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  1. AMPK Mediates Glucocorticoids Stress-Induced Downregulation of the Glucocorticoid Receptor in Cultured Rat Prefrontal Cortical Astrocytes.

    PubMed

    Yuan, Shi-Ying; Liu, Jue; Zhou, Jun; Lu, Wei; Zhou, Hai-Yun; Long, Li-Hong; Hu, Zhuang-Li; Ni, Lan; Wang, Yi; Chen, Jian-Guo; Wang, Fang

    2016-01-01

    Chronic stress induces altered energy metabolism and plays important roles in the etiology of depression, in which the glucocorticoid negative feedback is disrupted due to imbalanced glucocorticoid receptor (GR) functions. The mechanism underlying the dysregulation of GR by chronic stress remains elusive. In this study, we investigated the role of AMP-activated protein kinase (AMPK), the key enzyme regulating cellular energy metabolism, and related signaling pathways in chronic stress-induced GR dysregulation. In cultured rat cortical astrocytes, glucocorticoid treatment decreased the level, which was accompanied by the decreased expression of liver kinase B1 (LKB1) and reduced phosphorylation of AMPK. Glucocorticoid-induced effects were attenuated by glucocorticoid-inducible kinase 1 (SGK1) inhibitor GSK650394, which also inhibited glucocorticoid induced phosphorylation of Forkhead box O3a (FOXO3a). Furthermore, glucocorticoid-induced down-regulation of GR was mimicked by the inhibition of AMPK and abolished by the AMPK activators or the histone deacetylase 5 (HDAC5) inhibitors. In line with the role of AMPK in GR expression, AMPK activator metformin reversed glucocorticoid-induced reduction of AMPK phosphorylation and GR expression as well as behavioral alteration of rats. Taken together, these results suggest that chronic stress activates SGK1 and suppresses the expression of LKB1 via inhibitory phosphorylation of FOXO3a. Downregulated LKB1 contributes to reduced activation of AMPK, leading to the dephosphorylation of HDAC5 and the suppression of transcription of GR. PMID:27513844

  2. AMPK Mediates Glucocorticoids Stress-Induced Downregulation of the Glucocorticoid Receptor in Cultured Rat Prefrontal Cortical Astrocytes

    PubMed Central

    Lu, Wei; Zhou, Hai-Yun; Long, Li-Hong; Hu, Zhuang-Li; Ni, Lan; Wang, Yi; Chen, Jian-Guo; Wang, Fang

    2016-01-01

    Chronic stress induces altered energy metabolism and plays important roles in the etiology of depression, in which the glucocorticoid negative feedback is disrupted due to imbalanced glucocorticoid receptor (GR) functions. The mechanism underlying the dysregulation of GR by chronic stress remains elusive. In this study, we investigated the role of AMP-activated protein kinase (AMPK), the key enzyme regulating cellular energy metabolism, and related signaling pathways in chronic stress-induced GR dysregulation. In cultured rat cortical astrocytes, glucocorticoid treatment decreased the level, which was accompanied by the decreased expression of liver kinase B1 (LKB1) and reduced phosphorylation of AMPK. Glucocorticoid-induced effects were attenuated by glucocorticoid-inducible kinase 1 (SGK1) inhibitor GSK650394, which also inhibited glucocorticoid induced phosphorylation of Forkhead box O3a (FOXO3a). Furthermore, glucocorticoid-induced down-regulation of GR was mimicked by the inhibition of AMPK and abolished by the AMPK activators or the histone deacetylase 5 (HDAC5) inhibitors. In line with the role of AMPK in GR expression, AMPK activator metformin reversed glucocorticoid-induced reduction of AMPK phosphorylation and GR expression as well as behavioral alteration of rats. Taken together, these results suggest that chronic stress activates SGK1 and suppresses the expression of LKB1 via inhibitory phosphorylation of FOXO3a. Downregulated LKB1 contributes to reduced activation of AMPK, leading to the dephosphorylation of HDAC5 and the suppression of transcription of GR. PMID:27513844

  3. Hippocampal neuronal nitric oxide synthase mediates the stress-related depressive behaviors of glucocorticoids by downregulating glucocorticoid receptor.

    PubMed

    Zhou, Qi-Gang; Zhu, Li-Juan; Chen, Chen; Wu, Hai-Yin; Luo, Chun-Xia; Chang, Lei; Zhu, Dong-Ya

    2011-05-25

    The molecular mechanisms underlying the behavioral effects of glucocorticoids are poorly understood. We report here that hippocampal neuronal nitric oxide synthase (nNOS) is a crucial mediator. Chronic mild stress and glucocorticoids exposures caused hippocampal nNOS overexpression via activating mineralocorticoid receptor. In turn, hippocampal nNOS-derived nitric oxide (NO) significantly downregulated local glucocorticoid receptor expression through both soluble guanylate cyclase (sGC)/cGMP and peroxynitrite (ONOO(-))/extracellular signal-regulated kinase signal pathways, and therefore elevated hypothalamic corticotrophin-releasing factor, a peptide that governs the hypothalamic-pituitary-adrenal axis. More importantly, nNOS deletion or intrahippocampal nNOS inhibition and NO-cGMP signaling blockade (using NO scavenger or sGC inhibitor) prevented the corticosterone-induced behavioral modifications, suggesting that hippocampal nNOS is necessary for the role of glucocorticoids in mediating depressive behaviors. In addition, directly delivering ONOO(-) donor into hippocampus caused depressive-like behaviors. Our findings reveal a role of hippocampal nNOS in regulating the behavioral effects of glucocorticoids.

  4. Protective effects of a glucocorticoid on downregulation of pulmonary beta 2-adrenergic receptors in vivo.

    PubMed Central

    Mak, J C; Nishikawa, M; Shirasaki, H; Miyayasu, K; Barnes, P J

    1995-01-01

    We investigated the in vivo effects of a glucocorticoid on beta-agonist-induced downregulation of beta 1- and beta 2-adrenergic receptors (determined by [125I]iodocyanopindolol binding), mRNA expression (assessed by Northern blotting), and gene transcription (using nuclear run-on assays) in rat lung. Dexamethasone (Dex) (0.2 mg/kg/d, days 1-8) increased beta 1- and beta 2-receptor numbers by 70 and 69% above control, respectively, but did not change their mRNA expression. Isoproterenol (Iso) (0.96 mg/kg/d, days 2-8) decreased beta 1- and beta 2-receptor numbers by 48 and 51%, respectively, and also reduced mRNA expression by 69 and 57%, respectively. The combination of Dex and Iso resulted in no net change in beta 2-receptor number and its mRNA expression, although there was a significant reduction in beta 1-receptor number and mRNA expression. The mapping of beta 1- and beta 2-receptors by receptor autoradiography confirmed these findings over alveoli, epithelium, endothelium, and airway and vascular smooth muscle. We also measured the activation of the transcription factor, cyclic AMP response element binding protein (CREB) using an electrophoretic mobility shift assay. CREB-like DNA-binding activity was decreased after Iso treatment but this decrease was prevented after treatment with Dex. Nuclear run-on assays revealed that the transcription rate of the beta 1-receptor gene did not alter after Dex treatment, but was reduced after Iso treatment. The transcription rate of the beta 2-receptor gene was increased after Dex treatment by approximately twofold, but there was no change after Iso treatment. We conclude that glucocorticoids can prevent homologous downregulation of beta 2-receptor number and mRNA expression at the transcriptional level without affecting beta 1-receptors and that the transcription factor CREB may be involved in this phenomenon. Such an effect may have clinical implications for preventing the development of tolerance to beta 2-agonists in

  5. Icariin attenuates social defeat-induced down-regulation of glucocorticoid receptor in mice.

    PubMed

    Wu, Jinfeng; Du, Juan; Xu, Changqing; Le, Jingjing; Xu, Yizhe; Liu, Baojun; Dong, Jingcheng

    2011-04-01

    Icariin is a major constituent of flavonoids isolated from the herb Epimedium. It displays antidepressant-like activity in mice behavioral despair models and chronic mild stress models. In this study, a chronic social defeat protocol is used as a mouse model for depression, and the social avoidance effects of icariin administration are investigated. The data indicate that social defeat significantly reduces mice social interaction time and that icariin administered at 25 mg/kg and 50 mg/kg for 28 consecutive days produce remarkable increases in social interaction time. Impaired glucocorticoid receptor (GR) function is related to depression and normalization of GR function is closely associated with the recovery from depression. In this study, GR binding affinity and protein expression were evaluated by radioactive ligand and western blot, respectively. Our results demonstrate that both GR binding affinity and protein expression in the social defeat model are remarkably decreased and that icariin administration attenuates social defeat-induced GR down-regulation. In the present study, our data also show that icariin administration significantly inhibits social defeat-induced increases of corticosterone and IL-6 levels. The potential mechanisms of icariin induced GR modulation, such as effects on HPA-axis function, proinflammatory signaling pathway and membrane steroid transporters, need further study.

  6. Peroxisome proliferator-activated receptor {alpha} agonist-induced down-regulation of hepatic glucocorticoid receptor expression in SD rats

    SciTech Connect

    Chen Xiang; Li Ming; Sun Weiping; Bi Yan; Cai Mengyin; Liang Hua; Yu Qiuqiong; He Xiaoying; Weng Jianping

    2008-04-18

    It was reported that glucocorticoid production was inhibited by fenofibrate through suppression of type-1 11{beta}-hydroxysteroid dehydrogenase gene expression in liver. The inhibition might be a negative-feedback regulation of glucocorticoid receptor (GR) activity by peroxisome proliferator-activated receptor alpha (PPAR{alpha}), which is quickly induced by glucocorticoid in the liver. However, it is not clear if GR expression is changed by fenofibrate-induced PPAR{alpha} activation. In this study, we tested this possibility in the liver of Sprague-Dawley rats. GR expression was reduced by fenofibrate in a time- and does-dependent manner. The inhibition was observed in liver, but not in fat and muscle. The corticosterone level in the blood was increased significantly by fenofibrate. These effects of fenofibrate were abolished by PPAR{alpha} inhibitor MK886, suggesting that fenofibrate activated through PPAR{alpha}. In conclusion, inhibition of GR expression may represent a new molecular mechanism for the negative feedback regulation of GR activity by PPAR{alpha}.

  7. Rosiglitazone reverses memory decline and hippocampal glucocorticoid receptor down-regulation in an Alzheimer's disease mouse model

    SciTech Connect

    Escribano, Luis; Simon, Ana-Maria; Perez-Mediavilla, Alberto; Salazar-Colocho, Pablo; Rio, Joaquin Del; Frechilla, Diana

    2009-02-06

    Clinical trials with rosiglitazone, a potent agonist at peroxisome proliferator-activated receptor gamma (PPAR{gamma}) suggest an improvement of cognitive function in Alzheimer's disease (AD) patients. The mechanisms mediating this potential beneficial effect remain to be fully elucidated. In mice overexpressing mutant human amyloid precursor protein (hAPP), a model of AD, we found that memory impairment in the object recognition test was prevented and also reversed by chronic rosiglitazone treatment. Given the possible involvement of glucocorticoid receptors (GR) in the actions of PPAR{gamma}-ligands, we studied the effect of chronic rosiglitazone treatment on GR levels in the hippocampus of hAPP mice. An early down-regulation of GR, not related to elevated plasma corticosterone levels, was found in different hippocampal subfields of the transgenic mice and this decrease was prevented by rosiglitazone. In parallel with behavioural studies, rosiglitazone also normalized GR levels in older animals. This effect may contribute to explain the attenuation of memory decline by PPAR{gamma} activation in an AD mouse model.

  8. PCB disruption of the hypothalamus-pituitary-interrenal axis involves brain glucocorticoid receptor downregulation in anadromous Arctic charr

    USGS Publications Warehouse

    Aluru, N.; Jorgensen, E.H.; Maule, A.G.; Vijayan, M.M.

    2004-01-01

    We examined whether brain glucocorticoid receptor (GR) modulation by polychlorinated biphenyls (PCBs) was involved in the abnormal cortisol response to stress seen in anadromous Arctic charr (Salvelinus alpinus). Fish treated with Aroclor 1254 (0, 1, 10, and 100 mg/kg body mass) were maintained for 5 mo without feeding in the winter to mimic their seasonal fasting cycle, whereas a fed group with 0 and 100 mg/kg Aroclor was maintained for comparison. Fasting elevated plasma cortisol levels and brain GR content but depressed heat shock protein 90 (hsp90) and interrenal cortisol production capacity. Exposure of fasted fish to Aroclor 1254 resulted in a dose-dependent increase in brain total PCB content. This accumulation in fish with high PCB dose was threefold higher in fasted fish compared with fed fish. PCBs depressed plasma cortisol levels but did not affect in vitro interrenal cortisol production capacity in fasted charr. At high PCB dose, the brain GR content was significantly lower in the fasted fish and this corresponded with a lower brain hsp70 and hsp90 content. The elevation of plasma cortisol levels and upregulation of brain GR content may be an important adaptation to extended fasting in anadromous Arctic charr, and this response was disrupted by PCBs. Taken together, the hypothalamus-pituitary- interrenal axis is a target for PCB impact during winter emaciation in anadromous Arctic charr.

  9. Glucocorticoid receptor signaling in health and disease

    PubMed Central

    Kadmiel, Mahita; Cidlowski, John A.

    2013-01-01

    Glucocorticoids are steroid hormones regulated in a circadian and stres-associated manner to maintain various metabolic and homeostatic functions that are necessary for life. Synthetic glucocorticoids are widely prescribed drugs for many conditions including asthma, chronic obstructive pulmonary disease (COPD), and inflammatory disorders of the eye. Research in the last few years has begun to unravel the profound complexity of glucocorticoid signaling and has contributed remarkably to improved therapeutic strategies. Glucocorticoids signal through the glucocorticoid receptor, a member of the superfamily of nuclear receptors, in both genomic and non-genomic ways in almost every tissue in the human body. In this review, we will provide an update on glucocorticoid receptor signaling and highlight the role of GR signaling in physiological and pathophysiological conditions in the major organ systems in the human body. PMID:23953592

  10. Endothelial glucocorticoid receptor suppresses atherogenesis- Brief Report

    PubMed Central

    Zhang, Xinbo; Rotllan, Noemi; Feng, Yan; Zhou, Han; Fernández-Hernando, Carlos; Yu, Jun; Sessa, William C.

    2015-01-01

    Objective The purpose of this study was to determine the role of the endothelial glucocorticoid receptor in the pathogenesis of atherosclerosis. Approach and Results Control mice and mice lacking the endothelial glucocorticoid receptor were bred onto an Apoe knockout background and subjected to high-fat diet feeding for 12 weeks. Assessment of body weight and total cholesterol and triglycerides before and after the diet revealed no differences between the two groups of mice. However, mice lacking the endothelial glucocorticoid receptor developed more severe atherosclerotic lesions in the aorta, brachiocephalic artery and aortic sinus as well as a heightened inflammatory milieu as evidence by increased macrophage recruitment in the lesions. Conclusions These data suggest the endothelial glucocorticoid receptor is important for tonic inhibition of inflammation and limitation of atherosclerosis progression in this model. PMID:25810297

  11. Glucocorticoid Regulation of the Vitamin D Receptor

    PubMed Central

    Hidalgo, Alejandro A.; Trump, Donald L.; Johnson, Candace S.

    2010-01-01

    Many studies indicate calcitriol has potent anti-tumor activity in different types of cancers. However, high levels of vitamin D can produce hypercalcemia in some patients. Glucocorticoids are used to ameliorate hypercalcemia and to enhance calcitriol anti-tumor activity. Calcitriol in combination with the glucocorticoid dexamethasone (Dex) increased vitamin D receptor (VDR) protein levels and ligand binding in squamous cell carcinoma VII (SCC). In this study we found that both calcitriol and Dex induce VDR- and glucocorticoid receptor (GR)-mediated transcription respectively, indicating both hormone receptors are active in SCC. Pre-treatment with Dex increases VDR-mediated transcription at the human CYP24A1 promoter. Whereas, pre-treatment with other steroid hormones, including dihydrotestosterone and R1881, has no effect on VDR-mediated transcription. Real-time PCR indicates treatment with Dex increases Vdr transcripts in a time-dependent manner, suggesting Dex may directly regulate expression of Vdr. Numerous putative glucocorticoid response elements (GREs) were found in the Vdr gene. Chromatin immunoprecipitation (ChIP) assay demonstrated GR binding at several putative GREs located within the mouse Vdr gene. However, none of the putative GREs studied increase GR-mediated transcription in luciferase reporter assays. In an attempt to identify the response element responsible for Vdr transcript regulation, future studies will continue to analyze newly identified GREs more distal from the Vdr gene promoter. PMID:20398752

  12. Identification and Characterization of Glucocorticoid Receptors in Liver of Nude Mice.

    PubMed

    Chi, C.-W.; Liu, T.-Y.; Chiang, S.-H.; Cheng, S.-L.; Lin, C.-Y.; Ho, C.-K.

    1994-10-01

    Glucocorticoids regulate the expression of many liver-specific genes via glucocorticoid receptors. The presence of glucocorticoid receptors in liver has been reported in many mammalian species but not in nude mice. In the present study, we demonstrate the presence of specific glucocorticoid receptors in nude mouse liver. The binding of ligands to these receptors could be completely inhibited by RU486, and partially blocked by hydrocortisone and progesterone, whereas estrogen and testosterone had no effect. Hydrocortisone downregulated the level of glucocorticoid receptors in livers of nude mice and correspondingly enhanced the activities of tyrosine aminotransferase and gamma-glutamyltransferase. Our results indicate that glucocorticoid receptors in nude mouse liver are specific, fully functional, and present at levels 28.5-fold higher than in the liver of normal inbred mice. We suggest that the nude mouse is a valuable model for studies of hepatic glucocorticoid action and may provide a clue to a putative hepatic-thymic interaction. Copyright 1994 S. Karger AG, Basel

  13. Glucocorticoid receptor transformation and DNA binding

    SciTech Connect

    Tienrungroj, W.

    1986-01-01

    The overall goal is to probe the mechanism whereby glucocorticoid receptors are transformed from a non-DNA-binding form to their active DNA-binding form. The author has examined the effect of an endogenous inhibitor purified from rat liver cytosol on receptor binding to DNA. The inhibitor binds to transformed receptors in whole cytosol and prevent their binding to DNA. He also examined the role of sulfhydryl groups in determining the DNA binding activity of the transformed receptor and in determining the transformation process. Treatment of rat liver cytosol containing temperature-transformed, (/sup 3/H)dexamethasone-bound receptors at 0/sup 0/C with the sulfhydryl modifying reagent methyl methanethiosulfonate inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol. In addition, he has examined the relationship between receptor phosphorylation and DNA binding. Untransformed receptor complexes purified from cytosol prepared from mouse L cells grown in medium containing (/sup 32/P)orthophosphate contain two components, a 100 k-Da and a 90-kDa subunit, both of which are phosphoproteins. On transformation, the receptor dissociates from the 90-kDa protein. Transformation of the complex under cell free conditions does not result in a dephosphorylation of the 100-kDa steroid-binding protein. Transformed receptor that has been bound to DNA and purified by monoclonal antibody is still in a phosphorylated form. These results suggest that dephosphorylation is not required for receptor binding to DNA.

  14. AMPK Regulates Metabolic Actions of Glucocorticoids by Phosphorylating the Glucocorticoid Receptor through p38 MAPK

    PubMed Central

    Nader, Nancy; Ng, Sinnie Sin Man; Lambrou, George I.; Pervanidou, Panagiota; Wang, Yonghong; Chrousos, George P.; Kino, Tomoshige

    2010-01-01

    Glucocorticoids play central roles in the regulation of energy metabolism by shifting it toward catabolism, whereas AMP-activated protein kinase (AMPK) is the master regulator of energy homeostasis, sensing energy depletion and stimulating pathways of increasing fuel uptake and saving on peripheral supplies. We showed here that AMPK regulates glucocorticoid actions on carbohydrate metabolism by targeting the glucocorticoid receptor (GR) and modifying transcription of glucocorticoid-responsive genes in a tissue- and promoter-specific fashion. Activation of AMPK in rats reversed glucocorticoid-induced hepatic steatosis and suppressed glucocorticoid-mediated stimulation of glucose metabolism. Transcriptomic analysis in the liver suggested marked overlaps between the AMPK and glucocorticoid signaling pathways directed mostly from AMPK to glucocorticoid actions. AMPK accomplishes this by phosphorylating serine 211 of the human GR indirectly through phosphorylation and consequent activation of p38 MAPK and by altering attraction of transcriptional coregulators to DNA-bound GR. In human peripheral mononuclear cells, AMPK mRNA expression positively correlated with that of glucocorticoid-responsive glucocorticoid-inducible leucine zipper protein, which correlated also positively with the body mass index of subjects. These results indicate that the AMPK-mediated energy control system modulates glucocorticoid action at target tissues. Because increased action of glucocorticoids is associated with the development of metabolic disorders, activation of AMPK could be a promising target for developing pharmacological interventions to these pathologies. PMID:20660302

  15. Glucocorticoid receptors, in human alveolar macrophages and peripheral blood cells.

    PubMed Central

    Ozaki, T; Yasuoka, S; Nakayama, T; Tsubura, E

    1982-01-01

    The numbers of glucocorticoid receptors in human alveolar macrophages and peripheral blood cells were measured with 3H-prednisolone. Alveolar macrophages, which constituted 89.0 +/- 5.9% of broncho-alveolar cells, obtained by broncho-alveolar lavage from normal volunteers had much larger numbers of specific glucocorticoid receptors than peripheral blood cells. The numbers of glucocorticoid receptors in peripheral polymorphonuclear leucocytes, lymphocytes and lymphocyte subpopulations (B cells, T cells, TG cells and TnonG cells) were nearly equal. In patients with idiopathic pulmonary fibrosis, in whom alveolar macrophages amounted to over 85% of the broncho-alveolar cells, the number of glucocorticoid receptors in alveolar macrophages was significantly decreased, but the numbers in their peripheral blood cells were normal. This finding suggests that the number of glucocorticoid receptors in alveolar macrophages may change specifically during disorders of the lung. PMID:7075033

  16. A transgenic zebrafish model for monitoring glucocorticoid receptor activity.

    PubMed

    Krug, R G; Poshusta, T L; Skuster, K J; Berg, M R; Gardner, S L; Clark, K J

    2014-06-01

    Gene regulation resulting from glucocorticoid receptor and glucocorticoid response element interactions is a hallmark feature of stress response signaling. Imbalanced glucocorticoid production and glucocorticoid receptor activity have been linked to socioeconomically crippling neuropsychiatric disorders, and accordingly there is a need to develop in vivo models to help understand disease progression and management. Therefore, we developed the transgenic SR4G zebrafish reporter line with six glucocorticoid response elements used to promote expression of a short half-life green fluorescent protein following glucocorticoid receptor activation. Herein, we document the ability of this reporter line to respond to both chronic and acute exogenous glucocorticoid treatment. The green fluorescent protein expression in response to transgene activation was high in a variety of tissues including the brain, and provided single-cell resolution in the effected regions. The specificity of these responses is demonstrated using the partial agonist mifepristone and mutation of the glucocorticoid receptor. Importantly, the reporter line also modeled the temporal dynamics of endogenous stress response signaling, including the increased production of the glucocorticoid cortisol following hyperosmotic stress and the fluctuations of basal cortisol concentrations with the circadian rhythm. Taken together, these results characterize our newly developed reporter line for elucidating environmental or genetic modifiers of stress response signaling, which may provide insights to the neuronal mechanisms underlying neuropsychiatric disorders such as major depressive disorder.

  17. Glucocorticoid receptor antagonism reverts docetaxel resistance in human prostate cancer

    PubMed Central

    Kroon, Jan; Puhr, Martin; Buijs, Jeroen T; van der Horst, Geertje; Hemmer, Daniëlle M; Marijt, Koen A; Hwang, Ming S; Masood, Motasim; Grimm, Stefan; Storm, Gert; Metselaar, Josbert M; Meijer, Onno C; Culig, Zoran; van der Pluijm, Gabri

    2016-01-01

    Resistance to docetaxel is a major clinical problem in advanced prostate cancer (PCa). Although glucocorticoids (GCs) are frequently used in combination with docetaxel, it is unclear to what extent GCs and their receptor, the glucocorticoid receptor (GR), contribute to the chemotherapy resistance. In this study, we aim to elucidate the role of the GR in docetaxel-resistant PCa in order to improve the current PCa therapies. GR expression was analyzed in a tissue microarray of primary PCa specimens from chemonaive and docetaxel-treated patients, and in cultured PCa cell lines with an acquired docetaxel resistance (PC3-DR, DU145-DR, and 22Rv1-DR). We found a robust overexpression of the GR in primary PCa from docetaxel-treated patients and enhanced GR levels in cultured docetaxel-resistant human PCa cells, indicating a key role of the GR in docetaxel resistance. The capability of the GR antagonists (RU-486 and cyproterone acetate) to revert docetaxel resistance was investigated and revealed significant resensitization of docetaxel-resistant PCa cells for docetaxel treatment in a dose- and time-dependent manner, in which a complete restoration of docetaxel sensitivity was achieved in both androgen receptor (AR)-negative and AR-positive cell lines. Mechanistically, we demonstrated down-regulation of Bcl-xL and Bcl-2 upon GR antagonism, thereby defining potential treatment targets. In conclusion, we describe the involvement of the GR in the acquisition of docetaxel resistance in human PCa. Therapeutic targeting of the GR effectively resensitizes docetaxel-resistant PCa cells. These findings warrant further investigation of the clinical utility of the GR antagonists in the management of patients with advanced and docetaxel-resistant PCa. PMID:26483423

  18. Glucocorticoids decrease astrocyte numbers by reducing glucocorticoid receptor expression in vitro and in vivo.

    PubMed

    Unemura, Kazuhiro; Kume, Toshiaki; Kondo, Minami; Maeda, Yuki; Izumi, Yasuhiko; Akaike, Akinori

    2012-01-01

    Glucocorticoids are stress hormones released from the adrenal cortex and their concentration is controlled by the hypothalamic-pituitary-adrenal axis. In this study, we investigated the effect of glucocorticoids on the number of astrocytes and glucocorticoid receptor (GR) expression in vitro and in vivo. Proliferation of cultured astrocytes was reduced following treatment with corticosterone and dexamethasone for 72 h. Corticosterone and dexamethasone also reduced GR expression in astrocytes. RU486, a GR antagonist, inhibited the reduction in both astrocyte proliferation and GR expression. Furthermore, GR knockdown by siRNA inhibited astrocyte proliferation. We also examined the effect of excessive glucocorticoid release on GR expression and the number of astrocytes in vivo by administering adrenocorticotropic hormone to rats for 14 days. GR expression was reduced in the prefrontal cortex and hippocampus and the number of astrocytes was reduced in the frontal cortex. Overall, our results suggest that glucocorticoids decrease the number of astrocytes by reducing GR expression.

  19. Glucocorticoids and the non-steroidal selective glucocorticoid receptor modulator, compound A, differentially affect colon cancer-derived myofibroblasts.

    PubMed

    Drebert, Zuzanna; Bracke, Marc; Beck, Ilse M

    2015-05-01

    The glucocorticoid receptor functions as a ligand-dependent transcription factor that positively or negatively regulates the transcription of various specific target genes. Not only steroidal glucocorticoids can bind and activate the glucocorticoid receptor, but also the intensively examined non-steroidal selective glucocorticoid receptor modulators can do so, albeit with a select effector profile skewed to glucocorticoid receptor transrepression. Glucocorticoids are widely used to treat inflammatory afflictions, but also as anti-cancer therapies or adjuvants thereof. As the impact of glucocorticoids and selective glucocorticoid receptor modulators has scarcely been researched in this setting, we focused on colon cancer and its stromal environment, in particular the stromal myofibroblasts, which are known to influence cancer cells via paracrine signaling. In these myofibroblasts, the glucocorticoid dexamethasone is able to drive the glucocorticoid receptor into the nucleus and thus negatively regulates the expression of particular pro-inflammatory genes in TNFα-stimulated cells. The selective glucocorticoid receptor modulator compound A has an impaired ability to translocate GR, presumably underpinning its modest anti-inflammatory properties in these cells. Only dexamethasone, and not compound A, can upregulate the glucocorticoid receptor transactivation-dependent GILZ expression. Neither dexamethasone, nor compound A affects myofibroblast cell viability. However, compound A retards the growth of this myofibroblast cell line. Additionally, dexamethasone can inhibit the expression of Tenascin C, hepatocyte growth factor, and TGFβ, which are all factors known for their impact on colon cancer cell invasion, in a glucocorticoid receptor-dependent manner. In contrast, compound A can only slightly diminish the expression of just hepatocyte growth factor, and not tenascin C or TGFβ. Combined, our results expose new tumor microenvironment-modulating effects of

  20. NALP3 inflammasome up-regulation and CASP1 cleavage of the glucocorticoid receptor causes glucocorticoid resistance in leukemia cells

    PubMed Central

    Paugh, Steven W.; Bonten, Erik J.; Savic, Daniel; Ramsey, Laura B.; Thierfelder, William E.; Gurung, Prajwal; Malireddi, R. K. Subbarao; Actis, Marcelo; Mayasundari, Anand; Min, Jaeki; Coss, David R.; Laudermilk, Lucas T.; Panetta, John C.; McCorkle, J. Robert; Fan, Yiping; Crews, Kristine R.; Stocco, Gabriele; Wilkinson, Mark R.; Ferreira, Antonio M.; Cheng, Cheng; Yang, Wenjian; Karol, Seth E.; Fernandez, Christian A.; Diouf, Barthelemy; Smith, Colton; Hicks, J. Kevin; Zanut, Alessandra; Giordanengo, Audrey; Crona, Daniel; Bianchi, Joy J.; Holmfeldt, Linda; Mullighan, Charles G.; den Boer, Monique L.; Pieters, Rob; Jeha, Sima; Dunwell, Thomas L.; Latif, Farida; Bhojwani, Deepa; Carroll, William L.; Pui, Ching-Hon; Myers, Richard M.; Guy, R. Kiplin; Kanneganti, Thirumala-Devi; Relling, Mary V.; Evans, William E.

    2015-01-01

    Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and leukemia cell resistant to glucocorticoids confers a poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the sensitivity to prednisolone of primary leukemia cells from 444 newly diagnosed ALL patients, revealing significantly higher expression of caspase 1 (CASP1) and its activator NLRP3 in glucocorticoid resistant leukemia cells, due to significantly lower somatic methylation of CASP1 and NLRP3 promoters. Over-expression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1 overexpressing ALL. Our findings establish a new mechanism by which the NLRP3/CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on glucocorticoid transcriptional response suggests this mechanism could also modify glucocorticoid effects in other diseases. PMID:25938942

  1. Glucocorticoid and progesterone receptors in yolk sac placenta.

    PubMed

    Carbone, J P; Baldridge, R C; Magen, A B; Andrew, C L; Koszalka, T R; Brent, R L

    1986-01-01

    The parietal yolk sac (PYS) of the rat fetus at the 14th day of gestation contains glucocorticoid as well as progesterone receptors; both are present in the trophoblast cell layer. Following heat activation the receptors are capable of binding to deoxyribonucleic acid- (DNA-)cellulose. Glucocorticoid receptors, but not progesterone receptors, are also present in the visceral yolk sac (VYS) at the 14th day of gestation. Greater amounts (some 250 femtomoles/mg cytosol protein) of a glucocorticoid receptor are present in the VYS on the 17th day of gestation. The Kd is approximately 4 X 10(-9) M; following activation it also binds to DNA-cellulose. The elution pattern of the activated VYS receptor from diethylaminoethyl-(DEAE-)Sephadex, however, is similar to that found with kidney and colon rather than that of liver (i.e., it resembles corticosteroid binder IB rather than binder II) indicating a possible role in transport. Although the receptors are separate entities, progesterone competes as effectively as corticosterone for binding to the glucocorticoid receptors in both the PYS and and VYS, thus raising the question of the possible effect of changes in progesterone concentrations on the functioning of glucocorticoids during development.

  2. Glucocorticoid dysregulations and their clinical correlates. From receptors to therapeutics.

    PubMed

    Marques, Andrea H; Silverman, Marni N; Sternberg, Esther M

    2009-10-01

    Clinicians have long known that a substantial proportion of patients treated with high-dose glucocorticoids experience a variety of serious side effects, including metabolic syndrome, bone loss, and mood shifts, such as depressive symptomatology, manic or hypomanic symptoms, and even suicide. The reason for individual variability in expression or severity of these side effects is not clear. However, recent emerging literature is beginning to shed light on possible mechanisms of these effects. As an introduction to this volume, this chapter will review the basic biology of glucocorticoid release and molecular mechanisms of glucocorticoid receptor function, and will discuss how dysregulation of glucocorticoid action at all levels could contribute to such side effects. At the molecular level, glucocorticoid receptor polymorphisms may be associated either with receptor hypofunction or hyperfunction and could thus contribute to differential individual sensitivity to the effects of glucocorticoid treatment. Numerous factors regulate hypothalamic-pituitary-adrenal (HPA) axis responsiveness, which could also contribute to individual differences in glucocorticoid side effects. One of these is sex hormone status and the influence of estrogen and progesterone on HPA axis function and mood. Another is immune system activity, in which immune molecules, such as interleukins and cytokines, activate the HPA axis and alter brain function, including memory, cognition, and mood. The effects of cytokines in inducing sickness behaviors, which overlap with depressive symptomatology, could also contribute to individual differences in such symptomatology. Taken together, this knowledge will have important relevance for identifying at-risk patients to avoid or minimize such side effects when they are treated with glucocorticoids. A framework for assessment of patients is proposed that incorporates functional, physiological, and molecular biomarkers to identify subgroups of patients at risk

  3. Effects of suspension on tissue levels of glucocorticoid receptors

    NASA Technical Reports Server (NTRS)

    Steffen, J. M.

    1984-01-01

    Differential muscle responses can be simulated by hypokinetic/hypodynamic (H/H) suspension of rats with complete unloading of the hindlimb muscles. Since mechanism(s) underlying these atrophic effects were not clearly elucidated, experiments were initiated to investigate a possible role for glucocorticoids in the physiological and biochemical responses to H/H. The principal objective was to assess the potential for alterations in peripheral responsiveness to glucocorticoids in response to H/H. Studies have initially focused on the determination of tissue levels of glucocorticoid receptors as one index of hormonal sensitivity at the cellular level. Four hindlimb muscles (soleus, gastrocnemius, plantaris and EDL), previously demonstrated to exhibit differential responses to H/H, were investigated. Receptor levels in other glucocorticoid sensitive tissues (heart, liver, and kidney) were determined. Male rats (180-200g) were suspended for 7 or 14 days, sacrificed by cervical dislocation, and the tissues excised.

  4. Sensitivity of depression-like behavior to glucocorticoids and antidepressants is independent of forebrain glucocorticoid receptors

    PubMed Central

    Vincent, Melanie Y.; Hussain, Rifat J.; Zampi, Michael E.; Sheeran, Katherine; Solomon, Matia B.; Herman, James P.; Khan, Anum; Jacobson, Lauren

    2013-01-01

    The location of glucocorticoid receptors (GR) implicated in depression symptoms and antidepressant action remains unclear. Forebrain glucocorticoid receptor deletion on a C57B/6×129×CBA background (FBGRKO-T50) reportedly produces increased depression-like behavior and elevated glucocorticoids. We further hypothesized that forebrain GR deletion would reduce behavioral sensitivity to glucocorticoids and to antidepressants. We have tested this hypothesis in mice with calcium calmodulin kinase IIα-Cre-mediated forebrain GR deletion derived from a new founder on a pure C57BL/6 background (FBGRKO-T29-1). We measured immobility in forced swim or tail suspension tests after manipulating glucocorticoids or after dose response experiments with tricyclic or monoamine oxidase inhibitor antidepressants. Despite forebrain GR deletion that was at least as rapid and more extensive than reported in the mixed-strain FBGRKO-T50 mice (Boyle et al. 2005), and possibly because of their different founder, our FBGRKO-T29-1 mice did not exhibit increases in depression-like behavior or adrenocortical axis hormones. Nevertheless, FBGRKO-T29-1 mice were at least as sensitive as floxed GR controls to the depressive effects of glucocorticoids and the effects of two different classes of antidepressants. FBGRKO-T29-1 mice also unexpectedly exhibited increased mineralocorticoid receptor (MR) gene expression. Our results reinforce prior evidence that antidepressant action does not require forebrain GR, and suggest a correlation between the absence of depression-like phenotype and combined MR up-regulation and central amygdala GR deficiency. Our findings demonstrate that GR outside the areas targeted in FBGRKO-T29-1 mice are involved in the depressive effects of glucocorticoids, and leave open the possibility that these GR populations also contribute to antidepressant action. PMID:23727405

  5. Transcriptional regulation of kinases downstream of the T cell receptor: another immunomodulatory mechanism of glucocorticoids

    PubMed Central

    2014-01-01

    Background Glucocorticoids affect peripheral immune responses, including modulation of T-cell activation, differentiation, and apoptosis. The quantity and quality of T-cell receptor (TCR)-triggered intracellular signals modulate T-cell function. Thus, glucocorticoids may affect T cells by interfering with the TCR signaling cascade. The purpose of the study was to search for glucocorticoid-modulated kinases downstream of the TCR. Methods Gene modulation in lymphoid cells either treated with glucocorticoids or from glucocorticoid-treated mice was studied using a RNase protection assay, real-time PCR, and western blotting. The sensitivity of genetically modified thymocytes to glucocorticoid-induced apoptosis was studied by performing hypotonic propidium iodide staining and flow cytometry. The Student’s t-test was employed for statistical evaluation. Results We found that transcription of Itk, a non-receptor tyrosine kinase of the Tec family, was up-regulated in a mouse T-cell hybridoma by the synthetic glucocorticoid dexamethasone. In contrast, dexamethasone down-regulated the expression of Txk, a Tec kinase that functions redundantly with Itk, and Lck, the Src kinase immediately downstream of the TCR. We investigated the expression of Itk, Txk, and Lck in thymocytes and mature lymphocytes following in vitro and in vivo dexamethasone treatment at different time points and doses. Kinase expression was differentially modulated and followed distinct kinetics. Itk was up-regulated in all cell types and conditions tested. Txk was strongly up-regulated in mature lymphocytes but only weakly up-regulated or non-modulated in thymocytes in vitro or in vivo, respectively. Conversely, Lck was down-regulated in thymocytes, but not modulated or up-regulated in mature lymphocytes in the different experimental conditions. This complex behaviour correlates with the presence of both positive and negative glucocorticoid responsive elements (GRE and nGRE, respectively) in the Itk, Txk

  6. Autoradiographic localization of benzodiazepine receptor downregulation

    SciTech Connect

    Tietz, E.I.; Rosenberg, H.C.; Chiu, T.H.

    1986-01-01

    Regional differences in downregulation of brain benzodiazepine receptors were studied using a quantitative autoradiographic method. Rats were given a 4-week flurazepam treatment known to cause tolerance and receptor downregulation. A second group of rats was given a similar treatment, but for only 1 week. A third group was given a single acute dose of diazepam to produce a brain benzodiazepine-like activity equivalent to that found after the chronic treatment. Areas studied included hippocampal formation, cerebral cortex, superior colliculus, substantia nigra, dorsal geniculate nucleus, lateral amygdala and lateral hypothalamus. There was a regional variation in the degree of downregulation after 1 week of flurazepam treatment, ranging from 12% to 25%. Extending the flurazepam treatment to 4 weeks caused little further downregulation in those areas studied, except for the pars reticulata of the substantia nigra, which showed a 13% reduction in (/sup 3/H)flunitrazepam binding after 1 week and a 40% reduction after 4 weeks of treatment. In a few areas, such as the lateral hypothalamus, no significant change in binding was found after 4 weeks. Acute diazepam treatment caused no change in binding. This latter finding as well as results obtained during the development of the methodology show that downregulation was not an artifact due to residual drug content of brain slices. The regional variations in degree and rate of downregulation suggest areas that may be most important for benzodiazepine tolerance and dependence and may be related to the varying time courses for tolerance to different benzodiazepine actions.

  7. Reduced Glucocorticoid Receptor Expression Predicts Bladder Tumor Recurrence and Progression

    PubMed Central

    Ishiguro, Hitoshi; Kawahara, Takashi; Zheng, Yichun; Netto, George J.; Miyamoto, Hiroshi

    2015-01-01

    Objectives To assess the levels of glucocorticoid receptor (GR) expression in bladder tumors because the status and its prognostic value remain largely unknown. Methods We immunohistochemically stained for GR in bladder tumor and matched non-neoplastic bladder tissue specimens. Results Overall, GR was positive in 129 (87%) of 149 urothelial tumors, which was significantly (P = .026) lower than in non-neoplastic urothelium (90 [96%] of 94). Forty-two (79%) of 53 low-grade tumors vs 45 (47%) of 96 high-grade carcinomas (P < .001) and 61 (73%) of 84 non–muscle-invasive (NMI) tumors vs 26 (40%) of 65 muscle-invasive (MI) carcinomas (P < .001) were moderately to strongly immunoreactive for GR. Kaplan-Meier and log-rank tests revealed that loss or weak positivity of GR significantly or marginally correlated with recurrence of NMI tumors (P = .025), progression of MI tumors (P = .082), and cancer-specific survival of MI tumors (P = .067). Multivariate analysis identified low GR expression as a strong predictor for recurrence of NMI tumors (P = .034). Conclusions GR expression was downregulated in bladder tumors compared with nonneoplastic bladder tumors and in high-grade/MI tumors compared with low-grade/NMI tumors. Decreased expression of GR, as an independent prognosticator, predicted recurrence of NMI tumors. These results support experimental evidence suggesting an inhibitory role of GR signals in bladder cancer outgrowth. PMID:25015855

  8. Nestin Modulates Glucocorticoid Receptor Function by Cytoplasmic Anchoring

    PubMed Central

    Szalay, Beata; Hagel, Christian; Hohenberg, Heinrich; Deppert, Wolfgang; Bohn, Wolfgang

    2009-01-01

    Nestin is the characteristic intermediate filament (IF) protein of rapidly proliferating progenitor cells and regenerating tissue. Nestin copolymerizes with class III IF-proteins, mostly vimentin, into heteromeric filaments. Its expression is downregulated with differentiation. Here we show that a strong nestin expression in mouse embryo tissue coincides with a strong accumulation of the glucocorticoid receptor (GR), a key regulator of growth and differentiation in embryonic development. Microscopic studies on cultured cells show an association of GR with IFs composed of vimentin and nestin. Cells lacking nestin, but expressing vimentin, or cells expressing vimentin, but lacking nestin accumulate GR in the nucleus. Completing these networks with an exogenous nestin, respectively an exogenous vimentin restores cytoplasmic anchoring of GR to the IF system. Thus, heteromeric filaments provide the basis for anchoring of GR. The reaction pattern with phospho-GR specific antibodies and the presence of the chaperone HSC70 suggest that specifically the unliganded receptor is anchored to the IF system. Ligand addition releases GR from IFs and shifts the receptor into the nucleus. Suppression of nestin by specific shRNA abolishes anchoring of GR, induces its accumulation in the nucleus and provokes an irreversible G1/S cell cycle arrest. Suppression of GR prior to that of nestin prevents entry into the arrest. The data give evidence that nestin/vimentin specific anchoring modulates growth suppression by GR. We hypothesize that expression of nestin is a major determinant in suppression of anti-proliferative activity of GR in undifferentiated tissue and facilitates activation of this growth control in a precise tissue and differentiation dependent manner. PMID:19562035

  9. Aluminum fluoride inhibition of glucocorticoid receptor inactivation and transformation

    SciTech Connect

    Housley, P.R. )

    1990-04-10

    Fluoride, in the presence of aluminum ions, reversibly inhibits the temperature-mediated inactivation of unoccupied glucocorticoid receptors in cytosol preparations from mouse L cells. The effect is concentration-dependent, with virtually complete stabilization of specific glucocorticoid-binding capacity at 2 mM fluoride and 100 microM aluminum. These concentrations of aluminum and fluoride are ineffective when used separately. Aluminum fluoride also stabilizes receptors toward inactivation by gel filtration and ammonium sulfate precipitation. Aluminum fluoride prevents temperature-dependent transformation of steroid-receptor complexes to the DNA-binding state. Aluminum fluoride does not inhibit calf intestine alkaline phosphatase, and unoccupied receptors inactivated by this enzyme in the presence of aluminum fluoride can be completely reactivated by dithiothreitol. The effects of aluminum fluoride are due to stabilization of the complex between the glucocorticoid receptor and the 90-kDa mammalian heat-shock protein hsp90, which suggests that aluminum fluoride interacts directly with the receptor. Endogenous thermal inactivation of receptors in cytosol is not accompanied by receptor dephosphorylation. However, inactivation is correlated with dissociation of hsp90 from the unoccupied receptor. These results support the proposal that hsp90 is required for the receptor to bind steroid and dissociation of hsp90 is sufficient to inactivate the unoccupied receptor.

  10. Disuse atrophy, plasma corticosterone, and muscle glucocorticoid receptor levels

    NASA Technical Reports Server (NTRS)

    Steffen, J. M.; Musacchia, X. J.

    1987-01-01

    The effect of whole-body suspension on the time course and the extent of plasma corticosterone changes and the tissue sensitivity to glucocorticoids were investigated in rats subjected to seven days of whole-body suspension. Plasma corticosterone increased significantly on the first and the third days of suspension, but returned to control levels by day seven. Muscle glucocorticoid receptors exhibited a characteristic hormonal specificity (evaluated in competitive-displacement experiments). In controls, receptor site concentration in the slow-twitch soleus was comparable to that in the fast-twitch gastrocnemius and plantaris, but was significantly less than in the extensor; seven days of suspension resulted in significant differential effects on muscle receptor levels. The largest increase in receptor concentration was observed in the soleus in which it remained elevated after the receptor levels in other muscles returned to normal.

  11. Glucocorticoid activity detected by in vivo zebrafish assay and in vitro glucocorticoid receptor bioassay at environmental relevant concentrations.

    PubMed

    Chen, Qiyu; Jia, Ai; Snyder, Shane A; Gong, Zhiyuan; Lam, Siew Hong

    2016-02-01

    Glucocorticoids are pharmaceutical contaminants of emerging concern due to their incomplete removal during wastewater treatment, increased presence in aquatic environment and their biological potency. The zebrafish is a popular model for aquatic toxicology and environmental risk assessment. This study aimed to determine if glucocorticoids at environmental concentrations would perturb expression of selected glucocorticoid-responsive genes in zebrafish and to investigate their potentials as an in vivo zebrafish assay in complementing in vitro glucocorticoid receptor bioassay. The relative expression of eleven glucocorticoid-responsive genes in zebrafish larvae and liver of adult male zebrafish exposed to three representative glucocorticoids (dexamethasone, prednisolone and triamcinolone) was determined. The expression of pepck, baiap2 and pxr was up-regulated in zebrafish larvae and the expression of baiap2, pxr and mmp-2 was up-regulated in adult zebrafish exposed to glucocorticoids at concentrations equivalent to total glucocorticoids reported in environmental samples. The responsiveness of the specific genes were sufficiently robust in zebrafish larvae exposed to a complex environmental sample detected with in vitro glucocorticoid activity equivalent to 478 pM dexamethasone (DEX-EQ) and confirmed to contain low concentration (0.2 ng/L or less) of the targeted glucocorticoids, and possibly other glucocorticoid-active compounds. The findings provided in vivo relevance to the in vitro glucocorticoid activity and suggested that the environmental sample can perturb glucocorticoid-responsive genes in its original, or half the diluted, concentration as may be found in the environment. The study demonstrated the important complementary roles of in vivo zebrafish and in vitro bioassays coupled with analytical chemistry in monitoring environmental glucocorticoid contaminants.

  12. Renal tubular vasopressin receptors downregulated by dehydration

    SciTech Connect

    Steiner, M.; Phillips, M.I. )

    1988-03-01

    Receptors for arginine vasopressin (AVP) were characterized in tubular epithelial basolateral membranes (BL membranes) prepared from the kidneys of male Spraque-Dawley rats. Association of ({sup 3}H)AVP was rapid, reversible, and specific. Saturation studies revealed a single class of saturable binding sites with a maximal binding (B{sub max}) of 184 {plus minus} 15 fmol/mg protein. The V{sub 2} receptor antagonist was more than 3,700 times as effective in displacing ({sup 3}H)AVP than was the V{sub 1} antagonist. To investigate the physiological regulation of vasopressin receptors, the effects of elevated levels of circulating AVP on receptor characteristics were studied. Seventy-two-hour water deprivation significantly elevated plasma osmolality and caused an 11.5-fold increase in plasma (AVP). Scatchard analysis revealed a 38% decreased in the number of AVP receptors on the BL membranes from dehydrated animals. The high-affinity binding sites on the BL membranes fit the pharmacological profile for adenylate cyclase-linked vasopressin receptors (V{sub 2}), which mediate the antidiuretic action of the hormone. The authors conclude that physiologically elevated levels of AVP can downregulate vasopressin receptors in the kidney.

  13. Selective glucocorticoid receptor-activating adjuvant therapy in cancer treatments

    PubMed Central

    Sundahl, Nora; Clarisse, Dorien; Bracke, Marc; Offner, Fritz; Berghe, Wim Vanden; Beck, Ilse M.

    2016-01-01

    Although adverse effects and glucocorticoid resistance cripple their chronic use, glucocorticoids form the mainstay therapy for acute and chronic inflammatory disorders, and play an important role in treatment protocols of both lymphoid malignancies and as adjuvant to stimulate therapy tolerability in various solid tumors. Glucocorticoid binding to their designate glucocorticoid receptor (GR), sets off a plethora of cell-specific events including therapeutically desirable effects, such as cell death, as well as undesirable effects, including chemotherapy resistance, systemic side effects and glucocorticoid resistance. In this context, selective GR agonists and modulators (SEGRAMs) with a more restricted GR activity profile have been developed, holding promise for further clinical development in anti-inflammatory and potentially in cancer therapies. Thus far, the research into the prospective benefits of selective GR modulators in cancer therapy limped behind. Our review discusses how selective GR agonists and modulators could improve the therapy regimens for lymphoid malignancies, prostate or breast cancer. We summarize our current knowledge and look forward to where the field should move to in the future. Altogether, our review clarifies novel therapeutic perspectives in cancer modulation via selective GR targeting. PMID:27713909

  14. The role of glucocorticoid receptor (GR) polymorphisms in human erythropoiesis

    PubMed Central

    Varricchio, Lilian; Migliaccio, Anna Rita

    2014-01-01

    Glucocorticoids are endogenous steroid hormones that regulate several biological functions including proliferation, differentiation and apoptosis in numerous cell types in response to stress. Synthetic glucocorticoids, such as dexamethasone (Dex) are used to treat a variety of diseases ranging from allergy to depression. Glucocorticoids exert their effects by passively entering into cells and binding to a specific Glucocorticoid Receptor (GR) present in the cytoplasm. Once activated by its ligand, GR may elicit cytoplasmic (mainly suppression of p53), and nuclear (regulation of transcription of GR responsive genes), responses. Human GR is highly polymorphic and may encode > 260 different isoforms. This polymorphism is emerging as the leading cause for the variability of phenotype and response to glucocorticoid therapy observed in human populations. Studies in mice and clinical observations indicate that GR controls also the response to erythroid stress. This knowledge has been exploited in-vivo by using synthetic GR agonists for treatment of the erythropoietin-refractory congenic Diamond Blackfan Anemia and in-vitro to develop culture conditions that may theoretically generate red cells in numbers sufficient for transfusion. However, the effect exerted by GR polymorphism on the variability of the phenotype of genetic and acquired erythroid disorders observed in the human population is still poorly appreciated. This review will summarize current knowledge on the biological activity of GR and of its polymorphism in non-hematopoietic diseases and discuss the implications of these observations for erythropoiesis. PMID:25755906

  15. Identification of potential glucocorticoid receptor therapeutic targets in multiple myeloma

    PubMed Central

    Thomas, Alexandra L.; Coarfa, Cristian; Qian, Jun; Wilkerson, Joseph J.; Rajapakshe, Kimal; Krett, Nancy L.; Gunaratne, Preethi H.; Rosen, Steven T.

    2015-01-01

    Glucocorticoids (GC) are a cornerstone of combination therapies for multiple myeloma. However, patients ultimately develop resistance to GCs frequently based on decreased glucocorticoid receptor (GR) expression. An understanding of the direct targets of GC actions, which induce cell death, is expected to culminate in potential therapeutic strategies for inducing cell death by regulating downstream targets in the absence of a functional GR. The specific goal of our research is to identify primary GR targets that contribute to GC-induced cell death, with the ultimate goal of developing novel therapeutics around these targets that can be used to overcome resistance to GCs in the absence of GR. Using the MM.1S glucocorticoid-sensitive human myeloma cell line, we began with the broad platform of gene expression profiling to identify glucocorticoid-regulated genes further refined by combination treatment with phosphatidylinositol-3’-kinase inhibition (PI3Ki). To further refine the search to distinguish direct and indirect targets of GR that respond to the combination GC and PI3Ki treatment of MM.1S cells, we integrated 1) gene expression profiles of combination GC treatment with PI3Ki, which induces synergistic cell death; 2) negative correlation between genes inhibited by combination treatment in MM.1S cells and genes over-expressed in myeloma patients to establish clinical relevance and 3) GR chromatin immunoprecipitation with massively parallel sequencing (ChIP-Seq) in myeloma cells to identify global chromatin binding for the glucocorticoid receptor (GR). Using established bioinformatics platforms, we have integrated these data sets to identify a subset of candidate genes that may form the basis for a comprehensive picture of glucocorticoid actions in multiple myeloma. As a proof of principle, we have verified two targets, namely RRM2 and BCL2L1, as primary functional targets of GR involved in GC-induced cell death. PMID:26715915

  16. Glucocorticoids induce CCN5/WISP-2 expression and attenuate invasion in oestrogen receptor-negative human breast cancer cells.

    PubMed

    Ferrand, Nathalie; Stragier, Emilien; Redeuilh, Gérard; Sabbah, Michèle

    2012-10-01

    CCN5 (cysteine-rich 61/connective tissue growth factor/nephroblastoma overexpressed 5)/WISP-2 [WNT1 (wingless-type MMTV integration site family, member 1)-inducible signalling pathway protein 2] is an oestrogen-regulated member of the CCN family. CCN5 is a transcriptional repressor of genes associated with the EMT (epithelial-mesenchymal transition) and plays an important role in maintenance of the differentiated phenotype in ER (oestrogen receptor)-positive breast cancer cells. In contrast, CCN5 is undetectable in more aggressive ER-negative breast cancer cells. We now report that CCN5 is induced in ER-negative breast cancer cells such as MDA-MB-231 following glucocorticoid exposure, due to interaction of the endogenous glucocorticoid receptor with a functional glucocorticoid-response element in the CCN5 gene promoter. Glucocorticoid treatment of MDA-MB-231 cells is accompanied by morphological alterations, decreased invasiveness and attenuated expression of mesenchymal markers, including vimentin, cadherin 11 and ZEB1 (zinc finger E-box binding homeobox 1). Interestingly, glucocorticoid exposure did not increase CCN5 expression in ER-positive breast cancer cells, but rather down-regulated ER expression, thereby attenuating oestrogen pathway signalling. Taken together, our results indicate that glucocorticoid treatment of ER-negative breast cancer cells induces high levels of CCN5 expression and is accompanied by the appearance of a more differentiated and less invasive epithelial phenotype. These findings propose a novel therapeutic strategy for high-risk breast cancer patients.

  17. The glucocorticoid receptor: cause of or cure for obesity?

    PubMed

    John, Kezia; Marino, Joseph S; Sanchez, Edwin R; Hinds, Terry D

    2016-02-15

    Glucocorticoid hormones (GCs) are important regulators of lipid metabolism, promoting lipolysis with acute treatment but lipogenesis with chronic exposure. Conventional wisdom posits that these disparate outcomes are mediated by the classical glucocorticoid receptor GRα. There is insufficient knowledge of the GC receptors (GRα and GRβ) in metabolic conditions such as obesity and diabetes. We present acute models of GC exposure that induce lipolysis, such as exercise, as well as chronic-excess models that cause obesity and lipid accumulation in the liver, such as hepatic steatosis. Alternative mechanisms are then proposed for the lipogenic actions of GCs, including induction of GC resistance by the GRβ isoform, and promotion of lipogenesis by GC activation of the mineralocorticoid receptor (MR). Finally, the potential involvement of chaperone proteins in the regulation of adipogenesis is considered. This reevaluation may prove useful to future studies on the steroidal basis of adipogenesis and obesity. PMID:26714851

  18. Glucocorticoid receptor co-factors as therapeutic targets

    PubMed Central

    Simons, S. Stoney

    2010-01-01

    Summary Numerous transcriptional cofactors (e.g., coactivators, corepressors, and comodulators) are known to alter the maximal transcriptional activity (Amax) in gene induction and repression by steroid receptors in general and glucocorticoids in particular. However, recent data advance the earlier reports that these same factors also modify other parameters of glucocorticoid receptor transcriptional activity: the potency of agonists (or EC50) and the partial agonist activity of antisteroids (or PAA). In several instances, factors modulate the EC50 and/or PAA without changing Amax. Thus, studies of all three parameters reveal new factors acting at various stages of receptor action, thereby increasing the potential therapeutic targets for adjusting GR actions in pathological situations. PMID:20801081

  19. Estrogen-induced decrease of glucocorticoid receptor messenger ribonucleic acid concentration in rat anterior pituitary gland.

    PubMed

    Peiffer, A; Barden, N

    1987-06-01

    Using Northern blots and hybridization techniques, we have identified an approximately 6.5 kilobase glucocorticoid receptor mRNA species in rat anterior pituitary gland. Ovariectomy resulted in an approximately 2-fold increase in glucocorticoid receptor mRNA concentrations. This effect was maximal 8 days after surgery and glucocorticoid receptor mRNA levels remained elevated for at least up to 4 weeks. Administration of 17-beta-estradiol completely reversed the ovariectomy-induced increase in glucocorticoid receptor mRNA content of pituitary gland. Treatment of rats with corticosterone did not influence the ovariectomy-induced increase in glucocorticoid receptor mRNA content, indicating that this increase is not mediated via effects on circulating glucocorticoid levels or availability. In situ hybridization experiments confirmed the ovariectomy-induced increase in glucocorticoid receptor mRNA content and indicated that this action is widely distributed throughout the anterior pituitary gland.

  20. Abrogation of Glucocorticoid Receptor Dimerization Correlates with Dissociated Glucocorticoid Behavior of Compound A*

    PubMed Central

    Robertson, Steven; Allie-Reid, Fatima; Berghe, Wim Vanden; Visser, Koch; Binder, Anke; Africander, Donita; Vismer, Michael; De Bosscher, Karolien; Hapgood, Janet; Haegeman, Guy; Louw, Ann

    2010-01-01

    Compound A (CpdA), a dissociated glucocorticoid receptor modulator, decreases corticosteroid-binding globulin (CBG), adrenocorticotropic hormone (ACTH), and luteneinizing hormone levels in rats. Whether this is due to transcriptional regulation by CpdA is not known. Using promoter reporter assays we show that CpdA, like dexamethasone (Dex), directly transrepresses these genes. Results using a rat Cbg proximal-promoter reporter construct in BWTG3 and HepG2 cell lines support a glucocorticoid receptor (GR)-dependent transrepression mechanism for CpdA. However, CpdA, unlike Dex, does not result in transactivation via glucocorticoid-responsive elements within a promoter reporter construct even when GR is co-transfected. The inability of CpdA to result in transactivation via glucocorticoid-responsive elements is confirmed on the endogenous tyrosine aminotransferase gene, whereas transrepression ability is confirmed on the endogenous CBG gene. Consistent with a role for CpdA in modulating GR activity, whole cell binding assays revealed that CpdA binds reversibly to the GR, but with lower affinity than Dex, and influences association of [3H]Dex, but has no effect on dissociation. In addition, like Dex, CpdA causes nuclear translocation of the GR, albeit to a lesser degree. Several lines of evidence, including fluorescence resonance energy transfer, co-immunoprecipitation, and nuclear immunofluorescence studies of nuclear localization-deficient GR show that CpdA, unlike Dex, does not elicit ligand-induced GR dimerization. Comparison of the behavior of CpdA in the presence of wild type GR to that of Dex with a dimerization-deficient GR mutant (GRdim) strongly supports the conclusion that loss of dimerization is responsible for the dissociated behavior of CpdA. PMID:20037160

  1. Glucocorticoid receptor exhibits sexually dimorphic expression in the medaka brain.

    PubMed

    Kikuchi, Yukiko; Hosono, Kohei; Yamashita, Junpei; Kawabata, Yukika; Okubo, Kataaki

    2015-11-01

    The differential impact of stress on brain functions of males and females has been widely observed in vertebrates. Recent evidence suggests that stress-induced glucocorticoid signaling affects sexual differentiation and sex changes in teleost fish. These facts led us to postulate that there were sex differences in glucocorticoid signaling in the teleost brain that underlie some sex differences in their physiological and behavioral traits. Here we found sexually dimorphic expression of a glucocorticoid receptor gene (gr1) in the brain of medaka fish (Oryzias latipes), with females having greater expression in several preoptic and thalamic nuclei. Further, gr1 exhibits female-biased expression in neurons of the anterior parvocellular preoptic nucleus that produce the neuropeptides vasotocin and gonadotropin-releasing hormone 1 (these neuropeptides have been implicated in the regulation of neuroendocrine and behavioral functions). These findings suggest that glucocorticoids have a greater influence on physiology and behavior mediated by these neuropeptides in females than in males, which may contribute to sex differences in the brain's response to stress. PMID:26433060

  2. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    SciTech Connect

    Sato, Shoko; Shirakawa, Hitoshi; Tomita, Shuhei; Tohkin, Masahiro; Gonzalez, Frank J.; Komai, Michio

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.

  3. Glucocorticoid receptor gene expression in rat pituitary gland intermediate lobe following ovariectomy.

    PubMed

    Pfeiffer, A; Barden, N

    1988-02-01

    Using hybridization techniques and Northern blots we have identified a approximately 6.5 kb glucocorticoid receptor mRNA species in rat pituitary intermediate lobe. Glucocorticoid receptor mRNA concentrations, which are barely detectable or undetectable in normal animals, were greatly increased following ovariectomy. This ovariectomy-induced increase in glucocorticoid receptor mRNA content of the intermediate lobe, which was confirmed by in situ hybridization experiments, could be reversed by 17 beta-estradiol administration.

  4. The Transcriptomics of Glucocorticoid Receptor Signaling in Developing Zebrafish

    PubMed Central

    Nesan, Dinushan; Vijayan, Mathilakath M.

    2013-01-01

    Cortisol is the primary corticosteroid in teleosts that is released in response to stressor activation of the hypothalamus-pituitary-interrenal axis. The target tissue action of this hormone is primarily mediated by the intracellular glucocorticoid receptor (GR), a ligand-bound transcription factor. In developing zebrafish (Danio rerio) embryos, GR transcripts and cortisol are maternally deposited into the oocyte prior to fertilization and influence early embryogenesis. To better understand of the molecular mechanisms involved, we investigated changes in the developmental transcriptome prior to hatch, in response to morpholino oligonucleotide knockdown of GR using the Agilent zebrafish microarray platform. A total of 1313 and 836 mRNA transcripts were significantly changed at 24 and 36 hours post fertilization (hpf), respectively. Functional analysis revealed numerous developmental processes under GR regulation, including neurogenesis, eye development, skeletal and cardiac muscle formation. Together, this study underscores a critical role for glucocorticoid signaling in programming molecular events essential for zebrafish development. PMID:24348914

  5. Human Glucocorticoid Receptor β Regulates Gluconeogenesis and Inflammation in Mouse Liver

    PubMed Central

    He, Bo; Cruz-Topete, Diana; Oakley, Robert H.; Xiao, Xiao

    2015-01-01

    While in vitro studies have demonstrated that a glucocorticoid receptor (GR) splice isoform, β-isoform of human GR (hGRβ), acts as a dominant-negative inhibitor of the classic hGRα and confers glucocorticoid resistance, the in vivo function of hGRβ is poorly understood. To this end, we created an adeno-associated virus (AAV) to express hGRβ in the mouse liver under the control of the hepatocyte-specific promoter. Genome-wide expression analysis of mouse livers showed that hGRβ significantly increased the expression of numerous genes, many of which are involved in endocrine system disorders and the inflammatory response. Physiologically, hGRβ antagonized GRα's function and attenuated hepatic gluconeogenesis through downregulation of phosphoenolpyruvate carboxykinase (PEPCK) in wild-type (WT) mouse liver. Interestingly, however, hGRβ did not repress PEPCK in GR liver knockout (GRLKO) mice. In contrast, hGRβ regulates the expression of STAT1 in the livers of both WT and GRLKO mice. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays demonstrated that hGRβ binds to the intergenic glucocorticoid response element (GRE) of the STAT1 gene. Furthermore, treatment with RU486 inhibited the upregulation of STAT1 mediated by hGRβ. Finally, our array data demonstrate that hGRβ regulates unique components of liver gene expression in vivo by both GRα-dependent and GRα-independent mechanisms. PMID:26711253

  6. Alteration of the glucocorticoid receptor subcellular localization by non steroidal compounds.

    PubMed

    Prima, V; Depoix, C; Masselot, B; Formstecher, P; Lefebvre, P

    2000-01-01

    The glucocorticoid receptor (GR) engages transient or stable interactions with chaperones (hsp90, hsp70), co-chaperones (p60/hop, hsp40) and several other polypeptides such as immunophilins (Cyp40, FKBP59) and p23 to achieve a high affinity ligand binding state. This complex dissociates in response to hormonal stimuli and holo-GR translocates into the nucleus, where it regulates the activity of glucocorticoid-sensitive genes. GR activity is controlled through its ligand binding domain by steroids displaying either agonistic or antagonistic activity. An alternative approach to modulate GR activity is to target receptor-associated proteins (RAPs), and several non steroidal compounds binding to RAPs affect GR transcriptional activity. We have studied the effect of such drugs on the intracellular localization of a EGFP-GR fusion protein, which has wild type GR pharmacological properties. Agonist and antagonist binding induced nuclear translocation of GR, whereas rifampicin was found to be inactive in our system. Immunosuppressants FK506 and cyclosporin A were able to induce partial nuclear translocation of GR, suggesting that potentiation of glucocorticoid action by these compounds may also proceed through enhanced GR nuclear transfer. Short treatment of cells with the hsp90 inhibitor geldanamycin (GA) did not prevent nuclear translocation of GR. However, longer treatments, in parrallel to the inhibition of GR transcriptional activity, strongly perturbed GR subcellular localization concomitantly to the disruption of the actin network, and caused GR aggregation and down-regulation. The GA-induced transcriptional shutdown was also observed for other nuclear receptors which do not interact stably with hsp90. Thus RAP-binding compounds may exert their effects at least in part through perturbation of the GR cytosol to nucleus partitioning, and identify these proteins as valuable therapeutic targets to control nuclear receptor activity.

  7. Dopamine-dependent responses to morphine depend on glucocorticoid receptors

    PubMed Central

    Marinelli, Michela; Aouizerate, Bruno; Barrot, Michel; Le Moal, Michel; Piazza, Pier Vincenzo

    1998-01-01

    Previous work has shown that glucocorticoid hormones facilitate the behavioral and dopaminergic effects of morphine. In this study we examined the possible role in these effects of the two central corticosteroid receptor types: mineralocorticoid receptor (MR), and glucocorticoid receptor (GR). To accomplish this, specific antagonists of these receptors were infused intracerebroventricularly and 2 hr later we measured: (i) locomotor activity induced by a systemic injection of morphine (2 mg/kg); (ii) locomotor activity induced by an infusion of morphine (1 μg per side) into the ventral tegmental area, which is a dopamine-dependent behavioral response to morphine; (iii) morphine-induced dopamine release in the nucleus accumbens, a dopaminergic projection site mediating the locomotor and reinforcing effects of drugs of abuse. Blockade of MRs by spironolactone had no significant effects on locomotion induced by systemic morphine. In contrast, blockade of GRs by either RU38486 or RU39305, which is devoid of antiprogesterone effects, reduced the locomotor response to morphine, and this effect was dose dependent. GR antagonists also reduced the locomotor response to intraventral tegmental area morphine as well as the basal and morphine-induced increase in accumbens dopamine, as measured by microdialysis in freely moving rats. In contrast, spironolactone did not modify dopamine release. In conclusion, glucocorticoids, via GRs, facilitate the dopamine-dependent behavioral effects of morphine, probably by facilitating dopamine release. The possibility of decreasing the behavioral and dopaminergic effects of opioids by an acute administration of GR antagonists may open new therapeutic strategies for treatment of drug addiction. PMID:9636221

  8. The HIV-1 Virion-associated Protein Vpr Is a Coactivator of the Human Glucocorticoid Receptor

    PubMed Central

    Kino, Tomoshige; Gragerov, Alexander; Kopp, Jeffrey B.; Stauber, Roland H.; Pavlakis, George N.; Chrousos, George P.

    1999-01-01

    The HIV-1 virion-associated accessory protein Vpr affects both viral replication and cellular transcription, proliferation, and differentiation. We report that Vpr enhances the activity of glucocorticoids in lymphoid and muscle-derived cell lines by interacting directly with the glucocorticoid receptor and general transcription factors, acting as a coactivator. Vpr contains the signature motif LXXLL also present in cellular nuclear receptor coactivators, such as steroid receptor coactivator 1 and p300/CREB-binding protein, which mediates their interaction with the glucocorticoid and other nuclear hormone receptors. A mutant Vpr molecule with disruption of this coactivator signature motif lost its ability to influence transcription of glucocorticoid-responsive genes and became a dominant-negative inhibitor of Vpr, possibly by retaining its general transcription factor–binding activities. The glucocorticoid coactivator activity of Vpr may contribute to increased tissue glucocorticoid sensitivity in the absence of hypercortisolism and to the pathogenesis of AIDS. PMID:9874563

  9. Epigenetic and Glucocorticoid Receptor-Mediated Regulation of Glutathione Peroxidase 3 in Lung Cancer Cells

    PubMed Central

    An, Byung Chull; Jung, Nak-Kyun; Park, Chun Young; Oh, In-Jae; Choi, Yoo-Duk; Park, Jae-Il; Lee, Seung-won

    2016-01-01

    Glutathione peroxidase 3 (GPx3), an antioxidant enzyme, acts as a modulator of redox signaling, has immunomodulatory function, and catalyzes the detoxification of reactive oxygen species (ROS). GPx3 has been identified as a tumor suppressor in many cancers. Although hyper-methylation of the GPx3 promoter has been shown to down-regulate its expression, other mechanisms by which GPx3 expression is regulated have not been reported. The aim of this study was to further elucidate the mechanisms of GPx3 regulation. GPx3 gene analysis predicted the presence of ten glucocorticoid response elements (GREs) on the GPx3 gene. This result prompted us to investigate whether GPx3 expression is regulated by the glucocorticoid receptor (GR), which is implicated in tumor response to chemotherapy. The corticosteroid dexamethasone (Dex) was used to examine the possible relationship between GR and GPx3 expression. Dex significantly induced GPx3 expression in H1299, H1650, and H1975 cell lines, which exhibit low levels of GPx3 expression under normal conditions. The results of EMSA and ChIP-PCR suggest that GR binds directly to GRE 6 and 7, both of which are located near the GPx3 promoter. Assessment of GPx3 transcription efficiency using a luciferase reporter system showed that blocking formation of the GR-GRE complexes reduced luciferase activity by 7–8-fold. Suppression of GR expression by siRNA transfection also induced down-regulation of GPx3. These data indicate that GPx3 expression can be regulated independently via epigenetic or GR-mediated mechanisms in lung cancer cells, and suggest that GPx3 could potentiate glucocorticoid (GC)-mediated anti-inflammatory signaling in lung cancer cells. PMID:27484907

  10. Epigenetic and Glucocorticoid Receptor-Mediated Regulation of Glutathione Peroxidase 3 in Lung Cancer Cells.

    PubMed

    An, Byung Chull; Jung, Nak-Kyun; Park, Chun Young; Oh, In-Jae; Choi, Yoo-Duk; Park, Jae-Il; Lee, Seung-Won

    2016-08-31

    Glutathione peroxidase 3 (GPx3), an antioxidant enzyme, acts as a modulator of redox signaling, has immunomodulatory function, and catalyzes the detoxification of reactive oxygen species (ROS). GPx3 has been identified as a tumor suppressor in many cancers. Although hyper-methylation of the GPx3 promoter has been shown to down-regulate its expression, other mechanisms by which GPx3 expression is regulated have not been reported. The aim of this study was to further elucidate the mechanisms of GPx3 regulation. GPx3 gene analysis predicted the presence of ten glucocorticoid response elements (GREs) on the GPx3 gene. This result prompted us to investigate whether GPx3 expression is regulated by the glucocorticoid receptor (GR), which is implicated in tumor response to chemotherapy. The corticosteroid dexamethasone (Dex) was used to examine the possible relationship between GR and GPx3 expression. Dex significantly induced GPx3 expression in H1299, H1650, and H1975 cell lines, which exhibit low levels of GPx3 expression under normal conditions. The results of EMSA and ChIP-PCR suggest that GR binds directly to GRE 6 and 7, both of which are located near the GPx3 promoter. Assessment of GPx3 transcription efficiency using a luciferase reporter system showed that blocking formation of the GR-GRE complexes reduced luciferase activity by 7-8-fold. Suppression of GR expression by siRNA transfection also induced down-regulation of GPx3. These data indicate that GPx3 expression can be regulated independently via epigenetic or GR-mediated mechanisms in lung cancer cells, and suggest that GPx3 could potentiate glucocorticoid (GC)-mediated anti-inflammatory signaling in lung cancer cells. PMID:27484907

  11. Cancer cell-selective promoter recognition accompanies antitumor effect by glucocorticoid receptor-targeted gold nanoparticle.

    PubMed

    Sau, Samaresh; Agarwalla, Pritha; Mukherjee, Sudip; Bag, Indira; Sreedhar, Bojja; Pal-Bhadra, Manika; Patra, Chitta Ranjan; Banerjee, Rajkumar

    2014-06-21

    Nanoparticles, such as gold nanoparticles (GNP), upon convenient modifications perform multi tasks catering to many biomedical applications. However, GNP or any other type of nanoparticles is yet to achieve the feat of intracellular regulation of endogenous genes of choice such as through manipulation of a gene-promoter in a chromosome. As for gene modulation and delivery, GNP (or other nanoparticles) showed only limited gene therapy potential, which relied on the delivery of 'exogenous' genes invoking gene knockdown or replacement. Practically, there are no instances for the nanoparticle-mediated promoter regulation of 'endogenous' genes, more so, as a cancer selective phenomenon. In this regard, we report the development of a simple, easily modifiable GNP-formulation, which promoted/up-regulated the expression of a specific category of 'endogenous' genes, the glucocorticoid responsive genes. This genetic up-regulation was induced in only cancer cells by modified GNP-mediated transcriptional activation of its cytoplasmic receptor, glucocorticoid receptor (GR). Normal cells and their GR remained primarily unperturbed by this GNP-formulation. The most potent gene up-regulating GNP-formulation down-regulated a cancer-specific proliferative signal, phospho-Akt in cancer cells, which accompanied retardation of tumor growth in the murine melanoma model. We show that GR-targeted GNPs may find potential use in the targeting and modulation of genetic information in cancer towards developing novel anticancer therapeutics.

  12. Receptor-dependent mechanisms of glucocorticoid and dioxin-induced cleft palate

    SciTech Connect

    Pratt, R.M.

    1985-09-01

    Glucocorticoids (triamcinolone) and dioxins (TCDD) are highly specific teratogens in the mouse, in that cleft palate is the major malformation observed. Glucocorticoids and TCDD both readily cross the yolk sac and placenta and appear in the developing secondary palate. Structure-activity relationships for glucocorticoid- and TCDD-induced cleft palate suggest a receptor involvement. Receptors for glucocorticoids and TCDD are present in the palate and their levels in various mouse strains are highly correlated with their sensitivity to cleft palate induction. Receptors for glucocorticoids appear to be more prevalent in the palatal mesenchymal cells whereas those for TCDD are probably located in the palatal epithelial cells. Glucocorticoids exert their teratogenic effect on the palate by inhibiting the growth of the palatal mesenchymal cells whereas TCDD alters the terminal cell differentiation of the media palatal epithelial cells. 71 references.

  13. Membrane glucocorticoid receptor activation induces proteomic changes aligning with classical glucocorticoid effects.

    PubMed

    Vernocchi, Sara; Battello, Nadia; Schmitz, Stephanie; Revets, Dominique; Billing, Anja M; Turner, Jonathan D; Muller, Claude P

    2013-07-01

    Glucocorticoids exert rapid nongenomic effects by several mechanisms including the activation of a membrane-bound glucocorticoid receptor (mGR). Here, we report the first proteomic study on the effects of mGR activation by BSA-conjugated cortisol (Cort-BSA). A subset of target proteins in the proteomic data set was validated by Western blot and we found them responding to mGR activation by BSA-conjugated cortisol in three additional cell lines, indicating a conserved effect in cells originating from different tissues. Changes in the proteome of BSA-conjugated cortisol treated CCRF-CEM leukemia cells were associated with early and rapid pro-apoptotic, immune-modulatory and metabolic effects aligning with and possibly "priming" classical activities of the cytosolic glucocorticoid receptor (cGR). PCR arrays investigating target genes of the major signaling pathways indicated that the mGR does not exert its effects through the transcriptional activity of any of the most common kinases in these leukemic cells, but RhoA signaling emerged from our pathway analysis. All cell lines tested displayed very low levels of mGR on their surface. Highly sensitive and specific in situ proximity ligation assay visualized low numbers of mGR even in cells previously thought to be mGR negative. We obtained similar results when using three distinct anti-GR monoclonal antibodies directed against the N-terminal half of the cGR. This strongly suggests that the mGR and the cGR have a high sequence homology and most probably originate from the same gene. Furthermore, the mGR appears to reside in caveolae and its association with caveolin-1 (Cav-1) was clearly detected in two of the four cell lines investigated using double recognition proximity ligation assay. Our results indicate however that Cav-1 is not necessary for membrane localization of the GR since CCRF-CEM and Jurkat cells have a functional mGR, but did not express this caveolar protein. However, if expressed, this membrane protein

  14. Involvement of the Androgen and Glucocorticoid Receptors in Bladder Cancer.

    PubMed

    McBeth, Lucien; Grabnar, Maria; Selman, Steven; Hinds, Terry D

    2015-01-01

    Bladder cancer is encountered worldwide having been associated with a host of environmental and lifestyle risk factors. The disease has a male to female prevalence of 3 : 1. This disparity has raised the possibility of the androgen receptor (AR) pathway being involved in the genesis of the disease; indeed, research has shown that AR is involved in and is likely a driver of bladder cancer. Similarly, an inflammatory response has been implicated as a major player in bladder carcinogenesis. Consistent with this concept, recent work on anti-inflammatory glucocorticoid signaling points to a pathway that may impact bladder cancer. The glucocorticoid receptor- (GR-) α isoform has an important role in suppressing inflammatory processes, which may be attenuated by AR in the development of bladder cancer. In addition, a GR isoform that is inhibitory to GRα, GRβ, is proinflammatory and has been shown to induce cancer growth. In this paper, we review the evidence of inflammatory mediators and the relationship of AR and GR isoforms as they relate to the propensity for bladder cancer. PMID:26347776

  15. An epistatic ratchet constrains the direction of glucocorticoid receptor evolution

    SciTech Connect

    Bridgham, Jamie T.; Ortlund, Eric A.; Thornton, Joseph W.

    2010-10-28

    The extent to which evolution is reversible has long fascinated biologists. Most previous work on the reversibility of morphological and life-history evolution has been indecisive, because of uncertainty and bias in the methods used to infer ancestral states for such characters. Further, despite theoretical work on the factors that could contribute to irreversibility, there is little empirical evidence on its causes, because sufficient understanding of the mechanistic basis for the evolution of new or ancestral phenotypes is seldom available. By studying the reversibility of evolutionary changes in protein structure and function, these limitations can be overcome. Here we show, using the evolution of hormone specificity in the vertebrate glucocorticoid receptor as a case-study, that the evolutionary path by which this protein acquired its new function soon became inaccessible to reverse exploration. Using ancestral gene reconstruction, protein engineering and X-ray crystallography, we demonstrate that five subsequent 'restrictive' mutations, which optimized the new specificity of the glucocorticoid receptor, also destabilized elements of the protein structure that were required to support the ancestral conformation. Unless these ratchet-like epistatic substitutions are restored to their ancestral states, reversing the key function-switching mutations yields a non-functional protein. Reversing the restrictive substitutions first, however, does nothing to enhance the ancestral function. Our findings indicate that even if selection for the ancestral function were imposed, direct reversal would be extremely unlikely, suggesting an important role for historical contingency in protein evolution.

  16. Involvement of the Androgen and Glucocorticoid Receptors in Bladder Cancer

    PubMed Central

    McBeth, Lucien; Grabnar, Maria; Selman, Steven; Hinds, Terry D.

    2015-01-01

    Bladder cancer is encountered worldwide having been associated with a host of environmental and lifestyle risk factors. The disease has a male to female prevalence of 3 : 1. This disparity has raised the possibility of the androgen receptor (AR) pathway being involved in the genesis of the disease; indeed, research has shown that AR is involved in and is likely a driver of bladder cancer. Similarly, an inflammatory response has been implicated as a major player in bladder carcinogenesis. Consistent with this concept, recent work on anti-inflammatory glucocorticoid signaling points to a pathway that may impact bladder cancer. The glucocorticoid receptor- (GR-) α isoform has an important role in suppressing inflammatory processes, which may be attenuated by AR in the development of bladder cancer. In addition, a GR isoform that is inhibitory to GRα, GRβ, is proinflammatory and has been shown to induce cancer growth. In this paper, we review the evidence of inflammatory mediators and the relationship of AR and GR isoforms as they relate to the propensity for bladder cancer. PMID:26347776

  17. Glucocorticoid receptor-mediated induction of glutamine synthetase in skeletal muscle cells in vitro

    NASA Technical Reports Server (NTRS)

    Max, Stephen R.; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa; Konagaya, Masaaki

    1987-01-01

    The regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture is studied. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

  18. Glucocorticoid receptor isoforms alpha and beta in in vitro cytokine-induced glucocorticoid insensitivity.

    PubMed

    Torrego, Alfons; Pujols, Laura; Roca-Ferrer, Jordi; Mullol, Joaquim; Xaubet, Antoni; Picado, César

    2004-08-15

    We stimulated peripheral blood mononuclear cells from 14 healthy subjects, 14 patients with stable asthma, and 13 patients with unstable asthma with interleukin (IL)-2 and IL-4 to induce glucocorticoid insensitivity and we examined the relationship between insensitivity and the expression of glucocorticoid receptor (GR) isoforms. Results are expressed as IC(50) (nanomolar) values (means +/- SD) in proliferation assays and as 10(3) cDNA molecules per microgram of total RNA (means +/- SD) in real-time polymerase chain reaction analysis. Cells from patients with unstable asthma were less sensitive (316 +/- 7 nM) to dexamethasone antiproliferative effects than those from healthy control subjects (102 +/- 4 nM, p < 0.05) and patients with stable asthma (107 +/- 2 nM, p < 0.05). Coincubation with IL-2 and IL-4 repressed the inhibitory effect of dexamethasone on proliferation in all groups (unstable: 851 +/- 47 nM, p < 0.01; stable: 912 +/- 52 nM, p = 0.001; control subjects: 537 +/- 45 nM, p = 0.001). GR-alpha mRNA baseline expression was higher in patients with unstable asthma [(1.95 +/- 0.40) x 10(3) cDNA molecules/microg total RNA, p < 0.05] than in patients with stable asthma [(1.46 +/- 0.35) x 10(3) cDNA molecules/microg total RNA] and healthy subjects [(1.35 +/- 0.25) x 10(3) cDNA molecules/microg total RNA]. GR-beta mRNA was 600 times lower than GR-alpha in the three groups. Coincubation with IL-2 and IL-4 significantly increased GR-alpha mRNA expression in the three groups (p < 0.01), but caused no significant change in GR-beta mRNA. GR-alpha, but not GR-beta, protein was detected at baseline and after cytokine exposure. Our data do not support the hypothesis that increased GR-beta expression can contribute to cytokine-induced glucocorticoid insensitivity. PMID:15184204

  19. Glucocorticoid receptor beta increases migration of human bladder cancer cells.

    PubMed

    McBeth, Lucien; Nwaneri, Assumpta C; Grabnar, Maria; Demeter, Jonathan; Nestor-Kalinoski, Andrea; Hinds, Terry D

    2016-05-10

    Bladder cancer is observed worldwide having been associated with a host of environmental and lifestyle risk factors. Recent investigations on anti-inflammatory glucocorticoid signaling point to a pathway that may impact bladder cancer. Here we show an inverse effect on the glucocorticoid receptor (GR) isoform signaling that may lead to bladder cancer. We found similar GRα expression levels in the transitional uroepithelial cancer cell lines T24 and UMUC-3. However, the T24 cells showed a significant (p < 0.05) increased expression of GRβ compared to UMUC-3, which also correlated with higher migration rates. Knockdown of GRβ in the T24 cells resulted in a decreased migration rate. Mutational analysis of the 3' untranslated region (UTR) of human GRβ revealed that miR144 might positively regulate expression. Indeed, overexpression of miR144 increased GRβ by 3.8 fold. In addition, miR144 and GRβ were upregulated during migration. We used a peptide nucleic acid conjugated to a cell penetrating-peptide (Sweet-P) to block the binding site for miR144 in the 3'UTR of GRβ. Sweet-P effectively prevented miR144 actions and decreased GRβ expression, as well as the migration of the T24 human bladder cancer cells. Therefore, GRβ may have a significant role in bladder cancer, and possibly serve as a therapeutic target for the disease. PMID:27036026

  20. Characterization of a novel non-steroidal glucocorticoid receptor antagonist

    SciTech Connect

    Li, Qun-Yi; Zhang, Meng; Hallis, Tina M.; DeRosier, Therese A.; Yue, Jian-Min; Ye, Yang; Mais, Dale E.; Wang, Ming-Wei

    2010-01-15

    Selective antagonists of the glucocorticoid receptor (GR) are desirable for the treatment of hypercortisolemia associated with Cushing's syndrome, psychic depression, obesity, diabetes, neurodegenerative diseases, and glaucoma. NC3327, a non-steroidal small molecule with potent binding affinity to GR (K{sub i} = 13.2 nM), was identified in a high-throughput screening effort. As a full GR antagonist, NC3327 greatly inhibits the dexamethasone (Dex) induction of marker genes involved in hepatic gluconeogenesis, but has a minimal effect on matrix metalloproteinase 9 (MMP-9), a GR responsive pro-inflammatory gene. Interestingly, the compound recruits neither coactivators nor corepressors to the GR complex but competes with glucocorticoids for the interaction between GR and a coactivator peptide. Moreover, NC3327 does not trigger GR nuclear translocation, but significantly blocks Dex-induced GR transportation to the nucleus, and thus appears to be a 'competitive' GR antagonist. Therefore, the non-steroidal compound, NC3327, may represent a new class of GR antagonists as potential therapeutics for a variety of cortisol-related endocrine disorders.

  1. Glucocorticoid receptor beta increases migration of human bladder cancer cells

    PubMed Central

    McBeth, Lucien; Nwaneri, Assumpta C.; Grabnar, Maria; Demeter, Jonathan; Nestor-Kalinoski, Andrea; Hinds, Terry D.

    2016-01-01

    Bladder cancer is observed worldwide having been associated with a host of environmental and lifestyle risk factors. Recent investigations on anti-inflammatory glucocorticoid signaling point to a pathway that may impact bladder cancer. Here we show an inverse effect on the glucocorticoid receptor (GR) isoform signaling that may lead to bladder cancer. We found similar GRα expression levels in the transitional uroepithelial cancer cell lines T24 and UMUC-3. However, the T24 cells showed a significant (p < 0.05) increased expression of GRβ compared to UMUC-3, which also correlated with higher migration rates. Knockdown of GRβ in the T24 cells resulted in a decreased migration rate. Mutational analysis of the 3′ untranslated region (UTR) of human GRβ revealed that miR144 might positively regulate expression. Indeed, overexpression of miR144 increased GRβ by 3.8 fold. In addition, miR144 and GRβ were upregulated during migration. We used a peptide nucleic acid conjugated to a cell penetrating-peptide (Sweet-P) to block the binding site for miR144 in the 3′UTR of GRβ. Sweet-P effectively prevented miR144 actions and decreased GRβ expression, as well as the migration of the T24 human bladder cancer cells. Therefore, GRβ may have a significant role in bladder cancer, and possibly serve as a therapeutic target for the disease. PMID:27036026

  2. Cocaine downregulates beta-adrenergic receptors in pregnant sheep myometrium.

    PubMed

    Wang, F L; Gauvin, J M; Dombrowski, M P; Smith, Y R; Christopher, K A; Hurd, W W

    1996-01-01

    Cocaine abuse is associated with premature labor. Although cocaine is known to competitively inhibit beta-adrenergic receptor binding, cocaine's effect on receptor downregulation is uncertain. This study was designed to determine the in vitro effect of cocaine on downregulation of beta-adrenergic receptors in pregnant myometrium. Pregnant sheep myometrium was incubated with either cocaine, isoproterenol, or a cocaine metabolite, benzoylecgonine. Membrane fractions were assayed for beta-adrenergic receptors using (125I)-cyanopindolol and the beta 2-adrenergic antagonist ICI 118,551. We found that cocaine (10(-6) to 10(-4) mol/L), but not benzoylecgonine, downregulated both beta 1- and beta 2-adrenergic receptors, but did not further augment receptor downregulation by isoproterenol. The 46% decrease in beta-adrenergic receptors seen after exposure to cocaine was similar to the 53% decrease seen after isoproterenol. We hypothesize downregulation of beta-adrenergic receptors by cocaine may play a role in the association of cocaine abuse with premature labor.

  3. Gene Expression Control by Glucocorticoid Receptors during Innate Immune Responses

    PubMed Central

    Xavier, Andre Machado; Anunciato, Aparecida Kataryna Olimpio; Rosenstock, Tatiana Rosado; Glezer, Isaias

    2016-01-01

    Glucocorticoids (GCs) are potent anti-inflammatory compounds that have been extensively used in clinical practice for several decades. GC’s effects on inflammation are generally mediated through GC receptors (GRs). Signal transduction through these nuclear receptors leads to dramatic changes in gene expression programs in different cell types, typically due to GR binding to DNA or to transcription modulators. During the last decade, the view of GCs as exclusive anti-inflammatory molecules has been challenged. GR negative interference in pro-inflammatory gene expression was a landmark in terms of molecular mechanisms that suppress immune activity. In fact, GR can induce varied inhibitory molecules, including a negative regulator of Toll-like receptors pathway, or subject key transcription factors, such as NF-κB and AP-1, to a repressor mechanism. In contrast, the expression of some acute-phase proteins and other players of innate immunity generally requires GR signaling. Consequently, GRs must operate context-dependent inhibitory, permissive, or stimulatory effects on host defense signaling triggered by pathogens or tissue damage. This review aims to disclose how contradictory or comparable effects on inflammatory gene expression can depend on pharmacological approach (including selective GC receptor modulators; SEGRMs), cell culture, animal treatment, or transgenic strategies used as models. Although the current view of GR-signaling integrated many advances in the field, some answers to important questions remain elusive. PMID:27148162

  4. Epigenetic modulation of glucocorticoid receptors in posttraumatic stress disorder

    PubMed Central

    Labonté, B; Azoulay, N; Yerko, V; Turecki, G; Brunet, A

    2014-01-01

    Some individuals suffering from posttraumatic stress disorder (PTSD) exhibit lower basal salivary cortisol and higher glucocorticoid receptor (GR) sensitivity. Recent studies suggest that epigenetic mechanisms regulate the activity of cortisol and GR. As a means to combine and cross-validate those findings, we compared cortisol, GR expression and promoter methylation levels in peripheral T lymphocytes of healthy controls versus individuals endorsing a diagnosis of lifetime PTSD. Thirty subjects with lifetime (current or remitted) PTSD and 16 subjects never exposed to trauma were recruited. Salivary cortisol was collected at six time points over the course of a single weekday and analyzed utilizing a time-resolved fluorescence immunoassay. GR expression (GRtotal, 1B, 1C, 1F and 1H) was measured by quantitative RT-PCR. DNA methylation levels in human glucocorticoid receptor (hGR) 1B and 1C variant's promoter were quantified by epityper in T lymphocytes isolated by magnetic-assisted cell sorting. Individuals with lifetime PTSD have lower morning cortisol release, higher mRNA expression of hGRtotal, 1B, and 1C and lower overall methylation levels in hGR 1B and 1C promoters. Cortisol levels were inversely correlated with hGR 1B mRNA expression. Moreover, overall and CpG site-specific methylation levels were inversely correlated with hGRtotal and 1B mRNA expression. There was no difference between current and remitted PTSD across cortisol, GR expression mRNA and DNA methylation data. Traumatic events induce DNA methylation alterations in distinct promoters of hGR with transcriptional modifications that associate with hypoactive hypothalamus-pituitary-adrenal axis in individuals with PTSD. Our results also point toward an important role of hGR 1B variant in PTSD. PMID:24594779

  5. A naturally hypersensitive glucocorticoid receptor elicits a compensatory reduction of hypothalamus–pituitary–adrenal axis activity early in ontogeny

    PubMed Central

    Muráni, Eduard; Ponsuksili, Siriluck; Jaeger, Alexandra; Görres, Andreas; Tuchscherer, Armin; Wimmers, Klaus

    2016-01-01

    We comprehensively characterized the effects of a unique natural gain-of-function mutation in the glucocorticoid receptor (GR), GRAla610Val, in domestic pigs to expand current knowledge of the phenotypic consequences of GR hypersensitivity. Cortisol levels were consistently reduced in one-week-old piglets, at weaning and in peripubertal age, probably due to a reduced adrenal capacity to produce glucocorticoids (GC), which was indicated by an adrenocortical thinning in GRAla610Val carriers. Adrenocorticotrophic hormone (ACTH) levels were significantly reduced in one-week-old piglets only. Expression analyses in peripubertal age revealed significant downregulation of hypothalamic expression of CRH and AVP, the latter only in females, and upregulation of hepatic expression of SERPINA6, by GRAla610Val. Transcriptional repression of proinflammatory genes in peripheral blood mononuclear cells (PBMCs) from GRAla610Val carriers was more sensitive to dexamethasone treatment ex vivo. However, no significant effects on growth, body composition, blood chemistry or cell counts were observed under baseline conditions. These results suggest that GRAla610Val-induced GR hypersensitivity elicits a compensatory reduction in endogenous, bioactive glucocorticoid levels via readjustment of the hypothalamus–pituitary–adrenal (HPA) axis early in ontogeny to maintain an adequate response, but carriers are more sensitive to exogenous GC. Therefore, GRAla610Val pigs represent a valuable animal model to explore GR-mediated mechanisms of HPA axis regulation and responses to glucocorticoid-based drugs. PMID:27440422

  6. Adrenocortical LDL receptor function negatively influences glucocorticoid output.

    PubMed

    van der Sluis, Ronald J; Van Eck, Miranda; Hoekstra, Menno

    2015-09-01

    Over 50% of the cholesterol needed by adrenocortical cells for the production of glucocorticoids is derived from lipoproteins. However, the overall contribution of the different lipoproteins and associated uptake pathways to steroidogenesis remains to be determined. Here we aimed to show the importance of LDL receptor (LDLR)-mediated cholesterol acquisition for adrenal steroidogenesis in vivo. Female total body LDLR knockout mice with a human-like lipoprotein profile were bilaterally adrenalectomized and subsequently provided with one adrenal either expressing or genetically lacking the LDLR under their renal capsule to solely modulate adrenocortical LDLR function. Plasma total cholesterol levels and basal plasma corticosterone levels were identical in the two types of adrenal transplanted mice. Strikingly, restoration of adrenal LDLR function significantly reduced the ACTH-mediated stimulation of adrenal steroidogenesis (P<0.001), with plasma corticosterone levels that were respectively 44-59% lower (P<0.01) as compared to adrenal LDLR negative controls. In addition, LDLR positive adrenal transplanted mice exhibited a significant decrease (-39%; P<0.001) in their plasma corticosterone level under fasting stress conditions. Biochemical analysis did not show changes in the expression of genes involved in cholesterol mobilization. However, LDLR expressing adrenal transplants displayed a marked 62% reduction (P<0.05) in the transcript level of the key steroidogenic enzyme HSD3B2. In conclusion, our studies in a mouse model with a human-like lipoprotein profile provide the first in vivo evidence for a novel inhibitory role of the LDLR in the control of adrenal glucocorticoid production. PMID:26136384

  7. Cancer cell-selective promoter recognition accompanies antitumor effect by glucocorticoid receptor-targeted gold nanoparticle

    NASA Astrophysics Data System (ADS)

    Sau, Samaresh; Agarwalla, Pritha; Mukherjee, Sudip; Bag, Indira; Sreedhar, Bojja; Pal-Bhadra, Manika; Patra, Chitta Ranjan; Banerjee, Rajkumar

    2014-05-01

    Nanoparticles, such as gold nanoparticles (GNP), upon convenient modifications perform multi tasks catering to many biomedical applications. However, GNP or any other type of nanoparticles is yet to achieve the feat of intracellular regulation of endogenous genes of choice such as through manipulation of a gene-promoter in a chromosome. As for gene modulation and delivery, GNP (or other nanoparticles) showed only limited gene therapy potential, which relied on the delivery of `exogenous' genes invoking gene knockdown or replacement. Practically, there are no instances for the nanoparticle-mediated promoter regulation of `endogenous' genes, more so, as a cancer selective phenomenon. In this regard, we report the development of a simple, easily modifiable GNP-formulation, which promoted/up-regulated the expression of a specific category of `endogenous' genes, the glucocorticoid responsive genes. This genetic up-regulation was induced in only cancer cells by modified GNP-mediated transcriptional activation of its cytoplasmic receptor, glucocorticoid receptor (GR). Normal cells and their GR remained primarily unperturbed by this GNP-formulation. The most potent gene up-regulating GNP-formulation down-regulated a cancer-specific proliferative signal, phospho-Akt in cancer cells, which accompanied retardation of tumor growth in the murine melanoma model. We show that GR-targeted GNPs may find potential use in the targeting and modulation of genetic information in cancer towards developing novel anticancer therapeutics.Nanoparticles, such as gold nanoparticles (GNP), upon convenient modifications perform multi tasks catering to many biomedical applications. However, GNP or any other type of nanoparticles is yet to achieve the feat of intracellular regulation of endogenous genes of choice such as through manipulation of a gene-promoter in a chromosome. As for gene modulation and delivery, GNP (or other nanoparticles) showed only limited gene therapy potential, which relied

  8. Functional interaction between the glucocorticoid receptor and GANP/MCM3AP

    SciTech Connect

    Osman, Waffa; Laine, Sanna; Zilliacus, Johanna . E-mail: johanna.zilliacus@mednut.ki.se

    2006-10-06

    Glucocorticoids are widely used to treat inflammatory diseases but have a number of side effects that partly are connected to inhibition of cell proliferation. Glucocorticoids mediated their action by binding to the glucocorticoid receptor. In the present study, we have identified by two-hybrid screens the germinal center-associated protein (GANP) and MCM3-associated protein (MCM3AP), a splicing variant of GANP, as glucocorticoid receptor interacting proteins. GANP and MCM3AP can bind to the MCM3 protein involved in initiation of DNA replication. Glutathione-S-transferase-pull-down and co-immunoprecipitation assays showed that the C-terminal domain of GANP, encompassing MCM3AP, interacts with the ligand-binding domain of the glucocorticoid receptor. Characterization of the intracellular localization of GANP revealed that GANP is shuttling between the nucleus and the cytoplasm. Furthermore, we show that glucocorticoids are unable to inhibit DNA replication in HeLa cells overexpressing MCM3AP suggesting a role for both glucocorticoid receptor and GANP/MCM3AP in regulating cell proliferation.

  9. Cell-specific expression of the glucocorticoid receptor within granular convoluted tubules of the rat submaxillary gland

    SciTech Connect

    Antakly, T.; Zhang, C.X.; Sarrieau, A.; Raquidan, D. )

    1991-01-01

    The submaxillary gland, a heterogeneous tissue composed essentially of two functionally distinct cell types (tubular epithelial and acinar), offers an interesting system in which to study the mechanisms of steroid-dependent growth and differentiation. One cell type, the granular convoluted tubular (GCT) cell, secretes a large number of physiologically important polypeptides, including epidermal and nerve growth factors. Two steroids, androgens and glucocorticoids, greatly influence the growth, differentiation, and secretory activity of GCT cells. Because glucocorticoids can partially mimic or potentiate androgen effects, it has been thought that glucocorticoids act via androgen receptors. Since the presence of glucocorticoid receptors is a prerequisite for glucocorticoid action, we have investigated the presence and cellular distribution of glucocorticoid receptors within the rat submaxillary gland. Binding experiments using (3H)dexamethasone revealed the presence of high affinity binding sites in rat submaxillary tissue homogenates. Most of these sites were specifically competed by dexamethasone, corticosterone, and a pure glucocorticoid agonist RU 28362. Neither testosterone nor dihydrotestosterone competed for glucocorticoid binding. The cellular distribution of glucocorticoid receptors within the submaxillary gland was investigated by immunocytochemistry, using two highly specific glucocorticoid receptor antibodies. The receptor was localized in the GCT cells, but not in the acinar cells of rat and mouse submaxillary tissue sections. In GCT cells, the glucocorticoid receptor colocalized with several secretory polypeptides, including epidermal growth factor, nerve growth factor, alpha 2u-globulin, and atrial natriuretic factor.

  10. Down-regulation of rat mitochondrial branched-chain 2-oxoacid dehydrogenase kinase gene expression by glucocorticoids.

    PubMed Central

    Huang, Y S; Chuang, D T

    1999-01-01

    The mammalian mitochondrial branched-chain 2-oxoacid dehydrogenase (BCOD) complex is regulated by a reversible phosphorylation (inactivation)/dephosphorylation (activation) cycle. In the present study, the effects of glucocorticoids on the level of BCOD kinase mRNA were investigated in rat hepatoma cell lines (H4IIE and FTO-2B), as well as in the rat. In H4IIE cells, dexamethasone was found to significantly reduce steady-state concentrations of BCOD kinase mRNA after a 48 h culture, and this was correlated with a 2-fold increase in the dephosphorylated form of the BCOD complex. The half-life of the kinase mRNA in H4IIE cells was not affected by dexamethasone treatment. Therefore, the decrease in the steady-state kinase mRNA level resulting from dexamethasone treatment was not caused by changes in mRNA stability, which raised the possibility of regulation at the level of gene transcription. To identify the negative glucocorticoid-responsive element in the kinase promoter, nested deletion constucts in the 3.0 kb promoter region were examined in H4IIE cells cultured in the presence or absence of dexamethasone. No significant differences in promoter activity were observed on either transient or stable transfection. The data showed that the glucocorticoid-responsive element was located outside the 3. 0 kb promoter region. At the physiological level, hepatic BCOD kinase mRNA levels were reduced in rats injected intraperitoneally with dexamethasone. This effect was liver-specific, and was not detected in other tissues. These results suggest that the down-regulation of kinase gene expression by glucocorticoids is mediated through a liver-specific or -enriched transcription factor(s). PMID:10215586

  11. Liver X Receptors Regulate the Transcriptional Activity of the Glucocorticoid Receptor: Implications for the Carbohydrate Metabolism

    PubMed Central

    Nader, Nancy; Ng, Sinnie Sin Man; Wang, Yonghong; Abel, Brent S.; Chrousos, George P.; Kino, Tomoshige

    2012-01-01

    GLUCOCORTICOIDS are steroid hormones that strongly influence intermediary carbohydrate metabolism by increasing the transcription rate of glucose-6-phosphatase (G6Pase), a key enzyme of gluconeogenesis, and suppress the immune system through the glucocorticoid receptor (GR). The liver X receptors (LXRs), on the other hand, bind to cholesterol metabolites, heterodimerize with the retinoid X receptor (RXR), and regulate the cholesterol turnover, the hepatic glucose metabolism by decreasing the expression of G6Pase, and repress a set of inflammatory genes in immune cells. Since the actions of these receptors overlap with each other, we evaluated the crosstalk between the GR- and LXR-mediated signaling systems. Transient transfection-based reporter assays and gene silencing methods using siRNAs for LXRs showed that overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of certain glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion. Activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats. It also suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells, whereas endogenous, unliganded LXRs were required for dexamethasone-induced mRNA expression of phosphoenolpyruvate carboxylase. In microarray transcriptomic analysis of rat liver, GW3965 differentially regulated glucocorticoid-induced transcriptional activity of about 15% of endogenous glucocorticoid-responsive genes. To examine the mechanism through which activated LXRs attenuated GR transcriptional activity, we examined LXRα/RXRα binding to GREs. Endogenous LXRα/RXRα bound GREs and inhibited GR binding to these DNA sequences both in in vitro and in vivo chromatin immunoprecipitation assays, while their recombinant proteins did so on classic or G6Pase GREs in gel mobility shift assays. We propose that administration of

  12. RSUME Enhances Glucocorticoid Receptor SUMOylation and Transcriptional Activity

    PubMed Central

    Druker, Jimena; Liberman, Ana C.; Antunica-Noguerol, María; Gerez, Juan; Paez-Pereda, Marcelo; Rein, Theo; Iñiguez-Lluhí, Jorge A.; Holsboer, Florian

    2013-01-01

    Glucocorticoid receptor (GR) activity is modulated by posttranslational modifications, including phosphorylation, ubiquitination, and SUMOylation. The GR has three SUMOylation sites: lysine 297 (K297) and K313 in the N-terminal domain (NTD) and K721 within the ligand-binding domain. SUMOylation of the NTD sites mediates the negative effect of the synergy control motifs of GR on promoters with closely spaced GR binding sites. There is scarce evidence on the role of SUMO conjugation to K721 and its impact on GR transcriptional activity. We have previously shown that RSUME (RWD-containing SUMOylation enhancer) increases protein SUMOylation. We now demonstrate that RSUME interacts with the GR and increases its SUMOylation. RSUME regulates GR transcriptional activity and the expression of its endogenous target genes, FKBP51 and S100P. RSUME uncovers a positive role for the third SUMOylation site, K721, on GR-mediated transcription, demonstrating that GR SUMOylation acts positively in the presence of a SUMOylation enhancer. Both mutation of K721 and small interfering RNA-mediated RSUME knockdown diminish GRIP1 coactivator activity. RSUME, whose expression is induced under stress conditions, is a key factor in heat shock-induced GR SUMOylation. These results show that inhibitory and stimulatory SUMO sites are present in the GR and at higher SUMOylation levels the stimulatory one becomes dominant. PMID:23508108

  13. Correlations between the activities of DNA polymerase alpha and the glucocorticoid receptor.

    PubMed Central

    Schmidt, T J; Bollum, F J; Litwack, G

    1982-01-01

    Specific inhibitors and anti-DNA polymerase alpha IgG have been utilized to probe for similarities between cytoplasmic rat hepatic glucocorticoid receptors and DNA polymerase alpha [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7]. Rifamycin AF/013, an inhibitor of RNA and DNA polymerase activities, significantly inhibited the binding of activated [6,7-3H]-triamcinolone acetonide (TA) receptor complexes to DNA-cellulose. beta-Lapachone, an inhibitor of DNA polymerase alpha and reverse transcriptase activities, inhibited the specific binding of [6,7-3H]TA when preincubated with unbound receptors. Aphidicolin, another DNA polymerase alpha inhibitor, failed to inhibit any of the glucocorticoid-receptor functions tested. Two specific anti-DNA polymerase alpha IgGs interfered with glucocorticoid receptor functions as measured by their ability to inhibit the binding of [6,7-3H]TA to unbound receptors (85% maximal inhibition) and, to a lesser extent, to inhibit the binding of activated [6,7-3H]TA receptor complexes to DNA-cellulose (50% maximal inhibition). The anti-DNA polymerase alpha IgG and beta-lapachone failed to affect the binding of tritiated estradiol, progesterone, or 5 alpha-dihydrotestosterone to their receptors in appropriate rat target tissues or the binding of [1,2-3H]hydrocortisone to serum transcortin. The most obvious interpretation of these data is that cytoplasmic glucocorticoid receptors and DNA polymerase alpha share antigenic determinants. An alternative interpretation is that the polyclonal anti-DNA polymerase alpha antibody contains IgG molecules raised against calf thymus cytoplasmic activated glucocorticoid-receptor complexes that copurified with DNA polymerase alpha used as the antigen. Taken collectively, however, the antibody and inhibitor data suggest a relationship between DNA polymerase alpha and the glucocorticoid receptor. PMID:6812051

  14. Monoclonal antibodies against the rat liver glucocorticoid receptor.

    PubMed Central

    Okret, S; Wikström, A C; Wrange, O; Andersson, B; Gustafsson, J A

    1984-01-01

    Splenic cells from one BALB/c mouse and one C57/BL mouse, immunized with purified rat liver glucocorticoid receptor (GR), were fused with the mouse myeloma cell line Sp 2/0-Ag 14. Screening for production of anti-GR-antibodies by the hybridomas was carried out with an enzyme-linked immunosorbent assay, using partially purified rat liver GR as antigen. Further screening was by a second-antibody immunoprecipitation assay using [3H]triamcinolone acetonide-GR complex from rat liver cytosol as tracer. Hybridomas from 10 different microplate wells, positive in both assays, were successfully cloned by the limiting dilution method to monoclonality. The different origins of the monoclonal antibodies were confirmed by their various isoelectric points when analyzed by isoelectric focusing. Four of the monoclonal hybridoma cell lines secreted IgM antibodies; two, IgG1; three, IgG2a; and one, IgG2b. The GR-antibody complex was identified in glycerol density gradients by a shift of the 4S GR to an 8.5S or 19S GR-antibody complex when incubated with monoclonal IgG or IgM antibody, respectively. The 10 monoclonal antibodies recognized different determinants on the GR, all situated on that domain of the receptor that is separate from the ligand and DNA-binding domains. Also, the cross-reactivity to the mouse liver GR varied among the monoclonal antibodies. No cross-reactivity was observed to the human lymphocytic GR. NaDodSO4 electrophoresis of a 0.5% pure GR preparation followed by immunoblotting using one of the monoclonal antibodies identified a single peptide with a molecular weight of 94,000, identical to the purified rat liver GR. Images PMID:6200880

  15. Competitive inhibition of (TH)dexamethasone binding to mammary glucocorticoid receptor by leupeptin

    SciTech Connect

    Hsieh, L.C.C.; Su, C.; Markland, F.S. Jr.

    1987-03-01

    The inhibitory effect of leupeptin on (TH)dexamethasone binding to the glucocorticoid receptor from lactating goat mammary cytosol has been studied. Leupeptin (10 mM) caused a significant (about 35%) inhibition of (TH)dexamethasone binding to glucocorticoid receptor. Binding inhibition is further increased following filtration of unlabeled cytosolic receptor through a Bio-Gel A 0.5-m column. Binding inhibition was partially reversed by monothioglycerol at 10 mM concentration. A double reciprocal plot revealed that leupeptin appears to be a competitive inhibitor of (TH)dexamethasone binding to the glucocorticoid receptor. Low salt sucrose density gradient centrifugation revealed that the leupeptin-treated sample formed a slightly larger (approximately 9 S) receptor complex (leupeptin-free complex sediments at 8 S).

  16. Seasonal changes and regulation of the glucocorticoid receptor in the testis of the toad Rhinella arenarum.

    PubMed

    Regueira, Eleonora; Sassone, Alina Grisel; Scaia, María Florencia; Volonteri, María Clara; Ceballos, Nora Raquel

    2013-01-01

    Several studies indicate that wild free-living vertebrates seasonally regulate plasma glucocorticoids. However, not only glucocorticoids but also the amount of receptors is important in determining biological responses. In this context, seasonal regulation of glucocorticoid receptor (GR) is crucial to modulate the response to glucocorticoids. Rhinella arenarum is an anuran exhibiting seasonal variations in plasma glucocorticoids and also in the number of binding sites (B(max)) of the testicular cytosolic GR. In this work, we evaluated if the annual pattern of GR protein in the testis varies seasonally and, by an in vitro approach, the role of glucocorticoids, androgens, and melatonin in the regulation of the GR B(max) and protein level. For this purpose, testes were treated with two physiological concentrations of melatonin (40 and 200 pg/ml), with or without luzindole (melatonin-receptor antagonist); with testosterone, cyanoketone (inhibitor of steroidogenesis) or casodex (androgen-receptor antagonist); or with dexamethasone or RU486 (GR antagonist). After treatments, B(max) and protein level were determined by the binding of [(3)H]dexamethasone and Western blot, respectively. Results showed that GR protein decreases in the winter. The in vitro treatment with melatonin produced a biphasic effect on the B(max) with the lowest concentration decreasing this parameter by a receptor-mediated mechanism. However, melatonin had no effect on the GR protein level. Conversely, a high concentration of dexamethasone up-regulated the GR protein and androgens neither changed the B(max) nor the protein level. These findings suggest that seasonal changes in plasma melatonin and glucocorticoids modulate the effect of glucocorticoids in the testis of R. arenarum. PMID:23203422

  17. The transrepression arm of glucocorticoid receptor signaling is protective in mutant huntingtin-mediated neurodegeneration.

    PubMed

    Varadarajan, S; Breda, C; Smalley, J L; Butterworth, M; Farrow, S N; Giorgini, F; Cohen, G M

    2015-08-01

    The unfolded protein response (UPR) occurs following the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and orchestrates an intricate balance between its prosurvival and apoptotic arms to restore cellular homeostasis and integrity. However, in certain neurodegenerative diseases, the apoptotic arm of the UPR is enhanced, resulting in excessive neuronal cell death and disease progression, both of which can be overcome by modulating the UPR. Here, we describe a novel crosstalk between glucocorticoid receptor signaling and the apoptotic arm of the UPR, thus highlighting the potential of glucocorticoid therapy in treating neurodegenerative diseases. Several glucocorticoids, but not mineralocorticoids, selectively antagonize ER stress-induced apoptosis in a manner that is downstream of and/or independent of the conventional UPR pathways. Using GRT10, a novel selective pharmacological modulator of glucocorticoid signaling, we describe the importance of the transrepression arm of the glucocorticoid signaling pathway in protection against ER stress-induced apoptosis. Furthermore, we also observe the protective effects of glucocorticoids in vivo in a Drosophila model of Huntington's disease (HD), wherein treatment with different glucocorticoids diminished rhabdomere loss and conferred neuroprotection. Finally, we find that growth differentiation factor 15 has an important role downstream of glucocorticoid signaling in antagonizing ER stress-induced apoptosis in cells, as well as in preventing HD-mediated neurodegeneration in flies. Thus, our studies demonstrate that this novel crosstalk has the potential to be effectively exploited in alleviating several neurodegenerative disorders.

  18. Hormonal regulation of type II glucocorticoid receptor messenger ribonucleic acid in rat brain.

    PubMed

    Peiffer, A; Lapointe, B; Barden, N

    1991-10-01

    Differences in the regulation of type II glucocorticoid receptor (GR) mRNA levels in female rat brain regions involved in the control of the hypothalamic-pituitary-adrenal axis were studied by Northern blot analysis after chronic administration of corticosterone or dexamethasone to adrenalectomized (ADX), ovariectomized (OVX), and ADX/OVX animals. The effect of chronic estradiol or progesterone treatment of intact animals was also studied. Our results show that type II GR mRNA levels of ADX animals were significantly increased above control values in amygdala (140%) and hippocampus (196%), but not in hypothalamus. These increased transcript levels were down-regulated by corticosterone or dexamethasone, with the exception of those in the amygdala, where corticosterone had no effect. Ovariectomy significantly increased hypothalamic GR mRNA content (174%) over control values, and this increase was sensitive to dexamethasone. The combined effect of adrenalectomy/ovariectomy on GR mRNA levels was greater than that of adrenalectomy only in amygdala. Corticosterone increased amygdala transcript levels in OVX and ADX/OVX animals. Estradiol administration to intact animals raised the GR mRNA content of amygdala, while progesterone treatment had no effect on any of the brain regions studied. We conclude that there exists heterogeneity with respect to type II GR mRNA regulation by corticosterone and dexamethasone in brain regions of ADX female rats, and that certain limbic structures show greater sensitivity to these hormonal manipulations, suggesting a more prominent role in the regulation of the hypothalamic-pituitary-adrenal axis. Our results also suggest that circulating estrogens can influence the sensitivity of brain structures (i.e. hypothalamus and amygdala) to glucocorticoids by altering GR mRNA levels. These regions may represent integration sites at which gonadal steroids are able to alter stress hormone secretion.

  19. DHEA modulates the effect of cortisol on RACK1 expression via interference with the splicing of the glucocorticoid receptor

    PubMed Central

    Pinto, Antonella; Malacrida, Beatrice; Oieni, Jacopo; Serafini, Melania Maria; Davin, Annalisa; Galbiati, Valentina; Corsini, Emanuela; Racchi, Marco

    2015-01-01

    Background and Purpose Dehydroepiandrosterone (DHEA) is thought to be an anti-glucocorticoid hormone known to be fully functional in young people but deficient in aged humans. Our previous data suggest that DHEA not only counteracts the effect of cortisol on RACK1 expression, a protein required both for the correct functioning of immune cells and for PKC-dependent pathway activation, but also modulates the inhibitory effect of cortisol on LPS-induced cytokine production. The purpose of this study was to investigate the effect of DHEA on the splicing mechanism of the human glucocorticoid receptor (GR). Experimental Approach The THP1 monocytic cell line was used as a cellular model. Cytokine production was measured by specific elisa. Western blot and real-time RT-PCR were used, where appropriate, to determine the effect of DHEA on GRs, serine/arginine-rich proteins (SRp), and RACK1 protein and mRNA. Small-interfering RNA was used to down-regulate GRβ. Key Results DHEA induced a dose-related up-regulation of GRβ and GRβ knockdown completely prevented DHEA-induced RACK1 expression and modulation of cytokine release. Moreover, we showed that DHEA influenced the expression of some components of the SRps found within the spliceosome, the main regulators of the alternative splicing of the GR gene. Conclusions and Implications These data contribute to our understanding of the mechanism of action of DHEA and its effect on the immune system and as an anti-glucocorticoid agent. PMID:25626076

  20. Specific regulation of male rat liver cytosolic estrogen receptor by the modulator of the glucocorticoid receptor.

    PubMed

    Celiker, M Y; Haas, A; Saunders, D; Litwack, G

    1993-08-31

    Modulator is a novel low-molecular-weight organic compound that regulates activities of glucocorticoid and mineralocorticoid receptors as well as protein kinase C. In this study we show that male rat liver cytosolic estrogen receptor activation is inhibited by modulator in a dose-dependent manner. Fifty percent inhibition is obtained with 1 unit/ml modulator purified from bovine liver which is within the physiological concentration for modulator. However, sheep uterine cytosolic estrogen and androgen receptors are insensitive to regulation by modulator. Exogenous sodium molybdate treatment inhibits activation of all of these receptors of liver or uterus origin in an identical manner, further differentiating the effects of modulator and the molybdate anion. PMID:8363596

  1. Glucocorticoid receptor signaling in breast and prostate cancers: emergence as a therapeutic target

    PubMed Central

    Kach, Jacob; Conzen, Suzanne D; Szmulewitz, Russell Z

    2016-01-01

    Steroid receptors for androgens and estrogens have essential roles in prostate and breast cancers. Recently, glucocorticoid receptor (GR) activity has also been proposed as having an important role in these cancers. Underscoring the cooperative nature of nuclear receptor activity, data now suggest that GR function in prostate and breast cancers is dependent on the tumor’s concomitant androgen or estrogen receptor activity. PMID:26378243

  2. Glucocorticoid receptor-dependent inhibition of cellular proliferation in dexamethasone-resistant and hypersensitive rat hepatoma cell variants.

    PubMed Central

    Cook, P W; Swanson, K T; Edwards, C P; Firestone, G L

    1988-01-01

    Exposure of the Fu5 rat hepatoma cell line to glucocorticoids, such as dexamethasone and hydrocortisone, suppressed the growth rate and final density of cells grown in the presence of serum. This hormonal effect was proportional to receptor occupancy and affinity and, in addition, the glucocorticoid antagonist RU38486 prevented this response. Two classes of dexamethasone-resistant variants that failed to be growth inhibited were recovered from ethyl methylsulfonate-mutagenized populations by continuous culture in the presence of 1 microM dexamethasone. The first class, represented by the EDR3 subclone, was completely glucocorticoid unresponsive and failed to express receptor transcripts. The second class, represented by the EDR1, EDR5, and EDR7 subclones, possessed significant levels of glucocorticoid receptor but were only partially glucocorticoid responsive when stimulated with saturating levels of hormone. Introduction of functional glucocorticoid receptor genes into both classes of dexamethasone-resistant variants by a recombinant retrovirus expression vector restored glucocorticoid responsiveness and suppression of cell growth. A hypersensitive variant (BDS1), recovered by bromodeoxyuridine selection, was fully glucocorticoid responsive, and its inhibition of proliferation was more acutely regulated by dexamethasone. Taken together, our results established that the inhibition of proliferation in Fu5 rat hepatoma cells represents a new glucocorticoid response that requires the expression of a functional glucocorticoid receptor. Images PMID:3380086

  3. Down-regulated peroxisome proliferator-activated receptor γ (PPARγ) in lung epithelial cells promotes a PPARγ agonist-reversible proinflammatory phenotype in chronic obstructive pulmonary disease (COPD).

    PubMed

    Lakshmi, Sowmya P; Reddy, Aravind T; Zhang, Yingze; Sciurba, Frank C; Mallampalli, Rama K; Duncan, Steven R; Reddy, Raju C

    2014-03-01

    Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory condition and a leading cause of death, with no available cure. We assessed the actions in pulmonary epithelial cells of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear hormone receptor with anti-inflammatory effects, whose role in COPD is largely unknown. We found that PPARγ was down-regulated in lung tissue and epithelial cells of COPD patients, via both reduced expression and phosphorylation-mediated inhibition, whereas pro-inflammatory nuclear factor-κB (NF-κB) activity was increased. Cigarette smoking is the main risk factor for COPD, and exposing airway epithelial cells to cigarette smoke extract (CSE) likewise down-regulated PPARγ and activated NF-κB. CSE also down-regulated and post-translationally inhibited the glucocorticoid receptor (GR-α) and histone deacetylase 2 (HDAC2), a corepressor important for glucocorticoid action and whose down-regulation is thought to cause glucocorticoid insensitivity in COPD. Treating epithelial cells with synthetic (rosiglitazone) or endogenous (10-nitro-oleic acid) PPARγ agonists strongly up-regulated PPARγ expression and activity, suppressed CSE-induced production and secretion of inflammatory cytokines, and reversed its activation of NF-κB by inhibiting the IκB kinase pathway and by promoting direct inhibitory binding of PPARγ to NF-κB. In contrast, PPARγ knockdown via siRNA augmented CSE-induced chemokine release and decreases in HDAC activity, suggesting a potential anti-inflammatory role of endogenous PPARγ. The results imply that down-regulation of pulmonary epithelial PPARγ by cigarette smoke promotes inflammatory pathways and diminishes glucocorticoid responsiveness, thereby contributing to COPD pathogenesis, and further suggest that PPARγ agonists may be useful for COPD treatment.

  4. Overexpression of Glucocorticoid Receptor β Enhances Myogenesis and Reduces Catabolic Gene Expression.

    PubMed

    Hinds, Terry D; Peck, Bailey; Shek, Evan; Stroup, Steven; Hinson, Jennifer; Arthur, Susan; Marino, Joseph S

    2016-01-01

    Unlike the glucocorticoid receptor α (GRα), GR β (GRβ) has a truncated ligand-binding domain that prevents glucocorticoid binding, implicating GRα as the mediator of glucocorticoid-induced skeletal muscle loss. Because GRβ causes glucocorticoid resistance, targeting GRβ may be beneficial in impairing muscle loss as a result of GRα activity. The purpose of this study was to determine how the overexpression of GRβ affects myotube formation and dexamethasone (Dex) responsiveness. We measured GR isoform expression in C₂C12 muscle cells in response to Dex and insulin, and through four days of myotube formation. Next, lentiviral-mediated overexpression of GRβ in C₂C12 was performed, and these cells were characterized for cell fusion and myotube formation, as well as sensitivity to Dex via the expression of ubiquitin ligases. GRβ overexpression increased mRNA levels of muscle regulatory factors and enhanced proliferation in myoblasts. GRβ overexpressing myotubes had an increased fusion index. Myotubes overexpressing GRβ had lower forkhead box O3 (Foxo3a) mRNA levels and a blunted muscle atrophy F-box/Atrogen-1 (MAFbx) and muscle ring finger 1 (MuRF1) response to Dex. We showed that GRβ may serve as a pharmacological target for skeletal muscle growth and protection from glucocorticoid-induced catabolic signaling. Increasing GRβ levels in skeletal muscle may cause a state of glucocorticoid resistance, stabilizing muscle mass during exposure to high doses of glucocorticoids. PMID:26875982

  5. Overexpression of Glucocorticoid Receptor β Enhances Myogenesis and Reduces Catabolic Gene Expression

    PubMed Central

    Hinds, Terry D.; Peck, Bailey; Shek, Evan; Stroup, Steven; Hinson, Jennifer; Arthur, Susan; Marino, Joseph S.

    2016-01-01

    Unlike the glucocorticoid receptor α (GRα), GR β (GRβ) has a truncated ligand-binding domain that prevents glucocorticoid binding, implicating GRα as the mediator of glucocorticoid-induced skeletal muscle loss. Because GRβ causes glucocorticoid resistance, targeting GRβ may be beneficial in impairing muscle loss as a result of GRα activity. The purpose of this study was to determine how the overexpression of GRβ affects myotube formation and dexamethasone (Dex) responsiveness. We measured GR isoform expression in C2C12 muscle cells in response to Dex and insulin, and through four days of myotube formation. Next, lentiviral-mediated overexpression of GRβ in C2C12 was performed, and these cells were characterized for cell fusion and myotube formation, as well as sensitivity to Dex via the expression of ubiquitin ligases. GRβ overexpression increased mRNA levels of muscle regulatory factors and enhanced proliferation in myoblasts. GRβ overexpressing myotubes had an increased fusion index. Myotubes overexpressing GRβ had lower forkhead box O3 (Foxo3a) mRNA levels and a blunted muscle atrophy F-box/Atrogen-1 (MAFbx) and muscle ring finger 1 (MuRF1) response to Dex. We showed that GRβ may serve as a pharmacological target for skeletal muscle growth and protection from glucocorticoid-induced catabolic signaling. Increasing GRβ levels in skeletal muscle may cause a state of glucocorticoid resistance, stabilizing muscle mass during exposure to high doses of glucocorticoids. PMID:26875982

  6. [Gastroprotective action of corticotropin-releasing factor (CRF): involvement of glucocorticoids and CRF receptors type 2].

    PubMed

    Filaretova, L P; Bagaeva, T R; Morozova, O Iu

    2012-12-01

    The stress response involves the activation of two corticotropin-releasing factor (CRF) receptors types 1 and 2. The pituitary type 1 CRF receptors represent the primary receptors to activate the hypothalamic-pituitary-adrenocortical axis and, consequently, glucocorticoid production. Exogenous CRF induces an increase in glucocorticoid production and may protect the gastric mucosa against stress-induced injury. Here we examined contribution of glucocorticoids and CRF receptors type 2 to gastroprotective effect of exogenous CRF. Gastric injury was induced by 3 him-mobilization (at 10 degrees C) in conscious rats or 3.5 h gastric ischemia-reperfusion in anaesthetized rats. Intraperitoneal administration of CRF at the doses of 1.25 or 2.5 Mg/kg increased plasma corticosterone levels and suppressed the occurrence of gastric erosion induced by each stimulus. Metyrapone injected before CRF caused an inhibition of CRF-induced corticosterone response and prevented the protective effect of CRF on the gastric mucosa against erosion caused by immobilization (at 10 degrees C). However, metyrapone injection did not influence the protective effect of CRF on the gastric mucosa against ischemia-reperfusion-induced lesion. The protective effect of CRF on the gastric mucosa against ischemia-reperfusion-induced lesion was prevented by the nonselective CRF receptor antagonist astressin and selective type 2 CRF receptor antagonist astressin2-B. The results obtained suggest that exogenous CRF may protect the gastric mucosa against injury through involvement of glucocorticoids and also through CRF receptors type 2.

  7. Stress-induced sex differences: adaptations mediated by the glucocorticoid receptor.

    PubMed

    Bourke, Chase H; Harrell, Constance S; Neigh, Gretchen N

    2012-08-01

    Clinical evidence has indicated that women are more susceptible to stress-related and autoimmune disorders than men. Although females may be more susceptible to some disease states, males do not escape unscathed and are more susceptible to metabolic dysfunction. The hypothalamic-pituitary-axis plays a pivotal role in the sexually dimorphic effects of chronic stress through alterations in negative feedback. Recent evidence has implicated the glucocorticoid receptor and its co-chaperones in the etiology of psychiatric and somatic diseases. Gonadal hormones heavily interact with both glucocorticoid receptor expression and glucocorticoid receptor action either through direct or indirect effects on proteins in the chaperone and co-chaperone complex. Diverse systems including the hypothalamic-pituitary-axis, the immune system, and metabolism are affected differently in males and females, possibly through the glucocorticoid receptor system. New considerations of glucocorticoid regulation through the co-chaperone complex in the brain will be vital to the development of treatment strategies for men and women afflicted by neuropsychiatric and somatic disorders.

  8. Effect of cAMP signaling on expression of glucocorticoid receptor, Bim and Bad in glucocorticoid-sensitive and resistant leukemic and multiple myeloma cells.

    PubMed

    Dong, Hongli; Carlton, Michael E; Lerner, Adam; Epstein, Paul M

    2015-01-01

    Stimulation of cAMP signaling induces apoptosis in glucocorticoid-sensitive and resistant CEM leukemic and MM.1 multiple myeloma cell lines, and this effect is enhanced by dexamethasone in both glucocorticoid-sensitive cell types and in glucocorticoid-resistant CEM cells. Expression of the mRNA for the glucocorticoid receptor alpha (GR) promoters 1A3, 1B and 1C, expression of mRNA and protein for GR, and the BH3-only proapoptotic proteins, Bim and Bad, and the phosphorylation state of Bad were examined following stimulation of the cAMP and glucocorticoid signaling pathways. Expression levels of GR promoters were increased by cAMP and glucocorticoid signaling, but GR protein expression was little changed in CEM and decreased in MM.1 cells. Stimulation of these two signaling pathways induced Bim in CEM cells, induced Bad in MM.1 cells, and activated Bad, as indicated by its dephosphorylation on ser112, in both cell types. This study shows that leukemic and multiple myeloma cells, including those resistant to glucocorticoids, can be induced to undergo apoptosis by stimulating the cAMP signaling pathway, with enhancement by glucocorticoids, and the mechanism by which this occurs may be related to changes in Bim and Bad expression, and in all cases, to activation of Bad.

  9. A Natural Mutation in Helix 5 of the Ligand Binding Domain of Glucocorticoid Receptor Enhances Receptor-Ligand Interaction

    PubMed Central

    Reyer, Henry; Ponsuksili, Siriluck; Kanitz, Ellen; Pöhland, Ralf; Wimmers, Klaus; Murani, Eduard

    2016-01-01

    The glucocorticoid receptor (GR) is a central player in the neuroendocrine stress response; it mediates feedback regulation of the hypothalamus-pituitary-adrenal (HPA) axis and physiological actions of glucocorticoids in the periphery. Despite intensive investigations of GR in the context of receptor-ligand interaction, only recently the first naturally occurring gain-of-function substitution, Ala610Val, of the ligand binding domain was identified in mammals. We showed that this mutation underlies a major quantitative trait locus for HPA axis activity in pigs, reducing cortisol production by about 40–50 percent. To unravel the molecular mechanisms behind this gain of function, receptor-ligand interactions were evaluated in silico, in vitro and in vivo. In accordance with previously observed phenotypic effects, the mutant Val610 GR showed significantly increased activation in response to glucocorticoid and non-glucocorticoid steroids, and, as revealed by GR-binding studies in vitro and in pituitary glands, enhanced ligand binding. Concordantly, the protein structure prediction depicted reduced binding distances between the receptor and ligand, and altered interactions in the ligand binding pocket. Consequently, the Ala610Val substitution opens up new structural information for the design of potent GR ligands and to examine effects of the enhanced GR responsiveness to glucocorticoids on the entire organism. PMID:27736993

  10. General effect of endotoxin on glucocorticoid receptors in mammalian tissues

    SciTech Connect

    Stith, R.D.; McCallum, R.E.

    1986-01-01

    Considering the ubiquitous nature of glucocorticoid actions and the fact that endotoxin inhibits glucocorticoid action in the liver, we proposed to examine whether endotoxin affected extrahepatic actions of glucocorticoids. Fasted C57BL/6J mice were injected intraperitoneally with endotoxin (LD50) at 0800 and were killed 6 h later. Control mice were injected with an equal volume of saline. /sup 3/H-dexamethasone binding, measured by a new cytosol exchange assay utilizing molybdate plus dithiothreitol, in liver, kidney, skeletal muscle, spleen, lung, and heart tissue was significantly lower in treated than in control mice. The equilibrium dissociation constants were not significantly different, but the number of available binding sites in each tissue was reduced by endotoxin treatment. Phosphoenolpyruvate carboxykinase activity was significantly reduced in liver but not in kidney. Endotoxin treatment lowered glycogen content in liver but not in skeletal muscle. The reduction observed in the a form of liver glycogen synthase due to endotoxin was not seen in skeletal muscle glycogen synthase a. These data support the proposal that endotoxin or a mediator of its action inhibits systemic glucocorticoid action. The results also emphasize the central role of the liver in the metabolic disturbances of the endotoxin-treated mouse.

  11. Ligand-independent activation of the glucocorticoid receptor by ursodeoxycholic acid: Repression of IFN-{gamma}-induced MHC class II gene expression via a glucocorticoid receptor-dependent pathway

    SciTech Connect

    Tanaka, Hirotoshi; Makino, Yuichi; Miura, Takanori

    1996-02-15

    The therapeutic effectiveness of ursodeoxycholic acid (UDCA) for various autoimmune liver diseases strongly indicates that UDCA possesses immunomodulatory activities. Experimental evidence also supports this notion, since, for example, UDCA has been shown to suppress secretion of IL-2, IL-4, and IFN-{gamma} from activated T lymphocytes, and Ig production from B lymphocytes. To investigate the mechanical background of UDCA-mediated immunomodulation, we asked whether UDCA interacts with the intracellular signal transduction pathway, especially whether it is involved in immunosuppressive glucocorticoid hormone action. For this purpose, we used a cloned Chinese hamster ovary cell line, CHOpMTGR, in which glucocorticoid receptor cDNA was stably integrated. In immunocytochemical analysis, we found that treatment with UDCA promoted the nuclear translocation of the glucocorticoid receptor in a ligand-independent fashion, which was further confirmed by immunoprecipitation assays. Moreover, the translocated glucocorticoid receptor demonstrated sequence-specific DNA binding activity. Transient transfection experiments revealed that treatment of the cells with UDCA marginally enhanced glucocorticoid-responsive gene expression. We also showed that UDCA suppressed IFN-{gamma}-mediated induction of MHC class II gene expression via the glucocorticoid receptor-mediated pathway. Together, UDCA-dependent promotion of translocation of the glucocorticoid receptor may be associated with, at least in part, its immunomodulatory action through glucocorticoid receptor-mediated gene regulation. 68 refs., 8 figs.

  12. The Effect of Mineralocorticoid and Glucocorticoid Receptor Antagonism on Autobiographical Memory Recall and Amygdala Response to Implicit Emotional Stimuli

    PubMed Central

    Preskorn, Sheldon H.; Victor, Teresa; Misaki, Masaya; Bodurka, Jerzy; Drevets, Wayne C.

    2016-01-01

    Background: Acutely elevated cortisol levels in healthy humans impair autobiographical memory recall and alter hemodynamic responses of the amygdala to emotionally valenced stimuli. It is hypothesized that the effects of the cortisol on cognition are influenced by the ratio of mineralocorticoid receptor to glucocorticoid receptor occupation. The current study examined the effects of acutely blocking mineralocorticoid receptors and glucocorticoid receptors separately on 2 processes known to be affected by altering levels of cortisol: the specificity of autobiographical memory recall, and the amygdala hemodynamic response to sad and happy faces. Methods: We employed a within-subjects design in which 10 healthy male participants received placebo, the mineralocorticoid receptor antagonist spironolactone (600mg) alone, and the glucocorticoid receptor antagonist mifepristone (600mg) alone in a randomized, counter-balanced order separated by 1-week drug-free periods. Results: On autobiographical memory testing, mineralocorticoid receptor antagonism impaired, while glucocorticoid receptor antagonism improved, recall relative to placebo, as evinced by changes in the percent of specific memories recalled. During fMRI, the amygdala hemodynamic response to masked sad faces was greater under both mineralocorticoid receptor and glucocorticoid receptor antagonism relative to placebo, while the response to masked happy faces was attenuated only during mineralocorticoid receptor antagonism relative to placebo. Conclusions: These data suggest both mineralocorticoid receptor and glucocorticoid receptor antagonism (and potentially any deviation from the normal physiological mineralocorticoid receptor/glucocorticoid receptor ratio achieved under the circadian pattern) enhances amygdala-based processing of sad stimuli and may shift the emotional processing bias away from the normative processing bias and towards the negative valence. In contrast, autobiographical memory was enhanced by

  13. Chronic stress accelerates ligature-induced periodontitis by suppressing glucocorticoid receptor-α signaling

    PubMed Central

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

    2016-01-01

    Periodontitis is a common chronic inflammatory disease. Recent studies have shown that chronic stress (CS) might modulate periodontal disease, but there are few models of CS-induced periodontitis, and the underlying mechanisms are unclear. The present study established a rat model of periodontitis associated with CS induced by nylon thread ligatures. The severity of periodontitis was evaluated in this model by radiographic and pathological examination. The inflammatory reaction indicated by the elevated serum levels of interleukin (IL)-1β, IL-6 and IL-8 was assessed by enzyme-linked immunosorbent assay. Toll-like receptor-4 (TLR4) and glucocorticoid receptor-α (GR-α) expressions were detected by reverse transcriptase-PCR and western blotting. Open-field tests and serum corticosterone were used to evaluate CS. The results showed that CS induced behavioral changes and increased corticosterone levels of the animals with periodontitis. CS stimulation markedly increased alveolar bone loss, periodontal pocket depth and the number of plaques. It also enhanced the inflammatory reaction. These results suggest that CS accelerated the ligature-induced pathological changes associated with periodontitis. Further analysis of the mechanisms involved showed that GR-α expression was significantly downregulated in periodontal tissues of the animals undergoing CS. Blocking GR-α signaling in lipopolysaccharide and corticosteroid-treated human periodontal ligament fibroblast cells in vitro significantly upregulated the expression of p-Akt (protein kinase B) and TLR4, promoted nuclear factor-κB activity and increased levels of IL-1β, IL-6 and IL-8. This research suggests that CS might accelerate the pathological progression of periodontitis by a GR-α signaling-mediated inflammatory response and that this may be a potential therapeutic target for the treatment of periodontal disease, particularly in patients with CS. PMID:27012709

  14. Chronic stress accelerates ligature-induced periodontitis by suppressing glucocorticoid receptor-α signaling.

    PubMed

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

    2016-03-25

    Periodontitis is a common chronic inflammatory disease. Recent studies have shown that chronic stress (CS) might modulate periodontal disease, but there are few models of CS-induced periodontitis, and the underlying mechanisms are unclear. The present study established a rat model of periodontitis associated with CS induced by nylon thread ligatures. The severity of periodontitis was evaluated in this model by radiographic and pathological examination. The inflammatory reaction indicated by the elevated serum levels of interleukin (IL)-1β, IL-6 and IL-8 was assessed by enzyme-linked immunosorbent assay. Toll-like receptor-4 (TLR4) and glucocorticoid receptor-α (GR-α) expressions were detected by reverse transcriptase-PCR and western blotting. Open-field tests and serum corticosterone were used to evaluate CS. The results showed that CS induced behavioral changes and increased corticosterone levels of the animals with periodontitis. CS stimulation markedly increased alveolar bone loss, periodontal pocket depth and the number of plaques. It also enhanced the inflammatory reaction. These results suggest that CS accelerated the ligature-induced pathological changes associated with periodontitis. Further analysis of the mechanisms involved showed that GR-α expression was significantly downregulated in periodontal tissues of the animals undergoing CS. Blocking GR-α signaling in lipopolysaccharide and corticosteroid-treated human periodontal ligament fibroblast cells in vitro significantly upregulated the expression of p-Akt (protein kinase B) and TLR4, promoted nuclear factor-κB activity and increased levels of IL-1β, IL-6 and IL-8. This research suggests that CS might accelerate the pathological progression of periodontitis by a GR-α signaling-mediated inflammatory response and that this may be a potential therapeutic target for the treatment of periodontal disease, particularly in patients with CS.

  15. PPARα blocks glucocorticoid receptor α-mediated transactivation but cooperates with the activated glucocorticoid receptor α for transrepression on NF-κB

    PubMed Central

    Bougarne, Nadia; Paumelle, Réjane; Caron, Sandrine; Hennuyer, Nathalie; Mansouri, Roxane; Gervois, Philippe; Staels, Bart; Haegeman, Guy; De Bosscher, Karolien

    2009-01-01

    Glucocorticoid receptor α (GRα) and peroxisome proliferator-activated receptor α (PPARα) are transcription factors with clinically important immune-modulating properties. Either receptor can inhibit cytokine gene expression, mainly through interference with nuclear factor κB (NF-κB)-driven gene expression. The present work aimed to investigate a functional cross-talk between PPARα- and GRα-mediated signaling pathways. Simultaneous activation of PPARα and GRα dose-dependently enhances transrepression of NF-κB-driven gene expression and additively represses cytokine production. In sharp contrast and quite unexpectedly, PPARα agonists inhibit the expression of classical glucocorticoid response element (GRE)-driven genes in a PPARα-dependent manner, as demonstrated by experiments using PPARα wild-type and knockout mice. The underlying mechanism for this transcriptional antagonism relies on a PPARα-mediated interference with the recruitment of GRα, and concomitantly of RNA polymerase II, to GRE-driven gene promoters. Finally, the biological relevance of this phenomenon is underscored by the observation that treatment with the PPARα agonist fenofibrate prevents glucocorticoid-induced hyperinsulinemia of mice fed a high-fat diet. Taken together, PPARα negatively interferes with GRE-mediated GRα activity while potentiating its antiinflammatory effects, thus providing a rationale for combination therapy in chronic inflammatory disorders. PMID:19376972

  16. CB1 receptor mediates the effects of glucocorticoids on AMPK activity in the hypothalamus.

    PubMed

    Scerif, Miski; Füzesi, Tamás; Thomas, Julia D; Kola, Blerina; Grossman, Ashley B; Fekete, Csaba; Korbonits, Márta

    2013-10-01

    AMP-activated protein kinase (AMPK), a regulator of cellular and systemic energy homeostasis, can be influenced by several hormones. Tissue-specific alteration of AMPK activity by glucocorticoids may explain the increase in appetite, the accumulation of lipids in adipose tissues, and the detrimental cardiac effects of Cushing's syndrome. Endocannabinoids are known to mediate the effects of various hormones and to influence AMPK activity. Cannabinoids have central orexigenic and direct peripheral metabolic effects via the cannabinoid receptor type 1 (CB1). In our preliminary experiments, WT mice received implants of a corticosterone-containing pellet to establish a mouse model of Cushing's syndrome. Subsequently, WT and Cb1 (Cnr1)-knockout (CB1-KO) littermates were treated with corticosterone and AMPK activity in the hypothalamus, various adipose tissues, liver and cardiac tissue was measured. Corticosterone-treated CB1-KO mice showed a lack of weight gain and of increase in hypothalamic and hepatic AMPK activity. In adipose tissues, baseline AMPK activity was higher in CB1-KO mice, but a glucocorticoid-induced drop was observed, similar to that observed in WT mice. Cardiac AMPK levels were reduced in CB1-KO mice, but while WT mice showed significantly reduced AMPK activity following glucocorticoid treatment, CB1-KO mice showed a paradoxical increase. Our findings indicate the importance of the CB1 receptor in the central orexigenic effect of glucocorticoid-induced activation of hypothalamic AMPK activity. In the periphery adipose tissues, changes may occur independently of the CB1 receptor, but the receptor appears to alter the responsiveness of the liver and myocardial tissues to glucocorticoids. In conclusion, our data suggest that an intact cannabinoid pathway is required for the full metabolic effects of chronic glucocorticoid excess.

  17. CB1 receptor mediates the effects of glucocorticoids on AMPK activity in the hypothalamus.

    PubMed

    Scerif, Miski; Füzesi, Tamás; Thomas, Julia D; Kola, Blerina; Grossman, Ashley B; Fekete, Csaba; Korbonits, Márta

    2013-10-01

    AMP-activated protein kinase (AMPK), a regulator of cellular and systemic energy homeostasis, can be influenced by several hormones. Tissue-specific alteration of AMPK activity by glucocorticoids may explain the increase in appetite, the accumulation of lipids in adipose tissues, and the detrimental cardiac effects of Cushing's syndrome. Endocannabinoids are known to mediate the effects of various hormones and to influence AMPK activity. Cannabinoids have central orexigenic and direct peripheral metabolic effects via the cannabinoid receptor type 1 (CB1). In our preliminary experiments, WT mice received implants of a corticosterone-containing pellet to establish a mouse model of Cushing's syndrome. Subsequently, WT and Cb1 (Cnr1)-knockout (CB1-KO) littermates were treated with corticosterone and AMPK activity in the hypothalamus, various adipose tissues, liver and cardiac tissue was measured. Corticosterone-treated CB1-KO mice showed a lack of weight gain and of increase in hypothalamic and hepatic AMPK activity. In adipose tissues, baseline AMPK activity was higher in CB1-KO mice, but a glucocorticoid-induced drop was observed, similar to that observed in WT mice. Cardiac AMPK levels were reduced in CB1-KO mice, but while WT mice showed significantly reduced AMPK activity following glucocorticoid treatment, CB1-KO mice showed a paradoxical increase. Our findings indicate the importance of the CB1 receptor in the central orexigenic effect of glucocorticoid-induced activation of hypothalamic AMPK activity. In the periphery adipose tissues, changes may occur independently of the CB1 receptor, but the receptor appears to alter the responsiveness of the liver and myocardial tissues to glucocorticoids. In conclusion, our data suggest that an intact cannabinoid pathway is required for the full metabolic effects of chronic glucocorticoid excess. PMID:23884964

  18. Glucocorticoid hormones downregulate histidine decarboxylase mRNA and enzyme activity in rat lung.

    PubMed

    Zahnow, C A; Panula, P; Yamatodani, A; Millhorn, D E

    1998-08-01

    Histidine decarboxylase (HDC) is the primary enzyme regulating histamine biosynthesis. Histamine contributes to the pathogenesis of chronic inflammatory disorders such as asthma. Because glucocorticoids are effective in the treatment of asthma, we examined the effects of 6 h of exogenously administered dexamethasone (0.5-3,000 microg/kg ip), corticosterone (0.2-200 mg/kg ip), or endogenously elevated corticosterone (via exposure of rats to 10% oxygen) on HDC expression in the rat lung. HDC transcripts were decreased approximately 73% with dexamethasone treatment, 57% with corticosterone treatment, and 50% with exposure to 10% oxygen. Likewise, HDC enzyme activity was decreased 80% by treatment with dexamethasone and corticosterone and 60% by exposure to 10% oxygen. Adrenalectomy prevented the decreases in HDC mRNA and enzyme activity observed in rats exposed to 10% oxygen, suggesting that the adrenal gland is necessary for the mediation of hypoxic effects on HDC gene expression. These results demonstrate that corticosteroids initiate a process that leads to the decrease of HDC mRNA levels and enzyme activity in rat lung. PMID:9700103

  19. Methylation of the Glucocorticoid Receptor Gene Promoter in Preschoolers: Links with Internalizing Behavior Problems

    ERIC Educational Resources Information Center

    Parade, Stephanie H.; Ridout, Kathryn K.; Seifer, Ronald; Armstrong, David A.; Marsit, Carmen J.; McWilliams, Melissa A.; Tyrka, Audrey R.

    2016-01-01

    Accumulating evidence suggests that early adversity is linked to methylation of the glucocorticoid receptor (GR) gene, "NR3C1," which is a key regulator of the hypothalamic-pituitary-adrenal axis. Yet no prior work has considered the contribution of methylation of "NR3C1" to emerging behavior problems and psychopathology in…

  20. Differences in colocalization of corticosteroid-binding globulin and glucocorticoid receptor immunoreactivity in the rat brain.

    PubMed

    Sivukhina, Elena; Schäfer, Hans H; Jirikowski, Gustav F

    2013-05-01

    Endocrine regulation of central and systemic stress response as well as learning and memory are in part controlled by systemic glucocorticoid levels. So far steroids have been thought to act on the brain predominantly through nuclear receptors. However, some brain systems known to respond to glucocorticoids seem to be devoid of the respective receptor proteins (GR). It is likely that known central actions of adrenal steroids may also be mediated by non-genomic actions involving intrinsic binding globulins. In recent studies we described the intrinsic expression of corticosteroid-binding globulin (CBG) in rat, mouse and human brains. Here we report an immunohistochemical mapping study on the colocalization of CBG and of GR in the rat brain. In the nucleus accumbens, septum, hippocampus, globus pallidus, medial and basolateral amygdale nuclei, magnocellular preoptic nuclei, diagonal band of Broca high intensity of CBG immunoreactivity was accompanied by weak or moderate GR staining, and vice versa. In the caudate putamen, bed nucleus of stria terminalis, septohypothalamic nucleus and parvocellular subdivision of the paraventricular nucleus strong GR immunoreactivity was observed, but CBG was almost undetectable. In contrast, throughout the supraoptic nucleus and magnocellular subdivision of the paraventricular nucleus numerous strongly CBG-positive cells were observed, devoid of specific GR immunoreactivity. It is most likely that CBG in the brain may be involved in the response to changing systemic glucocorticoid levels in addition to known nuclear and membrane corticosteroid receptors, or in glucocorticoid responsive regions devoid of these receptors.

  1. GLUCOCORTICOID RECEPTOR REGULATION IN THE RAT EMBRYO: A POTENTIAL SITE FOR DEVELOPMENTAL TOXICITY?

    EPA Science Inventory

    Glucocorticoid receptor regulation in the rat embryo: a potential site for developmental toxicity?

    Ghosh B, Wood CR, Held GA, Abbott BD, Lau C.

    National Research Council, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA.

  2. Synthesis and structure-activity relationships of novel indazolyl glucocorticoid receptor partial agonists.

    PubMed

    Gilmore, John L; Sheppeck, James E; Wang, Jim; Dhar, T G Murali; Cavallaro, Cullen; Doweyko, Arthur M; Mckay, Lorraine; Cunningham, Mark D; Habte, Sium F; Nadler, Steven G; Dodd, John H; Somerville, John E; Barrish, Joel C

    2013-10-01

    SAR was used to further develop an indazole class of non-steroidal glucocorticoid receptor agonists aided by a GR LBD (ligand-binding domain)-agonist co-crystal structure described in the accompanying paper. Progress towards discovering a dissociated GR agonist guided by human in vitro assays biased the optimization of this compound series towards partial agonists that possessed excellent selectivity against other nuclear hormone receptors. PMID:23916594

  3. Antidepressants regulate glucocorticoid receptor messenger RNA concentrations in primary neuronal cultures.

    PubMed

    Pepin, M C; Beaulieu, S; Barden, N

    1989-07-01

    Increased cortisol secretion, caused by hyperactivity of the brain-pituitary-adrenal axis, and non-suppression of cortisol secretion following dexamethasone administration are two characteristics frequently associated with major depression or the depressed phase of bipolar illness. Antidepressants, irrespective of their selective inhibitory actions on the re-uptake of serotonin or of norepinephrine, modify glucocorticoid receptor messenger RNA concentrations in primary cultures of rat hypothalamic or amygdaloid neurons in a biphasic manner, with predominant stimulatory effects. This suggests a mechanism whereby antidepressants, by restoring the sensitivity of the limbic-hypothalamic system to glucocorticoid feedback inhibition, reverse the hyperactivity of the brain-pituitary-adrenal axis.

  4. miR-29c induction contributes to downregulation of vascular extracellular matrix proteins by glucocorticoids

    PubMed Central

    Chuang, Tsai-Der; Pearce, William J.

    2015-01-01

    Maternal undernutrition increases maternal glucocorticoids (GCs) and alters microRNA expression in offspring. Given that the mechanisms of GC action on vascular development are not clear, this study examined the influence of GCs on microRNA 29c (miR-29c) and its predicted targets in primary rat aorta smooth muscle cells (RAOSMCs). Dexamethasone (Dex) and corticosterone (Cor) time-dependently increased miR-29c expression and reduced collagen type III (Col3A1), collagen type IV (Col4A5), elastin (ELN), and matrix metalloproteinase-2 (MMP2) protein in RAOSMCs. These effects were blocked by mifepristone. These genes were also targeted by miR-29c, as confirmed by a significant decrease in luciferase reporter activity of Col3A1 (34%), Col4A5 (45%), ELN (17%), and MMP2 (28%). In cells transfected with reporter plasmids, including the 3′-untranslated region of genes targeted by miR-29c, treatment with Dex or Cor also resulted in decreases in luciferase activity. Gain or loss of function of miR-29c significantly altered mRNA expression of Col3A1 (26% and 26%, respectively), Col4A5 (28% and 32%, respectively), and MMP2 (24% and 14%, respectively) but did not affect ELN. Gain or loss of function of miR-29c also significantly altered protein levels of Col3A1 (51% and 16%, respectively), Col4A5 (56% and 22%, respectively), ELN (53% and 71%, respectively), and MMP2 (28% and 53%, respectively). Coincubation of anti-miR-29c with Dex or Cor partially attenuated the effects of these steroids on protein expression of Col3A1 (25% and 24%, respectively), Col4A5 (26% and 44%, respectively), ELN (31% and 55%, respectively), and MMP2 (46% and 26%, respectively) in RAOSMCs compared with anti-miR negative controls. Our results demonstrate that GCs regulate the expression of Col3A1, Col4A5, ELN, and MMP2, at least in part, through induction of miR-29c. PMID:26017148

  5. miR-29c induction contributes to downregulation of vascular extracellular matrix proteins by glucocorticoids.

    PubMed

    Chuang, Tsai-Der; Pearce, William J; Khorram, Omid

    2015-07-15

    Maternal undernutrition increases maternal glucocorticoids (GCs) and alters microRNA expression in offspring. Given that the mechanisms of GC action on vascular development are not clear, this study examined the influence of GCs on microRNA 29c (miR-29c) and its predicted targets in primary rat aorta smooth muscle cells (RAOSMCs). Dexamethasone (Dex) and corticosterone (Cor) time-dependently increased miR-29c expression and reduced collagen type III (Col3A1), collagen type IV (Col4A5), elastin (ELN), and matrix metalloproteinase-2 (MMP2) protein in RAOSMCs. These effects were blocked by mifepristone. These genes were also targeted by miR-29c, as confirmed by a significant decrease in luciferase reporter activity of Col3A1 (34%), Col4A5 (45%), ELN (17%), and MMP2 (28%). In cells transfected with reporter plasmids, including the 3'-untranslated region of genes targeted by miR-29c, treatment with Dex or Cor also resulted in decreases in luciferase activity. Gain or loss of function of miR-29c significantly altered mRNA expression of Col3A1 (26% and 26%, respectively), Col4A5 (28% and 32%, respectively), and MMP2 (24% and 14%, respectively) but did not affect ELN. Gain or loss of function of miR-29c also significantly altered protein levels of Col3A1 (51% and 16%, respectively), Col4A5 (56% and 22%, respectively), ELN (53% and 71%, respectively), and MMP2 (28% and 53%, respectively). Coincubation of anti-miR-29c with Dex or Cor partially attenuated the effects of these steroids on protein expression of Col3A1 (25% and 24%, respectively), Col4A5 (26% and 44%, respectively), ELN (31% and 55%, respectively), and MMP2 (46% and 26%, respectively) in RAOSMCs compared with anti-miR negative controls. Our results demonstrate that GCs regulate the expression of Col3A1, Col4A5, ELN, and MMP2, at least in part, through induction of miR-29c.

  6. miR-29c induction contributes to downregulation of vascular extracellular matrix proteins by glucocorticoids.

    PubMed

    Chuang, Tsai-Der; Pearce, William J; Khorram, Omid

    2015-07-15

    Maternal undernutrition increases maternal glucocorticoids (GCs) and alters microRNA expression in offspring. Given that the mechanisms of GC action on vascular development are not clear, this study examined the influence of GCs on microRNA 29c (miR-29c) and its predicted targets in primary rat aorta smooth muscle cells (RAOSMCs). Dexamethasone (Dex) and corticosterone (Cor) time-dependently increased miR-29c expression and reduced collagen type III (Col3A1), collagen type IV (Col4A5), elastin (ELN), and matrix metalloproteinase-2 (MMP2) protein in RAOSMCs. These effects were blocked by mifepristone. These genes were also targeted by miR-29c, as confirmed by a significant decrease in luciferase reporter activity of Col3A1 (34%), Col4A5 (45%), ELN (17%), and MMP2 (28%). In cells transfected with reporter plasmids, including the 3'-untranslated region of genes targeted by miR-29c, treatment with Dex or Cor also resulted in decreases in luciferase activity. Gain or loss of function of miR-29c significantly altered mRNA expression of Col3A1 (26% and 26%, respectively), Col4A5 (28% and 32%, respectively), and MMP2 (24% and 14%, respectively) but did not affect ELN. Gain or loss of function of miR-29c also significantly altered protein levels of Col3A1 (51% and 16%, respectively), Col4A5 (56% and 22%, respectively), ELN (53% and 71%, respectively), and MMP2 (28% and 53%, respectively). Coincubation of anti-miR-29c with Dex or Cor partially attenuated the effects of these steroids on protein expression of Col3A1 (25% and 24%, respectively), Col4A5 (26% and 44%, respectively), ELN (31% and 55%, respectively), and MMP2 (46% and 26%, respectively) in RAOSMCs compared with anti-miR negative controls. Our results demonstrate that GCs regulate the expression of Col3A1, Col4A5, ELN, and MMP2, at least in part, through induction of miR-29c. PMID:26017148

  7. G protein β interacts with the glucocorticoid receptor and suppresses its transcriptional activity in the nucleus

    PubMed Central

    Kino, Tomoshige; Tiulpakov, Anatoly; Ichijo, Takamasa; Chheng, Ly; Kozasa, Tohru; Chrousos, George P.

    2005-01-01

    Extracellular stimuli that activate cell surface receptors modulate glucocorticoid actions via as yet unclear mechanisms. Here, we report that the guanine nucleotide-binding protein (G protein)–coupled receptor-activated WD-repeat Gβ interacts with the glucocorticoid receptor (GR), comigrates with it into the nucleus and suppresses GR-induced transactivation of the glucocorticoid-responsive genes. Association of Gγ with Gβ is necessary for this action of Gβ. Both endogenous and enhanced green fluorescent protein (EGFP)–fused Gβ2 and Gγ2 proteins were detected in the nucleus at baseline, whereas a fraction of EGFP-Gβ2 and DsRed2-GR comigrated to the nucleus or the plasma membrane, depending on the exposure of cells to dexamethasone or somatostatin, respectively. Gβ2 was associated with GR/glucocorticoid response elements (GREs) in vivo and suppressed activation function-2–directed transcriptional activity of the GR. We conclude that the Gβγ complex interacts with the GR and suppresses its transcriptional activity by associating with the transcriptional complex formed on GR-responsive promoters. PMID:15955845

  8. Stress and corticosteroids regulate rat hippocampal mitochondrial DNA gene expression via the glucocorticoid receptor.

    PubMed

    Hunter, Richard G; Seligsohn, Ma'ayan; Rubin, Todd G; Griffiths, Brian B; Ozdemir, Yildirim; Pfaff, Donald W; Datson, Nicole A; McEwen, Bruce S

    2016-08-01

    Glucocorticoids (GCs) are involved in stress and circadian regulation, and produce many actions via the GC receptor (GR), which is classically understood to function as a nuclear transcription factor. However, the nuclear genome is not the only genome in eukaryotic cells. The mitochondria also contain a small circular genome, the mitochondrial DNA (mtDNA), that encodes 13 polypeptides. Recent work has established that, in the brain and other systems, the GR is translocated from the cytosol to the mitochondria and that stress and corticosteroids have a direct influence on mtDNA transcription and mitochondrial physiology. To determine if stress affects mitochondrially transcribed mRNA (mtRNA) expression, we exposed adult male rats to both acute and chronic immobilization stress and examined mtRNA expression using quantitative RT-PCR. We found that acute stress had a main effect on mtRNA expression and that expression of NADH dehydrogenase 1, 3, and 6 (ND-1, ND-3, ND-6) and ATP synthase 6 (ATP-6) genes was significantly down-regulated. Chronic stress induced a significant up-regulation of ND-6 expression. Adrenalectomy abolished acute stress-induced mtRNA regulation, demonstrating GC dependence. ChIP sequencing of GR showed that corticosterone treatment induced a dose-dependent association of the GR with the control region of the mitochondrial genome. These findings demonstrate GR and stress-dependent transcriptional regulation of the mitochondrial genome in vivo and are consistent with previous work linking stress and GCs with changes in the function of brain mitochondria. PMID:27457949

  9. Ontogeny of hippocampal corticosteroid receptors: effects of antenatal glucocorticoids in human and mouse.

    PubMed

    Noorlander, C W; De Graan, P N E; Middeldorp, J; Van Beers, J J B C; Visser, G H A

    2006-12-20

    Women at risk for preterm delivery are treated with synthetic glucocorticoids (GCs) to enhance fetal lung maturation. GCs can bind to two intracellular receptors, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), which function as transcription factors. Both are highly expressed in the hippocampus. Several studies have focused on adverse side effects of antenatal GC treatment. However, relatively little is known about the ontogeny of GR and MR, especially in human. Therefore, we studied the ontogeny of both receptors in the human and mouse hippocampus and investigated the effects of antenatal dexamethasone (dex) treatment, a synthetic glucocorticoid, on MR and GR mRNA levels during hippocampal development. The results demonstrate that MR mRNA was first expressed in mouse hippocampus at embryonic day (E)15.5, at the timepoint when dex was administered. In contrast, GR mRNA expression was first observed after birth at postnatal day (P)5. However, in the human hippocampus both receptors are expressed at 24 weeks of gestation, when antenatal GCs are administered in clinical practice. Quantitative in situ hybridization demonstrated that MR mRNA levels were reduced only shortly after dex treatment at E16, but were unaffected from E18 onwards. These findings indicate that a single antenatal dex administration at E15.5 transiently affects MR mRNA levels in the mouse hippocampus. No effect of antenatal dex treatment was found on the human hippocampus at the third trimester of pregnancy. These data on the prenatal ontogeny of both corticosteroid receptors in the human hippocampus is important for understanding the significance of fetal glucocorticoid or stress exposure and its potential effects on health and disease.

  10. Blocking Mineralocorticoid Receptors Impairs, Blocking Glucocorticoid Receptors Enhances Memory Retrieval in Humans

    PubMed Central

    Rimmele, Ulrike; Besedovsky, Luciana; Lange, Tanja; Born, Jan

    2013-01-01

    Memory retrieval is impaired at very low as well as very high cortisol levels, but not at intermediate levels. This inverted-U-shaped relationship between cortisol levels and memory retrieval may originate from different roles of the mineralocorticoid (MR) and glucocorticoid receptor (GR) that bind cortisol with distinctly different affinity. Here, we examined the role of MRs and GRs in human memory retrieval using specific receptor antagonists. In two double-blind within-subject, cross-over designed studies, young healthy men were asked to retrieve emotional and neutral texts and pictures (learnt 3 days earlier) between 0745 and 0915 hours in the morning, either after administration of 400 mg of the MR blocker spironolactone vs placebo (200 mg at 2300 hours and 200 mg at 0400 hours, Study I) or after administration of the GR blocker mifepristone vs placebo (200 mg at 2300 hours, Study II). Blockade of MRs impaired free recall of both texts and pictures particularly for emotional material. In contrast, blockade of GRs resulted in better memory retrieval for pictures, with the effect being more pronounced for neutral than emotional materials. These findings indicate indeed opposing roles of MRs and GRs in memory retrieval, with optimal retrieval at intermediate cortisol levels likely mediated by high MR but concurrently low GR activation. PMID:23303058

  11. Effect of long term dexamethasone treatment on the glucocorticoid receptor

    SciTech Connect

    Silva, C.M.; DeLorenzo, T.M.; Cidlowski, J.A.

    1986-05-01

    The ability of dexamethasone(dex) to induce alkaline phosphatase activity was found to decrease with chronic hormone exposure. In order to better understand this adaptive resistance, the structure of the receptor from control cells and cells under long term dex (10/sup -6/M) treatment was analyzed. Native isoelectric focusing showed that receptor from dex treated cells focused at more basic pI than receptor from control cells. Denaturing two-dimensional gel analysis resulted in the characteristic 4-5 spots of (/sup 3/H)dexamethasone mesylate (DM) binding of receptor from control cells, but no (/sup 3/H)DM binding could be seen for receptor from dex treated cells. In order to study DNA-binding characteristics, gels were renatured, transferred to nitrocellulose and probed with (/sup 32/P)MMTV-GRE. Receptor from control cells showed 5 spots of DNA-binding at 101 kDa molecular weight and a pI range of 7.42 to 7.32. However, receptor from dex treated cells showed less intense DNA-binding which occurred only at the more basic range of pIs (7.42 to 7.39). Furthermore, no nuclear receptor sites could be measured in the dex treated cells, whereas 20,000 sites were measured in control cells. Even after being taken off hormone treatment for 12 days, cells could regenerate only 50% of their receptors. In conclusion, this system is conducive to studying the mechanism of receptor regulation.

  12. Role of the hinge region of glucocorticoid receptor for HEXIM1-mediated transcriptional repression

    SciTech Connect

    Yoshikawa, Noritada; Shimizu, Noriaki; Sano, Motoaki; Ohnuma, Kei; Iwata, Satoshi; Hosono, Osamu; Fukuda, Keiichi; Morimoto, Chikao

    2008-06-20

    We previously reported that HEXIM1 (hexamethylene bisacetamide-inducible protein 1), which suppresses transcription elongation via sequestration of positive transcription elongation factor b (P-TEFb) using 7SK RNA as a scaffold, directly associates with glucocorticoid receptor (GR) to suppress glucocorticoid-inducible gene activation. Here, we revealed that the hinge region of GR is essential for its interaction with HEXIM1, and that oxosteroid receptors including GR show sequence homology in their hinge region and interact with HEXIM1, whereas the other members of nuclear receptors do not. We also showed that HEXIM1 suppresses GR-mediated transcription in two ways: sequestration of P-TEFb by HEXIM1 and direct interaction between GR and HEXIM1. In contrast, peroxisome proliferator-activated receptor {gamma}-dependent gene expression is negatively modulated by HEXIM1 solely via sequestration of P-TEFb. We, therefore, conclude that HEXIM1 may act as a gene-selective transcriptional regulator via direct interaction with certain transcriptional regulators including GR and contribute to fine-tuning of, for example, glucocorticoid-mediated biological responses.

  13. Gonadal steroids exert facilitating and "buffering" effects on glucocorticoid-mediated transcriptional regulation of corticotropin-releasing hormone and corticosteroid receptor genes in rat brain.

    PubMed

    Patchev, V K; Almeida, O F

    1996-11-01

    Gonadal steroids profoundly influence several brain functions and are apparently responsible for gender-specific differences in the regulation of hypothalamic-pituitary-adrenal (HPA) secretions. In this study, we examined the so-called "activational" effects of gonadal steroids on the glucocorticoid-mediated regulation of the gene transcription of corticotropin-releasing hormone (CRH) and corticosteroid receptors in brain areas of relevance for the control of pituitary-adrenal secretion. The efficacy of adrenalectomy (ADX) and chronic treatment with high doses of corticosterone (B) to regulate the gene transcription of CRH and corticosteroid receptors in the hypothalamic paraventricular nucleus (PVN) and hippocampus was studied in male and female rats under the conditions of deprivation of gonadectomy (GDX) and replacement with different gonadal steroids, such as estradiol (E2), progesterone (P), and dihydrotestosterone (DHT). In both sexes, ADX alone or in combination with GDX increased, and B treatment suppressed, the steady-state levels of CRH and corticosteroid receptor mRNAs, whereas GDX alone failed to affect any of the parameters studied. Administration of gonadal hormones to steroid-deprived (ADX/GDX) animals partially attenuated the upregulation of mRNAs encoding corticosteroid receptors in the hippocampus. Supplementation with gonadal steroids modified the effects of B on the gene transcription of CRH and corticosteroid receptors. Whereas P alone or in combination with E2 counteracted the B-induced downregulation of GR and CRH gene transcription in females, DHT and E2 administration further potentiated the effects of B on these parameters in a sex-specific manner. Taken together, the results indicate that gonadal steroids have minor influence on MR, GR, and CRH gene transcription under basal conditions, exert "glucocorticoid-like" effects on the transcription of corticosteroid receptors in the hippocampus of steroid-deprived animals, and interact with

  14. Environmental variability and the evolution of the glucocorticoid receptor (Nr3c1) in African starlings.

    PubMed

    Hofmeister, Natalie R; Rubenstein, Dustin R

    2016-10-01

    One of the primary ways that organisms cope with environmental change is through regulation of the hypothalamo-pituitary-adrenal (HPA) axis, the neuroendocrine system that controls reactions to stress. Variation in genes regulating the HPA axis - particularly the glucocorticoid receptor - may facilitate adaptation to changing climatic conditions by altering expression. Here we examine signatures of selection on the glucocorticoid receptor gene (Nr3c1) in African starlings that inhabit a range of environments, including those with variable climatic conditions. To investigate potential adaptive mechanisms underlying the vertebrate stress response, we sequence the Nr3c1 gene in 27 species of African starlings. Although we find some evidence of positive selection, substitution rate is negatively correlated with variance in precipitation. This suggests climatic cycling in sub-Saharan Africa may have resulted in lower substitution rates to maintain a successful coping strategy. When environmental conditions fluctuate rapidly, variation in the strength of purifying selection can explain evolutionary rate variation. PMID:27500971

  15. Homodimerization of glucocorticoid receptor from single cells investigated using fluorescence correlation spectroscopy and microwells.

    PubMed

    Oasa, Sho; Sasaki, Akira; Yamamoto, Johtaro; Mikuni, Shintaro; Kinjo, Masataka

    2015-08-01

    Glucocorticoid receptor α (GR) binds to the promoter regions of target genes as a homodimer and activates its transcriptional process. Though the homodimerization is thought to be the initial and essential process, the dissociation constant for homodimerization of GR remains controversial. To quantify homodimerization of (enhanced green fluorescence protein) EGFP-(glucocorticoid receptor) GR, the particle brightness in lysates from single cell was estimated for the fraction of homodimeric EGFP-GR using fluorescence correlation spectroscopy and microwells. Fitting the data with a bimolecular reaction model, the dissociation constant was determined. Moreover slow-diffusion complex was observed. These results suggest that EGFP-GR forms not only a monomer-dimer equivalent state but also a large-molecular-weight complex. PMID:26183204

  16. Sequence-specific DNA binding by glucocorticoid receptor "zinc finger peptides".

    PubMed

    Archer, T K; Hager, G L; Omichinski, J G

    1990-10-01

    Steroid hormone receptors can activate or repress transcription from responsive loci by binding to DNA. We have examined the mechanism of DNA binding by individually synthesizing the putative "zinc finger peptides" from the rat glucocorticoid receptor. Atomic absorption studies show that the peptides will bind zinc on an equimolar basis, and circular dichroism experiments demonstrate a significant alteration in secondary structure in the presence of zinc. The results from a series of experiments establish that metal ion is required for binding to DNA and that the amino-terminal zinc finger shows a significantly greater affinity for glucocorticoid response element-containing DNA over control DNA. These observations indicate that a single synthetic "zinc finger peptide" is able to bind to DNA in a sequence-specific manner. PMID:2120703

  17. Membrane glucocorticoid receptors are localised in the extracellular matrix and signal through the MAPK pathway in mammalian skeletal muscle fibres

    PubMed Central

    Boncompagni, Simona; Arthurton, Lewis; Akujuru, Eugene; Pearson, Timothy; Steverding, Dietmar; Protasi, Feliciano; Mutungi, Gabriel

    2015-01-01

    A number of studies have previously proposed the existence of glucocorticoid receptors on the plasma membrane of many cell types, including skeletal muscle fibres. However, their exact localisation and the cellular signalling pathway(s) they utilise to communicate with the rest of the cell are still poorly understood. In this study, we investigated the localisation and the mechanism(s) underlying the non-genomic physiological functions of these receptors in mouse skeletal muscle cells. The results show that the receptors were localised in the cytoplasm in myoblasts, in the nucleus in myotubes, in the extracellular matrix, in satellite cells and in the proximity of mitochondria in adult muscle fibres. Also, they bound laminin in a glucocorticoid-dependent manner. Treating small skeletal muscle fibre bundles with the synthetic glucocorticoid beclomethasone dipropionate increased the phosphorylation (= activation) of extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. This occurred within 5 min and depended on the fibre type and the duration of the treatment. It was also abolished by the glucocorticoid receptor inhibitor, mifepristone, and a monoclonal antibody against the receptor. From these results we conclude that the non-genomic/non-canonical physiological functions of glucocorticoids, in adult skeletal muscle fibres, are mediated by a glucocorticoid receptor localised in the extracellular matrix, in satellite cells and close to mitochondria, and involve activation of the mitogen-activated protein kinase pathway. PMID:25846902

  18. Discovery of novel dihydro-9,10-ethano-anthracene carboxamides as glucocorticoid receptor modulators.

    PubMed

    Yang, Bingwei V; Vaccaro, Wayne; Doweyko, Arthur M; Doweyko, Lidia M; Huynh, Tram; Tortolani, David; Nadler, Steven G; McKay, Lorraine; Somerville, John; Holloway, Deborah A; Habte, Sium; Weinstein, David S; Barrish, Joel C

    2009-04-15

    A series of dihydro-9,10-ethano-anthracene-11-carboxamides as novel glucocorticoid receptor modulators is reported. SAR exploration identified compounds from this series displaying a promising dissociation profile in discriminating between transrepression and transactivation activities. 17a is a partial agonist of GR-mediated transactivation which elicits potent and efficacious transrepression in reporter gene assays. A hypothetical binding mode is provided which accounts for the induction of functional activity by a bridgehead methyl group. PMID:19321341

  19. Discovery of potent and selective nonsteroidal indazolyl amide glucocorticoid receptor agonists.

    PubMed

    Sheppeck, James E; Gilmore, John L; Xiao, Hai-Yun; Dhar, T G Murali; Nirschl, David; Doweyko, Arthur M; Sack, Jack S; Corbett, Martin J; Malley, Mary F; Gougoutas, Jack Z; Mckay, Lorraine; Cunningham, Mark D; Habte, Sium F; Dodd, John H; Nadler, Steven G; Somerville, John E; Barrish, Joel C

    2013-10-01

    Modification of a phenolic lead structure based on lessons learned from increasing the potency of steroidal glucocorticoid agonists lead to the discovery of exceptionally potent, nonsteroidal, indazole GR agonists. SAR was developed to achieve good selectivity against other nuclear hormone receptors with the ultimate goal of achieving a dissociated GR agonist as measured by human in vitro assays. The specific interactions by which this class of compounds inhibits GR was elucidated by solving an X-ray co-crystal structure. PMID:23953070

  20. Renal sodium retention in cirrhotic rats depends on glucocorticoid-mediated activation of mineralocorticoid receptor due to decreased renal 11beta-HSD-2 activity.

    PubMed

    Thiesson, Helle C; Jensen, Boye L; Bistrup, Claus; Ottosen, Peter D; McNeilly, Alison D; Andrew, Ruth; Seckl, Jonathan; Skøtt, Ole

    2007-01-01

    Downregulation of the renal glucocorticoid-metabolizing enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD-2) during liver cirrhosis may allow activation of the mineralocorticoid receptor (MR) by glucocorticoids and contribute to sodium retention. We tested this hypothesis in male Wistar rats with decompensated liver cirrhosis and ascites 7 wk after bile duct ligation (BDL). Renal 11beta-HSD-2 mRNA, protein, and activity were significantly decreased in decompensated rats. The urinary Na(+)/K(+) ratio was reduced by 40%. Renal epithelial sodium channel (ENaC) mRNA and immunostaining were only slightly affected. Complete metabolic studies, including fecal excretion, showed that the BDL rats had avid renal sodium retention. Treatment of the BDL rats with dexamethasone suppressed endogenous glucocorticoid production, normalized total sodium balance and renal sodium excretion, and reduced ascites formation to the same degree as direct inhibition of MR with K-canrenoate. Total potassium balance was negative in the BDL rats, whereas renal potassium excretion was unchanged. In the distal colon, expression of ENaC was increased in BDL rats. Fecal potassium excretion was increased in cirrhotic rats, and this was corrected by treatment with K-canrenoate but not dexamethasone. We conclude that development of sodium retention and decompensation in cirrhotic rats is associated with downregulation of renal 11beta-HSD-2 activity and inappropriate activation of renal sodium reabsorption by endogenous glucocorticoids. In addition, the overall potassium loss in the BDL model is due to increased fecal potassium excretion, which is associated with upregulation of ENaC in distal colon. PMID:16917017

  1. Cytokine-Induced Loss of Glucocorticoid Function: Effect of Kinase Inhibitors, Long-Acting β2-Adrenoceptor Agonist and Glucocorticoid Receptor Ligands

    PubMed Central

    Rider, Christopher F.; Shah, Suharsh; Miller-Larsson, Anna; Giembycz, Mark A.; Newton, Robert

    2015-01-01

    Acting on the glucocorticoid receptor (NR3C1), glucocorticoids are widely used to treat inflammatory diseases. However, glucocorticoid resistance often leads to suboptimal asthma control. Since glucocorticoid-induced gene expression contributes to glucocorticoid activity, the aim of this study was to use a 2×glucocorticoid response element (GRE) reporter and glucocorticoid-induced gene expression to investigate approaches to combat cytokine-induced glucocorticoid resistance. Pre-treatment with tumor necrosis factor-α (TNF) or interleukin-1β inhibited dexamethasone-induced mRNA expression of the putative anti-inflammatory genes RGS2 and TSC22D3, or just TSC22D3, in primary human airway epithelial and smooth muscle cells, respectively. Dexamethasone-induced DUSP1 mRNA was unaffected. In human bronchial epithelial BEAS-2B cells, dexamethasone-induced TSC22D3 and CDKN1C expression (at 6 h) was reduced by TNF pre-treatment, whereas DUSP1 and RGS2 mRNAs were unaffected. TNF pre-treatment also reduced dexamethasone-dependent 2×GRE reporter activation. This was partially reversed by PS-1145 and c-jun N-terminal kinase (JNK) inhibitor VIII, inhibitors of IKK2 and JNK, respectively. However, neither inhibitor affected TNF-dependent loss of dexamethasone-induced CDKN1C or TSC22D3 mRNA. Similarly, inhibitors of the extracellular signal-regulated kinase, p38, phosphoinositide 3-kinase or protein kinase C pathways failed to attenuate TNF-dependent repression of the 2×GRE reporter. Fluticasone furoate, fluticasone propionate and budesonide were full agonists relative to dexamethasone, while GSK9027, RU24858, des-ciclesonide and GW870086X were partial agonists on the 2×GRE reporter. TNF reduced reporter activity in proportion with agonist efficacy. Full and partial agonists showed various degrees of agonism on RGS2 and TSC22D3 expression, but were equally effective at inducing CDKN1C and DUSP1, and did not affect the repression of CDKN1C or TSC22D3 expression by TNF. Finally

  2. ATP hydrolysis is essential for Bag-1M-mediated inhibition of the DNA binding by the glucocorticoid receptor

    SciTech Connect

    Hong, Wei; Chen, Linfeng; Liu, Yunde; Gao, Weizhen

    2009-12-04

    The 70-kDa heat shock protein (Hsp70) is involved in providing the appropriate conformation of various nuclear hormone receptors, including the glucocorticoid receptor (GR). The Bcl-2 associated athanogene 1M (Bag-1M) is known to downregulate the DNA binding by the GR. Also, Bag-1M interacts with the ATPase domain of Hsp70 to modulate the release of the substrate from Hsp70. In this study, we demonstrate that ATP hydrolysis enhances Bag-1M-mediated inhibition of the DNA binding by the GR. However, the inhibitory effect of Bag-1M was abolished when the intracellular ATP was depleted. In addition, a Bag-1M mutant lacking the interaction with Hsp70 did not influence the GR to bind DNA, suggesting the interaction of Bag-1M with Hsp70 in needed for its negative effect. These results indicate that ATP hydrolysis is essential for Bag-1M-mediated inhibition of the DNA binding by the GR and Hsp70 is a mediator for this process.

  3. Chronic stress alters glucocorticoid receptor and mineralocorticoid receptor mRNA expression in the European starling (Sturnus vulgaris) brain.

    PubMed

    Dickens, M; Romero, L M; Cyr, N E; Dunn, I C; Meddle, S L

    2009-10-01

    Although the glucocorticoid response to acute short-term stress is an adaptive physiological mechanism that aids in the response to and survival of noxious stimuli, chronic stress is associated with a negative impact on health. In wild-caught European starlings (Sturnus vulgaris), chronic stress alters the responsiveness of hypothalamic-pituitary-adrenal (HPA) axis as measured by the acute corticosterone response. In the present study, we investigated potential underlying neuroendocrine mechanisms by comparing glucocorticoid receptor and mineralocorticoid receptor mRNA expression in the brains of chronically and nonchronically-stressed starlings. Hypothalamic paraventricular nucleus, but not hippocampal, glucocorticoid receptor mRNA expression in chronically-stressed birds was significantly lower compared to controls, suggesting changes in the efficacy of corticosterone negative feedback. In addition, chronically-stressed birds showed a significant decrease in hippocampal MR mRNA expression. Together, these results suggest that chronic stress changes the brain physiology of wild birds and provides important information for the understanding of the underlying mechanisms that result in dysregulation of the HPA axis in wild animals by chronic stress. PMID:19686439

  4. An affective disorder in zebrafish with mutation of the glucocorticoid receptor

    PubMed Central

    Ziv, Limor; Muto, Akira; Schoonheim, Peter J.; Meijsing, Sebastiaan H.; Strasser, Daniel; Ingraham, Holly A.; Schaaf, Marcel J.M.; Yamamoto, Keith R.; Baier, Herwig

    2012-01-01

    Upon binding of cortisol, the glucocorticoid receptor (GR) regulates the transcription of specific target genes, including those that encode the stress hormones corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH). Dysregulation of the stress axis is a hallmark of major depression in human patients. However, it is still unclear how glucocorticoid signaling is linked to affective disorders. We identified an adult-viable zebrafish mutant in which the negative feedback on the stress response is disrupted, due to abolition of all transcriptional activity of GR. As a consequence, cortisol is elevated, but unable to signal through GR. When placed into an unfamiliar aquarium (‘novel tank’), mutant fish become immobile (‘freeze’), show reduced exploratory behavior and do not habituate to this stressor upon repeated exposure. Addition of the antidepressant fluoxetine to the holding water and social interactions restore normal behavior, followed by a delayed correction of cortisol levels. Fluoxetine does not affect overall transcription of CRH, the mineralocorticoid receptor (MR), the serotonin transporter Serta or GR itself. Fluoxetine, however, suppresses the stress-induced upregulation of MR and Serta in both wildtype fish and mutants. Our studies show a conserved, protective function of glucocorticoid signaling in the regulation of emotional behavior and reveal novel molecular aspects of how chronic stress impacts vertebrate brain physiology and behavior. Importantly, the zebrafish model opens up the possibility of high-throughput drug screens in search of new classes of antidepressants. PMID:22641177

  5. The antidepressant fluoxetine normalizes the nuclear glucocorticoid receptor evoked by psychosocial stress

    NASA Astrophysics Data System (ADS)

    Mitić, M.; Simić, I.; Djordjević, J.; Radojčić, M. B.; Adžić, M.

    2011-12-01

    Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis has been implicated in the pathophysiology of depression and stress disorders. Glucocorticoids, key regulators of the stress response, exert diverse effects on cellular processes in the hippocampus. Beside non-genomic pathways, glucocorticoid effects are mediated through activation of the glucocorticoid receptor (GR), a ligand activated transcriptional factor that belongs to the nuclear hormone receptor superfamily. We analysed the GR protein levels both in the cytoplasmic and nuclear compartments of the hippocampus of Wistar rats exposed to chronic psychosocial isolation stress upon chronic fluoxetine (FLU) treatment. Under chronic stress, corticosterone levels (CORT) were decreased compared to the control, and treatment with FLU did not change its level in the stressed rats. At the molecular level, FLU normalized the level of nuclear GR protein in the hippocampus of the stressed rats. Discrepancy between normalization of nuclear GR in the hippocampus and lack of normalization of HPA axis activity judged by CORT, suggests that other brain structures such as the amygdale and prefrontal cortex that also regulate HPA axis activity, seem not to be normalized by the FLU treatment used in our study.

  6. An affective disorder in zebrafish with mutation of the glucocorticoid receptor.

    PubMed

    Ziv, L; Muto, A; Schoonheim, P J; Meijsing, S H; Strasser, D; Ingraham, H A; Schaaf, M J M; Yamamoto, K R; Baier, H

    2013-06-01

    Upon binding of cortisol, the glucocorticoid receptor (GR) regulates the transcription of specific target genes, including those that encode the stress hormones corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone. Dysregulation of the stress axis is a hallmark of major depression in human patients. However, it is still unclear how glucocorticoid signaling is linked to affective disorders. We identified an adult-viable zebrafish mutant in which the negative feedback on the stress response is disrupted, due to abolition of all transcriptional activity of GR. As a consequence, cortisol is elevated, but unable to signal through GR. When placed into an unfamiliar aquarium ('novel tank'), mutant fish become immobile ('freeze'), show reduced exploratory behavior and do not habituate to this stressor upon repeated exposure. Addition of the antidepressant fluoxetine to the holding water and social interactions restore normal behavior, followed by a delayed correction of cortisol levels. Fluoxetine does not affect the overall transcription of CRH, the mineralocorticoid receptor (MR), the serotonin transporter (Serta) or GR itself. Fluoxetine, however, suppresses the stress-induced upregulation of MR and Serta in both wild-type fish and mutants. Our studies show a conserved, protective function of glucocorticoid signaling in the regulation of emotional behavior and reveal novel molecular aspects of how chronic stress impacts vertebrate brain physiology and behavior. Importantly, the zebrafish model opens up the possibility of high-throughput drug screens in search of new classes of antidepressants. PMID:22641177

  7. Doubling the Size of the Glucocorticoid Receptor Ligand Binding Pocket by Deacylcortivazol

    SciTech Connect

    Suino-Powell, Kelly; Xu, Yong; Zhang, Chenghai; Tao, Yong-guang; Tolbert, W. David; Simons, Jr., S. Stoney; Xu, H. Eric

    2010-03-08

    A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 {angstrom}{sup 3}, effectively doubling the size of the GR dexamethasone-binding pocket of 540 {angstrom}{sup 3} and yet leaving the structure of the coactivator binding site intact. DAC occupies only {approx}50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.

  8. Glucocorticoid receptor gene haplotype structure and steroid therapy outcome in IBD patients

    PubMed Central

    Mwinyi, Jessica; Wenger, Christa; Eloranta, Jyrki J; Kullak-Ublick, Gerd A

    2010-01-01

    AIM: To study whether the glucocorticoid receptor (GR/NR3C1) gene haplotypes influence the steroid therapy outcome in inflammatory bowel disease (IBD). METHODS: We sequenced all coding exons and flanking intronic sequences of the NR3C1 gene in 181 IBD patients, determined the single nucleotide polymorphisms, and predicted the NR3C1 haplotypes. Furthermore, we investigated whether certain NR3C1 haplotypes are significantly associated with steroid therapy outcomes. RESULTS: We detected 13 NR3C1 variants, which led to the formation of 17 different haplotypes with a certainty of > 95% in 173 individuals. The three most commonly occurring haplotypes were included in the association analysis of the influence of haplotype on steroid therapy outcome or IBD activity. None of the NR3C1 haplotypes showed statistically significant association with glucocorticoid therapy success. CONCLUSION: NR3C1 haplotypes are not related to steroid therapy outcome. PMID:20712049

  9. Evolution of corticosteroid specificity for human, chicken, alligator and frog glucocorticoid receptors.

    PubMed

    Katsu, Yoshinao; Kohno, Satomi; Oka, Kaori; Baker, Michael E

    2016-09-01

    We investigated the evolution of the response of human, chicken, alligator and frog glucocorticoid receptors (GRs) to dexamethasone, cortisol, cortisone, corticosterone, 11-deoxycorticosterone, 11-deoxycortisol and aldosterone. We find significant differences among these vertebrates in the transcriptional activation of their full length GRs by these steroids, indicating that there were changes in the specificity of the GR for steroids during the evolution of terrestrial vertebrates. To begin to study the role of interactions between different domains on the GR in steroid sensitivity and specificity for terrestrial GRs, we investigated transcriptional activation of truncated GRs containing their hinge domain and ligand binding domain (LBD) fused to a GAL4 DNA binding domain (GAL4-DBD). Compared to corresponding full length GRs, transcriptional activation of GAL4-DBD_GR-hinge/LBD constructs required higher steroid concentrations and displayed altered steroid specificity, indicating that interactions between the hinge/LBD and other domains are important in glucocorticoid activation of these terrestrial GRs. PMID:27317937

  10. The heat shock protein 70 cochaperone hip enhances functional maturation of glucocorticoid receptor.

    PubMed

    Nelson, Gregory M; Prapapanich, Viravan; Carrigan, Patricia E; Roberts, Patricia J; Riggs, Daniel L; Smith, David F

    2004-07-01

    Multiple molecular chaperones interact with steroid receptors to promote functional maturation and stability of receptor complexes. The heat shock protein (Hsp)70 cochaperone Hip has been identified in conjunction with Hsp70, Hsp90, and the Hsp70/Hsp90 cochaperone Hop/Sti1p in receptor complexes during an intermediate stage of receptor assembly, but a functional requirement for Hip in the receptor assembly process has not been established. Because the budding yeast Saccharomyces cerevisiae contains orthologs for most of the receptor-associated chaperones yet lacks an orthologous Hip gene, we exploited the well-established yeast model for steroid receptor function to ask whether Hip can alter steroid receptor function in vivo. Introducing human Hip into yeast enhances hormone-dependent activation of a reporter gene by glucocorticoid receptor (GR). Because Hip does not similarly enhance signaling by mineralocorticoid, progesterone, or estrogen receptors, a general effect on transcription can be excluded. Instead, Hip promotes functional maturation of GR without increasing steady-state levels of GR protein. Unexpectedly, Hip binding to Hsp70 is not critical for boosting GR responsiveness to hormone. In conclusion, Hip functions by a previously unrecognized mechanism to promote the efficiency of GR maturation in cells.

  11. Modelling the glucocorticoid receptor and producing therapeutic agents with anti-inflammatory effects but reduced side-effects.

    PubMed

    McMaster, Andrew; Ray, David William

    2007-03-01

    Glucocorticoid hormones exert a wide spectrum of metabolic and immunological effects. They are synthesized from a cholesterol precursor and are structurally related to the other steroid hormones, progesterone, aldosterone and oestrogen. They act through the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily. The GR is an intracellular receptor; the hydrophobic ligand accesses its receptor by diffusion across the plasma membrane. The ligand-activated GR translocates to the nucleus to regulate expression of its target genes. The GR, in common with the rest of the receptor family, can be functionally divided into an N-terminal transcription activation domain, a central DNA binding domain and a C-terminal ligand binding domain, which also includes a second transactivation domain. Although synthetic glucocorticoids are the most potent anti-inflammatory agents known, their use is limited owing to the range and severity of their side-effects. The structure of the ligand binding domain of the glucocorticoid receptor has now been solved, and a series of studies has shown that even subtle changes to the ligand structure alter the final conformation of the ligand-receptor complex, with consequences for further protein recruitment and for the function of the receptor. This, coupled with the successful development of selective oestrogen receptor agonists, has led to concerted efforts to find selective GR ligands, with preserved beneficial anti-inflammatory activity, but reduced side-effect profile. Current efforts have identified several useful tool compounds, and further molecules are in development in several pharmaceutical companies.

  12. Development of glucocorticoid receptor regulation in the rat forebrain: Implications for adverse effects of glucocorticoids in preterm infants

    EPA Science Inventory

    Glucocorticoids are the consensus treatment to avoid respiratory distress in preterm infants but there is accumulating evidence that these agents evoke long-term neurobehavioral deficits. Earlier, we showed that the developing rat forebrain is far more sensitive to glucocorticoi...

  13. Assessing the Dynamics of Nuclear Glucocorticoid-Receptor Complex: Adding Flexibility to Gene Expression Modeling1

    PubMed Central

    Hazra, Anasuya; DuBois, Debra C.; Almon, Richard R.; Jusko, William J.

    2014-01-01

    A retrospective analysis was performed to modify our fourth-generation pharmacodynamic model for glucocorticoid receptor (GR) dynamics with incorporation of more physiological features. This modified model was developed by integrating previously reported free cytosolic GR and GR mRNA data following single (10, 50 mg/kg) and dual (50 mg/kg at 0 and 24 hr) intravenous doses of methylprednisolone (MPL) in adrenalectomized (ADX) male Wistar rats with several in vitro studies describing real-time kinetics of the transfer of rat steroid-receptor complex from the cell cytosol to the nucleus. Additionally, free hepatic cytosolic GR and its mRNA data from a chronic infusion dosing study of MPL (0.1 and 0.3 mg/kg/hr) in male ADX Wistar rats were used to verify the predictability of the model. Incorporation of information regarding in vitro receptor kinetics allowed us to describe the receptor-mediated pharmacogenomic effects of MPL for a larger variety of genes in rat liver from microarray studies. These included early responsive gene like CCAAT/enhancer binding protein-β (CEBP-β), a transcription factor, as well as the later responsive gene for tyrosine aminotransferase (TAT), a classical biomarker of glucocorticoid (GC) genomic effects. This more mechanistic model of GR dynamics can be applied to characterize profiles for a greater number of genes in liver. PMID:17285360

  14. Glucocorticoid receptor ligand binding domain is sufficient for the modulation of glucocorticoid induction properties by homologous receptors, coactivator transcription intermediary factor 2, and Ubc9.

    PubMed

    Cho, Sehyung; Kagan, Benjamin L; Blackford, John A; Szapary, Daniele; Simons, S Stoney

    2005-02-01

    Several factors modulate the position of the dose-response curve of steroid receptor-agonist complexes and the partial agonist activity of antagonist complexes, thereby causing differential gene activation by circulating hormones and unequal gene repression during endocrine therapies with antisteroids. We now ask whether the modulatory activity of three factors (homologous receptor, coactivator transcription intermediary factor 2, and Ubc9) requires the same or different domains of glucocorticoid receptors (GRs). In all cases, we find that neither the amino terminal half of the receptor, which contains the activation function-1 activation domain, nor the DNA binding domain is required. This contrasts with the major role of activation function-1 in determining the amount of gene expression and partial agonist activity of antisteroids with most steroid receptors. However, the situation is more complicated with Ubc9, where GR N-terminal sequences prevent the actions of Ubc9, but not added GR or transcription intermediary factor 2, at low GR concentrations. Inhibition is relieved by deletion of these sequences or by replacement with the comparable region of progesterone receptors but not by overexpression of the repressive sequences. These results plus the binding of C-terminal GR sequences to the suppressive N-terminal domain implicate an intramolecular mechanism for the inhibition of Ubc9 actions at low GR concentrations. A shift from noncooperative to cooperative steroid binding at high GR concentrations suggests that conformational changes reposition the inhibitory N-terminal sequence to allow Ubc9 interaction with elements of the ligand binding domain. Collectively, these results indicate a dominant role of GR C-terminal sequences in the modulation of the dose-response curve and partial agonist activity of GR complexes. They also reveal mechanistic differences both among individual modulators and between the ability of the same factors to regulate the total amount

  15. Androgen receptor silences thioredoxin-interacting protein and competitively inhibits glucocorticoid receptor-mediated apoptosis in pancreatic β-Cells.

    PubMed

    Harada, Naoki; Katsuki, Takahiro; Takahashi, Yuji; Masuda, Tatsuya; Yoshinaga, Mariko; Adachi, Tetsuya; Izawa, Takeshi; Kuwamura, Mitsuru; Nakano, Yoshihisa; Yamaji, Ryoichi; Inui, Hiroshi

    2015-06-01

    Androgen receptor (AR) is known to bind to the same cis-element that glucocorticoid receptor (GR) binds to. However, the effects of androgen signaling on glucocorticoid signaling have not yet been elucidated. Here, we investigated the effects of testosterone on dexamethasone (DEX, a synthetic glucocorticoid)-induced apoptosis of pancreatic β-cells, which might be involved in the pathogenesis of type 2 diabetes mellitus in males. We used INS-1 #6 cells, which were isolated from the INS-1 pancreatic β-cell line and which express high levels of AR. Testosterone and dihydrotestosterone inhibited apoptosis induced by DEX in INS-1 #6 cells. AR knockdown and the AR antagonist hydroxyflutamide each diminished the anti-apoptotic effects of testosterone. AR was localized in the nucleus of both INS-1 #6 cells and pancreatic β-cells of male rats. Induction of thioredoxin-interacting protein (TXNIP) is known to cause pro-apoptotic effects in β-cells. Testosterone suppressed the DEX-induced increase of TXNIP at the transcriptional level. A Chromatin immunoprecipitation assays showed that both AR and GR competitively bound to the TXNIP promoter in ligand-dependent manners. Recombinant DNA-binding domain of AR bound to the same cis-element of the TXNIP promoter that GR binds to. Our results show that AR and GR competitively bind to the same cis-element of TXNIP promoter as a silencer and enhancer, respectively. These results indicate that androgen signaling functionally competes with glucocorticoid signaling in pancreatic β-cell apoptosis. PMID:25639671

  16. Endocrine-Disrupting Effects of Pesticides through Interference with Human Glucocorticoid Receptor.

    PubMed

    Zhang, Jianyun; Zhang, Jing; Liu, Rui; Gan, Jay; Liu, Jing; Liu, Weiping

    2016-01-01

    Many pesticides have been identified as endocrine-disrupting chemicals (EDCs) due to their ability to bind sex-steroid hormone receptors. However, little attention has been paid to the ability of pesticides to interfere with other steroid hormone receptors such as glucocorticoid receptor (GR) that plays a critical role in metabolic, endocrine, immune, and nervous systems. In this study, the glucocorticoidic and antiglucocorticoidic effects of 34 pesticides on human GR were investigated using luciferase reporter gene assay. Surprisingly, none of the test chemicals showed GR agonistic activity, but 12 chemicals exhibited apparent antagonistic effects. Bifenthrin, λ-cyhalothrin, cypermethrin, resmethrin, o,p'-DDT, p,p'-DDT, methoxychlor, ethiofencarb, and tolylfluanid showed remarkable GR antagonistic properties with RIC20 values lower than 10(-6) M. The disruption of glucocorticoid-responsive genes in H4IIE and J774A.1 cells was further evaluated on these 12 GR antagonists. In H4IIEcells, four organochlorine insecticides, bifenthrin, and 3-PBA decreased cortisol-induced PEPCK gene expression, while o,p'-DDT and methoxychlor inhibited cortisol-stimulated Arg and TAT gene expression. Cypermethrin and tolyfluanid attenuated cortisol-induced TAT expression. In J774A.1 cells, λ-cyhalothrin, resmethrin, 3-PBA, o,p'-DDT, p,p'-DDT, p,p'-DDE, methoxychlor- and tolylfluanid-reduced cortisol-stimulated GILZ expression. Furthermore, molecular docking simulation indicated that different interactions may stabilize the binding between molecules and GR. Our findings suggest that comprehensive screening and evaluation of GR antagonists and agonists should be considered to better understand the health and ecological risks of man-made chemicals such as pesticides.

  17. Endocrine-Disrupting Effects of Pesticides through Interference with Human Glucocorticoid Receptor.

    PubMed

    Zhang, Jianyun; Zhang, Jing; Liu, Rui; Gan, Jay; Liu, Jing; Liu, Weiping

    2016-01-01

    Many pesticides have been identified as endocrine-disrupting chemicals (EDCs) due to their ability to bind sex-steroid hormone receptors. However, little attention has been paid to the ability of pesticides to interfere with other steroid hormone receptors such as glucocorticoid receptor (GR) that plays a critical role in metabolic, endocrine, immune, and nervous systems. In this study, the glucocorticoidic and antiglucocorticoidic effects of 34 pesticides on human GR were investigated using luciferase reporter gene assay. Surprisingly, none of the test chemicals showed GR agonistic activity, but 12 chemicals exhibited apparent antagonistic effects. Bifenthrin, λ-cyhalothrin, cypermethrin, resmethrin, o,p'-DDT, p,p'-DDT, methoxychlor, ethiofencarb, and tolylfluanid showed remarkable GR antagonistic properties with RIC20 values lower than 10(-6) M. The disruption of glucocorticoid-responsive genes in H4IIE and J774A.1 cells was further evaluated on these 12 GR antagonists. In H4IIEcells, four organochlorine insecticides, bifenthrin, and 3-PBA decreased cortisol-induced PEPCK gene expression, while o,p'-DDT and methoxychlor inhibited cortisol-stimulated Arg and TAT gene expression. Cypermethrin and tolyfluanid attenuated cortisol-induced TAT expression. In J774A.1 cells, λ-cyhalothrin, resmethrin, 3-PBA, o,p'-DDT, p,p'-DDT, p,p'-DDE, methoxychlor- and tolylfluanid-reduced cortisol-stimulated GILZ expression. Furthermore, molecular docking simulation indicated that different interactions may stabilize the binding between molecules and GR. Our findings suggest that comprehensive screening and evaluation of GR antagonists and agonists should be considered to better understand the health and ecological risks of man-made chemicals such as pesticides. PMID:26647222

  18. Insights on Glucocorticoid Receptor Activity Modulation through the Binding of Rigid Steroids

    PubMed Central

    Presman, Diego M.; Alvarez, Lautaro D.; Levi, Valeria; Eduardo, Silvina; Digman, Michelle A.; Martí, Marcelo A.; Veleiro, Adriana S.; Burton, Gerardo; Pecci, Adali

    2010-01-01

    Background The glucocorticoid receptor (GR) is a transcription factor that regulates gene expression in a ligand-dependent fashion. This modular protein is one of the major pharmacological targets due to its involvement in both cause and treatment of many human diseases. Intense efforts have been made to get information about the molecular basis of GR activity. Methodology/Principal Findings Here, the behavior of four GR-ligand complexes with different glucocorticoid and antiglucocorticoid properties were evaluated. The ability of GR-ligand complexes to oligomerize in vivo was analyzed by performing the novel Number and Brightness assay. Results showed that most of GR molecules form homodimers inside the nucleus upon ligand binding. Additionally, in vitro GR-DNA binding analyses suggest that ligand structure modulates GR-DNA interaction dynamics rather than the receptor's ability to bind DNA. On the other hand, by coimmunoprecipitation studies we evaluated the in vivo interaction between the transcriptional intermediary factor 2 (TIF2) coactivator and different GR-ligand complexes. No correlation was found between GR intranuclear distribution, cofactor recruitment and the homodimerization process. Finally, Molecular determinants that support the observed experimental GR LBD-ligand/TIF2 interaction were found by Molecular Dynamics simulation. Conclusions/Significance The data presented here sustain the idea that in vivo GR homodimerization inside the nucleus can be achieved in a DNA-independent fashion, without ruling out a dependent pathway as well. Moreover, since at least one GR-ligand complex is able to induce homodimer formation while preventing TIF2 coactivator interaction, results suggest that these two events might be independent from each other. Finally, 21-hydroxy-6,19-epoxyprogesterone arises as a selective glucocorticoid with potential pharmacological interest. Taking into account that GR homodimerization and cofactor recruitment are considered essential

  19. Antenatal hypoxia induces epigenetic repression of glucocorticoid receptor and promotes ischemic-sensitive phenotype in the developing heart.

    PubMed

    Xiong, Fuxia; Lin, Thant; Song, Minwoo; Ma, Qingyi; Martinez, Shannalee R; Lv, Juanxiu; MataGreenwood, Eugenia; Xiao, Daliao; Xu, Zhice; Zhang, Lubo

    2016-02-01

    Large studies in humans and animals have demonstrated a clear association of an adverse intrauterine environment with an increased risk of cardiovascular disease later in life. Yet mechanisms remain largely elusive. The present study tested the hypothesis that gestational hypoxia leads to promoter hypermethylation and epigenetic repression of the glucocorticoid receptor (GR) gene in the developing heart, resulting in increased heart susceptibility to ischemia and reperfusion injury in offspring. Hypoxic treatment of pregnant rats from day 15 to 21 of gestation resulted in a significant decrease of GR exon 14, 15, 16, and 17 transcripts, leading to down-regulation of GR mRNA and protein in the fetal heart. Functional cAMP-response elements (CREs) at -4408 and -3896 and Sp1 binding sites at -3425 and -3034 were identified at GR untranslated exon 1 promoters. Hypoxia significantly increased CpG methylation at the CREs and Sp1 binding sites and decreased transcription factor binding to GR exon 1 promoter, accounting for the repression of the GR gene in the developing heart. Of importance, treatment of newborn pups with 5-aza-2'-deoxycytidine reversed hypoxia-induced promoter methylation, restored GR expression and prevented hypoxia-mediated increase in ischemia and reperfusion injury of the heart in offspring. The findings demonstrate a novel mechanism of epigenetic repression of the GR gene in fetal stress-mediated programming of ischemic-sensitive phenotype in the heart. PMID:26779948

  20. Novel Antidepressant-Like Activity of Propolis Extract Mediated by Enhanced Glucocorticoid Receptor Function in the Hippocampus

    PubMed Central

    Kim, Young Han; Park, Wan-Soon; Ahn, Won Gyeong; Park, Ok Kyu; Kwon, Seung-Hae; Morita, Kyoji; Shim, Insop; Her, Song

    2013-01-01

    Propolis is a natural product made by honeybees that has been widely used in folk medicine with a broad spectrum of biological activities. To investigate the antidepressant-like activity of propolis extract, CD-1 mice were administered an ethanol extract of propolis (50, 100, or 200 mg/kg, p.o.) prior to the behavioral test. The propolis extract-treated group showed a dose-dependent decrease in immobility time in the FST and tail suspension test without altering locomotor activity. Propolis extract decreased the limbic hypothalamic-pituitary-adrenal axis response to the FST as indicated by an attenuated corticosterone response and decreased in c-fos immunoreactive neurons in the hippocampal dentate gyrus. Western blot analysis revealed a reduction in hippocampal glucocorticoid receptor (GR) expression following the FST, which was reversed by propolis extract. Propolis extract also increased pGR(S220)/(S234) ratio by a differential phosphorylation in S220 and S234. FST-induced downregulation of cAMP-responsive element binding protein phosphorylation at S133 (pCREB) was restored by propolis extract, showing a strong and positive relationship between pCREB and pGR(S220)/(S234) ratio. These findings suggest that the propolis extract potentiates antidepressant-like activity by enhancing GR function which is one of the therapeutic mechanisms of antidepressant; thus, propolis extract may provide a novel therapy for depression. PMID:23853655

  1. Differential Expression of Glucocorticoid Receptor Noncoding RNA Repressor Gas5 in Autoimmune and Inflammatory Diseases.

    PubMed

    Mayama, T; Marr, A K; Kino, T

    2016-08-01

    Glucocorticoids have strong regulatory actions on the immune system and act as potent therapeutic compounds for autoimmune and inflammatory diseases. We previously reported that the long noncoding RNA growth arrest-specific 5 (Gas5), which accumulates inside the cells in response to cellular starvation/growth arrest, functions as a potent repressor of the glucocorticoid receptor (GR) through its RNA "glucocorticoid response element (GRE)". To evaluate potential roles of Gas5 in immune-related disorders, we examined Gas5 RNA levels in various autoimmune, inflammatory, and infectious diseases using the microarray data available in the Gene Expression Omnibus. We found that Gas5 levels were altered in whole blood or leukocytes of the patients with rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and sarcoidosis. Gas5 levels were also altered in infectious diseases, such as by the human immunodeficiency virus type-1 and influenza virus, and bacterial sepsis. In our experimental analysis using mice, Gas5 levels were kept at high basal levels and did not respond to fasting in immune organs, such as spleen and thymus, while its levels in metabolic organs, including liver, fat, and skeletal muscles, were low at baseline and were highly elevated upon this treatment, possibly through suppression of the mTOR pathway. These results suggest that Gas5 plays a role in the regulation of immune functions and pathogenesis/pathophysiology of autoimmune, inflammatory, and infectious diseases in part through modulation of the GR transcriptional activity via its decoy RNA "GRE". Changes in the Gas5 levels may also influence disease response to immunosuppressive glucocorticoid therapy. PMID:27214311

  2. Forebrain glucocorticoid receptor gene deletion attenuates behavioral changes and antidepressant responsiveness during chronic stress.

    PubMed

    Jacobson, Lauren

    2014-10-01

    Stress is an important risk factor for mood disorders. Stress also stimulates the secretion of glucocorticoids, which have been found to influence mood. To determine the role of forebrain glucocorticoid receptors (GR) in behavioral responses to chronic stress, the present experiments compared behavioral effects of repeated social defeat in mice with forebrain GR deletion and in floxed GR littermate controls. Repeated defeat produced alterations in forced swim and tail suspension immobility in floxed GR mice that did not occur in mice with forebrain GR deletion. Defeat-induced changes in immobility in floxed GR mice were prevented by chronic antidepressant treatment, indicating that these behaviors were dysphoria-related. In contrast, although mice with forebrain GR deletion exhibited antidepressant-induced decreases in tail suspension immobility in the absence of stress, this response did not occur in mice with forebrain GR deletion after defeat. There were no marked differences in plasma corticosterone between genotypes, suggesting that behavioral differences depended on forebrain GR rather than on abnormal glucocorticoid secretion. Defeat-induced gene expression of the neuronal activity marker c-fos in the ventral hippocampus, paraventricular thalamus and lateral septum correlated with genotype-related differences in behavioral effects of defeat, whereas c-fos induction in the nucleus accumbens and central and basolateral amygdala correlated with genotype-related differences in behavioral responses to antidepressant treatment. The dependence of both negative (dysphoria-related) and positive (antidepressant-induced) behaviors on forebrain GR is consistent with the contradictory effects of glucocorticoids on mood, and implicates these or other forebrain regions in these effects.

  3. Forebrain glucocorticoid receptor gene deletion attenuates behavioral changes and antidepressant responsiveness during chronic stress

    PubMed Central

    Jacobson, Lauren

    2014-01-01

    Stress is an important risk factor for mood disorders. Stress also stimulates the secretion of glucocorticoids, which have been found to influence mood. To determine the role of forebrain glucocorticoid receptors (GR) in behavioral responses to chronic stress, the present experiments compared behavioral effects of repeated social defeat in mice with forebrain GR deletion and in floxed GR littermate controls. Repeated defeat produced alterations in forced swim and tail suspension immobility in floxed GR mice that did not occur in mice with forebrain GR deletion. Defeat-induced changes in immobility in floxed GR mice were prevented by chronic antidepressant treatment, indicating that these behaviors were dysphoria-related. In contrast, although mice with forebrain GR deletion exhibited antidepressant-induced decreases in tail suspension immobility in the absence of stress, this response did not occur in mice with forebrain GR deletion after defeat. There were no marked differences in plasma corticosterone between genotypes, suggesting that behavioral differences depended on forebrain GR rather than on abnormal glucocorticoid secretion. Defeat-induced gene expression of the neuronal activity marker c-fos in the ventral hippocampus, paraventricular thalamus and lateral septum correlated with genotype-related differences in behavioral effects of defeat, whereas c-fos induction in the nucleus accumbens and central and basolateral amygdala correlated with genotype-related differences in behavioral responses to antidepressant treatment. The dependence of both negative (dysphoria-related) and positive (antidepressant-induced) behaviors on forebrain GR is consistent with the contradictory effects of glucocorticoids on mood, and implicates these or other forebrain regions in these effects. PMID:25168761

  4. Gender differences in response of hippocampus to chronic glucocorticoid stress: role of glutamate receptors.

    PubMed

    Liu, Howard H; Payne, H Ross; Wang, Bin; Brady, Scott T

    2006-04-01

    Glucocorticoids (GC) play critical roles in the pathophysiological reactions to environmental stress. In brain, morphological changes were examined in hippocampal CA3 neurons with 2 weeks of chronic elevation of GC in male and female mice. Molecular correlates and underlying mechanisms paralleling these morphologic changes in hippocampus were investigated. Although the hippocampal neurons in the CA3 area in male mice atrophy with chronically elevated GC, female mice show minimal morphological changes with comparable GC regimens. These sexual morphological differences correlate with differences in the postsynaptic dense protein (PSD95) as well as the spectrum of glutamate receptors induced by GC treatment in male and female mice, including NMDA, AMPA, and KA receptors. These findings suggest that synaptic receptor composition is adapted to the unique physiological requirements of males and females and illuminate underlying mechanisms of GC/stress responses in the brain.

  5. Do smoking intensity-related differences in vigilance indicate altered glucocorticoid receptor sensitivity?

    PubMed

    Reuter, Martin; Hennig, Juergen; Netter, Petra

    2004-03-01

    The relationship of critical flicker fusion frequency (CFF) and a pharmacologically induced cortisol suppression by means of dexamethasone (DEX) and metyrapone (MET) was investigated during nicotine deprivation in a between-subjects design in 60 male smokers divided into light, medium and heavy smokers. DEX reduced vigilance in medium smokers and improved it in heavy smokers compared to placebo, whereas MET was more detrimental in heavy smokers. The hypothesis was put forward that the intensity of nicotine consumption is related to differences in glucocorticoid and mineralocorticoid receptor sensitivity.

  6. Discovery of acylurea isosteres of 2-acylaminothiadiazole in the azaxanthene series of glucocorticoid receptor agonists.

    PubMed

    Gong, Hua; Yang, Michael; Xiao, Zili; Doweyko, Arthur M; Cunningham, Mark; Wang, Jinhong; Habte, Sium; Holloway, Deborah; Burke, Christine; Shuster, David; Gao, Ling; Carman, Julie; Somerville, John E; Nadler, Steven G; Salter-Cid, Luisa; Barrish, Joel C; Weinstein, David S

    2014-08-01

    Acylureas and acyclic imides are found to be excellent isosteres for 2-acylamino-1,3,4-thiadiazole in the azaxanthene-based series of glucocorticoid receptor (GR) agonists. The results reported herein show that primary acylureas maintain high affinity and selectivity for GR while providing improved CYP450 inhibition and pharmacokinetic profile over 2-acylamino-1,3,4-thiadiazoles. General methods for synthesis of a variety of acylureas and acyclic imides from a carboxylic acid were utilized and are described. PMID:24980053

  7. Dimethyl-diphenyl-propanamide derivatives as nonsteroidal dissociated glucocorticoid receptor agonists.

    PubMed

    Yang, Bingwei V; Weinstein, David S; Doweyko, Lidia M; Gong, Hua; Vaccaro, Wayne; Huynh, Tram; Xiao, Hai-Yun; Doweyko, Arthur M; McKay, Lorraine; Holloway, Deborah A; Somerville, John E; Habte, Sium; Cunningham, Mark; McMahon, Michele; Townsend, Robert; Shuster, David; Dodd, John H; Nadler, Steven G; Barrish, Joel C

    2010-12-01

    A series of 2,2-dimethyl-3,3-diphenyl-propanamides as novel glucocorticoid receptor modulators is reported. SAR exploration led to the identification of 4-hydroxyphenyl propanamide derivatives displaying good agonist activity in GR-mediated transrepression assays and reduced agonist activity in GR-mediated transactivation assays. Compounds 17 and 30 showed anti-inflammatory activity comparable to prednisolone in the rat carrageenan-induced paw edema model, with markedly decreased side effects with regard to increases in blood glucose and expression of hepatic tyrosine aminotransferase. A hypothetical binding mode accounting for the induction of the functional activity by a 4-hydroxyl group is proposed. PMID:21073190

  8. A Novel Point Mutation in Helix 10 of the Human Glucocorticoid Receptor Causes Generalized Glucocorticoid Resistance by Disrupting the Structure of the Ligand-Binding Domain

    PubMed Central

    Nader, Nancy; Bachrach, Bert E.; Hurt, Darrell E.; Gajula, Sonia; Pittman, Amy; Lescher, Rachel; Kino, Tomoshige

    2010-01-01

    Context: Generalized glucocorticoid resistance syndrome is a rare familial or sporadic condition characterized by partial insensitivity to glucocorticoids, caused by mutations in the glucocorticoid receptor (GR) gene. Most of the reported cases are adults, demonstrating symptoms associated with mineralocorticoid and/or adrenal androgen excess caused by compensatively increased secretion of the adrenocorticotropic hormone. Patient: We identified a new 2-yr-old female case of generalized glucocorticoid resistance syndrome. The patient (TJ) presented with a generalized seizure associated with hypoglycemia and hypokalemia. She also had hypertension and premature pubarche, whereas dexamethasone effectively suppressed these clinical manifestations. Results: The patient’s GR gene had a heterozygotic mutation (G→A) at nucleotide position 2141 (exon 8), which resulted in substitution of arginine by glutamine at amino acid position 714 in the ligand-binding domain (LBD) of the GRα. Molecular analysis revealed that the mutant receptor had significantly impaired transactivation activity with a 2-fold reduction in affinity to ligand. It showed attenuated transactivation of the activation function (AF)-2 and reduced binding to a p160 nuclear receptor coactivator. Computer-based structural analysis revealed that replacement of arginine by glutamine at position 714 transmitted a conformational change to the LBD and the AF-2 transactivation surface, resulting in a decreased binding affinity to ligand and to the LXXLL coactivator motif. Conclusions: Dexamethasone treatment is effective in controlling the premature pubarche, hypoglycemia, hypertension, and hypokalemia in this child case, wherein arginine 714 plays a key role in the proper formation of the ligand-binding pocket and the AF-2 surface of the GRα LBD. PMID:20335448

  9. Cannabinoid CB1 receptor mediates glucocorticoid effects on hormone secretion induced by volume and osmotic changes.

    PubMed

    Ruginsk, S G; Uchoa, E T; Elias, L L K; Antunes-Rodrigues, J

    2012-02-01

    The present study provides the first in vivo evidence that the cannabinoid CB(1) receptor mediates the effects of dexamethasone on hormone release induced by changes in circulating volume and osmolality. Male adult rats were administered with the CB(1) receptor antagonist rimonabant (10 mg/Kg, p.o.), followed or not in 1 hour by dexamethasone (1 mg/Kg, i.p.). Extracellular volume expansion (EVE, 2 mL/100 g of body weight, i.v.) was performed 2 hours after dexamethasone or vehicle treatment using either isotonic (I-EVE, 0.15 mol/L) or hypertonic (H-EVE, 0.30 mol/L) NaCl solution. Five minutes after EVE, animals were decapitated and trunk blood was collected for all plasma measurements. Rimonabant potentiated oxytocin (OT) secretion induced by H-EVE and completely reversed the inhibitory effects of dexamethasone in response to the same stimulus. These data suggest that glucocorticoid modulation of OT release is mediated by the CB(1) receptor. Although dexamethasone did not affect vasopressin (AVP) secretion induced by H-EVE, the administration of rimonabant potentiated AVP release in response to the same stimulus, supporting the hypothesis that the CB(1) receptor regulates AVP secretion independently of glucocorticoid-mediated signalling. Dexamethasone alone did not affect atrial natriuretic peptide (ANP) release stimulated by I-EVE or H-EVE. However, pretreatment with rimonabant potentiated ANP secretion induced by H-EVE, suggesting a possible role for the CB(1) receptor in the control of peripheral factors that modulate cardiovascular function. Rimonabant also reversed the inhibitory effects of dexamethasone on H-EVE-induced corticosterone secretion, reinforcing the hypothesis that the CB(1) receptor may be involved in the negative feedback exerted by glucocorticoids on the activity of the hypothalamic-pituitary-adrenal axis. Collectively, the results of the present study indicate that the CB(1) receptor modulates neurohypophyseal hormone secretion and

  10. Noncoding RNA Gas5 Is a Growth Arrest and Starvation-Associated Repressor of the Glucocorticoid Receptor

    PubMed Central

    Kino, Tomoshige; Hurt, Darrell E.; Ichijo, Takamasa; Nader, Nancy; Chrousos, George P.

    2010-01-01

    The availability of nutrients influences cellular growth and survival by affecting gene transcription. Glucocorticoids also influence gene transcription and have diverse activities on cell growth, energy expenditure, and survival. We found that the growth arrest-specific 5 (Gas5) noncoding RNA, which is abundant in cells whose growth has been arrested due to lack of nutrients or growth factors, sensitized cells to apoptosis by suppressing glucocorticoid-mediated induction of several responsive genes, including the one encoding cellular inhibitor of apoptosis 2. Gas5 bound to the DNA-binding domain of the glucocorticoid receptor (GR) by acting as a decoy “glucocorticoid response element (GRE)”, thus, competing with DNA GREs for binding to the GR. We conclude that Gas5 is a ribo-repressor of the GR, influencing cell survival and metabolic activities during starvation by modulating the transcriptional activity of the GR. PMID:20124551

  11. Glucocorticoid regulation of insulin receptor and substrate IRS-1 tyrosine phosphorylation in rat skeletal muscle in vivo.

    PubMed Central

    Giorgino, F; Almahfouz, A; Goodyear, L J; Smith, R J

    1993-01-01

    To test the hypothesis that glucocorticoid-induced insulin resistance might originate from abnormalities in insulin receptor signaling, we investigated the effects of glucocorticoids on in vivo tyrosine phosphorylation of the insulin receptor and the insulin receptor substrate IRS-1 in rat skeletal muscle. Male Sprague-Dawley rats were treated with cortisone (100 mg/kg for 5 d) and compared to pair-fed controls. Cortisone treatment of rats resulted in both hyperglycemia and hyperinsulinemia. Anesthetized animals were injected with 10 U/kg insulin via cardiac puncture and, after 2 min, hindlimb muscles were removed, snap-frozen, and homogenized in SDS. Protein tyrosine phosphorylation was studied by immunoblotting with phosphotyrosine antibody. Insulin receptors and substrate IRS-1 were identified and quantified with specific antibodies. Cortisone treatment increased the amount of insulin receptor protein by 36%, but decreased the total level of receptor tyrosine phosphorylation (69 +/- 4% of control, P < 0.05). The decreased level of receptor phosphorylation was explained by a reduced number of receptors containing phosphorylated tyrosine residues (64.6 +/- 5% of control, P < 0.05). Glucocorticoid excess decreased skeletal muscle IRS-1 content by 50%, but did not significantly alter the total level of IRS-1 tyrosine phosphorylation. The apparent M(r) of IRS-1 was reduced by approximately 10 kD. Treatment with protein phosphatase-2A reduced IRS-1 M(r) in control but not in glucocorticoid-treated muscle indicating that the lower M(r) likely results from lower phosphoserine and/or phosphothreonine content. To investigate the role of hyperinsulinemia in the glucocorticoid response, rats were made insulin-deficient with streptozotocin (100 mg/kg, i.p.). Subsequent treatment with cortisone for 5 d had no effects on insulin levels, tyrosine phosphorylation of insulin receptors or IRS-1, or the M(r) of IRS-1. In conclusion, glucocorticoid-treated skeletal muscle is

  12. Glucocorticoid Receptor Activity Contributes to Resistance to Androgen-Targeted Therapy in Prostate Cancer

    PubMed Central

    Isikbay, Masis; Otto, Kristen; Kregel, Steven; Kach, Jacob; Cai, Yi; Vander Griend, Donald J.; Conzen, Suzanne D.

    2015-01-01

    Despite new treatments for castrate-resistant prostate cancer (CRPC), the prognosis of patients with CRPC remains bleak due to acquired resistance to androgen receptor (AR)-directed therapy. The glucocorticoid receptor (GR) and AR share several transcriptional targets, including the anti-apoptotic genes serum and glucocorticoid-regulated kinase 1 (SGK1) and Map kinase phosphatase 1 (MKP1)/dual specificity phosphatase 1 (DUSP1). Because GR expression increases in a subset of primary prostate cancer (PC) following androgen deprivation therapy, we sought to determine whether GR activation can contribute to resistance to AR-directed therapy. We studied CWR-22Rv1 and LAPC4 AR/GR-expressing PC cell lines following treatment with combinations of the androgen R1881, AR antagonist MDV3100, GR agonist dexamethasone, GR antagonists mifepristone and CORT 122928, or the SGK1 inhibitor GSK650394. Cell lines stably expressing GR (NR3C1)-targeted shRNA or ectopic SGK1-Flag were also studied in vivo. GR activation diminished the effects of the AR antagonist MDV3100 on tumor cell viability. In addition, GR activation increased prostate-specific antigen (PSA) secretion and induced SGKI and MKP1/DUSP gene expression. Glucocorticoid-mediated cell viability was diminished by a GR antagonist or by co-treatment with the SGK1 inhibitor GSK650394. In vivo, GR depletion delayed castrate-resistant tumor formation, while SGK1-Flag-overexpressing PC xenografts displayed accelerated castrate-resistant tumor initiation, supporting a role for SGK1 in GR-mediated CRPC progression. We studied several PC models before and following treatment with androgen blockade and found that increased GR expression and activity contributed to tumor-promoting PC cell viability. Increased GR-regulated SGK1 expression appears, at least in part, to mediate enhanced PC cell survival. Therefore, GR and/or SGK1 inhibition may be useful adjuncts to AR blockade for treating CRPC. PMID:24615402

  13. Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor

    SciTech Connect

    Carroll, Sean Michael; Ortlund, Eric A; Thornton, Joseph W.

    2012-03-16

    Understanding the genetic, structural, and biophysical mechanisms that caused protein functions to evolve is a central goal of molecular evolutionary studies. Ancestral sequence reconstruction (ASR) offers an experimental approach to these questions. Here we use ASR to shed light on the earliest functions and evolution of the glucocorticoid receptor (GR), a steroid-activated transcription factor that plays a key role in the regulation of vertebrate physiology. Prior work showed that GR and its paralog, the mineralocorticoid receptor (MR), duplicated from a common ancestor roughly 450 million years ago; the ancestral functions were largely conserved in the MR lineage, but the functions of GRs - reduced sensitivity to all hormones and increased selectivity for glucocorticoids - are derived. Although the mechanisms for the evolution of glucocorticoid specificity have been identified, how reduced sensitivity evolved has not yet been studied. Here we report on the reconstruction of the deepest ancestor in the GR lineage (AncGR1) and demonstrate that GR's reduced sensitivity evolved before the acquisition of restricted hormone specificity, shortly after the GR-MR split. Using site-directed mutagenesis, X-ray crystallography, and computational analyses of protein stability to recapitulate and determine the effects of historical mutations, we show that AncGR1's reduced ligand sensitivity evolved primarily due to three key substitutions. Two large-effect mutations weakened hydrogen bonds and van der Waals interactions within the ancestral protein, reducing its stability. The degenerative effect of these two mutations is extremely strong, but a third permissive substitution, which has no apparent effect on function in the ancestral background and is likely to have occurred first, buffered the effects of the destabilizing mutations. Taken together, our results highlight the potentially creative role of substitutions that partially degrade protein structure and function and

  14. mu-Opioid receptor downregulation contributes to opioid tolerance in vivo.

    PubMed

    Stafford, K; Gomes, A B; Shen, J; Yoburn, B C

    2001-01-01

    The present study examined the contribution of downregulation of mu-opioid receptors to opioid tolerance in an intact animal model. Mice were implanted subcutaneously with osmotic minipumps that infused etorphine (50-250 microg/kg/day) for 7 days. Other mice were implanted subcutaneously with a morphine pellet (25 mg) or a morphine pellet plus an osmotic minipump that infused morphine (5-40 mg/kg/day) for 7 days. Controls were implanted with an inert placebo pellet. At the end of treatment, pumps and pellets were removed, and saturation binding studies were conducted in whole brain ([3H]DAMGO) or morphine and etorphine analgesic ED(50)s were determined (tail-flick). Morphine tolerance increased linearly with the infusion dose of morphine (ED(50) shift at highest infusion dose, 4.76). No significant downregulation of mu-receptors in whole brain was observed at the highest morphine treatment dose. Etorphine produced dose-dependent downregulation of mu-opioid receptor density and tolerance (ED(50) shift at highest infusion dose, 6.97). Downregulation of mu-receptors only occurred at the higher etorphine infusion doses (> or =150 microg/kg/day). Unlike morphine tolerance, the magnitude of etorphine tolerance was a nonlinear function of the dose and increased markedly at infusion doses that produced downregulation. These results suggest that mu-opioid receptor downregulation contributes to opioid tolerance in vivo. Therefore, opioid tolerance appears to rely upon both "receptor density-dependent" and " receptor density-independent" mechanisms.

  15. Effects of histamine H1 receptor signaling on glucocorticoid receptor activity. Role of canonical and non-canonical pathways

    PubMed Central

    Zappia, Carlos Daniel; Granja-Galeano, Gina; Fernández, Natalia; Shayo, Carina; Davio, Carlos; Fitzsimons, Carlos P.; Monczor, Federico

    2015-01-01

    Histamine H1 receptor (H1R) antagonists and glucocorticoid receptor (GR) agonists are used to treat inflammatory conditions such as allergic rhinitis, atopic dermatitis and asthma. Consistent with the high morbidity levels of such inflammatory conditions, these receptors are the targets of a vast number of approved drugs, and in many situations their ligands are co-administered. However, this drug association has no clear rationale and has arisen from clinical practice. We hypothesized that H1R signaling could affect GR-mediated activity, impacting on its transcriptional outcome. Indeed, our results show a dual regulation of GR activity by the H1R: a potentiation mediated by G-protein βγ subunits and a parallel inhibitory effect mediated by Gαq-PLC pathway. Activation of the H1R by its full agonists resulted in a composite potentiating effect. Intriguingly, inactivation of the Gαq-PLC pathway by H1R inverse agonists resulted also in a potentiation of GR activity. Moreover, histamine and clinically relevant antihistamines synergized with the GR agonist dexamethasone to induce gene transactivation and transrepression in a gene-specific manner. Our work provides a delineation of molecular mechanisms underlying the widespread clinical association of antihistamines and GR agonists, which may contribute to future dosage optimization and reduction of well-described side effects associated with glucocorticoid administration. PMID:26635083

  16. Distribution and Abundance of Glucocorticoid and Mineralocorticoid Receptors throughout the Brain of the Great Tit (Parus major).

    PubMed

    Senft, Rebecca A; Meddle, Simone L; Baugh, Alexander T

    2016-01-01

    The glucocorticoid stress response, regulated by the hypothalamic-pituitary-adrenal (HPA) axis, enables individuals to cope with stressors through transcriptional effects in cells expressing the appropriate receptors. The two receptors that bind glucocorticoids-the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR)-are present in a variety of vertebrate tissues, but their expression in the brain is especially important. Neural receptor patterns have the potential to integrate multiple behavioral and physiological traits simultaneously, including self-regulation of glucocorticoid secretion through negative feedback processes. In the present work, we quantified the expression of GR and MR mRNA throughout the brain of a female great tit (Parus major), creating a distribution map encompassing 48 regions. This map, the first of its kind for P. major, demonstrated a widespread but not ubiquitous distribution of both receptor types. In the paraventricular nucleus of the hypothalamus (PVN) and the hippocampus (HP)-the two brain regions that we sampled from a total of 25 birds, we found high GR mRNA expression in the former and, unexpectedly, low MR mRNA in the latter. We examined the covariation of MR and GR levels in these two regions and found a strong, positive relationship between MR in the PVN and MR in the HP and a similar trend for GR across these two regions. This correlation supports the idea that hormone pleiotropy may constrain an individual's behavioral and physiological phenotype. In the female song system, we found moderate GR in hyperstriatum ventrale, pars caudalis (HVC), and moderate MR in robust nucleus of the arcopallium (RA). Understanding intra- and interspecific patterns of glucocorticoid receptor expression can inform us about the behavioral processes (e.g. song learning) that may be sensitive to stress and stimulate future hypotheses concerning the relationships between receptor expression, circulating hormone concentrations and

  17. Distribution and Abundance of Glucocorticoid and Mineralocorticoid Receptors throughout the Brain of the Great Tit (Parus major).

    PubMed

    Senft, Rebecca A; Meddle, Simone L; Baugh, Alexander T

    2016-01-01

    The glucocorticoid stress response, regulated by the hypothalamic-pituitary-adrenal (HPA) axis, enables individuals to cope with stressors through transcriptional effects in cells expressing the appropriate receptors. The two receptors that bind glucocorticoids-the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR)-are present in a variety of vertebrate tissues, but their expression in the brain is especially important. Neural receptor patterns have the potential to integrate multiple behavioral and physiological traits simultaneously, including self-regulation of glucocorticoid secretion through negative feedback processes. In the present work, we quantified the expression of GR and MR mRNA throughout the brain of a female great tit (Parus major), creating a distribution map encompassing 48 regions. This map, the first of its kind for P. major, demonstrated a widespread but not ubiquitous distribution of both receptor types. In the paraventricular nucleus of the hypothalamus (PVN) and the hippocampus (HP)-the two brain regions that we sampled from a total of 25 birds, we found high GR mRNA expression in the former and, unexpectedly, low MR mRNA in the latter. We examined the covariation of MR and GR levels in these two regions and found a strong, positive relationship between MR in the PVN and MR in the HP and a similar trend for GR across these two regions. This correlation supports the idea that hormone pleiotropy may constrain an individual's behavioral and physiological phenotype. In the female song system, we found moderate GR in hyperstriatum ventrale, pars caudalis (HVC), and moderate MR in robust nucleus of the arcopallium (RA). Understanding intra- and interspecific patterns of glucocorticoid receptor expression can inform us about the behavioral processes (e.g. song learning) that may be sensitive to stress and stimulate future hypotheses concerning the relationships between receptor expression, circulating hormone concentrations and

  18. Acute Restraint Stress Enhances Hippocampal Endocannabinoid Function via Glucocorticoid Receptor Activation

    PubMed Central

    Wang, Meina; Hill, Matthew N.; Zhang, Longhua; Gorzalka, Boris B.; Hillard, Cecilia J.; Alger, Bradley E.

    2012-01-01

    Exposure to behavioral stress normally triggers a complex, multi-level response of the hypothalamic-pituitary-adrenal (HPA) axis that helps maintain homeostatic balance. Although the endocannabinoid (eCB) system (ECS) is sensitive to chronic stress, few studies have directly addressed its response to acute stress. Here we show that acute restraint stress enhances eCB-dependent modulation of GABA release measured by whole-cell voltage clamp of inhibitory post-synaptic currents (IPSCs) in rat hippocampal CA1 pyramidal cells in vitro. Both Ca2+-dependent, eCB-mediated depolarization-induced suppression of inhibition (DSI), and muscarinic cholinergic receptor (mAChR) mediated eCB mobilization are enhanced following acute stress exposure. DSI enhancement is dependent on the activation of glucocorticoid receptors (GRs) and is mimicked by both in vivo and in vitro corticosterone treatment. This effect does not appear to involve cyclooxygenase-2 (COX-2), an enzyme that can degrade eCBs; however, treatment of hippocampal slices with the L-type calcium (Ca2+) channel inhibitor, nifedipine, reverses while an agonist of these channels mimics the effect of in vivo stress. Finally, we find that acute stress produces a delayed (by 30 min) increase in the hippocampal content of 2-arachidonoylglycerol, the eCB responsible for DSI. These results support the hypothesis that the ECS is a biochemical effector of glucocorticoids in the brain, linking stress with changes in synaptic strength. PMID:21890595

  19. Glucocorticoid receptor-mediated repression of gonadotropin-releasing hormone promoter activity in GT1 hypothalamic cell lines.

    PubMed

    Chandran, U R; Attardi, B; Friedman, R; Dong, K W; Roberts, J L; DeFranco, D B

    1994-03-01

    The synthesis and release of GnRH within a specific subset of neurons in the hypothalamus, which serves as the primary drive to the hypothalamic-pituitary-gonadal (HPG) axis, is subject to various levels of control. Although a number of direct synaptic connections to GnRH-containing neurons have been identified, which presumably provide some regulatory inputs, the mechanisms responsible for hormonal regulation of GnRH synthesis and release mediated by either cell surface or intracellular receptors remain controversial. The recent demonstration that a subset of GnRH-containing neurons in the rat hypothalamus possesses immunoreactive glucocorticoid receptors (GR) implies that this class of steroid hormones could exert a direct effect to regulate the functioning of these neurons and perhaps the HPG axis. We used the GT1-3 and GT1-7 cell lines of immortalized GnRH-secreting hypothalamic neurons as a model to study the direct effects of glucocorticoids on GnRH gene expression. We demonstrated that these cell lines possess GR that bind the synthetic glucocorticoid, dexamethasone, in vitro with high affinity (Kd = 2-3 nM). These receptors are functional, as indicated by their ability to activate transcription from exogenously introduced heterologous glucocorticoid-responsive promoters. Furthermore, dexamethasone represses both the endogenous mouse GnRH gene, decreasing steady state levels of GnRH mRNA, and the transcriptional activity of transfected rat GnRH promoter-reporter gene vectors. Glucocorticoid repression of rat GnRH promoter activity appears to be mediated by sequences contained within the promoter proximal 459 basepairs and not be influenced by the relative basal activity of the GnRH promoter. Thus, our results provide the first direct demonstration of glucocorticoid repression of transcription in a hypothalamic cell line and suggest that GR acting directly within GnRH neurons could be at least partly responsible for negative regulation of the HPG axis by

  20. Hippocampal glucocorticoid receptor activation enhances voltage-dependent Ca2+ conductances: relevance to brain aging.

    PubMed Central

    Kerr, D S; Campbell, L W; Thibault, O; Landfield, P W

    1992-01-01

    Glucocorticoids (GCs) activate several biochemical/molecular processes in the hippocampus through two receptor types. In addition, GCs influence cognitive behaviors and hippocampal neural activity and can also increase the rate of aging-dependent cell loss in the hippocampus. However, the ionic mechanisms through which GCs modulate hippocampal neuronal function are not well understood. We report here direct evidence that activation of cytosolic steroid receptors, specifically of the type II GC receptor, can enhance voltage-dependent Ca2+ conductances in brain neurons. Ca2+ current was assessed by current-clamp measures of Ca2+ action potentials and by sharp electrode voltage-clamp analyses of voltage-sensitive currents in cesium-, tetrodotoxin-, and tetraethylammonium-treated CA1 neurons in hippocampal slices. Both Ca2+ action potentials and voltage-activated Ca2+ currents (N- and L-like) were increased by 2-hr exposure to the synthetic GC receptor agonist, RU 28362. This effect of RU 28362 was blocked by coincubation with cycloheximide, indicating that the GC receptor-Ca2+ channel interaction depends on de novo protein synthesis. Dysregulated calcium homeostasis is also viewed as a candidate mechanism in brain aging. Thus, present results are consistent with the hypothesis that excessive GC-receptor activation and resultant increased Ca2+ influx may be two sequential phases of a brain-aging process that results initially in impairment of function and eventually in neuronal loss. PMID:1528857

  1. Activated Glucocorticoid Receptor Interacts with the INHAT Component Set/TAF-Iβ and Releases it from a Glucocorticoid-responsive Gene Promoter, Relieving Repression: Implications for the Pathogenesis of Glucocorticoid Resistance in Acute Undifferentiated Leukemia with Set-Can Translocation

    PubMed Central

    Ichijo, Takamasa; Chrousos, George P.; Kino, Tomoshige

    2008-01-01

    SUMMARY Set/template-activating factor (TAF)-Iβ, part of the Set-Can oncogene product found in acute undifferentiated leukemia, is a component of the inhibitor of acetyltransferases (INHAT) complex. Set/TAF-Iβ interacted with the DNA-binding domain of the glucocorticoid receptor (GR) in yeast two-hybrid screening, and repressed GR-induced transcriptional activity of a chromatin-integrated glucocorticoid-responsive and a natural promoter. Set/TAF-Iβ was co-precipitated with glucocorticoid response elements (GREs) of these promoters in the absence of dexamethasone, while addition of the hormone caused dissociation of Set/TAF-Iβ from and attraction of the p160-type coactivator GRIP1 to the promoter GREs. Set-Can fusion protein, on the other hand, did not interact with GR, was constitutively co-precipitated with GREs and suppressed GRIP1-induced enhancement of GR transcriptional activity and histone acetylation. Thus, Set/TAF-Iβ acts as a ligand-activated GR-responsive transcriptional repressor, while Set-Can does not retain physiologic responsiveness to ligand-bound GR, possibly contributing to the poor responsiveness of Set-Can-harboring leukemic cells to glucocorticoids. PMID:18096310

  2. Thymic involution in the suspended rat - Adrenal hypertrophy and glucocorticoid receptor content

    NASA Technical Reports Server (NTRS)

    Steffen, J. M.; Musacchia, X. J.

    1986-01-01

    The relationship between thymic involution and adrenal hypertrophy is studied. The thymus, adrenal glands, and tissue water content are evaluated in male Sprague rats suspended in antiorthostatic (AO) or orthostatic (O) positions. A 50 percent decrease in the wet weight of the thymus and hypertrophy of the adrenal glands are observed during the seven days of AO suspension. After seven days of recovery the thymus weight is increased to control level; however, the hypertrophy of the adrenal glands remains unchanged. Thymic and renal responses in O postioned rats are similar to AO reactions. Thymic glucocorticoid (GC) receptor concentrations in the rats are analyzed; a 20 percent decrease in GC receptor site concentration, which is related to thymic involution, is detected in both AO and O rats. It is concluded that there is a temporal correlation between thymic involution and adrenal hypertrophy, which is not affected by AO positioning, and thymic involution is not associated with an increased sensitivity to GC.

  3. Glucocorticoid receptor isoforms direct distinct mitochondrial programs to regulate ATP production

    PubMed Central

    Morgan, David J.; Poolman, Toryn M.; Williamson, Andrew J. K.; Wang, Zichen; Clark, Neil R.; Ma’ayan, Avi; Whetton, Anthony D.; Brass, Andrew; Matthews, Laura C.; Ray, David W.

    2016-01-01

    The glucocorticoid receptor (GR), a nuclear receptor and major drug target, has a highly conserved minor splice variant, GRγ, which differs by a single arginine within the DNA binding domain. GRγ, which comprises 10% of all GR transcripts, is constitutively expressed and tightly conserved through mammalian evolution, suggesting an important non-redundant role. However, to date no specific role for GRγ has been reported. We discovered significant differences in subcellular localisation, and nuclear-cytoplasmic shuttling in response to ligand. In addition the GRγ transcriptome and protein interactome was distinct, and with a gene ontology signal for mitochondrial regulation which was confirmed using Seahorse technology. We propose that evolutionary conservation of the single additional arginine in GRγ is driven by a distinct, non-redundant functional profile, including regulation of mitochondrial function. PMID:27226058

  4. Are receptor concentrations correlated across tissues within individuals? A case study examining glucocorticoid and mineralocorticoid receptor binding.

    PubMed

    Lattin, Christine R; Keniston, Daniel E; Reed, J Michael; Romero, L Michael

    2015-04-01

    Hormone receptors are a necessary (although not sufficient) part of the process through which hormones like corticosterone create physiological responses. However, it is currently unknown to what extent receptor concentrations across different target tissues may be correlated within individual animals. In this study, we examined this question using a large dataset of radioligand binding data for glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs) in 13 different tissues in the house sparrow (Passer domesticus) (n=72). Our data revealed that individual house sparrows tended to exhibit higher or lower receptor binding across all tissues, which could be part of what creates the physiological and behavioral syndromes associated with different hormonal profiles. However, although statistically significant, the correlations between tissues were very weak. Thus, when each tissue was independently regressed on receptor concentrations in the other tissues, multivariate analysis revealed significant relationships only for sc fat (for GR) and whole brain, hippocampus, kidney, omental fat, and sc fat (for MR). We also found significant pairwise correlations only between receptor concentrations in brain and hippocampus, and brain and kidney (both for MR). This research reveals that although there are generalized individual consistencies in GR and MR concentrations, possibly due to such factors as hormonal regulation and genetic effects, the ability of 2 different tissues to respond to the same hormonal signal appears to be affected by additional factors that remain to be identified.

  5. Down-regulation of phospholipase C-beta1 following chronic muscarinic receptor activation.

    PubMed

    Sorensen, S D; Linseman, D A; Fisher, S K

    1998-04-01

    To determine whether prolonged activation of a phospholipase C-coupled receptor can lead to a down-regulation of its effector enzyme, SH-SY5Y neuroblastoma cells were incubated for 24 h with the muscarinic receptor agonist, oxotremorine-M. Under these conditions, significant reductions (46-53%) in muscarinic cholinergic receptor density, G(alphaq/11) and phospholipase C-beta1 (but not the beta3-or gamma1 isoforms) were observed. These results suggest that a selective down-regulation of phospholipase C-beta1 may play a role in adaptation to chronic muscarinic receptor activation. PMID:9617763

  6. Differences between brain structures in nuclear translocation and DNA binding of the glucocorticoid receptor during stress and the circadian cycle.

    PubMed

    Kitchener, Pierre; Di Blasi, Francesco; Borrelli, Emiliana; Piazza, Pier Vincenzo

    2004-04-01

    Glucocorticoid receptors (GRs) are transcription factors that, upon activation by glucocorticoids, translocate to the cell nucleus, and bind to specific response elements (GREs) in the promoter region of target genes. We analysed stress- and circadian-induced changes in nuclear translocation and GRE binding of GRs in the hippocampus and the prefrontal cortex of the rat brain. Nuclear translocation and binding to GRE were measured in nuclear extracts by Western blot and gel shift, respectively. When glucocorticoid levels were low, as during the light period of the circadian cycle, nuclear GRs and GRE binding were almost undetectable. However, the increase in glucocorticoid levels observed during the dark phase of the circadian cycle or after stress induced a massive nuclear translocation of GRs and GRE binding. These effects were corticosterone-dependent because they were suppressed by adrenalectomy and restored by the injection of corticosterone. Furthermore, GR translocation and GRE binding were of higher amplitude or lasted longer in the hippocampus than in the prefrontal cortex. By contrast, extracellular levels of glucocorticoids, measured by microdialysis in freely moving animals, were identical in the two structures. These results suggest that specific intracellular regulations of GR activity contribute to differentiate the effects of glucocorticoids in different regions of the brain. PMID:15078557

  7. Do persistent organic pollutants interact with the stress response? Individual compounds, and their mixtures, interaction with the glucocorticoid receptor.

    PubMed

    Wilson, Jodie; Berntsen, Hanne Friis; Zimmer, Karin Elisabeth; Verhaegen, Steven; Frizzell, Caroline; Ropstad, Erik; Connolly, Lisa

    2016-01-22

    Persistent organic pollutants (POPs) are toxic substances, highly resistant to environmental degradation, which can bio-accumulate and have long-range atmospheric transport potential (UNEP, 2001). The majority of studies on endocrine disruption have focused on interferences on the sexual steroid hormones and so have overlooked disruption to glucocorticoid hormones. Here the endocrine disrupting potential of individual POPs and their mixtures has been investigated in vitro to identify any disruption to glucocorticoid nuclear receptor transcriptional activity. POP mixtures were screened for glucocorticoid receptor (GR) translocation using a GR redistribution assay (RA) on a CellInsight™ NXT high content screening (HCS) platform. A mammalian reporter gene assay (RGA) was then used to assess the individual POPs, and their mixtures, for effects on glucocorticoid nuclear receptor transactivation. POP mixtures did not induce GR translocation in the GR RA or produce an agonist response in the GR RGA. However, in the antagonist test, in the presence of cortisol, an individual POP, p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE), was found to decrease glucocorticoid nuclear receptor transcriptional activity to 72.5% (in comparison to the positive cortisol control). Enhanced nuclear transcriptional activity, in the presence of cortisol, was evident for the two lowest concentrations of perfluorodecanoic acid (PFOS) potassium salt (0.0147mg/ml and 0.0294mg/ml), the two highest concentrations of perfluorodecanoic acid (PFDA) (0.0025mg/ml and 0.005mg/ml) and the highest concentration of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) (0.0000858mg/ml). It is important to gain a better understanding of how POPs can interact with GRs as the disruption of glucocorticoid action is thought to contribute to complex diseases. PMID:26599974

  8. Do persistent organic pollutants interact with the stress response? Individual compounds, and their mixtures, interaction with the glucocorticoid receptor.

    PubMed

    Wilson, Jodie; Berntsen, Hanne Friis; Zimmer, Karin Elisabeth; Verhaegen, Steven; Frizzell, Caroline; Ropstad, Erik; Connolly, Lisa

    2016-01-22

    Persistent organic pollutants (POPs) are toxic substances, highly resistant to environmental degradation, which can bio-accumulate and have long-range atmospheric transport potential (UNEP, 2001). The majority of studies on endocrine disruption have focused on interferences on the sexual steroid hormones and so have overlooked disruption to glucocorticoid hormones. Here the endocrine disrupting potential of individual POPs and their mixtures has been investigated in vitro to identify any disruption to glucocorticoid nuclear receptor transcriptional activity. POP mixtures were screened for glucocorticoid receptor (GR) translocation using a GR redistribution assay (RA) on a CellInsight™ NXT high content screening (HCS) platform. A mammalian reporter gene assay (RGA) was then used to assess the individual POPs, and their mixtures, for effects on glucocorticoid nuclear receptor transactivation. POP mixtures did not induce GR translocation in the GR RA or produce an agonist response in the GR RGA. However, in the antagonist test, in the presence of cortisol, an individual POP, p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE), was found to decrease glucocorticoid nuclear receptor transcriptional activity to 72.5% (in comparison to the positive cortisol control). Enhanced nuclear transcriptional activity, in the presence of cortisol, was evident for the two lowest concentrations of perfluorodecanoic acid (PFOS) potassium salt (0.0147mg/ml and 0.0294mg/ml), the two highest concentrations of perfluorodecanoic acid (PFDA) (0.0025mg/ml and 0.005mg/ml) and the highest concentration of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) (0.0000858mg/ml). It is important to gain a better understanding of how POPs can interact with GRs as the disruption of glucocorticoid action is thought to contribute to complex diseases.

  9. DNA binding triggers tetramerization of the glucocorticoid receptor in live cells.

    PubMed

    Presman, Diego M; Ganguly, Sourav; Schiltz, R Louis; Johnson, Thomas A; Karpova, Tatiana S; Hager, Gordon L

    2016-07-19

    Transcription factors dynamically bind to chromatin and are essential for the regulation of genes. Although a large percentage of these proteins appear to self-associate to form dimers or higher order oligomers, the stoichiometry of DNA-bound transcription factors has been poorly characterized in vivo. The glucocorticoid receptor (GR) is a ligand-regulated transcription factor widely believed to act as a dimer or a monomer. Using a unique set of imaging techniques coupled with a cell line containing an array of DNA binding elements, we show that GR is predominantly a tetramer when bound to its target DNA. We find that DNA binding triggers an interdomain allosteric regulation within the GR, leading to tetramerization. We therefore propose that dynamic changes in GR stoichiometry represent a previously unidentified level of regulation in steroid receptor activation. Quaternary structure analysis of other members of the steroid receptor family (estrogen, androgen, and progesterone receptors) reveals variation in oligomerization states among this family of transcription factors. Because GR's oligomerization state has been implicated in therapy outcome, our findings open new doors to the rational design of novel GR ligands and redefine the quaternary structure of steroid receptors. PMID:27382178

  10. Modifications to glucocorticoid and progesterone receptors alter cell fate in breast cancer.

    PubMed

    Leehy, Katherine A; Regan Anderson, Tarah M; Daniel, Andrea R; Lange, Carol A; Ostrander, Julie H

    2016-04-01

    Steroid hormone receptors (SRs) are heavily posttranslationally modified by the reversible addition of a variety of molecular moieties, including phosphorylation, acetylation, methylation, SUMOylation, and ubiquitination. These rapid and dynamic modifications may be combinatorial and interact (i.e. may be sequential, complement, or oppose each other), creating a vast array of uniquely modified receptor subspecies that allow for diverse receptor behaviors that enable highly sensitive and context-dependent hormone action. For example, in response to hormone or growth factor membrane-initiated signaling events, posttranslational modifications (PTMs) to SRs alter protein-protein interactions that govern the complex process of promoter or gene-set selection coupled to transcriptional repression or activation. Unique phosphorylation events allow SRs to associate or disassociate with specific cofactors that may include pioneer factors and other tethering partners, which specify the resulting transcriptome and ultimately change cell fate. The impact of PTMs on SR action is particularly profound in the context of breast tumorigenesis, in which frequent alterations in growth factor-initiated signaling pathways occur early and act as drivers of breast cancer progression toward endocrine resistance. In this article, with primary focus on breast cancer relevance, we review the mechanisms by which PTMs, including reversible phosphorylation events, regulate the closely related SRs, glucocorticoid receptor and progesterone receptor, allowing for precise biological responses to ever-changing hormonal stimuli.

  11. HDAC6 regulates glucocorticoid receptor signaling in serotonin pathways with critical impact on stress resilience.

    PubMed

    Espallergues, Julie; Teegarden, Sarah L; Veerakumar, Avin; Boulden, Janette; Challis, Collin; Jochems, Jeanine; Chan, Michael; Petersen, Tess; Deneris, Evan; Matthias, Patrick; Hahn, Chang-Gyu; Lucki, Irwin; Beck, Sheryl G; Berton, Olivier

    2012-03-28

    Genetic variations in certain components of the glucocorticoid receptor (GR) chaperone complex have been associated with the development of stress-related affective disorders and individual variability in therapeutic responses to antidepressants. Mechanisms that link GR chaperoning and stress susceptibility are not well understood. Here, we show that the effects of glucocorticoid hormones on socioaffective behaviors are critically regulated via reversible acetylation of Hsp90, a key component of the GR chaperone complex. We provide pharmacological and genetic evidence indicating that the cytoplasmic lysine deacetylase HDAC6 controls Hsp90 acetylation in the brain, and thereby modulates Hsp90-GR protein-protein interactions, as well as hormone- and stress-induced GR translocation, with a critical impact on GR downstream signaling and behavior. Pet1-Cre-driven deletion of HDAC6 in serotonin neurons, the densest HDAC6-expressing cell group in the mouse brain, dramatically reduced acute anxiogenic effects of the glucocorticoid hormone corticosterone in the open-field, elevated plus maze, and social interaction tests. Serotonin-selective depletion of HDAC6 also blocked the expression of social avoidance in mice exposed to chronic social defeat and concurrently prevented the electrophysiological and morphological changes induced, in serotonin neurons, by this murine model of traumatic stress. Together, these results identify HDAC6 inhibition as a potential new strategy for proresilience and antidepressant interventions through regulation of the Hsp90-GR heterocomplex and focal prevention of GR signaling in serotonin pathways. Our data thus uncover an alternate mechanism by which pan-HDAC inhibitors may regulate stress-related behaviors independently of their action on histones.

  12. Postnatal epigenetic modification of glucocorticoid receptor gene in preterm infants: a prospective cohort study

    PubMed Central

    Kantake, Masato; Yoshitake, Hiroshi; Ishikawa, Hitoshi; Araki, Yoshihiko; Shimizu, Toshiaki

    2014-01-01

    Objective To examine the environmental effects on cytosine methylation of preterm infant's DNA, because early life experiences are considered to influence the physiological and mental health of an individual through epigenetic modification of DNA. Design A prospective cohort study, comparison of epigenetic differences in the glucocorticoid receptor (GR) gene between healthy term and preterm infants. Setting Neonatal Intensive Care Unit in a Japanese University Hospital. Participants A cohort of 40 (20 term and 20 preterm) infants was recruited on the day of birth, and peripheral blood was obtained from each infant at birth and on postnatal day 4. Main outcome measures The methylation rates in the 1-F promoter region of the GR gene using the Mquant method. Results The methylation rate increased significantly between postnatal days 0 and 4 in preterm infants but remained stable in term infants. Thus, the methylation rate was significantly higher in preterm than in term infants at postnatal day 4. Several perinatal parameters were significantly correlated with this change in the methylation rate. Logistic regression analysis revealed that methylation rates at postnatal day 4 predicted the occurrence of later complications that required glucocorticoid administration during the neonatal period. No gene polymorphism was detected within the GR promoter region analysed. Conclusions Although further large-scale studies are needed to detect the environmental factors that explain the difference in epigenetic modification among infants after birth, our data show that the postnatal environment influences epigenetic programming of GR expression through methylation of the GR gene promoter in premature infants, which may result in relative glucocorticoid insufficiency during the postnatal period. PMID:25023132

  13. Glucocorticoids facilitate the transcription from the human cytomegalovirus major immediate early promoter in glucocorticoid receptor- and nuclear factor-I-like protein-dependent manner

    SciTech Connect

    Inoue-Toyoda, Maki; Kato, Kohsuke; Nagata, Kyosuke; Yoshikawa, Hiroyuki

    2015-02-27

    Human cytomegalovirus (HCMV) is a common and usually asymptomatic virus agent in healthy individuals. Initiation of HCMV productive infection depends on expression of the major immediate early (MIE) genes. The transcription of HCMV MIE genes is regulated by a diverse set of transcription factors. It was previously reported that productive HCMV infection is triggered probably by elevation of the plasma hydroxycorticoid level. However, it is poorly understood whether the transcription of MIE genes is directly regulated by glucocorticoid. Here, we found that the dexamethasone (DEX), a synthetic glucocorticoid, facilitates the transcription of HCMV MIE genes through the MIE promoter and enhancer in a glucocorticoid receptor (GR)-dependent manner. By competitive EMSA and reporter assays, we revealed that an NF-I like protein is involved in DEX-mediated transcriptional activation of the MIE promoter. Thus, this study supports a notion that the increased level of hydroxycorticoid in the third trimester of pregnancy reactivates HCMV virus production from the latent state. - Highlights: • DEX facilitates the transcription from the HCMV MIE promoter. • GR is involved in DEX-dependent transcription from the HCMV MIE promoter. • A 17 bp repeat is responsible for the HCMV MIE promoter activation by DEX. • An NF-I-like protein is involved in the HCMV MIE promoter activation by DEX.

  14. Glucocorticoid receptors in the prefrontal cortex regulate dopamine efflux to stress via descending glutamatergic feedback to the ventral tegmental area.

    PubMed

    Butts, Kelly A; Phillips, Anthony G

    2013-09-01

    Enhanced dopamine (DA) efflux in the medial prefrontal cortex (mPFC) is a well-documented response to acute stress. We have previously shown that glucocorticoid receptors in the mPFC regulate stress-evoked DA efflux but the underlying mechanism is unknown. DA neurons in the ventral tegmental area (VTA) receive excitatory input from and send reciprocal projections to the mPFC. We hypothesize that blockade of prefrontal glucocorticoid receptors can reduce activity of descending glutamatergic input to the VTA, thereby attenuating stress-evoked DA efflux in the mPFC. Using in vivo microdialysis, we demonstrate that acute tail-pinch stress leads to a significant increase in glutamate efflux in the VTA. Blockade of prefrontal glucocorticoid receptors with the selective antagonist CORT 108297 attenuates stress-evoked glutamate efflux in the VTA together with DA efflux in the mPFC. Furthermore, blockade of ionotrophic glutamate receptors in the VTA attenuates stress-evoked DA efflux in the mPFC. We also examine the possible role of glucocorticoid-induced synthesis and release of endocannabinoids acting presynaptically via cannabinoid CB1 receptors to inhibit GABA release onto prefrontal pyramidal cells, thus enhancing descending glutamatergic input to the VTA leading to an increase in mPFC DA efflux during stress. However, administration of the cannabinoid CB1 receptor antagonist into the mPFC does not attenuate stress-evoked DA efflux in the mPFC. Taken together, our data indicate that glucocorticoids act locally within the mPFC to modulate mesocortical DA efflux by potentiation of glutamatergic drive onto DA neurons in the VTA. PMID:23590841

  15. The activity of the glucocorticoid receptor is regulated by SUMO conjugation to FKBP51.

    PubMed

    Antunica-Noguerol, M; Budziñski, M L; Druker, J; Gassen, N C; Sokn, M C; Senin, S; Aprile-Garcia, F; Holsboer, F; Rein, T; Liberman, A C; Arzt, E

    2016-10-01

    FK506-binding protein 51 (FKBP51) regulates the activity of the glucocorticoid receptor (GR), and is therefore a key mediator of the biological actions of glucocorticoids. However, the understanding of the molecular mechanisms that govern its activity remains limited. Here, we uncover a novel regulatory switch for GR activity by the post-translational modification of FKBP51 with small ubiquitin-like modifier (SUMO). The major SUMO-attachment site, lysine 422, is required for FKBP51-mediated inhibition of GR activity in hippocampal neuronal cells. Importantly, impairment of SUMO conjugation to FKBP51 impacts on GR-dependent neuronal signaling and differentiation. We demonstrate that SUMO conjugation to FKBP51 is enhanced by the E3 ligase PIAS4 and by environmental stresses such as heat shock, which impact on GR-dependent transcription. SUMO conjugation to FKBP51 regulates GR hormone-binding affinity and nuclear translocation by promoting FKBP51 interaction within the GR complex. SUMOylation-deficient FKBP51 fails to interact with Hsp90 and GR thus facilitating the recruitment of the closely related protein, FKBP52, which enhances GR transcriptional activity. Moreover, we show that the modification of FKBP51 with SUMO modulates its binding to Hsp90. Our data establish SUMO conjugation as a novel regulatory mechanism in the Hsp90 cochaperone activity of FKBP51 with a functional impact on GR signaling in a neuronal context.

  16. Astragaloside IV inhibits microglia activation via glucocorticoid receptor mediated signaling pathway

    PubMed Central

    Liu, Hong-Shuai; Shi, Hai-Lian; Huang, Fei; Peterson, Karin E.; Wu, Hui; Lan, Yun-Yi; Zhang, Bei-Bei; He, Yi-Xin; Woods, Tyson; Du, Min; Wu, Xiao-Jun; Wang, Zheng-Tao

    2016-01-01

    Inhibition of microglia activation may provide therapeutic treatment for many neurodegenerative diseases. Astragaloside IV (ASI) with anti-inflammatory properties has been tested as a therapeutic drug in clinical trials of China. However, the mechanism of ASI inhibiting neuroinflammation is unknown. In this study, we showed that ASI inhibited microglia activation both in vivo and in vitro. It could enhance glucocorticoid receptor (GR)-luciferase activity and facilitate GR nuclear translocation in microglial cells. Molecular docking and TR-FRET GR competitive binding experiments demonstrated that ASI could bind to GR in spite of relative low affinity. Meanwhile, ASI modulated GR-mediated signaling pathway, including dephosphorylation of PI3K, Akt, I κB and NF κB, therefore, decreased downstream production of proinflammatory mediators. Suppression of microglial BV-2 activation by ASI was abrogated by GR inhibitor, RU486 or GR siRNA. Similarly, RU486 counteracted the alleviative effect of ASI on microgliosis and neuronal injury in vivo. Our findings demonstrated that ASI inhibited microglia activation at least partially by activating the glucocorticoid pathway, suggesting its possible therapeutic potential for neuroinflammation in neurological diseases. PMID:26750705

  17. Greater Glucocorticoid Receptor Activation in Hippocampus of Aged Rats Sensitizes Microglia

    PubMed Central

    Barrientos, Ruth M.; Thompson, Vanessa M.; Kitt, Meagan M.; Amat, Jose; Hale, Matthew W.; Frank, Matthew G.; Crysdale, Nicole Y.; Stamper, Christopher E.; Hennessey, Patrick A.; Watkins, Linda R.; Spencer, Robert L.; Lowry, Christopher A.; Maier, Steven F.

    2014-01-01

    Healthy aging individuals are more likely to suffer profound memory impairments following an immune challenge than are younger adults. These challenges produce a brain inflammatory response that is exaggerated with age. Sensitized microglia found in the normal aging brain are responsible for this amplified response, which in turn interferes with processes involved in memory formation. Here, we examine factors that may lead aging to sensitize microglia. Aged rats exhibited higher CORT levels in the hippocampus, but not in plasma, throughout the daytime (diurnal inactive phase). These elevated hippocampal CORT levels were associated with increased hippocampal 11β-HSD1 protein expression, the enzyme that catalyzes glucocorticoid formation, and greater hippocampal glucocorticoid receptor (GR) activation. Intracisternal administration of mifepristone, a GR antagonist, effectively reduced immune-activated proinflammatory responses, specifically from hippocampal microglia, and prevented E. coli-induced memory impairments in aged rats. Voluntary exercise as a therapeutic intervention significantly reduced total hippocampal GR expression. These data strongly suggest that increased GR activation in the aged hippocampus plays a critical role in sensitizing microglia. PMID:25559333

  18. Glucocorticoid receptors in the nucleus of the solitary tract (NTS) decrease endocrine and behavioral stress responses.

    PubMed

    Ghosal, Sriparna; Bundzikova-Osacka, Jana; Dolgas, C Mark; Myers, Brent; Herman, James P

    2014-07-01

    Stress activates the hypothalamo-pituitary-adrenal (HPA) axis, leading to adrenocortical secretion of glucocorticoids. The magnitude and duration of the HPA axis response is mediated in large part by the glucocorticoid receptor (GR). The nucleus of the solitary tract (NTS) abundantly expresses the GR and is a key brain region for processing autonomic and endocrine stress responses. This study tests the hypothesis that GR within the NTS plays an important role in inhibiting stress-induced endocrine and behavioral responses. Cohorts of rats received bilateral micropellet (30 μg) implantations of crystalline corticosterone, mifepristone (a GR antagonist) or cholesterol (control) directed into the region of the NTS, and were subsequently subjected to either acute psychogenic (restraint) stress or chronic variable stress (CVS). We found that NTS GR antagonism increased acute stress-induced corticosterone levels, whereas GR activation within the NTS attenuated this response. Following CVS, basal and 15 min post-restraint plasma corticosterone levels were increased by NTS GR antagonism, which was associated with an increase in Fos immunoreactivity within the PVN. Using the elevated plus maze (EPM) and forced swim test (FST), we assessed the effect of NTS GR inhibition on anxiety- and depression-like behaviors, respectively. GR inhibition within the NTS decreased open arm exploratory behavior in the EPM and increased immobility in the FST relative to controls. Together, the findings reveal a novel role of NTS GR signaling for inhibiting both endocrine and behavioral responses to stress.

  19. Neuronal nitric oxide synthase is an endogenous negative regulator of glucocorticoid receptor in the hippocampus.

    PubMed

    Liu, Meng-ying; Zhu, Li-Juan; Zhou, Qi-Gang

    2013-07-01

    The hippocampus is rich in both glucocorticoid receptor (GR) and neuronal nitric oxide synthase (nNOS). But the relationship between the two molecules under physiological states remains unrevealed. Here, we report that nNOS knockout mice display increased GR expression in the hippocampus. Both systemic administration of 7-Nitroindazole (7-NI), a selective nNOS activity inhibitor, and selective infusion of 7-NI into the hippocampus resulted in an increase in GR expression in the hippocampus. Moreover, KCl exposure, which can induce overexpression of nNOS, resulted in a decrease in GR protein level in cultured hippocampal neurons. Moreover, blockade of nNOS activity in the hippocampus leads to decreased corticosterone (CORT, glucocorticoids in rodents) concentration in the plasma and reduced corticotrophin-releasing factor expression in the hypothalamus. The results indicate that nNOS is an endogenous inhibitor of GR in the hippocampus and that nNOS in the hippocampus may participate in the modulation of Hypothalamic-Pituitary-Adrenal axis activity via GR.

  20. Greater glucocorticoid receptor activation in hippocampus of aged rats sensitizes microglia.

    PubMed

    Barrientos, Ruth M; Thompson, Vanessa M; Kitt, Meagan M; Amat, Jose; Hale, Matthew W; Frank, Matthew G; Crysdale, Nicole Y; Stamper, Christopher E; Hennessey, Patrick A; Watkins, Linda R; Spencer, Robert L; Lowry, Christopher A; Maier, Steven F

    2015-03-01

    Healthy aging individuals are more likely to suffer profound memory impairments following an immune challenge than are younger adults. These challenges produce a brain inflammatory response that is exaggerated with age. Sensitized microglia found in the normal aging brain are responsible for this amplified response, which in turn interferes with processes involved in memory formation. Here, we examine factors that may lead aging to sensitize microglia. Aged rats exhibited higher corticosterone levels in the hippocampus, but not in plasma, throughout the daytime (diurnal inactive phase). These elevated hippocampal corticosterone levels were associated with increased hippocampal 11β-hydroxysteroid dehydrogenase type 1 protein expression, the enzyme that catalyzes glucocorticoid formation and greater hippocampal glucocorticoid receptor (GR) activation. Intracisternal administration of mifepristone, a GR antagonist, effectively reduced immune-activated proinflammatory responses, specifically from hippocampal microglia and prevented Escherichia coli-induced memory impairments in aged rats. Voluntary exercise as a therapeutic intervention significantly reduced total hippocampal GR expression. These data strongly suggest that increased GR activation in the aged hippocampus plays a critical role in sensitizing microglia.

  1. Control of energy balance by hypothalamic gene circuitry involving two nuclear receptors, neuron-derived orphan receptor 1 and glucocorticoid receptor.

    PubMed

    Kim, Sun-Gyun; Lee, Bora; Kim, Dae-Hwan; Kim, Juhee; Lee, Seunghee; Lee, Soo-Kyung; Lee, Jae W

    2013-10-01

    Nuclear receptors (NRs) regulate diverse physiological processes, including the central nervous system control of energy balance. However, the molecular mechanisms for the central actions of NRs in energy balance remain relatively poorly defined. Here we report a hypothalamic gene network involving two NRs, neuron-derived orphan receptor 1 (NOR1) and glucocorticoid receptor (GR), which directs the regulated expression of orexigenic neuropeptides agouti-related peptide (AgRP) and neuropeptide Y (NPY) in response to peripheral signals. Our results suggest that the anorexigenic signal leptin induces NOR1 expression likely via the transcription factor cyclic AMP response element-binding protein (CREB), while the orexigenic signal glucocorticoid mobilizes GR to inhibit NOR1 expression by antagonizing the action of CREB. Also, NOR1 suppresses glucocorticoid-dependent expression of AgRP and NPY. Consistently, relative to wild-type mice, NOR1-null mice showed significantly higher levels of AgRP and NPY and were less responsive to leptin in decreasing the expression of AgRP and NPY. These results identify mutual antagonism between NOR1 and GR to be a key rheostat for peripheral metabolic signals to centrally control energy balance.

  2. Disruption of the glucocorticoid receptor assembly with heat shock protein 90 by a peptidic antiglucocorticoid.

    PubMed

    Dao-Phan, H P; Formstecher, P; Lefebvre, P

    1997-06-01

    Association of glucocorticoid (GR) and progesterone (PR) receptors with a set of molecular chaperones, including the 90-kDa heat shock protein (hsp90), is a dynamic process required for proper folding and maintaining these nuclear receptors under a transcriptionally inactive, ligand-responsive state. Mutational studies of the chicken hsp90 complementary DNA suggested that three regions of this protein (A, B, and Z) interact with the hormone-binding domain of GR, whereas region A is dispensable for hsp90 binding to PR. We found that this 69-amino acid region can be narrowed down to a 35-mer alpha-helical, acidic peptide, which is by itself able to inhibit hsp90 association to GR translated in vitro. The hsp90-free GR did not bind ligand, but was devoid of any specific DNA-binding activity, and higher peptide concentrations specifically inhibited the binding of activated GR to DNA. When overexpressed in cultured cells, this peptide acted as an antiglucocorticoid and inhibited the antiactivating protein-1 activity and the ligand-dependent nuclear transfer of GR. None of these effects, either in vivo and in vitro, was observed for PR. The region from residue 232 to residue 265 of hsp90 is, therefore, a domain critical for its association to GR, an association that is a prerequisite for receptor transcriptional activity. More importantly, these results demonstrate that targeting specific protein/protein interaction interfaces is a powerful means to specifically modulate nuclear receptor signaling pathways in a ligand-independent manner.

  3. Thymic involution in the suspended rat model for weightlessness - Decreased glucocorticoid receptor concentration

    NASA Technical Reports Server (NTRS)

    Steffen, J. M.; Musacchia, X. J.

    1984-01-01

    Hindlimb muscle atrophy, thymic involution and adrenal hypertrophy in rats during spaceflight can be simulated using suspension models. Skeletal muscle and thymus are sensitive to gluco-corticoids (GC), and previous studies have demonstrated that muscle atrophy in suspended rats is associated with increased GC receptor concentration. The objectives were to confirm thymic involution during suspension, and determine if involution correlated with increased GC receptor concentration. Seven days of antiorthostatic (AO) suspension of rats produced a significant (P less than 0.001) reduction in thymic wet weight not associated with an alteration of percent water content. GC receptor concentration (pmol/mg protein) decreased 20 percent (P less than 0.025) in thymus glands from 7 day AO suspended rats. Suspension, therefore, is associated with involution of the thymus, but this is not dependent upon AO positioning. Thymus GC receptor concentrations were depressed in 7-day suspended rats, in contrast with previous observations on skeletal muscle, suggesting that different mechanisms may underlie these responses.

  4. The first intron of the human growth hormone gene contains a binding site for glucocorticoid receptor.

    PubMed Central

    Moore, D D; Marks, A R; Buckley, D I; Kapler, G; Payvar, F; Goodman, H M

    1985-01-01

    Glucocorticoid receptor (GCR) protein stimulates transcription from a variety of cellular genes. We show here that GCR partially purified from rat liver binds specifically to a site within the first intron of the human growth hormone (hGH) gene, approximately 100 base pairs downstream from the start of hGH transcription. GCR binding is selectively inhibited by methylation of two short, symmetrically arranged clusters of guanine residues within this site. A cloned synthetic 24-base-pair deoxyoligonucleotide containing the predicted GCR binding sequence interacts specifically with GCR. The hGH binding site shares sequence homology with a GCR binding site upstream from the human metallothionein II gene and a subset of GCR binding sites from mouse mammary tumor virus. All of these binding sites for this eukaryotic transcriptional regulatory protein show remarkable similarity in overall geometry to the binding sites for several prokaryotic transcriptional regulatory proteins. Images PMID:2983311

  5. Enhanced conditioned approach responses in transgenic mice with impaired glucocorticoid receptor function.

    PubMed

    Steckler, T; Holsboer, F

    1999-07-01

    The long-term consequences of impaired glucocorticoid receptor (GR) function on reward-related learning were studied in transgenic mice with impaired GR function in a series of experiments taxing conditioned and unconditioned approach responses to stimuli predictive of food. There was a double-dissociation in that transgenic mice with impaired GR activity showed enhanced conditioned exploration in situations when stimuli predicted reward, while free-feeding food consumption over 24 h was reduced. Previous experiments have shown altered accumbens dopaminergic activity in these animals. In line with these findings, we observed an enhanced behavioural stimulation of transgenic mice following administration of d-amphetamine (2 mg/kg). This suggests that the increase in preparatory responses in transgenic mice may be mediated via an enhanced accumbens dopaminergic activity, possibly secondary to alterations in other brain systems. PMID:10403023

  6. Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity.

    PubMed

    Schöne, Stefanie; Jurk, Marcel; Helabad, Mahdi Bagherpoor; Dror, Iris; Lebars, Isabelle; Kieffer, Bruno; Imhof, Petra; Rohs, Remo; Vingron, Martin; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H

    2016-09-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes.

  7. Function changing mutations in glucocorticoid receptor evolution correlate with their relevance to mode coupling.

    PubMed

    Kav, Batuhan; Öztürk, Murat; Kabakçιoğlu, Alkan

    2016-05-01

    Nonlinear effects in protein dynamics are expected to play role in function, particularly of allosteric nature, by facilitating energy transfer between vibrational modes. A recently proposed method focusing on the non-Gaussian shape of the configurational population near equilibrium projects this information onto real space in order to identify the aminoacids relevant to function. We here apply this method to three ancestral proteins in glucocorticoid receptor (GR) family and show that the mutations that restrict functional activity during GR evolution correlate significantly with locations that are highlighted by the nonlinear contribution to the near-native configurational distribution. Our findings demonstrate that the analysis of nonlinear effects in protein dynamics can be harnessed into a predictive tool for functional site determination. PMID:26873882

  8. Mechanisms for the Evolution of a Derived Function in the Ancestral Glucocorticoid Receptor

    PubMed Central

    Carroll, Sean Michael; Ortlund, Eric A.; Thornton, Joseph W.

    2011-01-01

    Understanding the genetic, structural, and biophysical mechanisms that caused protein functions to evolve is a central goal of molecular evolutionary studies. Ancestral sequence reconstruction (ASR) offers an experimental approach to these questions. Here we use ASR to shed light on the earliest functions and evolution of the glucocorticoid receptor (GR), a steroid-activated transcription factor that plays a key role in the regulation of vertebrate physiology. Prior work showed that GR and its paralog, the mineralocorticoid receptor (MR), duplicated from a common ancestor roughly 450 million years ago; the ancestral functions were largely conserved in the MR lineage, but the functions of GRs—reduced sensitivity to all hormones and increased selectivity for glucocorticoids—are derived. Although the mechanisms for the evolution of glucocorticoid specificity have been identified, how reduced sensitivity evolved has not yet been studied. Here we report on the reconstruction of the deepest ancestor in the GR lineage (AncGR1) and demonstrate that GR's reduced sensitivity evolved before the acquisition of restricted hormone specificity, shortly after the GR–MR split. Using site-directed mutagenesis, X-ray crystallography, and computational analyses of protein stability to recapitulate and determine the effects of historical mutations, we show that AncGR1's reduced ligand sensitivity evolved primarily due to three key substitutions. Two large-effect mutations weakened hydrogen bonds and van der Waals interactions within the ancestral protein, reducing its stability. The degenerative effect of these two mutations is extremely strong, but a third permissive substitution, which has no apparent effect on function in the ancestral background and is likely to have occurred first, buffered the effects of the destabilizing mutations. Taken together, our results highlight the potentially creative role of substitutions that partially degrade protein structure and function and

  9. Post-training glucocorticoid receptor activation during Pavlovian conditioning reduces Pavlovian-instrumental transfer in rats.

    PubMed

    Pielock, Steffi M; Sommer, Susanne; Hauber, Wolfgang

    2013-03-01

    Considerable evidence suggests that glucocorticoid receptor activation can enhance memory consolidation in Pavlovian learning tasks. For instance, post-training injections of the synthetic glucocorticoid receptor agonist dexamethasone increased conditioned responding to reward-predictive Pavlovian stimuli. Here we explored whether post-training dexamethasone injections can enhance appetitive Pavlovian learning and amplify the ability of Pavlovian stimuli to invigorate instrumental behaviour, a phenomenon termed Pavlovian-instrumental transfer (PIT). Animals were given 8 training days with two sessions per day, an instrumental training session in the morning and a Pavlovian training session in the afternoon. Dexamethasone or vehicle injections were administered daily immediately after Pavlovian training sessions. In a subsequent transfer test, we measured the general PIT effect, i.e. the enhancement of lever pressing for expected reward during presentation of an appetitive Pavlovian stimulus predictive for the same reward. Repeated high-dose (1.2 mg/kg, i.p.) dexamethasone injections elicited pronounced body weight loss, markedly reduced instrumental performance and left Pavlovian learning unaltered, whereas repeated low-dose (3 μg/kg, i.p.) dexamethasone injections inhibited body weight gain, slightly reduced instrumental performance and left Pavlovian learning unaltered during training. Importantly, in rats subjected to high- and low-dose dexamethasone injections, the overall response rates and the PIT effect were reduced in the transfer test. Thus, dexamethasone given after Pavlovian training was not able to amplify the invigorating effects of Pavlovian stimuli on instrumental action. Considerable evidence suggests that body weight changes after repeated low- and high-dose dexamethasone treatment as observed here are associated with muscle atrophy that could impair response capabilities. However, our data suggest that impaired response capabilities are not a

  10. Dissection of Glucocorticoid Receptor-mediated Inhibition of the Hypothalamic-pituitary-adrenal Axis by Gene Targeting in Mice

    PubMed Central

    Laryea, Gloria; Muglia, Lisa; Arnett, Melinda; Muglia, Louis J.

    2014-01-01

    Negative feedback regulation of glucocorticoid (GC) synthesis and secretion occurs through the function of glucocorticoid receptor (GR) at sites in the hypothalamic-pituitary-adrenal (HPA) axis, as well as in brain regions such as the hippocampus, prefrontal cortex, and sympathetic nervous system. This function of GRs in negative feedback coordinates basal glucocorticoid secretion and stress-induced increases in secretion that integrate GC production with the magnitude and duration of the stressor. This review describes the effects of GR loss along major sites of negative feedback including the entire brain, the paraventricular nucleus of the hypothalamus (PVN), and the pituitary. In genetic mouse models, we evaluate circadian regulation of the HPA axis, stress-stimulated neuroendocrine response and behavioral activity, as well as the integrated response of organism metabolism. Our analysis provides information on contributions of region-specific GR-mediated negative feedback to provide insight in understanding HPA axis dysregulation and the pathogenesis of psychiatric and metabolic disorders. PMID:25256348

  11. FK506 binding to the 56-kilodalton immunophilin (Hsp56) in the glucocorticoid receptor heterocomplex has no effect on receptor folding or function.

    PubMed

    Hutchison, K A; Scherrer, L C; Czar, M J; Ning, Y; Sanchez, E R; Leach, K L; Deibel, M R; Pratt, W B

    1993-04-20

    It has recently been reported that the hsp56 component of glucocorticoid receptor heterocomplexes is an immunophilin of the FK506 binding class [Yem, A. W., Tomasselli, A. G., Heinrikson, R. L., Zurcher-Neely, H., Ruff, V. A., Johnson, R. A., & Deibel, M. R. (1992) J. Biol. Chem. 267, 2868-2871; Tai, P. K., Albers, M. W., Chang, H., Faber, L. E., & Schreiber, S. L. (1992) Science 256, 1315-1318]. The existence of binding proteins for these two potent groups of immunosuppressants in the same molecular complex compels us to ask whether FK506 affects glucocorticoid receptor function. We show here that hsp56 is a component of the native L-cell glucocorticoid receptor heterocomplex and that [3H]FK506 binds to the immunopurified, untransformed receptor complex. However, at concentrations in excess of those required to occupy all of its binding sites on hsp56, FK506 does not affect the steroid binding activity of the receptor nor does it stabilize or dissociate the receptor-hsp90 complex. FK506 does not affect steroid-mediated hsp90 dissociation from the receptor in vitro, and it does not affect steroid-mediated nuclear transfer of the receptor or steroid-mediated transcriptional enhancement from a reporter in intact cells. When immunopurified mouse glucocorticoid receptor is reconstituted into a heat shock protein complex by rabbit reticulocyte lysate, hsp56 is present in the reconstituted complex in addition to hsp90 and hsp70. FK506, however, does not affect reconstitution of the complex or return of the receptor to the steroid binding state, a change of conformation that occurs upon receptor association with hsp90.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Glucocorticoid Receptor Mutants Demonstrate Increased Motility Inside the Nucleus of Living Cells: Time of Fluorescence Recovery After Photobleaching (FRAP) Is an Integrated Measure of Receptor Function

    PubMed Central

    Kino, Tomoshige; Liou, Szu-Heng; Charmandari, Evangelia; Chrousos, George P

    2004-01-01

    Natural mutations of the human glucocorticoid receptor (GR) isoform α cause the glucocorticoid resistance syndrome. Mutant receptors may have abnormal interactions with the ligand, target DNA sequences, and/or multiple intracellular proteins, as well as aberrant nucleocytoplasmic trafficking. Using fluorescence recovery after photobleaching (FRAP) analysis, all GR pathologic mutant receptors examined, as well as 2 synthetic GR mutants lacking the activation function (AF)-1 or the lig-and-binding domain (and hence the AF-2), had defective transcriptional activity and dynamic motility defects inside the nucleus of living cells. In the presence of dexamethasone, these mutants displayed a curtailed 50% recovery time (t1/2) after photobleaching and, hence, significantly increased intranuclear motility and decreased “chromatin retention.” The t1/2 values of the mutants correlated positively with their transcriptional activities and depended on the GR domain affected. GRβ, a natural splice variant of the GR gene, also demonstrated a shorter t1/2 than GRα. The motility responsiveness of the natural and artificial mutant receptors examined, and of GRβ, to the proteasomal inhibitor MG-132 also depended on the mutant domain. Thus, mutant glucocorticoid receptors possess dynamic motility defects in the nucleus, possibly caused by their inability to properly interact with all key partner nuclear molecules necessary for full activation of glucocorticoid-responsive genes. PMID:16307173

  13. Role of the Ada adaptor complex in gene activation by the glucocorticoid receptor.

    PubMed Central

    Henriksson, A; Almlöf, T; Ford, J; McEwan, I J; Gustafsson, J A; Wright, A P

    1997-01-01

    We have shown that the Ada adaptor complex is important for the gene activation capacity of the glucocorticoid receptor in yeast. The recently isolated human Ada2 protein also increases the potency of the receptor protein in mammalian cells. The Ada pathway is of key significance for the tau1 core transactivation domain (tau1c) of the receptor, which requires Ada for activity in vivo and in vitro. Ada2 can be precipitated from nuclear extracts by a glutathione S-transferase-tau1 fusion protein coupled to agarose beads, and a direct interaction between Ada2 and tau1c can be shown by using purified proteins. This interaction is strongly reduced by a mutation in tau1c that reduces transactivation activity. Mutations affecting the Ada complex do not reverse transcriptional squelching by the tau1 domain, as they do for the VP16 transactivation domain, and thus these powerful acidic activators differ in at least some important aspects of gene activation. Mutations that reduce the activity of the tau1c domain in wild-type yeast strains cause similar reductions in ada mutants that contain little or no Ada activity. Thus, gene activation mechanisms, in addition to the Ada pathway, are involved in the activity of the tau1c domain. PMID:9154805

  14. The glucocorticoid properties of the synthetic steroid pregna-1,4-diene-11beta-ol-3,20-dione (deltaHOP) are not entirely correlated with the steroid binding to the glucocorticoid receptor.

    PubMed

    Vicent, G P; Pecci, A; Ghini, A A; Piwien-Pilipuk, G; Veleiro, A S; Burton, G; Lantos, C P; Galigniana, M D

    1999-03-25

    The natural steroid 11beta-hydroxyprogesterone is not only a modulator of 11beta-hydroxy-steroid dehydrogenase activity, but also an efficient inducer of tyrosine aminotransferase activity in hepatocytes. In contrast with the low affinity for the mineralocorticoid receptor. 11beta-hydroxyprogesterone binds well to both the glucocorticoid receptor and the carrier protein transcortin. It is accepted that the introduction of a 1:ene double bond into 3-keto 4:ene steroids increases the glucocorticoid potency, so that 3-keto-1,4:diene steroids show improved chemical stability and are more potent glucocorticoids than their respective 4:ene analogs. The steroid pregna-1,4-diene-11beta-ol-3,20-dione (deltaHOP) had previously been described as an anti-inflamatory compound and an inhibitor of macromolecular biosynthesis in thymocytes and lymphocytes. In such studies, deltaHOP also exhibited some particular glucocorticoid properties which made it attractive as a tool for the study of the mechanism of action of glucocorticoids. In the present paper we show that deltaHOP possesses some classical biological actions of glucocorticoids such as deposition of glycogen in rat liver, induction of TAT activity in hepatocytes, and inhibition of the uptake of leucine and thymidine by thymocytes. It also exhibits minimal sodium-retaining properties. Consistent with these biological effects, deltaHOP shows a 70 times lower relative binding affinity for the mineralocortioid receptor than aldosterone, but a reasonable affinity for the glucocorticoid receptor, and is as efficient as dexamethasone in dissociating the 90 kDa heat shock protein from the glucocorticoid receptor heterocomplex. However, the inhibition of the uptake of amino acids and nucleotides observed in the presence of deltaHOP is not efficiently blocked when thymocytes are coincubated in the presence of steroids with known antiglucocorticoid activity. deltaHOP is similarly inefficient in inducing chloramphenicol

  15. Targeted Ablation Reveals a Novel Role of FKBP52 in Gene-Specific Regulation of Glucocorticoid Receptor Transcriptional Activity

    PubMed Central

    Wolf, Irene M.; Periyasamy, Sumudra; Hinds, Terry; Yong, Weidong; Shou, Weinian; Sanchez, Edwin R.

    2009-01-01

    FKBP52 is a tetratricopeptide repeat (TPR) protein with peptidyl-prolyl isomerase activity and is found in steroid receptor complexes, including glucocorticoid receptor (GR). It is generally accepted that FKBP52 has a stimulatory effect on GR transcriptional activity. However, the mechanism by which FKBP52 controls GR is not yet clear, with reports showing effects on GR hormone-binding affinity and/or hormone-induced nuclear translocation. To address this issue, we have generated mice with targeted ablation of the FKBP52 gene. To date, no overt defects of GR-regulated physiology have been found in these animals, demonstrating that FKBP52 is not an essential regulator of global GR activity. To better assess the impact of FKBP52 on GR, mouse embryonic fibroblasts (MEFs) were generated from wild-type (WT) and FKBP52-deficient (KO) animals. Analysis of GR activity at reporter genes showed an approximate 70% reduction of activity in 52KO MEF cells, with no effect of FKBP52 loss on thyroid receptor. Interestingly, GR activity at endogenous genes was not globally affected in 52KO cells, with reduced activity at GILZ and FKBP51, but not at SGK and p21. Thus, FKBP52 appears to be a gene-specific modulator of GR. To investigate the mechanism of this action, analyses of GR heterocomplex composition, hormone-binding affinity, and ability to undergo hormone-induced nuclear translocation and DNA-binding were performed. Interestingly, no effect of FKBP52 loss was found for any of these GR properties, suggesting that the main function of FKBP52 is a heretofore-unknown ability to control GR activity at target genes. Lastly, loss of FKBP52 did not affect the ability of GR to undergo hormone-induced autologous down-regulation, showing that FKBP52 does not contribute to all branches of GR signaling. The implications of these results to the potential actions of FKBP52 on GR activity in vivo are discussed. PMID:19073255

  16. ATP-dependent release of glucocorticoid receptors from the nuclear matrix.

    PubMed Central

    Tang, Y; DeFranco, D B

    1996-01-01

    Glucocorticoid receptors (GRs) have the capacity to shuttle between the nuclear and cytoplasmic compartments, sharing that trait with other steroid receptors and unrelated nuclear proteins of diverse function. Although nuclear import of steroid receptors, like that of nearly all other karyophilic proteins examined to date, requires ATP, there appear to be different energetic requirements for export of proteins, including steroid receptors, from nuclei. In an attempt to reveal which steps, if any, in the nuclear export pathway utilized by steroid receptors require ATP, we have used indirect immunofluorescence to visualize GRs within cells subjected to a reversible ATP depletion. Under conditions which lead to >95% depletion of cellular ATP levels within 90 min, GRs remain localized within nuclei and do not efflux into the cytoplasm. Under analogous conditions of ATP depletion, transfected progesterone receptors are also retained within nuclei. Importantly, GRs which accumulate within nuclei of ATP-depleted cells are distinguished from nuclear receptors in metabolically active cells by their resistance to in situ extraction with a hypotonic, detergent-containing buffer. GRs in ATP-depleted cells are not permanently trapped in this nuclear compartment, as nuclear receptors rapidly regain their capacity to be extracted upon restoration of cellular ATP, even in the absence of de novo protein synthesis. More extensive extraction of cells with high salt and detergent, coupled with DNase I digestion, established that a significant fraction of GRs in ATP-depleted cells are associated with an RNA-containing nuclear matrix. Quantitative Western blot (immunoblot) analysis confirmed the dramatic increase in GR binding to the nuclear matrix of ATP-depleted cells, while confocal microscopy revealed that GRs are bound to the matrix throughout all planes of the nucleus. ATP depletion does not lead to wholesale collapse of nuclear proteins onto the matrix, as the interaction of a

  17. Effects of antenatal synthetic glucocorticoid on glucocorticoid receptor binding, DNA methylation, and genome-wide mRNA levels in the fetal male hippocampus.

    PubMed

    Crudo, Ariann; Petropoulos, Sophie; Suderman, Matthew; Moisiadis, Vasilis G; Kostaki, Alisa; Hallett, Michael; Szyf, Moshe; Matthews, Stephen G

    2013-11-01

    The endogenous glucocorticoid (GC) surge in late gestation plays a vital role in maturation of several organ systems. For this reason, pregnant women at risk of preterm labor are administered synthetic glucocorticoids (sGCs) to promote fetal lung development. Animal studies have shown that fetal sGC exposure can cause life-long changes in endocrine and metabolic function. We have previously shown that antenatal sGC treatment is associated with alterations in global DNA methylation and modifications to the hippocampal methylome and acetylome. In this study, we hypothesized that: 1) there are changes in the transcriptional landscape of the fetal hippocampus in late gestation, associated with the endogenous cortisol surge; 2) fetal sGC exposure alters genome-wide transcription in the hippocampus; and 3) these changes in transcription are associated with modified glucocorticoid receptor (GR) DNA binding and DNA methylation. sGC was administered as 2 courses on gestational days (GD) 40, 41, 50, and 51, and the hippocampi of fetal guinea pigs were examined before (GD52) and after (GD65) the endogenous cortisol surge (Term ∼GD67). We also analyzed fetal hippocampi 24 hours and 14 days following maternal sGC injections (n = 3-4/group). Genome-wide modification of transcription and GR DNA binding occurred in late gestation, in parallel with the normal GC surge. Further, sGC exposure had a substantial impact on the hippocampal transcriptome, GR-DNA binding, and DNA methylation at 24 hours and 14 days following the final sGC treatment. These data support the hypothesis that GC exposure in late gestation plays a significant role in modifying the transcriptional and epigenetic landscape of the developing fetal hippocampus and that substantial effects are evident for at least 2 weeks after sGC exposure.

  18. Reduction of glucocorticoid receptor ligand binding by the 11-beta hydroxysteroid dehydrogenase type 2 inhibitor, Thiram.

    PubMed

    Garbrecht, Mark R; Krozowski, Zygmunt S; Snyder, Jeanne M; Schmidt, Thomas J

    2006-10-01

    Endogenous and synthetic glucocorticoids (GCs), such as cortisol and dexamethasone (Dex), modulate airway inflammation, regulate the production of surfactant by lung epithelial cells, and influence fetal lung maturation. The 11-beta hydroxysteroid dehydrogenase type 2 (HSD2) enzyme catalyzes the oxidation of bioactive cortisol and Dex to their 11-keto metabolites. Thiram (tetramethylthiuram disulfide) specifically inhibits HSD2 activity by oxidizing cysteine residues located in the cofactor binding domain of the enzyme. During studies performed to define a potential role for HSD2 in modulating GC action in human lung epithelial cells, we observed that exposure of intact human lung epithelial cells (NCI-H441) to 50 microM Thiram significantly attenuated the down-stream effects of Dex (100 nM) on the expression of two GC-sensitive genes, pulmonary surfactant proteins A and B. This observation appeared to be inconsistent with simple inhibition of HSD2 activity. Although Thiram inhibited HSD2 oxidase activity in a dose-dependent manner without affecting HSD2 protein expression, Thiram also reduced specific binding of [3H]-Dex to the glucocorticoid receptor (GR). Pre-treatment of cells with 1 mM dithiothreitol (DTT), a thiol-reducing agent, completely blocked the inhibitory effect of Thiram on ligand binding. These results are suggestive that Thiram may alter the ligand-binding domain of the GR by oxidizing critical thiol-containing amino acid residues. Taken collectively, these data demonstrate that attenuated down-stream GC signaling, via decreased binding of ligand to the GR, is a novel cellular effect of Thiram exposure in human lung epithelial cells.

  19. Forkhead box A3 mediates glucocorticoid receptor function in adipose tissue.

    PubMed

    Ma, Xinran; Xu, Lingyan; Mueller, Elisabetta

    2016-03-22

    Glucocorticoids (GCs) are widely prescribed anti-inflammatory agents, but their chronic use leads to undesirable side effects such as excessive expansion of adipose tissue. We have recently shown that the forkhead box protein A3 (Foxa3) is a calorie-hoarding factor that regulates the selective enlargement of epididymal fat depots and suppresses energy expenditure in a nutritional- and age-dependent manner. It has been demonstrated that Foxa3 levels are elevated in adipose depots in response to high-fat diet regimens and during the aging process; however no studies to date have elucidated the mechanisms that control Foxa3's expression in fat. Given the established effects of GCs in increasing visceral adiposity and in reducing thermogenesis, we assessed the existence of a possible link between GCs and Foxa3. Computational prediction analysis combined with molecular studies revealed that Foxa3 is regulated by the glucocorticoid receptor (GR) in preadipocytes, adipocytes, and adipose tissues and is required to facilitate the binding of the GR to its target gene promoters in fat depots. Analysis of the long-term effects of dexamethasone treatment in mice revealed that Foxa3 ablation protects mice specifically against fat accretion but not against other pathological side effects elicited by this synthetic GC in tissues such as liver, muscle, and spleen. In conclusion our studies provide the first demonstration, to our knowledge, that Foxa3 is a direct target of GC action in adipose tissues and point to a role of Foxa3 as a mediator of the side effects induced in fat tissues by chronic treatment with synthetic steroids.

  20. Novel Mechanism of Steroid Action in Skin through Glucocorticoid Receptor Monomers

    PubMed Central

    Radoja, Nadezda; Komine, Mayumi; Jho, Sang H.; Blumenberg, Miroslav; Tomic-Canic, Marjana

    2000-01-01

    Glucocorticoids (GCs), important regulators of epidermal growth, differentiation, and homeostasis, are used extensively in the treatment of skin diseases. Using keratin gene expression as a paradigm of epidermal physiology and pathology, we have developed a model system to study the molecular mechanism of GCs action in skin. Here we describe a novel mechanism of suppression of transcription by the glucocorticoid receptor (GR) that represents an example of customizing a device for transcriptional regulation to target a specific group of genes within the target tissue, in our case, epidermis. We have shown that GCs repress the expression of the basal-cell-specific keratins K5 and K14 and disease-associated keratins K6, K16, and K17 but not the differentiation-specific keratins K3 and K10 or the simple epithelium-specific keratins K8, K18, and K19. We have identified the negative recognition elements (nGREs) in all five regulated keratin gene promoters. Detailed footprinting revealed that the function of nGREs is to instruct the GR to bind as four monomers. Furthermore, using cotransfection and antisense technology we have found that, unlike SRC-1 and GRIP-1, which are not involved in the GR complex that suppresses keratin genes, histone acetyltransferase and CBP are. In addition, we have found that GR, independently from GREs, blocks the induction of keratin gene expression by AP1. We conclude that GR suppresses keratin gene expression through two independent mechanisms: directly, through interactions of keratin nGREs with four GR monomers, as well as indirectly, by blocking the AP1 induction of keratin gene expression. PMID:10825196

  1. Hepatic growth hormone and glucocorticoid receptor signaling in body growth, steatosis and metabolic liver cancer development.

    PubMed

    Mueller, Kristina M; Themanns, Madeleine; Friedbichler, Katrin; Kornfeld, Jan-Wilhelm; Esterbauer, Harald; Tuckermann, Jan P; Moriggl, Richard

    2012-09-25

    Growth hormone (GH) and glucocorticoids (GCs) are involved in the control of processes that are essential for the maintenance of vital body functions including energy supply and growth control. GH and GCs have been well characterized to regulate systemic energy homeostasis, particular during certain conditions of physical stress. However, dysfunctional signaling in both pathways is linked to various metabolic disorders associated with aberrant carbohydrate and lipid metabolism. In liver, GH-dependent activation of the transcription factor signal transducer and activator of transcription (STAT) 5 controls a variety of physiologic functions within hepatocytes. Similarly, GCs, through activation of the glucocorticoid receptor (GR), influence many important liver functions such as gluconeogenesis. Studies in hepatic Stat5 or GR knockout mice have revealed that they similarly control liver function on their target gene level and indeed, the GR functions often as a cofactor of STAT5 for GH-induced genes. Gene sets, which require physical STAT5-GR interaction, include those controlling body growth and maturation. More recently, it has become evident that impairment of GH-STAT5 signaling in different experimental models correlates with metabolic liver disease, ranging from hepatic steatosis to hepatocellular carcinoma (HCC). While GH-activated STAT5 has a protective role in chronic liver disease, experimental disruption of GC-GR signaling rather seems to ameliorate metabolic disorders under metabolic challenge. In this review, we focus on the current knowledge about hepatic GH-STAT5 and GC-GR signaling in body growth, metabolism, and protection from fatty liver disease and HCC development.

  2. Forkhead box A3 mediates glucocorticoid receptor function in adipose tissue

    PubMed Central

    Ma, Xinran; Xu, Lingyan; Mueller, Elisabetta

    2016-01-01

    Glucocorticoids (GCs) are widely prescribed anti-inflammatory agents, but their chronic use leads to undesirable side effects such as excessive expansion of adipose tissue. We have recently shown that the forkhead box protein A3 (Foxa3) is a calorie-hoarding factor that regulates the selective enlargement of epididymal fat depots and suppresses energy expenditure in a nutritional- and age-dependent manner. It has been demonstrated that Foxa3 levels are elevated in adipose depots in response to high-fat diet regimens and during the aging process; however no studies to date have elucidated the mechanisms that control Foxa3’s expression in fat. Given the established effects of GCs in increasing visceral adiposity and in reducing thermogenesis, we assessed the existence of a possible link between GCs and Foxa3. Computational prediction analysis combined with molecular studies revealed that Foxa3 is regulated by the glucocorticoid receptor (GR) in preadipocytes, adipocytes, and adipose tissues and is required to facilitate the binding of the GR to its target gene promoters in fat depots. Analysis of the long-term effects of dexamethasone treatment in mice revealed that Foxa3 ablation protects mice specifically against fat accretion but not against other pathological side effects elicited by this synthetic GC in tissues such as liver, muscle, and spleen. In conclusion our studies provide the first demonstration, to our knowledge, that Foxa3 is a direct target of GC action in adipose tissues and point to a role of Foxa3 as a mediator of the side effects induced in fat tissues by chronic treatment with synthetic steroids. PMID:26957608

  3. Enhancement of stress resilience through Hdac6-mediated regulation of glucocorticoid receptor chaperone dynamics

    PubMed Central

    Jochems, Jeanine; Teegarden, Sarah L; Chen, Yong; Boulden, Janette; Challis, Collin; Ben-Dor, Gabriel A; Kim, Sangwon F; Berton, Olivier

    2014-01-01

    Background Acetylation of Hsp90 regulates downstream hormone signaling via the glucocorticoid receptor (GR), but the role of this molecular mechanism in stress homeostasis remains poorly understood. We tested whether acetylation of Hsp90 in the brain predicts and modulates the behavioral sequelae of a mouse model of social stress. Methods Mice subjected to chronic social defeat stress (CSDS) were stratified into resilient and vulnerable subpopulations. HPA axis function was probed using a DEX/CRF test. Hsp90 acetylation, Hsp90-GR interactions and GR translocation were measured in the dorsal raphe nucleus (DRN). To manipulate Hsp90 acetylation, we pharmacologically inhibited Hdac6, a known deacetylase of Hsp90 or overexpressed a point-mutant that mimics the hyperacetylated state of Hsp90 at lysine K294 Results Lower acetylated Hsp90, higher GR-Hsp90 association and enhanced GR translocation were observed in DRN of vulnerable mice after CSDS. Administration of ACY-738, an Hdac6-selective inhibitor, led to Hsp90 hyperacetylation in brain and in neuronal culture. In cell-based assays, ACY-738 increased the relative association of Hsp90 with FKBP51 versus FKBP52 and inhibited hormone-induced GR translocation. This effect was replicated by overexpressing the acetylation-mimic point-mutant of Hsp90. In vivo, ACY-738 promoted resilience to CSDS and serotonin-selective viral overexpression of the acetylation-mimic mutant of Hsp90 in raphe neurons reproduced the behaviroral effect of ACY-738. Conclusions Hyperacetylation of Hsp90 is a predictor and causal molecular determinant of stress resilience in mice. Brain-penetrant Hdac6 inhibitors increase Hsp90 acetylation and modulate GR chaperone dynamics offering a promising strategy to curtail deleterious socioaffective effects of stress and glucocorticoids. PMID:25442004

  4. A novel point mutation in the ligand-binding domain (LBD) of the human glucocorticoid receptor (hGR) causing generalized glucocorticoid resistance: the importance of the C terminus of hGR LBD in conferring transactivational activity.

    PubMed

    Charmandari, Evangelia; Raji, Annaswamy; Kino, Tomoshige; Ichijo, Takamasa; Tiulpakov, Anatoly; Zachman, Keith; Chrousos, George P

    2005-06-01

    Glucocorticoid resistance is a rare, familial or sporadic condition characterized by partial end-organ insensitivity to glucocorticoids. The clinical spectrum of the condition is broad, ranging from completely asymptomatic to severe hyperandrogenism and/or mineralocorticoid excess. The molecular basis of glucocorticoid resistance has been ascribed to mutations in the human glucocorticoid receptor-alpha (hGRalpha) gene, which impair one or more of the molecular mechanisms of GR action, thus altering tissue sensitivity to glucocorticoids. We identified a new case of generalized glucocorticoid resistance in a young woman who presented with a long-standing history of fatigue, anxiety, hyperandrogenism, and hypertension. The disease was caused by a novel, heterozygous mutation (T-->C) at nucleotide position 2318 (exon 9) of the hGRalpha gene, which resulted in substitution of leucine by proline at amino acid position 773 in the ligand-binding domain of the receptor. We systematically investigated the molecular mechanisms through which the natural hGRalphaL773P mutant impaired glucocorticoid signal transduction. Compared with the wild-type hGRalpha, hGRalphaL773P demonstrated a 2-fold reduction in the ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter, exerted a dominant negative effect on the wild-type receptor, had a 2.6-fold reduction in the affinity for ligand, showed delayed nuclear translocation (30 vs. 12 min), and, although it preserved its ability to bind to DNA, displayed an abnormal interaction with the GR-interacting protein 1 coactivator in vitro. We conclude that the carboxyl terminus of the ligand-binding domain of hGRalpha is extremely important in conferring transactivational activity by altering multiple functions of this composite transcription factor. PMID:15769988

  5. Enhanced in vitro refolding of soluble human glucocorticoid-induced TNF receptor-related ligand.

    PubMed

    Kovács, Erika; Szilágyi, László; Koncz, Gábor; Lányi, Szabolcs; Ábrahám, Beáta

    2013-06-01

    The glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily. Attachment of GITR to its ligand (GITRL) regulates diverse biological functions, including cell proliferation, differentiation, and survival. In this study, the extracellular region of human GITRL (hGITRL) was cloned, expressed, and purified. The coding sequence of the extracellular region of hGITRL was isolated from human brain cDNA and inserted in pET20b vector. The hGITRL was expressed in Escherichia coli BL21 (DE3) Star at 37 and 25 °C. The majority of the protein was found in inclusion bodies. We identified three important factors for efficient refolding of hGITRL: a ratio of GSH/GSSG, pH, and addition of polyethylene glycol. The renaturated protein was purified by Ni-NTA chromatography. The overall yield of the expression and refolding was higher than 50 mg/l E. coli culture grown at 37 °C. Size exclusion chromatography showed that hGITRL exists as mixture of various multimeric forms in solution. We tested the association of recombinant hGITRL with THP-1 and U937 cell lines and its activity to promote extracellular signal-regulated protein kinase phosphorylation. The results showed that the recombinant protein was biologically active.

  6. Blockade of glucocorticoid receptors improves cutaneous wound healing in stressed mice.

    PubMed

    de Almeida, Taís Fontoura; de Castro Pires, Taiza; Monte-Alto-Costa, Andréa

    2016-02-01

    Stress is an important condition of modern life. The successful wound healing requires the execution of three major overlapping phases: inflammation, proliferation, and remodeling, and stress can disturb this process. Chronic stress impairs wound healing through the activation of the hypothalamic-pituitary-adrenal axis, and the glucocorticoids (GCs) hormones have been shown to delay wound closure. Therefore, the aim of this study was to investigate the effects of a GC receptor antagonist (RU486) treatment on cutaneous healing in chronically stressed mice. Male mice were submitted to rotational stress, whereas control animals were not subjected to stress. Stressed and control animals were treated with RU486. A full-thickness excisional lesion was generated, and seven days later, lesions were recovered. The RU486 treatment improves wound healing since contraction takes place earlier in RU486-treated in comparison to non-treated mice, and the RU486 treatment also improves the angiogenesis in Stress+RU486 mice when compared to stressed animals. The Stress+RU486 group showed a decrease in inflammatory cell infiltration and in hypoxia-inducible factor-1α and inducible nitric oxide synthase expression; meanwhile, there was an increase in myofibroblasts quantity. In conclusion, blockade of GC receptors with RU486 partially ameliorates stress-impaired wound healing, suggesting that stress inhibits healing through more than one functional pathway.

  7. Blockade of glucocorticoid receptors improves cutaneous wound healing in stressed mice

    PubMed Central

    de Almeida, Taís Fontoura; de Castro Pires, Taiza

    2016-01-01

    Stress is an important condition of modern life. The successful wound healing requires the execution of three major overlapping phases: inflammation, proliferation, and remodeling, and stress can disturb this process. Chronic stress impairs wound healing through the activation of the hypothalamic–pituitary–adrenal axis, and the glucocorticoids (GCs) hormones have been shown to delay wound closure. Therefore, the aim of this study was to investigate the effects of a GC receptor antagonist (RU486) treatment on cutaneous healing in chronically stressed mice. Male mice were submitted to rotational stress, whereas control animals were not subjected to stress. Stressed and control animals were treated with RU486. A full-thickness excisional lesion was generated, and seven days later, lesions were recovered. The RU486 treatment improves wound healing since contraction takes place earlier in RU486-treated in comparison to non-treated mice, and the RU486 treatment also improves the angiogenesis in Stress+RU486 mice when compared to stressed animals. The Stress+RU486 group showed a decrease in inflammatory cell infiltration and in hypoxia-inducible factor-1α and inducible nitric oxide synthase expression; meanwhile, there was an increase in myofibroblasts quantity. In conclusion, blockade of GC receptors with RU486 partially ameliorates stress-impaired wound healing, suggesting that stress inhibits healing through more than one functional pathway. PMID:26515142

  8. Glucocorticoid receptor (GR) {beta} has intrinsic, GR{alpha}-independent transcriptional activity

    SciTech Connect

    Kino, Tomoshige; Manoli, Irini; Kelkar, Sujata; Wang, Yonghong; Su, Yan A.; Chrousos, George P.

    2009-04-17

    The human glucocorticoid receptor (GR) gene produces C-terminal GR{beta} and GR{alpha} isoforms through alternative use of specific exons 9{beta} and {alpha}, respectively. We explored the transcriptional activity of GR{beta} on endogenous genes by developing HeLa cells stably expressing EGFP-GR{beta} or EGFP. Microarray analyses revealed that GR{beta} had intrinsic gene-specific transcriptional activity, regulating mRNA expression of a large number of genes negatively or positively. Majority of GR{beta}-responsive genes was distinct from those modulated by GR{alpha}, while GR{beta} and GR{alpha} mutually modulated each other's transcriptional activity in a subpopulation of genes. We did not observe in HCT116 cells nuclear translocation of GR{beta} and activation of this receptor by RU 486, a synthetic steroid previously reported to bind GR{beta} and to induce nuclear translocation. Our results indicate that GR{beta} has intrinsic, GR{alpha}-independent, gene-specific transcriptional activity, in addition to its previously reported dominant negative effect on GR{alpha}-induced transactivation of GRE-driven promoters.

  9. Glucocorticoid receptor dysfunction: consequences for the pathophysiology and treatment of mood disorders

    PubMed Central

    Abraham, Aju; Watson, Stuart; Young, Allan H

    2003-01-01

    Background: Hypothalamic-pituitary-adrenal (HPA) axis dysfunction in mood disorders is one of the most robust findings in biological psychiatry. However, considerable debate surrounds the nature of the core abnormality, its cause, consequences and treatment implications. Aims: To review the evidence for the role of HPA axis dysfunction in the pathophysiology of mood disorders with particular reference to corticosteroid receptor pathology. Methods: A selective review of the published literature in this field, focusing on human studies. Results: The nature of basal HPA axis dysregulation described in both manic and depressed bipolars appears to be similar to those described in MDD. But studies using the dexamethasone/ corticotropin releasing hormone (dex/CRH) test and dexamethasone suppression test (DST) have shown that HPA axis dysfunction is more prevalent in bipolar than in unipolar disorder. There is robust evidence for corticotropin releasing hormone (CRH) hyperdrive and glucocorticoid receptor (GR) dysfunction in mood disorders, with increasing evidence for disorders within the AVP system. Conclusion: HPA axis dysfunction is prevalent in patients with mood disorder, particularly those with psychotic disorders and bipolar affective disorder. This may be secondary to genetic factors, early life adversities or both. Dysfunction of GR may be the underlying abnormality and preliminary findings suggest that it is a potential target for novel therapies. Declaration of interest: None PMID:21206827

  10. Heterocyclic glucocorticoid receptor modulators with a 2,2-dimethyl-3-phenyl-N-(thiazol or thiadiazol-2-yl)propanamide core.

    PubMed

    Xiao, Hai-Yun; Wu, Dauh-Rurng; Sheppeck, James E; Habte, Sium F; Cunningham, Mark D; Somerville, John E; Barrish, Joel C; Nadler, Steven G; Dhar, T G Murali

    2013-10-15

    A series of heterocyclic glucocorticoid receptor (GR) modulators with 2,2-dimethyl-3-phenyl-N-(thiazol or thiadiazol-2-yl)propanamide core are described. Structure-activity relationships suggest a combination of H-bond acceptor and a 4-fluorophenyl moiety as being important structural components contributing to the glucocorticoid receptor binding and functional activity for this series of GR modulators. PMID:24011644

  11. Down-regulation of insulin receptors is related to insulin internalization

    SciTech Connect

    Geiger, D.; Carpentier, J.L.; Gorden, P.; Orci, L. )

    1989-11-01

    In the present study, we have tested the influence of inhibition of endocytosis by hypertonic medium on the regulation of cell surface insulin receptors. We show that active internalization of {sup 125}I-insulin is markedly inhibited by hypertonic media and that, in parallel, cell surface invaginations are significantly diminished. These two events are accompanied by a marked inhibition of cell surface insulin receptor down-regulation. These data provide further strong evidence that receptor-mediated endocytosis is the major mechanism by which insulin receptors are regulated at the surface of target cells.

  12. EP4 receptor stimulation down-regulates human eosinophil function.

    PubMed

    Luschnig-Schratl, Petra; Sturm, Eva M; Konya, Viktoria; Philipose, Sonia; Marsche, Gunther; Fröhlich, Eleonore; Samberger, Claudia; Lang-Loidolt, Doris; Gattenlöhner, Stefan; Lippe, Irmgard Th; Peskar, Bernhard A; Schuligoi, Rufina; Heinemann, Akos

    2011-11-01

    Accumulation of eosinophils in tissue is a hallmark of allergic inflammation. Here we observed that a selective agonist of the PGE(2) receptor EP4, ONO AE1-329, potently attenuated the chemotaxis of human peripheral blood eosinophils, upregulation of the adhesion molecule CD11b and the production of reactive oxygen species. These effects were accompanied by the inhibition of cytoskeletal rearrangement and Ca(2+) mobilization. The involvement of the EP4 receptor was substantiated by a selective EP4 antagonist, which reversed the inhibitory effects of PGE(2) and the EP4 agonist. Selective kinase inhibitors revealed that the inhibitory effect of EP4 stimulation on eosinophil migration depended upon activation of PI 3-kinase and PKC, but not cAMP. Finally, we found that EP4 receptors are expressed by human eosinophils, and are also present on infiltrating leukocytes in inflamed human nasal mucosa. These data indicate that EP4 agonists might be a novel therapeutic option in eosinophilic diseases.

  13. Hepatic glucocorticoid and α1- and β2-adrenergic receptors in calves change during neonatal maturation and are related to energy regulation.

    PubMed

    Schäff, C T; Rohrbeck, D; Steinhoff-Wagner, J; Kanitz, E; Sauerwein, H; Bruckmaier, R M; Hammon, H M

    2015-02-01

    Catecholamines and glucocorticoids are involved in fetal maturation of organ systems to prepare the fetus for extrauterine life. Calves, especially when born preterm, depend on function of the adrenergic system and the glucocorticoid axis to adapt energy metabolism for the neonatal period. We tested the hypothesis that hepatic glucocorticoid and α1- and β2-adrenergic receptors in neonatal calves are involved in adaptation of energy metabolism around birth and that respective binding capacities depend on stage of maturation during the neonatal period. Calves (n=7 per group) were delivered by section preterm (PT, 9d before term) or were born at term (full-term, FT; spontaneous vaginal delivery), or spontaneously born and fed colostrum for 4d (FTC). Blood samples were taken immediately after birth and before and 2h after feeding at 24h after birth (PT, FT) or on d 4 of life (FTC) to determine metabolic and endocrine changes. After slaughter at 26h after birth (PT, FT) or on d 4 of life (FTC), liver tissue was obtained to measure hepatic binding capacity of glucocorticoid and α1- and β2-adrenergic receptors. Maximal binding capacity and binding affinity were calculated by saturation binding assays using [(3)H]-prazosin and [(3)H]-CGP-12177 for determination of α1- and β2-adrenergic receptors, respectively, and [(3)H]-dexamethasone for determination of glucocorticoid receptor in liver. Additional liver samples were taken to measure mRNA abundance of glucocorticoid and α1- and β2-adrenergic receptors, of key enzymes and factors related to hepatic lipid metabolism, and of insulin-like growth factor 1 (IGF1). Plasma concentrations of β-hydroxybutyrate and leptin changed with time, and leptin concentrations were affected by stage of maturation. The binding capacities for hepatic glucocorticoid and β2-adrenergic receptors as well as gene expression of IGF1 were greater in FTC than in FT and PT, and binding affinity for β2-adrenergic receptor was lowest in PT. The

  14. Influences of maternal and paternal PTSD on epigenetic regulation of the glucocorticoid receptor gene in Holocaust survivor offspring

    PubMed Central

    Desarnaud, Frank; Bader, Heather N.; Makotkine, Iouri; Flory, Janine D.; Bierer, Linda M.; Meaney, Michael J.

    2014-01-01

    Objective Differential effects of maternal and paternal PTSD have been observed in adult offspring of Holocaust survivors in both glucocorticoid receptor sensitivity and vulnerability to psychiatric disorder. The current study examined the relative influences of maternal and paternal PTSD on DNA methylation of the exon 1F promoter of the glucocorticoid receptor gene (NR3C1) in peripheral blood mononuclear cells (PBMCs), and its relationship to glucocorticoid receptor sensitivity, in Holocaust offspring. Method Adult offspring with at least one Holocaust survivor parent (n=80), and demographically similar participants without parental Holocaust exposure or PTSD (n=15) completed clinical interviews, self-report measures, and biological procedures. Blood samples were collected for analysis of glucocorticoid receptor gene exon 1F (GR-1F) promoter methylation and cortisol levels in response to low-dose dexamethasone, and two-way analysis of covariance was performed using maternal and paternal PTSD as main effects. Hierarchical-clustering analysis was used to permit visualization of maternal vs. paternal PTSD effects on clinical variables. Results A significant interaction demonstrated that in the absence of maternal PTSD, offspring with paternal PTSD showed higher GR-1F promoter methylation, whereas offspring with both maternal and paternal PTSD showed lower methylation. Lower GR-1F promoter methylation was significantly associated with greater post-dexamethasone cortisol suppression. The clustering analysis confirmed that maternal and paternal PTSD effects were differentially associated with clinical indicators. Conclusions This is the first study to demonstrate alterations of GR-1F promoter methylation in relation to parental PTSD and neuroendocrine outcomes. The moderation of paternal PTSD effects by maternal PTSD suggests different mechanisms for the intergenerational transmission of trauma-related vulnerabilities. PMID:24832930

  15. PPAR-α and glucocorticoid receptor synergize to promote erythroid progenitor self-renewal.

    PubMed

    Lee, Hsiang-Ying; Gao, Xiaofei; Barrasa, M Inmaculada; Li, Hu; Elmes, Russell R; Peters, Luanne L; Lodish, Harvey F

    2015-06-25

    Many acute and chronic anaemias, including haemolysis, sepsis and genetic bone marrow failure diseases such as Diamond-Blackfan anaemia, are not treatable with erythropoietin (Epo), because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production. Treatment of these anaemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently, we showed that glucocorticoids specifically stimulate self-renewal of an early erythroid progenitor, burst-forming unit erythroid (BFU-E), and increase the production of terminally differentiated erythroid cells. Here we show that activation of the peroxisome proliferator-activated receptor α (PPAR-α) by the PPAR-α agonists GW7647 and fenofibrate synergizes with the glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures of both mouse fetal liver BFU-Es and mobilized human adult CD34(+) peripheral blood progenitors, with a new and effective culture system being used for the human cells that generates normal enucleated reticulocytes. Although Ppara(-/-) mice show no haematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis, PPAR-α agonists facilitate recovery of wild-type but not Ppara(-/-) mice from PHZ-induced acute haemolytic anaemia. We also show that PPAR-α alleviates anaemia in a mouse model of chronic anaemia. Finally, both in control and corticosteroid-treated BFU-E cells, PPAR-α co-occupies many chromatin sites with GR; when activated by PPAR-α agonists, additional PPAR-α is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPAR-α agonists in stimulating self-renewal of early erythroid

  16. PPARα and glucocorticoid receptor synergize to promote erythroid progenitor self-renewal

    PubMed Central

    Lee, Hsiang-Ying; Gao, Xiaofei; Barrasa, M. Inmaculada; Li, Hu; Elmes, Russell R.; Peters, Luanne L.; Lodish, Harvey F.

    2015-01-01

    Summary Many acute and chronic anemias, including hemolysis, sepsis, and genetic bone marrow failure diseases such as Diamond-Blackfan Anemia (DBA), are not treatable with erythropoietin (Epo), because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production 1,2,3–5,6,7,8,9. Treatment of these anemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently we showed that glucocorticoids specifically stimulate self-renewal of the early erythroid progenitor, the burst-forming unit erythroid (BFU-E), and increase the production of terminally differentiated erythroid cells 10,11. Here we demonstrate that activation of the peroxisome proliferator-activated receptor alpha (PPARα) by PPARα agonists, GW7647 and fenofibrate, synergizes with glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures both of mouse fetal liver BFU-Es and of mobilized human adult CD34+ peripheral blood progenitors, the latter employing a new and effective culture system that generates normal enucleated reticulocytes. While PPARα−/− mice show no hematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis, PPARα agonists facilitate recovery of wild-type mice, but not PPARα−/− mice, from PHZ-induced acute hemolytic anemia. We also showed that PPARα alleviates anemia in a mouse model of chronic anemia. Finally, both in control and corticosteroid-treated BFU-E cells PPARα co-occupies many chromatin sites with GR; when activated by PPARα agonists, additional PPARα is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPARα agonists in stimulating self

  17. PPAR-α and glucocorticoid receptor synergize to promote erythroid progenitor self-renewal.

    PubMed

    Lee, Hsiang-Ying; Gao, Xiaofei; Barrasa, M Inmaculada; Li, Hu; Elmes, Russell R; Peters, Luanne L; Lodish, Harvey F

    2015-06-25

    Many acute and chronic anaemias, including haemolysis, sepsis and genetic bone marrow failure diseases such as Diamond-Blackfan anaemia, are not treatable with erythropoietin (Epo), because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production. Treatment of these anaemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently, we showed that glucocorticoids specifically stimulate self-renewal of an early erythroid progenitor, burst-forming unit erythroid (BFU-E), and increase the production of terminally differentiated erythroid cells. Here we show that activation of the peroxisome proliferator-activated receptor α (PPAR-α) by the PPAR-α agonists GW7647 and fenofibrate synergizes with the glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures of both mouse fetal liver BFU-Es and mobilized human adult CD34(+) peripheral blood progenitors, with a new and effective culture system being used for the human cells that generates normal enucleated reticulocytes. Although Ppara(-/-) mice show no haematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis, PPAR-α agonists facilitate recovery of wild-type but not Ppara(-/-) mice from PHZ-induced acute haemolytic anaemia. We also show that PPAR-α alleviates anaemia in a mouse model of chronic anaemia. Finally, both in control and corticosteroid-treated BFU-E cells, PPAR-α co-occupies many chromatin sites with GR; when activated by PPAR-α agonists, additional PPAR-α is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPAR-α agonists in stimulating self-renewal of early erythroid

  18. Molecular regulation of urea cycle function by the liver glucocorticoid receptor

    PubMed Central

    Okun, Jürgen G.; Conway, Sean; Schmidt, Kathrin V.; Schumacher, Jonas; Wang, Xiaoyue; de Guia, Roldan; Zota, Annika; Klement, Johanna; Seibert, Oksana; Peters, Achim; Maida, Adriano; Herzig, Stephan; Rose, Adam J.

    2015-01-01

    Objective One of the major side effects of glucocorticoid (GC) treatment is lean tissue wasting, indicating a prominent role in systemic amino acid metabolism. In order to uncover a novel aspect of GCs and their intracellular-receptor, the glucocorticoid receptor (GR), on metabolic control, we conducted amino acid and acylcarnitine profiling in human and mouse models of GC/GR gain- and loss-of-function. Methods Blood serum and tissue metabolite levels were determined in Human Addison's disease (AD) patients as well as in mouse models of systemic and liver-specific GR loss-of-function (AAV-miR-GR) with or without dexamethasone (DEX) treatments. Body composition and neuromuscular and metabolic function tests were conducted in vivo and ex vivo, the latter using precision cut liver slices. Results A serum metabolite signature of impaired urea cycle function (i.e. higher [ARG]:[ORN + CIT]) was observed in human (CTRL: 0.45 ± 0.03, AD: 1.29 ± 0.04; p < 0.001) and mouse (AAV-miR-NC: 0.97 ± 0.13, AAV-miR-GR: 2.20 ± 0.19; p < 0.001) GC/GR loss-of-function, with similar patterns also observed in liver. Serum urea levels were consistently affected by GC/GR gain- (∼+32%) and loss (∼−30%) -of-function. Combined liver-specific GR loss-of-function with DEX treatment revealed a tissue-autonomous role for the GR to coordinate an upregulation of liver urea production rate in vivo and ex vivo, and prevent hyperammonaemia and associated neuromuscular dysfunction in vivo. Liver mRNA expression profiling and GR-cistrome mining identified Arginase I (ARG1) a urea cycle gene targeted by the liver GR. Conclusions The liver GR controls systemic and liver urea cycle function by transcriptional regulation of ARG1 expression. PMID:26500844

  19. Glucocorticoid Receptor Activation Inhibits Chemotherapy-induced Cell Death in High-grade Serous Ovarian Carcinoma

    PubMed Central

    Stringer-Reasor, Erica M.; Baker, Gabrielle M.; Skor, Maxwell N.; Kocherginsky, Masha; Lengyel, Ernst; Fleming, Gini F.; Conzen, Suzanne D.

    2015-01-01

    Objectives To test the hypothesis that glucocorticoid receptor (GR) activation increases resistance to chemotherapy in high-grade serous ovarian cancer (HGS-OvCa) and that treatment with a GR antagonist will improve sensitivity to chemotherapy. Methods GR expression was assessed in OvCa cell lines by qRT-PCR and Western blot analysis and in xenografts and primary human tumors using immunohistochemistry (IHC). We also examined the effect of GR activation versus inhibition on chemotherapy-induced cytotoxicity in OvCa cell lines and in a xenograft model. Results With the exception of IGROV-1 cells, all OvCa cell lines tested had detectable GR expression by Western blot and qRT-PCR analysis. Twenty-five out of the 27 human primary HGS-OvCas examined expressed GR by IHC. No cell line expressed detectable progesterone receptor (PR) or androgen receptor (AR) by Western blot analysis. In vitro assays showed that in GR-positive HeyA8 and SKOV3 cells, dexamethasone (100 nM) treatment upregulated the pro-survival genes SGK1 and MKP1/DUSP1 and inhibited carboplatin/gemcitabine-induced cell death. Concurrent treatment with two GR antagonists, either mifepristone (100 nM) or CORT125134 (100 nM), partially reversed these effects. There was no anti-apoptotic effect of dexamethasone on chemotherapy-induced cell death in IGROV-1 cells, which did not have detectable GR protein. Mifepristone treatment alone was not cytotoxic in any cell line. HeyA8 OvCa xenograft studies demonstrated that adding mifepristone to carboplatin/gemcitabine increased tumor shrinkage by 48% compared to carboplatin/gemcitabine treatment alone (P=0.0004). Conclusions These results suggest that GR antagonism sensitizes GR+ OvCa to chemotherapy-induced cell death through inhibition of GR-mediated cell survival pathways. PMID:26115975

  20. Effects of bilobalide on anxiety, spatial learning, memory and levels of hippocampal glucocorticoid receptors in male Kunming mice.

    PubMed

    Ma, Leige; Wang, Shiyan; Tai, Fadao; Yuan, Gang; Wu, Ruiyong; Liu, Xiao; Wei, Bin; Yang, Xiangping

    2012-12-15

    With various constituents in ginkgo biloba extract, the detailed internal mechanism underlying a reduction in anxiety and improvements to learning and memory from GBE is not well understood. The present study investigated whether bilobalide, an important constituent in GBE, could affect anxiety, spatial learning and memory in male mice and whether hippocampal glucocorticoid receptor expression is associated with alteration in these behaviors. Mice were treated orally either with sesame seed oil or one of three dosages of bilobalide (2.5, 5.0 and 10.0 mg/kg) daily until testing. A series of behavioral tests showed that repeated bilobalide treatment decreased levels of anxiety-like behavior and increased locomotor activity in open field and elevated plus maze tests. Treatment with bilobalide also shortened the time taken to find the platform in a Morris water-maze test. Mice exposed to bilobalide showed higher and dose-dependent levels of glucocorticoid receptor expression in the hippocampus. These results suggest that bilobalide reduces anxiety levels and enhances spatial learning and memory, possibly through an increase in hippocampal glucocorticoid receptor expression. This finding sheds light on the mechanisms underlying the effect of ginkgo biloba extract on behavior and also provides an important candidate drug in treatment of anxiety, depression, hypomnesia and amnesia.

  1. Systems Biology and Birth Defects Prevention: Blockade of the Glucocorticoid Receptor Prevents Arsenic-Induced Birth Defects

    PubMed Central

    Ahir, Bhavesh K.; Sanders, Alison P.; Rager, Julia E.

    2013-01-01

    Background: The biological mechanisms by which environmental metals are associated with birth defects are largely unknown. Systems biology–based approaches may help to identify key pathways that mediate metal-induced birth defects as well as potential targets for prevention. Objectives: First, we applied a novel computational approach to identify a prioritized biological pathway that associates metals with birth defects. Second, in a laboratory setting, we sought to determine whether inhibition of the identified pathway prevents developmental defects. Methods: Seven environmental metals were selected for inclusion in the computational analysis: arsenic, cadmium, chromium, lead, mercury, nickel, and selenium. We used an in silico strategy to predict genes and pathways associated with both metal exposure and developmental defects. The most significant pathway was identified and tested using an in ovo whole chick embryo culture assay. We further evaluated the role of the pathway as a mediator of metal-induced toxicity using the in vitro midbrain micromass culture assay. Results: The glucocorticoid receptor pathway was computationally predicted to be a key mediator of multiple metal-induced birth defects. In the chick embryo model, structural malformations induced by inorganic arsenic (iAs) were prevented when signaling of the glucocorticoid receptor pathway was inhibited. Further, glucocorticoid receptor inhibition demonstrated partial to complete protection from both iAs- and cadmium-induced neurodevelopmental toxicity in vitro. Conclusions: Our findings highlight a novel approach to computationally identify a targeted biological pathway for examining birth defects prevention. PMID:23458687

  2. Kaposi's sarcoma-associated herpesvirus-encoded LANA associates with glucocorticoid receptor and enhances its transcriptional activities

    SciTech Connect

    Togi, Sumihito; Nakasuji, Misa; Muromoto, Ryuta; Ikeda, Osamu; Okabe, Kanako; Kitai, Yuichi; Kon, Shigeyuki; Oritani, Kenji; Matsuda, Tadashi

    2015-07-31

    Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA), which interacts with cellular proteins, plays a central role in modification of viral and/or cellular gene expression. Here, we show that LANA associates with glucocorticoid receptor (GR), and that LANA enhances the transcriptional activity of GR. Co-immunoprecipitation revealed a physical interaction between LANA and GR in transiently transfected 293T and HeLa cells. In human B-lymphoma cells, LANA overexpression enhanced GR activity and cell growth suppression following glucocorticoid stimulation. Furthermore, confocal microscopy showed that activated GR was bound to LANA and accumulated in the nucleus, leading to an increase in binding of activated GR to the glucocorticoid response element of target genes. Taken together, KSHV-derived LANA acts as a transcriptional co-activator of GR. Our results might suggest a careful use of glucocorticoids in the treatment of patients with KSHV-related malignancies such as Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman disease. - Highlights: • KSHV-LANA enhances the transcriptional activity of GR in 293T and HeLa cells. • KSHV-LANA physically associates with GR. • KSHV-LANA enhances GR activation and cell growth suppression in human B-lymphocytes. • KSHV-LANA influences the nuclear retention and DNA binding activity of GR.

  3. Metformin suppresses CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating aryl hydrocarbon receptor expression

    SciTech Connect

    Do, Minh Truong; Kim, Hyung Gyun; Tran, Thi Thu Phuong; Khanal, Tilak; Choi, Jae Ho; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-10-01

    Induction of cytochrome P450 (CYP) 1A1 and CYP1B1 by environmental xenobiotic chemicals or endogenous ligands through the activation of the aryl hydrocarbon receptor (AhR) has been implicated in a variety of cellular processes related to cancer, such as transformation and tumorigenesis. Here, we investigated the effects of the anti-diabetes drug metformin on expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and inducible conditions. Our results indicated that metformin down-regulated the expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced conditions. Down-regulation of AhR expression was required for metformin-mediated decreases in CYP1A1 and CYP1B1 expression, and the metformin-mediated CYP1A1 and CYP1B1 reduction is irrelevant to estrogen receptor α (ERα) signaling. Furthermore, we found that metformin markedly down-regulated Sp1 protein levels in breast cancer cells. The use of genetic and pharmacological tools revealed that metformin-mediated down-regulation of AhR expression was mediated through the reduction of Sp1 protein. Metformin inhibited endogenous AhR ligand-induced CYP1A1 and CYP1B1 expression by suppressing tryptophan-2,3-dioxygenase (TDO) expression in MCF-7 cells. Finally, metformin inhibits TDO expression through a down-regulation of Sp1 and glucocorticoid receptor (GR) protein levels. Our findings demonstrate that metformin reduces CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating AhR signaling. Metformin would be able to act as a potential chemopreventive agent against CYP1A1 and CYP1B1-mediated carcinogenesis and development of cancer. - Graphical abstract: Schematic of the CYP1A1 and CYP1B1 gene regulation by metformin. - Highlights: • Metformin inhibits CYP1A1 and CYP1B1 expression. • Metformin down-regulates the AhR signaling. • Metformin reduces Sp1 protein expression. • Metformin suppresses TDO expression.

  4. Adult-Onset Hypothyroidism Enhances Fear Memory and Upregulates Mineralocorticoid and Glucocorticoid Receptors in the Amygdala

    PubMed Central

    Montero-Pedrazuela, Ana; Fernández-Lamo, Iván; Alieva, María; Pereda-Pérez, Inmaculada; Venero, César; Guadaño-Ferraz, Ana

    2011-01-01

    Hypothyroidism is the most common hormonal disease in adults, which is frequently accompanied by learning and memory impairments and emotional disorders. However, the deleterious effects of thyroid hormones deficiency on emotional memory are poorly understood and often underestimated. To evaluate the consequences of hypothyroidism on emotional learning and memory, we have performed a classical Pavlovian fear conditioning paradigm in euthyroid and adult-thyroidectomized Wistar rats. In this experimental model, learning acquisition was not impaired, fear memory was enhanced, memory extinction was delayed and spontaneous recovery of fear memory was exacerbated in hypothyroid rats. The potentiation of emotional memory under hypothyroidism was associated with an increase of corticosterone release after fear conditioning and with higher expression of glucocorticoid and mineralocorticoid receptors in the lateral and basolateral nuclei of the amygdala, nuclei that are critically involved in the circuitry of fear memory. Our results demonstrate for the first time that adult-onset hypothyroidism potentiates fear memory and also increases vulnerability to develop emotional memories. Furthermore, our findings suggest that enhanced corticosterone signaling in the amygdala is involved in the pathophysiological mechanisms of fear memory potentiation. Therefore, we recommend evaluating whether inappropriate regulation of fear in patients with post-traumatic stress and other mental disorders is associated with abnormal levels of thyroid hormones, especially those patients refractory to treatment. PMID:22039511

  5. Glucocorticoid receptor gene methylation and HPA-axis regulation in adolescents. The TRAILS study.

    PubMed

    van der Knaap, Lisette J; Oldehinkel, Albertine J; Verhulst, Frank C; van Oort, Floor V A; Riese, Harriëtte

    2015-08-01

    Early life adversity and psychopathology are thought to be linked through HPA-axis deregulation. Changes in methylation levels of stress reactivity genes such as the glucocorticoid receptor gene (NR3C1) can be induced by adversity. Higher NR3C1 methylation levels have been associated with a reduced NR3C1 expression, possibly leading to impaired negative feedback regulation of the HPA-axis. In this study we tested whether methylation levels of NR3C1 were associated with HPA-axis regulation, operationalized as cortisol responses. In 361 adolescents (mean age 16.1, SD=0.6), salivary cortisol samples were collected before, during, and after a social stress task, from which response measures (cortisol activation and recovery) were calculated. Higher NR3C1 methylation levels were associated with a flattened cortisol recovery slope, indicating a delayed recovery time. Cortisol response activation was not associated with NR3C1 methylation. These results suggest that methylation of NR3C1 may impair negative feedback of the HPA-axis in adolescents.

  6. Expression of the glucocorticoid receptor in breast cancer-associated fibroblasts

    PubMed Central

    Catteau, Xavier; Simon, Philippe; Buxant, Frédéric; Noël, Jean-Christophe

    2016-01-01

    Cancer- associated fibroblasts (CAFs) are actively involved in breast carcinoma. Our previous study demonstrated that the majority of these CAFs were smooth muscle actin (SMA) positive and were therefore termed peritumoral myofibroblast (PMY). Glucocorticoid, linked or not with its receptor (GR), has been postulated to serve a major role in normal breast and breast carcinoma; however, their role in CAFs remains poorly understood. The aim of the present study was to assess the presence of GR in breast CAFs and particularly in PMY in 56 cases of invasive breast carcinoma in correlation with clinicopathological parameters, by immunohistochemistry. GR was observed in CAFs in 51 cases (91%) and were more frequent in luminal A subtype (19/19 cases; 100%). The stromal expression was statistically correlated with the tumor grade (P=0.03), the Ki-67 index (P=0.003) and the presence of GR in the epithelial component (P=0.01). The demonstration of a frequent expression of GR in breast CAFs may serve as an interesting target for future therapeutics for the regulation of the tumoral breast microenvironment. PMID:27699028

  7. The Central Nervous System Regulates Embryonic HSPC Production via Stress-Responsive Glucocorticoid Receptor Signaling.

    PubMed

    Kwan, Wanda; Cortes, Mauricio; Frost, Isaura; Esain, Virginie; Theodore, Lindsay N; Liu, Sarah Y; Budrow, Nadine; Goessling, Wolfram; North, Trista E

    2016-09-01

    Hematopoietic stem and progenitor cell (HSPC) specification is regulated by numerous defined factors acting locally within the hemogenic niche; however, it is unclear whether production can adapt to fluctuating systemic needs. Here we show that the CNS controls embryonic HSPC numbers via the hypothalamic-pituitary-adrenal/interrenal (HPA/I) stress response axis. Exposure to serotonin or the reuptake inhibitor fluoxetine increased runx1 expression and Flk1(+)/cMyb(+) HSPCs independent of peripheral innervation. Inhibition of neuronal, but not peripheral, tryptophan hydroxlyase (Tph) persistently reduced HSPC number. Consistent with central HPA/I axis induction and glucocorticoid receptor (GR) activation, GR agonists enhanced, whereas GR loss diminished, HSPC formation. Significantly, developmental hypoxia, as indicated by Hif1α function, induced the HPA/I axis and cortisol production. Furthermore, Hif1α-stimulated HSPC enhancement was attenuated by neuronal tph or GR loss. Our data establish that embryonic HSC production responds to physiologic stress via CNS-derived serotonin synthesis and central feedback regulation to control HSC numbers. PMID:27424782

  8. Glucocorticoid receptors in the locus coeruleus mediate sleep disorders caused by repeated corticosterone treatment

    PubMed Central

    Wang, Zi-Jun; Zhang, Xue-Qiong; Cui, Xiang-Yu; Cui, Su-Ying; Yu, Bin; Sheng, Zhao-Fu; Li, Sheng-Jie; Cao, Qing; Huang, Yuan-Li; Xu, Ya-Ping; Zhang, Yong-He

    2015-01-01

    Stress induced constant increase of cortisol level may lead to sleep disorder, but the mechanism remains unclear. Here we described a novel model to investigate stress mimicked sleep disorders induced by repetitive administration of corticosterone (CORT). After 7 days treatment of CORT, rats showed significant sleep disturbance, meanwhile, the glucocorticoid receptor (GR) level was notably lowered in locus coeruleus (LC). We further discovered the activation of noradrenergic neuron in LC, the suppression of GABAergic neuron in ventrolateral preoptic area (VLPO), the remarkable elevation of norepinephrine in LC, VLPO and hypothalamus, as well as increase of tyrosine hydroxylase in LC and decrease of glutamic acid decarboxylase in VLPO after CORT treatment. Microinjection of GR antagonist RU486 into LC reversed the CORT-induced sleep changes. These results suggest that GR in LC may play a key role in stress-related sleep disorders and support the hypothesis that repeated CORT treatment may decrease GR levels and induce the activation of noradrenergic neurons in LC, consequently inhibit GABAergic neurons in VLPO and result in sleep disorders. Our findings provide novel insights into the effect of stress-inducing agent CORT on sleep and GRs' role in sleep regulation. PMID:25801728

  9. Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity

    PubMed Central

    Schöne, Stefanie; Jurk, Marcel; Helabad, Mahdi Bagherpoor; Dror, Iris; Lebars, Isabelle; Kieffer, Bruno; Imhof, Petra; Rohs, Remo; Vingron, Martin; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H.

    2016-01-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes. PMID:27581526

  10. A ligand-specific kinetic switch regulates glucocorticoid receptor trafficking and function

    PubMed Central

    Trebble, Peter J.; Woolven, James M.; Saunders, Ken A.; Simpson, Karen D.; Farrow, Stuart N.; Matthews, Laura C.; Ray, David W.

    2013-01-01

    Summary The ubiquitously expressed glucocorticoid receptor (GR) is a major drug target for inflammatory disease, but issues of specificity and target tissue sensitivity remain. We now identify high potency, non-steroidal GR ligands, GSK47867A and GSK47869A, which induce a novel conformation of the GR ligand-binding domain (LBD) and augment the efficacy of cellular action. Despite their high potency, GSK47867A and GSK47869A both induce surprisingly slow GR nuclear translocation, followed by prolonged nuclear GR retention, and transcriptional activity following washout. We reveal that GSK47867A and GSK47869A specifically alter the GR LBD structure at the HSP90-binding site. The alteration in the HSP90-binding site was accompanied by resistance to HSP90 antagonism, with persisting transactivation seen after geldanamycin treatment. Taken together, our studies reveal a new mechanism governing GR intracellular trafficking regulated by ligand binding that relies on a specific surface charge patch within the LBD. This conformational change permits extended GR action, probably because of altered GR–HSP90 interaction. This chemical series may offer anti-inflammatory drugs with prolonged duration of action due to altered pharmacodynamics rather than altered pharmacokinetics. PMID:23687373

  11. Action control is mediated by prefrontal BDNF and glucocorticoid receptor binding.

    PubMed

    Gourley, Shannon L; Swanson, Andrew M; Jacobs, Andrea M; Howell, Jessica L; Mo, Michelle; Dileone, Ralph J; Koleske, Anthony J; Taylor, Jane R

    2012-12-11

    Stressor exposure biases decision-making strategies from those based on the relationship between actions and their consequences to others restricted by stimulus-response associations. Chronic stressor exposure also desensitizes glucocorticoid receptors (GR) and diminishes motivation to acquire food reinforcement, although causal relationships are largely not established. We show that a history of chronic exposure to the GR ligand corticosterone or acute posttraining GR blockade with RU38486 makes rodents less able to perform actions based on their consequences. Thus, optimal GR binding is necessary for the consolidation of new response-outcome learning. In contrast, medial prefrontal (but not striatal) BDNF can account for stress-related amotivation, in that selective medial prefrontal cortical Bdnf knockdown decreases break-point ratios in a progressive-ratio task. Knockdown also increases vulnerability to RU38486. Despite the role of BDNF in dendritic spine reorganization, deep-layer spine remodeling does not obviously parallel progressive-ratio response patterns, but treatment with the Na(+)-channel inhibitor riluzole reverses corticosteroid-induced motivational deficits and restores prefrontal BDNF expression after corticosterone. We argue that when prefrontal neurotrophin systems are compromised, and GR-mediated hypothalamic-pituitary-adrenal axis feedback is desensitized (as in the case of chronic stress hormone exposure), amotivation and inflexible maladaptive response strategies that contribute to stress-related mood disorders result. PMID:23185000

  12. Morphine conditioned place preference depends on glucocorticoid receptors in both hippocampus and nucleus accumbens.

    PubMed

    Dong, Zhifang; Han, Huili; Wang, Meina; Xu, Lin; Hao, Wei; Cao, Jun

    2006-01-01

    Learned association between drugs of abuse and context is essential for the formation of drug conditioned place preference (CPP), which is believed to engage many brain regions including hippocampus and nucleus accumbens (NAc). The underlying mechanisms are not fully understood. Here, we examined whether glucocorticoid receptors (GRs) of hippocampus and NAc influenced the formation of morphine CPP in Sprague Dawley rats. We found that systemic or intrahippocampal infused DMSO vehicle (DMSO 20% in saline) 30 min before daily morphine (10 mg/kg, s.c.) conditioning did not affect the formation of morphine CPP. In contrast, systemic administration (5 mg/kg, s.c.) or intrahippocampal infusion (0, 0.1, 1.0, 10, 20 microg per side) of the GR antagonist RU38486 blocked or impaired the formation of CPP in a dose-dependent manner, respectively. Furthermore, intra-NAc infused RU38486 (10 microg per side) but not DMSO vehicle also prevented the formation of CPP. These results demonstrate that both the GRs of hippocampus and NAc are necessary for the formation of morphine CPP, suggesting a neural network function of the GRs in forming the opiate-associated memory.

  13. Glucocorticoid receptor-PPARα axis in fetal mouse liver prepares neonates for milk lipid catabolism

    PubMed Central

    Rando, Gianpaolo; Tan, Chek Kun; Khaled, Nourhène; Montagner, Alexandra; Leuenberger, Nicolas; Bertrand-Michel, Justine; Paramalingam, Eeswari; Guillou, Hervé; Wahli, Walter

    2016-01-01

    In mammals, hepatic lipid catabolism is essential for the newborns to efficiently use milk fat as an energy source. However, it is unclear how this critical trait is acquired and regulated. We demonstrate that under the control of PPARα, the genes required for lipid catabolism are transcribed before birth so that the neonatal liver has a prompt capacity to extract energy from milk upon suckling. The mechanism involves a fetal glucocorticoid receptor (GR)-PPARα axis in which GR directly regulates the transcriptional activation of PPARα by binding to its promoter. Certain PPARα target genes such as Fgf21 remain repressed in the fetal liver and become PPARα responsive after birth following an epigenetic switch triggered by β-hydroxybutyrate-mediated inhibition of HDAC3. This study identifies an endocrine developmental axis in which fetal GR primes the activity of PPARα in anticipation of the sudden shifts in postnatal nutrient source and metabolic demands. DOI: http://dx.doi.org/10.7554/eLife.11853.001 PMID:27367842

  14. Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity.

    PubMed

    Schöne, Stefanie; Jurk, Marcel; Helabad, Mahdi Bagherpoor; Dror, Iris; Lebars, Isabelle; Kieffer, Bruno; Imhof, Petra; Rohs, Remo; Vingron, Martin; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H

    2016-01-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes. PMID:27581526

  15. Expression of the glucocorticoid receptor in breast cancer-associated fibroblasts

    PubMed Central

    Catteau, Xavier; Simon, Philippe; Buxant, Frédéric; Noël, Jean-Christophe

    2016-01-01

    Cancer- associated fibroblasts (CAFs) are actively involved in breast carcinoma. Our previous study demonstrated that the majority of these CAFs were smooth muscle actin (SMA) positive and were therefore termed peritumoral myofibroblast (PMY). Glucocorticoid, linked or not with its receptor (GR), has been postulated to serve a major role in normal breast and breast carcinoma; however, their role in CAFs remains poorly understood. The aim of the present study was to assess the presence of GR in breast CAFs and particularly in PMY in 56 cases of invasive breast carcinoma in correlation with clinicopathological parameters, by immunohistochemistry. GR was observed in CAFs in 51 cases (91%) and were more frequent in luminal A subtype (19/19 cases; 100%). The stromal expression was statistically correlated with the tumor grade (P=0.03), the Ki-67 index (P=0.003) and the presence of GR in the epithelial component (P=0.01). The demonstration of a frequent expression of GR in breast CAFs may serve as an interesting target for future therapeutics for the regulation of the tumoral breast microenvironment.

  16. Amygdala kindling increases fear responses and decreases glucocorticoid receptor mRNA expression in hippocampal regions.

    PubMed

    Kalynchuk, Lisa E; Meaney, Michael J

    2003-12-01

    Amygdala kindling dramatically increases fearful behavior in rats. Because kindling-induced fear increases in magnitude as rats receive more stimulations, kindling provides an excellent model for studying the nature and neural mechanisms of fear sensitization. In the present experiment, we studied whether the development of kindling-induced fear is related to changes in glucocorticoid receptor (GR) mRNA expression in various brain regions. Rats received 20, 60 or 100 amygdala kindling stimulations or 100 sham stimulations. One day after the final stimulation, their fearful behavior was assessed in an unfamiliar open field. Then, the rats were sacrificed and their brains were processed for in situ hybridization of GR mRNA expression. We found that compared with the sham-stimulated rats, the rats that received 60 or 100 kindling stimulations were significantly more fearful in the open field and also had significantly less GR mRNA expression in the dentate gyrus and CA1 subfield of the hippocampus. Importantly, the changes in fearful behavior were significantly correlated with the changes in GR mRNA expression. These results suggest that alterations in GR mRNA expression in hippocampal regions may play a role in the development of kindling-induced fear.

  17. Maternal undernutrition programs tissue-specific epigenetic changes in the glucocorticoid receptor in adult offspring.

    PubMed

    Begum, Ghazala; Davies, Alison; Stevens, Adam; Oliver, Mark; Jaquiery, Anne; Challis, John; Harding, Jane; Bloomfield, Frank; White, Anne

    2013-12-01

    Epidemiological data indicate that an adverse maternal environment during pregnancy predisposes offspring to metabolic syndrome with increased obesity, and type 2 diabetes. The mechanisms are still unclear although epigenetic modifications are implicated and the hypothalamus is a likely target. We hypothesized that maternal undernutrition (UN) around conception in sheep would lead to epigenetic changes in hypothalamic neurons regulating energy balance in the offspring, up to 5 years after the maternal insult. We found striking evidence of decreased glucocorticoid receptor (GR) promoter methylation, decreased histone lysine 27 trimethylation, and increased histone H3 lysine 9 acetylation in hypothalami from male and female adult offspring of UN mothers. These findings are entirely compatible with the increased GR mRNA and protein observed in the hypothalami. The increased GR predicted the decreased hypothalamic proopiomelanocortin expression and increased obesity that we observed in the 5-year-old adult males. The epigenetic and expression changes in GR were specific to the hypothalamus. Hippocampal GR mRNA and protein were decreased in UN offspring, whereas pituitary GR was altered in a sex-specific manner. In peripheral polymorphonuclear leukocytes there were no changes in GR methylation or protein, indicating that this epigenetic analysis did not predict changes in the brain. Overall, these results suggest that moderate changes in maternal nutrition, around the time of conception, signal life-long and tissue-specific epigenetic alterations in a key gene regulating energy balance in the hypothalamus.

  18. Yokukansan normalizes glucocorticoid receptor protein expression in oligodendrocytes of the corpus callosum by regulating microRNA-124a expression after stress exposure.

    PubMed

    Shimizu, Shoko; Tanaka, Takashi; Tohyama, Masaya; Miyata, Shingo

    2015-05-01

    Stressful events are known to down-regulate expression levels of glucocorticoid receptors (GRs) in the brain. Recently, we reported that stressed mice with elevated plasma levels of corticosterone exhibit morphological changes in the oligodendrocytes of nerve fiber bundles, such as those in the corpus callosum. However, little is known about the molecular mechanism of GR expression regulation in oligodendrocytes after stress exposure. A previous report has suggested that GR protein levels might be regulated by microRNA (miR)-18 and/or -124a in the brain. In this study, we aimed to elucidate the GR regulation mechanism in oligodendrocytes and evaluate the effects of yokukansan (YKS), a Kampo medicine, on GR protein regulation. Acute exposure to stress increased plasma corticosterone levels, decreased GR protein expression, and increased miR-124a expression in the corpus callosum of adult male mice, though the GR mRNA and miR-18 expression levels were not significant changes. YKS normalized the stress-induced changes in the plasma corticosterone, GR protein, and miR124a expression levels. An oligodendrocyte primary culture study also showed that YKS down-regulated miR-124a, but not miR-18, expression levels in dexamethasone-treated cells. These results suggest that the down-regulation of miR124a expression might be involved in the normalization of stress-induced decreases in GR protein in oligodendrocytes by YKS. This effect may imply the molecular mechanisms underlying the ameliorative effects of YKS on psychological symptoms and stress-related behaviors.

  19. Expression of mineralocorticoid and glucocorticoid receptors in preautonomic neurons of the rat paraventricular nucleus.

    PubMed

    Chen, Jian; Gomez-Sanchez, Celso E; Penman, Alan; May, Paul J; Gomez-Sanchez, Elise

    2014-03-01

    Activation of mineralocorticoid receptors (MR) of the hypothalamic paraventricular nucleus (PVN) increases sympathetic excitation. To determine whether MR and glucocorticoid receptors (GR) are expressed in preautonomic neurons of the PVN and how they relate to endogenous aldosterone levels in healthy rats, retrograde tracer was injected into the intermediolateral cell column at T4 to identify preautonomic neurons in the PVN. Expression of MR, GR, 11-β hydroxysteroid dehydrogenase1 and 2 (11β-HSD1, 2), and hexose-6-phosphate dehydrogenase (H6PD) required for 11β-HSD1 reductase activity was assessed by immunohistochemistry. RT-PCR and Western blot analysis were used to determine MR gene and protein expression. Most preautonomic neurons were in the caudal mediocellular region of PVN, and most expressed MR; none expressed GR. 11β-HSD1, but not 11β-HSD2 nor H6PD immunoreactivity, was detected in the PVN. In rats with chronic low or high sodium intakes, the low-sodium diet was associated with significantly higher plasma aldosterone, MR mRNA and protein expression, and c-Fos immunoreactivity within labeled preautonomic neurons. Plasma corticosterone and sodium and expression of tonicity-responsive enhancer binding protein in the PVN did not differ between groups, suggesting osmotic adaptation to the altered sodium intake. These results suggest that MR within preautonomic neurons in the PVN directly participate in the regulation of sympathetic nervous system drive, and aldosterone may be a relevant ligand for MR in preautonomic neurons of the PVN under physiological conditions. Dehydrogenase activity of 11β-HSD1 occurs in the absence of H6PD, which regenerates NADP(+) from NADPH and may increase MR gene expression under physiological conditions. PMID:24381176

  20. Role of the glucocorticoid receptor in the recurrence of primary nephrotic syndrome

    PubMed Central

    LIANG, YUMEI; CHEN, YINYIN; CHEN, YING; GONG, YUTING

    2015-01-01

    The present study aimed to investigate the changes in the expression levels of the glucocorticoid receptor (GR) and its subtypes in patients with recurrent renal syndrome. In addition, the effects of tumour necrosis factor α (TNF-α) and a TNF-α monoclonal antibody on these receptors in peripheral blood mononuclear cells (PBMCs) isolated from the patients was analysed. Furthermore, a new treatment method for recurrent renal syndrome was explored. The serum levels of TNF-α in the normal (A), stable renal syndrome (B) and renal syndrome recurrence (C) groups of patients were determined by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expression levels of GR, GRα and GRβ were determined by ELISA, western blot analysis and quantitative polymerase chain reaction in PBMC cultures from the three groups in the absence of intervention (blank control) and following stimulation with methylprednisolone, TNF-α and/or TNF-α monoclonal antibody. Group C exhibited higher expression levels of TNF-α and GRβ but a lower level of GRα expression (P<0.05) compared with the other groups. Regardless of methylprednisolone intervention, the expression levels of GR and GRβ in the three groups following stimulation by TNF-α were significantly higher compared with those in the respective blank control, whereas in group C, the GRα expression levels following TNF-α treatment were lower compared with those in the control group (P<0.05). The treatment of group C with TNF-α monoclonal antibodies resulted in higher GRα expression but lower GRβ expression compared with those in the blank control (P<0.05). The change in the ratios of the GR subtypes may be associated with renal syndrome recurrence. TNF-α may be involved in renal syndrome relapse by changing the levels of GR as well as the proportion of the GR subtypes. TNF-α monoclonal antibodies may mitigate the changes in the ratios of these subtypes. PMID:26622525

  1. Continuous blockade of brain glucocorticoid receptors facilitates spatial learning and memory in rats.

    PubMed

    Oitzl, M S; Fluttert, M; Sutanto, W; de Kloet, E R

    1998-12-01

    Previously, a corticosterone surge associated with a learning task was shown to facilitate cognitive processes through brain glucocorticoid receptors (GR) while chronic overexposure to this stress hormone impaired cognition. In the present study we tested the hypothesis that opposing effects on learning and memory might also occur after either phasic or continuous blockade of brain GR by intracerebroventricular (i.c.v.) administration of the GR antagonist RU38486 (aGR). We used a Morris water maze procedure to assess spatial learning and memory abilities in male Wistar rats. The effect of phasic brain GR blockade was studied following daily pretraining administration of 10 and 100 ng/microL aGR i.c.v. on 3 consecutive days. This repetitive aGR treatment impaired spatial learning and memory dose-dependently in comparison with vehicle controls. For continuous brain GR blockade, animals received an i.c.v., infusion of aGR (10 and 100 ng/0.5 microL per h or vehicle) over 10 days. Infusion of 100 ng aGR per hour resulted in a long-lasting facilitation of spatial performance. The 10 ng aGR infusion also caused initially a facilitating effect, which was, however, transient and performance became impaired during retest. Possible anxiolytic properties of the drugs were excluded in view of the animals' behaviour in the elevated plus maze. Both doses of aGR infusion reduced the number of mineralocorticoid receptors in the hippocampus, but only the high dose of aGR resulted in a significant reduction of available GR sites. In conclusion, continuous administration of GR antagonist improves cognitive function, while phasic blockade of brain GR function causes a cognitive deficit.

  2. Downregulation of transferrin receptor surface expression by intracellular antibody

    SciTech Connect

    Peng Jilin; Wu Sha; Zhao Xiaoping; Wang Min; Li Wenhan; Shen Xin; Liu Jing; Lei Ping; Zhu Huifen; Shen Guanxin . E-mail: guanxin_shen@yahoo.com.cn

    2007-03-23

    To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 {+-} 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors.

  3. Mixed-model QSAR at the glucocorticoid receptor: predicting the binding mode and affinity of psychotropic drugs.

    PubMed

    Spreafico, Morena; Ernst, Beat; Lill, Markus A; Smiesko, Martin; Vedani, Angelo

    2009-01-01

    The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily that affects immune response, development, and metabolism in target tissues. Glucocorticoids are widely used to treat diverse pathophysiological conditions, but their clinical applicability is limited by side effects. A prediction of the binding affinity toward the GR would be beneficial for identifying glucocorticoid-mediated adverse effects triggered by drugs or chemicals. By identifying the binding mode to the GR using flexible docking (software Yeti) and quantifying the binding affinity through multidimensional QSAR (software Quasar), we validated a model family based on 110 compounds, representing four different chemical classes. The correlation with the experimental data (cross-validated r(2)=0.702; predictive r(2)=0.719) suggests that our approach is suited for predicting the binding affinity of related compounds toward the GR. After challenging the model by a series of scramble tests, a consensus approach (software Raptor), and a prediction set, it was incorporated into our VirtualToxLab and used to simulate and quantify the interaction of 24 psychotropic drugs with the GR.

  4. A Novel Point Mutation in the Amino Terminal Domain of the Human Glucocorticoid Receptor (hGR) Gene Enhancing hGR-Mediated Gene Expression

    PubMed Central

    Charmandari, Evangelia; Ichijo, Takamasa; Jubiz, William; Baid, Smita; Zachman, Keith; Chrousos, George P.; Kino, Tomoshige

    2008-01-01

    Context: Interindividual variations in glucocorticoid sensitivity have been associated with manifestations of cortisol excess or deficiency and may be partly explained by polymorphisms in the human glucocorticoid receptor (hGR) gene. We studied a 43-yr-old female, who presented with manifestations consistent with tissue-selective glucocorticoid hypersensitivity. We detected a novel, single, heterozygous nucleotide (G → C) substitution at position 1201 (exon 2) of the hGR gene, which resulted in aspartic acid to histidine substitution at amino acid position 401 in the amino-terminal domain of the hGRα. We investigated the molecular mechanisms of action of the natural mutant receptor hGRαD401H. Methods-Results: Compared with the wild-type hGRα, the mutant receptor hGRαD401H demonstrated a 2.4-fold increase in its ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to dexamethasone but had similar affinity for the ligand (dissociation constant = 6.2 ± 0.6 vs. 6.1 ± 0.6 nm) and time to nuclear translocation (14.75 ± 0.25 vs. 14.25 ± 1.13 min). The mutant receptor hGRαD401H did not exert a dominant positive or negative effect upon the wild-type receptor, it preserved its ability to bind to glucocorticoid response elements, and displayed a normal interaction with the glucocorticoid receptor-interacting protein 1 coactivator. Conclusions: The mutant receptor hGRαD401H enhances the transcriptional activity of glucocorticoid-responsive genes. The presence of the D401H mutation may predispose subjects to obesity, hypertension, and other manifestations of the metabolic syndrome. PMID:18827003

  5. Downregulation of kinin B1 receptor function by B2 receptor heterodimerization and signaling.

    PubMed

    Zhang, Xianming; Brovkovych, Viktor; Zhang, Yongkang; Tan, Fulong; Skidgel, Randal A

    2015-01-01

    Signaling through the G protein-coupled kinin receptors B1 (kB1R) and B2 (kB2R) plays a critical role in inflammatory responses mediated by activation of the kallikrein-kinin system. The kB2R is constitutively expressed and rapidly desensitized in response to agonist whereas kB1R expression is upregulated by inflammatory stimuli and it is resistant to internalization and desensitization. Here we show that the kB1R heterodimerizes with kB2Rs in co-transfected HEK293 cells and natively expressing endothelial cells, resulting in significant internalization and desensitization of the kB1R response in cells pre-treated with kB2R agonist. However, pre-treatment of cells with kB1R agonist did not affect subsequent kB2R responses. Agonists of other G protein-coupled receptors (thrombin, lysophosphatidic acid) had no effect on a subsequent kB1R response. The loss of kB1R response after pretreatment with kB2R agonist was partially reversed with kB2R mutant Y129S, which blocks kB2R signaling without affecting endocytosis, or T342A, which signals like wild type but is not endocytosed. Co-endocytosis of the kB1R with kB2R was dependent on β-arrestin and clathrin-coated pits but not caveolae. The sorting pathway of kB1R and kB2R after endocytosis differed as recycling of kB1R to the cell surface was much slower than that of kB2R. In cytokine-treated human lung microvascular endothelial cells, pre-treatment with kB2R agonist inhibited kB1R-mediated increase in transendothelial electrical resistance (TER) caused by kB1R stimulation (to generate nitric oxide) and blocked the profound drop in TER caused by kB1R activation in the presence of pyrogallol (a superoxide generator). Thus, kB1R function can be downregulated by kB2R co-endocytosis and signaling, suggesting new approaches to control kB1R signaling in pathological conditions. PMID:25289859

  6. Down-regulation of the chemokine receptor CCR5 by activation of chemotactic formyl peptide receptor in human monocytes.

    PubMed

    Shen, W; Li, B; Wetzel, M A; Rogers, T J; Henderson, E E; Su, S B; Gong, W; Le, Y; Sargeant, R; Dimitrov, D S; Oppenheim, J J; Wang, J M

    2000-10-15

    Interactions between cell surface receptors are important regulatory elements in the complex host responses to infections. In this study, it is shown that a classic chemotactic factor, the bacterial chemotactic peptide N-formyl-methionyl-leucylphenyl-alanine (fMLF), rapidly induced a protein-kinase-C-mediated serine phosphorylation and down-regulation of the chemokine receptor CCR5, which serves as a major human immunodeficiency virus (HIV)-1 coreceptor. The fMLF binding to its receptor, formyl peptide receptor (FPR), resulted in significant attenuation of cell responses to CCR5 ligands and in inhibition of HIV-1-envelope-glycoprotein-mediated fusion and infection of cells expressing CD4, CCR5, and FPR. The finding that the expression and function of CCR5 can be regulated by peptides that use an unrelated receptor may provide a novel approach to the design of anti-inflamatory and antiretroviral agents. (Blood. 2000;96:2887-2894)

  7. Alpha-melanocyte-stimulating hormone down-regulates CXC receptors through activation of neutrophil elastase.

    PubMed

    Manna, Sunil K; Sarkar, Abira; Sreenivasan, Yashin

    2006-03-01

    Considering the role of interleukin-8 (IL-8) in a large number of acute and chronic inflammatory diseases, the regulation of IL-8-mediated biological responses is important. Alpha-melanocyte-stimulating hormone (alpha-MSH), a tridecapeptide, inhibits most forms of inflammation by an unknown mechanism. In the present study, we have found that alpha-MSH interacts predominantly with melanocortin-1 receptors and inhibits several IL-8-induced biological responses in macrophages and neutrophils. It down-regulated receptors for IL-8 but not for TNF, IL-4, IL-13 or TNF-related apoptosis-inducing ligand (TRAIL) in neutrophils. It down-regulated CXCR type 1 and 2 but not mRNA levels. alpha-MSH did not inhibit IL-8 binding in purified cell membrane or affinity-purified CXCR. IL-8 or anti-CXCR Ab protected against alpha-MSH-mediated inhibition of IL-8 binding. The level of neutrophil elastase, a specific serine protease, but not cathepsin G or proteinase 3 increased in alpha-MSH-treated cells, and restoration of CXCR by specific neutrophil elastase or serine protease inhibitors indicates the involvement of elastase in alpha-MSH-induced down-regulation of CXCR. These studies suggest that alpha-MSH inhibits IL-8-mediated biological responses by down-regulating CXCR through induction of serine protease and that alpha-MSH acts as a potent immunomodulator in neutrophil-driven inflammatory distress. PMID:16479540

  8. HIV-induced kidney cell injury: role of ROS-induced downregulated vitamin D receptor

    PubMed Central

    Salhan, Divya; Husain, Mohammad; Subrati, Ashaan; Goyal, Rohan; Singh, Tejinder; Rai, Partab; Malhotra, Ashwani

    2012-01-01

    Reactive oxygen species (ROS) have been demonstrated to contribute to HIV-induced tubular cell injury. We hypothesized that HIV-induced ROS generation may be causing tubular cell injury through downregulation of vitamin D receptor (VDR) and associated downstream effects. In the present study, HIV not only downregulated tubular cell VDR expression but also inflicted DNA injury. On the other hand, EB-1089, a VDR agonist (VD), inhibited both downregulation of VDR and tubular cell DNA injury in the HIV milieu. H2O2 (an O− donor) directly downregulated tubular cell VDR, whereas catalase, a free radical scavenger, inhibited HIV-induced downregulation of tubular cell VDR expression. HIV also stimulated the tubular cell renin-angiotensin system (RAS) through downregulation of VDR. Because losartan (an ANG II blolcker) partially inhibited HIV-induced tubular cell ROS generation while ANG II directly stimulated tubular cell ROS generation, it appears that HIV-induced ROS production was partly contributed by the RAS activation. VD not only inhibited HIV-induced RAS activation but also attenuated tubular cell ROS generation. Tubular cells displayed double jeopardy in the HIV milieu induction of double-strand breaks and attenuated DNA repair; additionally, in the HIV milieu, tubular cells exhibited enhanced expression of phospho-p53 and associated downstream signaling. A VDR agonist and an ANG II blocker not only preserved expression of tubular cell DNA repair proteins but also inhibited induction of double-strand breaks. In in vivo studies, renal cortical sections of Tg26 mice displayed attenuated expression of VDR both in podocytes and tubular cells. In addition, renal cortical sections of Tg26 mice displayed enhanced oxidative stress-induced kidney cell DNA damage. These findings indicated that HIV-induced tubular cell downregulation of VDR contributed to the RAS activation and associated tubular cell DNA damage. However, both VD and RAS blockade provided protection

  9. Mapping amino acids of the measles virus hemagglutinin responsible for receptor (CD46) downregulation.

    PubMed

    Bartz, R; Brinckmann, U; Dunster, L M; Rima, B; Ter Meulen, V; Schneider-Schaulies, J

    1996-10-01

    We compared the amino acid sequences of groups of receptor (CD46) downregulating and nondownregulating measles virus (MV) hemagglutinins (Hs) and identified seven group-specific differences as candidates for the mediation of the observed differential effects. Using site-directed mutagenesis, we mutated the chosen amino acids of the H of MV-strain WTF (WTF-H), a nondownregulating H, and Introduced the corresponding amino acids of Edmonston-H (Edm-H), a downregulating H. We identified four amino acids, 211G, 243R, 451V, and 481Y, which influenced the downregulative function when introduced into WTF-H. The double mutation 451V and 481Y in WTF-H led to a degree of CD46 downregulation comparable to that of Edm-H. Conversely, introducing amino acids 451E and 481N into Edm-H resulted in a loss of the downregulative function. These results indicate that these amino acids play a decisive role in the H-CD46 interaction. PMID:8862431

  10. Activation of farnesoid X receptor downregulates visfatin and attenuates diabetic nephropathy.

    PubMed

    Zhou, Baoshang; Feng, Bing; Qin, Zhexue; Zhao, Youguang; Chen, Yu; Shi, Zhengmin; Gong, Yi; Zhang, Jing; Yuan, Fahuan; Mu, Jiao

    2016-01-01

    Visfatin, a recently discovered adipocytokine, has been shown to have an important role in the pathogenesis of diabetic nephropathy (DN). The farnesoid X receptor (FXR), a ligand-activated nuclear receptor, plays a protective role in DN. The regulation between FXR and visfatin and their interaction in DN has not been well established. In this study, we reported that FXR agonist GW4064 reduced high glucose induced human mesangial cells (HMCs) inflammation, fibrosis and proliferation by downregulating visfatin expression, which can be blunted by exogenous visfatin treatment. Moreover, luciferase reporter assay showed FXR regulated visfatin transcription activity probably by binding to the -1607 bp and -1192 bp region of the visfatin promoter. In vivo study also showed that GW4064 ameliorated the progression of DN in db/db mice with a decreased visfatin expression. These findings suggest that FXR activation delayed the progression of diabetic nephropathy and this effect is through downregulating visfatin.

  11. Promoter Methylation of Glucocorticoid Receptor Gene Is Associated with Subclinical Atherosclerosis: a Monozygotic Twin Study

    PubMed Central

    Zhao, Jinying; An, Qiang; Goldberg, Jack; Quyyumi, Arshed A.; Vaccarino, Viola

    2015-01-01

    Objective Endothelial dysfunction assessed by brachial artery flow-mediated dilation (FMD) is a marker of early atherosclerosis. Glucocorticoid receptor gene (NR3C1) regulates many biological processes, including stress response, behavioral, cardiometabolic and immunologic functions. Genetic variants in NR3C1 have been associated with atherosclerosis and related risk factors. This study investigated the association of NR3C1 promoter methylation with FMD, independent of genetic and family-level environmental factors. Methods We studied 84 middle-aged, male-male monozygotic twin pairs recruited from the Vietnam Era Twin Registry. Brachial artery FMD was measured by ultrasound. DNA methylation levels at 22 CpG residues in the NR3C1 exon 1F promoter region were quantified by bisulfite pyrosequencing in genomic DNA isolated from peripheral blood leukocytes. Co-twin control analyses were conducted to examine the association of methylation variation with FMD, adjusting for smoking, physical activity, body mass index, lipids, blood pressure, fasting glucose, and depressive symptoms. Multiple testing was corrected using the false discovery rate. Results Mean methylation level across the 22 studied CpG sites was 2.02%. Methylation alterations at 12 out of the 22 CpG residues were significantly associated with FMD. On average, a 1% increase in the intra-pair difference in mean DNA methylation was associated with 2.83% increase in the intra-pair difference in FMD (95% CI: 1.46-4.20; P <0.0001) after adjusting for risk factors and multiple testing. Conclusion Methylation variation in NR3C1 exon 1F promoter significantly influences subclinical atherosclerosis, independent of genetic, early family environmental and other risk factors. PMID:26186654

  12. Glucocorticoid receptor impairment enhances impulsive responding in transgenic mice performing on a simultaneous visual discrimination task.

    PubMed

    Steckler, T; Sauvage, M; Holsboer, F

    2000-07-01

    Transgenic mice with impaired glucocorticoid receptor (GR) function were tested for their ability to learn and perform a series of simultaneous visual discriminations which allowed a dissociation between accuracy of discrimination from those of motivation and behavioural disinhibition. Animals were first trained on an operant five-choice simultaneous discrimination autoshaping procedure, followed by a continuous reinforcement schedule on that task. Subsequently, the number of choices was limited to two and data were analysed according to the mathematical methods of signal detection theory (SDT). The effects of GR-antisense expression on accuracy when different rates of responding were required were studied under different fixed ratio response requirements (FR1-FR10). Autoshaping was retarded in transgenic animals and accuracy was impaired in both the five-choice and the two-choice discrimination tasks, although transgenic mice showed clear evidence for learning. Under conditions of low response requirements, transgenic mice showed increased response and cognitive biases, but reduced perceptual bias, and a behavioural disinhibition, characterized by a reduction in errors of omission, decreased response latencies and increased number of responses during the inter-trial interval. Increasing the response requirement improved performance in transgenic animals as reflected by enhanced accuracy. Moreover, transgenics were less susceptible to the deleterious effects of higher response requirements, as indicated by relatively unaffected bias measures in this group, while bias increased in controls. These results indicate that altered performance in GR-antisense transgenic animals cannot simply be interpreted as a mnemonic deficit, but that altered motivation and enhanced impulsive responding may account for some of these impairments.

  13. Disrupting Hypothalamic Glucocorticoid Receptors Causes HPA Axis Hyperactivity and Excess Adiposity

    PubMed Central

    Laryea, Gloria; Schütz, Günther

    2013-01-01

    The glucocorticoid receptor (GR) regulates hypothalamic-pituitary-adrenal (HPA) axis activity during the stress response. The paraventricular nucleus (PVN) is a major site of negative feedback to coordinate the degree of the HPA axis activity with the magnitude of the exposed stressor. To define the function of endogenous PVN GR, we used Cre-loxP technology to disrupt different GR exons in Sim1-expressing neurons of the hypothalamus. GR exon 2-deleted mice (Sim1Cre-GRe2Δ) demonstrated 43% loss of PVN GR compared with an 87% GR loss in exon 3-deleted mice (Sim1Cre-GRe3Δ). Sim1Cre-GRe3Δ mice display stunted growth at birth but develop obesity in adulthood and display impaired stress-induced glucose release. We observed elevated basal and stress-induced corticosterone levels in Sim1Cre-GRe3Δ mice, compared with control and Sim1Cre-GRe2Δ mice, and impaired dexamethasone suppression, indicating an inability to negatively regulate corticosterone secretion. Sim1Cre-GRe3Δ mice also showed increased CRH mRNA in the PVN, increased basal plasma ACTH levels, and reduced locomotor behavior. We observed no differences in Sim1Cre-GRe2Δ mice compared with control mice in any measure. Our behavioral data suggest that GR deletion in Sim1-expressing neurons has no effect on anxiety or despair-like behavior under basal conditions. We conclude that loss of PVN GR results in severe HPA axis hyperactivity and Cushing's syndrome-like phenotype but does not affect anxiety and despair-like behaviors. PMID:23979842

  14. Crosstalk in Inflammation: The Interplay of Glucocorticoid Receptor-Based Mechanisms and Kinases and Phosphatases

    PubMed Central

    Beck, Ilse M. E.; Vanden Berghe, Wim; Vermeulen, Linda; Yamamoto, Keith R.; Haegeman, Guy; De Bosscher, Karolien

    2009-01-01

    Glucocorticoids (GCs) are steroidal ligands for the GC receptor (GR), which can function as a ligand-activated transcription factor. These steroidal ligands and derivatives thereof are the first line of treatment in a vast array of inflammatory diseases. However, due to the general surge of side effects associated with long-term use of GCs and the potential problem of GC resistance in some patients, the scientific world continues to search for a better understanding of the GC-mediated antiinflammatory mechanisms. The reversible phosphomodification of various mediators in the inflammatory process plays a key role in modulating and fine-tuning the sensitivity, longevity, and intensity of the inflammatory response. As such, the antiinflammatory GCs can modulate the activity and/or expression of various kinases and phosphatases, thus affecting the signaling efficacy toward the propagation of proinflammatory gene expression and proinflammatory gene mRNA stability. Conversely, phosphorylation of GR can affect GR ligand- and DNA-binding affinity, mobility, and cofactor recruitment, culminating in altered transactivation and transrepression capabilities of GR, and consequently leading to a modified antiinflammatory potential. Recently, new roles for kinases and phosphatases have been described in GR-based antiinflammatory mechanisms. Moreover, kinase inhibitors have become increasingly important as antiinflammatory tools, not only for research but also for therapeutic purposes. In light of these developments, we aim to illuminate the integrated interplay between GR signaling and its correlating kinases and phosphatases in the context of the clinically important combat of inflammation, giving attention to implications on GC-mediated side effects and therapy resistance. PMID:19890091

  15. Glucocorticoid receptors and extinction retention deficits in the single prolonged stress model.

    PubMed

    Knox, D; Nault, T; Henderson, C; Liberzon, I

    2012-10-25

    Single prolonged stress (SPS) is a rodent model of post traumatic stress disorder that is comprised of serial application of restraint (r), forced swim (fs), and ether (eth) followed by a 7-day quiescent period. SPS induces extinction retention deficits and it is believed that these deficits are caused by the combined stressful effect of serial exposure to r, fs, and eth. However, this hypothesis remains untested. Neurobiological mechanisms by which SPS induces extinction retention deficits are unknown, but SPS enhances glucocorticoid receptor (GR) expression in the hippocampus, which is critical for contextual modulation of extinction retrieval. Upregulation of GRs in extinction circuits may be a mechanism by which SPS induces extinction retention deficits, but this hypothesis has not been examined. In this study, we systematically altered the stressors that constitute SPS (i.e. r, fs, eth), generating a number of partial SPS (p-SPS) groups, and observed the effects SPS and p-SPSs had on extinction retention and GR levels in the hippocampus and prefrontal cortex (PFC). PFC GRs were assayed, because regions of the PFC are critical for maintaining extinction. We predicted that only exposure to full SPS would result in extinction retention deficits and enhance hippocampal and PFC GR levels. Only exposure to full SPS induced extinction retention deficits. Hippocampal and PFC GR expression was enhanced by SPS and most p-SPSs, however hippocampal GR expression was significantly larger following the full SPS exposure than all other conditions. Our findings suggest that the combined stressful effect of serial exposure to r, fs, and eth results in extinction retention deficits. The results also suggest that simple enhancements in GR expression in the hippocampus and PFC are insufficient to result in extinction retention deficits, but raise the possibility that a threshold-enhancement in hippocampal GR expression contributes to SPS-induced extinction retention deficits.

  16. The Anticancer Plant Triterpenoid, Avicin D, Regulates Glucocorticoid Receptor Signaling: Implications for Cellular Metabolism

    PubMed Central

    Haridas, Valsala; Xu, Zhi-Xiang; Kitchen, Doug; Jiang, Anna; Michels, Peter; Gutterman, Jordan U.

    2011-01-01

    Avicins, a family of apoptotic triterpene electrophiles, are known to regulate cellular metabolism and energy homeostasis, by targeting the mitochondria. Having evolved from “ancient hopanoids,” avicins bear a structural resemblance with glucocorticoids (GCs), which are the endogenous regulators of metabolism and energy balance. These structural and functional similarities prompted us to compare the mode of action of avicin D with dexamethasone (Dex), a prototypical GC. Using cold competition assay, we show that Avicin D competes with Dex for binding to the GC receptor (GR), leading to its nuclear translocation. In contrast to Dex, avicin-induced nuclear translocation of GR does not result in transcriptional activation of GC-dependent genes. Instead we observe a decrease in the expression of GC-dependent metabolic proteins such as PEPCK and FASN. However, like Dex, avicin D treatment does induce a transrepressive effect on the pro-inflammatory transcription factor NF-κB. While avicin's ability to inhibit NF-κB and its downstream targets appear to be GR-dependent, its pro-apoptotic effects were independent of GR expression. Using various deletion mutants of GR, we demonstrate the requirement of both the DNA and ligand binding domains of GR in mediating avicin D's transrepressive effects. Modeling of avicin-GR interaction revealed that avicin molecule binds only to the antagonist confirmation of GR. These findings suggest that avicin D has properties of being a selective GR modulator that separates transactivation from transrepression. Since the gene-activating properties of GR are mainly linked to its metabolic effects, and the negative interference with the activity of transcription factors to its anti-inflammatory and immune suppressive effects, the identification of such a dissociated GR ligand could have great potential for therapeutic use. PMID:22132201

  17. The anticancer plant triterpenoid, avicin D, regulates glucocorticoid receptor signaling: implications for cellular metabolism.

    PubMed

    Haridas, Valsala; Xu, Zhi-Xiang; Kitchen, Doug; Jiang, Anna; Michels, Peter; Gutterman, Jordan U

    2011-01-01

    Avicins, a family of apoptotic triterpene electrophiles, are known to regulate cellular metabolism and energy homeostasis, by targeting the mitochondria. Having evolved from "ancient hopanoids," avicins bear a structural resemblance with glucocorticoids (GCs), which are the endogenous regulators of metabolism and energy balance. These structural and functional similarities prompted us to compare the mode of action of avicin D with dexamethasone (Dex), a prototypical GC. Using cold competition assay, we show that Avicin D competes with Dex for binding to the GC receptor (GR), leading to its nuclear translocation. In contrast to Dex, avicin-induced nuclear translocation of GR does not result in transcriptional activation of GC-dependent genes. Instead we observe a decrease in the expression of GC-dependent metabolic proteins such as PEPCK and FASN. However, like Dex, avicin D treatment does induce a transrepressive effect on the pro-inflammatory transcription factor NF-κB. While avicin's ability to inhibit NF-κB and its downstream targets appear to be GR-dependent, its pro-apoptotic effects were independent of GR expression. Using various deletion mutants of GR, we demonstrate the requirement of both the DNA and ligand binding domains of GR in mediating avicin D's transrepressive effects. Modeling of avicin-GR interaction revealed that avicin molecule binds only to the antagonist confirmation of GR. These findings suggest that avicin D has properties of being a selective GR modulator that separates transactivation from transrepression. Since the gene-activating properties of GR are mainly linked to its metabolic effects, and the negative interference with the activity of transcription factors to its anti-inflammatory and immune suppressive effects, the identification of such a dissociated GR ligand could have great potential for therapeutic use.

  18. Effects of the Social Environment and Stress on Glucocorticoid Receptor Gene Methylation: A Systematic Review.

    PubMed

    Turecki, Gustavo; Meaney, Michael J

    2016-01-15

    The early-life social environment can induce stable changes that influence neurodevelopment and mental health. Research focused on early-life adversity revealed that early-life experiences have a persistent impact on gene expression and behavior through epigenetic mechanisms. The hypothalamus-pituitary-adrenal axis is sensitive to changes in the early-life environment that associate with DNA methylation of a neuron-specific exon 17 promoter of the glucocorticoid receptor (GR) (Nr3c1). Since initial findings were published in 2004, numerous reports have investigated GR gene methylation in relationship to early-life experience, parental stress, and psychopathology. We conducted a systematic review of this growing literature, which identified 40 articles (13 animal and 27 human studies) published since 2004. The majority of these examined the GR exon variant 1F in humans or the GR17 in rats, and 89% of human studies and 70% of animal studies of early-life adversity reported increased methylation at this exon variant. All the studies investigating exon 1F/17 methylation in conditions of parental stress (one animal study and seven human studies) also reported increased methylation. Studies examining psychosocial stress and psychopathology had less consistent results, with 67% of animal studies reporting increased exon 17 methylation and 17% of human studies reporting increased exon 1F methylation. We found great consistency among studies investigating early-life adversity and the effect of parental stress, even if the precise phenotype and measures of social environment adversity varied among studies. These results are encouraging and warrant further investigation to better understand correlates and characteristics of these associations.

  19. Tannic Acid Down-Regulates the Angiotensin Type 1 Receptor Through a MAPK-Dependent Mechanism

    PubMed Central

    Yesudas, Rekha; Gumaste, Upendra; Snyder, Russell

    2012-01-01

    In the present study, we investigated the effects of tannic acid (TA), a hydrolysable polyphenol, on angiotensin type 1 receptor (AT1R) expression in continuously passaged rat liver epithelial cells. Under normal conditions, exposure of cells to TA resulted in the down-regulation of AT1R-specific binding in concentrations ranging from 12.5–100 μg/ml (7.34–58.78 μm) over a time period of 2–24 h with no change in receptor affinity to angiotensin II (AngII). The inhibitory effect of TA on AT1R was specific and reversible. In TA-treated cells, we observed a significant reduction in AngII-mediated intracellular calcium signaling, a finding consistent with receptor down-regulation. Under similar conditions, TA down-regulated AT1R mRNA expression without changing the rate of mRNA degradation, suggesting that TA's effect is mediated through transcriptional inhibition. Cells expressing recombinant AT1R without the native promoter show no change in receptor expression, whereas a pCAT reporter construct possessing the rat AT1R promoter was significantly reduced in activity. Furthermore, TA induced the phosphorylation of MAPK p42/p44. Pretreatment of the cells with a MAPK kinase (MEK)-specific inhibitor PD98059 prevented TA-induced MAPK phosphorylation and down-regulation of the AT1R. Moreover, there was no reduction in AngII-mediated intracellular calcium release upon MEK inhibition, suggesting that TA's observed inhibitory effect is mediated through MEK/MAPK signaling. Our findings demonstrate, for the first time, that TA inhibits AT1R gene expression and cellular response, suggesting the observed protective effects of dietary polyphenols on cardiovascular conditions may be, in part, through inhibition of AT1R expression. PMID:22322600

  20. Immunochemical analysis of the glucocorticoid receptor: identification of a third domain separate from the steroid-binding and DNA-binding domains.

    PubMed Central

    Carlstedt-Duke, J; Okret, S; Wrange, O; Gustafsson, J A

    1982-01-01

    The glucocorticoid-receptor complex can be subdivided into three separate domains by limited proteolysis with trypsin or alpha-chymotrypsin. The following characteristics can be separated: steroid-binding activity (domain A), DNA-binding activity (domain B), and immunoactivity (domain C). We have previously reported the separation of the steroid-binding domain from the DNA-binding domain by limited proteolysis of the receptor with trypsin. In this paper, we report the detection by immunochemical analysis of a third domain of the glucocorticoid receptor, which does not bind hormone. Immunoactivity was detected by using specific antiglucocorticoid receptor antibodies raised in rabbits against purified rat liver glucocorticoid receptor and the assay used was an enzyme-linked immunosorbent assay. After digestion with alpha-chymotrypsin, the immunoactive region of the receptor (domain C) was separated from the other two domains (A and B). The immunoactive fragment was found to have a Stokes radius of 2.6 nm. Further digestion with alpha-chymotrypsin resulted in separation of the immunoactive fragment to give a fragment having a Stokes radius of 1.4 nm. The immunoactive domain could be separated from the half of the glucocorticoid receptor containing the steroid-binding and the DNA-binding domains (Stokes radius, 3.3 nm), by limited proteolysis of the receptor by alpha-chymotrypsin followed by gel filtration or chromatography on DNA-cellulose. PMID:6181503

  1. Glucocorticoid receptor-mediated cell cycle arrest is achieved through distinct cell-specific transcriptional regulatory mechanisms.

    PubMed Central

    Rogatsky, I; Trowbridge, J M; Garabedian, M J

    1997-01-01

    Glucocorticoids inhibit proliferation of many cell types, but the events leading from the activated glucocorticoid receptor (GR) to growth arrest are not understood. Ectopic expression and activation of GR in human osteosarcoma cell lines U2OS and SAOS2, which lack endogenous receptors, result in a G1 cell cycle arrest. GR activation in U2OS cells represses expression of the cyclin-dependent kinases (CDKs) CDK4 and CDK6 as well as their regulatory partner, cyclin D3, leading to hypophosphorylation of the retinoblastoma protein (Rb). We also demonstrate a ligand-dependent reduction in the expression of E2F-1 and c-Myc, transcription factors involved in the G1-to-S-phase transition. Mitogen-activated protein kinase, CDK2, cyclin E, and the CDK inhibitors (CDIs) p27 and p21 are unaffected by receptor activation in U2OS cells. The receptor's N-terminal transcriptional activation domain is not required for growth arrest in U2OS cells. In Rb-deficient SAOS2 cells, however, the expression of p27 and p21 is induced upon receptor activation. Remarkably, in SAOS2 cells that express a GR deletion derivative lacking the N-terminal transcriptional activation domain, induction of CDI expression is abolished and the cells fail to undergo ligand-dependent cell cycle arrest. Similarly, murine S49 lymphoma cells, which, like SAOS2 cells, lack Rb, require the N-terminal activation domain for growth arrest and induce CDI expression upon GR activation. These cell-type-specific differences in receptor domains and cellular targets linking GR activation to cell cycle machinery suggest two distinct regulatory mechanisms of GR-mediated cell cycle arrest: one involving transcriptional repression of G1 cyclins and CDKs and the other involving enhanced transcription of CDIs by the activated receptor. PMID:9154817

  2. Luman/CREB3 recruitment factor regulates glucocorticoid receptor activity and is essential for prolactin-mediated maternal instinct.

    PubMed

    Martyn, Amanda C; Choleris, Elena; Gillis, Daniel J; Armstrong, John N; Amor, Talya R; McCluggage, Adam R R; Turner, Patricia V; Liang, Genqing; Cai, Kimberly; Lu, Ray

    2012-12-01

    The hypothalamic-pituitary-adrenal (HPA) axis is a major part of the neuroendocrine system in animal responses to stress. It is known that the HPA axis is attenuated at parturition to prevent detrimental effects of glucocorticoid secretion including inhibition of lactation and maternal responsiveness. Luman/CREB3 recruitment factor (LRF) was identified as a negative regulator of CREB3 which is involved in the endoplasmic reticulum stress response. Here, we report a LRF gene knockout mouse line that has a severe maternal behavioral defect. LRF(-/-) females lacked the instinct to tend pups; 80% of their litters died within 24 h, while most pups survived if cross-fostered. Prolactin levels were significantly repressed in lactating LRF(-/-) dams, with glucocorticoid receptor (GR) signaling markedly augmented. In cell culture, LRF repressed transcriptional activity of GR and promoted its protein degradation. LRF was found to colocalize with the known GR repressor, RIP140/NRIP1, which inhibits the activity by GR within specific nuclear punctates that are similar to LRF nuclear bodies. Furthermore, administration of prolactin or the GR antagonist RU486 restored maternal responses in mutant females. We thus postulate that LRF plays a critical role in the attenuation of the HPA axis through repression of glucocorticoid stress signaling during parturition and the postpartum period.

  3. Luman/CREB3 Recruitment Factor Regulates Glucocorticoid Receptor Activity and Is Essential for Prolactin-Mediated Maternal Instinct

    PubMed Central

    Martyn, Amanda C.; Choleris, Elena; Gillis, Daniel J.; Armstrong, John N.; Amor, Talya R.; McCluggage, Adam R. R.; Turner, Patricia V.; Liang, Genqing; Cai, Kimberly

    2012-01-01

    The hypothalamic-pituitary-adrenal (HPA) axis is a major part of the neuroendocrine system in animal responses to stress. It is known that the HPA axis is attenuated at parturition to prevent detrimental effects of glucocorticoid secretion including inhibition of lactation and maternal responsiveness. Luman/CREB3 recruitment factor (LRF) was identified as a negative regulator of CREB3 which is involved in the endoplasmic reticulum stress response. Here, we report a LRF gene knockout mouse line that has a severe maternal behavioral defect. LRF−/− females lacked the instinct to tend pups; 80% of their litters died within 24 h, while most pups survived if cross-fostered. Prolactin levels were significantly repressed in lactating LRF−/− dams, with glucocorticoid receptor (GR) signaling markedly augmented. In cell culture, LRF repressed transcriptional activity of GR and promoted its protein degradation. LRF was found to colocalize with the known GR repressor, RIP140/NRIP1, which inhibits the activity by GR within specific nuclear punctates that are similar to LRF nuclear bodies. Furthermore, administration of prolactin or the GR antagonist RU486 restored maternal responses in mutant females. We thus postulate that LRF plays a critical role in the attenuation of the HPA axis through repression of glucocorticoid stress signaling during parturition and the postpartum period. PMID:23071095

  4. Synergistic Action of GA-Binding Protein and Glucocorticoid Receptor in Transcription from the Mouse Mammary Tumor Virus Promoter

    PubMed Central

    Aurrekoetxea-Hernández, Koldo; Buetti, Elena

    2000-01-01

    B lymphocytes are among the first cells to be infected by mouse mammary tumor virus (MMTV), and they play a crucial role in its life cycle. To study transcriptional regulation of MMTV in B cells, we have analyzed two areas of the long terminal repeat (LTR) next to the glucocorticoid receptor binding site, fp1 (at position −139 to −146 from the cap site) and fp2 (at −157 to −164). Both showed B-cell-specific protection in DNase I in vitro footprinting assays and contain binding sites for Ets transcription factors, a large family of proteins involved in cell proliferation and differentiation and oncogenic transformation. In gel retardation assays, fp1 and fp2 bound the heterodimeric Ets factor GA-binding protein (GABP) present in B-cell nuclear extracts, which was identified by various criteria: formation of dimers and tetramers, sensitivity to pro-oxidant conditions, inhibition of binding by specific antisera, and comigration of complexes with those formed by recombinant GABP. Mutations which prevented complex formation in vitro abolished glucocorticoid-stimulated transcription from an MMTV LTR linked to a reporter gene in transiently transfected B-cell lines, whereas they did not affect the basal level. Exogenously expressed GABP resulted in an increased level of hormone response of the LTR reporter plasmid and produced a synergistic effect with the coexpressed glucocorticoid receptor, indicating cooperation between the two. This is the first example of GABP cooperation with a steroid receptor, providing the opportunity for studying the integration of their intracellular signaling pathways. PMID:10799572

  5. The glucocorticoid receptor is a key regulator of the decision between self-renewal and differentiation in erythroid progenitors.

    PubMed Central

    Wessely, O; Deiner, E M; Beug, H; von Lindern, M

    1997-01-01

    During development and in regenerating tissues such as the bone marrow, progenitor cells constantly need to make decisions between proliferation and differentiation. We have used a model system, normal erythroid progenitors of the chicken, to determine the molecular players involved in making this decision. The molecules identified comprised receptor tyrosine kinases (c-Kit and c-ErbB) and members of the nuclear hormone receptor superfamily (thyroid hormone receptor and estrogen receptor). Here we identify the glucocorticoid receptor (GR) as a key regulator of erythroid progenitor self-renewal (i.e. continuous proliferation in the absence of differentiation). In media lacking a GR ligand or containing a GR antagonist, erythroid progenitors failed to self-renew, even if c-Kit, c-ErbB and the estrogen receptor were activated simultaneously. To induce self-renewal, the GR required the continuous presence of an activated receptor tyrosine kinase and had to cooperate with the estrogen receptor for full activity. Mutant analysis showed that DNA binding and a functional AF-2 transactivation domain are required for proliferation stimulation and differentiation arrest. c-myb was identified as a potential target gene of the GR in erythroblasts. It could be demonstrated that delta c-Myb, an activated c-Myb protein, can functionally replace the GR. PMID:9029148

  6. A role for glucocorticoid-signaling in depression-like behavior of gastrin-releasing peptide receptor knock-out mice.

    PubMed

    Monje, Francisco J; Kim, Eun-Jung; Cabatic, Maureen; Lubec, Gert; Herkner, Kurt R; Pollak, Daniela D

    2011-08-01

    Abstract Background. The gastrin-releasing peptide receptor (GRPR) is highly expressed in the limbic system, where it importantly regulates emotional functions and in the suprachiasmatic nucleus, where it is central for the photic resetting of the circadian clock. Mice lacking GRPR presented with deficient light-induced phase shift in activity as well altered emotional learning and amygdala function. The effect of GRPR deletion on depression-like behavior and its molecular signature in the amygdala, however, has not yet been evaluated. Methods. GRPR knock-out mice (GRPR-KO) were tested in the forced-swim test and the sucrose preference test for depression-like behavior. Gene expression in the basolateral nucleus of the amygdala was evaluated by micorarray analysis subsequent to laser-capture microdissection-assisted extraction of mRNA. The expression of selected genes was confirmed by RT-PCR. Results. GRPR-KO mice were found to present with increased depression-like behavior. Microarray analysis revealed down-regulation of several glucocorticoid-responsive genes in the basolateral amygdala. Acute administration of dexamethasone reversed the behavioral phenotype and alterations in gene expression. Discussion. We propose that deletion of GRPR leads to the induction of depression-like behavior which is paralleled by dysregulation of amygdala gene expression, potentially resulting from deficient light-induced corticosterone release in GRPR-KO.

  7. Discovery of Compound A – a selective activator of the glucocorticoid receptor with anti-inflammatory and anti-cancer activity

    PubMed Central

    Lesovaya, Ekaterina; Yemelyanov, Alexander; Swart, Amanda C.; Swart, Pieter; Haegeman, Guy; Budunova, Irina

    2015-01-01

    Glucocorticoids are among the most effective anti-inflammatory drugs, and are widely used for cancer therapy. Unfortunately, chronic treatment with glucocorticoids results in multiple side effects. Thus, there was an intensive search for selective glucocorticoid receptor (GR) activators (SEGRA), which retain therapeutic potential of glucocorticoids, but with fewer adverse effects. GR regulates gene expression by transactivation (TA), by binding as homodimer to gene promoters, or transrepression (TR), via diverse mechanisms including negative interaction between monomeric GR and other transcription factors. It is well accepted that metabolic and atrophogenic effects of glucocorticoids are mediated by GR TA. Here we summarized the results of extensive international collaboration that led to discovery and characterization of Compound A (CpdA), a unique SEGRA with a proven “dissociating” GR ligand profile, preventing GR dimerization and shifting GR activity towards TR both in vitro and in vivo. We outlined here the unusual story of compound's discovery, and presented a comprehensive overview of CpdA ligand properties, its anti-inflammatory effects in numerous animal models of inflammation and autoimmune diseases, as well as its anti-cancer effects. Finally, we presented mechanistic analysis of CpdA and glucocorticoid effects in skin, muscle, bone, and regulation of glucose and fat metabolism to explain decreased CpdA side effects compared to glucocorticoids. Overall, the results obtained by our and other laboratories underline translational potential of CpdA and its derivatives for treatment of inflammation, autoimmune diseases and cancer. PMID:26436695

  8. Direct interaction of the tau 1 transactivation domain of the human glucocorticoid receptor with the basal transcriptional machinery.

    PubMed Central

    McEwan, I J; Wright, A P; Dahlman-Wright, K; Carlstedt-Duke, J; Gustafsson, J A

    1993-01-01

    We have used a yeast (Saccharomyces cerevisiae) cell free transcription system to study protein-protein interactions involving the tau 1 transactivation domain of the human glucocorticoid receptor that are important for transcriptional transactivation by the receptor. Purified tau 1 specifically inhibited transcription from a basal promoter derived from the CYC1 gene and from the adenovirus 2 major late core promoter in a concentration-dependent manner. This inhibition or squelching was correlated with the transactivation activity of tau 1. Recombinant yeast TATA-binding protein (yTFIID), although active in vitro, did not specifically reverse the inhibitory effect of tau 1. In addition, no specific interaction between tau 1 and yTFIID could be shown in vitro by affinity chromatography. Taken together, these results indicate that the tau 1 transactivation domain of the human glucocorticoid receptor interacts directly with the general transcriptional apparatus through some target protein(s) that is distinct from the TATA-binding factor. Furthermore, this assay can be used to identify interacting factors, since after phosphocellulose chromatography of a whole-cell yeast extract, a fraction that contained an activity which selectively counteracted the squelching effect of tau 1 was found. Images PMID:8417339

  9. Biochemical characterization of nuclear receptors for vitamin D{sub 3} and glucocorticoids in prostate stroma cell microenvironment

    SciTech Connect

    Hidalgo, Alejandro A.; Montecinos, Viviana P.; Paredes, Roberto; Godoy, Alejandro S.; McNerney, Eileen M.; Tovar, Heribelt; Pantoja, Diego; Johnson, Candace; Trump, Donald; Onate, Sergio A.

    2011-08-19

    Highlights: {yields} Fibroblasts from benign and carcinoma-associated stroma were biochemically characterized for VDR and GR function as transcription factors in prostate stroma cell microenvironment. {yields} Decreased SRC-1/CBP coactivators recruitment to VDR and GR may result in hormone resistance to 1,25D{sub 3} in stromal cell microenvironment prostate cancer. {yields} 1a,25-Dyhidroxyvitamin D{sub 3} (1,25D{sub 3}) and glucocorticoids, either alone or in combination, may not be an alternative for 'some' advanced prostate cancers that fails androgen therapies. -- Abstract: The disruption of stromal cell signals in prostate tissue microenvironment influences the development of prostate cancer to androgen independence. 1{alpha},25-Dihydroxyvitamin D{sub 3} (1,25D{sub 3}) and glucocorticoids, either alone or in combination, have been investigated as alternatives for the treatment of advanced prostate cancers that fails androgen therapies. The effects of glucocorticoids are mediated by the intracellular glucocorticoid receptor (GR). Similarly, the effect of 1,25D{sub 3} is mediated by the 1,25D{sub 3} nuclear receptor (VDR). In this study, fibroblasts from benign- (BAS) and carcinoma-associated stroma (CAS) were isolated from human prostates to characterize VDR and GR function as transcription factors in prostate stroma. The VDR-mediated transcriptional activity assessed using the CYP24-luciferase reporter was limited to 3-fold induction by 1,25D{sub 3} in 9 out of 13 CAS (70%), as compared to >10-fold induction in the BAS clinical sample pair. Expression of His-tagged VDR (Ad-his-VDR) failed to recover the low transcriptional activity of the luciferase reporter in 7 out of 9 CAS. Interestingly, expression of Ad-his-VDR successfully recovered receptor-mediated induction in 2 out of the 9 CAS analyzed, suggesting that changes in the receptor protein itself was responsible for decreased response and resistance to 1,25D{sub 3} action. Conversely, VDR

  10. Modulation of c-Jun N-Terminal Kinase Signaling and Specific Glucocorticoid Receptor Phosphorylation in the Treatment of Major Depression

    PubMed Central

    Jovicic, Milica J.; Lukic, Iva; Radojcic, Marija; Adzic, Miroslav; Maric, Nadja P.

    2015-01-01

    Glucocorticoid resistance is a common finding in major depressive disorder. Increased glucocorticoid receptor (GR) phosphorylation at serine 226 is associated with increased glucocorticoid resistance. Previously we have demonstrated that depressed patients exhibit higher levels of GR phosphorylated at serine 226 compared to healthy controls. The enzyme that is involved in this specific GR phosphorylation is c-Jun N-Terminal Kinase (JNK). We propose that modulation of glucocorticoid phosphorylation at serine 226, by targeting JNK signaling pathway, could be a potential strategy for antidepressant treatment. We base this assumption on the results of previous research that examined GR phosphorylation and JNK signaling in animal models and human studies. We also discuss the potential challenges in targeting JNK signaling pathway in depression. PMID:26052031

  11. Reduction of hepatic and adipose tissue glucocorticoid receptor expression with antisense oligonucleotides improves hyperglycemia and hyperlipidemia in diabetic rodents without causing systemic glucocorticoid antagonism.

    PubMed

    Watts, Lynnetta M; Manchem, Vara Prasad; Leedom, Thomas A; Rivard, Amber L; McKay, Robert A; Bao, Dingjiu; Neroladakis, Teri; Monia, Brett P; Bodenmiller, Diane M; Cao, Julia Xiao-Chun; Zhang, Hong Yan; Cox, Amy L; Jacobs, Steven J; Michael, M Dodson; Sloop, Kyle W; Bhanot, Sanjay

    2005-06-01

    Glucocorticoids (GCs) increase hepatic gluconeogenesis and play an important role in the regulation of hepatic glucose output. Whereas systemic GC inhibition can alleviate hyperglycemia in rodents and humans, it results in adrenal insufficiency and stimulation of the hypothalamic-pituitary-adrenal axis. In the present study, we used optimized antisense oligonucleotides (ASOs) to cause selective reduction of the glucocorticoid receptor (GCCR) in liver and white adipose tissue (WAT) and evaluated the resultant changes in glucose and lipid metabolism in several rodent models of diabetes. Treatment of ob/ob mice with GCCR ASOs for 4 weeks resulted in approximately 75 and approximately 40% reduction in GCCR mRNA expression in liver and WAT, respectively. This was accompanied by approximately 65% decrease in fed and approximately 30% decrease in fasted glucose levels, a 60% decrease in plasma insulin concentration, and approximately 20 and 35% decrease in plasma resistin and tumor necrosis factor-alpha levels, respectively. Furthermore, GCCR ASO reduced hepatic glucose production and inhibited hepatic gluconeogenesis in liver slices from basal and dexamethasone-treated animals. In db/db mice, a similar reduction in GCCR expression caused approximately 40% decrease in fed and fasted glucose levels and approximately 50% reduction in plasma triglycerides. In ZDF and high-fat diet-fed streptozotocin-treated (HFD-STZ) rats, GCCR ASO treatment caused approximately 60% reduction in GCCR expression in the liver and WAT, which was accompanied by a 40-70% decrease in fasted glucose levels and a robust reduction in plasma triglyceride, cholesterol, and free fatty acids. No change in circulating corticosterone levels was seen in any model after GCCR ASO treatment. To further demonstrate that GCCR ASO does not cause systemic GC antagonism, normal Sprague-Dawley rats were challenged with dexamethasone after treating with GCCR ASO. Dexamethasone increased the expression of GC

  12. Reduction of hepatic and adipose tissue glucocorticoid receptor expression with antisense oligonucleotides improves hyperglycemia and hyperlipidemia in diabetic rodents without causing systemic glucocorticoid antagonism.

    PubMed

    Watts, Lynnetta M; Manchem, Vara Prasad; Leedom, Thomas A; Rivard, Amber L; McKay, Robert A; Bao, Dingjiu; Neroladakis, Teri; Monia, Brett P; Bodenmiller, Diane M; Cao, Julia Xiao-Chun; Zhang, Hong Yan; Cox, Amy L; Jacobs, Steven J; Michael, M Dodson; Sloop, Kyle W; Bhanot, Sanjay

    2005-06-01

    Glucocorticoids (GCs) increase hepatic gluconeogenesis and play an important role in the regulation of hepatic glucose output. Whereas systemic GC inhibition can alleviate hyperglycemia in rodents and humans, it results in adrenal insufficiency and stimulation of the hypothalamic-pituitary-adrenal axis. In the present study, we used optimized antisense oligonucleotides (ASOs) to cause selective reduction of the glucocorticoid receptor (GCCR) in liver and white adipose tissue (WAT) and evaluated the resultant changes in glucose and lipid metabolism in several rodent models of diabetes. Treatment of ob/ob mice with GCCR ASOs for 4 weeks resulted in approximately 75 and approximately 40% reduction in GCCR mRNA expression in liver and WAT, respectively. This was accompanied by approximately 65% decrease in fed and approximately 30% decrease in fasted glucose levels, a 60% decrease in plasma insulin concentration, and approximately 20 and 35% decrease in plasma resistin and tumor necrosis factor-alpha levels, respectively. Furthermore, GCCR ASO reduced hepatic glucose production and inhibited hepatic gluconeogenesis in liver slices from basal and dexamethasone-treated animals. In db/db mice, a similar reduction in GCCR expression caused approximately 40% decrease in fed and fasted glucose levels and approximately 50% reduction in plasma triglycerides. In ZDF and high-fat diet-fed streptozotocin-treated (HFD-STZ) rats, GCCR ASO treatment caused approximately 60% reduction in GCCR expression in the liver and WAT, which was accompanied by a 40-70% decrease in fasted glucose levels and a robust reduction in plasma triglyceride, cholesterol, and free fatty acids. No change in circulating corticosterone levels was seen in any model after GCCR ASO treatment. To further demonstrate that GCCR ASO does not cause systemic GC antagonism, normal Sprague-Dawley rats were challenged with dexamethasone after treating with GCCR ASO. Dexamethasone increased the expression of GC

  13. Downregulation of the CXCR4 receptor inhibits cervical carcinoma metastatic behavior in vitro and in vivo

    PubMed Central

    SEKUŁA, MAŁGORZATA; MIEKUS, KATARZYNA; MAJKA, MARCIN

    2014-01-01

    Cervical carcinoma is frequently diagnosed among women, particularly in low and middle income countries. In this study, we investigated the role of the SDF-1/CXCR4 axis during cervical carcinoma growth and progression in vitro and in vivo. Downregulation of CXCR4 receptor using an RNA interference system led to almost complete inhibition of the receptor expression, activation and function. CXCR4 receptor silencing led to decreased ability to signal, to induce migration and to form holoclone-like colonies, with no influence on viability/proliferation of the cells. CXCR4-deficient cells had also significantly lower levels of MMP-9. Interestingly, downregulation of CXCR4 expression resulted in reduced tumor growth in vivo. Tumors generated by CXCR4-deficient cells had also lower expression of the proliferation marker Ki-67 and decreased ability to engraft into lungs and spleen. Taken together, our results indicate that CXCR4 receptor may play an important role during cervical carcinoma invasion. In our study CXCR4 influenced invasive properties of cervical carcinoma cells both in vitro and in vivo. PMID:24728301

  14. Tolloid-like 1 is negatively regulated by stress and glucocorticoids.

    PubMed

    Tamura, Goichiro; Olson, Dawne; Miron, Joel; Clark, Timothy G

    2005-12-14

    Glucocorticoids affect a variety of tissues to enable the organism to adapt to the stress. Hippocampal neurons contain glucocorticoid receptors and respond to elevated glucocorticoid levels by down-regulating the HPA axis. Chronically, however, stress is deleterious to hippocampal neurons. Chronically elevated levels of glucocorticoids result in a decrease in the number of dendritic spines, reduced axonal growth and synaptogenesis, and decreased neurogenesis in the hippocampus. Tolloid-like 1 (Tll-1) is a metalloprotease that potentiates the activity of the bone morphogenetic proteins (BMPs). Neurogenesis in the hippocampus of both developing and adult mammals requires BMPs. In this study, we demonstrate that Tll-1 expression is increased in mice that have increased neurogenesis. The Tll-1 promoter contains glucocorticoid response elements which are capable of binding to purified glucocorticoid receptor. Glucocorticoids decrease Tll-1 expression in vitro. Finally, prenatal stress leads to a decrease in Tll-1 mRNA expression in the hippocampus of adult female mice that is not observed in adult male mice indicating that Tll-1 expression is differentially regulated in males and females. The results of this study indicate that Tll-1 is responsive to glucocorticoids and this mechanism might influence neurogenesis in the hippocampus.

  15. Interactions of the ubiquitous octamer-binding transcription factor-1 with both the signal transducer and activator of transcription 5 and the glucocorticoid receptor mediate prolactin and glucocorticoid-induced β-casein gene expression in mammary epithelial cells.

    PubMed

    Qian, Xi; Zhao, Feng-Qi

    2013-03-01

    Regulation of milk protein gene expression by lactogenic hormones (prolactin and glucocorticoids) provides an attractive model for studying the mechanisms by which protein and steroid hormones synergistically regulate gene expression. β-Casein is one of the major milk proteins and its expression in mammary epithelial cells is stimulated by lactogenic hormones. The signal transducer and activator of transcription 5 and glucocorticoid receptor are essential downstream mediators of prolactin and glucocorticoid signaling, respectively. Previous studies have shown that mutating the octamer-binding site of the β-casein gene proximal promoter dramatically reduces the hormonal induction of the promoter activity. However, little is known about the underlying molecular mechanisms. In this report, we show that lactogenic hormones rapidly induce the binding of octamer-binding transcription factor-1 to the β-casein promoter and this induction is not mediated by either increasing the expression of octamer-binding transcription factor-1 or inducing its translocation to the nucleus. Rather, lactogenic hormones induce physical interactions between the octamer-binding transcription factor-1, signal transducer and activator of transcription 5, and glucocorticoid receptor to form a ternary complex, and these interactions enhance or stabilize the binding of these transcription factors to the promoter. Abolishing these interactions significantly reduces the hormonal induction of β-casein gene transcription. Thus, our study indicates that octamer-binding transcription factor-1 may serve as a master regulator that facilitates the DNA binding of both signal transducer and activator of transcription 5 and glucocorticoid receptor in hormone-induced β-casein expression, and defines a novel mechanism of regulation of tissue-specific gene expression by the ubiquitous octamer-binding transcription factor-1.

  16. Down-regulation of rat kidney calcitonin receptors by salmon calcitonin infusion evidence by autoradiography

    SciTech Connect

    Bouizar, Z.; Rostene, W.H.; Milhaud, G.

    1987-08-01

    In treating age-related osteoporosis and Paget disease of bone, it is of major importance to avoid an escape phenomenon that would reduce effectiveness of the treatment. The factors involved in the loss of therapeutic efficacy with administration of large pharmacological doses of the hormone require special consideration. Down-regulation of the hormone receptors could account for the escape phenomenon. Specific binding sites for salmon calcitonin (sCT) were characterized and localized by autoradiography on rat kidney sections incubated with /sup 125/I-labeled sCT. Autoradiograms demonstrated a heterogeneous distribution of /sup 125/I-labeled sCT binding sites in the kidney, with high densities in both the superficial layer of the cortex and the outer medulla. Infusion of different doses of unlabeled sCT by means of Alzet minipumps for 7 days produced rapid changes in plasma calcium, phosphate, and magnesium levels, which were no longer observed after 2 or 6 days of treatment. Besides, infusion of high doses of sCT induced down-regulation of renal sCT binding sites located mainly in the medulla, where calcitonin (CT) has been shown to exert it physiological effects on water and ion reabsorption. These data suggest that the resistance to high doses of sCT often observed during long-term treatment of patients may be the consequence of not only bone-cell desensitization but also down-regulation of CT-sensitive kidney receptor sites.

  17. Short-day aggression is independent of changes in cortisol or glucocorticoid receptors in male Siberian hamsters (Phodopus sungorus).

    PubMed

    Scotti, Melissa-Ann L; Rendon, Nikki M; Greives, Timothy J; Romeo, Russell D; Demas, Gregory E

    2015-06-01

    Testosterone mediates aggression in many vertebrates. In some species, aggression remains high during the non-breeding season (e.g., winter), when testosterone levels are low. In Siberian hamsters (Phodopus sungorus), we have demonstrated photoperiodic changes in aggression with hamsters housed in short, "winter-like" days displaying significantly more territorial aggression than long-day animals, despite low levels of testosterone. The mechanisms by which photoperiod regulates aggression, however, remain largely unknown. Adrenocortical hormones (e.g., glucocorticoids) have been implicated in mediating seasonal aggression; circulating concentrations of these hormones have been correlated with aggression in some species. The goal of this study was to examine the role of cortisol and glucocorticoid receptors in mediating photoperiodic changes in aggression in male Siberian hamsters. Males were housed in long or short days and treated with either exogenous cortisol or vehicle. Circulating levels of cortisol, adrenal cortisol content, and aggression were quantified. Lastly, photoperiodic effects on glucocorticoid receptor (GR) protein levels were quantified in limbic brain regions associated with aggression, including medial prefrontal cortex, amygdala, and hippocampus. Short-day hamsters were more aggressive than long-day hamsters, however cortisol treatment did not affect aggression. Photoperiod had no effect on serum or adrenal cortisol or GR levels in the brain regions examined. Taken together, these data suggest that increases in cortisol levels do not cause increases associated with short-day aggression, and further that GR protein levels are not associated with photoperiodic changes in aggression. The results of this study contribute to our understanding of the role of adrenocortical steroids in mediating seasonal aggression.

  18. The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test.

    PubMed

    Solomon, Matia B; Wulsin, Aynara C; Rice, Taylor; Wick, Dayna; Myers, Brent; McKlveen, Jessica; Flak, Jonathan N; Ulrich-Lai, Yvonne; Herman, James P

    2014-04-01

    Pre-clinical and clinical studies have employed treatment with glucocorticoid receptor (GR) antagonists in an attempt to limit the deleterious behavioral and physiological effects of excess glucocorticoids. Here, we examined the effects of GR antagonists on neuroendocrine and behavioral stress responses, using two compounds: mifepristone, a GR antagonist that is also a progesterone receptor antagonist, and CORT 108297, a specific GR antagonist lacking anti-progestin activity. Given its well-documented impact on neuroendocrine and behavioral stress responses, imipramine (tricyclic antidepressant) served as a positive control. Male rats were treated for five days with mifepristone (10mg/kg), CORT 108297 (30mg/kg and 60mg/kg), imipramine (10mg/kg) or vehicle and exposed to forced swim test (FST) or restraint stress. Relative to vehicle, imipramine potently suppressed adrenocorticotropin hormone (ACTH) responses to FST and restraint exposure. Imipramine also decreased immobility in the FST, consistent with antidepressant actions. Both doses of CORT 108297 potently suppressed peak corticosterone responses to FST and restraint stress. However, only the higher dose of CORT 108297 (60mg/kg) significantly decreased immobility in the FST. In contrast, mifepristone induced protracted secretion of corticosterone in response to both stressors, and modestly decreased immobility in the FST. Taken together, the data indicate distinct effects of each compound on neuroendocrine stress responses and also highlight dissociation between corticosterone responses and immobility in the FST. Within the context of the present study, our data suggest that CORT 108297 may be an attractive alternative for mitigating neuroendocrine and behavioral states associated with excess glucocorticoid secretion.

  19. Expression of glucocorticoid receptor, mineralocorticoid receptor, and 11beta-hydroxysteroid dehydrogenase 1 and 2 in the fetal and postnatal ovine hippocampus: ontogeny and effects of prenatal glucocorticoid exposure.

    PubMed

    Sloboda, Deborah M; Moss, Timothy J M; Li, Shaofu; Matthews, Stephen G; Challis, John R G; Newnham, John P

    2008-05-01

    To determine the expression of glucocorticoid metabolizing and action genes in the hippocampus of fetal, neonatal, and adult sheep. Pregnant ewes (or their fetuses) received intramuscular injections of saline or betamethasone (BETA, 0-5 mg/kg) at 104, 111, 118, and/or 125 days of gestation (dG). Hippocampal tissue was collected prior to (75, 84, and 101 dG), during (109 and 116 dG), or after (121, 132, and 146 dG; 6 and 12 postnatal weeks; 3.5 years of age) saline or BETA injections. Hippocampal glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11beta-hydroxysteroid dehydrogenase (11betaHSD)1 and 11betaHSD2 mRNA levels were determined using qRT-PCR. Control animals late in gestation demonstrated a decrease in mRNA encoding GR and 11betaHSD1, whereas 11betaHSD2 was undetectable, consistent with a damping of the negative feedback influence of circulating or locally produced cortisol on the hypothalamic-pituitary-adrenal (HPA) axis. BETA-administration had transient effects on fetal GR and MR, and early in postnatal life (12 weeks of age) 11betaHSD1 mRNA was increased. Hippocampal MR mRNA was elevated in adult offspring exposed to either one or four doses of maternal BETA (P<0.001). Four courses of maternal BETA increased 11betaHSD2 (P<0.05) but not 11betaHSD1 mRNA levels. Late in gestation a reduction in hippocampal GR and 11betaHSD1 mRNA suggests lessening of glucocorticoid negative feedback, facilitating increased preterm HPA activity and parturition. Adult offspring of BETA-treated mothers demonstrated increased MR and 11betaHSD2 mRNA, therefore it appears that exposure of fetus to high levels of synthetic glucocorticoids may have long-lasting effects on the hippocampal expression of HPA-related genes into adulthood.

  20. Down-regulation of the G-proteins Gq alpha and G11 alpha by transfected human M3 muscarinic acetylcholine receptors in Chinese hamster ovary cells is independent of receptor down-regulation.

    PubMed Central

    van de Westerlo, E; Yang, J; Logsdon, C; Williams, J A

    1995-01-01

    Chinese hamster ovary cells stably transfected with human M3 muscarinic acetylcholine receptors show a 40-50% reduction in the immunoreactive G-proteins Gq alpha and G11 alpha when stimulated with the cholinergic agonist carbachol. This effect is seen after 9 h, is maximal after 24 h, and occurs over a range of carbachol concentrations that activate phosphoinositide hydrolysis in these cells. The effect is specific for Gq alpha family proteins as Gs alpha was slightly increased after carbachol treatment and G13 alpha was unchanged. Using a urea gel system, we were able to resolve Gq alpha and G11 alpha, both of which were down-regulated by carbachol. An M3 receptor mutant, with C-terminal threonines changed to alanines as described previously, binds ligand and activates phosphoinositide hydrolysis normally but is not down-regulated in response to carbachol. This receptor, however, induces Gq alpha/G11 alpha down-regulation similarly to wild-type M3 receptors, indicating that G-protein down-regulation is not directly coupled to receptor down-regulation. Thus down-regulation of Gq alpha and G11 alpha may contribute to heterologous desensitization particularly at longer times of agonist exposure. Images Figure 1 Figure 4 PMID:7654194

  1. Corticotropin-releasing hormone, proopiomelanocortin, and glucocorticoid receptor gene expression in adrenocorticotropin-producing tumors in vitro.

    PubMed Central

    Suda, T; Tozawa, F; Dobashi, I; Horiba, N; Ohmori, N; Yamakado, M; Yamada, M; Demura, H

    1993-01-01

    To differentiate between ectopic ACTH syndrome and Cushing's disease, gene expression of corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), and glucocorticoid receptor was examined in 10 pituitary adenomas (Cushing's disease) and in 10 ectopic ACTH-producing tumors. CRH increased plasma ACTH levels in all patients with Cushing's disease and in five patients with ectopic ACTH syndrome whose tumors contained CRH and CRH mRNA. In five CRH nonresponders, CRH was not detected in tumors that contained no CRH mRNA or that contained only long-size CRH mRNA. Dexamethasone (Dex) decreased plasma ACTH levels in all patients with Cushing's disease and in three patients with ectopic ACTH-producing bronchial carcinoid. These tumors contained glucocorticoid receptor mRNA. CRH increased and Dex decreased ACTH release and POMC mRNA levels in pituitary adenoma and bronchial carcinoid cells. PMA increased POMC mRNA levels only in carcinoid cells. These results reveal characteristics of ectopic ACTH-producing tumors: long-size CRH mRNA and PMA-induced POMC gene expression. In addition, there are two ectopic ACTH syndrome subtypes: tumors containing ACTH with CRH (CRH responder) and tumors without CRH. Dex decreases ACTH release and POMC mRNA levels in some bronchial carcinoids. Therefore, CRH and Dex tests have limited usefulness in differentiating between Cushing's disease and ectopic ACTH syndrome. Images PMID:8254033

  2. Prenatal Stress, Fearfulness, and the Epigenome: Exploratory Analysis of Sex Differences in DNA Methylation of the Glucocorticoid Receptor Gene.

    PubMed

    Ostlund, Brendan D; Conradt, Elisabeth; Crowell, Sheila E; Tyrka, Audrey R; Marsit, Carmen J; Lester, Barry M

    2016-01-01

    Exposure to stress in utero is a risk factor for the development of problem behavior in the offspring, though precise pathways are unknown. We examined whether DNA methylation of the glucocorticoid receptor gene, NR3C1, was associated with experiences of stress by an expectant mother and fearfulness in her infant. Mothers reported on prenatal stress and infant temperament when infants were 5 months old (n = 68). Buccal cells for methylation analysis were collected from each infant. Prenatal stress was not related to infant fearfulness or NR3C1 methylation in the sample as a whole. Exploratory sex-specific analysis revealed a trend-level association between prenatal stress and increased methylation of NR3C1 exon 1F for female, but not male, infants. In addition, increased methylation was significantly associated with greater fearfulness for females. Results suggest an experience-dependent pathway to fearfulness for female infants via epigenetic modification of the glucocorticoid receptor gene. Future studies should examine prenatal stress in a comprehensive fashion while considering sex differences in epigenetic processes underlying infant temperament. PMID:27462209

  3. Prenatal Stress, Fearfulness, and the Epigenome: Exploratory Analysis of Sex Differences in DNA Methylation of the Glucocorticoid Receptor Gene

    PubMed Central

    Ostlund, Brendan D.; Conradt, Elisabeth; Crowell, Sheila E.; Tyrka, Audrey R.; Marsit, Carmen J.; Lester, Barry M.

    2016-01-01

    Exposure to stress in utero is a risk factor for the development of problem behavior in the offspring, though precise pathways are unknown. We examined whether DNA methylation of the glucocorticoid receptor gene, NR3C1, was associated with experiences of stress by an expectant mother and fearfulness in her infant. Mothers reported on prenatal stress and infant temperament when infants were 5 months old (n = 68). Buccal cells for methylation analysis were collected from each infant. Prenatal stress was not related to infant fearfulness or NR3C1 methylation in the sample as a whole. Exploratory sex-specific analysis revealed a trend-level association between prenatal stress and increased methylation of NR3C1 exon 1F for female, but not male, infants. In addition, increased methylation was significantly associated with greater fearfulness for females. Results suggest an experience-dependent pathway to fearfulness for female infants via epigenetic modification of the glucocorticoid receptor gene. Future studies should examine prenatal stress in a comprehensive fashion while considering sex differences in epigenetic processes underlying infant temperament. PMID:27462209

  4. PDE4 inhibitors augment levels of glucocorticoid receptor in B cell chronic lymphocytic leukemia but not in normal circulating hematopoietic cells

    PubMed Central

    Meyers, John A.; Taverna, Josephine; Chaves, Jorge; Makkinje, Anthony; Lerner, Adam

    2009-01-01

    Purpose Type 4 cAMP phosphodiesterase (PDE4) inhibitors, compounds that activate cAMP-mediated signaling by inhibiting cAMP catabolism, potentiate glucocorticoid-mediated apoptosis in chronic lymphocytic leukemia (CLL) cells but the mechanism by which this occurs is unknown. In this study, we sought to address whether PDE4 inhibitors alter expression of glucocorticoid receptor (GRα) in CLL cells. Experimental Design CLL cells or normal hematopoietic cells were treated with PDE4 inhibitors followed by analysis of GRα transcript and protein by real-time PCR and Western analysis. Results PDE4 inhibitors up-regulate glucocorticoid receptor transcript levels in CLL cells but not normal circulating T cells, B cells, monocytes or neutrophils. As GRα transcript half-life does not vary in CLL cells treated with the prototypic PDE4 inhibitor rolipram, the four-fold increase in GRα mRNA levels observed within four hours of rolipram treatment appears to result from an increase in transcription. Rolipram treatment increases levels of transcripts derived from the 1A3 promoter to a greater extent than the 1B promoter. Treatment of CLL cells with cilomilast and roflumilast, two PDE4 inhibitors previously studied in clinical trials also augments GRα transcript levels and glucocorticoid-mediated apoptosis. Washout studies demonstrate that simultaneous treatment with both drug classes irreversibly augments apoptosis over the same time frame that glucocorticoid receptor up-regulation occurs. While treatment of CLL cells with glucocorticoids reduces basal GRα transcript levels in a dose-related manner, co-treatment with rolipram maintained GRα transcript levels above baseline. Conclusion Our results suggest that PDE4 inhibitors may sensitize CLL cells to glucocorticoid-induced apoptosis by augmenting GRα expression. PMID:17699872

  5. Effects of a Glucocorticoid Receptor Agonist, Dexamethasone, on Fathead Minnow Reproduction and Development

    EPA Science Inventory

    Few studies have examined the effects of synthetic glucocorticoids on the reproductive axis of fish, despite the fact that these chemicals are therapeutically prescribed anti-inflammatory agents that are abundantly produced and consumed. To generate data to assess potential risk ...

  6. Effects of a Glucocorticoid Receptor Agonist, Dexamethasone, on Fathead Minnow Reproduction, Growth, and Development.

    EPA Science Inventory

    Few studies have examined the effects of synthetic glucocorticoids on the reproductive axis of fish, despite the fact that these chemicals are therapeutically prescribed anti-inflammatory agents that are abundantly produced and consumed. To generate data to assess potential risk ...

  7. Downregulation of LH receptor mRNA in the rat uterus.

    PubMed

    Kasahara, Yoshimitsu; Kitahara, Yoshikazu; Nakamura, Kazuto; Minegishi, Takashi

    2012-05-01

    We detected luteinizing hormone receptor (LHR) mRNA in the immature rat uterus by northern blotting and downregulation of this receptor mRNA after pregnant mare serum gonadotropin (PMSG)-human chorionic gonadotropin (hCG) treatment. After administration of hCG, the mRNA levels in the rat uterus declined to an extremely low level from Days 1 to 3 and then rebounded and reached higher than pretreatment values at Day 4. At Day 5 the levels were 3-fold higher than the control levels. The cultured uterus displayed an hCG concentration-dependent increase in cAMP production in the medium. Immunohistochemical experiments showed that these receptor proteins were expressed in the epithelial cells of the endometrium. These results suggest that functional LHRs are present in the immature rat uterus and are downregulated by signals resulting from hCG treatment. These data may support the idea that LH acts on the uterus to inhibit contraction at ovulation. Although the precise role of the LHR in the uterus remains unknown, this study may provide a model with which to investigate the regulation of LHR.

  8. Molecular basis of the interaction specificity between the human glucocorticoid receptor and its endogenous steroid ligand cortisol.

    PubMed

    von Langen, Johannes; Fritzemeier, Karl-Heinrich; Diekmann, Stephan; Hillisch, Alexander

    2005-06-01

    We analyzed the binding of five steroids to the human glucocorticoid receptor (hGR) experimentally as well as theoretically. In vitro, we measured the binding affinity of aldosterone, cortisol, estradiol, progesterone, and testosterone to hGR in competition with the ligand dexamethasone. The binding affinity relative to the endogenous ligand cortisol (100%) is reduced for progesterone (22%) and aldosterone (20%) and is very weak for testosterone (1.5%) and estradiol (0.2%). In parallel, we constructed a homology model of the hGR ligand-binding domain (LBD) based on the crystal structure of the human progesterone receptor (hPR). After docking the five steroids into the hGR model ligand-binding pocket, we performed five separate 4-ns molecular dynamics (MD) simulations with these complexes in order to study the complex structures. We calculated the binding affinities with two different approaches (MM/PBSA, FlexX) and compared them with the values of the experimentally determined relative binding affinities. Both theoretical methods allowed discrimination between strongly and weakly binding ligands and recognition of cortisol as the endogenous ligand of the hGR in silico. Cortisol binds most strongly due to a nearly perfect steric and electrostatic complementarity with the hGR binding pocket. Chemically similar ligands such as estradiol, testosterone, and progesterone also fit into the hGR binding pocket, but they are unable to form all those contacts with the amino acids of the protein that are necessary to yield a stable, transcriptionally active receptor conformation. Our analysis thus explains the selectivity of the human glucocorticoid receptor for its endogenous ligand cortisol at a molecular level. PMID:15883974

  9. Selective and interactive down-regulation of mu- and delta-opioid receptors in human neuroblastoma SK-N-SH cells.

    PubMed

    Baumhaker, Y; Gafni, M; Keren, O; Sarne, Y

    1993-08-01

    Human neuroblastoma SK-N-SH cells, which contain both mu- and delta-opioid receptors, were grown under conditions that provided a mu:delta ratio of 1.5:1. Both receptors were down-regulated after 72 hr of exposure to 100 nM etorphine. Selective down-regulation was demonstrated using selective opioid agonists; the mu agonist Tyr-D-Ala2-Gly-(Me)Phe4-Gly-ol down-regulated mu- but not delta-opioid receptors, whereas prolonged exposure to the selective delta agonist D-Pen2,D-Pen5-enkephalin resulted in delta- but not mu-opioid receptor down-regulation. Morphine, which binds mu- as well as delta-opioid receptors, down-regulated both receptor subtypes. NG108-15 cells, which contain delta receptors exclusively, were also tested. NG108-15 cells did not exhibit delta-opioid receptor down-regulation when exposed to morphine. The discrepancy between the effect of chronic morphine treatment on delta receptors in SK-N-SH cells and in NG108-15 cells raised the question of whether the coexistence of mu receptors in the former allowed morphine to down-regulate delta receptors. The role of mu-opioid receptors in morphine-induced delta receptor down-regulation was studied by using the irreversible mu antagonist beta-funaltrexamine. Pretreatment of SK-N-SH cells with beta-funaltrexamine prevented down-regulation of delta receptors in response to chronic exposure to morphine but did not affect down-regulation of delta receptors in response to D-Pen2,D-Pen5-enkephalin. The experimental data indicate that morphine-induced delta-opioid receptor down-regulation is dependent on the presence of functional mu receptors in the same cell.

  10. Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications

    PubMed Central

    Nader, Nancy; Chrousos, George P.; Kino, Tomoshige

    2009-01-01

    Glucocorticoids, end products of the hypothalamic-pituitary-adrenal axis, influence functions of virtually all organs and tissues through the glucocorticoid receptor (GR). Circulating levels of glucocorticoids fluctuate naturally in a circadian fashion and regulate the transcriptional activity of GR in target tissues. The basic helix-loop-helix protein CLOCK, a histone acetyltransferase (HAT), and its heterodimer partner BMAL1 are self-oscillating transcription factors that generate circadian rhythms in both the central nervous system and periphery. We found that CLOCK/BMAL1 repressed GR-induced transcriptional activity in a HAT-activity- dependent fashion. In serum-shock-synchronized cells, transactivational activity of GR, accessed by mRNA expression of an endogenous-responsive gene, fluctuated spontaneously in a circadian fashion in reverse phase with CLOCK/BMAL1 mRNA expression. CLOCK and GR interacted with each other physically, and CLOCK suppressed binding of GR to its DNA recognition sequences by acetylating multiple lysine residues located in its hinge region. These findings indicate that CLOCK/BMAL1 functions as a reverse-phase negative regulator of glucocorticoid action in target tissues, possibly by antagonizing biological actions of diurnally fluctuating circulating glucocorticoids. Further, these results suggest that a peripheral target tissue circadian rhythm indirectly influences the functions of every organ and tissue inside the body through modulation of the ubiquitous and diverse actions of glucocorticoids.—Nader, N., Chrousos, G. P., Kino, T. Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications. PMID:19141540

  11. Glucocorticoids differentially regulate expression of duodenal and renal calbindin-D9k through glucocorticoid receptor-mediated pathway in mouse model.

    PubMed

    Lee, Geun-Shik; Choi, Kyung-Chul; Jeung, Eui-Bae

    2006-02-01

    Dexamethasone (Dex) is a member of the glucocorticoids (GCs), and is broadly used as an anti-inflammatory medication. Continuous administration with GCs induces adverse effects and suffering in humans (i.e., osteoporosis) due to negative calcium balance derived from low re- and absorption in the duodenum and kidney. A cytosolic calcium-binding protein, calbindin-D9k (CaBP-9k), is dominantly expressed in the renal and intestinal tissues involved in calcium re- and absorption and plays an active role in calcium transport. In the present study, we employed adrenalectomized (ADX) and sham-treated (Sham) male mice to examine the effect of Dex on CaBP-9k gene expression in the duodenum and kidney. Dex significantly reduced the levels of duodenal CaBP-9k mRNA and protein, and it restored ADX-induced decrease in renal CaBP-9k protein compared with the level of Sham control. Dex treatment increased calcium and phosphate levels in the sera of both Sham and ADX mice. In a time course experiment, Dex significantly decreased duodenal CaBP-9k at the transcriptional and translational levels at 3 days, whereas it temporarily increased CaBP-9k mRNA and protein levels at 12 and 24 h. Altered CaBP-9k expression by Dex was completely reversed by mifepristone, an antagonist for the GC receptor (GR). In addition, duodenal CaBP-9k and GR were colocalized on the enterocyte (duodenocyte), supporting a role for GR in regulating CaBP-9k. In ovariectomized (OVX) and ADX female mice daily treated with Dex for 3 days, duodenal CaBP-9k was expressed at the same level as in male mice. Also, no cross-activity of progesterone and Dex on their receptors was observed. Taken together, these results indicate that mouse CaBP-9k gene may be regulated by Dex in a tissue-specific manner, and reduced duodenal CaBP-9k via the GR pathway may take part in negative calcium absorption of GC-induced osteoporosis, whereas renal CaBP-9k may not be involved in the regulation of calcium homeostasis. PMID:16219669

  12. Effects of formaldehyde exposure on anxiety-like and depression-like behavior, cognition, central levels of glucocorticoid receptor and tyrosine hydroxylase in mice.

    PubMed

    Li, Yani; Song, Zhuoyi; Ding, Yujuan; Xin, Ye; Wu, Tong; Su, Tao; He, Rongqiao; Tai, Fadao; Lian, Zhenmin

    2016-02-01

    Formaldehyde exposure is toxic to the brains of mammals, but the mechanism remains unclear. We investigated the effects of inhaled formaldehyde on anxiety, depression, cognitive capacity and central levels of glucocorticoid receptor and tyrosine hydroxylase in mice. After exposure to 0, 1 or 2 ppm gaseous formaldehyde for one week, we measured anxiety-like behavior using open field and elevated plus-maze tests, depression-like behavior using a forced swimming test, learning and memory using novel object recognition tests, levels of glucocorticoid receptors in the hippocampus and tyrosine hydroxylase in the Arc, MPOA, ZI and VTA using immuhistochemistry. We found that inhalation of 1 ppm formaldehyde reduced levels of anxiety-like behavior. Inhalation of 2 ppm formaldehyde reduced body weight, but increased levels of depression-like behavior, impaired novel object recognition, and lowered the numbers of glucocorticoid receptor immonureactive neurons in the hippocampus and tyrosine hydroxylase immonureactive neurons in the ventral tegmental area and the zona incerta, medial preoptic area. Different concentrations of gaseous formaldehyde result in different effects on anxiety, depression-like behavior and cognition ability which may be associated with alterations in hippocampal glucocorticoid receptors and brain tyrosine hydroxylase levels.

  13. The opposite roles of glucocorticoid and α1-adrenergic receptors in stress triggered apoptosis of rat Leydig cells.

    PubMed

    Andric, Silvana A; Kojic, Zvezdana; Bjelic, Maja M; Mihajlovic, Aleksandar I; Baburski, Aleksandar Z; Sokanovic, Srdjan J; Janjic, Marija M; Stojkov, Natasa J; Stojilkovic, Stanko S; Kostic, Tatjana S

    2013-01-01

    The stress-induced initiation of proapoptotic signaling in Leydig cells is relatively well defined, but the duration of this signaling and the mechanism(s) involved in opposing the stress responses have not been addressed. In this study, immobilization stress (IMO) was applied for 2 h daily, and animals were euthanized immediately after the first (IMO1), second (IMO2), and 10th (IMO10) sessions. In IMO1 and IMO2 rats, serum corticosterone and adrenaline were elevated, whereas serum androgens and mRNA transcription of insulin-like factor-3 in Leydig cells were inhibited. Reduced oxygen consumption and the mitochondrial membrane potential coupled with a leak of cytochrome c from mitochondria and increased caspase-9 expression, caspase-3 activity, and number of apoptotic Leydig cells was also observed. Corticosterone and adrenaline were also elevated in IMO10 rats but were accompanied with a partial recovery of androgen secretion and normalization of insulin-like factor-3 transcription coupled with increased cytochrome c expression, abolition of proapoptotic signaling, and normalization of the apoptotic events. Blockade of intratesticular glucocorticoid receptors diminished proapoptotic effects without affecting antiapoptotic effects, whereas blockade of intratesticular α(1)-adrenergic receptors diminished the antiapoptotic effects without affecting proapoptotic effects. These results confirmed a critical role of glucocorticoids in mitochondria-dependent apoptosis and showed for the first time the relevance of stress-induced upregulation of α(1)-adrenergic receptor expression in cell apoptotic resistance to repetitive IMOs. The opposite role of two hormones in control of the apoptotic rate in Leydig cells also provides a rationale for a partial recovery of androgen production in chronically stressed animals.

  14. The effect of CD4 receptor downregulation and its downstream signaling molecules on HIV-1 latency

    SciTech Connect

    Kim, Kyung-Chang; Kim, Hyeon Guk; Roh, Tae-Young; Park, Jihwan; Jung, Kyung-Min; Lee, Joo-Shil; Choi, Sang-Yun; Kim, Sung Soon; Choi, Byeong-Sun

    2011-01-14

    Research highlights: {yields} CD4 receptors were downregulated on the surface of HIV-1 latently infected cells. {yields} CD4 downstream signaling molecules were suppressed in HIV-1 latently infected cells. {yields} HIV-1 progeny can be reactivated by induction of T-cell activation signal molecules. {yields} H3K4me3 and H3K9ac were highly enriched in CD4 downstream signaling molecules. {yields} HIV-1 latency can be maintained by the reduction of downstream signaling molecules. -- Abstract: HIV-1 can establish a latent infection in memory CD4 + T cells to evade the host immune response. CD4 molecules can act not only as the HIV-1 receptor for entry but also as the trigger in an intracellular signaling cascade for T-cell activation and proliferation via protein tyrosine kinases. Novel chronic HIV-1-infected A3.01-derived (NCHA) cells were used to examine the involvement of CD4 downstream signaling in HIV-1 latency. CD4 receptors in NCHA cells were dramatically downregulated on its surface but were slightly decreased in whole-cell lysates. The expression levels of CD4 downstream signaling molecules, including P56{sup Lck}, ZAP-70, LAT, and c-Jun, were sharply decreased in NCHA cells. The lowered histone modifications of H3K4me3 and H3K9ac correlated with the downregulation of P56{sup Lck}, ZAP-70, and LAT in NCHA cells. AP-1 binding activity was also reduced in NCHA cells. LAT and c-Jun suppressed in NCHA cells were highly induced after PMA treatment. In epigenetic analysis, other signal transduction molecules which are associated with active and/or latent HIV-1 infection showed normal states in HIV-1 latently infected cells compared to A3.01 cells. In conclusion, we demonstrated that the HIV-1 latent state is sustained by the reduction of downstream signaling molecules via the downregulation of CD4 and the attenuated activity of transcription factor as AP-1. The HIV-1 latency model via T-cell deactivation may provide some clues for the development of the new

  15. The Cochaperone SGTA (Small Glutamine-rich Tetratricopeptide Repeat-containing Protein Alpha) Demonstrates Regulatory Specificity for the Androgen, Glucocorticoid, and Progesterone Receptors*

    PubMed Central

    Paul, Atanu; Garcia, Yenni A.; Zierer, Bettina; Patwardhan, Chaitanya; Gutierrez, Omar; Hildenbrand, Zacariah; Harris, Diondra C.; Balsiger, Heather A.; Sivils, Jeffrey C.; Johnson, Jill L.; Buchner, Johannes; Chadli, Ahmed; Cox, Marc B.

    2014-01-01

    Steroid hormone receptors are ligand-dependent transcription factors that require the ordered assembly of multichaperone complexes for transcriptional activity. Although heat shock protein (Hsp) 90 and Hsp70 are key players in this process, multiple Hsp70- and Hsp90-associated cochaperones associate with receptor-chaperone complexes to regulate receptor folding and activation. Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) was recently characterized as an Hsp70 and Hsp90-associated cochaperone that specifically regulates androgen receptor activity. However, the specificity of SGTA for additional members of the steroid hormone receptor superfamily and the mechanism by which SGTA regulates receptor activity remain unclear. Here we report that SGTA associates with and specifically regulates the androgen, glucocorticoid, and progesterone receptors and has no effect on the mineralocorticoid and estrogen receptors in both yeast and mammalian cell-based reporter assays. In both systems, SGTA knockdown/deletion enhances receptor activity, whereas SGTA overexpression suppresses receptor activity. We demonstrate that SGTA binds directly to Hsp70 and Hsp90 in vitro with similar affinities yet predominately precipitates with Hsp70 from cell lysates, suggesting a role for SGTA in early, Hsp70-mediated folding. Furthermore, SGTA expression completely abrogates the regulation of receptor function by FKBP52 (52-kDa FK506-binding protein), which acts at a later stage of the chaperone cycle. Taken together, our data suggest a role for SGTA at distinct steps in the chaperone-dependent modulation of androgen, glucocorticoid, and progesterone receptor activity. PMID:24753260

  16. Identification of a selective glucocorticoid receptor ligand for the treatment of chronic inflammation in type 2 diabetes mellitus.

    PubMed

    Tan, Haifeng; Wang, Wei; Yin, Xiangang; Li, Yao; Yin, Rui

    2014-10-01

    The present study aimed to identify a new selective glucocorticoid receptor (GR) ligand for the treatment of chronic inflammation in type 2 diabetes mellitus. The IN Cell Analyzer 1000 platform was employed to screen for compounds that may promote GR nuclear translocation. A mammalian two-hybrid system and transactivation assay-were used to analyze the selected GR ligands and evaluate their activities for GR transcription and the recruitment of co-activators. A novel selective GR ligand, compound Q40, was identified that was able to promote GR nuclear translocation in a short period of time. It increased the ability of GR to recruit co-activators in a concentration-dependent manner, but had no positive effect on GR transcriptional activity. In conclusion, an increase in the expression levels of gluconeogeneic genes, induced by the transcriptional activation of GR, is the predisposing factor most commonly associated with the side-effects of glucocorticoids. The results suggest that compound Q40 is a ligand of the GR and exerts an agonistic action on the recruitment of co-activators without sugar dysmetabolism-related side-effects. Thus, compound Q40 has the potential to be used as an anti-inflammatory adjuvant therapy with minimal side-effects in patients with type 2 diabetes mellitus.

  17. Prenatal Exposure to Arsenic and Cadmium Impacts Infectious Disease-Related Genes within the Glucocorticoid Receptor Signal Transduction Pathway

    PubMed Central

    Rager, Julia E.; Yosim, Andrew; Fry, Rebecca C.

    2014-01-01

    There is increasing evidence that environmental agents mediate susceptibility to infectious disease. Studies support the impact of prenatal/early life exposure to the environmental metals inorganic arsenic (iAs) and cadmium (Cd) on increased risk for susceptibility to infection. The specific biological mechanisms that underlie such exposure-mediated effects remain understudied. This research aimed to identify key genes/signal transduction pathways that associate prenatal exposure to these toxic metals with changes in infectious disease susceptibility using a Comparative Genomic Enrichment Method (CGEM). Using CGEM an infectious disease gene (IDG) database was developed comprising 1085 genes with known roles in viral, bacterial, and parasitic disease pathways. Subsequently, datasets collected from human pregnancy cohorts exposed to iAs or Cd were examined in relationship to the IDGs, specifically focusing on data representing epigenetic modifications (5-methyl cytosine), genomic perturbations (mRNA expression), and proteomic shifts (protein expression). A set of 82 infection and exposure-related genes was identified and found to be enriched for their role in the glucocorticoid receptor signal transduction pathway. Given their common identification across numerous human cohorts and their known toxicological role in disease, the identified genes within the glucocorticoid signal transduction pathway may underlie altered infectious disease susceptibility associated with prenatal exposures to the toxic metals iAs and Cd in humans. PMID:25479081

  18. Glucocorticoid receptor (GR) immunohistochemical expression is correlated with cell cycle-related molecules in human colon cancer.

    PubMed

    Theocharis, Stamatios; Kouraklis, Gregorios; Margeli, Alexandra; Agapitos, Emmanuel; Ninos, Sotirios; Karatzas, Gabriel; Koutselinis, Antonios

    2003-09-01

    The aim of this study was to examine glucocorticoid receptor (GR) immunohistochemical expression in colon cancer histopathological specimens and to correlate it with clinicopathological parameters, tumor proliferative capacity, cell cycle-related molecule expression, and patients' survival. Primary tumoral samples from 91 colon cancer patients were immunostained for the detection of GR, cyclins D1 and E, Rb protein (pRb), p16, p21, and Ki-67, using the streptavidin-biotin-peroxidase technique. GR expression was correlated with tumor histopathological characteristics and proliferative capacity, cell cycle-related molecule expression, and patients' survival. GR positivity was prominent in 44 of 91 (48%) colon cancer cases and was positively correlated with the expression of cell cycle-related molecules pRb (P = 0.008) and p16 (P = 0.002), while lack of correlation was noted with cyclins D1 and E and p21. GR expression was not correlated with tumor location, grade of differentiation, Dukes' stage, lymph node and liver metastasis, venous invasion, tumor proliferative capacity (evident by Ki-67-labeling status) and patient survival. Our findings support evidence for GR participation in the biological mechanisms underlying the carcinogenic evolution in the colon, implying the use of glucocorticoids as an adjuvant treatment for cell cycle modulation in colon cancer cells.

  19. Dopamine D2-Receptor Antagonists Down-Regulate CYP1A1/2 and CYP1B1 in the Rat Liver.

    PubMed

    Harkitis, P; Daskalopoulos, E P; Malliou, F; Lang, M A; Marselos, M; Fotopoulos, A; Albucharali, G; Konstandi, M

    2015-01-01

    Dopaminergic systems regulate the release of several hormones including growth hormone (GH), thyroid hormones, insulin, glucocorticoids and prolactin (PRL) that play significant roles in the regulation of various Cytochrome P450 (CYP) enzymes. The present study investigated the role of dopamine D2-receptor-linked pathways in the regulation of CYP1A1, CYP1A2 and CYP1B1 that belong to a battery of genes controlled by the Aryl Hydrocarbon Receptor (AhR) and play a crucial role in the metabolism and toxicity of numerous environmental toxicants. Inhibition of dopamine D2-receptors with sulpiride (SULP) significantly repressed the constitutive and benzo[a]pyrene (B[a]P)-induced CYP1A1, CYP1A2 and CYP1B expression in the rat liver. The expression of AhR, heat shock protein 90 (HSP90) and AhR nuclear translocator (ARNT) was suppressed by SULP in B[a]P-treated livers, whereas the AhRR expression was increased by the drug suggesting that the SULP-mediated repression of the CYP1 inducibility is due to inactivation of the AhR regulatory system. At signal transduction level, the D2-mediated down-regulation of constitutive CYP1A1/2 and CYP1B1 expression appears to be mediated by activation of the insulin/PI3K/AKT pathway. PRL-linked pathways exerting a negative control on various CYPs, and inactivation of the glucocorticoid-linked pathways that positively control the AhR-regulated CYP1 genes, may also participate in the SULP-mediated repression of both, the constitutive and induced CYP1 expression. The present findings indicate that drugs acting as D2-dopamine receptor antagonists can modify several hormone systems that regulate the expression of CYP1A1, CYP1A2 and CYP1B1, and may affect the toxicity and carcinogenicity outcome of numerous toxicants and pre-carcinogenic substances. Therefore, these drugs could be considered as a part of the strategy to reduce the risk of exposure to environmental pollutants and pre-carcinogens. PMID:26466350

  20. Dopamine D2-Receptor Antagonists Down-Regulate CYP1A1/2 and CYP1B1 in the Rat Liver.

    PubMed

    Harkitis, P; Daskalopoulos, E P; Malliou, F; Lang, M A; Marselos, M; Fotopoulos, A; Albucharali, G; Konstandi, M

    2015-01-01

    Dopaminergic systems regulate the release of several hormones including growth hormone (GH), thyroid hormones, insulin, glucocorticoids and prolactin (PRL) that play significant roles in the regulation of various Cytochrome P450 (CYP) enzymes. The present study investigated the role of dopamine D2-receptor-linked pathways in the regulation of CYP1A1, CYP1A2 and CYP1B1 that belong to a battery of genes controlled by the Aryl Hydrocarbon Receptor (AhR) and play a crucial role in the metabolism and toxicity of numerous environmental toxicants. Inhibition of dopamine D2-receptors with sulpiride (SULP) significantly repressed the constitutive and benzo[a]pyrene (B[a]P)-induced CYP1A1, CYP1A2 and CYP1B expression in the rat liver. The expression of AhR, heat shock protein 90 (HSP90) and AhR nuclear translocator (ARNT) was suppressed by SULP in B[a]P-treated livers, whereas the AhRR expression was increased by the drug suggesting that the SULP-mediated repression of the CYP1 inducibility is due to inactivation of the AhR regulatory system. At signal transduction level, the D2-mediated down-regulation of constitutive CYP1A1/2 and CYP1B1 expression appears to be mediated by activation of the insulin/PI3K/AKT pathway. PRL-linked pathways exerting a negative control on various CYPs, and inactivation of the glucocorticoid-linked pathways that positively control the AhR-regulated CYP1 genes, may also participate in the SULP-mediated repression of both, the constitutive and induced CYP1 expression. The present findings indicate that drugs acting as D2-dopamine receptor antagonists can modify several hormone systems that regulate the expression of CYP1A1, CYP1A2 and CYP1B1, and may affect the toxicity and carcinogenicity outcome of numerous toxicants and pre-carcinogenic substances. Therefore, these drugs could be considered as a part of the strategy to reduce the risk of exposure to environmental pollutants and pre-carcinogens.

  1. Dopamine D2-Receptor Antagonists Down-Regulate CYP1A1/2 and CYP1B1 in the Rat Liver

    PubMed Central

    Harkitis, P.; Lang, M. A.; Marselos, M.; Fotopoulos, A.; Albucharali, G.; Konstandi, M.

    2015-01-01

    Dopaminergic systems regulate the release of several hormones including growth hormone (GH), thyroid hormones, insulin, glucocorticoids and prolactin (PRL) that play significant roles in the regulation of various Cytochrome P450 (CYP) enzymes. The present study investigated the role of dopamine D2-receptor-linked pathways in the regulation of CYP1A1, CYP1A2 and CYP1B1 that belong to a battery of genes controlled by the Aryl Hydrocarbon Receptor (AhR) and play a crucial role in the metabolism and toxicity of numerous environmental toxicants. Inhibition of dopamine D2-receptors with sulpiride (SULP) significantly repressed the constitutive and benzo[a]pyrene (B[a]P)-induced CYP1A1, CYP1A2 and CYP1B expression in the rat liver. The expression of AhR, heat shock protein 90 (HSP90) and AhR nuclear translocator (ARNT) was suppressed by SULP in B[a]P-treated livers, whereas the AhRR expression was increased by the drug suggesting that the SULP-mediated repression of the CYP1 inducibility is due to inactivation of the AhR regulatory system. At signal transduction level, the D2-mediated down-regulation of constitutive CYP1A1/2 and CYP1B1 expression appears to be mediated by activation of the insulin/PI3K/AKT pathway. PRL-linked pathways exerting a negative control on various CYPs, and inactivation of the glucocorticoid-linked pathways that positively control the AhR-regulated CYP1 genes, may also participate in the SULP-mediated repression of both, the constitutive and induced CYP1 expression. The present findings indicate that drugs acting as D2-dopamine receptor antagonists can modify several hormone systems that regulate the expression of CYP1A1, CYP1A2 and CYP1B1, and may affect the toxicity and carcinogenicity outcome of numerous toxicants and pre-carcinogenic substances. Therefore, these drugs could be considered as a part of the strategy to reduce the risk of exposure to environmental pollutants and pre-carcinogens. PMID:26466350

  2. Inhibition of dehydration-induced water intake by glucocorticoids is associated with activation of hypothalamic natriuretic peptide receptor-A in rat.

    PubMed

    Liu, Chao; Guan, Jing; Kang, Yunxiao; Xiu, Heming; Chen, Ying; Deng, Bao; Liu, Kunshen

    2010-12-20

    Atrial natriuretic peptide (ANP) provides a potent defense mechanism against volume overload in mammals. Its primary receptor, natriuretic peptide receptor-A (NPR-A), is localized mostly in the kidney, but also is found in hypothalamic areas involved in body fluid volume regulation. Acute glucocorticoid administration produces potent diuresis and natriuresis, possibly by acting in the renal natriuretic peptide system. However, chronic glucocorticoid administration attenuates renal water and sodium excretion. The precise mechanism underlying this paradoxical phenomenon is unclear. We assume that chronic glucocorticoid administration may activate natriuretic peptide system in hypothalamus, and cause volume depletion by inhibiting dehydration-induced water intake. Volume depletion, in turn, compromises renal water excretion. To test this postulation, we determined the effect of dexamethasone on dehydration-induced water intake and assessed the expression of NPR-A in the hypothalamus. The rats were deprived of water for 24 hours to have dehydrated status. Prior to free access to water, the water-deprived rats were pretreated with dexamethasone or vehicle. Urinary volume and water intake were monitored. We found that dexamethasone pretreatment not only produced potent diuresis, but dramatically inhibited the dehydration-induced water intake. Western blotting analysis showed the expression of NPR-A in the hypothalamus was dramatically upregulated by dexamethasone. Consequently, cyclic guanosine monophosphate (the second messenger for the ANP) content in the hypothalamus was remarkably increased. The inhibitory effect of dexamethasone on water intake presented in a time- and dose-dependent manner, which emerged at least after 18-hour dexamethasone pretreatment. This effect was glucocorticoid receptor (GR) mediated and was abolished by GR antagonist RU486. These results indicated a possible physiologic role for glucocorticoids in the hypothalamic control of water intake

  3. Role of Pro-637 and Gln-642 in human glucocorticoid receptors and Ser-843 and Leu-848 in mineralocorticoid receptors in their differential responses to cortisol and aldosterone.

    PubMed

    Mani, Orlando; Nashev, Lyubomir G; Livelo, Christopher; Baker, Michael E; Odermatt, Alex

    2016-05-01

    Mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) are descended from a common ancestral corticoid receptor. The basis for specificities of human MR for aldosterone and human GR for glucocorticoids, such as cortisol, bearing 17α-hydroxyl-groups, is incompletely understood. Differences in MR at S843 and L848 and GR at the corresponding P637 and Q642 have been proposed as important in their different responses to glucocorticoids with 17α-hydroxyl-groups. We investigated the impact of these residues on binding affinity (Ki) and transcriptional activation (EC50) of mutants MR-S843P, MR-L848Q and MR-S843P/L848Q and mutants GR-P637S, GR-Q642L and GR-P637S/Q642L in the presence of different corticosteroids. Aldosterone, cortisol and corticosterone had similar affinities for wild-type MR and all mutants, while dexamethasone had increased affinity for the three mutants. However, transactivation of MR-S843P and MR-S843P/L848Q by all four steroids was significantly lower than for wild-type MR. In contrast, transactivation of MR-L848Q tended to be 3-fold higher for cortisol and corticosterone and increased 7-fold for dexamethasone, indicating that MR-L848Q has an increased response to glucocorticoids, while retaining a strong response to aldosterone. Compared to wild-type GR, GR-P637S and GR-Q642L had increased affinities and significantly increased transcriptional activity with aldosterone and corticosterone, and GR-P637S had similar transcriptional activity with cortisol and dexamethasone, while GR-Q642L and GR-P637S/Q642L had a significant decrease in transcriptional activity with cortisol and dexamethasone. 3D-models of these MR and GR mutants revealed that dexamethasone and aldosterone, respectively, fit nicely into the steroid-binding pocket, consistent with the affinity of dexamethasone for MR mutants and aldosterone for GR mutants. PMID:26907965

  4. Molecular docking and 3D-QSAR studies on the glucocorticoid receptor antagonistic activity of hydroxylated polychlorinated biphenyls.

    PubMed

    Liu, S; Luo, Y; Fu, J; Zhou, J; Kyzas, G Z

    2016-01-01

    The glucocorticoid receptor (GR) antagonistic activities of hydroxylated polychlorinated biphenyls (HO-PCBs) were recently characterised. To further explore the interactions between HO-PCBs and the GR, and to elucidate structural characteristics that influence the GR antagonistic activity of HO-PCBs, molecular docking and three-dimensional quantitative structure-activity relationship (3D-QSAR) studies were performed. Comparative molecular similarity indices analysis (CoMSIA) was performed using both ligand- and receptor-based alignment schemes. Results generated from the receptor-based model were found to be more satisfactory, with q(2) of 0.632 and r(2) of 0.931 compared with those from the ligand-based model. Some internal validation strategies (e.g. cross-validation analysis, bootstrapping analysis and Y-randomisation) and an external validation method were used respectively to further assess the stability and predictive ability of the derived model. Graphical interpretation of the model provided some insights into the structural features that affected the GR antagonistic activity of HO-PCBs. Molecular docking studies revealed that some key residues were critical for ligand-receptor interactions by forming hydrogen bonds (Glu540) and hydrophobic interactions with ligands (Ile539, Val543 and Trp577). Although CoMSIA sometimes depends on the alignment of the molecules, the information provided is beneficial for predicting the GR antagonistic activities of HO-PCB homologues and is helpful for understanding the binding mechanisms of HO-PCBs to GR. PMID:26848875

  5. Glucocorticoid-dependent induction of interleukin-6 receptor expression in human hepatocytes facilitates interleukin-6 stimulation of amino acid transport.

    PubMed Central

    Fischer, C P; Bode, B P; Takahashi, K; Tanabe, K K; Souba, W W

    1996-01-01

    OBJECTIVE: The authors studied the effects of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) on glutamine and alanine transport in isolated human hepatocytes. They also evaluated the role of dexamethasone in modulating this response and its effects on the expression of the plasma membrane high-affinity IL-6 receptor. SUMMARY BACKGROUND DATA: Animal studies indicate that cytokines are important mediators of the increased hepatic amino acid uptake that occurs during cancer and sepsis, but studies in human tissues are lacking. The control of transport by cytokines and cytokine receptor expression in the liver may provide a mechanism by which hepatocytes can modulate amino acid availability during catabolic disease states. METHODS: Human hepatocytes were isolated from wedge biopsy specimens and plated in 24-well trays. Interleukin-6 and TNF-alpha, in combination with the synthetic glucocorticoid dexamethasone, were added to hepatocytes in culture, and the transport of radiolabeled glutamine and alanine was measured. Fluorescent-activated cell sorter (FACS) analysis was used to study the effects of dexamethasone on IL-6 receptor number in the well-differentiated human hepatoma HepG2. RESULTS: Both IL-6 and TNF-alpha exerted a small stimulatory effect on alanine and glutamine transport. Dexamethasone alone did not alter transport rates, but pretreatment of cells augmented the effects of both cytokines on carrier-mediated amino acid uptake. Dexamethasone pretreatment and a combination of IL-6 and TNF-alpha resulted in a greater than twofold increase in transport activity. Fluorescent-activated cell sorter analysis demonstrated that dexamethasone induced a threefold increase in the expression of high-affinity IL-6 receptors. CONCLUSIONS: Interleukin-6 and TNF-alpha work coordinately with glucocorticoids to stimulate amino acid uptake in human hepatocytes. Dexamethasone exerts a permissive effect on cytokine-mediated increases in transport by increasing IL

  6. The helix 1-3 loop in the glucocorticoid receptor LBD is a regulatory element for FKBP cochaperones.

    PubMed

    Cluning, Carmel; Ward, Bryan K; Rea, Sarah L; Arulpragasam, Ajanthy; Fuller, Peter J; Ratajczak, Thomas

    2013-07-01

    The heat-shock protein 90 (Hsp90) cochaperone FK506-binding protein 52 (FKBP52) upregulates, whereas FKBP51 inhibits, hormone binding and nuclear targeting of the glucocorticoid receptor (GR). Decreased cortisol sensitivity in the guinea pig is attributed to changes within the helix 1 to helix 3 (H1-H3) loop of the guinea pig GR (gpGR) ligand-binding domain. It has been proposed that this loop serves as a contact point for FKBP52 and/or FKBP51 with receptor. We examined the role of the H1-H3 loop in GR activation by FKBP52 using a Saccharomyces cerevisiae model. The activity of rat GR (rGR) containing the gpGR H1-H3 loop substitutions was still potentiated by FKBP52, confirming the loop is not involved in primary FKBP52 interactions. Additional assays also excluded a role for other intervening loops between ligand-binding domain helices in direct interactions with FKBP52 associated with enhanced receptor activity. Complementary studies in FKBP51-deficient mouse embryo fibroblasts and HEK293 cells demonstrated that substitution of the gpGR H1-H3 loop residues into rGR dramatically increased receptor repression by FKBP51 without enhancing receptor-FKBP51 interaction and did not alter recruitment of endogenous Hsp90 and the p23 cochaperone to receptor complexes. FKBP51 suppression of the mutated rGR did not require FKBP51 peptidylprolyl cis-trans isomerase activity and was not disrupted by mutation of the FK1 proline-rich loop thought to mediate reciprocal FKBP influences on receptor activity. We conclude that the gpGR-specific mutations within the H1-H3 loop confer global changes within the GR-Hsp90 complex that favor FKBP51 repression over FKBP52 potentiation, thus identifying the loop as an important target for GR regulation by the FKBP cochaperones. PMID:23686112

  7. CD200 restrains macrophage attack on oligodendrocyte precursors via toll-like receptor 4 downregulation.

    PubMed

    Hayakawa, Kazuhide; Pham, Loc-Duyen D; Seo, Ji Hae; Miyamoto, Nobukazu; Maki, Takakuni; Terasaki, Yasukazu; Sakadžić, Sava; Boas, David; van Leyen, Klaus; Waeber, Christian; Kim, Kyu-Won; Arai, Ken; Lo, Eng H

    2016-04-01

    There are numerous barriers to white matter repair after central nervous system injury and the underlying mechanisms remain to be fully understood. In this study, we propose the hypothesis that inflammatory macrophages in damaged white matter attack oligodendrocyte precursor cells via toll-like receptor 4 signaling thus interfering with this endogenous progenitor recovery mechanism. Primary cell culture experiments demonstrate that peritoneal macrophages can attack and digest oligodendrocyte precursor cells via toll-like receptor 4 signaling, and this phagocytosis of oligodendrocyte precursor cells can be inhibited by using CD200-Fc to downregulate toll-like receptor 4. In an in vivo model of white matter ischemia induced by endothelin-1, treatment with CD200-Fc suppressed toll-like receptor 4 expression in peripherally circulating macrophages, thus restraining macrophage phagocytosis of oligodendrocyte precursor cells and leading to improved myelination. Taken together, these findings suggest that deleterious macrophage effects may occur after white matter ischemia, whereby macrophages attack oligodendrocyte precursor cells and interfere with endogenous recovery responses. Targeting this pathway with CD200 may offer a novel therapeutic approach to amplify endogenous oligodendrocyte precursor cell-mediated repair of white matter damage in mammalian brain.

  8. Indole Glucocorticoid Receptor Antagonists Active in a Model of Dyslipidemia Act via a Unique Association with an Agonist Binding Site.

    PubMed

    Luz, John G; Carson, Matthew W; Condon, Bradley; Clawson, David; Pustilnik, Anna; Kohlman, Daniel T; Barr, Robert J; Bean, James S; Dill, M Joelle; Sindelar, Dana K; Maletic, Milan; Coghlan, Michael J

    2015-08-27

    To further elucidate the structural activity correlation of glucocorticoid receptor (GR) antagonism, the crystal structure of the GR ligand-binding domain (GR LBD) complex with a nonsteroidal antagonist, compound 8, was determined. This novel indole sulfonamide shows in vitro activity comparable to known GR antagonists such as mifepristone, and notably, this molecule lowers LDL (-74%) and raises HDL (+73%) in a hamster model of dyslipidemia. This is the first reported crystal structure of the GR LBD bound to a nonsteroidal antagonist, and this article provides additional elements for the design and pharmacology of clinically relevant nonsteroidal GR antagonists that may have greater selectivity and fewer side effects than their steroidal counterparts. PMID:26218343

  9. Novel synthesis of the hexahydroimidazo[1,5b]isoquinoline scaffold: application to the synthesis of glucocorticoid receptor modulators.

    PubMed

    Xiao, Hai-Yun; Wu, Dauh-Rurng; Malley, Mary F; Gougoutas, Jack Z; Habte, Sium F; Cunningham, Mark D; Somerville, John E; Dodd, John H; Barrish, Joel C; Nadler, Steven G; Dhar, T G Murali

    2010-02-11

    The first stereoselective synthesis of the hexahydroimidazo[1,5b]isoquinoline (HHII) scaffold as a surrogate for the steroidal A-B ring system is described. The structure-activity relationships of the analogs derived from this scaffold show that the basic imidazole moiety is tolerated by the glucocorticoid receptor (GR) in terms of binding affinity, although the partial agonist activity in the transrepressive assays depends on the substitution pattern on the B-ring. More importantly, most compounds in the HHII series bearing a tertiary alcohol moiety on the B-ring are either inactive or significantly less active in inducing GR-mediated transactivation, thus displaying a "dissociated" pharmacology in vitro. PMID:20047280

  10. Lipopolysaccharide induces a downregulation of adiponectin receptors in-vitro and in-vivo

    PubMed Central

    Hall, Alison; Leuwer, Martin; Trayhurn, Paul

    2015-01-01

    Background. Adipose tissue contributes to the inflammatory response through production of cytokines, recruitment of macrophages and modulation of the adiponectin system. Previous studies have identified a down-regulation of adiponectin in pathologies characterised by acute (sepsis and endotoxaemia) and chronic inflammation (obesity and type-II diabetes mellitus). In this study, we investigated the hypothesis that LPS would reduce adiponectin receptor expression in a murine model of endotoxaemia and in adipoocyte and myocyte cell cultures. Methods. 25 mg/kg LPS was injected intra-peritoneally into C57BL/6J mice, equivalent volumes of normal saline were used in control animals. Mice were killed at 4 or 24 h post injection and tissues harvested. Murine adipocytes (3T3-L1) and myocytes (C2C12) were grown in standard culture, treated with LPS (0.1 µg/ml–10 µg/ml) and harvested at 4 and 24 h. RNA was extracted and qPCR was conducted according to standard protocols and relative expression was calculated. Results. After LPS treatment there was a significant reduction after 4 h in gene expression of adipo R1 in muscle and peri-renal fat and of adipo R2 in liver, peri-renal fat and abdominal wall subcutaneous fat. After 24 h, significant reductions were limited to muscle. Cell culture extracts showed varied changes with reduction in adiponectin and adipo R2 gene expression only in adipocytes. Conclusions. LPS reduced adiponectin receptor gene expression in several tissues including adipocytes. This reflects a down-regulation of this anti-inflammatory and insulin-sensitising pathway in response to LPS. The trend towards base line after 24 h in tissue depots may reflect counter-regulatory mechanisms. Adiponectin receptor regulation differs in the tissues investigated. PMID:26618091

  11. Ritonavir binds to and downregulates estrogen receptors: Molecular mechanism of promoting early atherosclerosis

    SciTech Connect

    Xiang, Jin; Wang, Ying; Su, Ke; Liu, Min; Hu, Peng-Chao; Ma, Tian; Li, Jia-Xi; Wei, Lei; Zheng, Zhongliang; Yang, Fang

    2014-10-01

    Estrogenic actions are closely related to cardiovascular disease. Ritonavir (RTV), a human immunodeficiency virus (HIV) protease inhibitor, induces atherosclerosis in an estrogen-related manner. However, how RTV induce pathological phenotypes through estrogen pathway remains unclear. In this study, we found that RTV increases thickness of coronary artery walls of Sprague Dawley rats and plasma free fatty acids (FFA) levels. In addition, RTV could induce foam cell formation, downregulate both estrogen receptor α (ERα) and ERβ expression, upregulate G protein-coupled estrogen receptor (GPER) expression, and all of them could be partially blocked by 17β-estradiol (E2), suggesting RTV acts as an antagonist for E2. Computational modeling shows a similar interaction with ERα between RTV and 2-aryl indoles, which are highly subtype-selective ligands for ERα. We also found that RTV directly bound to ERα and selectively inhibited the nuclear localization of ERα, and residue Leu536 in the hydrophobic core of ligand binding domain (LBD) was essential for the interaction with RTV. In addition, RTV did not change the secondary structure of ERα-LBD like E2, which explained how ERα lost the capacity of nuclear translocation under the treatment of RTV. All of the evidences suggest that ritonavir acts as an antagonist for 17β-estradiol in regulating α subtype estrogen receptor function and early events of atherosclerosis. - Graphical abstract: RTV directly binds to ERα and Leu536 in the hydrophobic core of ligand binding domain is essential for the interaction. - Highlights: • RTV increases the thickness of rat coronary artery wall and foam cell formation. • RTV downregulates the expression of ERα and ERβ. • RTV inhibits ERα promoter activity. • RTV directly binds to ERα and the key amino acid is Leu536. • RTV inhibits the nuclear translocation of ERα and GPER.

  12. Dioxin mediates downregulation of the reduced folate carrier transport activity via the arylhydrocarbon receptor signalling pathway

    SciTech Connect

    Halwachs, Sandra; Lakoma, Cathleen; Gebhardt, Rolf; Schaefer, Ingo; Seibel, Peter; Honscha, Walther

    2010-07-15

    Dioxins such as 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) are common environmental contaminants known to regulate several genes via activation of the transcription factor aryl hydrocarbon receptor (AhR) associated with the development of numerous adverse biological effects. However, comparatively little is known about the molecular mechanisms by which dioxins display their toxic effects in vertebrates. The 5' untranslated region of the hepatocellular Reduced folate carrier (Rfc1; Slc19a1) exhibits AhR binding sites termed dioxin responsive elements (DRE) that have as yet only been found in the promoter region of prototypical TCDD target genes. Rfc1 mediated transport of reduced folates and antifolate drugs such as methotrexate (MTX) plays an essential role in physiological folate homeostasis and MTX cancer chemotherapy. In order to determine whether this carrier represents a target gene of dioxins we have investigated the influence of TCDD on functional Rfc1 activity in rat liver. Pre-treatment of rats with TCDD significantly diminished hepatocellular Rfc1 uptake activity in a time- and dose-dependent manner. In further mechanistic studies we demonstrated that this reduction was due to TCDD-dependent activation of the AhR signalling pathway. We additionally showed that binding of the activated receptor to DRE motifs in the Rfc1 promoter resulted in downregulation of Rfc1 gene expression and reduced carrier protein levels. As downregulation of pivotal Rfc1 activity results in functional folate deficiency associated with an elevated risk of cardiovascular diseases or carcinogenesis, our results indicate that deregulation of this essential transport pathway represents a novel regulatory mechanism how dioxins display their toxic effects through the Ah receptor.

  13. Glucocorticoid receptor knockdown decreases the antioxidant protection of B16 melanoma cells: an endocrine system-related mechanism that compromises metastatic cell resistance to vascular endothelium-induced tumor cytotoxicity.

    PubMed

    Obrador, Elena; Valles, Soraya L; Benlloch, María; Sirerol, J Antoni; Pellicer, José A; Alcácer, Javier; Coronado, Javier Alcácer-F; Estrela, José M

    2014-01-01

    We previously reported an interorgan system in which stress-related hormones (corticosterone and noradrenaline), interleukin-6, and glutathione (GSH) coordinately regulate metastatic growth of highly aggressive B16-F10 melanoma cells. Corticosterone, at levels measured in tumor-bearing mice, also induces apoptotic cell death in metastatic cells with low GSH content. In the present study we explored the potential role of glucocorticoids in the regulation of metastatic cell death/survival during the early stages of organ invasion. Glucocorticoid receptor (GCR) knockdown decreased the expression and activity of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting step in GSH synthesis, in metastatic cells in vivo independent of the tumor location (liver, lung, or subcutaneous). The decrease in γ-GCS activity was associated with lower intracellular GSH levels. Nrf2- and p53-dependent down-regulation of γ-GCS was associated with a decrease in the activities of superoxide dismutase 1 and 2, catalase, glutathione peroxidase, and glutathione reductase, but not of the O2--generating NADPH oxidase. The GCR knockdown-induced decrease in antioxidant protection caused a drastic decrease in the survival of metastatic cells during their interaction with endothelial cells, both in vitro and in vivo; only 10% of cancer cells attached to the endothelium survived compared to 90% survival observed in the controls. This very low rate of metastatic cell survival was partially increased (up to 52%) in vivo by inoculating B16-F10 cells preloaded with GSH ester, which enters the cell and delivers free GSH. Taken together, our results indicate that glucocorticoid signaling influences the survival of metastatic cells during their interaction with the vascular endothelium.

  14. The mouse glucocorticoid receptor: mapping of functional domains by cloning, sequencing and expression of wild-type and mutant receptor proteins.

    PubMed Central

    Danielsen, M; Northrop, J P; Ringold, G M

    1986-01-01

    We have isolated mouse glucocorticoid receptor (GR) cDNAs which, when expressed in transfected mammalian cells, produce a fully functional GR protein. Sequence analysis reveals an open reading frame of 2349 bp which could encode a protein of approximately 86,000 daltons. We have also isolated two receptor cDNAs from mouse S49 nuclear transfer-deficient (nt-) cells which encode mutant forms of the receptor protein. One cDNA encodes a protein that is unable to bind hormone and represents the endogenous hormone binding deficient receptor recently discovered in S49 cells. The lesion in this receptor is due to a single amino acid substitution (Glu-546 to Gly). The second cDNA from nt- cells produces a receptor protein that is able to bind hormone but has reduced nuclear binding. This cDNA, therefore, encodes for the S49 nt- receptor which has been shown to have reduced affinity for DNA. The lesion maps to a single amino acid substitution (Arg-484 to His) located in a highly Cys, Lys, Arg-rich region of the protein previously implicated in DNA binding. Our studies provide unambiguous identification of receptor domains and specific amino acids critical for the hormone and DNA binding properties of this transcriptional regulatory protein. Contained within the first 106 amino acids of the mouse GR is a stretch of nine glutamines with two prolines which are related to the family of transcribed repetitive elements, opa, found in Drosophila melanogaster. A truncated receptor lacking these 106 amino acids is functionally indistinguishable from the wild-type receptor. Images Fig. 2. Fig. 5. Fig. 6. Fig. 7. PMID:3780669

  15. Randomized placebo-controlled study comparing a leukotriene receptor antagonist and a nasal glucocorticoid in seasonal allergic rhinitis.

    PubMed

    Pullerits, T; Praks, L; Skoogh, B E; Ani, R; Lötvall, J

    1999-06-01

    Allergic rhinitis is an inflammatory disorder associated with local leukotriene release during periods of symptoms. Therefore, it has been suggested that antileukotrienes may be beneficial in the treatment of this disease. Leukotriene receptor antagonists have recently become available for asthma treatment, but little is known of their effects on allergic rhinitis. We have evaluated the effects of the leukotriene receptor antagonist zafirlukast versus placebo in patients with allergic rhinitis during the grass pollen season, using the nasal glucocorticoid beclomethasone dipropionate (BDP) as a positive treatment control. Thirty-three patients with seasonal allergic rhinitis were in a double-blind, double-dummy fashion randomized to treatments with oral zafirlukast (20 mg twice a day), intranasal beclomethasone dipropionate (200 microg twice a day), or placebo. The treatment was initiated 3 wk prior to the expected beginning of the grass pollen season. Patients completed a daily symptom-score list for sneezing, rhinorrhea, nasal itch, and nasal blockage during the 50-d treatment period. Nasal biopsies for quantification of local tissue eosinophilia (immunohistochemistry; EG2) were taken 1 mo before initiation of treatment and immediately after the peak of grass pollen season. Patients receiving treatment with zafirlukast had degrees of nasal symptoms similar to those in the placebo group, whereas the BDP group had significantly less symptoms compared with both treatments (p = 0.01 and p = 0.005, respectively). The numbers of activated eosinophils in the nasal tissue increased significantly during the pollen season in both the zafirlukast and the placebo groups, but not in the BDP group. These results obtained with a limited number of patients do not support any clinical efficacy of regular treatment with an oral antileukotriene in seasonal allergic rhinitis but rather favor the use of a nasal glucocorticoid. PMID:10351924

  16. Expression and differential effects of the activation of glucocorticoid receptors in mouse gonadotropin-releasing hormone neurons.

    PubMed

    Dondi, Donatella; Piccolella, Margherita; Messi, Elio; Demissie, Marek; Cariboni, Anna; Selleri, Silvia; Piva, Flavio; Samara, Athina; Consalez, G Giacomo; Maggi, Roberto

    2005-01-01

    Prenatal exposure of rodents to glucocorticoids (Gc) affects the sexual development of the offspring, possibly interfering with the differentiation of the hypothalamic-pituitary-gonadal axis. Glucocorticoid receptors (GR) are present on gonadotropin-releasing hormone (GnRH) neurons in the rat hypothalamus, suggesting a direct effect of Gc in the control of the synthesis and/or release of the hormone. In this study, we demonstrate the colocalization of immunoreactive GR with GnRH in a subpopulation of mouse hypothalamic GnRH neurons, confirming the possible involvement of Gc in mouse GnRH neuronal physiology. Receptor-binding assay, RT-PCR, immunocytochemistry, and immunoblotting experiments carried out in GN11 immortalized GnRH neurons show the presence of GR even in the more immature mouse GnRH neurons and confirm the expression of GR in GT1-7 mature GnRH cells. In GN11 cells, the activation of GR with dexamethasone produces nuclear translocation, but does not lead to the inhibition of GnRH gene expression already reported in GT1-7 cells. Long-term exposure of GN11 cells to dexamethasone induces an epithelial-like phenotype with a reorganization of F-actin in stress fibers. Finally, we found that Gc treatment significantly decreases the migratory activity in vitro and the levels of phosphorylated focal adhesion kinase of GN11 immature neurons. In conclusion, these data indicate that GR are expressed in mouse hypothalamic GnRH neurons in vivo as well as in the immature GN11 GnRH neurons in vitro. Moreover, the effects of the GR activation in GN11 and in GT1-7 cells may be related to the neuronal maturational stage of the two cell lines, suggesting a differential role of Gc in neuronal development.

  17. Glucocorticoids antagonize cAMP-induced Star transcription in Leydig cells through the orphan nuclear receptor NR4A1.

    PubMed

    Martin, Luc J; Tremblay, Jacques J

    2008-09-01

    It is well established that stress, either physical or psychosocial, causes a decrease in testosterone production by Leydig cells. Glucocorticoids (Gc) are the main mediators of stress response and they convey their repressive effect on Leydig cells through the glucocorticoid receptor (GR). So far, various mechanisms have been proposed to explain the mechanism of action of Gc on Leydig cell steroidogenesis including repression of genes involved in testosterone biosynthesis. Several steroidogenic genes, including steroidogenic acute regulatory (STAR) protein, have been shown to be repressed by Gc in a GR-dependent manner but the underlying mechanisms remain to be fully elucidated. Here, we found that dexamethasone (Dex), a potent synthetic Gc, partly antagonizes the cAMP-dependent stimulation of the mouse Star promoter in MA-10 Leydig cells as revealed by transient transfection assays. This repression requires an element located at -95 bp previously implicated in the activation of the Star promoter by the nuclear receptors, NR4A1 and NR5A1. Dex was found to inhibit NR4A1-dependent transactivation of the Star promoter in Leydig cells by decreasing NR4A1, but not NR5A1, recruitment to the proximal Star promoter as determined by chromatin immunoprecipitation assay. Western blots revealed that Dex did not affect NR4A1 or NR5A1 expression in response to cAMP. These data suggest that NR4A1 would be associated with the GR in a transcriptionally inactive complex as previously demonstrated in pituitary corticotrope cells. Thus, our data provide new molecular insights into the stress-mediated suppression of testosterone production in testicular Leydig cells.

  18. Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma

    PubMed Central

    Friesen, Claudia; Hormann, Inis; Roscher, Mareike; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf; Debatin, Klaus-Michael; Miltner, Erich

    2014-01-01

    Glioblastoma are the most frequent and malignant human brain tumors, having a very poor prognosis. The enhanced radio- and chemoresistance of glioblastoma and the glioblastoma stem cells might be the main reason why conventional therapies fail. The second messenger cyclic AMP (cAMP) controls cell proliferation, differentiation, and apoptosis. Downregulation of cAMP sensitizes tumor cells for anti-cancer treatment. Opioid receptor agonists triggering opioid receptors can activate inhibitory Gi proteins, which, in turn, block adenylyl cyclase activity reducing cAMP. In this study, we show that downregulation of cAMP by opioid receptor activation improves the effectiveness of anti-cancer drugs in treatment of glioblastoma. The µ-opioid receptor agonist D,L-methadone sensitizes glioblastoma as well as the untreatable glioblastoma stem cells for doxorubicin-induced apoptosis and activation of apoptosis pathways by reversing deficient caspase activation and deficient downregulation of XIAP and Bcl-xL, playing critical roles in glioblastomas’ resistance. Blocking opioid receptors using the opioid receptor antagonist naloxone or increasing intracellular cAMP by 3-isobutyl-1-methylxanthine (IBMX) strongly reduced opioid receptor agonist-induced sensitization for doxorubicin. In addition, the opioid receptor agonist D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux, whereas doxorubicin increased opioid receptor expression in glioblastomas. Furthermore, opioid receptor activation using D,L-methadone inhibited tumor growth significantly in vivo. Our findings suggest that opioid receptor activation triggering downregulation of cAMP is a promising strategy to inhibit tumor growth and to improve the effectiveness of anti-cancer drugs in treatment of glioblastoma and in killing glioblastoma stem cells. PMID:24626197

  19. Gestational dietary betaine supplementation suppresses hepatic expression of lipogenic genes in neonatal piglets through epigenetic and glucocorticoid receptor-dependent mechanisms.

    PubMed

    Cai, Demin; Wang, Junjian; Jia, Yimin; Liu, Haoyu; Yuan, Mengjie; Dong, Haibo; Zhao, Ruqian

    2016-01-01

    Methyl donors play critical roles in nutritional programming through epigenetic regulation of gene expression. Here we fed gestational sows with control or betaine-supplemented diets (3g/kg) throughout the pregnancy to explore the effects of maternal methyl-donor nutrient on neonatal expression of hepatic lipogenic genes. Betaine-exposed piglets demonstrated significantly lower liver triglyceride content associated with down-regulated hepatic expression of lipogenic genes acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD) and sterol regulatory element-binding protein-1c. Moreover, s-adenosyl methionine to s-adenosyl homocysteine ratio was elevated in the liver of betaine-exposed piglets, which was accompanied by DNA hypermethylation on FAS and SCD gene promoters and more enriched repression histone mark H3K27me3 on SCD gene promoter. Furthermore, glucocorticoid receptor (GR) binding to SCD gene promoter was diminished along with reduced serum cortisol and liver GR protein content in betaine-exposed piglets. GR-mediated SCD gene regulation was confirmed in HepG2 cells in vitro. Dexamethasone (Dex) drastically increased the luciferase activity of porcine SCD promoter, while the deletion of GR response element on SCD promoter significantly attenuated Dex-mediated SCD transactivation. In addition, miR-let-7e, miR-1285 and miR-124a, which respectively target porcine SCD, ACC and GR, were significantly up-regulated in the liver of betaine-exposed piglets, being in accordance with decreased protein content of these three genes. Taken together, our results suggest that maternal dietary betaine supplementation during gestation attenuates hepatic lipogenesis in neonatal piglets via epigenetic and GR-mediated mechanisms.

  20. The acceleration of amygdala kindling epileptogenesis by chronic low-dose corticosterone involves both mineralocorticoid and glucocorticoid receptors.

    PubMed

    Kumar, Gaurav; Couper, Abbie; O'Brien, Terence J; Salzberg, Michael R; Jones, Nigel C; Rees, Sandra M; Morris, Margaret J

    2007-08-01

    We have previously demonstrated that low-dose corticosterone (CS) administration, used as a model of the effect of chronic stress, accelerates epileptogenesis in the electrical amygdala kindling rat model of temporal lobe epilepsy (TLE). This current study examined the relative contributions to this effect of mineralocorticoid (MR) and glucocorticoid (GR) subtypes of glucocorticoid receptors. Female non-epileptic wistar rats 10-13 weeks of age were implanted with a bipolar electrode into the left amygdala. Five treatment groups were subjected to rapid amygdala kindling: water-control (n=9), CS treated (6 mg/100 ml added to drinking water; n=9), CS+spironolactone (MR antagonist, 50 mg/kg sc; n=9), CS+mifepristone (GR antagonist, 25 mg/kg sc; n=9), and CS+both antagonists (n=7). Rats were injected with vehicle or the relevant antagonist twice daily for the entire kindling period. Experimental groups differed significantly in the number of stimulations required to reach the 'fully kindled state' (Racine, 1972) ANOVA, F(4,38)=2.73, p=0.04). Amygdala kindling was accelerated in the CS-treated group compared with water controls (mean stimulations for full kindling: 45.2 vs. 86.5, p<0.01). This acceleration was inhibited by both the MR and GR antagonist treatments (mean stimulations: 69.6 and 70.4, p=0.04 and 0.04 vs. CS group, respectively), with the kindling rates in these groups not significantly different from water-treated subjects (p=0.26 and 0.29, respectively). The kindling rates in the MR and GR antagonist treatment groups did not significantly differ from each other (p=0.93), nor from the combined treatment group (mean stimulations: 62.8, p=0.59 and 0.54, respectively). This study demonstrates that activation of both high-affinity (MR) and low-affinity (GR) glucocorticoid receptors are involved in mediating CS-induced acceleration of amygdala kindling epileptogenesis.

  1. Receptor usage and differential downregulation of CD46 by measles virus wild-type and vaccine strains.

    PubMed

    Schneider-Schaulies, J; Schnorr, J J; Brinckmann, U; Dunster, L M; Baczko, K; Liebert, U G; Schneider-Schaulies, S; ter Meulen, V

    1995-04-25

    Recently, two cell surface molecules, CD46 and moesin, have been found to be functionally associated with measles virus (MV) infectivity of cells. We investigated the receptor usage of MV wild-type, subacute sclerosing panencephalitis, and vaccine strains and their effect on the down-regulation of CD46 after infection. We found that the infection of human cell lines with all 19 MV strains tested was inhibitable with antibodies against CD46. In contrast, not all strains of MV led to the downregulation of CD46 following infection. The group of CD46 non-downregulating strains comprised four lymphotropic wild-type isolates designated AB, DF, DL, and WTF. Since the downregulation of CD46 is caused by interaction with newly synthesized MV hemagglutinin (MV-H), we tested the capability of recombinant MV-H proteins to downregulate CD46. Recombinant MV-H proteins of MV strains Edmonston, Halle, and CM led to the down-regulation of CD46, whereas those of DL and WTF did not. This observed differential downregulation by different MV strains has profound consequences, since lack of CD46 on the cell surface leads to susceptibility of cells to complement lysis. These results suggest that lymphotropic wild-type strains of MV which do not downregulate CD46 may have an advantage for replication in vivo. The relatively weak immune response against attenuated vaccine strains of MV compared with wild-type strains might be related to this phenomenon. PMID:7732009

  2. Response to metal stress of Nicotiana langsdorffii plants wild-type and transgenic for the rat glucocorticoid receptor gene.

    PubMed

    Fuoco, Roger; Bogani, Patrizia; Capodaglio, Gabriele; Del Bubba, Massimo; Abollino, Ornella; Giannarelli, Stefania; Spiriti, Maria Michela; Muscatello, Beatrice; Doumett, Saer; Turetta, Clara; Zangrando, Roberta; Zelano, Vincenzo; Buiatti, Marcello

    2013-05-01

    Recently our findings have shown that the integration of the gene coding for the rat gluco-corticoid receptor (GR receptor) in Nicotiana langsdorffii plants induced morphophysiological effects in transgenic plants through the modification of their hormonal pattern. Phytohormones play a key role in plant responses to many different biotic and abiotic stresses since a modified hormonal profile up-regulates the activation of secondary metabolites involved in the response to stress. In this work transgenic GR plants and isogenic wild type genotypes were exposed to metal stress by treating them with 30ppm cadmium(II) or 50ppm chromium(VI). Hormonal patterns along with changes in key response related metabolites were then monitored and compared. Heavy metal up-take was found to be lower in the GR plants. The transgenic plants exhibited higher values of S-abscisic acid (S-ABA) and 3-indole acetic acid (IAA), salicylic acid and total polyphenols, chlorogenic acid and antiradical activity, compared to the untransformed wild type plants. Both Cd and Cr treatments led to an increase in hormone concentrations and secondary metabolites only in wild type plants. Analysis of the results suggests that the stress responses due to changes in the plant's hormonal system may derive from the interaction between the GR receptor and phytosteroids, which are known to play a key role in plant physiology and development.

  3. Glucocorticoid Receptor Transcriptional Activation via the BRG1-Dependent Recruitment of TOP2β and Ku70/86.

    PubMed

    Trotter, Kevin W; King, Heather A; Archer, Trevor K

    2015-08-01

    BRG1, the central ATPase of the human SWI/SNF complex, is critical for biological functions, including nuclear receptor (NR)-regulated transcription. Analysis of BRG1 mutants demonstrated that functional motifs outside the ATPase domain are important for transcriptional activity. In the course of experiments examining protein interactions mediated through these domains, Ku70 (XRCC6) was found to associate with a BRG1 fragment encompassing the conserved helicase-SANT-associated (HSA) and BRK domains of BRG1. Subsequent transcriptional activation assays and chromatin immunoprecipitation studies showed that Ku70/86 and components of the topoisomerase IIβ (TOP2β)/poly(ADP ribose) polymerase 1 (PARP1) complex are necessary for NR-mediated SWI/SNF-dependent transcriptional activation from endogenous promoters. In addition to establishing Ku-BRG1 binding and TOP2β/PARP1 recruitment by nuclear receptor transactivation, we demonstrate that the transient appearance of glucocorticoid receptor (GR)/BRG1-dependent, TOP2β-mediated double-strand DNA breaks is required for efficient GR-stimulated transcription. Taken together, these results suggest that a direct interaction between Ku70/86 and BRG1 brings together SWI/SNF remodeling capabilities and TOP2β activity to enhance the transcriptional response to hormone stimulation.

  4. Antileukemia Effect of Ciclopirox Olamine Is Mediated by Downregulation of Intracellular Ferritin and Inhibition β-Catenin-c-Myc Signaling Pathway in Glucocorticoid Resistant T-ALL Cell Lines

    PubMed Central

    Zhang, Ge; Gu, Ling; Zhang, Yanle; Gao, Ju; Wei, Yuquan

    2016-01-01

    Ciclopirox olamine (CPX) is an antifungal drug that has been reported to have antitumor effects. In this study we investigated the antileukemia effects and the possible mechanisms of CPX on glucocorticoid (GC)-resistant T-cell acute lymphoblastic leukemia (T-ALL) cell lines. The results indicated that CPX inhibited the growth of GC-resistant T-ALL cells in a time- and dose-dependent manner, and this effect was closely correlated with the downregulation of intracellular ferritin. CPX induced cell cycle arrest at G1 phase by upregulation of cyclin-dependent kinase (CDK) inhibitor of p21 and downregulation of the expressions of cyclin D, retinoblastoma protein (Rb), and phosphorylated Rb (pRb). CPX also enhanced apoptotic cell death by downregulation of anti-apoptotic proteins such as Bcl-2, Bcl-xL, and Mcl-1. More importantly, CPX demonstrated a strong synergistic antileukemia effect with GC and this effect was mediated, at least in part, by inhibition of the β-catenin-c-Myc signaling pathway. These findings suggest that CPX could be a promising antileukemia drug, and modulation of the intracellular ferritin expression might be an effective method in the treatment of ALL. Therefore, integrating CPX into the current GC-containing ALL protocols could lead to the improvement of the outcome of ALL, especially GC-resistant ALL. PMID:27551974

  5. Antileukemia Effect of Ciclopirox Olamine Is Mediated by Downregulation of Intracellular Ferritin and Inhibition β-Catenin-c-Myc Signaling Pathway in Glucocorticoid Resistant T-ALL Cell Lines.

    PubMed

    Wu, Jianrong; Liu, Huajun; Zhang, Ge; Gu, Ling; Zhang, Yanle; Gao, Ju; Wei, Yuquan; Ma, Zhigui

    2016-01-01

    Ciclopirox olamine (CPX) is an antifungal drug that has been reported to have antitumor effects. In this study we investigated the antileukemia effects and the possible mechanisms of CPX on glucocorticoid (GC)-resistant T-cell acute lymphoblastic leukemia (T-ALL) cell lines. The results indicated that CPX inhibited the growth of GC-resistant T-ALL cells in a time- and dose-dependent manner, and this effect was closely correlated with the downregulation of intracellular ferritin. CPX induced cell cycle arrest at G1 phase by upregulation of cyclin-dependent kinase (CDK) inhibitor of p21 and downregulation of the expressions of cyclin D, retinoblastoma protein (Rb), and phosphorylated Rb (pRb). CPX also enhanced apoptotic cell death by downregulation of anti-apoptotic proteins such as Bcl-2, Bcl-xL, and Mcl-1. More importantly, CPX demonstrated a strong synergistic antileukemia effect with GC and this effect was mediated, at least in part, by inhibition of the β-catenin-c-Myc signaling pathway. These findings suggest that CPX could be a promising antileukemia drug, and modulation of the intracellular ferritin expression might be an effective method in the treatment of ALL. Therefore, integrating CPX into the current GC-containing ALL protocols could lead to the improvement of the outcome of ALL, especially GC-resistant ALL. PMID:27551974

  6. Dysregulation of Ack1 inhibits down-regulation of the EGF receptor

    SciTech Connect

    Grovdal, Lene Melsaether; Johannessen, Lene E.; Rodland, Marianne Skeie; Madshus, Inger Helene; Stang, Espen

    2008-04-01

    The protein tyrosine kinase Ack1 has been linked to cancer when over-expressed. Ack1 has also been suggested to function in clathrin-mediated endocytosis and in down-regulation of the epidermal growth factor (EGF) receptor (EGFR). We have studied the intracellular localization of over-expressed Ack1 and found that Ack1 co-localizes with the EGFR upon EGF-induced endocytosis in cells with moderate over-expression of Ack. This co-localization is mainly observed in early endosomes. Furthermore, we found that over-expression of Ack1 retained the EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. Down-regulation of Ack1 in HeLa cells resulted in reduced rate of {sup 125}I-EGF internalization, whereas internalization of {sup 125}I-transferrin was not affected. In cells where Ack1 had been knocked down by siRNA, recycling of internalized {sup 125}I-EGF was increased, while degradation of {sup 125}I-EGF was inhibited. Together, these data suggest that Ack1 is involved in an early step of EGFR desensitization.

  7. Potent anti-prostate cancer agents derived from a novel androgen receptor down-regulating agent.

    PubMed

    Purushottamachar, Puranik; Khandelwal, Aakanksha; Vasaitis, Tadas S; Bruno, Robert D; Gediya, Lalji K; Njar, Vincent C O

    2008-04-01

    The search for novel androgen receptor (AR) down-regulating agents by catalyst HipHop pharmacophore modeling led to the discovery of some lead molecules. Unexpectedly, the effect of these leads on human prostate cancer LNCaP cell viability did not correlate with the ability of the compounds to cause down-regulation of AR protein expression. Through rational synthetic optimization of the lead compound (BTB01434), we have discovered a series of novel substituted diaryl molecules as potent anti-prostate cancer agents. Some compounds (1-6) were shown to be extremely potent inhibitors of LNCaP cell viability with GI(50) values in the nanomolar range (1.45-83 nM). The most potent compound (4-methylphenyl)[(4-methylphenyl)sulfonyl]amine (5) with a GI(50) value of 1.45 nM is 27,000 times more potent than our lead compound BTB01434 (GI(50)=39.8 microM). In addition, some of the compounds exhibited modest anti-androgenic activities and one was also a potent inhibitor (GI(50)=850 nM) of PC-3 (AR-null) cell growth. A clear structure-activity relationship (SAR) has been established for activity against LNCaP cells, where potent molecules possess two substituted/unsubstituted aromatic rings connected through a sulfonamide linker. These novel compounds are strong candidates for development for the treatment of hormone-sensitive and importantly hormone-refractory prostate cancers in humans. PMID:18316193

  8. Potent anti-prostate cancer agents derived from a novel androgen receptor down-regulating agent.

    PubMed

    Purushottamachar, Puranik; Khandelwal, Aakanksha; Vasaitis, Tadas S; Bruno, Robert D; Gediya, Lalji K; Njar, Vincent C O

    2008-04-01

    The search for novel androgen receptor (AR) down-regulating agents by catalyst HipHop pharmacophore modeling led to the discovery of some lead molecules. Unexpectedly, the effect of these leads on human prostate cancer LNCaP cell viability did not correlate with the ability of the compounds to cause down-regulation of AR protein expression. Through rational synthetic optimization of the lead compound (BTB01434), we have discovered a series of novel substituted diaryl molecules as potent anti-prostate cancer agents. Some compounds (1-6) were shown to be extremely potent inhibitors of LNCaP cell viability with GI(50) values in the nanomolar range (1.45-83 nM). The most potent compound (4-methylphenyl)[(4-methylphenyl)sulfonyl]amine (5) with a GI(50) value of 1.45 nM is 27,000 times more potent than our lead compound BTB01434 (GI(50)=39.8 microM). In addition, some of the compounds exhibited modest anti-androgenic activities and one was also a potent inhibitor (GI(50)=850 nM) of PC-3 (AR-null) cell growth. A clear structure-activity relationship (SAR) has been established for activity against LNCaP cells, where potent molecules possess two substituted/unsubstituted aromatic rings connected through a sulfonamide linker. These novel compounds are strong candidates for development for the treatment of hormone-sensitive and importantly hormone-refractory prostate cancers in humans.

  9. Tanshinone IIA attenuates atherosclerosis in ApoE(-/-) mice through down-regulation of scavenger receptor expression.

    PubMed

    Tang, Fu-Tian; Cao, Yuan; Wang, Tie-Qiao; Wang, Li-Jing; Guo, Jiao; Zhou, Xiao-Shi; Xu, Suo-Wen; Liu, Wei-Hua; Liu, Pei-Qing; Huang, He-Qing

    2011-01-10

    This study is designed to investigate the protection of tanshinone IIA (TSIIA) against atherosclerosis in apolipoprotein E deficient (ApoE(-/-)) mice and to explore the mechanisms by focusing on the expressions of scavenger receptors, scavenger receptor-A (SR-A) and CD36. The in vivo study demonstrated that TSIIA (10-90mg/kg) inhibited the atherosclerotic lesions, down-regulated the CD68 protein expression in lesion and decreased the contents of cholesterol in aortas of ApoE(-/-) mice. In addition, TSIIA reduced the serum levels of oxidized LDL (oxLDL) and down-regulated the mRNA expression of CD36, SR-A and peroxisome proliferator-activated receptor gamma (PPARγ) in aortas. The in vitro study showed that TSIIA (0.1-10μM) decreased cholesterol level and DiI-oxLDL uptake in mouse peritoneal macrophages treated with oxLDL (50μg/ml). In addition, TSIIA down-regulated the mRNA and protein expression of CD36 but not that of SR-A in oxLDL treated macrophages. TSIIA also down-regulated the mRNA expression of PPARγ in oxLDL treated macrophages. Furthermore, TSIIA reduced the mRNA expression of CD36 in macrophages treated with PPARγ agonist 15d-PGJ(2) (2μM) or troglitazone (50μM), whereas both 15d-PGJ(2) (0.5-1.5μM) and troglitazone (5-20μM) dose-dependently abolished the down-regulation of CD36 expression by TSIIA in oxLDL treated macrophages. These results suggest that TSIIA attenuates the atherosclerotic lesion in ApoE(-/-) mice, which might be attributed to the properties of both anti-oxidation and down-regulation of scavenger receptors. Furthermore, antagonism of PPARγ might be involved in the down-regulation of CD36 by TSIIA.

  10. Downregulation of androgen receptor is strongly associated with diabetes in triple negative breast cancer patients

    PubMed Central

    Collina, Francesca; Cerrone, Margherita; Peluso, Valentina; Laurentiis, Michelino De; Caputo, Roberta; Cecio, Rossella De; Liguori, Giuseppina; Botti, Gerardo; Cantile, Monica; Bonito, Maurizio Di

    2016-01-01

    Developing of personalized therapies for Triple Negative Breast Cancer (TNBC) requires a more detailed knowledge of its biology and a correct stratification of molecular subtypes. Androgen Receptor (AR) is expressed in a large part of TNBCs but its prognostic role in this Breast Cancer (BC) subtype is highly debated. In this study, we analyzed AR expression in a series of 238 TNBCs and correlated its expression with clinical-pathological features, survival, and metabolic profile. We showed a consistent association between AR expression and a better prognosis of TNBC patients, while its downregulation appeared strongly associated with diabetic disease. Since a recent prospective study reported a lower BC risk in diabetic women treated with drugs able to reduce circulating levels of glucose compared with non-diabetic woman, and in vitro studies showed that AR level are regulated directly by hyperglycemia, we speculate on the perspective of new integrated therapies for TNBC.

  11. Downregulation of androgen receptor is strongly associated with diabetes in triple negative breast cancer patients

    PubMed Central

    Collina, Francesca; Cerrone, Margherita; Peluso, Valentina; Laurentiis, Michelino De; Caputo, Roberta; Cecio, Rossella De; Liguori, Giuseppina; Botti, Gerardo; Cantile, Monica; Bonito, Maurizio Di

    2016-01-01

    Developing of personalized therapies for Triple Negative Breast Cancer (TNBC) requires a more detailed knowledge of its biology and a correct stratification of molecular subtypes. Androgen Receptor (AR) is expressed in a large part of TNBCs but its prognostic role in this Breast Cancer (BC) subtype is highly debated. In this study, we analyzed AR expression in a series of 238 TNBCs and correlated its expression with clinical-pathological features, survival, and metabolic profile. We showed a consistent association between AR expression and a better prognosis of TNBC patients, while its downregulation appeared strongly associated with diabetic disease. Since a recent prospective study reported a lower BC risk in diabetic women treated with drugs able to reduce circulating levels of glucose compared with non-diabetic woman, and in vitro studies showed that AR level are regulated directly by hyperglycemia, we speculate on the perspective of new integrated therapies for TNBC. PMID:27648143

  12. Downregulation of androgen receptor is strongly associated with diabetes in triple negative breast cancer patients.

    PubMed

    Collina, Francesca; Cerrone, Margherita; Peluso, Valentina; Laurentiis, Michelino De; Caputo, Roberta; Cecio, Rossella De; Liguori, Giuseppina; Botti, Gerardo; Cantile, Monica; Bonito, Maurizio Di

    2016-01-01

    Developing of personalized therapies for Triple Negative Breast Cancer (TNBC) requires a more detailed knowledge of its biology and a correct stratification of molecular subtypes. Androgen Receptor (AR) is expressed in a large part of TNBCs but its prognostic role in this Breast Cancer (BC) subtype is highly debated. In this study, we analyzed AR expression in a series of 238 TNBCs and correlated its expression with clinical-pathological features, survival, and metabolic profile. We showed a consistent association between AR expression and a better prognosis of TNBC patients, while its downregulation appeared strongly associated with diabetic disease. Since a recent prospective study reported a lower BC risk in diabetic women treated with drugs able to reduce circulating levels of glucose compared with non-diabetic woman, and in vitro studies showed that AR level are regulated directly by hyperglycemia, we speculate on the perspective of new integrated therapies for TNBC. PMID:27648143

  13. Hydrogen peroxide down-regulates inositol 1,4,5-trisphosphate receptor content through proteasome activation.

    PubMed

    Martín-Garrido, A; Boyano-Adánez, M C; Alique, M; Calleros, L; Serrano, I; Griera, M; Rodríguez-Puyol, D; Griendling, K K; Rodríguez-Puyol, M

    2009-11-15

    Hydrogen peroxide (H(2)O(2)) is implicated in the regulation of signaling pathways leading to changes in vascular smooth muscle function. Contractile effects produced by H(2)O(2) are due to the phosphorylation of myosin light chain kinase triggered by increases in intracellular calcium (Ca(2+)) from intracellular stores or influx of extracellular Ca(2+). One mechanism for mobilizing such stores involves the phosphoinositide pathway. Inositol 1,4,5-trisphosphate (IP(3)) mobilizes intracellular Ca(2+) by binding to a family of receptors (IP(3)Rs) on the endoplasmic-sarcoplasmic reticulum that act as ligand-gated Ca(2+) channels. IP(3)Rs can be rapidly ubiquitinated and degraded by the proteasome, causing a decrease in cellular IP(3)R content. In this study we show that IP(3)R(1) and IP(3)R(3) are down-regulated when vascular smooth muscle cells (VSMC) are stimulated by H(2)O(2), through an increase in proteasome activity. Moreover, we demonstrate that the decrease in IP(3)R by H(2)O(2) is accompanied by a reduction in calcium efflux induced by IP(3) in VSMC. Also, we observed that angiotensin II (ANGII) induces a decrease in IP(3)R by activation of NADPH oxidase and that preincubation with H(2)O(2) decreases ANGII-mediated calcium efflux and planar cell surface area in VSMC. The decreased IP(3) receptor content observed in cells was also found in aortic rings, which exhibited a decreased ANGII-dependent contraction after treatment with H(2)O(2). Altogether, these results suggest that H(2)O(2) mediates IP(3)R down-regulation via proteasome activity.

  14. Response Element Composition Governs Correlations between Binding Site Affinity and Transcription in Glucocorticoid Receptor Feed-forward Loops.

    PubMed

    Sasse, Sarah K; Zuo, Zheng; Kadiyala, Vineela; Zhang, Liyang; Pufall, Miles A; Jain, Mukesh K; Phang, Tzu L; Stormo, Gary D; Gerber, Anthony N

    2015-08-01

    Combinatorial gene regulation through feed-forward loops (FFLs) can bestow specificity and temporal control to client gene expression; however, characteristics of binding sites that mediate these effects are not established. We previously showed that the glucocorticoid receptor (GR) and KLF15 form coherent FFLs that cooperatively induce targets such as the amino acid-metabolizing enzymes AASS and PRODH and incoherent FFLs exemplified by repression of MT2A by KLF15. Here, we demonstrate that GR and KLF15 physically interact and identify low affinity GR binding sites within glucocorticoid response elements (GREs) for PRODH and AASS that contribute to combinatorial regulation with KLF15. We used deep sequencing and electrophoretic mobility shift assays to derive in vitro GR binding affinities across sequence space. We applied these data to show that AASS GRE activity correlated (r(2) = 0.73) with predicted GR binding affinities across a 50-fold affinity range in transfection assays; however, the slope of the linear relationship more than doubled when KLF15 was expressed. Whereas activity of the MT2A GRE was even more strongly (r(2) = 0.89) correlated with GR binding site affinity, the slope of the linear relationship was sharply reduced by KLF15, consistent with incoherent FFL logic. Thus, GRE architecture and co-regulator expression together determine the functional parameters that relate GR binding site affinity to hormone-induced transcriptional responses. Utilization of specific affinity response functions and GR binding sites by FFLs may contribute to the diversity of gene expression patterns within GR-regulated transcriptomes. PMID:26088140

  15. Activation of glucocorticoid receptors in Müller glia is protective to retinal neurons and suppresses microglial reactivity

    PubMed Central

    Gallina, Donika; Zelinka, Christopher Paul; Cebulla, Colleen; Fischer, Andy J.

    2015-01-01

    Reactive microglia and macrophages are prevalent in damaged retinas. Glucocorticoid signaling is known to suppress inflammation and the reactivity of microglia and macrophages. In the vertebrate retina, the glucocorticoid receptor (GCR) is known to be activated and localized to the nuclei of Müller glia (Gallina et al., 2014). Accordingly, we investigated how signaling through GCR influences the survival of neurons using the chick retina in vivo as a model system. We applied intraocular injections of GCR agonist or antagonist, assessed microglial reactivity, and the survival of retinal neurons following different damage paradigms. Microglial reactivity was increased in retinas from eyes that were injected with vehicle, and this reactivity was decreased by GCR-agonist dexamethasone (Dex) and increased by GCR-antagonist RU486. We found that activation of GCR suppresses the reactivity of microglia and inhibited the loss of retinal neurons resulting from excitotoxicity. We provide evidence that the protection-promoting effects of Dex were maintained when the microglia were selectively ablated. Similarly, intraocular injections of Dex protected ganglion cells from colchicine-treatment and protected photoreceptors from damage caused by retinal detachment. We conclude that activation of GCR promotes the survival of ganglion cells in colchicine-damaged retinas, promotes the survival of amacrine and bipolar cells in excitotoxin-damaged retinas, and promotes the survival of photoreceptors in detached retinas. We propose that suppression of microglial reactivity is secondary to activation of GCR in Müller glia, and this mode of signaling is an effective means to lessen the damage and vision loss resulting from different types of retinal damage. PMID:26272753

  16. Expression of eight glucocorticoid receptor isoforms in the human preterm placenta vary with fetal sex and birthweight

    PubMed Central

    Saif, Z.; Hodyl, N.A.; Stark, M.J.; Fuller, P.J.; Cole, T.; Lu, N.; Clifton, V.L.

    2016-01-01

    Introduction Administration of betamethasone to women at risk of preterm delivery is known to be associated with reduced fetal growth via alterations in placental function and possibly direct effects on the fetus. The placental glucocorticoid receptor (GR) is central to this response and recent evidence suggests there are numerous isoforms for GR in term placentae. In this study we have questioned whether GR isoform expression varies in preterm placentae in relation to betamethasone exposure, fetal sex and birthweight. Methods Preterm (24–36 completed weeks of gestation, n = 55) and term placentae (>37 completed weeks of gestation, n = 56) were collected at delivery. Placental GR expression was examined using Western Blot and analysed in relation to gestational age at delivery, fetal sex, birthweight and beta-methasone exposure. Data was analysed using non-parametric tests. Results Eight known isoforms of the GR were detected in the preterm placenta and include GRα (94 kDa), GRβ (91 kDa), GRα C (81 kDa) GR P (74 kDa) GR A (65 kDa), GRα D1–3 (50–55 kDa). Expression varied between preterm and term placentae with a greater expression of GRα C in preterm placentae relative to term placentae. The only sex differences in preterm placentae was that GRα D2 expression was higher in males than females. There were no alterations in preterm placental GR expression in association with betamethasone exposure. Discussion GRα C is the isoform involved in glucocorticoid induced apoptosis and suggests that its predominance in preterm placentae may contribute to the pathophysiology of preterm birth. PMID:25990415

  17. Modulation of Glucocorticoid Receptor Nuclear Translocation in Neurons by Immunophilins FKBP51 and FKBP52: Implications for Major Depressive Disorder

    PubMed Central

    Tatro, Erick T.; Everall, Ian P.; Kaul, Marcus; Achim, Cristian L.

    2009-01-01

    Mood disorders associated with dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis are common psychiatric conditions. The glucocorticoid receptor (GR) is a steroid-activated nuclear receptor that, upon binding to cortisol, translocates to the nucleus where it targets genes related to neuronal metabolism and plasticity. In patients suffering from major depressive disorder (MDD), hypercortisolemia is a common finding. In the current study we investigated the molecular events associated with the FK506 binding proteins (FKBP) -52 and -51 response to cortisol exposure in neuronal cell cultures and their effect on GR translocation. We noted that FK506 altered nuclear localization of the GR and inhibited expression of GR-responsive genes. Furthermore, si-RNA knockdown of FKBP4 gene, coding for the immunophilin FKBP52, inhibited cortisol-activated GR nuclear translocation, while knockdown of FKBP5, coding for immunophilin FKBP51, was associated with increased baseline GR nuclear localization. We propose that immunophilins are modulators of the cortisol-HPA axis response to stress and related chronic brain disorders. PMID:19545546

  18. Glucocorticoid-induced TNF receptor family-related protein ligand regulates the migration of monocytes to the inflamed intestine.

    PubMed

    Liao, Gongxian; van Driel, Boaz; Magelky, Erica; O'Keeffe, Michael S; de Waal Malefyt, Rene; Engel, Pablo; Herzog, Roland W; Mizoguchi, Emiko; Bhan, Atul K; Terhorst, Cox

    2014-01-01

    Glucocorticoid-induced TNF receptor family-related protein (GITR) regulates the function of both T cells and antigen-presenting cells (APCs), while the function of GITR ligand (GITR-L) is largely unknown. Here we evaluate the role of GITR-L, whose expression is restricted to APCs, in the development of enterocolitis. On injecting naive CD4(+) T cells, GITR-L(-/-)Rag(-/-) mice develop a markedly milder colitis than Rag(-/-) mice, which correlates with a 50% reduction of Ly6C(+)CD11b(+)MHCII(+) macrophages in the lamina propria and mesenteric lymph nodes. The same result was observed in αCD40-induced acute colitis and during peritonitis, suggesting an altered monocyte migration. In line with these observations, the number of nondifferentiated monocytes was approximately 3-fold higher in the spleen of GITR-L(-/-)Rag(-/-) mice than in Rag(-/-) mice after αCD40 induction. Consistent with the dynamic change in the formation of an active angiotensin II type 1 receptor (AT1) dimer in GITR-L(-/-) splenic monocytes during intestinal inflammation, the migratory capability of splenic monocytes from GITR-L-deficient mice was impaired in an in vitro transwell migration assay. Conversely, αGITR-L reduces the number of splenic Ly6C(hi) monocytes, concomitantly with an increase in AT1 dimers. We conclude that GITR-L regulates the number of proinflammatory macrophages in sites of inflammation by controlling the egress of monocytes from the splenic reservoir.

  19. Identification and molecular characterization of cellular factors required for glucocorticoid receptor-mediated mRNA decay

    PubMed Central

    Park, Ok Hyun; Park, Joori; Yu, Mira; An, Hyoung-Tae; Ko, Jesang; Kim, Yoon Ki

    2016-01-01

    Glucocorticoid (GC) receptor (GR) has been shown recently to bind a subset of mRNAs and elicit rapid mRNA degradation. However, the molecular details of GR-mediated mRNA decay (GMD) remain unclear. Here, we demonstrate that GMD triggers rapid degradation of target mRNAs in a translation-independent and exon junction complex-independent manner, confirming that GMD is mechanistically distinct from nonsense-mediated mRNA decay (NMD). Efficient GMD requires PNRC2 (proline-rich nuclear receptor coregulatory protein 2) binding, helicase ability, and ATM-mediated phosphorylation of UPF1 (upstream frameshift 1). We also identify two GMD-specific factors: an RNA-binding protein, YBX1 (Y-box-binding protein 1), and an endoribonuclease, HRSP12 (heat-responsive protein 12). In particular, using HRSP12 variants, which are known to disrupt trimerization of HRSP12, we show that HRSP12 plays an essential role in the formation of a functionally active GMD complex. Moreover, we determine the hierarchical recruitment of GMD factors to target mRNAs. Finally, our genome-wide analysis shows that GMD targets a variety of transcripts, implicating roles in a wide range of cellular processes, including immune responses. PMID:27798850

  20. A transition in transcriptional activation by the glucocorticoid and retinoic acid receptors at the tumor stage of dermal fibrosarcoma development.

    PubMed Central

    Vivanco, M D; Johnson, R; Galante, P E; Hanahan, D; Yamamoto, K R

    1995-01-01

    In transgenic mice harboring the bovine papillomavirus genome, fibrosarcomas arise along an experimentally accessible pathway in which normal dermal fibroblasts progress through two pre-neoplastic stages, mild and aggressive fibromatosis, followed by a final transition to the tumor stage. We found that the glucocorticoid receptor (GR) displays only modest transcriptional regulatory activity in cells derived from the three non-tumor stages, whereas it is highly active in fibrosarcoma cells. Upon inoculation into mice, the aggressive fibromatosis cells progress to tumor cells that have high GR activity; thus, the increased transcriptional regulatory activity of GR correlates with the cellular transition to the tumor stage. The intracellular levels of GR, as well as its hormone-dependent nuclear translocation and specific DNA binding activities, are unaltered throughout the progression. Strikingly, the low GR activity observed in the pre-neoplastic stages cannot be overcome by exogenous GR introduced by co-transfection. Moreover, comparisons of primary embryo fibroblasts and their transformed derivatives revealed a similar pattern--modest GR activity, unresponsive to overexpressed GR protein, in the normal cells was strongly increased in the transformed cells. Likewise, the retinoic acid receptor (RAR) displayed similar differential activity in the fibrosarcoma pathway. Thus, the oncogenic transformation of fibroblasts, and likely other cell types, is accompanied by a striking increase in the activities of transcriptional regulators such as GR and RAR. We suggest that normal primary cells have a heretofore unrecognized capability to limit the magnitude of induction of gene expression. Images PMID:7774580

  1. Cortisol stimulates proliferation and apoptosis in the late gestation fetal heart: differential effects of mineralocorticoid and glucocorticoid receptors

    PubMed Central

    Feng, Xiaodi; Reini, Seth A.; Richards, Elaine; Wood, Charles E.

    2013-01-01

    We have previously found that modest chronic increases in maternal cortisol result in an enlarged fetal heart. To explore the mechanisms of this effect, we used intrapericardial infusions of a mineralocorticoid receptor (MR) antagonist (canrenoate) or of a glucocorticoid receptor (GR) antagonist (mifepristone) in the fetus during maternal infusion of cortisol (1 mg·kg−1·day−1). We have shown that the MR antagonist blocked the increase in fetal heart weight and in wall thickness resulting from maternal cortisol infusion. In the current study we extended those studies and found that cortisol increased Ki67 staining in both ventricles, indicating cell proliferation, but also increased active caspase-3 staining in cells of the conduction pathway in the septum and subendocardial layers of the left ventricle, suggesting increased apoptosis in Purkinje fibers. The MR antagonist blocked the increase in cell proliferation, whereas the GR antagonist blocked the increased apoptosis in Purkinje fibers. We also found evidence of activation of caspase-3 in c-kit-positive cells, suggesting apoptosis in stem cell populations in the ventricle. These studies suggest a potentially important role of corticosteroids in the terminal remodeling of the late gestation fetal heart and suggest a mechanism for the cardiac enlargement with excess corticosteroid exposure. PMID:23785077

  2. Drosophila S6 Kinase Like Inhibits Neuromuscular Junction Growth by Downregulating the BMP Receptor Thickveins

    PubMed Central

    Zhao, Guoli; Wu, Yingga; Du, Li; Li, Wenhua; Xiong, Ying; Yao, Aiyu; Wang, Qifu; Zhang, Yong Q.

    2015-01-01

    Synaptic connections must be precisely controlled to ensure proper neural circuit formation. In Drosophila melanogaster, bone morphogenetic protein (BMP) promotes growth of the neuromuscular junction (NMJ) by binding and activating the BMP ligand receptors wishful thinking (Wit) and thickveins (Tkv) expressed in motor neurons. We report here that an evolutionally conserved, previously uncharacterized member of the S6 kinase (S6K) family S6K like (S6KL) acts as a negative regulator of BMP signaling. S6KL null mutants were viable and fertile but exhibited more satellite boutons, fewer and larger synaptic vesicles, larger spontaneous miniature excitatory junctional potential (mEJP) amplitudes, and reduced synaptic endocytosis at the NMJ terminals. Reducing the gene dose by half of tkv in S6KL mutant background reversed the NMJ overgrowth phenotype. The NMJ phenotypes of S6KL mutants were accompanied by an elevated level of Tkv protein and phosphorylated Mad, an effector of the BMP signaling pathway, in the nervous system. In addition, Tkv physically interacted with S6KL in cultured S2 cells. Furthermore, knockdown of S6KL enhanced Tkv expression, while S6KL overexpression downregulated Tkv in cultured S2 cells. This latter effect was blocked by the proteasome inhibitor MG132. Our results together demonstrate for the first time that S6KL regulates synaptic development and function by facilitating proteasomal degradation of the BMP receptor Tkv. PMID:25748449

  3. The long-acting β2-adrenoceptor agonist, indacaterol, enhances glucocorticoid receptor-mediated transcription in human airway epithelial cells in a gene- and agonist-dependent manner

    PubMed Central

    Joshi, T; Johnson, M; Newton, R; Giembycz, M A

    2015-01-01

    Background and Purpose Inhaled glucocorticoid (ICS)/long-acting β2-adrenoceptor agonist (LABA) combination therapy is a recommended treatment option for patients with moderate/severe asthma in whom adequate control cannot be achieved by an ICS alone. Previously, we discovered that LABAs can augment dexamethasone-inducible gene expression and proposed that this effect may explain how these two drugs interact to deliver superior clinical benefit. Herein, we extended that observation by analysing, pharmacodynamically, the effect of the LABA, indacaterol, on glucocorticoid receptor (GR)-mediated gene transcription induced by seven ligands with intrinsic activity values that span the spectrum of full agonism to antagonism. Experimental Approach BEAS-2B human airway epithelial cells stably transfected with a 2× glucocorticoid response element luciferase reporter were used to model gene transcription together with an analysis of several glucocorticoid-inducible genes. Key Results Indacaterol augmented glucocorticoid-induced reporter activation in a manner that was positively related to the intrinsic activity of the GR agonist. This effect was demonstrated by an increase in response maxima without a change in GR agonist affinity or efficacy. Indacaterol also enhanced glucocorticoid-inducible gene expression. However, the magnitude of this effect was dependent on both the GR agonist and the gene of interest. Conclusions and Implications These data suggest that indacaterol activates a molecular rheostat, which increases the transcriptional competency of GR in an agonist- and gene-dependent manner without apparently changing the relationship between fractional GR occupancy and response. These findings provide a platform to rationally design ICS/LABA combination therapy that is based on the generation of agonist-dependent gene expression profiles in target and off-target tissues. PMID:25598440

  4. Estrogen receptor beta inhibits transcriptional activity of hypoxia inducible factor-1 through the downregulation of arylhydrocarbon receptor nuclear translocator

    PubMed Central

    2011-01-01

    Introduction Estrogen receptor (ER) β is predicted to play an important role in prevention of breast cancer development and metastasis. We have shown previously that ERβ inhibits hypoxia inducible factor (HIF)-1α mediated transcription, but the mechanism by which ERβ works to exert this effect is not understood. Methods Vascular endothelial growth factor (VEGF) was measured in conditioned medium by enzyme-linked immunosorbent assays. Reverse transcription polymerase chain reaction (RT-PCR), Western blotting, immunoprecipitation, luciferase assays and chromatin immunoprecipitation (ChIP) assays were used to ascertain the implication of ERβ on HIF-1 function. Results In this study, we found that the inhibition of HIF-1 activity by ERβ expression was correlated with ERβ's ability to degrade aryl hydrocarbon receptor nuclear translocator (ARNT) via ubiquitination processes leading to the reduction of active HIF-1α/ARNT complexes. HIF-1 repression by ERβ was rescued by overexpression of ARNT as examined by hypoxia-responsive element (HRE)-driven luciferase assays. We show further that ERβ attenuated the hypoxic induction of VEGF mRNA by directly decreasing HIF-1α binding to the VEGF gene promoter. Conclusions These results show that ERβ suppresses HIF-1α-mediated transcription via ARNT down-regulation, which may account for the tumour suppressive function of ERβ. PMID:21435239

  5. Glucocorticoids and renal Na+ transport: implications for hypertension and salt sensitivity

    PubMed Central

    Hunter, Robert W; Ivy, Jessica R; Bailey, Matthew A

    2014-01-01

    The clinical manifestations of glucocorticoid excess include central obesity, hyperglycaemia, dyslipidaemia, electrolyte abnormalities and hypertension. A century on from Cushing's original case study, these cardinal features are prevalent in industrialized nations. Hypertension is the major modifiable risk factor for cardiovascular and renal disease and reflects underlying abnormalities of Na+ homeostasis. Aldosterone is a master regulator of renal Na+ transport but here we argue that glucocorticoids are also influential, particularly during moderate excess. The hypothalamic–pituitary–adrenal axis can affect renal Na+ homeostasis on multiple levels, systemically by increasing mineralocorticoid synthesis and locally by actions on both the mineralocorticoid and glucocorticoid receptors, both of which are expressed in the kidney. The kidney also expresses both of the 11β-hydroxysteroid dehydrogenase (11βHSD) enzymes. The intrarenal generation of active glucocorticoid by 11βHSD1 stimulates Na+ reabsorption; failure to downregulate the enzyme during adaption to high dietary salt causes salt-sensitive hypertension. The deactivation of glucocorticoid by 11βHSD2 underpins the regulatory dominance for Na+ transport of mineralocorticoids and defines the ‘aldosterone-sensitive distal nephron’. In summary, glucocorticoids can stimulate renal transport processes conventionally attributed to the renin–angiotensin–aldosterone system. Importantly, Na+ and volume homeostasis do not exert negative feedback on the hypothalamic–pituitary–adrenal axis. These actions are therefore clinically relevant and may contribute to the pathogenesis of hypertension in conditions associated with elevated glucocorticoid levels, such as the metabolic syndrome and chronic stress. PMID:24535442

  6. Induction of 11β-HSD 1 and activation of distinct mineralocorticoid receptor- and glucocorticoid receptor-dependent gene networks in decidualizing human endometrial stromal cells.

    PubMed

    Kuroda, Keiji; Venkatakrishnan, Radha; Salker, Madhuri S; Lucas, Emma S; Shaheen, Fozia; Kuroda, Masako; Blanks, Andrew; Christian, Mark; Quenby, Siobhan; Brosens, Jan J

    2013-02-01

    The actions of glucocorticoids at the feto-maternal interface are not well understood. Here, we show that decidualization of human endometrial stromal cells (HESCs) in response to progesterone and cAMP signaling is associated with a strong induction of 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) expression and enzyme activity. Decidualization also triggered a gradual decrease in glucocorticoid receptor (GR) expression and reciprocal increase in mineralocorticoid receptor (MR) levels. Gene expression profiling of differentiating HESCs after small interfering RNA (siRNA)-mediated knockdown of either GR or MR identified 239 and 167 significantly regulated genes, respectively. Interestingly, GR-repressed genes were enriched for Krüppel-associated box domain containing zinc-finger proteins, transcriptional repressors involved in heterochromatin formation. In agreement, GR knockdown was sufficient to enhance trimethylated H3K9 levels in decidualizing cells. Conversely, we identified several MR-dependent genes implicated in lipid droplet biogenesis and retinoid metabolism. For example, the induction in differentiating HESCs of DHRS3, encoding a highly conserved enzyme that catalyzes the oxidation/reduction of retinoids and steroids, was enhanced by aldosterone, attenuated in response to MR knockdown, and abolished upon treatment with the MR antagonist RU26752. Furthermore, we demonstrate that decidualization is associated with dynamic changes in the abundance and distribution of cytoplasmic lipid droplets, the formation of which was blocked by RU26752. In summary, progesterone drives local cortisol biosynthesis by decidual cells through induction of 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1), leading to transcriptional regulation of distinct GR and MR gene networks involved in epigenetic programming and lipid and retinoid metabolism, respectively. PMID:23275455

  7. Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG.

    PubMed

    Kyakumoto, S; Kurokawa, R; Ota, M

    1990-07-12

    Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor

  8. An overlapping set of genes is regulated by both NFIB and the glucocorticoid receptor during lung maturation

    PubMed Central

    2014-01-01

    Background Lung maturation is a late fetal developmental event in both mice and humans. Because of this, lung immaturity is a serious problem in premature infants. Disruption of genes for either the glucocorticoid receptor (Nr3c1) or the NFIB transcription factors results in perinatal lethality due to lung immaturity. In both knockouts, the phenotype includes excess cell proliferation, failure of saccularization and reduced expression of markers of epithelial differentiation. This similarity suggests that the two genes may co-regulate a specific set of genes essential for lung maturation. Results We analyzed the roles of these two transcription factors in regulating transcription using ChIP-seq data for NFIB, and RNA expression data and motif analysis for both. Our new ChIP-seq data for NFIB in lung at E16.5 shows that NFIB binds to a NFI motif. This motif is over-represented in the promoters of genes that are under-expressed in Nfib-KO mice at E18.5, suggesting an activator role for NFIB. Using available microarray data from Nr3c1-KO mice, we further identified 52 genes that are under-expressed in both Nfib and Nr3c1 knockouts, an overlap which is 13.1 times larger than what would be expected by chance. Finally, we looked for enrichment of 738 recently published transcription factor motifs in the promoters of these putative target genes and found that the NFIB and glucocorticoid receptor motifs were among the most enriched, suggesting that a subset of these genes may be directly activated by Nfib and Nr3c1. Conclusions Our data provide the first evidence for Nfib and Nr3c1 co-regulating genes related to lung maturation. They also establish that the in vivo DNA-binding specificity of NFIB is the same as previously seen in vitro, and highly similar to that of the other NFI-family members NFIA, NFIC and NFIX. PMID:24661679

  9. Effects of selective and non-selective glucocorticoid receptor II antagonists on rapid-onset diabetes in young rats.

    PubMed

    Beaudry, Jacqueline L; Dunford, Emily C; Teich, Trevor; Zaharieva, Dessi; Hunt, Hazel; Belanoff, Joseph K; Riddell, Michael C

    2014-01-01

    The blockade of glucocorticoid (GC) action through antagonism of the glucocorticoid receptor II (GRII) has been used to minimize the undesirable effects of chronically elevated GC levels. Mifepristone (RU486) is known to competitively block GRII action, but not exclusively, as it antagonizes the progesterone receptor. A number of new selective GRII antagonists have been developed, but limited testing has been completed in animal models of overt type 2 diabetes mellitus. Therefore, two selective GRII antagonists (C113176 and C108297) were tested to determine their effects in our model of GC-induced rapid-onset diabetes (ROD). Male Sprague-Dawley rats (∼ six weeks of age) were placed on a high-fat diet (60%), surgically implanted with pellets containing corticosterone (CORT) or wax (control) and divided into five treatment groups. Each group was treated with either a GRII antagonist or vehicle for 14 days after surgery: CORT pellets (400 mg/rat) + antagonists (80 mg/kg/day); CORT pellets + drug vehicle; and wax pellets (control) + drug vehicle. After 10 days of CORT treatment, body mass gain was increased with RU486 (by ∼20% from baseline) and maintained with C113176 administration, whereas rats given C108297 had similar body mass loss (∼15%) to ROD animals. Fasting glycemia was elevated in the ROD animals (>20 mM), normalized completely in animals treated with RU486 (6.2±0.1 mM, p<0.05) and improved in animals treated with C108297 and C113176 (14.0±1.6 and 8.8±1.6 mM, p<0.05 respectively). Glucose intolerance was normalized with RU486 treatment, whereas acute insulin response was improved with RU486 and C113176 treatment. Also, peripheral insulin resistance was attenuated with C113176 treatment along with improved levels of β-cell function while C108297 antagonism only provided modest improvements. In summary, C113176 is an effective agent that minimized some GC-induced detrimental metabolic effects and may provide an alternative to the effective, but non

  10. Effects of Selective and Non-Selective Glucocorticoid Receptor II Antagonists on Rapid-Onset Diabetes in Young Rats

    PubMed Central

    Beaudry, Jacqueline L.; Dunford, Emily C.; Teich, Trevor; Zaharieva, Dessi; Hunt, Hazel; Belanoff, Joseph K.; Riddell, Michael C.

    2014-01-01

    The blockade of glucocorticoid (GC) action through antagonism of the glucocorticoid receptor II (GRII) has been used to minimize the undesirable effects of chronically elevated GC levels. Mifepristone (RU486) is known to competitively block GRII action, but not exclusively, as it antagonizes the progesterone receptor. A number of new selective GRII antagonists have been developed, but limited testing has been completed in animal models of overt type 2 diabetes mellitus. Therefore, two selective GRII antagonists (C113176 and C108297) were tested to determine their effects in our model of GC-induced rapid-onset diabetes (ROD). Male Sprague-Dawley rats (∼ six weeks of age) were placed on a high-fat diet (60%), surgically implanted with pellets containing corticosterone (CORT) or wax (control) and divided into five treatment groups. Each group was treated with either a GRII antagonist or vehicle for 14 days after surgery: CORT pellets (400 mg/rat) + antagonists (80 mg/kg/day); CORT pellets + drug vehicle; and wax pellets (control) + drug vehicle. After 10 days of CORT treatment, body mass gain was increased with RU486 (by ∼20% from baseline) and maintained with C113176 administration, whereas rats given C108297 had similar body mass loss (∼15%) to ROD animals. Fasting glycemia was elevated in the ROD animals (>20 mM), normalized completely in animals treated with RU486 (6.2±0.1 mM, p<0.05) and improved in animals treated with C108297 and C113176 (14.0±1.6 and 8.8±1.6 mM, p<0.05 respectively). Glucose intolerance was normalized with RU486 treatment, whereas acute insulin response was improved with RU486 and C113176 treatment. Also, peripheral insulin resistance was attenuated with C113176 treatment along with improved levels of β-cell function while C108297 antagonism only provided modest improvements. In summary, C113176 is an effective agent that minimized some GC-induced detrimental metabolic effects and may provide an alternative to the effective, but non

  11. Short-term withdrawal from developmental exposure to cocaine activates the glucocorticoid receptor and alters spine dynamics.

    PubMed

    Caffino, Lucia; Giannotti, Giuseppe; Malpighi, Chiara; Racagni, Giorgio; Fumagalli, Fabio

    2015-10-01

    Although glucocorticoid receptors (GRs) contribute to the action of cocaine, their role following developmental exposure to the psychostimulant is still unknown. To address this issue, we exposed adolescent male rats to cocaine (20mg/kg/day) from post-natal day (PND) 28 to PND 42 and sacrificed them at PND 45 or 90. We studied the medial prefrontal cortex (mPFC), a brain region that is still developing during adolescence. In PND 45 rats we found enhanced GR transcription and translation as well as increased trafficking toward the nucleus of the receptor, with no alteration in plasma corticosterone levels. We also showed reduced expression of the GR co-chaperone FKBP51, that normally keeps the receptor in the cytoplasm, and increased expression of Src1, which cooperates in the activation of GR transcriptional activity, revealing that short withdrawal alters the finely tuned mechanisms regulating GR action. Since activation of GRs regulate dendritic spine morphology, we next investigated spine dynamics in cocaine-withdrawn rats. We found that PSD95, cofilin and F-actin, molecules regulating spine actin network, are reduced in the mPFC of PND 45 rats suggesting reduced spine density, confirmed by confocal imaging. Further, formation of filopodia, i.e. the inactive spines, is enhanced suggesting the formation of non-functional spines. Of note, no changes were found in molecules related to GR machinery or spine dynamics following long-term abstinence, i.e. in adult rats (PND 90). These findings demonstrate that short withdrawal promotes plastic changes in the developing brain via the dysregulation of the GR system and alterations in the spine network. PMID:26004981

  12. Brief neonatal maternal separation alters extinction of conditioned fear and corticolimbic glucocorticoid and NMDA receptor expression in adult rats.

    PubMed

    Wilber, Aaron A; Southwood, Christopher J; Wellman, Cara L

    Neonatal maternal separation alters adult HPA axis responsiveness to stress, adult emotionality, and glucocorticoid receptor (GR) concentrations in forebrain regions such as hippocampus. To investigate effects of neonatal maternal separation on emotion regulation and its neural substrates, we assessed acquisition and extinction of conditioned fear in adult rats that underwent neonatal maternal separation. Corticolimbic structures including basolateral amygdala and medial prefrontal cortex are critical for acquisition and extinction of conditioned fear, and such learning is N-methyl-D-aspartic acid (NMDA) receptor-dependent. Thus, we used immunohistochemistry to assess expression of the GR and the NR1 subunit of the NMDA receptor in basolateral amygdala and medial prefrontal cortex. On postnatal days 2-14, pups underwent control rearing or maternal separation for 15 min per day. Fear conditioning and extinction in adulthood were then assessed in male rats. Rats received five tone-alone habituation trials, then seven tone/footshock pairings. After 1 h, rats received tone-alone extinction trials to criterion, and 15 recall of extinction trials the next day. Brains were processed for immunohistochemical labeling of GR and NR1, and staining was quantified. Brief maternal separation did not alter acquisition or initial extinction, but impaired extinction recall. Brief maternal separation did not alter GR or NR1 expression in basolateral amygdala. However, brief maternal separation increased GR and decreased NR1 expression specifically in the infralimbic region of medial prefrontal cortex, consistent with work implicating this area in extinction recall. Thus, brief maternal separation impaired extinction recall and altered GR and NR1 expression in its neural substrate in adults.

  13. Associations of glucocorticoid receptor and corticosteroid-binding globulin gene polymorphisms on fat mass and fat mass distribution in prepubertal obese children.

    PubMed

    Barat, Pascal; Corcuff, Jean-Benoît; Tauber, Maïté; Moisan, Marie-Pierre

    2012-12-01

    Previous studies conducted in adult obese patients have shown that glucocorticoid receptor and corticosteroid-binding globulin gene polymorphisms influence cortisol-driven obesity and metabolic parameters. We investigated the impact of these polymorphisms in prepubertal obese children that were thoroughly examined for hypothalamic-pituitary-adrenal axis activity and for metabolic and obesity parameters. Obese children carrier of the allele G of the BclI polymorphism within glucocorticoid receptor gene tend to present a higher percentage of fat mass as well as a decreased cortisol suppression after low-dose dexamethasone as found in adult studies. Additionally, these allele G carriers show a strong correlation between truncal fat mass distribution and cortisol response to a standardized lunch, whereas this correlation is weak in allele C carriers. No differences were found for obesity or metabolic parameters between genotypes at the corticosteroid-binding globulin locus. However, allele 90 carriers present increased 24-h free urinary cortisol. Overall, this study provides new data showing the influence of glucocorticoid receptor and corticosteroid-binding globulin genes in obesity and/or cortisol action in prepubertal obese children.

  14. Blocking glucocorticoid receptors at adolescent age prevents enhanced freezing between repeated cue-exposures after conditioned fear in adult mice raised under chronic early life stress.

    PubMed

    Arp, J Marit; Ter Horst, Judith P; Loi, Manila; den Blaauwen, Jan; Bangert, Eline; Fernández, Guillén; Joëls, Marian; Oitzl, Melly S; Krugers, Harm J

    2016-09-01

    Early life adversity can have long-lasting impact on learning and memory processes and increase the risk to develop stress-related psychopathologies later in life. In this study we investigated (i) how chronic early life stress (ELS) - elicited by limited nesting and bedding material from postnatal day 2 to 9 - affects conditioned fear in adult mice and (ii) whether these effects can be prevented by blocking glucocorticoid receptors (GRs) at adolescent age. In adult male and female mice, ELS did not affect freezing behavior to the first tone 24h after training in an auditory fear-conditioning paradigm. Exposure to repeated tones 24h after training also resulted in comparable freezing behavior in ELS and control mice, both in males and females. However, male (but not female) ELS compared to control mice showed significantly more freezing behavior between the tone-exposures, i.e. during the cue-off periods. Intraperitoneal administration of the GR antagonist RU38486 during adolescence (on postnatal days 28-30) fully prevented enhanced freezing behavior during the cue-off period in adult ELS males. Western blot analysis revealed no effects of ELS on hippocampal expression of glucocorticoid receptors, neither at postnatal day 28 nor at adult age, when mice were behaviorally tested. We conclude that ELS enhances freezing behavior in adult mice in a potentially safe context after cue-exposure, which can be normalized by brief blockade of glucocorticoid receptors during the critical developmental window of adolescence. PMID:27246249

  15. Influenza Virus Infection Induces the Nuclear Relocalization of the Hsp90 Co-Chaperone p23 and Inhibits the Glucocorticoid Receptor Response

    PubMed Central

    Chase, Geoffrey; dos Santos Afonso, Emmanuel; Picard, Didier; Schwemmle, Martin; Naffakh, Nadia

    2011-01-01

    The genomic RNAs of influenza A viruses are associated with the viral polymerase subunits (PB1, PB2, PA) and nucleoprotein (NP), forming ribonucleoprotein complexes (RNPs). Transcription/replication of the viral genome occurs in the nucleus of infected cells. A role for Hsp90 in nuclear import and assembly of newly synthetized RNA-polymerase subunits has been proposed. Here we report that the p23 cochaperone of Hsp90, which plays a major role in glucocorticoid receptor folding and function, associates with influenza virus polymerase. We show that p23 is not essential for viral multiplication in cultured cells but relocalizes to the nucleus in influenza virus-infected cells, which may alter some functions of p23 and Hsp90. Moreover, we show that influenza virus infection inhibits glucocorticoid receptor-mediated gene transactivation, and that this negative effect can occur through a p23-independent pathway. Viral-induced inhibition of the glucocorticoid receptor response might be of significant importance regarding the physiopathology of influenza infections in vivo. PMID:21853119

  16. Cannabinoid receptors activation and glucocorticoid receptors deactivation in the amygdala prevent the stress-induced enhancement of a negative learning experience.

    PubMed

    Ramot, Assaf; Akirav, Irit

    2012-05-01

    The enhancement of emotional memory is clearly important as emotional stimuli are generally more significant than neutral stimuli for surviving and reproduction purposes. Yet, the enhancement of a negative emotional memory following exposure to stress may result in dysfunctional or intrusive memory that underlies several psychiatric disorders. Here we examined the effects of stress exposure on a negative emotional learning experience as measured by a decrease in the magnitude of the expected quantity of reinforcements in an alley maze. In contrast to other fear-related negative experiences, reward reduction is more associated with frustration and is assessed by measuring the latency to run the length of the alley to consume the reduced quantity of reward. We also examined whether the cannabinoid receptors agonist WIN55,212-2 (5 μg/side) and the glucocorticoid receptors (GRs) antagonist RU-486 (10 ng/side) administered into the rat basolateral amygdala (BLA) could prevent the stress-induced enhancement. We found that intra-BLA RU-486 or WIN55,212 before stress exposure prevented the stress-induced enhancement of memory consolidation for reduction in reward magnitude. These findings suggest that cannabinoid receptors and GRs in the BLA are important modulators of stress-induced enhancement of emotional memory.

  17. Coactivator TIF1β Interacts with Transcription Factor C/EBPβ and Glucocorticoid Receptor To Induce α1-Acid Glycoprotein Gene Expression

    PubMed Central

    Chang, Ching-Jin; Chen, Ya-Ling; Lee, Sheng-Chung

    1998-01-01

    The transcription of the α1-acid glycoprotein gene is induced by inflammatory cytokines and glucocorticoids. C/EBPβ is a major transcription factor involved in the induction of the agp gene by some cytokines. In this report, we have identified a novel transcriptional intermediary factor, TIF1β, which could enhance the transcription of the agp gene by the glucocorticoid receptor (GR) and C/EBPβ. TIF1β belongs to a subgroup of RING (really interesting new gene) finger proteins that contain a RING finger preceding two B box-type fingers and a putative coiled-coil domain (RBCC domain). Immunoprecipitation experiments showed that the interaction between GR and TIF1β is ligand independent. The overexpression of the TIF1β gene enhances GR-regulated expression in a ligand- and glucocorticoid-responsive element (GRE)-dependent manner. TIF1β can also augment C/EBPβ-mediated activity on wild-type and GRE-mutated agp genes, but this augmentation is diminished when all three C/EBPβ-binding elements are mutated. Functional and biochemical characterizations indicated that the bZIP domain of C/EBPβ and the RBCC domain, plant homeodomain finger, and bromodomain of TIF1β are crucial for the interactions of these proteins. Taken together, these results suggest that TIF1β serves as a converging mediator of signal transduction pathways of glucocorticoids and some inflammatory cytokines. PMID:9742105

  18. Measles virus spread by cell-cell contacts: uncoupling of contact-mediated receptor (CD46) downregulation from virus uptake.

    PubMed

    Firsching, R; Buchholz, C J; Schneider, U; Cattaneo, R; ter Meulen, V; Schneider-Schaulies, J

    1999-07-01

    CD46, which serves as a receptor for measles virus (MV; strain Edmonston), is rapidly downregulated from the cell surface after contact with viral particles or infected cells. We show here that the same two CD46 complement control protein (CCP) domains responsible for primary MV attachment mediate its downregulation. Optimal downregulation efficiency was obtained with CD46 recombinants containing CCP domains 1 and 2, whereas CCP 1, alone and duplicated, induced a slight downregulation. Using persistently infected monocytic/promyelocytic U937 cells which release very small amounts of infectious virus, and uninfected HeLa cells as contact partners, we then showed that during contact the formation of CD46-containing patches and caps precedes CD46 internalization. Nevertheless, neither substances inhibiting capping nor the fusion-inhibiting peptide Z-D-Phe-L-Phe-Gly-OH (FIP) blocked CD46 downregulation. Thus, CD46 downregulation can be uncoupled from fusion and subsequent virus uptake. Interestingly, in that system cell-cell contacts lead to a remarkably efficient infection of the target cells which is only partially inhibited by FIP. The finding that the contact of an infected with uninfected cells results in transfer of infectious viral material without significant (complete) fusion of the donor with the recipient cell suggests that microfusion events and/or FIP-independent mechanisms may mediate the transfer of MV infectivity from cell to cell. PMID:10364272

  19. Glucocorticoid Receptors, Brain-Derived Neurotrophic Factor, Serotonin and Dopamine Neurotransmission are Associated with Interferon-Induced Depression

    PubMed Central

    Udina, M; Navinés, R; Egmond, E; Oriolo, G; Langohr, K; Gimenez, D; Valdés, M; Gómez-Gil, E; Grande, I; Gratacós, M; Kapczinski, F; Artigas, F; Vieta, E; Solà, R

    2016-01-01

    Background: The role of inflammation in mood disorders has received increased attention. There is substantial evidence that cytokine therapies, such as interferon alpha (IFN-alpha), can induce depressive symptoms. Indeed, proinflammatory cytokines change brain function in several ways, such as altering neurotransmitters, the glucocorticoid axis, and apoptotic mechanisms. This study aimed to evaluate the impact on mood of initiating IFN-alpha and ribavirin treatment in a cohort of patients with chronic hepatitis C. We investigated clinical, personality, and functional genetic variants associated with cytokine-induced depression. Methods: We recruited 344 Caucasian outpatients with chronic hepatitis C, initiating IFN-alpha and ribavirin therapy. All patients were euthymic at baseline according to DSM-IV-R criteria. Patients were assessed at baseline and 4, 12, 24, and 48 weeks after treatment initiation using the Patient Health Questionnaire (PHQ), the Hospital Anxiety and Depression Scale (HADS), and the Temperament and Character Inventory (TCI). We genotyped several functional polymorphisms of interleukin-28 (IL28B), indoleamine 2,3-dioxygenase (IDO-1), serotonin receptor-1A (HTR1A), catechol-O-methyl transferase (COMT), glucocorticoid receptors (GCR1 and GCR2), brain-derived neurotrophic factor (BDNF), and FK506 binding protein 5 (FKBP5) genes. A survival analysis was performed, and the Cox proportional hazards model was used for the multivariate analysis. Results: The cumulative incidence of depression was 0.35 at week 24 and 0.46 at week 48. The genotypic distributions were in Hardy-Weinberg equilibrium. Older age (p = 0.018, hazard ratio [HR] per 5 years = 1.21), presence of depression history (p = 0.0001, HR = 2.38), and subthreshold depressive symptoms at baseline (p = 0.005, HR = 1.13) increased the risk of IFN-induced depression. So too did TCI personality traits, with high scores on fatigability (p = 0.0037, HR = 1.17), impulsiveness (p = 0.0200 HR = 1

  20. Mu-opioid receptor down-regulation and tolerance are not equally dependent upon G-protein signaling.

    PubMed

    Gomes, Benedict A; Shen, Ji; Stafford, Kristi; Patel, Minesh; Yoburn, Byron C

    2002-05-01

    In the present study, the contribution of pertussis toxin (PTX)-sensitive G(i/o)-proteins to opioid tolerance and mu-opioid receptor down-regulation in the mouse were examined. Mice were injected once intracerebroventricularly and intrathecally with PTX (0.1 microg/site). Controls were treated with saline. On the 10th day following PTX treatment, continuous subcutaneous infusion of etorphine (150 or 200 microg/kg/day) or morphine (40 mg/kg/day+25 mg slow-release pellet) was begun. Control mice were implanted with inert placebo pellets. Pumps and pellets were removed 3 days later, and mice were tested for morphine analgesia or mu-opioid receptor density was determined in the whole brain, spinal cord, and midbrain. Both infusion doses of etorphine produced significant tolerance (ED50 shift=approximately 4-6-fold) and down-regulation of mu-opioid receptors (approximately 20-35%). Morphine treatment also produced significant tolerance (ED50 shift= approximately 5-8-fold), but no mu-opioid receptor down-regulation. PTX dramatically reduced the acute potency of morphine and blocked the further development of tolerance by both etorphine and morphine treatments. However, PTX had no effect on etorphine-induced mu-opioid receptor down-regulation in brain, cord, or midbrain. These results suggest that PTX-sensitive G-proteins have a minimal role in agonist-induced mu-opioid receptor density regulation in vivo, but are critical in mediating acute and chronic functional effects of opioids such as analgesia and tolerance.

  1. Celastrol suppresses invasion of colon and pancreatic cancer cells through the downregulation of expression of CXCR4 chemokine receptor.

    PubMed

    Yadav, Vivek R; Sung, Bokyung; Prasad, Sahdeo; Kannappan, Ramaswamy; Cho, Sung-Gook; Liu, Mingyao; Chaturvedi, Madan M; Aggarwal, Bharat B

    2010-12-01

    Although metastasis accounts for >90% of cancer-related deaths, no therapeutic that targets this process has yet been approved. Because the chemokine receptor CXCR4 is one of the targets closely linked with tumor metastasis, inhibitors of this receptor have the potential to abrogate metastasis. In the current report, we demonstrate that celastrol can downregulate the CXCR4 expression on breast cancer MCF-7 cells stably transfected with HER2, an oncogene known to induce the chemokine receptor. Downregulation of CXCR4 by the triterpenoid was not cell type-specific as downregulation occurred in colon cancer, squamous cell carcinoma, and pancreatic cancer cells. Decrease in CXCR4 expression was not due to proteolysis as neither proteasome inhibitors nor lysosomal stabilization had any effect. Quantitative reverse transcription polymerase chain reaction analysis revealed that downregulation of CXCR4 messenger RNA (mRNA) by celastrol occurred at the translational level. Chromatin immunoprecipitation analysis revealed regulation at the transcriptional level as well. Abrogation of the chemokine receptor by celastrol or by gene-silencing was accompanied by suppression of invasiveness of colon cancer cells induced by CXCL12, the ligand for CXCR4. This effect was not cell type-specific as celastrol also abolished invasiveness of pancreatic tumor cells, and this effect again correlated with the disappearance of both the CXCR4 mRNA and CXCR4 protein. Other triterpenes, such as withaferin A and gedunin, which are known to inhibit Hsp90, did not downregulate CXCR4 expression, indicating that the effects were specific to celastrol. Overall, these results show that celastrol has potential in suppressing invasion and metastasis of cancer cells by down-modulation of CXCR4 expression. PMID:20798912

  2. DOWNREGULATION OF HYPOTHALAMIC INSULIN RECEPTOR EXPRESSION ELICITS DEPRESSIVE-LIKE BEHAVIORS IN RATS

    PubMed Central

    Grillo, Claudia A.; Piroli, Gerardo G.; Kaigler, Kris F.; Wilson, Steven P.; Wilson, Marlene A.; Reagan, Lawrence P.

    2011-01-01

    Ongoing epidemiological studies estimate that greater than 60% of the adult US population may be categorized as either overweight or obese. There is a growing appreciation that the complications of obesity extend to the central nervous system (CNS) and may result in increased risk for neurological co-morbidities like depressive illness. One potential mechanistic mediator linking obesity and depressive illness is the adipocyte derived hormone leptin. We previously demonstrated that lentivirus-mediated downregulation of hypothalamic insulin receptors increases body weight, adiposity and plasma leptin levels, which is consistent with features of the metabolic syndrome. Using this novel model of obesity, we examined performance in the forced swim test (FST), the sucrose preference test and the elevated plus maze (EPM), approaches that are often used as measures of depressive-like and anxiety-like behaviors, in rats that received third ventricular injections of either an insulin receptor antisense lentivirus (hypo-IRAS) or a control lentivirus (hypo-Con). Hypo-IRAS rats exhibited significant increases in immobility time and corresponding decreases in active behaviors in the FST and exhibited anhedonia as measured by decreased sucrose intake compared to hypo-Con rats. Hypo-IRAS rats also exhibited increases in anxiety-like behaviors in the EPM. Plasma, hippocampal and amygdalar brain-derived neurotrophic factor (BDNF) levels were reduced in hypo-IRAS rats, suggesting that the obesity/hyperleptinemic phenotype may elicit this behavioral phenotype through modulation of neurotrophic factor expression. Collectively, these data support the hypothesis for an increased risk for mood disorders in obesity, which may be related to decreased expression of hippocampal and amygdalar BDNF. PMID:21458499

  3. Genetic and epigenetic variation of the glucocorticoid receptor (NR3C1) in placenta and infant neurobehavior.

    PubMed

    Bromer, Cailey; Marsit, Carmen J; Armstrong, David A; Padbury, James F; Lester, Barry

    2013-11-01

    The intrauterine environment can impact the developing infant by altering the function of the placenta through changes to the epigenetic regulatory features of this tissue. Genetic variation, too, may impact infant development or may modify the relationship between epigenetic alterations and infant outcomes. To examine the associations of these variations with early life infant neurodevelopment, we examined the extent of DNA methylation of the glucocorticoid receptor gene (NR3C1) promoter and a common single nucleotide polymorphism in the promoter region in a series of 186 placentas from healthy newborn infants. We associated these molecular features with specific summary measures from the NICU Network Neurobehavioral Scales. After controlling for genotype and confounders, we identified significant associations of NR3C1 methylation with infant quality of movement (p = .05) and with infant attention (p = .05), and a potential interaction between methylation and genotype on infant attention score. These results suggest that epigenetic alteration of the NR3C1 gene in the placentas of genetically susceptible infants can have impacts on neurodevelopment which may have lifelong impact on neurobehavioral and mental health outcomes. Further research is needed to more precisely define these relationships and the interaction between epigenetic alterations and genetic variations on infant health.

  4. Glucocorticoid receptor gene (NR3C1) promoter is hypermethylated in Thai females with major depressive disorder.

    PubMed

    Nantharat, M; Wanitchanon, T; Amesbutr, M; Tammachote, R; Praphanphoj, V

    2015-12-29

    Major depressive disorder (MDD) has been associated with the stress response in the brain, which is controlled by the hypothalamic-pituitary-adrenal (HPA) axis. This HPA negative feedback mechanism acts via the activation of glucocorticoid receptor, which is encoded by the NR3C1 gene. The methylation status at the promoter of NR3C1 has been correlated with traumatic experiences in early life, which develop into mental disorder. The aim of this study was to examine the potential associations between the methylation status of NR3C1 promoter, gene expression, blood plasma cortisol levels, and adulthood MDD. The study was conducted with 29 MDD patients (9 males, 20 females) and 33 normal individuals (7 males, 26 females). Bisulfite pyrosequencing on 7 CpG dinucleotides in the region showed significantly higher methylation levels at the CpG7 in MDD patients. When separated by gender, the methylation levels differed significantly in females, but not in males. No significant differences between NR3C1 gene expression level and plasma cortisol levels of MDD patients and normal controls were observed. These data suggest that higher levels of methylation at the NR3C1 promoter may be associated with MDD in a gender-specific manner.

  5. Novel antidepressant candidate RO-05 modulated glucocorticoid receptors activation and FKBP5 expression in chronic mild stress model in rats.

    PubMed

    Xing, Y; Hou, J; Meng, Q; Yang, M; Kurihara, H; Tian, J

    2015-04-01

    In this study, a novel TRI (triple reuptake inhibitors) antidepressant candidate RO-05 (4-[1-[1-(benzoyloxy)cyclohexyl]-2-(dimethylamino)ethyl]-phenyl benzoate) was investigated in TST (tail suspension test), FST (forced swimming test) and CMS (chronic mild stress) model. Results showed RO-05 significantly decreased the immobility time in FST and TST at 4.5-, 9-, 18-mg/kg in rats and 9-, 18-, 36-mg/kg in mice. Chronic administration of 18-mg/kg RO-05 improved the behavioral index, anhedonia and normalized the hyperactivity of HPA (hypothalamic-pituitary-adrenal axis) of CMS rats. We further investigated the possible mechanisms of RO-05 in the CMS model. Eighteen milligrams per kilogram of RO-05 chronic administration significantly reversed the increase of mRNA and protein expression of FKBP5 in the CMS rat hippocampus, which facilitated the activation of GR- (glucocorticoid receptor) and GR-responsive gene Foxo1 expression. RO-05 also elevated the expression of BDNF (brain-derived neurotrophic factor) in CMS rat hippocampus. In summary, our results indicated that RO-05 is a promising antidepressant candidate. The possible antidepressant mechanisms of RO-05 were the modulation of FKBP5 expression, GR activation, corresponding inhibition of HPA axis hyperactivity, and the increase of BDNF expression.

  6. Glucocorticoid-induced TNF receptor expression by T cells is reciprocally regulated by NF-kappaB and NFAT.

    PubMed

    Zhan, Yifan; Gerondakis, Steve; Coghill, Elise; Bourges, Dorothee; Xu, Yuekang; Brady, Jamie L; Lew, Andrew M

    2008-10-15

    Although the transcription factor Foxp3 is implicated in regulating glucocorticoid-induced TNF receptor (GITR) expression in the T regulatory cell lineage, little is known about how GITR is transcriptionally regulated in conventional T cells. In this study, we provide evidence that TCR-mediated GITR expression depends on the ligand affinity and the maturity of conventional T cells. A genetic dissection of GITR transcriptional control revealed that of the three transcription factors downstream of the classical NF-kappaB pathway (RelA, cRel, and NF-kappaB1), RelA is a critical positive regulator of GITR expression, although cRel and NF-kappaB1 also play a positive regulatory role. Consistent with this finding, inhibiting NF-kappaB using Bay11-7082 reduces GITR up-regulation. In contrast, NFAT acts as a negative regulator of GITR expression. This was evidenced by our findings that agents suppressing NFAT activity (e.g., cyclosporin A and FK506) enhanced TCR-mediated GITR expression, whereas agents enhancing NFAT activity (e.g., lithium chloride) suppressed TCR-mediated GITR up-regulation. Critically, the induction of GITR was found to confer protection to conventional T cells from TCR-mediated apoptosis. We propose therefore that two major transcriptional factors activated downstream of the TCR, namely, NF-kappaB and NFAT, act reciprocally to balance TCR-mediated GITR expression in conventional T cells, an outcome that appears to influence cell survival.

  7. Methylation of the Glucocorticoid Receptor (NR3C1) in Placenta Is Associated with Infant Cry Acoustics

    PubMed Central

    Sheinkopf, Stephen J.; Righi, Giulia; Marsit, Carmen J.; Lester, Barry M.

    2016-01-01

    Epigenetic mechanisms regulating expression of the glucocorticoid receptor gene (NR3C1) promoter may influence behavioral and biological aspects of stress response in human infants. Acoustic features of infant crying are an indicator of neurobehavioral and neurological status not yet investigated in relation to epigenetic mechanisms. We examined NR3C1 methylation in placental tissue from a series of 120 healthy newborn infants in relation to a detailed set of acoustic features extracted from newborn infant cries. We identified significant associations of NR3C1 methylation with energy variation in infants' cries as well as with the presence of very high fundamental frequency in cry utterances. The presence of high fundamental frequency in cry (above 1 kHz) has been linked to poor vocal tract control, poor regulation of stress response, and may be an indicator or poor neurobehavioral integrity. Thus, these results add to evidence linking epigenetic alteration of the NR3C1 gene in the placenta to neurodevelopmental features in infants. PMID:27313516

  8. Glucocorticoid Receptor (NR3C1) Variants Associate with the Muscle Strength and Size Response to Resistance Training.

    PubMed

    Ash, Garrett I; Kostek, Matthew A; Lee, Harold; Angelopoulos, Theodore J; Clarkson, Priscilla M; Gordon, Paul M; Moyna, Niall M; Visich, Paul S; Zoeller, Robert F; Price, Thomas B; Devaney, Joseph M; Gordish-Dressman, Heather; Thompson, Paul D; Hoffman, Eric P; Pescatello, Linda S

    2016-01-01

    Glucocorticoid receptor (NR3C1) polymorphisms associate with obesity, muscle strength, and cortisol sensitivity. We examined associations among four NR3C1 polymorphisms and the muscle response to resistance training (RT). European-American adults (n = 602, 23.8±0.4yr) completed a 12 week unilateral arm RT program. Maximum voluntary contraction (MVC) assessed isometric strength (kg) and MRI assessed biceps size (cm2) pre- and post-resistance training. Subjects were genotyped for NR3C1 -2722G>A, -1887G>A, -1017T>C, and +363A>G. Men carrying the -2722G allele gained less relative MVC (17.3±1.2vs33.5±6.1%) (p = 0.010) than AA homozygotes; men with -1887GG gained greater relative MVC than A allele carriers (19.6±1.4vs13.2±2.3%) (p = 0.016). Women carrying the -1017T allele gained greater relative size (18.7±0.5vs16.1±0.9%) (p = 0.016) than CC homozygotes. We found sex-specific NR3C1 associations with the muscle strength and size response to RT. Future studies should investigate whether these associations are partially explained by cortisol's actions in muscle tissue as they interact with sex differences in cortisol production. PMID:26821164

  9. Histone acetylation characterizes chromatin presetting by NF1 and Oct1 and enhances glucocorticoid receptor binding to the MMTV promoter

    SciTech Connect

    Astrand, Carolina; Belikov, Sergey; Wrange, Orjan

    2009-09-10

    Transcription from the mouse mammary tumor virus (MMTV) promoter is induced by the glucocorticoid receptor (GR). This switch was reconstituted in Xenopus oocytes. Previously, we showed that Nuclear Factor 1 (NF1) and Octamer Transcription Factor 1 (Oct1) bind constitutively to the MMTV promoter and thereby induce translational nucleosome positioning representing an intermediary, i.e. preset, state of nucleosome organization. Here we further characterize this NF1 and Oct1 induced preset chromatin in relation to the inactive and the hormone-activated state. The preset chromatin exhibits increased histone acetylation but does not cause dissociation of histone H1 as oppose to the hormone-activated state. Furthermore, upon hormone induction the preset MMTV chromatin displays an enhanced and prolonged GR binding capacity and transcription during an intrinsic and time-dependent silencing of the injected template. The silencing process correlates with a reduced histone acetylation. However, a histone deacetylase inhibitor, trichostatin A (TSA), does not counteract silencing in spite of its distinct stimulation of GR-DNA binding. The latter indicates the importance of histone acetylation to maintain DNA access for inducible factor binding. We discuss how constitutively bound factors such as NF1 and Oct1 may participate in the maintenance of tissue specificity of hormone responsive genes.

  10. Genetic and epigenetic variation of the glucocorticoid receptor (NR3C1) in placenta and infant neurobehavior.

    PubMed

    Bromer, Cailey; Marsit, Carmen J; Armstrong, David A; Padbury, James F; Lester, Barry

    2013-11-01

    The intrauterine environment can impact the developing infant by altering the function of the placenta through changes to the epigenetic regulatory features of this tissue. Genetic variation, too, may impact infant development or may modify the relationship between epigenetic alterations and infant outcomes. To examine the associations of these variations with early life infant neurodevelopment, we examined the extent of DNA methylation of the glucocorticoid receptor gene (NR3C1) promoter and a common single nucleotide polymorphism in the promoter region in a series of 186 placentas from healthy newborn infants. We associated these molecular features with specific summary measures from the NICU Network Neurobehavioral Scales. After controlling for genotype and confounders, we identified significant associations of NR3C1 methylation with infant quality of movement (p = .05) and with infant attention (p = .05), and a potential interaction between methylation and genotype on infant attention score. These results suggest that epigenetic alteration of the NR3C1 gene in the placentas of genetically susceptible infants can have impacts on neurodevelopment which may have lifelong impact on neurobehavioral and mental health outcomes. Further research is needed to more precisely define these relationships and the interaction between epigenetic alterations and genetic variations on infant health. PMID:22714792

  11. Glucocorticoid Receptor (NR3C1) Variants Associate with the Muscle Strength and Size Response to Resistance Training.

    PubMed

    Ash, Garrett I; Kostek, Matthew A; Lee, Harold; Angelopoulos, Theodore J; Clarkson, Priscilla M; Gordon, Paul M; Moyna, Niall M; Visich, Paul S; Zoeller, Robert F; Price, Thomas B; Devaney, Joseph M; Gordish-Dressman, Heather; Thompson, Paul D; Hoffman, Eric P; Pescatello, Linda S

    2016-01-01

    Glucocorticoid receptor (NR3C1) polymorphisms associate with obesity, muscle strength, and cortisol sensitivity. We examined associations among four NR3C1 polymorphisms and the muscle response to resistance training (RT). European-American adults (n = 602, 23.8±0.4yr) completed a 12 week unilateral arm RT program. Maximum voluntary contraction (MVC) assessed isometric strength (kg) and MRI assessed biceps size (cm2) pre- and post-resistance training. Subjects were genotyped for NR3C1 -2722G>A, -1887G>A, -1017T>C, and +363A>G. Men carrying the -2722G allele gained less relative MVC (17.3±1.2vs33.5±6.1%) (p = 0.010) than AA homozygotes; men with -1887GG gained greater relative MVC than A allele carriers (19.6±1.4vs13.2±2.3%) (p = 0.016). Women carrying the -1017T allele gained greater relative size (18.7±0.5vs16.1±0.9%) (p = 0.016) than CC homozygotes. We found sex-specific NR3C1 associations with the muscle strength and size response to RT. Future studies should investigate whether these associations are partially explained by cortisol's actions in muscle tissue as they interact with sex differences in cortisol production.

  12. Glucocorticoid Receptor (NR3C1) Variants Associate with the Muscle Strength and Size Response to Resistance Training

    PubMed Central

    Ash, Garrett I.; Kostek, Matthew A.; Lee, Harold; Angelopoulos, Theodore J.; Gordon, Paul M.; Moyna, Niall M.; Visich, Paul S.; Zoeller, Robert F.; Price, Thomas B.; Devaney, Joseph M.; Gordish-Dressman, Heather; Thompson, Paul D.; Hoffman, Eric P.; Pescatello, Linda S.

    2016-01-01

    Glucocorticoid receptor (NR3C1) polymorphisms associate with obesity, muscle strength, and cortisol sensitivity. We examined associations among four NR3C1 polymorphisms and the muscle response to resistance training (RT). European-American adults (n = 602, 23.8±0.4yr) completed a 12 week unilateral arm RT program. Maximum voluntary contraction (MVC) assessed isometric strength (kg) and MRI assessed biceps size (cm2) pre- and post-resistance training. Subjects were genotyped for NR3C1 -2722G>A, -1887G>A, -1017T>C, and +363A>G. Men carrying the -2722G allele gained less relative MVC (17.3±1.2vs33.5±6.1%) (p = 0.010) than AA homozygotes; men with -1887GG gained greater relative MVC than A allele carriers (19.6±1.4vs13.2±2.3%) (p = 0.016). Women carrying the -1017T allele gained greater relative size (18.7±0.5vs16.1±0.9%) (p = 0.016) than CC homozygotes. We found sex-specific NR3C1 associations with the muscle strength and size response to RT. Future studies should investigate whether these associations are partially explained by cortisol’s actions in muscle tissue as they interact with sex differences in cortisol production. PMID:26821164

  13. Epigenetic changes in the hypothalamic proopiomelanocortin and glucocorticoid receptor genes in the ovine fetus after periconceptional undernutrition.

    PubMed

    Stevens, Adam; Begum, Ghazala; Cook, Alice; Connor, Kristin; Rumball, Christopher; Oliver, Mark; Challis, John; Bloomfield, Frank; White, Anne

    2010-08-01

    Maternal food restriction is associated with the development of obesity in offspring. This study examined how maternal undernutrition in sheep affects the fetal hypothalamic glucocorticoid receptor (GR) and the appetite-regulating neuropeptides, proopiomelanocortin (POMC) and neuropeptide Y, which it regulates. In fetuses from ewes undernourished from -60 to +30 d around conception, there was increased histone H3K9 acetylation (1.63-fold) and marked hypomethylation (62% decrease) of the POMC gene promoter but no change in POMC expression. In the same group, acetylation of histone H3K9 associated with the hypothalamic GR gene was increased 1.60-fold and the GR promoter region was hypomethylated (53% decrease). In addition, there was a 4.7-fold increase in hypothalamic GR expression but no change in methylation of GR gene expression in the anterior pituitary or hippocampus. Interestingly, hypomethylation of both POMC and GR promoter markers in fetal hypothalami was also identified after maternal undernutrition from -60 to 0 d and -2 to +30 d. In comparison, the Oct4 gene, was hypermethylated in both control and underfed groups. Periconceptional undernutrition is therefore associated with marked epigenetic changes in hypothalamic genes. Increase in GR expression in the undernourished group may contribute to fetal programming of a predisposition to obesity, via altered GR regulation of POMC and neuropeptide Y. These epigenetic changes in GR and POMC in the hypothalamus may also predispose the offspring to altered regulation of food intake, energy expenditure, and glucose homeostasis later in life.

  14. The antidepressant desipramine is an arrestin-biased ligand at the α(2A)-adrenergic receptor driving receptor down-regulation in vitro and in vivo.

    PubMed

    Cottingham, Christopher; Chen, Yunjia; Jiao, Kai; Wang, Qin

    2011-10-14

    The neurobiological mechanisms of action underlying antidepressant drugs remain poorly understood. Desipramine (DMI) is an antidepressant classically characterized as an inhibitor of norepinephrine reuptake. Available evidence, however, suggests a mechanism more complex than simple reuptake inhibition. In the present study, we have characterized the direct interaction between DMI and the α(2A)-adrenergic receptor (α(2A)AR), a key regulator of noradrenergic neurotransmission with altered expression and function in depression. DMI alone was found to be sufficient to drive receptor internalization acutely and a robust down-regulation of α(2A)AR expression and signaling following prolonged stimulation in vitro. These effects are achieved through arrestin-biased regulation of the receptor, as DMI selectively induces recruitment of arrestin but not activation of heterotrimeric G proteins. Meanwhile, a physiologically relevant concentration of endogenous agonist (norepinephrine) was unable to sustain a down-regulation response. Prolonged in vivo administration of DMI resulted in significant down-regulation of synaptic α(2A)AR expression, a response that was lost in arrestin3-null animals. We contend that direct DMI-driven arrestin-mediated α(2A)AR down-regulation accounts for the therapeutically desirable but mechanistically unexplained adaptive alterations in receptor expression associated with this antidepressant. Our results provide novel insight into both the pharmacology of this antidepressant drug and the targeting of the α(2A)AR in depression.

  15. Muscarinic Receptors Control Frequency Tuning Through the Downregulation of an A-Type Potassium Current

    PubMed Central

    Ellis, Lee D.; Krahe, Rüdiger; Bourque, Charles W.; Dunn, Robert J.; Chacron, Maurice J.

    2016-01-01

    The functional role of cholinergic input in the modulation of sensory responses was studied using a combination of in vivo and in vitro electrophysiology supplemented by mathematical modeling. The electrosensory system of weakly electric fish recognizes different environmental stimuli by their unique alteration of a self-generated electric field. Variations in the patterns of stimuli are primarily distinguished based on their frequency. Pyramidal neurons in the electrosensory lateral line lobe (ELL) are often tuned to respond to specific input frequencies. Alterations in the tuning of the pyramidal neurons may allow weakly electric fish to preferentially select for certain stimuli. Here we show that muscarinic receptor activation in vivo enhances the excitability, burst firing, and subsequently the response of pyramidal cells to naturalistic sensory input. Through a combination of in vitro electrophysiology and mathematical modeling, we reveal that this enhanced excitability and bursting likely results from the down-regulation of an A-type potassium current. Further, we provide an explanation of the mechanism by which these currents can mediate frequency tuning. PMID:17615127

  16. Exposure to cigarette smoke downregulates β2-adrenergic receptor expression and upregulates inflammation in alveolar macrophages.

    PubMed

    Wang, Wei; Li, Xiaoguang; Xu, Jie

    2015-01-01

    Cigarette smoke-triggered inflammation is important in the pathophysiology of chronic obstructive pulmonary disease (COPD). β2-Adrenergic receptor (β2-AR) is abundantly expressed on inflammatory cells, which is associated with inflammation regulation. To observe alterations in inflammation, pathological changes in lung tissues, and detect changes in β2-AR expression, rats were exposed for 4 months to cigarette smoke. Pathological changes were observed in lung tissue sections. The levels of inflammatory mediators tumor necrosis factor (TNF)-α, interleukin (IL)-1β in bronchoalveolar lavage fluid (BALF), and lung tissues were measured using enzyme-linked immunosorbent assay (ELISA). Nuclear factor (NF)-κB activity was detected by electrophoretic mobility shift assay (EMSA). Exposure to this regimen of cigarette smoke induced peribronchial and perivascular lymphocytic aggregates and parenchymal accumulation of macrophages in rats. EMSA demonstrated that smoke exposure enhanced NF-κB activation in rats' alveolar macrophages (AMs). Compared with the control group, smoke exposure induced a notable increase in TNF-α and IL-1β in BALF, lung tissues, and a decrease of β2-AR expression of AMs. The expression of β2-AR from AMs was inversely correlated with TNF-α and IL-1β levels of BALF. These data demonstrated that chronic smoke-triggered lung inflammation was accompanied by down-regulation of β2-AR in rat lungs' AMs.

  17. Dysregulation of Hypothalamo-Pituitary-Adrenocortical Axis in Overweight Female Diabetic Subjects is Associated with Downregulation of Corticosteroid Receptors and 11β-HSD1 in the Brain.

    PubMed

    Li, S; Liao, Y; Wang, L; Huang, R; Yue, J; Xu, H; Zhou, H; Lou, Z; Hu, Y; Liu, W

    2016-03-01

    The objective of this work was to assess hypothalamo-pituitary-adrenocortical (HPA) axis dysregulation in overweight diabetic women and investigate the possible mechanism using overweight diabetic rats. Twenty-two overweight diabetic women were recruited alongside 34 lean and 23 overweight healthy women serving as controls. Dexamethasone suppression test (0.25 mg DST) and low dose adrenocorticotropic hormone (ACTH) stimulation assay were used to evaluate the HPA axis activity. Then, high fat diet (HF) and STZ-induced diabetic rats were utilized to investigate the possible mechanism. After measurement of corticosterone circadian patterns and dexamethasone suppression levels, mRNA amounts of mineralocorticoid receptors (MR), glucocorticoid receptors (GR), and 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) were determined by real time PCR at hippocampus, hypothalamus, and pituitary levels. Overweight diabetic women showed impaired HPA axis with negative feedback efficacy (suppression ratio F-DEX%: 0.52±0.06% vs. 0.49±0.06% vs. 0.14±0.08%), as well as increased adrenal cortisol secretion response to low dose ACTH stimulation. Interestingly, F-DEX% was negatively correlated with BMI (r=- 0.323, p=0.003), waist circumference (r=- 0.319, p=0.004), and HbA1c (r=- 0.334, p=0.002). Stepwise linear regression analysis showed F-DEX% was significantly related to HbA1c level (β=- 0.328, p=0.007) after adjusting for other covariates (age, BMI, waist circumference, SBP, TC, TG, and HOMA-IR). Furthermore, 11β-HSD1, MR, and GR mRNA expression levels were reduced at pituitary level while GR expression was downregulated at hippocampus level in HF and HF+STZ rats. In conclusion, hyperactive HPA axis in overweight diabetic subjects may be associated with downregulation of 11β-HSD1, MR, and GR in the brain. PMID:26212138

  18. Reversal of islet GIP receptor down-regulation and resistance to GIP by reducing hyperglycemia in the Zucker rat

    SciTech Connect

    Piteau, Shalea; Olver, Amy; Kim, Su-Jin; Winter, Kyle; Pospisilik, John Andrew; Lynn, Francis; Manhart, Susanne; Demuth, Hans-Ulrich; Speck, Madeleine; Pederson, Raymond A.; McIntosh, Christopher H.S.

    2007-11-03

    In type 2 diabetes (T2DM) {beta}-cell responsiveness to glucose-dependent insulinotropic polypeptide (GIP) is reduced. In a model of T2DM, the VDF Zucker rat, GIP receptor mRNA and protein levels were shown to be down-regulated. Possible restoration of responsiveness to GIP in Zucker rats by reducing hyperglycemia has been examined. ZDF rats with extreme hyperglycemia demonstrated greater islet GIP receptor mRNA down-regulation (94.3 {+-} 3.8%) than ZF rats (48.8 {+-} 22.8%). GIP receptor mRNA levels in ZDF rats returned to 83.0 {+-} 17.9% of lean following normalization of hyperglycemia by phlorizin treatment and pancreas perfusions demonstrated markedly improved GIP responsiveness. Treatment of VDF rats with a DP IV inhibitor (P32/98) resulted in improved glucose tolerance and restored sensitivity to GIP in isolated pancreata. These findings support the proposal that GIP receptor down-regulation in rodent T2DM is secondary to chronic hyperglycemia and that normalization of glycemia can restore GIP sensitivity.

  19. SUMO-Mediated Inhibition of Glucocorticoid Receptor Synergistic Activity Depends on Stable Assembly at the Promoter But Not on DAXX

    PubMed Central

    Holmstrom, Sam R.; Chupreta, Sergey; So, Alex Yick-Lun; Iñiguez-Lluhí, Jorge A.

    2008-01-01

    Multiple transcription factors, including members of the nuclear receptor family, harbor one or more copies of a short regulatory motif that limits synergistic transactivation in a context-dependent manner. These synergy control (SC) motifs exert their effects by serving as sites for posttranslational modification by small ubiquitin-like modifier (SUMO) proteins. By analyzing the requirements for both synergy control and SUMOylation in the glucocorticoid receptor (GR), we find that an intact ligand-binding domain and an engaged DNA- binding domain dimerization interface are necessary for effective synergy control. However, these features, which promote stable assembly of GR-DNA complexes, are required downstream of SUMOylation because their disruption or deletion does not interfere with SUMO modification. Remarkably, in the absence of these features, sensitivity to the effects of SUMOylation can be restored simply by stabilization of DNA interactions through a heterologous DNA binding domain. The data indicate that stable interaction with DNA is an important prerequisite for SUMO-dependent transcriptional inhibition. Analysis of genomic regions occupied by GR indicates that the effects of SC motif SUMOylation are most evident at multiple, near-ideal GR binding sites and that SUMOylation selectively affects the induction of linked endogenous genes. Although the SUMO-binding protein DAXX has been proposed to mediate the inhibitory effects of GR SUMOylation, we find that inhibition by DAXX is independent of GR SUMOylation. Furthermore, neither expression nor knockdown of DAXX influences SUMO effects on GR. We therefore propose that stable binding of GR to multiple sites on DNA allows for the SUMO-dependent recruitment of inhibitory factors distinct from DAXX. PMID:18562626

  20. Structural bisphenol analogues differentially target steroidogenesis in murine MA-10 Leydig cells as well as the glucocorticoid receptor.

    PubMed

    Roelofs, Maarke J E; van den Berg, Martin; Bovee, Toine F H; Piersma, Aldert H; van Duursen, Majorie B M

    2015-03-01

    Although much information on the endocrine activity of bisphenol A (BPA) is available, a proper human hazard assessment of analogues that are believed to have a less harmful toxicity profile is lacking. Here the possible effects of BPA, bisphenol F (BPF), bisphenol S (BPS), as well as the brominated structural analogue and widely used flame retardant tetrabromobisphenol A (TBBPA) on human glucocorticoid and androgen receptor (GR and AR) activation were assessed. BPA, BPF, and TBBPA showed clear GR and AR antagonism with IC50 values of 67 μM, 60 μM, and 22 nM for GR, and 39 μM, 20 μM, and 982 nM for AR, respectively, whereas BPS did not affect receptor activity. In addition, murine MA-10 Leydig cells exposed to the bisphenol analogues were assessed for changes in secreted steroid hormone levels. Testicular steroidogenesis was altered by all bisphenol analogues tested. TBBPA effects were more directed towards the male end products and induced testosterone synthesis, while BPF and BPS predominantly increased the levels of progestagens that are formed in the beginning of the steroidogenic pathway. The MA-10 Leydig cell assay shows added value over the widely used H295R steroidogenesis assay because of its fetal-like characteristics and specificity for the physiologically more relevant testicular Δ4 steroidogenic pathway. Therefore, adding an in vitro assay covering fetal testicular steroidogenesis, such as the MA-10 cell line, to the panel of tests used to screen potential endocrine disruptors, is highly recommendable. PMID:25576683

  1. Role of hippocampal β-adrenergic and glucocorticoid receptors in the novelty-induced enhancement of fear extinction.

    PubMed

    Liu, Jian-Feng; Yang, Chang; Deng, Jia-Hui; Yan, Wei; Wang, Hui-Min; Luo, Yi-Xiao; Shi, Hai-Shui; Meng, Shi-Qiu; Chai, Bai-Sheng; Fang, Qin; Chai, Ning; Xue, Yan-Xue; Sun, Jia; Chen, Chen; Wang, Xue-Yi; Wang, Ji-Shi; Lu, Lin

    2015-05-27

    Fear extinction forms a new memory but does not erase the original fear memory. Exposure to novelty facilitates transfer of short-term extinction memory to long-lasting memory. However, the underlying cellular and molecular mechanisms are still unclear. Using a classical contextual fear-conditioning model, we investigated the effect of novelty on long-lasting extinction memory in rats. We found that exposure to a novel environment but not familiar environment 1 h before or after extinction enhanced extinction long-term memory (LTM) and reduced fear reinstatement. However, exploring novelty 6 h before or after extinction had no such effect. Infusion of the β-adrenergic receptor (βAR) inhibitor propranolol and glucocorticoid receptor (GR) inhibitor RU486 into the CA1 area of the dorsal hippocampus before novelty exposure blocked the effect of novelty on extinction memory. Propranolol prevented activation of the hippocampal PKA-CREB pathway, and RU486 prevented activation of the hippocampal extracellular signal-regulated kinase 1/2 (Erk1/2)-CREB pathway induced by novelty exposure. These results indicate that the hippocampal βAR-PKA-CREB and GR-Erk1/2-CREB pathways mediate the extinction-enhancing effect of novelty exposure. Infusion of RU486 or the Erk1/2 inhibitor U0126, but not propranolol or the PKA inhibitor Rp-cAMPS, into the CA1 before extinction disrupted the formation of extinction LTM, suggesting that hippocampal GR and Erk1/2 but not βAR or PKA play critical roles in this process. These results indicate that novelty promotes extinction memory via hippocampal βAR- and GR-dependent pathways, and Erk1/2 may serve as a behavioral tag of extinction.

  2. Structural bisphenol analogues differentially target steroidogenesis in murine MA-10 Leydig cells as well as the glucocorticoid receptor.

    PubMed

    Roelofs, Maarke J E; van den Berg, Martin; Bovee, Toine F H; Piersma, Aldert H; van Duursen, Majorie B M

    2015-03-01

    Although much information on the endocrine activity of bisphenol A (BPA) is available, a proper human hazard assessment of analogues that are believed to have a less harmful toxicity profile is lacking. Here the possible effects of BPA, bisphenol F (BPF), bisphenol S (BPS), as well as the brominated structural analogue and widely used flame retardant tetrabromobisphenol A (TBBPA) on human glucocorticoid and androgen receptor (GR and AR) activation were assessed. BPA, BPF, and TBBPA showed clear GR and AR antagonism with IC50 values of 67 μM, 60 μM, and 22 nM for GR, and 39 μM, 20 μM, and 982 nM for AR, respectively, whereas BPS did not affect receptor activity. In addition, murine MA-10 Leydig cells exposed to the bisphenol analogues were assessed for changes in secreted steroid hormone levels. Testicular steroidogenesis was altered by all bisphenol analogues tested. TBBPA effects were more directed towards the male end products and induced testosterone synthesis, while BPF and BPS predominantly increased the levels of progestagens that are formed in the beginning of the steroidogenic pathway. The MA-10 Leydig cell assay shows added value over the widely used H295R steroidogenesis assay because of its fetal-like characteristics and specificity for the physiologically more relevant testicular Δ4 steroidogenic pathway. Therefore, adding an in vitro assay covering fetal testicular steroidogenesis, such as the MA-10 cell line, to the panel of tests used to screen potential endocrine disruptors, is highly recommendable.

  3. Glucocorticoid receptor impairment alters CNS responses to a psychological stressor: an in vivo microdialysis study in transgenic mice.

    PubMed

    Linthorst, A C; Flachskamm, C; Barden, N; Holsboer, F; Reul, J M

    2000-01-01

    To study the consequences of impaired functioning of the glucocorticoid receptor (GR) for behavioural, neuroendocrine and neurochemical responses to a psychological stressor, a transgenic mouse expressing antisense RNA against GR was used. Previous studies on these transgenic mice have shown that impairment of GR evolves in disturbed neuroendocrine regulation and certain behavioural responses to stress. Here we investigated putative disturbances on the level of brain neurotransmission in GR-impaired (GR-i) mice using an in vivo microdialysis method. Through a microdialysis probe in the hippocampus, serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and free corticosterone [as an index of hypothalamic-pituitary-adrenocortical (HPA) axis activity] were monitored. Moreover, specific behaviours (e.g. grooming, eating/drinking, sniffing, nest building and locomotion) displayed by the mice during collection of the dialysates were scored. Measurement of dialysate concentrations of corticosterone on days 1 and 3 after insertion of the microdialysis probe showed that the free levels of this glucocorticoid were significantly lower in GR-i mice toward the evening. On day 2 after insertion of the microdialysis probe, baseline values of dialysate corticosterone, 5-HT and 5-HIAA were assessed, after which mice were exposed to a rat placed into their home cage. The rat and mouse were separated by a Plexiglas wall. A positive correlation between baseline hippocampal extracellular levels of 5-HT and 5-HIAA and the time spent performing active behaviours was observed in both genotypes. The main active behaviour performed at the baseline was grooming behaviour. During the rat exposure period, control mice remained mostly sitting and/or lying with their eyes fixed on the rat. Moreover, they showed a profound rise in free corticosterone levels. In contrast, GR-i mice displayed significantly more activities along the separation wall and a trend toward more grooming behaviour, but no

  4. Selective Non-Steroidal Glucocorticoid Receptor Agonists Attenuate Inflammation but Do Not Impair Intestinal Epithelial Cell Restitution In Vitro

    PubMed Central

    Reuter, Kerstin C.; Loitsch, Stefan M.; Dignass, Axel U.; Steinhilber, Dieter; Stein, Jürgen

    2012-01-01

    Introduction Despite the excellent anti-inflammatory and immunosuppressive action of glucocorticoids (GCs), their use for the treatment of inflammatory bowel disease (IBD) still carries significant risks in terms of frequently occurring severe side effects, such as the impairment of intestinal tissue repair. The recently-introduced selective glucocorticoid receptor (GR) agonists (SEGRAs) offer anti-inflammatory action comparable to that of common GCs, but with a reduced side effect profile. Methods The in vitro effects of the non-steroidal SEGRAs Compound A (CpdA) and ZK216348, were investigated in intestinal epithelial cells and compared to those of Dexamethasone (Dex). GR translocation was shown by immunfluorescence and Western blot analysis. Trans-repressive effects were studied by means of NF-κB/p65 activity and IL-8 levels, trans-activation potency by reporter gene assay. Flow cytometry was used to assess apoptosis of cells exposed to SEGRAs. The effects on IEC-6 and HaCaT cell restitution were determined using an in vitro wound healing model, cell proliferation by BrdU assay. In addition, influences on the TGF-β- or EGF/ERK1/2/MAPK-pathway were evaluated by reporter gene assay, Western blot and qPCR analysis. Results Dex, CpdA and ZK216348 were found to be functional GR agonists. In terms of trans-repression, CpdA and ZK216348 effectively inhibited NF-κB activity and IL-8 secretion, but showed less trans-activation potency. Furthermore, unlike SEGRAs, Dex caused a dose-dependent inhibition of cell restitution with no effect on cell proliferation. These differences in epithelial restitution were TGF-β-independent but Dex inhibited the EGF/ERK1/2/MAPK-pathway important for intestinal epithelial wound healing by induction of MKP-1 and Annexin-1 which was not affected by CpdA or ZK216348. Conclusion Collectively, our results indicate that, while their anti-inflammatory activity is comparable to Dex, SEGRAs show fewer side effects with respect to wound healing

  5. The clinical use of glucocorticoids.

    PubMed

    Kehrl, J H; Fauci, A S

    1983-01-01

    Glucocorticoids are potent anti-inflammatory agents and their administration results in a wide range of effects on inflammatory and immunologically mediated disease processes. The precise mechanisms by which glucocorticoids impair the human immune response are unknown. Intracytoplasmic glucocorticoids specific receptors are important in the specificity of glucocorticoid actions. Glucocorticoid administration results in neutrophillia, monocytopenia, lymphopenia, and eosinopenia. A principle mechanism whereby glucocorticoids limit inflammation is by limiting the access of leukocytes, particularly neutrophils, to inflammatory sites. Neutrophil function is relatively refractory while monocyte and T-cell function is more easily impaired. A variety of glucocorticoid preparations are available for use, and appreciation of their relative potency and plasma half-lives is essential for designing therapeutic regimens. High doses and frequent administration of glucocorticoids are necessary in order to induce a remission in patients with flagrantly active disease. Once a remission is induced, the glucocorticoid regimen should be adjusted to attain maximal therapeutic benefit with minimal adverse effects. Alternate day dosage regimens can often be used to maintain a remission.

  6. Insights from the predicted interactions of plant derived compounds to the gluco-corticoid receptor as an alternative to dexa-methasone

    PubMed Central

    Sarmah, Rajeev

    2012-01-01

    Dexamethasone (DEX) an anti-inflamatory 9-fluoro-glucocorticoid, activates the cytosolic glucocorticoid receptor (GR) binding to its Ligand Binding Domain (LBD). The GR-ligand complex then translocates to the nucleus and binds to the Glucocorticoid Response Element (GRE) resulting up-regulation of target gene expression of anti-inflamatory proteins. DEX is one of the most effective ligand for GR activation but comply to side effects. Therefore, alternative for DEX – plant metabolites of Calotropis sp and Swertia chirata were screened using docking appraoch. These plants compounds were selected because; parts of these plants are widely used againsts inflamation, allergy, asthma etc. Three metabolites of Swertia chirata namely Gentianine (GENT), Xanthone (XANT) and Swerchirin (SWER) are found to be occupying the same binding pocket in the LBD of the GR (PDB ID 1M2Z). The binding affinity as reflected by binding energies of GENT-1M2Z, XANT-1M2Z and SWER-1M2Z are -5.6, -6.7 and -6.7, and all the output parameter of the respective compounds positively correlates with that of DEX-1M2Z with r = 0.9, 0.6 and 0.6 respectively indicating similar GR activation function. Visualization analysis of the models clearly indicates that GENT and SWER may be GR activators. Rest of the compounds mostly docked onto the surface of the receptor molecule. PMID:23275688

  7. Insights from the predicted interactions of plant derived compounds to the gluco-corticoid receptor as an alternative to dexa-methasone.

    PubMed

    Sarmah, Rajeev

    2012-01-01

    Dexamethasone (DEX) an anti-inflamatory 9-fluoro-glucocorticoid, activates the cytosolic glucocorticoid receptor (GR) binding to its Ligand Binding Domain (LBD). The GR-ligand complex then translocates to the nucleus and binds to the Glucocorticoid Response Element (GRE) resulting up-regulation of target gene expression of anti-inflamatory proteins. DEX is one of the most effective ligand for GR activation but comply to side effects. Therefore, alternative for DEX - plant metabolites of Calotropis sp and Swertia chirata were screened using docking appraoch. These plants compounds were selected because; parts of these plants are widely used againsts inflamation, allergy, asthma etc. Three metabolites of Swertia chirata namely Gentianine (GENT), Xanthone (XANT) and Swerchirin (SWER) are found to be occupying the same binding pocket in the LBD of the GR (PDB ID 1M2Z). The binding affinity as reflected by binding energies of GENT-1M2Z, XANT-1M2Z and SWER-1M2Z are -5.6, -6.7 and -6.7, and all the output parameter of the respective compounds positively correlates with that of DEX-1M2Z with r = 0.9, 0.6 and 0.6 respectively indicating similar GR activation function. Visualization analysis of the models clearly indicates that GENT and SWER may be GR activators. Rest of the compounds mostly docked onto the surface of the receptor molecule.

  8. Adult glucocorticoid exposure leads to transcriptional and DNA methylation changes in nuclear steroid receptors in the hippocampus and kidney of mouse male offspring.

    PubMed

    Petropoulos, Sophie; Matthews, Stephen G; Szyf, Moshe

    2014-02-01

    Synthetic glucocorticoids (sGCs) are commonly prescribed for the management of inflammatory and endocrine disorders. However, nothing is known regarding the effects of sGC on adult germline methylome and whether these effects can be transmitted to the next generation. We hypothesized that administration of sGC to adult male mice alters DNA methylation in mature sperm and modifies the transcription and methylation of steroid receptors in male F1 offspring. Adult C57BL/6 males (n = 10/group) were injected on five consecutive days with 1 mg/kg sGC (i.e., dexamethasone) or vehicle and euthanized 35 or 60 days after initial treatment or bred with control females (60 days postinitial treatment; n = 5/group). A significant increase in global non-CpG methylation was observed in F0 sperm 60 days following sGC treatment. In the hippocampus and kidney of Postnatal Day 50 (PND50) and PND240 male offspring derived from fathers exposed to sGC, significant differences in mineralocorticoid receptor (Nr3c2; Mr), estrogen alpha receptor (Nr3a1; Ers1), and glucocorticoid receptor (Nr3c1; Gr) expression were observed. Furthermore, significant demethylation in regulatory regions of Mr, Gr, and Esr1 was observed in the PND50 kidney derived from fathers exposed to sGC. This is the first demonstration that paternal pharmacological exposure to sGC can alter the expression and DNA methylation of nuclear steroid receptors in brain and somatic tissues of offspring. These findings provide proof of principle that adult male exposure to sGC can affect DNA methylation and gene expression in offspring, indicating the possibility that adult experiences that evoke increases in endogenous glucocorticoid (i.e., stress) might have similar effects.

  9. Glucocorticoid-remediable aldosteronism.

    PubMed

    Halperin, Florencia; Dluhy, Robert G

    2011-06-01

    Glucocorticoid-remediable aldosteronism (GRA) is a hereditary form of primary hyperaldosteronism and the most common monogenic cause of hypertension. A chimeric gene duplication leads to ectopic aldosterone synthase activity in the cortisol-producing zona fasciculata of the adrenal cortex, under the regulation of adrenocorticotropin (ACTH). Hypertension typically develops in childhood, and may be refractory to standard therapies. Hypokalemia is uncommon in the absence of treatment with diuretics. The discovery of the genetic basis of the disorder has permitted the development of accurate diagnostic testing. Glucocorticoid suppression of ACTH is the mainstay of treatment; alternative treatments include mineralocorticoid receptor antagonists.

  10. Homologous down-regulation of the insulin receptor is associated with increased receptor biosynthesis in cultured human lymphocytes (IM-9 line)

    SciTech Connect

    Rouiller, D.G.; Gorden, P.

    1987-01-01

    Cultured IM-9 lymphocytes were preincubated with 1 ..mu..M insulin, a condition resulting in a 56% reduction in cell surface insulin receptors. Cellular proteins were then metabolically labeled, and the radioactivity incorporated into the insulin proreceptor and receptor mature subunits was measured over a 4-hr chase period. As early as 30 min of chase, incorporation into the proreceptor was 28 +/- 6% higher in down-regulated cells than in control cells. By 1 hr of chase, the difference reached 41 +/- 14% for the proreceptor and 84 +/- 28% for the ..cap alpha.. subunit, values returned to normal by 2 hr. At 4 hr of chase, labeling of the ..cap alpha.. subunit of down-regulated cells was diminished 36 +/- 9% below control. The increased biosynthetic rate of the proreceptor was more prominent when the chase medium contained 25 ..mu..M monensin, an inhibitor of processing of the proreceptor into mature subunits. Similar effects occurred whether (/sup 3/H)mannose or (/sup 3/H)lysine was used as biosynthetic marker. The effect was specific for the insulin receptor. These data demonstrate that insulin receptor homologous down-regulation is associated with increased proreceptor biosynthesis and processing into mature subunits. This might represent a cellular mechanism compensating for insulin-induced receptor loss.

  11. Ligand-induced down-regulation of the cannabinoid 1 receptor is mediated by the G-protein-coupled receptor-associated sorting protein GASP1.

    PubMed

    Martini, Lene; Waldhoer, Maria; Pusch, Margareta; Kharazia, Viktor; Fong, Jamie; Lee, Josephine H; Freissmuth, Clarissa; Whistler, Jennifer L

    2007-03-01

    The cannabinoid 1 receptor (CB1R) is one of the most abundant seven transmembrane (7TM) spanning/G-protein-coupled receptors in the central nervous system and plays an important role in pain transmission, feeding, and the rewarding effects of cannabis. Tolerance to cannabinoids has been widely observed after long-term use, with concomitant receptor desensitization and/or down-regulation depending on the brain region studied. Several CB1R agonists promote receptor internalization after activation, but the postendocytic sorting of the receptor has not been studied in detail. Utilizing human embryonic kidney (HEK293) cells stably expressing the CB1R and primary cultured neurons expressing endogenous CB1R, we show that treatment with cannabinoid agonists results in CB1R degradation after endocytosis and that the G-protein-coupled receptor-associated sorting protein GASP1 plays a major role in the postendocytic sorting process. Thus, these results may identify a molecular mechanism underlying tolerance and receptor down-regulation after long-term use of cannabinoids.

  12. Distribution of beta-adrenergic receptors in failing human myocardium. Implications for mechanisms of down-regulation

    SciTech Connect

    Murphree, S.S.; Saffitz, J.E.

    1989-06-01

    The density of beta-adrenergic receptors is reduced in crude membranes prepared from failing human myocardium. We used quantitative autoradiography of radioligand binding sites in intact tissue slices to determine whether the total tissue content of receptors is reduced and to characterize the transmural distribution of receptors in cardiac myocytes and the coronary vasculature in hearts obtained from nine cardiac transplant patients with severe congestive failure. Binding of (125Iodo)cyanopindolol to transmural slices of human myocardium was rapid, saturable, stereoselective, and displaceable by agonists and antagonists with an appropriate rank order of potency. Binding isotherms in four normal and nine failing ventricles showed a significant reduction in the total tissue content of beta-receptors in failing myocardium (38.3 +/- 2.0 fmol/mg protein) compared with normal tissue (52.4 +/- 1.7 fmol/mg protein, p = 0.038). In the normal ventricles, the greatest receptor density was observed autoradiographically in myocytic regions of the subendocardium. Receptor density of the coronary arterioles was approximately 70% of that in adjacent myocytic regions. The density of binding sites in both myocytic regions and arterioles was diminished in all regions of the failing ventricles, but down-regulation was due primarily to a selective reduction of beta-receptors of subendocardial myocytes (63 +/- 5% of subepicardial receptor density vs. 115 +/- 6% in controls, p less than 0.0001). These observations indicate that down-regulation occurs nonuniformly in the transmural distribution and thus is likely not related simply to elevated circulating catecholamine levels.

  13. Behavioral and neurochemical characterization of TrkB-dependent mechanisms of agomelatine in glucocorticoid receptor-impaired mice.

    PubMed

    Boulle, F; Velthuis, H; Koedam, K; Steinbusch, H W; van den Hove, D L A; Kenis, G; Gabriel, C; Mocaer, E; Franc, B; Rognan, D; Mongeau, R; Lanfumey, L

    2016-01-01

    Growing evidence indicates that impairment of the stress response, in particular the negative feedback regulation mechanism exerted by the hypothalamo-pituitary-adrenal (HPA) axis, might be responsible for the hippocampal atrophy observed in depressed patients. Antidepressants, possibly through the activation of BDNF signaling, may enhance neuroplasticity and restore normal hippocampal functions. In this context, glucocorticoid receptor-impaired (GR-i) mice-a transgenic mouse model of reduced GR-induced negative feedback regulation of the HPA axis-were used to investigate the role of BDNF/TrkB signaling in the behavioral and neurochemical effects of the new generation antidepressant drug, agomelatine. GR-i mice exhibited marked alterations in depressive-like and anxiety-like behaviors, together with a decreased cell proliferation and altered levels of neuroplastic and epigenetic markers in the hippocampus. GR-i mice and their wild-type littermates were treated for 21 days with vehicle, agomelatine (50mg/kg/day; i.p) or the TrkB inhibitor Ana-12 (0.5mg/kg/day, i.p) alone, or in combination with agomelatine. Chronic treatment with agomelatine resulted in antidepressant-like effects in GR-i mice and reversed the deficit in hippocampal cell proliferation and some of the alterations of mRNA plasticity markers in GR-i mice. Ana-12 blocked the effect of agomelatine on motor activity as well as its ability to restore a normal hippocampal cell proliferation and expression of neurotrophic factors. Altogether, our findings indicate that agomelatine requires TrkB signaling to reverse some of the molecular and behavioral alterations caused by HPA axis impairment. PMID:26653128

  14. Development and validation of an immunohistochemistry assay to assess glucocorticoid receptor expression for clinical trials of mifepristone in breast cancer

    PubMed Central

    Baker, Gabrielle M; Murphy, Tiffany; Block, Thaddeus; Nguyen, Dat; Lynch, Frank J

    2015-01-01

    Background Glucocorticoid receptor (GR) activity has been associated with chemotherapy resistance and poor outcomes in patients with triple negative breast cancer (TNBC). The aim of this study was to develop an immunohistochemistry (IHC) assay to assess GR expression in archival formalin-fixed, paraffin-embedded human invasive breast carcinoma samples. Methods An optimized GR assay protocol was developed using rabbit monoclonal antibody to GR clone D8H2. Precision and reproducibility of the GR IHC assay was determined by conducting multiple staining runs of four invasive breast carcinoma samples using replicate serial sections. Assay sensitivity was examined in 50 TNBC samples (>10 mm) obtained from a tumor bank, and 43 paired TNBC samples from a tissue microarray (TMA) (1.5 mm). GR positivity was assessed using a percent scoring approach with a ≥10% cutoff for nuclear staining of tumor cells at any intensity. Analysis of the paired TMA cores was performed by averaging the scores of the two cores for each case. Results Equivalent cellular patterns of GR reactivity were observed in all replicates from the multiple staining runs; coefficients of variation did not exceed 4.7% for average H-scores greater than 3.4, thus meeting the criteria for assay precision and reproducibility (coefficient of variation ≤20%). GR expression in TNBC single-tissue samples and TMA cores was characterized as mostly nuclear, with some concurrent cytoplasmic reactivity. Eighty-four percent of the 49 evaluable TNBC samples and 60% of the 42 evaluable paired TMA samples were positive for GR expression. Conclusion A robust and reproducible GR IHC assay was successfully developed for use in invasive breast carcinoma tissues. Differences in GR expression between larger single tissues and smaller TMA cores illustrate the heterogeneity of the disease, as well as potential intra-tumoral heterogeneity. This assay is currently being utilized in clinical trials of mifepristone, a GR antagonist

  15. Epigenetic modifications of the glucocorticoid receptor gene are associated with the vulnerability to psychopathology in childhood maltreatment.

    PubMed

    Radtke, K M; Schauer, M; Gunter, H M; Ruf-Leuschner, M; Sill, J; Meyer, A; Elbert, T

    2015-01-01

    Stress, particularly when experienced early in life, can have profound implications for mental health. Previous research covering various tissues such as the brain, suggests that the detrimental impact of early-life stress (ELS) on mental health is mediated via epigenetic modifications including DNA methylation. Genes of the hypothalamic-pituitary-adrenal axis--in particular, the glucocorticoid receptor (hGR) gene--stand out as key targets for ELS. Even though the link between hGR methylation and either ELS or psychopathology is fairly well established, the mutually dependent relationships between ELS, DNA methylation and psychopathology remain to be uncovered. The specific psychopathology an individual might develop in the aftermath of stressful events can be highly variable, however, most studies investigating hGR methylation and psychopathology suffer from being limited to a single symptom cluster of mental disorders. Here, we screened volunteers for childhood maltreatment and analyzed whether it associates with hGR methylation in lymphocytes and a range of measures of psychological ill-health. hGR methylation in lymphocytes most likely reflects methylation patterns found in the brain and thus provides valuable insights into the etiology of psychopathology. We find the interaction between childhood maltreatment and hGR methylation to be strongly correlated with an increased vulnerability to psychopathology providing evidence of epigenome × environment interactions. Furthermore, our results indicate an additive effect of childhood maltreatment and hGR methylation in predicting borderline personality disorder (BPD)-associated symptoms, suggesting that the combination of both ELS and DNA methylation that possibly represents unfavorable events experienced even earlier in life poses the risk for BPD. PMID:26080088

  16. [ASSOCIATION OF CYCLIC CITRULLINATED PEPTIDE ANTIBODIES LEVEL WITH RHEUMATOID ARTHRITIS ACTIVITY BASED ON GLUCOCORTICOID RECEPTOR GENE BBL1 POLYMORPHISM].

    PubMed

    Prystupa, L; Savchenko, O; Koroza, S

    2015-10-01

    The ambiguity of facts on connection between glucocorticoid receptor gene (GR) Bcl1 polymorphism in rheumatoid arthritis (RA) and its activity as well as lack of facts on its association with serological variants of the desease, makes ir reasonable to investigate its connections between cyclic citrullinated peptide antibodiss (ACCP) concentration and clinico-laboratorial parameters of RA (DAS 28 desease activity score, C-reactive protein concentration (CRP) and erythrocyte sedimentation rate (ESR) level based on GR gene Bcl1 polymorphism. Study involved 161 RA patients aged over 40 as well as 96 healthy individuals. Routine examination of RA diagnostics, anthropometric and molecular genetic methods were used in the research. Statistical analysis of the results was performed using SPSS-17 program. It has been proved that there is no significant difference in GR gene Bcl1 polymorphism distribution based on DAS 28 RA desease activity score, ACCP concentration and ESR level. However, we have found out that G/G genotype bearers have positive correlation relationship between ACCP titre and RA activity by laboratorial parameters (CRP, ESR),DAS 28 score and rheumatoid factor (RF) which has not been found in C/C and C/G genotype bearing patients. The above indicates the association of G/G genotype by GR gene Bcl1 polymorphism with clinico-laboratorial parameters of RA inflammatory activity. In course of the study we have identified the existance of correlation relationship between ACCP concentration and DAS 28 score of RA activity, CRP concentration and ESR level in individuals bearing G/G gene by GR gene Bcl11 polymorphism gene. The association between GR gene Bcl1 polymorphism and clinico-laboratorial parameters of RA inflammatory activity has not been found. PMID:26483373

  17. Association of N-terminal domain polymorphisms of the porcine glucocorticoid receptor with carcass composition and meat quality traits.

    PubMed

    Reyer, Henry; Ponsuksili, Siriluck; Wimmers, Klaus; Murani, Eduard

    2014-02-01

    The glucocorticoid receptor (GR) is a ubiquitously acting transcription factor that is responsible for mediating the physiological response to stress and adaptation to environmental conditions. Genetic variation of a GR gene (NR3C1) may therefore contribute to multiple phenotypic alterations and influence relevant traits of animal production. Here, we examined effects of two non-synonymous mutations of the porcine NR3C1, leading to amino acid exchanges p.Glu13Asp (c.39A>C) and p.Val19Leu (c.55G>C) in the N-terminal domain of the GR, on meat quality and carcass composition. In addition, we explored their influence on transcriptional activity of GR in vitro. A commercial crossbreed Pietrain × (German Large White × German Landrace) herd (n = 545) in which genotypes and relevant traits had been collected was used to perform the association analysis. The single nucleotide polymorphism (SNP) c.55G>C was significantly associated with conductivity and meat color scores. These effects were highly consistent considering the physiological relationship between these traits. Association analysis of SNP c.39A>C also revealed significant effects on closely connected meat quality traits. In addition, SNP c.55G>C showed association with carcass traits, mainly those related to muscle deposition. The molecular mechanism of action of both amino acid substitutions remains obscure because neither showed significant influence on transcriptional activity of GR. Our study emphasizes NR3C1 as an important candidate gene for muscle-related traits in pigs, but further work is necessary to clarify the molecular background of the identified associations.

  18. Differential effects of antidepressants on glucocorticoid receptors in human primary blood cells and human monocytic U-937 cells.

    PubMed

    Heiske, Andreas; Jesberg, Jutta; Krieg, Jürgen-Christian; Vedder, Helmut

    2003-04-01

    A number of data support the assumption that antidepressants (ADs) normalize the altered function of the hypothalamic-pituitary-adrenocortical (HPA) system involved in the pathophysiology of depressive disorder via direct effects on glucocorticoid receptors (GRs). In the present study, we examined the tricyclic ADs desipramine (DESI) and imipramine (IMI), the noradrenaline reuptake inhibitor maprotiline (MAPRO), and the noradrenergic and specific serotonergic AD (NaSSA) mirtazapine (MIR) for their effects on GR expression in primary human leukocytes and in monocytic U-937 cells. Semiquantitative RT-PCR indicated that the ADs exert differential effects on GR-mRNA levels in both primary human leukocytes and U-937 cells: whereas MAPRO and IMI did not induce pronounced changes in GR-mRNA levels, DESI and MIR significantly decreased the amounts of GR-mRNA in both cell systems. Further characterization of the effects of MIR revealed a time dependency of the regulation with an initial increase of GR-mRNA levels above control levels after 2.5 h of treatment and a decrease after 4, 24, and 48 h of incubation. A dose-response analysis demonstrated maximal effects of MIR at a concentration of 10(-7) M. Immunohistochemical studies showed that MIR increased the GR protein levels in a time-dependent manner and that this upregulation appeared earlier by additional treatment with dexamethasone (DEX). A translocation of the GR protein from the cytoplasm to the nucleus was induced between 24 and 48 h of treatment with MIR and MIR/DEX, respectively. Taken together, our data further support the assumption that ADs influence the neuroendocrine and immune system via effects on cellular GRs. PMID:12655328

  19. The effect of sex and irritable bowel syndrome on HPA axis response and peripheral glucocorticoid receptor expression

    PubMed Central

    Videlock, Elizabeth J.; Shih, Wendy; Adeyemo, Mopelola; Mahurkar-Joshi, Swapna; Presson, Angela P.; Polytarchou, Christos; Alberto, Melissa; Iliopoulos, Dimitrios; Mayer, Emeran A.; Chang, Lin

    2016-01-01

    Background and aims Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis has been reported in irritable bowel syndrome (IBS). Enhanced HPA axis response has been associated with reduced glucocorticoid receptor (GR) mediated negative feedback inhibition. We aimed to study the effects of IBS status, sex, or presence of early adverse life events (EAL) on the cortisol response to corticotropin-releasing factor (CRF) and adrenocorticotropic hormone (ACTH), and on GR mRNA expression in peripheral blood mononuclear cells (PBMCs). Methods Rome III+ IBS patients and healthy controls underwent CRF (1 μg/kg ovine) and ACTH (250 μg) stimulation tests with serial plasma ACTH and cortisol levels measured (n = 116). GR mRNA levels were measured using quantitative PCR (n = 143). Area under the curve (AUC) and linear mixed effects models were used to compare ACTH and cortisol response measured across time between groups. Results There were divergent effects of IBS on the cortisol response to ACTH by sex. In men, IBS was associated with an increased AUC (p = 0.009), but in women AUC was blunted in IBS (p = 0.006). Men also had reduced GR mRNA expression (p = 0.007). Cumulative exposure to EALs was associated with an increased HPA response. Lower GR mRNA was associated with increased pituitary HPA response and increased severity of overall symptoms and abdominal pain in IBS. Conclusion This study highlights the importance of considering sex in studies of IBS and the stress response in general. Our findings also provide support for PBMC GR mRNA expression as a peripheral marker of central HPA response. PMID:27038676

  20. Characterization of Inhibitors of Glucocorticoid Receptor Nuclear Translocation: A Model of Cytoplasmic Dynein-Mediated Cargo Transport

    PubMed Central

    Daghestani, Hikmat N.; Zhu, Guangyu; Shinde, Sunita N.; Brodsky, Jeffrey L.

    2012-01-01

    Abstract Agonist-induced glucocorticoid receptor [GR] transport from the cytoplasm to the nucleus was used as a model to identify dynein-mediated cargo transport inhibitors. Cell-based screening of the library of pharmacologically active compound (LOPAC)-1280 collection identified several small molecules that stalled the agonist-induced transport of GR-green fluorescent protein (GFP) in a concentration-dependent manner. Fluorescent images of microtubule organization, nuclear DNA staining, expression of GR-GFP, and its subcellular distribution were inspected and quantified by image analysis to evaluate the impact of compounds on cell morphology, toxicity, and GR transport. Given the complexity of the multi-protein complex involved in dynein-mediated cargo transport and the variety of potential mechanisms for interruption of that process, we therefore developed and validated a panel of biochemical assays to investigate some of the more likely intracellular target(s) of the GR transport inhibitors. Although the apomorphine enantiomers exhibited the most potency toward the ATPase activities of cytoplasmic dynein, myosin, and the heat-shock proteins (HSPs), their apparent lack of specificity made them unattractive for further study in our quest. Other molecules appeared to be nonspecific inhibitors that targeted reactive cysteines of proteins. Ideally, specific retrograde transport inhibitors would either target dynein itself or one of the other important proteins associated with the transport process. Although the hits from the cell-based screen of the LOPAC-1280 collection did not exhibit this desired profile, this screening platform provided a promising phenotypic system for the discovery of dynein/HSP modulators. PMID:21919741

  1. Haploinsufficiency for NR3C1, the gene encoding the glucocorticoid receptor, in blastic plasmacytoid dendritic cell neoplasms.

    PubMed

    Emadali, Anouk; Hoghoughi, Neda; Duley, Samuel; Hajmirza, Azadeh; Verhoeyen, Els; Cosset, Francois-Loic; Bertrand, Philippe; Roumier, Christophe; Roggy, Anne; Suchaud-Martin, Céline; Chauvet, Martine; Bertrand, Sarah; Hamaidia, Sieme; Rousseaux, Sophie; Josserand, Véronique; Charles, Julie; Templier, Isabelle; Maeda, Takahiro; Bruder-Costa, Juliana; Chaperot, Laurence; Plumas, Joel; Jacob, Marie-Christine; Bonnefoix, Thierry; Park, Sophie; Gressin, Remy; Tensen, Cornelis P; Mecucci, Cristina; Macintyre, Elizabeth; Leroux, Dominique; Brambilla, Elisabeth; Nguyen-Khac, Florence; Luquet, Isabelle; Penther, Dominique; Bastard, Christian; Jardin, Fabrice; Lefebvre, Christine; Garnache, Francine; Callanan, Mary B

    2016-06-16

    Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive leukemia for which knowledge on disease mechanisms and effective therapies are currently lacking. Only a handful of recurring genetic mutations have been identified and none is specific to BPDCN. In this study, through molecular cloning in an index case that presented a balanced t(3;5)(q21;q31) and molecular cytogenetic analyses in a further 46 cases, we identify monoallelic deletion of NR3C1 (5q31), encoding the glucocorticoid receptor (GCR), in 13 of 47 (28%) BPDCN patients. Targeted deep sequencing in 36 BPDCN cases, including 10 with NR3C1 deletion, did not reveal NR3C1 point mutations or indels. Haploinsufficiency for NR3C1 defined a subset of BPDCN with lowered GCR expression and extremely poor overall survival (P = .0006). Consistent with a role for GCR in tumor suppression, functional analyses coupled with gene expression profiling identified corticoresistance and loss-of-EZH2 function as major downstream consequences of NR3C1 deletion in BPDCN. Subsequently, more detailed analyses of the t(3;5)(q21;q31) revealed fusion of NR3C1 to a long noncoding RNA (lncRNA) gene (lincRNA-3q) that encodes a novel, nuclear, noncoding RNA involved in the regulation of leukemia stem cell programs and G1/S transition, via E2F. Overexpression of lincRNA-3q was a consistent feature of malignant cells and could be abrogated by bromodomain and extraterminal domain (BET) protein inhibition. Taken together, this work points to NR3C1 as a haploinsufficient tumor suppressor in a subset of BPDCN and identifies BET inhibition, acting at least partially via lncRNA blockade, as a novel treatment option in BPDCN.

  2. Dexamethasone stimulated gene expression in peripheral blood indicates glucocorticoid-receptor hypersensitivity in job-related exhaustion.

    PubMed

    Menke, Andreas; Arloth, Janine; Gerber, Markus; Rex-Haffner, Monika; Uhr, Manfred; Holsboer, Florian; Binder, Elisabeth B; Holsboer-Trachsler, Edith; Beck, Johannes

    2014-06-01

    Work-related stress can lead to various health problems ranging from job-related exhaustion to psychiatric and somatic diseases. Biomarkers of job-related exhaustion could help to improve our understanding of the biological mechanisms and might be useful to guide prevention and treatment strategies. The present study included 12 male cases suffering from job-related exhaustion and 12 matched healthy controls. Severity of exhaustion was assessed with the Maslach Burnout Inventory (MBI) and the Shirom-Melamed Burnout Measure (SMBM). Whole genome expression profiles derived from whole blood cells (baseline and following glucocorticoid-receptor (GR) stimulation with 1.5mg dexamethasone p.o.) and corresponding plasma cortisol levels were analyzed. All cases participated in regular aerobic exercise for 12 consecutive weeks and were then re-assessed at follow-up for exhaustion symptoms as well as for cortisol levels and gene expression profiles. At baseline, we found increased basal cortisol levels and an enhanced suppression of plasma cortisol concentrations following dexamethasone in cases suffering from job-related exhaustion. Gene expression analysis revealed that 1.6-fold more transcripts were significantly regulated by dexamethasone in cases as compared to controls. At follow-up after 12 weeks of regular exercise training which was accompanied by significantly improved exhaustion severity scores, cortisol levels and gene expression profiles of cases normalized to the levels observed in controls. In conclusion, we detected GR-induced neuroendocrine and gene expression changes in cases suffering from job-related exhaustion which are in line with an increased sensitivity of GR function. This GR dysregulation normalized with symptom recovery.

  3. Predicting the probability of successful efficacy of a dissociated agonist of the glucocorticoid receptor from dose-response analysis.

    PubMed

    Conrado, Daniela J; Krishnaswami, Sriram; Shoji, Satoshi; Kolluri, Sheela; Hey-Hadavi, Judith; McCabe, Dorothy; Rojo, Ricardo; Tammara, Brinda K

    2016-06-01

    PF-04171327 is a dissociated agonist of the glucocorticoid receptor (DAGR) being developed to retain anti-inflammatory efficacy while reducing unwanted effects. Our aim was to conduct a longitudinal dose-response analysis to identify the DAGR doses with efficacy similar to or greater than prednisone 10 mg once daily (QD). The data included were from a Phase 2, randomized, double-blind, parallel-group study in 323 subjects with active rheumatoid arthritis on a background of methotrexate. Subjects received DAGR 1, 5, 10 or 15 mg, prednisone 5 or 10 mg, or placebo QD for 8 weeks. The Disease Activity Score 28-4 calculated using C-Reactive Protein (DAS28-4 CRP) was the efficacy endpoint utilized in this dose-response model. For DAGR, the maximum effect (Emax) on DAS28-4 CRP was estimated to be -1.2 points (95 % CI -1.7, -0.84), and the evaluated dose range provided 31-87 % of the Emax; for prednisone 5 and 10 mg, the estimated effects were -0.27 (95 % CI -0.55, 0.006) and -0.94 point (95 % CI -1.3, -0.59), respectively. Stochastic simulations indicated that the DAGR 1, 5, 10 and 15 mg have probabilities of 0.9, 29, 54 and 62 %, respectively, to achieve efficacy greater than prednisone 10 mg at week 8. DAGR 9 mg estimated probability was 50 % suggesting that DAGR ≥9 mg QD has an effect on DAS28-4 CRP comparable to or greater than prednisone 10 mg QD. This work informs dose selection for late-stage confirmatory trials. PMID:27178257

  4. Haploinsufficiency for NR3C1, the gene encoding the glucocorticoid receptor, in blastic plasmacytoid dendritic cell neoplasms

    PubMed Central

    Emadali, Anouk; Hoghoughi, Neda; Duley, Samuel; Hajmirza, Azadeh; Verhoeyen, Els; Cosset, Francois-Loic; Bertrand, Philippe; Roumier, Christophe; Roggy, Anne; Suchaud-Martin, Céline; Chauvet, Martine; Bertrand, Sarah; Hamaidia, Sieme; Rousseaux, Sophie; Josserand, Véronique; Charles, Julie; Templier, Isabelle; Maeda, Takahiro; Bruder-Costa, Juliana; Chaperot, Laurence; Plumas, Joel; Jacob, Marie-Christine; Bonnefoix, Thierry; Park, Sophie; Gressin, Remy; Tensen, Cornelis P.; Mecucci, Cristina; Macintyre, Elizabeth; Leroux, Dominique; Brambilla, Elisabeth; Nguyen-Khac, Florence; Luquet, Isabelle; Penther, Dominique; Bastard, Christian; Jardin, Fabrice; Lefebvre, Christine; Garnache, Francine

    2016-01-01

    Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive leukemia for which knowledge on disease mechanisms and effective therapies are currently lacking. Only a handful of recurring genetic mutations have been identified and none is specific to BPDCN. In this study, through molecular cloning in an index case that presented a balanced t(3;5)(q21;q31) and molecular cytogenetic analyses in a further 46 cases, we identify monoallelic deletion of NR3C1 (5q31), encoding the glucocorticoid receptor (GCR), in 13 of 47 (28%) BPDCN patients. Targeted deep sequencing in 36 BPDCN cases, including 10 with NR3C1 deletion, did not reveal NR3C1 point mutations or indels. Haploinsufficiency for NR3C1 defined a subset of BPDCN with lowered GCR expression and extremely poor overall survival (P = .0006). Consistent with a role for GCR in tumor suppression, functional analyses coupled with gene expression profiling identified corticoresistance and loss-of-EZH2 function as major downstream consequences of NR3C1 deletion in BPDCN. Subsequently, more detailed analyses of the t(3;5)(q21;q31) revealed fusion of NR3C1 to a long noncoding RNA (lncRNA) gene (lincRNA-3q) that encodes a novel, nuclear, noncoding RNA involved in the regulation of leukemia stem cell programs and G1/S transition, via E2F. Overexpression of lincRNA-3q was a consistent feature of malignant cells and could be abrogated by bromodomain and extraterminal domain (BET) protein inhibition. Taken together, this work points to NR3C1 as a haploinsufficient tumor suppressor in a subset of BPDCN and identifies BET inhibition, acting at least partially via lncRNA blockade, as a novel treatment option in BPDCN. PMID:27060168

  5. Differential regulation of glucocorticoid receptor expression in distinct columns of periaqueductal grey in rats with behavioural disability following nerve injury.

    PubMed

    Mor, David; Keay, Kevin A

    2013-10-01

    Neuropathic pain is diagnosed primarily by sensory dysfunction, which includes both spontaneous, and stimulus-evoked pain. Clinical evaluation highlights the disabilities which characterise this condition for most patients. Chronic constriction injury of the sciatic nerve (CCI) evokes sensory dysfunction characteristic of neuropathic pain. Approximately, 30 % of CCI rats show disabilities similar to those identified in clinical evaluation of neuropathic pain patients, these include: altered social behaviours; sleep disturbances; and endocrine dysfunction. The periaqueductal grey (PAG) is a nodal point in the brain circuits which regulate these functions, and undergoes a distinct set of neural and glial adaptations following CCI, in rats with disabilities. CCI increases corticosterone, which through its actions at the glucocorticoid receptor (GR), can trigger cellular adaptation. GR expression in PAG was quantified using qRT-PCR, Western blotting and immunohistochemical analyses and nerve-injured rats, with and without disabilities, were compared. Our data showed that the PAG of disabled rats has significantly increased expression of GR mRNA and protein. Further, this increased protein expression reflects contrasting patterns of change in GR expression in PAG subregions. The dorsolateral PAG had significant increases in the number of GR-immunoreactive (GR-IR) cells and the caudal lateral and ventrolateral PAG each had significant reductions in the number of GR-IR cells. These regional increases and decreases correlated with the degree of disability, as indicated by the degree of change in social behaviours. Our results suggest a role for altered PAG, GR-corticosterone interactions and their resultant cellular consequences in the expression of disabilities in a subpopulation of nerve-injured rats.

  6. Epigenetic modifications of the glucocorticoid receptor gene are associated with the vulnerability to psychopathology in childhood maltreatment

    PubMed Central

    Radtke, K M; Schauer, M; Gunter, H M; Ruf-Leuschner, M; Sill, J; Meyer, A; Elbert, T

    2015-01-01

    Stress, particularly when experienced early in life, can have profound implications for mental health. Previous research covering various tissues such as the brain, suggests that the detrimental impact of early-life stress (ELS) on mental health is mediated via epigenetic modifications including DNA methylation. Genes of the hypothalamic–pituitary–adrenal axis—in particular, the glucocorticoid receptor (hGR) gene—stand out as key targets for ELS. Even though the link between hGR methylation and either ELS or psychopathology is fairly well established, the mutually dependent relationships between ELS, DNA methylation and psychopathology remain to be uncovered. The specific psychopathology an individual might develop in the aftermath of stressful events can be highly variable, however, most studies investigating hGR methylation and psychopathology suffer from being limited to a single symptom cluster of mental disorders. Here, we screened volunteers for childhood maltreatment and analyzed whether it associates with hGR methylation in lymphocytes and a range of measures of psychological ill-health. hGR methylation in lymphocytes most likely reflects methylation patterns found in the brain and thus provides valuable insights into the etiology of psychopathology. We find the interaction between childhood maltreatment and hGR methylation to be strongly correlated with an increased vulnerability to psychopathology providing evidence of epigenome × environment interactions. Furthermore, our results indicate an additive effect of childhood maltreatment and hGR methylation in predicting borderline personality disorder (BPD)-associated symptoms, suggesting that the combination of both ELS and DNA methylation that possibly represents unfavorable events experienced even earlier in life poses the risk for BPD. PMID:26080088

  7. Predicting the probability of successful efficacy of a dissociated agonist of the glucocorticoid receptor from dose-response analysis.

    PubMed

    Conrado, Daniela J; Krishnaswami, Sriram; Shoji, Satoshi; Kolluri, Sheela; Hey-Hadavi, Judith; McCabe, Dorothy; Rojo, Ricardo; Tammara, Brinda K

    2016-06-01

    PF-04171327 is a dissociated agonist of the glucocorticoid receptor (DAGR) being developed to retain anti-inflammatory efficacy while reducing unwanted effects. Our aim was to conduct a longitudinal dose-response analysis to identify the DAGR doses with efficacy similar to or greater than prednisone 10 mg once daily (QD). The data included were from a Phase 2, randomized, double-blind, parallel-group study in 323 subjects with active rheumatoid arthritis on a background of methotrexate. Subjects received DAGR 1, 5, 10 or 15 mg, prednisone 5 or 10 mg, or placebo QD for 8 weeks. The Disease Activity Score 28-4 calculated using C-Reactive Protein (DAS28-4 CRP) was the efficacy endpoint utilized in this dose-response model. For DAGR, the maximum effect (Emax) on DAS28-4 CRP was estimated to be -1.2 points (95 % CI -1.7, -0.84), and the evaluated dose range provided 31-87 % of the Emax; for prednisone 5 and 10 mg, the estimated effects were -0.27 (95 % CI -0.55, 0.006) and -0.94 point (95 % CI -1.3, -0.59), respectively. Stochastic simulations indicated that the DAGR 1, 5, 10 and 15 mg have probabilities of 0.9, 29, 54 and 62 %, respectively, to achieve efficacy greater than prednisone 10 mg at week 8. DAGR 9 mg estimated probability was 50 % suggesting that DAGR ≥9 mg QD has an effect on DAS28-4 CRP comparable to or greater than prednisone 10 mg QD. This work informs dose selection for late-stage confirmatory trials.

  8. The atypical antipsychotics clozapine and olanzapine promote down-regulation and display functional selectivity at human 5-HT7 receptors

    PubMed Central

    Andressen, K W; Manfra, O; Brevik, C H; Ulsund, A H; Vanhoenacker, P; Levy, F O; Krobert, K A

    2015-01-01

    Background and Purpose Classically, ligands of GPCRs have been classified primarily upon their affinity and efficacy to activate a signal transduction pathway. Recent reports indicate that the efficacy of a particular ligand can vary depending on the receptor-mediated response measured (e.g. activating G proteins, other downstream responses, internalization). Previously, we reported that inverse agonists induce both homo- and heterologous desensitization, similar to agonist stimulation, at the Gs-coupled 5-HT7 receptor. The primary objective of this study was to determine whether different inverse agonists at the 5-HT7 receptor also induce internalization and/or degradation of 5-HT7 receptors. Experimental Approach HEK293 cells expressing 5-HT7(a, b or d) receptors were pre-incubated with 5-HT, clozapine, olanzapine, mesulergine or SB269970 and their effects upon receptor density, AC activity, internalization, recruitment of β-arrestins and lysosomal trafficking were measured. Key Results The agonist 5-HT and three out of four inverse agonists tested increased internalization independently of β-arrestin recruitment. Among these, only the atypical antipsychotics clozapine and olanzapine promoted lysosomal sorting and reduced 5-HT7 receptor density (∼60% reduction within 24 h). Inhibition of lysosomal degradation with chloroquine blocked the clozapine- and olanzapine-induced down-regulation of 5-HT7 receptors. Incubation with SB269970 decreased both 5-HT7(b) constitutive internalization and receptor density but increased 5-HT7(d) receptor density, indicating differential ligand regulation among the 5-HT7 splice variants. Conclusions and Implications Taken together, we found that various ligands differentially activate regulatory processes governing receptor internalization and degradation in addition to signal transduction. Thus, these data extend our understanding of functional selectivity at the 5-HT7 receptor. PMID:25884989

  9. Downregulation of the T-Cell Receptor by Human Immunodeficiency Virus Type 2 Nef Does Not Protect against Disease Progression▿

    PubMed Central

    Feldmann, Jérôme; Leligdowicz, Aleksandra; Jaye, Assan; Dong, Tao; Whittle, Hilton; Rowland-Jones, Sarah L.

    2009-01-01

    Chronic immune activation is thought to play a major role in human immunodeficiency virus (HIV) pathogenesis, but the relative contributions of multiple factors to immune activation are not known. One proposed mechanism to protect against immune activation is the ability of Nef proteins from some HIV and simian immunodeficiency virus strains to downregulate the T-cell receptor (TCR)-CD3 complex of the infected cell, thereby reducing the potential for deleterious activation. HIV type 1 (HIV-1) Nef has lost this property. In contrast to HIV-1, HIV-2 infection is characterized by a marked disparity in the disease course, with most individuals maintaining a normal life span. In this study, we examined the relationship between the ability of HIV-2 Nef proteins to downregulate the TCR and immune activation, comparing progressors and nonprogressors. Representative Nef variants were isolated from 28 HIV-2-infected individuals. We assessed their abilities to downregulate the TCR from the surfaces of CD4 T cells. In the same individuals, the activation of peripheral lymphocytes was evaluated by measurement of the expression levels of HLA-DR and CD38. We observed a striking correlation of the TCR downregulation efficiency of HIV-2 Nef variants with immune activation in individuals with a low viral load. This strongly suggests that Nef expression can influence the activation state of the immune systems of infected individuals. However, the efficiency of TCR downregulation by Nef was not reduced in progressing individuals, showing that TCR downregulation does not protect against progression in HIV-2 infection. PMID:19812166

  10. Glucocorticoid receptor haploinsufficiency causes hypertension and attenuates hypothalamic-pituitary-adrenal axis and blood pressure adaptions to high-fat diet.

    PubMed

    Michailidou, Z; Carter, R N; Marshall, E; Sutherland, H G; Brownstein, D G; Owen, E; Cockett, K; Kelly, V; Ramage, L; Al-Dujaili, E A S; Ross, M; Maraki, I; Newton, K; Holmes, M C; Seckl, J R; Morton, N M; Kenyon, C J; Chapman, K E

    2008-11-01

    Glucocorticoid hormones are critical to respond and adapt to stress. Genetic variations in the glucocorticoid receptor (GR) gene alter hypothalamic-pituitary-adrenal (HPA) axis activity and associate with hypertension and susceptibility to metabolic disease. Here we test the hypothesis that reduced GR density alters blood pressure and glucose and lipid homeostasis and limits adaption to obesogenic diet. Heterozygous GR(betageo/+) mice were generated from embryonic stem (ES) cells with a gene trap integration of a beta-galactosidase-neomycin phosphotransferase (betageo) cassette into the GR gene creating a transcriptionally inactive GR fusion protein. Although GR(betageo/+) mice have 50% less functional GR, they have normal lipid and glucose homeostasis due to compensatory HPA axis activation but are hypertensive due to activation of the renin-angiotensin-aldosterone system (RAAS). When challenged with a high-fat diet, weight gain, adiposity, and glucose intolerance were similarly increased in control and GR(betageo/+) mice, suggesting preserved control of intermediary metabolism and energy balance. However, whereas a high-fat diet caused HPA activation and increased blood pressure in control mice, these adaptions were attenuated or abolished in GR(betageo/+) mice. Thus, reduced GR density balanced by HPA activation leaves glucocorticoid functions unaffected but mineralocorticoid functions increased, causing hypertension. Importantly, reduced GR limits HPA and blood pressure adaptions to obesogenic diet. PMID:18697839

  11. BAY 11-7085 induces glucocorticoid receptor activation and autophagy that collaborate with apoptosis to induce human synovial fibroblast cell death

    PubMed Central

    Relic, Biserka; Charlier, Edith; Deroyer, Celine; Malaise, Olivier; Neuville, Sophie; Desoroux, Aline; Gillet, Philippe; de Seny, Dominique; Malaise, Michel G.

    2016-01-01

    Inhibition of proapoptotic pathways in synovial fibroblasts is one of the major causes of synovial proliferation and hyperplasia in rheumatic diseases. We have shown previously that NF-κB inhibitor BAY 11-7085, through inactivation of PPAR-γ, induces apoptosis in human synovial fibroblasts. In this work we showed that BAY 11-7085 induced autophagy that preceded BAY 11-7085-induced apoptosis. Of interest, BAY 11-7085 induced Serine 211 phosphorylation and degradation of glucocorticoid receptor (GR). Glucocorticoid prednisolone induced both activation and degradation of GR, as well as autophagy in synovial fibroblasts. BAY 11-7085-induced cell death was significantly decreased with glucocorticoid inhibitor mifepristone and with inhibitors of autophagy. Both BAY 11-7085-induced autophagy and GR activation were down regulated with PPAR-γ agonist, 15d-PGJ2 and MEK/ERK inhibitor UO126. Inhibition of autophagy markedly decreased endogenous and BAY 11-7085-induced ERK phosphorylation, suggesting a positive feed back loop between ERK activation and autophagy in synovial fibroblasts. Co-transfection of MEK1 with PPAR-γ1 in HEK293 cells caused known inhibitory phosphorylation of PPAR-γ1 (Serine 112) and enhanced GR degradation, in the absence or presence of prednisolone. Furthermore, GR was both phosphorylated on Serine 211 and down regulated in synovial fibroblasts during serum starvation induced autophagy. These results showed that GR activation and PPAR-γ inactivation mediated BAY 11-7085-induced autophagy. PMID:26993765

  12. Blockage of glucocorticoid receptors during memory acquisition, retrieval and reconsolidation prevents the expression of morphine-induced conditioned place preferences in mice.

    PubMed

    Fan, Yao-Dong; Niu, Hai-Chen; Huma, Tanzeel; Li, Ling; Wang, Gui-Mei; Xu, Li-Qi; Ren, He; Ma, Yuan-Ye; Yu, Hua-Lin

    2013-01-01

    Association between the reward caused by consuming drugs and the context in which they are consumed is essential in the formation of morphine-induced conditioned place preference (CPP). Glucocorticoid receptor (GRs) activation in different regions of the brain affects reward-based reinforcement and memory processing. A wide array of studies have demonstrated that blockage of GRs in some brain areas can have an effect on reward-related memory; however, to date there have been no systematic studies about the involvement of glucocorticoids (GCs) in morphine-related reward memory. Here, we used the GR antagonist RU38486 to investigate how GRs blockage affects the sensitization and CPP behavior during different phases of reward memory included acquisition, retrieval and reconsolidation. Interestingly, our results showed RU38486 has the ability to impair the acquisition, retrieval and reconsolidation of reward-based memory in CPP and sensitization behavior. But RU38486 by itself cannot induce CPP or conditioned place aversion (CPA) behavior. Our data provide a much more complete picture of the potential effects that glucocorticoids have on the reward memory of different phases and inhibit the sensitization behavior.

  13. The caffeine-binding adenosine A2A receptor induces age-like HPA-axis dysfunction by targeting glucocorticoid receptor function.

    PubMed

    Batalha, Vânia L; Ferreira, Diana G; Coelho, Joana E; Valadas, Jorge S; Gomes, Rui; Temido-Ferreira, Mariana; Shmidt, Tatiana; Baqi, Younis; Buée, Luc; Müller, Christa E; Hamdane, Malika; Outeiro, Tiago F; Bader, Michael; Meijsing, Sebastiaan H; Sadri-Vakili, Ghazaleh; Blum, David; Lopes, Luísa V

    2016-01-01

    Caffeine is associated with procognitive effects in humans by counteracting overactivation of the adenosine A2A receptor (A2AR), which is upregulated in the human forebrain of aged and Alzheimer's disease (AD) patients. We have previously shown that an anti-A2AR therapy reverts age-like memory deficits, by reestablishment of the hypothalamic-pituitary-adrenal (HPA) axis feedback and corticosterone circadian levels. These observations suggest that A2AR over-activation and glucocorticoid dysfunction are key events in age-related hippocampal deficits; but their direct connection has never been explored. We now show that inducing A2AR overexpression in an aging-like profile is sufficient to trigger HPA-axis dysfunction, namely loss of plasmatic corticosterone circadian oscillation, and promotes reduction of GR hippocampal levels. The synaptic plasticity and memory deficits triggered by GR in the hippocampus are amplified by A2AR over-activation and were rescued by anti-A2AR therapy; finally, we demonstrate that A2AR act on GR nuclear translocation and GR-dependent transcriptional regulation. We provide the first demonstration that A2AR is a major regulator of GR function and that this functional interconnection may be a trigger to age-related memory deficits. This supports the idea that the procognitive effects of A2AR antagonists, namely caffeine, on Alzheimer's and age-related cognitive impairments may rely on its ability to modulate GR actions.

  14. The caffeine-binding adenosine A2A receptor induces age-like HPA-axis dysfunction by targeting glucocorticoid receptor function.

    PubMed

    Batalha, Vânia L; Ferreira, Diana G; Coelho, Joana E; Valadas, Jorge S; Gomes, Rui; Temido-Ferreira, Mariana; Shmidt, Tatiana; Baqi, Younis; Buée, Luc; Müller, Christa E; Hamdane, Malika; Outeiro, Tiago F; Bader, Michael; Meijsing, Sebastiaan H; Sadri-Vakili, Ghazaleh; Blum, David; Lopes, Luísa V

    2016-01-01

    Caffeine is associated with procognitive effects in humans by counteracting overactivation of the adenosine A2A receptor (A2AR), which is upregulated in the human forebrain of aged and Alzheimer's disease (AD) patients. We have previously shown that an anti-A2AR therapy reverts age-like memory deficits, by reestablishment of the hypothalamic-pituitary-adrenal (HPA) axis feedback and corticosterone circadian levels. These observations suggest that A2AR over-activation and glucocorticoid dysfunction are key events in age-related hippocampal deficits; but their direct connection has never been explored. We now show that inducing A2AR overexpression in an aging-like profile is sufficient to trigger HPA-axis dysfunction, namely loss of plasmatic corticosterone circadian oscillation, and promotes reduction of GR hippocampal levels. The synaptic plasticity and memory deficits triggered by GR in the hippocampus are amplified by A2AR over-activation and were rescued by anti-A2AR therapy; finally, we demonstrate that A2AR act on GR nuclear translocation and GR-dependent transcriptional regulation. We provide the first demonstration that A2AR is a major regulator of GR function and that this functional interconnection may be a trigger to age-related memory deficits. This supports the idea that the procognitive effects of A2AR antagonists, namely caffeine, on Alzheimer's and age-related cognitive impairments may rely on its ability to modulate GR actions. PMID:27510168

  15. The caffeine-binding adenosine A2A receptor induces age-like HPA-axis dysfunction by targeting glucocorticoid receptor function

    PubMed Central

    Batalha, Vânia L.; Ferreira, Diana G.; Coelho, Joana E.; Valadas, Jorge S.; Gomes, Rui; Temido-Ferreira, Mariana; Shmidt, Tatiana; Baqi, Younis; Buée, Luc; Müller, Christa E.; Hamdane, Malika; Outeiro, Tiago F.; Bader, Michael; Meijsing, Sebastiaan H.; Sadri-Vakili, Ghazaleh; Blum, David; Lopes, Luísa V.

    2016-01-01

    Caffeine is associated with procognitive effects in humans by counteracting overactivation of the adenosine A2A receptor (A2AR), which is upregulated in the human forebrain of aged and Alzheimer’s disease (AD) patients. We have previously shown that an anti-A2AR therapy reverts age-like memory deficits, by reestablishment of the hypothalamic-pituitary-adrenal (HPA) axis feedback and corticosterone circadian levels. These observations suggest that A2AR over-activation and glucocorticoid dysfunction are key events in age-related hippocampal deficits; but their direct connection has never been explored. We now show that inducing A2AR overexpression in an aging-like profile is sufficient to trigger HPA-axis dysfunction, namely loss of plasmatic corticosterone circadian oscillation, and promotes reduction of GR hippocampal levels. The synaptic plasticity and memory deficits triggered by GR in the hippocampus are amplified by A2AR over-activation and were rescued by anti-A2AR therapy; finally, we demonstrate that A2AR act on GR nuclear translocation and GR-dependent transcriptional regulation. We provide the first demonstration that A2AR is a major regulator of GR function and that this functional interconnection may be a trigger to age-related memory deficits. This supports the idea that the procognitive effects of A2AR antagonists, namely caffeine, on Alzheimer’s and age-related cognitive impairments may rely on its ability to modulate GR actions. PMID:27510168

  16. Downregulation of the CXCR4 receptor inhibits cervical carcinoma metastatic behavior in vitro and in vivo.

    PubMed

    Sekuła, Małgorzata; Miekus, Katarzyna; Majka, Marcin

    2014-06-01

    Cervical carcinoma is frequently diagnosed among women, particularly in low and middle income countries. In this study, we investigated the role of the SDF-1/CXCR4 axis during cervical carcinoma growth and progression in vitro and in vivo. Downregulation of CXCR4 receptor using an RNA interference system led to almost complete inhibition of the receptor expression, activation and function. CXCR4 receptor silencing led to decreased ability to signal, to induce migration and to form holoclone-like colonies, with no influence on viability/proliferation of the cells. CXCR4-deficient cells had also significantly lower levels of MMP-9. Interestingly, downregulation of CXCR4 expression resulted in reduced tumor growth in vivo. Tumors generated by CXCR4-deficient cells had also lower expression of the proliferation marker Ki‑67 and decreased ability to engraft into lungs and spleen. Taken together, our results indicate that CXCR4 receptor may play an important role during cervical carcinoma invasion. In our study CXCR4 influenced invasive properties of cervical carcinoma cells both in vitro and in vivo.

  17. Morphine-induced desensitization and down-regulation at mu-receptors in 7315C pituitary tumor cells

    SciTech Connect

    Puttfarcken, P.S.; Cox, B.M. )

    1989-01-01

    Pituitary 7315c tumor cells maintained in culture were treated with varying concentrations of morphine from 10 nM to 300 {mu}M, for periods of five or forty-eight hours. The ability of the mu-opioid receptor agonist, DAMGO, to inhibit forskolin-stimulated adenylyl cyclase in washed membrane preparations from the treated cells was compared with its activity in membranes from cells incubated in the absence of added morphine. In the same membrane preparations, the number and affinity of mu-opioid receptors was estimated by measurements of ({sup 3}H)diprenorphine binding. After 5 hr of treatment with morphine concentrations of 100 nM or higher, a significant reduction in inhibition of adenylyl cyclase by DAMGO was observed. Little further loss of agonist activity was observed when the incubations were extended to 48 hr. After 5 hr of morphine treatment, there was no change in either the number of receptors, or their affinity for ({sup 3}H)diprenorphine. However after 48 hr of morphine treatment, greater than 25% reductions in receptor number were apparent with morphine pretreatment concentrations of 10 {mu}M or higher. These results suggest that opioid tolerance in this system is primarily associated with a reduced ability of agonist-occupied receptor to activate the effector system. Receptor down-regulation was not necessary for loss of agonist response, although a reduction in receptor number occurred after exposure to high concentrations of morphine for periods longer than 5hr.

  18. Delineation of a small region within the major transactivation domain of the human glucocorticoid receptor that mediates transactivation of gene expression.

    PubMed Central

    Dahlman-Wright, K; Almlöf, T; McEwan, I J; Gustafsson, J A; Wright, A P

    1994-01-01

    Previous deletion analysis localized the major transactivation function of the human glucocorticoid receptor to a 185-amino acid segment close to the N terminus of the receptor protein. This region was named tau 1 [Hollenberg, S. M. & Evans, R. M. (1988) Cell 55, 899-906]. To delineate the smallest active region within tau 1, we have systematically tested the transactivation capacity of deletion derivatives of the tau 1 domain, fused to the glucocorticoid receptor DNA-binding domain, in yeast cells. Internal scanning deletions suggested that residues near the C terminus of tau 1 are most important for activity. Deletions of N-terminal and C-terminal sequences identified a 41-amino acid "core" region near the C terminus of tau 1 that is crucial for tau 1 function. Small peptide fragments containing the tau 1 core region are competent for transactivation, while regions outside the tau 1 core are not active. We have previously demonstrated that the intact tau 1 domain squelches the activity of a minimal promoter in vivo and in vitro, suggesting involvement of interactions with a component/components of the basal transcription machinery in the mechanism of transactivation. This activity was maintained in the tau 1 core-containing segments. Images PMID:8127854

  19. An analysis of glucocorticoid receptor-mediated gene expression in BEAS-2B human airway epithelial cells identifies distinct, ligand-directed, transcription profiles with implications for asthma therapeutics

    PubMed Central

    Joshi, T; Johnson, M; Newton, R; Giembycz, M

    2015-01-01

    Background and Purpose International asthma guidelines recommend that inhaled glucocorticoids be used as a monotherapy in all patients with mild to moderate disease because of their ability to suppress airways inflammation. Current evidence suggests that the therapeutic benefit of glucocorticoids is due to the transactivation and transrepression of anti-inflammatory and pro-inflammatory genes respectively. However, the extent to which clinically relevant glucocorticoids are equivalent in their ability to modulate gene expression is unclear. Experimental Approach A pharmacodynamics investigation of glucocorticoid receptor (GR)-mediated gene transactivation in BEAS-2B human airway epithelial cells was performed using a glucocorticoid response element luciferase reporter coupled with an analysis of glucocorticoid-inducible genes encoding proteins with anti-inflammatory and adverse-effect potential. Key Results Using transactivation as a functionally relevant output, a given glucocorticoid displayed a unique, gene expression ‘fingerprint’ where intrinsic efficacy and GR density were essential determinants. We showed that depending on the gene selected for analysis, a given glucocorticoid can behave as an antagonist, partial agonist, full agonist or even ‘super agonist’. In the likely event that different, tissue-dependent gene expression profiles are reproduced in vivo, then the anti-inflammatory and adverse-effect potential of many glucocorticoids currently available as asthma therapeutics may not be equivalent. Conclusions and Implications The generation of gene expression ‘fingerprints’ in target and off-target human tissues could assist the rational design of GR agonists with improved therapeutic ratios. This approach could identify compounds that are useful in the management of severe asthma and other inflammatory disorders where systemic exposure is desirable. PMID:25393397

  20. The non-ligand binding beta-isoform of the human glucocorticoid receptor (hGR beta): tissue levels, mechanism of action, and potential physiologic role.

    PubMed Central

    de Castro, M.; Elliot, S.; Kino, T.; Bamberger, C.; Karl, M.; Webster, E.; Chrousos, G. P.

    1996-01-01

    BACKGROUND: Alternative splicing of the transcripts of the human glucocorticoid receptor gene results in two mutually exclusive products, the classic, ligand-binding glucocorticoid receptor (hGR alpha), and a dominant negative non-ligand-binding isoform, hGR beta. MATERIALS AND METHODS: We examined the existence of and quantified both hGR alpha and hGR beta isoforms in a panel of human tissues, as well as in intact and fractionated HeLa cells, using specific quantitative Western blots and/or immunocytochemistry. We studied the potential interactions of hGR beta with heat shock protein (hsp) 90 and/or hGR alpha using cross-immunoadsorption/precipitation procedures followed by Western blots. RESULTS: For the first time, we demonstrated the natural existence of the hGR beta protein, which was widely expressed in human tissues. The ratio of immunoreactive hGR alpha to hGR beta varied from 0.2 to 1.0 among different tissues, and was approximately 0.2 in HeLa cells. In the latter, both isoforms were distributed in the cytoplasm and nucleus in the absence of the hormonal ligand, and translocated into the nucleus after addition of dexamethasone. The cytosolic and nuclear hGR alpha-to-hGR beta ratio remained the same before and after dexamethasone exposure, suggesting that upon activation the two isoforms translocated into the nucleus in equal proportions. hGR alpha- and hGR beta-specific antibodies cross-adsorbed and precipitated cytosolic and nuclear glucocorticoid hGR alpha and hGR beta, respectively, as well as hsp90, suggesting that hGR alpha and hGR beta are in complex with hsp90 and/or each other. CONCLUSIONS: The hGR beta protein is widely expressed throughout the human body and present mostly in the cytoplasm of human cells, in complex with hsp90 and other proteins. In the presence of glucocorticoid, hGR beta probably heterodimerizes with ligand-bound hGR alpha and translocates into the nucleus to act as a dominant negative inhibitor of the classic receptor. Images

  1. Transcriptional down-regulation of epidermal growth factor receptors by nerve growth factor treatment of PC12 cells.

    PubMed

    Shibutani, M; Lazarovici, P; Johnson, A C; Katagiri, Y; Guroff, G

    1998-03-20

    Treatment of PC12 cells with nerve growth factor leads to a decrease in the number of epidermal growth factor receptors on the cell membrane. The mRNA for the epidermal growth factor receptor decreases in a comparable fashion. This decrease appears due to a decrease in the transcription of the epidermal growth factor receptor gene because first, there is no difference in the stability of the epidermal growth factor receptor mRNA, second, newly transcribed epidermal growth factor receptor mRNA is decreased in nerve growth factor-differentiated cells, and third, constructs containing the promoter region of the epidermal growth factor receptor gene are transcribed much less readily in nerve growth factor-differentiated cells than in untreated cells. The decreases in mRNA are not seen in the p140(trk)-deficient variant PC12nnr5 cells nor in cells containing either dominant-negative Ras or dominant-negative Src. Treatment with nerve growth factor also increases the cellular content of GCF2, a putative transcription factor inhibitory for the transcription of the epidermal growth factor receptor gene. The increase in GCF2, like the decrease in the epidermal growth factor receptor mRNA, is not seen in PC12nnr5 cells nor in cells expressing either dominant-negative Ras or dominant-negative Src. The results suggest that nerve growth factor-induced down-regulation of the epidermal growth factor receptor is under transcriptional control, is p140(trk)-, Ras-, and Src-dependent, and may involve transcriptional repression by GCF2.

  2. Analysis of BCLI, N363S and ER22/23EK Polymorphisms of the Glucocorticoid Receptor Gene in Adrenal Incidentalomas

    PubMed Central

    Reimondo, Giuseppe; Chiodini, Iacopo; Puglisi, Soraya; Pia, Anna; Morelli, Valentina; Kastelan, Darko; Cannavo, Salvatore; Berchialla, Paola; Giachino, Daniela; Perotti, Paola; Cuccurullo, Alessandra; Paccotti, Piero; Beck-Peccoz, Paolo; De Marchi, Mario; Terzolo, Massimo

    2016-01-01

    Context Patients with adrenal incidentalomas (AI) may experience detrimental consequences due to a minimal cortisol excess sustained by adrenal adenoma. SNPs of the glucocorticoid receptor gene (NR3C1) modulate individual sensitivity to glucocorticoids and may interfere with the clinical presentation. Objective To compare the frequency of N363S, ER22/23EK and BclI SNPs in patients with AI with the general population and to evaluate whether these SNPs are linked to consequences of cortisol excess. Setting Multicentric, retrospective analysis of patients referred from 2010 to 2014 to 4 centers (Orbassano, Milano, Messina [Italy] and Zagreb [Croatia]). Patients 411 patients with AI; 153 males and 258 females and 186 from blood donors. Main outcomes measures All patients and controls were genotyped for BclI, N363S and ER22/23EK and SNPs frequency was associated with clinical and hormonal features. Results SNP frequency was: SNP frequency was: N363S 5.4% (MAF 0.027), BclI 54.7% (MAF 0.328), ER22/23EK 4.4% (MAF 0.022), without any significant difference between patients and controls. N363S was more frequent in hypertensive patients (p = 0.03) and was associated with hypertension (p = 0.015) in patients with suppressed cortisol after the 1-mg DST. Conclusions Our results demonstrate that SNPs of the glucocorticoid receptor gene do not play a pathogenetic role for AI. The impact of any single SNP on the phenotypic expression of minimal cortisol excess is limited and their analysis does not provide additional data that may be exploited for patient management. PMID:27649075

  3. Design and x-ray crystal structures of high-potency nonsteroidal glucocorticoid agonists exploiting a novel binding site on the receptor

    SciTech Connect

    Biggadike, Keith; Bledsoe, Randy K.; Coe, Diane M.; Cooper, Tony W.J.; House, David; Iannone, Marie A.; Macdonald, Simon J.F.; Madauss, Kevin P.; McLay, Iain M.; Shipley, Tracy J.; Taylor, Simon J.; Tran, Thuy B.; Uings, Iain J.; Weller, Victoria; Williams, Shawn P.

    2010-09-17

    Crystallography and computer modeling have been used to exploit a previously unexplored channel in the glucocorticoid receptor (GR). Highly potent, nonsteroidal indazole amides showing excellent complementarity to the channel were designed with the assistance of the computational technique AlleGrow. The accuracy of the design process was demonstrated through crystallographic structural determination of the GR ligand-binding domain-agonist complex of the D-prolinamide derivative 11. The utility of the channel was further exemplified through the design of a potent phenylindazole in which structural motifs, seen to interact with the traditional GR ligand pocket, were abandoned and replaced by interactions within the new channel. Occupation of the channel was confirmed with a second GR crystal structure of this truncated D-alaninamide derivative 13. Compound 11 displays properties compatible with development as an intranasal solution formulation, whereas oral bioavailability has been demonstrated with a related truncated exemplar 14. Data with the pyrrolidinone amide 12 demonstrate the potential for further elaboration within the 'meta' channel to deliver compounds with selectivity for the desired transrepressive activity of glucocorticoids. The discovery of these interactions with this important receptor offers significant opportunities for the design of novel GR modulators.

  4. DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome, gene 1) selectively inhibits transactivation but not transrepression mediated by the glucocorticoid receptor in a LXXLL-dependent manner.

    PubMed

    Zhou, Junguo; Oakley, Robert H; Cidlowski, John A

    2008-07-01

    The glucocorticoid receptor (GR) mediates virtually all actions of glucocorticoids, and the nature and magnitude of a cell's response to these steroids are determined primarily by hormone concentration and GR signaling capacity. DAX-1 (dosagesensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome, gene 1) is an orphan nuclear receptor that functions as a corepressor, and deletion or mutation of DAX-1 causes a decrease in glucocorticoid production. However it is unclear whether DAX-1 also alters GR function as a transcription factor. Here, we demonstrate that DAX-1 acts as a novel selective GR modulator. It specifically inhibits ligand-dependent GR transactivation with little effect on GR-mediated transrepression. As demonstrated by coimmunoprecipitation and glutathione- S-transferase pull-down assays, DAX-1 physically interacts with GR, but this interaction does not influence either ligand-induced GR nuclear translocation or subsequent GR association with glucocorticoid-responsive elements. Instead, DAX-1 competes with coactivators such as GR-interacting protein 1 for binding to the receptor. Specifically, suppression of GR transactivation is mediated by the N-terminal half of DAX-1, and in particular the LXXLL motifs. Thus we demonstrate that DAX-1 directly modulates GR signaling in addition to affecting glucocorticoid hormone levels. PMID:18417736

  5. Pharmacological Inhibition of O-GlcNAcase Does Not Increase Sensitivity of Glucocorticoid Receptor-Mediated Transrepression

    PubMed Central

    Stivers, Peter J.; Harmonay, Lauren; Hicks, Alexandra; Mehmet, Huseyin; Morris, Melody; Robinson, Gain M.; Strack, Peter R.; Savage, Mary J.; Zaller, Dennis M.; Zwierzynski, Izabela; Brandish, Philip E.

    2015-01-01

    Glucocorticoid signaling regulates target genes by multiple mechanisms, including the repression of transcriptional activities of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) though direct protein-protein interactions and subsequent O-GlcNAcylation of RNA polymerase II (pol II). Recent studies have shown that overexpression of O-linked β-N-acetylglucosamine transferase (OGT), which adds an O-linked β-N-acetylglucosamine (O-GlcNAc) group to the C-terminal domain of RNA pol II, increases the transrepression effects of glucocorticoids (GC). As O-GlcNAcase (OGA) is an enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, we hypothesized that the potentiation of GC effects following OGT overexpression could be similarly observed via the direct inhibition of OGA, inhibiting O-GlcNAc removal from pol II. Here we show that despite pharmacological evidence of target engagement by a selective small molecule inhibitor of OGA, there is no evidence for a sensitizing effect on glucocorticoid-mediated effects on TNF-α promoter activity, or gene expression generally, in human cells. Furthermore, inhibition of OGA did not potentiate glucocorticoid–induced apoptosis in several cancer cell lines. Thus, despite evidence for O-GlcNAc modification of RNA pol II in GR-mediated transrepression, our data indicate that pharmacological inhibition of OGA does not potentiate or enhance glucocorticoid-mediated transrepression. PMID:26670328

  6. Corticotropin-releasing factor receptor subtype 2 in human colonic mucosa: Down-regulation in ulcerative colitis

    PubMed Central

    Chatzaki, Ekaterini; Anton, Peter A; Million, Mulugeta; Lambropoulou, Maria; Constantinidis, Theodoros; Kolios, George; Taché, Yvette; Grigoriadis, Dimitri E

    2013-01-01

    AIM: To assess corticotropin-releasing factor receptor 2 (CRF2) expression in the colon of healthy subjects and patients with ulcerative colitis (UC). METHODS: We examined CRF2 gene and protein expression in the distal/sigmoid colonic mucosal biopsies from healthy subjects and patients with UC (active or disease in remission), human immunodeficiency virus (HIV) and functional bowel disease (FBD) by reverse transcription-polymerase chain reaction and immunofluorescence. RESULTS: Gene expression of CRF2 was demonstrated in the normal human colonic biopsies, but not in the human colorectal adenocarcinoma cell line Caco2. Receptor protein localization showed immunoreactive CRF2 receptors in the lamina propria and in the epithelial cells of the distal/sigmoid biopsy samples. Interestingly, CRF2 immunoreactivity was no longer observed in epithelial cells of patients with mild-moderately active UC and disease in remission, while receptor protein expression did not change in the lamina propria. No differences in CRF2 expression profile were observed in distal/sigmoid intestinal biopsies from HIV infection and FBD patients, showing no signs of inflammation. CONCLUSION: The down-regulation of the CRF2 receptor in the distal/sigmoid biopsies of UC patients is indicative of change in CRF2 signalling associated with the process of inflammation. PMID:23539366

  7. The herpes simplex virus receptor nectin-1 is down-regulated after trans-interaction with glycoprotein D

    SciTech Connect

    Stiles, Katie M.; Milne, Richard S.B.; Cohen, Gary H.; Eisenberg, Roselyn J.; Krummenacher, Claude

    2008-03-30

    During herpes simplex virus (HSV) entry, membrane fusion occurs either on the cell surface or after virus endocytosis. In both cases, binding of glycoprotein D (gD) to a receptor such as nectin-1 or HVEM is required. In this study, we co-cultured cells expressing gD with nectin-1 expressing cells to investigate the effects of gD on nectin-1 at cell contacts. After overnight co-cultures with gD expressing cells, there was a down-regulation of nectin-1 in B78H1-C10, SY5Y, A431 and HeLa cells, which HSV enters by endocytosis. In contrast, on Vero cells, which HSV enters at the plasma membrane, nectin-1 was not down-regulated. Further analysis of B78H1-derived cells showed that nectin-1 down-regulation corresponds to the ability of gD to bind nectin-1 and is achieved by internalization and low-pH-dependent degradation of nectin-1. Moreover, gD is necessary for virion internalization in B78H1 cells expressing nectin-1. These data suggest that the determinants of gD-mediated internalization of nectin-1 may direct HSV to an endocytic pathway during entry.

  8. Effect of Lipopolysaccharide on Glucocorticoid Receptor Function in Control Nasal Mucosa Fibroblasts and in Fibroblasts from Patients with Chronic Rhinosinusitis with Nasal Polyps and Asthma

    PubMed Central

    Fernández-Bertolín, Laura; Mullol, Joaquim; Fuentes-Prado, Mireya; Roca-Ferrer, Jordi; Alobid, Isam; Picado, César; Pujols, Laura

    2015-01-01

    Background Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease of the upper airways frequently associated with asthma. Bacterial infection is a feature of CRSwNP that can aggravate the disease and the response to glucocorticoid treatment. Objective We examined whether the bacterial product lipopolysaccharide (LPS) reduces glucocorticoid receptor (GR) function in control nasal mucosa (NM) fibroblasts and in nasal polyp (NP) fibroblasts from patients with CRSwNP and asthma. Methods NP (n = 12) and NM fibroblasts (n = 10) were in vitro pre-incubated with LPS (24 hours) prior to the addition of dexamethasone. Cytokine/chemokine secretion was measured by ELISA and Cytometric Bead Array. GRα, GRβ, mitogen-activated protein-kinase phosphatase-1 (MKP-1) and glucocorticoid-induced leucine zipper (GILZ) expression was measured by RT-PCR and immunoblotting, GRα nuclear translocation by immunocytochemistry, and GRβ localization by immunoblotting. The role of MKP-1 and GILZ on dexamethasone-mediated cytokine inhibition was analyzed by small interfering RNA silencing. Results Pre-incubation of nasal fibroblasts with LPS enhanced the secretion of IL-6, CXCL8, RANTES, and GM-CSF induced by FBS. FBS-induced CXCL8 secretion wa