene expression through a direct interaction with Smad, a transducer of TGF-beta signaling. Transient transfection studies demonstrate that SHP represses Smad3-induced transcription. In vivo and in vitro protein interaction assays revealed that SHP directly interacts with Smad2 and Smad3 but not with Smad4. Mapping of domains mediating the interaction between SHP and Smad3 showed that the entire N-terminal domain (1-159 amino acids) of SHP and the linker domain of Smad3 are involved in this interaction. In vitro glutathione S-transferase pulldown competition experiments revealed the SHP-mediated repression of Smad3 transactivation through competition with its co-activator p300. SHP also inhibits the activation of endogenous TGF-beta-responsive gene promoters, the p21, Smad7, and plasminogen activator inhibitor-1 (PAI-1) promoters. Moreover, adenovirus-mediated overexpression of SHP decreases PAI-1 mRNA levels, and down-regulation of SHP by a small interfering RNA increases both the transactivation of Smad3 and the PAI-1 mRNA levels. Finally, the PAI-1 gene is expressed in SHP(-/-) mouse hepatocytes at a higher level than in normal hepatocytes. Taken together, these data indicate that SHP is a novel co-regulator of Smad3, and this study provides new insights into regulation of TGF-beta signaling.

  12. Transactivation of ErbB Family of Receptor Tyrosine Kinases Is Inhibited by Angiotensin-(1-7) via Its Mas Receptor

    PubMed Central

    Akhtar, Saghir; Chandrasekhar, Bindu; Attur, Sreeja; Dhaunsi, Gursev S.; Yousif, Mariam H. M.; Benter, Ibrahim F.

    2015-01-01

    Transactivation of the epidermal growth factor receptor (EGFR or ErbB) family members, namely EGFR and ErbB2, appears important in the development of diabetes-induced vascular dysfunction. Angiotensin-(1–7) [Ang-(1–7)] can prevent the development of hyperglycemia-induced vascular complications partly through inhibiting EGFR transactivation. Here, we investigated whether Ang-(1–7) can inhibit transactivation of ErbB2 as well as other ErbB receptors in vivo and in vitro. Streptozotocin-induced diabetic rats were chronically treated with Ang-(1–7) or AG825, a selective ErbB2 inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in cultured vascular smooth muscle cells (VSMCs). Ang-(1–7) or AG825 treatment inhibited diabetes-induced phosphorylation of ErbB2 receptor at tyrosine residues Y1221/22, Y1248, Y877, as well as downstream signaling via ERK1/2, p38 MAPK, ROCK, eNOS and IkB-α in the mesenteric vascular bed. In VSMCs cultured in high glucose (25 mM), Ang-(1–7) inhibited src-dependent ErbB2 transactivation that was opposed by the selective Mas receptor antagonist, D-Pro7-Ang-(1–7). Ang-(1–7) via Mas receptor also inhibited both Angiotensin II- and noradrenaline/norephinephrine-induced transactivation of ErbB2 and/or EGFR receptors. Further, hyperglycemia-induced transactivation of ErbB3 and ErbB4 receptors could be attenuated by Ang-(1–7) that could be prevented by D-Pro7-Ang-(1–7) in VSMC. These data suggest that Ang-(1–7) via its Mas receptor acts as a pan-ErbB inhibitor and might represent a novel general mechanism by which Ang-(1–7) exerts its beneficial effects in many disease states including diabetes-induced vascular complications. PMID:26536590

  13. The BET bromodomain inhibitor JQ1 activates HIV latency through antagonizing Brd4 inhibition of Tat-transactivation

    PubMed Central

    Li, Zichong; Guo, Jia; Wu, Yuntao; Zhou, Qiang

    2013-01-01

    Latent HIV reservoirs are the primary hurdle to eradication of infection. Identification of agents, pathways and molecular mechanisms that activate latent provirus may, in the presence of highly active antiretroviral therapy, permit clearance of infected cells by the immune system. Promoter-proximal pausing of RNA polymerase (Pol) II is a major rate-limiting step in HIV gene expression. The viral Tat protein recruits human Super Elongation Complex (SEC) to paused Pol II to overcome this limitation. Here, we identify the bromodomain protein Brd4 and its inhibition of Tat-transactivation as a major impediment to latency reactivation. Brd4 competitively blocks the Tat–SEC interaction on HIV promoter. The BET bromodomain inhibitor JQ1 dissociates Brd4 from the HIV promoter to allow Tat recruitment of SEC to stimulate HIV elongation. JQ1 synergizes with another latency activator prostratin, which promotes Pol II loading onto the viral promoter. Because JQ1 activates viral latency without inducing global T cell activation, this and other closely related compounds and their antagonization of Brd4 to promote Tat–SEC interaction merit further investigations as effective agents/strategies for eliminating latent HIV. PMID:23087374

  14. Overexpression of ERβ is sufficient to inhibit hypoxia-inducible factor-1 transactivation

    SciTech Connect

    Park, Choa; Lee, YoungJoo

    2014-07-18

    Highlights: • We examined the effect of ERβ specific ligand on HIF-1 inhibition. • DPN down-regulates the ARNT protein levels in PC3 cells. • DPN did not show additional effect in ERβ transfected MCF-7 cells. • Our study shows that unliganded ERβ is sufficient to inhibit HIF-1 in systems of overexpression. - Abstract: Estrogen receptor (ER) β is predicted to play an important role in the prevention of breast cancer development and progression. We have previously shown that ERβ suppresses hypoxia inducible factor (HIF)-1-mediated transcription through aryl hydrocarbon receptor nuclear translocator (ARNT) degradation via ubiquitination processes. In this study, we attempted to examine the effect of ERβ specific ligand on HIF-1 inhibition in ERβ positive PC3 cells and ERβ transfected MCF-7 cells. ERβ specific agonist diarylpropionitrile (DPN) stimulated estrogen response element (ERE)-luciferase activity in a similar fashion to estradiol in PC3 cells. We observed that DPN down-regulates the ARNT protein levels leading to an attenuation of hypoxia-induced hypoxia response element (HRE)-driven luciferase reporter gene activation in PC3 cells. Treatment of DPN reduced vascular endothelial growth factor (VEGF) expression and co-treatment with ERβ specific antagonist PHTPP abrogated the effect in PC3 cells. We then examined the effect of DPN in ERβ transfected MCF-7 cells. HIF-1 transcriptional activity repression by ERβ was not further reduced by DPN, as examined by HRE-driven luciferase assays. Expression of ERβ significantly decreased VEGF secretion and ARNT expression under hypoxic conditions. However, DPN did not additionally affect this suppression in MCF-7 cells transfected with ERβ. This result shows that unliganded ERβ is sufficient to inhibit HIF-1 in systems of overexpression.

  15. Inhibition of {beta}-catenin-mediated transactivation by flavanone in AGS gastric cancer cells

    SciTech Connect

    Park, Chi Hoon; Hahm, Eun Ryeong; Lee, Ju Hyung; Jung, Kyung Chae; Yang, Chul Hak . E-mail: chulyang@plaza.snu.ac.kr

    2005-06-17

    Recently, data which prove that Wnt pathway activation may be an early event in multistep carcinogenesis in the stomach have been accumulating. We examined the effect of flavanone against {beta}-catenin/Tcf signaling in AGS gastric cancer cells. Reporter gene assay showed that flavanone inhibited {beta}-catenin/Tcf signaling efficiently. In addition, the inhibition of {beta}-catenin/Tcf signaling by flavanone in HEK293 cells transiently transfected with constitutively mutant {beta}-catenin gene, whose product is not phosphorylated by GSK3{beta}, indicates that its inhibitory mechanism was related to {beta}-catenin itself or downstream components. To investigate the precise inhibitory mechanism, we performed immunofluorescence, Western blot, and EMSA. As a result, our data revealed that there is no change of {beta}-catenin distribution and of nuclear {beta}-catenin levels through flavanone. In addition, the binding of Tcf complexes to DNA is not influenced by flavanone. The {beta}-catenin/Tcf transcriptional target gene cyclinD1 was downregulated by flavanone. These data suggest that flavanone inhibits the transcription of {beta}-catenin/Tcf responsive genes, by modulating Tcf activity without disrupting {beta}-catenin/Tcf complex formation.

  16. CREB trans-activation of disruptor of telomeric silencing-1 mediates forskolin inhibition of CTGF transcription in mesangial cells.

    PubMed

    Yu, Zhiyuan; Kong, Qun; Kone, Bruce C

    2010-03-01

    Connective tissue growth factor (CTGF) participates in diverse fibrotic processes including glomerulosclerosis. The adenylyl cyclase agonist forskolin inhibits CTGF expression in mesangial cells by unclear mechanisms. We recently reported that the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) suppresses CTGF gene expression in collecting duct cells (J Clin Invest 117: 773-783, 2007) and HEK 293 cells (J Biol Chem In press). In the present study, we characterized the involvement of Dot1 in mediating the inhibitory effect of forskolin on CTGF transcription in mouse mesangial cells. Overexpression of Dot1 or treatment with forskolin dramatically suppressed basal CTGF mRNA levels and CTGF promoter-luciferase activity, while hypermethylating H3K79 in chromatin associated with the CTGF promoter. siRNA knockdown of Dot1 abrogated the inhibitory effect of forskolin on CTGF mRNA expression. Analysis of the Dot1 promoter sequence identified a CREB response element (CRE) at -384/-380. Overexpression of CREB enhanced forskolin-stimulated Dot1 promoter activity. A constitutively active CREB mutant (CREB-VP16) strongly induced Dot1 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. Mutation of the -384/-380 CRE resulted in 70% lower levels of Dot1 promoter activity. ChIP assays confirmed CREB binding to the Dot1 promoter in chromatin. We conclude that forskolin stimulates CREB-mediated trans-activation of the Dot1 gene, which leads to hypermethylation of histone H3K79 at the CTGF promoter, and inhibition of CTGF transcription. These data are the first to describe regulation of the Dot1 gene, and disclose a complex network of genetic and epigenetic controls on CTGF transcription.

  17. Brk/PTK6 Sustains Activated EGFR Signaling through Inhibiting EGFR Degradation and Transactivating EGFR

    PubMed Central

    Li, X; Lu, Y; Liang, K; Hsu, J -M.; Albarracin, C; Mills, G B; Hung, M-C; Fan, Z

    2011-01-01

    Epidermal growth factor receptor (EGFR)-mediated cell signaling is critical for mammary epithelial cell growth and survival; however, targeting EGFR has shown no or only minimal therapeutic benefit in patients with breast cancer. Here, we report a novel regulatory mechanism of EGFR signaling that may explain the low response rates. We found that breast tumor kinase (Brk)/protein-tyrosine kinase 6 (PTK6), a nonreceptor protein tyrosine kinase highly expressed in most human breast tumors, interacted with EGFR and sustained ligand-induced EGFR signaling. We demonstrate that Brk inhibits ligand-induced EGFR degradation through uncoupling activated EGFR from Cbl-mediated EGFR ubiquitination. In addition, upon activation by EGFR, Brk directly phosphorylated Y845 in the EGFR kinase domain, thereby further potentiating EGFR kinase activity. Experimental elevation of Brk conferred resistance of breast cancer cells to cetuximab (an EGFR-blocking antibody)-induced inhibition of cell signaling and proliferation, whereas knockdown of Brk sensitized the cells to cetuximab by inducing apoptosis. Our findings reveal a previously unknown role of Brk in EGFR-targeted therapy. PMID:22231447

  18. Brk/PTK6 sustains activated EGFR signaling through inhibiting EGFR degradation and transactivating EGFR.

    PubMed

    Li, X; Lu, Y; Liang, K; Hsu, J-M; Albarracin, C; Mills, G B; Hung, M-C; Fan, Z

    2012-10-04

    Epidermal growth factor receptor (EGFR)-mediated cell signaling is critical for mammary epithelial cell growth and survival; however, targeting EGFR has shown no or only minimal therapeutic benefit in patients with breast cancer. Here, we report a novel regulatory mechanism of EGFR signaling that may explain the low response rates. We found that breast tumor kinase (Brk)/protein-tyrosine kinase 6 (PTK6), a nonreceptor protein-tyrosine kinase highly expressed in most human breast tumors, interacted with EGFR and sustained ligand-induced EGFR signaling. We demonstrate that Brk inhibits ligand-induced EGFR degradation through uncoupling activated EGFR from casitas B-lineage lymphoma-mediated EGFR ubiquitination. In addition, upon activation by EGFR, Brk directly phosphorylated Y845 in the EGFR kinase domain, thereby further potentiating EGFR kinase activity. Experimental elevation of Brk conferred resistance of breast cancer cells to cetuximab (an EGFR-blocking antibody)-induced inhibition of cell signaling and proliferation, whereas knockdown of Brk sensitized the cells to cetuximab by inducing apoptosis. Our findings reveal a previously unknown role of Brk in EGFR-targeted therapy.

  19. A peptide nucleic acid-aminosugar conjugate targeting transactivation response element of HIV-1 RNA genome shows a high bioavailability in human cells and strongly inhibits tat-mediated transactivation of HIV-1 transcription.

    PubMed

    Das, Indrajit; Désiré, Jérôme; Manvar, Dinesh; Baussanne, Isabelle; Pandey, Virendra N; Décout, Jean-Luc

    2012-07-12

    The 6-aminoglucosamine ring of the aminoglycoside antibiotic neomycin B (ring II) was conjugated to a 16-mer peptide nucleic acid (PNA) targeting HIV-1 TAR RNA. For this purpose, we prepared the aminoglucosamine monomer 15 and attached it to the protected PNA prior to its cleavage from the solid support. We found that the resulting PNA-aminoglucosamine conjugate is stable under acidic conditions, efficiently taken up by the human cells and fairly distributed in both cytosol and nucleus without endosomal entrapment because cotreatment with endosome-disrupting agent had no effect on its cellular distribution. The conjugate displayed very high target specificity in vitro and strongly inhibited Tat mediated transactivation of HIV-1 LTR transcription in a cell culture system. The unique properties of this new class of PNA conjugate suggest it to be a potential candidate for therapeutic application.

  20. Growth differentiation factor (GDF)-15 blocks norepinephrine-induced myocardial hypertrophy via a novel pathway involving inhibition of epidermal growth factor receptor transactivation.

    PubMed

    Xu, Xin-ye; Nie, Ying; Wang, Fang-fang; Bai, Yan; Lv, Zhi-zhen; Zhang, You-yi; Li, Zi-jian; Gao, Wei

    2014-04-04

    Accumulating evidence suggests that growth differentiation factor 15 (GDF-15) is associated with the severity and prognosis of various cardiovascular diseases. However, the effect of GDF-15 on the regulation of cardiac remodeling is still poorly understood. In this present study, we demonstrate that GDF-15 blocks norepinephrine (NE)-induced myocardial hypertrophy through a novel pathway involving inhibition of EGFR transactivation. Both in vivo and in vitro assay indicate that NE was able to stimulate the synthesis of GDF-15. The up-regulation of GDF-15 feedback inhibits NE-induced myocardial hypertrophy, including quantitation of [(3)H]leucine incorporation, protein/DNA ratio, cell surface area, and ANP mRNA level. Further research shows that GDF-15 could inhibit the phosphorylation of EGF receptor and downstream kinases (AKT and ERK1/2) induced by NE. Clinical research also shows that serum GDF-15 levels in hypertensive patients were significant higher than in healthy volunteers and were positively correlated with the thickness of the posterior wall of the left ventricle, interventricular septum, and left ventricular mass, as well as the serum level of norepinephrine. In conclusion, NE induces myocardial hypertrophy and up-regulates GDF-15, and this up-regulation of GDF-15 negatively regulates NE-induced myocardial hypertrophy by inhibiting EGF receptor transactivation following NE stimulation.

  1. PIAS3 induction of PRB sumoylation represses PRB transactivation by destabilizing its retention in the nucleus

    PubMed Central

    Man, Jiang-Hong; Li, Hui-Yan; Zhang, Pei-Jing; Zhou, Tao; He, Kun; Pan, Xin; Liang, Bing; Li, Ai-Ling; Zhao, Jie; Gong, Wei-Li; Jin, Bao-Feng; Xia, Qing; Yu, Ming; Shen, Bei-Fen; Zhang, Xue-Min

    2006-01-01

    Progesterone receptor (PR) plays a critical role in cell proliferation and differentiation, and its transcriptional activity is known to be modulated by cofactor proteins. In the present study, we demonstrated that in the presence of progesterone, protein inhibitor of activated STAT-3 (PIAS3) significantly inhibited the PR transcriptional activity and the expression of progesterone-responsive genes. Reduction of endogenous PIAS3 by PIAS3 small-interfering RNA enhanced PR transactivation in a ligand-dependent manner. PIAS3 interacted with PR both in vitro and in vivo and the interaction was enhanced by progesterone. Furthermore, our findings suggested that PIAS3 strongly induced PRB sumoylation at three sites, Lys-7, Lys-388 and Lys-531. In addition, novel roles in PRB nuclear retention and transactivation were identified for these sites. Our data also suggested that PIAS3 was recruited in a largely hormone-dependent manner in response to a progesterone-responsive promoter. Finally, we demonstrated that PIAS3 inhibited the DNA-binding activity of PR and influenced its nuclear export as well as PR transactivation. Taken together, these data strongly suggested that PIAS3 played an important physiological role in PR function. PMID:17020914

  2. Lysine 246 of the vitamin D receptor is crucial for ligand-dependent interaction with coactivators and transcriptional activity.

    PubMed

    Jiménez-Lara, A M; Aranda, A

    1999-05-07

    Mutant K246A in the predicted helix 3 of the ligand-binding domain, as well as mutants L417S and E420Q in helix 12, which contains the core ligand-dependent transcriptional activation domain (AF-2), were generated to examine AF-2 activity of the vitamin D receptor (VDR). These mutations abolished vitamin D-dependent transactivation. In addition, VDR mediates a ligand-dependent repression of the response of the retinoic acid receptor beta2 promoter to retinoic acid, and the helix 3 and helix 12 mutants were unable to mediate transrepression. Furthermore, the VDR mutants, but not the native receptor, enhanced phorbol ester induction of the activator protein-1-containing collagenase promoter. The helix 3 and helix 12 mutations strikingly reduced the ability of VDR to interact with the coactivators steroid receptor coactivator-1, ACTR, and the CREB-binding protein. As a consequence, overexpression of steroid receptor coactivator-1 increased vitamin D-dependent transactivation by VDR but not by the K246A mutant. These results indicate that the lysine 246 participates, together with residues in helix 12, in the recruitment of coactivators and that AF-2 activity is involved both in ligand-dependent transactivation and in transrepression by VDR.

  3. Drosophila Polycomb-group regulated chromatin inhibits the accessibility of a trans-activator to its target DNA.

    PubMed Central

    Zink, D; Paro, R

    1995-01-01

    The genes of the Polycomb-group (Pc-G) are responsible for maintaining the inactive expression state of homeotic genes. They act through specific cis-regulatory DNA elements termed PREs (Pc-G Response Elements). Multimeric complexes containing the Pc-G proteins are thought to induce heterochromatin-like structures, which stably and heritably inactivate transcription. We have tested the functional role of the FAB fragment, a PRE of the bithorax complex. We find that this element behaves as an orientation dependent silencer, capable of inducing mosaic gene expression on neighboring genes. Transgenic fly lines were constructed containing a PRE adjacent to a reporter gene inducible by the yeast GAL4 trans-activator. The competition between the activator and Pc-G-containing chromatin was visualized on polytene chromosomes using immunocytochemistry. The Pc-G protein Polycomb and GAL4 have mutually exclusive binding patterns, supporting the notion that Pc-G-induced chromatin structures can prevent activators from binding to their target sequences. However, this antagonistic function can be overcome by high doses of GAL4, even in the absence of DNA replication. Images PMID:8521823

  4. Vaccinia Virus Protein C6 Inhibits Type I IFN Signalling in the Nucleus and Binds to the Transactivation Domain of STAT2

    PubMed Central

    Stuart, Jennifer H.; Sumner, Rebecca P.; Lu, Yongxu

    2016-01-01

    The type I interferon (IFN) response is a crucial innate immune signalling pathway required for defense against viral infection. Accordingly, the great majority of mammalian viruses possess means to inhibit this important host immune response. Here we show that vaccinia virus (VACV) strain Western Reserve protein C6, is a dual function protein that inhibits the cellular response to type I IFNs in addition to its published function as an inhibitor of IRF-3 activation, thereby restricting type I IFN production from infected cells. Ectopic expression of C6 inhibits the induction of interferon stimulated genes (ISGs) in response to IFNα treatment at both the mRNA and protein level. C6 inhibits the IFNα-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway at a late stage, downstream of STAT1 and STAT2 phosphorylation, nuclear translocation and binding of the interferon stimulated gene factor 3 (ISGF3) complex to the interferon stimulated response element (ISRE). Mechanistically, C6 associates with the transactivation domain of STAT2 and this might explain how C6 inhibits the type I IFN signalling very late in the pathway. During virus infection C6 reduces ISRE-dependent gene expression despite the presence of the viral protein phosphatase VH1 that dephosphorylates STAT1 and STAT2. The ability of a cytoplasmic replicating virus to dampen the immune response within the nucleus, and the ability of viral immunomodulators such as C6 to inhibit multiple stages of the innate immune response by distinct mechanisms, emphasizes the intricacies of host-pathogen interactions and viral immune evasion. PMID:27907166

  5. Cooperation of Spi-1/PU.1 with an activated erythropoietin receptor inhibits apoptosis and Epo-dependent differentiation in primary erythroblasts and induces their Kit ligand-dependent proliferation.

    PubMed Central

    Quang, C T; Wessely, O; Pironin, M; Beug, H; Ghysdael, J

    1997-01-01

    Spi-1/PU.1 is a myeloid- and B-cell specific transcription factor which is also involved in Friend virus-induced murine erythroleukemia. The pre-leukemic phase of Friend erythroleukemia results from activation of the erythropoietin receptor (EpoR) by the spleen focus forming virus (SFFV) envelope glycoprotein, followed by the emergence of leukemic clones characterized by overexpression of Spi-1 and mutation of the p53 tumor suppressor gene. We developed a heterologous system to analyze the contribution of these alterations to the induction of primary erythroblast transformation. Avian erythroblasts expressing the activated mouse EpoR(R129C) differentiated into erythrocytes in response to hEpo. Expression of Spi-1 in these cells inhibited this ability to differentiate and rescued the cells from the apoptotic cell death program normally induced upon hEpo withdrawal. Although devoid of any effect by itself, a mutant p53 cooperated with Spi-1 and EpoR(R129C) to reinforce both phenotypes. Analysis of erythroblasts co-expressing Spi-1 and the wild-type mouse EpoR showed that differentiation arrest and inhibition of apoptosis depended on specific cooperation between Spi-1 and EpoR(R129C). This cooperation was also required to induce the sustained proliferation of differentiation-blocked erythroblasts in response to ligand activation of the endogenous tyrosine kinase receptor c-Kit. These results show that Spi-1/PU.1 requires signals emanating from specific cytokine and growth factor receptors to affect the survival, proliferation and differentiation control of primary erythroblasts. They also suggest that the function of Spi-1/PU.1 in the late phase of Friend leukemia requires specific signaling from the gp55-modified EpoR generated during the early phase of the disease. PMID:9312023

  6. Nickel(II) Complex of Polyhydroxybenzaldehyde N4-Thiosemicarbazone Exhibits Anti-Inflammatory Activity by Inhibiting NF-κB Transactivation

    PubMed Central

    Loh, Sheng Wei; Looi, Chung Yeng; Hassandarvish, Pouya; Phan, Alicia Yi Ling; Wong, Won Fen; Wang, Hao; Paterson, Ian C.; Ea, Chee Kwee; Mustafa, Mohd Rais; Maah, Mohd Jamil

    2014-01-01

    Background The biological properties of thiosemicarbazone have been widely reported. The incorporation of some transition metals such as Fe, Ni and Cu to thiosemicarbazone complexes is known to enhance its biological effects. In this study, we incorporated nickel(II) ions into thiosemicarbazone with N4-substitution groups H3L (H; H3L1, CH3; H3L2, C6H5; H3L3 and C2H5; H3L4) and examined its potential anti-inflammatory activity. Methodology/Principal Findings Four ligands (1–4) and their respective nickel-containing complexes (5–8) were synthesized and characterized. The compounds synthesized were tested for their effects on NF-κB nuclear translocation, pro-inflammatory cytokines secretion and NF-κB transactivation activity. The active compound was further evaluated on its ability to suppress carrageenan-induced acute inflammation in vivo. A potential binding target of the active compound was also predicted by molecular docking analysis. Conclusions/Significance Among all synthesized compounds tested, we found that complex [Ni(H2L1)(PPh3)]Cl (5) (complex 5), potently inhibited IκBα degradation and NF-κB p65 nuclear translocation in LPS-stimulated RAW264.7 cells as well as TNFα-stimulated HeLa S3 cells. In addition, complex 5 significantly down-regulated LPS- or TNFα-induced transcription of NF-κB target genes, including genes that encode the pro-inflammatory cytokines TNFα, IFNβ and IL6. Luciferase reporter assays confirmed that complex 5 inhibited the transactivation activity of NF-κB. Furthermore, the anti-inflammatory effect of complex 5 was also supported by its suppressive effect on carrageenan-induced paw edema formation in wild type C57BL/6 mice. Interestingly, molecular docking study showed that complex 5 potentially interact with the active site of IKKβ. Taken together, we suggest complex 5 as a novel NF-κB inhibitor with potent anti-inflammatory effects. PMID:24977407

  7. Role of the essential yeast protein PSU1 in p6anscriptional enhancement by the ligand-dependent activation function AF-2 of nuclear receptors.

    PubMed Central

    Gaudon, C; Chambon, P; Losson, R

    1999-01-01

    Nuclear receptors (NRs) can function as ligandinducible transregulators in both mammalian and yeast cells, indicating that important features of transcriptional control have been conserved throughout evolution. We report here the isolation and characterization of an essential yeast protein of unknown function, PSU1, which exhibits properties expected for a co-activator/mediator of the ligand-dependent activation function AF-2 present in the ligand-binding domain (LBD, region E) of NRs. PSU1 interacts in a ligand-dependent manner with the LBD of several NRs, including retinoic acid (RARalpha), retinoid X (RXRalpha), thyroid hormone (TRalpha), vitamin D3 (VDR) and oestrogen (ERalpha) receptors. Importantly, both in yeast and in vitro, these interactions require the integrity of the AF-2 activating domain. When tethered to a heterologous DNA-binding domain, PSU1 can activate transcription on its own. By using yeast reporter cells that express PSU1 conditionally, we show that PSU1 is required for transactivation by the AF-2 of ERalpha. Taken together these data suggest that in yeast, PSU1 is involved in ligand-dependent transactivation by NRs. Sequence analysis revealed that in addition to a highly conserved motif found in a family of MutT-related proteins, PSU1 contains several alpha-helical leucine-rich motifs sharing the consensus sequence LLxPhiL (x, any amino acid; Phi, hydrophobic amino acid) in regions that elicit either transactivation or NR-binding activity. PMID:10205176

  8. Melatonin inhibits MMP-9 transactivation and renal cell carcinoma metastasis by suppressing Akt-MAPKs pathway and NF-κB DNA-binding activity.

    PubMed

    Lin, Yung-Wei; Lee, Liang-Ming; Lee, Wei-Jiunn; Chu, Chih-Ying; Tan, Peng; Yang, Yi-Chieh; Chen, Wei-Yu; Yang, Shun-Fa; Hsiao, Michael; Chien, Ming-Hsien

    2016-04-01

    Renal cell carcinoma (RCC) is the most lethal of all urological malignancies because of its potent metastasis potential. Melatonin exerts multiple tumor-suppressing activities through antiproliferative, proapoptotic, and anti-angiogenic actions and has been tested in clinical trials. However, the antimetastastic effect of melatonin and its underlying mechanism in RCC are unclear. In this study, we demonstrated that melatonin at the pharmacologic concentration (0.5-2 mm) considerably reduced the migration and invasion of RCC cells (Caki-1 and Achn). Furthermore, we found that melatonin suppressed metastasis of Caki-1 cells in spontaneous and experimental metastasis animal models. Mechanistic investigations revealed that melatonin transcriptionally inhibited MMP-9 by reducing p65- and p52-DNA-binding activities. Moreover, the Akt-mediated JNK1/2 and ERK1/2 signaling pathways were involved in melatonin-regulated MMP-9 transactivation and cell motility. Clinical samples revealed an inverse correlation between melatonin receptor 1A (MTNR1A) and MMP-9 expression in normal kidney and RCC tissues. In addition, a higher survival rate was found in MTNR1A(high) /MMP-9(low) patients than in MTNR1A(low) /MMP-9(high) patients. Overall, our results provide new insights into the role of melatonin-induced molecular regulation in suppressing RCC metastasis and suggest that melatonin has potential therapeutic applications for metastastic RCC.

  9. Triptonide Effectively Inhibits Wnt/β-Catenin Signaling via C-terminal Transactivation Domain of β-catenin

    PubMed Central

    Chinison, Jessica; Aguilar, Jose S.; Avalos, Alan; Huang, Ying; Wang, Zhijun; Cameron, D. Joshua; Hao, Jijun

    2016-01-01

    Abnormal activation of canonical Wnt/β-catenin signaling is implicated in many diseases including cancer. As a result, therapeutic agents that disrupt this signaling pathway have been highly sought after. Triptonide is a key bioactive small molecule identified in a traditional Chinese medicine named Tripterygium wilfordii Hook F., and it has a broad spectrum of biological functions. Here we show that triptonide can effectively inhibit canonical Wnt/β-catenin signaling by targeting the downstream C-terminal transcription domain of β-catenin or a nuclear component associated with β-catenin. In addition, triptonide treatment robustly rescued the zebrafish “eyeless” phenotype induced by GSK-3β antagonist 6-bromoindirubin-30-oxime (BIO) for Wnt signaling activation during embryonic gastrulation. Finally, triptonide effectively induced apoptosis of Wnt-dependent cancer cells, supporting the therapeutic potential of triptonide. PMID:27596363

  10. Inhibition of aryl hydrocarbon receptor transactivation and DNA adduct formation by CYP1 isoform-selective metabolic deactivation of benzo[a]pyrene

    SciTech Connect

    Endo, Kaori; Uno, Shigeyuki; Seki, Taiichiro; Ariga, Toyohiko; Kusumi, Yoshiaki; Mitsumata, Masako; Yamada, Sachiko; Makishima, Makoto

    2008-07-15

    Benzo[a]pyrene (BaP), a polyaromatic hydrocarbon produced by the combustion of cigarettes and coke ovens, is a known procarcinogen. BaP activates the aryl hydrocarbon receptor (AhR) and induces the expression of a battery of genes, including CYP1A1, which metabolize BaP to toxic compounds. The possible role of CYP1 enzymes in mediating BaP detoxification or metabolic activation remains to be elucidated. In this study, we assessed the effects of CYP1 enzymes (CYP1A1, CYP1A2 and CYP1B1) on BaP-induced AhR transactivation and DNA adduct formation in HEK293 cells and HepG2 cells. Transfection of CYP1A1 and CYP1B1, but not CYP1A2, suppressed BaP-induced activation of AhR. Expression of CYP1A1 and CYP1A2, but not CYP1B1, inhibited DNA adduct formation in BaP-treated HepG2 cells. These results indicate that CYP1A1 and CYP1B1 play a role in deactivation of BaP on AhR and that CYP1A1 and CYP1A2 are involved in BaP detoxification by suppressing DNA adduct formation. BaP treatment did not induce DNA adduct formation in HEK293 cells, even after transfection of CYP1 enzymes, suggesting that expression of CYP1 enzymes is not sufficient for DNA adduct formation. Lower expression of epoxide hydrolase and higher expression of glutathione S-transferase P1 (GSTP1) and GSTM1/M2 were observed in HEK293 cells compared with HepG2 cells. Dynamic expression of CYP1A1, CYP1A2 and CYP1B1 along with expression of other enzymes such as epoxide hydrolase and phase II enzymes may determine the detoxification or metabolic activation of BaP.

  11. Inflammatory stimuli inhibit glucocorticoid-dependent transactivation in human pulmonary epithelial cells: rescue by long-acting beta2-adrenoceptor agonists.

    PubMed

    Rider, Christopher F; King, Elizabeth M; Holden, Neil S; Giembycz, Mark A; Newton, Robert

    2011-09-01

    By repressing inflammatory gene expression, glucocorticoids are the most effective treatment for chronic inflammatory diseases such as asthma. However, in some patients with severe disease, or who smoke or suffer from chronic obstructive pulmonary disease, glucocorticoids are poorly effective. Although many investigators focus on defects in the repression of inflammatory gene expression, glucocorticoids also induce (transactivate) the expression of numerous genes to elicit anti-inflammatory effects. Using human bronchial epithelial (BEAS-2B) and pulmonary (A549) cells, we show that cytokines [tumor necrosis factor α (TNFα) and interleukin 1β], mitogens [fetal calf serum (FCS) and phorbol ester], cigarette smoke, and a G(q)-linked G protein-coupled receptor agonist attenuate simple glucocorticoid response element (GRE)-dependent transcription. With TNFα and FCS, this effect was not overcome by increasing concentrations of dexamethasone, budesonide, or fluticasone propionate. Thus, the maximal ability of the glucocorticoid to promote GRE-dependent transcription was reduced, and this was shown additionally for the glucocorticoid-induced gene p57(KIP2). The long-acting β(2)-adrenoceptor agonists (LABAs) formoterol fumarate and salmeterol xinafoate enhanced simple GRE-dependent transcription to a level that could not be achieved by glucocorticoid alone. In the presence of TNFα or FCS, which repressed glucocorticoid responsiveness, these LABAs restored glucocorticoid-dependent transcription to levels that were achieved by glucocorticoid alone. Given the existence of genes, such as p57(KIP2), which may mediate anti-inflammatory actions of glucocorticoids, we propose that repression of transactivation represents a mechanism for glucocorticoid resistance and for understanding the clinical benefit of LABAs as an add-on therapy in asthma and chronic obstructive pulmonary disease.

  12. Sigma-1 receptor activation inhibits osmotic swelling of rat retinal glial (Müller) cells by transactivation of glutamatergic and purinergic receptors.

    PubMed

    Vogler, Stefanie; Winters, Helge; Pannicke, Thomas; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas

    2016-01-01

    Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Sigma (σ) receptor activation is known to have neuroprotective effects in the retina. Here, we show that the nonselective σ receptor agonist ditolylguanidine, and the selective σ1 receptor agonist PRE-084, inhibit the osmotic swelling of Müller cell somata induced by superfusion of rat retinal slices with a hypoosmotic solution containing barium ions. In contrast, PRE-084 did not inhibit the osmotic swelling of bipolar cell somata. The effects of σ receptor agonists on the Müller cell swelling were abrogated in the presence of blockers of metabotropic glutamate and purinergic P2Y1 receptors, respectively, suggesting that σ receptor activation triggers activation of a glutamatergic-purinergic signaling cascade which is known to prevent the osmotic Müller cell swelling. The swelling-inhibitory effect of 17β-estradiol was prevented by the σ1 receptor antagonist BD1047, suggesting that the effect is mediated by σ1 receptor activation. The data may suggest that the neuroprotective effect of σ receptor activation in the retina is in part mediated by prevention of the cytotoxic swelling of retinal glial cells.

  13. Upregulating endogenous genes by an RNA-programmable artificial transactivator

    PubMed Central

    Fimiani, Cristina; Goina, Elisa; Mallamaci, Antonello

    2015-01-01

    To promote expression of endogenous genes ad libitum, we developed a novel, programmable transcription factor prototype. Kept together via an MS2 coat protein/RNA interface, it includes a fixed, polypeptidic transactivating domain and a variable RNA domain that recognizes the desired gene. Thanks to this device, we specifically upregulated five genes, in cell lines and primary cultures of murine pallial precursors. Gene upregulation was small, however sufficient to robustly inhibit neuronal differentiation. The transactivator interacted with target gene chromatin via its RNA cofactor. Its activity was restricted to cells in which the target gene is normally transcribed. Our device might be useful for specific applications. However for this purpose, it will require an improvement of its transactivation power as well as a better characterization of its target specificity and mechanism of action. PMID:26152305

  14. NF2 loss promotes oncogenic RAS-induced thyroid cancers via YAP-dependent transactivation of RAS proteins and sensitizes them to MEK inhibition

    PubMed Central

    Garcia-Rendueles, Maria E.R.; Ricarte-Filho, Julio C.; Untch, Brian R.; Landa, Iňigo; Knauf, Jeffrey A.; Voza, Francesca; Smith, Vicki E.; Ganly, Ian; Taylor, Barry S.; Persaud, Yogindra; Oler, Gisele; Fang, Yuqiang; Jhanwar, Suresh C.; Viale, Agnes; Heguy, Adriana; Huberman, Kety H.; Giancotti, Filippo; Ghossein, Ronald; Fagin, James A.

    2015-01-01

    Ch22q LOH is preferentially associated with RAS mutations in papillary and in poorly differentiated thyroid cancer (PDTC). The 22q tumor suppressor NF2, encoding merlin, is implicated in this interaction because of its frequent loss of function in human thyroid cancer cell lines. Nf2 deletion or Hras mutation are insufficient for transformation, whereas their combined disruption leads to murine PDTC with increased MAPK signaling. Merlin loss induces RAS signaling in part through inactivation of Hippo, which activates a YAP-TEAD transcriptional program. We find that the three RAS genes are themselves YAP-TEAD1 transcriptional targets, providing a novel mechanism of promotion of RAS-induced tumorigenesis. Moreover, pharmacological disruption of YAP-TEAD with verteporfin blocks RAS transcription and signaling, and inhibits cell growth. The increased MAPK output generated by NF2 loss in RAS-mutant cancers may inform therapeutic strategies, as it generates greater dependency on the MAPK pathway for viability. PMID:26359368

  15. Targeting Ligand-Dependent and Ligand-Independent Androgen Receptor Signaling in Prostate Cancer

    DTIC Science & Technology

    2013-10-01

    sub 10nM range efficacy. Our primary objective was to establish a series of compounds blocking the AR ligand-dependent and ligand-independent gene ...of AR driven genes to be more comprehensive and more in line with what is currently known about AR-driven signaling in prostate cancer. We have...developed a robust panel of genes for AR signaling that is reflective of the clinical findings in both ligand dependent and ligand-independent androgen

  16. Multiple, Ligand-Dependent Routes from the Active Site of Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Winn, Peter J.; Wade, Rebecca C.

    2012-02-13

    The active site of liver-specific, drug-metabolizing cytochrome P450 (CYP) monooxygenases is deeply buried in the protein and is connected to the protein surface through multiple tunnels, many of which were found open in different CYP crystal structures. It has been shown that different tunnels could serve as ligand passage routes in different CYPs. However, it is not understood whether one CYP uses multiple routes for substrate access and product release and whether these routes depend on ligand properties. From 300 ns of molecular dynamics simulations of CYP2C9, the second most abundant CYP in the human liver we found four main ligand exit routes, the occurrence of each depending on the ligand type and the conformation of the F-G loop, which is likely to be affected by the CYP-membrane interaction. A non-helical F-G loop favored exit towards the putative membrane-embedded region. Important protein features that direct ligand exit include aromatic residues that divide the active site and whose motions control access to two pathways. The ligands interacted with positively charged residues on the protein surface through hydrogen bonds that appear to select for acidic substrates. The observation of multiple, ligand-dependent routes in a CYP aids understanding of how CYP mutations affect drug metabolism and provides new possibilities for CYP inhibition.

  17. Role of ligand-dependent GR phosphorylation and half-life in determination of ligand-specific transcriptional activity.

    PubMed

    Avenant, Chanel; Ronacher, Katharina; Stubsrud, Elisabeth; Louw, Ann; Hapgood, Janet P

    2010-10-07

    A central question in glucocorticoid mechanism of action via the glucocorticoid receptor (GR) is what determines ligand-selective transcriptional responses. Using a panel of 12 GR ligands, we show that the extent of GR phosphorylation at S226 and S211, GR half-life and transcriptional response, occur in a ligand-selective manner. While GR phosphorylation at S226 was shown to inhibit maximal transcription efficacy, phosphorylation at S211 is required for maximal transactivation, but not for transrepression efficacy. Both ligand-selective GR phosphorylation and half-life correlated with efficacy for transactivation and transrepression. For both expressed and endogenous GR, in two different cell lines, agonists resulted in the greatest extent of phosphorylation and the greatest extent of GR downregulation, suggesting a link between these functions. However, using phosphorylation-deficient GR mutants we established that phosphorylation of the GR at S226 or S211 does not determine the rank order of ligand-selective GR transactivation. These results are consistent with a model whereby ligand-selective GR phosphorylation and half-life are a consequence of upstream events, such as ligand-specific GR conformations, which are maintained in the phosphorylation mutants.

  18. H11-H12 loop retinoic acid receptor mutants exhibit distinct trans-activating and trans-repressing activities in the presence of natural or synthetic retinoids.

    PubMed

    Lefebvre, B; Mouchon, A; Formstecher, P; Lefebvre, P

    1998-06-30

    Retinoids, such as the naturally occurring all-trans-retinoic acid (atRA) and synthetic ligand CD367 modulate ligand-dependent transcription through retinoic acid receptors (RARs). Retinoid binding to RAR is believed to trigger structural transitions in the ligand-binding domain (LBD), leading to helix H1 and helix H12 repositioning and coactivator recruitment and corepressor release. Here, we carried out a detailed mutagenesis analysis of the H11-H12 loop (designated the L box) to study its contribution to hRARalpha activation process. Point mutations that reduced transactivation by atRA also reduced atRA-induced transrepression of AP1 transcription, correlating ligand-induced activation and repression. However, a correlation was not observed with these mutations when tested with another ligand CD367, a synthetic agonist with binding properties identical to those of atRA. Transcription was strongly inhibited in the presence of CD367 for some mutants, thus leading to an inverse agonist activity of this ligand. None of these mutations significantly altered binding affinity for either ligand, indicating that altered transcription was not caused by altered ligand binding by these mutations. Although simple correlations with transcriptional activities were not found, these mutations were also characterized by altered ligand-induced structural transitions, which were distinct for the atRA-hRARalpha or CD367-hRARalpha complexes. These results indicate that amino acids in the L box are involved in specifying trans-repressive and trans-activating properties of the hRARalpha, and support the notion that different agonists induce distinct conformations in the LBD of the receptor.

  19. EGFR Transactivation by Peptide G Protein-Coupled Receptors in Cancer.

    PubMed

    Moody, Terry W; Nuche-Berenguer, Bernardo; Nakamura, Taichi; Jensen, Robert T

    2016-01-01

    Lung cancer kills approximately 1.3 million citizens in the world annually. The tyrosine kinase inhibitors (TKI) erlotinib and gefitinib are effective anti-tumor agents especially in lung cancer patients with epidermal growth factor receptor (EGFR) mutations. The goal is to increase the potency of TKI in lung cancer patients with wild type EGFR. G protein-coupled receptors (GPCR) transactivate the wild type EGFR in lung cancer cells. The GPCR can be activated by peptide agonists causing phosphatidylinositol turnover or stimulation of adenylylcyclase. Recently, nonpeptide antagonists were found to inhibit the EGFR transactivation caused by peptides. Nonpeptide antagonists for bombesin (BB), neurotensin (NTS) and cholecystokinin (CCK) inhibit lung cancer growth and increase the cytotoxicity of gefitinib. The results suggest that GPCR transactivation of the EGFR may play an important role in cancer cell proliferation.

  20. The Shine-Dalgarno sequence of riboswitch-regulated single mRNAs shows ligand-dependent accessibility bursts

    PubMed Central

    Rinaldi, Arlie J.; Lund, Paul E.; Blanco, Mario R.; Walter, Nils G.

    2016-01-01

    In response to intracellular signals in Gram-negative bacteria, translational riboswitches—commonly embedded in messenger RNAs (mRNAs)—regulate gene expression through inhibition of translation initiation. It is generally thought that this regulation originates from occlusion of the Shine-Dalgarno (SD) sequence upon ligand binding; however, little direct evidence exists. Here we develop Single Molecule Kinetic Analysis of RNA Transient Structure (SiM-KARTS) to investigate the ligand-dependent accessibility of the SD sequence of an mRNA hosting the 7-aminomethyl-7-deazaguanine (preQ1)-sensing riboswitch. Spike train analysis reveals that individual mRNA molecules alternate between two conformational states, distinguished by ‘bursts' of probe binding associated with increased SD sequence accessibility. Addition of preQ1 decreases the lifetime of the SD's high-accessibility (bursting) state and prolongs the time between bursts. In addition, ligand-jump experiments reveal imperfect riboswitching of single mRNA molecules. Such complex ligand sensing by individual mRNA molecules rationalizes the nuanced ligand response observed during bulk mRNA translation. PMID:26781350

  1. Ligand-Independent Activation of Platelet-Derived Growth Factor Receptor β during Human Immunodeficiency Virus-Transactivator of Transcription and Cocaine-Mediated Smooth Muscle Hyperplasia.

    PubMed

    Dalvi, Pranjali N; Gupta, Vijayalaxmi G; Griffin, Brooke R; O'Brien-Ladner, Amy; Dhillon, Navneet K

    2015-09-01

    Our previous study supports an additive effect of cocaine to human immunodeficiency virus infection in the development of pulmonary arteriopathy through enhancement of proliferation of pulmonary smooth muscle cells (SMCs), while also suggesting involvement of platelet-derived growth factor receptor (PDGFR) activation in the absence of further increase in PDGF-BB ligand. Redox-related signaling pathways have been shown to regulate tyrosine kinase receptors independent of ligand binding, so we hypothesized that simultaneous treatment of SMCs with transactivator of transcription (Tat) and cocaine may be able to indirectly activate PDGFR through modulation of reactive oxygen species (ROS) without the need for PDGF binding. We found that blocking the binding of ligand using suramin or monoclonal IMC-3G3 antibody significantly reduced ligand-induced autophosphorylation of Y1009 without affecting ligand-independent transphosphorylation of Y934 residue on PDGFRβ in human pulmonary arterial SMCs treated with both cocaine and Tat. Combined treatment of human pulmonary arterial SMCs with cocaine and Tat resulted in augmented production of superoxide radicals and hydrogen peroxide when compared with either treatment alone. Inhibition of this ROS generation prevented cocaine- and Tat-mediated Src activation and transphosphorylation of PDGFRβ at Y934 without any changes in phosphorylation of Y1009, in addition to attenuation of smooth muscle hyperplasia. Furthermore, pretreatment with an Src inhibitor, PP2, also suppressed cocaine- and Tat-mediated enhanced Y934 phosphorylation and smooth muscle proliferation. Finally, we report total abrogation of cocaine- and Tat-mediated synergistic increase in cell proliferation on inhibition of both ligand-dependent and ROS/Src-mediated ligand-independent phosphorylation of PDGFRβ.

  2. Ligand-Dependent Modulation of G Protein Conformation Alters Drug Efficacy.

    PubMed

    Furness, Sebastian George Barton; Liang, Yi-Lynn; Nowell, Cameron James; Halls, Michelle Louise; Wookey, Peter John; Dal Maso, Emma; Inoue, Asuka; Christopoulos, Arthur; Wootten, Denise; Sexton, Patrick Michael

    2016-10-20

    G protein-coupled receptor (GPCR) signaling, mediated by hetero-trimeric G proteins, can be differentially controlled by agonists. At a molecular level, this is thought to occur principally via stabilization of distinct receptor conformations by individual ligands. These distinct conformations control subsequent recruitment of transducer and effector proteins. Here, we report that ligand efficacy at the calcitonin GPCR (CTR) is also correlated with ligand-dependent alterations to G protein conformation. We observe ligand-dependent differences in the sensitivity of the G protein ternary complex to disruption by GTP, due to conformational differences in the receptor-bound G protein hetero-trimer. This results in divergent agonist-dependent receptor-residency times for the hetero-trimeric G protein and different accumulation rates for downstream second messengers. This study demonstrates that factors influencing efficacy extend beyond receptor conformation(s) and expands understanding of the molecular basis for how G proteins control/influence efficacy. This has important implications for the mechanisms that underlie ligand-mediated biased agonism. VIDEO ABSTRACT.

  3. Transrepression and transactivation potencies of inhaled glucocorticoids.

    PubMed

    Dirks, N L; Li, S; Huth, B; Hochhaus, G; Yates, C R; Meibohm, B

    2008-12-01

    The anti-inflammatory activity of inhaled glucocorticoids is primarily mediated through transrepression of pro-inflammatory transcription factors such as AP-1 and NF-kappaB, while systemic side effects are largely attributed to transactivation via glucocorticoid response elements (GRE) in the promoter region of responsive genes. The objective of this study is to investigate whether inhaled corticosteroids exhibit differences in their transactivation and transrepression potencies. A549 human alveolar epithelial type II like cells, stably transfected with a reporter plasmid containing an AP-1, NF-kappaB or GRE induced secreted alkaline phosphatase reporter gene (SEAP), were exposed to a panel of concentrations of the six inhaled and three systemic glucocorticoids. Glucocorticoid-induced changes in SEAP expression were quantified by chemiluminescence. For eight glucocorticoids (budesonide, desisobutyryl-cicle-sonide, dexamethasone, flunisolide, fluocortolone, fluticasone propionate, mometasone furoate, prednisolone) the EC50 for NF-kappaB mediated transrepression was significantly larger than that for both transactivation and transrepression via AP-1. For the remaining glucocorticoid (triamcinolone acetonide), it was greater than that for transactivation. It is concluded that, within the studied cell system, inhaled corticosteroids did not exhibit preferential transrepression, but had higher potencies for transactivation than for transrepression via NF-kappaB and had differential potencies for the two transrepression pathways.

  4. Epidermal Growth Factor Receptor Transactivation: Mechanisms, Pathophysiology, and Potential Therapies in the Cardiovascular System.

    PubMed

    Forrester, Steven J; Kawai, Tatsuo; O'Brien, Shannon; Thomas, Walter; Harris, Raymond C; Eguchi, Satoru

    2016-01-01

    Epidermal growth factor receptor (EGFR) activation impacts the physiology and pathophysiology of the cardiovascular system, and inhibition of EGFR activity is emerging as a potential therapeutic strategy to treat diseases including hypertension, cardiac hypertrophy, renal fibrosis, and abdominal aortic aneurysm. The capacity of G protein-coupled receptor (GPCR) agonists, such as angiotensin II (AngII), to promote EGFR signaling is called transactivation and is well described, yet delineating the molecular processes and functional relevance of this crosstalk has been challenging. Moreover, these critical findings are dispersed among many different fields. The aim of our review is to highlight recent advancements in defining the signaling cascades and downstream consequences of EGFR transactivation in the cardiovascular renal system. We also focus on studies that link EGFR transactivation to animal models of the disease, and we discuss potential therapeutic applications.

  5. Conformational dynamics of ligand-dependent alternating access in LeuT.

    PubMed

    Kazmier, Kelli; Sharma, Shruti; Quick, Matthias; Islam, Shahidul M; Roux, Benoît; Weinstein, Harel; Javitch, Jonathan A; McHaourab, Hassane S

    2014-05-01

    The leucine transporter (LeuT) from Aquifex aeolicus is a bacterial homolog of neurotransmitter/sodium symporters (NSSs) that catalyze reuptake of neurotransmitters at the synapse. Crystal structures of wild-type and mutants of LeuT have been interpreted as conformational states in the coupled transport cycle. However, the mechanistic identities inferred from these structures have not been validated, and the ligand-dependent conformational equilibrium of LeuT has not been defined. Here, we used distance measurements between spin-label pairs to elucidate Na(+)- and leucine-dependent conformational changes on the intracellular and extracellular sides of the transporter. The results identify structural motifs that underlie the isomerization of LeuT between outward-facing, inward-facing and occluded states. The conformational changes reported here present a dynamic picture of the alternating-access mechanism of LeuT and NSSs that is different from the inferences reached from currently available structural models.

  6. Ligand dependent restoration of human TLR3 signaling and death in p53 mutant cells

    PubMed Central

    Menendez, Daniel; Lowe, Julie M.; Snipe, Joyce; Resnick, Michael A.

    2016-01-01

    Diversity within the p53 transcriptional network can arise from a matrix of changes that include target response element sequences and p53 expression level variations. We previously found that wild type p53 (WT p53) can regulate expression of most innate immune-related Toll-like-receptor genes (TLRs) in human cells, thereby affecting immune responses. Since many tumor-associated p53 mutants exhibit change-of-spectrum transactivation from various p53 targets, we examined the ability of twenty-five p53 mutants to activate endogenous expression of the TLR gene family in p53 null human cancer cell lines following transfection with p53 mutant expression vectors. While many mutants retained the ability to drive TLR expression at WT levels, others exhibited null, limited, or change-of-spectrum transactivation of TLR genes. Using TLR3 signaling as a model, we show that some cancer-associated p53 mutants amplify cytokine, chemokine and apoptotic responses after stimulation by the cognate ligand poly(I:C). Furthermore, restoration of WT p53 activity for loss-of-function p53 mutants by the p53 reactivating drug RITA restored p53 regulation of TLR3 gene expression and enhanced DNA damage-induced apoptosis via TLR3 signaling. Overall, our findings have many implications for understanding the impact of WT and mutant p53 in immunological responses and cancer therapy. PMID:27533082

  7. Sox5 induces epithelial to mesenchymal transition by transactivation of Twist1

    SciTech Connect

    Pei, Xin-Hong; Lv, Xin-Quan; Li, Hui-Xiang

    2014-03-28

    Highlights: • Depletion of Sox5 inhibits breast cancer proliferation, migration, and invasion. • Sox5 transactivates Twist1 expression. • Sox5 induces epithelial to mesenchymal transition through transactivation of Twist1 expression. - Abstract: The epithelial to mesenchymal transition (EMT), a highly conserved cellular program, plays an important role in normal embryogenesis and cancer metastasis. Twist1, a master regulator of embryonic morphogenesis, is overexpressed in breast cancer and contributes to metastasis by promoting EMT. In exploring the mechanism underlying the increased Twist1 in breast cancer cells, we found that the transcription factor SRY (sex-determining region Y)-box 5(Sox5) is up-regulation in breast cancer cells and depletion of Sox5 inhibits breast cancer cell proliferation, migration, and invasion. Furthermore, depletion of Sox5 in breast cancer cells caused a dramatic decrease in Twist1 and chromosome immunoprecipitation assay showed that Sox5 can bind directly to the Twist1 promoter, suggesting that Sox5 transactivates Twist1 expression. We further demonstrated that knockdown of Sox5 up-regulated epithelial phenotype cell biomarker (E-cadherin) and down-regulated mesenchymal phenotype cell biomarkers (N-cadherin, Vimentin, and Fibronectin 1), resulting in suppression of EMT. Our study suggests that Sox5 transactivates Twist1 expression and plays an important role in the regulation of breast cancer progression.

  8. Ligand-dependent localization and function of ORP-VAP complexes at membrane contact sites.

    PubMed

    Weber-Boyvat, Marion; Kentala, Henriikka; Peränen, Johan; Olkkonen, Vesa M

    2015-05-01

    Oxysterol-binding protein/OSBP-related proteins (ORPs) constitute a conserved family of sterol/phospholipid-binding proteins with lipid transporter or sensor functions. We investigated the spatial occurrence and regulation of the interactions of human OSBP/ORPs or the S. cerevisiae orthologs, the Osh (OSBP homolog) proteins, with their endoplasmic reticulum (ER) anchors, the VAMP-associated proteins (VAPs), by employing bimolecular fluorescence complementation and pull-down set-ups. The ORP-VAP interactions localize frequently at distinct subcellular sites, shown in several cases to represent membrane contact sites (MCSs). Using established ORP ligand-binding domain mutants and pull-down assays with recombinant proteins, we show that ORP liganding regulates the ORP-VAP association, alters the subcellular targeting of ORP-VAP complexes, or modifies organelle morphology. There is distinct protein specificity in the effects of the mutants on subcellular targeting of ORP-VAP complexes. We provide evidence that complexes of human ORP2 and VAPs at ER-lipid droplet interfaces regulate the hydrolysis of triglycerides and lipid droplet turnover. The data suggest evolutionarily conserved, complex ligand-dependent functions of ORP-VAP complexes at MCSs, with implications for cellular lipid homeostasis and signaling.

  9. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

    PubMed

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

    2015-04-28

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

  10. A Potent HER3 Monoclonal Antibody That Blocks Both Ligand-Dependent and -Independent Activities: Differential Impacts of PTEN Status on Tumor Response.

    PubMed

    Xiao, Zhan; Carrasco, Rosa A; Schifferli, Kevin; Kinneer, Krista; Tammali, Ravinder; Chen, Hong; Rothstein, Ray; Wetzel, Leslie; Yang, Chunning; Chowdhury, Partha; Tsui, Ping; Steiner, Philipp; Jallal, Bahija; Herbst, Ronald; Hollingsworth, Robert E; Tice, David A

    2016-04-01

    HER3/ERBB3 is a kinase-deficient member of the EGFR family receptor tyrosine kinases (RTK) that is broadly expressed and activated in human cancers. HER3 is a compelling cancer target due to its important role in activation of the oncogenic PI3K/AKT pathway. It has also been demonstrated to confer tumor resistance to a variety of cancer therapies, especially targeted drugs against EGFR and HER2. HER3 can be activated by its ligand (heregulin/HRG), which induces HER3 heterodimerization with EGFR, HER2, or other RTKs. Alternatively, HER3 can be activated in a ligand-independent manner through heterodimerization with HER2 in HER2-amplified cells. We developed a fully human mAb against HER3 (KTN3379) that efficiently suppressed HER3 activity in both ligand-dependent and independent settings. Correspondingly, KTN3379 inhibited tumor growth in divergent tumor models driven by either ligand-dependent or independent mechanisms in vitro and in vivo Most intriguingly, while investigating the mechanistic underpinnings of tumor response to KTN3379, we discovered an interesting dichotomy in that PTEN loss, a frequently occurring oncogenic lesion in a broad range of cancer types, substantially blunted the tumor response in HER2-amplified cancer, but not in the ligand-driven cancer. To our knowledge, this represents the first study ascertaining the impact of PTEN loss on the antitumor efficacy of a HER3 mAb. KTN3379 is currently undergoing a phase Ib clinical trial in patients with advanced solid tumors. Our current study may help us optimize patient selection schemes for KTN3379 to maximize its clinical benefits. Mol Cancer Ther; 15(4); 689-701. ©2016 AACR.

  11. Ligand-dependent conformational equilibria of serum albumin revealed by tryptophan fluorescence quenching.

    PubMed Central

    Chadborn, N; Bryant, J; Bain, A J; O'Shea, P

    1999-01-01

    Ligand-dependent structural changes in serum albumin are suggested to underlie its role in physiological solute transport and receptor-mediated cellular selection. Evidence of ligand-induced (oleic acid) structural changes in serum albumin are shown in both time-resolved and steady-state fluorescence quenching and anisotropy measurements of tryptophan 214 (Trp214). These studies were augmented with column chromatography separations. It was found that both the steady-state and time-resolved Stern-Volmer collisional quenching studies of Trp214 with acrylamide pointed to the existence of an oleate-dependent structural transformation. The bimolecular quenching rate constant of defatted human serum albumin, 1.96 x 10(9) M-1 s-1, decreased to 0.94 x 10(9) M-1 s-1 after incubation with oleic acid (9:1). Furthermore, Stern-Volmer quenching studies following fractionation of the structural forms by hydrophobic interaction chromatography were in accordance with this interpretation. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information of motion within the protein together with the whole protein molecule. Characteristic changes in these motions were observed after the binding of oleate to albumin. The addition of oleate was accompanied by an increase in the rotational diffusion time of the albumin molecule from approximately 22 to 33.6 ns. Within the body of the protein, however, the rotational diffusion time for Trp214 exhibited a slight decrease from 191 to 182 ps and was accompanied by a decrease in the extent of the angular motion of Trp214, indicating a transition after oleate binding to a more spatially restricted but less viscous environment. PMID:10096914

  12. ERK2 phosphorylation of EBNA1 serine 383 residue is important for EBNA1-dependent transactivation

    PubMed Central

    Lee, Eun Kyung; Choi, Eun Young; Kang, Myung-Soo

    2016-01-01

    Functional inhibition of Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) can cause the death of EBV infected cells. In this study, a bioinformatics tool predicted the existence of putative extracellular signal-regulated kinase (ERK) docking and substrate consensus sites on EBNA1, suggesting that ERK2 could bind to and phosphorylate EBNA1. In accordance, ERK2 was found to phosphorylate EBNA1 serine 383 in a reaction suppressed by H20 (a structural congener of the ERK inhibitor), U0126 (an inhibitor of MEK kinase), and mutations at substrate (S383A) or putative ERK docking sites. Wild-type (S383) and phosphomimetic (S383D) EBNA1 demonstrated comparable transactivation function, which was suppressed by H20 or U0126. In contrast, non-phosphorylated EBNA1 mutants displayed significantly impaired transactivation activity. ERK2 knock-down by siRNA, or treatment with U0126 or H20 repressed EBNA1-dependent transactivation. Collectively, these data indicate that blocking ERK2-directed phosphorylation can suppress EBNA1-transactivation function in latent EBV-infected cells, validating ERK2 as a drug target for EBV-associated disorders. PMID:27009860

  13. Pin1 promotes GR transactivation by enhancing recruitment to target genes.

    PubMed

    Poolman, Toryn M; Farrow, Stuart N; Matthews, Laura; Loudon, Andrew S; Ray, David W

    2013-10-01

    The glucocorticoid receptor (GR) is a ligand activated transcription factor, serving to regulate both energy metabolism and immune functions. Factors that influence cellular sensitivity to glucocorticoids (GC) are therefore of great interest. The N-terminal of the GR contains numerous potential proline-directed phosphorylation sites, some of which can regulate GR transactivation. Unrestricted proline isomerisation can be inhibited by adjacent serine phosphorylation and requires a prolyl isomerise, Pin1. Pin1 therefore determines the functional outcome of proline-directed kinases acting on the GR, as cis/trans isomers are distinct pools with different interacting proteins. We show that Pin1 mediates GR transactivation, but not GR trans-repression. Two N-terminal GR serines, S203 and S211, are targets for Pin1 potentiation of GR transactivation, establishing a direct link between Pin1 and the GR. We also demonstrate GC-activated co-recruitment of GR and Pin1 to the GILZ gene promoter. The Pin1 effect required both its WW and catalytic domains, and GR recruitment to its GRE was Pin1-dependent. Therefore, Pin1 is a selective regulator of GR transactivation, acting through N-terminal phospho-serine residues to regulate GR recruitment to its target sites in the genome. As Pin1 is dysregulated in disease states, this interaction may contribute to altered GC action in inflammatory conditions.

  14. G-protein alpha-s and -12 subunits are involved in androgen-stimulated PI3K activation and androgen receptor transactivation in prostate cancer cells

    PubMed Central

    Liu, Jianjun; Youn, Hyewon; Yang, Jun; Du, Ningchao; Liu, Jihong; Liu, Hongwei; Li, Benyi

    2011-01-01

    BACKGROUND The androgen receptor (AR) is a ligand-dependent transcription factor that mediates androgenic hormone action in cells. We recently demonstrated the involvement of phosphoinositide 3-OH kinase (PI3K) p110beta in AR transactivation and gene expression. In this study, we determined the upstream signals that lead to PI3K/p110beta activation and AR transactivation after androgen stimulation. METHODS Human prostate cancer LAPC-4 and 22Rv1 cell lines were used for the experiments. AR transactivation was assessed using an androgen responsive element-driven luciferase (ARE-LUC) assay. Cell proliferation was examined using BrdU incorporation and MTT assays. Target genes were silenced using small interfering RNA (siRNA) approach. Gene expression was evaluated at the mRNA level (real-time RT-PCR) and protein level (Western blot). PI3K kinase activities were measured using immunoprecipitation-based in vitro kinase assay. The AR-DNA binding activity was determined using Chromatin-immunoprecipitation (ChIP) assay. RESULTS First, at the cellular plasma membrane, disrupting the integrity of caveolae microdomain with methyl-β- cyclodextrin (M-β-CD) abolished androgen-induced AR transactivation and gene expression. Then, knocking down caveolae structural proteins caveolin-1 or -2 with the gene-specific siRNAs significantly reduced androgen-induced AR transactivation. Next, silencing Gαs and Gα12 genes but not other G-proteins blocked androgen-induced AR transactivation and cell proliferation. Consistently, overexpression of Gαs or Gα12 active mutants enhanced androgen-induced AR transactivation, of which Gαs active mutant sensitized the AR to castration-level of androgen (R1881). Most interestingly, knocking down Gαs but not Gα12 subunit significantly suppressed androgen-stimulated PI3K p110beta activation. However, chromatin-immunoprecipitation (ChIP) analysis revealed that both Gαs or Gα12 subunits are involved in androgen-induced AR interaction with the AR

  15. Crosstalk between the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and the vitamin D receptor (VDR) in human breast cancer cells: PPAR{gamma} binds to VDR and inhibits 1{alpha},25-dihydroxyvitamin D{sub 3} mediated transactivation

    SciTech Connect

    Alimirah, Fatouma; Peng, Xinjian; Yuan, Liang; Mehta, Rajeshwari R.; Knethen, Andreas von; Choubey, Divaker; Mehta, Rajendra G.

    2012-11-15

    Heterodimerization and cross-talk between nuclear hormone receptors often occurs. For example, estrogen receptor alpha (ER{alpha}) physically binds to peroxisome proliferator-activated receptor gamma (PPAR{gamma}) and inhibits its transcriptional activity. The interaction between PPAR{gamma} and the vitamin D receptor (VDR) however, is unknown. Here, we elucidate the molecular mechanisms linking PPAR{gamma} and VDR signaling, and for the first time we show that PPAR{gamma} physically associates with VDR in human breast cancer cells. We found that overexpression of PPAR{gamma} decreased 1{alpha},25-dihydroxyvitamin D{sub 3} (1,25D{sub 3}) mediated transcriptional activity of the vitamin D target gene, CYP24A1, by 49% and the activity of VDRE-luc, a vitamin D responsive reporter, by 75% in T47D human breast cancer cells. Deletion mutation experiments illustrated that helices 1 and 4 of PPAR{gamma}'s hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPAR{gamma}'s AF2 domain attenuated its repressive action on 1,25D{sub 3} transactivation, indicating that this domain is integral in inhibiting VDR signaling. PPAR{gamma} was also found to compete with VDR for their binding partner retinoid X receptor alpha (RXR{alpha}). Overexpression of RXR{alpha} blocked PPAR{gamma}'s suppressive effect on 1,25D{sub 3} action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPAR{gamma} and VDR pathways. -- Highlights: PPAR{gamma}'s role on 1{alpha},25-dihydroxyvitamin D{sub 3} transcriptional activity is examined. Black-Right-Pointing-Pointer PPAR{gamma} physically binds to VDR and inhibits 1{alpha},25-dihydroxyvitamin D{sub 3} action. Black-Right-Pointing-Pointer PPAR{gamma}'s hinge and ligand binding domains are important for this inhibitory effect. Black-Right-Pointing-Pointer PPAR{gamma} competes with VDR for the availability of their binding partner, RXR{alpha}.

  16. ATF2 impairs glucocorticoid receptor–mediated transactivation in human CD8+ T cells

    PubMed Central

    Li, Ling-bo; Leung, Donald Y. M.; Strand, Matthew J.

    2007-01-01

    Chronic inflammatory diseases often have residual CD8+ T-cell infiltration despite treatment with systemic corticosteroids, which suggests divergent steroid responses between CD4+ and CD8+ cells. To examine steroid sensitivity, dexamethasone (DEX)–induced histone H4 lysine 5 (K5) acetylation and glucocorticoid receptor α (GCRα) translocation were evaluated. DEX treatment for 6 hours significantly induced histone H4 K5 acetylation in normal CD4+ cells (P = .001) but not in CD8+ cells. DEX responses were functionally impaired in CD8+ compared with CD4+ cells when using mitogen-activated protein kinase phosphatase (1 hour; P = .02) and interleukin 10 mRNA (24 hours; P = .004) induction as a readout of steroid-induced transactivation. Normal DEX-induced GCRα nuclear translocation and no significant difference in GCRα and GCRβ mRNA expression were observed in both T-cell types. In addition, no significant difference in SRC-1, p300, or TIP60 expression was found. However, activating transcription factor-2 (ATF2) expression was significantly lower in CD8+ compared with CD4+ cells (P = .009). Importantly, inhibition of ATF2 expression by small interfering RNA in CD4+ cells resulted in inhibition of DEX-induced transactivation in CD4+ cells. The data indicate refractory steroid-induced transactivation but similar steroid-induced transrepression of CD8+ cells compared with CD4+ cells caused by decreased levels of the histone acetyltransferase ATF2. PMID:17525285

  17. Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

    PubMed Central

    Cattaneo, Fabio; Guerra, Germano; Parisi, Melania; De Marinis, Marta; Tafuri, Domenico; Cinelli, Mariapia; Ammendola, Rosario

    2014-01-01

    G protein-coupled receptors (GPCRs) are seven transmembrane-spanning proteins belonging to a large family of cell-surface receptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK) occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS) are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca2+ from endoplasmic reticulum leads to the activation of protein kinase C (PKC) isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors. Herein, we

  18. Biofunction-assisted aptasensors based on ligand-dependent 3' processing of a suppressor tRNA in a wheat germ extract.

    PubMed

    Ogawa, Atsushi; Tabuchi, Junichiro

    2015-06-28

    We have developed a novel type of biofunction-assisted aptasensor that harnesses ligand-dependent 3' processing of a premature amber suppressor tRNA and the subsequent amber suppression of a reporter gene in a wheat germ extract.

  19. Correction: Biofunction-assisted aptasensors based on ligand-dependent 3' processing of a suppressor tRNA in a wheat germ extract.

    PubMed

    Ogawa, Atsushi; Tabuchi, Junichiro

    2015-08-14

    Correction for 'Biofunction-assisted aptasensors based on ligand-dependent 3' processing of a suppressor tRNA in a wheat germ extract' by Atsushi Ogawa et al., Org. Biomol. Chem., 2015, 13, 6681-6685.

  20. Rational design of artificial riboswitches based on ligand-dependent modulation of internal ribosome entry in wheat germ extract and their applications as label-free biosensors.

    PubMed

    Ogawa, Atsushi

    2011-03-01

    Riboswitches are RNA elements in mRNA that control gene expression in cis in response to their specific ligands. Because artificial riboswitches make it possible to regulate any gene with an arbitrary molecule, they are expected to function as biosensors, in which the output is easily detectable protein expression. I report herein a fully rational design strategy for artificially constructing novel riboswitches that work in a eukaryotic cell-free translation system (wheat germ extract). In these riboswitches, translation mediated by an internal ribosome entry site (IRES) is promoted only in the presence of a specific ligand (ON), while it is inhibited in the absence of the ligand (OFF). The first rationally designed riboswitch, which is regulated by theophylline, showed a high switching efficiency and dependency on theophylline. In addition, based on the design of the theophylline-dependent riboswitch, other three kinds of riboswitches controlled by FMN, tetracycline, and sulforhodamine B, were constructed only by calculating the ΔG value of one stem-loop structure. The rational design strategy described herein is therefore useful for easily producing various ligand-dependent riboswitches, which are available as biosensors for detecting their ligands.

  1. Functional differences between the Oct2 transactivation domains determine the transactivation potential of individual Oct2 isoforms.

    PubMed Central

    Annweiler, A; Zwilling, S; Wirth, T

    1994-01-01

    The lymphocyte specific transcription factor Oct2 is involved in mediating the B-cell specific transcriptional activity of the octamer motif. Mutational analyses in the context of the complete Oct2 protein had indicated that Oct2 contains two transactivation domains. These two domains appeared to be redundant for activation from a promoter proximal position, whereas stimulation from a remote enhancer position specifically required the C-terminal transactivation domain and an additional B-cell restricted activity. We have generated fusion proteins between the DNA binding domain of the yeast Gal4 transcription factor and individual Oct2 protein domains to analyze their transactivation potential separately. We show that both N- and C-terminal domains can stimulate transcription from a promoter proximal position independently. However, only the C-terminal transactivation domain activates from a distance and it can only do so in B-cells. The C-terminal transactivation domain represents a composite transactivation domain. Whereas removal of just 9 aminoacids from the extreme C-terminus lead to a complete inactivation of this domain deletions from the other side resulted in a gradual loss of activity. We also characterized the transactivation potential of different N-terminal regions of Oct2 generated by alternative splicing. We show that the N-terminus of one of the isoforms, Oct2.3, contains a negative regulatory domain (NRD), which can inactivate the neighbouring glutamine-rich transactivation in cis. The presence of this NRD affects the overall phosphorylation state of the Oct2 protein. This result suggests that the mechanism of inactivation might involve differential protein phosphorylation. Images PMID:7937153

  2. Cleavage of the transactivation-inhibitory domain of p63 by caspases enhances apoptosis.

    PubMed

    Sayan, Berna S; Sayan, A Emre; Yang, Ai Li; Aqeilan, Rami I; Candi, Eleonora; Cohen, Gerald M; Knight, Richard A; Croce, Carlo M; Melino, Gerry

    2007-06-26

    p63 is a p53-related transcription factor. Utilization of two different promoters and alternative splicing at the C terminus lead to generation of six isoforms. The alpha isoforms of TAp63 and DeltaNp63 contain a transactivation-inhibitory (TI) domain at the C termini, which can bind to the transactivation (TA) domain and inhibit its transcriptional activity. Consequently, TAp63alpha can directly inhibit its activity through an intramolecular interaction; similarly, DeltaNp63alpha can inhibit the activity of the active TAp63 isoforms through an intermolecular interaction. In this work, we demonstrate that after induction of apoptosis, the TI domain of the p63alpha isoforms is cleaved by activated caspases. Cleavage of DeltaNp63alpha relieves its inhibitory effect on the transcriptionally active p63 proteins, and the cleavage of TAp63alpha results in production of a TAp63 protein with enhanced transcriptional activity. In agreement with these data, generation of the N-terminal TAp63 fragment has a role in apoptosis because stable cell lines expressing wild-type TAp63 are more sensitive to apoptosis compared with cells expressing the noncleavable mutant. We also used a model system in which TAp63 expression was induced by trichostatin-A treatment in HCT116 cells. Trichostatin-A sensitized these cells to apoptosis, and this sensitization was associated with cleavage of up-regulated p63.

  3. TGF{beta} induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

    SciTech Connect

    Ebi, Masahide; Kataoka, Hiromi; Shimura, Takaya; Kubota, Eiji; Hirata, Yoshikazu; Mizushima, Takashi; Mizoshita, Tsutomu; Tanaka, Mamoru; Mabuchi, Motoshi; Tsukamoto, Hironobu; Tanida, Satoshi; Kamiya, Takeshi; Higashiyama, Shigeki; Joh, Takashi

    2010-11-19

    Research highlights: {yields} TGF{beta} induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. {yields} TGF{beta} induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. {yields} TGF{beta} enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. {yields} Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGF{beta}. {yields} ADAM17 may play a crucial role in this TGF{beta}-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGF{beta}) is known to potently inhibit cell growth. Loss of responsiveness to TGF{beta} inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGF{beta} and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGF{beta}. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGF{beta} was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGF{beta} was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGF{beta}-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGF{beta} induced shedding of proHB-EGF allowing HB-EGF-CTF to

  4. EGFR trans-activation mediates pleiotrophin-induced activation of Akt and Erk in cultured osteoblasts.

    PubMed

    Fan, Jian-Bo; Liu, Wei; Yuan, Kun; Zhu, Xin-Hui; Xu, Da-Wei; Chen, Jia-Jia; Cui, Zhi-Ming

    2014-05-09

    Pleiotrophin (Ptn) plays an important role in bone growth through regulating osteoblasts' functions. The underlying signaling mechanisms are not fully understood. In the current study, we found that Ptn induced heparin-binding epidermal growth factor (HB-EGF) release to trans-activate EGF-receptor (EGFR) in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, Ptn activated Akt and Erk signalings in cultured osteoblasts. The EGFR inhibitor AG1478 as well as the monoclonal antibody against HB-EGF (anti-HB-EGF) significantly inhibited Ptn-induced EGFR activation and Akt and Erk phosphorylations in MC3T3-E1 cells and primary osteoblasts. Further, EGFR siRNA depletion or dominant negative mutation suppressed also Akt and Erk activation in MC3T3-E1 cells. Finally, we observed that Ptn increased alkaline phosphatase (ALP) activity and inhibited dexamethasone (Dex)-induced cell death in both MC3T3-E1 cells and primary osteoblasts, such effects were alleviated by AG1478 or anti-HB-EGF. Together, these results suggest that Ptn-induced Akt/Erk activation and some of its pleiotropic functions are mediated by EGFR trans-activation in cultured osteoblasts.

  5. Suppression of estrogen receptor-alpha transactivation by thyroid transcription factor-2 in breast cancer cells

    SciTech Connect

    Park, Eunsook; Gong, Eun-Yeung; Romanelli, Maria Grazia; Lee, Keesook

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer TTF-2 was expressed in mammary glands and breast cancer cells. Black-Right-Pointing-Pointer TTF-2 repressed ER{alpha} transactivation. Black-Right-Pointing-Pointer TTF-2 inhibited the proliferation of breast cancer cells. -- Abstract: Estrogen receptors (ERs), which mediate estrogen actions, regulate cell growth and differentiation of a variety of normal tissues and hormone-responsive tumors through interaction with cellular factors. In this study, we show that thyroid transcription factor-2 (TTF-2) is expressed in mammary gland and acts as ER{alpha} co-repressor. TTF-2 inhibited ER{alpha} transactivation in a dose-dependent manner in MCF-7 breast cancer cells. In addition, TTF-2 directly bound to and formed a complex with ER{alpha}, colocalizing with ER{alpha} in the nucleus. In MCF-7/TTF-2 stable cell lines, TTF-2 repressed the expression of endogenous ER{alpha} target genes such as pS2 and cyclin D1 by interrupting ER{alpha} binding to target promoters and also significantly decreased cell proliferation. Taken together, these data suggest that TTF-2 may modulate the function of ER{alpha} as a corepressor and play a role in ER-dependent proliferation of mammary cells.

  6. Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

    PubMed

    Sugiyama, Y; Sasajima, J; Mizukami, Y; Koizumi, K; Kawamoto, T; Ono, Y; Karasaki, H; Tanabe, H; Fujiya, M; Kohgo, Y

    2016-06-01

    The hedgehog pathway is known to promote proliferation of pancreatic ductal adenocarcinoma (PDA) and has been shown to restrain tumor progression. To understand how hedgehog causes these effects, we sought to carefully examine protein expression of hedgehog signaling components during different tumor stages. Genetically engineered mice, Pdx1-Cre;LSL-KrasG12D and Pdx1-Cre;LSL-KrasG12D;p53lox/+, were utilized to model distinct phases of tumorigenesis, pancreatic intraepithelial neoplasm (PanIN) and PDA. Human pancreatic specimens of intraductal papillary mucinous neoplasm (IPMN) and PDA were also employed. PanIN and IPMN lesions highly express Sonic Hedgehog, at a level that is slightly higher than that observed in PDA. GLI2 protein is also expressed in both PanIN/IPMN and PDA. Although there was no difference in the nuclear staining, the cytoplasmic GLI2 level in PDA was modest in comparison to that in PanIN/IPMN. Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDA was significantly attenuated. There were no differences in expression of Patched1 at early and late stages. Finally, a strong correlation between Sonic Hedgehog and GLI2 staining was found in both human and murine pancreatic tumors. The results indicate that the GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms.

  7. Transcription activation by the siderophore sensor Btr is mediated by ligand-dependent stimulation of promoter clearance

    PubMed Central

    Gaballa, Ahmed; MacLellan, Shawn; Helmann, John D.

    2012-01-01

    Bacterial transcription factors often function as DNA-binding proteins that selectively activate or repress promoters, although the biochemical mechanisms vary. In most well-understood examples, activators function by either increasing the affinity of RNA polymerase (RNAP) for the target promoter, or by increasing the isomerization of the initial closed complex to the open complex. We report that Bacillus subtilis Btr, a member of the AraC family of activators, functions principally as a ligand-dependent activator of promoter clearance. In the presence of its co-activator, the siderophore bacillibactin (BB), the Btr:BB complex enhances productive transcription, while having only modest effects on either RNAP promoter association or the production of abortive transcripts. Btr binds to two direct repeat sequences adjacent to the −35 region; recognition of the downstream motif is most important for establishing a productive interaction between the Btr:BB complex and RNAP. The resulting Btr:BB dependent increase in transcription enables the production of the ferric-BB importer to be activated by the presence of its cognate substrate. PMID:22210890

  8. Anti-inflammatory and PPAR transactivational properties of flavonoids from the roots of Sophora flavescens.

    PubMed

    Quang, Tran Hong; Ngan, Nguyen Thi Thanh; Minh, Chau Van; Kiem, Phan Van; Tai, Bui Huu; Nhiem, Nguyen Xuan; Thao, Nguyen Phuong; Luyen, Bui Thi Thuy; Yang, Seo Young; Kim, Young Ho

    2013-09-01

    Anti-inflammatory and peroxisome proliferator-activated receptors (PPARs) transactivational effects of nine compounds (1 - 9) from the roots of Sophora flavescens were evaluated using NF-κB-luciferase, reverse transcriptase polymerase chain reaction, peroxisome proliferator response element (PPRE)-luciferase, and GAL-4-PPAR chimera assays. Compounds 4 and 8 significantly inhibited TNFα-induced NF-κB transcriptional activity in HepG2 cells in a dose-dependent manner, with IC₅₀ values of 4.0 and 4.4 μM, respectively. Furthermore, the transcriptional inhibitory function of these compounds was confirmed by a decrease in cyclooxgenase 2 and inducible nitric oxide synthase gene expression levels in HepG2 cells. Compounds 1, 3, 5, 6, 8, and 9 significantly activated the transcription of PPARs in a dose-dependent manner, with EC₅₀ values ranging from 1.1 to 13.0 μM. Compounds 1, 3, 5, 6, 8, and 9 exhibited dose-dependent PPARα transactivational activity, with EC₅₀ values in a range of 0.9 - 16.0 μM. Compounds 1, 3, 8, and 9 also significantly upregulated PPARγ activity in a dose-dependent manner, with EC₅₀ values of 10.5, 6.6, 15.7, and 1.6 μM, whereas compounds 1, 8, and 9 demonstrated transactivational PPARβ(δ) effects with EC₅₀ values of 11.4, 10.3, and 1.5 μM, respectively. These results provide a scientific rationale for the use of the roots of S. flavescens and warrant further studies to develop new agents for the prevention and treatment of inflammatory and metabolic diseases.

  9. Nine-amino-acid transactivation domain: establishment and prediction utilities.

    PubMed

    Piskacek, Simona; Gregor, Martin; Nemethova, Maria; Grabner, Martin; Kovarik, Pavel; Piskacek, Martin

    2007-06-01

    Here we describe the establishment and prediction utilities for a novel nine-amino-acid transactivation domain, 9aa TAD, that is common to the transactivation domains of a large number of yeast and animal transcription factors. We show that the 9aa TAD motif is required for the function of the transactivation domain of Gal4 and the related transcription factors Oaf1 and Pip2. The 9aa TAD possesses an autonomous transactivation activity in yeast and mammalian cells. Using sequence alignment and experimental data we derived a pattern that can be used for 9aa TAD prediction. The pattern allows the identification of 9aa TAD in other Gal4 family members or unrelated yeast, animal, and viral transcription factors. Thus, the 9aa TAD represents the smallest known denominator for a broad range of transcription factors. The wide occurrence of the 9aa TAD suggests that this domain mediates conserved interactions with general transcriptional cofactors. A computational search for the 9aa TAD is available online from National EMBnet-Node Austria at http://www.at.embnet.org/toolbox/9aatad/.

  10. Gastrointestinal hormones stimulate growth of Foregut Neuroendocrine Tumors by transactivating the EGF receptor.

    PubMed

    Di Florio, Alessia; Sancho, Veronica; Moreno, Paola; Delle Fave, Gianfranco; Jensen, Robert T

    2013-03-01

    Foregut neuroendocrine tumors [NETs] usually pursuit a benign course, but some show aggressive behavior. The treatment of patients with advanced NETs is marginally effective and new approaches are needed. In other tumors, transactivation of the EGF receptor (EGFR) by growth factors, gastrointestinal (GI) hormones and lipids can stimulate growth, which has led to new treatments. Recent studies show a direct correlation between NET malignancy and EGFR expression, EGFR inhibition decreases basal NET growth and an autocrine growth effect exerted by GI hormones, for some NETs. To determine if GI hormones can stimulate NET growth by inducing transactivation of EGFR, we examined the ability of EGF, TGFα and various GI hormones to stimulate growth of the human foregut carcinoid,BON, the somatostatinoma QGP-1 and the rat islet tumor,Rin-14B-cell lines. The EGFR tyrosine-kinase inhibitor, AG1478 strongly inhibited EGF and the GI hormones stimulated cell growth, both in BON and QGP-1 cells. In all the three neuroendocrine cell lines studied, we found EGF, TGFα and the other growth-stimulating GI hormones increased Tyr(1068) EGFR phosphorylation. In BON cells, both the GI hormones neurotensin and a bombesin analogue caused a time- and dose-dependent increase in EGFR phosphorylation, which was strongly inhibited by AG1478. Moreover, we found this stimulated phosphorylation was dependent on Src kinases, PKCs, matrix metalloproteinase activation and the generation of reactive oxygen species. These results raise the possibility that disruption of this signaling cascade by either EGFR inhibition alone or combined with receptor antagonists may be a novel therapeutic approach for treatment of foregut NETs/PETs.

  11. Gastrointestinal hormones stimulate growth of Foregut Neuroendocrine Tumors by transactivating the EGF receptor

    PubMed Central

    Di Florio, Alessia; Sancho, Veronica; Moreno, Paola; Fave, Gianfranco Delle; Jensen, Robert T.

    2012-01-01

    Foregut Neuroendocrine Tumors[NETs] usually pursuit a benign course, but some show aggressive behavior. The treatment of patients with advanced NETs is marginally effective and new approaches are needed. In other tumors, transactivation of the EGF receptor(EGFR) by growth factors, gastrointestinal(GI) hormones and lipids can stimulate growth, which has led to new treatments. Recent studies show a direct correlation between NET malignancy and EGFR expression, EGFR inhibition decreases basal NET growth and an autocrine growth effect exerted by GI hormones, for some NETs. To determine if GI hormones can stimulate NET growth by inducing transactivation of EGFR, we examined the ability of EGF, TGFα and various GI hormones to stimulate growth of the human foregut carcinoid, BON, the somatostatinoma QGP-1 and the rat islet tumor, Rin-14B-cell lines. The EGFR tyrosine-kinase inhibitor, AG1478 strongly inhibited EGF and the GI hormones stimulated cell growth, both in BON and QGP-1 cells. In all the three neuroendocrine cell lines studied, we found EGF, TGFα and the other growth-stimulating GI hormones increased Tyr1068 EGFR phosphorylation. In BON cells, both the GI hormones neurotensin and a bombesin analogue caused a time- and dose-dependent increase in EGFR phosphorylation, which was strongly inhibited by AG1478. Moreover, we found this stimulated phosphorylation was dependent on Src kinases, PKCs, matrix metalloproteinase activation and the generation of reactive oxygen species. These results raise the possibility that disruption of this signaling cascade by either EGFR inhibition alone or combined with receptor antagonists may be a novel therapeutic approach for treatment of foregut NETs/PETs. PMID:23220008

  12. Ligand-Dependent Disorder of Loop Observed in Extended-Spectrum SHV-Type beta-Lactamase

    SciTech Connect

    J Sampson; W Ke; C Bethel; S Pagadala; M Nottingham; R Bonomo; J Buynak; F van den Akker

    2011-12-31

    Among Gram-negative bacteria, resistance to {beta}-lactams is mediated primarily by {beta}-lactamases (EC 3.2.6.5), periplasmic enzymes that inactivate {beta}-lactam antibiotics. Substitutions at critical amino acid positions in the class A {beta}-lactamase families result in enzymes that can hydrolyze extended-spectrum cephalosporins, thus demonstrating an 'extended-spectrum' {beta}-lactamase (ESBL) phenotype. Using SHV ESBLs with substitutions in the {Omega} loop (R164H and R164S) as target enzymes to understand this enhanced biochemical capability and to serve as a basis for novel {beta}-lactamase inhibitor development, we determined the spectra of activity and crystal structures of these variants. We also studied the inactivation of the R164H and R164S mutants with tazobactam and SA2-13, a unique {beta}-lactamase inhibitor that undergoes a distinctive reaction chemistry in the active site. We noted that the reduced K{sub i} values for the R164H and R164S mutants with SA2-13 are comparable to those with tazobactam (submicromolar). The apo enzyme crystal structures of the R164H and R164S SHV variants revealed an ordered {Omega} loop architecture that became disordered when SA2-13 was bound. Important structural alterations that result from the binding of SA2-13 explain the enhanced susceptibility of these ESBL enzymes to this inhibitor and highlight ligand-dependent {Omega} loop flexibility as a mechanism for accommodating and hydrolyzing {beta}-lactam substrates.

  13. A functional selectivity mechanism at the serotonin-2A GPCR involves ligand-dependent conformations of intracellular loop 2.

    PubMed

    Perez-Aguilar, Jose Manuel; Shan, Jufang; LeVine, Michael V; Khelashvili, George; Weinstein, Harel

    2014-11-12

    With recent progress in determination of G protein-coupled receptor (GPCR) structure with crystallography, a variety of other experimental approaches (e.g., NMR spectroscopy, fluorescent-based assays, mass spectrometry techniques) are also being used to characterize state-specific and ligand-specific conformational states. MD simulations offer a powerful complementary approach to elucidate the dynamic features associated with ligand-specific GPCR conformations. To shed light on the conformational elements and dynamics of the important aspect of GPCR functional selectivity, we carried out unbiased microsecond-length MD simulations of the human serotonin 2A receptor (5-HT(2A)R) in the absence of ligand and bound to four distinct serotonergic agonists. The 5-HT(2A)R is a suitable system to study the structural features involved in the ligand-dependent conformational heterogeneity of GPCRs because it is well-characterized experimentally and exhibits a strong agonist-specific phenotype in that some 5-HT(2A)R agonists induce LSD-like hallucinations, while others lack this psychoactive property entirely. Here we report evidence for structural and dynamic differences in 5-HT(2A)R interacting with such pharmacologically distinct ligands, hallucinogens, and nonhallucinogens obtained from all-atom MD simulations. Differential ligand binding contacts were identified for structurally similar hallucinogens and nonhallucinogens and found to correspond to different conformations in the intracellular loop 2 (ICL2). From the different ICL2 conformations, functional selective phenotypes are suggested through effects on dimerization and/or distinct direct interaction with effector proteins. The findings are presented in the context of currently proposed hallucinogenesis mechanisms, and ICL2 is proposed as a fine-tuning selective switch that can differentiates modes of 5-HT(2A)R activation.

  14. A functional selectivity mechanism at the serotonin-2A GPCR involves ligand-dependent conformations of intracellular loop 2

    SciTech Connect

    Perez-Aguilar, Jose Manuel; Shan, Jufang; LeVine, Michael V.; Khelashvili, George; Weinstein, Harel

    2014-10-14

    With recent progress in determination of G protein-coupled receptor (GPCR) structure with crystallography, a variety of other experimental approaches (e.g., NMR spectroscopy, fluorescent-based assays, mass spectrometry techniques) are also being used to characterize state-specific and ligand-specific conformational states. MD simulations offer a powerful complementary approach to elucidate the dynamic features associated with ligand-specific GPCR conformations. To shed light on the conformational elements and dynamics of the important aspect of GPCR functional selectivity, we carried out unbiased microsecond-length MD simulations of the human serotonin 2A receptor (5-HT2AR) in the absence of ligand and bound to four distinct serotonergic agonists. The 5-HT2AR is a suitable system to study the structural features involved in the ligand-dependent conformational heterogeneity of GPCRs because it is well-characterized experimentally and exhibits a strong agonist-specific phenotype in that some 5-HT2AR agonists induce LSD-like hallucinations, while others lack this psychoactive property entirely. Here we report evidence for structural and dynamic differences in 5-HT2AR interacting with such pharmacologically distinct ligands, hallucinogens, and nonhallucinogens obtained from all-atom MD simulations. Differential ligand binding contacts were identified for structurally similar hallucinogens and nonhallucinogens and found to correspond to different conformations in the intracellular loop 2 (ICL2). From the different ICL2 conformations, functional selective phenotypes are suggested through effects on dimerization and/or distinct direct interaction with effector proteins. Lastly, the findings are presented in the context of currently proposed hallucinogenesis mechanisms, and ICL2 is proposed as a fine-tuning selective switch that can differentiates modes of 5-HT2AR activation.

  15. A functional selectivity mechanism at the serotonin-2A GPCR involves ligand-dependent conformations of intracellular loop 2

    DOE PAGES

    Perez-Aguilar, Jose Manuel; Shan, Jufang; LeVine, Michael V.; ...

    2014-10-14

    With recent progress in determination of G protein-coupled receptor (GPCR) structure with crystallography, a variety of other experimental approaches (e.g., NMR spectroscopy, fluorescent-based assays, mass spectrometry techniques) are also being used to characterize state-specific and ligand-specific conformational states. MD simulations offer a powerful complementary approach to elucidate the dynamic features associated with ligand-specific GPCR conformations. To shed light on the conformational elements and dynamics of the important aspect of GPCR functional selectivity, we carried out unbiased microsecond-length MD simulations of the human serotonin 2A receptor (5-HT2AR) in the absence of ligand and bound to four distinct serotonergic agonists. The 5-HT2ARmore » is a suitable system to study the structural features involved in the ligand-dependent conformational heterogeneity of GPCRs because it is well-characterized experimentally and exhibits a strong agonist-specific phenotype in that some 5-HT2AR agonists induce LSD-like hallucinations, while others lack this psychoactive property entirely. Here we report evidence for structural and dynamic differences in 5-HT2AR interacting with such pharmacologically distinct ligands, hallucinogens, and nonhallucinogens obtained from all-atom MD simulations. Differential ligand binding contacts were identified for structurally similar hallucinogens and nonhallucinogens and found to correspond to different conformations in the intracellular loop 2 (ICL2). From the different ICL2 conformations, functional selective phenotypes are suggested through effects on dimerization and/or distinct direct interaction with effector proteins. Lastly, the findings are presented in the context of currently proposed hallucinogenesis mechanisms, and ICL2 is proposed as a fine-tuning selective switch that can differentiates modes of 5-HT2AR activation.« less

  16. Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion.

    PubMed

    JavanMoghadam, Sonia; Weihua, Zhang; Hunt, Kelly K; Keyomarsi, Khandan

    2016-06-17

    Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G2/M phases, whereas the G1 phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G2/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G2/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G2/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.

  17. Functional interaction between the HIV transactivator Tat and the transcriptional coactivator PC4 in T cells.

    PubMed

    Holloway, A F; Occhiodoro, F; Mittler, G; Meisterernst, M; Shannon, M F

    2000-07-14

    The human immunodeficiency virus (HIV) transactivator Tat is a potent activator of transcription from the HIV long terminal repeat and is essential for efficient viral gene expression and replication. Tat has been shown to interact with components of the basal transcription machinery and transcriptional activators. Here we identify the cellular coactivator PC4 as a Tat-interacting protein using the yeast two-hybrid system and confirmed this interaction both in vitro and in vivo by coimmunoprecipitation. We found that this interaction has a functional outcome in that PC4 overexpression enhanced activation of the HIV long terminal repeat in transient transfection studies in a Tat-dependent manner. The domains of PC4 and Tat required for the interaction were mapped. In vitro binding studies showed that the basic transactivation-responsive binding domain of Tat is required for the interaction with PC4. The minimum region of PC4 required for Tat binding was amino acids 22-91, whereas mutation of the lysine-rich domain between amino acids 22 and 43 prevented interaction with Tat. Tat-PC4 interactions may be controlled by phosphorylation, because phosphorylation of PC4 by casein kinase II inhibited interactions with Tat both in vivo and in vitro. We propose that PC4 may be involved in linking Tat to the basal transcription machinery.

  18. Tyrosine kinase receptor transactivation associated to G protein-coupled receptors.

    PubMed

    Almendro, Vanessa; García-Recio, Susana; Gascón, Pedro

    2010-09-01

    G protein-coupled receptors (GPCRs) comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth, cell differentiation and oncogenesis among others. Some of the effects of GPCRs are known to be mediated by the activation of MAPK pathways. Several GPCRs are also able to transactivate receptors with tyrosine kinase activity (TKR) such as EGFR and HER2 and thus to control DNA synthesis and cell proliferation. The interaction between these receptors not only plays an important physiological role but its disregulation can induce pathological states such as cancer. For this reason, the crosstalk between these two types of receptors can be considered a possible mechanism for cell transformation, tumor progression, reactivation of the metastatic disease, and the acquisition of resistance to therapies targeting TKR receptors. The transactivation of some TKRs by GPCRs is related to the lost of response of TKRs to inhibitors of TK activity, mainly by the activation of the c-Src protein which can directly phosphorylate and activate the cytoplasmic domain of a TKR. For these reason, the dual inhibition of GPCRs and TKRs in some types of cancer has been proposed as a better strategy to kill tumor cells. Increased understanding of the mechanisms that interconnect the two pathways regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies.

  19. The application of molecular modelling in the safety assessment of chemicals: A case study on ligand-dependent PPARγ dysregulation.

    PubMed

    Al Sharif, Merilin; Tsakovska, Ivanka; Pajeva, Ilza; Alov, Petko; Fioravanzo, Elena; Bassan, Arianna; Kovarich, Simona; Yang, Chihae; Mostrag-Szlichtyng, Aleksandra; Vitcheva, Vessela; Worth, Andrew P; Richarz, Andrea-N; Cronin, Mark T D

    2016-02-04

    The aim of this paper was to provide a proof of concept demonstrating that molecular modelling methodologies can be employed as a part of an integrated strategy to support toxicity prediction consistent with the mode of action/adverse outcome pathway (MoA/AOP) framework. To illustrate the role of molecular modelling in predictive toxicology, a case study was undertaken in which molecular modelling methodologies were employed to predict the activation of the peroxisome proliferator-activated nuclear receptor γ (PPARγ) as a potential molecular initiating event (MIE) for liver steatosis. A stepwise procedure combining different in silico approaches (virtual screening based on docking and pharmacophore filtering, and molecular field analysis) was developed to screen for PPARγ full agonists and to predict their transactivation activity (EC50). The performance metrics of the classification model to predict PPARγ full agonists were balanced accuracy=81%, sensitivity=85% and specificity=76%. The 3D QSAR model developed to predict EC50 of PPARγ full agonists had the following statistical parameters: q(2)cv=0.610, Nopt=7, SEPcv=0.505, r(2)pr=0.552. To support the linkage of PPARγ agonism predictions to prosteatotic potential, molecular modelling was combined with independently performed mechanistic mining of available in vivo toxicity data followed by ToxPrint chemotypes analysis. The approaches investigated demonstrated a potential to predict the MIE, to facilitate the process of MoA/AOP elaboration, to increase the scientific confidence in AOP, and to become a basis for 3D chemotype development.

  20. NLRC5: a newly discovered MHC class I transactivator (CITA).

    PubMed

    Meissner, Torsten B; Li, Amy; Kobayashi, Koichi S

    2012-06-01

    Major histocompatibility complex (MHC) class I and class II are crucial for the function of the human adaptive immune system. An NLR protein, CIITA (MHC class II transactivator), is a master regulator of MHC class II gene expression as well as of some of the genes involved in MHC class II antigen presentation. It has recently been discovered that another member of the NLR protein family, NLRC5, transcriptionally activates MHC class I genes, and thus acts as "CITA" (MHC class I transactivator), a counterpart to CIITA. In addition to MHC class I genes, NLRC5 can induce the expression of β2M, TAP1 and LMP2, essential components of MHC class I antigen presentation. These findings indicate that NLRC5 and CIITA are transcriptional regulators that orchestrate the concerted expression of critical components in the MHC class I and MHC class II pathways, respectively.

  1. Identification of protein interaction antagonists using the repressed transactivator two-hybrid system.

    PubMed

    Joshi, Phalgun B; Hirst, Martin; Malcolm, Tom; Parent, Jennifer; Mitchell, David; Lund, Karen; Sadowski, Ivan

    2007-05-01

    The repressed transactivator (RTA) yeast two-hybrid system was developed to enable genetic identification of interactions with transcriptional activator proteins. We have devised modifications of this system that enable its use in screening for inhibitors of protein interactions from small molecule compound libraries. We show that inhibition of protein interactions can be measured by monitoring growth in selective medium containing 3-aminotriazole (3-AT) and using this assay have identified inhibitors of four independent protein interactions in screens with a 23,000 small molecule compound library. Compounds found to inhibit one of the tested interactions between FKBP12 and the transforming growth factor beta receptor (TGFbeta-R) were validated in vivo and found to inhibit calcineurin-dependent signaling in T cells. One of these compounds was also found to cause elevated basal expression of a TGFbeta-R/SMAD-dependent reporter gene. These results demonstrate the capability of the RTA small molecule screening assay for discovery of potentially novel therapeutic compounds.

  2. GPR54 (KISS1R) transactivates EGFR to promote breast cancer cell invasiveness.

    PubMed

    Zajac, Mateusz; Law, Jeffrey; Cvetkovic, Dragana Donna; Pampillo, Macarena; McColl, Lindsay; Pape, Cynthia; Di Guglielmo, Gianni M; Postovit, Lynne M; Babwah, Andy V; Bhattacharya, Moshmi

    2011-01-01

    Kisspeptins (Kp), peptide products of the Kisspeptin-1 (KISS1) gene are endogenous ligands for a G protein-coupled receptor 54 (GPR54). Previous findings have shown that KISS1 acts as a metastasis suppressor in numerous cancers in humans. However, recent studies have demonstrated that an increase in KISS1 and GPR54 expression in human breast tumors correlates with higher tumor grade and metastatic potential. At present, whether or not Kp signaling promotes breast cancer cell invasiveness, required for metastasis and the underlying mechanisms, is unknown. We have found that kisspeptin-10 (Kp-10), the most potent Kp, stimulates the invasion of human breast cancer MDA-MB-231 and Hs578T cells using Matrigel-coated Transwell chamber assays and induces the formation of invasive stellate structures in three-dimensional invasion assays. Furthermore, Kp-10 stimulated an increase in matrix metalloprotease (MMP)-9 activity. We also found that Kp-10 induced the transactivation of epidermal growth factor receptor (EGFR). Knockdown of the GPCR scaffolding protein, β-arrestin 2, inhibited Kp-10-induced EGFR transactivation as well as Kp-10 induced invasion of breast cancer cells via modulation of MMP-9 secretion and activity. Finally, we found that the two receptors associate with each other under basal conditions, and FRET analysis revealed that GPR54 interacts directly with EGFR. The stability of the receptor complex formation was increased upon treatment of cells by Kp-10. Taken together, our findings suggest a novel mechanism by which Kp signaling via GPR54 stimulates breast cancer cell invasiveness.

  3. Role of EGFR transactivation in angiotensin II signaling to extracellular regulated kinase in preglomerular smooth muscle cells.

    PubMed

    Andresen, Bradley T; Linnoila, Jenny J; Jackson, Edwin K; Romero, Guillermo G

    2003-03-01

    Angiotensin (Ang) II promotes the phosphorylation of extracellular regulated kinase (ERK); however, the mechanisms leading to Ang II-induced ERK phosphorylation are debated. The currently accepted theory involves transactivation of epidermal growth factor receptor (EGFR). We have shown that generation of phosphatidic acid (PA) is required for the recruitment of Raf to membranes and the activation of ERK by multiple agonists, including Ang II. In the present report, we confirm that phospholipase D-dependent generation of PA is required for Ang II-mediated phosphorylation of ERK in Wistar-Kyoto and spontaneously hypertensive rat preglomerular smooth muscle cells (PGSMCs). However, EGF stimulation does not activate phospholipase D or generate PA. These observations indicate that EGF recruits Raf to membranes via a mechanism that does not involve PA, and thus, Ang II-mediated phosphorylation of ERK is partially independent of EGFR-mediated signaling cascades. We hypothesized that phosphoinositide-3-kinase (PI3K) can also act to recruit Raf to membranes; therefore, inhibition of PI3K should inhibit EGF signaling to ERK. Wortmannin, a PI3K inhibitor, inhibited EGF-mediated phosphorylation of ERK (IC50, approximately 14 nmol/L). To examine the role of the EGFR in Ang II-mediated phosphorylation of ERK we utilized 100 nmol/L wortmannin to inhibit EGFR signaling to ERK and T19N RhoA to block Ang II-mediated ERK phosphorylation. Wortmannin treatment inhibited EGF-mediated but not Ang II-mediated phosphorylation of ERK. Furthermore, T19N RhoA inhibited Ang II-mediated ERK phosphorylation, whereas T19N RhoA had significantly less effect on EGF-mediated ERK phosphorylation. We conclude that transactivation of the EGFR is not primarily responsible for Ang II-mediated activation of ERK in PGSMCs.

  4. Novel mutants of NAB corepressors enhance activation by Egr transactivators.

    PubMed Central

    Svaren, J; Sevetson, B R; Golda, T; Stanton, J J; Swirnoff, A H; Milbrandt, J

    1998-01-01

    The NGFI-A binding corepressors NAB1 and NAB2 interact with a conserved domain (R1 domain) within the Egr1/NGFI-A and Egr2/Krox20 transactivators, and repress the transcription of Egr target promoters. Using a novel adaptation of the yeast two-hybrid screen, we have identified several point mutations in NAB corepressors that interfere with their ability to bind to the Egr1 R1 domain. Surprisingly, NAB proteins bearing some of these mutations increased Egr1 activity dramatically. The mechanism underlying the unexpected behavior of these mutants was elucidated by the discovery that NAB conserved domain 1 (NCD1) not only binds to Egr proteins but also mediates multimerization of NAB molecules. The activating mutants exert a dominant negative effect on NAB repression by multimerizing with native NAB proteins and preventing binding of endogenous NAB proteins with Egr transactivators. To examine NAB repression of a native Egr target gene, we show that NAB2 represses Egr2/Krox20-mediated activation of the bFGF/FGF-2 promoter, and that repression is reversed by coexpression of dominant negative NAB2. Because of their specific ability to alleviate NAB repression of Egr target genes, the dominant negative NAB mutants will be useful in elucidating the mechanism and function of NAB corepressors. PMID:9774344

  5. The Chicken Ovalbumin Upstream Promoter-Transcription Factor II Negatively Regulates the Transactivation of Androgen Receptor in Prostate Cancer Cells

    PubMed Central

    Song, Chin-Hee; Lee, Hyun Joo; Park, Eunsook; Lee, Keesook

    2012-01-01

    Androgen receptor (AR) is involved in the development and progression of prostate cancers. However, the mechanisms by which this occurs remain incompletely understood. In previous reports, chicken ovalbumin upstream promoter-transcription factor II (COUP-TF II) has been suggested to play a role in the development of cancers. In the present study, we explored a putative role of COUP-TF II in prostate cancers by investigating its effect on cell proliferation and a cross-talk between COUP-TF II and AR. Overexpression of COUP-TF II results in the inhibition of androgen-dependent proliferation of prostate cancer cells. Further studies show that COUP-TF II functions as a corepressor of AR. It represses AR transactivation on target promoters containing the androgen response element (ARE) in a dose-dependent manner. In addition, COUP-TF II interacts physically with AR in vitro and in vivo. It binds to both the DNA binding domain (DBD) and the ligand-binding domain (LBD) of AR and disrupts the N/C terminal interaction of AR. Furthermore, COUP-TF II competes with coactivators such as ARA70, SRC-1, and GRIP1 to modulate AR transactivation as well as inhibiting the recruitment of AR to its ARE-containing target promoter. Taken together, our findings suggest that COUP-TF II is a novel corepressor of AR, and provide an insight into the role of COUP-TF II in prostate cancers. PMID:23145053

  6. trans-Activation of a globin promoter in nonerythroid cells.

    PubMed Central

    Evans, T; Felsenfeld, G

    1991-01-01

    We show that expression in fibroblasts of a single cDNA, encoding the erythroid DNA-binding protein Eryf1 (GF-1, NF-E1), very efficiently activates transcription of a chicken alpha-globin promoter, trans-Activation in these cells occurred when Eryf1 bound to a single site within a minimal globin promoter. In contrast, efficient activation in erythroid cells required multiple Eryf1 binding sites. Our results indicate that mechanisms exist that are capable of modulating the trans-acting capabilities of Eryf1 in a cell-specific manner, without affecting DNA binding. The response of the minimal globin promoter to Eryf1 in fibroblasts was at least as great as for optimal constructions in erythroid cells. Therefore, the assay provides a very simple and sensitive system with which to study gene activation by a tissue-specific factor. Images PMID:1990287

  7. ADAM17 Transactivates EGFR Signaling during Embryonic Eyelid Closure

    PubMed Central

    Hassemer, Eryn L.; Endres, Bradley; Toonen, Joseph A.; Ronchetti, Adam; Dubielzig, Richard; Sidjanin, Duska J.

    2013-01-01

    Purpose. During mammalian embryonic eyelid closure ADAM17 has been proposed to play a role as a transactivator of epidermal growth factor receptor (EGFR) signaling by shedding membrane bound EGFR ligands. However, ADAM17 also sheds numerous other ligands, thus implicating ADAM17 in additional molecular pathways. The goal of this study was to experimentally establish the role of ADAM17 and determine ADAM17-mediated pathways essential for the embryonic eyelid closure. Methods. Wild-type (WT) and woe mice, carrying a hypomorphic mutation in Adam17, were evaluated using H&E and scanning electron microscopy. Expressions of ADAM17, EGFR, and the phosphorylated form EGFR-P were evaluated using immunohistochemistry. BrdU and TUNEL assays were used to evaluate cell proliferation and apoptosis, respectively. In vitro scratch assays of primary cultures were used to evaluate cell migration. Clinical and histologic analyses established if the hypermorphic EgfrDsk5 allele can rescue the woe embryonic eyelid closure. Results. woe mice exhibited a failure to develop the leading edge of the eyelid and consequently failure of the embryonic eyelid closure. Expression of ADAM17 was identified in the eyelid epithelium in the cells of the leading edge. ADAM17 is essential for epithelial cell migration, but does not play a role in proliferation and apoptosis. EGFR was expressed in both WT and woe eyelid epithelium, but the phosphorylated EGFR-P form was detected only in WT. The EgfrDsk5 allele rescued woe eyelid closure defects, but also rescued woe anterior segment defects and the absence of meibomian glands. Conclusions. We provide in vivo genetic evidence that the role of ADAM17 during embryonic eyelid closure is to transactivate EGFR signaling. PMID:23211830

  8. Idiopathic pulmonary fibrosis fibroblasts become resistant to Fas ligand-dependent apoptosis via the alteration of decoy receptor 3.

    PubMed

    Im, Jintaek; Kim, Kyutae; Hergert, Polla; Nho, Richard Seonghun

    2016-09-01

    Idiopathic pulmonary fibrosis (IPF) is an irreversible lethal lung disease with an unknown etiology. IPF patients' lung fibroblasts express inappropriately high Akt activity, protecting them in response to an apoptosis-inducing type I collagen matrix. FasL, a ligand for Fas, is known to be increased in the lung tissues of patients with IPF, implicated with the progression of IPF. Expression of Decoy Receptor3 (DcR3), which binds to FasL, thereby subsequently suppressing the FasL-Fas-dependent apoptotic pathway, is frequently altered in various human disease. However, the role of DcR3 in IPF fibroblasts in regulating their viability has not been examined. We found that enhanced DcR3 expression exists in the majority of IPF fibroblasts on collagen matrices, resulting in the protection of IPF fibroblasts from FasL-induced apoptosis. Abnormally high Akt activity suppresses GSK-3β function, thereby accumulating the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) in the nucleus, increasing DcR3 expression in IPF fibroblasts. This alteration protects IPF cells from FasL-induced apoptosis on collagen. However, the inhibition of Akt or NFATc1 decreases DcR3 mRNA and protein levels, which sensitizes IPF fibroblasts to FasL-mediated apoptosis. Furthermore, enhanced DcR3 and NFATc1 expression is mainly present in myofibroblasts in the fibroblastic foci of lung tissues derived from IPF patients. Our results showed that when IPF cells interact with collagen matrix, aberrantly activated Akt increases DcR3 expression via GSK-3β-NFATc1 and protects IPF cells from the FasL-dependent apoptotic pathway. These findings suggest that the inhibition of DcR3 function may be an effective approach for sensitizing IPF fibroblasts in response to FasL, limiting the progression of lung fibrosis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  9. A previously functional tetracycline-regulated transactivator fails to target gene expression to the bone

    PubMed Central

    2011-01-01

    Background The tetracycline-controlled transactivator system is a powerful tool to control gene expression in vitro and to generate consistent and conditional transgenic in vivo model organisms. It has been widely used to study gene function and to explore pathological mechanisms involved in human diseases. The system permits the regulation of the expression of a target gene, both temporally and quantitatively, by the application of tetracycline or its derivative, doxycycline. In addition, it offers the possibility to restrict gene expression in a spatial fashion by utilizing tissue-specific promoters to drive the transactivator. Findings In this study, we report our problems using a reverse tetracycline-regulated transactivator (rtTA) in a transgenic mouse model system for the bone-specific expression of the Hutchinson-Gilford progeria syndrome mutation. Even though prior studies have been successful utilizing the same rtTA, expression analysis of the transactivator revealed insufficient activity for regulating the transgene expression in our system. The absence of transactivator could not be ascribed to differences in genetic background because mice in a mixed genetic background and in congenic mouse lines showed similar results. Conclusions The purpose of this study is to report our negative experience with previously functional transactivator mice, to raise caution in the use of tet-based transgenic mouse lines and to reinforce the need for controls to ensure the stable functionality of generated tetracycline-controlled transactivators over time. PMID:21835026

  10. Zinc Finger and X-Linked Factor (ZFX) Binds to Human SET Transcript 2 Promoter and Transactivates SET Expression.

    PubMed

    Xu, Siliang; Duan, Ping; Li, Jinping; Senkowski, Tristan; Guo, Fengbiao; Chen, Haibin; Romero, Alberto; Cui, Yugui; Liu, Jiayin; Jiang, Shi-Wen

    2016-10-20

    SET (SE Translocation) protein carries out multiple functions including those for protein phosphatase 2A (PP2A) inhibition, histone modification, DNA repair, and gene regulation. SET overexpression has been detected in brain neurons of patients suffering Alzheimer's disease, follicle theca cells of Polycystic Ovary Syndrome (PCOS) patients, and ovarian cancer cells, indicating that SET may play a pathological role for these disorders. SET transcript 2, produced by a specific promoter, represents a major transcript variant in different cell types. In this study, we characterized the transcriptional activation of human SET transcript 2 promoter in HeLa cells. Promoter deletion experiments and co-transfection assays indicated that ZFX, the Zinc finger and X-linked transcription factor, was able to transactivate the SET promoter. A proximal promoter region containing four ZFX-binding sites was found to be critical for the ZFX-mediated transactivation. Mutagenesis study indicated that the ZFX-binding site located the closest to the transcription start site accounted for most of the ZFX-mediated transactivity. Manipulation of ZFX levels by overexpression or siRNA knockdown confirmed the significance and specificity of the ZFX-mediated SET promoter activation. Chromatin immunoprecipitation results verified the binding of ZFX to its cognate sites in the SET promoter. These findings have led to identification of ZFX as an upstream factor regulating SET gene expression. More studies are required to define the in vivo significance of this mechanism, and specifically, its implication for several benign and malignant diseases related to SET dysregulation.

  11. In situ autoradiography and ligand-dependent tyrosine kinase activity reveal insulin receptors and insulin-like growth factor I receptors in prepancreatic chicken embryos.

    PubMed Central

    Girbau, M; Bassas, L; Alemany, J; de Pablo, F

    1989-01-01

    We previously reported specific cross-linking of 125I-labeled insulin and 125I-labeled insulin-like growth factor I (IGF-I) to the alpha subunit of their respective receptors in chicken embryos of 20 somites and older. To achieve adequate sensitivity and localize spatially the receptors in younger embryos, we adapted an autoradiographic technique using whole-mounted chicken blastoderms. Insulin receptors and IGF-I receptors were expressed and could be localized as early as gastrulation, before the first somite is formed. Relative density was analyzed by a computer-assisted image system, revealing overall slightly higher binding of IGF-I than of insulin. Structures rich in both types of receptors were predominantly of ectodermal origin: Hensen's node in gastrulating embryos and neural folds, neural tube and optic vesicles during neurulation. The signal transduction capability of the receptors in early organogenesis was assessed by their ability to phosphorylate the exogenous substrate poly(Glu80Tyr20). Ligand-dependent tyrosine phosphorylation was demonstrable with both insulin and IGF-I in glycoprotein-enriched preparations from embryos at days 2 through 6 of embryogenesis. There was a developmentally regulated change in ligand-dependent tyrosine kinase activity, with a sharp increase from day 2 to day 4, in contrast with a small increase in the ligand binding. Binding of 125I-labeled IGF-I was, with the solubilized receptors, severalfold higher than binding of 125I-labeled insulin. However, the insulin-dependent phosphorylation was as high as the IGF-I-dependent phosphorylation at each developmental stage. Images PMID:2548191

  12. Biochemical Analysis of Pathogenic Ligand-Dependent FGFR2 Mutations Suggests Distinct Pathophysiological Mechanisms for Craniofacial and Limb Abnormalities in Human Skeletal Disorders

    SciTech Connect

    Ibrahimi,O.; Zhang, F.; Eliseenkova, A.; Itoh, N.; Linhardt, R.; Mohammadi, M.

    2004-01-01

    Gain-of-function missense mutations in FGF receptor 2 (FGFR2) are responsible for a variety of craniosynostosis syndromes including Apert syndrome (AS), Pfeiffer syndrome (PS) and Crouzon syndrome (CS). Unlike the majority of FGFR2 mutations, S252W and P253R AS mutations and a D321A PS mutation retain ligand-dependency and are also associated with severe limb pathology. In addition, a recently identified ligand-dependent S252L/A315S double mutation in FGFR2 was shown to cause syndactyly in the absence of craniosynostosis. Here, we analyze the effect of the canonical AS mutations, the D321A PS mutation and the S252L/A315S double mutation on FGFR2 ligand binding affinity and specificity using surface plasmon resonance. Both AS mutations and the D321A PS mutation, but not the S252L/A315S double mutation, increase the binding affinity of FGFR2c to multiple FGFs expressed in the cranial suture. Additionally, all four pathogenic mutations also violate FGFR2c ligand binding specificity and enable this receptor to bind FGF10. Based on our data, we propose that an increase in mutant FGFR2c binding to multiple FGFs results in craniosynostosis, whereas binding of mutant FGFR2c to FGF10 results in severe limb pathology. Structural and biophysical analysis shows that AS mutations in FGFR2b also enhance and violate FGFR2b ligand binding affinity and specificity, respectively. We suggest that elevated AS mutant FGFR2b signaling may account for the dermatological manifestations of AS.

  13. Acetylation reduces SOX9 nuclear entry and ACAN gene transactivation in human chondrocytes.

    PubMed

    Bar Oz, Michal; Kumar, Ashok; Elayyan, Jinan; Reich, Eli; Binyamin, Milana; Kandel, Leonid; Liebergall, Meir; Steinmeyer, Juergen; Lefebvre, Veronique; Dvir-Ginzberg, Mona

    2016-06-01

    Changes in the content of aggrecan, an essential proteoglycan of articular cartilage, have been implicated in the pathophysiology of osteoarthritis (OA), a prevalent age-related, degenerative joint disease. Here, we examined the effect of SOX9 acetylation on ACAN transactivation in the context of osteoarthritis. Primary chondrocytes freshly isolated from degenerated OA cartilage displayed lower levels of ACAN mRNA and higher levels of acetylated SOX9 compared with cells from intact regions of OA cartilage. Degenerated OA cartilage presented chondrocyte clusters bearing diffused immunostaining for SOX9 compared with intact cartilage regions. Primary human chondrocytes freshly isolated from OA knee joints were cultured in monolayer or in three-dimensional alginate microbeads (3D). SOX9 was hypo-acetylated in 3D cultures and displayed enhanced binding to a -10 kb ACAN enhancer, a result consistent with higher ACAN mRNA levels than in monolayer cultures. It also co-immunoprecipitated with SIRT1, a major deacetylase responsible for SOX9 deacetylation. Finally, immunofluorescence assays revealed increased nuclear localization of SOX9 in primary chondrocytes treated with the NAD SIRT1 cofactor, than in cells treated with a SIRT1 inhibitor. Inhibition of importin β by importazole maintained SOX9 in the cytoplasm, even in the presence of NAD. Based on these data, we conclude that deacetylation promotes SOX9 nuclear translocation and hence its ability to activate ACAN.

  14. Endocannabinoids regulate interneuron migration and morphogenesis by transactivating the TrkB receptor.

    PubMed

    Berghuis, Paul; Dobszay, Marton B; Wang, Xinyu; Spano, Sabrina; Ledda, Fernanda; Sousa, Kyle M; Schulte, Gunnar; Ernfors, Patrik; Mackie, Ken; Paratcha, Gustavo; Hurd, Yasmin L; Harkany, Tibor

    2005-12-27

    In utero exposure to Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the active component from marijuana, induces cognitive deficits enduring into adulthood. Although changes in synaptic structure and plasticity may underlie Delta(9)-THC-induced cognitive impairments, the neuronal basis of Delta(9)-THC-related developmental deficits remains unknown. Using a Boyden chamber assay, we show that agonist stimulation of the CB(1) cannabinoid receptor (CB(1)R) on cholecystokinin-expressing interneurons induces chemotaxis that is additive with brain-derived neurotrophic factor (BDNF)-induced interneuron migration. We find that Src kinase-dependent TrkB receptor transactivation mediates endocannabinoid (eCB)-induced chemotaxis in the absence of BDNF. Simultaneously, eCBs suppress the BDNF-dependent morphogenesis of interneurons, and this suppression is abolished by Src kinase inhibition in vitro. Because sustained prenatal Delta(9)-THC stimulation of CB(1)Rs selectively increases the density of cholecystokinin-expressing interneurons in the hippocampus in vivo, we conclude that prenatal CB(1)R activity governs proper interneuron placement and integration during corticogenesis. Moreover, eCBs use TrkB receptor-dependent signaling pathways to regulate subtype-selective interneuron migration and specification.

  15. The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18.

    PubMed Central

    Le Douarin, B; Zechel, C; Garnier, J M; Lutz, Y; Tora, L; Pierrat, P; Heery, D; Gronemeyer, H; Chambon, P; Losson, R

    1995-01-01

    Nuclear receptors (NRs) bound to response elements mediate the effects of cognate ligands on gene expression. Their ligand-dependent activation function, AF-2, presumably acts on the basal transcription machinery through intermediary proteins/mediators. We have isolated a mouse nuclear protein, TIF1, which enhances RXR and RAR AF-2 in yeast and interacts in a ligand-dependent manner with several NRs in yeast and mammalian cells, as well as in vitro. Remarkably, these interactions require the amino acids constituting the AF-2 activating domain conserved in all active NRs. Moreover, the oestrogen receptor (ER) AF-2 antagonist hydroxytamoxifen cannot promote ER-TIF1 interaction. We propose that TIF1, which contains several conserved domains found in transcriptional regulatory proteins, is a mediator of ligand-dependent AF-2. Interestingly, the TIF1 N-terminal moiety is fused to B-raf in the mouse oncoprotein T18. Images PMID:7744009

  16. The paradigm of G protein receptor transactivation: a mechanistic definition and novel example.

    PubMed

    Little, Peter J; Burch, Micah L; Al-aryahi, Sefaa; Zheng, Wenhua

    2011-03-22

    Seven transmembrane G protein-coupled receptors are among the most common in biology and they transduce cellular signals from a plethora of hormones. As well as their own well-characterized signaling pathways, they can also transactivate tyrosine kinase growth factor receptors to greatly expand their own cellular repertoire of responses. Recent data in vascular smooth muscle cells have expanded the breadth of transactivation to include serine/threonine kinase receptors, specifically the receptor for transforming growth factor beta (TGF-beta). Stimulation with endothelin and thrombin leads to the rapid formation of C-terminal phosphorylated Smad2, which is the immediate product of activation of the TGF-beta receptor. Additionally, it appears that no definition of transactivation based on mechanism is available, so we have provided a definition involving temporal aspects and the absence of de novo protein synthesis. The phenomenon of transactivation is an important biochemical mechanism and potential drug target in multiple diseases.

  17. Pigment-cell-specific genes from fibroblasts are transactivated after chromosomal transfer into melanoma cells

    SciTech Connect

    Powers, T.P.; Davidson, R.L.; Shows, T.B.

    1994-02-01

    Human and mouse fibroblast chromosomes carrying tyrosinase or b-locus genes were introduced, by microcell hybridization, into pigmented Syrian hamster melanoma cells, and the microcell hybrids were tested for transactivation of the fibroblast tyrosinase and b-locus genes. By using species-specific PCR amplification to distinguish fibroblast and melanoma cDNAs, it was demonstrated that the previously silent fibroblast tyrosinase and b-locus genes were transactivated following chromosomal transfer into pigmented melanoma cells. However, transactivation of the mouse fibroblast tyrosinase gene was unstable in microcell hybrid subclones and possibly dependent on a second fibroblast locus that could have segregated in the subclones. This second locus was not necessary for transactivation of the fibroblast b-locus gene, thus demonstrating noncoordinate transactivation of fibroblast tyrosinase and b-locus genes. Transactivation of the fibroblast tyrosinase gene in microcell hybrids apparently is dependent on the absence of a putative fibroblast extinguisher locus for tyrosinase gene expression, which presumably is responsible for the extinction of pigmentation in hybrids between karyotypically complete fibroblasts and melanoma cells. 46 refs., 5 figs., 2 tabs.

  18. A functional siRNA screen identifies genes modulating angiotensin II-mediated EGFR transactivation

    PubMed Central

    George, Amee J.; Purdue, Brooke W.; Gould, Cathryn M.; Thomas, Daniel W.; Handoko, Yanny; Qian, Hongwei; Quaife-Ryan, Gregory A.; Morgan, Kylie A.; Simpson, Kaylene J.; Thomas, Walter G.; Hannan, Ross D.

    2013-01-01

    Summary The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR) to mediate cellular growth, however, the molecular mechanisms involved have not yet been resolved. To address this, we performed a functional siRNA screen of the human kinome in human mammary epithelial cells that demonstrate a robust AT1R–EGFR transactivation. We identified a suite of genes encoding proteins that both positively and negatively regulate AT1R–EGFR transactivation. Many candidates are components of EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, have not been previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but this did not occur following direct stimulation of the EGFR with EGF, indicating that these proteins function between the activated AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely to be required for AT1R–EGFR transactivation. CHKA also mediated EGFR transactivation in response to another G protein-coupled receptor (GPCR) ligand, thrombin, indicating a pervasive role for CHKA in GPCR–EGFR crosstalk. Our study reveals the power of unbiased, functional genomic screens to identify new signalling mediators important for tissue remodelling in cardiovascular disease and cancer. PMID:24046455

  19. Runx2 Trans-Activation Mediated by the Msx2-Interacting Nuclear Target Requires Homeodomain Interacting Protein Kinase-3

    PubMed Central

    Sierra, Oscar L.; Towler, Dwight A.

    2010-01-01

    Runt-related transcription factor 2 (Runx2) and muscle segment homeobox homolog 2-interacting nuclear target (MINT) (Spen homolog) are transcriptional regulators critical for mammalian development. MINT enhances Runx2 activation of osteocalcin (OC) fibroblast growth factor (FGF) response element in an FGF2-dependent fashion in C3H10T1/2 cells. Although the MINT N-terminal RNA recognition motif domain contributes, the muscle segment homeobox homolog 2-interacting domain is sufficient for Runx2 activation. Intriguingly, Runx1 cannot replace Runx2 in this assay. To better understand this Runx2 signaling cascade, we performed structure-function analysis of the Runx2-MINT trans-activation relationship. Systematic truncation and domain swapping in Runx1:Runx2 chimeras identified that the unique Runx2 activation domain 3 (AD3), encompassed by residues 316–421, conveys MINT+FGF2 trans-activation in transfection assays. Ala mutagenesis of Runx2 Ser/Thr residues identified that S301 and T326 in AD3 are necessary for full MINT+FGF2 trans-activation. Conversely, phosphomimetic Asp substitution of these AD3 Ser/Thr residues enhanced activation by MINT. Adjacent Pro residues implicated regulation by a proline-directed protein kinase (PDPK). Systematic screening with PDPK inhibitors identified that the casein kinase-2/homeodomain-interacting protein kinase (HIPK)/dual specificity tyrosine phosphorylation regulated kinase inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), but not ERK, c-Jun N-terminal kinase, p38MAPK, or other casein kinase-2 inhibitors, abrogated Runx2-, MINT-, and FGF2-activation. Systematic small interfering RNA-mediated silencing of DMAT-inhibited PDPKs revealed that HIPK3 depletion reduced MINT+FGF2-dependent activation of Runx2. HIPK3 and Runx2 coprecipitate after in vitro transcription-translation, and recombinant HIPK3 recognizes Runx2 AD3 as kinase substrate. Furthermore, DMAT treatment and HIPK3 RNAi inhibited MINT+FGF2 activation of

  20. Polycomb recruitment at the Class II transactivator gene.

    PubMed

    Boyd, Nathaniel H; Morgan, Julie E; Greer, Susanna F

    2015-10-01

    The Class II Transactivator (CIITA) is the master regulator of Major Histocompatibility Class II (MHC II) genes. Transcription of CIITA through the IFN-γ inducible CIITA promoter IV (CIITA pIV) during activation is characterized by a decrease in trimethylation of histone H3 lysine 27 (H3K27me3), catalyzed by the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2). While EZH2 is the known catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and is present at the inactive CIITA pIV, the mechanism of PRC2 recruitment to mammalian promoters remains unknown. Here we identify two DNA-binding proteins, which interact with and regulate PRC2 recruitment to CIITA pIV. We demonstrate Yin Yang 1 (YY1) and Jumonji domain containing protein 2 (JARID2) are binding partners along with EZH2 in mammalian cells. Upon IFN-γ stimulation, YY1 dissociates from CIITA pIV while JARID2 binding to CIITA pIV increases, suggesting novel roles for these proteins in regulating expression of CIITA pIV. Knockdown of YY1 and JARID2 yields decreased binding of EZH2 and H3K27me3 at CIITA pIV, suggesting important roles for YY1 and JARID2 at CIITA pIV. JARID2 knockdown also results in significantly elevated levels of CIITA mRNA upon IFN-γ stimulation. This study is the first to identify novel roles of YY1 and JARID2 in the epigenetic regulation of the CIITA pIV by recruitment of PRC2. Our observations indicate the importance of JARID2 in CIITA pIV silencing, and also provide a novel YY1-JARID2-PRC2 regulatory complex as a possible explanation of differential PRC2 recruitment at inducible versus permanently silenced genes.

  1. Ligand-dependent responses of the silkworm prothoracicotropic hormone receptor, Torso, are maintained by unusual intermolecular disulfide bridges in the transmembrane region.

    PubMed

    Konogami, Tadafumi; Yang, Yiwen; Ogihara, Mari H; Hikiba, Juri; Kataoka, Hiroshi; Saito, Kazuki

    2016-03-01

    The insect membrane-protein, Torso, is a member of the receptor-tyrosine-kinase family, and is activated by its ligand, prothoracicotropic hormone (PTTH). Although PTTH is one of the most important regulators of insect development, the mechanism of Torso activation by the hormone has remained elusive. In this study, using heterologous expression in cultured Drosophila S2 cells, we detected ligand-independent dimerization of silkworm Torso, and found that the receptor molecules in the dimer were linked by intermolecular disulfide bridges. By examining the oligomerization states of several truncation and substitution mutants of Torso, atypical cysteine residues in the transmembrane region were identified as being responsible for the intermolecular linkage in the dimer. The replacement of all of the cysteines in the region with phenylalanines abolished the disulfide-bond-mediated dimerization; however, non-covalent dimerization of the mutant was detected using a cross-linking reagent, both with and without ligand stimulation. This non-covalent dimerization caused apparent receptor autophosphorylation independently of the ligand stimulation, but did not promote the ERK phosphorylation in the downstream signaling pathway. The unique Torso structure with the intermolecular disulfide bridges in the transmembrane region is necessary to maintain the ligand-dependent receptor functions of autophosphorylation and downstream activation.

  2. Kinetic analysis of aptazyme-regulated gene expression in a cell-free translation system: modeling of ligand-dependent and -independent expression.

    PubMed

    Kobori, Shungo; Ichihashi, Norikazu; Kazuta, Yasuaki; Matsuura, Tomoaki; Yomo, Tetsuya

    2012-08-01

    Aptazymes are useful as RNA-based switches of gene expression responsive to several types of compounds. One of the most important properties of the switching ability is the signal/noise (S/N) ratio, i.e., the ratio of gene expression in the presence of ligand to that in the absence of ligand. The present study was performed to gain a quantitative understanding of how the aptazyme S/N ratio is determined by factors involved in gene expression, such as transcription, RNA self-cleavage, RNA degradation, protein translation, and their ligand dependencies. We performed switching of gene expression using two on-switch aptazymes with different properties in a cell-free translation system, and constructed a kinetic model that quantitatively describes the dynamics of RNA and protein species involved in switching. Both theoretical and experimental analyses consistently demonstrated that factors determining both the absolute value and the dynamics of the S/N ratio are highly dependent on the routes of translation in the absence of ligand: translation from the ligand-independently cleaved RNA or leaky translation from the noncleaved RNA. The model obtained here is useful to assess the factors that restrict the S/N ratio and to improve aptazymes more efficiently.

  3. Detection of anabolic steroid abuse using a yeast transactivation system.

    PubMed

    Zierau, Oliver; Lehmann, Sylvi; Vollmer, Günter; Schänzer, Willhelm; Diel, Patrick

    2008-10-01

    The classical analytical method for detection of anabolic steroid abuse is gas chromatography followed by mass spectrometry (GC/MS). However, even molecules with a chemical structure typical for this class of substances, are sometimes not identified in routine screening by GC/MS when their precise chemical structure is still unknown. A supplementary approach to identify anabolic steroid abuse could be a structure-independent identification of anabolic steroids based on their biological activity. To test the suitability of such a system, we have analyzed the yeast androgen receptor (AR) reporter gene system to identify anabolic steroids in human urine samples. Analysis of different anabolic steroids dissolved in buffer demonstrated that the yeast reporter gene system is able to detect a variety of different anabolic steroids and their metabolites with high specificity, including the so-called 'designer steroid' tetrahydrogestrinone. In contrast, other non-androgenic steroids, like glucocordicoids, progestins, mineralocordicoids and estrogens had a low potency to stimulate transactivation. To test whether the system would also allow the detection of androgens in urine, experiments with spiked urine samples were performed. The androgen reporter gene in yeast responds very sensitive to 5alpha-dihydrotestosterone (DHT), even at high urine concentrations. To examine whether the test system would also be able to detect anabolic steroids in the urine of anabolic steroid abusers, anonymous urine samples previously characterized by GCMS were analyzed with the reporter gene assay. Even when the concentration of the anabolic metabolites was comparatively low in some positive samples it was possible to identify the majority of positive samples by their biological activity. In conclusion, our results demonstrate that the yeast reporter gene system detects anabolic steroids and corresponding metabolites with high sensitivity even in urine of anabolic steroid abusing athletes

  4. Characterization of the novel progestin gestodene by receptor binding studies and transactivation assays.

    PubMed

    Fuhrmann, U; Slater, E P; Fritzemeier, K H

    1995-01-01

    Gestodene is a novel progestin used in oral contraceptives with an increased separation of progestogenic versus androgenic activity and a distinct antimineralocorticoid activity. This specific pharmacological profile of gestodene is defined by its pattern of binding affinities to a variety of steroid hormone receptors. In the present study the affinity of gestodene to the progesterone receptor (PR), the androgen receptor (AR), the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR) and the estrogen receptor (ER) was re-evaluated by steroid binding assays and compared to those obtained for 3-keto-desogestrel and progesterone. The two synthetic progestins displayed identical high affinity to rabbit PR and similar marked binding to rat AR and GR, while progesterone showed high affinity to PR but only low binding to AR and GR. Furthermore, 3-keto-desogestrel exhibited almost no binding to MR, whereas gestodene, similar to progesterone, showed marked affinity to this receptor. In addition to receptor binding studies, transactivation assays were carried out to investigate the effects of gestodene on AR-, GR- and MR-mediated induction of transcription. In contrast to progesterone, which showed antiandrogenic activity, gestodene and 3-keto-desogestrel both exhibited androgenic activity. Furthermore, all three progestins exhibited weak GR-mediated antagonistic activity. In contrast to progesterone, which showed almost no glucocorticoid activity, gestodene and 3-keto-desogestrel showed weak glucocorticoid action. In addition, gestodene inhibited the aldosterone-induced reporter gene transcription, similar to progesterone, whereas unlike progesterone, gestodene did not induce reporter gene transcription. 3-Keto-desogestrel showed neither antimineralocorticoid nor mineralocorticoid action.

  5. Secretion of the human T cell leukemia virus type I transactivator protein tax.

    PubMed

    Alefantis, Timothy; Mostoller, Kate; Jain, Pooja; Harhaj, Edward; Grant, Christian; Wigdahl, Brian

    2005-04-29

    Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis. The HTLV-I protein Tax is well known as a transcriptional transactivator and inducer of cellular transformation. However, it is also known that extracellular Tax induces the production and release of cytokines, such as tumor necrosis factor-alpha and interleukin-6, which have adverse effects on cells of the central nervous system. The cellular process by which Tax exits the cell into the extracellular environment is currently unknown. In most cell types, Tax has been shown to localize primarily to the nucleus. However, Tax has also been found to accumulate in the cytoplasm. The results contained herein begin to characterize the process of Tax secretion from the cell. Specifically, cytoplasmic Tax was demonstrated to localize to organelles associated with the cellular secretory process including the endoplasmic reticulum and Golgi complex. Additionally, it was demonstrated that full-length Tax was secreted from both baby hamster kidney cells and a human kidney tumor cell line, suggesting that Tax enters the secretory pathway in a leaderless manner. Tax secretion was partially inhibited by brefeldin A, suggesting that Tax migrated from the endoplasmic reticulum to the Golgi complex. In addition, combined treatment of Tax-transfected BHK-21 cells with phorbol myristate acetate and ionomycin resulted in a small increase in the amount of Tax secreted, suggesting that a fraction of cytoplasmic Tax was present in the regulated secretory pathway. These studies begin to provide a link between Tax localization to the cytoplasm, the detection of Tax in the extracellular environment, its possible role as an extracellular effector molecule, and a potential role in neurodegenerative disease associated with HTLV-I infection.

  6. Serine 249 phosphorylation by ATM protein kinase regulates hepatocyte nuclear factor-1α transactivation.

    PubMed

    Zhao, Long; Chen, Hui; Zhan, Yi-Qun; Li, Chang-Yan; Ge, Chang-Hui; Zhang, Jian-Hong; Wang, Xiao-Hui; Yu, Miao; Yang, Xiao-Ming

    2014-07-01

    Hepatocyte nuclear factor-1 alpha (HNF1α) exerts important effects on gene expression in multiple tissues. Several studies have directly or indirectly supported the role of phosphorylation processes in the activity of HNF1α. However, the molecular mechanism of this phosphorylation remains largely unknown. Using microcapillary liquid chromatography MS/MS and biochemical assays, we identified a novel phosphorylation site in HNF1α at Ser249. We also found that the ATM protein kinase phosphorylated HNF1α at Ser249 in vitro in an ATM-dependent manner and that ATM inhibitor KU55933 treatment inhibited phosphorylation of HNF1α at Ser249 in vivo. Coimmunoprecipitation assays confirmed the association between HNF1α and ATM. Moreover, ATM enhanced HNF1α transcriptional activity in a dose-dependent manner, whereas the ATM kinase-inactive mutant did not. The use of KU55933 confirmed our observation. Compared with wild-type HNF1α, a mutation in Ser249 resulted in a pronounced decrease in HNF1α transactivation, whereas no dominant-negative effect was observed. The HNF1αSer249 mutant also exhibited normal nuclear localization but decreased DNA-binding activity. Accordingly, the functional studies of HNF1αSer249 mutant revealed a defect in glucose metabolism. Our results suggested that ATM regulates the activity of HNF1α by phosphorylation of serine 249, particularly in glucose metabolism, which provides valuable insights into the undiscovered mechanisms of ATM in the regulation of glucose homeostasis.

  7. Stimulation of Inducible Nitric Oxide by Hepatitis B Virus Transactivator Protein HBx Requires MTA1 Coregulator*

    PubMed Central

    Bui-Nguyen, Tri M.; Pakala, Suresh B.; Sirigiri, Divijendranatha Reddy; Martin, Emil; Murad, Ferid; Kumar, Rakesh

    2010-01-01

    Nitric oxide has been implicated in the pathogenesis of inflammatory disorders, including hepatitis B virus-associated hepatocellular carcinoma. Transactivator protein HBx, a major regulator of cellular responses of hepatitis B virus, is known to induce the expression of MTA1 (metastasis-associated protein 1) coregulator via NF-κB signaling in hepatic cells. However, the underlying mechanism of HBx regulation of the inducible nitric-oxide synthase (iNOS) pathway remains unknown. Here we provide evidence that MTA1 is a positive regulator of iNOS transcription and plays a mechanistic role in HBx stimulation of iNOS expression and activity. We found that the HBx-MTA1 complex is recruited onto the human iNOS promoter in an NF-κB-dependent manner. Pharmacological inhibition of the NF-κB signaling prevented the ability of HBx to stimulate the transcription, the expression, and the activity of iNOS; nevertheless, these effects could be substantially rescued by MTA1 dysregulation. We further discovered that HBx-mediated stimulation of MTA1 is paralleled by the suppression of miR-661, a member of the small noncoding RNAs, recently shown to target MTA1. We observed that miR-661 controls of MTA1 expression contributed to the expression and activity of iNOS in HBx-expressing HepG2 cells. Accordingly, depletion of MTA1 by either miR-661 or siRNA in HBx-expressing cells severely impaired the ability of HBx to modulate the endogenous levels of iNOS and nitrite production. Together, these findings reveal an inherent role of MTA1 in HBx regulation of iNOS expression and consequently its function in the liver cancer cells. PMID:20022949

  8. I-mfa domain proteins specifically interact with HTLV-1 Tax and repress its transactivating functions

    SciTech Connect

    Kusano, Shuichi; Yoshimitsu, Makoto; Hachiman, Miho; Ikeda, Masanori

    2015-12-15

    The I-mfa domain proteins HIC (also known as MDFIC) and I-mfa (also known as MDFI) are candidate tumor suppressor genes that are involved in cellular and viral transcriptional regulation. Here, we show that HIC and I-mfa directly interact with human T-cell leukemia virus type-1 (HTLV-1) Tax protein in vitro. In addition, HIC and I-mfa repress Tax-dependent transactivation of an HTLV-1 long terminal repeat (LTR) reporter construct in COS-1, Jurkat and high-Tax-producing HTLV-1-infected T cells. HIC also interacts with Tax through its I-mfa domain in vivo and represses Tax-dependent transactivation of HTLV-1 LTR and NF-κB reporter constructs in an interaction-dependent manner. Furthermore, we show that HIC decreases the nuclear distribution and stimulates the proteasomal degradation of Tax. These data reveal that HIC specifically interacts with HTLV-1 Tax and negatively regulates Tax transactivational activity by altering its subcellular distribution and stability. - Highlights: • I-mfa domain proteins, HIC and I-mfa, specifically interact with HTLV-1 Tax. • HIC and I-mfa repress the Tax-dependent transactivation of HTLV-1 LTR. • HIC represses the Tax-dependent transactivation of NF-κΒ. • HIC decreases the nuclear distribution of Tax. • HIC stimulates the proteasomal degradation of Tax.

  9. The nucleotide-binding domain of NLRC5 is critical for nuclear import and transactivation activity

    SciTech Connect

    Meissner, Torsten B.; Li, Amy; Liu, Yuen-Joyce; Gagnon, Etienne; Kobayashi, Koichi S.

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer NLRC5 requires an intact NLS for its function as MHC class I transactivator. Black-Right-Pointing-Pointer Nuclear presence of NLRC5 is required for MHC class I induction. Black-Right-Pointing-Pointer Nucleotide-binding controls nuclear import and transactivation activity of NLRC5. -- Abstract: Major histocompatibility complex (MHC) class I and class II are crucial for the function of the human adaptive immune system. A member of the NLR (nucleotide-binding domain, leucine-rich repeat) protein family, NLRC5, has recently been identified as a transcriptional regulator of MHC class I and related genes. While a 'master regulator' of MHC class II genes, CIITA, has long been known, NLRC5 specifically associates with and transactivates the proximal promoters of MHC class I genes. In this study, we analyzed the molecular requirements of NLRC5 nuclear import and transactivation activity. We show that NLRC5-mediated MHC class I gene induction requires an intact nuclear localization signal and nuclear distribution of NLRC5. In addition, we find that the nucleotide-binding domain (NBD) of NLRC5 is critical not only for nuclear translocation but also for the transactivation of MHC class I genes. Changing the cellular localization of NLRC5 is likely to immediately impact MHC class I expression as well as MHC class I-mediated antigen presentation. NLRC5 may thus provide a promising target for the modulation of MHC class I antigen presentation, especially in the setting of transplant medicine.

  10. Endogenous endothelin 1 mediates angiotensin II-induced hypertrophy in electrically paced cardiac myocytes through EGFR transactivation, reactive oxygen species and NHE-1.

    PubMed

    Correa, María V; Nolly, Mariela B; Caldiz, Claudia I; de Cingolani, Gladys E Chiappe; Cingolani, Horacio E; Ennis, Irene L

    2014-09-01

    Emerging evidence supports a key role for endothelin-1 (ET-1) and the transactivation of the epidermal growth factor receptor (EGFR) in angiotensin II (Ang II) action. We aim to determine the potential role played by endogenous ET-1, EGFR transactivation and redox-dependent sodium hydrogen exchanger-1 (NHE-1) activation in the hypertrophic response to Ang II of cardiac myocytes. Electrically paced adult cat cardiomyocytes were placed in culture and stimulated with 1 nmol l(-1) Ang II or 5 nmol l(-1) ET-1. Ang II increased ~45 % cell surface area (CSA) and ~37 % [(3)H]-phenylalanine incorporation, effects that were blocked not only by losartan (Los) but also by BQ123 (AT1 and ETA receptor antagonists, respectively). Moreover, Ang II significantly increased ET-1 messenger RNA (mRNA) expression. ET-1 similarly increased myocyte CSA and protein synthesis, actions prevented by the reactive oxygen species scavenger MPG or the NHE-1 inhibitor cariporide (carip). ET-1 increased the phosphorylation of the redox-sensitive ERK1/2-p90(RSK) kinases, main activators of the NHE-1. This effect was prevented by MPG and the antagonist of EGFR, AG1478. Ang II, ET-1 and EGF increased myocardial superoxide production (187 ± 9 %, 149 ± 8 % and 163.7 ± 6 % of control, respectively) and AG1478 inhibited these effects. Interestingly, Los inhibited only Ang II whilst BQ123 cancelled both Ang II and ET-1 actions, supporting the sequential and unidirectional activation of AT1, ETA and EGFR. Based on the present evidence, we propose that endogenous ET-1 mediates the hypertrophic response to Ang II by a mechanism that involves EGFR transactivation and redox-dependent activation of the ERK1/2-p90(RSK) and NHE-1 in adult cardiomyocytes.

  11. Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

    SciTech Connect

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N.; Nishida, Clinton R.; de Montellano, Paul R. Ortiz

    2015-02-10

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.

  12. Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

    DOE PAGES

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; ...

    2015-02-10

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop withmore » various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

  13. Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography*

    PubMed Central

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N.; Nishida, Clinton R.; de Montellano, Paul R. Ortiz

    2015-01-01

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. PMID:25670859

  14. Endothelin B Receptors on Primary Chicken Müller Cells and the Human MIO-M1 Müller Cell Line Activate ERK Signaling via Transactivation of Epidermal Growth Factor Receptors

    PubMed Central

    Harun-Or-Rashid, Mohammad; Konjusha, Dardan; Galindo-Romero, Caridad

    2016-01-01

    Injury to the eye or retina triggers Müller cells, the major glia cell of the retina, to dedifferentiate and proliferate. In some species they attain retinal progenitor properties and have the capacity to generate new neurons. The epidermal growth factor receptor (EGFR) system and extracellular signal-regulated kinase (ERK) signaling are key regulators of these processes in Müller cells. The extracellular signals that modulate and control these processes are not fully understood. In this work we studied whether endothelin receptor signaling can activate EGFR and ERK signaling in Müller cells. Endothelin expression is robustly upregulated at retinal injury and endothelin receptors have been shown to transactivate EGFRs in other cell types. We analyzed the endothelin signaling system in chicken retina and cultured primary chicken Müller cells as well as the human Müller cell line MIO-M1. The Müller cells were stimulated with receptor agonists and treated with specific blockers to key enzymes in the signaling pathway or with siRNAs. We focused on endothelin receptor mediated transactivation of EGFRs by using western blot analysis, quantitative reverse transcriptase PCR and immunocytochemistry. The results showed that chicken Müller cells and the human Müller cell line MIO-M1 express endothelin receptor B. Stimulation by the endothelin receptor B agonist IRL1620 triggered phosphorylation of ERK1/2 and autophosphorylation of (Y1173) EGFR. The effects could be blocked by Src-kinase inhibitors (PP1, PP2), EGFR-inhibitor (AG1478), EGFR-siRNA and by inhibitors to extracellular matrix metalloproteinases (GM6001), consistent with a Src-kinase mediated endothelin receptor response that engage ligand-dependent and ligand-independent EGFR activation. Our data suggest a mechanism for how injury-induced endothelins, produced in the retina, may modulate the Müller cell responses by Src-mediated transactivation of EGFRs. The data give support to a view in which endothelins

  15. Limited temperature-sensitive transactivation by mutant adenovirus type 2 E1a proteins.

    PubMed Central

    Fahnestock, M L; Lewis, J B

    1989-01-01

    A series of linker-scanning and deletion mutations was generated in the transactivating domain of the larger, 289-amino-acid-residue E1a protein of adenovirus type 2. Mutant genes were recombined into virus to assay the ability of the variant E1a proteins to activate expression of an E1a-dependent viral gene during infection. Results of assays performed at 32, 37, and 40 degrees C indicated that at least 2 of the 10 mutants tested showed limited temperature sensitivity for transactivation. Images PMID:2523001

  16. Phosphorylation of Serine422 increases the stability and transactivation activities of human Osterix.

    PubMed

    Xu, Yuexin; Yao, Bing; Shi, Kaikai; Lu, Jianlei; Jin, Yucui; Qi, Bing; Li, Hongwei; Pan, Shiyang; Chen, Li; Ma, Changyan

    2015-03-24

    Osterix (Osx) is an essential regulator for osteoblast differentiation and bone formation. Although phosphorylation has been reported to be involved in the regulation of Osx activity, the precise underlying mechanisms remain to be elucidated. Here we identified S422 as a novel phosphorylation site of Osx and demonstrated that GSK-3β interacted and co-localized with Osx. GSK-3β increased the stability and transactivation activity of Osx through phosphorylation of the newly identified site. These findings expanded our understanding of the mechanisms of posttranslational regulation of Osx and the role of GSK-3β in the control of Osx transactivation activity.

  17. Identifying p53 Transactivation Domain 1-Specific Inhibitors to Alleviate the Side Effects of Prostate Cancer Therapy

    DTIC Science & Technology

    2015-12-01

    cancer. 15. SUBJECT TERMS p53 inhibitor, p53 transactivation mutants 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES...gene regulatory elements. Keywords p53 inhibitor, p53 transactivation mutants , p53 transcriptional activation domain inhibitor Overall Project

  18. Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA

    PubMed Central

    Liguori, Alessia; Malito, Enrico; Lo Surdo, Paola; Fagnocchi, Luca; Cantini, Francesca; Haag, Andreas F.; Brier, Sébastien; Pizza, Mariagrazia; Delany, Isabel; Bottomley, Matthew J.

    2016-01-01

    Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in

  19. Gentian violet induces wtp53 transactivation in cancer cells.

    PubMed

    Garufi, Alessia; D'Orazi, Valerio; Arbiser, Jack L; D'Orazi, Gabriella

    2014-04-01

    Recent studies suggest that gentian violet (GV) may have anticancer activity by inhibiting for instance NADPH oxidases (Nox genes) whose overexpression is linked to tumor progression. Nox1 overexpression has been shown to inhibit transcriptional activity of the oncosuppressor p53, impairing tumor cell response to anticancer drugs. The tumor suppressor p53 is a transcription factor that, upon cellular stress, is activated to induce target genes involved in tumor cell growth inhibition and apoptosis. Thus, its activation is important for efficient tumor eradication. In this study, we examined the effect of GV on wild-type (wt) p53 activity in cancer cells. We found that GV was able to overcome the inhibitory effect of the NADPH oxidase Nox1 on p53 transcriptional activity. For the first time we show that GV was able to directly induce p53/DNA binding and transcriptional activity. In vitro, GV markedly induced cancer cell death and apoptotic marker PARP cleavage in wtp53-carrying cells. GV-induced cell death was partly inhibited in cells deprived of p53, suggesting that the anticancer activity of GV may partly depend on p53 activation. GV is US Food and Drug Administration approved for human use and may, therefore, have therapeutic potential in the management of cancer through p53 activation.

  20. Gentian violet induces wtp53 transactivation in cancer cells

    PubMed Central

    GARUFI, ALESSIA; D’ORAZI, VALERIO; ARBISER, JACK L.; D’ORAZI, GABRIELLA

    2014-01-01

    Recent studies suggest that gentian violet (GV) may have anticancer activity by inhibiting for instance NADPH oxidases (Nox genes) whose overexpression is linked to tumor progression. Nox1 overexpression has been shown to inhibit transcriptional activity of the oncosuppressor p53, impairing tumor cell response to anticancer drugs. The tumor suppressor p53 is a transcription factor that, upon cellular stress, is activated to induce target genes involved in tumor cell growth inhibition and apoptosis. Thus, its activation is important for efficient tumor eradication. In this study, we examined the effect of GV on wild-type (wt) p53 activity in cancer cells. We found that GV was able to overcome the inhibitory effect of the NADPH oxidase Nox1 on p53 transcriptional activity. For the first time we show that GV was able to directly induce p53/DNA binding and transcriptional activity. In vitro, GV markedly induced cancer cell death and apoptotic marker PARP cleavage in wtp53-carrying cells. GV-induced cell death was partly inhibited in cells deprived of p53, suggesting that the anticancer activity of GV may partly depend on p53 activation. GV is US Food and Drug Administration approved for human use and may, therefore, have therapeutic potential in the management of cancer through p53 activation. PMID:24535435

  1. Sox10 is an active nucleocytoplasmic shuttle protein, and shuttling is crucial for Sox10-mediated transactivation.

    PubMed

    Rehberg, Stephan; Lischka, Peter; Glaser, Gabi; Stamminger, Thomas; Wegner, Michael; Rosorius, Olaf

    2002-08-01

    Sox10 belongs to a family of transcription regulators characterized by a DNA-binding domain known as the HMG box. It plays fundamental roles in neural crest development, peripheral gliogenesis, and terminal differentiation of oligodendrocytes. In accord with its function as transcription factor, Sox10 contains two nuclear localization signals and is most frequently detected in the nucleus. In this study, we report that Sox10 is an active nucleocytoplasmic shuttle protein, competent of both entering and exiting the nucleus. We identified a functional Rev-type nuclear export signal within the DNA-binding domain of Sox10. Mutational inactivation of this nuclear export signal or treatment of cells with the CRM1-specific export inhibitor leptomycin B inhibited nuclear export and consequently nucleocytoplasmic shuttling of Sox10. Importantly, the inhibition of the nuclear export of Sox10 led to decreased transactivation of transfected reporters and endogenous target genes, arguing that continuous nucleocytoplasmic shuttling is essential for the function of Sox10. To our knowledge this is the first time that nuclear export has been reported and shown to be functionally relevant for any Sox protein.

  2. Variability of Inducible Expression across the Hematopoietic System of Tetracycline Transactivator Transgenic Mice

    PubMed Central

    Takiguchi, Megumi; Dow, Lukas E.; Prier, Julia E.; Carmichael, Catherine L.; Kile, Benjamin T.; Turner, Stephen J.; Lowe, Scott W.; Huang, David C. S.; Dickins, Ross A.

    2013-01-01

    The tetracycline (tet)-regulated expression system allows for the inducible overexpression of protein-coding genes, or inducible gene knockdown based on expression of short hairpin RNAs (shRNAs). The system is widely used in mice, however it requires robust expression of a tet transactivator protein (tTA or rtTA) in the cell type of interest. Here we used an in vivo tet-regulated fluorescent reporter approach to characterise inducible gene/shRNA expression across a range of hematopoietic cell types of several commonly used transgenic tet transactivator mouse strains. We find that even in strains where the tet transactivator is expressed from a nominally ubiquitous promoter, the efficiency of tet-regulated expression can be highly variable between hematopoietic lineages and between differentiation stages within a lineage. In some cases tet-regulated reporter expression differs markedly between cells within a discrete, immunophenotypically defined population, suggesting mosaic transactivator expression. A recently developed CAG-rtTA3 transgenic mouse displays intense and efficient reporter expression in most blood cell types, establishing this strain as a highly effective tool for probing hematopoietic development and disease. These findings have important implications for interpreting tet-regulated hematopoietic phenotypes in mice, and identify mouse strains that provide optimal tet-regulated expression in particular hematopoietic progenitor cell types and mature blood lineages. PMID:23326559

  3. Transactivation of lung lysozyme expression by Ets family member ESE-1.

    PubMed

    Lei, Wanli; Jaramillo, Richard J; Harrod, Kevin S

    2007-11-01

    Epithelial-specific Ets (ESE) transcription factors, consisting of ESE-1, ESE-2, and ESE-3, are constitutively expressed in distinct epithelia of mucosal tissues, including the lung. Each ESE member exhibits alternative splicing and yields at least two isoforms (a and b) with transcriptional targets largely unidentified. The studies described herein define a novel role for ESE transcription factors in transactivation of the human lysozyme gene (LYZ), an essential component of innate defense in lung epithelia. Of the six ESE isoforms, ESE-1a and ESE-1b transactivated LYZ promoter in reporter gene assays, whereas only ESE-1b dramatically upregulated transcription of endogenous LYZ in both nonpulmonary and pulmonary epithelial cells. Importantly, ESE-1a and ESE-1b could transactivate the LYZ promoter in cultured primary airway epithelial cells. ESE-2 and ESE-3 isoforms were unable to substantially transactivate the lysozyme promoter or upregulate transcription of endogenous LYZ. Two functional consensus Ets sites located in the proximal 130-bp LYZ promoter were responsive to ESE-1b as identified by site-directed mutagenesis and DNA binding assays. Short hairpin RNA attenuation of endogenous ESE-1b mRNA levels in lung epithelia resulted in decreased LYZ transcription. Furthermore, ESE-1 antibody specifically enriched the 130-bp proximal LYZ promoter in chromatin immunoprecipitation analyses. These findings define a novel role for ESE transcription factors in regulating lung innate defense and suggest distinct regulatory functions for ESE family members.

  4. Proteomic analysis of the herpes simplex virus 1 virion protein 16 transactivator protein in infected cells.

    PubMed

    Suk, Hyung; Knipe, David M

    2015-06-01

    The herpes simplex virus 1 virion protein 16 (VP16) tegument protein forms a transactivation complex with the cellular proteins host cell factor 1 (HCF-1) and octamer-binding transcription factor 1 (Oct-1) upon entry into the host cell. VP16 has also been shown to interact with a number of virion tegument proteins and viral glycoprotein H to promote viral assembly, but no comprehensive study of the VP16 proteome has been performed at early times postinfection. We therefore performed a proteomic analysis of VP16-interacting proteins at 3 h postinfection. We confirmed the interaction of VP16 with HCF-1 and a large number of cellular Mediator complex proteins, but most surprisingly, we found that the major viral protein associating with VP16 is the infected cell protein 4 (ICP4) immediate-early (IE) transactivator protein. These results raise the potential for a new function for VP16 in associating with the IE ICP4 and playing a role in transactivation of early and late gene expression, in addition to its well-documented function in transactivation of IE gene expression.

  5. Transactivation of bad by vorinostat-induced acetylated p53 enhances doxorubicin-induced cytotoxicity in cervical cancer cells.

    PubMed

    Lee, Sook-Jeong; Hwang, Sung-Ook; Noh, Eun Joo; Kim, Dong-Uk; Nam, Miyoung; Kim, Jong Hyeok; Nam, Joo Hyun; Hoe, Kwang-Lae

    2014-02-14

    Vorinostat (VOR) has been reported to enhance the cytotoxic effects of doxorubicin (DOX) with fewer side effects because of the lower DOX dosage in breast cancer cells. In this study, we investigated the novel mechanism underlying the synergistic cytotoxic effects of VOR and DOX co-treatment in cervical cancer cells HeLa, CaSki and SiHa cells. Co-treatment with VOR and DOX at marginal doses led to the induction of apoptosis through caspase-3 activation, poly (ADP-ribose) polymerase cleavage and DNA micronuclei. Notably, the synergistic growth inhibition induced by the co-treatment was attributed to the upregulation of the pro-apoptotic protein Bad, as the silencing of Bad expression using small interfering RNA (siRNA) abolished the phenomenon. As siRNA against p53 did not result in an increase in acetylated p53 and the consequent upregulation of Bad, the observed Bad upregulation was mediated by acetylated p53. Moreover, a chromatin immunoprecipitation analysis showed that the co-treatment of HeLa cells with VOR and DOX increased the recruitment of acetylated p53 to the bad promoter, with consequent bad transactivation. Conversely, C33A cervical cancer cells containing mutant p53 co-treated with VOR and DOX did not exhibit Bad upregulation, acetylated p53 induction or consequent synergistic growth inhibition. Together, the synergistic growth inhibition of cervical cancer cell lines induced by co-treatment with VOR and DOX can be attributed to the upregulation of Bad, which is induced by acetylated p53. These results show for the first time that the acetylation of p53, rather than histones, is a mechanism for the synergistic growth inhibition induced by VOR and DOX co-treatments.

  6. Epidermal growth factor receptor transactivation is implicated in IL-6-induced proliferation and ERK1/2 activation in non-transformed prostate epithelial cells.

    PubMed

    Poncet, Nadège; Guillaume, Johann; Mouchiroud, Guy

    2011-03-01

    Epidermal growth factor receptor (EGF-R) is a receptor tyrosine kinase that can be activated by molecules other than its cognate ligands. This form of crosstalk called transactivation is frequently observed in both physiological and pathological cellular responses, yet it involves various mechanisms. Using the RWPE-1 cell line as a model of non-transformed prostate epithelial progenitor cells, we observed that interleukin-6 (IL-6) is able to promote cell proliferation and ERK1/2 activation provided that EGF-R kinase activity is not impaired. Treatment with GM6001, a general matrix metalloprotease inhibitor, indicated that IL-6 activates EGF-R through cleavage and release of membrane-anchored EGF-R ligands. Several inhibitors were used to test implication of "a disintegrin and metalloprotease" ADAM10 and ADAM17. GW280264X that targets both ADAM10 and ADAM17 blocked IL-6-induced proliferation and ERK1/2 phosphorylation with same potency as GM6001. However, ADAM10 inhibitor GI254023X and ADAM17 inhibitor TAPI-2 were less efficient in inhibiting response of RWPE-1 cells to IL-6, indicating possible cooperation of ADAM17 with ADAM10 or other metalloproteases. Accordingly, our findings suggest that IL-6 stimulates shedding of EGF-R ligands and transactivation of EGF-R in normal prostate epithelial cells, which may be an important mechanism to promote cell proliferation in inflammatory prostate.

  7. A1 adenosine receptor–stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation

    PubMed Central

    Prakasam, H. Sandeep; Gallo, Luciana I.; Li, Hui; Ruiz, Wily G.; Hallows, Kenneth R.; Apodaca, Gerard

    2014-01-01

    Despite the importance of ADAM17-dependent cleavage in normal biology and disease, the physiological cues that trigger its activity, the effector pathways that promote its function, and the mechanisms that control its activity, particularly the role of phosphorylation, remain unresolved. Using native bladder epithelium, in some cases transduced with adenoviruses encoding small interfering RNA, we observe that stimulation of apically localized A1 adenosine receptors (A1ARs) triggers a Gi-Gβγ-phospholipase C-protein kinase C (PKC) cascade that promotes ADAM17-dependent HB-EGF cleavage, EGFR transactivation, and apical exocytosis. We further show that the cytoplasmic tail of rat ADAM17 contains a conserved serine residue at position 811, which resides in a canonical PKC phosphorylation site, and is phosphorylated in response to A1AR activation. Preventing this phosphorylation event by expression of a nonphosphorylatable ADAM17S811A mutant or expression of a tail-minus construct inhibits A1AR-stimulated, ADAM17-dependent HB-EGF cleavage. Furthermore, expression of ADAM17S811A in bladder tissues impairs A1AR-induced apical exocytosis. We conclude that adenosine-stimulated exocytosis requires PKC- and ADAM17-dependent EGFR transactivation and that the function of ADAM17 in this pathway depends on the phosphorylation state of Ser-811 in its cytoplasmic domain. PMID:25232008

  8. The N-terminal nuclear localization sequences of liver X receptors alpha and beta bind to importin alpha and are essential for both nuclear import and transactivating functions.

    PubMed

    Miller, Anna; Crumbley, Christine; Prüfer, Kirsten

    2009-04-01

    Liver X receptors (LXRs) alpha and beta are nuclear receptors, which form obligate heterodimers with the retinoid X receptor (RXR). The LXRs regulate both redundantly and non-redundantly the transcription of genes controlling cholesterol metabolism and transport as well as lipogenesis. Previously, we showed that mutations in putative N-terminal nuclear localization sequences (NLSs) within both LXRs inhibit nuclear import. Through in vitro studies, we show here that these NLSs bind importin alpha and are both necessary and sufficient for the nuclear import of LXRs. Imaging, transactivation, and electro-mobility shift experiments show that RXR rescues the nuclear import of the LXRalpha NLS mutant yet does not restore its transcriptional activity despite intact DNA binding. In contrast, RXR partially rescues the import of the LXRbeta NLS mutant, but has no effect on its transcriptional activity due to the loss of DNA binding. Experiments with NLS mutant RXR confirmed that RXR may dominate the nuclear import of the RXR/LXRalpha heterodimer, whereas LXRbeta dominates the nuclear import of the RXR/LXRbeta heterodimer. Intriguingly, our data indicate differences between LXRalpha and LXRbeta in their interaction with RXR and in the role their NLSs play in transactivating functions independent of nuclear import.

  9. Physical and functional interaction of the Epstein-Barr virus BZLF1 transactivator with the retinoic acid receptors RAR alpha and RXR alpha.

    PubMed Central

    Sista, N D; Barry, C; Sampson, K; Pagano, J

    1995-01-01

    Epstein-Barr virus (EBV) reactivation, indicated by induction of EBV early antigens from latently infected lymphoid cell lines by phorbol esters, is inhibited by retinoic acid (RA). Viral reactivation, which is triggered by the immediate-early BZLF-1 (Z) viral gene product, is repressed by retinoic acid receptors (RARs) RAR alpha and RXR alpha. These proteins negatively regulate Z-mediated transactivation of the promoter for an EBV early gene product, early antigen-diffuse (EaD). Here we confirm a direct physical interaction between the AP1-like protein Z and RXR alpha and map the domains of interaction in the Z protein and RXR alpha. The domain required for homodimerization of Z is separate from that required for its interaction with RXR alpha. Z also has the effect of repressing activation of an RAR-responsive cellular promoter (BRE). Point mutants in the dimerization domain of Z unable to interact with RXR alpha do not repress RXR alpha-mediated transactivation of BRE, the promoter for RAR beta, which suggests that interaction between the two proteins is required for this repressor effect. The domain of RXR alpha required for interaction with Z has been mapped, and is again separate from that required for homodimerization. These results indicate that a 'cross-coupling' or direct interaction between Z and RAR alpha and RXR alpha can modulate the reactivation of latent EBV infection and suggest that, reciprocally, the viral protein Z may influence cellular regulatory pathways. Images PMID:7784177

  10. Sharpin promotes hepatocellular carcinoma progression via transactivation of Versican expression

    PubMed Central

    Tanaka, Y; Tateishi, K; Nakatsuka, T; Kudo, Y; Takahashi, R; Miyabayashi, K; Yamamoto, K; Asaoka, Y; Ijichi, H; Tateishi, R; Shibahara, J; Fukayama, M; Ishizawa, T; Hasegawa, K; Kokudo, N; Koike, K

    2016-01-01

    Sharpin (Shank-associated RH domain-interacting protein, also known as SIPL1) is a multifunctional molecule that participates in various biological settings, including nuclear factor-κB signaling activation and tumor suppressor gene inhibition. Sharpin is upregulated in various types of cancers, including hepatocellular carcinoma (HCC), and is implicated in tumor progression. However, the exact roles of Sharpin in tumorigenesis and tumor progression remain largely unknown. Here we report novel mechanisms of HCC progression through Sharpin overexpression. In our study, Sharpin was upregulated in human HCC tissues. Increased Sharpin expression enhanced hepatoma cell invasion, whereas decrease in Sharpin expression by RNA interference inhibited invasion. Microarray analysis identified that Versican, a chondroitin sulfate proteoglycan that plays crucial roles in tumor progression and invasion, was also upregulated in Sharpin-expressing stable cells. Versican expression increased in the majority of HCC tissues and knocking down of Versican greatly attenuated hepatoma cell invasion. Sharpin expression resulted in a significant induction of Versican transcription synergistically with Wnt/β-catenin pathway activation. Furthermore, Sharpin-overexpressing cells had high tumorigenic properties in vivo. These results demonstrate that Sharpin promotes Versican expression synergistically with the Wnt/β-catenin pathway, potentially contributing to HCC development. A Sharpin/Versican axis could be an attractive therapeutic target for this currently untreatable cancer. PMID:27941932

  11. The 9aaTAD Transactivation Domains: From Gal4 to p53

    PubMed Central

    Havelka, Marek; Rezacova, Martina

    2016-01-01

    The family of the Nine amino acid Transactivation Domain, 9aaTAD family, comprises currently over 40 members. The 9aaTAD domains are universally recognized by the transcriptional machinery from yeast to man. We had identified the 9aaTAD domains in the p53, Msn2, Pdr1 and B42 activators by our prediction algorithm. In this study, their competence to activate transcription as small peptides was proven. Not surprisingly, we elicited immense 9aaTAD divergence in hundreds of identified orthologs and numerous examples of the 9aaTAD species' convergence. We found unforeseen similarity of the mammalian p53 with yeast Gal4 9aaTAD domains. Furthermore, we identified artificial 9aaTAD domains generated accidentally by others. From an evolutionary perspective, the observed easiness to generate 9aaTAD transactivation domains indicates the natural advantage for spontaneous generation of transcription factors from DNA binding precursors. PMID:27618436

  12. c-Jun transactivates Puma gene expression to promote osteoarthritis.

    PubMed

    Lu, Huading; Hou, Gang; Zhang, Yongkai; Dai, Yuhu; Zhao, Huiqing

    2014-05-01

    Osteoarthritis (OA) is a chronic degenerative joint disorder in which genetic, hormonal, mechanical and ageing factors affect its progression. Current studies are focusing on chondrocytes as a key mediator of OA at a cellular level. however, the mechanism underlying chondrocyte apoptosis remains unclear. PUMA is a pro-apoptotic member of the BH3-only subgroup of the Bcl-2 family and is involved in a large number of physiological and pathological processes. In the present study, we examined whether PUMA has a role in IL-1β-induced apoptosis and whether the c-Jun N-terminal kinase (JNK)/c-Jun pathway mediates the induction of PUMA, thus contributing to chondrocyte apoptosis. The results demonstrated an increase in PUMA protein and mRNA levels in cultured mouse chondrocytes following 4 h of IL-1β treatment. Furthermore, this upregulation of PUMA was critical for chondrocyte apoptosis as knockdown of PUMA using PUMA-specific siRNA significantly reduced apoptosis in cultured cells. Upon pharmacological inhibition of the JNK/c-Jun pathway with CE11004 or SP600125, the expression of PUMA was notably suppressed with a concomitant decrease in apoptosis observed in IL-1β-treated chondrocytes. Also, immunohistochemical studies revealed that the PUMA and c-Jun proteins were upregulated in chondrocytes from the articular cartilage of OA patients. Together, these data suggest a role for PUMA and the JNK/c-Jun pathway in the regulation of chondrocyte apoptosis during OA.

  13. Transactivation of Epidermal Growth Factor Receptor by G Protein-Coupled Receptors: Recent Progress, Challenges and Future Research

    PubMed Central

    Wang, Zhixiang

    2016-01-01

    Both G protein-coupled receptors (GPCRs) and receptor-tyrosine kinases (RTKs) regulate large signaling networks, control multiple cell functions and are implicated in many diseases including various cancers. Both of them are also the top therapeutic targets for disease treatment. The discovery of the cross-talk between GPCRs and RTKs connects these two vast signaling networks and complicates the already complicated signaling networks that regulate cell signaling and function. In this review, we focus on the transactivation of epidermal growth factor receptor (EGFR), a subfamily of RTKs, by GPCRs. Since the first report of EGFR transactivation by GPCR, significant progress has been made including the elucidation of the mechanisms underlying the transactivation. Here, we first provide a basic picture for GPCR, EGFR and EGFR transactivation by GPCR. We then discuss the progress made in the last five years and finally provided our view of the future challenge and future researches needed to overcome these challenges. PMID:26771606

  14. Transactivation of Epidermal Growth Factor Receptor by G Protein-Coupled Receptors: Recent Progress, Challenges and Future Research.

    PubMed

    Wang, Zhixiang

    2016-01-12

    Both G protein-coupled receptors (GPCRs) and receptor-tyrosine kinases (RTKs) regulate large signaling networks, control multiple cell functions and are implicated in many diseases including various cancers. Both of them are also the top therapeutic targets for disease treatment. The discovery of the cross-talk between GPCRs and RTKs connects these two vast signaling networks and complicates the already complicated signaling networks that regulate cell signaling and function. In this review, we focus on the transactivation of epidermal growth factor receptor (EGFR), a subfamily of RTKs, by GPCRs. Since the first report of EGFR transactivation by GPCR, significant progress has been made including the elucidation of the mechanisms underlying the transactivation. Here, we first provide a basic picture for GPCR, EGFR and EGFR transactivation by GPCR. We then discuss the progress made in the last five years and finally provided our view of the future challenge and future researches needed to overcome these challenges.

  15. EPAS1 trans-activation during hypoxia requires p42/p44 MAPK.

    PubMed

    Conrad, P W; Freeman, T L; Beitner-Johnson, D; Millhorn, D E

    1999-11-19

    Hypoxia is a common environmental stress that regulates gene expression and cell function. A number of hypoxia-regulated transcription factors have been identified and have been shown to play critical roles in mediating cellular responses to hypoxia. One of these is the endothelial PAS-domain protein 1 (EPAS1/HIF2-alpha/HLF/HRF). This protein is 48% homologous to hypoxia-inducible factor 1-alpha (HIF1-alpha). To date, virtually nothing is known about the signaling pathways that lead to either EPAS1 or HIF1-alpha activation. Here we show that EPAS1 is phosphorylated when PC12 cells are exposed to hypoxia and that p42/p44 MAPK is a critical mediator of EPAS1 activation. Pretreatment of PC12 cells with the MEK inhibitor, PD98059, completely blocked hypoxia-induced trans-activation of a hypoxia response element (HRE) reporter gene by transfected EPAS1. Likewise, expression of a constitutively active MEK1 mimicked the effects of hypoxia on HRE reporter gene expression. However, pretreatment with PD98059 had no effect on EPAS1 phosphorylation during hypoxia, suggesting that MAPK targets other proteins that are critical for the trans-activation of EPAS1. We further show that hypoxia-induced trans-activation of EPAS1 is independent of Ras. Finally, pretreatment with calmodulin antagonists nearly completely blocked both the hypoxia-induced phosphorylation of MAPK and the EPAS1 trans-activation of HRE-Luc. These results demonstrate that the MAPK pathway is a critical mediator of EPAS1 activation and that activation of MAPK and EPAS1 occurs through a calmodulin-sensitive pathway and not through the GTPase, Ras. These results are the first to identify a specific signaling pathway involved in EPAS1 activation.

  16. Crucial Roles for Interactions between MLL3/4 and INI1 in Nuclear Receptor Transactivation

    PubMed Central

    Lee, Seunghee; Kim, Dae-Hwan; Goo, Young Hwa; Lee, Young Chul; Lee, Soo-Kyung; Lee, Jae W.

    2009-01-01

    Nuclear receptor (NR) transactivation involves multiple coactivators, and the molecular basis for how these are functionally integrated needs to be determined to fully understand the NR action. Activating signal cointegrator-2 (ASC-2), a transcriptional coactivator of many NRs and transcription factors, forms a steady-state complex, ASCOM (for ASC-2 complex), which contains histone H3-lysine-4 (H3K4) methyltransferase MLL3 or its paralog MLL4. Here, we show that ASCOM requires a functional cross talk with the ATPase-dependent chromatin remodeling complex Swi/Snf for efficient NR transactivation. Our results reveal that ASCOM and Swi/Snf are tightly colocalized in the nucleus and that ASCOM and Swi/Snf promote each other’s binding to NR target genes. We further show that the C-terminal SET domain of MLL3 and MLL4 directly interacts with INI1, an integral subunit of Swi/Snf. Our mutational analysis demonstrates that this interaction underlies the mutual facilitation of ASCOM and Swi/Snf recruitment to NR target genes. Importantly, this study uncovers a specific protein-protein interaction as a novel venue to couple two distinct enzymatic coactivator complexes during NR transactivation. PMID:19221051

  17. I-mfa domain proteins specifically interact with HTLV-1 Tax and repress its transactivating functions.

    PubMed

    Kusano, Shuichi; Yoshimitsu, Makoto; Hachiman, Miho; Ikeda, Masanori

    2015-12-01

    The I-mfa domain proteins HIC (also known as MDFIC) and I-mfa (also known as MDFI) are candidate tumor suppressor genes that are involved in cellular and viral transcriptional regulation. Here, we show that HIC and I-mfa directly interact with human T-cell leukemia virus type-1 (HTLV-1) Tax protein in vitro. In addition, HIC and I-mfa repress Tax-dependent transactivation of an HTLV-1 long terminal repeat (LTR) reporter construct in COS-1, Jurkat and high-Tax-producing HTLV-1-infected T cells. HIC also interacts with Tax through its I-mfa domain in vivo and represses Tax-dependent transactivation of HTLV-1 LTR and NF-κB reporter constructs in an interaction-dependent manner. Furthermore, we show that HIC decreases the nuclear distribution and stimulates the proteasomal degradation of Tax. These data reveal that HIC specifically interacts with HTLV-1 Tax and negatively regulates Tax transactivational activity by altering its subcellular distribution and stability.

  18. Androgen receptor transactivation assay using green fluorescent protein as a reporter.

    PubMed

    Beck, Verena; Reiter, Evelyne; Jungbauer, Alois

    2008-02-15

    For screening of a large number of samples for androgenic activity, a robust system with minimal handling is required. The coding sequence for human androgen receptor (AR) was inserted into expression plasmid YEpBUbi-FLAG1, resulting in the plasmid YEpBUbiFLAG-AR, and the estrogen response element (ERE) on the reporter vector YRpE2 was replaced by an androgen response element (ARE), resulting in the plasmid YRpE2-ARE. Thus, a fully functional transactivation assay system with beta-galactosidase as a reporter gene could be created. Furthermore, green fluorescent protein (GFP) was introduced as an alternative reporter gene that resulted in a simplification of the whole assay procedure. For evaluation of both reporter systems, seven steroidal compounds with known AR agonistic properties (5 alpha-dihydrotestosterone, testosterone, androstenedione, 17 alpha-methyltestosterone, progesterone, epitestosterone, and d-norgestrel) were tested, and their potencies obtained in the different assays were compared. Furthermore, potencies from the transactivation assays were compared with IC(50) values obtained in radioligand binding assays. The newly developed androgen receptor transactivation assay is a useful tool for characterizing compounds with androgenic activity.

  19. Transactivation of the parathyroid hormone promoter by specificity proteins and the nuclear factor Y complex.

    PubMed

    Alimov, Alexander P; Park-Sarge, Ok-Kyong; Sarge, Kevin D; Malluche, Hartmut H; Koszewski, Nicholas J

    2005-08-01

    We previously identified a highly conserved specificity protein 1 (Sp1) DNA element in mammalian PTH promoters that acted as an enhancer of gene transcription and bound Sp1 and Sp3 proteins present in parathyroid gland nuclear extracts. More recently, a nuclear factor (NF)-Y element (NF-Y(prox)) was also described by our group, which was located approximately 30 bp downstream from the Sp1 site in the human PTH (hPTH) promoter and by itself acted as a weak enhancer of gene transcription. We now report that Sp proteins and NF-Y can synergistically enhance transcription of a minimal hPTH promoter construct. Positioning of the Sp1 DNA element appears to be critical for this synergism because deviations of one half of a helical turn caused an approximate 60% decrease in transactivation. Finally, examination of the bovine PTH (bPTH) promoter also revealed Sp1/NF-Y synergism, in conjunction with the identification of an analogous NF-Y binding site similarly positioned downstream from the bPTH Sp1 element. In summary, synergistic transactivation of the hPTH and bPTH promoters is observed by Sp proteins and the NF-Y complex. The conservation of this transactivation in the human and bovine promoters suggests that this may be a principle means of enhancing PTH gene transcription.

  20. Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells

    PubMed Central

    Chen, Fang-Fang; Yen, Zen-Zen; Lindemann, Nils; Meyer, Steffen; Spehr, Johannes; van den Heuvel, Joop

    2016-01-01

    The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS. PMID:26934632

  1. Divergent Transactivation of Maize Storage Protein Zein Genes by the Transcription Factors Opaque2 and OHPs

    PubMed Central

    Yang, Jun; Ji, Chen; Wu, Yongrui

    2016-01-01

    Maize transcription factors (TFs) opaque2 (O2) and the O2 heterodimerizing proteins (OHP1 and OHP2) originated from an ancient segmental duplication. The 22-kDa (z1C) and 19-kDa (z1A, z1B, and z1D) α-zeins are the most abundant storage proteins in maize endosperm. O2 is known to regulate α-zein gene expression, but its target motifs in the 19-kDa α-zein gene promoters have not been identified. The mechanisms underlying the regulation of α-zein genes by these TFs are also not well understood. In this study, we found that the O2 binding motifs in the α-zein gene promoters are quite flexible, with ACGT being present in the z1C and z1A promoters and a variant, ACAT, being present in the z1B and z1D promoters. OHPs recognized and transactivated all of the α-zein promoters, although to much lower levels than did O2. In the presence of O2, the suppression of OHPs did not cause a significant reduction in the transcription of α-zein genes, but in the absence of O2, OHPs were critical for the expression of residual levels of α-zeins. These findings demonstrated that O2 is the primary TF and that OHPs function as minor TFs in this process. This relationship is the converse of that involved in 27-kDa γ-zein gene regulation, indicating that the specificities of O2 and the OHPs for regulating zein genes diverged after gene duplication. The prolamine-box binding factor by itself has limited transactivation activity, but it promotes the binding of O2 to O2 motifs, resulting in the synergistic transactivation of α-zein genes. PMID:27474726

  2. Delta-1 Activation of Notch-1 Signaling Results in HES-1 Transactivation

    PubMed Central

    Jarriault, Sophie; Le Bail, Odile; Hirsinger, Estelle; Pourquié, Olivier; Logeat, Frédérique; Strong, Clare F.; Brou, Christel; Seidah, Nabil G.; Israël, Alain

    1998-01-01

    The Notch receptor is involved in many cell fate determination events in vertebrates and invertebrates. It has been shown in Drosophila melanogaster that Delta-dependent Notch signaling activates the transcription factor Suppressor of Hairless, leading to an increased expression of the Enhancer of Split genes. Genetic evidence has also implicated the kuzbanian gene, which encodes a disintegrin metalloprotease, in the Notch signaling pathway. By using a two-cell coculture assay, we show here that vertebrate Dl-1 activates the Notch-1 cascade. Consistent with previous data obtained with active forms of Notch-1 a HES-1-derived promoter construct is transactivated in cells expressing Notch-1 in response to Dl-1 stimulation. Impairing the proteolytic maturation of the full-length receptor leads to a decrease in HES-1 transactivation, further supporting the hypothesis that only mature processed Notch is expressed at the cell surface and activated by its ligand. Furthermore, we observed that Dl-1-induced HES-1 transactivation was dependent both on Kuzbanian and RBP-J activities, consistent with the involvement of these two proteins in Notch signaling in Drosophila. We also observed that exposure of Notch-1-expressing cells to Dl-1 results in an increased level of endogenous HES-1 mRNA. Finally, coculture of Dl-1-expressing cells with myogenic C2 cells suppresses differentiation of C2 cells into myotubes, as previously demonstrated for Jagged-1 and Jagged-2, and also leads to an increased level of endogenous HES-1 mRNA. Thus, Dl-1 behaves as a functional ligand for Notch-1 and has the same ability to suppress cell differentiation as the Jagged proteins do. PMID:9819428

  3. Characterization of a nuclear export signal within the human T cell leukemia virus type I transactivator protein Tax.

    PubMed

    Alefantis, Timothy; Barmak, Kate; Harhaj, Edward W; Grant, Christian; Wigdahl, Brian

    2003-06-13

    Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis. The HTLV-I transactivator protein Tax plays an integral role in the etiology of adult T cell leukemia, as expression of Tax in T lymphocytes has been shown to result in immortalization. In addition, Tax is known to interface with numerous transcription factor families, including activating transcription factor/cAMP response element-binding protein and nuclear factor-kappaB, requiring Tax to localize to both the nucleus and cytoplasm. In this report, the nucleocytoplasmic localization of Tax was examined in Jurkat, HeLa, and U-87 MG cells. The results reported herein indicate that Tax contains a leucine-rich nuclear export signal (NES) that, when fused to green fluorescent protein (GFP), can direct nuclear export via the CRM-1 pathway, as determined by leptomycin B inhibition of nuclear export. However, cytoplasmic localization of full-length Tax was not altered by treatment with leptomycin B, suggesting that native Tax utilizes another nuclear export pathway. Additional support for the presence of a functional NES has also been shown because the NES mutant Tax(L200A)-GFP localized to the nuclear membrane in the majority of U-87 MG cells. Evidence has also been provided suggesting that the Tax NES likely exists as a conditionally masked signal because the truncation mutant TaxDelta214-GFP localized constitutively to the cytoplasm. These results suggest that Tax localization may be directed by specific changes in Tax conformation or by specific interactions with cellular proteins leading to changes in the availability of the Tax NES and nuclear localization signal.

  4. Suppression of RelA/p65 transactivation activity by a lignoid manassantin isolated from Saururus chinensis.

    PubMed

    Lee, Jeong-Hyung; Hwang, Bang Yeon; Kim, Kyung-Sook; Nam, Jeong Beom; Hong, Young Soo; Lee, Jung Joon

    2003-11-15

    In our search for NF-kappaB inhibitors from natural resources, we have previously identified two structurally related dilignans, manassantin A and B as specific inhibitors of NF-kappaB activation from Saururus chinensis. However, their molecular mechanism of action remains unclear. We here demonstrate that manassantins A and B are potent inhibitors of NF-kappaB activation by the suppression of transciptional activity of RelA/p65 subunit of NF-kappaB. These compounds significantly inhibited the induced expression of NF-kappaB reporter gene by LPS or TNF-alpha in a dose-dependent manner. However, these compounds did not prevent the DNA-binding activity of NF-kappaB assessed by electrophoretic mobility shift assay as well as the induced-degradation of IkappaB-alpha protein by LPS or TNF-alpha. Further analysis revealed that manassantins A and B dose-dependently suppressed not only the induced NF-kappaB activation by overexpression of RelA/p65, but also transactivation activity of RelA/p65. Furthermore, treatment of cells with these compounds prevented the TNF-alpha-induced expression of anti-apoptotic NF-kappaB target genes Bfl-1/A1, a prosurvival Bcl-2 homologue, and resulted in sensitizing HT-1080 cells to TNF-alpha-induced cell death. Similarly, these compounds also suppressed the LPS-induced inducible nitric oxide synthase expression and nitric oxide production. Taken together, manassantins A and B could be valuable candidate for the intervention of NF-kappaB-dependent pathological condition such as inflammation and cancer.

  5. DNA-binding-defective mutants of the Epstein-Barr virus lytic switch activator Zta transactivate with altered specificities.

    PubMed Central

    Flemington, E K; Lytle, J P; Cayrol, C; Borras, A M; Speck, S H

    1994-01-01

    The Epstein-Barr virus BRLF1 and BZLF1 genes are the first viral genes transcribed upon induction of the viral lytic cycle. The protein products of both genes (referred to here as Rta and Zta, respectively) activate expression of other viral genes, thereby initiating the lytic cascade. Among the viral antigens expressed upon induction of the lytic cycle, however, Zta is unique in its ability to disrupt viral latency; expression of the BZLF1 gene is both necessary and sufficient for triggering the viral lytic cascade. We have previously shown that Zta can activate its own promoter (Zp), through binding to two Zta recognition sequences (ZIIIA and ZIIIB). Here we describe mutant Zta proteins that do not bind DNA (referred to as Zta DNA-binding mutants [Zdbm]) but retain the ability to transactivate Zp. Consistent with the inability of these mutants to bind DNA, transactivation of Zp by Zdbm is not dependent on the Zta recognition sequences. Instead, transactivation by Zdbm is dependent upon promoter elements that bind cellular factors. An examination of other viral and cellular promoters identified promoters that are weakly responsive or unresponsive to Zdbm. An analysis of a panel of artificial promoters containing one copy of various promoter elements demonstrated a specificity for Zdbm activation that is distinct from that of Zta. These results suggest that non-DNA-binding forms of some transactivators retain the ability to transactivate specific target promoters without direct binding to DNA. Images PMID:8164660

  6. Mutations in the amino-terminal domain of the human androgen receptor may be associated with partial androgen insensitivity and impaired transactivation in vitro.

    PubMed

    Holterhus, P-M; Werner, R; Struve, D; Hauffa, B P; Schroeder, C; Hiort, O

    2005-09-01

    The majority of genetic variations in the androgen receptor (AR) gene are point mutations leading to impairment of the DNA- or hormone-binding domains. The N-terminus encoded by the first exon of the AR-gene usually harbors disruptive mutations associated with complete androgen insensitivity syndrome (CAIS) while missense mutations related with partial androgen insensitivity syndrome (PAIS) are seemingly rare. We present a 46,XY male with scrotal hypospadias in whom we detected a S432 F point mutation within the N-terminus. Transient transfections of an AR expression plasmid carrying the S432 F mutation using Chinese Hamster Ovary (CHO) cells revealed a significant partial reduction in transactivation of the co-transfected androgen responsive (ARE) (2)TATA luciferase reporter gene thus confirming PAIS. In two further 46, XY patients with slight to moderate virilization defects, we detected an S411 N mutation, and a 9 base pair deletion leading to the loss of amino acids 409 to 411 (L-A-S), respectively. These mutations did not compromise AR-function under the chosen experimental settings. The S432 F-patient supports particular significance of the AR-N-terminus for mild forms of AIS while the functional role of the two further mutations remains unclear. The N-terminus is a species-specific AR-domain possibly also involved in contributing to target tissue selectivity of AR-actions via mediating co-regulator interactions. Therefore, mild molecular defects of the AR-N-terminus may not necessarily inhibit general transactivation properties using currently established reporter gene models.

  7. Transactivation of the Receptor-tyrosine Kinase Ephrin Receptor A2 Is Required for the Low Molecular Weight Hyaluronan-mediated Angiogenesis That Is implicated in Tumor Progression*

    PubMed Central

    Lennon, Frances E; Mirzapoiazova, Tamara; Mambetsariev, Nurbek; Mambetsariev, Bolot; Salgia, Ravi; Singleton, Patrick A.

    2014-01-01

    Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, understanding the mechanism(s) by which angiogenesis occurs can have important therapeutic implications in numerous malignancies. We and others have demonstrated that low molecular weight hyaluronan (LMW-HA, ∼2500 Da) promotes endothelial cell (EC) barrier disruption and angiogenesis. However, the mechanism(s) by which this occurs is poorly defined. Our data indicate that treatment of human EC with LMW-HA induced CD44v10 association with the receptor-tyrosine kinase, EphA2, transactivation (tyrosine phosphorylation) of EphA2, and recruitment of the PDZ domain scaffolding protein, PATJ, to the cell periphery. Silencing (siRNA) CD44, EphA2, PATJ, or Dbs (RhoGEF) expression blocked LMW-HA-mediated angiogenesis (EC proliferation, migration, and tubule formation). In addition, silencing EphA2, PATJ, Src, or Dbs expression blocked LMW-HA-mediated RhoA activation. To translate our in vitro findings, we utilized a novel anginex/liposomal targeting of murine angiogenic endothelium with either CD44 or EphA2 siRNA and observed inhibition of LMW-HA-induced angiogenesis in implanted Matrigel plugs. Taken together, these results indicate LMW-HA-mediated transactivation of EphA2 is required for PATJ and Dbs membrane recruitment and subsequent RhoA activation required for angiogenesis. These results suggest that targeting downstream effectors of LMW-HA could be a useful therapeutic intervention for angiogenesis-associated diseases including tumor progression. PMID:25023279

  8. The carboxyl terminus of the chemokine receptor CCR3 contains distinct domains which regulate chemotactic signaling and receptor down-regulation in a ligand-dependent manner.

    PubMed

    Sabroe, Ian; Jorritsma, Annelies; Stubbs, Victoria E L; Xanthou, Georgina; Jopling, Louise A; Ponath, Paul D; Williams, Timothy J; Murphy, Philip M; Pease, James E

    2005-04-01

    The chemokine receptor CCR3 regulates the chemotaxis of leukocytes implicated in allergic disease, such as eosinophils. Incubation of eosinophils with CCL11, CCL13 or CCL5 resulted in a rapid decrease of cell-surface CCR3 which was replicated using CCR3 transfectants. Progressive truncation of the CCR3 C terminus by 15 amino acids produced three constructs, Delta340, Delta325 and Delta310. Delta340 and Delta325 were able to bind CCL11 with affinities similar to wild-type CCR3. Delta340 transfectants exhibited enhanced migration and reduced receptor down-regulation in response to CCL11 and CCL13. Delta325 transfectants displayed chemotactic responses to CCL11 and CCL13 similar to wild-type CCR3, and had impaired down-regulation when stimulated with CCL13 but not CCL11. In contrast, neither the Delta325 nor Delta340 truncation affected chemotaxis or receptor down-regulation induced by CCL5. Delta310 transfectants bound CCL11 poorly and were biologically inactive. Inhibitors of p38 mitogen-activated protein kinase and PI3-kinase antagonized eosinophil shape change responses and chemotaxis of transfectants to CCL11 and CCL13. In contrast, shape change but not chemotaxis was sensitive to inhibition of the extracellular signal-regulated kinase kinase pathway suggesting differential regulation of the two responses. Thus, the CCR3 C terminus contains distinct domains responsible for the regulation of receptor desensitization and for coupling to chemotactic responses.

  9. Enhancement of alkaloid production in opium and California poppy by transactivation using heterologous regulatory factors.

    PubMed

    Apuya, Nestor R; Park, Joon-Hyun; Zhang, Liping; Ahyow, Maurice; Davidow, Patricia; Van Fleet, Jennifer; Rarang, Joel C; Hippley, Matthew; Johnson, Thomas W; Yoo, Hye-Dong; Trieu, Anthony; Krueger, Shannon; Wu, Chuan-yin; Lu, Yu-ping; Flavell, Richard B; Bobzin, Steven C

    2008-02-01

    Genes encoding regulatory factors isolated from Arabidopsis, soybean and corn have been screened to identify those that modulate the expression of genes encoding for enzymes involved in the biosynthesis of morphinan alkaloids in opium poppy (Papaver somniferum) and benzophenanthridine alkaloids in California poppy (Eschscholzia californica). In opium poppy, the over-expression of selected regulatory factors increased the levels of PsCOR (codeinone reductase), Ps4'OMT (S-adenosyl-l-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase) and Ps6OMT [(R,S)-norcoclaurine 6-O-methyltransferase] transcripts by 10- to more than 100-fold. These transcriptional activations translated into an enhancement of alkaloid production in opium poppy of up to at least 10-fold. In California poppy, the transactivation effect of regulatory factor WRKY1 resulted in an increase of up to 60-fold in the level of EcCYP80B1 [(S)-N-methylcoclaurine 3'-hydroxylase] and EcBBE (berberine bridge enzyme) transcripts. As a result, the accumulations of selected alkaloid intermediates were enhanced up to 30-fold. The transactivation effects of other regulatory factors led to the accumulation of the same intermediates. These regulatory factors also led to the production of new alkaloids in California poppy callus culture.

  10. A versatile cis-blocking and trans-activation strategy for ribozyme characterization

    PubMed Central

    Kennedy, Andrew B.; Liang, Joe C.; Smolke, Christina D.

    2013-01-01

    Synthetic RNA control devices that use ribozymes as gene-regulatory components have been applied to controlling cellular behaviors in response to environmental signals. Quantitative measurement of the in vitro cleavage rate constants associated with ribozyme-based devices is essential for advancing the molecular design and optimization of this class of gene-regulatory devices. One of the key challenges encountered in ribozyme characterization is the efficient generation of full-length RNA from in vitro transcription reactions, where conditions generally lead to significant ribozyme cleavage. Current methods for generating full-length ribozyme-encoding RNA rely on a trans-blocking strategy, which requires a laborious gel separation and extraction step. Here, we develop a simple two-step gel-free process including cis-blocking and trans-activation steps to support scalable generation of functional full-length ribozyme-encoding RNA. We demonstrate our strategy on various types of natural ribozymes and synthetic ribozyme devices, and the cleavage rate constants obtained for the RNA generated from our strategy are comparable with those generated through traditional methods. We further develop a rapid, label-free ribozyme cleavage assay based on surface plasmon resonance, which allows continuous, real-time monitoring of ribozyme cleavage. The surface plasmon resonance-based characterization assay will complement the versatile cis-blocking and trans-activation strategy to broadly advance our ability to characterize and engineer ribozyme-based devices. PMID:23155065

  11. Modeling the accessible conformations of the intrinsically unstructured transactivation domain of p53.

    PubMed

    Lowry, David F; Stancik, Amber; Shrestha, Ranjay Mann; Daughdrill, Gary W

    2008-05-01

    Internuclear distances derived from paramagnetic relaxation enhancement (PRE) data were used to restrain molecular dynamics simulations of the intrinsically unstructured transactivation domain of the tumor suppressor protein, p53. About 1000 structures were simulated using ensemble averaging of replicate molecules to compensate for the inherent bias in the PRE-derived distances. Gyration radii measurements on these structures show that the p53 transactivation domain (p53TAD) is statistically predominantly in a partially collapsed state that is unlike the open structure that is found for p53TAD bound to either the E3 ubiquitin ligase, MDM2, or the 70 kDa subunit of replication protein A, RPA70. Contact regions that potentially mediate the collapse were identified and found to consist of mostly hydrophobic residues. The identified contact regions preferentially place the MDM2 and RPA70 binding regions in close proximity. We show that our simulations thoroughly sample the available range of conformations and that a fraction of the molecules are in an open state that would be competent for binding either MDM2 or RPA70. We also show that the Stokes radius estimated from the average gyration radius of the ensemble is in good agreement with the value determined using size exclusion chromatography. Finally, the presence of a persistent loop localized to a PXP motif was identified. Serine residues flanking the PXP motif become phosphorylated in response to DNA damage, and we postulate that this will perturb the equilibrium population to more open conformations.

  12. Acetylation of FOXM1 is essential for its transactivation and tumor growth stimulation

    PubMed Central

    Lv, Cuicui; Zhao, Ganye; Sun, Xinpei; Wang, Pan; Xie, Nan; Luo, Jianyuan; Tong, Tanjun

    2016-01-01

    Forkhead box transcription factor M1 (FOXM1) plays crucial roles in a wide array of biological processes, including cell proliferation and differentiation, the cell cycle, and tumorigenesis by regulating the expression of its target genes. Elevated expression of FOXM1 is frequently observed in a multitude of malignancies. Here we show that FOXM1 can be acetylated by p300/CBP at lysines K63, K422, K440, K603 and K614 in vivo. This modification is essential for its transactivation on the target genes. Acetylation of FOXM1 increases during the S phase and remains high throughout the G2 and M phases, when FOXM1 transcriptional activity is required. We find that the acetylation-deficient FOXM1 mutant is less active and exhibits significantly weaker tumorigenic activities compared to wild-type FOXM1. Mechanistically, the acetylation of FOXM1 enhances its transcriptional activity by increasing its DNA binding affinity, protein stability, and phosphorylation sensitivity. In addition, we demonstrate that NAD-dependent histone deacetylase SIRT1 physically binds to and deacetylates FOXM1 in vivo. The deacetylation of FOXM1 by SIRT1 attenuates its transcriptional activity and decreases its protein stability. Together, our findings demonstrate that the reversible acetylation of FOXM1 by p300/CBP and SIRT1 modulates its transactivation function. PMID:27542221

  13. NLRC5/MHC class I transactivator is a target for immune evasion in cancer

    PubMed Central

    Yoshihama, Sayuri; Roszik, Jason; Downs, Isaac; Meissner, Torsten B.; Vijayan, Saptha; Chapuy, Bjoern; Sidiq, Tabasum; Shipp, Margaret A.; Lizee, Gregory A.; Kobayashi, Koichi S.

    2016-01-01

    Cancer cells develop under immune surveillance, thus necessitating immune escape for successful growth. Loss of MHC class I expression provides a key immune evasion strategy in many cancers, although the molecular mechanisms remain elusive. MHC class I transactivator (CITA), known as “NLRC5” [NOD-like receptor (NLR) family, caspase recruitment (CARD) domain containing 5], has recently been identified as a critical transcriptional coactivator of MHC class I gene expression. Here we show that the MHC class I transactivation pathway mediated by CITA/NLRC5 constitutes a target for cancer immune evasion. In all the 21 tumor types we examined, NLRC5 expression was highly correlated with the expression of MHC class I, with cytotoxic T-cell markers, and with genes in the MHC class I antigen-presentation pathway, including LMP2/LMP7, TAP1, and β2-microglobulin. Epigenetic and genetic alterations in cancers, including promoter methylation, copy number loss, and somatic mutations, were most prevalent in NLRC5 among all MHC class I-related genes and were associated with the impaired expression of components of the MHC class I pathway. Strikingly, NLRC5 expression was significantly associated with the activation of CD8+ cytotoxic T cells and patient survival in multiple cancer types. Thus, NLRC5 constitutes a novel prognostic biomarker and potential therapeutic target of cancers. PMID:27162338

  14. Transactivation of anthocyanin biosynthetic genes following transfer of B regulatory genes into maize tissues.

    PubMed Central

    Goff, S A; Klein, T M; Roth, B A; Fromm, M E; Cone, K C; Radicella, J P; Chandler, V L

    1990-01-01

    The C1, B and R genes regulating the maize anthocyanin biosynthetic pathway encode tissue-specific regulatory proteins with similarities to transcriptional activators. The C1 and R regulatory genes are usually responsible for pigmentation of seed tissues, and the B-Peru allele of B, but not the B-I allele, can substitute for R function in the seed. In this study, members of the B family of regulatory genes were delivered to intact maize tissues by high velocity microprojectiles. In colorless r aleurones or embryos, the introduction of the B-Peru genomic clone or the expressed cDNAs of B-Peru or B-I resulted in anthocyanin-producing cells. Luciferase produced from the Bronze1 anthocyanin structural gene promoter was induced 100-fold when co-introduced with the expressed B-Peru or B-I cDNAs. This quantitative transactivation assay demonstrates that the proteins encoded by these two B alleles are equally able to transactivate the Bronze1 promoter. Analogous results were obtained using embryogenic callus cells. These observations suggest that one major contribution towards tissue-specific anthocyanin synthesis controlled by the various alleles of the B and R genes is the differential expression of functionally similar proteins. Images Fig. 2. PMID:2369901

  15. NLRC5/MHC class I transactivator is a target for immune evasion in cancer.

    PubMed

    Yoshihama, Sayuri; Roszik, Jason; Downs, Isaac; Meissner, Torsten B; Vijayan, Saptha; Chapuy, Bjoern; Sidiq, Tabasum; Shipp, Margaret A; Lizee, Gregory A; Kobayashi, Koichi S

    2016-05-24

    Cancer cells develop under immune surveillance, thus necessitating immune escape for successful growth. Loss of MHC class I expression provides a key immune evasion strategy in many cancers, although the molecular mechanisms remain elusive. MHC class I transactivator (CITA), known as "NLRC5" [NOD-like receptor (NLR) family, caspase recruitment (CARD) domain containing 5], has recently been identified as a critical transcriptional coactivator of MHC class I gene expression. Here we show that the MHC class I transactivation pathway mediated by CITA/NLRC5 constitutes a target for cancer immune evasion. In all the 21 tumor types we examined, NLRC5 expression was highly correlated with the expression of MHC class I, with cytotoxic T-cell markers, and with genes in the MHC class I antigen-presentation pathway, including LMP2/LMP7, TAP1, and β2-microglobulin. Epigenetic and genetic alterations in cancers, including promoter methylation, copy number loss, and somatic mutations, were most prevalent in NLRC5 among all MHC class I-related genes and were associated with the impaired expression of components of the MHC class I pathway. Strikingly, NLRC5 expression was significantly associated with the activation of CD8(+) cytotoxic T cells and patient survival in multiple cancer types. Thus, NLRC5 constitutes a novel prognostic biomarker and potential therapeutic target of cancers.

  16. The homeoprotein Alx3 contains discrete functional domains and exhibits cell-specific and selective monomeric binding and transactivation.

    PubMed

    Pérez-Villamil, Beatriz; Mirasierra, Mercedes; Vallejo, Mario

    2004-09-03

    Alx3 is a paired class aristaless-like homeoprotein expressed during embryonic development. Transcriptional transactivation by aristaless-like proteins has been associated with cooperative dimerization upon binding to artificially generated DNA consensus sequences known as P3 sites, but natural target sites in genes regulated by Alx3 are unknown. We report the cloning of a cDNA encoding the rat homolog of Alx3, and we characterize the protein domains that are important for transactivation, dimerization, and binding to DNA. Two proline-rich domains located amino-terminal to the homeodomain (Pro1 and Pro2) are necessary for Alx3-dependent transactivation, whereas another one (Pro3) located in the carboxyl terminus is dispensable but contributes to enhance the magnitude of the response. We confirmed that transcriptional activity of Alx3 from a P3 site correlates with cooperative dimerization upon binding to DNA. However, Alx3 was found to bind selectively to non-P3-related TAAT-containing sites present in the promoter of the somatostatin gene in a specific manner that depends on the nuclear protein environment. Cell-specific transactivation elicited by Alx3 from these sites could not be predicted from in vitro DNA-binding experiments by using recombinant Alx3. In addition, transactivation did not depend on cooperative dimerization upon binding to cognate somatostatin DNA sites. Our data indicate that the paradigm according to which Alx3 must act homodimerically via cooperative binding to P3-like sites is insufficient to explain the mechanism of action of this homeoprotein to regulate transcription of natural target genes. Instead, Alx3 undergoes restrictive or permissive interactions with nuclear proteins that determine its binding to and transactivation from TAAT target sites selected in a cell-specific manner.

  17. Epstein-Barr Virus Latent Membrane Protein LMP-2A Is Sufficient for Transactivation of the Human Endogenous Retrovirus HERV-K18 Superantigen

    PubMed Central

    Sutkowski, Natalie; Chen, Gang; Calderon, German; Huber, Brigitte T.

    2004-01-01

    Superantigens are microbial proteins that strongly stimulate T cells. We described previously that the Epstein-Barr virus (EBV) transactivates a superantigen encoded by the human endogenous retrovirus, HERV-K18. We now report that the transactivation is dependent upon the EBV latent cycle proteins. Moreover, LMP-2A is sufficient for induction of HERV-K18 superantigen activity. PMID:15220463

  18. Piroxicam and c-phycocyanin prevent colon carcinogenesis by inhibition of membrane fluidity and canonical Wnt/β-catenin signaling while up-regulating ligand dependent transcription factor PPARγ.

    PubMed

    Saini, Manpreet Kaur; Sanyal, Sankar Nath

    2014-06-01

    The colon cancer tissues from DMH treated rats exhibited higher membrane potential, fluidity and changed lipid order as examined by Merocyanine 540 and 1,6-diphenyl-1,3,5-hexatriene, respectively. A transition from gel to liquid crystalline state was observed by Laurdan fluorescence and also reduced fluorescence quenching of NBD-PE as contributed in the decreased membrane lipid phase separation. With piroxicam, a traditional NSAID and c-phycocyanin, a biliprotein from Spirulina platensis, these effects were normalized. An augmented intracellular Ca(+2) had contributed to the drug mediated apoptosis which is supported by an elevated calpain-9 expression. Histopathologically, a large pool of secreted acid/neutral mucopolysaccrides as well as the presence of blood vessels and dysplastic crypts signifies invasive mucinous adenocarcinoma while both the drugs reduced these neoplastic alterations. Wnt/β-catenin pathway was also found to be up-regulated which served as a crucial indicator for cancer cell growth. A concomitant down regulation of PPARγ was noted in DMH treatment which is associated with tumor progression. The expression of PPARα and δ, the other two isoforms of PPAR family was also modulated. We conclude that piroxicam and c-phycocyanin exert their anti-neoplastic effects via regulating membrane properties, raising calpain-9 and PPARγ expression while suppressing Wnt/β-catenin signaling in experimental colon carcinogenesis.

  19. Use of cryopreserved transiently transfected cells in high-throughput pregnane X receptor transactivation assay.

    PubMed

    Zhu, Zhengrong; Puglisi, Jaime; Connors, David; Stewart, Jeremy; Herbst, John; Marino, Anthony; Sinz, Michael; O'Connell, Jonathan; Banks, Martyn; Dickinson, Kenneth; Cacace, Angela

    2007-03-01

    Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r(2) > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.

  20. Enhancement of gene transactivation activity of androgen receptor by hepatitis B virus X protein

    SciTech Connect

    Zheng Yanyan; Chen Wenling; Ma, W.-L. Maverick; Chang Chawnshang; Ou, J.-H. James . E-mail: jamesou@hsc.usc.edu

    2007-07-05

    Hepatitis B virus (HBV) X protein (HBx) is a regulatory protein that is required for efficient replication of HBV in its natural host. In this report, we demonstrate by co-immunoprecipitation experiments that HBx can physically bind to the androgen receptor (AR), which is a nuclear hormone receptor that is expressed in many different tissues including the liver. This observation is further supported by confocal microscopy, which reveals that HBx can alter the subcellular localization of the AR both in the presence and in the absence of dihydrotestosterone (DHT). Further studies indicate that HBx can enhance the gene transactivation activity of AR by enhancing its DNA binding activity in a DHT-dependent manner. However, HBx does not remain associated with AR on the DNA. As AR can regulate the expression of a number of cellular genes, our results raise the possibility that HBV pathogenesis may be mediated in part via the interaction between HBx and AR.

  1. Identification of transcriptional regulatory nodes in soybean defense networks using transient co-transactivation assays

    PubMed Central

    Wang, Yongli; Wang, Hui; Ma, Yujie; Du, Haiping; Yang, Qing; Yu, Deyue

    2015-01-01

    Plant responses to major environmental stressors, such as insect feeding, not only occur via the functions of defense genes but also involve a series of regulatory factors. Our previous transcriptome studies proposed that, in addition to two defense-related genes, GmVSPβ and GmN:IFR, a high proportion of transcription factors (TFs) participate in the incompatible soybean-common cutworm interaction networks. However, the regulatory mechanisms and effects of these TFs on those induced defense-related genes remain unknown. In the present work, we isolated and identified 12 genes encoding MYB, WRKY, NAC, bZIP, and DREB TFs from a common cutworm-induced cDNA library of a resistant soybean line. Sequence analysis of the promoters of three co-expressed genes, including GmVSPα, GmVSPβ, and GmN:IFR, revealed the enrichment of various TF-binding sites for defense and stress responses. To further identify the regulatory nodes composed of these TFs and defense gene promoters, we performed extensive transient co-transactivation assays to directly test the transcriptional activity of the 12 TFs binding at different levels to the three co-expressed gene promoters. The results showed that all 12 TFs were able to transactivate the GmVSPβ and GmN:IFR promoters. GmbZIP110 and GmMYB75 functioned as distinct regulators of GmVSPα/β and GmN:IFR expression, respectively, while GmWRKY39 acted as a common central regulator of GmVSPα/β and GmN:IFR expression. These corresponding TFs play crucial roles in coordinated plant defense regulation, which provides valuable information for understanding the molecular mechanisms involved in insect-induced transcriptional regulation in soybean. More importantly, the identified TFs and suitable promoters can be used to engineer insect-resistant plants in molecular breeding studies. PMID:26579162

  2. Identification of transcriptional regulatory nodes in soybean defense networks using transient co-transactivation assays.

    PubMed

    Wang, Yongli; Wang, Hui; Ma, Yujie; Du, Haiping; Yang, Qing; Yu, Deyue

    2015-01-01

    Plant responses to major environmental stressors, such as insect feeding, not only occur via the functions of defense genes but also involve a series of regulatory factors. Our previous transcriptome studies proposed that, in addition to two defense-related genes, GmVSPβ and GmN:IFR, a high proportion of transcription factors (TFs) participate in the incompatible soybean-common cutworm interaction networks. However, the regulatory mechanisms and effects of these TFs on those induced defense-related genes remain unknown. In the present work, we isolated and identified 12 genes encoding MYB, WRKY, NAC, bZIP, and DREB TFs from a common cutworm-induced cDNA library of a resistant soybean line. Sequence analysis of the promoters of three co-expressed genes, including GmVSPα, GmVSPβ, and GmN:IFR, revealed the enrichment of various TF-binding sites for defense and stress responses. To further identify the regulatory nodes composed of these TFs and defense gene promoters, we performed extensive transient co-transactivation assays to directly test the transcriptional activity of the 12 TFs binding at different levels to the three co-expressed gene promoters. The results showed that all 12 TFs were able to transactivate the GmVSPβ and GmN:IFR promoters. GmbZIP110 and GmMYB75 functioned as distinct regulators of GmVSPα/β and GmN:IFR expression, respectively, while GmWRKY39 acted as a common central regulator of GmVSPα/β and GmN:IFR expression. These corresponding TFs play crucial roles in coordinated plant defense regulation, which provides valuable information for understanding the molecular mechanisms involved in insect-induced transcriptional regulation in soybean. More importantly, the identified TFs and suitable promoters can be used to engineer insect-resistant plants in molecular breeding studies.

  3. Hypo- and Hypermorphic FOXC1 Mutations in Dominant Glaucoma: Transactivation and Phenotypic Variability

    PubMed Central

    Medina-Trillo, Cristina; Sánchez-Sánchez, Francisco; Aroca-Aguilar, José-Daniel; Ferre-Fernández, Jesús-José; Morales, Laura; Méndez-Hernández, Carmen-Dora; Blanco-Kelly, Fiona; Ayuso, Carmen; García-Feijoo, Julián; Escribano, Julio

    2015-01-01

    Dominant glaucoma, a heterogeneous, infrequent and irreversible optic neuropathy, is often associated with elevated intraocular pressure and early-onset. The role of FOXC1 in this type of glaucoma was investigated in twelve Spanish probands via nucleotide variation screening of its proximal promoter and unique exon. Functional evaluations of the identified variants included analyses of the transcriptional activity, protein stability, DNA binding ability and subcellular localization. Four different mutations that were identified in four probands (33.3%) were associated with remarkable phenotypic variability and were functionally classified as either hypermorphic (p.Y47X, p.Q106X and p.G447_G448insDG) or hypomorphic (p.I126S) alleles. To the best of our knowledge, three of the variants are novel (p.Y47X, p.I126S and p.G447_G448insDG) and, in addition, hypermorphic FOXC1 mutations are reported herein for the first time. The presence of an intact N-terminal activation domain in the truncated proteins p.Y47X and p.Q106X may underlie their associated transactivation hyperactivity by a gain-of-function mechanism involving dysregulated protein-protein interactions. Similarly, altered molecular interactions may also lead to increased p.G447_G448insDG activity. In contrast, the partial loss-of-function associated with p.I126S was due to impaired protein stability, DNA binding, protein phosphorylation and subcellular distribution. These results support that moderate and variable FOXC1 transactivation changes are associated with moderate goniodysgenesis, dominant glaucoma and remarkable phenotypic variability. PMID:25786029

  4. Hypo- and hypermorphic FOXC1 mutations in dominant glaucoma: transactivation and phenotypic variability.

    PubMed

    Medina-Trillo, Cristina; Sánchez-Sánchez, Francisco; Aroca-Aguilar, José-Daniel; Ferre-Fernández, Jesús-José; Morales, Laura; Méndez-Hernández, Carmen-Dora; Blanco-Kelly, Fiona; Ayuso, Carmen; García-Feijoo, Julián; Escribano, Julio

    2015-01-01

    Dominant glaucoma, a heterogeneous, infrequent and irreversible optic neuropathy, is often associated with elevated intraocular pressure and early-onset. The role of FOXC1 in this type of glaucoma was investigated in twelve Spanish probands via nucleotide variation screening of its proximal promoter and unique exon. Functional evaluations of the identified variants included analyses of the transcriptional activity, protein stability, DNA binding ability and subcellular localization. Four different mutations that were identified in four probands (33.3%) were associated with remarkable phenotypic variability and were functionally classified as either hypermorphic (p.Y47X, p.Q106X and p.G447_G448insDG) or hypomorphic (p.I126S) alleles. To the best of our knowledge, three of the variants are novel (p.Y47X, p.I126S and p.G447_G448insDG) and, in addition, hypermorphic FOXC1 mutations are reported herein for the first time. The presence of an intact N-terminal activation domain in the truncated proteins p.Y47X and p.Q106X may underlie their associated transactivation hyperactivity by a gain-of-function mechanism involving dysregulated protein-protein interactions. Similarly, altered molecular interactions may also lead to increased p.G447_G448insDG activity. In contrast, the partial loss-of-function associated with p.I126S was due to impaired protein stability, DNA binding, protein phosphorylation and subcellular distribution. These results support that moderate and variable FOXC1 transactivation changes are associated with moderate goniodysgenesis, dominant glaucoma and remarkable phenotypic variability.

  5. c-Maf increases apoptosis in peripheral CD8 cells by transactivating Caspase 6

    PubMed Central

    Peng, Siying; Wu, Hailong; Mo, Yin-Yuan; Watabe, Kounosuke; Pauza, Mary E

    2009-01-01

    In addition to transactivation of interleukin-4 (IL-4), cellular muscular aponeurotic fibrosarcoma (c-Maf) enhances CD4 cell apoptosis by limiting Bcl-2 expression. The CD8 cells also express c-Maf and peripheral CD8 cell numbers are reduced in c-Maf transgenic mice, suggesting that c-Maf may influence CD8 cell survival in a manner similar to CD4 cells. Here we confirm that, similar to CD4 cells, c-Maf enhances CD8 cell susceptibility to apoptosis induced by multiple stimuli, independent of IL-4. However, unlike CD4 cells, c-Maf enhancement of apoptosis is independent of Bcl-2, suggesting that c-Maf uses other mechanisms to regulate CD8 cell apoptosis. Real-time reverse transcription–polymerase chain reaction reveals that the pro-apoptotic gene Caspase 6 is upregulated in c-Maf transgenic CD8 cells, suggesting that Caspase 6 is a novel c-Maf target gene. Luciferase reporter assays and site-directed mutagenesis reveal a functional c-Maf recognition element (MARE) within the first intron of Caspase 6. Binding of c-Maf to the MARE site is detectable by chromatin immunoprecipitation using non-transgenic T-cell lysates, so c-Maf can interact with the Caspase 6 MARE site in normal T cells. Furthermore, caspase 6 activity is increased among CD8 cells from c-Maf transgenic mice following T-cell receptor engagement. As expected, activity of the downstream caspases 3 and 7 is also increased. Consistent with the ability of caspase 6 to participate in positive feedback loops, cytochrome c release and caspase 8 activation are also increased. Together these results indicated that c-Maf increases CD8 cell sensitivity to apoptotic stimuli, at least in part, by direct transactivation of Caspase 6, providing increased substrate for Caspase 6-dependent apoptosis pathways. PMID:19476513

  6. Altered-function p53 missense mutations identified in breast cancers can have subtle effects on transactivation.

    PubMed

    Jordan, Jennifer J; Inga, Alberto; Conway, Kathleen; Edmiston, Sharon; Carey, Lisa A; Wu, Lin; Resnick, Michael A

    2010-05-01

    Mutations of the sequence-specific master regulator p53 that alter transactivation function from promoter response elements (RE) could result in changes in the strength of gene activation or spectra of genes regulated. Such mutations in this tumor suppressor might lead to dramatic phenotypic changes and diversification of cell responses to stress. We have determined "functional fingerprints" of sporadic breast cancer-related p53 mutants, many of which are also associated with familial cancer proneness such as the Li-Fraumeni syndrome and germline BRCA1/2 mutant-associated cancers. The ability of p53, wild-type and mutants, to transactivate from 11 human target REs has been assessed at variable expression levels using a cellular, isogenomic yeast model system that allows for the rapid analysis of p53 function using a qualitative and a quantitative reporter. Among 50 missense mutants, 29 were classified as loss of function. The remaining 21 retained transactivation toward at least one RE. At high levels of galactose-induced p53 expression, 12 of 21 mutants that retain transactivation seemed similar to wild-type. When the level of galactose was reduced, transactivation defects could be revealed, suggesting that some breast cancer-related mutants can have subtle changes in transcription. These findings have been compared with clinical data from an ongoing neoadjuvant chemotherapy treatment trial for locally advanced breast tumors. The functional and nonfunctional missense mutations may distinguish tumors in terms of demographics, appearance, and relapse, implying that heterogeneity in the functionality of specific p53 mutations could affect clinical behavior and outcome.

  7. Creb1-Mecp2-mCpG Complex Transactivates Postnatal Murine Neuronal Glucose Transporter Isoform 3 Expression

    PubMed Central

    Chen, Yongjun; Shin, Bo-Chul; Thamotharan, Shanthie

    2013-01-01

    The murine neuronal facilitative glucose transporter isoform 3 (Glut3) is developmentally regulated, peaking in expression at postnatal day (PN)14. In the present study, we characterized a canonical CpG island spanning the 5′-flanking region of the glut3 gene. Methylation-specific PCR and bisulfite sequencing identified methylation of this CpG (mCpG) island of the glut3 gene, frequency of methylation increasing 2.5-fold with a 1.6-fold increase in DNA methyl transferase 3a concentrations noted with advancing postnatal age (PN14 vs PN3). 5′-flanking region of glut3-luciferase reporter transient transfection in HT22 hippocampal neurons demonstrated that mCpGs inhibit glut3 transcription. Contrary to this biological function, glut3 expression rises synchronously with mCpGs in PN14 vs PN3 neurons. Chromatin immunoprecipitation (IP) revealed that methyl-CpG binding protein 2 (Mecp2) bound the glut3-mCpGs. Depending on association with specific coregulators, Mecp2, a dual regulator of gene transcription, may repress or activate a downstream gene. Sequential chromatin IP uncovered the glut3-mCpGs to bind Mecp2 exponentially upon recruitment of Creb1 rather than histone deacetylase 1. Co-IP and coimmunolocalization confirmed that Creb1 associated with Mecp2 and cotransfection with glut3-mCpG in HT22 cells enhanced glut3 transcription. Separate 5-aza-2′-deoxycytidine pretreatment or in combination with trichostatin A reduced mCpG and specific small interference RNAs targeting Mecp2 and Creb1 separately or together depleting Mecp2 and/or Creb1 binding of glut3-mCpGs reduced glut3 expression in HT22 cells. We conclude that Glut3 is a methylation-sensitive neuronal gene that recruits Mecp2. Recruitment of Creb1-Mecp2 by glut3-mCpG contributes towards transactivation, formulating an escape from mCpG-induced gene suppression, and thereby promoting developmental neuronal glut3 gene transcription and expression. PMID:23493374

  8. SUMO modification of a heterochromatin histone demethylase JMJD2A enables viral gene transactivation and viral replication

    PubMed Central

    Yang, Wan-Shan; Campbell, Mel

    2017-01-01

    Small ubiquitin-like modifier (SUMO) modification of chromatin has profound effects on transcription regulation. By using Kaposi’s sarcoma associated herpesvirus (KSHV) as a model, we recently demonstrated that epigenetic modification of viral chromatin by SUMO-2/3 is involved in regulating gene expression and viral reactivation. However, how this modification orchestrates transcription reprogramming through targeting histone modifying enzymes remains largely unknown. Here we show that JMJD2A, the first identified Jumonji C domain-containing histone demethylase, is the histone demethylase responsible for SUMO-2/3 enrichment on the KSHV genome during viral reactivation. Using in vitro and in vivo SUMOylation assays, we found that JMJD2A is SUMOylated on lysine 471 by KSHV K-bZIP, a viral SUMO-2/3-specific E3 ligase, in a SUMO-interacting motif (SIM)-dependent manner. SUMOylation is required for stabilizing chromatin association and gene transactivation by JMJD2A. These finding suggest that SUMO-2/3 modification plays an essential role in the epigenetic regulatory function of JMJD2A. Consistently, hierarchical clustering analysis of RNA-seq data showed that a SUMO-deficient mutant of JMJD2A was more closely related to JMJD2A knockdown than to wild-type. Our previous report demonstrated that JMJD2A coated and maintained the “ready to activate” status of the viral genome. Consistent with our previous report, a SUMO-deficient mutant of JMJD2A reduced viral gene expression and virion production. Importantly, JMJD2A has been implicated as an oncogene in various cancers by regulating proliferation. We therefore further analyzed the role of SUMO modification of JMJD2A in regulating cell proliferation. Interestingly, the SUMO-deficient mutant of JMJD2A failed to rescue the proliferation defect of JMJD2A knockdown cells. Emerging specific inhibitors of JMJD2A have been generated for evaluation in cancer studies. Our results revealed that SUMO conjugation mediates an

  9. Transactivation of progestin- and estrogen-responsive promoters by 19-nor progestins in African Green Monkey Kidney CV1 cells.

    PubMed

    Pasapera, A M; Gutiérrez-Sagal, R; García-Becerra, R; Ulloa-Aguirre, A; Savouret, J F

    2001-12-01

    New and more potent progestins and antiprogestins suitable for reproductive therapy and contraception are currently the target of intensive research. The design of such drugs has been hampered by the complex technology required for screening these compounds at the molecular level. To solve this problem, we developed an in vitro cell system that allows detection of the progestagenic effects of a given compound using a PRE2-TATA-CAT reporter vector transiently introduced in a cell line stably transfected with the rabbit progesterone receptor (PR). The African Green Monkey Kidney CV1 (AGMK-CV1) cell line was chosen because these cells do not express endogenous steroid receptors; the selected clone stably expressing the rabbit PR has been maintained in our laboratory for more than 2 yr without detectable losses in PR content and progestagenic response. The presence and function of the PR were assessed by immunohistochemical and saturation analyses as well as by monitoring transactivation of the PRE2-TATA-CAT reporter gene. In this cell line, the PR is expressed at a concentration of 0.170 fmol/mg of protein, and the receptor is localized within the cell nucleus in either the presence or absence of the potent synthetic progestin R5020. This PR-expressing cell system allowed study of the in vitro progestational activity of several 19-nor progestins. The antiprogestin RU486 inhibited CAT activity induced by R5020; norethisterone (NET), levonorgestrel (LNG), and gestodene (GSD) induced PRE2-TATA-CAT activity at concentrations similar to those of R5020, whereas NET A-ring-reduced metabolites induced CAT activity at an extent lower than (5alpha-NET) or similar (3beta,5alpha-NET) to that of the precursor compound. The PRE2-TATA-CAT induction by 17beta-estradiol was also analyzed and no crossreactivity was detected. However, when the ERE-VitA2-TK-CAT (estrogen-responsive element-vitellogenin A2-thymidine kinase promoter-CAT) reporter vector and the estradiol receptor alpha or

  10. Full trans-activation mediated by the immediate-early protein of equine herpesvirus 1 requires a consensus TATA box, but not its cognate binding sequence.

    PubMed

    Kim, Seong K; Shakya, Akhalesh K; O'Callaghan, Dennis J

    2016-01-04

    The immediate-early protein (IEP) of equine herpesvirus 1 (EHV-1) has extensive homology to the IEP of alphaherpesviruses and possesses domains essential for trans-activation, including an acidic trans-activation domain (TAD) and binding domains for DNA, TFIIB, and TBP. Our data showed that the IEP directly interacted with transcription factor TFIIA, which is known to stabilize the binding of TBP and TFIID to the TATA box of core promoters. When the TATA box of the EICP0 promoter was mutated to a nonfunctional TATA box, IEP-mediated trans-activation was reduced from 22-fold to 7-fold. The IEP trans-activated the viral promoters in a TATA motif-dependent manner. Our previous data showed that the IEP is able to repress its own promoter when the IEP-binding sequence (IEBS) is located within 26-bp from the TATA box. When the IEBS was located at 100 bp upstream of the TATA box, IEP-mediated trans-activation was very similar to that of the minimal IE(nt -89 to +73) promoter lacking the IEBS. As the distance from the IEBS to the TATA box decreased, IEP-mediated trans-activation progressively decreased, indicating that the IEBS located within 100 bp from the TATA box sequence functions as a distance-dependent repressive element. These results indicated that IEP-mediated full trans-activation requires a consensus TATA box of core promoters, but not its binding to the cognate sequence (IEBS).

  11. Recruitment of hepatocyte nuclear factor 4 into specific intranuclear compartments depends on tyrosine phosphorylation that affects its DNA-binding and transactivation potential.

    PubMed Central

    Ktistaki, E; Ktistakis, N T; Papadogeorgaki, E; Talianidis, I

    1995-01-01

    Hepatocyte nuclear factor 4 (HNF-4) is a prominent member of the family of liver-enriched transcription factors, playing a role in the expression of a large number of liver-specific genes. We report here that HNF-4 is a phosphoprotein and that phosphorylation at tyrosine residue(s) is important for its DNA-binding activity and, consequently, for its transactivation potential both in cell-free systems and in cultured cells. Tyrosine phosphorylation did not affect the transport of HNF-4 from the cytoplasm to the nucleus but had a dramatic effect on its subnuclear localization. HNF-4 was concentrated in distinct nuclear compartments, as evidenced by in situ immunofluorescence and electron microscopy. This compartmentalization disappeared when tyrosine phosphorylation was inhibited by genistein. The correlation between the intranuclear distribution of HNF-4 and its ability to activate endogenous target genes demonstrates a phosphorylation signal-dependent pathway in the regulation of transcription factor activity. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:7568236

  12. Structural and functional relationships of the steroid hormone receptors’ N-terminal transactivation domain

    PubMed Central

    Kumar, Raj; Litwack, Gerald

    2009-01-01

    Steroid hormone receptors are members of a family of ligand inducible transcription factors, and regulate the transcriptional activation of target genes by recruiting coregulatory proteins to the pre-initiation machinery. The binding of these coregulatory proteins to the steroid hormone receptors is often mediated through their two activation functional domains, AF1, which resides in the N-terminal domain, and the ligand-dependent AF2, which is localized in the C-terminal ligand binding domain. Compared to other important functional domains of the steroid hormone receptors, our understanding of the mechanisms of action of the AF1 are incomplete, in part, due to the fact that, in solution, AF1 is intrinsically disordered (ID). However, recent studies have shown that AF1 must adopt a functionally active and folded conformation for its optimal activity under physiological conditions. In this review, we summarize and discuss current knowledge regarding the molecular mechanisms of AF1-mediated gene activation, focusing on AF1 conformation and coactivator binding. We further propose models for the binding/folding of the AF1 domains of the steroid hormone receptors and their protein-protein interactions. The population of ID AF1 can be visualized as a collection of many different conformations, some of which may be assuming the proper functional folding for other critical target binding partners that result in ultimate assembly of AF1:coactivator complexes and subsequent gene regulation. Knowledge of the mechanisms involved therein will significantly help in understanding how signals from a steroid to a specific target gene are conveyed. PMID:19666041

  13. TAR RNA binding properties and relative transactivation activities of human immunodeficiency virus type 1 and 2 Tat proteins.

    PubMed Central

    Rhim, H; Rice, A P

    1993-01-01

    Using gel shift assays, we found that the human immunodeficiency virus type 1 (HIV-1) Tat protein (Tat-1) bound both HIV-1 and HIV-2 TAR RNAs with similar high affinities. In contrast, the HIV-2 Tat protein (Tat-2) bound only TAR-2 RNA with high affinity. We conclude that the weak in vivo activity of Tat-2 on the HIV-1 long terminal repeat that has been observed previously is likely the result of low affinity for TAR-1 RNA. Additionally, TAR-2 RNA was found to contain multiple specific binding sites for Tat proteins. GAL4-Tat fusion proteins were analyzed to compare the relative transactivation activities of Tat-1 and Tat-2 in the absence of requirements for binding to TAR RNAs. The GAL4-Tat-2 protein was found to transactivate synthetic promoters containing GAL4 binding sites at levels severalfold higher than did the GAL4-Tat-1 protein. Images PMID:8419640

  14. EPI-001, A Compound Active against Castration-Resistant Prostate Cancer, Targets Transactivation Unit 5 of the Androgen Receptor.

    PubMed

    De Mol, Eva; Fenwick, R Bryn; Phang, Christopher T W; Buzón, Victor; Szulc, Elzbieta; de la Fuente, Alex; Escobedo, Albert; García, Jesús; Bertoncini, Carlos W; Estébanez-Perpiñá, Eva; McEwan, Iain J; Riera, Antoni; Salvatella, Xavier

    2016-09-16

    Castration-resistant prostate cancer is the lethal condition suffered by prostate cancer patients that become refractory to androgen deprivation therapy. EPI-001 is a recently identified compound active against this condition that modulates the activity of the androgen receptor, a nuclear receptor that is essential for disease progression. The mechanism by which this compound exerts its inhibitory activity is however not yet fully understood. Here we show, by using high resolution solution nuclear magnetic resonance spectroscopy, that EPI-001 selectively interacts with a partially folded region of the transactivation domain of the androgen receptor, known as transactivation unit 5, that is key for the ability of prostate cells to proliferate in the absence of androgens, a distinctive feature of castration-resistant prostate cancer. Our results can contribute to the development of more potent and less toxic novel androgen receptor antagonists for treating this disease.

  15. EPI-001, a compound active against castration-resistant prostate cancer, targets transactivation unit 5 of the androgen receptor

    PubMed Central

    De Mol, Eva; Fenwick, R. Bryn; Phang, Christopher T. W.; Buzón, Victor; Szulc, Elzbieta; de la Fuente, Alex; Escobedo, Albert; García, Jesús; Bertoncini, Carlos W.; Estébanez-Perpiñá, Eva; McEwan, Iain J.; Riera, Antoni; Salvatella, Xavier

    2016-01-01

    Castration-resistant prostate cancer is the lethal condition suffered by prostate cancer patients that become refractory to androgen deprivation therapy. EPI-001 is a recently identified compound active against this condition that modulates the activity of the androgen receptor, a nuclear receptor that is essential for disease progression. The mechanism by which this compound exerts its inhibitory activity is however not yet fully understood. Here we show, by using high resolution solution nuclear magnetic resonance spectroscopy, that EPI-001 selectively interacts with a partially folded region of the transactivation domain of the androgen receptor, known as transactivation unit 5, that is key for the ability of prostate cells to proliferate in the absence of androgens, a distinctive feature of castration-resistant prostate cancer. Our results can contribute to the development of more potent and less toxic novel androgen receptor antagonists for treating this disease. PMID:27356095

  16. Transactivation domains are not functionally conserved between vertebrate and invertebrate serum response factors.

    PubMed

    Avila, Sonia; Casero, Marie-Carmen; Fernandez-Cantón, Rocío; Sastre, Leandro

    2002-08-01

    The transcription factor serum response factor (SRF) regulates expression of growth factor-dependent genes and muscle-specific genes in vertebrates. Homologous factors regulate differentiation of some ectodermic tissues in invertebrates. To explore the molecular basis of these different physiological functions, the functionality of human, Drosophila melanogaster and Artemia franciscana SRFs in mammalian cells has been compared in this article. D. melanogaster and, to a lesser extend, A. franciscana SRF co-expression represses the activity of strong SRF-dependent promoters, such as those of the mouse c-fos and A. franciscana actin 403 genes. Domain-exchange experiments showed that these results can be explained by the absence of a transactivation domain, functional in mammalian cells, in D. melanogaster and A. franciscana SRFs. Both invertebrate SRFs can dimerize with endogenous mouse SRF through the conserved DNA-binding and dimerization domain. Co-expression of human and A. franciscana SRFs activate expression of weaker SRF-dependent promoters, such as those of the human cardiac alpha-actin gene or an A. franciscana actin 403 promoter where the SRF-binding site has been mutated. Mapping of A. franciscana SRF domains involved in transcriptional activation has shown that the conserved DNA-binding and dimerization domain is neccessary, but not sufficient, for promoter activation in mammalian cells.

  17. Transactivation of the proximal promoter of human oxytocin gene by TR4 orphan receptor

    SciTech Connect

    Wang, C.-P.; Lee, Y.-F.; Chang, C.; Lee, H.-J. . E-mail: hjlee@mail.ndhu.edu.tw

    2006-12-08

    The human testicular receptor 4 (TR4) shares structural homology with members of the nuclear receptor superfamily. Some other members of this superfamily were able to regulate the transcriptional activity of the human oxytocin (OXT) promoter by binding to the first DR0 regulatory site. However, little investigation was conducted systematically in the study of the second dDR4 site of OXT proximal promoter, and the relationship between the first and the second sites of OXT promoter. Here, we demonstrated for the first time that TR4 could increase the proximal promoter activity of the human OXT gene via DR0, dDR4, and OXT (both DR0 and dDR4) elements, respectively. TR4 might induce OXT gene expression through the OXT element in a dose-dependent manner. However, there is no synergistic effect between DR0 and dDR4 elements during TR4 transactivation. Taken together, these results suggested that TR4 should be one of important regulators of OXT gene expression.

  18. Abnormal XPD-induced nuclear receptor transactivation in DNA repair disorders: trichothiodystrophy and xeroderma pigmentosum.

    PubMed

    Zhou, Xiaolong; Khan, Sikandar G; Tamura, Deborah; Ueda, Takahiro; Boyle, Jennifer; Compe, Emmanuel; Egly, Jean-Marc; DiGiovanna, John J; Kraemer, Kenneth H

    2013-08-01

    XPD (ERCC2) is a DNA helicase involved in nucleotide excision repair and in transcription as a structural bridge tying the transcription factor IIH (TFIIH) core with the cdk-activating kinase complex, which phosphorylates nuclear receptors. Mutations in XPD are associated with several different phenotypes, including trichothiodystrophy (TTD), with sulfur-deficient brittle hair, bone defects, and developmental abnormalities without skin cancer, xeroderma pigmentosum (XP), with pigmentary abnormalities and increased skin cancer, or XP/TTD with combined features, including skin cancer. We describe the varied clinical features and mutations in nine patients examined at the National Institutes of Health who were compound heterozygotes for XPD mutations but had different clinical phenotypes: four TTD, three XP, and two combined XP/TTD. We studied TFIIH-dependent transactivation by nuclear receptor for vitamin D (VDR) and thyroid in cells from these patients. The vitamin D stimulation ratio of CYP24 and osteopontin was associated with specific pairs of mutations (reduced in 5, elevated in 1) but not correlated with distinct clinical phenotypes. Thyroid receptor stimulation ratio for KLF9 was not significantly different from normal. XPD mutations frequently were associated with abnormal VDR stimulation in compound heterozygote patients with TTD, XP, or XP/TTD.

  19. Several different upstream promoter elements can potentiate transactivation by the BPV-1 E2 protein.

    PubMed Central

    Ham, J; Dostatni, N; Arnos, F; Yaniv, M

    1991-01-01

    The enhancer and upstream promoter regions of RNA polymerase II transcribed genes modulate the rate of transcription initiation and establish specific patterns of gene expression. Both types of region consist of clusters of DNA binding sites for nuclear proteins. To determine how efficiently the same factor can activate transcription when acting as an enhancer or promoter factor, we have studied transactivation by the BPV-1 E2 protein, a papillomavirus transcriptional regulator. By cotransfecting a BPV-1 E2 expression vector and a series of reporter plasmids containing well-defined chimeric promoters we have found that whilst E2 can strongly stimulate complex promoters such as that of the HSV tk gene, it does not efficiently activate constructions containing only a TATA box and initiation site. We show that insertion of upstream promoter elements, but not of spacer DNA, between E2 binding sites and the TATA box greatly increases E2 activation. This effect was observed with more than one type of upstream promoter element, is not related to the strength of the promoter and is unlikely to result from co-operative DNA binding by E2 and the transcription factors tested. These results would suggest that E2 has the properties of an enhancer rather than promoter factor and that in certain cases promoter and enhancer factors may affect different steps in the process of transcriptional activation. Images PMID:1655407

  20. Downregulation of class II transactivator (CIITA) expression by synthetic cannabinoid CP55,940.

    PubMed

    Gongora, Celine; Hose, Stacey; O'Brien, Terrence P; Sinha, Debasish

    2004-01-30

    Cannabinoid receptors are known to be expressed in microglia; however, their involvement in specific aspects of microglial immune function has not been demonstrated. Many effects of cannabinoids are mediated by two G-protein coupled receptors, designated CB1 and CB2. We have shown that the CB1 receptor is expressed in microglia that also express MHC class II antigen (J. Neuroimmunol. 82 (1998) 13-21). In our present study, we have analyzed the effect of cannabinoid agonist CP55,940 on MHC class II expression on the surface of IFN-gamma induced microglial cells by flow cytometry. CP55,940 blocked the class II MHC expression induced by IFN-gamma. It has been shown that the regulation of class II MHC genes occurs primarily at the transcriptional level, and a non-DNA binding protein, class II transactivator (CIITA), has been shown to be the master activator for class II transcription. We find that mRNA levels of CIITA are increased in IFN-gamma induced EOC 20 microglial cells and that this increase is almost entirely eliminated by the cannabinoid agonist CP55,940. These data suggests that cannabinoids affect MHC class II expression through actions on CIITA at the transcriptional level.

  1. Trans-activation of TRPV1 by D1R in mouse dorsal root ganglion neurons.

    PubMed

    Lee, Dong Woo; Cho, Pyung Sun; Lee, Han Kyu; Lee, Sang Hoon; Jung, Sung Jun; Oh, Seog Bae

    2015-10-02

    TRPV1, a ligand-gated ion channel expressed in nociceptive sensory neurons is modulated by a variety of intracellular signaling pathways. Dopamine is a neurotransmitter that plays important roles in motor control, cognition, and pain modulation in the CNS, and acts via a variety of dopamine receptors (D1R-D5R), a class of GPCRs. Although nociceptive sensory neurons express D1-like receptors, very little is known about the effect of dopamine on TRPV1 in the peripheral nervous system. Therefore, in this study, we examined the effects of D1R activation on TRPV1 in mouse DRG neurons using Ca(2+) imaging and immunohistochemical analysis. The D1R agonist SKF-38393 induced reproducible Ca(2+) responses via Ca(2+) influx through TRPV1 rather than Ca(2+) mobilization from intracellular Ca(2+) stores. Immunohistochemical analysis revealed co-expression of D1R and TRPV1 in mouse DRG neurons. The PLC-specific inhibitor blocked the SKF-38393-induced Ca(2+) response, whereas the PKC, DAG lipase, AC, and PKA inhibitors had no effect on the SKF-38393-induced Ca(2+) response. Taken together, our results suggest that the SKF-38393-induced Ca(2+) response results from the direct activation of TRPV1 by a PLC/DAG-mediated membrane-delimited pathway. These results provide evidence that the trans-activation of TRPV1 following D1R activation may contribute to the modulation of pain signaling in nociceptive sensory neurons.

  2. Targeting reverse tetracycline-dependent transactivator to murine mammary epithelial cells that express the progesterone receptor.

    PubMed

    Mukherjee, Atish; Soyal, Selma M; Fernandez-Valdivia, Rodrigo; DeMayo, Francesco J; Lydon, John P

    2007-10-01

    Through an established gene-targeting strategy, reverse tetracycline-dependent transactivator (rtTA) was targeted downstream of the murine progesterone receptor (PR) promoter. Mice were generated in which one (PR(+/rtTA)) or both (PR(rtTA/rtTA)) PR alleles harbor the rtTA insertion. The PR(+/rtTA) and PR(rtTA/rtTA) knockins exhibit phenotypes identical to the normal and the progesterone receptor knockout mouse, respectively. Crossed with the TZA reporter, which carries the TetO-LacZ responder transgene, the PR(+/rtTA)/TZA and PR(rtTA/rtTA)/TZA bigenics exhibit doxycycline-induced beta-galactosidase activity specifically in progesterone responsive target tissues such as the mammary gland, uterus, ovary, and pituitary gland. In the case of the PR(+/rtTA)/TZA mammary epithelium, dual immunofluorescence demonstrated that PR expression and doxycycline-induced beta-galactosidase activity colocalized; beta-galactosidase was not detected in the absence of doxycycline. Although both the PR(+/rtTA) and PR(rtTA/rtTA) knockins represent innovative animal models with which to further query progesterone's mechanism of action in vivo, the PR(rtTA/rtTA) mouse in particular promises to provide unique insight into the paracrine mechanism of action, which underpins progesterone's involvement in mammary morphogenesis with obvious implications for extending our understanding of this steroid's role in breast cancer progression.

  3. BFV activates the NF-kappaB pathway through its transactivator (BTas) to enhance viral transcription

    SciTech Connect

    Wang Jian; Tan Juan; Zhang Xihui; Guo Hongyan; Zhang Qicheng; Guo Tingting; Geng Yunqi; Qiao Wentao

    2010-05-10

    Multiple families of viruses have evolved sophisticated strategies to regulate nuclear factor-kappaB (NF-kappaB) signaling, which plays a pivotal role in diverse cellular events, including virus-host interactions. In this study, we report that bovine foamy virus (BFV) is able to activate the NF-kappaB pathway through the action of its transactivator, BTas. Both cellular IKKbeta and IkappaBalpha also participate in this activation. In addition, we demonstrate that BTas induces the processing of p100, which implies that BTas can activate NF-kappaB through a noncanonical pathway as well. Co-immunoprecipitation analysis shows that BTas interacts with IKK catalytic subunits (IKKalpha and IKKbeta), which may be responsible for regulation of IKK kinase activity and persistent NF-kappaB activation. Furthermore, our results indicate that the level of BTas-mediated LTR transcription correlates with the activity of cellular NF-kappaB. Together, this study suggests that BFV activates the NF-kappaB pathway through BTas to enhance viral transcription.

  4. SIP1/NHERF2 enhances estrogen receptor alpha transactivation in breast cancer cells

    PubMed Central

    Meneses-Morales, Ivan; Tecalco-Cruz, Angeles C.; Barrios-García, Tonatiuh; Gómez-Romero, Vania; Trujillo-González, Isis; Reyes-Carmona, Sandra; García-Zepeda, Eduardo; Méndez-Enríquez, Erika; Cervantes-Roldán, Rafael; Pérez-Sánchez, Víctor; Recillas-Targa, Félix; Mohar-Betancourt, Alejandro; León-Del-Río, Alfonso

    2014-01-01

    The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that possesses two activating domains designated AF-1 and AF-2 that mediate its transcriptional activity. The role of AF-2 is to recruit coregulator protein complexes capable of modifying chromatin condensation status. In contrast, the mechanism responsible for the ligand-independent AF-1 activity and for its synergistic functional interaction with AF-2 is unclear. In this study, we have identified the protein Na+/H+ Exchanger RegulatoryFactor 2 (NHERF2) as an ERα-associated coactivator that interacts predominantly with the AF-1 domain of the nuclear receptor. Overexpression of NHERF2 in breast cancer MCF7 cells produced an increase in ERα transactivation. Interestingly, the presence of SRC-1 in NHERF2 stably overexpressing MCF7 cells produced a synergistic increase in ERα activity. We show further that NHERF2 interacts with ERα and SRC-1 in the promoter region of ERα target genes. The binding of NHERF2 to ERα in MCF7 cells increased cell proliferation and the ability of MCF7 cells to form tumors in a mouse model. We analyzed the expression of NHERF2 in breast cancer tumors finding a 2- to 17-fold increase in its mRNA levels in 50% of the tumor samples compared to normal breast tissue. These results indicate that NHERF2 is a coactivator of ERα that may participate in the development of estrogen-dependent breast cancer tumors. PMID:24771346

  5. Mulberroside A suppresses PXR-mediated transactivation and gene expression of P-gp in LS174T cells.

    PubMed

    Li, Yuhua; Huang, Ling; Sun, Jiahong; Wei, Xiaohua; Wen, Jinhua; Zhong, Guoping; Huang, Min; Bi, Huichang

    2016-12-05

    Mulberroside A (Mul A) is the main bioactive constituents of Sangbaipi, which is officially listed in the Chinese Pharmacopoeia. The pregnane X receptor (PXR) has been recognized as the critical mediator of human P-glycoprotein (P-gp) gene transactivation. In this study, the effect of Mul A on PXR-mediated transactivation and gene expression of P-gp was investigated. It was found that Mul A significantly suppressed PXR-mediated P-gp luciferase activity induced by rifampicin (Rif). Furthermore, Rif induced an elevation of P-gp expression and transport activity, which was apparently suppressed by Mul A. However, Mul A did not suppress the P-gp luciferase activity, P-gp expression, and function in the absence of Rif. These findings suggest that Mul A suppresses PXR-mediated transactivation and P-gp expression induced by Rif. This should be taken into consideration to predict any potential herb-drug interactions when Mul A or Sangbaipi are co-administered with Rif or other PXR agonist drugs.

  6. Screening of endocrine disrupting chemicals with MELN cells, an ER-transactivation assay combined with cytotoxicity assessment.

    PubMed

    Berckmans, P; Leppens, H; Vangenechten, C; Witters, H

    2007-10-01

    There is growing concern that some chemicals can cause endocrine disrupting effects to wild animals and humans. Therefore a rapid and reliable screening assay to assess the activity of endocrine disrupting chemicals (EDCs) is required. These EDCs can act at multiple sites. Most studied mechanism is direct interaction with the hormone receptors, e.g. estrogen receptor. In this study the luciferase reporter gene assay using transgenic human MELN cells was used. Since cytotoxicity of the chemicals can decrease the luminescent signal in the transactivation assays, a cytotoxicity assay must be implemented. Mostly the neutral red (NR) assay is performed in parallel with the estrogenicity assay. To increase the reliability and cost-efficiency of the test, a method to measure estrogenicity and cytotoxicity in the same cell culture plate instead of in parallel plates was developed and evaluated. Therefore the NR-assay was compared with the CytoTox-ONE homogeneous membrane integrity assay. The latter measures LDH (lactate dehydrogenase) leakage based on a fluorometric method. For all compounds tested, the CytoTox-ONE test showed comparable curves and EC50-values to those obtained by the NR-assay. So the CytoTox-ONE kit, which seemed more sensitive than measurements of LDH-leakage based on a colorimetric method, is recommended to test cytotoxicity to MELN cells, with the advantage to use the same cells for ER-transactivation measurements. The chemicals tested in the optimised MELN assay showed estrogenic potencies comparable to those reported for several other transactivation assays.

  7. Cholecystokinin 1 receptor modulates the MEKK1-induced c-Jun trans-activation: structural requirements of the receptor

    PubMed Central

    Ibarz, Géraldine; Oiry, Catherine; Carnazzi, Eric; Crespy, Philippe; Escrieut, Chantal; Fourmy, Daniel; Galleyrand, Jean Claude; Gagne, Didier; Martinez, Jean

    2006-01-01

    In cells overexpressing active MEKK1 to enhance c-Jun trans-activation, expression of rat cholecystokinin 1 receptor increased the activity of c-Jun while in the same experimental conditions overexpression of mouse cholecystokinin 1 receptor repressed it. This differential trans-activation is specific, since it was not observed for either the other overexpressed kinases (MEK, PKA) or for other transcription factors (ATF2, ELK-1, CREB). This differential behaviour was also detected in a human colon adenocarcinoma cell-line naturally producing high levels of endogenous MEKK1. This differential behaviour between the two receptors on the MEKK1-induced c-Jun trans-activation was independent of the activation state of JNK, of the phosphorylation level of c-Jun and of its ability to bind its specific DNA responsive elements. Two amino acids (Val43 and Phe50 in the mouse cholecystokinin 1 receptor, replaced by Leu43 and Ileu50 in the rat cholecystokinin 1 receptor) localized in the first transmembrane domain were found to play a crucial role in this differential behaviour. MEKK1 probably activates a transcriptional partner of c-Jun whose activity is maintained or increased in the presence of the rat cholecystokinin 1 receptor but repressed in the presence of the mouse cholecystokinin 1 receptor. PMID:16491099

  8. Cholecystokinin 1 receptor modulates the MEKK1-induced c-Jun trans-activation: structural requirements of the receptor.

    PubMed

    Ibarz, Géraldine; Oiry, Catherine; Carnazzi, Eric; Crespy, Philippe; Escrieut, Chantal; Fourmy, Daniel; Galleyrand, Jean Claude; Gagne, Didier; Martinez, Jean

    2006-04-01

    In cells overexpressing active MEKK1 to enhance c-Jun trans-activation, expression of rat cholecystokinin 1 receptor increased the activity of c-Jun while in the same experimental conditions overexpression of mouse cholecystokinin 1 receptor repressed it. This differential trans-activation is specific, since it was not observed for either the other overexpressed kinases (MEK, PKA) or for other transcription factors (ATF2, ELK-1, CREB). This differential behaviour was also detected in a human colon adenocarcinoma cell-line naturally producing high levels of endogenous MEKK1. This differential behaviour between the two receptors on the MEKK1-induced c-Jun trans-activation was independent of the activation state of JNK, of the phosphorylation level of c-Jun and of its ability to bind its specific DNA responsive elements. Two amino acids (Val43 and Phe50 in the mouse cholecystokinin 1 receptor, replaced by Leu43 and Ileu50 in the rat cholecystokinin 1 receptor) localized in the first transmembrane domain were found to play a crucial role in this differential behaviour. MEKK1 probably activates a transcriptional partner of c-Jun whose activity is maintained or increased in the presence of the rat cholecystokinin 1 receptor but repressed in the presence of the mouse cholecystokinin 1 receptor.

  9. Suppression of RNA Silencing by a Geminivirus Nuclear Protein, AC2, Correlates with Transactivation of Host Genes†

    PubMed Central

    Trinks, Daniela; Rajeswaran, R.; Shivaprasad, P. V.; Akbergenov, Rashid; Oakeley, Edward J.; Veluthambi, K.; Hohn, Thomas; Pooggin, Mikhail M.

    2005-01-01

    Bipartite geminiviruses encode a small protein, AC2, that functions as a transactivator of viral transcription and a suppressor of RNA silencing. A relationship between these two functions had not been investigated before. We characterized both of these functions for AC2 from Mungbean yellow mosaic virus-Vigna (MYMV). When transiently expressed in plant protoplasts, MYMV AC2 strongly transactivated the viral promoter; AC2 was detected in the nucleus, and a split nuclear localization signal (NLS) was mapped. In a model Nicotiana benthamiana plant, in which silencing can be triggered biolistically, AC2 reduced local silencing and prevented its systemic spread. Mutations in the AC2 NLS or Zn finger or deletion of its activator domain abolished both these effects, suggesting that suppression of silencing by AC2 requires transactivation of host suppressor(s). In line with this, in Arabidopsis protoplasts, MYMV AC2 or its homologue from African cassava mosaic geminivirus coactivated >30 components of the plant transcriptome, as detected with Affymetrix ATH1 GeneChips. Several corresponding promoters cloned from Arabidopsis were strongly induced by both AC2 proteins. These results suggest that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes discovered here may code for components of an endogenous network that controls silencing. PMID:15681452

  10. Suppression of RNA silencing by a geminivirus nuclear protein, AC2, correlates with transactivation of host genes.

    PubMed

    Trinks, Daniela; Rajeswaran, R; Shivaprasad, P V; Akbergenov, Rashid; Oakeley, Edward J; Veluthambi, K; Hohn, Thomas; Pooggin, Mikhail M

    2005-02-01

    Bipartite geminiviruses encode a small protein, AC2, that functions as a transactivator of viral transcription and a suppressor of RNA silencing. A relationship between these two functions had not been investigated before. We characterized both of these functions for AC2 from Mungbean yellow mosaic virus-Vigna (MYMV). When transiently expressed in plant protoplasts, MYMV AC2 strongly transactivated the viral promoter; AC2 was detected in the nucleus, and a split nuclear localization signal (NLS) was mapped. In a model Nicotiana benthamiana plant, in which silencing can be triggered biolistically, AC2 reduced local silencing and prevented its systemic spread. Mutations in the AC2 NLS or Zn finger or deletion of its activator domain abolished both these effects, suggesting that suppression of silencing by AC2 requires transactivation of host suppressor(s). In line with this, in Arabidopsis protoplasts, MYMV AC2 or its homologue from African cassava mosaic geminivirus coactivated >30 components of the plant transcriptome, as detected with Affymetrix ATH1 GeneChips. Several corresponding promoters cloned from Arabidopsis were strongly induced by both AC2 proteins. These results suggest that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes discovered here may code for components of an endogenous network that controls silencing.

  11. The mechanism of transactivation regulation due to polymorphic short tandem repeats (STRs) using IGF1 promoter as a model

    PubMed Central

    Chen, Holly Y.; Ma, Suk Ling; Huang, Wei; Ji, Lindan; Leung, Vincent H. K.; Jiang, Honglin; Yao, Xiaoqiang; Tang, Nelson L. S.

    2016-01-01

    Functional short tandem repeats (STR) are polymorphic in the population, and the number of repeats regulates the expression of nearby genes (known as expression STR, eSTR). STR in IGF1 promoter has been extensively studied for its association with IGF1 concentration in blood and various clinical traits and represents an important eSTR. We previously used an in-vitro luciferase reporter model to examine the interaction between STRs and SNPs in IGF1 promoter. Here, we further explored the mechanism how the number of repeats of the STR regulates gene transcription. An inverse correlation between the number of repeats and the extent of transactivation was found in a haplotype consisting of three promoter SNPs (C-STR-T-T). We showed that these adjacent SNPs located outside the STR were required for the STR to function as eSTR. The C allele of rs35767 provides a binding site for CCAAT/enhancer-binding-protein δ (C/EBPD), which is essential for the gradational transactivation property of eSTR and FOXA3 may also be involved. Therefore, we propose a mechanism in which the gradational transactivation by the eSTR is caused by the interaction of one or more transcriptional complexes located outside the STR, rather than by direct binding to a repeat motif of the STR. PMID:27910883

  12. Non-transactivational, dual pathways for LPA-induced Erk1/2 activation in primary cultures of brown pre-adipocytes

    SciTech Connect

    Holmstroem, Therese E.; Mattsson, Charlotte L.; Wang, Yanling; Iakovleva, Irina; Petrovic, Natasa; Nedergaard, Jan

    2010-10-01

    In many cell types, G-protein-coupled receptor (GPCR)-induced Erk1/2 MAP kinase activation is mediated via receptor tyrosine kinase (RTK) transactivation, in particular via the epidermal growth factor (EGF) receptor. Lysophosphatidic acid (LPA), acting via GPCRs, is a mitogen and MAP kinase activator in many systems, and LPA can regulate adipocyte proliferation. The mechanism by which LPA activates the Erk1/2 MAP kinase is generally accepted to be via EGF receptor transactivation. In primary cultures of brown pre-adipocytes, EGF can induce Erk1/2 activation, which is obligatory and determinant for EGF-induced proliferation of these cells. Therefore, we have here examined whether LPA, via EGF transactivation, can activate Erk1/2 in brown pre-adipocytes. We found that LPA could induce Erk1/2 activation. However, the LPA-induced Erk1/2 activation was independent of transactivation of EGF receptors (or PDGF receptors) in these cells (whereas in transformed HIB-1B brown adipocytes, the LPA-induced Erk1/2 activation indeed proceeded via EGF receptor transactivation). In the brown pre-adipocytes, LPA instead induced Erk1/2 activation via two distinct non-transactivational pathways, one G{sub i}-protein dependent, involving PKC and Src activation, the other, a PTX-insensitive pathway, involving PI3K (but not Akt) activation. Earlier studies showing LPA-induced Erk1/2 activation being fully dependent on RTK transactivation have all been performed in cell lines and transfected cells. The present study implies that in non-transformed systems, RTK transactivation may not be involved in the mediation of GPCR-induced Erk1/2 MAP kinase activation.

  13. Lysophosphatidic Acid-induced ERK Activation and Chemotaxis in MC3T3-E1 Preosteoblasts are Independent of EGF Receptor Transactivation

    SciTech Connect

    Karagiosis, Sue A.; Chrisler, William B.; Bollinger, Nikki; Karin, Norman J.

    2009-06-01

    Growing evidence indicates that bone-forming osteoblasts and their progenitors are target cells for the lipid growth factor lysophosphatidic acid (LPA) which is produced by degranulating platelets at sites of injury. LPA is a potent inducer of bone cell migration, proliferation and survival in vitro and an attractive candidate to facilitate preosteoblast chemotaxis during skeletal regeneration in vivo, but the intracellular signaling pathways mediating the effects of this lipid on bone cells are not defined. In this study we measured the ability of LPA to stimulate extracellular signal-related kinase (ERK1/2) in MC3T3-E1 preosteoblastic cells and determined the contribution of this pathway to LPA-stimulated chemotaxis. LPA-treated cells exhibited a bimodal activation of ERK1/2 with maximal phosphorylation at 5 and 60 minutes. The kinetics of ERK1/2 phosphorylation were not coupled to Ras activation or LPA-induced elevations in cytosolic Ca2+. While LPA is coupled to the transactivation of the EGF receptor in many cell types, LPA-stimulated ERK1/2 activation in MC3T3-E1 cells was unaffected by inhibition of EGF receptor function. ERK isoforms rapidly accumulated at nuclear sites in LPA-treated cells, a process that was blocked if ERK1/2 phosphorylation was prevented with the MEK1 inhibitor U0126. Blocking ERK1/2 phosphorylation with U0126 also diminished MC3T3-E1 cell migration and altered the normal disassembly of LPA-induced stress fibers, while the inhibition of EGF receptor function had no effect on LPA-coupled preosteoblast motility. Our results identify ERK1/2 activation as a mediatora mediator of LPA-stimulated MC3T3-E1 cell migration that may be relevant to preosteoblast motility during bone repair in vivo.

  14. A Hoxb13-driven Reverse Tetracycline Transactivator system for conditional gene expression in the prostate

    PubMed Central

    Rao, Varsha; Heard, Jamie C.; Ghaffari, Helya; Wali, Aminah; Mutton, Laura N.; Bieberich, Charles J.

    2013-01-01

    BACKGROUND Genetically engineered mouse models play important roles in analyses of prostate development and pathobiology. While constitutive genetic gain-and loss-of-function models have contributed significantly to our understanding of molecular events driving these processes, the availability of a tightly regulated inducible expression system could extend the utility of transgenic approaches. Here, we describe the development of a Tet-regulatory system that employs Hoxb13 transcriptional control elements to direct reverse tetracycline transactivator (rtTA) expression in the prostate. METHODS Using recombineering technology, the rtTA gene was placed under Hoxb13 cis-regulatory transcriptional control in the context of a 218-kb bacterial artificial chromosome. F1 offspring carrying the Hoxb13-rtTA transgene were bred to a Tetracycline operator–Histone 2B-Green Fluorescent Protein (TetO-H2BGFP) responder line. Detailed reporter gene expression analyses, including doxycycline (Dox) induction and withdrawal kinetics, were performed in Hoxb13-rtTA|TetO-H2BGFP double transgenic adult mice and embryos. RESULTS Dox-dependent GFP expression was observed exclusively in the prostate and distal colon epithelia of double transgenic mice. Reporter gene mRNA was detected in the prostate within 6 hr of Dox exposure, and was extinguished within 24 hr after Dox withdrawal. Furthermore, Dox-induced reporter gene expression persisted after castration. CONCLUSIONS The Hoxb13-rtTA transgenic system provides a powerful tool for conditional Tet operator-driven transgene expression in the normal prostate and during disease progression. Used in conjunction with other prostate pathology models, these mice will enable precise, temporally controlled analyses of gene function and can provide opportunities for detailed analyses of molecular events underlying prostate diseases. PMID:22297979

  15. Molecular signature of MT1-MMP: transactivation of the downstream universal gene network in cancer.

    PubMed

    Rozanov, Dmitri V; Savinov, Alexei Y; Williams, Roy; Liu, Kang; Golubkov, Vladislav S; Krajewski, Stan; Strongin, Alex Y

    2008-06-01

    Invasion-promoting MT1-MMP is directly linked to tumorigenesis and metastasis. Our studies led us to identify those genes, the expression of which is universally linked to MT1-MMP in multiple tumor types. Genome-wide expression profiling of MT1-MMP-overexpressing versus MT1-MMP-silenced cancer cells and a further data mining analysis of the preexisting expression database of 190 human tumors of 14 cancer types led us to identify 11 genes, the expression of which correlated firmly and universally with that of MT1-MMP (P < 0.00001). These genes included regulators of energy metabolism (NNT), trafficking and membrane fusion (SLCO2A1 and ANXA7), signaling and transcription (NR3C1, JAG1, PI3K delta, and CK2 alpha), chromatin rearrangement (SMARCA1), cell division (STK38/NDR1), apoptosis (DAPK1), and mRNA splicing (SNRPB2). Our subsequent extensive analysis of cultured cells, tumor xenografts, and cancer patient biopsies supported our data mining. Our results suggest that transcriptional reprogramming of the specific downstream genes, which themselves are associated with tumorigenesis, represents a distinctive "molecular signature" of the proteolytically active MT1-MMP. We suggest that the transactivation activity of MT1-MMP contributes to the promigratory cell phenotype, which is induced by this tumorigenic proteinase. The activated downstream gene network then begins functioning in unison with MT1-MMP to rework the signaling, transport, cell division, energy metabolism, and other critical cell functions and to commit the cell to migration, invasion, and, consequently, tumorigenesis.

  16. Dimerization and Transactivation Domains as Candidates for Functional Modulation and Diversity of Sox9

    PubMed Central

    Geraldo, Marcos Tadeu; Valente, Guilherme Targino; Nakajima, Rafael Takahiro; Martins, Cesar

    2016-01-01

    Sox9 plays an important role in a large variety of developmental pathways in vertebrates. It is composed of three domains: high-mobility group box (HMG box), dimerization (DIM) and transactivation (TAD). One of the main processes for regulation and variability of the pathways involving Sox9 is the self-gene expression regulation of Sox9. However, the subsequent roles of the Sox9 domains can also generate regulatory modulations. Studies have shown that TADs can bind to different types of proteins and its function seems to be influenced by DIM. Therefore, we hypothesized that both domains are directly associated and can be responsible for the functional variability of Sox9. We applied a method based on a broad phylogenetic context, using sequences of the HMG box domain, to ensure the homology of all the Sox9 copies used herein. The data obtained included 4,921 sequences relative to 657 metazoan species. Based on coevolutionary and selective pressure analyses of the Sox9 sequences, we observed coevolutions involving DIM and TADs. These data, along with the experimental data from literature, indicate a functional relationship between these domains. Moreover, DIM and TADs may be responsible for the functional plasticity of Sox9 because they are more tolerant for molecular changes (higher Ka/Ks ratio than the HMG box domain). This tolerance could allow a differential regulation of target genes or promote novel targets during transcriptional activation. In conclusion, we suggest that DIM and TADs functional association may regulate differentially the target genes or even promote novel targets during transcription activation mediated by Sox9 paralogs, contributing to the subfunctionalization of Sox9a and Sox9b in teleosts. PMID:27196604

  17. Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA*

    PubMed Central

    Narayanan, Aarthi; Iordanskiy, Sergey; Das, Ravi; Van Duyne, Rachel; Santos, Steven; Jaworski, Elizabeth; Guendel, Irene; Sampey, Gavin; Dalby, Elizabeth; Iglesias-Ussel, Maria; Popratiloff, Anastas; Hakami, Ramin; Kehn-Hall, Kylene; Young, Mary; Subra, Caroline; Gilbert, Caroline; Bailey, Charles; Romerio, Fabio; Kashanchi, Fatah

    2013-01-01

    Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 104–106 copies/ml TAR RNA in exosomes derived from infected culture supernatants and 103 copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS. PMID:23661700

  18. Differential usage of class II transactivator promoters PI and PIV during inflammation and injury in kidney.

    PubMed

    Takeuchi, Oki; Sims, Tasha N; Takei, Yutaka; Ramassar, Vido; Famulski, Konrad S; Halloran, Philip F

    2003-11-01

    Expression of class II transactivator (CIITA), the transcriptional regulator that controls all class II expression, is controlled in cell lines in vitro by three promoters: the dendritic cell promoter PI, the B cell promoter PIII, and the interferon-gamma (IFN-gamma)-inducible promoter, PIV. The authors examined the promoter usage in vivo in mouse kidney in the basal state and in response to IFN-gamma, endotoxin, allostimulation, and renal injury. Genetically modified mice were used to examine the dependency of each promoter on IFN-gamma and on the transcription factor interferon regulatory factor 1 (IRF-1). Usage of distinct CIITA promoters was monitored by real-time reverse transcriptase polymerase chain reaction (RT-PCR) using the unique sequences in the 5' end of the transcript from each promoter. Kidneys in both control mice and IFN-gamma knockouts expressed chiefly PI- and PIV-related products. Administration of recombinant IFN-gamma activated only promoter PIV. Endotoxin or allogeneic stimulation elevated the PIV-related mRNA, dependent on IFN-gamma and on IRF-1. Ischemic renal injury, however, increased the PI- and PIV-driven mRNA expression in wild-type but also in IFN-gamma-deficient mice. Thus the in vivo control of CIITA promoters in kidney is similar to that observed in vitro (i.e., basal-state usage of PI and IFN-gamma-dependent usage of PIV during inflammation), but it also shows additional levels of control: IFN-gamma-independent basal activity of PIV and IFN-gamma-independent induction of PIV during tissue injury.

  19. TFIIA and the transactivator Rap1 cooperate to commit TFIID for transcription initiation.

    PubMed

    Papai, Gabor; Tripathi, Manish K; Ruhlmann, Christine; Layer, Justin H; Weil, P Anthony; Schultz, Patrick

    2010-06-17

    Transcription of eukaryotic messenger RNA (mRNA) encoding genes by RNA polymerase II (Pol II) is triggered by the binding of transactivating proteins to enhancer DNA, which stimulates the recruitment of general transcription factors (TFIIA, B, D, E, F, H) and Pol II on the cis-linked promoter, leading to pre-initiation complex formation and transcription. In TFIID-dependent activation pathways, this general transcription factor containing TATA-box-binding protein is first recruited on the promoter through interaction with activators and cooperates with TFIIA to form a committed pre-initiation complex. However, neither the mechanisms by which activation signals are communicated between these factors nor the structural organization of the activated pre-initiation complex are known. Here we used cryo-electron microscopy to determine the architecture of nucleoprotein complexes composed of TFIID, TFIIA, the transcriptional activator Rap1 and yeast enhancer-promoter DNA. These structures revealed the mode of binding of Rap1 and TFIIA to TFIID, as well as a reorganization of TFIIA induced by its interaction with Rap1. We propose that this change in position increases the exposure of TATA-box-binding protein within TFIID, consequently enhancing its ability to interact with the promoter. A large Rap1-dependent DNA loop forms between the activator-binding site and the proximal promoter region. This loop is topologically locked by a TFIIA-Rap1 protein bridge that folds over the DNA. These results highlight the role of TFIIA in transcriptional activation, define a molecular mechanism for enhancer-promoter communication and provide structural insights into the pathways of intramolecular communication that convey transcription activation signals through the TFIID complex.

  20. Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA.

    PubMed

    Narayanan, Aarthi; Iordanskiy, Sergey; Das, Ravi; Van Duyne, Rachel; Santos, Steven; Jaworski, Elizabeth; Guendel, Irene; Sampey, Gavin; Dalby, Elizabeth; Iglesias-Ussel, Maria; Popratiloff, Anastas; Hakami, Ramin; Kehn-Hall, Kylene; Young, Mary; Subra, Caroline; Gilbert, Caroline; Bailey, Charles; Romerio, Fabio; Kashanchi, Fatah

    2013-07-05

    Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10(4)-10(6) copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10(3) copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.

  1. A small circular TAR RNA decoy specifically inhibits Tat-activated HIV-1 transcription.

    PubMed Central

    Bohjanen, P R; Colvin, R A; Puttaraju, M; Been, M D; Garcia-Blanco, M A

    1996-01-01

    Linear TAR RNA has previously been used as a decoy to inhibit HIV-1 transcription in vitro and HIV-1 replication in vivo. A 48 nucleotide circular RNA containing the stem, bulge and loop of the HIV-1 TAR element was synthesized using the self-splicing activity of a group I permuted intron-exon and was tested for its ability to function as a TAR decoy in vitro. This small circular TAR molecule was exceptionally stable in HeLa nuclear extracts, whereas a similar linear TAR molecule was rapidly degraded. The TAR circle bound specifically to Tfr38, a peptide containing the TAR-binding region of Tat. The ability of Tat to trans-activate transcription from the HIV-1 promoter in vitro was efficiently inhibited by circular TAR RNA but not by TAR circles that contained either bulge or loop mutations. TAR circles did not inhibit transactivation exclusively by binding to Tat since this inhibition was not reversed by adding excess Tat to the transcription reaction. Together, these data suggest that TAR circles act as decoys that inhibit transactivation by binding to Tat and at least one cellular factor. These data also demonstrate the utility of small circular RNA molecules as tools for biochemical studies. PMID:8871552

  2. Essential roles for ankyrin repeat and transactivation domains in induction of T-cell leukemia by notch1.

    PubMed

    Aster, J C; Xu, L; Karnell, F G; Patriub, V; Pui, J C; Pear, W S

    2000-10-01

    Notch receptors participate in a conserved signaling pathway that controls the development of diverse tissues and cell types, including lymphoid cells. Signaling is normally initiated through one or more ligand-mediated proteolytic cleavages that permit nuclear translocation of the intracellular portion of the Notch receptor (ICN), which then binds and activates transcription factors of the Su(H)/CBF1 family. Several mammalian Notch receptors are oncogenic when constitutively active, including Notch1, a gene initially identified based on its involvement in a (7;9) chromosomal translocation found in sporadic T-cell lymphoblastic leukemias and lymphomas (T-ALL). To investigate which portions of ICN1 contribute to transformation, we performed a structure-transformation analysis using a robust murine bone marrow reconstitution assay. Both the ankyrin repeat and C-terminal transactivation domains were required for T-cell leukemogenesis, whereas the N-terminal RAM domain and a C-terminal domain that includes a PEST sequence were nonessential. Induction of T-ALL correlated with the transactivation activity of each Notch1 polypeptide when fused to the DNA-binding domain of GAL4, with the exception of polypeptides deleted of the ankyrin repeats, which lacked transforming activity while retaining strong transactivation activity. Transforming polypeptides also demonstrated moderate to strong activation of the Su(H)/CBF1-sensitive HES-1 promoter, while polypeptides with weak or absent activity on this promoter failed to cause leukemia. These experiments define a minimal transforming region for Notch1 in T-cell progenitors and suggest that leukemogenic signaling involves recruitment of transcriptional coactivators to ICN1 nuclear complexes.

  3. Integrating the GPCR transactivation-dependent and biased signalling paradigms in the context of PAR1 signalling.

    PubMed

    Little, P J; Hollenberg, M D; Kamato, D; Thomas, W; Chen, J; Wang, T; Zheng, W; Osman, N

    2016-10-01

    Classically, receptor-mediated signalling was conceived as a linear process involving one agonist, a variety of potential targets within a receptor family (e.g. α- and β-adrenoceptors) and a second messenger (e.g. cAMP)-triggered response. If distinct responses were stimulated by the same receptor in different tissues (e.g. lipolysis in adipocytes vs. increased beating rate in the heart caused by adrenaline), the differences were attributed to different second messenger targets in the different tissues. It is now realized that an individual receptor can couple to multiple effectors (different G proteins and different β-arrestins), even in the same cell, to drive very distinct responses. Furthermore, tailored agonists can mould the receptor conformation to activate one signal pathway versus another by a process termed 'biased signalling'. Complicating issues further, we now know that activating one receptor can rapidly trigger the local release of agonists for a second receptor via a process termed 'transactivation'. Thus, the end response can represent a cooperative signalling process involving two or more receptors linked by transactivation. This overview, with a focus on the GPCR, protease-activated receptor-1, integrates both of these processes to predict the complex array of responses that can arise when biased receptor signalling also involves the receptor transactivation process. The therapeutic implications of this signalling matrix are also briefly discussed. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

  4. Comparison of 7α-methyl-19-nortestosterone effectiveness alone or combined with progestins on androgen receptor mediated-transactivation.

    PubMed

    García-Becerra, Rocío; Ordaz-Rosado, David; Noé, Gabriela; Chávez, Bertha; Cooney, Austin J; Larrea, Fernando

    2012-02-01

    7α-methyl-19-nortestosterone (MENT) is an androgen with potent gonadotropin inhibitory activity and prostate-sparing effects. These attributes give MENT advantages over testosterone as a male contraceptive, but, as in the case of testosterone, a partial dose-dependent suppression of spermatogenesis has been observed. Combination of testosterone or MENT with synthetic progestins improves the rate of azoospermia; however, it is unknown whether these combinations affect hormone androgenicity or exert synergistic effects via progestational or androgenic interaction. Herein, using transactivation assays, we examined the ability of MENT alone or combined with several 19-nor-derived synthetic progestins to activate androgen receptor (AR)-dependent gene transcription. In addition, the capability of 7α-methyl-estradiol (7α-methyl-E(2)), an aromatized metabolite of MENT, to transactivate gene transcription via estrogen receptor α (ERα; ESR1) or ERβ (ESR2) was also investigated. As expected, MENT induced gene transactivation through either the progesterone receptor (PGR) or the AR. MENT was as efficient as progesterone in activating PGR-mediated reporter gene expression, but it was ten times more potent than testosterone and dihydrotestoterone in activating of AR-driven gene expression. The addition of increasing concentrations of other 19-nortestosterone derivatives (norethisterone or levonorgestrel) did not affect, in a significant manner, the ability of MENT to activate AR-dependent reporter gene transcription. The same results were obtained with different cell lines. 7α-Methyl-E(2) resulted in potent estrogen activity via both ER subtypes with efficiency similar to natural E(2). These results suggest that the addition of 19-nortestosterone-derived progestins, as a hormonal adjuvant in male fertility strategies for effective spermatogenic suppression, does not display any detrimental effect that would interfere with MENT androgenic transcriptional activity.

  5. Glutamine Repeat Variants in Human RUNX2 Associated with Decreased Femoral Neck BMD, Broadband Ultrasound Attenuation and Target Gene Transactivation

    PubMed Central

    Morrison, Nigel A.; Stephens, Alexandre A.; Osato, Motomi; Polly, Patsie; Tan, Timothy C.; Yamashita, Namiko; Doecke, James D.; Pasco, Julie; Fozzard, Nicolette; Jones, Graeme; Ralston, Stuart H.; Prince, Richard L.; Nicholson, Geoff C.

    2012-01-01

    RUNX2 is an essential transcription factor required for skeletal development and cartilage formation. Haploinsufficiency of RUNX2 leads to cleidocranial displaysia (CCD) a skeletal disorder characterised by gross dysgenesis of bones particularly those derived from intramembranous bone formation. A notable feature of the RUNX2 protein is the polyglutamine and polyalanine (23Q/17A) domain coded by a repeat sequence. Since none of the known mutations causing CCD characterised to date map in the glutamine repeat region, we hypothesised that Q-repeat mutations may be related to a more subtle bone phenotype. We screened subjects derived from four normal populations for Q-repeat variants. A total of 22 subjects were identified who were heterozygous for a wild type allele and a Q-repeat variant allele: (15Q, 16Q, 18Q and 30Q). Although not every subject had data for all measures, Q-repeat variants had a significant deficit in BMD with an average decrease of 0.7SD measured over 12 BMD-related parameters (p = 0.005). Femoral neck BMD was measured in all subjects (−0.6SD, p = 0.0007). The transactivation function of RUNX2 was determined for 16Q and 30Q alleles using a reporter gene assay. 16Q and 30Q alleles displayed significantly lower transactivation function compared to wild type (23Q). Our analysis has identified novel Q-repeat mutations that occur at a collective frequency of about 0.4%. These mutations significantly alter BMD and display impaired transactivation function, introducing a new class of functionally relevant RUNX2 mutants. PMID:22912713

  6. Human immunodeficiency virus trans-activator of transcription peptide detection via ribonucleic acid aptamer on aminated diamond biosensor

    NASA Astrophysics Data System (ADS)

    Rahim Ruslinda, A.; Wang, Xianfen; Ishii, Yoko; Ishiyama, Yuichiro; Tanabe, Kyosuke; Kawarada, Hiroshi

    2011-09-01

    The potential of ribonucleic acid (RNA) as both informational and ligand binding molecule have opened a scenario in the development of biosensors. An aminated diamond-based RNA aptasensor is presented for human immunodeficiency virus (HIV) trans-activator of transcription (Tat) peptide protein detection that not only gives a labeled or label-free detection method but also provides a reusable platform for a simple, sensitive, and selective detection of proteins. The immobilized procedure was based on the binding interaction between positively charged amine terminated diamond and the RNA aptamer probe molecules with the negatively charged surface carboxylic compound linker molecule such as terephthalic acid.

  7. Epstein-Barr virus nuclear antigen 2 transactivates the long terminal repeat of human immunodeficiency virus type 1.

    PubMed

    Scala, G; Quinto, I; Ruocco, M R; Mallardo, M; Ambrosino, C; Squitieri, B; Tassone, P; Venuta, S

    1993-05-01

    Human immunodeficiency virus type 1 (HIV-1)-infected subjects show a high incidence of Epstein-Barr virus (EBV) infection. This suggests that EBV may function as a cofactor that affects HIV-1 activation and may play a major role in the progression of AIDS. To test this hypothesis, we generated two EBV-negative human B-cell lines that stably express the EBNA2 gene of EBV. These EBNA2-positive cell lines were transiently transfected with plasmids that carry either the wild type or deletion mutants of the HIV-1 long terminal repeat (LTR) fused to the chloramphenicol acetyltransferase (CAT) gene. There was a consistently higher HIV-1 LTR activation in EBNA2-expressing cells than in control cells, which suggested that EBNA2 proteins could activate the HIV-1 promoter, possibly by inducing nuclear factors binding to HIV-1 cis-regulatory sequences. To test this possibility, we used CAT-based plasmids carrying deletions of the NF-kappa B (pNFA-CAT), Sp1 (pSpA-CAT), or TAR (pTAR-CAT) region of the HIV-1 LTR and retardation assays in which nuclear proteins from EBNA2-expressing cells were challenged with oligonucleotides encompassing the NF-kappa B or Sp1 region of the HIV-1 LTR. We found that both the NF-kappa B and the Sp1 sites of the HIV-1 LTR are necessary for EBNA2 transactivation and that increased expression resulted from the induction of NF-kappa B-like factors. Moreover, experiments with the TAR-deleted pTAR-CAT and with the tat-expressing pAR-TAT plasmids indicated that endogenous Tat-like proteins could participate in EBNA2-mediated activation of the HIV-1 LTR and that EBNA2 proteins can synergize with the viral tat transactivator. Transfection experiments with plasmids expressing the EBNA1, EBNA3, and EBNALP genes did not cause a significant HIV-1 LTR activation. Thus, it appears that among the latent EBV genes tested, EBNA2 was the only EBV gene active on the HIV-1 LTR. The transactivation function of EBNA2 was also observed in the HeLa epithelial cell line

  8. Ets transcription factors bind and transactivate the core promoter of the von Willebrand factor gene.

    PubMed

    Schwachtgen, J L; Janel, N; Barek, L; Duterque-Coquillaud, M; Ghysdael, J; Meyer, D; Kerbiriou-Nabias, D

    1997-12-18

    von Willebrand factor (vWF) gene expression is restricted to endothelial cells and megakaryocytes. Previous results demonstrated that basal transcription of the human vWF gene is mediated through a promoter located between base pairs -89 and +19 (cap site: +1) which is functional in endothelial and non endothelial cells. Two DNA repeats TTTCCTTT correlating with inverted consensus binding sites for the Ets family of transcription factors are present in the -56/-36 sequence. In order to analyse whether these DNA elements are involved in transcription, human umbilical vein endothelial cells (HUVEC), bovine calf pulmonary endothelial cell line (CPAE), HeLa and COS cells were transfected with constructs containing deletions of the -89/+19 fragment, linked to the chloramphenicol acetyl transferase (CAT) reporter gene. The -60/+19 region exhibits significant promoter activity in HUVEC and CPAE cells only. The -42/+19 fragment is not active. Mutations of the -60/+19 promoter fragment in the 5' (-56/-49) Ets binding site abolish transcription in endothelial cells whereas mutations in the 3' (-43/-36) site does not. The -60/-33 fragment forms three complexes with proteins from HUVEC nuclear extracts in electrophoretic mobility shift assay which are dependent on the presence of the 5' Ets binding site. Binding of recombinant Ets-1 protein to the -60/-33 fragment gives a complex which also depends on the 5' site. The -60/+19 vWF gene core promoter is transactivated in HeLa cells by cotransfecting with Ets-1 or Erg (Ets-related gene) expression plasmids. In contrast to the wild type construct, transcription of the 5' site mutants is not increased by these expressed proteins. The results indicate that the promoter activity of the -60/+19 region of the vWF gene depends on transcription factors of the Ets family of which several members like Ets-1, Ets-2 and Erg are expressed in endothelium. Cotransfection of Ets-1 and Erg expression plasmids is sufficient to induce the -60/+19 v

  9. Transforming growth factor-β1 induces EMT by the transactivation of epidermal growth factor signaling through HA/CD44 in lung and breast cancer cells

    PubMed Central

    LI, LINGMEI; QI, LISHA; LIANG, ZHIJIE; SONG, WANGZHAO; LIU, YANXUE; WANG, YALEI; SUN, BAOCUN; ZHANG, BIN; CAO, WENFENG

    2015-01-01

    Epithelial-mesenchymal transition (EMT), a process closely related to tumor development, is regulated by a variety of signaling pathways and growth factors, such as transforming growth factor-β1 (TGF-β1) and epidermal growth factor (EGF). Hyaluronan (HA) has been shown to induce EMT through either TGF-β1 or EGF signaling and to be a regulator of the crosstalk between these two pathways in fibroblasts. In this study, in order to clarify whether HA has the same effect in tumor cells, we utilized the lung cancer cell line, A549, and the breast cancer cell line, MCF-7, and found that the effects of stimulation with TGF-β1 were more potent than those of EGF in regulating the expression of EMT-associated proteins and in enhancing cell migration and invasion. In addition, we observed that TGF-β1 activated EGF receptor (EGFR) and its downstream AKT and extracellular signal-regulated kinase (ERK) pathways. Furthermore, we found that TGF-β1 upregulated the expression of hyaluronan synthases (HAS1, HAS2 and HAS3) and promoted the expression of CD44, a cell surface receptor for HA, which interacts with EGFR, resulting in the activation of the downstream AKT and ERK pathways. Conversely, treatment with 4-methylumbelliferone (4-MU; an inhibitor of HAS) prior to stimulation with TGF-β1, inhibited the expression of CD44 and EGFR, abolished the interaction between CD44 and EGFR. Furthermore, the use of shRNA targeting CD44 impaired the expression of EGFR, deactivated the AKT and ERK pathways, reversed EMT and decreased the migration and invasion ability of cells. In conclusion, our data demonstrate that TGF-β1 induces EMT by the transactivation of EGF signaling through HA/CD44 in lung and breast cancer cells. PMID:26005723

  10. Transactivation of the proenkephalin gene promoter by the Tax sub 1 protein of human T-cell lymphotropic virus type I

    SciTech Connect

    Joshi, J.B. ); Dave, H.P.G. )

    1992-02-01

    Human T-cell lymphotropic virus type I (HTLV-I), an etiologic agent for adult T-cell leukemia, is strongly associated with certain neurological diseases. The HTLV-I genome encodes a protein, Tax{sub 1}, that transactivates viral gene transcription. CD4-positive T helper lymphocytes express the proenkephalin gene, and enkephalins have been implicated as neuroimmunomodulators. The authors have investigated the effect of Tax{sub 1} on the proenkephalin gene promoter in C6 rat glioma cells and demonstrated its transactivation. Analysis using 5{prime} deletion mutants of the promoter region showed that sequences upstream of base pair - 190 are necessary for maximal transactivation. Forskolin, a cAMP modulator, synergistically increased Tax{sub 1}-mediated transactivation of the proenkephalin promoter. Neither Tax{sub 1} transactivation alone nor Tax{sub 1}/cAMP synergism exclusively involved cAMP-responsive elements. Endogenous proenkephalin gene expression increased in Tax{sub 1}-expressing C6 cells. Since HTLV-I infects lymphocytes, which express proenkephalin mRNA, Tax{sub 1} transregulation of proenkephalin expression may provide bidirectional communication between the nervous and immune systems in HTLV-I-related diseases.

  11. Corepressive Action of CBP on Androgen Receptor Transactivation in Pericentric Heterochromatin in a Drosophila Experimental Model System▿ †

    PubMed Central

    Zhao, Yue; Takeyama, Ken-ichi; Sawatsubashi, Shun; Ito, Saya; Suzuki, Eriko; Yamagata, Kaoru; Tanabe, Masahiko; Kimura, Shuhei; Fujiyama, Sally; Ueda, Takashi; Murata, Takuya; Matsukawa, Hiroyuki; Shirode, Yuko; Kouzmenko, Alexander P.; Li, Feng; Tabata, Testuya; Kato, Shigeaki

    2009-01-01

    Ligand-bound nuclear receptors (NR) activate transcription of the target genes. This activation is coupled with histone modifications and chromatin remodeling through the function of various coregulators. However, the nature of the dependence of a NR coregulator action on the presence of the chromatin environment at the target genes is unclear. To address this issue, we have developed a modified position effect variegation experimental model system that includes an androgen-dependent reporter transgene inserted into either a pericentric heterochromatin region or a euchromatic region of Drosophila chromosome. Human androgen receptor (AR) and its constitutively active truncation mutant (AR AF-1) were transcriptionally functional in both chromosomal regions. Predictably, the level of AR-induced transactivation was lower in the pericentric heterochromatin. In genetic screening for AR AF-1 coregulators, Drosophila CREB binding protein (dCBP) was found to corepress AR transactivation at the pericentric region whereas it led to coactivation in the euchromatic area. Mutations of Sir2 acetylation sites or deletion of the CBP acetyltransferase domain abrogated dCBP corepressive action for AR at heterochromatic areas in vivo. Such a CBP corepressor function for AR was observed in the transcriptionally silent promoter of an AR target gene in cultured mammalian cells. Thus, our findings suggest that the action of NR coregulators may depend on the state of chromatin at the target loci. PMID:19075001

  12. The membrane-topogenic vectorial behaviour of Nrf1 controls its post-translational modification and transactivation activity

    PubMed Central

    Zhang, Yiguo; Hayes, John D.

    2013-01-01

    The integral membrane-bound Nrf1 transcription factor fulfils important functions in maintaining cellular homeostasis and organ integrity, but how it is controlled vectorially is unknown. Herein, creative use of Gal4-based reporter assays with protease protection assays (GRAPPA), and double fluorescence protease protection (dFPP), reveals that the membrane-topogenic vectorial behaviour of Nrf1 dictates its post-translational modification and transactivation activity. Nrf1 is integrated within endoplasmic reticulum (ER) membranes through its NHB1-associated TM1 in cooperation with other semihydrophobic amphipathic regions. The transactivation domains (TADs) of Nrf1, including its Asn/Ser/Thr-rich (NST) glycodomain, are transiently translocated into the ER lumen, where it is glycosylated in the presence of glucose to become a 120-kDa isoform. Thereafter, the NST-adjoining TADs are partially repartitioned out of membranes into the cyto/nucleoplasmic side, where Nrf1 is subject to deglycosylation and/or proteolysis to generate 95-kDa and 85-kDa isoforms. Therefore, the vectorial process of Nrf1 controls its target gene expression. PMID:23774320

  13. IDX-1: a new homeodomain transcription factor expressed in rat pancreatic islets and duodenum that transactivates the somatostatin gene.

    PubMed Central

    Miller, C P; McGehee, R E; Habener, J F

    1994-01-01

    We describe the cloning from a rat islet somatostatin-producing cell line of a 1.4 kb cDNA encoding a new homeoprotein, IDX-1 (islet/duodenum homeobox-1), with close sequence similarity to the Drosophila melanogaster homeobox protein Antennapedia (Antp) and the Xenopus laevis endoderm-specific homeoprotein XlHbox8. Analyses of IDX-1 mRNA and protein in rat tissues show that IDX-1 is expressed in pancreatic islets and ducts and in the duodenum. In electrophoretic mobility shift assays IDX-1 binds to three sites in the 5' flanking region of the rat somatostatin gene. In co-transfection experiments IDX-1 transactivates reporter constructs containing somatostatin promoter sequences, and mutation of the IDX-1 binding sites attenuates transactivation. Reverse transcription-polymerase chain reaction of islet RNA using degenerate amplimers for mRNAs encoding homeoproteins indicates that IDX-1 is the most abundant of 12 different Antp-like homeodomain mRNAs expressed in adult rat islets. The pattern of expression, relative abundance and transcriptional regulatory activity suggests that IDX-1 may be involved in the regulation of islet hormone genes and in cellular differentiation in the endocrine pancreas and the duodenum. Images PMID:7907546

  14. The membrane-topogenic vectorial behaviour of Nrf1 controls its post-translational modification and transactivation activity.

    PubMed

    Zhang, Yiguo; Hayes, John D

    2013-01-01

    The integral membrane-bound Nrf1 transcription factor fulfils important functions in maintaining cellular homeostasis and organ integrity, but how it is controlled vectorially is unknown. Herein, creative use of Gal4-based reporter assays with protease protection assays (GRAPPA), and double fluorescence protease protection (dFPP), reveals that the membrane-topogenic vectorial behaviour of Nrf1 dictates its post-translational modification and transactivation activity. Nrf1 is integrated within endoplasmic reticulum (ER) membranes through its NHB1-associated TM1 in cooperation with other semihydrophobic amphipathic regions. The transactivation domains (TADs) of Nrf1, including its Asn/Ser/Thr-rich (NST) glycodomain, are transiently translocated into the ER lumen, where it is glycosylated in the presence of glucose to become a 120-kDa isoform. Thereafter, the NST-adjoining TADs are partially repartitioned out of membranes into the cyto/nucleoplasmic side, where Nrf1 is subject to deglycosylation and/or proteolysis to generate 95-kDa and 85-kDa isoforms. Therefore, the vectorial process of Nrf1 controls its target gene expression.

  15. New insights into the anti-inflammatory mechanisms of glucocorticoids: an emerging role for glucocorticoid-receptor-mediated transactivation.

    PubMed

    Vandevyver, Sofie; Dejager, Lien; Tuckermann, Jan; Libert, Claude

    2013-03-01

    Glucocorticoids are anti-inflammatory drugs that are widely used for the treatment of numerous (autoimmune) inflammatory diseases. They exert their actions by binding to the glucocorticoid receptor (GR), a member of the nuclear receptor family of transcription factors. Upon ligand binding, the GR translocates to the nucleus, where it acts either as a homodimeric transcription factor that binds glucocorticoid response elements (GREs) in promoter regions of glucocorticoid (GC)-inducible genes, or as a monomeric protein that cooperates with other transcription factors to affect transcription. For decades, it has generally been believed that the undesirable side effects of GC therapy are induced by dimer-mediated transactivation, whereas its beneficial anti-inflammatory effects are mainly due to the monomer-mediated transrepressive actions of GR. Therefore, current research is focused on the development of dissociated compounds that exert only the GR monomer-dependent actions. However, many recent reports undermine this dogma by clearly showing that GR dimer-dependent transactivation is essential in the anti-inflammatory activities of GR. Many of these studies used GR(dim/dim) mutant mice, which show reduced GR dimerization and hence cannot control inflammation in several disease models. Here, we review the importance of GR dimers in the anti-inflammatory actions of GCs/GR, and hence we question the central dogma. We summarize the contribution of various GR dimer-inducible anti-inflammatory genes and question the use of selective GR agonists as therapeutic agents.

  16. The serine 106 residue within the N-terminal transactivation domain is crucial for Oct4 function in mice.

    PubMed

    Mitani, Atsushi; Fukuda, Atsushi; Miyashita, Toshiyuki; Umezawa, Akihiro; Akutsu, Hidenori

    2017-03-07

    Pou5f1/Oct4 is a key transcription factor for the induction of pluripotency and totipotency in preimplantation mouse embryos. In mice, loss or gain of function experiments have demonstrated an important role for Oct4 in preimplantation and developmental ability. In this study, using mouse preimplantation embryos as a model for the evaluation of Oct4 function, we constructed Oct4 overexpression embryos with various mutations at the N-terminal transactivation domain. Developmental competency and molecular biological phenotypes depended on the type of mutation. The replacement of serine 106 with alanine resulted in more severe phenotypes similar to that of wild type Oct4, indicating that this alteration using alanine is negligible for Oct4 function. In contrast, we found that Oct4-specific antibodies could not recognize Oct4 protein when this residue was replaced by aspartic acid (Oct4-S106D). Oct4-S106D overexpressing embryos did not show developmental arrest and aberrant chromatin structure. Thus, these results demonstrated that the Ser-106 residue within the N-terminal transactivation domain is crucial for Oct4 function and suggested that this mutation might affect Oct4 protein conformation.

  17. Low levels of the reverse transactivator fail to induce target transgene expression in vascular smooth muscle cells.

    PubMed

    Viceconte, Nikenza; McKenna, Tomás; Eriksson, Maria

    2014-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disease with multiple features that are suggestive of premature aging. Most patients with HGPS carry a mutation on one of their copies of the LMNA gene. The LMNA gene encodes the lamin A and lamin C proteins, which are the major proteins of the nuclear lamina. The organs of the cardiovascular system are amongst those that are most severely affected in HGPS, undergoing a progressive depletion of vascular smooth muscle cells, and most children with HGPS die in their early teens from cardio-vascular disease and other complications from atherosclerosis. In this study, we developed a transgenic mouse model based on the tet-ON system to increase the understanding of the molecular mechanisms leading to the most lethal aspect of HGPS. To induce the expression of the most common HGPS mutation, LMNA c.1824C>T; p.G608G, in the vascular smooth muscle cells of the aortic arch and thoracic aorta, we used the previously described reverse tetracycline-controlled transactivator, sm22α-rtTA. However, the expression of the reverse sm22α-transactivator was barely detectable in the arteries, and this low level of expression was not sufficient to induce the expression of the target human lamin A minigene. The results from this study are important because they suggest caution during the use of previously functional transgenic animal models and emphasize the importance of assessing transgene expression over time.

  18. Low Levels of the Reverse Transactivator Fail to Induce Target Transgene Expression in Vascular Smooth Muscle Cells

    PubMed Central

    Viceconte, Nikenza; McKenna, Tomás; Eriksson, Maria

    2014-01-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a genetic disease with multiple features that are suggestive of premature aging. Most patients with HGPS carry a mutation on one of their copies of the LMNA gene. The LMNA gene encodes the lamin A and lamin C proteins, which are the major proteins of the nuclear lamina. The organs of the cardiovascular system are amongst those that are most severely affected in HGPS, undergoing a progressive depletion of vascular smooth muscle cells, and most children with HGPS die in their early teens from cardio-vascular disease and other complications from atherosclerosis. In this study, we developed a transgenic mouse model based on the tet-ON system to increase the understanding of the molecular mechanisms leading to the most lethal aspect of HGPS. To induce the expression of the most common HGPS mutation, LMNA c.1824C>T; p.G608G, in the vascular smooth muscle cells of the aortic arch and thoracic aorta, we used the previously described reverse tetracycline-controlled transactivator, sm22α-rtTA. However, the expression of the reverse sm22α-transactivator was barely detectable in the arteries, and this low level of expression was not sufficient to induce the expression of the target human lamin A minigene. The results from this study are important because they suggest caution during the use of previously functional transgenic animal models and emphasize the importance of assessing transgene expression over time. PMID:25090270

  19. Random mutagenesis of the human immunodeficiency virus type-1 trans-activator of transcription (HIV-1 Tat).

    PubMed Central

    Siderovski, D P; Matsuyama, T; Frigerio, E; Chui, S; Min, X; Erfle, H; Sumner-Smith, M; Barnett, R W; Mak, T W

    1992-01-01

    A new method is described for the direct construction of randomly mutagenized genes by applying the polymerase chain reaction (PCR) to an oligonucleotide synthesized using doped nucleotide reservoirs. We have demonstrated the utility of this method by generating a library of mutant HIV-1 tat genes. Several arbitrarily selected, inactive tat clones were sequenced to evaluate the extent of the mutagenesis. Moreover, fourteen recombinants encoding varying levels of transcriptional trans-activator activity were isolated by transient transfection of sub-library pools into a HeLa cell line bearing an HIV-LTR-chloramphenicol acetyltransferase (CAT) reporter gene. Sequence data revealed a spectrum of alterations including nucleotide substitutions, insertions, and deletions, suggesting that mutations arose from both the doped DNA synthesis and the subsequent PCR 'rescue' of full-length product. Sequence comparison between inactive and active Tat clones revealed a selection pressure against amino-acid substitutions within the N-terminal domains of Tat, indicating the importance of this region to trans-activation competence. In addition, single and double missense mutations within the basic-rich, TAR RNA-binding domain were seen to be tolerated within active Tat clones. Images PMID:1437550

  20. Transactivation of ErbB receptors by leptin in the cardiovascular system: mechanisms, consequences and target for therapy.

    PubMed

    Bełtowski, Jerzy; Jazmroz-Wiśniewska, Anna

    2014-01-01

    Many experimental and clinical studies have demonstrated that elevated leptin concentration in patients with obesity/metabolic syndrome contributes to the pathogenesis of cardiovascular disorders including arterial hypertension, atherosclerosis, restenosis after coronary angioplasty and myocardial hypertrophy. Receptor tyrosine kinases belonging to the ErbB family, especially ErbB1 (epidermal growth factor receptor) and ErbB2 are abundantly expressed in the blood vessels and the heart. EGFR is activated not only by its multiple peptide ligands but also by many other factors including angiotensin II, endothelin-1, norepinephrine, thrombin and prorenin; the phenomenon referred to as "transactivation". Augmented EGFR signaling contributes to abnormalities of vascular tone and renal sodium handling as well as vascular remodeling and myocardial hypertrophy through various intracellular mechanisms, in particular extracellular signal-regulated kinases (ERK) and phosphoinositide 3-kinase (PI3K). Recent experimental studies indicate that chronically elevated leptin transactivates the EGFR through the mechanisms requiring reactive oxygen species and cytosolic tyrosine kinase, c-Src. In addition, hyperleptinemia increases ErbB2 activity in the arterial wall. Stimulation of EGFR and ErbB2 downstream signaling pathways such as ERK and PI3K in the vascular wall and the kidney may contribute to the increase in vascular tone, enhanced tubular sodium reabsorption as well as vascular and renal lesions in hyperleptinemic obese subjects.

  1. Activation of human papillomavirus type 18 gene expression by herpes simplex virus type 1 viral transactivators and a phorbol ester

    SciTech Connect

    Gius, D.; Laimins, L.A.

    1989-02-01

    Several viral trans-activators and a tumor promoter were examined for the ability to activate human papillomavirus type 18 (HPV-18) gene expression. A plasmid containing the HPV-18 noncoding region placed upstream of the chloramphenicol acetyltransferase reporter gene was cotransfected with different herpes simplex virus type 1 (HSV-1) genes into several cell lines. Both HSV-1 TIF and ICPO activated HPV-18 expression; however, activation by TIF was observed only in epithelial cells, while ICPO stimulated expression in a wide variety of cells. The element activated by both TIF and ICOP was mapped to a 229-base-pair fragment which also contains an HPV-18 epithelial cell-preferred enhancer. The inclusion of a papillomavirus E2 trans-activator with TIF and ICOP further increased HPV-18 expression. In contrast, the HSV-1 ICP4 and ICP27 genes, as well as the human T-cell lymphotropic virus type I and human immunodeficiency virus type 1 tat genes, were found to have no effect on HPV-18 expression. In transient assays, the addition of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also activated HPV-18 expression. The region of HPV-18 activated by TPA was localized to a sequence which is homologous to other TPA-responsive elements.

  2. Identification of Novel Kaposi's Sarcoma-Associated Herpesvirus Orf50 Transcripts: Discovery of New RTA Isoforms with Variable Transactivation Potential.

    PubMed

    Wakeman, Brian S; Izumiya, Yoshihiro; Speck, Samuel H

    2017-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that has been associated with primary effusion lymphoma and multicentric Castleman's disease, as well as its namesake Kaposi's sarcoma. As a gammaherpesvirus, KSHV is able to acutely replicate, enter latency, and reactivate from this latent state. A key protein involved in both acute replication and reactivation from latency is the replication and transcriptional activator (RTA) encoded by the gene Orf50 RTA is a known transactivator of multiple viral genes, allowing it to control the switch between latency and virus replication. We report here the identification of six alternatively spliced Orf50 transcripts that are generated from four distinct promoters. These newly identified promoters are shown to be transcriptionally active in 293T (embryonic kidney), Vero (African-green monkey kidney epithelial), 3T12 (mouse fibroblast), and RAW 264.7 (mouse macrophage) cell lines. Notably, the newly identified Orf50 transcripts are predicted to encode four different isoforms of the RTA which differ by 6 to 10 residues at the amino terminus of the protein. We show the global viral transactivation potential of all four RTA isoforms and demonstrate that all isoforms can transcriptionally activate an array of KSHV promoters to various levels. The pattern of transcriptional activation appears to support a transcriptional interference model within the Orf50 region, where silencing of previously expressed isoforms by transcription initiation from upstream Orf50 promoters has the potential to modulate the pattern of viral gene activation.

  3. Transactivation Domain of Human c-Myc Is Essential to Alleviate Poly(Q)-Mediated Neurotoxicity in Drosophila Disease Models.

    PubMed

    Raj, Kritika; Sarkar, Surajit

    2017-03-18

    Polyglutamine (poly(Q)) disorders, such as Huntington's disease (HD) and spinocerebellar ataxias, represent a group of neurological disorders which arise due to an atypically expanded poly(Q) tract in the coding region of the affected gene. Pathogenesis of these disorders inside the cells begins with the assembly of these mutant proteins in the form of insoluble inclusion bodies (IBs), which progressively sequester several vital cellular transcription factors and other essential proteins, and finally leads to neuronal dysfunction and apoptosis. We have shown earlier that targeted upregulation of Drosophila myc (dmyc) dominantly suppresses the poly(Q) toxicity in Drosophila. The present study examines the ability of the human c-myc proto-oncogene and also identifies the specific c-Myc isoform which drives the mitigation of poly(Q)-mediated neurotoxicity, so that it could be further substantiated as a potential drug target. We report for the first time that similar to dmyc, tissue-specific induced expression of human c-myc also suppresses poly(Q)-mediated neurotoxicity by an analogous mechanism. Among the three isoforms of c-Myc, the rescue potential was maximally manifested by the full-length c-Myc2 protein, followed by c-Myc1, but not by c-MycS which lacks the transactivation domain. Our study suggests that strategies focussing on the transactivation domain of c-Myc could be a very useful approach to design novel drug molecules against poly(Q) disorders.

  4. Fruit-juice concentrate of Asian plum inhibits growth signals of vascular smooth muscle cells induced by angiotensin II.

    PubMed

    Utsunomiya, Hirotoshi; Takekoshi, Susumu; Gato, Nobuki; Utatsu, Hisao; Motley, Evangeline D; Eguchi, Kunie; Fitzgerald, Trinita G; Mifune, Mizuo; Frank, Gerald D; Eguchi, Satoru

    2002-12-27

    Bainiku-ekisu, the fruit-juice concentrate of the Oriental plum (Prunus mume) has recently been shown to improve human blood fluidity. We have shown that angiotensin II (AngII) stimulates growth of vascular smooth muscle cells (VSMCs) through epidermal growth factor (EGF) receptor transactivation that involves reactive oxygen species (ROS) production. To better understanding the possible cardiovascular protective effect of Bainiku-ekisu, we have studied whether Bainiku-ekisu inhibits AngII-induced growth promoting signals in VSMCs. Bainiku-ekisu markedly inhibited AngII-induced EGF receptor transactivation. H(2)O(2)-induced EGF receptor transactivation was also inhibited by Bainiku-ekisu. Thus, Bainiku-ekisu markedly inhibited AngII-induced extracellular signal-regulated kinase (ERK) activation. However, EGF-induced ERK activation was not affected by Bainiku-ekisu. AngII stimulated leucine uptake in VSMCs that was significantly inhibited by Bainiku-ekisu. Also, Bainiku-ekisu possesses a potent antioxidant activity. Since the activation of EGF receptor, ERK and the production of ROS play central roles in mediating AngII-induced vascular remodeling, these data suggest that Bainiku-ekisu could exert a powerful cardiovascular protective effect with regard to cardiovascular diseases.

  5. Transactivation Assays to Assess Canine and Rodent Pregnane X Receptor (PXR) and Constitutive Androstane Receptor (CAR) Activation

    PubMed Central

    Pinne, Marija; Ponce, Elsa; Raucy, Judy L.

    2016-01-01

    The pregnane X receptor (PXR/SXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are nuclear receptors (NRs) involved in the regulation of many genes including cytochrome P450 enzymes (CYPs) and transporters important in metabolism and uptake of both endogenous substrates and xenobiotics. Activation of these receptors can lead to adverse drug effects as well as drug-drug interactions. Depending on which nuclear receptor is activated will determine which adverse effect could occur, making identification important. Screening for NR activation by New Molecular Entities (NMEs) using cell-based transactivation assays is the singular high throughput method currently available for identifying the activation of a particular NR. Moreover, screening for species-specific NR activation can minimize the use of animals in drug development and toxicology studies. With this in mind, we have developed in vitro transactivation assays to identify compounds that activate canine and rat PXR and CAR3. We found differences in specificity for canine and rat PXR, with the best activator for canine PXR being 10 μM SR12813 (60.1 ± 3.1-fold) and for rat PXR, 10 μM dexamethasone (60.9 ± 8.4 fold). Of the 19 test agents examined, 10 and 9 significantly activated rat and canine PXR at varying degrees, respectively. In contrast, 5 compounds exhibited statistically significant activation of rat CAR3 and 4 activated the canine receptor. For canine CAR3, 50 μM artemisinin proved to be the best activator (7.3 ± 1.8 and 10.5 ± 2.2 fold) while clotrimazole (10 μM) was the primary activator of the rat variant (13.7 ± 0.8 and 26.9 ± 1.3 fold). Results from these studies demonstrated that cell-based transactivation assays can detect species-specific activators and revealed that PXR was activated by at least twice as many compounds as was CAR3, suggesting that there are many more agonists for PXR than CAR. PMID:27732639

  6. The hepatitis B virus X protein increases the cellular level of TATA-binding protein, which mediates transactivation of RNA polymerase III genes

    SciTech Connect

    Wang, Horng-Dar; Johnson, D.L.; Yuh, Chio-Hwa

    1995-12-01

    This report decribes the mechanism by which the hepatitis B virus X gene product induces RNA polymerase III genes. The RNA pol III transcription system serves as model for understanding the mechanism of X in the transactivation of cellular genes in both Drosophila and rat cell lines. 53 refs., 7 figs., 1 tab.

  7. Conserved structural motifs at the C-terminus of baculovirus protein IE0 are important for its functions in transactivation and supporting hr5-mediated DNA replication.

    PubMed

    Luria, Neta; Lu, Liqun; Chejanovsky, Nor

    2012-05-01

    IE0 and IE1 are transactivator proteins of the most studied baculovirus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). IE0 is a 72.6 kDa protein identical to IE1 with the exception of its 54 N-terminal amino acid residues. To gain some insight about important structural motifs of IE0, we expressed the protein and C‑terminal mutants of it under the control of the Drosophila heat shock promoter and studied the transactivation and replication functions of the transiently expressed proteins. IE0 was able to promote replication of a plasmid bearing the hr5 origin of replication of AcMNPV in transient transfections with a battery of eight plasmids expressing the AcMNPV genes dnapol, helicase, lef-1, lef-2, lef-3, p35, ie-2 and lef-7. IE0 transactivated expression of the baculovirus 39K promoter. Both functions of replication and transactivation were lost after introduction of selected mutations at the basic domain II and helix-loop-helix conserved structural motifs in the C-terminus of the protein. These IE0 mutants were unable to translocate to the cell nucleus. Our results point out the important role of some structural conserved motifs to the proper functioning of IE0.

  8. The number of positively charged amino acids in the basic domain of Tat is critical for trans-activation and complex formation with TAR RNA.

    PubMed Central

    Delling, U; Roy, S; Sumner-Smith, M; Barnett, R; Reid, L; Rosen, C A; Sonenberg, N

    1991-01-01

    The basic domain of Tat is required for trans-activation of viral gene expression. We have performed scanning peptide studies to demonstrate that only this domain is capable of binding to the TAR RNA stem-loop. Strikingly, the basic domain of the other human immunodeficiency virus trans-acting factor, Rev, but no other region, is also capable of binding to TAR. Peptide derivatives of Tat do not require the highly conserved glutamine residue at position 54 for TAR binding, since it may be substituted or deleted. In addition, the two lysine residues may be replaced by arginines. Analysis of binding and trans-activation demonstrated that homopolymers of arginine can completely substitute for the basic domain. Such homopolymers have high affinity for wild-type TAR RNA and lower affinity for mutant TAR. Homopolymers of six to nine arginines substituting for the basic domain of Tat enable full trans-activation in vivo. Homopolymers of at least seven arginines are required for detectable in vitro complex formation, although approximately 30% trans-activation is achieved with a mutant Tat containing only five arginines. Images PMID:2068104

  9. A transcriptional enhancer sequence of HTLV-I is responsible for trans-activation mediated by p40 chi HTLV-I.

    PubMed Central

    Fujisawa, J; Seiki, M; Sato, M; Yoshida, M

    1986-01-01

    Human T-cell leukemia virus type I (HTLV-I) contains a unique sequence pX that is located between env and the 3' long terminal repeat (LTR) and codes for three pX proteins, p40 chi, pp27 chi-III and pp21 chi-III. One of these proteins, p40 chi, was previously shown to activate transcription from the LTR in a trans-acting manner, which suggested that it activated some cellular genes involved in leukemogenesis. In this study, the sequences in the LTR responsible for this trans-activation were analyzed. Construction of deletion mutants of the LTR in pLTR-CAT and measurement of their activities in trans-activated expression of the CAT gene showed that sequences upstream of the TATA box were responsible for the trans-activation mediated by p40 chi. The active unit was identified as an enhancer sequence containing direct repeats by inserting it into an enhancer-minus SV40 promoter. Thus, it was concluded that an enhancer sequence in HTLV-I LTR is responsible, at least in part, for transcriptional trans-activation mediated by the viral product p40 chi. Images Fig.2. Fig.4. PMID:3011423

  10. Sonic Hedgehog modulates EGFR dependent proliferation of neural stem cells during late mouse embryogenesis through EGFR transactivation

    PubMed Central

    Reinchisi, Gisela; Parada, Margarita; Lois, Pablo; Oyanadel, Claudia; Shaughnessy, Ronan; Gonzalez, Alfonso; Palma, Verónica

    2013-01-01

    Sonic Hedgehog (Shh/GLI) and EGFR signaling pathways modulate Neural Stem Cell (NSC) proliferation. How these signals cooperate is therefore critical for understanding normal brain development and function. Here we report a novel acute effect of Shh signaling on EGFR function. We show that during late neocortex development, Shh mediates the activation of the ERK1/2 signaling pathway in Radial Glial cells (RGC) through EGFR transactivation. This process is dependent on metalloprotease activity and accounts for almost 50% of the EGFR-dependent mitogenic response of late NSCs. Furthermore, in HeLa cancer cells, a well-known model for studying the EGFR receptor function, Shh also induces cell proliferation involving EGFR activation, as reflected by EGFR internalization and ERK1/2 phosphorylation. These findings may have important implications for understanding the mechanisms that regulate NSC proliferation during neurogenesis and may lead to novel approaches to the treatment of tumors. PMID:24133411

  11. Sonic Hedgehog modulates EGFR dependent proliferation of neural stem cells during late mouse embryogenesis through EGFR transactivation.

    PubMed

    Reinchisi, Gisela; Parada, Margarita; Lois, Pablo; Oyanadel, Claudia; Shaughnessy, Ronan; Gonzalez, Alfonso; Palma, Verónica

    2013-01-01

    Sonic Hedgehog (Shh/GLI) and EGFR signaling pathways modulate Neural Stem Cell (NSC) proliferation. How these signals cooperate is therefore critical for understanding normal brain development and function. Here we report a novel acute effect of Shh signaling on EGFR function. We show that during late neocortex development, Shh mediates the activation of the ERK1/2 signaling pathway in Radial Glial cells (RGC) through EGFR transactivation. This process is dependent on metalloprotease activity and accounts for almost 50% of the EGFR-dependent mitogenic response of late NSCs. Furthermore, in HeLa cancer cells, a well-known model for studying the EGFR receptor function, Shh also induces cell proliferation involving EGFR activation, as reflected by EGFR internalization and ERK1/2 phosphorylation. These findings may have important implications for understanding the mechanisms that regulate NSC proliferation during neurogenesis and may lead to novel approaches to the treatment of tumors.

  12. Recombinant production of Epstein-Barr virus BZLF1 trans-activator and characterization of its DNA-binding specificity.

    PubMed

    Lim, Chun Shen; Goh, Siang Ling; Krishnan, Gopala; Ng, Ching Ching

    2014-03-01

    This paper describes the recombinant production of a biologically active Epstein-Barr virus BZLF1 trans-activator, i.e., Z-encoded broadly reactive activator (ZEBRA), that recognized specific DNA motifs. We used auto-induction for histidine-tagged BZLF1 expression in Escherichia coli and immobilized cobalt affinity membrane chromatography for protein purification under native conditions. We obtained the purified BZLF1 at a yield of 5.4mg per gram of wet weight cells at 75% purity, in which 27% of the recombinant BZLF1 remained biologically active. The recombinant BZLF1 bound to oligonucleotides containing ZEBRA response elements, either AP-1 or ZIIIB, but not a ZIIIB mutant. The recombinant BZLF1 showed a specific DNA-binding activity which could be useful for functional studies.

  13. Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses.

    PubMed Central

    Pruss, G; Ge, X; Shi, X M; Carrington, J C; Bowman Vance, V

    1997-01-01

    Synergistic viral diseases of higher plants are caused by the interaction of two independent viruses in the same host and are characterized by dramatic increases in symptoms and in accumulation of one of the coinfecting viruses. In potato virus X (PVX)/potyviral synergism, increased pathogenicity and accumulation of PVX are mediated by the expression of potyviral 5' proximal sequences encoding P1, the helper component proteinase (HC-Pro), and a fraction of P3. Here, we report that the same potyviral sequence (termed P1/HC-Pro) enhances the pathogenicity and accumulation of two other heterologous viruses: cucumber mosaic virus and tobacco mosaic virus. In the case of PVX-potyviral synergism, we show that the expression of the HC-Pro gene product, but not the RNA sequence itself, is sufficient to induce the increase in PVX pathogenicity and that both P1 and P3 coding sequences are dispensable for this aspect of the synergistic interaction. In protoplasts, expression of the potyviral P1/HC-Pro region prolongs the accumulation of PVX (-) strand RNA and transactivates expression of a reporter gene from a PVX subgenomic promoter. Unlike the synergistic enhancement of PVX pathogenicity, which requires only expression of HC-Pro, the enhancement of PVX (-) strand RNA accumulation in protoplasts is significantly greater when the entire P1/HC-Pro sequence is expressed. These results indicate that the potyviral P1/HC-Pro region affects a step in disease development that is common to a broad range of virus infections and suggest a mechanism involving transactivation of viral replication. PMID:9212462

  14. Effect of p40tax trans-activator of human T cell lymphotropic virus type I on expression of autoantigens.

    PubMed

    Banki, K; Ablonczy, E; Nakamura, M; Perl, A

    1994-03-01

    The possibility of a retroviral etiology has long been raised in a number of autoimmune disorders. More recently, Sjögren's syndrome and rheumatoid arthritis were noted in transgenic mice carrying the tax gene of human T cell leukemia virus type I (HTLV-I). To evaluate the involvement of HTLV-I Tax in autoimmunity, its effect on expression of autoantigens was investigated. A metallothionein promoter-driven p40tax expression plasmid, pMAXRHneo-1, was stably transfected into Molt4 and Jurkat cells and the p40tax protein was induced with CdCl2. trans-Activation or trans-repression of autoantigens by HTLV-I Tax was studied by Western blot analysis utilizing autoantigen-specific murine monoclonal and rabbit polyvalent antibodies as well as sera from 161 autoimmune patients. Induction of p40tax of HTLV-I had no significant effect on levels of expression of common autoantigens U1 snRNP, Sm, Ro, La, HSP-70, topoisomerase I/Scl70, PCNA, and HRES-1. Expression of two potentially novel autoantigens, 44 and 46 kDa, was induced by p40tax as detected by sera of progressive systemic sclerosis patients, BAK and VAR. By contrast, expression of 24- and 34-kDa proteins was suppressed in response to induction of p40tax as detected by sera of systemic lupus erythematosus patients PUS and HOR. Because none of these patients were infected by HTLV-I, a protein functionally similar to p40tax may be involved in eliciting autoantigen expression and a subsequent autoantibody response in a minority of patients with PSS and SLE. Sera of autoimmune patients may also be utilized to detect novel proteins trans-activated or trans-repressed by p40tax of HTLV-I.

  15. The viral transactivator HBx protein exhibits a high potential for regulation via phosphorylation through an evolutionarily conserved mechanism

    PubMed Central

    2012-01-01

    Background Hepatitis B virus (HBV) encodes an oncogenic factor, HBx, which is a multifunctional protein that can induce dysfunctional regulation of signaling pathways, transcription, and cell cycle progression, among other processes, through interactions with target host factors. The subcellular localization of HBx is both cytoplasmic and nuclear. This dynamic distribution of HBx could be essential to the multiple roles of the protein at different stages during HBV infection. Transactivational functions of HBx may be exerted both in the nucleus, via interaction with host DNA-binding proteins, and in the cytoplasm, via signaling pathways. Although there have been many studies describing different pathways altered by HBx, and its innumerable binding partners, the molecular mechanism that regulates its different roles has been difficult to elucidate. Methods In the current study, we took a bioinformatics approach to investigate whether the viral protein HBx might be regulated via phosphorylation by an evolutionarily conserved mechanism. Results We found that the phylogenetically conserved residues Ser25 and Ser41 (both within the negative regulatory domain), and Thr81 (in the transactivation domain) are predicted to be phosphorylated. By molecular 3D modeling of HBx, we further show these residues are all predicted to be exposed on the surface of the protein, making them easily accesible to these types of modifications. Furthermore, we have also identified Yin Yang sites that might have the potential to be phosphorylated and O-β-GlcNAc interplay at the same residues. Conclusions Thus, we propose that the different roles of HBx displayed in different subcellular locations might be regulated by an evolutionarily conserved mechanism of posttranslational modification, via phosphorylation. PMID:23079056

  16. Trans-activation function of a 3 prime truncated X gene-cell fusion product from integrated hepatitis B virus DNA in chronic hepatitis tissues

    SciTech Connect

    Takada, Shinako; Koike, Katsuro )

    1990-08-01

    To investigate the expression and transactivation function of the X gene in integrated hepatitis B virus (HBV) DNA from chronic hepatitis tissues, a series of transfectants containing cloned integrated HBV DNAs was made and analyzed for X mRNA expression and trans-activation activity by using a chloramphenicol acetyltransferase assay. Most of the integrated HBV DNAs expressed X mRNA and encoded a product with trans-activation activity in spite of the loss of the 3{prime} end region of the X gene due to integration. From cDNA cloning and sequence analysis of X mRNA transcribed from native or integrated HBV DNA, the X protein was found to be translated from the X open reading frame without splicing. For integrated HBV DNA, transcription was extended to a cellular flanking DNA and an X gene-cell fusion transcript was terminated by using a cellular poly(A) signal. The amino acid sequence deduced from an X-cell fusion transcript indicated truncation of the carboxyl-terminal five amino acids, but the upstream region of seven amino acids conserved among hepadnaviruses was retained in the integrated HBV DNA, suggesting that this conserved region is essential for the transactivation function of the X protein. These findings support the following explanation for hepatocarcinogenesis by HBV DNA integration: the expression of a cellular oncogene(s) is transactivated at the time of chronic infection by the increasing amounts of the integrated HBV gene product(s), such as the X-cell fusion product.

  17. Anti-TAR Polyamide Nucleotide Analog Conjugated with a Membrane-Permeating Peptide Inhibits Human Immunodeficiency Virus Type 1 Production

    PubMed Central

    Kaushik, Neerja; Basu, Amartya; Palumbo, Paul; Myers, Rene L.; Pandey, Virendra N.

    2002-01-01

    The emergence of drug-resistant variants has posed a significant setback against effective antiviral treatment for human immunodeficiency virus (HIV) infections. The choice of a nonmutable region of the viral genome such as the conserved transactivation response element (TAR element) in the 5′ long terminal repeat (LTR) may potentially be an effective target for drug development. We have earlier demonstrated that a polyamide nucleotide analog (PNA) targeted to the TAR hairpin element, when transfected into cells, can effectively inhibit Tat-mediated transactivation of HIV type 1 (HIV-1) LTR (T. Mayhood et al., Biochemistry 39:11532-11539, 2000). Here we show that this anti-TAR PNA (PNATAR), upon conjugation with a membrane-permeating peptide vector (transportan) retained its affinity for TAR in vitro similar to the unconjugated analog. The conjugate was efficiently internalized into the cells when added to the culture medium. Examination of the functional efficacy of the PNATAR-transportan conjugate in cell culture using luciferase reporter gene constructs resulted in a significant inhibition of Tat-mediated transactivation of HIV-1 LTR. Furthermore, PNATAR-transportan conjugate substantially inhibited HIV-1 production in chronically HIV-1-infected H9 cells. The mechanism of this inhibition appeared to be regulated at the level of transcription. These results demonstrate the efficacy of PNATAR-transportan as a potential anti-HIV agent. PMID:11907228

  18. In situ distinction between steroid receptor binding and transactivation at a target gene.

    PubMed Central

    McDonnell, D P; Nawaz, Z; O'Malley, B W

    1991-01-01

    We have developed a DNA interference assay in the yeast Saccharomyces cerevisiae that is designed to indicate the intracellular DNA-binding status of the estrogen receptor. The assay utilizes a promoter containing multiple copies of a GAL4-estrogen receptor binding sequence. This element is designed so that either an estrogen receptor or a GAL4 molecule, but not both, can occupy it simultaneously. The assay is extremely sensitive, and at concentrations of estrogen receptor below that required for maximal transcriptional activation of its target estrogen response element, a quantitative inhibition of GAL4-mediated transcription is seen. Inhibition occurs thought the disruption of complex cooperative interactions among the GAL4 molecules in this reporter. The data obtained from our experiments show that at low concentrations of receptor, hormone is required to promote DNA binding. Overexpression of receptor leads to occupation of the estrogen receptor element in the absence of ligand. In contrast, this latter receptor form will not activate transcription. Our results are consistent with a two-step process for receptor activation. Ligand first causes dissociation of receptor from an inhibitory complex within the cell and produces a DNA-binding form. Second, it converts receptor to a transcriptionally competent form. With use of this yeast model system, these two steps can be distinguished in situ. PMID:1875926

  19. Inactivation of p53 by HTLV type 1 and HTLV type 2 Tax trans-activators.

    PubMed

    Mahieux, R; Pise-Masison, C A; Nicot, C; Green, P; Hall, W W; Brady, J N

    2000-11-01

    Human T cell lymphotropic virus type II (HTLV-2) was originally isolated from a patient with a hairy T cell leukemia. It has been associated with rare cases of CD8(+) T lymphoproliferative disorders, and has a controversial role as a pathogen. The loss of p53 function, as a consequence of mutation or inactivation, increases the chances of genetic damage. Indeed, the importance of p53 as a tumor suppressor is evident from the fact that over 60% of all human cancers have a mutant or inactive p53. p53 status has been extensively studied in HTLV-1-infected cell lines. Interestingly, despite the fact that p53 mutations have been found in only a minority of cells, the p53 functions were found to be impaired. We have analyzed the functional activity of the p53 tumor suppressor in cells transformed with HTLV-2 subtypes A and B. As with HTLV-1-infected cells, abundant levels of the p53 protein are detected in HTLV-2 virus-infected cell lines. Using p53 reporter plasmid or induction of p53-responsive genes in response to gamma-irradiation, the p53 was found to be transcriptionally inhibited in HTLV-2-infected cells. Interestingly, although Tax-2A and-2B inactivate p53, the Tax-2A protein appears to inhibit p53 function less efficiently than either Tax-1 or Tax-2B in T cells, but not in fibroblasts.

  20. Isolation and Characterization of Ischemia-Derived Astrocytes (IDAs) with Ability to Transactivate Quiescent Astrocytes

    PubMed Central

    Villarreal, Alejandro; Rosciszewski, Gerardo; Murta, Veronica; Cadena, Vanesa; Usach, Vanina; Dodes-Traian, Martin M.; Setton-Avruj, Patricia; Barbeito, Luis H.; Ramos, Alberto J.

    2016-01-01

    Reactive gliosis involving activation and proliferation of astrocytes and microglia, is a widespread but largely complex and graded glial response to brain injury. Astroglial population has a previously underestimated high heterogeneity with cells differing in their morphology, gene expression profile, and response to injury. Here, we identified a subset of reactive astrocytes isolated from brain focal ischemic lesions that show several atypical characteristics. Ischemia-derived astrocytes (IDAs) were isolated from early ischemic penumbra and core. IDA did not originate from myeloid precursors, but rather from pre-existing local progenitors. Isolated IDA markedly differ from primary astrocytes, as they proliferate in vitro with high cell division rate, show increased migratory ability, have reduced replicative senescence and grow in the presence of macrophages within the limits imposed by the glial scar. Remarkably, IDA produce a conditioned medium that strongly induced activation on quiescent primary astrocytes and potentiated the neuronal death triggered by oxygen-glucose deprivation. When re-implanted into normal rat brains, eGFP-IDA migrated around the injection site and induced focal reactive gliosis. Inhibition of gamma secretases or culture on quiescent primary astrocytes monolayers facilitated IDA differentiation to astrocytes. We propose that IDA represent an undifferentiated, pro-inflammatory, highly replicative and migratory astroglial subtype emerging from the ischemic microenvironment that may contribute to the expansion of reactive gliosis. Main Points: Ischemia-derived astrocytes (IDA) were isolated from brain ischemic tissue IDA show reduced replicative senescence, increased cell division and spontaneous migration IDA potentiate death of oxygen-glucose deprived cortical neurons IDA propagate reactive gliosis on quiescent astrocytes in vitro and in vivo Inhibition of gamma secretases facilitates IDA differentiation to astrocytes PMID:27313509

  1. Binding and inhibition of the ternary complex factor Elk-4/Sap1 by the adapter protein Dok-4.

    PubMed

    Hooker, Erika; Baldwin, Cindy; Roodman, Victoria; Batra, Anupam; Takano, Tomoko; Lemay, Serge

    2017-03-08

    The adapter protein Dok-4 has been reported as both activator and inhibitor of Erk and Elk-1, but lack of knowledge about the identity of its partner molecules has precluded any mechanistic insight into these seemingly conflicting properties. We report that Dok-4 interacts with the transactivation domain of Elk-4  through an atypical PTB domain-mediated interaction. Dok-4 possesses a nuclear export signal and can relocalize Elk-4 from nucleus to cytosol, whereas Elk-4 possesses two nuclear localization signals that restrict interaction with Dok-4. Elk-4 protein, unlike Elk-1, is highly unstable in the presence of Dok-4, through both a an interaction-dependent mechanism and a PH domain-dependent but interaction-independent mechanism. This is reversed by proteasome inhibition, depletion of endogenous Dok-4 or lysine-to-arginine mutation of putative Elk-4 ubiquitination sites. Finally, Elk-4 transactivation is potently inhibited by Dok-4 overexpression but enhanced by Dok-4 knockdown in MDCK renal tubular cells, which correlates with  increased basal and EGF-induced expression of Egr-1, Fos and cylcinD1 mRNA and cell proliferation despite reduced Erk activation. Thus, Dok-4 can target Elk-4 activity through multiple mechanisms including binding of the transactivation domain, nuclear exclusion and protein destabilization, without a requirement for inhibition of Erk.

  2. Interleukin-1-receptor-associated kinase 2 (IRAK2)-mediated interleukin-1-dependent nuclear factor kappaB transactivation in Saos2 cells requires the Akt/protein kinase B kinase.

    PubMed Central

    Cenni, Vittoria; Sirri, Alessandra; De Pol, Anto; Maraldi, Nadir Mario; Marmiroli, Sandra

    2003-01-01

    The post-receptor pathway that leads to nuclear factor kappaB (NF-kappaB) activation begins with the assembly of a membrane-proximal complex among the interleukin 1 (IL-1) receptors and the adaptor molecules, myeloid differentiation protein 88 (MyD88), IL-1-receptor-associated kinases (IRAKs) and tumour-necrosis-factor-receptor-associated factor 6. Eventually, phosphorylation of the inhibitor of NF-kappaB (IkappaB) by the IkappaB kinases releases NF-kappaB, which translocates to the nucleus and modulates gene expression. In this paper, we report that IRAK2 and MyD88, but not IRAK1, interact physically with Akt, as demonstrated by co-immunoprecipitation and pull-down experiments. Interestingly, the association of Akt with recombinant IRAK2 is decreased by stimulation with IL-1, and is favoured by pre-treatment with phosphatase. Likewise, Akt association with IRAK2 is increased considerably by overexpression of PTEN (phosphatase and tensin homologue deleted on chromosome 10), while it is completely abrogated by overexpression of phosphoinositide-dependent protein kinase 1. These data indicate that Akt takes part in the formation of the signalling complex that conveys the signal from the IL-1 receptors to NF-kappaB, a step that is much more membrane-proximal than was reported previously. We also demonstrate that Akt activity is necessary for IL-1-dependent NF-kappaB transactivation, since a kinase-defective mutant of Akt impairs IRAK2- and MyD88-dependent, but not IRAK1-dependent, NF-kappaB activity, as monitored by a gene reporter assay. Accordingly, IRAK2 failed to trigger inducible nitric oxide synthase and IL-1beta production in cells expressing dominant-negative Akt. However, NF-kappaB binding to DNA was not affected by inhibition of Akt, indicating that Akt regulates NF-kappaB at a level distinct from the dissociation of p65 from IkappaBalpha and its translocation to the nucleus, possibly involving phosphorylation of the p65 transactivation domain. PMID:12906710

  3. Quercetin-induced downregulation of phospholipase D1 inhibits proliferation and invasion in U87 glioma cells

    SciTech Connect

    Park, Mi Hee; Min, Do Sik

    2011-09-09

    Highlights: {yields} Quercetin, a bioactive flavonoid, suppresses expression and enzymatic activity of phospholipase D1. {yields} Quercetin abolishes NFkB-induced phospholipase D1 expression via inhibition of NFkB transactivation. {yields} Quercetin-induced suppression of phospholipase D1 inhibits invasion and proliferation of human glioma cells. -- Abstract: Phospholipase D (PLD) has been recognized as a regulator of cell proliferation and tumorigenesis, but little is known about the molecules regulating PLD expression. Thus, the identification of small molecules inhibiting PLD expression would be an important advance in PLD-mediated physiology. Quercetin, a ubiquitous bioactive flavonoid, is known to inhibit proliferation and induce apoptosis in a variety of cancer cells. In the present study, we examined the effect of quercetin on the expression of PLD in U87 glioma cells. Quercetin significantly suppressed the expression of PLD1 at the transcriptional level. Moreover, quercetin abolished the protein expression of PLD1 in a time and dose-dependent manner, as well as inhibited PLD activity. Quercetin suppressed NF{kappa}B-induced PLD1 expression via inhibition of NFkB transactivation. Furthermore, quercetin inhibited activation and invasion of metalloproteinase-2 (MMP-2), a key modulator of glioma cell invasion, induced by phosphatidic acid (PA), a product of PLD activity. Taken together these data demonstrate that quercetin abolishes PLD1 expression and subsequently inhibits invasion and proliferation of glioma cells.

  4. Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat

    SciTech Connect

    Kenney, S.; Kamine, J.; Markovitz, D.; Fenrick, R.; Pagano, J.

    1988-03-01

    Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, the authors demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.

  5. A novel tetracycline-controlled transactivator-transrepressor system enables external control of oncolytic adenovirus replication.

    PubMed

    Fechner, H; Wang, X; Srour, M; Siemetzki, U; Seltmann, H; Sutter, A P; Scherübl, H; Zouboulis, C C; Schwaab, R; Hillen, W; Schultheiss, H-P; Poller, W

    2003-09-01

    The use of restricted replication-competent adenoviruses (RRCAs) inducing tumor cell-specific lysis is a promising approach in cancer gene therapy. However, the use of RRCAs in humans carries considerable risk, since after injection into the patient, further regulation or inhibition of virus replication from the outside is impossible. Therefore, we have developed a novel system allowing external pharmacological control of RRCA replication. We show here that a tumor-selective E1B-deleted RRCA can be tightly regulated by use of doxycycline (dox)-controlled adenoviral E1A gene expression, which in turn determines vector replication. RRCA replication is switched on by addition and switched off by withdrawal of dox. The system results in efficient tumor cell killing after induction by dox, whereas cells are unaffected by the uninduced system. It was also employed for efficient external control of transgene expression from cotransfected replication-deficient adenovectors. Furthermore, the use of a liver cell-specific human alpha1-antitrypsin (hAAT)-promoter driving a tetracycline-controlled transcriptional silencer allowed specific protection of cells with hAAT-promoter activity in the absence of dox in vitro and in vivo, delineating a new principle of 'tissue protective' gene therapy. The concept of external control of RRCAs may help to improve the safety of cancer gene therapy.

  6. c-Jun N-terminal kinase - c-Jun pathway transactivates Bim to promote osteoarthritis.

    PubMed

    Ye, Zhiqiang; Chen, Yuxian; Zhang, Rongkai; Dai, Haitao; Zeng, Chun; Zeng, Hua; Feng, Hui; Du, Gengheng; Fang, Hang; Cai, Daozhang

    2014-02-01

    Osteoarthritis (OA) is a chronic degenerative joint disorder. Previous studies have shown abnormally increased apoptosis of chondrocytes in patients and animal models of OA. TNF-α and nitric oxide have been reported to induce chondrocyte ageing; however, the mechanism of chondrocyte apoptosis induced by IL-1β has remained unclear. The aim of this study is to identify the role of the c-Jun N-terminal kinase (JNK) - c-Jun pathway in regulating induction of Bim, and its implication in chondrocyte apoptosis. This study showed that Bim is upregulated in chondrocytes obtained from the articular cartilage of OA patients and in cultured mouse chondrocytes treated with IL-1β. Upregulation of Bim was found to be critical for chondrocyte apoptosis induced by IL-1β, as revealed by the genetic knockdown of Bim, wherein apoptosis was greatly reduced in the chondrocytes. Moreover, activation of the JNK-c-Jun pathway was observed under IL-1β treatment, as indicated by the increased expression levels of c-Jun protein. Suppression of the JNK-c-Jun pathway, using chemical inhibitors and RNA interference, inhibited the Bim upregulation induced by IL-1β. These findings suggest that the JNK-c-Jun pathway is involved in the upregulation of Bim during OA and that the JNK-c-Jun-Bim pathway is vital for chondrocyte apoptosis.

  7. Toll-Like receptor-3 mediates HIV-1 transactivation via NFκB and JNK pathways and histone acetylation, but prolonged activation suppresses Tat and HIV-1 replication

    PubMed Central

    Bhargavan, Biju; Woollard, Shawna M.; Kanmogne, Georgette D.

    2016-01-01

    TLR3 has been implicated in the pathogenesis of several viral infections, including SIV- and HIV-1-induced inflammation and AIDS. However the molecular mechanisms of these TLR3-mediated effects are not known, and it is not known whether HIV interacts with cellular TLR3 to affect disease process. Here we investigate the effects of TLR3 ligands on HIV-1 transactivation using both primary human macrophages and cells containing integrated copies of the HIV-1 promoter. We demonstrate that TLR3 activation induced upregulation of transcription factors such as c-Jun, CCAAT/enhancer-binding protein alpha (CEBPA), signal transducer and activator of transcription (STAT)-1, STAT-2, RELB, and nuclear factor kappa-B1 (NFκB1), most of which are known to regulate the HIV promoter activity. We also demonstrate that TLR3 activation increased HIV-1 transactivation via the c-Jun N-terminal kinase (JNK) and NFκB pathways. This was associated with epigenetics modifications, including decreased histone deacetylase activity, increased histone acetyl transferase (HAT) activity, and increased acetylation of histones H3 and H4 at lysine residues in the nucleosome-0 and nucleosome-1 of the HIV-1 promoter. However, prolonged TLR3 activation decreased HIV-1 transactivation, decreased HAT activity and Tat transcription, and suppressed viral replication. Overall, data suggests TLR3 can acts as viral sensor to mediate viral transactivation, cellular signaling, innate immune response, and inflammation in HIV-infected humans. Our study provides novel insights into the molecular basis for these TLR3-mediated effects. PMID:26569339

  8. GPCR responses in vascular smooth muscle can occur predominantly through dual transactivation of kinase receptors and not classical Gαq protein signalling pathways.

    PubMed

    Little, Peter J

    2013-05-30

    GPCR signalling is well known to proceed through several linear pathways involving activation of G proteins and their downstream signalling pathways such as activation of phospholipase C. In addition, GPCRs signal via transactivation of Protein Tyrosine Kinase receptors such as that for Epidermal Growth Factor (EGF) and Platelet-Derived Growth Factor (PDGF) where GPCR agonists mediate increase levels of phosphorylated Erk (pErk) the immediate downstream product of the activation of EGF receptor. It has recently been shown that this paradigm can be extended to include the GPCR transactivation of a Protein Serine/Threonine Kinase receptor, specifically the Transforming Growth Factor β Type I receptor (also known as Alk V) (TβRI) in which case GPCR activation leads to the formation of carboxy terminal polyphosphorylated Smad2 (phosphoSmad2) being the immediate downstream product of the activation of TβRI. Growth factor and hormone regulation of proteoglycan synthesis in vascular smooth muscle cells represent one component of an in vitro model of atherosclerosis because modified proteoglycans show enhanced binding to lipoproteins as the initiating step in atherosclerosis. In the example of proteoglycan synthesis stimulated by GPCR agonists such as thrombin and endothelin-1, the transactivation pathways for the EGF receptor and TβRI are both active and together account for essentially all of the response to the GPCRs. In contrast, signalling downstream of GPCRs such as increased inositol 1,4,5 trisphosphate (IP3) and intracellular calcium do not have any effect on GPCR stimulated proteoglycan synthesis. These data lead to the conclusion that dual transactivation pathways for protein tyrosine and serine/threonine kinase receptors may play a far greater role in GPCR signalling than currently recognised.

  9. Epidermal Growth Factor Receptor Transactivation by the Cannabinoid Receptor (CB1) and Transient Receptor Potential Vanilloid 1 (TRPV1) Induces Differential Responses in Corneal Epithelial Cells

    DTIC Science & Technology

    2010-01-01

    inhibitors of proteolytic release of heparin bound EGF ( HB -EGF). CB1- induced Ca2þ transients were reduced during exposure to either the CB1 antagonist...blockage eliminated this response. Furthermore, EGFR transactivation was abolished by inhibitors of proteolytic release of heparin bound EGF ( HB -EGF...IL-8 or IL-6 Chemiluminescent Immunoassay ; R&D Systems, Minneapolis, MN). The cells were washed with basic medium and then exposed to CPZ, or AM251

  10. Pulling a Ligase out of a “HAT”: pCAF Mediates Ubiquitination of the Class II Transactivator

    PubMed Central

    2017-01-01

    The Class II Transactivator (CIITA) is essential to the regulation of Major Histocompatibility Class II (MHC II) genes transcription. As the “master regulator” of MHC II transcription, CIITA regulation is imperative and requires various posttranslational modifications (PTMs) in order to facilitate its role. Previously we identified various ubiquitination events on CIITA. Monoubiquitination is important for CIITA transactivity, while K63 linked ubiquitination is involved in crosstalk with ERK1/2 phosphorylation, where together they mediate cellular movement from the cytoplasm to nuclear region. Further, CIITA is also modified by degradative K48 polyubiquitination. However, the E3 ligase responsible for these modifications was unknown. We show CIITA ubiquitination and transactivity are enhanced with the histone acetyltransferase (HAT), p300/CBP associated factor (pCAF), and the E3 ligase region within pCAF is necessary for both. Additionally, pCAF mediated ubiquitination is independent of pCAF's HAT domain, and acetylation deficient CIITA is K48 polyubiquitinated and degraded in the presence of pCAF. Lastly, we identify the histone acetyltransferase, pCAF, as the E3 ligase responsible for CIITA's ubiquitination. PMID:28286521

  11. [Retracted] Nuclear import of prototype foamy virus transactivator Bel1 is mediated by KPNA1, KPNA6 and KPNA7.

    PubMed

    Duan, Jihui; Tang, Zhiqin; Mu, Hong; Zhang, Guojun

    2017-03-01

    We would like to retract the article entitled "Nuclear import of prototype foamy virus transactivator Bel1 is mediated by KPNA1, KPNA6 and KPNA7" published in International Journal of Molecular Medicine 38: 339-406, 2016. The results presented in Fig. 6 have been demonstrated to be unreproducible, and we cannot provide an explanation for this. Furthermore, we have recently identified that the cell cultures used in our experiments were partly contaminated with Mycoplasma, which could have contributed to the irreproducibility of the results. In addition, we are currently in dispute with a colleague who has contributed towards this study, but does not wish to be included as a named author on the paper. We are therefore going to retract this article. All the authors unanimously agree to the retraction of this paper, and we deeply apologize to readers and editors for any inconvenience caused by this retraction. [the original article was published in the International Journal of Molecular Medicine 38: 399-406, 2016; DOI: 10.3892/ijmm.2016.2635].

  12. Methylation of Gata3 protein at Arg-261 regulates transactivation of the Il5 gene in T helper 2 cells.

    PubMed

    Hosokawa, Hiroyuki; Kato, Miki; Tohyama, Hiroyuki; Tamaki, Yuuki; Endo, Yusuke; Kimura, Motoko Y; Tumes, Damon John; Motohashi, Shinichiro; Matsumoto, Masaki; Nakayama, Keiichi I; Tanaka, Tomoaki; Nakayama, Toshinori

    2015-05-22

    Gata3 acts as a master regulator for T helper 2 (Th2) cell differentiation by inducing chromatin remodeling of the Th2 cytokine loci, accelerating Th2 cell proliferation, and repressing Th1 cell differentiation. Gata3 also directly transactivates the interleukin-5 (Il5) gene via additional mechanisms that have not been fully elucidated. We herein identified a mechanism whereby the methylation of Gata3 at Arg-261 regulates the transcriptional activation of the Il5 gene in Th2 cells. Although the methylation-mimicking Gata3 mutant retained the ability to induce IL-4 and repress IFNγ production, the IL-5 production was selectively impaired. We also demonstrated that heat shock protein (Hsp) 60 strongly associates with the methylation-mimicking Gata3 mutant and negatively regulates elongation of the Il5 transcript by RNA polymerase II. Thus, arginine methylation appears to play a pivotal role in the organization of Gata3 complexes and the target gene specificity of Gata3.

  13. Mutations in the zinc fingers of ADR1 that change the specificity of DNA binding and transactivation.

    PubMed Central

    Thukral, S K; Morrison, M L; Young, E T

    1992-01-01

    ADR1 is a yeast transcription factor that contains two zinc fingers of the Cys-2-His-2 (C2H2) class. Mutations that change the specificity of DNA binding of ADR1 to its target site, upstream activation sequence 1 (UAS1), have been identified at three positions in the first zinc finger. Mutations Arg-115 to Gln, His-118 to Thr, and Arg-121 to Asn led to new specificities of DNA binding at adjacent positions 10, 9, and 8 (3'-GAG-5') in UAS1. Arg-115 is at the finger tip, and His-118 and Arg-121 are at positions 3 and 6, respectively, in the alpha helix of finger 1. One double mutant displayed the binding specificity expected from the properties of its constituent new-specificity mutations. Mutations in the second finger that allowed its binding site to be identified through loss-of-contact phenotypes were made. These mutations imply a tail-to-tail orientation of the two ADR1 monomers on their adjacent binding sites. Finger 1 is aligned on UAS1 in an amino-to-carboxyl-terminal orientation along the guanine-rich strand in a 3'-to-5' direction. One of the ADR1 mutants was functional in vivo with both its cognate binding site and wild-type UAS1, but the other two mutants were defective in transactivation despite their ability to bind with high affinity to their cognate binding sites. Images PMID:1588970

  14. Transactivator of transcription (TAT) peptide- chitosan functionalized multiwalled carbon nanotubes as a potential drug delivery vehicle for cancer therapy.

    PubMed

    Dong, Xia; Liu, Lanxia; Zhu, Dunwan; Zhang, Hailing; Leng, Xigang

    2015-01-01

    Carbon nanotube (CNT)-based drug delivery vehicles might find great potential in cancer therapy via the combination of chemotherapy with photothermal therapy due to the strong optical absorbance of CNTs in the near-infrared region. However, the application of CNTs in cancer therapy was considerably constrained by their lack of solubility in aqueous medium, as well as the cytotoxicity caused by their hydrophobic surface. Intracellular delivery efficiency is another factor determining the application potential of CNTs in cancer therapy. In the present study, low-molecular-weight chitosan conjugated with transactivator of transcription (TAT) peptide was used for noncovalent functionalization of multiwalled carbon nanotubes (MWCNTs), aiming at providing a more efficient drug delivery vehicle for cancer therapy. The TAT-chitosan-conjugated MWCNTs (MWCNTs-TC) were further investigated for their water solubility, cytotoxicity, cell-penetrating capability, and accumulation in tumor. It was found that MWCNTs-TC were essentially nontoxic with satisfying water solubility, and they were more efficient in terms of cancer-targeted intracellular transport both in vitro and in vivo as compared with chitosan-modified MWCNTs (MWCNTs-CS), suggesting the great application potential of MWCNTs-TC in cancer therapy.

  15. Improvement of the reverse tetracycline transactivator by single amino acid substitutions that reduce leaky target gene expression to undetectable levels.

    PubMed

    Roney, Ian J; Rudner, Adam D; Couture, Jean-François; Kærn, Mads

    2016-06-21

    Conditional gene expression systems that enable inducible and reversible transcriptional control are essential research tools and have broad applications in biomedicine and biotechnology. The reverse tetracycline transcriptional activator is a canonical system for engineered gene expression control that enables graded and gratuitous modulation of target gene transcription in eukaryotes from yeast to human cell lines and transgenic animals. However, the system has a tendency to activate transcription even in the absence of tetracycline and this leaky target gene expression impedes its use. Here, we identify single amino-acid substitutions that greatly enhance the dynamic range of the system in yeast by reducing leaky transcription to undetectable levels while retaining high expression capacity in the presence of inducer. While the mutations increase the inducer concentration required for full induction, additional sensitivity-enhancing mutations can compensate for this effect and confer a high degree of robustness to the system. The novel transactivator variants will be useful in applications where tight and tunable regulation of gene expression is paramount.

  16. Analysis of sequences involved in IE2 transactivation of a baculovirus immediate-early gene promoter and identification of a new regulatory motif.

    PubMed

    Shippam-Brett, C E; Willis, L G; Theilmann, D A

    2001-05-01

    Opep-2 is a unique baculovirus early gene that has only been identified in the Orgyia pseudotsugata multiple capsid nucleopolyhedrovirus (OpMNPV). Previous analyses have shown this gene is expressed at very early times post-infection (p.i.) but is shut down by 36-48 h p.i. The promoter of opep-2 therefore, represents a class of early genes that is temporally regulated. In this study, a detailed analysis of the opep-2 promoter is performed to analyze the role individual motifs play in early gene expression. A new 13 base pair regulatory element was identified and shown to be essential in controlling high-level expression of this gene. In addition, mutational analysis revealed that GATA and CACGTG motifs, which have been shown to bind cellular factors in Sf9 and Ld652Y cells, played minor roles in influencing opep-2 expression in the absence of other viral factors. The OpMNPV transactivator IE2 causes a significant activation of the opep-2 promoter. Cotransfection of an extensive number of promoter deletions and mutations did not show any sequence specificity for IE2 transactivation. This is the first detailed analysis of the sequence requirements for IE2 transactivation, and these results suggest that IE2 does not bind directly to specific elements in the opep-2 promoter.

  17. A major transactivator of varicella-zoster virus, the immediate-early protein IE62, contains a potent N-terminal activation domain.

    PubMed Central

    Perera, L P; Mosca, J D; Ruyechan, W T; Hayward, G S; Straus, S E; Hay, J

    1993-01-01

    Accumulating evidence indicates that the product of the putative immediate-early gene ORF62 (IE62) activates varicella-zoster virus (VZV) genes thought to represent all three kinetic classes, namely, immediate-early (alpha), early (beta), and late (gamma) classes, of VZV genes as well as a variety heterologous gene promoters. However, the mechanism(s) by which IE62 protein mediates transactivation of these diverse VZV and heterologous gene promoters remains to be elucidated. In this study, by using yeast GAL4 protein chimeras, the coding regions of VZV ORF62 possessing activation domains have been assessed. We demonstrate that the VZV IE62 protein contains a potent activation domain in the N-terminal portion of the molecule, encoded within the first 86 codons of ORF62. The predicted secondary structure profile and the acid-base composition of this IE62 domain resemble those of other transregulatory proteins whose activation is mediated through acidic, hydrophobic elements. In addition, we show that deletion of this activation domain from the 1,310-residue native IE62 protein results in ablation of the transactivator function of IE62. We also present evidence that the mutant IE62 protein lacking the activation domain, though devoid of transactivation ability, was still capable of interfering with the activation of target promoters by the native, full-length IE62. Images PMID:8392592

  18. Ligand-dependent regulation of the activity of the orphan nuclear receptor, small heterodimer partner (SHP), in the repression of bile acid biosynthetic CYP7A1 and CYP8B1 genes.

    PubMed

    Miao, Ji; Choi, Sung-E; Seok, Sun Mi; Yang, Linda; Zuercher, William J; Xu, Yong; Willson, Timothy M; Xu, H Eric; Kemper, Jongsook Kim

    2011-07-01

    Small heterodimer partner (SHP) plays important roles in diverse biological processes by directly interacting with transcription factors and inhibiting their activities. SHP has been designated an orphan nuclear receptor, but whether its activity can be modulated by ligands has been a long-standing question. Recently, retinoid-related molecules, including 4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3Cl-AHPC), were shown to bind to SHP and enhance apoptosis. We have examined whether 3Cl-AHPC acts as an agonist and increases SHP activity in the repression of bile acid biosynthetic CYP7A1 and CYP8B1 genes and delineated the underlying mechanisms. Contrary to this expectation, micromolar concentrations of 3Cl-AHPC increased CYP7A1 expression but indirectly via p38 kinase signaling. Nanomolar concentrations, however, repressed CYP7A1 expression and decreased bile acid levels in HepG2 cells, and little repression was observed when SHP was down-regulated by small hairpin RNA. Mechanistic studies revealed that 3Cl-AHPC bound to SHP, increased the interaction of SHP with liver receptor homologue (LRH)-1, a hepatic activator for CYP7A1 and CYP8B1 genes, and with repressive cofactors, Brahma, mammalian Sin3a, and histone deacetylase-1, and, subsequently, increased the occupancy of SHP and these cofactors at the promoters. Mutation of Leu-100, predicted to contact 3Cl-AHPC within the SHP ligand binding pocket by molecular modeling, severely impaired the increased interaction with LRH-1, and repression of LRH-1 activity mediated by 3Cl-AHPC. 3Cl-AHPC repressed SHP metabolic target genes in a gene-specific manner in human primary hepatocytes and HepG2 cells. These data suggest that SHP may act as a ligand-regulated receptor in metabolic pathways. Modulation of SHP activity by synthetic ligands may be a useful therapeutic strategy.

  19. Pumpkin seed extract: Cell growth inhibition of hyperplastic and cancer cells, independent of steroid hormone receptors.

    PubMed

    Medjakovic, Svjetlana; Hobiger, Stefanie; Ardjomand-Woelkart, Karin; Bucar, Franz; Jungbauer, Alois

    2016-04-01

    Pumpkin seeds have been known in folk medicine as remedy for kidney, bladder and prostate disorders since centuries. Nevertheless, pumpkin research provides insufficient data to back up traditional beliefs of ethnomedical practice. The bioactivity of a hydro-ethanolic extract of pumpkin seeds from the Styrian pumpkin, Cucurbita pepo L. subsp. pepo var. styriaca, was investigated. As pumpkin seed extracts are standardized to cucurbitin, this compound was also tested. Transactivational activity was evaluated for human androgen receptor, estrogen receptor and progesterone receptor with in vitro yeast assays. Cell viability tests with prostate cancer cells, breast cancer cells, colorectal adenocarcinoma cells and a hyperplastic cell line from benign prostate hyperplasia tissue were performed. As model for non-hyperplastic cells, effects on cell viability were tested with a human dermal fibroblast cell line (HDF-5). No transactivational activity was found for human androgen receptor, estrogen receptor and progesterone receptor, for both, extract and cucurbitin. A cell growth inhibition of ~40-50% was observed for all cell lines, with the exception of HDF-5, which showed with ~20% much lower cell growth inhibition. Given the receptor status of some cell lines, a steroid-hormone receptor independent growth inhibiting effect can be assumed. The cell growth inhibition for fast growing cells together with the cell growth inhibition of prostate-, breast- and colon cancer cells corroborates the ethnomedical use of pumpkin seeds for a treatment of benign prostate hyperplasia. Moreover, due to the lack of androgenic activity, pumpkin seed applications can be regarded as safe for the prostate.

  20. Antidepressants activate the lysophosphatidic acid receptor LPA(1) to induce insulin-like growth factor-I receptor transactivation, stimulation of ERK1/2 signaling and cell proliferation in CHO-K1 fibroblasts.

    PubMed

    Olianas, Maria C; Dedoni, Simona; Onali, Pierluigi

    2015-06-15

    Different lines of evidence indicate that the lysophosphatidic acid (LPA) receptor LPA1 is involved in neurogenesis, synaptic plasticity and anxiety-related behavior, but little is known on whether this receptor can be targeted by neuropsychopharmacological agents. The present study investigated the effects of different antidepressants on LPA1 signaling. We found that in Chinese hamster ovary (CHO)-K1 fibroblasts expressing endogenous LPA1 tricyclic and tetracyclic antidepressants and fluoxetine induced the phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) and CREB. This response was antagonized by either LPA1 blockade with Ki16425 and AM966 or knocking down LPA1 with siRNA. Antidepressants induced ERK1/2 phosphorylation in human embryonic kidney (HEK)-293 cells overexpressing LPA1, but not in wild-type cells. In PathHunter™ assay measuring receptor-β-arrestin interaction, amitriptyline, mianserin and fluoxetine failed to induce activation of LPA2 and LPA3 stably expressed in CHO-K1 cells. ERK1/2 stimulation by antidepressants and LPA was suppressed by pertussis toxin and inhibition of Src, phosphatidylinositol-3 kinase and insulin-like growth factor-I receptor (IGF-IR) activities. Antidepressants and LPA induced tyrosine phosphorylation of IGF-IR and insulin receptor-substrate-1 through LPA1 and Src. Prolonged exposure of CHO-K1 fibroblasts to either mianserin, mirtazapine or LPA enhanced cell proliferation as indicated by increased [(3)H]-thymidine incorporation and Ki-67 immunofluorescence. This effect was inhibited by blockade of LPA1- and ERK1/2 activity. These data provide evidence that different antidepressants induce LPA1 activation, leading to receptor tyrosine kinase transactivation, stimulation of ERK1/2 signaling and enhanced cell proliferation.

  1. Sex steroids inhibit osmotic swelling of retinal glial cells.

    PubMed

    Neumann, Florian; Wurm, Antje; Linnertz, Regina; Pannicke, Thomas; Iandiev, Ianors; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas

    2010-04-01

    Osmotic swelling of glial cells may contribute to the development of retinal edema. We investigated whether sex steroids inhibit the swelling of glial somata in acutely isolated retinal slices and glial cells of the rat. Superfusion of retinal slices or cells from control animals with a hypoosmolar solution did not induce glial swelling, whereas glial swelling was observed in slices of postischemic and diabetic retinas. Progesterone, testosterone, estriol, and 17beta-estradiol prevented glial swelling with half-maximal effects at approximately 0.3, 0.6, 6, and 20 microM, respectively. The effect of progesterone was apparently mediated by transactivation of metabotropic glutamate receptors, P2Y1, and adenosine A1 receptors. The data suggest that sex steroids may inhibit cytotoxic edema in the retina.

  2. Cynaropicrin from Cynara scolymus L. suppresses photoaging of skin by inhibiting the transcription activity of nuclear factor-kappa B.

    PubMed

    Tanaka, Yuka Tsuda; Tanaka, Kiyotaka; Kojima, Hiroyuki; Hamada, Tomoji; Masutani, Teruaki; Tsuboi, Makoto; Akao, Yukihiro

    2013-01-15

    Aging of skin is characterized by skin wrinkling, laxity, and pigmentation induced by several environmental stress factors. Histological changes during the photoaging of skin include hyperproliferation of keratinocytes and melanocytes causing skin wrinkles and pigmentation. Nuclear factor kappa B (NF-κB) is one of the representative transcription factors active in conjunction with inflammation. NF-κB is activated by stimulation such as ultraviolet rays and inflammatory cytokines and induces the expression of various genes such as those of basic fibroblast growth factor (bFGF) and matrix metalloprotease-1 (MMP-1). We screened several plant extracts for their possible inhibitory effect on the transcriptional activity of NF-κB. One of them, an extract from Cynara scolymus L., showed a greatest effect on the suppression of NF-κB transactivation. As a result, we found that cynaropicrin, which is a sesquiterpene lactone, inhibited the NF-κB-mediated transactivation of bFGF and MMP-1. Furthermore, it was confirmed that in an in vivo mouse model cynaropicrin prevented skin photoaging processes leading to the hyperproliferation of keratinocytes and melanocytes. These findings taken together indicate that cynaropicrin is an effective antiphotoaging agent that acts by inhibiting NF-κB-mediated transactivation.

  3. Nuclear import of prototype foamy virus transactivator Bel1 is mediated by KPNA1, KPNA6 and KPNA7.

    PubMed

    Duan, Jihui; Tang, Zhiqin; Mu, Hong; Zhang, Guojun

    2016-08-01

    Bel1, a transactivator of the prototype foamy virus (PFV), plays pivotal roles in the replication of PFV. Previous studies have demonstrated that Bel1 bears a nuclear localization signal (NLS); however, its amino acid sequence remains unclear and the corresponding adaptor importins have not yet been identified. In this study, we inserted various fragments of Bel1 into an EGFP-GST fusion protein and investigated their subcellular localization by fluorescence microscopy. We found that the 215PRQKRPR221 fragment, which accords with the consensus sequence K(K/R)X(K/R) of the monopartite NLS, directed the nuclear translocation of Bel1. Point mutation experiments revealed that K218, R219 and R221 were essential for the nuclear localization of Bel1. The results of GST pull-down assay revealed that the Bel1 peptide 215-221, which bears the NLS, interacted with the nucleocytoplasmic transport receptors, karyopherin alpha 1 (importin alpha 5) (KPNA1), karyopherin alpha 6 (importin alpha 7) (KPNA6) and karyopherin alpha 7 (importin alpha 8) (KPNA7). Finally, in vitro nuclear import assays demonstrated that KPNA1, KPNA6 or KPNA7, along with other necessary nuclear factors, caused Bel1 to localize to the nucleus. Thus, the findings of our study indicate that KPNA1, KPNA6 and KPNA7 are involved in Bel1 nuclear distribution.

  4. Modulation of the disordered conformational ensembles of the p53 transactivation domain by cancer-associated mutations.

    PubMed

    Ganguly, Debabani; Chen, Jianhan

    2015-04-01

    Intrinsically disordered proteins (IDPs) are frequently associated with human diseases such as cancers, and about one-fourth of disease-associated missense mutations have been mapped into predicted disordered regions. Understanding how these mutations affect the structure-function relationship of IDPs is a formidable task that requires detailed characterization of the disordered conformational ensembles. Implicit solvent coupled with enhanced sampling has been proposed to provide a balance between accuracy and efficiency necessary for systematic and comparative assessments of the effects of mutations as well as post-translational modifications on IDP structure and interaction. Here, we utilize a recently developed replica exchange with guided annealing enhanced sampling technique to calculate well-converged atomistic conformational ensembles of the intrinsically disordered transactivation domain (TAD) of tumor suppressor p53 and several cancer-associated mutants in implicit solvent. The simulations are critically assessed by quantitative comparisons with several types of experimental data that provide structural information on both secondary and tertiary levels. The results show that the calculated ensembles reproduce local structural features of wild-type p53-TAD and the effects of K24N mutation quantitatively. On the tertiary level, the simulated ensembles are overly compact, even though they appear to recapitulate the overall features of transient long-range contacts qualitatively. A key finding is that, while p53-TAD and its cancer mutants sample a similar set of conformational states, cancer mutants could introduce both local and long-range structural modulations to potentially perturb the balance of p53 binding to various regulatory proteins and further alter how this balance is regulated by multisite phosphorylation of p53-TAD. The current study clearly demonstrates the promise of atomistic simulations for detailed characterization of IDP conformations, and

  5. Transcriptional transactivation functions localized to the glucocorticoid receptor N terminus are necessary for steroid induction of lymphocyte apoptosis.

    PubMed Central

    Dieken, E S; Miesfeld, R L

    1992-01-01

    Genetic studies have suggested that transcriptional regulation of specific target genes (by either induction or repression) is the molecular basis of glucocorticoid-mediated lymphocyte apoptosis. To examine the role of transcriptional regulation more directly, we developed a complementation assay utilizing stable transfection of wild-type (wt) and mutant (nti) glucocorticoid receptor (GR) cDNA constructs into a GR-deficient S49 murine cell line (7r). Our data confirm that the level of functional GR is rate limiting for S49 apoptosis and moreover that the GR amino terminus (N terminus), which as been deleted from the nti GR, is absolutely required for complementation in this system. Surprisingly, we found that at physiological levels of receptor, expression of the nti GR in cells containing wt GR results in enhanced dexamethasone sensitivity rather than a dominant negative phenotype. One interpretation of these data is that DNA binding by wt-nti heterodimers may be functionally similar to that of wt-wt homodimers, indicating that GRE occupancy by at least one transactivation domain may be sufficient to induce the hormonal response. To determine whether acidic activating sequences such as those localized to the GR N terminus are important in the induction of lymphocyte apoptosis, we tested the activity of a chimeric receptor in which we replaced the entire GR N terminus with sequences from the herpes simplex virus VP16 protein. Our results demonstrate that 7r cells expressing VP-GR fusions are indeed steroid sensitive, strongly supporting the idea that S49 apoptosis is dependent on transcriptional regulation of specific genes which respond to acidic activating domains, implying that induction, rather than repression, may be the critical initiating event. Images PMID:1310148

  6. The ubiquitin ligase Cullin5SOCS2 regulates NDR1/STK38 stability and NF-κB transactivation

    PubMed Central

    Paul, Indranil; Batth, Tanveer S.; Iglesias-Gato, Diego; Al-Araimi, Amna; Al-Haddabi, Ibrahim; Alkharusi, Amira; Norstedt, Gunnar; Olsen, Jesper V.; Zadjali, Fahad; Flores-Morales, Amilcar

    2017-01-01

    SOCS2 is a pleiotropic E3 ligase. Its deficiency is associated with gigantism and organismal lethality upon inflammatory challenge. However, mechanistic understanding of SOCS2 function is dismal due to our unawareness of its protein substrates. We performed a mass spectrometry based proteomic profiling upon SOCS2 depletion and yield quantitative data for ~4200 proteins. Through this screen we identify a novel target of SOCS2, the serine-threonine kinase NDR1. Over-expression of SOCS2 accelerates turnover, while its knockdown stabilizes, endogenous NDR1 protein. SOCS2 interacts with NDR1 and promotes its degradation through K48-linked ubiquitination. Functionally, over-expression of SOCS2 antagonizes NDR1-induced TNFα-stimulated NF-κB activity. Conversely, depletion of NDR1 rescues the effect of SOCS2-deficiency on TNFα-induced NF-κB transactivation. Using a SOCS2−/− mice model of colitis we show that SOCS2-deficiency is pro-inflammatory and negatively correlates with NDR1 and nuclear p65 levels. Lastly, we provide evidence to suggest that NDR1 acts as an oncogene in prostate cancer. To the best of our knowledge, this is the first report of an identified E3 ligase for NDR1. These results might explain how SOCS2-deficiency leads to hyper-activation of NF-κB and downstream pathological implications and posits that SOCS2 induced degradation of NDR1 may act as a switch in restricting TNFα-NF-κB pathway. PMID:28216640

  7. A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation

    PubMed Central

    Chen, Yupeng; Zhang, Lirong; Estarás, Conchi; Choi, Seung H.; Moreno, Luis; Karn, Jonathan; Moresco, James J.; Yates, John R.

    2014-01-01

    HIV-1 Tat stimulates transcription elongation by recruiting the P-TEFb (positive transcription elongation factor-b) (CycT1:CDK9) C-terminal domain (CTD) kinase to the HIV-1 promoter. Here we show that Tat transactivation also requires the Ssu72 CTD Ser5P (S5P)-specific phosphatase, which mediates transcription termination and intragenic looping at eukaryotic genes. Importantly, HIV-1 Tat interacts directly with Ssu72 and strongly stimulates its CTD phosphatase activity. We found that Ssu72 is essential for Tat:P-TEFb-mediated phosphorylation of the S5P-CTD in vitro. Interestingly, Ssu72 also stimulates nascent HIV-1 transcription in a phosphatase-dependent manner in vivo. Chromatin immunoprecipitation (ChIP) experiments reveal that Ssu72, like P-TEFb and AFF4, is recruited by Tat to the integrated HIV-1 proviral promoter in TNF-α signaling 2D10 T cells and leaves the elongation complex prior to the termination site. ChIP-seq (ChIP combined with deep sequencing) and GRO-seq (genome-wide nuclear run-on [GRO] combined with deep sequencing) analysis further reveals that Ssu72 predominantly colocalizes with S5P–RNAPII (RNA polymerase II) at promoters in human embryonic stem cells, with a minor peak in the terminator region. A few genes, like NANOG, also have high Ssu72 at the terminator. Ssu72 is not required for transcription at most cellular genes but has a modest effect on cotranscriptional termination. We conclude that Tat alters the cellular function of Ssu72 to stimulate viral gene expression and facilitate the early S5P–S2P transition at the integrated HIV-1 promoter. PMID:25319827

  8. Temperature inducible β-sheet structure in the transactivation domains of retroviral regulatory proteins of the Rev family

    NASA Astrophysics Data System (ADS)

    Thumb, Werner; Graf, Christine; Parslow, Tristram; Schneider, Rainer; Auer, Manfred

    1999-11-01

    The interaction of the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev with cellular cofactors is crucial for the viral life cycle. The HIV-1 Rev transactivation domain is functionally interchangeable with analog regions of Rev proteins of other retroviruses suggesting common folding patterns. In order to obtain experimental evidence for similar structural features mediating protein-protein contacts we investigated activation domain peptides from HIV-1, HIV-2, VISNA virus, feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) by CD spectroscopy, secondary structure prediction and sequence analysis. Although different in polarity and hydrophobicity, all peptides showed a similar behavior with respect to solution conformation, concentration dependence and variations in ionic strength and pH. Temperature studies revealed an unusual induction of β-structure with rising temperatures in all activation domain peptides. The high stability of β-structure in this region was demonstrated in three different peptides of the activation domain of HIV-1 Rev in solutions containing 40% hexafluoropropanol, a reagent usually known to induce α-helix into amino acid sequences. Sequence alignments revealed similarities between the polar effector domains from FIV and EIAV and the leucine rich (hydrophobic) effector domains found in HIV-1, HIV-2 and VISNA. Studies on activation domain peptides of two dominant negative HIV-1 Rev mutants, M10 and M32, pointed towards different reasons for the biological behavior. Whereas the peptide containing the M10 mutation (L 78E 79→D 78L 79) showed wild-type structure, the M32 mutant peptide (L 78L 81L 83→A 78A 81A 83) revealed a different protein fold to be the reason for the disturbed binding to cellular cofactors. From our data, we conclude, that the activation domain of Rev proteins from different viral origins adopt a similar fold and that a β-structural element is involved in binding to a

  9. Modulation of the Disordered Conformational Ensembles of the p53 Transactivation Domain by Cancer-Associated Mutations

    PubMed Central

    Ganguly, Debabani; Chen, Jianhan

    2015-01-01

    Intrinsically disordered proteins (IDPs) are frequently associated with human diseases such as cancers, and about one-fourth of disease-associated missense mutations have been mapped into predicted disordered regions. Understanding how these mutations affect the structure-function relationship of IDPs is a formidable task that requires detailed characterization of the disordered conformational ensembles. Implicit solvent coupled with enhanced sampling has been proposed to provide a balance between accuracy and efficiency necessary for systematic and comparative assessments of the effects of mutations as well as post-translational modifications on IDP structure and interaction. Here, we utilize a recently developed replica exchange with guided annealing enhanced sampling technique to calculate well-converged atomistic conformational ensembles of the intrinsically disordered transactivation domain (TAD) of tumor suppressor p53 and several cancer-associated mutants in implicit solvent. The simulations are critically assessed by quantitative comparisons with several types of experimental data that provide structural information on both secondary and tertiary levels. The results show that the calculated ensembles reproduce local structural features of wild-type p53-TAD and the effects of K24N mutation quantitatively. On the tertiary level, the simulated ensembles are overly compact, even though they appear to recapitulate the overall features of transient long-range contacts qualitatively. A key finding is that, while p53-TAD and its cancer mutants sample a similar set of conformational states, cancer mutants could introduce both local and long-range structural modulations to potentially perturb the balance of p53 binding to various regulatory proteins and further alter how this balance is regulated by multisite phosphorylation of p53-TAD. The current study clearly demonstrates the promise of atomistic simulations for detailed characterization of IDP conformations, and

  10. In vitro screening of inhibition of PPAR-γ activity as a first step in identification of potential breast carcinogens.

    PubMed

    Kopp, T I; Lundqvist, J; Petersen, R K; Oskarsson, A; Kristiansen, K; Nellemann, C; Vogel, U

    2015-11-01

    Alcohol consumption and increased estrogen levels are major risk factors for breast cancer, and peroxisome proliferator-activated receptor γ (PPAR-γ) plays an important role in alcohol-induced breast cancer. PPAR-γ activity is inhibited by ethanol, leading to increased aromatase activity and estrogen biosynthesis ultimately leading to breast cancer. If other organic solvents inhibit PPAR-γ activity, they should also lead to increased oestrogen biosynthesis and thus be potential breast carcinogens. Ten commonly used hydrophilic organic solvents were first tested in a cell-based screening assay for inhibitory effects on PPAR-γ transactivation. The chemicals shown to inhibit PPAR-γ were tested with vectors encoding PPAR-γ with deleted AB domains and only the ligand-binding domain to rule out unspecific toxicity. Next, the effects on biosynthesis of estradiol, testosterone and oestrone sulphate were measured in the H295R steroidogenesis assay after incubation with the chemicals. Ethylene glycol, ethyl acetate, and dimethyl sulphoxide inhibited PPAR-γ transactivation in a dose-dependent manner. The inhibitory effect on PPAR-γ was specific for PPAR-γ since the AB domain of PPAR-γ was required for the inhibitory effect. In the second step, ethylene glycol significantly increased production of oestradiol by 19% (p < 0.05) and ethyl acetate inhibited production of testosterone (p < 0.05). We here show that screening of 10 commonly used organic solvents for the ability to inhibit PPAR-γ transactivation followed by a well-established steroidogenesis assay for production of sex hormones in exposed H295 R cells may provide a screening tool for potential breast carcinogens. This initial screening thus identified ethylene glycol and possibly ethyl acetate as potential breast carcinogens.

  11. Transcriptional repressor NIR interacts with the p53-inhibiting ubiquitin ligase MDM2.

    PubMed

    Heyne, Kristina; Förster, Juliane; Schüle, Roland; Roemer, Klaus

    2014-04-01

    NIR (novel INHAT repressor) can bind to p53 at promoters and inhibit p53-mediated gene transactivation by blocking histone acetylation carried out by p300/CBP. Like NIR, the E3 ubiquitin ligase MDM2 can also bind and inhibit p53 at promoters. Here, we present data indicating that NIR, which shuttles between the nucleolus and nucleoplasm, not only binds to p53 but also directly to MDM2, in part via the central acidic and zinc finger domain of MDM2 that is also contacted by several other nucleolus-based MDM2/p53-regulating proteins. Like some of these, NIR was able to inhibit the ubiquitination of MDM2 and stabilize MDM2; however, unlike these nucleolus-based MDM2 regulators, NIR did not inhibit MDM2 to activate p53. Rather, NIR cooperated with MDM2 to repress p53-induced transactivation. This cooperative repression may at least in part involve p300/CBP. We show that NIR can block the acetylation of p53 and MDM2. Non-acetylated p53 has been documented previously to more readily associate with inhibitory MDM2. NIR may thus help to sustain the inhibitory p53:MDM2 complex, and we present evidence suggesting that all three proteins can indeed form a ternary complex. In sum, our findings suggest that NIR can support MDM2 to suppress p53 as a transcriptional activator.

  12. Transactivation of cellular genes involved in nucleotide metabolism by the regulatory IE1 protein of murine cytomegalovirus is not critical for viral replicative fitness in quiescent cells and host tissues.

    PubMed

    Wilhelmi, Vanessa; Simon, Christian O; Podlech, Jürgen; Böhm, Verena; Däubner, Torsten; Emde, Simone; Strand, Dennis; Renzaho, Angélique; Lemmermann, Niels A W; Seckert, Christof K; Reddehase, Matthias J; Grzimek, Natascha K A

    2008-10-01

    Despite its high coding capacity, murine CMV (mCMV) does not encode functional enzymes for nucleotide biosynthesis. It thus depends on cellular enzymes, such as ribonucleotide reductase (RNR) and thymidylate synthase (TS), to be supplied with deoxynucleoside triphosphates (dNTPs) for its DNA replication. Viral transactivation of these cellular genes in quiescent cells of host tissues is therefore a parameter of viral fitness relevant to pathogenicity. Previous work has shown that the IE1, but not the IE3, protein of mCMV transactivates RNR and TS gene promoters and has revealed an in vivo attenuation of the mutant virus mCMV-DeltaIE1. It was attractive to propose the hypothesis that lack of transactivation by IE1 and a resulting deficiency in the supply of dNTPs are the reasons for growth attenuation. Here, we have tested this hypothesis with the mutant virus mCMV-IE1-Y165C expressing an IE1 protein that selectively fails to transactivate RNR and TS in quiescent cells upon transfection while maintaining the capacity to disperse repressive nuclear domains (ND10). Our results confirm in vivo attenuation of mCMV-DeltaIE1, as indicated by a longer doubling time in host organs, whereas mCMV-IE1-Y165C replicated like mCMV-WT and the revertant virus mCMV-IE1-C165Y. Notably, the mutant virus transactivated RNR and TS upon infection of quiescent cells, thus indicating that IE1 is not the only viral transactivator involved. We conclude that transactivation of cellular genes of dNTP biosynthesis is ensured by redundancy and that attenuation of mCMV-DeltaIE1 results from the loss of other critical functions of IE1, with its function in the dispersal of ND10 being a promising candidate.

  13. Differential transactivation by orphan nuclear receptor NOR1 and its fusion gene product EWS/NOR1: possible involvement of poly(ADP-ribose) polymerase I, PARP-1.

    PubMed

    Ohkura, Naganari; Nagamura, Yuko; Tsukada, Toshihiko

    2008-10-15

    In extraskeletal myxoid chondrosarcoma, a chromosomal translocation creates a gene fusion between EWS and an orphan nuclear receptor, NOR1. The resulting fusion protein EWS/NOR1 has been believed to lead to malignant transformation by functioning as a transactivator for NOR1-target genes. By comparing the gene expression profiles of NOR1- and EWS/NOR1-overexpressing cells, we found that they largely shared up-regulated genes, but no significant correlation was observed with respect to the transactivation levels of each gene. In addition, the proteins associated with NOR1 and EWS/NOR1 were mostly the same in these cells. The results suggest that these proteins differentially transactivate overlapping target genes through a similar transcriptional machinery. To clarify the mechanisms underlying the transcriptional divergence between NOR1 and EWS/NOR1, we searched for alternatively associated proteins, and identified poly(ADP-ribose) polymerase I (PARP-1) as an NOR1-specific binding protein. Consistent with its binding properties, PARP-1 acted as a transcriptional repressor of NOR1, but not EWS/NOR1, in a luciferase reporter assay employing PARP-1(-/-) fibroblasts. Interestingly, suppressive activity of PARP-1 was observed in a DNA response element-specific manner, and in a subtype-specific manner toward the NR4A family (Nur77, Nurr1, and NOR1), suggesting that PARP-1 plays a role in the diversity of transcriptional regulation mediated by the NR4A family in normal cells. Altogether, our findings suggest that NOR1 and EWS/NOR1 regulate overlapping target genes differently by utilizing associated proteins, including PARP-1; and that EWS/NOR1 may acquire oncogenic activities by avoiding (or gaining) transcription factor-specific modulation by the associated proteins.

  14. NCOA3 is a selective co-activator of estrogen receptor α-mediated transactivation of PLAC1 in MCF-7 breast cancer cells

    PubMed Central

    2013-01-01

    Background The placenta-specific 1 (PLAC1) gene encodes a membrane-associated protein which is selectively expressed in the placental syncytiotrophoblast and in murine fetal tissues during embryonic development. In contrast to its transcriptional repression in all other adult normal tissues, PLAC1 is frequently activated and highly expressed in a variety of human cancers, in particular breast cancer, where it associates with estrogen receptor α (ERα) positivity. In a previous study, we showed that ERα-signaling in breast cancer cells transactivates PLAC1 expression in a non-classical pathway. As the members of the p160/nuclear receptor co-activator (NCOA) family, NCOA1, NCOA2 and NCOA3 are known to be overexpressed in breast cancer and essentially involved in estrogen-mediated cancer cell proliferation we asked if these proteins are involved in the ERα-mediated transactivation of PLAC1 in breast cancer cells. Methods Applying quantitative real-time RT-PCR (qRT-PCR), Western Blot analysis and chromatin immunoprecipitation, we analyzed the involvement of NCOA1, NCOA2, NCOA3 in the ERα-mediated transactivation of PLAC1 in the breast cancer cell lines MCF-7 and SK-BR-3. RNAi-mediated silencing of NCOA3, qRT-PCR, Western blot analysis and ERα activation assays were used to examine the role of NCOA3 in the ERα-mediated regulation of PLAC1 in further detail. Transcript expression of NCOA3 and PLAC1 in 48 human breast cancer samples was examined by qRT-PCR and statistical analysis was performed using Student’s t-test. Results We detected selective recruitment of NCOA3 but not NCOA1 or NCOA2 to the PLAC1 promoter only in ERα-positive MCF-7 cells but not in ERα-negative SK-BR-3 breast cancer cells. In addition, we demonstrate that silencing of NCOA3 results in a remarkable decrease of PLAC1 expression levels in MCF-7 cells which cannot be restored by treatment with estradiol (E2). Moreover, significant higher transcript levels of PLAC1 were found only in ER

  15. A naturally occurring mutation in the human androgen receptor of a subject with complete androgen insensitivity confers binding and transactivation by estradiol.

    PubMed

    Bonagura, Thomas W; Deng, Min; Brown, Terry R

    2007-01-15

    The clinical phenotype of complete androgen insensitivity (CAIS) was associated with a mutation in the human androgen receptor (hAR) gene encoding the amino acid substitution, M745I, in the hAR protein. Transcriptional activation of hAR(M745I) by the synthetic androgen, methyltrienolone (R1881), was reduced compared to wild-type (wt) hAR. The transcriptional co-activator, androgen receptor associated protein 70 (ARA70), failed to enhance transactivation of hAR(M745I) at lower concentrations of R1881 (0.01-0.1 nM), whereas the p160 co-activators, SRC-1 and TIF2, stimulated activity. Transcriptional activity of hAR(M745I) was stimulated by 1 or 10 nM R1881 and activity was further enhanced by co-expression of ARA70 similar to that of the hAR(wt). Transcriptional activity of hAR(wt) was minimally stimulated by estradiol (E2) without or with co-expression of ARA70, whereas 10 or 100 nM E2 increased transactivation by hAR(M745I) of the androgen-responsive MMTV-luciferase reporter gene by 10-fold and activity was further enhanced by ARA70. Increasing concentrations of E2 competed more effectively for binding of R1881 to hAR(M745I) than to hAR(wt), indicative of the preferential binding of E2 to the mutant hAR. Partial tryptic digestion of hAR wt and M745I revealed that activation of the mutant protein was reduced in the presence of R1881. By contrast, tryptic digestion showed that the mutant hAR was activated by the binding of E2. In conclusion, the clinical phenotype of CAIS resulted from a hAR gene mutation encoding hAR(M745I) with reduced binding and transactivation by androgens, but the novel properties of enhanced affinity for and increased transactivation by estradiol.

  16. Transactivation of the P2 promoter of parathyroid hormone-related protein by human T-cell lymphotropic virus type I Tax1: evidence for the involvement of transcription factor Ets1.

    PubMed Central

    Dittmer, J; Gitlin, S D; Reid, R L; Brady, J N

    1993-01-01

    Expression of the parathyroid hormone-related protein (PTHrP), a protein that plays a primary role in the development of the humoral hypercalcemia of malignancy, is regulated by two distinct promoters, P1 and P2. PTHrP is overexpressed in lymphocytes from adult T-cell leukemia patients. We now demonstrate that in the human T-cell lymphotropic virus type I-transformed cell line MT-2, RNA synthesis is initiated primarily at the P2 promoter. Furthermore, in cotransfection experiments, Tax1 transactivates the P2 promoter 10- to 12-fold. By using deletion and site-specific point mutations, we have identified a promoter-proximal sequence (positions -72 to -40) which is important for Tax1 transactivation. The PTHrP promoter-proximal element contains two potential overlapping Ets1 binding sites, EBS I and EBS II. Gel shift analysis demonstrated that Ets1 binds specifically to both EBS I and EBS II. Mutation of the consensus GGAA core motif in EBS I abolished binding and Tax1 transactivation in Jurkat T lymphocytes. In Ets1-deficient cells, cotransfection of Tax1 and Ets1 expression plasmids stimulates PTHrP promoter activity. In the absence of Ets1, minimal transactivation of the PTHrP promoter is observed. These data suggest that Ets1 binds to EBS I and cooperates with Tax1 to transactivate the PTHrP P2 promoter. Images PMID:8371355

  17. Retinoic acid receptors inhibit AP1 activation by regulating extracellular signal-regulated kinase and CBP recruitment to an AP1-responsive promoter.

    PubMed

    Benkoussa, Madjid; Brand, Céline; Delmotte, Marie-Hélène; Formstecher, Pierre; Lefebvre, Philippe

    2002-07-01

    Retinoids exhibit antineoplastic activities that may be linked to retinoid receptor-mediated transrepression of activating protein 1 (AP1), a heterodimeric transcription factor composed of fos- and jun-related proteins. Here we show that transcriptional activation of an AP1-regulated gene through the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway (MAPK(ERK)) is characterized, in intact cells, by a switch from a fra2-junD dimer to a junD-fosB dimer loading on its promoter and by simultaneous recruitment of ERKs, CREB-binding protein (CBP), and RNA polymerase II. All-trans-retinoic acid (atRA) receptor (RAR) was tethered constitutively to the AP1 promoter. AP1 transrepression by retinoic acid was concomitant to glycogen synthase kinase 3 activation, negative regulation of junD hyperphosphorylation, and to decreased RNA polymerase II recruitment. Under these conditions, fra1 loading to the AP1 response element was strongly increased. Importantly, CBP and ERKs were excluded from the promoter in the presence of atRA. AP1 transrepression by retinoids was RAR and ligand dependent, but none of the functions required for RAR-mediated transactivation was necessary for AP1 transrepression. These results indicate that transrepressive effects of retinoids are mediated through a mechanism unrelated to transcriptional activation, involving the RAR-dependent control of transcription factors and cofactor assembly on AP1-regulated promoters.

  18. Retinoic Acid Receptors Inhibit AP1 Activation by Regulating Extracellular Signal-Regulated Kinase and CBP Recruitment to an AP1-Responsive Promoter

    PubMed Central

    Benkoussa, Madjid; Brand, Céline; Delmotte, Marie-Hélène; Formstecher, Pierre; Lefebvre, Philippe

    2002-01-01

    Retinoids exhibit antineoplastic activities that may be linked to retinoid receptor-mediated transrepression of activating protein 1 (AP1), a heterodimeric transcription factor composed of fos- and jun-related proteins. Here we show that transcriptional activation of an AP1-regulated gene through the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway (MAPKERK) is characterized, in intact cells, by a switch from a fra2-junD dimer to a junD-fosB dimer loading on its promoter and by simultaneous recruitment of ERKs, CREB-binding protein (CBP), and RNA polymerase II. All-trans-retinoic acid (atRA) receptor (RAR) was tethered constitutively to the AP1 promoter. AP1 transrepression by retinoic acid was concomitant to glycogen synthase kinase 3 activation, negative regulation of junD hyperphosphorylation, and to decreased RNA polymerase II recruitment. Under these conditions, fra1 loading to the AP1 response element was strongly increased. Importantly, CBP and ERKs were excluded from the promoter in the presence of atRA. AP1 transrepression by retinoids was RAR and ligand dependent, but none of the functions required for RAR-mediated transactivation was necessary for AP1 transrepression. These results indicate that transrepressive effects of retinoids are mediated through a mechanism unrelated to transcriptional activation, involving the RAR-dependent control of transcription factors and cofactor assembly on AP1-regulated promoters. PMID:12052862

  19. Inhibition of the Epstein-Barr virus lytic cycle by moronic acid.

    PubMed

    Chang, Fang-Rong; Hsieh, Yi-Chung; Chang, Yung-Fu; Lee, Kuo-Hsiung; Wu, Yang-Chang; Chang, Li-Kwan

    2010-03-01

    Epstein-Barr virus (EBV) expresses two transcription factors, Rta and Zta, during the immediate-early stage of the lytic cycle to activate the transcription of viral lytic genes. Our immunoblotting and flow cytometry analyses find that moronic acid, found in galls of Rhus chinensis and Brazilian propolis, at 10microM inhibits the expression of Rta, Zta, and an EBV early protein, EA-D, after lytic induction with sodium butyrate. This study also finds that moronic acids inhibits the capacity of Rta to activate a promoter that contains an Rta-response element, indicating that moronic acid interferes with the function of Rta. On the other hand, moronic acid does not appear to influence with the transactivation function of Zta. Therefore, the lack of expression of Zta and EA-D after moronic acid treatment is attributable to the inhibition of the transactivation functions of Rta. Because the expression of Zta, EA-D and many EBV lytic genes depends on Rta, the treatment of P3HR1 cells with moronic acid substantially reduces the numbers of EBV particles produced by the cells after lytic induction. This study suggests that moronic acid is a new structural lead for anti-EBV drug development.

  20. Defective MHC class II expression in an MHC class II deficiency patient is caused by a novel deletion of a splice donor site in the MHC class II transactivator gene.

    PubMed

    Peijnenburg, A; Van den Berg, R; Van Eggermond, M J; Sanal, O; Vossen, J M; Lennon, A M; Alcaïde-Loridan, C; Van den Elsen, P J

    2000-01-01

    MHC class II deficiency patients are mutated for transcription factors that regulate the expression of major histocompatibility complex (MHC) class II genes. Four complementation groups (A-D) are defined and the gene defective in group A has been shown to encode the MHC class II transactivator (CIITA). Here, we report the molecular characterization of a new MHC class II deficiency patient, ATU. Cell fusion experiments indicated that ATU belongs to complementation group A. Subsequent mutation analysis revealed that the CIITA mRNA lacked 84 nucleotides. This deletion was the result of the absence of a splice donor site in the CIITA gene of ATU. As a result of this novel homozygous genomic deletion, ATU CIITA failed to transactivate MHC class II genes. Furthermore, this truncated CIITA of ATU did not display a dominant negative effect on CIITA-mediated transactivation of various isotypic MHC class II promoters.

  1. Induction of apoptosis by the transactivating domains of the hepatitis B virus X gene leads to suppression of oncogenic transformation of primary rat embryo fibroblasts.

    PubMed

    Schuster, R; Gerlich, W H; Schaefer, S

    2000-02-24

    Epidemiology shows a clear correlation between chronic infection with the hepatitis B virus (HBV) and development of hepatocellular carcinoma (HCC). The potential role of the transactivating hepatitis B virus X protein (HBx) in transformation by HBV is controversial. Here we report that HBx suppresses transformation of primary rat embryo fibroblasts (REFs). Cooperating oncogenes like c-Ha-ras and c-myc transform REF very efficiently but cotransfection with HBx suppressed transformation of REFs down to 5%. Similarly, transfection of HBx together with the cooperating oncogenes Ha-ras and SV40 LTAg or c-Ha-ras and mutant p53 reduced the number of foci to 13%. Comparable results were obtained with HBx in the context of the whole HBV. Suppression of focus formation in REF could be partly relieved by cotransfection of apoptosis inhibitors Bcl-2 or E1B. However, cotransfection of apoptosis inhibitors crmA and p35 did not influence the proapoptotic functions of HBx. Thus, HBx may specifically activate the Bcl-2 sensitive pathway leading to apoptosis. Experiments with 13 HBx linker scanning mutants revealed that the domains necessary for HBx dependent transactivation overlap with the domains needed for the apoptotic/growth arrest functions of HBx.

  2. Deregulation of Rb-E2F1 Axis Causes Chromosomal Instability by Engaging the Transactivation Function of Cdc20–Anaphase-Promoting Complex/Cyclosome

    PubMed Central

    Nath, Somsubhra; Chowdhury, Abhishek; Dey, Sanjib; Roychoudhury, Anirban; Ganguly, Abira; Bhattacharyya, Dibyendu

    2014-01-01

    The E2F family of transcription factors regulates genes involved in various aspects of the cell cycle. Beyond the well-documented role in G1/S transition, mitotic regulation by E2F has also been reported. Proper mitotic progression is monitored by the spindle assembly checkpoint (SAC). The SAC ensures bipolar separation of chromosomes and thus prevents aneuploidy. There are limited reports on the regulation of the SAC by E2F. Our previous work identified the SAC protein Cdc20 as a novel transcriptional regulator of the mitotic ubiquitin carrier protein UbcH10. However, none of the Cdc20 transcription complex proteins have any known DNA binding domain. Here we show that an E2F1-DP1 heterodimer is involved in recruitment of the Cdc20 transcription complex to the UBCH10 promoter and in transactivation of the gene. We further show that inactivation of Rb can facilitate this transactivation process. Moreover, this E2F1-mediated regulation of UbcH10 influences mitotic progression. Deregulation of this pathway results in premature anaphase, chromosomal abnormalities, and aneuploidy. We conclude that excess E2F1 due to Rb inactivation recruits the complex of Cdc20 and the anaphase-promoting complex/cyclosome (Cdc20-APC/C) to deregulate the expression of UBCH10, leading to chromosomal instability in cancer cells. PMID:25368385

  3. The LIM homeobox transcription factor Lhx2 is required to specify the retina field and synergistically cooperates with Pax6 for Six6 trans-activation.

    PubMed

    Tétreault, Nicolas; Champagne, Marie-Pier; Bernier, Gilbert

    2009-03-15

    In mammals, a limited set of homeobox-containing transcription factors are expressed in the presumptive eye field and required to initiate eye development. How these factors interact together at the genetic and molecular level to coordinate this developmental process is poorly understood. We found that the Lhx2 and Pax6 transcription factors operate in a concerted manner during retinal development to promote transcriptional activation of the Six6 homeobox-gene in primitive and mature retinal progenitors. Lhx2 demarcates the presumptive retina field at the neural plate stage and Lhx2 inactivation delays initiation of Rx, Six3 and Pax6 expression in this domain. The later expressed Six6 is properly activated in the pituitary/hypothalamic axis of Lhx2(-/-) embryos, but expression fails to be initiated in the optic vesicle. Lhx2 and Pax6 associate with the chromatin at several regions of Six6 in vivo and cooperate for trans-activation of Six6 regulatory elements in vitro. In retinal progenitor/stem cells, both Lhx2 and Pax6 are genetically required for proper Six6 expression and forced co-expression of Lhx2 and Pax6 can synergistically trans-activate the Six6 locus. Our work reveals how two master regulators of eye development coordinate their action to sequentially promote tissue-specific transcriptional initiation and full activation of a retinal determinant gene.

  4. The mungbean yellow mosaic begomovirus transcriptional activator protein transactivates the viral promoter-driven transgene and causes toxicity in transgenic tobacco plants.

    PubMed

    Rajeswaran, Rajendran; Sunitha, Sukumaran; Shivaprasad, Padubidri V; Pooggin, Mikhail M; Hohn, Thomas; Veluthambi, Karuppannan

    2007-12-01

    The Begomovirus transcriptional activator protein (TrAP/AC2/C2) is a multifunctional protein which activates the viral late gene promoters, suppresses gene silencing, and determines pathogenicity. To study TrAP-mediated transactivation of a stably integrated gene, we generated transgenic tobacco plants with a Mungbean yellow mosaic virus (MYMV) AV1 late gene promoter-driven reporter gene and supertransformed them with the MYMV TrAP gene driven by a strong 35S promoter. We obtained a single supertransformed plant with an intact 35S-TrAP gene that activated the reporter gene 2.5-fold. However, 10 of the 11 supertransformed plants did not have the TrAP region of the T-DNA, suggesting the likely toxicity of TrAP in plants. Upon transformation of wild-type tobacco plants with the TrAP gene, six of the seven transgenic plants obtained had truncated T-DNAs which lacked TrAP. One plant, which had the intact TrAP gene, did not express TrAP. The apparent toxic effect of the TrAP transgene was abolished by mutations in its nuclear-localization signal or zinc-finger domain and by deletion of its activation domain. Therefore, all three domains of TrAP, which are required for transactivation and suppression of gene silencing, also are needed for its toxic effect.

  5. Elements of the maize A1 promoter required for transactivation by the anthocyanin B/C1 or phlobaphene P regulatory genes.

    PubMed Central

    Tuerck, J A; Fromm, M E

    1994-01-01

    The extensive genetic and molecular characterization of the flavonoid pathway's structural and regulatory genes has provided some of the most detailed knowledge of gene interactions in plants. In maize flavonoid biosynthesis, the A1 gene is independently regulated in the anthocyanin and phlobaphene pathways. Anthocyanin production requires the expression of the C1 or PI and R or B regulatory genes, whereas phlobaphene production requires only the P regulatory gene. By deletion analysis of the A1 promoter, we show that the sequences between -123 and -88 are critical for activation by anthocyanin and phlobaphene regulatory genes. Linker-scanner mutations indicated that the -123 to -100 region is more important for transactivation by the P protein. The -98 to -88 region is more important for B/C1 transactivation and shows a strong homology with the region of the Bz1 anthocyanin structural gene promoter shown to be activated by B/C1 and not by P. We identified a 14-bp consensus sequence that is also present in the promoters of three other genes in the anthocyanin pathway, and we propose a model for how the flavonoid regulatory proteins interact with the promoters of the structural genes. PMID:7827497

  6. Subcellular localisation of BAG-1 and its regulation of vitamin D receptor-mediated transactivation and involucrin expression in oral keratinocytes: Implications for oral carcinogenesis

    SciTech Connect

    Lee, San San; Crabb, Simon J.; Janghra, Nari; Carlberg, Carsten; Williams, Ann C.; Cutress, Ramsey I.; Packham, Graham; Hague, Angela

    2007-09-10

    In oral cancers, cytoplasmic BAG-1 overexpression is a marker of poor prognosis. BAG-1 regulates cellular growth, differentiation and survival through interactions with diverse proteins, including the vitamin D receptor (VDR), a key regulator of keratinocyte growth and differentiation. BAG-1 is expressed ubiquitously in human cells as three major isoforms of 50 kDa (BAG-1L), 46 kDa (BAG-1M) and 36 kDa (BAG-1S) from a single mRNA. In oral keratinocytes BAG-1L, but not BAG-1M and BAG-1S, enhanced VDR transactivation in response to 1{alpha},25-dihydroxyvitamin D{sub 3.} BAG-1L was nucleoplasmic and nucleolar, whereas BAG-1S and BAG-1M were cytoplasmic and nucleoplasmic in localisation. Having identified the nucleolar localisation sequence in BAG-1L, we showed that mutation of this sequence did not prevent BAG-1L from potentiating VDR activity. BAG-1L also potentiated transactivation of known vitamin-D-responsive gene promoters, osteocalcin and 24-hydroxylase, and enhanced VDR-dependent transcription and protein expression of the keratinocyte differentiation marker, involucrin. These results demonstrate endogenous gene regulation by BAG-1L by potentiating nuclear hormone receptor function and suggest a role for BAG-1L in 24-hydroxylase regulation of vitamin D metabolism and the cellular response of oral keratinocytes to 1{alpha},25-dihydroxyvitamin D{sub 3}. By contrast to the cytoplasmic BAG-1 isoforms, BAG-1L may act to suppress tumorigenesis.

  7. Synergistic trans-activation of the human C-reactive protein promoter by transcription factor HNF-1 binding at two distinct sites.

    PubMed Central

    Toniatti, C; Demartis, A; Monaci, P; Nicosia, A; Ciliberto, G

    1990-01-01

    The promoter region of the human C-reactive protein (CRP) gene comprises two distinct regions (APREs, for Acute Phase Responsive Elements) each one containing information necessary and sufficient for liver specific and IL-6 inducible expression in human hepatoma Hep3B cells. In this paper we show that both APREs contain a low affinity binding site for the liver specific transcription factor HNF-1/LF-B1. The two sites are separated by approximately 80 bp. Mutations in either of the two sites abolish inducible expression. The same effect is specifically obtained in cotransfection competition experiments when the human albumin HNF-1 site is used as competitor. However, HNF-1 is not the intranuclear mediator of IL-6 because synthetic promoters formed by multimerized copies of different HNF-1 binding sites are not transcriptionally activated by this cytokine. An expression vector encoding full length HNF-1 is capable of trans-activating transcription from the wild-type CRP promoter but not from mutants which have lost the ability to bind HNF-1. Moreover, the level of trans-activation observed with the natural promoter containing both HNF-1 binding sites is far greater than the level of mutated variants containing only one of the two sites. This result strongly suggests that two HNF-1 molecules bound simultaneously to sites distant from each other can act synergistically to activate gene expression. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 7. PMID:2265613

  8. Blockade of T-cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells.

    PubMed Central

    Martínez-Martínez, S; Gómez del Arco, P; Armesilla, A L; Aramburu, J; Luo, C; Rao, A; Redondo, J M

    1997-01-01

    Dithiocarbamates (DTCs) have recently been reported as powerful inhibitors of NF-kappaB activation in a number of cell types. Given the role of this transcription factor in the regulation of gene expression in the inflammatory response, NF-kappaB inhibitors have been suggested as potential therapeutic drugs for inflammatory diseases. We show here that DTCs inhibited both interleukin 2 (IL-2) synthesis and membrane expression of antigens which are induced during T-cell activation. This inhibition, which occurred with a parallel activation of c-Jun transactivating functions and expression, was reflected by transfection experiments at the IL-2 promoter level, and involved not only the inhibition of NF-kappaB-driven reporter activation but also that of nuclear factor of activated T cells (NFAT). Accordingly, electrophoretic mobility shift assays (EMSAs) indicated that pyrrolidine DTC (PDTC) prevented NF-kappaB, and NFAT DNA-binding activity in T cells stimulated with either phorbol myristate acetate plus ionophore or antibodies against the CD3-T-cell receptor complex and simultaneously activated the binding of AP-1. Furthermore, PDTC differentially targeted both NFATp and NFATc family members, inhibiting the transactivation functions of NFATp and mRNA induction of NFATc. Strikingly, Western blotting and immunocytochemical experiments indicated that PDTC promoted a transient and rapid shuttling of NFATp and NFATc, leading to their accelerated export from the nucleus of activated T cells. We propose that the activation of an NFAT kinase by PDTC could be responsible for the rapid shuttling of the NFAT, therefore transiently converting the sustained transactivation of this transcription factor that occurs during lymphocyte activation, and show that c-Jun NH2-terminal kinase (JNK) can act by directly phosphorylating NFATp. In addition, the combined inhibitory effects on NFAT and NF-KB support a potential use of DTCs as immunosuppressants. PMID:9343406

  9. Direct inhibition of the NOTCH transcription factor complex.

    PubMed

    Moellering, Raymond E; Cornejo, Melanie; Davis, Tina N; Del Bianco, Cristina; Aster, Jon C; Blacklow, Stephen C; Kung, Andrew L; Gilliland, D Gary; Verdine, Gregory L; Bradner, James E

    2009-11-12

    Direct inhibition of transcription factor complexes remains a central challenge in the discipline of ligand discovery. In general, these proteins lack surface involutions suitable for high-affinity binding by small molecules. Here we report the design of synthetic, cell-permeable, stabilized alpha-helical peptides that target a critical protein-protein interface in the NOTCH transactivation complex. We demonstrate that direct, high-affinity binding of the hydrocarbon-stapled peptide SAHM1 prevents assembly of the active transcriptional complex. Inappropriate NOTCH activation is directly implicated in the pathogenesis of several disease states, including T-cell acute lymphoblastic leukaemia (T-ALL). The treatment of leukaemic cells with SAHM1 results in genome-wide suppression of NOTCH-activated genes. Direct antagonism of the NOTCH transcriptional program causes potent, NOTCH-specific anti-proliferative effects in cultured cells and in a mouse model of NOTCH1-driven T-ALL.

  10. Direct inhibition of the NOTCH transcription factor complex

    PubMed Central

    Moellering, Raymond E.; Cornejo, Melanie; Davis, Tina N.; Del Bianco, Cristina; Aster, Jon C.; Blacklow, Stephen C.; Kung, Andrew L.; Gilliland, D. Gary; Verdine, Gregory L.; Bradner, James E.

    2010-01-01

    Direct inhibition of transcription factor complexes remains a central challenge in the discipline of ligand discovery. In general, these proteins lack surface involutions suitable for high-affinity binding by small molecules. Here we report the design of synthetic, cell-permeable, stabilized α-helical peptides that target a critical protein–protein interface in the NOTCH transactivation complex. We demonstrate that direct, high-affinity binding of the hydrocarbon-stapled peptide SAHM1 prevents assembly of the active transcriptional complex. Inappropriate NOTCH activation is directly implicated in the pathogenesis of several disease states, including T-cell acute lymphoblastic leukaemia (T-ALL). The treatment of leukaemic cells with SAHM1 results in genome-wide suppression of NOTCH-activated genes. Direct antagonism of the NOTCH transcriptional program causes potent, NOTCH-specific anti-proliferative effects in cultured cells and in a mouse model of NOTCH1-driven T-ALL. PMID:19907488

  11. Fluoxetine regulates cell growth inhibition of interferon-α.

    PubMed

    Lin, Yu-Min; Yu, Bu-Chin; Chiu, Wen-Tai; Sun, Hung-Yu; Chien, Yu-Chieh; Su, Hui-Chen; Yen, Shu-Yang; Lai, Hsin-Wen; Bai, Chyi-Huey; Young, Kung-Chia; Tsao, Chiung-Wen

    2016-10-01

    Fluoxetine, a well-known anti-depression agent, may act as a chemosensitizer to assist and promote cancer therapy. However, how fluoxetine regulates cellular signaling to enhance cellular responses against tumor cell growth remains unclear. In the present study, addition of fluoxetine promoted growth inhibition of interferon-alpha (IFN-α) in human bladder carcinoma cells but not in normal uroepithelial cells through lessening the IFN-α-induced apoptosis but switching to cause G1 arrest, and maintaining the IFN-α-mediated reduction in G2/M phase. Activations and signal transducer and transactivator (STAT)-1 and peroxisome proliferator-activated receptor alpha (PPAR-α) were involved in this process. Chemical inhibitions of STAT-1 or PPAR-α partially rescued bladder carcinoma cells from IFN-α-mediated growth inhibition via blockades of G1 arrest, cyclin D1 reduction, p53 downregulation and p27 upregulation in the presence of fluoxetine. However, the functions of both proteins were not involved in the control of fluoxetine over apoptosis and maintained the declined G2/M phase of IFN-α. These results indicated that activation of PPAR-α and STAT-1 participated, at least in part, in growth inhibition of IFN-α in the presence of fluoxetine.

  12. Calcipotriol Targets LRP6 to Inhibit Wnt Signaling in Pancreatic Cancer

    PubMed Central

    Arensman, Michael D.; Nguyen, Phillip; Kershaw, Kathleen M.; Lay, Anna R.; Ostertag-Hill, Claire A.; Sherman, Mara H.; Downes, Michael; Liddle, Christopher; Evans, Ronald M.; Dawson, David W.

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy in need of more effective treatment approaches. One potential therapeutic target is Wnt/β-catenin signaling, which plays important roles in PDAC tumor initiation and progression. Among Wnt inhibitors with suitable in vivo biological activity is vitamin D, which is known to antagonize Wnt/β-catenin signaling in colorectal cancer and have anti-tumor activity in PDAC. For this study the relationship between vitamin D signaling, Wnt/β-catenin activity and tumor cell growth in PDAC was investigated through the use of calcipotriol, a potent non-hypercalcemic vitamin D analog. PDAC tumor cell growth inhibition by calcipotriol was positively correlated with vitamin D receptor (VDR) expression and Wnt/β-catenin activity. Furthermore, vitamin D and Wnt signaling activity were found to be reciprocally linked through feedback regulation. Calcipotriol inhibited autocrine Wnt/β-catenin signaling in PDAC cell lines in parallel with decreased protein levels of the low density lipoprotein receptor-related protein 6 (LRP6), a requisite co-receptor for ligand-dependent canonical Wnt signaling. Decrease in LRP6 protein seen with calcipotriol was mediated through a novel mechanism involving transcriptional upregulation of low-density lipoprotein receptor adaptor protein 1 (LDLRAP1). Finally, changes in LRP6 or LDLRAP1 expression directly altered Wnt reporter activity, supporting their roles as regulators of ligand-dependent Wnt/β-catenin signaling. Implications This study provides a novel biochemical target through which vitamin D signaling exerts inhibitory effects on Wnt/β-catenin signaling, as well as potential biomarkers for predicting and following tumor response to vitamin D-based therapy. PMID:26224368

  13. The Wedelolactone Derivative Inhibits Estrogen Receptor-Mediated Breast, Endometrial, and Ovarian Cancer Cells Growth

    PubMed Central

    Xu, Defeng; Lin, Tzu-Hua; Cheng, Max A.; Chen, Lu-Min; Chang, Chawnshang; Yeh, Shuyuan

    2014-01-01

    Estrogen and estrogen receptor (ER)-mediated signaling pathways play important roles in the etiology and progression of human breast, endometrial, and ovarian cancers. Attenuating ER activities by natural products and their derivatives is a relatively practical strategy to control and reduce breast, endometrial, and ovarian cancer risk. Here, we found 3-butoxy-1,8,9-trihydroxy-6H-benzofuro[3,2-c]benzopyran-6-one (BTB), a new derivative of wedelolactone, could effectively inhibit the 17-estradiol (E2)-induced ER transactivation and suppress the growth of breast cancer as well as endometrial and ovarian cancer cells. Our results indicate that 2.5 μM BTB effectively suppresses ER-positive, but not ER-negative, breast, endometrial, and ovarian cancer cells. Furthermore, our data indicate that BTB can modulate ER transactivation and suppress the expression of E2-mediated ER target genes (Cyclin D1, E2F1, and TERT) in the ER-positive MCF-7, Ishikawa, and SKOV-3 cells. Importantly, this BTB mediated inhibition of ER activity is selective since BTB does not suppress the activities of other nuclear receptors, including glucocorticoid receptor and progesterone receptor, suggesting that BTB functions as a selective ER signaling inhibitor with the potential to treat breast, endometrial, and ovarian cancers. PMID:25221777

  14. Anyalysis of Msx1 and Msx2 Transactivation Function in the Context of the Heat Shock 70 (Hspa1b) Gene Promoter

    PubMed Central

    Zhuang, Fengfeng; Nguyen, Manuel P.; Shuler, Charles; Liu, Yi-Hsin

    2009-01-01

    Previous studies have shown that Msx proteins control gene transcription predominantly through repression mechanisms. However, gene expression studies using either the gain-of-function or the loss-of-function mutants revealed many gene targets whose expression require functional Msx proteins. To date, investigations into the mechanisms of Msx-dependent trans-activation have been hindered by the lack of a responsive promoter. Here, we demonstrated the usefulness of the mouse Hspa1b promoter in probing Msx-dependent mechanisms of gene activation. We showed that Msx protein activates Hspa1b promoter via its C-terminal domain. The activation absolutely depends on the HSEs and physical interactions between Msx proteins and Heat shock factors may play a contributing role. PMID:19338779

  15. The cavity in the hydrophobic core of Myb DNA-binding domain is reserved for DNA recognition and trans-activation.

    PubMed

    Ogata, K; Kanei-Ishii, C; Sasaki, M; Hatanaka, H; Nagadoi, A; Enari, M; Nakamura, H; Nishimura, Y; Ishii, S; Sarai, A

    1996-02-01

    The DNA-binding domain of Myb consists of three imperfect repeats, R1, R2 and R3, each containing a helix-turn-helix motif variation. Among these repeats, R2 has distinct characteristics with high thermal instability. The NMR structure analysis found a cavity inside the hydrophobic core of R2 but not in R1 or R3. Here, we show that R2 has slow conformational fluctuations, and that a cavity-filling mutation which stabilizes the R2 structure significantly reduces specific Myb DNA-binding activity and trans-activation. Structural observations of the free and DNA-complexed stages suggest that the implied inherent conformational flexibility of R2, associated with the presence of the cavity, could be important for DNA recognition by Myb.

  16. Wide-line NMR and DSC studies on intrinsically disordered p53 transactivation domain and its helically pre-structured segment

    PubMed Central

    Tompa, Peter; Han, Kyou-Hoon; Bokor, Mónika; Kamasa, Pawel; Tantos, Ágnes; Fritz, Beáta; Kim, Do-Hyoung; Lee, Chewook; Verebélyi, Tamás; Tompa, Kálmán

    2016-01-01

    Wide-line 1H NMR intensity and differential scanning calorimetry measurements were carried out on the intrinsically disordered 73-residue full transactivation domain (TAD) of the p53 tumor suppressor protein and two peptides: one a wild type p53 TAD peptide with a helix pre-structuring property, and a mutant peptide with a disabled helix-forming propensity. Measurements were carried out in order to characterize their water and ion binding characteristics. By quantifying the number of hydrate water molecules, we provide a microscopic description for the interactions of water with a wild-type p53 TAD and two p53 TAD peptides. The results provide direct evidence that intrinsically disordered proteins (IDPs) and a less structured peptide not only have a higher hydration capacity than globular proteins, but are also able to bind a larger amount of charged solute ions. [BMB Reports 2016; 49(9): 497-501] PMID:27418282

  17. Versatile reporter systems show that transactivation by human T-cell leukemia virus type 1 Tax occurs independently of chromatin remodeling factor BRG1.

    PubMed

    Zhang, Ling; Liu, Meihong; Merling, Randall; Giam, Chou-Zen

    2006-08-01

    Potent activation of human T-cell leukemia virus type 1 (HTLV-1) gene expression is mediated by the virus-encoded transactivator protein Tax and three imperfect 21-bp repeats in the viral long terminal repeats. Each 21-bp repeat contains a cAMP-responsive-element core flanked by 5' G-rich and 3' C-rich sequences. Tax alone does not bind DNA. Rather, it interacts with basic domain-leucine zipper transcription factors CREB and ATF-1 to form ternary complexes with the 21-bp repeats. In the context of the ternary complexes, Tax contacts the G/C-rich sequences and recruits transcriptional coactivators CREB-binding protein (CBP)/p300 to effect potent transcriptional activation. Using an easily transduced and chromosomally integrated reporter system derived from a self-inactivating lentivirus vector, we showed in a BRG1- and BRM1-deficient adrenal carcinoma cell line, SW-13, that Tax- and 21-bp repeat-mediated transactivation does not require BRG1 or BRM1 and is not enhanced by BRG1. With a similar reporter system, we further demonstrated that Tax- and tumor necrosis factor alpha-induced NF-kappaB activation occurs readily in SW-13 cells in the absence of BRG1 and BRM1. These results suggest that the assembly of stable multiprotein complexes containing Tax, CREB/ATF-1, and CBP/p300 on the 21-bp repeats is the principal mechanism employed by Tax to preclude nucleosome formation at the HTLV-1 enhancer/promoter. This most likely bypasses the need for BRG1-containing chromatin-remodeling complexes. Likewise, recruitment of CBP/p300 by NF-kappaB may be sufficient to disrupt histone-DNA interaction for the initiation of transcription.

  18. Activation of the human keratinocyte B1 bradykinin receptor induces expression and secretion of metalloproteases 2 and 9 by transactivation of epidermal growth factor receptor.

    PubMed

    Matus, Carola E; Ehrenfeld, Pamela; Pavicic, Francisca; González, Carlos B; Concha, Miguel; Bhoola, Kanti D; Burgos, Rafael A; Figueroa, Carlos D

    2016-09-01

    The B1 bradykinin receptor (BDKRB1) is a component of the kinin cascade localized in the human skin. Some of the effects produced by stimulation of BDKRB1 depend on transactivation of epidermal growth factor receptor (EGFR), but the mechanisms involved in this process have not been clarified yet. The primary purpose of this study was to determine the effect of a BDKRB1 agonist on wound healing in a mouse model and the migration and secretion of metalloproteases 2 and 9 from human HaCaT keratinocytes and delineate the signalling pathways that triggered their secretion. Although stimulation of BDKRB1 induces weak chemotactic migration of keratinocytes and wound closure in an in vitro scratch-wound assay, the BDKRB1 agonist improved wound closure in a mouse model. BDKRB1 stimulation triggers synthesis and secretion of both metalloproteases, effects that depend on the activity of EGFR and subsequent phosphorylation of ERK1/2 and p38 mitogen-activated protein kinases and PI3K/Akt. In the mouse model, immunoreactivity for both gelatinases was concentrated around wound borders. EGFR transactivation by BDKRB1 agonist involves Src kinases family and ADAM17. In addition to extracellular matrix degradation, metalloproteases 2 and 9 regulate cell migration and differentiation, cell functions that are associated with the role of BDKRB1 in keratinocyte differentiation. Considering that BDKRB1 is up-regulated by inflammation and/or by cytokines that are abundant in the inflammatory milieu, more stable BDKRB1 agonists may be of therapeutic value to modulate wound healing.

  19. Serine 350 of human pregnane X receptor is crucial for its heterodimerization with retinoid X receptor alpha and transactivation of target genes in vitro and in vivo.

    PubMed

    Wang, Yue-Ming; Chai, Sergio C; Lin, Wenwei; Chai, Xiaojuan; Elias, Ayesha; Wu, Jing; Ong, Su Sien; Pondugula, Satyanarayana R; Beard, Jordan A; Schuetz, Erin G; Zeng, Su; Xie, Wen; Chen, Taosheng

    2015-08-15

    The human pregnane X receptor (hPXR), a member of the nuclear receptor superfamily, senses xenobiotics and controls the transcription of genes encoding drug-metabolizing enzymes and transporters. The regulation of hPXR's transcriptional activation of its target genes is important for xenobiotic detoxification and endobiotic metabolism, and hPXR dysregulation can cause various adverse drug effects. Studies have implicated the putative phosphorylation site serine 350 (Ser(350)) in regulating hPXR transcriptional activity, but the mechanism of regulation remains elusive. Here we investigated the transactivation of hPXR target genes in vitro and in vivo by hPXR with a phosphomimetic mutation at Ser(350) (hPXR(S350D)). The S350D phosphomimetic mutation reduced the endogenous expression of cytochrome P450 3A4 (an hPXR target gene) in HepG2 and LS180 cells. Biochemical assays and structural modeling revealed that Ser(350) of hPXR is crucial for formation of the hPXR-retinoid X receptor alpha (RXRα) heterodimer. The S350D mutation abrogated heterodimerization in a ligand-independent manner, impairing hPXR-mediated transactivation. Further, in a novel humanized transgenic mouse model expressing the hPXR(S350D) transgene, we demonstrated that the S350D mutation alone is sufficient to impair hPXR transcriptional activity in mouse liver. This transgenic mouse model provides a unique tool to investigate the regulation and function of hPXR, including its non-genomic function, in vivo. Our finding that phosphorylation regulates hPXR activity has implications for development of novel hPXR antagonists and for safety evaluation during drug development.

  20. Estrogen Receptor β (ERβ1) Transactivation Is Differentially Modulated by the Transcriptional Coregulator Tip60 in a cis-Acting Element-dependent Manner*

    PubMed Central

    Lee, Ming-Tsung; Leung, Yuet-Kin; Chung, Irving; Tarapore, Pheruza; Ho, Shuk-Mei

    2013-01-01

    Estrogen receptor (ER) β1 and ERα have overlapping and distinct functions despite their common use of estradiol as the physiological ligand. These attributes are explained in part by their differential utilization of coregulators and ligands. Although Tip60 has been shown to interact with both receptors, its regulatory role in ERβ1 transactivation has not been defined. In this study, we found that Tip60 enhances transactivation of ERβ1 at the AP-1 site but suppresses its transcriptional activity at the estrogen-response element (ERE) site in an estradiol-independent manner. However, different estrogenic compounds can modify the Tip60 action. The corepressor activity of Tip60 at the ERE site is abolished by diarylpropionitrile, genistein, equol, and bisphenol A, whereas its coactivation at the AP-1 site is augmented by fulvestrant (ICI 182,780). GRIP1 is an important tethering mediator for ERs at the AP-1 site. We found that coexpression of GRIP1 synergizes the action of Tip60. Although Tip60 is a known acetyltransferase, it is unable to acetylate ERβ1, and its coregulatory functions are independent of its acetylation activity. In addition, we showed the co-occupancy of ERβ1 and Tip60 at ERE and AP-1 sites of ERβ1 target genes. Tip60 differentially regulates the endogenous expression of the target genes by modulating the binding of ERβ1 to the cis-regulatory regions. Thus, we have identified Tip60 as the first dual-function coregulator of ERβ1. PMID:23857583

  1. Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells.

    PubMed

    Kunihiro, Marie; Fujii, Hideki; Miyagi, Takuya; Takahashi, Yoshiaki; Tanaka, Reiko; Fukushima, Takuya; Ansari, Aftab A; Tanaka, Yuetsu

    2016-07-11

    The environmental factors that lead to the reactivation of human T cell leukemia virus type-1 (HTLV-I) in latently infected T cells in vivo remain unknown. It has been previously shown that heat shock (HS) is a potent inducer of HTLV-I viral protein expression in long-term cultured cell lines. However, the precise HTLV-I protein(s) and mechanisms by which HS induces its effect remain ill-defined. We initiated these studies by first monitoring the levels of the trans-activator (Tax) protein induced by exposure of the HTLV-I infected cell line to HS. HS treatment at 43 °C for 30 min for 24 h led to marked increases in the level of Tax antigen expression in all HTLV-I-infected T cell lines tested including a number of HTLV-I-naturally infected T cell lines. HS also increased the expression of functional HTLV-I envelope gp46 antigen, as shown by increased syncytium formation activity. Interestingly, the enhancing effect of HS was partially inhibited by the addition of the heat shock protein 70 (HSP70)-inhibitor pifithlin-μ (PFT). In contrast, the HSP 70-inducer zerumbone (ZER) enhanced Tax expression in the absence of HS. These data suggest that HSP 70 is at least partially involved in HS-mediated stimulation of Tax expression. As expected, HS resulted in enhanced expression of the Tax-inducible host antigens, such as CD83 and OX40. Finally, we confirmed that HS enhanced the levels of Tax and gp46 antigen expression in primary human CD4⁺ T cells isolated from HTLV-I-infected humanized NOD/SCID/γc null (NOG) mice and HTLV-I carriers. In summary, the data presented herein indicate that HS is one of the environmental factors involved in the reactivation of HTLV-I in vivo via enhanced Tax expression, which may favor HTLV-I expansion in vivo.

  2. Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells

    PubMed Central

    Kunihiro, Marie; Fujii, Hideki; Miyagi, Takuya; Takahashi, Yoshiaki; Tanaka, Reiko; Fukushima, Takuya; Ansari, Aftab A.; Tanaka, Yuetsu

    2016-01-01

    The environmental factors that lead to the reactivation of human T cell leukemia virus type-1 (HTLV-I) in latently infected T cells in vivo remain unknown. It has been previously shown that heat shock (HS) is a potent inducer of HTLV-I viral protein expression in long-term cultured cell lines. However, the precise HTLV-I protein(s) and mechanisms by which HS induces its effect remain ill-defined. We initiated these studies by first monitoring the levels of the trans-activator (Tax) protein induced by exposure of the HTLV-I infected cell line to HS. HS treatment at 43 °C for 30 min for 24 h led to marked increases in the level of Tax antigen expression in all HTLV-I-infected T cell lines tested including a number of HTLV-I-naturally infected T cell lines. HS also increased the expression of functional HTLV-I envelope gp46 antigen, as shown by increased syncytium formation activity. Interestingly, the enhancing effect of HS was partially inhibited by the addition of the heat shock protein 70 (HSP70)-inhibitor pifithlin-μ (PFT). In contrast, the HSP 70-inducer zerumbone (ZER) enhanced Tax expression in the absence of HS. These data suggest that HSP 70 is at least partially involved in HS-mediated stimulation of Tax expression. As expected, HS resulted in enhanced expression of the Tax-inducible host antigens, such as CD83 and OX40. Finally, we confirmed that HS enhanced the levels of Tax and gp46 antigen expression in primary human CD4+ T cells isolated from HTLV-I-infected humanized NOD/SCID/γc null (NOG) mice and HTLV-I carriers. In summary, the data presented herein indicate that HS is one of the environmental factors involved in the reactivation of HTLV-I in vivo via enhanced Tax expression, which may favor HTLV-I expansion in vivo. PMID:27409630

  3. Anti-Inflammatory and PPAR Transactivational Effects of Oleanane-Type Triterpenoid Saponins from the Roots of Pulsatilla koreana

    PubMed Central

    Li, Wei; Yan, Xi Tao; Sun, Ya Nan; Ngan, Thi Thanh; Shim, Sang Hee; Kim, Young Ho

    2014-01-01

    In this study, 23 oleanane-type triterpenoid saponins were isolated from a methanol extract of the roots of Pulsatilla koreana. The NF-κB inhibitory activity of the isolated compounds was measured in TNFα-treated HepG2 cells using a luciferase reporter system. Compounds 19–23 inhibited TNFα-stimulated NF-κB activation in a dose-dependent manner, with IC50 values ranging from 0.75–8.30 μM. Compounds 19 and 20 also inhibited the TNFα-induced expression of iNOS and ICAM-1 mRNA. Moreover, effect of the isolated compounds on PPARs transcriptional activity was assessed. Compounds 7–11 and 19–23 activated PPARs the transcriptional activity significantly in a dose-dependent manner, with EC50 values ranging from 0.9–10.8 μM. These results suggest the presence of potent anti-inflammatory components in P. koreana, and will facilitate the development of novel anti-inflammatory agents. PMID:25143813

  4. The IFN-γ-induced Transcriptional Program of the CIITA Gene is Inhibited by Statins

    PubMed Central

    Lee, Sun Jung; Qin, Hongwei; Benveniste, Etty N.

    2009-01-01

    Summary Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors that exert anti-inflammatory effects. IFN-γ induction of class II MHC expression, which requires the class II transactivator (CIITA), is inhibited by statins, however, the molecular basis for suppression is undetermined. We describe that statins inhibit IFN-γ-induced class II MHC expression by suppressing CIITA gene expression, which is dependent on the HMG-CoA reductase pathway. In addition, CIITA expression is inhibited by GGTI-298 or Clostridium difficile Toxin A, specific inhibitors of Rho family protein prenylation, indicating the involvement of small GTPases. Rac1 is involved in IFN-γ inducible expression of CIITA, and statins inhibit IFN-γ-induced Rac1 activation, contributing to the inhibitory effect of statins. IFN-γ induction of the CIITA gene is regulated by the transcription factors STAT-1α, IRF-1 and USF-1. We previously reported that statins inhibit constitutive STAT-1α expression. IRF-1, a STAT-1 dependent gene, is also inhibited by statins. Therefore, statin treatment results in decreased recruitment of STAT-1α and IRF-1 to the endogenous CIITA pIV promoter. The recruitment of USF-1 to CIITA pIV is also reduced by statins, as is the recruitment of RNA Polymerase II, p300 and Brg-1. These data indicate that statins inhibit the transcriptional program of the CIITA gene. PMID:18601229

  5. p53 inhibits DNA replication in vitro in a DNA-binding-dependent manner.

    PubMed Central

    Miller, S D; Farmer, G; Prives, C

    1995-01-01

    The p53 tumor suppressor gene product is a sequence-specific DNA-binding protein that is necessary for the G1 arrest of many cell types. Consistent with its role as a cell cycle checkpoint factor, p53 has been shown to be capable of both transcriptional activation and repression. Here we show a new potential role for p53 as a DNA-binding-dependent regulator of DNA replication. Constructs containing multiple copies of the ribosomal gene cluster (RGC) p53 binding site cloned on the late side of the polyomavirus origin were used in in vitro replication assays. In the presence of p53, the replication of these constructs was strongly inhibited, while the replication of constructs containing a mutant version of the RGC site was not affected by p53. Several tumor-derived mutant p53 proteins were unable to inhibit replication of the construct with wild-type RGC sites. Additionally, the transactivator GAL4-VP16 was unable to inhibit replication of a construct containing GAL4 binding sites adjacent to the polyomavirus origin. We also show that the inhibition by p53 can occur from sites cloned as far as 600 bp from the origin. Preincubation experiments suggest that p53 inhibits replication at a step mediated by ATP, possibly by inhibiting the binding of polyomavirus T antigen to the core origin. The presence of an endogenous p53 binding site in the polyomavirus origin suggests potential mechanisms for the observed inhibition. PMID:8524220

  6. Troglitazone inhibits vascular smooth muscle cell growth and intimal hyperplasia.

    PubMed Central

    Law, R E; Meehan, W P; Xi, X P; Graf, K; Wuthrich, D A; Coats, W; Faxon, D; Hsueh, W A

    1996-01-01

    Vascular smooth muscle cell (VSMC) proliferation and migration are responses to arterial injury that are highly important to the processes of restenosis and atherosclerosis. In the arterial balloon injury model in the rat, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are induced in the vessel wall and regulate these VSMC activities. Novel insulin sensitizing agents, thiazolidinediones, have been demonstrated to inhibit insulin and epidermal growth factor-induced growth of VSMCs. We hypothesized that these agents might also inhibit the effect of PDGF and bFGF on cultured VSMCs and intimal hyperplasia in vivo. Troglitazone (1 microM), a member of the thiazolidinedione class, produced a near complete inhibition of both bFGF-induced DNA synthesis as measured by bromodeoxyuridine incorporation (6.5+/-3.9 vs. 17.6+/-4.3% cells labeled, P < 0.05) and c-fos induction. This effect was associated with an inhibition (by 73+/-4%, P < 0.01) by troglitazone of the transactivation of the serum response element, which regulates c-fos expression. Inhibition of c-fos induction by troglitazone appeared to occur via a blockade of the MAP kinase pathway at a point downstream of MAP kinase activation by MAP kinase kinase. At this dose, troglitazone also inhibited PDGF-BB-directed migration of VSMC (by 70+/-6%, P < 0.01). These in vitro effects were operative in vivo. Quantitative image analysis revealed that troglitazone-treated rats had 62% (P < 0.001) less neointima/media area ratio 14 d after balloon injury of the aorta compared with injured rats that received no troglitazone. These results suggest troglitazone is a potent inhibitor of VSMC proliferation and migration and, thus, may be a useful agent to prevent restenosis and possibly atherosclerosis. PMID:8878442

  7. Resveratrol inhibits LXRα-dependent hepatic lipogenesis through novel antioxidant Sestrin2 gene induction

    SciTech Connect

    Jin, So Hee; Yang, Ji Hye; Shin, Bo Yeon; Seo, Kyuhwa; Shin, Sang Mi; Cho, Il Je; Ki, Sung Hwan

    2013-08-15

    Liver X receptor-α (LXRα), a member of the nuclear receptor superfamily of ligand-activated transcription factors, regulates de novo fatty acid synthesis that leads to stimulate hepatic steatosis. Although, resveratrol has beneficial effects on metabolic disease, it is not known whether resveratrol affects LXRα-dependent lipogenic gene expression. This study investigated the effect of resveratrol in LXRα-mediated lipogenesis and the underlying molecular mechanism. Resveratrol inhibited the ability of LXRα to activate sterol regulatory element binding protein-1c (SREBP-1c) and thereby inhibited target gene expression in hepatocytes. Moreover, resveratrol decreased LXRα–RXRα DNA binding activity and LXRE-luciferase transactivation. Resveratrol is known to activate Sirtuin 1 (Sirt1) and AMP-activated protein kinase (AMPK), although its precise mechanism of action remains controversial. We found that the ability of resveratrol to repress T0901317-induced SREBP-1c expression was not dependent on AMPK and Sirt1. It is well established that hepatic steatosis is associated with antioxidant and redox signaling. Our data showing that expression of Sestrin2 (Sesn2), which is a novel antioxidant gene, was significantly down-regulated in the livers of high-fat diet-fed mice. Moreover, resveratrol up-regulated Sesn2 expression, but not Sesn1 and Sesn3. Sesn2 overexpression repressed LXRα-activated SREBP-1c expression and LXRE-luciferase activity. Finally, Sesn2 knockdown using siRNA abolished the effect of resveratrol in LXRα-induced FAS luciferase gene transactivation. We conclude that resveratrol affects Sesn2 gene induction and contributes to the inhibition of LXRα-mediated hepatic lipogenesis. - Highlights: • We investigated the effect of resveratrol in LXRα-mediated lipogenesis. • Resveratrol attenuated the ability of the LXRα-mediated lipogenic gene expression. • Resveratrol’s effects on T090-induced lipogenesis is not dependent on Sirt1 or AMPK.

  8. Autocrine growth inhibition by transforming growth factor β-1 (TGFβ-1) in human neuroendocrine tumour cells

    PubMed Central

    Wimmel, A; Wiedenmann, B; Rosewicz, S

    2003-01-01

    Background and aim: The role of transforming growth factor β-1 (TGFβ-1) in neuroendocrine tumour biology is currently unknown. We therefore examined the expression and biological significance of TGFβ signalling components in neuroendocrine tumours (NETs) of the gastroenteropancreatic (GEP) tract. Methods: Expression of TGFβ-1 and its receptors, Smads and Smad regulated proteins, was examined in surgically resected NET specimens and human NET cell lines by immunohistochemistry, reverse transcriptase-polymerase chain reaction, immunoblotting, and ELISA. Activation of TGFβ-1 dependent promoters was tested by transactivation assays. Growth regulation was evaluated by cell numbers, soft agar assays, and cell cycle analysis using flow cytometry. The role of endogenous TGFβ was assessed by a TGFβ neutralising antibody and stable transfection of a dominant negative TGFβR II receptor construct. Results: Coexpression of TGFβ-1 and its receptors TGFβR I and TGFβR II was detected in 67% of human NETs and in all three NET cell lines examined. NET cell lines expressed the TGFβ signal transducers Smad 2, 3, and 4. In two of the three cell lines, TGFβ-1 treatment resulted in transactivation of a TGFβ responsive reporter construct as well as inhibition of c-myc and induction of p21(WAF1) expression. TGFβ-1 inhibited anchorage dependent and independent growth in a time and dose dependent manner in TGFβ-1 responsive cell lines. TGFβ-1 mediated growth inhibition was due to G1 arrest without evidence of induction of apoptosis. Functional inactivation of endogenous TGFβ revealed the existence of an autocrine antiproliferative loop in NET cells. Conclusions: Neuroendocrine tumour cells of the gastroenteropancreatic tract are subject to paracrine and autocrine growth inhibition by TGFβ-1, which may account in part for the low proliferative index of this tumour entity. PMID:12912863

  9. Cobaltous chloride and hypoxia inhibit aryl hydrocarbon receptor-mediated responses in breast cancer cells.

    PubMed

    Khan, Shaheen; Liu, Shengxi; Stoner, Matthew; Safe, Stephen

    2007-08-15

    The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-ERalpha crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1alpha or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NFkappaB which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 h, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 h, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 h enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide.

  10. COBALTOUS CHLORIDE AND HYPOXIA INHIBIT ARYL HYDROCARBON RECEPTOR-MEDIATED RESPONSES IN BREAST CANCER CELLS

    PubMed Central

    Khan, Shaheen; Liu, Shengxi; Stoner, Matthew; Safe, Stephen

    2007-01-01

    The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-Erα crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1α or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NFκB which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 hr, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 hr, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 hr enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide. PMID:17599377

  11. Cobaltous chloride and hypoxia inhibit aryl hydrocarbon receptor-mediated responses in breast cancer cells

    SciTech Connect

    Khan, Shaheen; Liu Shengxi; Stoner, Matthew; Safe, Stephen

    2007-08-15

    The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-ER{alpha} crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1{alpha} or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NF{kappa}B which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 h, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 h, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 h enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide.

  12. Statins prevent NF-kappaB transactivation independently of the IKK-pathway in human endothelial cells.

    PubMed

    Hölschermann, Hans; Schuster, Daniel; Parviz, Behnoush; Haberbosch, Werner; Tillmanns, Harald; Muth, Heidrun

    2006-04-01

    Statins have been linked to a wide range of vascular benefits, many of them are likely to be due to attenuation of chronic vascular inflammation. Nuclear factor kappaB (NF-kappaB) is one of the key regulators of transcription of a variety of genes involved in immune and inflammatory responses. Therefore, we investigated the effect of statins on TNF-alpha-induced NF-kappaB signaling in human endothelial cells (EC). ECs were pre-incubated for 16 h with cerivastatin (10(-9) to 10(-7) M) or vehicle in the presence or absence of mevalonate, followed by stimulation with 20 ng/ml TNF-alpha. Statin-treatment prevented TNF-alpha-induced NF-kappaB binding activity, nuclear translocation of the NF-kappaB p65 subunit, as well as NF-kappaB controlled tissue factor (TF) gene transcription in cultured EC. IkappaBalpha phosphorylation and IkappaBalpha degradation, however, still occurred in statin-treated cells. TNF-alpha also activated phosphatidylinositol (PI)3-kinase, as reflected by phosphorylation of Akt. Statin treatment of cells abrogated TNF-alpha-induced Akt phosphorylation and p65 nuclear translocation. As observed with statins, inhibition of PI3-kinase activity by Ly294002 also blocked TNF-alpha-induced p65 translocation, but did not prevent IkappaBalpha phosphorylation nor IkappaBalpha degradation. These studies demonstrate that TNF-alpha-induced NF-kappaB activation is abrogated by statin treatment in HUVEC independently of the classical IKK-pathway but via inhibition of PI3-kinase/Akt signaling.

  13. Ser-634 and Ser-636 of Kaposi’s Sarcoma-Associated Herpesvirus RTA are Involved in Transactivation and are Potential Cdk9 Phosphorylation Sites

    PubMed Central

    Tsai, Wan-Hua; Wang, Pei-Wen; Lin, Shu-Yu; Wu, I-Lin; Ko, Ying-Chieh; Chen, Yu-Lian; Li, Mengtao; Lin, Su-Fang

    2011-01-01

    The replication and transcription activator (RTA) of Kaposi’s sarcoma-associated herpesvirus (KSHV), K-RTA, is a lytic switch protein that moderates the reactivation process of KSHV latency. By mass spectrometric analysis of affinity purified K-RTA, we showed that Thr-513 or Thr-514 was the primary in vivo phosphorylation site. Thr-513 and Thr-514 are proximal to the nuclear localization signal (527KKRK530) and were previously hypothesized to be target sites of Ser/Thr kinase hKFC. However, substitutions of Thr with Ala at 513 and 514 had no effect on K-RTA subcellular localization or transactivation activity. By contrast, replacement of Ser with Ala at Ser-634 and Ser-636 located in a Ser/Pro-rich region of K-RTA, designated as S634A/S636A, produced a polypeptide with ∼10 kDa shorter in molecular weight and reduced transactivation in a luciferase reporter assay relative to the wild type. In contrast to prediction, the decrease in molecular weight was not due to lack of phosphorylation because the overall Ser and Thr phosphorylation state in K-RTA and S634A/S636A were similar, excluding that Ser-634 or Ser-636 motif served as docking sites for consecutive phosphorylation. Interestingly, S634A/S636A lost ∼30% immuno-reactivity to MPM2, an antibody specific to pSer/pThr-Pro motif, indicating that 634SPSP637 motif was in vivo phosphorylated. By in vitro kinase assay, we showed that K-RTA is a substrate of CDK9, a Pro-directed Ser/Thr kinase central to transcriptional regulation. Importantly, the capability of K-RTA in associating with endogenous CDK9 was reduced in S634A/S636A, which suggested that Ser-634 and Ser-636 may be involved in CDK9 recruitment. In agreement, S634A/S636A mutant exhibited ∼25% reduction in KSHV lytic cycle reactivation relative to that by the wild type K-RTA. Taken together, our data propose that Ser-634 and Ser-636 of K-RTA are phosphorylated by host transcriptional kinase CDK9 and such a process contributes to a full

  14. Roles of PU.1 in monocyte- and mast cell-specific gene regulation: PU.1 transactivates CIITA pIV in cooperation with IFN-gamma.

    PubMed

    Ito, Tomonobu; Nishiyama, Chiharu; Nakano, Nobuhiro; Nishiyama, Makoto; Usui, Yoshihiko; Takeda, Kazuyoshi; Kanada, Shunsuke; Fukuyama, Kanako; Akiba, Hisaya; Tokura, Tomoko; Hara, Mutsuko; Tsuboi, Ryoji; Ogawa, Hideoki; Okumura, Ko

    2009-07-01

    Over-expression of PU.1, a myeloid- and lymphoid-specific transcription factor belonging to the Ets family, induces monocyte-specific gene expression in mast cells. However, the effects of PU.1 on each target gene and the involvement of cytokine signaling in PU.1-mediated gene expression are largely unknown. In the present study, PU.1 was over-expressed in two different types of bone marrow-derived cultured mast cells (BMMCs): BMMCs cultured with IL-3 plus stem cell factor (SCF) and BMMCs cultured with pokeweed mitogen-stimulated spleen-conditioned medium (PWM-SCM). PU.1 over-expression induced expression of MHC class II, CD11b, CD11c and F4/80 on PWM-SCM-cultured BMMCs, whereas IL-3/SCF-cultured BMMCs expressed CD11b and F4/80, but not MHC class II or CD11c. When IFN-gamma was added to the IL-3/SCF-based medium, PU.1 transfectant acquired MHC class II expression, which was abolished by antibody neutralization or in Ifngr(-/-) BMMCs, through the induction of expression of the MHC class II transactivator, CIITA. Real-time PCR detected CIITA mRNA driven by the fourth promoter, pIV, and chromatin immunoprecipitation indicated direct binding of PU.1 to pIV in PU.1-over-expressing BMMCs. PU.1-over-expressing cells showed a marked increase in IL-6 production in response to LPS stimulation in both IL-3/SCF and PWM-SCM cultures. These results suggest that PU.1 overproduction alone is sufficient for both expression of CD11b and F4/80 and for amplification of LPS-induced IL-6 production. However, IFN-gamma stimulation is essential for PU.1-mediated transactivation of CIITA pIV. Reduced expression of mast cell-related molecules and transcription factors GATA-1/2 and up-regulation of C/EBPalpha in PU.1 transfectants indicate that enforced PU.1 suppresses mast cell-specific gene expression through these transcription factors.

  15. Inhibition of NF-IL6 activity by manassantin B, a dilignan isolated from Saururus chinensis, in phorbol myristate acetate-stimulated U937 promonocytic cells.

    PubMed

    Son, Kyung-No; Song, In-sung; Shin, Yong-Hyun; Pai, Tong-Kun; Chung, Dae-Kyun; Baek, Nam-In; Lee, Jung Joon; Kim, Jiyoung

    2005-08-31

    Mannasantin B, a dilignan structurally related to manssantin A, is an inhibitor of NF-kappaB transactivation. In the present study, we found that it inhibited PMA-induced expression of IL-1beta, IL-1beta mRNA, and IL-1beta promoter activity in U937 cells with IC50 values of about 50 nM. It also inhibited NF-IL6- and NF-kappaB-induced activation of IL-1beta, with IC50 values of 78 nM and 1.6 microM, respectively, revealing a potent inhibitory effect on NF-IL6. Electrophoretic mobility shift assays showed that manassantin B had an inhibitory effect on DNA binding by NF-IL6, but not by NF-kappaB. Further analysis revealed that transactivation by NF-IL6 was also inhibited. Our results indicate that manassantin B suppresses expression of IL-1beta in promonocytic cells by inhibiting not only NF-kappaB but also NF-IL6 activity. Furthermore, our observations suggest that manassantin B may be clinically useful as a potent inhibitor of NF-IL6 activity.

  16. Lycopene and other carotenoids inhibit estrogenic activity of 17beta-estradiol and genistein in cancer cells.

    PubMed

    Hirsch, Keren; Atzmon, Andrea; Danilenko, Michael; Levy, Joseph; Sharoni, Yoav

    2007-08-01

    Epidemiological evidence suggests that carotenoids prevent several types of cancer, including mammary and endometrial cancers. On the other hand, such studies have also shown that estrogens are the most important risk factors for these cancer types. Genistein, the phytoestrogen mainly found in soy, also shows significant estrogenic activity when tested at concentrations found in human blood. The aim of this study was to determine whether carotenoids inhibit signaling of steroidal estrogen and phytoestrogen which could explain their cancer preventive activity. Similar to the known effect of 17beta-estradiol (E(2)), treatment of breast (T47D and MCF-7) and endometrial (ECC-1) cancer cells with phytoestrogens induced cell proliferation, cell-cycle progression and transactivation of the estrogen response element (ERE). However, each of the tested carotenoids (lycopene, phytoene, phytofluene, and beta-carotene) inhibited cancer cell proliferation induced by either E(2) or genistein. The inhibition of cell growth by lycopene was accompanied by slow down of cell-cycle progression from G1 to S phase. Moreover, the carotenoids inhibited estrogen-induced transactivation of ERE that was mediated by both estrogen receptors (ERs) ERalpha and ERbeta. The possibility that this inhibition results from competition of carotenoid-activated transcription systems on a limited pool of shared coactivators with the ERE transcription system was tested. Although cotransfection of breast and endometrial cancer cells with four different coactivators (SRC-1, SRC-2, SRC-3, and DRIP) strongly stimulated ERE reporter gene activity, it did not oppose the inhibitory effect of carotenoids. These results suggest that dietary carotenoids inhibit estrogen signaling of both 17beta-estradiol and genistein, and attenuate their deleterious effect in hormone-dependent malignancies.

  17. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a

    PubMed Central

    Seredynski, Aurore L.; Balthazart, Jacques; Ball, Gregory F.

    2015-01-01

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER–mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. SIGNIFICANCE STATEMENT The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  18. A poliovirus 2A(pro) mutant unable to cleave 3CD shows inefficient viral protein synthesis and transactivation defects.

    PubMed Central

    Ventoso, I; Carrasco, L

    1995-01-01

    Four poliovirus mutants with modifications of tyrosine 88 in 2A(pro) were generated and introduced into the cloned poliovirus genome. Mutants Y88P and Y88L were nonviable, mutant Y88F showed a wild-type (WT) phenotype, and mutant Y88S showed a delayed cytopathic effect and formed small plaques in HeLa cells. Growth of Y88S in HeLa cells was restricted, giving rise to about 20% of the PFU production of the WT poliovirus. The 2A (Y88S) mutant synthesized significantly lower levels of viral proteins in HeLa cells than did the WT poliovirus, while the kinetics of p220 cleavage were identical for both viruses. Strikingly, the 2A (Y88S) mutant was unable to cleave 3CD, as shown by analysis of poliovirus proteins labeled with [35S]methionine or immunoblotted with a specific anti-3C serum. The ability of the Y88S mutant to form infectious virus and cleave 3CD can be complemented by the WT poliovirus. Synthesis of viral RNA was diminished in the Y88S mutant but less than the inhibition of translation of viral RNA. Experiments in which guanidine was used to inhibit poliovirus RNA synthesis suggest that the primary defect of the Y88S mutant virus is at the level of poliovirus RNA translation, while viral genome replication is much less affected. Transfection of HeLa cells infected with the WT poliovirus with a luciferase mRNA containing the poliovirus 5' untranslated sequence gives rise to a severalfold increase in luciferase activity. This enhanced translation of leader-luc mRNA was not observed when the transfected cells were infected with the 2A (Y88S) mutant. Moreover, cotransfection with mRNA encoding WT poliovirus 2A(pro) enhanced translation of leader-luc mRNA. This enhancement was much lower upon transfection with mRNA encoding 2A(Y88S), 2A(Y88L), or 2A(Y88P). These findings support the view that 2A(pro) itself, rather than the 3C' and/or 3D' products, is necessary for efficient translation of poliovirus RNA in HeLa cells. PMID:7666528

  19. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a.

    PubMed

    Seredynski, Aurore L; Balthazart, Jacques; Ball, Gregory F; Cornil, Charlotte A

    2015-09-23

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER-mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. Significance statement: The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  20. The C-terminal domain of the nuclear factor I-B2 isoform is glycosylated and transactivates the WAP gene in the JEG-3 cells

    SciTech Connect

    Mukhopadhyay, Sudit S. . E-mail: suditmukhopadhy@yahoo.com; Rosen, Jeffrey M. . E-mail: jrosen@bcm.tmc.edu

    2007-07-06

    The transcription factor nuclear factor I (NFI) has been shown previously both in vivo and in vitro to be involved in the cooperative regulation of whey acidic protein (WAP) gene transcription along with the glucocorticoid receptor and STAT5. In addition, one of the specific NFI isoforms, NFI-B2, was demonstrated in transient co-transfection experiments in JEG cells, which lack endogenous NFI, to be preferentially involved in the cooperative regulation of WAP gene expression. A comparison of the DNA-binding specificities of the different NFI isoforms only partially explained their differential ability to activate the WAP gene transcription. Here, we analyzed the transactivation regions of two NFI isoforms by making chimeric proteins between the NFI-A and B isoforms. Though, their DNA-binding specificities were not altered as compared to the corresponding wild-type transcription factors, the C-terminal region of the NFI-B isoform was shown to preferentially activate WAP gene transcription in cooperation with GR and STAT5 in transient co-transfection assays in JEG-3 cells. Furthermore, determination of serine and threonine-specific glycosylation (O-linked N-acetylglucosamine) of the C-terminus of the NFI-B isoform suggested that the secondary modification by O-GlcNAc might play a role in the cooperative regulation of WAP gene transcription by NFI-B2 and STAT5.

  1. Deubiquitylation of hepatitis B virus X protein (HBx) by ubiquitin-specific peptidase 15 (USP15) increases HBx stability and its transactivation activity

    PubMed Central

    Su, Zhi-Jun; Cao, Jia-Shou; Wu, Yan-Fang; Chen, Wan-Nan; Lin, Xinjian; Wu, Yun-Li; Lin, Xu

    2017-01-01

    Hepatitis B virus X protein (HBx) plays important roles in viral replication and the development of hepatocellular carcinoma. HBx is a rapid turnover protein and ubiquitin-proteasome pathway has been suggested to influence HBx stability as treatment with proteasome inhibitors increases the levels of HBx protein and causes accumulation of the polyubiquitinated forms of HBx. Deubiquitinases (DUBs) are known to act by removing ubiquitin moieties from proteins and thereby reverse their stability and/or activity. However, no information is available regarding the involvement of DUBs in regulation of ubiquitylation-dependent proteasomal degradation of HBx protein. This study identified the deubiquitylating enzyme USP15 as a critical regulator of HBx protein level. USP15 was found to directly interact with HBx via binding to the HBx region between amino acid residues 51 and 80. USP15 increased HBx protein levels in a dose-dependent manner and siRNA-mediated knockdown of endogenous USP15 reduced HBx protein levels. Increased HBx stability and steady-state level by USP15 were attributable to reduced HBx ubiquitination and proteasomal degradation. Importantly, the transcriptional transactivation function of HBx is enhanced by overexpression of USP15. These results suggest that USP15 plays an essential role in stabilizing HBx and subsequently affects the biological function of HBx. PMID:28074857

  2. Invariant chain+ N2a neuroblastoma cells stably expressing the class II MHC transactivator CIITA fail to stimulate anti-tumor immunity.

    PubMed

    Rickard, Steve; Ono, Santa Jeremy

    2008-12-01

    A promising cancer treatment strategy involves stimulation of anti-tumor immune responses. CD4(+) T cell responses are particularly desirable, as they enhance CD8(+) T cell activity and provide immune memory. The major histocompatibility complex (MHC) class II transactivator CIITA can be used to stimulate expression of MHC II on tumor cells, thereby promoting CD4(+) T cell activation. In this study, N2a neuroblastoma cells were stably transfected with CIITA. N2aCIITA cells displayed increased expression of MHC I, MHC II and invariant chain; CD80 and CD86 were expressed by neither the parental N2a cells nor by the N2aCIITA cells. All mice injected with N2aCIITA cells developed tumors. Furthermore, no increase in the numbers of T cells, natural killer cells, macrophages, or eosinophils was observed in the spleens or tumors of mice injected with N2aCIITA cells, compared to tissues from mice injected with the parental N2a cells. This absence of an anti-tumor immune response despite MHC II expression is likely due to the presence of invariant chain, in support of the MHCII(+)/Ii(-) paradigm.

  3. Whole-genome analysis of genetic recombination of hepatitis delta virus: molecular domain in delta antigen determining trans-activating efficiency.

    PubMed

    Chao, Mei; Lin, Chia-Chi; Lin, Feng-Ming; Li, Hsin-Pai; Iang, Shan-Bei

    2015-12-01

    Hepatitis delta virus (HDV) is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity and is replicated by host RNA polymerase. HDV RNA recombination was previously demonstrated in patients and in cultured cells by analysis of a region corresponding to the C terminus of the delta antigen (HDAg), the only viral-encoded protein. Here, a whole-genome recombination map of HDV was constructed using an experimental system in which two HDV-1 sequences were co-transfected into cultured cells and the recombinants were analysed by sequencing of cloned reverse transcription-PCR products. Fifty homologous recombinants with 60 crossovers mapping to 22 junctions were identified from 200 analysed clones. Small HDAg chimeras harbouring a junction newly detected in the recombination map were then constructed. The results further indicated that the genome-replication level of HDV was sensitive to the sixth amino acid within the N-terminal 22 aa of HDAg. Therefore, the recombination map established in this study provided a tool for not only understanding HDV RNA recombination, but also elucidating the related mechanisms, such as molecular elements responsible for the trans-activation levels of the small HDAg.

  4. Transcription initiation in vivo without classical transactivators: DNA kinks flanking the core promoter of the housekeeping yeast adenylate kinase gene, AKY2, position nucleosomes and constitutively activate transcription.

    PubMed

    Angermayr, Michaela; Oechsner, Ulrich; Gregor, Kerstin; Schroth, Gary P; Bandlow, Wolfhard

    2002-10-01

    The housekeeping gene of the major adenylate kinase in Saccharomyces cerevisiae (AKY2, ADK1) is constitutively transcribed at a moderate level. The promoter has been dissected in order to define elements that effect constitutive transcription. Initiation of mRNA synthesis at the AKY2 promoter is shown to be mediated by a non-canonic core promoter, (TA)(6). Nucleotide sequences 5' of this element only marginally affect transcription suggesting that promoter activation can dispense with transactivators and essentially involves basal transcription. We show that the core promoter of AKY2 is constitutively kept free of nucleosomes. Analyses of permutated AKY2 promoter DNA revealed the presence of bent DNA. DNA structure analysis by computer and by mutation identified two kinks flanking an interstitial stretch of 65 bp of moderately bent core promoter DNA. Kinked DNA is likely incompatible with packaging into nucleosomes and responsible for positioning nucleosomes at the flanks allowing unimpeded access of the basal transcription machinery to the core promoter. The data show that in yeast, constitutive gene expression can dispense with classical transcriptional activator proteins, if two prerequisites are met: (i) the core promoter is kept free of nucleosomes; this can be due to structural properties of the DNA as an alternative to chromatin remodeling factors; and (ii) the core promoter is pre-bent to allow a high rate of basal transcription initiation.

  5. Role of acylglycerol kinase in LPA-induced IL-8 secretion and transactivation of epidermal growth factor-receptor in human bronchial epithelial cells

    PubMed Central

    Kalari, Satish; Zhao, Yutong; Spannhake, Ernst Wm.; Berdyshev, Evgeny V.; Natarajan, Viswanathan

    2009-01-01

    LPA (lysophosphatidic acid) is a potent bioactive phospholipid, which regulates a number of diverse cellular responses through G protein-coupled LPA receptors. Intracellular LPA is generated by the phosphorylation of monoacylglycerol by acylglycerol kinase (AGK); however, the role of intracellular LPA in signaling and cellular responses remains to be elucidated. Here, we investigated signaling pathways of IL-8 secretion mediated by AGK and intracellular LPA in human bronchial epithelial cells (HBEpCs). Expression of AGK in HBEpCs was detected by real-time PCR, and overexpressed AGK was mainly localized in mitochondria as determined by immunofluorescence and confocal microscopy. Overexpression of lentiviral AGK wild type increased intracellular LPA production (∼1.8-fold), enhanced LPA-mediated IL-8 secretion, and stimulated tyrosine phosphorylation epidermal growth factor-receptor (EGF-R). Furthermore, downregulation of native AGK by AGK small interfering RNA decreased intracellular LPA levels (∼2-fold) and attenuated LPA-induced p38 MAPK, JNK, and NF-κB activation, tyrosine phosphorylation of EGF-R, and IL-8 secretion. These results suggest that native AGK regulates LPA-mediated IL-8 secretion involving MAPKs, NF-κB, and transactivation of EGF-R. Thus AGK may play an important role in innate immunity and airway remodeling during inflammation. PMID:19112101

  6. Coexisting adult polyglucosan body disease with frontotemporal lobar degeneration with transactivation response DNA-binding protein-43 (TDP-43)-positive neuronal inclusions.

    PubMed

    Farmer, Jill G; Crain, Barbara J; Harris, Brent T; Turner, R Scott

    2013-01-01

    Frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) is one of the most common pathological findings associated with the clinical FTLD syndromes. However, molecular characterization with genetic sequencing and protein expression techniques are recognizing many new subtypes for FTLDs. FTLDs are diverse and new nomenclature schemes have been proposed based on the molecular defects that are being discovered ( Mackenzie et al., 2010 , Acta Neuropathologica, 119, 1). Adult polyglucosan body disease (APBD) is a very rare disorder associated with systemic neurological signs and symptoms including progressive dementia with executive dysfunction and motor neuron disease. We report the clinical course of an individual with a clinical FTLD and the as yet unreported findings of coexistent APBD with FTLD-U and transactivation response DNA-binding protein-43 (TDP-43)-positive inclusions at autopsy (or more accurately, FTLD-TDP). It is unclear if these distinct findings are coincidental in this individual, or if pathogenic pathways may intersect to promote these coexisting pathologies.

  7. Nuclear factor-I and activator protein-2 bind in a mutually exclusive way to overlapping promoter sequences and trans-activate the human growth hormone gene.

    PubMed Central

    Courtois, S J; Lafontaine, D A; Lemaigre, F P; Durviaux, S M; Rousseau, G G

    1990-01-01

    Transcription of the human growth hormone (hGH) gene and its regulation are controlled by trans-acting factors that bind to hGH gene promoter sequences. Several DNase I footprints have been described within 500 bp of this promoter, one of which (-289 to -267) has not yet been ascribed to a defined factor. By DNase I footprinting, gel mobility shift, and methylation interference assays with extracts from HeLa cells and GH-producing pituitary tumor (GC) cells, we show that this factor belongs to the NF-I family. When NF-I was competed out of the cell extracts, the trans-acting factor AP-2 bound to the same site as NF-I. AP-2 was present not only in HeLa cells, but also in GC cells albeit at a much lower concentration. Consistent with the mutually exclusive binding of NF-I and AP-2, their methylation interference patterns included four guanine residues that were crucial for binding of both NF-I and AP-2. Cell-free transcription from the hGH gene promoter showed that these two factors can transactivate this gene. Images PMID:2308836

  8. Transcription factor USF from duck erythrocytes transactivates expression of the histone H5 gene in vitro by interacting with an intragenic sequence.

    PubMed Central

    Düring, F; Gerhold, H; Seifart, K H

    1990-01-01

    The duck histone H5 gene contains a 12 base pair (bp) sequence motif within the coding region, which shows homology in 10 out of 12 bp with the consensus sequence of the USF binding site in the Ad2ML-promoter. The functional equivalent of transcription factor USF, partially purified from whole cell extracts of duck erythrocytes (EUSF), was shown to interact with this intragenic sequence. Electrophoretic mobility shift analyses revealed the selective formation of a complex between this protein fraction and the duck H5 gene. Footprint assays with DNase I delineated specific binding to the intragenic sequence outlined above. Moreover this protein fraction, containing EUSF, transactivates the expression of the duck H5 gene in vitro and elimination of the USF-consensus sequence leads to a loss of stimulation but retains the basic transcription of the gene. These results suggest an as yet unknown functional role of EUSF in the expression of the H5 gene of the duck. Images PMID:2320416

  9. The RNA polymerase I transactivator upstream binding factor requires its dimerization domain and high-mobility-group (HMG) box 1 to bend, wrap, and positively supercoil enhancer DNA.

    PubMed Central

    Putnam, C D; Copenhaver, G P; Denton, M L; Pikaard, C S

    1994-01-01

    Upstream binding factor (UBF) is an important transactivator of RNA polymerase I and is a member of a family of proteins that contain nucleic acid binding domains named high-mobility-group (HMG) boxes because of their similarity to HMG chromosomal proteins. UBF is a highly sequence-tolerant DNA-binding protein for which no binding consensus sequence has been identified. Therefore, it has been suggested that UBF may recognize preformed structural features of DNA, a hypothesis supported by UBF's ability to bind synthetic DNA cruciforms, four-way junctions, and even tRNA. We show here that full-length UBF can also bend linear DNA to mediate circularization of probes as small as 102 bp in the presence of DNA ligase. Longer probes in the presence of UBF become positively supercoiled when ligated, suggesting that UBF wraps the DNA in a right-handed direction, opposite the direction of DNA wrapping around a nucleosome. The dimerization domain and HMG box 1 are necessary and sufficient to circularize short probes and supercoil longer probes in the presence of DNA ligase. UBF's sequence tolerance coupled with its ability to bend and wrap DNA makes UBF an unusual eukaryotic transcription factor. However, UBF's ability to bend DNA might explain how upstream and downstream rRNA gene promoter domains interact. UBF-induced DNA wrapping could also be a mechanism by which UBF counteracts histone-mediated gene repression. Images PMID:7935371

  10. OsMYC2, an essential factor for JA-inductive sakuranetin production in rice, interacts with MYC2-like proteins that enhance its transactivation ability

    PubMed Central

    Ogawa, Satoshi; Miyamoto, Koji; Nemoto, Keiichirou; Sawasaki, Tatsuya; Yamane, Hisakazu; Nojiri, Hideaki; Okada, Kazunori

    2017-01-01

    Biosynthesis of sakuranetin, a flavonoid anti-fungal phytoalexin that occurs in rice, is highly dependent on jasmonic acid (JA) signalling and induced by a variety of environmental stimuli. We previously identified OsNOMT, which encodes naringenin 7-O-methyltransferase (NOMT); NOMT is a key enzyme for sakuranetin production. Although OsNOMT expression is induced by JA treatment, the regulation mechanism that activates the biosynthetic pathway of sakuranetin has not yet been elucidated. In this study, we show that JA-inducible basic helix-loop-helix transcriptional factor OsMYC2 drastically enhances the activity of the OsNOMT promoter and is essential for JA-inducible sakuranetin production. In addition, we identified 2 collaborators of OsMYC2, OsMYC2-like protein 1 and 2 (OsMYL1 and OsMYL2) that further activated the OsNOMT promoter in synergy with OsMYC2. Physical interaction of OsMYC2 with OsMYL1 and OsMYL2 further supported the idea that these interactions lead to the enhancement of the transactivation activity of OsMYC2. Our results indicate that JA signalling via OsMYC2 is reinforced by OsMYL1 and OsMYL2, resulting in the inductive production of sakuranetin during defence responses in rice. PMID:28067270

  11. Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

    PubMed Central

    Geng, Deyu; Zhang, Zhixia; Guo, Huarong

    2012-01-01

    p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase) and pRL-CMV-luc (CMV promoter linked to Renilla luciferase) into marine flatfish flounder gill (FG) cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C) and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation), but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl) phthalate (DEHP), a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner. PMID:25585933

  12. Stromal cell-derived factor-1{alpha} (SDF-1{alpha}/CXCL12) stimulates ovarian cancer cell growth through the EGF receptor transactivation

    SciTech Connect

    Porcile, Carola; Bajetto, Adriana . E-mail: bajetto@cba.unige.it; Barbieri, Federica; Barbero, Simone; Bonavia, Rudy; Biglieri, Marianna; Pirani, Paolo; Florio, Tullio . E-mail: florio@cba.unige.it; Schettini, Gennaro

    2005-08-15

    Ovarian cancer (OC) is the leading cause of death in gynecologic diseases in which there is evidence for a complex chemokine network. Chemokines are a family of proteins that play an important role in tumor progression influencing cell proliferation, angiogenic/angiostatic processes, cell migration and metastasis, and, finally, regulating the immune cells recruitment into the tumor mass. We previously demonstrated that astrocytes and glioblastoma cells express both the chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1), and that SDF-1{alpha} treatment induced cell proliferation, supporting the hypothesis that chemokines may play an important role in tumor cells' growth in vitro. In the present study, we report that CXCR4 and SDF-1 are expressed in OC cell lines. We demonstrate that SDF-1{alpha} induces a dose-dependent proliferation in OC cells, by the specific interaction with CXCR4 and a biphasic activation of ERK1/2 and Akt kinases. Our results further indicate that CXCR4 activation induces EGF receptor (EGFR) phosphorylation that in turn was linked to the downstream intracellular kinases activation, ERK1/2 and Akt. In addition, we provide evidence for cytoplasmic tyrosine kinase (c-Src) involvement in the SDF-1/CXCR4-EGFR transactivation. These results suggest a possible important 'cross-talk' between SDF-1/CXCR4 and EGFR intracellular pathways that may link signals of cell proliferation in ovarian cancer.

  13. Equine herpesvirus 1 gene 12, the functional homologue of herpes simplex virus VP16, transactivates via octamer sequences in the equine herpesvirus IE gene promoter.

    PubMed

    Elliott, G; O'Hare, P

    1995-10-20

    The HSV-1 transactivator of immediate-early (IE) gene expression, VP16, has several functional homologues among the alphaherpesviruses which have not yet been extensively studied in relation to their modes of action. To date, nothing is known of the exact sites or mechanism of interaction of the equine herpesvirus type 1 (EHV-1) homologue, the gene 12 protein, with the EHV-1 IE promoter. We show that the gene 12 protein utilises the promoter proximal region of the IE gene to induce activation and identify four potential octamer DNA binding sites within that region. Although there was divergence from its consensus, Oct-1 bound to each of these sites in an in vitro complex formation assay, and in the presence of the gene 12 product a second complex of slower migration, which was also dependent on Oct-1, was detected. When each site was inserted into a basal promoter, two conferred activation by gene 12 with a resulting increase in expression of up to 50-fold compared to basal levels. These results show that, despite the differences between the two proteins, the mechanism of interaction of the gene 12 protein with its target is analogous to that of VP16.

  14. Wnt/β-catenin pathway transactivates microRNA-150 that promotes EMT of colorectal cancer cells by suppressing CREB signaling.

    PubMed

    Guo, Yan-Hua; Wang, Lu-Qin; Li, Bin; Xu, Hui; Yang, Jian-Hua; Zheng, Li-Si; Yu, Peng; Zhou, Ai-Dong; Zhang, Yin; Xie, Shu-Juan; Liang, Zi-Rui; Zhang, Chen-Min; Zhou, Hui; Qu, Liang-Hu

    2016-07-05

    A hallmark of aberrant activation of the Wnt/β-catenin signaling pathway has been observed in most colorectal cancers (CRC), but little is known about the role of non-coding RNAs regulated by this pathway. Here, we found that miR-150 was the most significantly upregulated microRNA responsive to elevated of Wnt/β-catenin signaling activity in both HCT116 and HEK293T cells. Mechanistically, the β-catenin/LEF1 complex binds to the conserved TCF/LEF1-binding element in the miR-150 promoter and thereby transactivates its expression. Enforced expression of miR-150 in HCT116 cell line transformed cells into a spindle shape with higher migration and invasion activity. miR-150 markedly suppressed the CREB signaling pathway by targeting its core transcription factors CREB1 and EP300. Knockdown of CREB1 or EP300 and knockout of CREB1 by CRISPR/Cas9 phenocopied the epithelial-mesenchymal transition (EMT) observed in HCT116 cells in response to miR-150 overexpression. In summary, our data indicate that miR-150 is a novel Wnt effector that may significantly enhance EMT of CRC cells by targeting the CREB signaling pathway.

  15. Coupling of LETM1 up-regulation with oxidative phosphorylation and platelet-derived growth factor receptor signaling via YAP1 transactivation

    PubMed Central

    Lee, Jandee; Lee, Woo Kyung; Seol, Mi-Youn; Lee, Seul Gi; Kim, Daham; Kim, Hyunji; Park, Jongsun; Jung, Sang Geun; Chung, Woong Youn; Lee, Eun Jig; Jo, Young Suk

    2016-01-01

    Persistent cellular proliferation and metabolic reprogramming are essential processes in carcinogenesis. Here, we performed Gene Set Enrichment Analysis (GSEA) and found that that LETM1, a mitochondrial calcium transporter, is associated with cellular growth signals such as platelet-derived growth factor (PDGF) receptor signaling and insulin signaling pathways. These results were then verified by qRT-PCR and immnunoblotting. Mechanistically, up-regulation of LETM1 induced YAP1 nuclear accumulation, increasing the expression of PDGFB, PDGFRB and THBS4. Consistent with this, LETM1 silencing caused loss of YAP1 nuclear signal, decreasing the expression of PDGFB, PDGFRB and THBS4. Immunohistochemical staining consistently indicated a positive association between LETM1 up-regulation, YAP1 nuclear localization and high PDGFB expression. In clinical data analysis, LETM1 up-regulation in thyroid cancer was found to be related to aggressive tumor features such as lymphovascular invasion (LVI, P < 0.001) and lymph node metastasis (LNM, P = 0.011). Multivariate analysis demonstrated that LETM1 up-regulation increases the risk of LVI and LNM (OR = 3.455, 95% CI = 1.537–7.766 and OR = 3.043, 95% CI = 1.282–7.225, respectively). Collectively, these data suggest that up-regulation of LETM1 induces sustained activation of proliferative signaling pathways, such as PDGF signal pathway by AKT induced YAP1 transactivation, resulting in aggressive thyroid cancer phenotypes. PMID:27556512

  16. Genomic duplication and translocation of reactivation transactivator and bZIP-homolog genes is a conserved event in alcelaphine herpesvirus 1

    PubMed Central

    Myster, Françoise; van Beurden, Steven J.; Sorel, Océane; Suárez, Nicolás M.; Vanderplasschen, Alain; Davison, Andrew J.; Dewals, Benjamin G.

    2016-01-01

    Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus carried asymptomatically by wildebeest. Upon cross-species transmission, AlHV-1 induces malignant catarrhal fever (MCF), a fatal lymphoproliferative disease of ruminants, including cattle. The strain C500 has been cloned as an infectious, pathogenic bacterial artificial chromosome (BAC) that is used to study MCF. Although AlHV-1 infection can be established in cell culture, multiple passages in vitro cause a loss of virulence associated with rearrangements of the viral genome. Here, sequencing of the BAC clone showed that the long unique region (LUR) of the genome is nearly identical to that of the previously sequenced strain from which the BAC was derived, and identified the duplication and translocation of a region from within LUR, containing the entire coding sequences of ORF50-encoding reactivation transactivator Rta and A6-encoding bZIP protein genes. The duplicated region was further located to a position within the terminal repeat (TR) and its deletion resulted in lower ORF50 expression levels and reduced viral fitness. Finally, the presence of a similar but not identical duplication and translocation containing both genes was found in AlHV-1 strain WC11. These results indicate that selection pressure for enhanced viral fitness may drive the duplication of ORF50 and A6 in AlHV-1. PMID:27924936

  17. Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene.

    PubMed

    Waldburger, J M; Suter, T; Fontana, A; Acha-Orbea, H; Reith, W

    2001-08-20

    MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-gamma-induced MHCII expression on a wide variety of non-bone marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4(+) T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-gamma-activated cells of the macrophage lineage. pIV(-/-) mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.

  18. Long non-coding RNA NEAT1 is a transcriptional target of p53 and modulates p53-induced transactivation and tumor-suppressor function.

    PubMed

    Idogawa, Masashi; Ohashi, Tomoko; Sasaki, Yasushi; Nakase, Hiroshi; Tokino, Takashi

    2017-03-14

    p53 is one of the most important tumor suppressor genes, and the direct transcriptional targets of p53 must be explored to elucidate its functional mechanisms. Thus far, the p53 targets that have been primarily studied are protein-coding genes. Our previous study revealed that several long non-coding RNAs (lncRNAs) are direct transcriptional targets of p53, and knockdown of specific lncRNAs modulates p53-induced apoptosis. In this study, analysis of next-generation chromatin immunoprecipitation-sequencing (ChIP-seq) data for p53 revealed that the lncRNA NEAT1 is a direct transcriptional target of p53. The suppression of NEAT1 induction by p53 attenuates the inhibitory effect of p53 on cancer cell growth and also modulates gene transactivation, including that of many lncRNAs. Furthermore, low expression of NEAT1 is related to poor prognosis in several cancers. These results indicate that the induction of NEAT1 expression contributes to the tumor-suppressor function of p53 and suggest that p53 and NEAT1 constitute a transcriptional network contributing to various biological functions and tumor suppression.

  19. Novel mutations within the RFX-B gene and partial rescue of MHC and related genes through exogenous class II transactivator in RFX-B-deficient cells.

    PubMed

    Nagarajan, U M; Peijnenburg, A; Gobin, S J; Boss, J M; van den elsen, P J

    2000-04-01

    MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific regulatory factors. Fibroblasts derived from two recently identified bare lymphocyte syndrome patients, EBA and FZA, were found to contain novel mutations in the RFX-B gene. RFX-B encodes a component of the RFX transcription factor that functions in the assembly of multiple transcription factors on MHC class II promoters. Unlike RFX5- and RFXAP-deficient cells, transfection of exogenous class II transactivator (CIITA) into these RFX-B-deficient fibroblasts resulted in the induction of HLA-DR and HLA-DP and, to a lesser extent, HLA-DQ. Similarly, CIITA-mediated induction of MHC class I, beta2-microglobulin, and invariant chain genes was also found in these RFX-B-deficient fibroblasts. Expression of wild-type RFX-B completely reverted the noted deficiencies in these cells. Transfection of CIITA into Ramia cells, a B cell line that does not produce a stable RFX-B mRNA, resulted in induction of an MHC class II reporter, suggesting that CIITA overexpression may partially override the RFX-B defect.

  20. The Krüppel-like zinc finger protein GLIS3 transactivates neurogenin 3 for proper fetal pancreatic islet differentiation in mice

    PubMed Central

    Yang, Y.; Chang, B. H-J.; Yechoor, V.; Chen, W.; Li, L.; Tsai, M.-J.

    2011-01-01

    Aims/hypothesis Mutations in GLIS3, which encodes a Krüppel-like zinc finger transcription factor, were found to underlie sporadic neonatal diabetes. Inactivation of Glis3 by gene targeting in mice was previously shown to lead to neonatal diabetes, but the underlying mechanism remains largely unknown. We aimed to elucidate the mechanism of action of GLIS family zinc finger 3 (GLIS3) in Glis3−/− mice and to further decipher its action in in-vitro systems. Methods We created Glis3−/− mice and monitored the morphological and biochemical phenotype of their pancreatic islets at different stages of embryonic development. We combined these observations with experiments on Glis3 expressed in cultured cells, as well as in in vitro systems in the presence of other reconstituted components. Results In vivo and in vitro analyses placed Glis3 upstream of Neurog3, the endocrine pancreas lineage-defining transcription factor. We found that GLIS3 binds to specific GLIS3-response elements in the Neurog3 promoter, activating Neurog3 gene transcription both directly, and synergistically with hepatic nuclear factor 6 and forkhead box A2. Conclusions/interpretation These results indicate that GLIS3 controls fetal islet differentiation via direct transactivation of Neurog3, a perturbation that causes neonatal diabetes in mice. PMID:21786021

  1. Identification of small molecules that induce apoptosis in a Myc-dependent manner and inhibit Myc-driven transformation

    PubMed Central

    Mo, Hao; Henriksson, Marie

    2006-01-01

    The Myc transcription factor plays a central role in the regulation of cell cycle progression, apoptosis, angiogenesis, and cellular transformation. Myc is a potent oncoprotein that is deregulated in a wide variety of human tumors and is therefore an attractive target for novel cancer therapies. Using a cellular screening approach, we have identified low-molecular-weight compounds, Myc pathway response agents (MYRAs), that induce apoptosis in a c-Myc-dependent manner and inhibit Myc-driven cellular transformation. MYRA-A inhibits Myc transactivation and interferes with the DNA-binding activity of Myc family proteins but has no effect on the E-box-binding protein USF. In contrast, MYRA-B induces Myc-dependent apoptosis without affecting Myc transactivation or Myc/Max DNA binding. Our data show that cellular screening assays can be a powerful strategy for the identification of candidate substances that modulate the Myc pathway. These compounds can be useful tools for studying Myc function and may also be of therapeutic potential as leads for drug development. PMID:16606833

  2. Glucocorticoid receptor activation inhibits p53-induced apoptosis of MCF10Amyc cells via induction of protein kinase Cε.

    PubMed

    Aziz, Moammir H; Shen, Hong; Maki, Carl G

    2012-08-24

    Glucocorticoid receptor (GR) is a ligand-dependent transcription factor that can promote apoptosis or survival in a cell-specific manner. Activated GR has been reported to inhibit apoptosis in mammary epithelial cells and breast cancer cells by increasing pro-survival gene expression. In this study, activated GR inhibited p53-dependent apoptosis in MCF10A cells and human mammary epithelial cells that overexpress the MYC oncogene. Specifically, GR agonists hydrocortisone or dexamethasone inhibited p53-dependent apoptosis induced by cisplatin, ionizing radiation, or the MDM2 antagonist Nutlin-3. In contrast, the GR antagonist RU486 sensitized the cells to apoptosis by these agents. Apoptosis inhibition was associated with maintenance of mitochondrial membrane potential, diminished caspase-3 and -7 activation, and increased expression at both the mRNA and protein level of the anti-apoptotic PKC family member PKCε. Knockdown of PKCε via siRNA targeting reversed the protective effect of dexamethasone and restored apoptosis sensitivity. These data provide evidence that activated GR can inhibit p53-dependent apoptosis through induction of the anti-apoptotic factor PKCε.

  3. Xylosylation of the Notch receptor preserves the balance between its activation by trans-Delta and inhibition by cis-ligands in Drosophila.

    PubMed

    Lee, Tom V; Pandey, Ashutosh; Jafar-Nejad, Hamed

    2017-04-10

    The Drosophila glucoside xylosyltransferase Shams xylosylates Notch and inhibits Notch signaling in specific contexts including wing vein development. However, the molecular mechanisms underlying context-specificity of the shams phenotype is not known. Considering the role of Delta-Notch signaling in wing vein formation, we hypothesized that Shams might affect Delta-mediated Notch signaling in Drosophila. Using genetic interaction studies, we find that altering the gene dosage of Delta affects the wing vein and head bristle phenotypes caused by loss of Shams or by mutations in the Notch xylosylation sites. Clonal analysis suggests that loss of shams promotes Delta-mediated Notch activation. Further, Notch trans-activation by ectopically overexpressed Delta shows a dramatic increase upon loss of shams. In agreement with the above in vivo observations, cell aggregation and ligand-receptor binding assays show that shams knock-down in Notch-expressing cells enhances the binding between Notch and trans-Delta without affecting the binding between Notch and trans-Serrate and cell surface levels of Notch. Loss of Shams does not impair the cis-inhibition of Notch by ectopic overexpression of ligands in vivo or the interaction of Notch and cis-ligands in S2 cells. Nevertheless, removing one copy of endogenous ligands mimics the effects of loss shams on Notch trans-activation by ectopic Delta. This favors the notion that trans-activation of Notch by Delta overcomes the cis-inhibition of Notch by endogenous ligands upon loss of shams. Taken together, our data suggest that xylosylation selectively impedes the binding of Notch with trans-Delta without affecting its binding with cis-ligands and thereby assists in determining the balance of Notch receptor's response to cis-ligands vs. trans-Delta during Drosophila development.

  4. Transactivation of EGF receptor and ErbB2 protects intestinal epithelial cells from TNF-induced apoptosis.

    PubMed

    Yamaoka, Toshimitsu; Yan, Fang; Cao, Hanwei; Hobbs, Stuart S; Dise, Rebecca S; Tong, Wei; Polk, D Brent

    2008-08-19

    TNF is a pleiotropic cytokine that activates both anti- and proapoptotic signaling pathways, with cell fate determined by the balance between these two pathways. Activation of ErbB family members, including EGF receptor (EGFR/ErbB1), promotes cell survival and regulates several signals that overlap with those stimulated by TNF. This study was undertaken to determine the effects of TNF on EGFR and ErbB2 activation and intestinal epithelial cell survival. Mice, young adult mouse colon epithelial cells, and EGFR knockout mouse colon epithelial cells were treated with TNF. Activation of EGFR, ErbB2, Akt, Src, and apoptosis were determined in vivo and in vitro. TNF stimulated EGFR phosphorylation in young adult mouse colon epithelial cells, and loss of EGFR expression or inhibition of kinase activity increased TNF-induced apoptosis, which was prevented in WT but not by kinase-inactive EGFR expression. Similarly, TNF injection stimulated apoptosis in EGFR-kinase-defective mice (EGFR(wa2)) compared with WT mice. TNF also activated ErbB2, and loss of ErbB2 expression increased TNF-induced apoptosis. Furthermore, Src-kinase activity and the expression of both EGFR and ErbB2 were required for TNF-induced cell survival. Akt was shown to be a downstream target of TNF-activated EGFR and ErbB2. These findings demonstrate that EGFR and ErbB2 are critical mediators of TNF-regulated antiapoptotic signals in intestinal epithelial cells. Given evidence for TNF signaling in the development of colitis-associated carcinoma, this observation has significant implications for understanding the role of EGFR in maintaining intestinal epithelial cell homeostasis during cytokine-mediated inflammatory responses.

  5. SHARP1/DEC2 inhibits adipogenic differentiation by regulating the activity of C/EBP.

    PubMed

    Gulbagci, Neriman Tuba; Li, Li; Ling, Belinda; Gopinadhan, Suma; Walsh, Martin; Rossner, Moritz; Nave, Klaus-Armin; Taneja, Reshma

    2009-01-01

    SHARP1, a basic helix-loop-helix transcription factor, is expressed in many cell types; however, the mechanisms by which it regulates cellular differentiation remain largely unknown. Here, we show that SHARP1 negatively regulates adipogenesis. Although expression of the early marker CCAAT/enhancer binding protein beta (C/EBPbeta) is not altered, its crucial downstream targets C/EBPalpha and peroxisome proliferator-activated receptor gamma (PPARgamma) are downregulated by SHARP1. Protein interaction studies confirm that SHARP1 interacts with and inhibits the transcriptional activity of both C/EBPbeta and C/EBPalpha, and enhances the association of C/EBPbeta with histone deacetylase 1 (HDAC1). Consistently, in SHARP1-expressing cells, HDAC1 and the histone methyltransferase G9a are retained at the C/EBP regulatory sites on the C/EBPalpha and PPARgamma2 promoters during differentiation, resulting in inhibition of their expression. Interestingly, treatment with troglitazone results in displacement of HDAC1 and G9a, and rescues the differentiation defect of SHARP1-overexpressing cells. Our data indicate that SHARP1 inhibits adipogenesis through the regulation of C/EBP activity, which is essential for PPARgamma-ligand-dependent displacement of co-repressors from adipogenic promoters.

  6. Nongenomic signaling of the retinoid X receptor through binding and inhibiting Gq in human platelets

    PubMed Central

    Moraes, Leonardo A.; Swales, Karen E.; Wray, Jessica A.; Damazo, Amilcar; Gibbins, Jonathan M.; Warner, Timothy D.

    2007-01-01

    Retinoid X receptors (RXRs) are important transcriptional nuclear hormone receptors, acting as either homodimers or the binding partner for at least one fourth of all the known human nuclear receptors. Functional nongenomic effects of nuclear receptors are poorly understood; however, recently peroxisome proliferator-activated receptor (PPAR) γ, PPARβ, and the glucocorticoid receptor have all been found active in human platelets. Human platelets express RXRα and RXRβ. RXR ligands inhibit platelet aggregation and TXA2 release to ADP and the TXA2 receptors, but only weakly to collagen. ADP and TXA2 both signal via the G protein, Gq. RXR rapidly binds Gq but not Gi/z/o/t/gust in a ligand-dependent manner and inhibits Gq-induced Rac activation and intracellular calcium release. We propose that RXR ligands may have beneficial clinical actions through inhibition of platelet activation. Furthermore, our results demonstrate a novel nongenomic mode for nuclear receptor action and a functional cross-talk between G-protein and nuclear receptor signaling families. PMID:17213293

  7. MHC class II transactivator is an in vivo regulator of osteoclast differentiation and bone homeostasis co-opted from adaptive immunity.

    PubMed

    Benasciutti, Elisa; Mariani, Elisabetta; Oliva, Laura; Scolari, Maria; Perilli, Egon; Barras, Emmanuele; Milan, Enrico; Orfanelli, Ugo; Fazzalari, Nicola L; Campana, Lara; Capobianco, Annalisa; Otten, Luc; Particelli, Francesca; Acha-Orbea, Hans; Baruffaldi, Fabio; Faccio, Roberta; Sitia, Roberto; Reith, Walter; Cenci, Simone

    2014-02-01

    The molecular networks controlling bone homeostasis are not fully understood. The common evolution of bone and adaptive immunity encourages the investigation of shared regulatory circuits. MHC Class II Transactivator (CIITA) is a master transcriptional co-activator believed to be exclusively dedicated for antigen presentation. CIITA is expressed in osteoclast precursors, and its expression is accentuated in osteoporotic mice. We thus asked whether CIITA plays a role in bone biology. To this aim, we fully characterized the bone phenotype of two mouse models of CIITA overexpression, respectively systemic and restricted to the monocyte-osteoclast lineage. Both CIITA-overexpressing mouse models revealed severe spontaneous osteoporosis, as assessed by micro-computed tomography and histomorphometry, associated with increased osteoclast numbers and enhanced in vivo bone resorption, whereas osteoblast numbers and in vivo bone-forming activity were unaffected. To understand the underlying cellular and molecular bases, we investigated ex vivo the differentiation of mutant bone marrow monocytes into osteoclasts and immune effectors, as well as osteoclastogenic signaling pathways. CIITA-overexpressing monocytes differentiated normally into effector macrophages or dendritic cells but showed enhanced osteoclastogenesis, whereas CIITA ablation suppressed osteoclast differentiation. Increased c-fms and receptor activator of NF-κB (RANK) signaling underlay enhanced osteoclast differentiation from CIITA-overexpressing precursors. Moreover, by extending selected phenotypic and cellular analyses to additional genetic mouse models, namely MHC Class II deficient mice and a transgenic mouse line lacking a specific CIITA promoter and re-expressing CIITA in the thymus, we excluded MHC Class II expression and T cells from contributing to the observed skeletal phenotype. Altogether, our study provides compelling genetic evidence that CIITA, the molecular switch of antigen presentation

  8. Plasma Kallikrein Promotes Epidermal Growth Factor Receptor Transactivation and Signaling in Vascular Smooth Muscle through Direct Activation of Protease-activated Receptors*

    PubMed Central

    Abdallah, Rany T.; Keum, Joo-Seob; Lee, Mi-Hye; Wang, Bing; Gooz, Monika; Luttrell, Deirdre K.; Luttrell, Louis M.; Jaffa, Ayad A.

    2010-01-01

    The kallikrein-kinin system, along with the interlocking renin-angiotensin system, is a key regulator of vascular contractility and injury response. The principal effectors of the kallikrein-kinin system are plasma and tissue kallikreins, proteases that cleave high molecular weight kininogen to produce bradykinin. Most of the cellular actions of kallikrein (KK) are thought to be mediated by bradykinin, which acts via G protein-coupled B1 and B2 bradykinin receptors on VSMCs and endothelial cells. Here, we find that primary aortic vascular smooth muscle but not endothelial cells possess the ability to activate plasma prekallikrein. Surprisingly, exposing VSMCs to prekallikrein leads to activation of the ERK1/2 mitogen-activated protein kinase cascade via a mechanism that requires kallikrein activity but does not involve bradykinin receptors. In transfected HEK293 cells, we find that plasma kallikrein directly activates G protein-coupled protease-activated receptors (PARs) 1 and 2, which possess consensus kallikrein cleavage sites, but not PAR4. In vascular smooth muscles, KK stimulates ADAM (a disintegrin and metalloprotease) 17 activity via a PAR1/2 receptor-dependent mechanism, leading sequentially to release of the endogenous ADAM17 substrates, amphiregulin and tumor necrosis factor-α, metalloprotease-dependent transactivation of epidermal growth factor receptors, and metalloprotease and epidermal growth factor receptor-dependent ERK1/2 activation. These results suggest a novel mechanism of bradykinin-independent kallikrein action that may contribute to the regulation of vascular responses in pathophysiologic states, such as diabetes mellitus. PMID:20826789

  9. Akt-mediated transactivation of the S1P1 receptor in caveolin-enriched microdomains regulates endothelial barrier enhancement by oxidized phospholipids.

    PubMed

    Singleton, Patrick A; Chatchavalvanich, Santipongse; Fu, Panfeng; Xing, Junjie; Birukova, Anna A; Fortune, Jennifer A; Klibanov, Alexander M; Garcia, Joe G N; Birukov, Konstantin G

    2009-04-24

    Endothelial cell (EC) barrier dysfunction results in increased vascular permeability, leading to increased mass transport across the vessel wall and leukocyte extravasation, the key mechanisms in pathogenesis of tissue inflammation and edema. We have previously demonstrated that OxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) significantly enhances vascular endothelial barrier properties in vitro and in vivo and attenuates endothelial hyperpermeability induced by inflammatory and edemagenic agents via Rac and Cdc42 GTPase dependent mechanisms. These findings suggested potential important therapeutic value of barrier-protective oxidized phospholipids. In this study, we examined involvement of signaling complexes associated with caveolin-enriched microdomains (CEMs) in barrier-protective responses of human pulmonary ECs to OxPAPC. Immunoblotting from OxPAPC-treated ECs revealed OxPAPC-mediated rapid recruitment (5 minutes) to CEMs of the sphingosine 1-phosphate receptor (S1P(1)), the serine/threonine kinase Akt, and the Rac1 guanine nucleotide exchange factor Tiam1 and phosphorylation of caveolin-1, indicative of signaling activation in CEMs. Abolishing CEM formation (methyl-beta-cyclodextrin) blocked OxPAPC-mediated Rac1 activation, cytoskeletal reorganization, and EC barrier enhancement. Silencing (small interfering RNA) Akt expression blocked OxPAPC-mediated S1P(1) activation (threonine phosphorylation), whereas silencing S1P(1) receptor expression blocked OxPAPC-mediated Tiam1 recruitment to CEMs, Rac1 activation, and EC barrier enhancement. To confirm our in vitro results in an in vivo murine model of acute lung injury with pulmonary vascular hyperpermeability, we observed that selective lung silencing of caveolin-1 or S1P(1) receptor expression blocked OxPAPC-mediated protection from ventilator-induced lung injury. Taken together, these results suggest Akt-dependent transactivation of S1P(1) within CEMs is important for Ox

  10. Transactivation of epidermal growth factor receptor in vascular and renal systems in rats with experimental hyperleptinemia: role in leptin-induced hypertension.

    PubMed

    Jamroz-Wiśniewska, Anna; Wójcicka, Grazyna; Łowicka, Ewelina; Ksiazek, Marta; Bełtowski, Jerzy

    2008-04-15

    We examined the role of epidermal growth factor (EGF) receptor in the pathogenesis of leptin-induced hypertension in the rat. Leptin, administered in increasing doses (0.1-0.5 mg/kg/day) for 10 days, increased phosphorylation levels of non-receptor tyrosine kinase, c-Src, EGF receptor and extracellular signal-regulated kinases (ERK) in aorta and kidney, which was accompanied by the increase in plasma concentration and urinary excretion of isoprostanes and H2O2. Blood pressure and renal Na+,K+-ATPase activity were higher, whereas urinary sodium excretion was lower in animals receiving leptin. The effects of leptin on renal Na+,K+-ATPase, natriuresis and blood pressure were abolished by NADPH oxidase inhibitor, apocynin, Src kinase inhibitor, PP2, EGF receptor inhibitor, AG1478, protein farnesyltransferase inhibitor, manumycin A, and ERK inhibitor, PD98059. In contrast, inhibitors of insulin-like growth factor-1 and platelet-derived growth factor receptors, AG1024 and AG1295, respectively, only slightly reduced ERK phosphorylation and had no effect on blood pressure in rats receiving leptin. These data indicate that: (1) experimental hyperleptinemia is associated with oxidative stress and c-Src-dependent transactivation of the EGF receptor, which stimulates ERK in vascular wall and the kidney, (2) overactivity of EGF receptor-ERK pathway contributes to leptin-induced hypertension by stimulating renal Na+,K+-ATPase and reducing sodium excretion, (3) inhibitors of c-Src, EGF receptor and ERK may be considered as a novel therapy for hypertension associated with hyperleptinemia, e.g. in patients with obesity and metabolic syndrome.

  11. Protease-Activated Receptor 2 Promotes Pro-Atherogenic Effects through Transactivation of the VEGF Receptor 2 in Human Vascular Smooth Muscle Cells

    PubMed Central

    Indrakusuma, Ira; Romacho, Tania; Eckel, Jürgen

    2017-01-01

    Background: Obesity is associated with impaired vascular function. In the cardiovascular system, protease-activated receptor 2 (PAR2) exerts multiple functions such as the control of the vascular tone. In pathological conditions, PAR2 is related to vascular inflammation. However, little is known about the impact of obesity on PAR2 in the vasculature. Therefore, we explored the role of PAR2 as a potential link between obesity and cardiovascular diseases. Methods: C57BL/6 mice were fed with either a chow or a 60% high fat diet for 24 weeks prior to isolation of aortas. Furthermore, human coronary artery endothelial cells (HCAEC) and human coronary smooth muscle cells (HCSMC) were treated with conditioned medium obtained from in vitro differentiated primary human adipocytes. To investigate receptor interaction vascular endothelial growth factor receptor 2 (VEGFR2) was blocked by exposure to calcium dobesilate and a VEGFR2 neutralization antibody, before treatment with PAR2 activating peptide. Student's t-test or one-way were used to determine statistical significance. Results: Both, high fat diet and exposure to conditioned medium increased PAR2 expression in aortas and human vascular cells, respectively. In HCSMC, conditioned medium elicited proliferation as well as cyclooxygenase 2 induction, which was suppressed by the PAR2 antagonist GB83. Specific activation of PAR2 by the PAR2 activating peptide induced proliferation and cyclooxygenase 2 expression which were abolished by blocking the VEGFR2. Additionally, treatment of HCSMC with the PAR2 activating peptide triggered VEGFR2 phosphorylation. Conclusion: Under obesogenic conditions, where circulating levels of pro-inflammatory adipokines are elevated, PAR2 arises as an important player linking obesity-related adipose tissue inflammation to atherogenesis. We show for the first time that the underlying mechanisms of these pro-atherogenic effects involve a potential transactivation of the VEGFR2 by PAR2. PMID

  12. The human papillomavirus type 16 E7 gene product interacts with and trans-activates the AP1 family of transcription factors.

    PubMed Central

    Antinore, M J; Birrer, M J; Patel, D; Nader, L; McCance, D J

    1996-01-01

    The E7 gene product of human papillomavirus type 16 (HPV16) binds to the retinoblastoma gene product (pRb) and dissociates pRb-E2F complexes. However, the observation that the ability of E7 to bind pRb is not required for the HPV16-induced immortalization of primary keratinocytes prompted a search for other cellular factors bound by E7. Using a glutathione-S-transferase (GST) fusion protein system, we show that E7 complexes with AP1 transcription factors including c-Jun, JunB, JunD and c-Fos. The ability of E7 to complex with c-Jun in vivo is demonstrated by co-immunoprecipitation and the yeast two-hybrid system. An analysis of E7 point mutants in the GST system indicates that the E7 zinc-finger motif, but not the pRb binding domain, is involved in these interactions. Using c-Jun deletion mutants, E7 binding maps between amino acids 224 and 286 of c-Jun. E7 trans-activates c-Jun-induced transcription from a Jun responsive promoter, and this activity correlates with the ability of E7 mutants to bind Jun proteins. Finally, a transcriptionally inactive c-Jun deletion, which can bind E7, interferes with the E7-induced transformation of rat embryo fibroblasts in cooperation with an activated ras, indicating that the Jun-E7 interaction is physiologically relevant and that Jun factors may be targeted in the E7 transformation pathway. Images PMID:8617242

  13. Oxidative stress contributes to the enhanced expression of Gqα/PLCβ1 proteins and hypertrophy of VSMC from SHR: role of growth factor receptor transactivation.

    PubMed

    Atef, Mohammed Emehdi; Anand-Srivastava, Madhu B

    2016-03-01

    We showed previously that vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHRs) exhibit overexpression of Gqα/PLCβ1 proteins, which contribute to increased protein synthesis through the activation of MAP kinase signaling. Because oxidative stress has been shown to be increased in hypertension, the present study was undertaken to examine the role of oxidative stress and underlying mechanisms in enhanced expression of Gqα/PLCβ1 proteins and VSMC hypertrophy. Protein expression was determined by Western blotting, whereas protein synthesis and cell volume, markers for VSMC hypertrophy, were determined by [(3)H]-leucine incorporation and three-dimensional confocal imaging, respectively. The increased expression of Gqα/PLCβ1 proteins, increased protein synthesis, and augmented cell volume exhibited by VSMCs from SHRs were significantly attenuated by antioxidants N-acetyl-cysteine (NAC), a scavenger of superoxide anion, DPI, an inhibitor of NAD(P)H oxidase. In addition, PP2, AG1024, AG1478, and AG1295, inhibitors of c-Src, insulin-like growth factor receptor (IGFR), epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor (PDGFR), respectively, also attenuated the enhanced expression of Gqα/PLCβ1 proteins and enhanced protein synthesis in VSMCs from SHRs toward control levels. Furthermore, the levels of IGF-1R and EGFR proteins and not of PDGFR were also enhanced in VSMCs from SHRs, which were attenuated significantly by NAC, DPI, and PP2. In addition, NAC, DPI, and PP2 also attenuated the enhanced phosphorylation of IGF-1R, PDGFR, EGFR, c-Src, and EKR1/2 in VSMCs from SHRs. These data suggest that enhanced oxidative stress in VSMCs from SHRs activates c-Src, which through the transactivation of growth factor receptors and MAPK signaling contributes to enhanced expression of Gqα/PLCβ1 proteins and resultant VSMC hypertrophy.

  14. Copper depletion inhibits CoCl2-induced aggressive phenotype of MCF-7 cells via downregulation of HIF-1 and inhibition of Snail/Twist-mediated epithelial-mesenchymal transition.

    PubMed

    Li, Shun; Zhang, Jing; Yang, Hong; Wu, Chunhui; Dang, Xitong; Liu, Yiyao

    2015-07-15

    Copper, a strictly regulated trace element, is essential for many physiological processes including angiogenesis. Dysregulated angiogenesis has been associated with increased copper in tumors, and thus copper chelators have been used to inhibit tumor angiogenesis. However, it remains unclear whether copper has any effect on epithelial-mesenchymal transition (EMT). Using CoCl2-induced EMT of human breast carcinoma MCF-7 cells, we found that TEPA, a copper chelator, inhibited EMT-like cell morphology and cytoskeleton arrangement triggered by CoCl2; decreased the expression of vimentin and fibronectin, markers typical of EMT; inhibited HIF-1 activation and HIF1-α accumulation in nuclear; and down-regulated the expression of hypoxia-associated transcription factors, Snail and Twist1. Moreover, knockdown copper transport protein, Ctr1, also inhibited CoCl2-induced EMT and reversed the mesenchymal phenotype. In EMT6 xenograft mouse models, TEPA administration inhibited the tumor growth and increased mice survival. Immunohistochemical analysis of the xenograft further demonstrated that TEPA administration significantly inhibited tumor angiogenesis, down-regulated hypoxia-induced transcription factors, Snail and Twist1, leading to decreased transactivation of EMT-associated marker genes, vimentin and fibronectin. These results indicate that TEPA inhibits CoCl2-induced EMT most likely via HIF1-α-Snail/Twist signaling pathway, and copper depletion may be exploited as a therapeutic for breast cancer.

  15. Copper depletion inhibits CoCl2-induced aggressive phenotype of MCF-7 cells via downregulation of HIF-1 and inhibition of Snail/Twist-mediated epithelial-mesenchymal transition

    PubMed Central

    Li, Shun; Zhang, Jing; Yang, Hong; Wu, Chunhui; Dang, Xitong; Liu, Yiyao

    2015-01-01

    Copper, a strictly regulated trace element, is essential for many physiological processes including angiogenesis. Dysregulated angiogenesis has been associated with increased copper in tumors, and thus copper chelators have been used to inhibit tumor angiogenesis. However, it remains unclear whether copper has any effect on epithelial-mesenchymal transition (EMT). Using CoCl2-induced EMT of human breast carcinoma MCF-7 cells, we found that TEPA, a copper chelator, inhibited EMT-like cell morphology and cytoskeleton arrangement triggered by CoCl2; decreased the expression of vimentin and fibronectin, markers typical of EMT; inhibited HIF-1 activation and HIF1-α accumulation in nuclear; and down-regulated the expression of hypoxia-associated transcription factors, Snail and Twist1. Moreover, knockdown copper transport protein, Ctr1, also inhibited CoCl2-induced EMT and reversed the mesenchymal phenotype. In EMT6 xenograft mouse models, TEPA administration inhibited the tumor growth and increased mice survival. Immunohistochemical analysis of the xenograft further demonstrated that TEPA administration significantly inhibited tumor angiogenesis, down-regulated hypoxia-induced transcription factors, Snail and Twist1, leading to decreased transactivation of EMT-associated marker genes, vimentin and fibronectin. These results indicate that TEPA inhibits CoCl2-induced EMT most likely via HIF1-α-Snail/Twist signaling pathway, and copper depletion may be exploited as a therapeutic for breast cancer. PMID:26174737

  16. Inhibition of FGF/FGFR autocrine signaling in mesothelioma with the FGF ligand trap, FP-1039/GSK3052230

    PubMed Central

    Ganji, Gopinath; Barnette, Mary; Hoang, Bao; Tunstead, James; Skedzielewski, Tina; Alsaid, Hasan; Jucker, Beat M.; Minthorn, Elisabeth; Kumar, Rakesh; DeYoung, M. Phillip

    2016-01-01

    Fibroblast growth factor (FGF) ligand-dependent signaling has a fundamental role in cancer development and tumor maintenance. GSK3052230 (also known as FP-1039) is a soluble decoy receptor that sequesters FGFs and inhibits FGFR signaling. Herein, the efficacy of this molecule was tested in models of mesothelioma, a tumor type shown to express high levels of FGF2 and FGFR1. GSK3052230 demonstrated antiproliferative activity across a panel of mesothelioma cell lines and inhibited growth of tumor xenografts in mice. High expression of FGF2 and FGFR1 correlated well with response to FGF pathway inhibition. GSK3052230 inhibited MAPK signaling as evidenced by decreased phospho-ERK and phospho-S6 levels in vitro and in vivo. Additionally, dose-dependent and statistically-significant reductions in tumor vessel density were observed in GSK3052230-treated tumors compared to vehicle-treated tumors. These data support the role of GSK3052230 in effectively targeting FGF-FGFR autocrine signaling in mesothelioma, demonstrate its impact on tumor growth and angiogenesis, and provide a rationale for the current clinical evaluation of this molecule in mesothelioma patients. PMID:27223434

  17. Macrophage migration inhibitory factor interacts with HBx and inhibits its apoptotic activity

    SciTech Connect

    Zhang Shimeng; Lin Ruxian; Zhou Zhe; Wen Siyuan; Lin Li; Chen Suhong; Shan Yajun; Cong Yuwen; Wang Shengqi . E-mail: sqwang@nic.bmi.ac.cn

    2006-04-07

    HBx, a transcriptional transactivating protein of hepatitis B virus (HBV), is required for viral infection and has been implicated in virus-mediated liver oncogenesis. However, the precise molecular mechanism remains largely elusive. We used the yeast two-hybrid system to identify that HBx interacts with MIF directly. Macrophage migration inhibitory factor (MIF) is implicated in the regulation of inflammation, cell growth, and even tumor formation. The interaction between HBx and MIF was verified with co-immunoprecipitation, GST pull-down, and cellular colocalization. The expression of MIF was up-regulated in HBV particle producing cell 2.2.15 compared with HepG2 cell. Both HBx and MIF cause HepG2 cell G /G{sub 1} phase arrest, proliferation inhibition, and apoptosis. However, MIF can counteract the apoptotic effect of HBx. These results may provide evidence to explain the link between HBV infection and hepatocellular carcinoma.

  18. Ligand independent aryl hydrocarbon receptor inhibits lung cancer cell invasion by degradation of Smad4.

    PubMed

    Lee, Chen-Chen; Yang, Wen-Hao; Li, Ching-Hao; Cheng, Yu-Wen; Tsai, Chi-Hao; Kang, Jaw-Jou

    2016-07-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent-activated transcriptional factor that regulates the metabolism of xenobiotic and endogenous compounds. Although AhR plays a crucial role in air toxicant-induced carcinogenesis, AhR expression was shown to negatively regulate tumorigenesis. Therefore, in the present study, we investigated the effect of AhR without ligand treatment on cancer invasion in lung cancer cell lines. Lung cancer cells expressing lower levels of AhR showed higher invasion ability (H1299 cells) compared with cells expressing higher levels of AhR (A549 cells). Overexpression of AhR in H1299 cells inhibited the invasion ability. We found that vimentin expression was inhibited in AhR-overexpressing H1299 cells. Additionally, the expression of EMT-related transcriptional factors Snail and ID-1 decreased. Interestingly, we found that Smad4 degradation was induced in AhR-overexpressing H1299 cells. Our data showed that AhR could interact with Jun-activation domain binding protein (Jab1) and Smad4, which may cause degradation of Smad4 by the proteasome. Our data suggest that AhR affects the transforming growth factor-β signaling pathway by inducing Smad4 degradation by the proteasome and suppressing tumor metastasis via epithelial to mesenchymal transition reduction in lung cancer cells.

  19. Prostacyclin inhibits IFN-γ-stimulated cytokine expression by reduced recruitment of p300/CBP to STAT1 in a SOCS-1 independent manner

    PubMed Central

    Strassheim, Derek; Riddle, Suzzette R.; Burke, Danielle L.; Geraci, Mark W; Stenmark, Kurt R.

    2009-01-01

    Increasing evidence indicates that Pulmonary Arterial Hypertension (PAH) is a vascular inflammatory disease. Prostacyclin (PGI2) is widely used to treat PAH and is believed to benefit patients largely through vasodilatory effects. PGI2 is also increasingly believed to have anti-inflammatory effects; including decreasing leukocyte cytokine production, yet few mechanistic details exist to explain how these effects are mediated at the transcriptional level. Since activated monocytes are critical sources of MCP-1 and other cytokines in cardiovascular inflammation, we examined the effects of iloprost on IFN-γ and IL-6 stimulated cytokine production in human monocytes. We found iloprost inhibited IFN-γ and IL-6-induced MCP-1, IL-8, RANTES, and TNF-α production in monocytes indicating wide-ranging anti-inflammatory action. We found that activation of STAT1 was critical for IFN-γ-induced MCP-1 production and demonstrated that iloprost inhibited STAT1 activation by several actions: 1) iloprost inhibited the phosphorylation of STAT1-S727 in the transactivation domain (TAD), thereby reducing recruitment of the histone acetylase and co-activator CBP/p300 to STAT1; 2) iloprost selectively inhibited activation of janus kinase 2 (JAK2), but not JAK1, both responsible for activation STAT1 via phosphorylation of STAT1-Y701, resulting in reduced nuclear recruitment and activation of STAT1; 3) SOCS-1, which normally terminates IFN-γ-signaling, was not involved in iloprost-mediated inhibition of STAT1, indicating divergence from the classical pathway for terminating IFN-γ-signaling. We conclude that PGI2 exerts anti-inflammatory action by inhibiting STAT1 induced cytokine production, in part by targeting the transactivation domain induced recruitment of the histone acetylase CBP/p300. PMID:19915063

  20. Central nervous system-derived cells express a kappa B-binding activity that enhances human immunodeficiency virus type 1 transcription in vitro and facilitates TAR-independent transactivation by Tat.

    PubMed Central

    Taylor, J P; Pomerantz, R J; Raj, G V; Kashanchi, F; Brady, J N; Amini, S; Khalili, K

    1994-01-01

    The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent activator of long terminal repeat-directed transcription. While in most cell types, activation requires interaction of Tat with the unusual transcription element TAR, astrocytic glial cells support TAR-independent transactivation of HIV-1 transcription by Tat. This alternative pathway of Tat activation is mediated by the viral enhancer, a kappa B domain capable of binding the prototypical form of the transcription factor nuclear factor kappa B (NF-kappa B) present in many cell types, including T lymphocytes. Tat transactivation mediated by the kappa B domain is sufficient to allow replication of TAR-deleted mutant HIV-1 in astrocytes. The present study demonstrates the existence of kappa B-specific binding factors present in human glial astrocytes that differ from prototypical NF-kappa B. The novel astrocyte-derived kappa B-binding activity is retained on an HIV-1 Tat affinity column, while prototypical NF-kappa B from Jurkat T cells is not. In vitro transcription studies demonstrate that astrocyte-derived kappa B-binding factors activate transcription of the HIV-1 long terminal repeat and that this activation is dependent on the kappa B domain. Moreover, TAR-independent transactivation of HIV-1 transcription is reproduced in vitro in an astrocyte factor-dependent manner which correlates with kappa B-binding activity. The importance of the central nervous system-enriched kappa B transcription factor in the regulation of HIV-1 expression is discussed. Images PMID:8189531

  1. Residues R{sup 199}H{sup 200} of prototype foamy virus transactivator Bel1 contribute to its binding with LTR and IP promoters but not its nuclear localization

    SciTech Connect

    Ma, Qinglin; Tan, Juan; Cui, Xiaoxu; Luo, Di; Yu, Miao; Liang, Chen; Qiao, Wentao

    2014-01-20

    Prototype foamy virus encodes a transactivator called Bel1 that enhances viral gene transcription and is essential for PFV replication. Nuclear localization of Bel1 has been reported to rely on two proximal basic motifs R{sup 199}H{sup 200} and R{sup 221}R{sup 222}R{sup 223} that likely function together as a bipartite nuclear localization signal. In this study, we report that mutating R{sup 221}R{sup 222}R{sup 223}, but not R{sup 199}H{sup 200}, relocates Bel1 from the nucleus to the cytoplasm, suggesting an essential role for R{sup 221}R{sup 222}R{sup 223} in the nuclear localization of Bel1. Although not affecting the nuclear localization of Bel1, mutating R{sup 199}H{sup 200} disables Bel1 from transactivating PFV promoters. Results of EMSA reveal that the R{sup 199}H{sup 200} residues are vital for the binding of Bel1 to viral promoter DNA. Moreover, mutating R{sup 199}H{sup 200} in Bel1 impairs PFV replication to a much greater extent than mutating R{sup 221}R{sup 222}R{sup 223}. Collectively, our findings suggest that R{sup 199}H{sup 200} directly participate in Bel1 binding to viral promoter DNA and are indispensible for Bel1 transactivation activity. - Highlights: • The R{sup 221}R{sup 222}R{sup 223} residues are essential for the nuclear localization of Bel1. • Although not affecting the nuclear localization of Bel1, mutating R{sup 199}H{sup 200} disables Bel1 from transactivating PFV promoters. • The R{sup 199}H{sup 200} residues directly participate in Bel1 binding to viral promoter DNA. • Mutating R{sup 199}H{sup 200} in Bel1 impairs PFV replication to a much greater extent than mutating R{sup 221}R{sup 222}R{sup 223}.

  2. High-Level Clonal FGFR Amplification and Response to FGFR Inhibition in a Translational Clinical Trial

    PubMed Central

    Babina, Irina S.; Herrera-Abreu, Maria Teresa; Tarazona, Noelia; Peckitt, Clare; Kilgour, Elaine; Smith, Neil R.; Geh, Catherine; Rooney, Claire; Cutts, Ros; Campbell, James; Ning, Jian; Fenwick, Kerry; Swain, Amanda; Brown, Gina; Chua, Sue; Thomas, Anne; Johnston, Stephen R.D.; Ajaz, Mazhar; Sumpter, Katherine; Gillbanks, Angela; Watkins, David; Chau, Ian; Popat, Sanjay; Cunningham, David; Turner, Nicholas C.

    2017-01-01

    FGFR1 and FGFR2 are amplified in many tumor types, yet what determines response to FGFR inhibition in amplified cancers is unknown. In a translational clinical trial, we show that gastric cancers with high-level clonal FGFR2 amplification have a high response rate to the selective FGFR inhibitor AZD4547, whereas cancers with subclonal or low-level amplification did not respond. Using cell lines and patient-derived xenograft models, we show that high-level FGFR2 amplification initiates a distinct oncogene addiction phenotype, characterized by FGFR2-mediated transactivation of alternative receptor kinases, bringing PI3K/mTOR signaling under FGFR control. Signaling in low-level FGFR1-amplified cancers is more restricted to MAPK signaling, limiting sensitivity to FGFR inhibition. Finally, we show that circulating tumor DNA screening can identify high-level clonally amplified cancers. Our data provide a mechanistic understanding of the distinct pattern of oncogene addiction seen in highly amplified cancers and demonstrate the importance of clonality in predicting response to targeted therapy. Significance Robust single-agent response to FGFR inhibition is seen only in high-level FGFR-amplified cancers, with copy-number level dictating response to FGFR inhibition in vitro, in vivo, and in the clinic. High-level amplification of FGFR2 is relatively rare in gastric and breast cancers, and we show that screening for amplification in circulating tumor DNA may present a viable strategy to screen patients. PMID:27179038

  3. Two phenylalanines in the C-terminus of Epstein-Barr virus Rta protein reciprocally modulate its DNA binding and transactivation function

    SciTech Connect

    Chen, L.-W.; Raghavan, Vineetha; Chang, Pey-Jium; Shedd, Duane; Heston, Lee; Delecluse, Henri-Jacques; Miller, George

    2009-04-10

    The Rta (R transactivator) protein plays an essential role in the Epstein-Barr viral (EBV) lytic cascade. Rta activates viral gene expression by several mechanisms including direct and indirect binding to target viral promoters, synergy with EBV ZEBRA protein, and stimulation of cellular signaling pathways. We previously found that Rta proteins with C-terminal truncations of 30 aa were markedly enhanced in their capacity to bind DNA (Chen, L.W., Chang, P.J., Delecluse, H.J., and Miller, G., (2005). Marked variation in response of consensus binding elements for the Rta protein of Epstein-Barr virus. J. Virol. 79(15), 9635-9650.). Here we show that two phenylalanines (F600 and F605) in the C-terminus of Rta play a crucial role in mediating this DNA binding inhibitory function. Amino acids 555 to 605 of Rta constitute a functional DNA binding inhibitory sequence (DBIS) that markedly decreased DNA binding when transferred to a minimal DNA binding domain of Rta (aa 1-350). Alanine substitution mutants, F600A/F605A, abolished activity of the DBIS. F600 and F605 are located in the transcriptional activation domain of Rta. Alanine substitutions, F600A/F605A, decreased transcriptional activation by Rta protein, whereas aromatic substitutions, such as F600Y/F605Y or F600W/F605W, partially restored transcriptional activation. Full-length Rta protein with F600A/F605A mutations were enhanced in DNA binding compared to wild-type, whereas Rta proteins with F600Y/F605Y or F600W/F605W substitutions were, like wild-type Rta, relatively poor DNA binders. GAL4 (1-147)/Rta (416-605) fusion proteins with F600A/F605A mutations were diminished in transcriptional activation, relative to GAL4/Rta chimeras without such mutations. The results suggest that, in the context of a larger DBIS, F600 and F605 play a role in the reciprocal regulation of DNA binding and transcriptional activation by Rta. Regulation of DNA binding by Rta is likely to be important in controlling its different modes of

  4. The functional importance of a cap site-proximal region of the human prointerleukin 1 beta gene is defined by viral protein trans-activation.

    PubMed Central

    Hunninghake, G W; Monks, B G; Geist, L J; Monick, M M; Monroy, M A; Stinski, M F; Webb, A C; Dayer, J M; Auron, P E; Fenton, M J

    1992-01-01

    Prointerleukin 1 beta (IL-1 beta) is a cytokine that mediates a broad range of biological activities. Genomic sequences that regulate IL-1 beta transcription include both inducible regulatory elements located more than 2,700 bp upstream of the transcriptional start site (cap site) and proximal elements located near the TATA box of this gene. In this study, we focused on the identification and characterization of trans-acting nuclear regulatory proteins that bind to the cap site-proximal region of the human IL-1 beta gene. We identified a protein, termed NFIL-1 beta A (NF beta A), that binds to a highly conserved 12-bp DNA sequence (-49 to -38) located upstream of the TATA box motif in both the human and murine IL-1 beta genes. The IL-1 alpha gene, which lacks a TATA motif, does not possess an NF beta A-binding sequence within the promoter region, suggesting that NF beta A may selectively regulate IL-1 beta expression. Using electrophoretic mobility shift assays, we identified several distinct DNA-protein complexes that are expressed in a cell-type-specific manner. In monocytic cell lines, the relative abundance of these complexes varies rapidly following stimulation of the cells with phorbol esters or lipopolysaccharide. UV cross-linking analysis identified two distinct DNA-binding polypeptides that comprise distinct complexes. The functional role of NF beta A was assessed in transient transfection assays. These data indicate that NF beta A is required for both basal and inducible promoter activity in monocytic cells. Furthermore, the human cytomegalovirus immediate-early 1 gene product requires the presence of NF beta A in order to trans-activate the proximal IL-1 beta promoter in a monocytic cell line. We propose that NF beta A is a factor that mediates either direct or indirect activation by the immediate-early 1 gene product. The proximity of this essential factor to the TATA motif suggests a possible role in transcriptional initiation. Images PMID:1630455

  5. The Human Papillomavirus Type 16 E5 Oncoprotein Inhibits Epidermal Growth Factor Trafficking Independently of Endosome Acidification ▿

    PubMed Central

    Suprynowicz, Frank A.; Krawczyk, Ewa; Hebert, Jess D.; Sudarshan, Sawali R.; Simic, Vera; Kamonjoh, Christopher M.; Schlegel, Richard

    2010-01-01

    The human papillomavirus type 16 E5 oncoprotein (16E5) enhances acute, ligand-dependent activation of the epidermal growth factor receptor (EGFR) and concomitantly alkalinizes endosomes, presumably by binding to the 16-kDa “c” subunit of the V-ATPase proton pump (16K) and inhibiting V-ATPase function. However, the relationship between 16K binding, endosome alkalinization, and altered EGFR signaling remains unclear. Using an antibody that we generated against 16K, we found that 16E5 associated with only a small fraction of endogenous 16K in keratinocytes, suggesting that it was unlikely that E5 could significantly affect V-ATPase function by direct inhibition. Nevertheless, E5 inhibited the acidification of endosomes, as determined by a new assay using a biologically active, pH-sensitive fluorescent EGF conjugate. Since we also found that 16E5 did not alter cell surface EGF binding, the number of EGFRs on the cell surface, or the endocytosis of prebound EGF, we postulated that it might be blocking the fusion of early endosomes with acidified vesicles. Our studies with pH-sensitive and -insensitive fluorescent EGF conjugates and fluorescent dextran confirmed that E5 prevented endosome maturation (acidification and enlargement) by inhibiting endosome fusion. The E5-dependent defect in vesicle fusion was not due to detectable disruption of actin, tubulin, vimentin, or cytokeratin filaments, suggesting that membrane fusion was being directly affected rather than vesicle transport. Perhaps most importantly, while bafilomycin A1 (like E5) binds to 16K and inhibits endosome acidification, it did not mimic the ability of E5 to inhibit endosome enlargement or the trafficking of EGF. Thus, 16E5 alters EGF endocytic trafficking via a pH-independent inhibition of vesicle fusion. PMID:20686024

  6. The European flounder (Platichthys flesus) TP53 functions as a temperature-sensitive transcription factor which inhibits cell growth in yeast.

    PubMed

    Cachot, J; Flaman, J M; Frébourg, T; Leboulenger, F

    2004-01-07

    Numerous studies focus on biological roles of the TP53 tumor suppressor gene in mammals but little is known about the actual function of TP53 in lower vertebrates. In this study, we used an in vivo functional assay in yeast to address the transactivation capacity of the flounder TP53 protein. We showed that the flounder TP53 acts as a sequence-specific transcription factor which is able to transactivate various human promoters containing a p53-responsive element (RE). This transcriptional activity was completely abrogated in the Val147Glu TP53 mutant previously identified in two flounder hepatic hyperplasia. In addition, we showed that the wild-type (wt) flounder TP53 but not the Val147Glu mutant inhibits cell growth when expressed in yeast. We finally reported that transcription regulation and growth inhibition by the wild-type flounder TP53 is temperature-dependent. The flounder TP53 optimal temperature appeared lower than those reported for the Xenopus and human homologues.

  7. Epidermal growth factor increases coactivation of the androgen receptor in recurrent prostate cancer.

    PubMed

    Gregory, Christopher W; Fei, Xiaoyin; Ponguta, Liliana A; He, Bin; Bill, Heather M; French, Frank S; Wilson, Elizabeth M

    2004-02-20

    Growth of normal and neoplastic prostate is mediated by the androgen receptor (AR), a ligand-dependent transcription factor activated by high affinity androgen binding. The AR is highly expressed in recurrent prostate cancer cells that proliferate despite reduced circulating androgen. In this report, we show that epidermal growth factor (EGF) increases androgen-dependent AR transactivation in the recurrent prostate cancer cell line CWR-R1 through a mechanism that involves a post-transcriptional increase in the p160 coactivator transcriptional intermediary factor 2/glucocorticoid receptor interacting protein 1 (TIF2/GRIP1). Site-specific mutagenesis and selective MAPK inhibitors linked the EGF-induced increase in AR transactivation to phosphorylation of TIF2/GRIP1. EGF signaling increased the coimmunoprecipitation of TIF2 and AR. AR transactivation and its stimulation by EGF were reduced by small interfering RNA inhibition of TIF2/GRIP1 expression. The data indicate that EGF signaling through MAPK increases TIF2/GRIP1 coactivation of AR transactivation in recurrent prostate cancer.

  8. NcoA2-Dependent Inhibition of HIF-1α Activation Is Regulated via AhR.

    PubMed

    Tsai, Chi-Hao; Li, Ching-Hao; Liao, Po-Lin; Cheng, Yu-Wen; Lin, Cheng-Hui; Huang, Shih-Hsuan; Kang, Jaw-Jou

    2015-12-01

    High endogenous levels of aryl hydrocarbon receptor (AhR) contribute to hypoxia signaling pathway inhibition following exposure to the potent AhR ligand benzo[a]pyrene (B[a]P) and could alter cellular homeostasis and disease condition. Increasing evidence indicates that AhR might compete with AhR nuclear translocator (ARNT) for complex formation with hypoxia-inducible factor-1α (HIF-1α) for transactivation, which could alter several physiological variables. Nuclear receptor coactivator 2 (NcoA2) is a transcription coactivator that regulates transcription factor activation and inhibition of basic helix-loop-helix Per (Period)-ARNT-SIM (single-minded) (bHLH-PAS) family proteins, such as HIF-1α, ARNT, and AhR, through protein-protein interactions. In this study, we demonstrated that both hypoxia and hypoxia-mimic conditions decreased NcoA2 protein expression in HEK293T cells. Hypoxia response element (HRE) and xenobiotic-responsive element (XRE) transactivation also were downregulated with NcoA2 knockdown under hypoxic conditions. In addition, B[a]P significantly decreased NcoA2 protein expression be accompanied with AhR degradation. We next evaluated whether the absence of AhR could affect NcoA2 protein function under hypoxia-mimetic conditions. NcoA2 and HIF-1α nuclear localization decreased in both B[a]P-pretreated and AhR-knockdown HepG2 cells under hypoxia-mimic conditions. Interestingly, NcoA2 overexpression downregulated HRE transactivation by competing with HIF-1α and AhR to form protein complexes with ARNT. Both NcoA2 knockdown and overexpression inhibited endothelial cell tube formation in vitro. We also demonstrated using the in vivo plug assay that NcoA2-regulated vascularization decreased in mice. Taken together, these results revealed a biphasic role of NcoA2 between AhR and hypoxic conditions, thus providing a novel mechanism underlying the cross talk between AhR and hypoxia that affects disease development and progression.

  9. CP-31398 inhibits the growth of p53-mutated liver cancer cells in vitro and in vivo.

    PubMed

    He, Xing-Xing; Zhang, Yu-Nan; Yan, Jun-Wei; Yan, Jing-Jun; Wu, Qian; Song, Yu-Hu

    2016-01-01

    The tumor suppressor p53 is one of the most frequently mutated genes in hepatocellular carcinoma (HCC). Previous studies demonstrated that CP-31398 restored the native conformation of mutant p53 and trans-activated p53 downstream genes in tumor cells. However, the research on the application of CP-31398 to liver cancer has not been reported. Here, we investigated the effects of CP-31398 on the phenotype of HCC cells carrying p53 mutation. The effects of CP-31398 on the characteristic of p53-mutated HCC cells were evaluated through analyzing cell cycle, cell apoptosis, cell proliferation, and the expression of p53 downstream genes. In tumor xenografts developed by PLC/PRF/5 cells, the inhibition of tumor growth by CP-31398 was analyzed through gross morphology, growth curve, and the expression of p53-related genes. Firstly, we demonstrated that CP-31398 inhibited the growth of p53-mutated liver cancer cells in a dose-dependent and p53-dependent manner. Then, further study showed that CP-31398 re-activated wild-type p53 function in p53-mutated HCC cells, which resulted in inhibitive response of cell proliferation and an induction of cell-cycle arrest and apoptosis. Finally, in vivo data confirmed that CP-31398 blocked the growth of xenografts tumors through transactivation of p53-responsive downstream molecules. Our results demonstrated that CP-31398 induced desired phenotypic change of p53-mutated HCC cells in vitro and in vivo, which revealed that CP-31398 would be developed as a therapeutic candidate for HCC carrying p53 mutation.

  10. African Swine Fever Virus Blocks the Host Cell Antiviral Inflammatory Response through a Direct Inhibition of PKC-θ-Mediated p300 Transactivation▿

    PubMed Central

    Granja, Aitor G.; Sánchez, Elena G.; Sabina, Prado; Fresno, Manuel; Revilla, Yolanda

    2009-01-01

    During a viral infection, reprogramming of the host cell gene expression pattern is required to establish an adequate antiviral response. The transcriptional coactivators p300 and CREB binding protein (CBP) play a central role in this regulation by promoting the assembly of transcription enhancer complexes to specific promoters of immune and proinflammatory genes. Here we show that the protein A238L encoded by African swine fever virus counteracts the host cell inflammatory response through the control of p300 transactivation during the viral infection. We demonstrate that A238L inhibits the expression of the inflammatory regulators cyclooxygenase-2 (COX-2) and tumor necrosis factor alpha (TNF-α) by preventing the recruitment of p300 to the enhanceosomes formed on their promoters. Furthermore, we report that A238L inhibits p300 activity during the viral infection and that its amino-terminal transactivation domain is essential in the A238L-mediated inhibition of the inflammatory response. Importantly, we found that the residue serine 384 of p300 is required for the viral protein to accomplish its inhibitory function and that ectopically expressed PKC-θ completely reverts this inhibition, thus indicating that this signaling pathway is disrupted by A238L during the viral infection. Furthermore, we show here that A238L does not affect PKC-θ enzymatic activity, but the molecular mechanism of this viral inhibition relies on the lack of interaction between PKC-θ and p300. These findings shed new light on how viruses alter the host cell antiviral gene expression pattern through the blockade of the p300 activity, which represents a new and sophisticated viral mechanism to evade the inflammatory and immune defense responses. PMID:19004945

  11. The Rel/NF-κB pathway and transcription of immediate early genes in T cell activation are inhibited by microgravity

    PubMed Central

    Chang, Tammy T.; Walther, Isabelle; Li, Chai-Fei; Boonyaratanakornkit, Jim; Galleri, Grazia; Meloni, Maria Antonia; Pippia, Proto; Cogoli, Augusto; Hughes-Fulford, Millie

    2012-01-01

    This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in μg. Immunosuppression during spaceflight is a major barrier to safe, long-term human space habitation and travel. The goals of these experiments were to prove that μg was the cause of impaired T cell activation during spaceflight, as well as understand the mechanisms controlling early T cell activation. T cells from four human donors were stimulated with Con A and anti-CD28 on board the ISS. An on-board centrifuge was used to generate a 1g simultaneous control to isolate the effects of μg from other variables of spaceflight. Microarray expression analysis after 1.5 h of activation demonstrated that μg- and 1g-activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly, differentially down-regulated in μg. Importantly, several key immediate early genes were inhibited in μg. In particular, transactivation of Rel/NF-κB, CREB, and SRF gene targets were down-regulated. Expression of cREL gene targets were significantly inhibited, and transcription of cREL itself was reduced significantly in μg and upon anti-CD3/anti-CD28 stimulation in simulated μg. Analysis of gene connectivity indicated that the TNF pathway is a major early downstream effector pathway inhibited in μg and may lead to ineffective proinflammatory host defenses against infectious pathogens during spaceflight. Results from these experiments indicate that μg was the causative factor for impaired T cell activation during spaceflight by inhibiting transactivation of key immediate early genes. PMID:22750545

  12. Reversible cell cycle inhibition and premature aging features imposed by conditional expression of p16Ink4a

    PubMed Central

    Boquoi, Amelie; Arora, Sanjeevani; Chen, Tina; Litwin, Sam; Koh, James; Enders, Greg H

    2015-01-01

    The cyclin-dependent kinase (Cdk) inhibitor p16Ink4a (p16) is a canonical mediator of cellular senescence and accumulates in aging tissues, where it constrains proliferation of some progenitor cells. However, whether p16 induction in tissues is sufficient to inhibit cell proliferation, mediate senescence, and/or impose aging features has remained unclear. To address these issues, we generated transgenic mice that permit conditional p16 expression. Broad induction at weaning inhibited proliferation of intestinal transit-amplifying and Lgr5+ stem cells and rapidly imposed features of aging, including hair loss, skin wrinkling, reduced body weight and subcutaneous fat, an increased myeloid fraction in peripheral blood, poor dentition, and cataracts. Aging features were observed with multiple combinations of p16 transgenes and transactivators and were largely abrogated by a germline Cdk4 R24C mutation, confirming that they reflect Cdk inhibition. Senescence markers were not found, and de-induction of p16, even after weeks of sustained expression, allowed rapid recovery of intestinal cell proliferation and reversal of aging features in most mice. These results suggest that p16-mediated inhibition of Cdk activity is sufficient to inhibit cell proliferation and impose aging features in somatic tissues of mammals and that at least some of these aging features are reversible. PMID:25481981

  13. Tumor cell-specific inhibition of MYC function using small molecule inhibitors of the HUWE1 ubiquitin ligase

    PubMed Central

    Peter, Stefanie; Bultinck, Jennyfer; Myant, Kevin; Jaenicke, Laura A; Walz, Susanne; Müller, Judith; Gmachl, Michael; Treu, Matthias; Boehmelt, Guido; Ade, Carsten P; Schmitz, Werner; Wiegering, Armin; Otto, Christoph; Popov, Nikita; Sansom, Owen; Kraut, Norbert; Eilers, Martin

    2014-01-01

    Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARF-BP1, MULE) associates with both MYC and the MYC-associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high-throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC-dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC-activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells. See also: FX Schaub & JL Cleveland (December 2014) PMID:25253726

  14. Prolactin inhibits a major tumor-suppressive function of wild type BRCA1.

    PubMed

    Chen, Kuan-Hui Ethan; Walker, Ameae M

    2016-06-01

    Even though mutations in the tumor suppressor, BRCA1, markedly increase the risk of breast and ovarian cancer, most breast and ovarian cancers express wild type BRCA1. An important question is therefore how the tumor-suppressive function of normal BRCA1 is overcome during development of most cancers. Because prolactin promotes these and other cancers, we investigated the hypothesis that prolactin interferes with the ability of BRCA1 to inhibit the cell cycle. Examining six different cancer cell lines with wild type BRCA1, and making use of both prolactin and the growth-inhibiting selective prolactin receptor modulator, S179D PRL, we demonstrate that prolactin activation of Stat5 results in the formation of a complex between phospho-Stat5 and BRCA1. Formation of this complex does not interfere with nuclear translocation or binding of BRCA1 to the p21 promoter, but does interfere with the ability of BRCA1 to transactivate the p21 promoter. Overexpression of a dominant-negative Stat5 in prolactin-stimulated cells resulted in increased p21 expression. We conclude that prolactin inhibits a major tumor-suppressive function of BRCA1 by interfering with BRCA1's upregulation of expression of the cell cycle inhibitor, p21.

  15. Inhibition of ERK Oscillations by Ionizing Radiation and Reactive Oxygen Species

    SciTech Connect

    Shankaran, Harish; Chrisler, William B; Sontag, Ryan L; Weber, Thomas J

    2010-12-28

    The shuttling of activated protein kinases between the cytoplasm and nucleus is an essential feature of normal growth factor signaling cascades. Here we demonstrate that transforming growth factor alpha (TGFα) induces oscillations in extracellular signal regulated kinase (ERK) cytoplasmic-nuclear translocations in human keratinocytes. TGFα-dependent ERK oscillations mediated through the epidermal growth factor receptor (EGFR) are inhibited by low dose X-irradiation (10 cGy) and low concentrations of hydrogen peroxide (0.32–3.26 µM H2O2) used as a model reactive oxygen species (ROS). A fluorescent indicator dye (H2-DCFDA) was used to measure cellular ROS levels following X-irradiation, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and H2O2. X-irradiation did not generate significant ROS production while 0.32 µM H2O2 and TPA induced significant increases in ROS levels with H2O2 > TPA. TPA alone induced transactivation of the EGFR but did not induce ERK oscillations. TPA as a cotreatment did not inhibit TGFα-stimulated ERK oscillations but qualitatively altered TGFα-dependent ERK oscillation characteristics (amplitude, time-period). Collectively, these observations demonstrate that TGFα-induced ERK oscillations are inhibited by ionizing radiation/ROS and perturbed by epigenetic carcinogen in human keratinocytes. © 2010 Wiley-Liss, Inc.

  16. MYBPH inhibits NM IIA assembly via direct interaction with NMHC IIA and reduces cell motility

    SciTech Connect

    Hosono, Yasuyuki; Usukura, Jiro; Yamaguchi, Tomoya; Yanagisawa, Kiyoshi; Suzuki, Motoshi; Takahashi, Takashi

    2012-11-09

    Highlights: Black-Right-Pointing-Pointer MYBPH inhibits NMHC IIA assembly and cell motility. Black-Right-Pointing-Pointer MYBPH interacts to assembly-competent NM IIA. Black-Right-Pointing-Pointer MYBPH inhibits RLC and NMHC IIA, independent components of NM IIA. -- Abstract: Actomyosin filament assembly is a critical step in tumor cell migration. We previously found that myosin binding protein H (MYBPH) is directly transactivated by the TTF-1 lineage-survival oncogene in lung adenocarcinomas and inhibits phosphorylation of the myosin regulatory light chain (RLC) of non-muscle myosin IIA (NM IIA) via direct interaction with Rho kinase 1 (ROCK1). Here, we report that MYBPH also directly interacts with an additional molecule, non-muscle myosin heavy chain IIA (NMHC IIA), which was found to occur between MYBPH and the rod portion of NMHC IIA. MYBPH inhibited NMHC IIA assembly and reduced cell motility. Conversely, siMYBPH-induced increased motility was partially, yet significantly, suppressed by blebbistatin, a non-muscle myosin II inhibitor, while more profound effects were attained by combined treatment with siROCK1 and blebbistatin. Electron microscopy observations showed well-ordered paracrystals of NMHC IIA reflecting an assembled state, which were significantly less frequently observed in the presence of MYBPH. Furthermore, an in vitro sedimentation assay showed that a greater amount of NMHC IIA was in an unassembled state in the presence of MYBPH. Interestingly, treatment with a ROCK inhibitor that impairs transition of NM IIA from an assembly-incompetent to assembly-competent state reduced the interaction between MYBPH and NMHC IIA, suggesting that MYBPH has higher affinity to assembly-competent NM IIA. These results suggest that MYBPH inhibits RLC and NMHC IIA, independent components of NM IIA, and negatively regulates actomyosin organization at 2 distinct steps, resulting in firm inhibition of NM IIA assembly.

  17. Androgen receptor exon 1 mutation causes androgen insensitivity by creating phosphorylation site and inhibiting melanoma antigen-A11 activation of NH2- and carboxyl-terminal interaction-dependent transactivation.

    PubMed

    Lagarde, William H; Blackwelder, Amanda J; Minges, John T; Hnat, Andrew T; French, Frank S; Wilson, Elizabeth M

    2012-03-30

    Naturally occurring germ line mutations in the X-linked human androgen receptor (AR) gene cause incomplete masculinization of the external genitalia by disrupting AR function in males with androgen insensitivity syndrome. Almost all AR missense mutations that cause androgen insensitivity syndrome are located in the highly structured DNA and ligand binding domains. In this report we investigate the functional defect associated with an AR exon 1 missense mutation, R405S, that caused partial androgen insensitivity. The 46,XX heterozygous maternal carrier had a wild-type Arg-405 CGC allele but transmitted an AGC mutant allele coding for Ser-405. At birth, the 46,XY proband had a bifid scrotum, hypospadias, and micropenis consistent with clinical stage 3 partial androgen insensitivity. Androgen-dependent transcriptional activity of AR-R405S expressed in CV1 cells was less than wild-type AR and refractory in androgen-dependent AR NH(2)- and carboxyl interaction transcription assays that depend on the coregulator effects of melanoma antigen-A11. This mutation created a Ser-405 phosphorylation site evident by the gel migration of an AR-R405S NH(2)-terminal fragment as a double band that converted to the wild-type single band after treatment with λ-phosphatase. Detrimental effects of the R405S mutation were related to the proximity of the AR WXXLF motif (433)WHTLF(437) required for melanoma antigen-A11 and p300 to stimulate transcriptional activity associated with the AR NH(2)- and carboxyl-terminal interaction. We conclude that the coregulator effects of melanoma antigen-A11 on the AR NH(2)- and carboxyl-terminal interaction amplify the androgen-dependent transcriptional response to p300 required for normal human male sex development in utero.

  18. Transactivation activity of human, zebrafish, and rainbow trout aryl hydrocarbon receptors expressed in COS-7 cells: greater insight into species differences in toxic potency of polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners.

    PubMed

    Abnet, C C; Tanguay, R L; Heideman, W; Peterson, R E

    1999-08-15

    Transactivation assays were used to compare the potency and efficacy of polychlorinated dibenzo-p-dioxin (PCDD), dibenzofuran (PCDF), and biphenyl (PCB) congeners in activating aryl hydrocarbon receptors (AhRs) from rainbow trout (rtAhR2alpha and rtAhR2beta), zebrafish (zfAhR2), and human (huAhR), respectively. All AhRs were expressed with their species-specific AhR nuclear translocator (ARNT) in COS-7 cells. Transactivation activity was determined for two PCDD, two PCDF, and seven PCB congeners with each of the four AhR/ARNT pairs using prt1Aluc, a luciferase reporter driven by two dioxin-responsive enhancer elements (DREs) from the rainbow trout cyp1A gene. Maximal-fold induction, EC50, and relative potency values were calculated for congeners that exhibited dose-related activity in the assay. Of the four AhR/ARNT pairs tested with PCDD, PCDF, and non-ortho PCB congeners, three exhibited high activity (rainbow trout AhR2alpha, zebrafish AhR2, and human AhR), while rainbow trout AhR2beta had very weak or no activity. Comparisons between these AhRs showed that while mono-ortho PCBs were able to activate the human AhR, they were generally ineffective in activating rainbow trout and zebrafish AhR2s. This supports the hypothesis that structural differences between mammalian and fish AhRs may account for differences in relative potencies of the mono-ortho PCBs between mammals and fish. Another important finding was a significant difference in transactivation activity between the two rainbow trout AhR2 isoforms despite the fact that they are 95% identical at the amino acid level. For all PCDD, PCDF, and PCB agonists tested, rainbow trout AhR2alpha was significantly more active than AhR2beta. However, rainbow trout AhR2beta is active as a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-activated transcription factor, with enhancer elements from the mouse cyp1A gene. This suggests that AhR2beta may have evolved to serve a different physiological function than AhR2alpha in

  19. Saccadic Inhibition in Reading

    ERIC Educational Resources Information Center

    Reingold, Eyal M.; Stampe, Dave M.

    2004-01-01

    In 5 experiments, participants read text that was briefly replaced by a transient image for 33 ms at random intervals. A decrease in saccadic frequency, referred to as saccadic inhibition, occurred as early as 60-70 ms following the onset of abrupt changes in visual input. It was demonstrated that the saccadic inhibition was influenced by the…

  20. In brown adipocytes, adrenergically induced β{sub 1}-/β{sub 3}-(G{sub s})-, α{sub 2}-(G{sub i})- and α{sub 1}-(G{sub q})-signalling to Erk1/2 activation is not mediated via EGF receptor transactivation

    SciTech Connect

    Wang, Yanling; Fälting, Johanna M.; Mattsson, Charlotte L.; Holmström, Therése E.; Nedergaard, Jan

    2013-10-15

    Brown adipose tissue is unusual in that the neurotransmitter norepinephrine influences cell destiny in ways generally associated with effects of classical growth factors: regulation of cell proliferation, of apoptosis, and progression of differentiation. The norepinephrine effects are mediated through G-protein-coupled receptors; further mediation of such stimulation to e.g. Erk1/2 activation is in cell biology in general accepted to occur through transactivation of the EGF receptor (by external or internal pathways). We have examined here the significance of such transactivation in brown adipocytes. Stimulation of mature brown adipocytes with cirazoline (α{sub 1}-adrenoceptor coupled via G{sub q}), clonidine (α{sub 2} via G{sub i}) or CL316243 (β{sub 3} via G{sub s}) or via β{sub 1}-receptors significantly activated Erk1/2. Pretreatment with the EGF receptor kinase inhibitor AG1478 had, remarkably, no significant effect on Erk1/2 activation induced by any of these adrenergic agonists (although it fully abolished EGF-induced Erk1/2 activation), demonstrating absence of EGF receptor-mediated transactivation. Results with brown preadipocytes (cells in more proliferative states) were not qualitatively different. Joint stimulation of all adrenoceptors with norepinephrine did not result in synergism on Erk1/2 activation. AG1478 action on EGF-stimulated Erk1/2 phosphorylation showed a sharp concentration–response relationship (IC{sub 50} 0.3 µM); a minor apparent effect of AG1478 on norepinephrine-stimulated Erk1/2 phosphorylation showed nonspecific kinetics, implying caution in interpretation of partial effects of AG1478 as reported in other systems. Transactivation of the EGF receptor is clearly not a universal prerequisite for coupling of G-protein coupled receptors to Erk1/2 signalling cascades. - Highlights: • In brown adipocytes, norepinephrine regulates proliferation, apoptosis, differentiation. • EGF receptor transactivation is supposed to mediate GPCR

  1. Addiction of lung cancer cells to GOF p53 is promoted by up-regulation of epidermal growth factor receptor through multiple contacts with p53 transactivation domain and promoter

    PubMed Central

    Vaughan, Catherine A.; Pearsall, Isabella; Singh, Shilpa; Windle, Brad; Deb, Swati P.; Grossman, Steven R.; Yeudall, W. Andrew; Deb, Sumitra

    2016-01-01

    Human lung cancers harboring gain-of-function (GOF) p53 alleles express higher levels of the epidermal growth factor receptor (EGFR). We demonstrate that a number of GOF p53 alleles directly upregulate EGFR. Knock-down of p53 in lung cancer cells lowers EGFR expression and reduces tumorigenicity and other GOF p53 properties. However, addiction of lung cancer cells to GOF p53 can be compensated by overexpressing EGFR, suggesting that EGFR plays a critical role in addiction. Chromatin immunoprecipitation (ChIP) using lung cancer cells expressing GOF p53 alleles showed that GOF p53 localized to the EGFR promoter. The sequence where GOF p53 is found to interact by ChIP seq can act as a GOF p53 response element. The presence of GOF p53 on the EGFR promoter increased histone H3 acetylation, indicating a mechanism whereby GOF p53 enhances chromatin opening for improved access to transcription factors (TFs). ChIP and ChIP-re-ChIP with p53, Sp1 and CBP histone acetylase (HAT) antibodies revealed docking of GOF p53 on Sp1, leading to increased binding of Sp1 and CBP to the EGFR promoter. Up-regulation of EGFR can occur via GOF p53 contact at other novel sites in the EGFR promoter even when TAD-I is inactivated; these sites are used by both intact and TAD-I mutated GOF p53 and might reflect redundancy in GOF p53 mechanisms for EGFR transactivation. Thus, the oncogenic action of GOF p53 in lung cancer is highly dependent on transactivation of the EGFR promoter via a novel transcriptional mechanism involving coordinated interactions of TFs, HATs and GOF p53. PMID:26820293

  2. Efficient translation of distal cistrons of a polycistronic mRNA of a plant pararetrovirus requires a compatible interaction between the mRNA 3' end and the proteinaceous trans-activator.

    PubMed

    Edskes, H K; Kiernan, J M; Shepherd, R J

    1996-10-15

    Caulimoviruses, a type of plant pararetrovirus, employ a highly unusual mechanism to express the multiple cistrons of their pregenomic RNA. It involves translation of a polycistronic mRNA utilizing cis-acting viral RNA sequences and a transacting virus-encoded protein (P6). In addition to its role in polycistronic translation, the translational trans-activator protein P6 also activates its own expression from a monocistronic subgenomic RNA. Using Nicotiana Edwardsonii cell suspension protoplasts, we analyzed the ability of P6 proteins from three different caulimoviruses to activate viral RNA-based reporter constructs. Cis-acting elements present in figwort mosaic caulimovirus (FMV) are functional not only in the presence of the cognate P6 activator protein, but also in the presence of the heterologous activators from cauliflower mosaic caulimovirus (CaMV) and peanut chlorotic streak caulimovirus (PCISV). However, when 3' cis-acting elements essential for efficient polycistronic expression of FMV are replaced by their counterparts from PCISV, reporter gene expression is only observed in the presence of PCISV P6. Derepression of monocistronic reporter constructs tailed with FMV or CaMV 3' proximal sequences is less efficient in the presence of PCISV P6 than with either FMV or CaMV P6, but more efficient when the constructs contain a cognate PCISV 3' cis-element. Efficient expression of polycistronic and monocistronic caulimovirus mRNAs in plant cells thus requires compatible interactions between P6, a translational trans-activator, and its cognate cis-element at the 3' end of the mRNA.

  3. Proteasome inhibition reduces superantigen-mediated T cell activation and the severity of psoriasis in a SCID-hu model.

    PubMed

    Zollner, Thomas M; Podda, Maurizio; Pien, Christine; Elliott, Peter J; Kaufmann, Roland; Boehncke, Wolf-Henning

    2002-03-01

    There is increasing evidence that bacterial superantigens contribute to inflammation and T cell responses in psoriasis. Psoriatic inflammation entails a complex series of inductive and effector processes that require the regulated expression of various proinflammatory genes, many of which require NF-kappa B for maximal trans-activation. PS-519 is a potent and selective proteasome inhibitor based upon the naturally occurring compound lactacystin, which inhibits NF-kappa B activation by blocking the degradation of its inhibitory protein I kappa B. We report that proteasome inhibition by PS-519 reduces superantigen-mediated T cell-activation in vitro and in vivo. Proliferation was inhibited along with the expression of very early (CD69), early (CD25), and late T cell (HLA-DR) activation molecules. Moreover, expression of E-selectin ligands relevant to dermal T cell homing was reduced, as was E-selectin binding in vitro. Finally, PS-519 proved to be therapeutically effective in a SCID-hu xenogeneic psoriasis transplantation model. We conclude that inhibition of the proteasome, e.g., by PS-519, is a promising means to treat T cell-mediated disorders such as psoriasis.

  4. Inhibition of selectin binding

    DOEpatents

    Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Carolyn

    1999-10-05

    This invention provides a system for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, this system can be used to palliate certain inflammatory and immunological conditions.

  5. Inhibition of selectin binding

    DOEpatents

    Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Caroline

    2001-10-09

    This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

  6. Inhibition of selectin binding

    DOEpatents

    Nagy, Jon O.; Spevak, Wayne R.; Dasgupta, Falguni; Bertozzi, Caroline

    1999-01-01

    This invention provides compositions for inhibiting the binding between two cells, one expressing P- or L-selectin on the surface and the other expressing the corresponding ligand. A covalently crosslinked lipid composition is prepared having saccharides and acidic group on separate lipids. The composition is then interposed between the cells so as to inhibit binding. Inhibition can be achieved at an effective oligosaccharide concentration as low as 10.sup.6 fold below that of the free saccharide. Since selectins are involved in recruiting cells to sites of injury, these composition scan be used to palliate certain inflammatory and immunological conditions.

  7. Brown Pine Leaf Extract and Its Active Component Trans-Communic Acid Inhibit UVB-Induced MMP-1 Expression by Targeting PI3K.

    PubMed

    Huh, Won Bum; Kim, Jong-Eun; Kang, Young-Gyu; Park, Gaeun; Lim, Tae-gyu; Kwon, Jung Yeon; Song, Da Som; Jeong, Eun Hee; Lee, Charles C; Son, Joe Eun; Seo, Sang Gwon; Lee, Eunjung; Kim, Jong Rhan; Lee, Chang Yong; Park, Jun Seong; Lee, Ki Won

    2015-01-01

    Japanese red pine (Pinus densiflora) is widely present in China, Japan, and Korea. Its green pine leaves have traditionally been used as a food as well as a coloring agent. After being shed, pine leaves change their color from green to brown within two years, and although the brown pine leaves are abundantly available, their value has not been closely assessed. In this study, we investigated the potential anti-photoaging properties of brown pine leaves for skin. Brown pine leaf extract (BPLE) inhibited UVB-induced matrix metalloproteinase-1 (MMP-1) expression to a greater extent than pine leaf extract (PLE) in human keratinocytes and a human skin equivalent model. HPLC analysis revealed that the quantity of trans-communic acid (TCA) and dehydroabietic acid (DAA) significantly increases when the pine leaf color changes from green to brown. BPLE and TCA elicited reductions in UVB-induced MMP-1 mRNA expression and activator protein-1 (AP-1) transactivation by reducing DNA binding activity of phospho-c-Jun, c-fos and Fra-1. BPLE and TCA also inhibited UVB-induced Akt phosphorylation, but not mitogen activated protein kinase (MAPK), known regulators of AP-1 transactivation. We additionally found that BPLE and TCA inhibited phosphoinositide 3-kinase (PI3K), the upstream kinase of Akt, in vitro. In summary, both BPLE and its active component TCA exhibit protective effects against UVB-induced skin aging. Taken together, these findings underline the potential for BPLE and TCA to be utilized as anti-wrinkling agents and cosmetic ingredients, as they suppress UVB-induced MMP-1 expression.

  8. Brown Pine Leaf Extract and Its Active Component Trans-Communic Acid Inhibit UVB-Induced MMP-1 Expression by Targeting PI3K

    PubMed Central

    Park, Gaeun; Lim, Tae-gyu; Kwon, Jung Yeon; Song, Da Som; Jeong, Eun Hee; Lee, Charles C.; Son, Joe Eun; Seo, Sang Gwon; Lee, Eunjung; Kim, Jong Rhan; Lee, Chang Yong; Park, Jun Seong; Lee, Ki Won

    2015-01-01

    Japanese red pine (Pinus densiflora) is widely present in China, Japan, and Korea. Its green pine leaves have traditionally been used as a food as well as a coloring agent. After being shed, pine leaves change their color from green to brown within two years, and although the brown pine leaves are abundantly available, their value has not been closely assessed. In this study, we investigated the potential anti-photoaging properties of brown pine leaves for skin. Brown pine leaf extract (BPLE) inhibited UVB-induced matrix metalloproteinase-1 (MMP-1) expression to a greater extent than pine leaf extract (PLE) in human keratinocytes and a human skin equivalent model. HPLC analysis revealed that the quantity of trans-communic acid (TCA) and dehydroabietic acid (DAA) significantly increases when the pine leaf color changes from green to brown. BPLE and TCA elicited reductions in UVB-induced MMP-1 mRNA expression and activator protein-1 (AP-1) transactivation by reducing DNA binding activity of phospho-c-Jun, c-fos and Fra-1. BPLE and TCA also inhibited UVB-induced Akt phosphorylation, but not mitogen activated protein kinase (MAPK), known regulators of AP-1 transactivation. We additionally found that BPLE and TCA inhibited phosphoinositide 3-kinase (PI3K), the upstream kinase of Akt, in vitro. In summary, both BPLE and its active component TCA exhibit protective effects against UVB-induced skin aging. Taken together, these findings underline the potential for BPLE and TCA to be utilized as anti-wrinkling agents and cosmetic ingredients, as they suppress UVB-induced MMP-1 expression. PMID:26066652

  9. 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

  10. p-HPEA-EDA, a phenolic compound of virgin olive oil, activates AMP-activated protein kinase to inhibit carcinogenesis.

    PubMed

    Khanal, Prem; Oh, Won-Keun; Yun, Hyo Jeong; Namgoong, Gwang Mo; Ahn, Sang-Gun; Kwon, Seong-Min; Choi, Hoo-Kyun; Choi, Hong Seok

    2011-04-01

    Phenolic constituents of virgin olive oil are reported to have antitumor activity. However, the underlying molecular mechanisms and specific target proteins of virgin olive oil remain to be elucidated. Here, we report that dialdehydic form of decarboxymethyl ligstroside aglycone (p-HPEA-EDA), a phenolic compound of virgin olive oil, inhibits tumor promoter-induced cell transformation in JB6 Cl41 cells and suppress cyclooxygenase-2 (COX-2) and tumorigenicity by adenosine monophosphate-activated protein kinase (AMPK) activation in HT-29 cells. p-HPEA-EDA inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of extracellular signal-regulated kinases 1/2 and p90RSK in JB6 Cl41 cells, resulting in the inhibition of cell proliferation, activator protein-1 transactivation and cell transformation promoted by TPA. Moreover, p-HPEA-EDA strongly inhibited the cell viability and COX-2 expression by activation of AMPK activity in HT-29 cells, resulted from depletion of intracellular adenosine triphosphate. p-HPEA-EDA-induced activation of caspase-3 and poly-adenosine diphosphate-ribose polymerase, phosphorylation of p53 (Ser15) and DNA fragmentation in HT-29 cells, leading to apoptosis. Importantly, p-HPEA-EDA suppressed the colony formation of HT-29 cells in soft agar. In contrast, Compound C, an AMPK inhibitor, and Z-DEVD-FMK, a caspase-3 inhibitor, blocked the p-HPEA-EDA-inhibited colony formation in HT-29 cells. In vivo chorioallantoic membrane assay also showed that p-HPEA-EDA-inhibited tumorigenicity of HT-29 cells. These findings revealed that targeted activation of AMPK and inhibition of COX-2 expression by p-HPEA-EDA contribute to the chemopreventive and chemotherapeutic potential of virgin olive oil against colon cancer cells.

  11. Inhibition of Axl improves the targeted therapy against ALK-mutated neuroblastoma

    SciTech Connect

    Xu, Fei; Li, Hongling; Sun, Yong

    2014-11-28

    Highlights: • First reported Axl is co-expressed with ALK in neuroblastoma tissues and cell lines. • Axl activation promotes cell growth and impairs the efficiency of ALK inhibitor. • Further found silence of Axl leads to increased sensitivity to ALK inhibitors. • Axl inhibitor promotes the efficiency of targeted therapy in vitro and in vivo. • Axl activation should be considered in the clinical application of ALK inhibitors. - Abstract: Neuroblastoma (NB) patients harboring mutated ALK can be expected to potentially benefit from targeted therapy based on ALK tyrosine kinase inhibitor (TKI), such as crizotinib and ceritinib. However, the effect of the treatment varies with different individuals, although with the same genic changes. Axl receptor tyrosine kinase is expressed in a variety of human cancers, but little data are reported in NB, particularly in which carrying mutated ALK. In this study, we focus on the roles of Axl in ALK-mutated NB for investigating rational therapeutic strategy. We found that Axl is expressed in ALK-positive NB tissues and cell lines, and could be effectively activated by its ligand GAS6. Ligand-dependent Axl activation obviously rescued crizotinib-mediated suppression of cell proliferation in ALK-mutated NB cells. Genetic inhibition of Axl with specific small interfering RNA markedly increased the sensitivity of cells to ALK-TKIs. Furthermore, a small-molecule inhibitor of Axl significantly enhanced ALK-targeted therapy, as an increased frequency of apoptosis was observed in NB cells co-expressing ALK and Axl. Taken together, our results demonstrated that activation of Axl could lead to insensitivity to ALK inhibitors, and dual inhibition of ALK and Axl might be a potential therapeutic strategy against ALK-mutated NB.

  12. SUMOylation of the Farnesoid X Receptor (FXR) Regulates the Expression of FXR Target Genes*

    PubMed Central

    Balasubramaniyan, Natarajan; Luo, Yuhuan; Sun, An-Qiang; Suchy, Frederick J.

    2013-01-01

    The farnesoid X receptor (FXR) belongs to a family of ligand-activated transcription factors that regulate many aspects of metabolism including bile acid homeostasis. Here we show that FXR is covalently modified by the small ubiquitin-like modifier (Sumo1), an important regulator of cell signaling and transcription. Well conserved consensus sites at lysine 122 and 275 in the AF-1 and ligand binding domains, respectively, of FXR were subject to SUMOylation in vitro and in vivo. Chromatin immunoprecipitation (ChIP) analysis showed that Sumo1 was recruited to the bile salt export pump (BSEP), the small heterodimer partner (SHP), and the OSTα-OSTβ organic solute transporter loci in a ligand-dependent fashion. Sequential chromatin immunoprecipitation (ChIP-ReChIP) verified the concurrent binding of FXR and Sumo1 to the BSEP and SHP promoters. Overexpression of Sumo1 markedly decreased binding and/or recruitment of FXR to the BSEP and SHP promoters on ChIP-ReChIP. SUMOylation did not have an apparent effect on nuclear localization of FXR. Expression of Sumo1 markedly inhibited the ligand-dependent, transactivation of BSEP and SHP promoters by FXR/retinoid X receptor α (RXRα) in HepG2 cells. In contrast, mutations that abolished SUMOylation of FXR or siRNA knockdown of Sumo1 expression augmented the transactivation of BSEP and SHP promoters by FXR. Pathways for SUMOylation were significantly altered during obstructive cholestasis with differential Sumo1 recruitment to the promoters of FXR target genes. In conclusion, FXR is subject to SUMOylation that regulates its capacity to transactivate its target genes in normal liver and during obstructive cholestasis. PMID:23546875

  13. Celastrol Inhibits Tat-mediated Human Immunodeficiency Virus (HIV) Transcription and Replication

    PubMed Central

    Narayan, Vivek; Kodihalli, Ravindra C.; Chiaro, Chris; Cary, Daniele; Aggarwal, Bharat B.; Henderson, Andrew J.; Prabhu, K. Sandeep

    2011-01-01

    Current drugs used for anti-retroviral therapy against HIV have a narrow spectrum of activity, and more often have associated toxicities and severe side effects in addition to developing resistance. Thus, there is a need to develop new therapeutic strategies against HIV/AIDS to complement the already existing ones. Surprisingly, Tat, an early virus encoded protein required for the efficient transcription of the HIV genome, has not been developed as a target for small molecular therapeutics. We have previously described the ability of an endogenous Michael acceptor electrophile (MAE), 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), to inhibit Tat-dependent transcription, by targeting its cysteine (Cys) rich domain. In an effort to identify other MAEs possessing inhibitory activity against HIV-1 Tat, we tested a collection of plant-derived compounds with electrophilic properties, including curcumin, rosmarinic acid, and gambogic acid, for their ability to inhibit Tat. Celastrol (Cel), a triterpenoid MAE isolated from T. wilfordii, exhibited the highest inhibitory activity against Tat. Using biochemical techniques, we demonstrate that Cel by covalently modifying the cysteine thiols inhibits Tat transactivation function. Using circular dichroism (CD) spectroscopy, we show that alklylation of Tat brought about a change in the secondary structure of Tat, which inhibited the transcription elongation of the HIV proviral genome by effecting mechanisms other than Tat-TAR interaction. Our results demonstrate the underlying mechanism of anti-retroviral activity of the plant-derived MAEs, and suggest that Cel could serve as a lead compound to develop novel anti-viral therapeutics. PMID:21763500

  14. GIV/Girdin activates Gαi and inhibits Gαs via the same motif

    PubMed Central

    Gupta, Vijay; Bhandari, Deepali; Leyme, Anthony; Aznar, Nicolas; Midde, Krishna K.; Lo, I-Chung; Ear, Jason; Niesman, Ingrid; López-Sánchez, Inmaculada; Blanco-Canosa, Juan Bautista; von Zastrow, Mark; Garcia-Marcos, Mikel; Farquhar, Marilyn G.; Ghosh, Pradipta

    2016-01-01

    We previously showed that guanine nucleotide-binding (G) protein α subunit (Gα)-interacting vesicle-associated protein (GIV), a guanine-nucleotide exchange factor (GEF), transactivates Gα activity-inhibiting polypeptide 1 (Gαi) proteins in response to growth factors, such as EGF, using a short C-terminal motif. Subsequent work demonstrated that GIV also binds Gαs and that inactive Gαs promotes maturation of endosomes and shuts down mitogenic MAPK–ERK1/2 signals from endosomes. However, the mechanism and consequences of dual coupling of GIV to two G proteins, Gαi and Gαs, remained unknown. Here we report that GIV is a bifunctional modulator of G proteins; it serves as a guanine nucleotide dissociation inhibitor (GDI) for Gαs using the same motif that allows it to serve as a GEF for Gαi. Upon EGF stimulation, GIV modulates Gαi and Gαs sequentially: first, a key phosphomodification favors the assembly of GIV–Gαi complexes and activates GIV’s GEF function; then a second phosphomodification terminates GIV’s GEF function, triggers the assembly of GIV–Gαs complexes, and activates GIV’s GDI function. By comparing WT and GIV mutants, we demonstrate that GIV inhibits Gαs activity in cells responding to EGF. Consequently, the cAMP→PKA→cAMP response element-binding protein signaling axis is inhibited, the transit time of EGF receptor through early endosomes are accelerated, mitogenic MAPK–ERK1/2 signals are rapidly terminated, and proliferation is suppressed. These insights define a paradigm in G-protein signaling in which a pleiotropically acting modulator uses the same motif both to activate and to inhibit G proteins. Our findings also illuminate how such modulation of two opposing Gα proteins integrates downstream signals and cellular responses. PMID:27621449

  15. XPO1 (CRM1) inhibition represses STAT3 activation to drive a survivin-dependent oncogenic switch in triple-negative breast cancer.

    PubMed

    Cheng, Yan; Holloway, Michael P; Nguyen, Kevin; McCauley, Dilara; Landesman, Yosef; Kauffman, Michael G; Shacham, Sharon; Altura, Rachel A

    2014-03-01

    Inhibition of XPO1 (CRM1)-mediated nuclear export of multiple tumor suppressor proteins has been proposed as a novel cancer therapeutic strategy to turn off oncogenic signals and enhance tumor suppression. Survivin is a multifunctional protein with oncogenic properties when expressed in the cytoplasm that requires the XPO1-RanGTP complex for its nuclear export. We investigated the antitumor mechanisms of the drug-like selective inhibitors of nuclear export (SINE) XPO1 antagonists KPT-185, KPT-251 KPT-276, and KPT-330 in estrogen receptor-positive and triple-negative breast cancer (TNBC) cell lines and xenograft models of human breast tumors. KPT compounds significantly inhibited breast cancer cell growth and induced tumor cell death, both in vitro and in vivo. These drugs initially promoted survivin accumulation within tumor cell nuclei. However, their major in vitro effect was to decrease survivin cytoplasmic protein levels, correlating with the onset of apoptosis. XPO1 inhibition repressed Survivin transcription by inhibiting CREB-binding protein-mediated STAT3 acetylation, and blocking STAT3 binding to the Survivin promoter. In addition, caspase-3 was activated to cleave survivin, rendering it unavailable to bind X-linked inhibitor of apoptosis protein and block the caspase cascade. Collectively, these data demonstrate that XPO1 inhibition by SINE compounds represses STAT3 transactivation to block the selective oncogenic properties of survivin and supports their clinical use in TNBC.

  16. Potentiation of latent inhibition.

    PubMed

    Rodriguez, Gabriel; Hall, Geoffrey

    2008-07-01

    Rats were given exposure either to an odor (almond) or a compound of odor plus taste (almond plus saline), prior to training in which the odor served as the conditioned stimulus. It was found, for both appetitive and aversive procedures, that conditioning was retarded by preexposure (a latent inhibition effect), and the extent of the retardation was greater in rats preexposed to the compound (i.e., latent inhibition to the odor was potentiated by the presence of the taste). In contrast, the presence of the taste during conditioning itself overshadowed learning about the odor. We argue that the presence of the salient taste in compound with the odor enhances the rate of associative learning, producing a rapid loss in the associability of the odor. This loss of associability will generate both overshadowing and the potentiation of latent inhibition that is observed after preexposure to the compound.

  17. Chitin deacetylase product inhibition.

    PubMed

    Jaworska, Malgorzata M

    2011-02-01

    Chitin deacetylase is the only known enzyme catalyzing the hydrolysis of the acetamino linkage in the N-acetylglucosamine units of chitin and chitosan. This reaction can play an important role in enzymatic production of chitosan from chitin, or in enzymatic modification of chitosan, which has applications in medicine, pharmacy or plant protection. It was previously shown that acetic acid, a product of the deacetylation process, may act as an inhibitor of chitin deacetylase. Here we show the mechanism of inhibition of chitin deacetylase isolated from Absidia orchidis vel coerulea by acetic acid released during the deacetylation process. The process follows competitive inhibition with respect to acetic acid with an inhibition constant of K(i) = 0.286 mmol/L. These results will help to find the optimal system to carry out the enzymatic deacetylation process for industrial applications.

  18. HIV Tat protein inhibits hERG K+ channels: a potential mechanism of HIV infection induced LQTs.

    PubMed

    Bai, Yun-Long; Liu, Hui-Bin; Sun, Bo; Zhang, Ying; Li, Qi; Hu, Chao-Wei; Zhu, Jiu-Xin; Gong, Dong-Mei; Teng, Xue; Zhang, Qin; Yang, Bao-Feng; Dong, De-Li

    2011-11-01

    HIV-infected patients have a high prevalence of long QT syndrome (LQTs). hERG K(+) channel encoded by human ether-a-go-go related gene contributes to IKr K(+) currents responsible for the repolarization of cardiomyocytes. Inhibition of hERG K(+) channels leads to LQTs. HIV Tat protein, the virus transactivator protein, plays a pivotal role in AIDS. The aim of the present study is to examine the effects of HIV Tat protein on hERG K(+) channels stably expressed in HEK293 cells. The hERG K(+) currents were recorded by whole-cell patch-clamp technique and the hERG channel expression was measured by real-time PCR and Western blot techniques. HIV Tat protein at 200 ng/ml concentration showed no acute effect on hERG currents, but HIV Tat protein (200 ng/ml) incubation for 24 h significantly inhibited hERG currents. In HIV Tat incubated cells, the inactivation and the recovery time from inactivation of hERG channels were significantly changed. HIV Tat protein incubation (200 ng/ml) for 24h had no effect on the hERG mRNA expression, but dose-dependently inhibited hERG protein expression. The MTT assay showed that HIV Tat protein at 50 ng/ml and 200 ng/ml had no effect on the cell viability. HIV Tat protein increased reactive oxygen species (ROS) generation and the inhibition of hERG channel protein expression by HIV Tat protein was prevented by antioxidant tempol. HIV Tat protein in vivo treatment reduced IKr currents and prolonged action potential duration of guinea pig cardiomyocytes. We conclude that HIV Tat protein inhibits hERG K(+) currents through the inhibition of hERG protein expression, which might be the potential mechanism of HIV infection induced LQTs.

  19. Peptide bioregulators inhibit apoptosis.

    PubMed

    Khavinson, V K; Kvetnoii, I M

    2000-12-01

    The effects of peptide bioregulators epithalon and vilon on the dynamics of irradiation-induced apoptotic death of spleen lymphocytes in rats indicate that these agents inhibit physiologically programmed cell death. The antiapoptotic effect of vilon was more pronounced, which corroborates the concept on tissue-specific effect of peptide bioregulators.

  20. Serine proteases inhibiting cyanopeptides.

    PubMed

    Radau, G

    2000-08-01

    There are many compounds inhibiting serine proteases which play an important role in the human organism. This article reviews publications on the low-molecular weight, serine protease inhibitory cyanopeptides and reports on new developments in establishing structure-activity relationships.

  1. Reinforcement of inhibition

    PubMed Central

    Anger, Douglas

    1983-01-01

    A differential-reinforcement-of-other-behavior (DRO) schedule with trials and delayed reinforcement was investigated. Periodically a wheel was briefly available to rats, followed six seconds later by brief availability of a bar. Variable-ratio food reinforcement of wheel turns was adjusted to give 95% turns. After variable-ratio-five reinforcement of bar presses produced 100% pressing, then separate ratio schedules were used for presses following turns (turn presses) and presses following nonturns (nonturn presses). Increasing nonturn-press reinforcements decreased turns, even though total reinforcements increased. Reversal by decreasing nonturn-press reinforcements raised turns, though with hysteresis. Thus food reinforcement increased nonturns even though delayed six to ten seconds after nonturns, a delay that greatly reduces response reinforcement. Those and other results indicate that the turn decrease was not due to reinforcement of competing responses. Evidence against other alternatives, and the reduction of responding by increased reinforcement, indicate that the term inhibition is appropriate for the phenomenon reinforced. Response-specific inhibition appears appropriate for this particular kind, since its effects are more specific to particular responses than Pavlovian conditioned-inhibition. Response-specific inhibition seems best considered a behavioral output comparable to responses (e.g., both reinforcible) but with important properties different from responses (e.g., different reinforcement-delay gradients). PMID:16812315

  2. Ligand-dependent intersubunit association with the insulin receptor complex activates its intrinsic kinase activity

    SciTech Connect

    Boeni-Schnetzler, M.; Kaligian, A.; DelVecchio, R.; Pilch, P.F.

    1988-05-15

    Insulin receptor halves (..cap alpha beta..) were obtained upon selective reduction of the holoreceptor (..cap alpha../sub 2/..beta../sub 2/) and were isolated in concentrated form. Autophosphorylation of concentrated ..cap alpha beta.. receptor halves can be stimulated by insulin an average of 4.0-fold, whereas nonreduced holoreceptor can be stimulated 5.4-fold. If ..cap alpha beta.. half-receptors are immobilized on wheat germ agglutinin-agarose, no insulin-stimulated autophosphorylation is observed, whereas immobilized holoreceptor retains insulin responsiveness. Treatment of ..cap alpha beta.. half-receptors with glutathione in the presence of insulin results in reoxidation to the holoreceptor form (..cap alpha../sub 2/..beta../sub 2/) with an efficiency of 60-70% as visualized by immunoblotting, thus providing evidence that two ..cap alpha beta.. halves are in close physical proximity. This reoxidation reaction, which is evident prior to autophosphorylation, is rapid and strictly dependent on the presence of insulin, consistent with the hypothesis that insulin promotes the association of two ..cap alpha beta.. halves. Furthermore, the insulin-induced reoxidation reaction and the insulin-induced autophosphorylation show the same dose dependence suggesting that the noncovalent association of ..cap alpha beta.. half-receptors upon insulin binding is a prerequisite for insulin-stimulated autophosphorylation in concentrated ..gamma beta.. half-receptor preparations. If the ..cap alpha beta.. half-receptor forms are phosphorylated in the presence of an anti-phosphotyrosine antibody and separated from nonphosphorylated ..cap alpha beta.. receptors, we observe that the phosphorylated ..cap alpha beta.. receptor halves contain bound insulin.

  3. Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain.

    PubMed

    Patel, Dipali; Kopec, Jolanta; Fitzpatrick, Fiona; McCorvie, Thomas J; Yue, Wyatt W

    2016-04-06

    The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme.

  4. Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain

    PubMed Central

    Patel, Dipali; Kopec, Jolanta; Fitzpatrick, Fiona; McCorvie, Thomas J.; Yue, Wyatt W.

    2016-01-01

    The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme. PMID:27049649

  5. Rhodium-catalysed hydroacylation or reductive aldol reactions: a ligand dependent switch of reactivity.

    PubMed

    Osborne, James D; Willis, Michael C

    2008-10-28

    The pathway for the combination of enones and beta-S-substituted aldehydes using Rh-catalysis can be switched between a hydroacylation reaction or a reductive aldol reaction by simple choice of the phosphine ligand; this catalyst controlled switch allows access to new ketone hydroacylation products; useful 1,4-diketone intermediates for the synthesis of N-, S- and O-heterocycles.

  6. Targeting Ligand-Dependent and Ligand-Independent Androgen Receptor Signaling in Prostate Cancer

    DTIC Science & Technology

    2015-10-01

    Pugh M, Raj GV, Brown GD, D’Santos C, Robinson JL, Silva G, Launchbury R, Perou CM, Stingl J, Caldas C, Tilley WD, Carroll JS. Progesterone receptor... Peng Y, Raj GV, Yee D, Lange CA. Progesterone receptor-B enhances estrogen responsiveness of breast cancer cells via scaffolding PELP1- and estrogen

  7. Ligand-dependent genomic function of glucocorticoid receptor in triple-negative breast cancer.

    PubMed

    Chen, Zhong; Lan, Xun; Wu, Dayong; Sunkel, Benjamin; Ye, Zhenqing; Huang, Jiaoti; Liu, Zhihua; Clinton, Steven K; Jin, Victor X; Wang, Qianben

    2015-09-16

    Glucocorticoids (GCs) have been widely used as coadjuvants in the treatment of solid tumours, but GC treatment may be associated with poor pharmacotherapeutic response or prognosis. The genomic action of GC in these tumours is largely unknown. Here we find that dexamethasone (Dex, a synthetic GC)-regulated genes in triple-negative breast cancer (TNBC) cells are associated with drug resistance. Importantly, these GC-regulated genes are aberrantly expressed in TNBC patients and are associated with unfavourable clinical outcomes. Interestingly, in TNBC cells, Compound A (CpdA, a selective GR modulator) only regulates a small number of genes not involved in carcinogenesis and therapy resistance. Mechanistic studies using a ChIP-exo approach reveal that Dex- but not CpdA-liganded glucocorticoid receptor (GR) binds to a single glucocorticoid response element (GRE), which drives the expression of pro-tumorigenic genes. Our data suggest that development of safe coadjuvant therapy should consider the distinct genomic function between Dex- and CpdA-liganded GR.

  8. Identification of a ligand-dependent switch within a muscarinic receptor.

    PubMed

    Spalding, T A; Burstein, E S; Henderson, S C; Ducote, K R; Brann, M R

    1998-08-21

    G-protein-coupled receptors spontaneously switch between active and inactive conformations. Agonists stabilize the active conformation, whereas antagonists stabilize the inactive conformation. In a systematic search for residues that participate in receptor function, several regions of the m5 muscarinic receptor were randomly mutated and tested for their functional properties. Mutations spanning one face of transmembrane 6 (TM6) were found to induce high levels of receptor activity in the absence of agonists (constitutive activity). The same face of TM6 contained several residues crucial for receptor activation by agonists and one residue identified as a contact site for both agonists and antagonists. In addition, one mutation induced agonist-like responses from the receptor when exposed to classical antagonists. These results suggest that TM6 is a switch that defines the activation state of the receptor, and that ligand interactions with TM6 stabilize the receptor in either an active or an inactive conformation.

  9. Ligand-dependent structural variations in Escherichia coli F1 ATPase revealed by cryoelectron microscopy.

    PubMed Central

    Gogol, E P; Johnston, E; Aggeler, R; Capaldi, R A

    1990-01-01

    The Escherichia coli F1 ATPase, ECF1, has been examined by cryoelectron microscopy after reaction with Fab' fragments generated from monoclonal antibodies to the alpha and epsilon subunits. The enzyme-antibody complexes appeared triangular due to the superposition of three anti-alpha Fab' fragments on alternating densities of the hexagonally arranged alpha and beta subunits. The Fab' to the epsilon subunit superimposed on a beta subunit. A density was observed near the center of the structure in the internal cavity. The position of this central density with respect to peripheral sites was not fixed. Sorting of images of ECF1 labeled with the combination of three anti-alpha Fab' fragments plus an Fab' directed to the epsilon subunit gave three classes in each of which the central density was closest to a different beta subunit. The distribution of the central density among the three classes was measured for different ligand-binding conditions. When ATP was present in catalytic sites under conditions where there was no enzyme turnover (i.e., without Mg2+ present), there were approximately equal numbers of images in each of three classes. When ATP and Mg2+ were added and ATP hydrolysis was allowed to proceed, almost two-thirds of the images were in the class in which the central density was closest to the beta subunit superimposed by the epsilon subunit. We conclude that domains within the ECF1 structure, either the central mass or a domain including the epsilon subunit, move in the enzyme in response to ligand binding. We suggest that this movement is involved in coupling catalytic sites to the proton channel in the F0 part of the ATP synthase. Images PMID:2148209

  10. Dipyridylamide ligand dependent dimensionality in luminescent zinc 2,4-pyridinedicarboxylate coordination complexes

    NASA Astrophysics Data System (ADS)

    Wudkewych, Megan J.; LaDuca, Robert L.

    2016-09-01

    Zinc nitrate, 2,4-pyridinedicarboxylic acid (2,4-pdcH2), and a hydrogen-bonding capable dipyridylamide ligand were combined in aqueous solution and subjected to hydrothermal reaction conditions. Three new crystalline coordination complexes were generated; their dimensionality depends crucially on the dipyridylamide length and geometric disposition of the pyridyl nitrogen donors. The three new phases were structurally characterized via single-crystal X-ray diffraction. {[H23-pina][Zn(2,4-pdc)2(H2O)2]·H2O} (1, 3-pina = 3-pyridylisonicotinamide) is a salt with protonated dipyridylamide cations and coordination complex anions. {[Zn2(2,4-pdc)2(H2O)4(3-pna)]·3H2O}n (2, 3-pna = 3-pyridylnicotinamide) shows a system of two-fold interpenetrated ruffled (6,3) coordination polymer layers. {[Zn(2,4-pdc)(H2O)2(3-pmna)]n (3, 3-pmna = 3-pyridylmethylnicotinamide) manifests a simple 1D chain topology. Luminescence was observed for two of the zinc complexes; this behavior is attributed to π-π* or π-n molecular orbital transitions. Thermal decomposition properties of the new phases are also probed.

  11. Targeting Ligand Dependent and Ligand Independent Androgen Receptor Signaling in Prostate Cancer

    DTIC Science & Technology

    2014-10-01

    subunits interact under specific condi- tions, and the consequences of these interactions are fun- damental to drug development. Thus, it can be... drug therapy approaches are summarized in Table 2. Converting Peptides to Peptidomimetics Identifying PPI surfaces Identifying interaction motifs between... drugs that have potent effects in blocking the interaction between Runx1:CBFb found in acute myeloid lymphoma (102). Limitations of Peptidomimetics The

  12. Ligand-dependent dynamics of the active-site lid in bacterial dimethylarginine dimethylaminohydrolase.

    PubMed

    Rasheed, Masooma; Richter, Christine; Chisty, Liisa T; Kirkpatrick, John; Blackledge, Martin; Webb, Martin R; Driscoll, Paul C

    2014-02-18

    The dimethylarginine dimethylaminohydrolase (DDAH) enzyme family has been the subject of substantial investigation as a potential therapeutic target for the regulation of vascular tension. DDAH enzymes catalyze the conversion of asymmetric N(η),N(η)-dimethylarginine (ADMA) to l-citrulline. Here the influence of substrate and product binding on the dynamic flexibility of DDAH from Pseudomonas aeruginosa (PaDDAH) has been assessed. A combination of heteronuclear NMR spectroscopy, static and time-resolved fluorescence measurements, and atomistic molecular dynamics simulations was employed. A monodisperse monomeric variant of the wild-type enzyme binds the reaction product l-citrulline with a low millimolar dissociation constant. A second variant, engineered to be catalytically inactive by substitution of the nucleophilic Cys249 residue with serine, can still convert the substrate ADMA to products very slowly. This PaDDAH variant also binds l-citrulline, but with a low micromolar dissociation constant. NMR and molecular dynamics simulations indicate that the active site "lid", formed by residues Gly17-Asp27, exhibits a high degree of internal motion on the picosecond-to-nanosecond time scale. This suggests that the lid is open in the apo state and allows substrate access to the active site that is otherwise buried. l-Citrulline binding to both protein variants is accompanied by an ordering of the lid. Modification of PaDDAH with a coumarin fluorescence reporter allowed measurement of the kinetic mechanism of the PaDDAH reaction. A combination of NMR and kinetic data shows that the catalytic turnover of the enzyme is not limited by release of the l-citrulline product. The potential to develop the coumarin-PaDDAH adduct as an l-citrulline sensor is discussed.

  13. Orphan receptor TR3 enhances p53 transactivation and represses DNA double-strand break repair in hepatoma cells under ionizing radiation.

    PubMed

    Zhao, Bi-xing; Chen, Hang-zi; Du, Xiao-dan; Luo, Jie; He, Jian-ping; Wang, Rong-hao; Wang, Yuan; Wu, Rong; Hou, Ru-rong; Hong, Ming; Wu, Qiao

    2011-08-01

    In response to ionizing radiation (IR)-induced DNA double-strand breaks (DSB), cells elicit an evolutionarily conserved checkpoint response that induces cell cycle arrest and either DNA repair or apoptosis, thereby maintaining genomic stability. DNA-dependent protein kinase (DNA-PK) is a central enzyme involved in DSB repair for mammalian cells that comprises a DNA-PK catalytic subunit and the Ku protein, which act as regulatory elements. DNA-PK also functions as a signaling molecule to selectively regulate p53-dependent apoptosis in response to IR. Herein, we demonstrate that the orphan nuclear receptor TR3 suppresses DSB repair by blocking Ku80 DNA-end binding activity and promoting DNA-PK-induced p53 activity in hepatoma cells. We find that TR3 interacts with Ku80 and inhibits its binding to DNA ends, which then suppresses DSB repair. Furthermore, TR3 is a phosphorylation substrate for DNA-PK and interacts with DNA-PK catalytic subunit in a Ku80-independent manner. Phosphorylated TR3, in turn, enhances DNA-PK-induced phosphorylation and p53 transcription activity, thereby enhancing IR-induced apoptosis in hepatoma cells. Together, our findings reveal novel functions for TR3, not only in DSB repair regulation but also in IR-induced hepatoma cell apoptosis, and they suggest that TR3 is a potential target for cancer radiotherapy.

  14. Transactivation of TrkB by Sigma-1 receptor mediates cocaine-induced changes in dendritic spine density and morphology in hippocampal and cortical neurons

    PubMed Central

    Ka, Minhan; Kook, Yeon-Hee; Liao, Ke; Buch, Shilpa; Kim, Woo-Yang

    2016-01-01

    Cocaine is a highly addictive narcotic associated with dendritic spine plasticity in the striatum. However, it remains elusive whether cocaine modifies spines in a cell type-specific or region-specific manner or whether it alters different types of synapses in the brain. In addition, there is a paucity of data on the regulatory mechanism(s) involved in cocaine-induced modification of spine density. In the current study, we report that cocaine exposure differentially alters spine density, spine morphology, and the types of synapses in hippocampal and cortical neurons. Cocaine exposure in the hippocampus resulted in increased spine density, but had no significant effect on cortical neurons. Although cocaine exposure altered spine morphology in both cell types, the patterns of spine morphology were distinct for each cell type. Furthermore, we observed that cocaine selectively affects the density of excitatory synapses. Intriguingly, in hippocampal neurons cocaine-mediated effects on spine density and morphology involved sigma-1 receptor (Sig-1 R) and its downstream TrkB signaling, which were not the case in cortical neurons. Furthermore, pharmacological inhibition of Sig-1 R prevented cocaine-induced TrkB activation in hippocampal neurons. Our findings reveal a novel mechanism by which cocaine induces selective changes in spine morphology, spine density, and synapse formation, and could provide insights into the cellular basis for the cognitive impairment observed in cocaine addicts. PMID:27735948

  15. Inhibition of cyclin D1 expression by androgen receptor in breast cancer cells--identification of a novel androgen response element.

    PubMed

    Lanzino, Marilena; Sisci, Diego; Morelli, Catia; Garofalo, Cecilia; Catalano, Stefania; Casaburi, Ivan; Capparelli, Claudia; Giordano, Cinzia; Giordano, Francesca; Maggiolini, Marcello; Andò, Sebastiano

    2010-09-01

    Cyclin D1 gene (CCND1) is a critical mitogen-regulated cell-cycle control element whose transcriptional modulation plays a crucial role in breast cancer growth and progression. Here we demonstrate that the non-aromatizable androgen 5-α-dihydrotestosterone (DHT) inhibits endogenous cyclin D1 expression, as evidenced by reduction of cyclin D1 mRNA and protein levels, and decrease of CCND1-promoter activity, in MCF-7 cells. The DHT-dependent inhibition of CCND1 gene activity requires the involvement and the integrity of the androgen receptor (AR) DNA-binding domain. Site directed mutagenesis, DNA affinity precipitation assay, electrophoretic mobility shift assay and chromatin immunoprecipitation analyses indicate that this inhibitory effect is ligand dependent and it is mediated by direct binding of AR to an androgen response element (CCND1-ARE) located at -570 to -556-bp upstream of the transcription start site, in the cyclin D1 proximal promoter. Moreover, AR-mediated repression of the CCND1 involves the recruitment of the atypical orphan nuclear receptor DAX1 as a component of a multiprotein repressor complex also embracing the participation of Histone Deacetylase 1. In conclusion, identification of the CCND1-ARE allows defining cyclin D1 as a specific androgen target gene in breast and might contribute to explain the molecular basis of the inhibitory role of androgens on breast cancer cells proliferation.

  16. Expression of the rat sterol regulatory element-binding protein-1c gene in response to insulin is mediated by increased transactivating capacity of specificity protein 1 (Sp1).

    PubMed

    Deng, Xiong; Yellaturu, Chandrahasa; Cagen, Lauren; Wilcox, Henry G; Park, Edwards A; Raghow, Rajendra; Elam, Marshall B

    2007-06-15

    The induction of genes involved in lipid biosynthesis by insulin is mediated in part by the sterol regulatory element-binding protein-1c (SREBP-1c). SREBP-1c is directly regulated by insulin by transcriptional and post-transcriptional mechanisms. Previously, we have demonstrated that the insulin-responsive cis-acting unit of the rat SREBP-1c promoter is composed of several elements that include a sterol regulatory element, two liver X receptor elements, and a number of conserved GC boxes. Here we systematically dissected the role of these GC boxes and report that five bona fide Sp1-binding elements of the SREBP-1c promoter determine its basal and insulin-induced activation. Luciferase expression driven by the rat SREBP-1c promoter was accelerated by ectopic expression of Sp1, and insulin further enhanced the transactivation potential of Sp1. Introduction of a small interfering RNA against Sp1 reduced both basal and insulin-induced activation of the SREBP-1c promoter. We also found that Sp1 interacted with both SREBP-1c and LXRalpha proteins and that insulin promoted these interactions. Chromatin immunoprecipitation studies revealed that insulin facilitated the recruitment of the steroid receptor coactivator-1 to the SREBP-1c promoter. These studies identify a novel mechanism by which maximal activation of the rat SREBP-1c gene expression by insulin is mediated by Sp1 and its enhanced ability to interact with other transcriptional regulatory proteins.

  17. Nuclear Hormone Receptor Activity of Polybrominated Diphenyl Ethers and Their Hydroxylated and Methoxylated Metabolites in Transactivation Assays Using Chinese Hamster Ovary Cells

    PubMed Central

    Kojima, Hiroyuki; Takeuchi, Shinji; Uramaru, Naoto; Sugihara, Kazumi; Yoshida, Takahiko; Kitamura, Shigeyuki

    2009-01-01

    Background An increasing number of studies are reporting the existence of polybrominated diphenyl ethers (PBDEs) and their hydroxylated (HO) and methoxylated (MeO) metabolites in the environment and in tissues from wildlife and humans. Objective Our aim was to characterize and compare the agonistic and antagonistic activities of principle PBDE congeners and their HO and MeO metabolites against human nuclear hormone receptors. Methods We tested the hormone receptor activities of estrogen receptor α (ERα), ERβ, androgen receptor (AR), glucocorticoid receptor (GR), thyroid hormone receptor α1 (TRα1), and TRβ1 against PBDE congeners BDEs 15, 28, 47, 85, 99, 100, 153, and 209, four para-HO-PBDEs, and four para-MeO-PBDEs by highly sensitive reporter gene assays using Chinese hamster ovary cells. Results Of the 16 compounds tested, 6 and 2 showed agonistic activities in the ERα and ERβ assays, respectively, and 6 and 6 showed antagonistic activities in these assays. 4′-HO-BDE-17 showed the most potent estrogenic activity via ERα/β, and 4′-HO-BDE-49 showed the most potent anti estrogenic activity via ERα/β. In the AR assay, 13 compounds showed antagonistic activity, with 4′-HO-BDE-17 in particular inhibiting AR-mediated transcriptional activity at low concentrations in the order of 10−8 M. In the GR assay, seven compounds, including two HO-PBDEs and two MeO-PBDEs, showed weak antagonistic activity. In the TRα1 and TRβ1 assays, only 4-HO-BDE-90 showed weak antagonistic activity. Conclusions Taken together, these results suggest that PBDEs and their metabolites might have multiple endocrine-disrupting effects via nuclear hormone receptors, and para-HO-PBDEs, in particular, possess more potent receptor activities compared with those of the parent PBDEs and corresponding para-MeO-PBDEs. PMID:19672399

  18. Kisspeptin-10 inhibits cell migration in vitro via a receptor-GSK3 beta-FAK feedback loop in HTR8SVneo cells.

    PubMed

    Roseweir, A K; Katz, A A; Millar, R P

    2012-05-01

    Kisspeptin inhibits cancer cell metastasis and placental trophoblast cell migration. Kisspeptin gene expression in the placenta and circulating kisspeptin levels change during normal pregnancy and they are altered in preeclampsia. We therefore assessed the effect of kisspeptin-10 on the in vitro migration of a human placental cell line derived from first trimester extravillious trophoblasts (HTR8SVneo). HTR8SVneo cells specifically bound 125I-Kisspeptin-10 but kisspeptin-10 did not induce inositol phosphate production. Cell migration was inhibited by kisspeptin-10 with a maximal inhibition at 100nM. The signaling pathways involved in inhibition of cell migration were examined. Treatment with kisspeptin-10 elicited phosphorylation of GSK3 beta at Ser9 (which inhibits activity), with a 3-fold increase at 5 min. Transient phosphorylation of ERK1/2 and p38MAPK peaked at 10min. Phosphorylation of focal adhesion kinase (FAK) at Tyr925 increased 3-fold at 10 min. Inhibition of GSK3 beta correlated with release of beta-catenin into the cytoplasm. These signaling events were differentially blocked by inhibitors of G(q/11), Src, EGFR, PI(3)K, PKC and MEK. The data suggest that kisspeptin/GPR54 EGF-receptor transactivation leads to phosphorylation of ERK1/2, causing activation of p90rsk which in turn inhibits GSK3 beta via Ser9 phosphorylation. Inactivation of GSK3 beta results in release of beta-catenin into the cytoplasm, affecting cell-cell adhesion and Tyr925 phosphorylation of FAK, which increases phosphorylation of ERK1/2 via RAS/Raf-1 creating a feedback loop to enhance the effects on migration. These findings indicate that kisspeptin-10 inhibits the migration of human placental trophoblast-derived HTR8SVneo cells by stimulating complex ERK1/2-p90rsk-GSK3 beta-FAK feedback interactions.

  19. The immunosuppressant FTY720 inhibits tumor angiogenesis via the sphingosine 1-phosphate receptor 1.

    PubMed

    Schmid, Gerald; Guba, Markus; Ischenko, Ivan; Papyan, Armine; Joka, Mareile; Schrepfer, Sabine; Bruns, Christiane J; Jauch, Karl-Walter; Heeschen, Christopher; Graeb, Christian

    2007-05-01

    FTY720, a sphingosine 1-phosphate (S1P) analog, acts as an immunosuppressant through trapping of T cells in secondary lymphoid tissues. FTY720 was also shown to prevent tumor growth and to inhibit vascular permeability. The MTT proliferation assay illustrated that endothelial cells are more susceptible to the anti-proliferative effect of FTY720 than Lewis lung carcinoma (LLC1) cells. In a spheroid angiogenesis model, FTY720 potently inhibited the sprouting activity of VEGF-A-stimulated endothelial cells even at concentrations that apparently had no anti-proliferative effect. Mechanistically, the anti-angiogenic effect of the general S1P receptor agonist FTY720 was mimicked by the specific S1P1 receptor agonist SEW2871. Moreover, the anti-angiogenic effect of FTY720 was abrogated in the presence of CXCR4-neutralizing antibodies. This indicates that the effect was at least in part mediated by the S1P1 receptor and involved transactivation of the CXCR4 chemokine receptor. Additionally, we could illustrate in a coculture spheroid model, employing endothelial and smooth muscle cells (SMCs), that the latter confer a strong protective effect regarding the action of FTY720 upon the endothelial cells. In a subcutaneous LLC1 tumor model, the anti-angiogenic capacity translated into a reduced tumor size in syngeneic C57BL/6 mice. Consistently, in the Matrigel plug in vivo assay, 10 mg/kg/d FTY720 resulted in a strong inhibition of angiogenesis as demonstrated by a reduced capillary density. Thus, in organ transplant patients, FTY720 may prove efficacious in preventing graft rejection as well as tumor development.

  20. IRF-4-mediated CIITA transcription is blocked by KSHV encoded LANA to inhibit MHC II presentation.

    PubMed

    Cai, Qiliang; Banerjee, Shuvomoy; Cervini, Amanda; Lu, Jie; Hislop, Andrew D; Dzeng, Richard; Robertson, Erle S

    2013-10-01

    Peptides presentation to T cells by MHC class II molecules is of importance in initiation of immune response to a pathogen. The level of MHC II expression directly influences T lymphocyte activation and is often targeted by various viruses. Kaposi's sarcoma-associated herpesvirus (KSHV) encoded LANA is known to evade MHC class I peptide processing, however, the effect of LANA on MHC class II remains unclear. Here, we report that LANA down-regulates MHC II expression and presentation by inhibiting the transcription of MHC II transactivator (CIITA) promoter pIII and pIV in a dose-dependent manner. Strikingly, although LANA knockdown efficiently disrupts the inhibition of CIITA transcripts from its pIII and pIV promoter region, the expression of HLA-DQβ but no other MHC II molecules was significantly restored. Moreover, we revealed that the presentation of HLA-DQβ enhanced by LANA knockdown did not help LANA-specific CD4+ T cell recognition of PEL cells, and the inhibition of CIITA by LANA is independent of IL-4 or IFN-γ signaling but dependent on the direct interaction of LANA with IRF-4 (an activator of both the pIII and pIV CIITA promoters). This interaction dramatically blocked the DNA-binding ability of IRF-4 on both pIII and pIV promoters. Thus, our data implies that LANA can evade MHC II presentation and suppress CIITA transcription to provide a unique strategy of KSHV escape from immune surveillance by cytotoxic T cells.

  1. The immunosuppressive effect of Gamisanghyulyunbueum through inhibition of mitogen-activated protein kinase and nuclear factor activation in MOLT-4 cells.

    PubMed

    Shin, Hye-Young; Jeong, Hyun-Ja; Na, Ho-Jeong; Kim, Hong-Joon; Moon, Goo; Shin, Tae-Yong; Yang, Deok-Chun; Hong, Seung-Heon; Kim, Hyung-Min

    2005-07-01

    Gamisanghyulyunbueum (GSHYBE) has been used clinically to treat skin related disease in South Korea. We investigated GSHYBE-mediated changes in downstream T cell signal transduction. To determine the mechanism of inhibition, we have studied many of the major pathways in phytohemagglutinin (PHA)-activated T cell. We show that among the mitogen-activated protein kinase family activation of phosphorylation of extra cellular signal-regulated kinase 1/2 (ERK1/2, p44/42) and p38, but not c-jun NH2-terminal kinase is inhibited. In activated MOLT-4 cells, the nuclear localization of nuclear factor of activated T cells (NFATc) was blocked by GSHYBE (1 mg/ml). Also, degradation of inhibitor kappaB-alpha and transactivation by nuclear factor-kappaB (NF-kappaB)/Rel A were impaired by GSHYBE (1 mg/ml). Furthermore, interlukin (IL)-2, IL-4 and Interferen (IFN)-gamma secretion by PHA activated MOLT-4 cells and peripheral blood mononuclear cells (PBMC) were significantly diminishes following GSHYBE treatment (1 mg/ml). Also, oral administration of GSHYBE inhibited IL-2 secretion in skin allergic reaction. In conclusion, our data indicate that GSHYBE treatment of T cells inhibits ERK1/2 and p38 activation and nuclear translocation of NFATc, NF-kappaB, resulting in diminished secretion of IL-2.

  2. Adenovirus Vectors Block Human Immunodeficiency Virus–1 Replication in Human Alveolar Macrophages by Inhibition of the Long Terminal Repeat

    PubMed Central

    Kaner, Robert J.; Santiago, Francisco; Rahaghi, Franck; Michaels, Elizabeth; Moore, John P.; Crystal, Ronald G.

    2010-01-01

    Heterologous viruses may transactivate or suppress human immunodeficiency virus (HIV)–1 replication. An adenovirus type 5 gene transfer vector (Ad5) HIV-1 vaccine was recently evaluated in a clinical trial, without efficacy. In this context, it is relevant to ask what effect Ad vectors have on HIV-1 replication, particularly in cells that are part of the innate immune system. Infection of HIV-1–infected human alveolar macrophages (AMs) obtained from HIV-1+ individuals with an Ad vector containing no transgene (AdNull) resulted in dose-responsive inhibition of endogenous HIV-1 replication. HIV-1 replication in normal AMs infected with HIV-1 in vitro was inhibited by AdNull with a similar dose response. Ad reduced AM HIV-1 replication up to 14 days after HIV-1 infection. Fully HIV-1–infected AMs were treated with 3′-azido-3′-deoxythymidine, after which Ad infection still inhibited HIV-1 replication, suggesting a postentry step was affected. Substantial HIV-1 DNA was still produced after Ad infection, as quantified by TaqMan real-time PCR, suggesting that the replication block occurred after reverse transcription. AdNull blocked HIV-1 long terminal repeat (LTR) transcription, as assessed by an vesicular stomatitis virus G protein pseudotyped HIV-1 LTR luciferase construct. The formation of HIV-1 DNA integrated into the host chromosome was not inhibited by Ad, as quantified by a two-step TaqMan real-time PCR assay, implying a postintegration block to HIV-1 replication. These data indicate that Ad vectors are inhibitory to HIV-1 replication in human AMs based, in part, on their ability to inhibit LTR-driven transcription. PMID:19805482

  3. Endothelins Inhibit Osmotic Swelling of Rat Retinal Glial and Bipolar Cells by Activation of Growth Factor Signaling.

    PubMed

    Vogler, Stefanie; Grosche, Antje; Pannicke, Thomas; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas

    2016-10-01

    Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Here, we show that endothelin-1 (ET-1) dose-dependently inhibits the hypoosmotic swelling of Müller cells in freshly isolated retinal slices of control and diabetic rats, with a maximal inhibition at 100 nM. Osmotic Müller cell swelling was also inhibited by ET-2. The effect of ET-1 was mediated by activation of ETA and ETB receptors resulting in transactivation of metabotropic glutamate receptors, purinergic P2Y1, and adenosine A1 receptors. ET-1 (but not ET-2) also inhibited the osmotic swelling of bipolar cells in retinal slices, but failed to inhibit the swelling of freshly isolated bipolar cells. The inhibitory effect of ET-1 on the bipolar cell swelling in retinal slices was abrogated by inhibitors of the FGF receptor kinase (PD173074) and of TGF-β1 superfamily activin receptor-like kinase receptors (SB431542), respectively. Both Müller and bipolar cells displayed immunoreactivities of ETA and ETB receptor proteins. The data may suggest that neuroprotective effects of ETs in the retina are in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. ET-1 acts directly on Müller cells, while the inhibitory effect of ET-1 on bipolar cell swelling is indirectly mediated, via stimulation of the release of growth factors like bFGF and TGF-β1 from Müller cells.

  4. The aryl hydrocarbon receptor and estrogen receptor alpha differentially modulate nuclear factor erythroid-2-related factor 2 transactivation in MCF-7 breast cancer cells

    SciTech Connect

    Lo, Raymond; Matthews, Jason

    2013-07-15

    Nuclear factor erythroid-2-related factor 2 (NRF2; NFE2L2) plays an important role in mediating cellular protection against reactive oxygen species. NRF2 signaling is positively modulated by the aryl hydrocarbon receptor (AHR) but inhibited by estrogen receptor alpha (ERα). In this study we investigated the crosstalk among NRF2, AHR and ERα in MCF-7 breast cancer cells treated with the NRF2 activator sulforaphane (SFN), a dual AHR and ERα activator, 3,3′-diindolylmethane (DIM), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 17β-estradiol (E2). SFN-dependent increases in NADPH-dependent oxidoreductase 1 (NQO1) and heme oxygenase I (HMOX1) mRNA levels were significantly reduced after co-treatment with E2. E2-dependent repression of NQO1 and HMOX1 was associated with increased ERα but reduced p300 recruitment and reduced histone H3 acetylation at both genes. In contrast, DIM + SFN or TCDD + SFN induced NQO1 and HMOX1 mRNA expression to levels higher than SFN alone, which was prevented by RNAi-mediated knockdown of AHR. DIM + SFN but not TCDD + SFN also induced recruitment of ERα to NQO1 and HMOX1. However, the presence of AHR at NQO1 and HMOX1 restored p300 recruitment and histone H3 acetylation, thereby reversing the ERα-dependent repression of NRF2. Taken together, our study provides further evidence of functional interplay among NRF2, AHR and ERα signaling pathways through altered p300 recruitment to NRF2-regulated target genes. - Highlights: • We examined crosstalk among ERα, AHR, and NRF2 in MCF-7 breast cancer cells. • AHR enhanced the mRNA expression levels of two NRF2 target genes – HMOX1 and NQO1. • ERα repressed HMOX1 and NQO1 expression via decreased histone acetylation. • AHR prevented ERα-dependent repression of HMOX1 and NQO1.

  5. The Unfolded Protein Response and the Phosphorylations of Activating Transcription Factor 2 in the trans-Activation of il23a Promoter Produced by β-Glucans*

    PubMed Central

    Rodríguez, Mario; Domingo, Esther; Alonso, Sara; Frade, Javier García; Eiros, José; Crespo, Mariano Sánchez; Fernández, Nieves

    2014-01-01

    Current views on the control of IL-23 production focus on the regulation of il23a, the gene encoding IL-23 p19, by NF-κB in combination with other transcription factors. C/EBP homologous protein (CHOP), X2-Box-binding protein 1 (XBP1), activator protein 1 (AP1), SMAD, CCAAT/enhancer-binding protein (C/EBPβ), and cAMP-response element-binding protein (CREB) have been involved in response to LPS, but no data are available regarding the mechanism triggered by the fungal mimic and β-glucan-containing stimulus zymosan, which produces IL-23 and to a low extent the related cytokine IL-12 p70. Zymosan induced the mobilization of CHOP from the nuclear fractions to phagocytic vesicles. Hypha-forming Candida also induced the nuclear disappearance of CHOP. Assay of transcription factor binding to the il23a promoter showed an increase of Thr(P)-71–Thr(P)-69-activating transcription factor 2 (ATF2) binding in response to zymosan. PKC and PKA/mitogen- and stress-activated kinase inhibitors down-regulated Thr(P)-71–ATF2 binding to the il23a promoter and il23a mRNA expression. Consistent with the current concept of complementary phosphorylations on N-terminal Thr-71 and Thr-69 of ATF2 by ERK and p38 MAPK, MEK, and p38 MAPK inhibitors blunted Thr(P)-69–ATF2 binding. Knockdown of atf2 mRNA with siRNA correlated with inhibition of il23a mRNA, but it did not affect the expression of il12/23b and il10 mRNA. These data indicate the following: (i) zymosan decreases nuclear proapoptotic CHOP, most likely by promoting its accumulation in phagocytic vesicles; (ii) zymosan-induced il23a mRNA expression is best explained through coordinated κB- and ATF2-dependent transcription; and (iii) il23a expression relies on complementary phosphorylation of ATF2 on Thr-69 and Thr-71 dependent on PKC and MAPK activities. PMID:24982422

  6. Semantic processing and response inhibition.

    PubMed

    Chiang, Hsueh-Sheng; Motes, Michael A; Mudar, Raksha A; Rao, Neena K; Mansinghani, Sethesh; Brier, Matthew R; Maguire, Mandy J; Kraut, Michael A; Hart, John

    2013-11-13

    The present study examined functional MRI (fMRI) BOLD signal changes in response to object categorization during response selection and inhibition. Young adults (N=16) completed a Go/NoGo task with varying object categorization requirements while fMRI data were recorded. Response inhibition elicited increased signal change in various brain regions, including medial frontal areas, compared with response selection. BOLD signal in an area within the right angular gyrus was increased when higher-order categorization was mandated. In addition, signal change during response inhibition varied with categorization requirements in the left inferior temporal gyrus (lIT). lIT-mediated response inhibition when inhibiting the response only required lower-order categorization, but lIT mediated both response selection and inhibition when selecting and inhibiting the response required higher-order categorization. The findings characterized mechanisms mediating response inhibition associated with semantic object categorization in the 'what' visual object memory system.

  7. Sumoylation delays the ATF7 transcription factor subcellular localization and inhibits its transcriptional activity.

    PubMed

    Hamard, Pierre-Jacques; Boyer-Guittaut, Michaël; Camuzeaux, Barbara; Dujardin, Denis; Hauss, Charlotte; Oelgeschläger, Thomas; Vigneron, Marc; Kedinger, Claude; Chatton, Bruno

    2007-01-01

    Over the past few years, small ubiquitin-like modifier (SUMO) modification has emerged as an important regulator of diverse pathways and activities including protein localization and transcriptional regulation. We identified a consensus sumoylation motif (IKEE), located within the N-terminal activation domain of the ATF7 transcription factor and thus investigated the role of this modification. ATF7 is a ubiquitously expressed transcription factor, homologous to ATF2, that binds to CRE elements within specific promoters. This protein is able to heterodimerize with Jun or Fos proteins and its transcriptional activity is mediated by interaction with TAF12, a subunit of the general transcription factor TFIID. In the present article, we demonstrate that ATF7 is sumoylated in vitro (using RanBP2 as a E3-specific ligase) and in vivo. Moreover, we show that ATF7 sumoylation affects its intranuclear localization by delaying its entry into the nucleus. Furthermore, SUMO conjugation inhibits ATF7 transactivation activity by (i) impairing its association with TAF12 and (ii) blocking its binding-to-specific sequences within target promoters.

  8. Cell cycle-dependent inhibition of 53BP1 signaling by BRCA1

    PubMed Central

    Feng, Lin; Li, Nan; Li, Yujing; Wang, Jiadong; Gao, Min; Wang, Wenqi; Chen, Junjie

    2015-01-01

    DNA damage response mediator protein 53BP1 is a key regulator of non-homologous end-joining (NHEJ) repair. 53BP1 protects DNA broken ends from resection by recruiting two downstream factors, RIF1 (RAP1-interacting factor 1) and PTIP (Pax transactivation domain-interacting protein), to double-stranded breaks (DSBs) via ATM (ataxia telangiectasia mutated)-mediated 53BP1 phosphorylation, and competes with BRCA1-mediated homologous recombination (HR) repair in G1 phase. In contrast, BRCA1 antagonizes 53BP1-direct NHEJ repair in S/G2 phases. We and others have found that BRCA1 prevents the translocation of RIF1 to DSBs in S/G2 phases; however, the underlying mechanism remains unclear. Here we show that efficient ATM-dependent 53BP1 phosphorylation is restricted to the G1 phase of the cell cycle, as a consequence RIF1 and PTIP accumulation at DSB sites only occur in G1 phase. Mechanistically, both BRCT and RING domains of BRCA1 are required for the inhibition of 53BP1 phosphorylation in S and G2 phases. Thus, our findings reveal how BRCA1 antagonizes 53BP1 signaling to ensure that HR repair is the dominant repair pathway in S/G2 phases. PMID:27462418

  9. Fisetin induces Sirt1 expression while inhibiting early adipogenesis in 3T3-L1 cells.

    PubMed

    Kim, Sang Chon; Kim, Yoo Hoon; Son, Sung Wook; Moon, Eun-Yi; Pyo, Suhkneung; Um, Sung Hee

    2015-11-27

    Fisetin (3,7,3',4'-tetrahydroxyflavone) is a naturally found flavonol in many fruits and vegetables and is known to have anti-aging, anti-cancer and anti-viral effects. However, the effects of fisetin on early adipocyte differentiation and the epigenetic regulator controlling adipogenic transcription factors remain unclear. Here, we show that fisetin inhibits lipid accumulation and suppresses the expression of PPARγ in 3T3-L1 cells. Fisetin suppressed early stages of preadipocyte differentiation, and induced expression of Sirt1. Depletion of Sirt1 abolished the inhibitory effects of fisetin on intracellular lipid accumulation and on PPARγ expression. Mechanistically, fisetin facilitated Sirt1-mediated deacetylation of PPARγ and FoxO1, and enhanced the association of Sirt1 with the PPARγ promoter, leading to suppression of PPARγ transcriptional activity, thereby repressing adipogenesis. Lowering Sirt1 levels reversed the effects of fisetin on deacetylation of PPARγ and increased PPARγ transactivation. Collectively, our results suggest the effects of fisetin in increasing Sirt1 expression and in epigenetic control of early adipogenesis.

  10. Inhibition of G0/G1 Switch 2 Ameliorates Renal Inflammation in Chronic Kidney Disease.

    PubMed

    Matsunaga, Naoya; Ikeda, Eriko; Kakimoto, Keisuke; Watanabe, Miyako; Shindo, Naoya; Tsuruta, Akito; Ikeyama, Hisako; Hamamura, Kengo; Higashi, Kazuhiro; Yamashita, Tomohiro; Kondo, Hideaki; Yoshida, Yuya; Matsuda, Masaki; Ogino, Takashi; Tokushige, Kazutaka; Itcho, Kazufumi; Furuichi, Yoko; Nakao, Takaharu; Yasuda, Kaori; Doi, Atsushi; Amamoto, Toshiaki; Aramaki, Hironori; Tsuda, Makoto; Inoue, Kazuhide; Ojida, Akio; Koyanagi, Satoru; Ohdo, Shigehiro

    2016-11-01

    Chronic kidney disease (CKD) is a global health problem, and novel therapies to treat CKD are urgently needed. Here, we show that inhibition of G0/G1 switch 2 (G0s2) ameliorates renal inflammation in a mouse model of CKD. Renal expression of chemokine (C-C motif) ligand 2 (Ccl2) was increased in response to p65 activation in the kidneys of wild-type 5/6 nephrectomy (5/6Nx) mice. Moreover, 5/6Nx Clk/Clk mice, which carry homozygous mutations in the gene encoding circadian locomotor output cycles kaput (CLOCK), did not exhibit aggravation of apoptosis or induction of F4/80-positive cells. The renal expression of G0s2 in wild-type 5/6Nx mice was important for the transactivation of Ccl2 by p65. These pathologies were ameliorated by G0s2 knockdown. Furthermore, a novel small-molecule inhibitor of G0s2 expression was identified by high-throughput chemical screening, and the inhibitor suppressed renal inflammation in 5/6Nx mice. These findings indicated that G0s2 inhibitors may have applications in the treatment of CKD.

  11. A plant alkaloid, veratridine, potentiates cancer chemosensitivity by UBXN2A-dependent inhibition of an oncoprotein, mortalin-2

    PubMed Central

    Abdullah, Ammara; Sane, Sanam; Branick, Kate A.; Freeling, Jessica L.; Wang, Hongmin; Zhang, Dong; Rezvani, Khosrow

    2015-01-01

    Veratridine (VTD), an alkaloid derived from the Liliaceae plant shows anti-tumor effects; however, its molecular targets have not been thoroughly studied. Using a high-throughput drug screen, we found that VTD enhances transactivation of UBXN2A, resulting in upregulation of UBXN2A in the cytoplasm, where UBXN2A binds and inhibits the oncoprotein mortalin-2 (mot-2). VTD-treated cancer cells undergo cell death in UBXN2A- and mot-2-dependent manners. The cytotoxic function of VTD is grade-dependent, and the combined treatment with a sub-optimal dose of the standard chemotherapy, 5-Fluorouracil (5-FU) and etoposide, demonstrated a synergistic effect, resulting in higher therapeutic efficacy. VTD influences the CD44+ stem cells, possibly through UBXN2A-dependent inhibition of mot-2. The VTD-dependent expression of UBXN2A is a potential candidate for designing novel strategies for colon cancer treatment because: 1) In 50% of colon cancer patients, UBXN2A protein levels in tumor tissues are significantly lower than those in the adjacent normal tissues. 2) Cytoplasmic expression of the mot-2 protein is very low in non-cancerous cells; thus, VTD can produce tumor-specific toxicity while normal cells remain intact. 3) Finally, VTD or its modified analogs offer a valuable adjuvant chemotherapy strategy to improve the efficacy of 5-FU-based chemotherapy for colon cancer patients harboring WT-p53. PMID:26188124

  12. Brusatol inhibits HIF-1 signaling pathway and suppresses glucose uptake under hypoxic conditions in HCT116 cells

    PubMed Central

    Lu, Yapeng; Wang, Bo; Shi, Qian; Wang, Xueting; Wang, Dang; Zhu, Li

    2016-01-01

    Hypoxia-inducible factor-1 (HIF-1) is an important transcription factor that induces adaptive responses upon low oxygen conditions in human cancers and triggers off a poor prognostic outcome of conventional treatments. In this study, we discovered for the first time that brusatol (BRU), a quassinoid extracted from Brucea Esters, has the capability to inhibit HIF-1 signaling pathway. We found that BRU concentration-dependently down-regulated HIF-1α protein levels under hypoxia or CoCl2-induced mimic hypoxia in HCT116 cells without causing significant cytotoxicity. Besides, the transactivation activity of HIF-1 was suppressed by BRU under hypoxic conditions, as well as the expression of HIF-1 target genes, including VEGF, GLUT1, HK2 and LDHA. In addition, BRU can also decrease glucose consumption under hypoxia through inhibition of HIF-1 signaling pathway. Further studies revealed that the inhibitory effect of BRU on HIF-1 signaling pathway might be attributed to promoting degradation of HIF-1α. Interestingly, intracellular reactive oxygen species (ROS) levels and mitochondrial ROS level were both decreased by BRU treatment, indicating the involvment of mitochondrial ROS regulation in the action of BRU. Taken together, these results provided clear evidence for BRU-mediated HIF-1α regulation and suggested its therapeutic potential in colon tumors. PMID:27982118

  13. A plant alkaloid, veratridine, potentiates cancer chemosensitivity by UBXN2A-dependent inhibition of an oncoprotein, mortalin-2.

    PubMed

    Abdullah, Ammara; Sane, Sanam; Branick, Kate A; Freeling, Jessica L; Wang, Hongmin; Zhang, Dong; Rezvani, Khosrow

    2015-09-15

    Veratridine (VTD), an alkaloid derived from the Liliaceae plant shows anti-tumor effects; however, its molecular targets have not been thoroughly studied. Using a high-throughput drug screen, we found that VTD enhances transactivation of UBXN2A, resulting in upregulation of UBXN2A in the cytoplasm, where UBXN2A binds and inhibits the oncoprotein mortalin-2 (mot-2). VTD-treated cancer cells undergo cell death in UBXN2A- and mot-2-dependent manners. The cytotoxic function of VTD is grade-dependent, and the combined treatment with a sub-optimal dose of the standard chemotherapy, 5-Fluorouracil (5-FU) and etoposide, demonstrated a synergistic effect, resulting in higher therapeutic efficacy. VTD influences the CD44+ stem cells, possibly through UBXN2A-dependent inhibition of mot-2. The VTD-dependent expression of UBXN2A is a potential candidate for designing novel strategies for colon cancer treatment because: 1) In 50% of colon cancer patients, UBXN2A protein levels in tumor tissues are significantly lower than those in the adjacent normal tissues. 2) Cytoplasmic expression of the mot-2 protein is very low in non-cancerous cells; thus, VTD can produce tumor-specific toxicity while normal cells remain intact. 3) Finally, VTD or its modified analogs offer a valuable adjuvant chemotherapy strategy to improve the efficacy of 5-FU-based chemotherapy for colon cancer patients harboring WT-p53.

  14. Pharmacological inhibition of FTO.

    PubMed

    McMurray, Fiona; Demetriades, Marina; Aik, WeiShen; Merkestein, Myrte; Kramer, Holger; Andrew, Daniel S; Scudamore, Cheryl L; Hough, Tertius A; Wells, Sara; Ashcroft, Frances M; McDonough, Michael A; Schofield, Christopher J; Cox, Roger D

    2015-01-01

    In 2007, a genome wide association study identified a SNP in intron one of the gene encoding human FTO that was associated with increased body mass index. Homozygous risk allele carriers are on average three kg heavier than those homozygous for the protective allele. FTO is a DNA/RNA demethylase, however, how this function affects body weight, if at all, is unknown. Here we aimed to pharmacologically inhibit FTO to examine the effect of its demethylase function in vitro and in vivo as a first step in evaluating the therapeutic potential of FTO. We showed that IOX3, a known inhibitor of the HIF prolyl hydroxylases, decreased protein expression of FTO (in C2C12 cells) and reduced maximal respiration rate in vitro. However, FTO protein levels were not significantly altered by treatment of mice with IOX3 at 60 mg/kg every two days. This treatment did not affect body weight, or RER, but did significantly reduce bone mineral density and content and alter adipose tissue distribution. Future compounds designed to selectively inhibit FTO's demethylase activity could be therapeutically useful for the treatment of obesity.

  15. Pharmacological Inhibition of FTO

    PubMed Central

    McMurray, Fiona; Demetriades, Marina; Aik, WeiShen; Merkestein, Myrte; Kramer, Holger; Andrew, Daniel S.; Scudamore, Cheryl L.; Hough, Tertius A.; Wells, Sara; Ashcroft, Frances M.; McDonough, Michael A.; Schofield, Christopher J.; Cox, Roger D.

    2015-01-01

    In 2007, a genome wide association study identified a SNP in intron one of the gene encoding human FTO that was associated with increased body mass index. Homozygous risk allele carriers are on average three kg heavier than those homozygous for the protective allele. FTO is a DNA/RNA demethylase, however, how this function affects body weight, if at all, is unknown. Here we aimed to pharmacologically inhibit FTO to examine the effect of its demethylase function in vitro and in vivo as a first step in evaluating the therapeutic potential of FTO. We showed that IOX3, a known inhibitor of the HIF prolyl hydroxylases, decreased protein expression of FTO (in C2C12 cells) and reduced maximal respiration rate in vitro. However, FTO protein levels were not significantly altered by treatment of mice with IOX3 at 60 mg/kg every two days. This treatment did not affect body weight, or RER, but did significantly reduce bone mineral density and content and alter adipose tissue distribution. Future compounds designed to selectively inhibit FTO’s demethylase activity could be therapeutically useful for the treatment of obesity. PMID:25830347

  16. DNA-binding and transactivation properties of Pax-6: three amino acids in the paired domain are responsible for the different sequence recognition of Pax-6 and BSAP (Pax-5).

    PubMed Central

    Czerny, T; Busslinger, M

    1995-01-01

    Pax-6 is known to be a key regulator of vertebrate eye development. We have now isolated cDNA for an invertebrate Pax-6 protein from sea urchin embryos. Transcripts of this gene first appear during development at the gastrula stage and are later expressed at high levels in the tube foot of the adult sea urchin. The sea urchin Pax-6 protein is highly homologous throughout the whole protein to its vertebrate counterpart with the paired domain and homeodomain being virtually identical. Consequently, we found that the DNA-binding and transactivation properties of the sea urchin and mouse Pax-6 proteins are very similar, if not identical. A potent activation domain capable of stimulating transcription from proximal promoter and distal enhancer positions was localized within the C-terminal sequences of both the sea urchin and mouse Pax-6 proteins. The homeodomain of Pax-6 was shown to cooperatively dimerize on DNA sequences consisting of an inverted repeat of the TAAT motif with a preferred spacing of 3 nucleotides. The consensus recognition sequence of the Pax-6 paired domain deviates primarily only at one position from that of BSAP (Pax-5), and yet the two proteins exhibit largely different binding specificities for individual, naturally occurring sites. By creating Pax-6-BSAP fusion proteins, we were able to identify a short amino acid stretch in the N-terminal part of the paired domain which is responsible for these differences in DNA-binding specificity. Mutation of three Pax-6-specific residues in this region (at positions 42, 44, and 47 of the paired domain) to the corresponding amino acids of BSAP resulted in a complete switch of the DNA-binding specificity from Pax-6 to BSAP. These three amino acids were furthermore shown to discriminate between the Pax-6- and BSAP-specific nucleotide at the divergent position of the two consensus recognition sequences. PMID:7739566

  17. Trans-Activation between EphA and FGFR Regulates Self-Renewal and Differentiation of Mouse Embryonic Neural Stem/Progenitor Cells via Differential Activation of FRS2α.

    PubMed

    Sawada, Takahiro; Arai, Daiki; Jing, Xuefeng; Furushima, Kenryo; Chen, Qingfa; Kawakami, Kazuki; Yokote, Hideyuki; Miyajima, Masayasu; Sakaguchi, Kazushige

    2015-01-01

    Ephs and FGFRs belong to a superfamily of receptor tyrosine kinases, playing important roles in stem cell biology. We previously reported that EphA4 and FGFR form a heterodimer following stimulation with ligands, trans-activating each other and signaling through a docking protein, FRS2α, that binds to both receptors. Here, we investigated whether the interaction between EphA4 and FGFRs can be generalized to other Ephs and FGFRs, and, in addition, examined the downstream signal mediating their function in embryonic neural stem/progenitor cells. We revealed that various Ephs and FGFRs interact with each other through similar molecular domains. When neural stem/progenitor cells were stimulated with FGF2 and ephrin-A1, the signal transduced from the EphA4/FGFR/FRS2α complex enhanced self-renewal, while stimulation with ephrin-A1 alone induced neuronal differentiation. The downstream signal required for neuronal differentiation appears to be MAP kinase mainly linked to the Ras family of G proteins. MAP kinase activation was delayed and sustained, distinct from the transient activation induced by FGF2. Interestingly, this effect on neuronal differentiation required the presence of FGFRs. Specific FGFR inhibitor almost completely abolished the function of ephrin-A1 stimulation. These findings suggest that the ternary complex of EphA, FGFR and FRS2α formed by ligand stimulation regulates self-renewal and differentiation of mouse embryonic neural stem/progenitor cells by ligand-specific fine tuning of the downstream signal via FRS2α.

  18. Fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin transactivates aryl hydrocarbon receptor-responsive element III in the tyrosine hydroxylase immunoreactive neurons of the mouse midbrain.

    PubMed

    Tanida, Takashi; Tasaka, Ken; Akahoshi, Eiichi; Ishihara-Sugano, Mitsuko; Saito, Michiko; Kawata, Shigehisa; Danjo, Megumi; Tokumoto, Junko; Mantani, Youhei; Nagahara, Daichi; Tabuchi, Yoshiaki; Yokoyama, Toshifumi; Kitagawa, Hiroshi; Kawata, Mitsuhiro; Hoshi, Nobuhiko

    2014-02-01

    Fetal exposure to dioxins and related compounds is known to disrupt normal development of the midbrain dopaminergic system, which regulates behavior, cognition and emotion. The toxicity of these chemicals is mediated mainly by aryl hydrocarbon receptor (AhR) signaling. Previously, we identified a novel binding motif of AhR, the AhR-responsive element III (AHRE-III), in vitro. This motif is located upstream from the gene encoding tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine biosynthesis. To provide in vivo evidence, we investigated whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could regulate AHRE-III transcriptional activity in midbrain dopaminergic neurons. We produced transgenic mice with inserted constructs of the AHRE-III enhancers, TH gene promoter and the c-myc-tagged luciferase gene. Single oral administrations of TCDD (0-2000 ng kg⁻¹ body weight) to the transgenic dams markedly enhanced TH-immunoreactive (ir) intensity in the A9, A10 and A8 areas of their offspring at 3 days and 8 weeks of age. The offspring of dams treated with 200 ng kg⁻¹ TCDD exhibited significant increases in the numbers of TH- and double (TH and c-myc)-ir neurons in area A9 compared with controls at 8 weeks. These results show that fetal exposure to TCDD upregulates TH expression and increases TH-ir neurons in the midbrain. Moreover, the results suggest that TCDD directly transactivates the TH promoter via the AhR-AHRE-III-mediated pathway in area A9. Fetal exposure to TCDD caused stable upregulation of TH via the AhR-AHRE-III signaling pathway and overgrowth of TH-ir neurons in the midbrain, implying possible involvement in the etiology of neurodevelopmental disorders such as attention-deficit/hyperactivity disorder (ADHD).

  19. Activation of c-Jun N-Terminal Kinase 1 by UV Irradiation Is Inhibited by Wortmannin without Affecting c-jun Expression

    PubMed Central

    Fritz, G.; Kaina, B.

    1999-01-01

    Activation of c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases is an early response of cells upon exposure to DNA-damaging agents. JNK-mediated phosphorylation of c-Jun is currently understood to stimulate the transactivating potency of AP-1 (e.g., c-Jun/c-Fos; c-Jun/ATF-2), thereby increasing the expression of AP-1 target genes. Here we show that stimulation of JNK1 activity is not a general early response of cells exposed to genotoxic agents. Treatment of NIH 3T3 cells with UV light (UV-C) as well as with methyl methanesulfonate (MMS) caused activation of JNK1 and an increase in c-Jun protein and AP-1 binding activity, whereas antineoplastic drugs such as mafosfamide, mitomycin C, N-hydroxyethyl-N-chloroethylnitrosourea, and treosulfan did not elicit this response. The phosphatidylinositol 3-kinase inhibitor wortmannin specifically blocked the UV-stimulated activation of JNK1 but did not affect UV-driven activation of extracellular regulated kinase 2 (ERK2). To investigate the significance of JNK1 for transactivation of c-jun, we analyzed the effect of UV irradiation on c-jun expression under conditions of wortmannin-mediated inhibition of UV-induced stimulation of JNK1. Neither the UV-induced increase in c-jun mRNA, c-Jun protein, and AP-1 binding nor the activation of the collagenase and c-jun promoters was affected by wortmannin. In contrast, the mitogen-activated protein kinase/ERK kinase inhibitor PD98056, which blocked ERK2 but not JNK1 activation by UV irradiation, impaired UV-driven c-Jun protein induction and AP-1 binding. Based on the data, we suggest that JNK1 stimulation is not essential for transactivation of c-jun after UV exposure, whereas activation of ERK2 is required for UV-induced signaling leading to elevated c-jun expression. PMID:10022864

  20. Firefly luciferase inhibition.

    PubMed

    Leitão, João M M; Esteves da Silva, Joaquim C G

    2010-10-05

    Firefly luciferase (Luc) is the most studied of the luciferase enzymes and the mechanism and kinetics of the reactions catalyzed by this enzyme have been relatively well characterized. Luc catalyzes the bioluminescent reaction involving firefly luciferin (D-LH(2)), adenosine triphosphate (ATP), magnesium ion and molecular oxygen with the formation of an electronically excited species (oxyluciferin), inorganic pyrophosphate (PPi), carbon dioxide and adenosine monophosphate (AMP). Luc also catalyzes other non-luminescent reactions, which can interfere with the light production mechanism. Following electronic relaxation, the excited oxyluciferin emits radiation in the visible region of the electromagnetic spectrum (550-570 nm). Among the various possible compounds, several classes of inhibitory substances interfere with the activity of this enzyme: here, we consider substrate-related compounds, intermediates or products of the Luc catalyzed reactions, in addition to anesthetics and, fatty acids. This review summarizes the main inhibitors of Luc and the corresponding inhibition kinetic parameters.

  1. Anxiety and retrieval inhibition: support for an enhanced inhibition account.

    PubMed

    Nuñez, Mia; Gregory, Josh; Zinbarg, Richard E

    2017-02-01

    Retrieval inhibition of negative associations is important for exposure therapy for anxiety, but the relationship between memory inhibition and anxiety is not well understood-anxiety could either be associated with enhanced or deficient inhibition. The present study tested these two competing hypotheses by measuring retrieval inhibition of negative stimuli by related neutral stimuli. Non-clinically anxious undergraduates completed measures of trait and state anxiety and completed a retrieval induced forgetting task. Adaptive forgetting varied with state anxiety. Low levels of state anxiety were associated with no evidence for retrieval inhibition for either threatening or non-threatening categories. Participants in the middle tertile of state anxiety scores exhibited retrieval inhibition for non-threatening categories but not for threatening categories. Participants in the highest tertile of state anxiety, however, exhibited retrieval inhibition for both threatening and non-threatening categories with the magnitude of retrieval inhibition being greater for threatening than non-threatening categories. The data are in line with the avoidance aspect of the vigilance-avoidance theory of anxiety and inhibition. Implications for cognitive behavioural therapy practices are discussed.

  2. Identification of approved and investigational drugs that inhibit hypoxia-inducible factor-1 signaling

    PubMed Central

    Hsu, Chia-Wen; Huang, Ruili; Khuc, Thai; Shou, David; Bullock, Joshua; Grooby, Suzanne; Griffin, Sue; Zou, Chaozhong; Little, Annette; Astley, Holly; Xia, Menghang

    2016-01-01

    One of the requirements for tumor development is blood supply, most often driven by hypoxia-induced angiogenesis. Hypoxia induces the stabilization of hypoxia-inducible factor-1 alpha (HIF-1α), which induces expression of an angiogenic factor, vascular endothelial growth factor (VEGF). The purpose of this study is to validate a new screening platform combined with orthogonal assays to rapidly identify HIF-1 inhibitors and to evaluate the effectiveness of approved drugs on modulating HIF-1 signaling. We generated an endogenous HIF-1α–NanoLuc luciferase reporter allele in the human HCT116 colon cancer cell line using genome editing and screened a panel of small interfering RNAs (siRNAs) to 960 druggable targets and approximately 2,500 drugs on a quantitative high-throughput screening (qHTS) platform. Selected compounds were further investigated with secondary assays to confirm their anti-HIF activity and to study their mode of action. The qHTS assay identified over 300 drugs that inhibited HIF-1α-NanoLuc expression. The siRNA screening results supported the effectiveness of several target-specific inhibitors. Moreover, the identified HIF-1 inhibitors, such as mycophenolate mofetil, niclosamide, and trametinib, were able to suppress cancer cell proliferation and angiogenesis. Our study indicates that blocking the mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K) pathways effectively inhibits hypoxia-induced HIF-1α accumulation and HIF-1α transactivation and that proteasome inhibitors induce accumulation and decrease transcriptional activity of HIF-1α. These findings underline the importance of developing a battery of robust assay platforms and confirmation studies that focus on endogenous protein targets so that only relevant and reliable data will be taken into pre-clinical and clinical studies. PMID:26882567

  3. Inhibition of neddylation represses lipopolysaccharide-induced proinflammatory cytokine production in macrophage cells.

    PubMed

    Chang, Fang-Mei; Reyna, Sara M; Granados, Jose C; Wei, Sung-Jen; Innis-Whitehouse, Wendy; Maffi, Shivani K; Rodriguez, Edward; Slaga, Thomas J; Short, John D

    2012-10-12

    Cullin-RING E3 ligases (CRLs) are a class of ubiquitin ligases that control the proteasomal degradation of numerous target proteins, including IκB, and the activity of these CRLs are positively regulated by conjugation of a Nedd8 polypeptide onto Cullin proteins in a process called neddylation. CRL-mediated degradation of IκB, which normally interacts with and retains NF-κB in the cytoplasm, permits nuclear translocation and transactivation of the NF-κB transcription factor. Neddylation occurs through a multistep enzymatic process involving Nedd8 activating enzymes, and recent studies have shown that the pharmacological agent, MLN4924, can potently inhibit Nedd8 activating enzymes, thereby preventing neddylation of Cullin proteins and preventing the degradation of CRL target proteins. In macrophages, regulation of NF-κB signaling functions as a primary pathway by which infectious agents such as lipopolysaccharides (LPSs) cause the up-regulation of proinflammatory cytokines. Here we have analyzed the effects of MLN4924, and compared the effects of MLN4924