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Sample records for downregulates glucocorticoid receptor

  1. 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

  2. Downregulation of brain mineralocorticoid and glucocorticoid receptor by antisense oligodeoxynucleotide treatment fails to alter spatial navigation in rats.

    PubMed

    Engelmann, M; Landgraf, R; Lörscher, P; Conzelmann, C; Probst, J C; Holsboer, F; Reul, J M

    1998-11-13

    Adult male Brown Norway rats were long-term intracerebroventricularly (i.c.v.) infused with antisense oligodeoxynucleotides (18-mer, double endcapped phosphorothioate protected) targeting either mineralocorticoid or glucocorticoid receptor mRNA, or received the respective mixed bases sequence or vehicle. Mineralocorticoid receptor-mixed bases and glucocorticoid receptor-mixed bases oligodeoxynucleotide infusion (1 microg/0.5 microl/h) over a time period of seven days did not alter hippocampal mineralocorticoid receptor and glucocorticoid receptor binding when compared to vehicle treatment. In contrast, i.c.v. administration of mineralocorticoid receptor, as well as glucocorticoid receptor-antisense over the same time period resulted in a significantly reduced binding of mineralocorticoid receptor and glucocorticoid receptor in the hippocampus [mineralocorticoid receptor-antisense group approx. 72% of mineralocorticoid receptor-mixed bases and vehicle groups (100%); glucocorticoid receptor antisense group approx. 77% of glucocorticoid receptor-mixed bases and vehicle]. The specificity of these antisense effects is indicated by the finding that rats treated with mineralocorticoid receptor-antisense did not show any changes in glucocorticoid receptor and vice versa. Animals treated according to this infusion protocol and tested in the Morris water maze for their spatial navigation abilities failed to show significant differences among the groups. These data indicate that a reduction of hippocampal mineralocorticoid receptor or glucocorticoid receptor binding capacity by 20-30% does not interfere with spatial navigation.

  3. 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}.

  4. 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.

  5. Hippocampal microglial activation and glucocorticoid receptor down-regulation precipitate visceral hypersensitivity induced by colorectal distension in rats.

    PubMed

    Zhang, Gongliang; Zhao, Bing-Xue; Hua, Rong; Kang, Jie; Shao, Bo-Ming; Carbonaro, Theresa M; Zhang, Yong-Mei

    2016-03-01

    Visceral hypersensitivity is a common characteristic in patients suffering from irritable bowel syndrome (IBS) and other disorders with visceral pain. Although the pathogenesis of visceral hypersensitivity remains speculative due to the absence of pathological changes, the long-lasting sensitization in neuronal circuitry induced by early life stress may play a critical role beyond the digestive system even after complete resolution of the initiating event. The hippocampus integrates multiple sources of afferent inputs and sculpts integrated autonomic outputs for pain and analgesia regulation. Here, we examined the hippocampal mechanism in the pathogenesis of visceral hypersensitivity with a rat model induced by neonatal and adult colorectal distensions (CRDs). Neither neonatal nor adult CRD evoked behavioral abnormalities in adulthood; however, adult re-exposure to CRD induced persistent visceral hypersensitivity, depression-like behaviors, and spatial learning impairment in rats that experienced neonatal CRD. Rats that experienced neonatal and adult CRDs presented a decrease in hippocampal glucocorticoid receptor (GR) immunofluorescence staining and protein expression, and increases in hippocampal microglial activation and cytokine (IL-1β and TNF-α) accumulation. The decrease in hippocampal GR expression and increase in hippocampal IL-1β and TNF-α accumulation could be prevented by hippocampal local infusion of minocycline, a microglial inhibitor. These results suggest that neonatal CRD can increase the vulnerability of hippocampal microglia, and adult CRD challenge facilitates the hippocampal cytokine release from the sensitized microglia, which down-regulates hippocampal GR protein expression and, subsequently, precipitates visceral hypersensitivity.

  6. Selective Glucocorticoid Receptor modulators.

    PubMed

    De Bosscher, Karolien

    2010-05-31

    The ancient two-faced Roman god Janus is often used as a metaphor to describe the characteristics of the Glucocorticoid Receptor (NR3C1), which exhibits both a beneficial side, that serves to halt inflammation, and a detrimental side responsible for undesirable effects. However, recent developments suggest that the Glucocorticoid Receptor has many more faces with the potential to express a range of different functionalities, depending on factors that include the tissue type, ligand type, receptor variants, cofactor surroundings and target gene promoters. This behavior of the receptor has made the development of safer ligands, that trigger the expression program of only a desirable subset of genes, a real challenge. Thus more knowledge-based fundamental research is needed to ensure the design and development of selective Glucocorticoid Receptor modulators capable of reaching the clinic. Recent advances in the characterization of novel selective Glucocorticoid Receptor modulators, specifically in the context of anti-inflammatory strategies, will be described in this review.

  7. 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.

  8. A nonsteroidal glucocorticoid receptor antagonist.

    PubMed

    Miner, Jeffrey N; Tyree, Curtis; Hu, Junlian; Berger, Elaine; Marschke, Keith; Nakane, Masaki; Coghlan, Michael J; Clemm, Dave; Lane, Ben; Rosen, Jon

    2003-01-01

    Selective intracellular receptor antagonists are used clinically to ameliorate hormone-dependent disease states. Patients with Cushing's syndrome have high levels of the glucocorticoid, cortisol, and suffer significant consequences from this overexposure. High levels of this hormone are also implicated in exacerbating diabetes and the stress response. Selectively inhibiting this hormone may have clinical benefit in these disease states. To this end, we have identified the first selective, nonsteroidal glucocorticoid receptor (GR) antagonist. This compound is characterized by a tri-aryl methane core chemical structure. This GR-specific antagonist binds with nanomolar affinity to the GR and has no detectable binding affinity for the highly related receptors for mineralocorticoids, androgens, estrogens, and progestins. We demonstrate that this antagonist inhibits glucocorticoid-mediated transcriptional regulation. This compound binds competitively with steroids, likely occupying a similar site within the ligand-binding domain. Once bound, however, the compound fails to induce critical conformational changes in the receptor necessary for agonist activity.

  9. Up-regulation of the fetal baboon hypothalamo-pituitary-adrenal axis in intrauterine growth restriction: coincidence with hypothalamic glucocorticoid receptor insensitivity and leptin receptor down-regulation.

    PubMed

    Li, Cun; Ramahi, Emma; Nijland, Mark J; Choi, Jaeyhek; Myers, Dean A; Nathanielsz, Peter W; McDonald, Thomas J

    2013-07-01

    Intrauterine growth restriction (IUGR) is an important fetal developmental problem resulting from 2 broad causes: maternal undernutrition and/or decreased fetal nutrient delivery to the fetus via placental insufficiency. IUGR is often accompanied by up-regulation of the hypothalamo-pituitary-adrenal axis (HPAA). Sheep studies show fetal HPAA autonomy in late gestation. We hypothesized that IUGR, resulting from poor fetal nutrient delivery, up-regulates the fetal baboon HPAA in late gestation, driven by hypothalamo-pituitary glucocorticoid receptor (GR) insensitivity and decreased fetal leptin in peripheral plasma. Maternal baboons were fed as ad libitum controls or nutrient restricted to produce IUGR (fed 70% of the control diet) from 0.16 to 0.9 gestation. Peripheral ACTH, cortisol, and leptin were measured by immunoassays. CRH, arginine vasopressin (AVP), GR, leptin receptor (ObRb), and pro-opiomelanocortin peptide expression were determined immunohistochemically. IUGR fetal peripheral cortisol and ACTH, but not leptin, were increased (P < .05). IUGR increased CRH peptide expression, but not AVP, in the fetal hypothalamic paraventricular nucleus (PVN) and median eminence (P < .05). PVN ObRb peptide expression, but not GR, was decreased (P < .05) with IUGR. ObRb and pro-opiomelanocortin were robustly expressed in the anterior pituitary gland, but ∼1% of cells showed colocalization. We conclude that (1) CRH, not AVP, is the major releasing hormone driving ACTH and cortisol secretion during primate IUGR, (2) fetal HPAA activation was aided by GR insensitivity and decreased ObRb expression in the PVN, and (3) the anterior pituitary is not a site for ObRb effects on the HPAA.

  10. Regulation of triglyceride metabolism by glucocorticoid receptor

    PubMed Central

    2012-01-01

    Glucocorticoids are steroid hormones that play critical and complex roles in the regulation of triglyceride (TG) homeostasis. Depending on physiological states, glucocorticoids can modulate both TG synthesis and hydrolysis. More intriguingly, glucocorticoids can concurrently affect these two processes in adipocytes. The metabolic effects of glucocorticoids are conferred by intracellular glucocorticoid receptors (GR). GR is a transcription factor that, upon binding to glucocorticoids, regulates the transcriptional rate of specific genes. These GR primary target genes further initiate the physiological and pathological responses of glucocorticoids. In this article, we overview glucocorticoid-regulated genes, especially those potential GR primary target genes, involved in glucocorticoid-regulated TG metabolism. We also discuss transcriptional regulators that could act with GR to participate in these processes. This knowledge is not only important for the fundamental understanding of steroid hormone actions, but also are essential for future therapeutic interventions against metabolic diseases associated with aberrant glucocorticoid signaling, such as insulin resistance, dyslipidemia, central obesity and hepatic steatosis. PMID:22640645

  11. Xenobiotics and the Glucocorticoid Receptor.

    PubMed

    Gulliver, Linda S M

    2017-03-15

    Glucocorticoid Receptor (GR) is present in virtually every human cell type. Representing a nuclear receptor superfamily, GR has several different isoforms essentially acting as ligand-dependent transcription factors, regulating glucocorticoid-responsive gene expression in both a positive and a negative manner. Although the natural ligand of the Glucocorticoid Receptor, glucocorticoids (GC) represent only some of the multiple ligands for GR. Xenobiotics, ubiquitous in the environment, bind to GR and are also capable of activating or repressing GR gene expression, thereby modulating GR cell and tissue-specific downstream effects in a multitude of ways that include responses to inflammatory, allergic, metabolic, neoplastic and autoimmune processes. Many xenobiotics, if inadequately metabolized by xenobiotic metabolizing enzymes and not wholly eliminated, could have deleterious toxic effects with potentially lethal consequences. This review examines GR, the genomic and non-genomic actions of natural and synthetic GC and the body's handling of xenobiotic compounds, before reviewing what is presently known about GR's interactions with many of the more commonly encountered and some of the less well known GR-associated xenobiotics. GR promiscuity and crosstalk with other signaling pathways is discussed, alongside novel roles for GR that include mood disorder and addiction. A knowledge of GR interactions with xenobiotics is increasingly relevant when considering aging populations and the related prevalence of neoplastic disease, together with growing concerns around human exposure to mixtures of chemicals in the environment. Furthermore, escalating rates of obesity, Type 2 diabetes; autoimmune, allergy, addiction and mood disorder-related pathologies, require novel targeted interventions and GR appears a promising pharmacological candidate. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. 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

  13. 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

  14. [Glucocorticoid receptors: basis for the diverse clinical actions of glucocorticoids].

    PubMed

    Gehring, Ulrich

    2004-05-15

    Domain structure of the receptor polypeptide and association with accessory proteins: This review summarizes our present knowledge on the different forms of the glucocorticoid receptor emphasizing structure and functional significance. The nonactivated receptor resides in the cytoplasm. It contains the human receptor polypeptide of 777 amino acids as heteromeric complex in association with two molecules of the heat-shock protein hsp90 and one immunophilin. After binding the hormonal ligand, the receptor becomes activated by dissociation of these accessory proteins. The receptor functions as transcriptional regulator: The receptor polypeptide itself, complexed with hormone, moves on into the cell nucleus to there interact with chromatin and to affect transcriptional processes. By binding as homodimer to specific response elements on the DNA, the receptor functions as positive transcription factor causing increased expression of tissue-specific genes. Alternatively, the receptor interacts with transcription factors like AP-1 or NF-kappaB and inhibits their effects on actively transcribed genes. Pharmacological considerations: The pharmacological possibilities of influencing the diverse medical actions of glucocorticoids are discussed on the level of receptors.

  15. Glucocorticoid receptor in human respiratory epithelial cells.

    PubMed

    Pujolsa, Laura; Mullol, Joaquim; Picado, Cèsar

    2009-01-01

    Inhaled and intranasal glucocorticoids (GCs) are the most common and effective drugs for controlling symptoms and airway inflammation in respiratory diseases such as allergic rhinitis, chronic rhinosinusitis with/without nasal polyps, and asthma, and the respiratory epithelium is a primary target of GC anti-inflammatory actions. GC effects are mediated through the GC receptor (GR). In humans, one single GR gene gives rise to two main GR products, namely GRalpha and GRbeta, which are subject to translational and posttranslational modifications. GRalpha is expressed in virtually all human cells and tissues, including respiratory epithelial cells, and - at least in vitro - is downregulated by GC. GRalpha mediates the anti-inflammatory actions of GC by activating transcription of anti-inflammatory genes through binding of GRalpha to glucocorticoid response elements (GRE) located in the promoter region of target genes, repressing transcription of proinflammatory genes through direct interaction between GRalpha and proinflammatory transcription factors, such as AP-1 and NF-kappaB (transrepression), and also by destabilizing the mRNA of proinflammatory mediators. GRbeta acts as a dominant negative inhibitor of GRalpha-mediated transactivation and transrepression in certain in vitro studies with transfected cells. The GRbeta message is expressed at low levels in numerous tissues and its protein is mainly expressed in inflammatory cells, although it has also been detected in airway epithelial cells. Increased GRbeta expression has been reported in bronchial asthma and nasal polyposis, and after incubation of cells with certain proinflammatory stimuli. However, the role of GRbeta in modulating GC sensitivity in vivo has been highly debated and is as yet unclear.

  16. Distinct Glucocorticoid Receptor Transcriptional Regulatory Surfaces Mediate the Cytotoxic and Cytostatic Effects of Glucocorticoids

    PubMed Central

    Rogatsky, Inez; Hittelman, Adam B.; Pearce, David; Garabedian, Michael J.

    1999-01-01

    Glucocorticoids act through the glucocorticoid receptor (GR), which can function as a transcriptional activator or repressor, to elicit cytostatic and cytotoxic effects in a variety of cells. The molecular mechanisms regulating these events and the target genes affected by the activated receptor remain largely undefined. Using cultured human osteosarcoma cells as a model for the GR antiproliferative effect, we demonstrate that in U20S cells, GR activation leads to irreversible growth inhibition, apoptosis, and repression of Bcl2. This cytotoxic effect is mediated by GR’s transcriptional repression function, since transactivation-deficient mutants and ligands still bring about apoptosis and Bcl2 down-regulation. In contrast, the antiproliferative effect of GR in SAOS2 cells is reversible, does not result in apoptosis or repression of Bcl2, and is a function of the receptor’s ability to stimulate transcription. Thus, the cytotoxic versus cytostatic outcome of glucocorticoid treatment is cell context dependent. Interestingly, the cytostatic effect of glucocorticoids in SAOS2 cells involves multiple GR activation surfaces. GR mutants and ligands that disrupt individual transcriptional activation functions (activation function 1 [AF-1] and AF-2) or receptor dimerization fail to fully inhibit cellular proliferation and, remarkably, discriminate between the targets of GR’s cytostatic action, the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. Induction of p21Cip1 is agonist dependent and requires AF-2 but not AF-1 or GR dimerization. In contrast, induction of p27Kip1 is agonist independent, does not require AF-2 or AF-1, but depends on GR dimerization. Our findings indicate that multiple GR transcriptional regulatory mechanisms that employ distinct receptor surfaces are used to evoke either the cytostatic or cytotoxic response to glucocorticoids. PMID:10373553

  17. Molecular mechanisms of glucocorticoid receptor signaling.

    PubMed

    Labeur, Marta; Holsboer, Florian

    2010-01-01

    This review highlights the most recent findings on the molecular mechanisms of the glucocorticoid receptor (GR). Most effects of glucocorticoids are mediated by the intracellular GR which is present in almost every tissue and controls transcriptional activation via direct and indirect mechanisms. Nevertheless the glu-cocorticoid responses are tissue -and gene- specific. GR associates selectively with corticosteroid ligands produced in the adrenal gland in response to changes of humoral homeostasis. Ligand interaction with GR promotes either GR binding to genomic glucocorticoid response elements, in turn modulating gene transcription, or interaction of GR monomers with other transcription factors activated by other signalling pathways leading to transrepression. The GR regulates a broad spectrum of physiological functions, including cell differentiation, metabolism and inflammatory responses. Thus, disruption or dysregulation of GR function will result in severe impairments in the maintenance of homeostasis and the control of adaptation to stress.

  18. Tumor Necrosis Factor Inhibits Glucocorticoid Receptor Function in Mice

    PubMed Central

    Van Bogaert, Tom; Vandevyver, Sofie; Dejager, Lien; Van Hauwermeiren, Filip; Pinheiro, Iris; Petta, Ioanna; Engblom, David; Kleyman, Anna; Schütz, Günther; Tuckermann, Jan; Libert, Claude

    2011-01-01

    As glucocorticoid resistance (GCR) and the concomitant burden pose a worldwide problem, there is an urgent need for a more effective glucocorticoid therapy, for which insights into the molecular mechanisms of GCR are essential. In this study, we addressed the hypothesis that TNFα, a strong pro-inflammatory mediator in numerous inflammatory diseases, compromises the protective function of the glucocorticoid receptor (GR) against TNFα-induced lethal inflammation. Indeed, protection of mice by dexamethasone against TNFα lethality was completely abolished when it was administered after TNFα stimulation, indicating compromised GR function upon TNFα challenge. TNFα-induced GCR was further demonstrated by impaired GR-dependent gene expression in the liver. Furthermore, TNFα down-regulates the levels of both GR mRNA and protein. However, this down-regulation seems to occur independently of GC production, as TNFα also resulted in down-regulation of GR levels in adrenalectomized mice. These findings suggest that the decreased amount of GR determines the GR response and outcome of TNFα-induced shock, as supported by our studies with GR heterozygous mice. We propose that by inducing GCR, TNFα inhibits a major brake on inflammation and thereby amplifies the pro-inflammatory response. Our findings might prove helpful in understanding GCR in inflammatory diseases in which TNFα is intimately involved. PMID:21646349

  19. Lysine 419 targets human glucocorticoid receptor for proteasomal degradation.

    PubMed

    Wallace, Andrew D; Cao, Yan; Chandramouleeswaran, Sindhu; Cidlowski, John A

    2010-12-01

    Glucocorticoid receptors (GRs) are members of a highly conserved family of ligand dependent transcription factors which following hormone binding undergo homologous down-regulation reducing the levels of receptor protein. This decline in human GR (hGR) is due in part to a decrease in protein receptor stability that may limit cellular responsiveness to ligand. To examine the role of the proteasome protein degradation pathway in steroid-dependent hGR responsiveness, we utilized the proteasomal inhibitors MG-132, beta-lactone, and epoxomicin. HeLa cells and COS cells were treated with proteasome inhibitors in the presence of the GR agonist dexamethasone (Dex), or were pretreated with proteasomal inhibitor and then Dex. Dexamethasone induced glucocorticoid responsive reporter activity significantly over untreated controls, whereas cells treated with proteasomal inhibitors and Dex together showed 2-3-fold increase in activity. Protein sequence analysis of the hGR protein identified several candidate protein degradation motifs including a PEST element. Mutagenesis of this element at lysine 419 was done and mutant K419A hGR failed to undergo ligand dependent down-regulation. Mutant K419A hGR displayed 2-3-fold greater glucocorticoid responsive reporter activity in the presence of Dex than wild type hGR. These differences in transcriptional activity were not due to altered subcellular localization, since when the mutant K419A hGR was fused with the green fluorescent protein (GFP) it was found to move in and out of the nucleus similarly to wild type hGR. Together these results suggest that the proteasome and the identified PEST degradation motif limit steroid-dependent human glucocorticoid receptor signaling.

  20. From receptor balance to rational glucocorticoid therapy.

    PubMed

    de Kloet, E Ron

    2014-08-01

    Corticosteroids secreted as end product of the hypothalamic-pituitary-adrenal axis act like a double-edged sword in the brain. The hormones coordinate appraisal processes and decision making during the initial phase of a stressful experience and promote subsequently cognitive performance underlying the management of stress adaptation. This action exerted by the steroids on the initiation and termination of the stress response is mediated by 2 related receptor systems: mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs). The receptor types are unevenly distributed but colocalized in abundance in neurons of the limbic brain to enable these complementary hormone actions. This contribution starts from a historical perspective with the observation that phasic occupancy of GR during ultradian rhythmicity is needed to maintain responsiveness to corticosteroids. Then, during stress, initially MR activation enhances excitability of limbic networks that are engaged in appraisal and emotion regulation. Next, the rising hormone concentration occupies GR, resulting in reallocation of energy to limbic-cortical circuits with a role in behavioral adaptation and memory storage. Upon MR:GR imbalance, dysregulation of the hypothalamic-pituitary-adrenal axis occurs, which can enhance an individual's vulnerability. Imbalance is characteristic for chronic stress experience and depression but also occurs during exposure to synthetic glucocorticoids. Hence, glucocorticoid psychopathology may develop in susceptible individuals because of suppression of ultradian/circadian rhythmicity and depletion of endogenous corticosterone from brain MR. This knowledge generated from testing the balance hypothesis can be translated to a rational glucocorticoid therapy.

  1. Selenoprotein P Regulation by the Glucocorticoid Receptor

    PubMed Central

    Rock, Colleen; Moos, Philip J.

    2010-01-01

    Maintenance of the antioxidant activity of selenoproteins is one potential mechanism of the beneficial health effects of selenium. Selenoprotein P is the primary selenium distribution protein of the body as well as the major selenium containing protein in serum. The transcriptional regulation of selenoprotein P is of interest since the extrahepatic expression of this gene has demonstrated differentiation-dependent expression in development as well as under different disease states. SEPP1 displays patterned expression in numerous tissues during development and the loss of SEPP1 expression has been observed in malignancy. In addition, factors that influence inflammatory processes like cytokines and their regulators have been implicated in selenoprotein P transcriptional control. Herein, we identify a retinoid responsive element and describe a mechanism where the glucocorticoid receptor negatively regulates expression of selenoprotein P. Luciferase reporter assays and quantitative PCR were used to measure selenoprotein P transcription in engineered HEK-293 cells. When stimulated with ecdysone analogs, selenoprotein P expression was increased with the use of a fusion transcription factor that contains the glucocorticoid receptor DNA binding domain, an ecdysone ligand-binding domain, and a strong transactivation domain as well as the retinoid X receptor. The native glucocorticoid receptor inhibited selenoprotein P transactivation, and selenoprotein P was further attenuated in the presence of dexamethasone. Our results may provide insight into a potential mechanism by which selenium is redistributed during development, differentiation or under conditions of critical illness, where glucocorticoid levels are typically increased. PMID:19513589

  2. Glucocorticoid regulation of the vitamin D receptor.

    PubMed

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

    2010-07-01

    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 immuno-precipitation (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.

  3. 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

  4. Cell cycle regulation of glucocorticoid receptor function.

    PubMed Central

    Hsu, S C; Qi, M; DeFranco, D B

    1992-01-01

    Glucocorticoid receptor (GR) nuclear translocation, transactivation and phosphorylation were examined during the cell cycle in mouse L cell fibroblasts. Glucocorticoid-dependent transactivation of the mouse mammary tumor virus promoter was observed in G0 and S phase synchronized L cells, but not in G2 synchronized cells. G2 effects were selective on the glucocorticoid hormone signal transduction pathway, since glucocorticoid but not heavy metal induction of the endogenous Metallothionein-1 gene was also impaired in G2 synchronized cells. GRs that translocate to the nucleus of G2 synchronized cells in response to dexamethasone treatment were not efficiently retained there and redistributed to the cytoplasmic compartment. In contrast, GRs bound by the glucocorticoid antagonist RU486 were efficiently retained within nuclei of G2 synchronized cells. Inefficient nuclear retention was observed for both dexamethasone- and RU486-bound GRs in L cells that actively progress through G2 following release from an S phase arrest. Finally, site-specific alterations in GR phosphorylation were observed in G2 synchronized cells suggesting that cell cycle regulation of specific protein kinases and phosphatases could influence nuclear retention, recycling and transactivation activity of the GR. Images PMID:1505524

  5. Glucocorticoids relieve collectin-driven suppression of apoptotic cell uptake in murine alveolar macrophages through downregulation of SIRPα1

    PubMed Central

    McCubbrey, Alexandra L; Sonstein, Joanne; Ames, Theresa M.; Freeman, Christine M; Curtis, Jeffrey L

    2012-01-01

    The lung environment actively inhibits apoptotic cell (AC) uptake by alveolar macrophages (AMø) via lung collectin signaling through signal regulatory protein α (SIRPα). Even brief glucocorticoid treatment during maturation of human blood monocyte-derived or murine bone marrow-derived macrophages (Mø) increases their AC uptake. Whether glucocorticoids similarly impact differentiated tissue Mø and the mechanisms for this rapid response are unknown and important to define, given the widespread therapeutic use of inhaled glucocorticoids. We found that the glucocorticoid fluticasone rapidly and dose-dependently increased AC uptake by murine AMø without a requirement for protein synthesis. Fluticasone rapidly suppressed AMø expression of SIRPα mRNA and surface protein, and also activated a more delayed, translation-dependent upregulation of AC recognition receptors that was not required for the early increase in AC uptake. Consistent with a role for SIRPα suppression in rapid glucocorticoid action, murine peritoneal macrophages (PMø) that had not been exposed to lung collectins showed delayed but not rapid increase in AC uptake. However, pretreatment of PMø with the lung collectin surfactant protein D inhibited AC uptake and fluticasone treatment rapidly reversed this inhibition. Thus, glucocorticoids act not only by upregulating AC recognition receptors during Mø maturation but also via a novel rapid down-regulation of SIRPα expression by differentiated tissue Mø. Release of AMø from inhibition of AC uptake by lung collectins may in part explain the beneficial role of inhaled glucocorticoids in inflammatory lung diseases, especially emphysema, in which there is both increased lung parenchymal cell apoptosis and defective AC uptake by AMø. PMID:22615206

  6. Downregulation of cholesteryl ester transfer protein by glucocorticoids: a randomised study on HDL.

    PubMed

    Werumeus Buning, Jorien; Dimova, Lidya G; Perton, Frank G; Tietge, Uwe J F; van Beek, André P; Dullaart, Robin P F

    2017-07-01

    High density lipoprotein (HDL) cholesterol is not decreased in hypercortisolism despite high triglycerides, which may be ascribed to effects on the cholesteryl ester transfer protein (CETP) pathway. We explored if CETP mRNA expression is modulated by glucocorticoid treatment in vitro. Effects of doubling the hydrocortisone (HCT) replacement dose on plasma CETP activity, and HDL characteristics were tested in patients with secondary adrenal insufficiency. Human THP-1 macrophages were incubated with corticosterone in vitro in the presence or absence of a liver X receptor (LXR) agonist, followed by determination of CETP mRNA levels by quantitative real-time PCR. In addition, a randomised double-blind cross-over study was performed in 47 patients with secondary adrenal insufficiency (university medical setting; 10 weeks exposure to a higher HCT dose (0·4-0·6 mg/kg body weight) vs. 10 weeks of a lower HCT dose (0·2-0·3 mg/kg body weight). Corticosterone dose dependently decreased CETP mRNA in THP-1 macrophages. Corticosterone also decreased CETP mRNA expression after LXR pretreatment. In patients, CETP activity decreased with doubling of the HCT dose (P = 0·049), coinciding with an increase in HDL cholesterol, apolipoprotein A-I and the HDL cholesterol/apolipoprotein A-I ratio (reflecting HDL size; P < 0·01 for each). The increase in the HDL cholesterol/apolipoprotein A-I ratio was correlated with the decrease in plasma CETP activity (r = -0·442, P = 0·002). Glucocorticoids downregulate CETP gene expression in a human macrophage cell system. In line, a higher glucocorticoid replacement dose decreases plasma CETP activity in patients, thereby contributing to higher HDL cholesterol and an increase in estimated HDL size. © 2017 Stichting European Society for Clinical Investigation Journal Foundation.

  7. Glucocorticoid Receptor Expression in Peripheral WBCs of Critically Ill Children.

    PubMed

    Shibata, Audrey R Ogawa; Troster, Eduardo J; Wong, Hector R

    2015-06-01

    To characterize glucocorticoid receptor expression in peripheral WBCs of critically ill children using flow cytometry. Prospective observational cohort. A university-affiliated, tertiary PICU. Fifty-two critically ill children. Samples collected for measurement of glucocorticoid receptor expression and parallel cortisol levels. Subjects with cardiovascular failure had significantly lower glucocorticoid receptor expression both in CD4 lymphocytes (mean fluorescence intensity, 522 [354-787] vs 830 [511-1,219]; p = 0.036) and CD8 lymphocytes (mean fluorescence intensity, 686 [350-835] vs 946 [558-1,511]; p = 0.019) compared with subjects without cardiovascular failure. Subjects in the upper 50th percentile of Pediatric Risk of Mortality III scores and organ failure also had significantly lower glucocorticoid receptor expression in CD4 and CD8 lymphocytes. There was no linear correlation between cortisol concentrations and glucocorticoid receptor expression. Our study suggests that patients with shock and increased severity of illness have lower glucocorticoid receptor expression in CD4 and CD8 lymphocytes. Glucocorticoid receptor expression does not correlate well with cortisol levels. Future studies could focus on studying glucocorticoid receptor expression variability and isoform distribution in the pediatric critically ill population as well as on different strategies to optimize glucocorticoid response.

  8. In vitro glucocorticoid receptor binding and transcriptional activation by topically active glucocorticoids.

    PubMed

    Smith, C L; Kreutner, W

    1998-09-01

    Mometasone furoate (MF, CAS 83919-23-7, Sch 32088), budesonide (BUD, CAS 51372-29-3), fluticasone propionate (FP, CAS 80474-14-2), and triamcinolone acetonide (TA, CAS-76-25-5) are corticosteroids that are either currently available or under development for allergic rhinitis and asthma. The relative affinity of these drugs for the glucocorticoid receptor and their ability to stimulate glucocorticoid receptor-mediated transactivation of gene expression were analyzed. All of the test compounds had a higher affinity for the recombinant glucocorticoid receptor than the reference glucocorticoid receptor ligand, dexamethasone (DEX, CAS 50-02-2). In addition, all compounds showed greater potency than dexamethasone in stimulating transcription of a synthetic target gene regulated by a glucocorticoid response element. Of the compounds tested, mometasone furoate had the highest relative binding affinity for the glucocorticoid receptor, followed by fluticasone propionate, budesonide, and triamcinolone acetonide. Similarly, mometasone furoate was the most potent stimulator of glucocorticoid receptor-mediated transactivation of gene expression, followed by fluticasone propionate, tri-amcinolone acetonide, and budesonide. These in vitro studies provide a sensitive means to compare the potency of glucocorticoids and may reliably predict the in vivo topical potency of these drugs.

  9. Chronic restraint stress causes anxiety- and depression-like behaviors, downregulates glucocorticoid receptor expression, and attenuates glutamate release induced by brain-derived neurotrophic factor in the prefrontal cortex.

    PubMed

    Chiba, Shuichi; Numakawa, Tadahiro; Ninomiya, Midori; Richards, Misty C; Wakabayashi, Chisato; Kunugi, Hiroshi

    2012-10-01

    Stress and the resulting increase in glucocorticoid levels have been implicated in the pathophysiology of depressive disorders. We investigated the effects of chronic restraint stress (CRS: 6 hours × 28 days) on anxiety- and depression-like behaviors in rats and on the possible changes in glucocorticoid receptor (GR) expression as well as brain-derived neurotrophic factor (BDNF)-dependent neural function in the prefrontal cortex (PFC). We observed significant reductions in body weight gain, food intake and sucrose preference from 1 week after the onset of CRS. In the 5th week of CRS, we conducted open-field (OFT), elevated plus-maze (EPM) and forced swim tests (FST). We observed a decrease in the number of entries into open arms during the EPM (anxiety-like behavior) and increased immobility during the FST (depression-like behavior). When the PFC was removed after CRS and subject to western blot analysis, the GR expression reduced compared with control, while the levels of BDNF and its receptors remained unchanged. Basal glutamate concentrations in PFC acute slice which were measured by high performance liquid chromatography were not influenced by CRS. However, BDNF-induced glutamate release was attenuated after CRS. These results suggest that reduced GR expression and altered BDNF function may be involved in chronic stress-induced anxiety--and depression-like behaviors. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Glucocorticoid receptors in murine erythroleukaemic cells

    SciTech Connect

    Hammond, K.D.; Torrance, J.M.; DiDomenico, M.

    1987-01-01

    Glucocorticoid receptors in murine erythroleukaemic cells were studied in relation to hexamethylene bisacetamide (HMBA) induced differentiation. Specific binding of dexamethasone was measured. A single class of saturable, high affinity binding sites was demonstrated in intact cells; with cell homogenates or fractions binding was low and could not be reliably quantified. Receptor binding in whole cell suspensions was lower in cells which had been treated with HMBA (36.5 +/- 8.2 pmol/g protein) than in untreated controls (87.9 +/- 23.6 pmol/g protein); dissociation constants were similar in treated (2.7 nM) and untreated cells (2.5 nM). Dexamethasone, hydrocortisone, corticosterone and progesterone competed with tritium-labelled dexamethasone for receptor binding sites; cortisone, deoxycorticosterone and oestradiol had little effect.

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

    PubMed Central

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

    2015-01-01

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

  12. A transgenic zebrafish model for monitoring glucocorticoid receptor activity

    PubMed Central

    Krug, Randall G.; Poshusta, Tanya L.; Skuster, Kimberly J.; Berg, MaKayla R.; Gardner, Samantha L.; Clark, Karl J.

    2014-01-01

    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 socio-economically 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. PMID:24679220

  13. The glucocorticoid receptor: a revisited target for toxins.

    PubMed

    Marketon, Jeanette I Webster; Sternberg, Esther M

    2010-06-01

    The hypothalamic-pituitary-adrenal (HPA) axis activation and glucocorticoid responses are critical for survival from a number of bacterial, viral and toxic insults, demonstrated by the fact that removal of the HPA axis or GR blockade enhances mortality rates. Replacement with synthetic glucocorticoids reverses these effects by providing protection against lethal effects. Glucocorticoid resistance/insensitivity is a common problem in the treatment of many diseases. Much research has focused on the molecular mechanism behind this resistance, but an area that has been neglected is the role of infectious agents and toxins. We have recently shown that the anthrax lethal toxin is able to repress glucocorticoid receptor function. Data suggesting that the glucocorticoid receptor may be a target for a variety of toxins is reviewed here. These studies have important implications for glucocorticoid therapy.

  14. Glucocorticoid Receptor: Implications for Rheumatic Diseases “Glucocorticoids in Rheumatic Diseases”

    PubMed Central

    Kino, Tomoshige; Charmandari, Evangelia; Chrousos, George P.

    2013-01-01

    The glucocorticoid receptor (GR), a member of the nuclear receptor superfamily, mediates most of the known biologic effects of glucocorticoids. The human GR gene consists of 9 exons and expresses 2 alternative splicing isoforms, the GRα and GRβ. GRα is the classic receptor that binds to glucocorticoids and mediates most of the known actions of glucocorticoids, while GRβ does not bind to these hormones and exerts a dominant negative effect upon the GRα-induced transcriptional activity. Each of the two GR splice isoforms has 8 translational variants with specific transcriptional activity and tissue distribution. GRα consists of three subdomains, translocates from the cytoplasm into the nucleus upon binding to glucocorticoids, and regulates the transcriptional activity of numerous glucocorticoid-responsive genes either by binding to its cognate DNA sequences or by interacting with other transcription factors. In addition to these genomic actions, the GR also exerts rapid, non-genomic effects, which are possibly mediated by membrane-localized receptors or by translocation into the mitochondria. All these actions of the GR appear to play an important role in the regulation of the immune system. Specifically, the splicing variant GRβ may be involved in the pathogenesis of rheumatic diseases, while the circadian regulation of the GR activity via acetylation by the Clock transcription factor may have therapeutic implications for the preferential timing of glucocorticoid administration in autoimmune inflammatory disorders. PMID:22018181

  15. Decreased glucocorticoid receptor activity following glucocorticoid receptor antisense RNA gene fragment transfection.

    PubMed Central

    Pepin, M C; Barden, N

    1991-01-01

    Depression is often characterized by increased cortisol secretion caused by hyperactivity of the hypothalamic-pituitary-adrenal axis and by nonsuppression of cortisol secretion following dexamethasone administration. This hyperactivity of the hypothalamic-pituitary-adrenal axis could result from a reduced glucocorticoid receptor (GR) activity in neurons involved in its control. To investigate the effect of reduced neuronal GR levels, we have blocked cellular GR mRNA processing and/or translation by introduction of a complementary GR antisense RNA strand. Two cell lines were transfected with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under control of the mouse mammary tumor virus long terminal repeat (a glucocorticoid-inducible promoter). This gene construction permitted assay of the sensitivity of the cells to glucocorticoid hormones. Cells were also cotransfected with a plasmid containing 1,815 bp of GR cDNA inserted in the reverse orientation downstream from either a neurofilament gene promoter element or the Rous sarcoma virus promoter element. Northern (RNA) blot analysis demonstrated formation of GR antisense RNA strands. Measurement of the sensitivity of CAT activity to exogeneous dexamethasone showed that although dexamethasone increased CAT activity by as much as 13-fold in control incubations, expression of GR antisense RNA caused a 2- to 4-fold decrease in the CAT response to dexamethasone. Stable transfectants bearing the GR antisense gene fragment construction demonstrated a 50 to 70% decrease of functional GR levels compared with normal cells, as evidenced by a ligand-binding assay with the type II glucocorticoid receptor-specific ligand [3H]RU 28362. These results validate the use of antisense RNA to GR to decrease cellular response to glucocorticoids. Images PMID:1996114

  16. Expression of glucocorticoid receptors in the regenerating human skeletal muscle.

    PubMed

    Filipović, D; Pirkmajer, S; Mis, K; Mars, T; Grubic, Z

    2011-01-01

    Many stress conditions are accompanied by skeletal muscle dysfunction and regeneration, which is essentially a recapitulation of the embryonic development. However, regeneration usually occurs under conditions of hypothalamus-pituitary-adrenal gland axis activation and therefore increased glucocorticoid (GC) levels. Glucocorticoid receptor (GR), the main determinant of cellular responsiveness to GCs, exists in two isoforms (GRalpha and GRbeta) in humans. While the role of GRalpha is well characterized, GRbeta remains an elusive player in GC signalling. To elucidate basic characteristics of GC signalling in the regenerating human skeletal muscle we assessed GRalpha and GRbeta expression pattern in cultured human myoblasts and myotubes and their response to 24-hour dexamethasone (DEX) treatment. There was no difference in GRalpha mRNA and protein expression or DEX-mediated GRalpha down-regulation in myoblasts and myotubes. GRbeta mRNA level was very low in myoblasts and remained unaffected by differentiation and/or DEX. GRbeta protein could not be detected. These results indicate that response to GCs is established very early during human skeletal muscle regeneration and that it remains practically unchanged before innervation is established. Very low GRbeta mRNA expression and inability to detect GRbeta protein suggests that GRbeta is not a major player in the early stages of human skeletal muscle regeneration.

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

    PubMed

    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.

  18. Glucocorticoid receptors on and in a unicellular organism, Cryptobia salmositica.

    PubMed

    Li, Mao; Woo, Patrick T K

    2014-03-01

    This is the first report to our knowledge that demonstrates a functional steroid hormone receptor in a protozoon. The study used Cryptobia salmositica, a pathogenic haemoflagellate found in salmonid fishes. It has been previously shown that cortisol and dexamethasone (a synthetic glucocorticoid) enhanced the multiplication of C. salmositica under in vitro conditions indicating the presence of glucocorticoid receptors on/in the parasite. Also, the glucocorticoid receptor antagonist, mifepristone (RU486), inhibited the stimulatory effect of the two glucocorticoids on parasite multiplication. In the present study, we used an antibody (produced in a rabbit against glucocorticoid receptor protein) agglutination test and confocal microscopy with immunohistofluorescence staining to demonstrate cortisol-glucocorticoid receptor-like protein receptors on the plasma membrane and in the cytoplasm of the parasite. In two in vitro studies, the addition of 50ngml(-1) of RU486 was more effective in inhibiting parasite replication in cultures with 7,000parasitesml(-1) than in cultures with 14,000parasitesml(-1). Also, 100ngml(-1) of RU486/ml was more effective than 50ngml(-1) in inhibiting parasite multiplication in the 14,000 parasitesml(-1) cultures. These in vitro studies indicate that the number of binding sites on/in the parasite is finite. The findings may be important in future studies especially on steroid receptor signalling pathways and dissection of ligand-receptor interactions, and for evaluating the adaptations that develop in pathogens as part of the host-parasite interaction.

  19. Relationship between expression of glucocorticoid receptor isoforms and glucocorticoid resistance in immune thrombocytopenia.

    PubMed

    Liang, Yan; Song, Meng Meng; Liu, Shi Yan; Ma, Liang Liang

    2016-08-01

    Glucocorticoids (GCs) play an important role in the treatment of several hematological malignancies, such as immune thrombocytopenia (ITP). The glucocorticoid receptor (GR) mediates the effects of GCs. Five isoforms of GR mRNA were described: GRα, GRβ, GRγ, GRP, and GRA. GR levels are regulated by alternative splicing of GR mRNA. Several studies demonstrated that a lower GR expression was associated with poor GC response. This study investigated the expression of GR isoforms and the relationship between GC resistance in ITP. This study determined GRα/β/γ/P mRNA and GRα/β protein expression levels using SYBR Green Real-time PCR and Western blot, respectively, in 49 newly diagnosed ITP patients and 31 controls. The expression of GR isoform mRNA in ITP and controls showed the following trend: GRα > GRP > GRγ > GRβ. The expression of GRα, β mRNA and the total frequency of the four GR isoforms in ITP was significantly higher than in controls (P < 0.05). The expression of GRα mRNA and protein in the GC-resistant group was significantly lower than that in the GC-sensitive group and controls (P < 0.05). GRβ could not be detected at the protein level in our experimental conditions. GRα and GRP were the main GR isoforms responsible for the effects of GC, and GRα and GRP exhibited synergistic effects. The down-regulation of GRα levels may play an important role in GC resistance in ITP. The effects of GCs in ITP were not associated with changes in GRβ and GRγ.

  20. Endothelial glucocorticoid receptor suppresses atherogenesis--brief report.

    PubMed

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

    2015-04-01

    The purpose of this study was to determine the role of the endothelial glucocorticoid receptor in the pathogenesis of atherosclerosis. 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 2 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 evidenced by increased macrophage recruitment in the lesions. These data suggest that the endothelial glucocorticoid receptor is important for tonic inhibition of inflammation and limitation of atherosclerosis progression in this model. © 2015 American Heart Association, Inc.

  1. 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

  2. NALP3 inflammasome upregulation and CASP1 cleavage of the glucocorticoid receptor cause glucocorticoid resistance in leukemia cells.

    PubMed

    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

    2015-06-01

    Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and resistance to glucocorticoids in leukemia cells confers poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia cells from 444 patients newly diagnosed with ALL and found significantly higher expression of CASP1 (encoding caspase 1) and its activator NLRP3 in glucocorticoid-resistant leukemia cells, resulting from significantly lower somatic methylation of the CASP1 and NLRP3 promoters. Overexpression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished the 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 the glucocorticoid transcriptional response suggests that this mechanism could also modify glucocorticoid effects in other diseases.

  3. 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

  4. Characterization of glucocorticoid receptor on lymphocytes in Chinese patients with glucocorticoid-induced glaucoma.

    PubMed

    Zhuo, Y; Ge, J; Guo, Y

    1998-09-01

    We studied the pathogenesis of glucocorticoid-induced glucoma (GIG) through characterization of glucocorticoid receptor (GR) on lymphocytes in Chinese patients with GIG. By radioligand receptor binding followed by Scatchard analysis, the specific binding sites were characterized and quantitated for glucocorticoid receptors on peripheral blood lymphocytes obtained from patients with GIG and the control group. The binding sites we detected were as follows: 12.7 +/- 1.47 x 10(3) receptors per cell with a KD of 3.02 +/- 0.62 nmol/L in patients with GIG, 7.26 +/- 0.45 x 10(3) receptors per cell with a KD of 3.03 +/- 0.56 nmol/L in the control group. The statistical difference of receptors per cell is significant between two groups (p < 0.05), patients with GIG having more GR binding sites, while the difference of Kd is not significant (p > 0.05). The preliminary findings suggest that patients with GIG are more sensitive to glucocorticoid and the increase of binding sites of GR may be the receptor and molecular basis of the pathogenesis of GIG.

  5. [Modulation of glucocorticoid receptor interaction with non-steroidal drugs].

    PubMed

    Golikov, P P; Nikolaeva, N Iu

    1993-01-01

    The Scatchard analysis of the specific binding of triamcinolone 3H-acetonide (TA-3HA) to Type II glucocorticoid receptors of cytosol from the liver of female Wistar rats weighing 180-200 g has shown that emoxipin at concentrations of 1 and 2 mM and analgin at concentrations of 5 and 10 mM reduce the density of glucocorticoid receptors and the association constant of a hormone-receptor complex. Analgin, 5 mM, increases the dissociation velocity constant of TA-3HA 5 times the effect of unlabeled triamcinolone acetonide. Emoxipin, 1 mM, produces the same effect on the receptor dissociation velocity constant of TA-3HA as the unlabeled triamcinolone acetonide. The Berke analysis has established that emoxipin and analgin reduce glucocorticoid receptor interactions by uncompetitive inhibition.

  6. Glucocorticoids and atrial natriuretic factor receptors on vascular smooth muscle.

    PubMed

    Yasunari, K; Kohno, M; Murakawa, K; Yokokawa, K; Takeda, T

    1990-11-01

    The effect of glucocorticoids on the atrial natriuretic factor (ANF)-mediated formation of cyclic guanosine monophosphate (cGMP) by intact vascular smooth muscle cells (VSMC) was studied in rats. Cultured VSMC were obtained from the renal arteries of 14-week-old Wistar rats by the explant method. Micromolar concentrations of dexamethasone, given as pretreatment for 48 hours, suppressed the ANF-mediated response. The dexamethasone-induced suppression was detectable at 6 hours and reached a maximum 24 hours after administration in a dose-dependent manner. Inhibitors of protein synthesis blocked this effect of the glucocorticoid. The basal activity of guanylate cyclase in the dexamethasone-treated cells was lower than in the control cells. Other steroids having glucocorticoid action mimicked this suppression of the ANF-mediated response. This suppression was blocked by a glucocorticoid receptor antagonist. The results suggest that glucocorticoids suppress ANF-mediated cGMP formation by VSMC through glucocorticoid type II receptors and the induction of protein synthesis. Suppression of the ANF-mediated response may play a role in glucocorticoid-induced hypertension.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. Negative glucocorticoid receptor response elements and their role in glucocorticoid action.

    PubMed

    Dostert, A; Heinzel, T

    2004-01-01

    The glucocorticoid receptor (GR) belongs to the steroid hormone receptor subclass of nuclear receptors and controls physiological processes through activation and repression of specific target genes. The ligand-activated receptor dimer activates gene expression by binding to specific DNA sequences (glucocorticoid response element, GRE) in the promoter regions of glucocorticoid-regulated genes. In contrast to the regulation of these classical GREs, the repression of negatively regulated target genes is mediated by negative GREs (nGRE), composite GREs or by transrepression. Due to their broad therapeutic spectrum and superior therapeutic effects glucocorticoids (GCs) are the most effective drugs used for the treatment of acute and chronic inflammatory diseases. Unfortunately, long term systemic therapy with GCs is restricted due to their metabolic side effects. It is assumed that transrepression of transcription factors such as AP-1 and NF-kappa B is the main mechanism by which glucocorticoids mediate their anti-inflammatory activity, whereas the side effects of GCs are mainly mediated by GR-DNA-interaction either by activation or by negative regulation of gene expression. While trans-repression has been characterized in detail, the molecular mechanisms of DNA-dependent cis-repression remain unclear. In this review, we focus on current knowledge about nGRE-mediated target gene repression and the relevance and function of these genes for glucocorticoid action. Negative GREs contribute to the regulation of the hypothalamic-pituitary-adrenal (HPA) axis (POMC and CRH), bone (osteocalcin) and skin (keratins) function, inflammation (IL-1beta), angiogenesis (proliferin) and lactation (prolactin). The discovery of the underlying mechanisms, especially the comparison to positive GREs and trans-repression may help in the future to discover and analyze novel selective GR agonists.

  12. Glucocorticoid receptor activation and inactivation in cultured human lymphocytes.

    PubMed

    Wheeler, R H; Leach, K L; La Forest, A C; O'Toole, T E; Wagner, R; Pratt, W B

    1981-01-10

    Although glucocorticoids are not cytolytic for and do not inhibit the growth of the IM-9 line of cultured human lymphoblasts, these cells have a high steroid-binding capacity. We have used IM-9 cells in order to examine whether unoccupied glucocorticoid receptors are inactivated and activated in intact cells. when IM-9 cells are incubated in glucose-free medium in a nitrogen atmosphere, both their ability to bind triamcinolone acetonide and their ATP levels decline and, when glucose and oxygen are reintroduced, ATP levels and receptor activity return. The specific glucocorticoid-binding activity of cytosol prepared from cells exposed to various degrees of energy limitation is directly correlated with the ATP content. Receptor activation in intact cells is rapid and independent of protein synthesis. Cytosol prepared from inactivated cells cannot be activated by addition of ATP. The inactivation of glucocorticoid receptors that occurs when cytosol from normal IM-9 cells is incubated at 25 degrees C is inhibited by molybdate, vanadate, fluoride, ATP, and several other nucleotides. The experiments with intact human lymphoblasts suggest that assays of specific glucocorticoid-binding capacity do not necessarily reflect the cellular content of receptor protein.

  13. Novel selective glucocorticoid receptor agonists (SEGRAs) with a covalent warhead for long-lasting inhibition.

    PubMed

    Ryabtsova, Oksana; Joossens, Jurgen; Van Der Veken, Pieter; Vanden Berghe, Wim; Augustyns, Koen; De Winter, Hans

    2016-10-15

    The synthesis and in vitro properties of six analogues of the selective glucocorticoid receptor (GR) agonist GSK866, bearing a warhead for covalent linkage to the glucocorticoid receptor, is described.

  14. Specific glucocorticoid receptor binding to DNA reconstituted in a nucleosome.

    PubMed Central

    Perlmann, T; Wrange, O

    1988-01-01

    We have reconstituted a nucleosome with core histones from rat liver using a restriction fragment containing a sequence from the mouse mammary tumour virus (MTV) long terminal repeat (LTR). This sequence harbours glucocorticoid responsive elements (GREs) which mediate glucocorticoid hormone induction of transcription from the MTV promoter via glucocorticoid receptor (GR) binding. Exonuclease III and DNase I footprinting demonstrated that the reconstituted nucleosome was specifically located between positions -219 and -76. A nucleosome was previously shown to be located at a similar or identical position in the MTV promoter in situ and to be structurally altered upon glucocorticoid hormone induction. We demonstrated, by DNase I footprinting, that GR is able to bind sequence specifically to the DNA in the in vitro assembled nucleosome. No evidence for unfolding of the nucleosome was obtained, but the DNase I footprinting pattern demonstrated GR induced local alterations in the DNA. Images PMID:2846275

  15. Modulatory effects of unsaturated fatty acids on the binding of glucocorticoids to rat liver glucocorticoid receptors.

    PubMed

    Vallette, G; Vanet, A; Sumida, C; Nunez, E A

    1991-09-01

    Binding of the synthetic glucocorticoid dexamethasone to the rat liver cytosol glucocorticoid receptor was inhibited by physiological concentrations of nonesterified fatty acids as a function of increasing dose, degree of unsaturation, and chain length of the fatty acid. Polyunsaturated fatty acids were the most potent inhibitors. Scatchard analysis and Line-weaver-Burk plots of the binding data revealed that both the association constants and number of binding sites decreased and that polyunsaturated fatty acids inhibition was of a mixed non-competitive type. The dissociation rate constant of [3H]dexamethasone from glucocorticoid receptors was increased by up to 10 times in the presence of docosahexaenoic acid, whereas a competitive inhibitor like the glucocorticoid antagonist RU 38486 had no effect. Moreover, sucrose density gradient analysis showed that docosahexaenoic acid inhibited the binding of [3H] dexamethasone to both the 8.8S and 4S forms. The results strongly suggest that unsaturated fatty acids are interacting at a site on the receptor different from the hormone binding site and the heat shock protein and that by binding to a second site unsaturated fatty acids greatly change the conformation of the hormone binding site to reduce its affinity for the hormone, either partially or completely depending on the concentration and the class of the fatty acid.

  16. Interaction of rat liver glucocorticoid receptor with sodium tungstate.

    PubMed

    Murakami, N; Healy, S P; Moudgil, V K

    1982-06-15

    Effects of sodium tungstate on various properties of rat liver glucocorticoid receptor were examined at pH7 and pH 8. At pH 7, [3H]triamcinolone acetonide binding in rat liver cytosol preparations was completely blocked in the presence of 10--20 mM-sodium tungstate at 4 degrees C, whereas at 37 degrees C a 30 min incubation of cytosol receptor preparation with 1 mM-sodium tungstate reduced the loss of unoccupied receptor by 50%. At pH 8.0, tungstate presence during the 37 degrees C incubation maintained the steroid-binding capacity of unoccupied glucocorticoid receptor at control (4 degrees C) levels. In addition, heat-activation of cytosolic glucocorticoid-receptor complex was blocked by 1 mM- and 10 mM-sodium tungstate at pH 7 and pH 8 respectively. The DNA-cellulose binding by activated receptor was also inhibited completely and irreversibly by 5 mM-tungstate at pH 7, whereas at pH 8 no significant effect was observed with up to 20 mM-tungstate. The entire DNA-cellulose-bound glucocorticoid-receptor complex from control samples could be extracted by incubation with 1 mM- and 20 mM-tungstate at pH 7 and pH 8 respectively, and appeared to sediment as a 4.3--4.6 S molecule, both in 0.01 M- and 0.3 M-KCl-containing sucrose gradients. Tungstate effects are, therefore, pH-dependent and appear to involve an interaction with both the non-activated and the activated forms of the glucocorticoid receptor.

  17. Sepsis and glucocorticoids upregulate p300 and downregulate HDAC6 expression and activity in skeletal muscle.

    PubMed

    Alamdari, Nima; Smith, Ira J; Aversa, Zaira; Hasselgren, Per-Olof

    2010-08-01

    Muscle wasting during sepsis is in part regulated by glucocorticoids. In recent studies, treatment of cultured muscle cells in vitro with dexamethasone upregulated expression and activity of p300, a histone acetyl transferase (HAT), and reduced expression and activity of the histone deacetylases-3 (HDAC3) and -6, changes that favor hyperacetylation. Here, we tested the hypothesis that sepsis and glucocorticoids regulate p300 and HDAC3 and -6 in skeletal muscle in vivo. Because sepsis-induced metabolic changes are particularly pronounced in white, fast-twitch skeletal muscle, most experiments were performed in extensor digitorum longus muscles. Sepsis in rats upregulated p300 mRNA and protein levels, stimulated HAT activity, and reduced HDAC6 expression and HDAC activity. The sepsis-induced changes in p300 and HDAC expression were prevented by the glucocorticoid receptor antagonist RU38486. Treatment of rats with dexamethasone increased expression of p300 and HAT activity, reduced expression of HDAC3 and -6, and inhibited HDAC activity. Finally, treatment with the HDAC inhibitor trichostatin A resulted in increased muscle proteolysis and expression of the ubiquitin ligase atrogin-1. Taken together, our results suggest for the first time that sepsis-induced muscle wasting may be regulated by glucocorticoid-dependent hyperacetylation caused by increased p300 and reduced HDAC expression and activity. The recent development of pharmacological HDAC activators may provide a novel avenue to prevent and treat muscle wasting in sepsis and other catabolic conditions.

  18. Glucocorticoid receptors in Epstein-Barr virus-transformed lymphocytes from patients with glucocorticoid resistance and a glucocorticoid-resistant New World primate species.

    PubMed

    Tomita, M; Brandon, D D; Chrousos, G P; Vingerhoeds, A C; Foster, C M; Fowler, D; Loriaux, D L; Lipsett, M B

    1986-06-01

    Members of a previously reported family with glucocorticoid resistance and several New World primates have high plasma cortisol concentrations without any signs of glucocorticoid excess. The glucocorticoid receptor in circulating leukocytes and cultured skin fibroblasts from these patients and the animals is characterized by a decreased affinity for dexamethasone. On the other hand, the cell content of receptor is similar to that of corresponding tissues of normal humans. Detailed biochemical-biophysical studies of the glucocorticoid receptor in this familial syndrome and animal model became possible with the use of Epstein-Barr virus-transformed lymphocyte lines. Cell lines from patients with this syndrome and from the marmoset (Saguinus oedipus) contained decreased amounts of glucocorticoid receptors with concomitant decreases in nuclear receptor content compared to cultured Epstein-Barr virus-transformed lymphocytes from normal human subjects. This may reflect diminished induction of glucocorticoid receptor during viral transformation of cells from the patients and the animal model. Receptors from a severely affected glucocorticoid-resistant patient and the marmoset had decreased affinity for dexamethasone. Evidence for a mild affinity defect of the glucocorticoid receptor in a patient with asymptomatic glucocorticoid resistance was obtained by increased hormone-receptor dissociation at an elevated temperature. Thermal stability, mero-receptor formation, thermal activation of cytosolic receptor, and mol wt of receptors from all cell lines were normal. Only the receptors of the severely affected patient had a discernible defect in temperature-induced activation of intact cells. We conclude that the major detectable change in the receptor in both the patients and the animal model is the decreased affinity for glucocorticoid. Viral receptor induction is decreased in both patient and marmoset cells. The physiological relevance of this phenomenon is not known. Gross

  19. 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.

  20. FK506-Binding Protein 51 Regulates Nuclear Transport of the Glucocorticoid Receptor β and Glucocorticoid Responsiveness

    PubMed Central

    Zhang, Xinyu; Clark, Abbot F.; Yorio, Thomas

    2008-01-01

    PURPOSE A spliced variant of the human glucocorticoid receptor GRβ has been implicated in glucocorticoid responsiveness in glaucoma. Over-expression of the FK506-binding immunophilin FKBP51 also causes a generalized state of glucocorticoid resistance. In the present study, the roles of FKBP51 in the nuclear transport of GRβ and glucocorticoid responsiveness were investigated. METHODS Human trabecular meshwork cells (GTM3 and TM5) and HeLa cells were treated with dexamethasone (DEX) and FK506 and transfected with GRβ and FKBP51 expression vectors. Coimmunoprecipitation and Western blot analyses were performed to study interactions of FKBP51 and FKBP52 with GRα, GRβ, Hsp90, or dynein. The cells were transfected with a GRE-luciferase reporter to evaluate the effects of DEX and FK506 and the overexpression of GRβ and FKBP51 on glucocorticoid-mediated gene expression. RESULTS FKBP51 was involved in constitutive nuclear transport of both GRα and -β in the absence of ligands. FKBP52 appeared to be solely responsible for the nuclear transport of ligand-activated GRα. DEX stimulated the translocation of GRα but not GRβ. Overexpression of either GRβ or FKBP51 stimulated GRβ translocation and reduced DEX-induced luciferase in HeLa cells. FK506 did not alter DEX-induced translocation of GRα. However, FK506 increased the association of FKBP51 with GRβ and stimulated DEX-induced translocation of GRβ in normal TM cells, but not in glaucoma TM cells. Increased nuclear GRβ significantly inhibited glucocorticoid responsiveness in TM cells. CONCLUSIONS Nuclear transport of GRβ represents a novel mechanism through which FKBP51 alters GC sensitivity. GRβ and FKBP51 may be responsible for increased responsiveness in steroid-induced ocular hypertensive individuals as well as in patients with glaucoma. PMID:18326728

  1. FK506-binding protein 51 regulates nuclear transport of the glucocorticoid receptor beta and glucocorticoid responsiveness.

    PubMed

    Zhang, Xinyu; Clark, Abbot F; Yorio, Thomas

    2008-03-01

    A spliced variant of the human glucocorticoid receptor GRbeta has been implicated in glucocorticoid responsiveness in glaucoma. Over-expression of the FK506-binding immunophilin FKBP51 also causes a generalized state of glucocorticoid resistance. In the present study, the roles of FKBP51 in the nuclear transport of GRbeta and glucocorticoid responsiveness were investigated. Human trabecular meshwork cells (GTM3 and TM5) and HeLa cells were treated with dexamethasone (DEX) and FK506 and transfected with GRbeta and FKBP51 expression vectors. Coimmunoprecipitation and Western blot analyses were performed to study interactions of FKBP51 and FKBP52 with GRalpha, GRbeta, Hsp90, or dynein. The cells were transfected with a GRE-luciferase reporter to evaluate the effects of DEX and FK506 and the overexpression of GRbeta and FKBP51 on glucocorticoid-mediated gene expression. FKBP51 was involved in constitutive nuclear transport of both GRalpha and -beta in the absence of ligands. FKBP52 appeared to be solely responsible for the nuclear transport of ligand-activated GRalpha. DEX stimulated the translocation of GRalpha but not GRbeta. Overexpression of either GRbeta or FKBP51 stimulated GRbeta translocation and reduced DEX-induced luciferase in HeLa cells. FK506 did not alter DEX-induced translocation of GRalpha. However, FK506 increased the association of FKBP51 with GRbeta and stimulated DEX-induced translocation of GRbeta in normal TM cells, but not in glaucoma TM cells. Increased nuclear GRbeta significantly inhibited glucocorticoid responsiveness in TM cells. Nuclear transport of GRbeta represents a novel mechanism through which FKBP51 alters GC sensitivity. GRbeta and FKBP51 may be responsible for increased responsiveness in steroid-induced ocular hypertensive individuals as well as in patients with glaucoma.

  2. Discovery of orally available tetrahydroquinoline-based glucocorticoid receptor agonists.

    PubMed

    Hudson, Andrew R; Higuchi, Robert I; Roach, Steven L; Adams, Mark E; Vassar, Angela; Syka, Peter M; Mais, Dale E; Miner, Jeffrey N; Marschke, Keith B; Zhi, Lin

    2011-03-15

    A series of tetrahydroquinoline derivatives were synthesized and profiled for their ability to act as glucocorticoid receptor selective modulators. Structure-activity relationships of the tetrahydroquinoline B-ring lead to the discovery of orally available GR-selective agonists with high in vivo activity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. The glucocorticoid receptor: pivot of depression and of antidepressant treatment?

    PubMed

    Anacker, Christoph; Zunszain, Patricia A; Carvalho, Livia A; Pariante, Carmine M

    2011-04-01

    Hyperactivity of the hypothalamus-pituitary-adrenal (HPA) axis and increased levels of glucocorticoid hormones in patients with depression have mostly been ascribed to impaired feedback regulation of the HPA axis, possibly caused by altered function of the receptor for glucocorticoid hormones, the glucocorticoid receptor (GR). Antidepressants, in turn, ameliorate many of the neurobiological disturbances in depression, including HPA axis hyperactivity, and thereby alleviate depressive symptoms. There is strong evidence for the notion that antidepressants exert these effects by modulating the GR. Such modulations, however, can be manifold and range from regulation of receptor expression to post-translational modifications, which may result in differences in GR nuclear translocation and GR-dependent gene transcription. The idea that the therapeutic action of antidepressants is mediated, at least in part, by restoring GR function, is consistent with studies showing that decreased GR function contributes to HPA axis hyperactivity and to the development of depressive symptoms. Conversely, excessive glucocorticoid signalling, which requires an active GR, is associated with functional impairments in the depressed brain, especially in the hippocampus, where it results in reduced neurogenesis and impaired neuroplasticity. In this review, we will focus on the GR as a key player in the precipitation, development and resolution of depression. We will discuss potential explanations for the apparent controversy between glucocorticoid resistance and the detrimental effects of excessive glucocorticoid signalling. We will review some of the evidence for modulation of the GR by antidepressants and we will provide further insight into how antidepressants may regulate the GR to overcome depressive symptoms.

  4. Glomerular Glucocorticoid Receptors Expression and Clinicopathological Types of Childhood Nephrotic Syndrome.

    PubMed

    Gamal, Yasser; Badawy, Ahlam; Swelam, Salwa; Tawfeek, Mostafa S K; Gad, Eman Fathalla

    2017-02-01

    Glucocorticoids are primary therapy of idiopathic nephrotic syndrome (INS). However, not all children respond to steroid therapy. We assessed glomerular glucocorticoid receptor expression in fifty-one children with INS and its relation to response to steroid therapy and to histopathological type. Clinical, laboratory and glomerular expression of glucocorticoid receptors were compared between groups with different steroid response. Glomerular glucocorticoid expression was slightly higher in controls than in minimal change early responders, which in turn was significantly higher than in minimal change late responders. There was significantly lower glomerular glucocorticoid receptor expression in steroid-resistance compared to early responders, late responders and controls. Glomerular glucocorticoid expression was significantly higher in all minimal change disease (MCD) compared to focal segmental glomerulosclerosis. In INS, response to glucocorticoid is dependent on glomerular expression of receptors and peripheral expression. Evaluation of glomerular glucocorticoid receptor expression at time of diagnosis of NS can predict response to steroid therapy.

  5. 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.

  6. Loss of the podocyte glucocorticoid receptor exacerbates proteinuria after injury.

    PubMed

    Zhou, Han; Tian, Xuefei; Tufro, Alda; Moeckel, Gilbert; Ishibe, Shuta; Goodwin, Julie

    2017-08-29

    Nephrotic syndrome is a common disorder in adults and children whose etiology is largely unknown. Glucocorticoids remain the mainstay of therapy in most cases, though their mechanism of action remains poorly understood. Emerging evidence suggests that immunomodulatory therapies used in nephrotic syndrome directly target the podocytes. To study how steroids directly affect the podocytes in the treatment of proteinuria, we created a mouse model with podocyte-specific deletion of the glucocorticoid receptor. The podocyte-specific glucocorticoid receptor (GR) knockout mice had similar renal function and protein excretion compared to wild type. However, after glomerular injury induced by either LPS or nephrotoxic serum, the podocyte GR knockout mice demonstrated worsened proteinuria compared to wild type. Ultrastructural examination of podocytes confirmed more robust foot process effacement in the knockout animals. Expression of several key slit diaphragm protein was down regulated in pGR KO mice. Primary podocytes isolated from wild type and podocyte GR knockout mice showed similar actin stress fiber staining patterns in unstimulated conditions. Yet, when exposed to LPS, GR knockout podocytes demonstrated fewer stress fibers and impaired migration compared to wild type podocytes. We conclude that the podocyte glucocorticoid receptor is important for limiting proteinuria in settings of podocyte injury.

  7. 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.

  8. Properties of binding of partially purified glucocorticoid receptor from rat liver with glucocorticoids of different biopotencies.

    PubMed

    Izawa, M; Satoh, Y; Yoshida, A; Ichii, S

    1985-06-01

    To elucidate the relationship between binding parameters and biopotencies of glucocorticoids, we partially purified the receptor from the liver cytosol of rats in a dexamethasone-bound and unactivated form by precipitation with protamine sulfate, gel filtration and DEAE-cellulose chromatography (approximately 100-fold) and examined the interaction of the preparation with 3 glucocorticoids of different biopotencies (dexamethasone; Dex, corticosterone; Cort and prednisolone; Pred). The partially purified receptor (PPR) was stable at -20 degrees C for at least 2 months in the presence of bovine serum albumin, glycerol, molybdate and dithiothreitol. Treatment of the PPR with p-hydroxymercuribenzoate liberated the ligands and the treated PPR reassociated 3H-glucocorticoids efficiently following the addition of dithiothreitol. The reassociated PPR was bound to the DNA-cellulose after a brief heating. Metabolic activity on ligands and inactivation of the binding sites in the PPR were insignificant under the conditions used. Kd's were approximately 0.9, approximately 3 and approximately 6 nM for Dex, Cort and Pred, respectively (at 0 degree C). Relative binding affinity of ligands to the PPR which was estimated by competitions was higher in the order of triamcinolone acetonide greater than Dex greater than Cort greater than Pred greater than progesterone greater than cortexolone. Association of Dex and Cort was relatively rapid and significantly accelerated by raising the incubation temperature, while the association of Pred was slower and effects of the temperature was moderate. The rate of dissociations was also varied with ligands. The rate of dissociation of Dex was the lowest among the 3 ligands and was elevated by raising the temperature. Because the effect of temperature was more pronounced in the dissociation than in the association, apparent Ka's decreased at higher temperature. Thermodynamic examinations of glucocorticoid binding in the PPR revealed that the

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

    PubMed

    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.

  10. 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

  11. 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

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

    PubMed

    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.

  13. 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

  14. Glucocorticoid regulation of inflammation and its behavioral and metabolic correlates: from HPA axis to glucocorticoid receptor dysfunction

    PubMed Central

    Silverman, Marni N.; Sternberg, Esther M.

    2012-01-01

    Enhanced susceptibility to inflammatory and autoimmune disease can be related to impairments in HPA axis activity and associated hypocortisolism, or to glucocorticoid resistance resulting from impairments in local factors affecting glucocorticoid availability and function, including the glucocorticoid receptor (GR). The enhanced inflammation and hypercortisolism that typically characterize stress-related illnesses, such as depression, metabolic syndrome, cardiovascular disease or osteoporosis, may also be related to increased glucocorticoid resistance. This review focuses on impaired GR function as a molecular mechanism of glucocorticoid resistance. Both genetic and environmental factors can contribute to impaired GR function. The evidence that glucocorticoid resistance can be environmentally induced has important implications for management of stress-related inflammatory illnesses and underscores the importance of prevention and management of chronic stress. The simultaneous assessment of neural, endocrine, and immune biomarkers through various noninvasive methods will also be discussed. PMID:22823394

  15. Glucocorticoid-induced hypertension and cardiac injury: effects of mineralocorticoid and glucocorticoid receptor antagonism.

    PubMed

    Hattori, Takuya; Murase, Tamayo; Iwase, Erika; Takahashi, Keiji; Ohtake, Masafumi; Tsuboi, Koji; Ohtake, Mayuko; Miyachi, Masaaki; Murohara, Toyoaki; Nagata, Kohzo

    2013-02-01

    Glucocorticoids are widely administered for the treatment of various disorders, although their long-term use results in adverse effects associated with glucocorticoid excess. We investigated the pathophysiological roles of glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs) in the cardiac changes induced by exogenous corticosterone in rats. Corticosterone or vehicle was injected twice daily in rats from 8 to 12 weeks of age. The effects of the GR antagonist RU486, the MR antagonist spironolactone, or both agents on corticosterone action were also determined. Corticosterone induced hypertension, left ventricular (LV) fibrosis, and LV diastolic dysfunction. Neither RU486 nor spironolactone affected corticosterone-induced hypertension, whereas spironolactone, but not RU486, attenuated the effects of corticosterone on LV fibrosis and diastolic function. Corticosterone also increased cardiac oxidative stress and inflammation in a manner sensitive to spironolactone but not to RU486. The corticosterone-induced LV atrophy was not affected by either RU486 or spironolactone. Our results implicate MRs in the cardiac fibrosis and diastolic dysfunction, but not MRs or GRs in the cardiac atrophy, induced by corticosterone. Neither MRs nor GRs appear to contribute to corticosterone-induced hypertension.

  16. Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene

    SciTech Connect

    Karl, M.; Lamberts, S.W.J.; Detera-Wadleigh, S.D.; Encio, I.J.; Stratakis, C.A.; Hurley, D.M.; Accili, D.; Chrousos, G.P. Erasmus Univ. of Rotterdam )

    1993-03-01

    The clinical syndrome of generalized, compensated glucocorticoid resistance is characterized by increased cortisol secretion without clinical evidence of hyper- or hypocortisolism, and manifestations of androgen and/or mineralocorticoid excess. This condition results from partial failure of the glucocorticoid receptor (GR) to modulate transcription of its target genes. The authors studied the molecular mechanisms of this syndrome in a Dutch kindred, whose affected members had hypercortisolism and approximately half of normal GRs, and whose proband was a young woman with manifestations of hyperandrogenism. Using the polymerase chain reaction to amplify and sequence each of the nine exons of the GR gene [alpha], along with their 5[prime]- and 3[prime]-flanking regions, the authors identified a 4-base deletion at the 3[prime]-boundary of exon 6 in one GR allele ([Delta][sub 4]), which removed a donor splice site in all three affected members studied. In contrast, the sequence of exon 6 in the two unaffected siblings was normal. A single nucleotide substitution causing an amino acid substitution in the amino terminal domain of the GR (asparagine to serine, codon 363) was also discovered in exon 2 of the other allele (G[sub 1220]) in the proband, in one of her affected brothers and in her unaffected sister. This deletion in the glucocorticoid receptor gene was associated with the expression of only one allele and a decrease of GR protein by 50% in affected members of this glucocorticoid resistant family. The mutation identified in exon 2 did not segregate with the disease and appears to be of no functional significance. The presence of the null allele was apparently compensated for by increased cortisol production at the expense of concurrent hyperandrogenism. 40 refs., 3 figs.

  17. Glucocorticoids.

    PubMed

    Barnes, Peter J

    2014-01-01

    Glucocorticoids are the most effective anti-inflammatory treatment for allergic diseases, and inhaled glucocorticoids have now become the first-line treatment for asthma. Glucocorticoids were discovered in the 1940s as extracts of the adrenal cortex and this was followed by the isolation of adrenocorticotropic hormone (ACTH) from pituitary gland extracts. Cortisone and ACTH were found to be very beneficial in the treatment of rheumatoid arthritis and Kendall, Reichstein and Hench received the Nobel Prize in Physiology and Medicine for this work in 1950. Bordley and colleagues first showed that ACTH was very beneficial in the treatment of allergic diseases in 1949, but the use of systemic glucocorticoids was limited by side effects. Inhaled glucocorticoids were discovered from topical steroids developed for skin inflammation and beclomethasone dipropionate was introduced in 1972, initially in low doses but later in higher doses, and became the standard treatment for persistent asthma. Subsequently, inhaled glucocorticoids were combined with long-acting β2-agonists in combination inhalers for even greater therapeutic benefit. There is now a good understanding of the molecular basis for the anti-inflammatory effects of glucocorticoids in allergic diseases. The search for even safer glucocorticoids based on the dissociation of anti-inflammatory and side effect mechanisms is currently ongoing.

  18. Altered glucocorticoid receptor expression and function during mouse skin carcinogenesis.

    PubMed

    Budunova, I V; Carbajal, S; Kang, H; Viaje, A; Slaga, T J

    1997-03-01

    Glucocorticoids are the most potent inhibitors of tumor promotion in mouse skin, when applied with a promoting agent at the early stages of promotion. However, established skin papillomas become resistant to growth inhibition by glucocorticoids. Glucocorticoid control of cellular functions is mediated by the glucocorticoid receptor (GR), a well-known transcription factor. Here we present data on GR expression and function in mouse papillomas and squamous cell carcinomas. Tumors were produced in SENCAR mice by a 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate two-stage protocol. In early papillomas (after 15-20 wk of promotion), northern blotting revealed a decrease in the GR mRNA level that was confirmed by a binding assay. However, in late papillomas (after 30-40 wk of promotion), and especially in squamous cell carcinomas, the level of GR in both assays was similar to or higher than the GR level in normal epidermis. To test the functional capability of GR in tumors, we compared the effect of the synthetic glucocorticoid fluocinolone acetonide (FA) on keratinocyte proliferation and on expression of glucocorticoid-responsive genes in normal epidermis, hyperplastic skin surrounding tumors, and mouse skin papillomas. FA strongly inhibited DNA synthesis in keratinocytes in normal skin and tumor-surrounding skin but had no effect on DNA synthesis in papillomas. In addition, FA strongly induced metallothionein 1 expression and inhibited connexin 26 expression in skin but did not affect expression of these genes in tumors. These data suggest that alteration of both the expression and function of GR may be an important mechanism of tumor promotion in skin.

  19. The human glucocorticoid receptor: molecular basis of biologic function.

    PubMed

    Nicolaides, Nicolas C; Galata, Zoi; Kino, Tomoshige; Chrousos, George P; Charmandari, Evangelia

    2010-01-01

    The characterization of the subfamily of steroid hormone receptors has enhanced our understanding of how a set of hormonally derived lipophilic ligands controls cellular and molecular functions to influence development and help achieve homeostasis. The glucocorticoid receptor (GR), the first member of this subfamily, is a ubiquitously expressed intracellular protein, which functions as a ligand-dependent transcription factor that regulates the expression of glucocorticoid-responsive genes. The effector domains of the GR mediate transcriptional activation by recruiting coregulatory multi-subunit complexes that remodel chromatin, target initiation sites, and stabilize the RNA-polymerase II machinery for repeated rounds of transcription of target genes. This review summarizes the basic aspects of the structure and actions of the human (h) GR, and the molecular basis of its biologic functions.

  20. Evolution of hormone selectivity in glucocorticoid and mineralocorticoid receptors.

    PubMed

    Baker, Michael E; Funder, John W; Kattoula, Stephanie R

    2013-09-01

    Mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) are descended from an ancestral corticoid receptor (CR). To date, the earliest CR have been found in lamprey and hagfish, two jawless fish (cyclostomes) that evolved at the base of the vertebrate line. Lamprey CR has both MR and GR activity. Distinct orthologs of the GR and MR first appear in skates and sharks, which are cartilaginous fishes (Chondrichthyes). Aldosterone, the physiological mineralocorticoid in terrestrial vertebrates, first appears in lobe-finned fish, such as lungfish and coelacanth, forerunners of terrestrial vertebrates, but not in sharks, skates or ray-finned fish. Skate MR are transcriptionally activated by glucocorticoids, such as corticosterone and cortisol, as well as by mineralocorticoids such as deoxycorticosterone and (experimentally) aldosterone; skate GR have low affinity for all human corticosteroids and 1α-OH-corticosterone, which has been proposed to be biologically active glucocorticoid. In fish, cortisol is both physiological mineralocorticoid and glucocorticoid; in terrestrial vertebrates, cortisol or corticosterone are the physiological glucocorticoids acting through GR, and aldosterone via MR as the physiologic mineralocorticoid. MR have equally high affinity for cortisol, corticosterone and progesterone. We review this evolutionary process through an analysis of changes in sequence and structure of vertebrate GR and MR, identifying changes in these receptors in skates and lobe-fined fish important in allowing aldosterone to act as an agonist at epithelial MR and glucocorticoid specificity for GR. hMR and hGR have lost a key contact between helix 3 and helix 5 that was present in their common ancestor. A serine that is diagnostic for vertebrate MR, and absent in terrestrial and fish GR, is present in lamprey CR, skate MR and GR, but not in coelacanth GR, marking the transition of the GR from MR ancestor. Based on the response of the CR and skate MR and GR to

  1. Glucocorticoid hormone resistance during primate evolution: receptor-mediated mechanisms.

    PubMed Central

    Chrousos, G P; Renquist, D; Brandon, D; Eil, C; Pugeat, M; Vigersky, R; Cutler, G B; Loriaux, D L; Lipsett, M B

    1982-01-01

    The concentrations of total and protein-unbound plasma cortisol of New World monkeys are higher than those of Old World primates and prosimians. The urinary free-cortisol excretion also is increased markedly. However, there is no physiologic evidence of increased cortisol effect. These findings suggest end-organ resistance to glucocorticoids. This was confirmed by showing that the hypothalamic-pituitary adrenal axis is resistant to suppression by dexamethasone. To study this phenomenon, glucocorticoid receptors were examined in circulating mononuclear leukocytes and cultured skin fibroblasts from both New and Old World species. The receptor content is the same in all species, but the New World monkeys have a markedly decreased binding affinity for dexamethasone. Thus, the resistance of these species to the action of cortisol is due to the decreased binding affinity of the glucocorticoid receptor. This presumed mutation must have occurred after the bifurcation of Old and New World primates (approximately 60 x 10(6) yr ago) and before the diversion of the New World primates from each other (approximately 15 x 10(6) yr ago). Images PMID:6952251

  2. Glucocorticoid hormone resistance during primate evolution: receptor-mediated mechanisms.

    PubMed

    Chrousos, G P; Renquist, D; Brandon, D; Eil, C; Pugeat, M; Vigersky, R; Cutler, G B; Loriaux, D L; Lipsett, M B

    1982-03-01

    The concentrations of total and protein-unbound plasma cortisol of New World monkeys are higher than those of Old World primates and prosimians. The urinary free-cortisol excretion also is increased markedly. However, there is no physiologic evidence of increased cortisol effect. These findings suggest end-organ resistance to glucocorticoids. This was confirmed by showing that the hypothalamic-pituitary adrenal axis is resistant to suppression by dexamethasone. To study this phenomenon, glucocorticoid receptors were examined in circulating mononuclear leukocytes and cultured skin fibroblasts from both New and Old World species. The receptor content is the same in all species, but the New World monkeys have a markedly decreased binding affinity for dexamethasone. Thus, the resistance of these species to the action of cortisol is due to the decreased binding affinity of the glucocorticoid receptor. This presumed mutation must have occurred after the bifurcation of Old and New World primates (approximately 60 x 10(6) yr ago) and before the diversion of the New World primates from each other (approximately 15 x 10(6) yr ago).

  3. Glucocorticoids and dopamine-1 receptors on vascular smooth muscle cells.

    PubMed

    Yasunari, K; Kohno, M; Balmforth, A; Murakawa, K; Yokokawa, K; Kurihara, N; Takeda, T

    1989-06-01

    The effect of glucocorticoids on the dopamine (DA)-mediated cyclic adenosine monophosphate (cAMP) by intact vascular smooth muscle cells (VSMC) was studied in rats. Cultured VSMC were obtained from renal arteries of 14-week-old Wistar-Kyoto rats by explant method. Micromolar concentrations of dexamethasone (DEX) pretreatment for 48 hours potentiated DA-mediated response without any change of affinity constant. However, micromolar concentrations of aldosterone pretreatment for 48 hours had almost no effect on DA-mediated response. The DEX-induced facilitation began at 6 hours and reached maximum at 24 hours after DEX administration in a dose-dependent manner. Inhibitors of protein and RNA synthesis blocked this glucocorticoid effect. The basal activity of adenylate cyclase in DEX-treated cells was twofold higher than that in control cells. Treatment of VSMC with DEX increased cholera toxin-stimulated and forskolin-stimulated adenylate cyclase activity. However, pertussis toxin treatment did not augment or reduce the effect of DEX treatment. These results suggest that glucocorticoids increase DA-mediated cAMP formation by VSMC through glucocorticoid type II receptors and the induction of protein synthesis and that the activation of the catalytic unit may play some role in this facilitation.

  4. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Characterization of human glucocorticoid receptor complexes formed with DNA fragments containing or lacking glucocorticoid response elements

    SciTech Connect

    Tully, D.B.; Cidlowski, J.A. )

    1989-03-07

    Sucrose density gradient shift assays were used to study the interactions of human glucocorticoid receptors (GR) with small DNA fragments either containing or lacking glucocorticoid response element (GRE) DNA consensus sequences. When crude cytoplasmic extracts containing ({sup 3}H)triamcinolone acetonide (({sup 3}H)TA) labeled GR were incubated with unlabeled DNA under conditions of DNA excess, a GRE-containing DNA fragment obtained from the 5' long terminal repeat of mouse mammary tumor virus (MMTV LTR) formed a stable 12-16S complex with activated, but not nonactivated, ({sup 3}H)TA receptor. By contrast, if the cytosols were treated with calf thymus DNA-cellulose to deplete non-GR-DNA-binding proteins prior to heat activation, a smaller 7-10S complex was formed with the MMTV LTR DNA fragment. Activated ({sup 3}H)TA receptor from DNA-cellulose pretreated cytosols also interacted with two similarly sized fragments from pBR322 DNA. Stability of the complexes formed between GR and these three DNA fragments was strongly affected by even moderate alterations in either the salt concentration or the pH of the gradient buffer. Under all conditions tested, the complex formed with the MMTV LTR DNA fragment was more stable than the complexes formed with either of the pBR322 DNA fragments. Together these observations indicate that the formation of stable complexes between activated GR and isolated DNA fragments requires the presence of GRE consensus sequences in the DNA.

  6. 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.

  7. Glucocorticoid-induced glucocorticoid-receptor expression and promoter usage is not linked to glucocorticoid resistance in childhood ALL.

    PubMed

    Tissing, Wim J E; Meijerink, Jules P P; Brinkhof, Bas; Broekhuis, Mathilde J C; Menezes, Renee X; den Boer, Monique L; Pieters, Rob

    2006-08-01

    Glucocorticoid (GC) resistance is an adverse prognostic factor in childhood acute lymphoblastic leukemia (ALL), but little is known about causes of GC resistance. Up-regulation of the glucocorticoid receptor (GR) has been suggested as an essential step to the induction of apoptosis in leukemic cells. In this study we investigated whether baseline mRNA expression levels of the 5 different GR promoter transcripts (1A1, 1A2, 1A3, 1B, and 1C) or differences in the degree of regulation of the GR or GR promoter transcripts upon GC exposure are related to GC resistance. Therefore, mRNA levels of the 5 GR promoter transcripts and of the GR were measured by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR; Taqman) technology in primary ALL cells prior to and after 3, 8, and 24 hours of prednisolone exposure. GR expression is induced upon GC exposure in primary ALL patient samples, which is opposite to what is found in tissues in which GCs do not induce apoptosis. GC resistance in childhood ALL cannot be attributed to an inability of resistant cells to up-regulate the expression of the GR upon GC exposure, nor to differences in GR promoter usage (at baseline and upon GC exposure).

  8. Antiinflammatory glucocorticoid receptor ligand with reduced side effects exhibits an altered protein-protein interaction profile.

    PubMed

    Miner, Jeffrey N; Ardecky, Bob; Benbatoul, Khalid; Griffiths, Kimberly; Larson, Christopher J; Mais, Dale E; Marschke, Keith; Rosen, Jon; Vajda, Eric; Zhi, Lin; Negro-Vilar, Andres

    2007-12-04

    Glucocorticoids are commonly used antiinflammatory agents whose use is limited by side effects. We have developed a series of glucocorticoid receptor (GR) ligands that retain the strong antiinflammatory activity of conventional glucocorticoids with reduced side effects. We present a compound, LGD5552, that binds the receptor efficiently and strongly represses inflammatory gene expression. LGD5552 bound to GR activates gene expression somewhat differently than glucocorticoids. It activates some genes with an efficacy similar to that of the glucocorticoids. However, other glucocorticoid-activated genes are not regulated by LGD5552. These differences may be because of the more efficient binding of corepressor in the presence of LGD5552, compared with glucocorticoid agonists. This class of nonsteroidal, GR-dependent antiinflammatory drugs may offer a safer alternative to steroidal glucocorticoids in the treatment of inflammatory disease.

  9. Antiinflammatory glucocorticoid receptor ligand with reduced side effects exhibits an altered protein–protein interaction profile

    PubMed Central

    Miner, Jeffrey N.; Ardecky, Bob; Benbatoul, Khalid; Griffiths, Kimberly; Larson, Christopher J.; Mais, Dale E.; Marschke, Keith; Rosen, Jon; Vajda, Eric; Zhi, Lin; Negro-Vilar, Andres

    2007-01-01

    Glucocorticoids are commonly used antiinflammatory agents whose use is limited by side effects. We have developed a series of glucocorticoid receptor (GR) ligands that retain the strong antiinflammatory activity of conventional glucocorticoids with reduced side effects. We present a compound, LGD5552, that binds the receptor efficiently and strongly represses inflammatory gene expression. LGD5552 bound to GR activates gene expression somewhat differently than glucocorticoids. It activates some genes with an efficacy similar to that of the glucocorticoids. However, other glucocorticoid-activated genes are not regulated by LGD5552. These differences may be because of the more efficient binding of corepressor in the presence of LGD5552, compared with glucocorticoid agonists. This class of nonsteroidal, GR-dependent antiinflammatory drugs may offer a safer alternative to steroidal glucocorticoids in the treatment of inflammatory disease. PMID:18032610

  10. Epigenetic regulation of glucocorticoid receptor and infantile spasms.

    PubMed

    Yang, Guang; Zou, Li-Ping; Wang, Jing; Ding, Ying-Xue

    2011-02-01

    IS is one of the few seizure syndromes that can be alleviated by adrenocorticotropic hormone (ACTH) or glucocorticoids (GCs) that are considered effective drugs of choice. This indicates that, indeed, IS may be fundamentally different from most other seizure disorders owing to the dysregulation of the hypothalamic-hypophysial-adrenal axis. GCs have multiple critical effects on fetal development, especially in normal brain development. Most glucocorticoid effects are mediated by the glucocorticoid receptor (GR), a steroid-activated nuclear receptor that translocates to the nucleus upon binding to cortisol. In the nucleus, GR targets genes related to neuronal metabolism and plasticity. The GR has also been characterized as a critical checkpoint in the delicate hormonal control of energy homeostasis. Recent studies suggest a possible correlation between prenatal stress and the onset of infantile spasms. In this paper, we propose a hypothesis that connects the adverse events in early life with the onset of IS through methylation of the GR gene, which is an epigenetic mechanism.

  11. Discovery of selective glucocorticoid receptor modulators by multiplexed reporter screening

    PubMed Central

    Gerber, Anthony N.; Masuno, Kiriko; Diamond, Marc I.

    2009-01-01

    Glucocorticoids are widely used to suppress inflammation and treat various immune-mediated diseases. Some glucocorticoid receptor (GR)-regulated genes mediate the therapeutic response, whereas others cause debilitating side effects. To discover selective modulators of the GR response, we developed a high-throughput, multiplexed system to monitor regulation of 4 promoters simultaneously. An initial screen of 1,040 natural products and Food and Drug Administration-approved drugs identified modulators that caused GR to regulate only a subset of its target promoters. Some compounds selectively inhibited GR-mediated gene activation without altering the repression of cytokine expression by GR. This approach will facilitate identification of genes and small molecules that augment beneficial effects of GR and diminish deleterious ones. Our results have important implications for the development of GR modulators and the identification of cross-talk pathways that control selective GR gene regulation. PMID:19255438

  12. Exogenous nitric oxide activates the endothelial glucocorticoid receptor.

    PubMed

    Ji, Julie Y; Diamond, Scott L

    2004-05-21

    This study investigated the effect of exogenous nitric oxide (NO) on endothelial glucocorticoid receptor (GR) function. The NO donor diethylenetriamine NONOate (DETA, 50-500microM) caused concentration dependent nuclear localization of transfected chimeric green fluorescent protein GFP-GR and elevated expression of secreted alkaline phosphatase (SEAP) from a glucocorticoid response element (GRE) promoter construct in bovine aortic endothelial cells. Other weaker NO donors (S-nitroso-N-acetylpenicillamine and spermine NONOate) failed to induce GFP-GR nuclear localization, but all the NO donors activated GRE-SEAP expression, a response unaffected by the antioxidant N-acetyl-L-cysteine. Overall, exogenous NO from high concentration donors can directly activate GR, suggesting a potential feedback mechanism for NO to regulate endothelial inducible nitric oxide synthase (iNOS) expression.

  13. Brain-derived neurotrophic factor signaling rewrites the glucocorticoid transcriptome via glucocorticoid receptor phosphorylation.

    PubMed

    Lambert, W Marcus; Xu, Chong-Feng; Neubert, Thomas A; Chao, Moses V; Garabedian, Michael J; Jeanneteau, Freddy D

    2013-09-01

    Abnormal glucocorticoid and neurotrophin signaling has been implicated in numerous psychiatric disorders. However, the impact of neurotrophic signaling on glucocorticoid receptor (GR)-dependent gene expression is not understood. We therefore examined the impact of brain-derived neurotrophic factor (BDNF) signaling on GR transcriptional regulatory function by gene expression profiling in primary rat cortical neurons stimulated with the selective GR agonist dexamethasone (Dex) and BDNF, alone or in combination. Simultaneous treatment with BDNF and Dex elicited a unique set of GR-responsive genes associated with neuronal growth and differentiation and also enhanced the induction of a large number of Dex-sensitive genes. BDNF via its receptor TrkB enhanced the transcriptional activity of a synthetic GR reporter, suggesting a direct effect of BDNF signaling on GR function. Indeed, BDNF treatment induces the phosphorylation of GR at serine 155 (S155) and serine 287 (S287). Expression of a nonphosphorylatable mutant (GR S155A/S287A) impaired the induction of a subset of BDNF- and Dex-regulated genes. Mechanistically, BDNF-induced GR phosphorylation increased GR occupancy and cofactor recruitment at the promoter of a BDNF-enhanced gene. GR phosphorylation in vivo is sensitive to changes in the levels of BDNF and TrkB as well as stress. Therefore, BDNF signaling specifies and amplifies the GR transcriptome through a coordinated GR phosphorylation-dependent detection mechanism.

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

    PubMed

    He, Bo; Cruz-Topete, Diana; Oakley, Robert H; Xiao, Xiao; Cidlowski, John A

    2015-12-28

    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.

  15. 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.

  16. Triterpenes from Alisma orientalis act as androgen receptor agonists, progesterone receptor antagonists, and glucocorticoid receptor antagonists.

    PubMed

    Lin, Hsiang-Ru

    2014-08-01

    Alisma orientalis, a well-known traditional medicine, exerts numerous pharmacological effects including anti-diabetes, anti-hepatitis, and anti-diuretics but its bioactivity is not fully clear. Androgen receptor (AR), progesterone receptor (PR), and glucocorticoid receptor (GR) are three members of nuclear receptor superfamily that has been widely targeted for developing treatments for essential diseases including prostate cancer and breast cancer. In this study, two triterpenes, alisol M 23-acetate and alisol A 23-acetate from Alisma orientalis were determined whether they may act as androgen receptor (AR), progesterone receptor (PR), or glucocorticoid receptor (GR) modulators. Indeed, in the transient transfection reporter assays, alisol M 23-acetate and alisol A 23-acetate transactivated AR in dose-dependent manner, while they transrepressed the transactivation effects exerted by agonist-activated PR and GR. Through molecular modeling docking studies, they were shown to respectively interact with AR, PR, or GR ligand binding pocket fairly well. All these results indicate that alisol M 23-acetate and alisol A 23-acetate from Alisma orientalis might possess therapeutic effects through their modulation of AR, PR, and GR pathways. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Feed-forward inhibition of androgen receptor activity by glucocorticoid action in human adipocytes.

    PubMed

    Hartig, Sean M; He, Bin; Newberg, Justin Y; Ochsner, Scott A; Loose, David S; Lanz, Rainer B; McKenna, Neil J; Buehrer, Benjamin M; McGuire, Sean E; Marcelli, Marco; Mancini, Michael A

    2012-09-21

    We compared transcriptomes of terminally differentiated mouse 3T3-L1 and human adipocytes to identify cell-specific differences. Gene expression and high content analysis (HCA) data identified the androgen receptor (AR) as both expressed and functional, exclusively during early human adipocyte differentiation. The AR agonist dihydrotestosterone (DHT) inhibited human adipocyte maturation by downregulation of adipocyte marker genes, but not in 3T3-L1. It is interesting that AR induction corresponded with dexamethasone activation of the glucocorticoid receptor (GR); however, when exposed to the differentiation cocktail required for adipocyte maturation, AR adopted an antagonist conformation and was transcriptionally repressed. To further explore effectors within the cocktail, we applied an image-based support vector machine (SVM) classification scheme to show that adipocyte differentiation components inhibit AR action. The results demonstrate human adipocyte differentiation, via GR activation, upregulates AR but also inhibits AR transcriptional activity.

  18. Interaction between the trout mineralocorticoid and glucocorticoid receptors in vitro.

    PubMed

    Kiilerich, Pia; Triqueneaux, Gérard; Christensen, Nynne Meyn; Trayer, Vincent; Terrien, Xavier; Lombès, Marc; Prunet, Patrick

    2015-08-01

    The salmonid corticosteroid receptors (CRs), glucocorticoid receptors 1 and 2 (GR1 and GR2) and the mineralocorticoid receptor (MR) share a high degree of homology with regard to structure, ligand- and DNA response element-binding, and cellular co-localization. Typically, these nuclear hormone receptors homodimerize to confer transcriptional activation of target genes, but a few studies using mammalian receptors suggest some degree of heterodimerization. We observed that the trout MR confers a several fold lower transcriptional activity compared to the trout GRs. This made us question the functional relevance of the MR when this receptor is located in the same cells as the GRs and activated by cortisol. A series of co-transfection experiments using different glucocorticoid response elements (GREs) containing promoter-reporter constructs were carried out to investigate any possible interaction between the piscine CRs. Co-transfection of the GRs with the MR significantly reduced the total transcriptional activity even at low MR levels, suggesting interaction between these receptors. Co-transfection of GR1 or GR2 with the MR did not affect the subcellular localization of the GRs, and the MR-mediated inhibition seemed to be independent of specific activation or inhibition of the MR. Site-directed mutagenesis of the DNA-binding domain and dimerization interface of the MR showed that the inhibition was dependent on DNA binding but not necessarily on dimerization ability. Thus, we suggest that the interaction between MR and the GRs may regulate the cortisol response in cell types where the receptors co-localize and propose a dominant-negative role for the MR in cortisol-mediated transcriptional activity. © 2015 Society for Endocrinology.

  19. Melatonin inhibits glucocorticoid receptor nuclear translocation in mouse thymocytes.

    PubMed

    Presman, Diego M; Hoijman, Esteban; Ceballos, Nora R; Galigniana, Mario D; Pecci, Adali

    2006-11-01

    The antiapoptotic effect of melatonin (MEL) has been described in several systems. In particular, MEL inhibits glucocorticoid-mediated apoptosis. Our group previously demonstrated that in the thymus, MEL inhibits the release of Cytochrome C from mitochondria and the dexamethasone-dependent increase of bax mRNA levels. In this study we analyzed the ability of MEL to regulate the activation of the glucocorticoid receptor (GR) in mouse thymocytes. We found that even though the methoxyindole does not affect the ligand binding capacity of the receptor, it impairs the steroid-dependent nuclear translocation of the GR and also prevents transformation by blocking the dissociation of the 90-kDa heat shock protein. Coincubation of the methoxyindole with dexamethasone did not affect the expression of a reporter gene in GR-transfected Cos-7 cells or HC11 and L929 mouse cell lines that express Mel-1a and retinoid-related orphan receptor-alpha (RORalpha) receptors. Therefore, the antagonistic effect of MEL seems to be specific for thymocytes, in a Mel 1a- and RORalpha-independent manner. In summary, the present results suggest a novel mechanism for the antagonistic action of MEL on GR-mediated effects, which involves the inhibition of 90-kDa heat shock protein dissociation and the cytoplasmic retention of the GR.

  20. Glucocorticoid resistance in a multiple myeloma cell line is regulated by a transcription elongation block in the glucocorticoid receptor gene (NR3C1).

    PubMed

    Sánchez-Vega, Beatriz; Gandhi, Varsha

    2009-03-01

    Glucocorticoid (GC) effects are mediated by the glucocorticoid receptor (GR). Several studies have demonstrated that a lower number of receptors per cell were associated with poor GC response. The regulation of GR expression is complex; the levels of GR can be autologously regulated by its ligand and also by transcriptional, post-transcriptional and post-translational mechanisms. Using three human myeloma cell lines that parallel the development of GC resistance, this work describes the mechanism involved in the downregulation of GR expression. The decreased expression was neither due to mutations in the gene encoding GR, NR3C1, nor due to methylation of the promoters. A gradual decrease in NR3C1 transcripts was seen during the development of resistance, the level of expression of exon 1 to 2 RNA fragments remained the same in sensitive and resistant cell lines but a chromatin immunoprecipitation assay demonstrated that RNA polymerase II, detectable throughout exon 2 to 3 in the sensitive cells, was undetectable on exon 3 in the resistant variant, suggesting lower or no transcription at this site. These studies demonstrated that downregulation of NR3C1 mRNA in a resistant cell line involves a block to transcriptional elongation within intron B of NR3C1. This block may represent an important element in the regulation of GR expression.

  1. Glucocorticoids and their receptors: insights into specific roles in mitochondria.

    PubMed

    Lee, Sung-Ryul; Kim, Hyoung-Kyu; Song, In-Sung; Youm, Jaeboum; Dizon, Louise Anne; Jeong, Seung-Hun; Ko, Tae-Hee; Heo, Hye-Jin; Ko, Kyoung Soo; Rhee, Byoung Doo; Kim, Nari; Han, Jin

    2013-05-01

    Glucocorticoids (GCs) affect most physiological systems and are the most frequently used drugs for multiple disorders and organ transplantation. GC functions depend on a balance between circulating GC and cytoplasmic glucocorticoid receptor II (GR). Mitochondria individually enclose circular, double-stranded DNA that is expressed and replicated in response to nuclear-encoded factors imported from the cytoplasm. Fine-tuning and response to cellular demands should be coordinately regulated by the nucleus and mitochondria; thus mitochondrial-nuclear interaction is vital to optimal mitochondrial function. Elucidation of the direct and indirect effects of steroids, including GCs, on mitochondria is an important and emerging field of research. Mitochondria may also be under GC control because GRs are present in mitochondria, and glucocorticoid response elements (GREs) reside in the mitochondrial genome. Therefore, mitochondrial gene expression can be regulated by GCs via at least two different mechanisms: direct action on mitochondrial DNA and oxidative phosphorylation (OXPHOS) genes, or by an indirect effect through interaction with nuclear genes. In this review, we outline possible mechanisms of regulation of mitochondrial genes in response to GCs in view of translocation of the GR into mitochondria and the possible regulation of OXPHOS genes by GREs in the mitochondrial genome.

  2. Metabolic functions of glucocorticoid receptor in skeletal muscle.

    PubMed

    Kuo, Taiyi; Harris, Charles A; Wang, Jen-Chywan

    2013-11-05

    Glucocorticoids (GCs) exert key metabolic influences on skeletal muscle. GCs increase protein degradation and decrease protein synthesis. The released amino acids are mobilized from skeletal muscle to liver, where they serve as substrates for hepatic gluconeogenesis. This metabolic response is critical for mammals' survival under stressful conditions, such as fasting and starvation. GCs suppress insulin-stimulated glucose uptake and utilization and glycogen synthesis, and play a permissive role for catecholamine-induced glycogenolysis, thus preserving the level of circulating glucose, the major energy source for the brain. However, chronic or excess exposure of GCs can induce muscle atrophy and insulin resistance. GCs convey their signal mainly through the intracellular glucocorticoid receptor (GR). While GR can act through different mechanisms, one of its major actions is to regulate the transcription of its primary target genes through genomic glucocorticoid response elements (GREs) by directly binding to DNA or tethering onto other DNA-binding transcription factors. These GR primary targets trigger physiological and pathological responses of GCs. Much progress has been made to understand how GCs regulate protein and glucose metabolism. In this review, we will discuss how GR primary target genes confer metabolic functions of GCs, and the mechanisms governing the transcriptional regulation of these targets. Comprehending these processes not only contributes to the fundamental understanding of mammalian physiology, but also will provide invaluable insight for improved GC therapeutics.

  3. Recent advances in the molecular mechanisms determining tissue sensitivity to glucocorticoids: novel mutations, circadian rhythm and ligand-induced repression of the human glucocorticoid receptor

    PubMed Central

    2014-01-01

    Glucocorticoids are pleiotropic hormones, which are involved in almost every cellular, molecular and physiologic network of the organism, and regulate a broad spectrum of physiologic functions essential for life. The cellular response to glucocorticoids displays profound variability both in magnitude and in specificity of action. Tissue sensitivity to glucocorticoids differs among individuals, within tissues of the same individual and within the same cell. The actions of glucocorticoids are mediated by the glucocorticoid receptor, a ubiquitously expressed intracellular, ligand-dependent transcription factor. Multiple mechanisms, such as pre-receptor ligand metabolism, receptor isoform expression, and receptor-, tissue-, and cell type-specific factors, exist to generate diversity as well as specificity in the response to glucocorticoids. Alterations in the molecular mechanisms of glucocorticoid receptor action impair glucocorticoid signal transduction and alter tissue sensitivity to glucocorticoids. This review summarizes the recent advances in our understanding of the molecular mechanisms determining tissue sensitivity to glucocorticoids with particular emphasis on novel mutations and new information on the circadian rhythm and ligand-induced repression of the glucocorticoid receptor. PMID:25155432

  4. Proopiomelanocortin, glucocorticoid, and CRH receptor expression in human ACTH-secreting pituitary adenomas.

    PubMed

    Cassarino, Maria Francesca; Sesta, Antonella; Pagliardini, Luca; Losa, Marco; Lasio, Giovanni; Cavagnini, Francesco; Pecori Giraldi, Francesca

    2017-03-01

    ACTH-secreting pituitary tumors are by definition partially autonomous, i.e., secrete ACTH independent of physiological control. However, only few, small-sized studies on proopiomelanocortin (POMC) and its regulation by corticotropin-releasing hormone (CRH) or glucocorticoids are available. Objective of the present study was to report on constitutive and CRH- and dexamethasone-regulated POMC, CRH (CRH-R1), and glucocorticoid receptor (NR3C1) gene expression in a large series of human corticotrope adenomas. Fifty-three ACTH-secreting adenomas were incubated with 10 nM CRH or 10 nM dexamethasone for 24 h. POMC, CRH-R1, NR3C1, and its alpha and beta isoforms were quantified and medium ACTH measured. Constitutive POMC expression proved extremely variable, with macroadenomas exhibiting higher levels than microadenomas. POMC increased during CRH in most specimens; conversely, changes induced by dexamethasone were varied, ranging from decrease to paradoxical increase. No correlation between POMC and ACTH was detected in any experimental condition. CRH-R1 expression was not linked to the response to CRH while NR3C1 was expressed at greater levels in specimens who failed to inhibit during dexamethasone; glucocorticoid receptor α was the more abundant isoform and subject to down-regulation by dexamethasone. Our results demonstrate a considerable variability in POMC expression among tumors and no correlation between POMC and ACTH, suggesting that POMC peptide processing/transport plays a major role in modulating ACTH secretion. Further, CRH-R1 and NR3C1 expression were not linked to the expected ligand-induced outcome, indicating that receptor signaling rather than abundance determines corticotrope responses. Our findings pave the way to new avenues of research into Cushing's disease pathophysiology.

  5. 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

  6. Increased levels of glucocorticoid receptors and enhanced glucocorticoid receptor auto-regulation after hydrocortisone challenge in B-lymphoblastoids from patients with affective disorders.

    PubMed

    Henning, Uwe; Krieger, Klaus; Loeffler, Stefan; Rivas, Fabio; Orozco, Guillermo; de Castro, Manuel G; Rietschel, Marcella; Noethen, Markus M; Klimke, Ansgar

    2005-05-01

    The stress response is mediated by a negative feedback effect of glucocorticoids on corticosteroid receptors. Here, we examine the potential contribution of these receptors and their response to a glucocorticoid challenge to dysfunctions of the hypothalamic-pituitary-adrenal axis reported for patients with affective disorders. In a pilot-study, we established B-lymphoblastoid cell lines from patients suffering from affective disorders and healthy subjects and measured the quantity of glucocorticoid receptors at steady state conditions after 12-weeks cell culture. After short-term incubation with 0.1 microM hydrocortisone for 48 h, the decrease of glucocorticoid receptors was also investigated. After 12-weeks cell culture, we found a significantly higher number of cytosolic glucocorticoid receptors in B-lymphoblastoids from patients (B(max)=804.9+/-342.5 fmol/mg protein) compared to those from healthy subjects (B(max)=576.9+/-190.3 fmol/mg protein: p=0.045; t-test). The increase of the glucocorticoid receptor level in the group of patients could be attributed largely to the higher number of these receptors measured in B-lymphoblastoids of patients suffering from major depressive disorder. The in vitro regulation of glucocorticoid receptors in response to 0.1 microM hydrocortisone for 48 h resulted in a significantly larger decrease in cultures of B-lymphoblastoids derived from patients (to 32.9+/-7.5%) than in those from healthy subjects (to 45.8+/-8.2%). The stronger decrease of glucocorticoid receptors in the group of patients (p=0.0001; t-test) was independent of the duration of illness and medication, suggesting a trait-like characteristic of the response.

  7. Characterisation of glucocorticoid receptors in peripheral blood leukocytes of Carp, Cyprinus carpio L.

    PubMed

    Weyts, F A; Verburg-van Kemenade, B M; Flik, G

    1998-07-01

    Binding studies with [3H]cortisol revealed the presence of a single class of cortisol-binding sites on carp peripheral blood leukocytes (PBL). These binding sites showed high affinity (Kd of 3.8 nM) and low capacity (490 binding sites per cell), indicative of receptor binding. Affinity for cortisone was 254-fold lower than for cortisol. Affinity for the two synthetic glucocorticoids dexamethasone and triamcinolone acetonide (TA) was 4- and 10-fold higher than for cortisol, respectively. Further evidence for the GR character of the receptor came from results showing that cortisol induced apoptosis, which could be blocked by the glucocorticoid analogue RU486. A single meal of cortisol-containing food elevated plasma cortisol concentrations and decreased GR density in PBL, as measured 3 h later. The percentage of circulating B lymphocytes also decreased. Cortisol-induced redistribution of B lymphocytes from the blood, due to cortisol treatment, may explain the decrease of GR numbers in PBL, although downregulation of available GR remains possible.

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

    PubMed Central

    Pratt, R M

    1985-01-01

    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 medial palatal epithelial cells. PMID:2998748

  9. 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.

  10. Glucocorticoid receptor mediated suppression of natural killer cell activity: identification of associated deacetylase and corepressor molecules.

    PubMed

    Bush, Kristin A; Krukowski, Karen; Eddy, Justin L; Janusek, Linda Witek; Mathews, Herbert L

    2012-01-01

    Physical and psychological stressors reduce natural killer cell function. This reduction in cellular function results from stress-induced release of glucocorticoids. Glucocorticoids act upon natural killer cells to deacetylate and transrepress immune response genes through epigenetic processes. However, other than the glucocorticoid receptor, the proteins that participate in this process are not well described in natural killer cells. The purpose of this study was to identify the proteins associated with the glucocorticoid receptor that are likely epigenetic participants in this process. Treatment of natural killer cells with the synthetic glucocorticoid, dexamethasone, produced a significant time dependent reduction in natural killer cell activity as early as 8h post treatment. This reduction in natural killer cell activity was preceded by nuclear localization of the glucocorticoid receptor with histone deacetylase 1 and the corepressor, SMRT. Other class I histone deacetylases were not associated with the glucocorticoid receptor nor was the corepressor NCoR. These results demonstrate histone deacetylase 1 and SMRT to associate with the ligand activated glucocorticoid receptor within the nuclei of natural killer cells and to be the likely participants in the histone deacetylation and transrepression that accompanies glucocorticoid mediated reductions in natural killer cell function. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. 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.

  12. 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

  13. Overexpression of mineralocorticoid and transdominant glucocorticoid receptor blocks the impairing effects of glucocorticoids on memory.

    PubMed

    Ferguson, Deveroux; Sapolsky, Robert

    2008-01-01

    It is well established that mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) influence hippocampal-dependent spatial memory. MRs are saturated in the presence of low corticosterone (CORT) levels; consequently receptor protein levels play a rate-limiting role in regulating the positive effects of MR-mediated gene transcription. In this study, viral vector-mediated transgene expression was used to simultaneously manipulate both MR and GR signaling. This approach allowed us to investigate the effects of spatially restricted overexpression of MR and a negative transdominant GR (TD) in the dentate gyrus (DG) subfield of the hippocampus, on short term and long term spatial memory in animals overexpressing one copy of MR or TD, two copies of MR ("MR/MR"), or one copy of each ("MR/TD"). Expression of transgenes did not influence the acquisition (learning) phase of the Morris water maze task. However, we found an overall enhancing effect of MR/MR expression on short term memory performance. In addition, rats expressing TD and MR/TD blocked the high CORT-induced impairments on long term spatial memory retrieval. These findings illustrate the potential beneficial effects of increasing MR signaling or decreasing GR signaling to enhance specific aspects of cognitive function.

  14. The Human Glucocorticoid Receptor: Molecular Basis of Biologic Function

    PubMed Central

    Nicolaides, Nicolas C.; Galata, Zoi; Kino, Tomoshige; Chrousos, George P.; Charmandari, Evangelia

    2009-01-01

    The characterization of the subfamily of steroid hormone receptors has enhanced our understanding of how a set of hormonally derived lipophilic ligands controls cellular and molecular functions to influence development and help achieve homeostasis. The glucocorticopid receptor (GR), the first member of this subfamily, is a ubiquitously expressed intracellular protein, which functions as a ligand-dependent transcription factor that regulates the expression of glucocorticoid-responsive genes. The effector domains of the GR mediate transcriptional activation by recruiting coregulatory multi-subunit complexes that remodel chromatin, target initiation sites, and stabilize the RNA polymerase II machinery for repeated rounds of transcription of target genes. This review summarizes the basic aspects of the structure and of the human (h) GR, and the molecular basis of its biologic function. PMID:19818358

  15. 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.

  16. 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.

  17. HES1 Is a Master Regulator of Glucocorticoid Receptor-Dependent Gene Expression

    PubMed Central

    Revollo, Javier R.; Oakley, Robert H.; Lu, Nick Z.; Kadmiel, Mahita; Gandhavadi, Maheer; Cidlowski, John A.

    2014-01-01

    Hairy and enhancer of split-1 (HES1) is a basic helix-loop-helix transcription factor that is a key regulator of development and organogenesis. However, little is known about the role of HES1 after birth. Glucocorticoids, primary stress hormones that are essential for life, regulate numerous homeostatic processes that permit vertebrates to cope with physiological challenges. The molecular actions of glucocorticoids are mediated by glucocorticoid receptor-dependent regulation of nearly 25% of the genome. We now establish a genome wide molecular link between HES1 and glucocorticoid receptors that controls the ability of cells and animals to respond to stress. Glucocorticoid signaling rapidly and robustly silenced HES1 expression. This glucocorticoid-dependent repression of HES1 was necessary for the glucocorticoid receptor to regulate many of its target genes. Mice with conditional knockout of HES1 in the liver exhibited an expanded glucocorticoid receptor signaling profile and aberrant metabolic phenotype. Our results indicate that HES1 acts as a master repressor, the silencing of which is required for proper glucocorticoid signaling. PMID:24300895

  18. LGD-5552, an antiinflammatory glucocorticoid receptor ligand with reduced side effects, in vivo.

    PubMed

    López, Francisco J; Ardecky, Robert J; Bebo, Bruce; Benbatoul, Khalid; De Grandpre, Louise; Liu, Sha; Leibowitz, Mark D; Marschke, Keith; Rosen, Jon; Rungta, Deepa; Viveros, Humberto O; Yen, Wan-Ching; Zhi, Lin; Negro-Vilar, Andrés; Miner, Jeffrey N

    2008-05-01

    Treatment of inflammation is often accomplished through the use of glucocorticoids. However, their use is limited by side effects. We have examined the activity of a novel glucocorticoid receptor ligand that binds the receptor efficiently and strongly represses inflammatory gene expression. This compound has potent antiinflammatory activity in vivo and represses the transcription of the inflammatory cytokine monocyte chemoattractant protein-1 and induces the antiinflammatory cytokine IL-10. The compound demonstrates differential gene regulation, compared with commonly prescribed glucocorticoids, effectively inducing some genes and repressing others in a manner different from the glucocorticoid prednisolone. The separation between the antiinflammatory effects of LGD-5552 and the side effects commonly associated with glucocorticoid treatment suggest that this molecule differs significantly from prednisolone and other steroids and may provide a safer therapeutic window for inflammatory conditions now commonly treated with steroidal glucocorticoids.

  19. Rapid Nongenomic Glucocorticoid Actions in Male Mouse Hypothalamic Neuroendocrine Cells Are Dependent on the Nuclear Glucocorticoid Receptor

    PubMed Central

    Nahar, Jebun; Haam, Juhee; Chen, Chun; Jiang, Zhiying; Glatzer, Nicholas R.; Muglia, Louis J.; Dohanich, Gary P.; Herman, James P.

    2015-01-01

    Corticosteroids act classically via cognate nuclear receptors to regulate gene transcription; however, increasing evidence supports rapid, nontranscriptional corticosteroid actions via activation of membrane receptors. Using whole-cell patch clamp recordings in hypothalamic slices from male mouse genetic models, we tested for nongenomic glucocorticoid actions at glutamate and gamma aminobutyric acid (GABA) synapses in hypothalamic neuroendocrine cells, and for their dependence on the nuclear glucocorticoid receptor (GR). In enhanced green fluorescent protein-expressing CRH neurons of the paraventricular nucleus (PVN) and in magnocellular neurons of the PVN and supraoptic nucleus (SON), dexamethasone activated postsynaptic membrane-associated receptors and G protein signaling to elicit a rapid suppression of excitatory postsynaptic inputs, which was blocked by genetic deletion of type I cannabinoid receptors and a type I cannabinoid receptor antagonist. In magnocellular neurons, dexamethasone also elicited a rapid nitric oxide-dependent increase in inhibitory postsynaptic inputs. These data indicate a rapid, synapse-specific glucocorticoid-induced retrograde endocannabinoid signaling at glutamate synapses and nitric oxide signaling at GABA synapses. Unexpectedly, the rapid glucocorticoid effects on both excitatory and inhibitory synaptic transmission were lost with conditional deletion of GR in the PVN and SON in slices from a single minded-1-cre-directed conditional GR knockout mouse. Thus, the nongenomic glucocorticoid actions at glutamate and GABA synapses on PVN and SON neuroendocrine cells are dependent on the nuclear GR. The nuclear GR, therefore, is responsible for transducing the rapid steroid response at the membrane, or is either a critical component in the signaling cascade or regulates a critical component of the signaling cascade of a distinct membrane GR. PMID:26061727

  20. Glucocorticoid receptor signalling activates YAP in breast cancer

    PubMed Central

    Sorrentino, Giovanni; Ruggeri, Naomi; Zannini, Alessandro; Ingallina, Eleonora; Bertolio, Rebecca; Marotta, Carolina; Neri, Carmelo; Cappuzzello, Elisa; Forcato, Mattia; Rosato, Antonio; Mano, Miguel; Bicciato, Silvio; Del Sal, Giannino

    2017-01-01

    The Hippo pathway is an oncosuppressor signalling cascade that plays a major role in the control of cell growth, tissue homoeostasis and organ size. Dysregulation of the Hippo pathway leads to aberrant activation of the transcription co-activator YAP (Yes-associated protein) that contributes to tumorigenesis in several tissues. Here we identify glucocorticoids (GCs) as hormonal activators of YAP. Stimulation of glucocorticoid receptor (GR) leads to increase of YAP protein levels, nuclear accumulation and transcriptional activity in vitro and in vivo. Mechanistically, we find that GCs increase expression and deposition of fibronectin leading to the focal adhesion-Src pathway stimulation, cytoskeleton-dependent YAP activation and expansion of chemoresistant cancer stem cells. GR activation correlates with YAP activity in human breast cancer and predicts bad prognosis in the basal-like subtype. Our results unveil a novel mechanism of YAP activation in cancer and open the possibility to target GR to prevent cancer stem cells self-renewal and chemoresistance. PMID:28102225

  1. NFκB and glucocorticoid receptor activity in steroid resistance.

    PubMed

    Dawson, Charlotte; Dhanda, Ashwin; Conway-Campbell, Becky; Dimambro, Alexandra; Lightman, Stafford; Dayan, Colin

    2012-02-01

    Resistance to the anti-inflammatory and immunosuppressive effects of steroids is an important clinical problem that complicates the treatment of approximately 30% of patients with conditions for which steroids are normally first-line therapy. Previous studies have shown that steroid-resistant (SR) patients have more severe disease and higher levels of inflammatory cytokine production than steroid-sensitive (SS) patients, but the molecular mechanisms for this remain poorly understood. Peripheral blood mononuclear cells from healthy volunteers were tested for steroid resistance by their in vitro response to the anti-proliferative effects of dexamethasone. The SR cohort had high baseline levels of NFκB DNA binding activity, equivalent to that in phytohemagglutinin (PHA)-stimulated SS cells. In SR cells, dexamethasone exposure, but not PHA, increased binding of the p65 NFκB subunit to the κB promoter element. Glucocorticoid receptor (GR) was not detected at either the κB promoter element or the glucocorticoid response element (GRE), suggesting that it does not translocate to the nucleus in these cells. Conversely, in SS cells, baseline p65 DNA binding activity was low and significantly increased by PHA, but not by dexamethasone. Unlike in SR cells, GR was detected at the κB element and at the GRE. These findings suggest that in SR patients, steroids may be harmful by increasing NFκB activity which would exacerbate disease by increasing transcription of inflammatory cytokines.

  2. Role of Prefrontal Cortex Glucocorticoid Receptors in Stress and Emotion

    PubMed Central

    McKlveen, Jessica M.; Myers, Brent; Flak, Jonathan N.; Bundzikova, Jana; Solomon, Matia B.; Seroogy, Kim B.; Herman, James P.

    2013-01-01

    Background Stress-related disorders (e.g., depression) are associated with hypothalamic-pituitary-adrenocortical axis dysregulation and prefrontal cortex (PFC) dysfunction, suggesting a functional link between aberrant prefrontal corticosteroid signaling and mood regulation. Methods We used a virally mediated knockdown strategy (short hairpin RNA targeting the glucocorticoid receptor [GR]) to attenuate PFC GR signaling in the rat PFC. Adult male rats received bilateral microinjections of vector control or short hairpin RNA targeting the GR into the prelimbic (n = 44) or infralimbic (n = 52) cortices. Half of the animals from each injection group underwent chronic variable stress, and all were subjected to novel restraint. The first 2 days of chronic variable stress were used to assess depression- and anxiety-like behavior in the forced swim test and open field. Results The GR knockdown confined to the infralimbic PFC caused acute stress hyper-responsiveness, sensitization of stress responses after chronic variable stress, and induced depression-like behavior (increased immobility in the forced swim test). Knockdown of GR in the neighboring prelimbic PFC increased hypothalamic-pituitary-adrenocortical axis responses to acute stress and caused hyper-locomotion in the open field, but did not affect stress sensitization or helplessness behavior. Conclusions The data indicate a marked functional heterogeneity of glucocorticoid action in the PFC and highlight a prominent role for the infralimbic GR in appropriate stress adaptation, emotional control, and mood regulation. PMID:23683655

  3. 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.

  4. Is there a role for glucocorticoid receptor beta in asthma?

    PubMed Central

    Gagliardo, Rosalia; Vignola, Antonio M; Mathieu, Marc

    2001-01-01

    Glucocorticoids (GCs) are routinely used as anti-inflammatory drugs in the treatment of asthma. They act through binding to glucocorticoid receptor α (GRα), which represses numerous genes encoding pro-inflammatory mediators. A hormone binding deficient GR isoform named GRβ has been isolated in humans. When overexpressed by transfection, GRβ may function as a dominant negative modulator of GRα. However, to act as such, GRβ has to be more abundant than GRα, and conflicting data have been obtained concerning the relative levels of the two isoforms in cell lines and freshly isolated cells. Moreover, the dominant negative effect was not confirmed by independent laboratories. In GC-resistant asthmatics, GRβ was expressed by an increased number of peripheral blood mononuclear cells (PBMCs), airway T cells, and cells found in skin biopsies of tuberculin responses. However, the relative amounts of GRα and GRβ in these cells were not determined. In GC-dependent asthmatics, PBMCs expressed GRα predominantly. No cells containing higher levels of GRβ than GRα have yet been reported in asthmatics. Even if the existence of such cells is demonstrated, the role of GRβ in asthma will remain a matter of controversy because functional studies have given discrepant data. PMID:11686858

  5. Bifunctional Ligands Allow Deliberate Extrinsic Reprogramming of the Glucocorticoid Receptor

    PubMed Central

    Højfeldt, Jonas W.; Cruz-Rodríguez, Osvaldo; Imaeda, Yasuhiro; Van Dyke, Aaron R.; Carolan, James P.; Mapp, Anna K.

    2014-01-01

    Therapies based on conventional nuclear receptor ligands are extremely powerful, yet their broad and long-term use is often hindered by undesired side effects that are often part of the receptor's biological function. Selective control of nuclear receptors such as the glucocorticoid receptor (GR) using conventional ligands has proven particularly challenging. Because they act solely in an allosteric manner, conventional ligands are constrained to act via cofactors that can intrinsically partner with the receptor. Furthermore, effective means to rationally encode a bias for specific coregulators are generally lacking. Using the (GR) as a framework, we demonstrate here a versatile approach, based on bifunctional ligands, that extends the regulatory repertoire of GR in a deliberate and controlled manner. By linking the macrolide FK506 to a conventional agonist (dexamethasone) or antagonist (RU-486), we demonstrate that it is possible to bridge the intact receptor to either positively or negatively acting coregulatory proteins bearing an FK506 binding protein domain. Using this strategy, we show that extrinsic recruitment of a strong activation function can enhance the efficacy of the full agonist dexamethasone and reverse the antagonist character of RU-486 at an endogenous locus. Notably, the extrinsic recruitment of histone deacetylase-1 reduces the ability of GR to activate transcription from a canonical GR response element while preserving ligand-mediated repression of nuclear factor-κB. By providing novel ways for the receptor to engage specific coregulators, this unique ligand design approach has the potential to yield both novel tools for GR study and more selective therapeutics. PMID:24422633

  6. The Role of S-Palmitoylation of the Human Glucocorticoid Receptor (hGR) in Mediating the Nongenomic Glucocorticoid Actions.

    PubMed

    Nicolaides, Nicolas C; Kino, Tomoshige; Roberts, Michael L; Katsantoni, Eleni; Sertedaki, Amalia; Moutsatsou, Paraskevi; Psarra, Anna-Maria G; Chrousos, George P; Charmandari, Evangelia

    2017-01-01

    Many rapid nongenomic glucocorticoid actions are mediated by membrane-bound glucocorticoid receptors (GRs). S-palmitoylation is a lipid post-translational modification that mediates the membrane localization of some steroid receptors. A highly homologous amino acid sequence (663YLCM KTLLL671) is present in the ligand-binding domain of hGRα, suggesting that hGRα might also undergo S-palmitoylation. To investigate the role of the motif 663YLCMKTLLL671 in membrane localization of the hGRα and in mediating rapid nongenomic glucocorticoid signaling. We showed that the mutant receptors hGRαY663A, hGRαC665A and hGRαLL670/671AA, and the addition of the palmitoylation inhibitor 2-bromopalmitate did not prevent membrane localization of hGRα and co-localization with caveolin-1, and did not influence the biphasic activation of mitogen-activated protein kinase (MAPK) signaling pathway in the early time points. Finally, the hGRα was not shown to undergo S-palmitoylation. The motif 663YLCMKTLLL671 does not play a role in membrane localization of hGRα and does not mediate the nongenomic glucocorticoid actions.

  7. The glucocorticoid-glucocorticoid receptor signal transduction pathway, transforming growth factor-beta, and embryonic mouse lung development in vivo.

    PubMed

    Jaskoll, T; Choy, H A; Melnick, M

    1996-05-01

    Lung morphogenesis has been shown to be regulated by glucocorticoids (CORT). Because CORT has been primarily thought to affect fetal lung development, previous studies have focused on the role of CORT receptor (GR)-mediated regulation of fetal lung development. Although endogenous CORT increases during embryonic and fetal stages and exogenous CORT treatment in vivo and in vitro clearly accelerates embryonic lung development, little is known about the morphoregulatory role of the embryonic CORT-GR signal transduction pathway during lung development. In this study, we characterize the embryonic mouse CORT-GR pathway and demonstrate: stage-specific in situ patterns of GR immunolocalization; similarity in GR relative mobility with progressive (E13 --> E17) development; that embryonic GR can be activated to bind a GR response element (GRE); significantly increasing levels of functional GR with increasing lung maturation; and the presence of heat shock protein (hsp) 70 and hsp90 from early (E13) to late (E17) developmental stages. These results support the purported importance of the embryonic CORT-GR signal transduction pathway in progressive lung differentiation. To demonstrate that the embryonic CORT-GR directed pathway plays a role in lung development, early embryonic (E12) lungs were exposed to CORT in utero and surfactant-associated protein A (SP-A) expression was analyzed; CORT treatment up-regulates SP-A mRNA expression and spatiotemporal protein distribution. Finally, to determine whether CORT-GR-directed pulmonary morphogenesis in vivo involves the modulation of growth factors, we studied the effect of CORT on TGF-beta gene expression. Northern analysis of TGF-beta 1, TGF-beta 2, and TGF-beta 3 transcript levels in vivo indicates that CORT regulates the rate of lung morpho- and histodifferentiation by down-regulating TGF-beta 3 gene expression.

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

    PubMed

    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.

  9. 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

  10. Conservation analysis predicts in vivo occupancy of glucocorticoid receptor-binding sequences at glucocorticoid-induced genes.

    PubMed

    So, Alex Yick-Lun; Cooper, Samantha B; Feldman, Brian J; Manuchehri, Mitra; Yamamoto, Keith R

    2008-04-15

    The glucocorticoid receptor (GR) interacts with specific GR-binding sequences (GBSs) at glucocorticoid response elements (GREs) to orchestrate transcriptional networks. Although the sequences of the GBSs are highly variable among different GREs, the precise sequence within an individual GRE is highly conserved. In this study, we examined whether sequence conservation of sites resembling GBSs is sufficient to predict GR occupancy of GREs at genes responsive to glucocorticoids. Indeed, we found that the level of conservation of these sites at genes up-regulated by glucocorticoids in mouse C3H10T1/2 mesenchymal stem-like cells correlated directly with the extent of occupancy by GR. In striking contrast, we failed to observe GR occupancy of GBSs at genes repressed by glucocorticoids, despite the occurrence of these sites at a frequency similar to that of the induced genes. Thus, GR occupancy of the GBS motif correlates with induction but not repression, and GBS conservation alone is sufficient to predict GR occupancy and GRE function at induced genes.

  11. The Interactome of the Glucocorticoid Receptor and Its Influence on the Actions of Glucocorticoids in Combatting Inflammatory and Infectious Diseases

    PubMed Central

    Petta, Ioanna; Dejager, Lien; Ballegeer, Marlies; Lievens, Sam; Tavernier, Jan; Libert, Claude

    2016-01-01

    SUMMARY Glucocorticoids (GCs) have been widely used for decades as a first-line treatment for inflammatory and autoimmune diseases. However, their use is often hampered by the onset of adverse effects or resistance. GCs mediate their effects via binding to glucocorticoid receptor (GR), a transcription factor belonging to the family of nuclear receptors. An important aspect of GR's actions, including its anti-inflammatory capacity, involves its interactions with various proteins, such as transcription factors, cofactors, and modifying enzymes, which codetermine receptor functionality. In this review, we provide a state-of-the-art overview of the protein-protein interactions (PPIs) of GR that positively or negatively affect its anti-inflammatory properties, along with mechanistic insights, if known. Emphasis is placed on the interactions that affect its anti-inflammatory effects in the presence of inflammatory and microbial diseases. PMID:27169854

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

    PubMed

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

    2013-03-01

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

  13. Thiazolidinediones are Partial Agonists for the Glucocorticoid Receptor

    PubMed Central

    Matthews, L; Berry, A; Tersigni, M; D’Acquisto, F; Ianaro, A; Ray, D

    2014-01-01

    Although thiazolidinediones were designed as specific PPARγ-ligands there is evidence for some off-target effects mediated by a non-PPARγ mechanism. Previously we have shown that Rosiglitazone has anti-inflammatory actions not explicable by activation of PPARγ, but possibly by the glucocorticoid receptor (GR). Rosiglitazone induces nuclear translocation both of GR-GFP, and endogenous GR in HeLa and U20S cells but with slower kinetics than Dexamethasone. Rosiglitazone also induces GR phosphorylation (Ser211), a GR ligand-binding specific effect. Rosiglitazone drives luciferase expression from a simple GRE containing reporter gene in a GR-dependent manner (EC50 4μM), with a similar amplitude response to the partial GR agonist RU486. Rosiglitazone also inhibits Dexamethasone driven reporter gene activity (IC50 2.9μM) in a similar fashion to RU486, suggesting partial agonist activity. Importantly we demonstrate a similar effect in PPARγ-null cells suggesting both GR-dependence and PPARγ-independence. Rosiglitazone also activates a GAL4-GR chimera, driving a UAS promoter, demonstrating DNA template sequence independence, and furthermore enhanced SRC1-GR interaction, measured by a mammalian two-hybrid assay. Both Ciglitazone and Pioglitazone, structurally related to Rosiglitazone, show similar effects on the GR. The antiproliferative effect of Rosiglitazone is increased in U20S cells that overexpress GR, suggesting a biologically important GR-dependent component of Rosiglitazone action. Rosiglitazone is a partial GR agonist, affecting GR activation and trafficking to influence engagement of target genes and affect cell function. This novel mode of action may explain some off-target effects observed in vivo. Additionally, antagonism of glucocorticoid action may contribute to the anti-diabetic actions of Rosiglitazone. PMID:18801908

  14. Glucocorticoids regulate arrestin gene expression and redirect the signaling profile of G protein-coupled receptors.

    PubMed

    Oakley, Robert H; Revollo, Javier; Cidlowski, John A

    2012-10-23

    G protein-coupled receptors (GPCRs) compose the largest family of cell surface receptors and are the most common target of therapeutic drugs. The nonvisual arrestins, β-arrestin-1 and β-arrestin-2, are multifunctional scaffolding proteins that play critical roles in GPCR signaling. On binding of activated GPCRs at the plasma membrane, β-arrestins terminate G protein-dependent responses (desensitization) and stimulate β-arrestin-dependent signaling pathways. Alterations in the cellular complement of β-arrestin-1 and β-arrestin-2 occur in many human diseases, and their genetic ablation in mice has severe consequences. Surprisingly, however, the factors that control β-arrestin gene expression are poorly understood. We demonstrate that glucocorticoids differentially regulate β-arrestin-1 and β-arrestin-2 gene expression in multiple cell types. Glucocorticoids act via the glucocorticoid receptor (GR) to induce the synthesis of β-arrestin-1 and repress the expression of β-arrestin-2. Glucocorticoid-dependent regulation involves the recruitment of ligand-activated glucocorticoid receptors to conserved and functional glucocorticoid response elements in intron-1 of the β-arrestin-1 gene and intron-11 of the β-arrestin-2 gene. In human lung adenocarcinoma cells, the increased expression of β-arrestin-1 after glucocorticoid treatment impairs G protein-dependent activation of inositol phosphate signaling while enhancing β-arrestin-1-dependent stimulation of the MAPK pathway by protease activated receptor 1. These studies demonstrate that glucocorticoids redirect the signaling profile of GPCRs via alterations in β-arrestin gene expression, revealing a paradigm for cross-talk between nuclear and cell surface receptors and a mechanism by which glucocorticoids alter the clinical efficacy of GPCR-based drugs.

  15. 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

  16. Glucocorticoid receptor antagonism by cyproterone acetate and RU486.

    PubMed

    Honer, Christian; Nam, Kiyean; Fink, Cynthia; Marshall, Paul; Ksander, Gary; Chatelain, Ricardo E; Cornell, Wendy; Steele, Ronald; Schweitzer, Robert; Schumacher, Christoph

    2003-05-01

    The steroid compound cyproterone acetate was identified in a high-throughput screen for glucocorticoid receptor (GR) binding compounds. Cyproterone (Schering AG) is clinically used as an antiandrogen for inoperable prostate cancer, virilizing syndromes in women, and the inhibition of sex drive in men. Despite its progestin properties, cyproterone shares a similar pharmacological profile with the antiprogestin mifepristone (RU486; Roussel Uclaf SA). The binding affinities of cyproterone and RU486 for the GR and progesterone receptor were similar (K(d), 15-70 nM). Both compounds were characterized as competitive antagonists of dexamethasone without intrinsic transactivating properties in rat hepatocytes (K(i), 10-30 nM). In osteosarcoma cells, RU486 revealed a higher potency than cyproterone acetate to prevent responses to dexamethasone-induced GR transactivation and NF kappa B transrepression. Upon administration to Sprague-Dawley rats, both compounds were found to be orally bioavailable and to inhibit transactivation of liver GR. Molecular docking of cyproterone acetate and RU486 into the homology model for the GR ligand binding domain illustrated overlapping steroid scaffolds in the binding pocket. However, in contrast to RU486, cyproterone lacks a bulky side chain at position C11 beta that has been proposed to trigger active antagonism of nuclear receptors by displacing the C-terminal helix of the ligand-binding domain, thereby affecting activation function 2. Cyproterone may therefore inhibit transactivation of the GR by a molecular mechanism recently described as passive antagonism. New therapeutic profiles may result from compounds designed to selectively stabilize the inactive and active conformations of certain nuclear receptors.

  17. Glucocorticoid Receptor Accelerates, but Is Dispensable for, Adipogenesis.

    PubMed

    Park, Young-Kwon; Ge, Kai

    2017-01-15

    Dexamethasone (DEX), a synthetic ligand for glucocorticoid receptor (GR), is routinely used to stimulate adipogenesis in culture. GR-depleted preadipocytes show adipogenesis defects 1 week after induction of differentiation. However, it has remained unclear whether GR is required for adipogenesis in vivo By deleting GR in precursors of brown adipocytes, we found unexpectedly that GR is dispensable for brown adipose tissue development in mice. In culture, GR-deficient primary or immortalized white and brown preadipocytes showed severely delayed adipogenesis 1 week after induction of differentiation. However, when differentiation was extended to 3 weeks, GR-deficient preadipocytes showed levels of adipogenesis marker expression and lipid accumulation similar to those of the wild-type cells, indicating that DEX-bound GR accelerates, but is dispensable for, adipogenesis. Consistently, DEX accelerates, but is dispensable for, adipogenesis in culture. We show that DEX-bound GR accelerates adipogenesis by directly promoting the expression of adipogenic transcription factors CCAAT/enhancer-binding protein alpha (C/EBPα), C/EBPβ, C/EBPδ, KLF5, KLF9, and peroxisome proliferator-activated receptor γ (PPARγ) in the early phase of differentiation. Mechanistically, DEX-bound GR recruits histone H3K27 acetyltransferase CBP to promote activation of C/EBPβ-primed enhancers of adipogenic genes. These results clarify the role of GR in adipogenesis in vivo and demonstrate that DEX-mediated activation of GR accelerates, but is dispensable for, adipogenesis. Copyright © 2017 American Society for Microbiology.

  18. Historical contingency and its biophysical basis in glucocorticoid receptor evolution.

    PubMed

    Harms, Michael J; Thornton, Joseph W

    2014-08-14

    Understanding how chance historical events shape evolutionary processes is a central goal of evolutionary biology. Direct insights into the extent and causes of evolutionary contingency have been limited to experimental systems, because it is difficult to know what happened in the deep past and to characterize other paths that evolution could have followed. Here we combine ancestral protein reconstruction, directed evolution and biophysical analysis to explore alternative 'might-have-been' trajectories during the ancient evolution of a novel protein function. We previously found that the evolution of cortisol specificity in the ancestral glucocorticoid receptor (GR) was contingent on permissive substitutions, which had no apparent effect on receptor function but were necessary for GR to tolerate the large-effect mutations that caused the shift in specificity. Here we show that alternative mutations that could have permitted the historical function-switching substitutions are extremely rare in the ensemble of genotypes accessible to the ancestral GR. In a library of thousands of variants of the ancestral protein, we recovered historical permissive substitutions but no alternative permissive genotypes. Using biophysical analysis, we found that permissive mutations must satisfy at least three physical requirements--they must stabilize specific local elements of the protein structure, maintain the correct energetic balance between functional conformations, and be compatible with the ancestral and derived structures--thus revealing why permissive mutations are rare. These findings demonstrate that GR evolution depended strongly on improbable, non-deterministic events, and this contingency arose from intrinsic biophysical properties of the protein.

  19. 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.

  20. Dopamine DA1 receptors on vascular smooth muscle cells are regulated by glucocorticoid and sodium chloride.

    PubMed

    Yasunari, K; Kohno, M; Yokokawa, K; Horio, T; Takeda, T

    1994-09-01

    The modulation of dopamine DA1 receptors of cultured rat renal arterial smooth muscle cells by glucocorticoid and sodium chloride was studied. At a concentration of 10 nM, the synthetic glucocorticoid dexamethasone increased maximum receptor binding but had no effect on the dissociation constant. However, the maximum binding of [3H]Sch-23390 in cells treated with 100 mM sodium chloride did not change. However, the dissociation constant for DA1 receptor was increased by adding sodium chloride. The glucocorticoid effect on DA1 of arterial smooth muscle cells became apparent after hours of incubation in the presence of the steroid and was significantly inhibited by cycloheximide (10 micrograms/ml) or by the glucocorticoid receptor antagonist RU-38486, indicating that the effect required protein synthesis through glucocorticoid receptors. Treatment of cells with 1 microM dexamethasone for 24 h increased basal and DA1-stimulated adenylate cyclase activity. Basal adenylate cyclase was decreased by sodium chloride in a dose-dependent manner. These results suggest differential control of DA1 receptors on vascular smooth muscle cells by glucocorticoid or sodium chloride.

  1. 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.

  2. The glucocorticoid receptor in the distal nephron is not necessary for the development or maintenance of dexamethasone-induced hypertension

    PubMed Central

    Goodwin, Julie E.; Zhang, Junhui; Velazquez, Heino; Geller, David S.

    2010-01-01

    Glucocorticoids are used as a treatment for a variety of conditions and hypertension is a well-recognized side effect of their use. The mechanism of glucocorticoid-induced hypertension is incompletely understood and has traditionally been attributed to promiscuous activation of the mineralocorticoid receptor by cortisol. Multiple lines of evidence, however, point to the glucocorticoid receptor as an important mediator as well. We have developed a mouse model of glucocorticoid-induced hypertension, which is dependent on the glucocorticoid receptor. To determine the site(s) of glucocorticoid receptor action relevant to the development of hypertension, we studied glucocorticoid-induced hypertension in a mouse with a tissue-specific knockout of the glucocorticoid receptor in the distal nephron. Although knockout mice had similar body weight, nephron number and renal histology compared to littermate controls, their baseline blood pressure was mildly elevated. Nevertheless, distal nephron glucocorticoid receptor knockout mice and controls had a similar hypertensive response to dexamethasone. Urinary excretion of electrolytes, both before and after administration of glucocorticoid was also indistinguishable between the two groups. We conclude that the glucocorticoid receptor in the distal nephron is not necessary for the development or maintenance of dexamethasone-induced hypertension in our model. PMID:20188070

  3. Tetrahydroquinoline glucocorticoid receptor agonists: discovery of a 3-hydroxyl for improving receptor selectivity.

    PubMed

    Roach, Steven L; Higuchi, Robert I; Hudson, Andrew R; Adams, Mark E; Syka, Peter M; Mais, Dale E; Miner, Jeffrey N; Marschke, Keith B; Zhi, Lin

    2011-01-01

    We have previously disclosed a series of glucocorticoid receptor (GR) ligands derived from 6-indole-1,2,3,4-tetrahydroquinolines through structure-activity relationship (SAR) of the pendent C6-indole ring. In parallel with this effort, we now report SAR of the tetrahydroquinoline A-ring that identified the importance of a C3 hydroxyl in improving GR selectivity within a series of non-steroidal GR agonists. Copyright © 2010 Elsevier Ltd. All rights reserved.

  4. 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

  5. Acute stress enhances heterodimerization and binding of corticosteroid receptors at glucocorticoid target genes in the hippocampus.

    PubMed

    Mifsud, Karen R; Reul, Johannes M H M

    2016-10-04

    A stressful event results in secretion of glucocorticoid hormones, which bind to mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) in the hippocampus to regulate cognitive and affective responses to the challenge. MRs are already highly occupied by low glucocorticoid levels under baseline conditions, whereas GRs only become substantially occupied by stress- or circadian-driven glucocorticoid levels. Currently, however, the binding of MRs and GRs to glucocorticoid-responsive elements (GREs) within hippocampal glucocorticoid target genes under such physiological conditions in vivo is unknown. We found that forced swim (FS) stress evoked increased hippocampal RNA expression levels of the glucocorticoid-responsive genes FK506-binding protein 5 (Fkbp5), Period 1 (Per1), and serum- and glucocorticoid-inducible kinase 1 (Sgk1). Chromatin immunoprecipitation (ChIP) analysis showed that this stressor caused substantial gene-dependent increases in GR binding and surprisingly, also MR binding to GREs within these genes. Different acute challenges, including novelty, restraint, and FS stress, produced distinct glucocorticoid responses but resulted in largely similar MR and GR binding to GREs. Sequential and tandem ChIP analyses showed that, after FS stress, MRs and GRs bind concomitantly to the same GRE sites within Fkbp5 and Per1 but not Sgk1 Thus, after stress, MRs and GRs seem to bind to GREs as homo- and/or heterodimers in a gene-dependent manner. MR binding to GREs at baseline seems to be restricted, whereas after stress, GR binding may facilitate cobinding of MR. This study reveals that the interaction of MRs and GRs with GREs within the genome constitutes an additional level of complexity in hippocampal glucocorticoid action beyond expectancies based on ligand-receptor interactions.

  6. Acute stress enhances heterodimerization and binding of corticosteroid receptors at glucocorticoid target genes in the hippocampus

    PubMed Central

    2016-01-01

    A stressful event results in secretion of glucocorticoid hormones, which bind to mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) in the hippocampus to regulate cognitive and affective responses to the challenge. MRs are already highly occupied by low glucocorticoid levels under baseline conditions, whereas GRs only become substantially occupied by stress- or circadian-driven glucocorticoid levels. Currently, however, the binding of MRs and GRs to glucocorticoid-responsive elements (GREs) within hippocampal glucocorticoid target genes under such physiological conditions in vivo is unknown. We found that forced swim (FS) stress evoked increased hippocampal RNA expression levels of the glucocorticoid-responsive genes FK506-binding protein 5 (Fkbp5), Period 1 (Per1), and serum- and glucocorticoid-inducible kinase 1 (Sgk1). Chromatin immunoprecipitation (ChIP) analysis showed that this stressor caused substantial gene-dependent increases in GR binding and surprisingly, also MR binding to GREs within these genes. Different acute challenges, including novelty, restraint, and FS stress, produced distinct glucocorticoid responses but resulted in largely similar MR and GR binding to GREs. Sequential and tandem ChIP analyses showed that, after FS stress, MRs and GRs bind concomitantly to the same GRE sites within Fkbp5 and Per1 but not Sgk1. Thus, after stress, MRs and GRs seem to bind to GREs as homo- and/or heterodimers in a gene-dependent manner. MR binding to GREs at baseline seems to be restricted, whereas after stress, GR binding may facilitate cobinding of MR. This study reveals that the interaction of MRs and GRs with GREs within the genome constitutes an additional level of complexity in hippocampal glucocorticoid action beyond expectancies based on ligand–receptor interactions. PMID:27655894

  7. Improved androgen specificity of AR-EcoScreen by CRISPR based glucocorticoid receptor knockout.

    PubMed

    Zwart, Nick; Andringa, Dave; de Leeuw, Willem-Jan; Kojima, Hiroyuki; Iida, Mitsuru; Houtman, Corine J; de Boer, Jacob; Kool, Jeroen; Lamoree, Marja H; Hamers, Timo

    2017-08-11

    The AR-EcoScreen is a widely used reporter assay for the detection of androgens and anti-androgens. Endogenous expression of glucocorticoid receptors and their affinity for the androgen responsive element that drives reporter expression, however, makes the reporter cells sensitive to interference by glucocorticoids and less specific for (anti-)androgens. To create a glucocorticoid insensitive derivative of the AR-EcoScreen, CRISPR/Cas9 genome editing was used to develop glucocorticoid receptor knockout mutants by targeting various sites in the glucocorticoid gene. Two mutant cell lines were further characterized and validated against the unmodified AR-EcoScreen with a set of 19 environmentally relevant chemicals and a series of environmental passive sampler extracts with (anti-)androgenic activity. Sequencing of the targeted sites revealed premature stop codons following frame-shift mutations, leading to an absence of functional glucocorticoid receptor expression. The introduced mutations rendered cell lines insensitive to glucocorticoid activation and caused no significant difference in the responsiveness towards (anti-)androgens, compared to the unmodified AR-EcoScreen cells, allowing the selective, GR-independent, determination of (anti-)androgenicity in environmental passive sampler extracts. The increase in selectivity for (anti-)androgens improves reliability of the AR-EcoScreen and will provide higher accuracy in determining (anti-)androgenic potential when applied in toxicity screening and environmental monitoring of both single compounds and mixtures. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Deletion of Neurotrophin Signaling through the Glucocorticoid Receptor Pathway Causes Tau Neuropathology

    PubMed Central

    Arango-Lievano, Margarita; Peguet, Camille; Catteau, Matthias; Parmentier, Marie-Laure; Wu, Synphen; Chao, Moses V; Ginsberg, Stephen D.; Jeanneteau, Freddy

    2016-01-01

    Glucocorticoid resistance is a risk factor for Alzheimer’s disease (AD). Molecular and cellular mechanisms of glucocorticoid resistance in the brain have remained unknown and are potential therapeutic targets. Phosphorylation of glucocorticoid receptors (GR) by brain-derived neurotrophic factor (BDNF) signaling integrates both pathways for remodeling synaptic structure and plasticity. The goal of this study is to test the role of the BDNF-dependent pathway on glucocorticoid signaling in a mouse model of glucocorticoid resistance. We report that deletion of GR phosphorylation at BDNF-responding sites and downstream signaling via the MAPK-phosphatase DUSP1 triggers tau phosphorylation and dendritic spine atrophy in mouse cortex. In human cortex, DUSP1 protein expression correlates with tau phosphorylation, synaptic defects and cognitive decline in subjects diagnosed with AD. These findings provide evidence for a causal role of BDNF-dependent GR signaling in tau neuropathology and indicate that DUSP1 is a potential target for therapeutic interventions. PMID:27849045

  9. 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

  10. Historical contingency and its biophysical basis in glucocorticoid receptor evolution

    PubMed Central

    Harms, Michael J.; Thornton, Joseph W.

    2015-01-01

    Understanding how chance historical events shape evolutionary processes is a central goal of evolutionary biology1–7. Direct insights into the extent and causes of evolutionary contingency have been limited to experimental systems,7–9 because it is difficult to know what happened in the deep past and to characterize other paths that evolution could have followed. Here we combine ancestral protein reconstruction, directed evolution, and biophysical analysis to explore alternate “might-have-been” trajectories during the ancient evolution of a novel protein function. We previously found that the evolution of cortisol specificity in the ancestral glucocorticoid receptor (GR) was contingent on permissive substitutions, which had no apparent effect on receptor function but were necessary for GR to tolerate the large-effect mutations that caused the shift in specificity.6 Here we show that alternative mutations that could have permitted the historical function-switching substitutions are extremely rare in the ensemble of genotypes accessible to the ancestral GR. In a library of thousands of variants of the ancestral protein, we recovered historical permissive substitutions, but no alternate permissive genotypes. Using biophysical analysis, we found that permissive mutations must satisfy at least three physical requirements—they must stabilize specific local elements of the protein structure, maintain the correct energetic balance between functional conformations, and be compatible with the ancestral and derived structures—thus revealing why permissive mutations are rare. These findings demonstrate that GR evolution depended strongly on improbable, nondeterministic events, and this contingency arose from intrinsic biophysical properties of the protein. PMID:24930765

  11. Glucocorticoid receptor alters isovolumetric contraction and restrains cardiac fibrosis

    PubMed Central

    Richardson, Rachel V; Batchen, Emma J; Thomson, Adrian J W; Darroch, Rowan; Pan, Xinlu; Rog-Zielinska, Eva A; Wyrzykowska, Wiktoria; Scullion, Kathleen; Al-Dujaili, Emad A S; Diaz, Mary E; Moran, Carmel M; Kenyon, Christopher J; Gray, Gillian A

    2017-01-01

    Corticosteroids directly affect the heart and vasculature and are implicated in the pathogenesis of heart failure. Attention is focussed upon the role of the mineralocorticoid receptor (MR) in mediating pro-fibrotic and other adverse effects of corticosteroids upon the heart. In contrast, the role of the glucocorticoid receptor (GR) in the heart and vasculature is less well understood. We addressed this in mice with cardiomyocyte and vascular smooth muscle deletion of GR (SMGRKO mice). Survival of SMGRKO mice to weaning was reduced compared with that of littermate controls. Doppler measurements of blood flow across the mitral valve showed an elongated isovolumetric contraction time in surviving adult SMGRKO mice, indicating impairment of the initial left ventricular contractile phase. Although heart weight was elevated in both genders, only male SMGRKO mice showed evidence of pathological cardiomyocyte hypertrophy, associated with increased myosin heavy chain-β expression. Left ventricular fibrosis, evident in both genders, was associated with elevated levels of mRNA encoding MR as well as proteins involved in cardiac remodelling and fibrosis. However, MR antagonism with spironolactone from birth only modestly attenuated the increase in pro-fibrotic gene expression in SMGRKO mice, suggesting that elevated MR signalling is not the primary driver of cardiac fibrosis in SMGRKO mice, and cardiac fibrosis can be dissociated from MR activation. Thus, GR contributes to systolic function and restrains normal cardiac growth, the latter through gender-specific mechanisms. Our findings suggest the GR:MR balance is critical in corticosteroid signalling in specific cardiac cell types. PMID:28057868

  12. Purified glucocorticoid receptors bind selectively in vitro to a cloned DNA fragment whose transcription is regulated by glucocorticoids in vivo.

    PubMed

    Payvar, F; Wrange, O; Carlstedt-Duke, J; Okret, S; Gustafsson, J A; Yamamoto, K R

    1981-11-01

    Activated glucocorticoid receptor protein, purified to 40-60% homogeneity from rat liver extracts, binds selectively in vitro to a cloned fragment of murine mammary tumor virus (MTV) DNA. The DNA fragment tested contains about half of the sequences present in intact MTV DNA, and its rate of transcription, like that of the intact viral element, is strongly stimulated by glucocorticoids when it is introduced into the genome of a receptor-containing cell. In contrast, the receptor fails to bind selectively to DNA restriction fragments from E. coli plasmids pBR322 and RSF2124 or from bacteriophages lambda and T4. Preliminary experiments to localize regions within MTV DNA responsible for selective binding have revealed thus far one subfragment that fails to bind the receptor and one selectively bound subfragment that maps far downstream from the 5' terminus of the normal RNA transcript. These studies are consistent with the notion that steroid receptors may modulate rates of transcription by recognizing specific DNA sequences within or near the regulated genes.

  13. 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).

  14. Selective Androgen Receptor Downregulators (SARDs): A New Prostate Cancer Therapy

    DTIC Science & Technology

    2006-10-01

    used to down-regulate the AR include antisense oligonucleotides (9, 10), ribozyme treatments (11, 12), AR dominant negatives (13) and small...findings suggest that ICI may present a useful treatment option for patients with AR-dependent PCa. Unlike the ribozyme , antisense, siRNA, or dominant...of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme . Mol Endocrinol, 12: 1558

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

    PubMed Central

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

    2015-01-01

    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. PMID:25656655

  16. Extraction of DNA-cellulose-bound glucocorticoid-receptor complexes with sodium tungstate.

    PubMed

    Murakami, N; Moudgil, V K

    1981-09-04

    Glucocorticoid-receptor complex from rat liver cytosol, activated by warming at 23 degrees C or fractionation with (NH4)2SO4, was adsorbed over DNA-cellulose. This DNA-cellulose-bound [3H]triamcinolone acetonide-receptor complex was extracted in a dose-dependent manner by incubation with different concentrations of sodium tungstate. A 50% recovery of receptor was achieved with 5 mM sodium tungstate. Almost the entire glucocorticoid-receptor complex bound to DNA-cellulose could be extracted with 20 mM sodium tungstate. The [3H]triamcinolone acetonide released from DNA-cellulose following tungstate and molybdate treatment was found to be associated with a macromolecule, as seen by analysis on a Sephadex G-75 column. The glucocorticoid-receptor complex extracted by both the compounds sedimented as a 4 S entity of 5-20% sucrose gradients under low- and high-salt conditions. Addition of tungstate or molybdate to the preparations containing activated receptor had no effect on the sedimentation rate of receptor. However, addition of tungstate to non-activated receptor preparation caused aggregates of larger size. The tungstate-extracted glucocorticoid-receptor complex failed to rebind to DNA-cellulose even after extensive dialysis, whereas receptor in molybdate-extract retained its DNA-cellulose binding capacity.

  17. Evidence that the modulator of the glucocorticoid-receptor complex is the endogenous molybdate factor.

    PubMed Central

    Bodine, P V; Litwack, G

    1988-01-01

    We have recently purified the modulator of the glucocorticoid-receptor complex from rat liver. Purified modulator inhibits glucocorticoid-receptor complex activation and stabilizes the steroid-binding ability of the unoccupied glucocorticoid receptor. Since these activities are shared by exogenous sodium molybdate, modulator appears to be the endogenous factor that sodium molybdate mimics. In this report, we present additional evidence for the mechanism of action of purified modulator. (i) Molybdate and modulator inhibit receptor activation as measured by DNA-cellulose binding, DEAE-cellulose chromatography, and Sepharose 4B gel filtration. (ii) The ability of molybdate and modulator to inhibit receptor activation and stabilize the unoccupied receptor appears to be additive. (iii) Scatchard analysis of heat-destabilized unoccupied receptors indicates that the number of steroid-binding sites is reduced during destabilization, whereas the steroid dissociation constant remains unchanged. Molybdate and modulator stabilize the receptor by maintaining the number of steroid-binding sites. (iv) Molybdate and modulator do not inhibit alkaline phosphatase-induced destabilization of the unoccupied receptor. However, alkaline phosphatase-induced destabilization is reversed by the addition of dithiothreitol in the presence, but not in the absence, of molybdate or modulator. These results suggest that the mechanism of action for modulator is identical to that of sodium molybdate, and we propose that modulator is the endogenous molybdate factor for the glucocorticoid receptor. PMID:3422744

  18. Glucocorticoid receptor signaling contributes to constitutive activation of the noncanonical NF-κB pathway in term human placenta.

    PubMed

    Wang, Bingbing; Palomares, Kristy; Parobchak, Nataliya; Cece, John; Rosen, Max; Nguyen, Anh; Rosen, Todd

    2013-02-01

    Our recent study demonstrated that constitutively activated RelB/NF-κB2 positively regulates the CRH in the human placenta. In the current study, we explored the role of the glucocorticoid receptor (GR) signaling in constitutive activation of the noncanonical NF-κB pathway. A glucocorticoid response element (GRE) motif search suggests that both NF-κB inducing kinase (NIK) and RelB genes, which are key regulators of the noncanonical NF-κB pathway, have a putative GRE within their promoter, approximately 1 kb upstream from the transcription start site. By using chromatin immunoprecipitation assay we identified that the GR and phosphorylated GR at Ser211 were associated with the GREs of both NIK and RelB. Dexamethasone stimulated expression of NIK, RelB, NF-κB2 as well as CRH and cyclooxygenase-2 (COX-2). Repression of GR by short interfering RNA resulted in inhibition of NIK, RelB, NF-κB2, CRH, and COX-2. In addition, depletion of GR attenuated glucocorticoid-mediated up-regulation of NIK, RelB, NF-κB2, CRH, and COX-2. Furthermore, siRNA specifically targeting NIK down-regulated CRH and COX-2. Taken together, these results suggest that constitutive activation of the noncanonical NF-κB pathway in term human placenta is driven by the GR signaling, which in turn up-regulates placental CRH and other NF-κB-responsive genes.

  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. Structure and specific DNA binding of the rat liver glucocorticoid receptor.

    PubMed

    Gustafsson, J A; Carlstedt-Duke, J; Okret, S; Wikström, A C; Wrange, O; Payvar, F; Yamamoto, K

    1984-01-01

    During recent years major advances have been made in our understanding of glucocorticoid mechanism of action. This progress has been made possible by access to purified glucocorticoid receptor in significant amounts as well as by application of hybrid DNA technology within the field of glucocorticoid control of gene expression. Especially the mammary tumour virus genome has turned out to be a convenient experimental system suitable for such investigations. This paper summarizes some of the work carried out in our own laboratory, partially in collaboration with Dr Keith Yamamoto and his associates at the Department of Biochemistry and Biophysics, University of California, San Francisco, U.S.A.

  1. Selective modulation through the glucocorticoid receptor ameliorates muscle pathology in mdx mice.

    PubMed

    Huynh, Tony; Uaesoontrachoon, Kitipong; Quinn, James L; Tatem, Kathleen S; Heier, Christopher R; Van Der Meulen, Jack H; Yu, Qing; Harris, Mark; Nolan, Christopher J; Haegeman, Guy; Grounds, Miranda D; Nagaraju, Kanneboyina

    2013-10-01

    The over-expression of NF-κB signalling in both muscle and immune cells contribute to the pathology in dystrophic muscle. The anti-inflammatory properties of glucocorticoids, mediated predominantly through monomeric glucocorticoid receptor inhibition of transcription factors such as NF-κB (transrepression), are postulated to be an important mechanism for their beneficial effects in Duchenne muscular dystrophy. Chronic glucocorticoid therapy is associated with adverse effects on metabolism, growth, bone mineral density and the maintenance of muscle mass. These detrimental effects result from direct glucocorticoid receptor homodimer interactions with glucocorticoid response elements of the relevant genes. Compound A, a non-steroidal selective glucocorticoid receptor modulator, is capable of transrepression without transactivation. We confirm the in vitro NF-κB inhibitory activity of compound A in H-2K(b) -tsA58 mdx myoblasts and myotubes, and demonstrate improvements in disease phenotype of dystrophin deficient mdx mice. Compound A treatment in mdx mice from 18 days of post-natal age to 8 weeks of age increased the absolute and normalized forelimb and hindlimb grip strength, attenuated cathepsin-B enzyme activity (a surrogate marker for inflammation) in forelimb and hindlimb muscles, decreased serum creatine kinase levels and reduced IL-6, CCL2, IFNγ, TNF and IL-12p70 cytokine levels in gastrocnemius (GA) muscles. Compared with compound A, treatment with prednisolone, a classical glucocorticoid, in both wild-type and mdx mice was associated with reduced body weight, reduced GA, tibialis anterior and extensor digitorum longus muscle mass and shorter tibial lengths. Prednisolone increased osteopontin (Spp1) gene expression and osteopontin protein levels in the GA muscles of mdx mice and had less favourable effects on the expression of Foxo1, Foxo3, Fbxo32, Trim63, Mstn and Igf1 in GA muscles, as well as hepatic Igf1 in wild-type mice. In conclusion, selective

  2. Selective modulation through the glucocorticoid receptor ameliorates muscle pathology in mdx mice

    PubMed Central

    Huynh, Tony; Uaesoontrachoon, Kitipong; Quinn, James L; Tatem, Kathleen S; Heier, Christopher R; Van Der Meulen, Jack H; Yu, Qing; Harris, Mark; Nolan, Christopher J; Haegeman, Guy; Grounds, Miranda D; Nagaraju, Kanneboyina

    2014-01-01

    The over-expression of NF-κB signalling in both muscle and immune cells contribute to the pathology in dystrophic muscle. The anti-inflammatory properties of glucocorticoids, mediated predominantly through monomeric glucocorticoid receptor inhibition of transcription factors such as NF-κB (transrepression), are postulated to be an important mechanism for their beneficial effects in Duchenne muscular dystrophy. Chronic glucocorticoid therapy is associated with adverse effects on metabolism, growth, bone mineral density and the maintenance of muscle mass. These detrimental effects result from direct glucocorticoid receptor homodimer interactions with glucocorticoid response elements of the relevant genes. Compound A, a non-steroidal selective glucocorticoid receptor modulator, is capable of transrepression without transactivation. We confirm the in vitro NF-κB inhibitory activity of compound A in H-2Kb-tsA58 mdx myoblasts and myotubes, and demonstrate improvements in disease phenotype of dystrophin deficient mdx mice. Compound A treatment in mdx mice from 18 days of post-natal age to 8 weeks of age increased the absolute and normalized forelimb and hindlimb grip strength, attenuated cathepsin-B enzyme activity (a surrogate marker for inflammation) in forelimb and hindlimb muscles, decreased serum creatine kinase levels and reduced IL-6, CCL2, IFNγ, TNF and IL-12p70 cytokine levels in gastrocnemius (GA) muscles. Compared with compound A, treatment with prednisolone, a classical glucocorticoid, in both wild-type and mdx mice was associated with reduced body weight, reduced GA, tibialis anterior and extensor digitorum longus muscle mass and shorter tibial lengths. Prednisolone increased osteopontin (Spp1) gene expression and osteopontin protein levels in the GA muscles of mdx mice and had less favourable effects on the expression of Foxo1, Foxo3, Fbxo32, Trim63, Mstn and Igf1 in GA muscles, as well as hepatic Igf1 in wild-type mice. In conclusion, selective

  3. Epigenetic regulation of the glucocorticoid receptor in human brain associates with childhood abuse

    PubMed Central

    McGowan, Patrick O; Sasaki, Aya; D’Alessio, Ana C; Dymov, Sergiy; Labonté, Benoit; Szyf, Moshe; Turecki, Gustavo; Meaney, Michael J

    2010-01-01

    Maternal care influences hypothalamic-pituitary-adrenal (HPA) function in the rat through epigenetic programming of glucocorticoid receptor expression. In humans, childhood abuse alters HPA stress responses and increases the risk of suicide. We examined epigenetic differences in a neuron-specific glucocorticoid receptor (NR3C1) promoter between postmortem hippocampus obtained from suicide victims with a history of childhood abuse and those from either suicide victims with no childhood abuse or controls. We found decreased levels of glucocorticoid receptor mRNA, as well as mRNA transcripts bearing the glucocorticoid receptor 1F splice variant and increased cytosine methylation of an NR3C1 promoter. Patch-methylated NR3C1 promoter constructs that mimicked the methylation state in samples from abused suicide victims showed decreased NGFI-A transcription factor binding and NGFI-A–inducible gene transcription. These findings translate previous results from rat to humans and suggest a common effect of parental care on the epigenetic regulation of hippocampal glucocorticoid receptor expression. PMID:19234457

  4. Identifying a Mechanism for Crosstalk Between the Estrogen and Glucocorticoid Receptors | Center for Cancer Research

    Cancer.gov

    Estrogen has long been known to play important roles in the development and progression of breast cancer. Its receptor (ER), a member of the steroid receptor family, binds to estrogen response elements (EREs) in DNA and regulates gene transcription. More recently, another steroid receptor family member, the glucocorticoid receptor (GR), has been implicated in breast cancer progression, and ER/GR status is an important predictor of breast cancer outcome.

  5. Discovery and optimization of novel, non-steroidal glucocorticoid receptor modulators.

    PubMed

    Ray, Nicholas C; Clark, Robin D; Clark, David E; Williams, Karen; Hickin, H G; Crackett, Peter H; Dyke, Hazel J; Lockey, Peter M; Wong, Melanie; Devos, René; White, Anne; Belanoff, Joseph K

    2007-09-01

    A virtual screening approach comprising a 3-D similarity search based on known GR modulators was used to identify a novel series of non-steroidal glucocorticoid receptor (GR) antagonists. Optimization of the initial hit to provide potent compounds which exhibit good selectivity against other steroidal nuclear hormone receptors is described.

  6. MicroRNA-433 Dampens Glucocorticoid Receptor Signaling, Impacting Circadian Rhythm and Osteoblastic Gene Expression.

    PubMed

    Smith, Spenser S; Dole, Neha S; Franceschetti, Tiziana; Hrdlicka, Henry C; Delany, Anne M

    2016-10-07

    Serum glucocorticoids play a critical role in synchronizing circadian rhythm in peripheral tissues, and multiple mechanisms regulate tissue sensitivity to glucocorticoids. In the skeleton, circadian rhythm helps coordinate bone formation and resorption. Circadian rhythm is regulated through transcriptional and post-transcriptional feedback loops that include microRNAs. How microRNAs regulate circadian rhythm in bone is unexplored. We show that in mouse calvaria, miR-433 displays robust circadian rhythm, peaking just after dark. In C3H/10T1/2 cells synchronized with a pulse of dexamethasone, inhibition of miR-433 using a tough decoy altered the period and amplitude of Per2 gene expression, suggesting that miR-433 regulates rhythm. Although miR-433 does not directly target the Per2 3'-UTR, it does target two rhythmically expressed genes in calvaria, Igf1 and Hif1α. miR-433 can target the glucocorticoid receptor; however, glucocorticoid receptor protein abundance was unaffected in miR-433 decoy cells. Rather, miR-433 inhibition dramatically enhanced glucocorticoid signaling due to increased nuclear receptor translocation, activating glucocorticoid receptor transcriptional targets. Last, in calvaria of transgenic mice expressing a miR-433 decoy in osteoblastic cells (Col3.6 promoter), the amplitude of Per2 and Bmal1 mRNA rhythm was increased, confirming that miR-433 regulates circadian rhythm. miR-433 was previously shown to target Runx2, and mRNA for Runx2 and its downstream target, osteocalcin, were also increased in miR-433 decoy mouse calvaria. We hypothesize that miR-433 helps maintain circadian rhythm in osteoblasts by regulating sensitivity to glucocorticoid receptor signaling.

  7. 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 Central

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

    2014-01-01

    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. PMID:24368768

  8. 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

    2014-03-07

    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.

  9. 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

  10. New Insights in Glucocorticoid Receptor Signaling—More Than Just a Ligand-Binding Receptor

    PubMed Central

    Scheschowitsch, Karin; Leite, Jacqueline Alves; Assreuy, Jamil

    2017-01-01

    The clinical use of classical glucocorticoids (GC) is narrowed by the many side effects it causes and the resistance to GC observed in some diseases. Since the great majority of GC effects depend on the activation of a glucocorticoid receptor (GR), many research groups had focused to better understand the signaling pathways involving those receptors. Transgenic animal models and genetic modifications of the receptor brought a huge insight into GR mechanisms of action. This in turn opened a new window for the search of selective GR modulators that ideally may have agonistic and antagonistic combined effects and activate one specific signaling pathway, inducing mostly transrepression or transactivation mechanisms. Another important research field concerns to posttranslational modifications that affect the GR and consequently also affect its signaling and function. In this mini review, we discuss many of those aspects of GR signaling, as well as findings like the ligand-independent activation of GR, which add another layer of complexity in GR signaling pathways. Although several recent data have been added to the GR field, much work has yet to be done, especially to find out the biological relevance of those alternative GR signaling pathways. Improving the knowledge about alternative GR signaling pathways and understanding how these pathways intercommunicate and in which situations they are relevant might help to develop new strategies to take benefit of it and to improve GC or other compounds efficacy causing minimal side effects. PMID:28220107

  11. Selective prostacyclin receptor agonism augments glucocorticoid-induced gene expression in human bronchial epithelial cells.

    PubMed

    Wilson, Sylvia M; Shen, Pamela; Rider, Christopher F; Traves, Suzanne L; Proud, David; Newton, Robert; Giembycz, Mark A

    2009-11-15

    Prostacyclin receptor (IP-receptor) agonists display anti-inflammatory and antiviral activity in cell-based assays and in preclinical models of asthma and chronic obstructive pulmonary disease. In this study, we have extended these observations by demonstrating that IP-receptor activation also can enhance the ability of glucocorticoids to induce genes with anti-inflammatory activity. BEAS-2B bronchial epithelial cells stably transfected with a glucocorticoid response element (GRE) luciferase reporter were activated in a concentration-dependent manner by the glucocorticoid dexamethasone. An IP-receptor agonist, taprostene, increased cAMP in these cells and augmented luciferase expression at all concentrations of dexamethasone examined. Analysis of the concentration-response relationship that described this effect showed that taprostene increased the magnitude of transcription without affecting the potency of dexamethasone and was, thus, steroid-sparing in this simple system. RO3244794, an IP-receptor antagonist, and oligonucleotides that selectively silenced the IP-receptor gene, PTGIR, abolished these effects of taprostene. Infection of BEAS-2B GRE reporter cells with an adenovirus vector encoding a highly selective inhibitor of cAMP-dependent protein kinase (PKA) also prevented taprostene from enhancing GRE-dependent transcription. In BEAS-2B cells and primary cultures of human airway epithelial cells, taprostene and dexamethasone interacted either additively or cooperatively in the expression of three glucocorticoid-inducible genes (GILZ, MKP-1, and p57(kip2)) that have anti-inflammatory potential. Collectively, these data show that IP-receptor agonists can augment the ability of glucocorticoids to induce anti-inflammatory genes in human airway epithelial cells by activating a cAMP/PKA-dependent mechanism. This observation may have clinical relevance in the treatment of airway inflammatory diseases that are either refractory or respond suboptimally to

  12. Potent and multiple regulatory actions of microglial glucocorticoid receptors during CNS inflammation

    PubMed Central

    Carrillo-de Sauvage, M Á; Maatouk, L; Arnoux, I; Pasco, M; Sanz Diez, A; Delahaye, M; Herrero, M T; Newman, T A; Calvo, C F; Audinat, E; Tronche, F; Vyas, S

    2013-01-01

    In CNS, glucocorticoids (GCs) activate both GC receptor (GR) and mineralocorticoid receptor (MR), whereas GR is widely expressed, the expression of MR is restricted. However, both are present in the microglia, the resident macrophages of the brain and their activation can lead to pro- or anti-inflammatory effects. We have therefore addressed the specific functions of GR in microglia. In mice lacking GR in macrophages/microglia and in the absence of modifications in MR expression, intraparenchymal injection of lipopolysaccharide (LPS) activating Toll-like receptor 4 signaling pathway resulted in exacerbated cellular lesion, neuronal and axonal damage. Global inhibition of GR by RU486 pre-treatment revealed that microglial GR is the principal mediator preventing neuronal degeneration triggered by lipopolysaccharide (LPS) and contributes with GRs of other cell types to the protection of non-neuronal cells. In vivo and in vitro data show GR functions in microglial differentiation, proliferation and motility. Interestingly, microglial GR also abolishes the LPS-induced delayed outward rectifier currents by downregulating Kv1.3 expression known to control microglia proliferation and oxygen radical production. Analysis of GR transcriptional function revealed its powerful negative control of pro-inflammatory effectors as well as upstream inflammatory activators. Finally, we analyzed the role of GR in chronic unpredictable mild stress and aging, both known to prime or sensitize microglia in vivo. We found that microglial GR suppresses rather than mediates the deleterious effects of stress or aging on neuronal survival. Overall, the results show that microglial GR acts on several key processes limiting pro-inflammatory actions of activated microglia. PMID:24013726

  13. nti glucocorticoid receptor transcripts lack sequences encoding the amino-terminal transcriptional modulatory domain.

    PubMed Central

    Dieken, E S; Meese, E U; Miesfeld, R L

    1990-01-01

    Glucocorticoid induction of cell death (apoptosis) in mouse lymphoma S49 cells has long been studied as a molecular genetic model of steroid hormone action. To better understand the transcriptional control of glucocorticoid-induced S49 cell death, we isolated and characterized glucocorticoid receptor (GR) cDNA from two steroid-resistant nti S49 mutant cell lines (S49.55R and S49.143R) and the wild-type parental line (S49.A2). Our data reveal that nti GR transcripts encode intact steroid- and DNA-binding domains but lack 404 amino-terminal residues as a result of aberrant RNA splicing between exons 1 and 3. Results from transient cotransfection experiments into CV1 cells using nti receptor expression plasmids and a glucocorticoid-responsive reporter gene demonstrated that the truncated nti receptor exhibits a reduced transcriptional regulatory activity. Gene fusions containing portions of both the wild-type and the nti GR-coding sequences were constructed and used to functionally map the nti receptor mutation. We found that the loss of the modulatory domain alone is sufficient to cause the observed defect in nti transcriptional transactivation. These results support the proposal that glucocorticoid-induced S49 cell death requires GR sequences which have previously been shown to be required for transcriptional regulation, suggesting that steroid-regulated apoptosis is controlled at the level of gene expression. Images PMID:2388618

  14. Complex genomic interactions in the dynamic regulation of transcription by the glucocorticoid receptor.

    PubMed

    Miranda, Tina B; Morris, Stephanie A; Hager, Gordon L

    2013-11-05

    The glucocorticoid receptor regulates transcriptional output through complex interactions with the genome. These events require continuous remodeling of chromatin, interactions of the glucocorticoid receptor with chaperones and other accessory factors, and recycling of the receptor by the proteasome. Therefore, the cohort of factors expressed in a particular cell type can determine the physiological outcome upon treatment with glucocorticoid hormones. In addition, circadian and ultradian cycling of hormones can also affect GR response. Here we will discuss revision of the classical static model of GR binding to response elements to incorporate recent findings from single cell and genome-wide analyses of GR regulation. We will highlight how these studies have changed our views on the dynamics of GR recruitment and its modulation of gene expression.

  15. How glucocorticoid receptors modulate the activity of other transcription factors: a scope beyond tethering.

    PubMed

    Ratman, Dariusz; Vanden Berghe, Wim; Dejager, Lien; Libert, Claude; Tavernier, Jan; Beck, Ilse M; De Bosscher, Karolien

    2013-11-05

    The activity of the glucocorticoid receptor (GR), a nuclear receptor transcription factor belonging to subclass 3C of the steroid/thyroid hormone receptor superfamily, is typically triggered by glucocorticoid hormones. Apart from driving gene transcription via binding onto glucocorticoid response elements in regulatory regions of particular target genes, GR can also inhibit gene expression via transrepression, a mechanism largely based on protein:protein interactions. Hereby GR can influence the activity of other transcription factors, without contacting DNA itself. GR is known to inhibit the activity of a growing list of immune-regulating transcription factors. Hence, GCs still rule the clinic for treatments of inflammatory disorders, notwithstanding concomitant deleterious side effects. Although patience is a virtue when it comes to deciphering the many mechanisms GR uses to influence various signaling pathways, the current review is testimony of the fact that groundbreaking mechanistic work has been accumulating over the past years and steadily continues to grow.

  16. Discovery of betamethasone 17alpha-carbamates as dissociated glucocorticoid receptor modulators in the rat.

    PubMed

    Ali, Amjad; Balkovec, James M; Greenlee, Mark; Hammond, Milton L; Rouen, Greg; Taylor, Gayle; Einstein, Monica; Ge, Lan; Harris, Georgianna; Kelly, Terri M; Mazur, Paul; Pandit, Shilpa; Santoro, Joseph; Sitlani, Ayesha; Wang, Chuanlin; Williamson, Joann; Forrest, Michael J; Carballo-Jane, Ester; Luell, Silvi; Lowitz, Karen; Visco, Denise

    2008-08-15

    A series of betamethasone 17alpha-carbamates were designed, synthesized, and evaluated for their ability to dissociate the two main functions of the glucocorticoid receptor, that is, transactivation and transrepression, in rat cell lines. A number of alkyl substituted betamethasone 17alpha-carbamates were identified with excellent affinity for the glucocorticoid receptor (e.g., 7, GR IC(50) 5.1 nM) and indicated dissociated profiles in functional assays of transactivation (rat tyrosine aminotransferase, TAT, and rat glutamine synthetase, GS) and transrepression (human A549 cells, MMP-1 assay). Gratifyingly, the in-vivo profile of these compounds, for example, 7, also indicated potent anti-inflammatory activity with impaired effects on glucose, insulin, triglycerides, and body weight. Taken together, these results indicate that dissociated glucocorticoid receptor modulators can be identified in rodents.

  17. Multiple specific binding sites for purified glucocorticoid receptors on mammary tumor virus DNA.

    PubMed

    Payvar, F; Firestone, G L; Ross, S R; Chandler, V L; Wrange, O; Carlstedt-Duke, J; Gustafsson, J A; Yamamoto, K R

    1982-01-01

    Glucocorticoid hormones selectively stimulate the rate of transcription of integrated mammary tumor virus (MTV) sequences in infected rat hepatoma cells. Using two independent assays, we find that purified rat liver glucocorticoid receptor protein binds specifically to at least four widely separated regions on pure MTV proviral DNA. One of these specific binding domains, which itself contains at least two distinct receptor binding sites, resides within a fragment of viral DNA that maps 110-449 bp upstream of the promoter for MTV RNA synthesis. Three other binding domains lie downstream of the promoter and within the MTV primary transcription unit. Restriction fragments bearing separate binding domains have been introduced into cultured cells; transformants have been recovered in which the introduced fragments are expressed under glucocorticoid control. Thus, it appears that this assay will be useful for assessing the biological significance of the receptor binding sites detected in vitro.

  18. 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.

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

    PubMed Central

    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. PMID:26528184

  20. How does stress affect human being-a molecular dynamic simulation study on cortisol and its glucocorticoid receptor.

    PubMed

    Zhang, Dan; Tian, Geng

    2017-03-01

    Stress can be either positive or negative to human beings. Under stressful conditions, the mental and physical conditions of human can be affected. There exists certain relation between stress and illness. The cortisol and other glucocorticoids bind to the same receptor, which is called glucocorticoid receptor. Some evidences indicated that cortisol molecule binding to its glucocorticoid receptor was necessary for the stress response. Up to now, the structure-function relationships between cortisol molecule and its glucocorticoid receptor have not been deliberated from the atomic-level. In order to get a detailed understanding of the structure-function relationships between the cortisol molecule and glucocorticoids receptor, we have carried out molecular dynamic (MD) simulations on glucocorticoid receptor (Apo system) and cortisol with its glucocorticoid receptor complex (HCY system). On the basis of molecular dynamic simulations, a couple of key residues were identified, which were crucial for the binding of cortisol molecule. The results of binding free energy calculations are in good agreement with the experiment data. Our research gives clear insights from atomic-level into the structural-functional aspects of cortisol molecule and its glucocorticoid receptor, and also provides valuable information for the design of drug which can treat stress related illnesses.

  1. Purified glucocorticoid receptors bind selectively in vitro to a cloned DNA fragment that mediates a delayed secondary response to glucocorticoids in vivo.

    PubMed

    Hess, P; Meenakshi, T; Chan, G C; Carlstedt-Duke, J; Gustafsson, J A; Payvar, F

    1990-04-01

    We have identified and characterized a 206-base-pair region downstream from rat alpha 2u-globulin promoter that specifically mediates a delayed secondary response to glucocorticoids. Unlike positive primary glucocorticoid response elements (GREs), this regulatory element, termed delayed sGRE, dictates an inductive process preceded by a time lag of several hours and blocked by the protein synthesis inhibitor cycloheximide. Reminiscent of GREs and negative GREs (nGREs), a delayed sGRE confers hormonal regulation upon a linked heterologous promoter from a downstream position with respect to transcription start site and, remarkably, also interacts selectively with purified glucocorticoid receptor. These results imply that receptor binding to a delayed sGRE in vivo may mediate certain secondary responses to glucocorticoid hormones.

  2. Purified glucocorticoid receptors bind selectively in vitro to a cloned DNA fragment that mediates a delayed secondary response to glucocorticoids in vivo.

    PubMed Central

    Hess, P; Meenakshi, T; Chan, G C; Carlstedt-Duke, J; Gustafsson, J A; Payvar, F

    1990-01-01

    We have identified and characterized a 206-base-pair region downstream from rat alpha 2u-globulin promoter that specifically mediates a delayed secondary response to glucocorticoids. Unlike positive primary glucocorticoid response elements (GREs), this regulatory element, termed delayed sGRE, dictates an inductive process preceded by a time lag of several hours and blocked by the protein synthesis inhibitor cycloheximide. Reminiscent of GREs and negative GREs (nGREs), a delayed sGRE confers hormonal regulation upon a linked heterologous promoter from a downstream position with respect to transcription start site and, remarkably, also interacts selectively with purified glucocorticoid receptor. These results imply that receptor binding to a delayed sGRE in vivo may mediate certain secondary responses to glucocorticoid hormones. Images PMID:1690888

  3. 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.

  4. Glucocorticoid receptor activation impairs hippocampal plasticity by suppressing BDNF expression in obese mice

    PubMed Central

    Wosiski-Kuhn, Marlena; Erion, Joanna R.; Gomez-Sanchez, Elise P.; Gomez-Sanchez, Celso E.; Stranahan, Alexis M.

    2015-01-01

    Diabetes and obesity are associated with perturbation of adrenal steroid hormones and impairment of hippocampal plasticity, but the question of whether these conditions recruit glucocorticoid-mediated molecular cascades that are comparable to other stressors has yet to be fully addressed. We have used a genetic mouse model of obesity and diabetes with chronically elevated glucocorticoids to determine the mechanism for glucocorticoid-induced deficits in hippocampal synaptic function. Pharmacological inhibition of adrenal steroidogenesis attenuates structural and functional impairments by regulating plasticity among dendritic spines in the hippocampus of leptin receptor deficient (db/db) mice. Synaptic deficits evoked by exposure to elevated corticosterone levels in db/db mice are attributable to glucocorticoid receptor-mediated transrepression of AP-1 actions at BDNF promoters I and IV. db/db mice exhibit corticosterone-mediated reductions in brain-derived neurotrophic factor (BDNF), and a change in the ratio of TrkB to P75NTR that silences the functional response to BDNF stimulation. Lentiviral suppression of glucocorticoid receptor expression rescues behavioral and synaptic function in db/db mice, and also reinstates BDNF expression, underscoring the relevance of molecular mechanisms previously demonstrated after psychological stress to the functional alterations observed in obesity and diabetes. PMID:24636513

  5. Contribution of glucocorticoid-mineralocorticoid receptor pathway on the obesity-related adipocyte dysfunction.

    PubMed

    Hirata, Ayumu; Maeda, Norikazu; Nakatsuji, Hideaki; Hiuge-Shimizu, Aki; Okada, Takuya; Funahashi, Tohru; Shimomura, Iichiro

    2012-03-09

    Mineralocorticoid receptor (MR) blockade ameliorated insulin resistance with improvements in adipocytokine dysregulation, inflammation, and excess of reactive oxygen species (ROS) in obese adipose tissue and adipocytes, but its mechanism has not been clarified. The 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), producing active glucocorticoids, is highly expressed in adipocytes and glucocorticoids bind to MR with higher affinity than to glucocorticoid receptor (GR). We investigated whether glucocorticoids effect on adipocytokines and ROS through MR in adipocytes. In addition, fat distributions of MR and GR were investigated in human subjects. Corticoid receptors and their target genes were examined in adipose tissue of obese db/db mice. 3T3-L1 adipocytes were treated with glucocorticoids, H(2)O(2), MR antagonist eplerenone (EP), GR antagonist RU486 (RU), MR-siRNA, and/or N-acetylcysteine. Human adipose tissues were obtained from seven patients who underwent abdominal surgery. The mRNA levels of MR and its target gene were higher in db/db mice than in control db/m+mice. In 3T3-L1 adipocytes, glucocorticoids, similar to H(2)O(2), caused the dysregulation of mRNA levels of various genes related to adipocytokines and the increase of intracellular ROS. Such changes were rectified by MR blockade, not by GR antagonist. In human fat, MR mRNA level was increased in parallel with the increase of body mass index (BMI) and its increase was more significant in visceral fat, while there were no apparent correlations of GR mRNA level to BMI or fat distribution. Glucocorticoid-MR pathway may contribute to the obesity-related adipocytokine dysregulation and adipose ROS. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. 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

  7. 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.

  8. 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

  9. SIRT1 is a transcriptional enhancer of the glucocorticoid receptor acting independently to its deacetylase activity.

    PubMed

    Suzuki, Shigeru; Iben, James R; Coon, Steven L; Kino, Tomoshige

    2017-09-18

    Glucocorticoids have strong effects on diverse human activities through the glucocorticoid receptor (GR). Sirtuin 1 (SIRT1) is a NAD(+)-dependent histone deacetylase and promotes longevity by influencing intermediary metabolism and other regulatory activities including mitochondrial function. In this study, we examined the effects of SIRT1 on GR-mediated transcriptional activity. We found that SIRT1 enhanced GR-induced transcriptional activity on endogenous and exogenous glucocorticoid-responsive genes, whereas knockdown of SIRT1 attenuated it. This effect of SIRT1 was independent to its deacetylase activity, as the SIRT1 mutant defective in this activity (H363Y) enhanced GR transcriptional activity, and the compounds inhibiting or activating the SIRT1 deacetylase activity did not influence it. RNA-seq analysis revealed that SIRT1 knockdown influenced ∼30% of the glucocorticoid-responsive transcriptome for most of which it acted as an enhancer for positive/negative effects of this hormone. SIRT1 physically interacted with GR, and was attracted to GR-bound glucocorticoid response elements in a glucocorticoid-dependent fashion. SIRT1 cooperatively activated GR transcriptional activity with the PPARγ coactivator-1α also in its deacetylase activity-independent fashion. Thus, SIRT1 is a novel transcriptional enhancer of GR-induced transcriptional activity possibly by functioning as a scaffold for the transcriptional complex formed on GR. Published by Elsevier B.V.

  10. Peripheral CLOCK Regulates Target-Tissue Glucocorticoid Receptor Transcriptional Activity in a Circadian Fashion in Man

    PubMed Central

    Charmandari, Evangelia; Chrousos, George P.; Lambrou, George I.; Pavlaki, Aikaterini; Koide, Hisashi; Ng, Sinnie Sin Man; Kino, Tomoshige

    2011-01-01

    Context and Objective Circulating cortisol fluctuates diurnally under the control of the “master” circadian CLOCK, while the peripheral “slave” counterpart of the latter regulates the transcriptional activity of the glucocorticoid receptor (GR) at local glucocorticoid target tissues through acetylation. In this manuscript, we studied the effect of CLOCK-mediated GR acetylation on the sensitivity of peripheral tissues to glucocorticoids in humans. Design and Participants We examined GR acetylation and mRNA expression of GR, CLOCK-related and glucocorticoid-responsive genes in peripheral blood mononuclear cells (PBMCs) obtained at 8 am and 8 pm from 10 healthy subjects, as well as in PBMCs obtained in the morning and cultured for 24 hours with exposure to 3-hour hydrocortisone pulses every 6 hours. We used EBV-transformed lymphocytes (EBVLs) as non-synchronized controls. Results GR acetylation was higher in the morning than in the evening in PBMCs, mirroring the fluctuations of circulating cortisol in reverse phase. All known glucocorticoid-responsive genes tested responded as expected to hydrocortisone in non-synchronized EBVLs, however, some of these genes did not show the expected diurnal mRNA fluctuations in PBMCs in vivo. Instead, their mRNA oscillated in a Clock- and a GR acetylation-dependent fashion in naturally synchronized PBMCs cultured ex vivo in the absence of the endogenous glucocorticoid, suggesting that circulating cortisol might prevent circadian GR acetylation-dependent effects in some glucocorticoid-responsive genes in vivo. Conclusions Peripheral CLOCK-mediated circadian acetylation of the human GR may function as a target-tissue, gene-specific counter regulatory mechanism to the actions of diurnally fluctuating cortisol, effectively decreasing tissue sensitivity to glucocorticoids in the morning and increasing it at night. PMID:21980503

  11. Expression and nuclear translocation of glucocorticoid receptors in type 2 taste receptor cells.

    PubMed

    Parker, M Rockwell; Feng, Dianna; Chamuris, Brianna; Margolskee, Robert F

    2014-06-13

    Stress increases the secretion of glucocorticoids (GCs), potent steroid hormones that exert their effects on numerous target tissues by acting through glucocorticoid receptors (GRs). GC signaling significantly affects ingestive behavior and taste preferences in humans and rodent models, but far less is known about the hormonal modulation of the peripheral sensory system that detects and assesses nutrient content of foods. A previous study linked restraint stress in rats to diminished expression of mRNA for one subunit of the sweet taste receptor (Tas1r3) in taste tissue and reduced gustatory nerve excitation by sweet compounds. Using RT-PCR, we detected mRNAs for GRα in circumvallate taste papillae and in oral epithelium devoid of taste buds ("non-taste" tissue). Further, circumvallate tissue was significantly enriched in GR mRNA compared to non-taste tissue based on quantitative PCR. Histologically, GR protein was expressed in all taste bud populations examined (circumvallate, foliate and fungiform papillae). Using transgenic mice expressing green fluorescent protein, almost all (97%) Tas1r3-positive taste cells (sweet-/umami-sensitive) expressed GR compared to a significantly smaller percentage (89%) of TrpM5-positive taste cells (sweet-, umami- and bitter-sensitive). When mice (n=4) were restrain stressed, GR protein mobilized to the nucleus in Tas1r3-GFP taste cells (1.7-fold over controls). Our results suggest that GR can be activated in taste receptor cells and may play a role in specific taste qualities (e.g., sweet, umami, and bitter) to shape how the taste system responds to stress. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  12. 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…

  13. Human receptor kinetics and lung tissue retention of the enhanced-affinity glucocorticoid fluticasone furoate

    PubMed Central

    Valotis, Anagnostis; Högger, Petra

    2007-01-01

    Fluticasone furoate (FF) – USAN approved name, a new topically active glucocorticoid has been recently identified. The aim of this study was to characterise the binding affinity of this compound to the human lung glucocorticoid receptor in relation to other glucocorticoids. Additionally, we sought to determine the binding behaviour of fluticasone furoate to human lung tissue. The glucocorticoid receptor binding kinetics of fluticasone furoate revealed a remarkably fast association and a slow dissociation resulting in a relative receptor affinity (RRA) of 2989 ± 135 with reference to dexamethasone (RRA: 100 ± 5). Thus, the RRA of FF exceeds the RRAs of all currently clinically used corticosteroids such as mometasone furoate (MF; RRA 2244), fluticasone propionate (FP; RRA 1775), ciclesonide's active metabolite (RRA 1212 – rat receptor data) or budesonide (RRA 855). FP and FF displayed pronounced retention in human lung tissue in vitro. Lowest tissue binding was found for MF. There was no indication of instability or chemical modification of FF in human lung tissue. These advantageous binding attributes may contribute to a highly efficacious profile for FF as a topical treatment for inflammatory disorders of the respiratory tract. PMID:17650349

  14. 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.

  15. Tetrahydroquinolin-3-yl carbamate glucocorticoid receptor agonists with reduced PEPCK activation.

    PubMed

    Roach, Steven L; Higuchi, Robert I; Hudson, Andrew R; Vassar, Angie; Grant, Virginia H S; Lamer, Ryan; Hooper, Charlene; Rungta, Deepa; Syka, Peter M; Mais, Dale E; Marschke, Keith B; Zhi, Lin

    2011-03-15

    Continuing studies on tetrahydroquinoline glucocorticoid receptor anti-inflammatory agents lead to the identification of several tetrahydroquinolin-3-yl carbamates that exhibited steroid-like activity in in vitro transrepression assays with reduced transactivation of phosphoenol pyruvate carboxykinase (PEPCK), a key enzyme in the gluconeogenesis pathway. Copyright © 2011. Published by Elsevier Ltd.

  16. 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.

  17. 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…

  18. More than meets the dimer: What is the quaternary structure of the glucocorticoid receptor?

    PubMed Central

    Hager, Gordon L.

    2017-01-01

    ABSTRACT It is widely accepted that the glucocorticoid receptor (GR), a ligand-regulated transcription factor that triggers anti-inflammatory responses, binds specific response elements as a homodimer. Here, we will discuss the original primary data that established this model and contrast it with a recent report characterizing the GR–DNA complex as a tetramer. PMID:27764575

  19. Glucocorticoid receptor-mediated cis-repression of osteogenic genes requires BRM-SWI/SNF.

    PubMed

    Pico, Michael J; Hashemi, Sharareh; Xu, Fuhua; Nguyen, Kevin Hong; Donnelly, Robert; Moran, Elizabeth; Flowers, Stephen

    2016-12-01

    Glucocorticoids are an effective therapy for a variety of severe inflammatory and autoimmune disorders; however, the therapeutic use of glucocorticoids is severely limited by their negative side effects, particularly on osteogenesis. Glucocorticoids regulate transcription by binding to the glucocorticoid receptor (GR), which then binds the promoters of target genes to induce either activation or repression. The gene activation effects of nuclear hormone receptors broadly require the cooperation of the chromatin remodeling complex known as SWI/SNF, which is powered by an ATPase core. The well-studied SWI/SNF ATPase, BRG1, is required for gene activation by a spectrum of nuclear hormone receptors including GR. However, glucocorticoid-induced side effects specifically related to impaired osteogenesis are mostly linked with GR-mediated repression. We have considered whether cis-repression of osteogenic genes by GR may be mediated by a distinct subclass of SWI/SNF powered by the alternative ATPase, BRM. BRM does not have an essential role in mammalian development, but plays a repressor role in osteoblast differentiation and favors adipogenic lineage selection over osteoblast commitment, effects that mirror the repressor effects of GR. The studies reported here examine three key GR cis-repression gene targets, and show that GR association with these promoters is sharply reduced in BRM deficient cells. Each of these GR-targeted genes act in a different way. Bglap encodes osteocalcin, which contributes to normal maturation of osteoblasts from committed pre-osteoblasts. The Per3 gene product acts in uncommitted mesenchymal stem cells to influence the osteoblast/adipocyte lineage selection point. Fas ligand, encoded by FasL, is a means by which osteoblasts can modulate bone degradation by osteoclasts. Repression of each of these genes by glucocorticoid favors bone loss. The essential role of BRM in cooperation with GR at each of these control points offers a novel

  20. Angiopoietin-like 4 (ANGPTL4, fasting-induced adipose factor) is a direct glucocorticoid receptor target and participates in glucocorticoid-regulated triglyceride metabolism.

    PubMed

    Koliwad, Suneil K; Kuo, Taiyi; Shipp, Lauren E; Gray, Nora E; Backhed, Fredrik; So, Alex Yick-Lun; Farese, Robert V; Wang, Jen-Chywan

    2009-09-18

    Glucocorticoids are important regulators of lipid homeostasis, and chronically elevated glucocorticoid levels induce hypertriglyceridemia, hepatic steatosis, and visceral obesity. The occupied glucocorticoid receptor (GR) is a transcription factor. However, those genes regulating lipid metabolism under GR control are not fully known. Angiopoietin-like 4 (ANGPTL4, fasting-induced adipose factor), a protein inhibitor of lipoprotein lipase, is synthesized and secreted during fasting, when circulating glucocorticoid levels are physiologically increased. We therefore tested whether the ANGPTL4 gene (Angptl4) is transcriptionally controlled by GR. We show that treatment with the synthetic glucocorticoid dexamethasone increased Angptl4 mRNA levels in primary hepatocytes and adipocytes (2-3-fold) and in the livers and white adipose tissue of mice (approximately 4-fold). We tested the mechanism of this increase in H4IIE hepatoma cells and found that dexamethasone treatment increased the transcriptional rate of Angptl4. Using bioinformatics and chromatin immunoprecipitation, we identified a GR binding site within the rat Angptl4 sequence. A reporter plasmid containing this site was markedly activated by dexamethasone, indicative of a functional glucocorticoid response element. Dexamethasone treatment also increased histone H4 acetylation and DNase I accessibility in genomic regions near this site, further supporting that it is a glucocorticoid response element. Glucocorticoids promote the flux of triglycerides from white adipose tissue to liver. We found that mice lacking ANGPTL4 (Angptl4(-/-)) had reductions in dexamethasone-induced hypertriglyceridemia and hepatic steatosis, suggesting that ANGPTL4 is required for this flux. Overall, we establish that ANGPTL4 is a direct GR target that participates in glucocorticoid-regulated triglyceride metabolism.

  1. Gene expression of peripheral blood cells reveals pathways downstream of glucocorticoid receptor antagonism and nab-paclitaxel treatment

    PubMed Central

    Maranville, Joseph C; Nanda, Rita; Fleming, Gini F; Skor, Maxwell N; Di Rienzo, Anna; Conzen, Suzanne D

    2014-01-01

    Objectives While paclitaxel treatment is associated with leukopenia, the mechanisms that underlie this effect are not well-characterized. Additionally, despite the importance of glucocorticoid signaling in cancer treatment, the genomic effects of glucocorticoid receptor (GR) antagonism by mifepristone treatment in primary human cells have never been described. Methods As part of a randomized Phase 1 clinical trial, we used microarrays to profile gene expression in peripheral blood cells sampled from each of 4 patients at baseline, after placebo/nab-paclitaxel treatment (cycle 1), and after mifepristone/nab-paclitaxel treatment (cycle 2). Results We found that 63 genes were differentially-expressed following treatment with nab-paclitaxel, including multiple genes in the tubulin pathway. We also found 606 genes that were differentially expressed in response to mifepristone; genes down-regulated by mifepristone overlapped significantly with those previously identified as being up-regulated by dexamethasone. Conclusions These results provide insights into the mechanisms of paclitaxel and GR inhibition in peripheral blood cells. PMID:25000515

  2. Covalent affinity labeling, radioautography, and immunocytochemistry localize the glucocorticoid receptor in rat testicular Leydig cells

    SciTech Connect

    Stalker, A.; Hermo, L.; Antakly, T. )

    1989-12-01

    The presence and distribution of glucocorticoid receptors in the rat testis were examined by using 2 approaches: in vivo quantitative radioautography and immunocytochemistry. Radioautographic localization was made possible through the availability of a glucocorticoid receptor affinity label, dexamethasone 21-mesylate, which binds covalently to the glucocorticoid receptor, thereby preventing dissociation of the steroid-receptor complex. Adrenalectomized adult rats were injected with a tritiated (3H) form of this steroid into the testis and the tissue was processed for light-microscope radioautography. Silver grains were observed primarily over the Leydig cells of the interstitial space and to a lesser extent, over the cellular layers which make up the seminiferous epithelium, with no one cell type showing preferential labeling. To determine the specificity of the labeling, a 25- or 50-fold excess of unlabeled dexamethasone was injected simultaneously with the same dose of (3H)-dexamethasone 21-mesylate. In these control experiments, a marked reduction in label intensity was noted over the Leydig as well as tubular cells. Endocytic macrophages of the interstitium were non-specifically labeled, indicating uptake of the ligand possibly by fluid-phase endocytosis. A quantitative analysis of the label confirmed the presence of statistically significant numbers of specific binding sites for glucocorticoids in both Leydig cells and the cellular layers of the seminiferous epithelium; 86% of the label was found over Leydig cells, and only 14% over the cells of the seminiferous epithelium. These binding data were confirmed by light-microscope immunocytochemistry using a monoclonal antibody to the glucocorticoid receptor.

  3. Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling.

    PubMed

    Kuo, Taiyi; Lew, Michelle J; Mayba, Oleg; Harris, Charles A; Speed, Terence P; Wang, Jen-Chywan

    2012-07-10

    Glucocorticoids elicit a variety of biological responses in skeletal muscle, including inhibiting protein synthesis and insulin-stimulated glucose uptake and promoting proteolysis. Thus, excess or chronic glucocorticoid exposure leads to muscle atrophy and insulin resistance. Glucocorticoids propagate their signal mainly through glucocorticoid receptors (GR), which, upon binding to ligands, translocate to the nucleus and bind to genomic glucocorticoid response elements to regulate the transcription of nearby genes. Using a combination of chromatin immunoprecipitation sequencing and microarray analysis, we identified 173 genes in mouse C2C12 myotubes. The mouse genome contains GR-binding regions in or near these genes, and gene expression is regulated by glucocorticoids. Eight of these genes encode proteins known to regulate distinct signaling events in insulin/insulin-like growth factor 1 pathways. We found that overexpression of p85α, one of these eight genes, caused a decrease in C2C12 myotube diameters, mimicking the effect of glucocorticoids. Moreover, reducing p85α expression by RNA interference in C2C12 myotubes significantly compromised the ability of glucocorticoids to inhibit Akt and p70 S6 kinase activity and reduced glucocorticoid induction of insulin receptor substrate 1 phosphorylation at serine 307. This phosphorylation is associated with insulin resistance. Furthermore, decreasing p85α expression abolished glucocorticoid inhibition of protein synthesis and compromised glucocorticoid-induced reduction of cell diameters in C2C12 myotubes. Finally, a glucocorticoid response element was identified in the p85α GR-binding regions. In summary, our studies identified GR-regulated transcriptional networks in myotubes and showed that p85α plays a critical role in glucocorticoid-induced insulin resistance and muscle atrophy in C2C12 myotubes.

  4. Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling

    PubMed Central

    Kuo, Taiyi; Lew, Michelle J.; Mayba, Oleg; Harris, Charles A.; Speed, Terence P.; Wang, Jen-Chywan

    2012-01-01

    Glucocorticoids elicit a variety of biological responses in skeletal muscle, including inhibiting protein synthesis and insulin-stimulated glucose uptake and promoting proteolysis. Thus, excess or chronic glucocorticoid exposure leads to muscle atrophy and insulin resistance. Glucocorticoids propagate their signal mainly through glucocorticoid receptors (GR), which, upon binding to ligands, translocate to the nucleus and bind to genomic glucocorticoid response elements to regulate the transcription of nearby genes. Using a combination of chromatin immunoprecipitation sequencing and microarray analysis, we identified 173 genes in mouse C2C12 myotubes. The mouse genome contains GR-binding regions in or near these genes, and gene expression is regulated by glucocorticoids. Eight of these genes encode proteins known to regulate distinct signaling events in insulin/insulin-like growth factor 1 pathways. We found that overexpression of p85α, one of these eight genes, caused a decrease in C2C12 myotube diameters, mimicking the effect of glucocorticoids. Moreover, reducing p85α expression by RNA interference in C2C12 myotubes significantly compromised the ability of glucocorticoids to inhibit Akt and p70 S6 kinase activity and reduced glucocorticoid induction of insulin receptor substrate 1 phosphorylation at serine 307. This phosphorylation is associated with insulin resistance. Furthermore, decreasing p85α expression abolished glucocorticoid inhibition of protein synthesis and compromised glucocorticoid-induced reduction of cell diameters in C2C12 myotubes. Finally, a glucocorticoid response element was identified in the p85α GR-binding regions. In summary, our studies identified GR-regulated transcriptional networks in myotubes and showed that p85α plays a critical role in glucocorticoid-induced insulin resistance and muscle atrophy in C2C12 myotubes. PMID:22733784

  5. Transformation of glucocorticoid receptors bound to the antagonist RU 486: Effects of alkaline phosphatase

    SciTech Connect

    Gruol, D.J.; Wolfe, K.A. )

    1990-08-28

    RU 486 is a synthetic steroid that binds avidly to glucocorticoid receptors without promoting their transformation into activated transcription factors. A significant part of this behavior has been shown to be due to a failure of the RU 486 bound receptor to be efficiently released from a larger (sedimenting at 8-9 S) multimeric complex containing the 90-kDa heat shock protein. The studies have found that in vitro at 15{degree}C the RU 486-receptor was slowly released from the 8-9S complex and converted into a DNA binding protein by a process that could be blocked by sodium fluoride. Moreover, this transition was significantly accelerated by treatment with alkaline phosphatase. High-resolution anion-exchange chromatography showed that the profile of receptor subspecies released from the 8-9S complex was different for the RU 486 bound receptor when compared to the receptor occupied by the agonist triamcinolone acetonide. Production of the earliest eluting receptor form (peak A) was inhibited with RU 486. Treatment of the Ru 486-receptor with alkaline phosphatase increased the formation of the peak A subspecies as well as the capacity of receptor to bind DNA-cellulose. Taken together, the results indicate that phosphorylation of the receptor or a tightly bound factor contributes to defining the capacity with which individual steroids can promote dissociation of the 8-9S complex and conversion of the glucocorticoid receptor into a DNA-binding protein.

  6. Folding and stability of the ligand-binding domain of the glucocorticoid receptor

    PubMed Central

    McLaughlin, Stephen H.; Jackson, Sophie E.

    2002-01-01

    A complex pathway involving many molecular chaperones has been proposed for the folding, assembly, and maintenance of a high-affinity ligand-binding form of steroid receptors in vivo, including the glucocorticoid receptor. To better understand this intricate folding and assembly process, we studied the folding of the ligand-binding domain of the glucocorticoid receptor in vitro. We found that this domain can be refolded into a compact, highly structured state in vitro in the absence of chaperones. However, the presence of zwitterionic detergent is required to maintain the domain in a soluble form. In this state, the protein is dimeric and has considerable helical structure as shown by far-UV circular dichroism. Further investigation of the properties of this in vitro refolded state show that it is stable and resistant to denaturation by heat or low concentrations of chemical denaturants. A detailed analysis of the unfolding equilibria using three different structural probes demonstrated that this state unfolds via a highly populated dimeric intermediate state. Together, these data clearly show that the ligand-binding domain of the glucocorticoid receptor does not require chaperones for folding per se. However, this in vitro refolded state binds the ligand dexamethasone only weakly (Kd = 45 μM) compared to the in vivo assembled receptor (Kd = 3.4 nM). We suggest that the role of Hsp90 and associated chaperones is to bind to, and stabilize, a specific conformational state of the receptor which binds ligand with high affinity. PMID:12142447

  7. Effects of Maternal Dexamethasone Treatment Early in Pregnancy on Glucocorticoid Receptors in the Ovine Placenta

    PubMed Central

    Shang, H.; Meng, W.; Sloboda, D. M.; Li, S.; Ehrlich, L.; Plagemann, A.; Dudenhausen, J. W.; Henrich, W.; Newnham, J. P.; Challis, J. R. G.

    2015-01-01

    The effects of endogenous cortisol on binucleate cells (BNCs), which promote fetal growth, may be mediated by glucocorticoid receptors (GRs), and exposure to dexamethasone (DEX) in early pregnancy stages of placental development might modify this response. In this article, we have investigated the expression of GR as a determinant of these responses. Pregnant ewes carrying singleton fetuses (n = 119) were randomized to control (2 mL saline/ewe) or DEX-treated groups (intramuscular injections of 0.14 mg/kg ewe weight per 12 hours) at 40 to 41 days of gestation (dG). Placental tissue was collected at 50, 100, 125, and 140 dG. Total glucocorticoid receptor protein (GRt) was increased significantly by DEX at 50 and 125 dG in females only, but decreased in males at 125 dG as compared to controls. Glucocorticoid receptor α (GRα) protein was not changed after DEX treatment. Three BNC phenotypes were detected regarding GRα expression (++, +−, −−), DEX increased the proportion of (++) and decreased (−−) BNC at 140 dG. Effects were sex- and cell type dependent, modifying the responsiveness of the placenta to endogenous cortisol. We speculate that 3 maturational stages of BNCs exist and that the overall activity of BNCs is determined by the distribution of these 3 cell types, which may become altered through early pregnancy exposure to elevated glucocorticoids. PMID:25332218

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

    PubMed Central

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

    2016-01-01

    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. Glucocorticoid receptor monoclonal antibodies define the biological action of RU 38486 in intact B16 melanoma cells.

    PubMed

    Lindemeyer, R G; Robertson, N M; Litwack, G

    1990-12-15

    The mechanism of action of the synthetic glucocorticoid antagonist, RU 38486, has yet to be completely elucidated. Although RU 38486 is a potent antiglucocorticoid in vivo, several studies have indicated that it has some agonist activities in vitro, such as high-affinity steroid binding to the receptor, activation, and DNA binding. Nevertheless, these in vitro postbinding events do not lead to any known gene expression. To understand the action of the glucocorticoid antagonist RU 38486, we studied glucocorticoid receptor localization on a mouse melanoma cell line (B16C3) by indirect immunofluorescent staining techniques, using monoclonal antibodies to the glucocorticoid receptor. Our data in intact cells suggest that, unlike glucocorticoid agonists such as triamcinolone acetonide, and similar to the glucocorticoid antagonist cortexolone, RU 38486-bound receptors do not translocate to the nucleus and hence do not allow for transcription of glucocorticoid-regulated genes to occur. Passage through the nuclear membrane may be a rate-limiting step in the action of glucocorticoid antagonists, and translocation may in itself be an important regulatory mechanism of steroid hormone action.

  10. Identification of highly efficacious glucocorticoid receptor agonists with a potential for reduced clinical bone side effects.

    PubMed

    Harcken, Christian; Riether, Doris; Kuzmich, Daniel; Liu, Pingrong; Betageri, Raj; Ralph, Mark; Emmanuel, Michel; Reeves, Jonathan T; Berry, Angela; Souza, Donald; Nelson, Richard M; Kukulka, Alison; Fadra, Tazmeen N; Zuvela-Jelaska, Ljiljana; Dinallo, Roger; Bentzien, Jörg; Nabozny, Gerald H; Thomson, David S

    2014-02-27

    Synthesis and structure-activity relationship (SAR) of a series of nonsteroidal glucocorticoid receptor (GR) agonists are described. These compounds contain "diazaindole" moieties and display different transcriptional regulatory profiles in vitro and are considered "dissociated" between gene transrepression and transactivation. The lead optimization effort described in this article focused in particular on limiting the transactivation of genes which result in bone side effects and these were assessed in vitro in MG-63 osteosarcoma cells, leading to the identification of (R)-18 and (R)-21. These compounds maintained anti-inflammatory activity in vivo in collagen induced arthritis studies in mouse but had reduced effects on bone relevant parameters compared to the widely used synthetic glucocorticoid prednisolone 2 in vivo. To our knowledge, we are the first to report on selective glucocorticoid ligands with reduced bone loss in a preclinical in vivo model.

  11. Determinants of cell- and gene-specific transcriptional regulation by the glucocorticoid receptor.

    PubMed

    So, Alex Yick-Lun; Chaivorapol, Christina; Bolton, Eric C; Li, Hao; Yamamoto, Keith R

    2007-06-01

    The glucocorticoid receptor (GR) associates with glucocorticoid response elements (GREs) and regulates selective gene transcription in a cell-specific manner. Native GREs are typically thought to be composite elements that recruit GR as well as other regulatory factors into functional complexes. We assessed whether GR occupancy is commonly a limiting determinant of GRE function as well as the extent to which core GR binding sequences and GRE architecture are conserved at functional loci. We surveyed 100-kb regions surrounding each of 548 known or potentially glucocorticoid-responsive genes in A549 human lung cells for GR-occupied GREs. We found that GR was bound in A549 cells predominately near genes responsive to glucocorticoids in those cells and not at genes regulated by GR in other cells. The GREs were positionally conserved at each responsive gene but across the set of responsive genes were distributed equally upstream and downstream of the transcription start sites, with 63% of them >10 kb from those sites. Strikingly, although the core GR binding sequences across the set of GREs varied extensively around a consensus, the precise sequence at an individual GRE was conserved across four mammalian species. Similarly, sequences flanking the core GR binding sites also varied among GREs but were conserved at individual GREs. We conclude that GR occupancy is a primary determinant of glucocorticoid responsiveness in A549 cells and that core GR binding sequences as well as GRE architecture likely harbor gene-specific regulatory information.

  12. Determinants of Cell- and Gene-Specific Transcriptional Regulation by the Glucocorticoid Receptor

    PubMed Central

    So, Alex Yick-Lun; Chaivorapol, Christina; Bolton, Eric C; Li, Hao; Yamamoto, Keith R

    2007-01-01

    The glucocorticoid receptor (GR) associates with glucocorticoid response elements (GREs) and regulates selective gene transcription in a cell-specific manner. Native GREs are typically thought to be composite elements that recruit GR as well as other regulatory factors into functional complexes. We assessed whether GR occupancy is commonly a limiting determinant of GRE function as well as the extent to which core GR binding sequences and GRE architecture are conserved at functional loci. We surveyed 100-kb regions surrounding each of 548 known or potentially glucocorticoid-responsive genes in A549 human lung cells for GR-occupied GREs. We found that GR was bound in A549 cells predominately near genes responsive to glucocorticoids in those cells and not at genes regulated by GR in other cells. The GREs were positionally conserved at each responsive gene but across the set of responsive genes were distributed equally upstream and downstream of the transcription start sites, with 63% of them >10 kb from those sites. Strikingly, although the core GR binding sequences across the set of GREs varied extensively around a consensus, the precise sequence at an individual GRE was conserved across four mammalian species. Similarly, sequences flanking the core GR binding sites also varied among GREs but were conserved at individual GREs. We conclude that GR occupancy is a primary determinant of glucocorticoid responsiveness in A549 cells and that core GR binding sequences as well as GRE architecture likely harbor gene-specific regulatory information. PMID:17559307

  13. Endothelin-1 downregulates Mas receptor expression in human cardiomyocytes.

    PubMed

    Chen, Zhiheng; Tang, Yamei; Yang, Zuocheng; Liu, Shaojun; Liu, Yong; Li, Yan; He, Wei

    2013-09-01

    Endothelin-1 (ET-1) and the renin-angiotensin system (RAS) are involved in the pathogenesis of cardiac dysfunction. The Mas receptor is a functional binding site for angiotensin (Ang)‑(1-7), which is now considered a critical component of the RAS and exerts cardioprotective effects. To the best of our knowledge, the present study aimed to examine, for the first time, the effects of ET-1 on Mas expression in cultured human cardiomyocytes. Human cardiomyocytes were treated with ET-1 at different concentrations (1, 5, 10, 20 and 30 nM) for varied time periods (0.5, 1.5, 3, 4.5 or 6 h) with or without the transcription inhibitor actinomycin D, endothelin A (ETA) receptor blocker BQ123 and ETB receptor blocker BQ788, or different kinase inhibitors. ET-1 decreased the Mas mRNA level in a statistically significant dose- and time-dependent manner within 4.5 h, which was reflected in the dose-dependent downregulation of Mas promoter activity, Mas protein levels and Ang-(1-7) binding on the cell membrane. Actinomycin D (1 mg/ml), BQ123 (1 µM), p38 mitogen-activated protein kinase (MAPK) siRNA and inhibitor PD169316 (25 µM), completely eliminated the inhibitory effects of ET-1 on Mas expression in human cardiomyocytes. In conclusion, the present study demonstrated that ET-1 downregulates Mas expression at the transcription level in human cardiomyocytes via the ETA receptor by a p38 MAPK‑dependent mechanism. This study provides novel insights into the function of ET-1 and the Ang‑(1-7)/Mas axis in cardiac pathophysiology.

  14. Glucocorticoids promote hepatic cholestasis in mice by inhibiting the transcriptional activity of the farnesoid X receptor.

    PubMed

    Lu, Yan; Zhang, Zhijian; Xiong, Xuelian; Wang, Xiaolin; Li, Jin; Shi, Guojun; Yang, Jian; Zhang, Xianfeng; Zhang, Huijie; Hong, Jie; Xia, Xuefeng; Ning, Guang; Li, Xiaoying

    2012-12-01

    Glucocorticoids have potent anti-inflammatory effects, but also can cause insulin resistance, osteoporosis, and muscle wasting, preventing their long-term use. Glucocorticoids also have been associated with the development of hepatic cholestasis and gallstone disease, but little is known about their pathogenic mechanisms. We analyzed levels of bile acids (BAs) and glucocorticoids in serum samples from patients with Cushing disease and obese individuals (body mass index, >30). C57BL/6 mice were injected with dexamethasone and db/db obese mice were injected with glucocorticoid receptor (GR) antagonists or small hairpin RNAs. We analyzed farnesoid X receptor (FXR) signaling in HepG2 cells and cells from mice using immunoprecipitation, luciferase reporter, and glutathione-s-transferase and chromatin immunoprecipitation assays. We analyzed BA metabolism in FXR-/- mice and mice with reduced levels of the transcription factor C-terminal binding protein (CtBP). Serum levels of BAs were higher in patients with Cushing disease or obesity than in individuals with normal levels of glucocorticoids. Administration of dexamethasone promoted cholestasis and overproduction of BAs in C57BL/6 mice, but not in FXR-/- mice. GR antagonists, or injection of an adenoviral small hairpin RNA against GR, reduced features of hepatic cholestasis in db/db mice. The GR interacted with FXR to reduce its transcriptional activity by recruiting CtBP co-repressor complexes. Mice with reduced levels of CtBP were resistant to induction of hepatic cholestasis by dexamethasone. Glucocorticoids promote hepatic cholestasis in mice by recruiting CtBP co-repressor complexes to FXR and thereby blocking the transcriptional activity. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

  15. The ligand binding domain controls glucocorticoid receptor dynamics independent of ligand release.

    PubMed

    Meijsing, Sebastiaan H; Elbi, Cem; Luecke, Hans F; Hager, Gordon L; Yamamoto, Keith R

    2007-04-01

    Ligand binding to the glucocorticoid receptor (GR) results in receptor binding to glucocorticoid response elements (GREs) and the formation of transcriptional regulatory complexes. Equally important, these complexes are continuously disassembled, with active processes driving GR off GREs. We found that co-chaperone p23-dependent disruption of GR-driven transcription depended on the ligand binding domain (LBD). Next, we examined the importance of the LBD and of ligand dissociation in GR-GRE dissociation in living cells. We showed in fluorescence recovery after photobleaching studies that dissociation of GR from GREs is faster in the absence of the LBD. Furthermore, GR interaction with a target promoter revealed ligand-specific exchange rates. However, using covalently binding ligands, we demonstrated that ligand dissociation is not required for receptor dissociation from GREs. Overall, these studies showed that activities impinging on the LBD regulate GR exchange with GREs but that the dissociation of GR from GREs is independent from ligand dissociation.

  16. Binding of steroids to the progestin and glucocorticoid receptors analyzed by correspondence analysis.

    PubMed

    Ojasoo, T; Doré, J C; Gilbert, J; Raynaud, J P

    1988-06-01

    The relative binding affinities of over 30 steroids have been measured for the cytosol glucocorticoid receptor (GR) of thymus, liver, and hepatoma tissue culture cells and for progestin, androgen, and mineralocorticoid receptors. The data have been analyzed by correspondence analysis to reveal the singularities among the receptors of different hormonal classes, the similarities in GR of different origins, and the different specificities of the ligands. Additional data on new steroids have been injected into the system as well as results on a further parameter, namely the induction of tyrosine aminotransferase (TAT) activity, to illustrate the power and flexibility of the methodology. The analysis has confirmed previous correlations between GR binding and TAT response but also highlighted the antiglucocorticoid activity of progestins. This method should prove to be a substantial aid to the interpretation of increasingly complex data, in particular with regard to the action of existing and newly synthesized steroids on glucocorticoid systems of differential sensitivity.

  17. Oxandrolone blocks glucocorticoid signaling in an androgen receptor-dependent manner.

    PubMed

    Zhao, Jingbo; Bauman, William A; Huang, Ruojun; Caplan, Avrom J; Cardozo, Christopher

    2004-05-01

    The anabolic steroid oxandrolone is increasingly used to preserve or restore muscle mass in those with HIV infection or serious burns. These effects are mediated, in part, by the androgen receptor (AR). Anti-glucocorticoid effects have also been reported for some anabolic steroids, and the goal of our studies was to determine whether oxandrolone had a similar mechanism of action. Studies with in vitro translated glucocorticoid receptor (GR), however, showed no inhibition of cortisol binding by oxandrolone. Conversely, experiments in cell culture systems demonstrated significant antagonism of cortisol-induced transcriptional activation by oxandrolone in cells expressing both the AR and GR. Inhibition was not overcome by increased cortisol concentration, and no inhibition by oxandrolone was observed in cells expressing GR alone, confirming that non-competitive mechanisms were involved. AR-dependent repression of transcriptional activation by oxandrolone was also observed with the synthetic glucocorticoids dexamethasone and methylprednisolone. Furthermore, the AR antagonists 2-hydroxyflutamide and DDE also repressed GR transactivation in an AR-dependent manner. A mutant AR lacking a functional nuclear localization signal (AR(4RKM)) was active in oxandrolone-mediated repression of GR even though oxandrolone-bound AR(4RKM) failed to enter the nucleus and did not affect nuclear import of GR. These data indicate a novel action of oxandrolone to suppress glucocorticoid action via crosstalk between AR and GR.

  18. A glucocorticoid receptor in fetal mouse: its relationship to cleft palate formation.

    PubMed

    Hackney, J F

    1980-02-01

    Fetal mouse tissue was investigated for a glucocorticoid binding receptor which might be responsible for cleft palate formation. Fetal mouse heads contain a soluble component which binds the glucocorticoid triamcinolone acetonide in vitro with high affinity. This binding component is present in small finite amounts. Other glucocorticoids compete with triamcinolone acetonide for the binding site in a manner consistent with their potency ranking as cleft palate teratogens. Several mineralocorticoids and progestins also compete when administered in vitro but not when administered in vivo. Triamcinolone acetonide binding was determined in three mouse strains, A/J, C3H, and C57BL, which are listed in decreasing order of cleft palate susceptibility to cortisone. No positive correlation was found between cortisone cleft palate susceptibility and either triamcinolone acetonide binding affinity or binding amount in fetuses from these strains. Cleft palate dose response curves for triamcinolone acetonide were determined in these strains, but they were not parallel to each other as they were for cortisone. This suggests that triamcinolone acetonide may cause cleft palate by different mechanisms in these strains. Thus, fetal mouse tissue contains an apparent glucocorticoid receptors, but its relationship to cleft palate formation in mice is not clear.

  19. Single point estimation of glucocorticoid receptors in lymphocytes of normal subjects and of children under long term glucocorticoid treatment.

    PubMed

    Lapcík, P; Hampl, R; Bicíková, M

    1992-03-01

    A single point assay of glucocorticoid receptors (GR) in human lymphocytes based on the measurement of specific dexamethasone binding has been developed and compared with a common multi-point Scatchard analysis. The assay conditions-concentration of the ligand 20 nmol/l, incubation time 2 h and the cell count 2-6 mil. cells/tube in the assay volume 0.25 ml were found to be optimal. An attempt was also undertaken to use a cell harvester for the separation of cells from unbound ligand. Though specifically bound dexamethasone measured by whole-cell assay and that using cell harvester correlated well, almost by one order lower values obtained with the latter method render it non-applicable for receptor quantitation. The results from 9 healthy volunteers (average GR concentration 7131 +/- 1256 sites/cell) correlated excellently with those obtained by the Scatchard analysis. The single point assay has been also applied for determination of GH in 10 children treated with large doses of prednisone. The average values from healthy volunteers did not differ significantly from those found in these children, though much broader range was found in patients.

  20. Modulation of central glucocorticoid receptors in short- and long-term experimental hyperthyroidism.

    PubMed

    Nikolopoulou, Elena; Mytilinaios, Dimitrios; Calogero, Aldo E; Kamilaris, Themis C; Troupis, Theodore; Chrousos, George P; Johnson, Elizabeth O

    2015-08-01

    Hyperthyroidism is associated with a significant increase in circulating glucocorticoid levels and hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. The aim of this study was to examine whether the HPA axis hyperactivity observed in hyperthyroidism may be explained by a disturbed feedback inhibition of endogenous glucocorticoids through two specific intracellular receptors in the brain: the high affinity mineralocorticoid receptor (MR) and the lower affinity glucocorticoid receptor (GR). Cytosolic receptor binding and gene expression was assessed in rats with short (7 days) and long standing (60 days) eu- and hyperthyroidism. Glucocorticoid receptor number and binding affinity (Kd) in the hippocampus were measured using [(3)H2]-dexamethasone radioreceptor assay. In situ hybridization was employed to examine the effects of hyperthyroidism on the GR and MR mRNA levels in the hippocampus and the pituitary. Both short- and long-term hyperthyroid rats showed pronounced reduction in the concentration of cytosolic GR in the hippocampus, without changes in binding affinity or changes in GR expression. In contrast, GR mRNA in the pituitary increased after 7 days and decreased after 60 days of thyroxin treatment. MR mRNA was moderately affected. Hyperthyroidism is associated with significant decreases in hippocampal GR levels supporting the hypothesis that hyperactivity of the HPA axis observed in experimentally induced hyperthyroidism may be attributed, at least in part, to decreased negative feedback at the level of the hippocampus. These findings further support the notion that a central locus is principally responsible for the hyperactivity of the HPA axis observed in hyperthyroidism.

  1. 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.

  2. Glucocorticoid receptor antagonism disrupts the reconsolidation of social reward-related memories in rats.

    PubMed

    Achterberg, E J Marijke; Trezza, Viviana; Vanderschuren, Louk J M J

    2014-06-01

    Reconsolidation is the process whereby consolidated memories are destabilized upon retrieval and restabilized to persist for later use. Although the neurobiology of the reconsolidation of both appetitive and aversive memories has been intensively investigated, reconsolidation of memories of physiologically relevant social rewards has received little attention. Social play, the most characteristic social behaviour displayed by young mammals, is highly rewarding, illustrated by the fact that it can induce conditioned place preference (CPP). Here, we investigated the role of signalling mechanisms implicated in memory processes, including reconsolidation, namely glucocorticoid, mineralocorticoid, NMDA glutamatergic and CB1 cannabinoid receptors, in the reconsolidation of social play-induced CPP in rats. Systemic treatment with the glucocorticoid receptor antagonist mifepristone before, but not immediately after, retrieval disrupted the reconsolidation of social play-induced CPP. Mifepristone did not affect social play-induced CPP in the absence of memory retrieval. Treatment with the NMDA receptor antagonist MK-801 modestly affected the reconsolidation of social play-induced CPP. However, the reconsolidation of social play-induced CPP was not affected by treatment with the mineralocorticoid and CB1 cannabinoid receptor antagonists spironolactone and rimonabant, respectively. We conclude that glucocorticoid neurotransmission mediates the reconsolidation of social reward-related memories in rats. These data indicate that the neural mechanisms of the reconsolidation of social reward-related memories only partially overlap with those underlying the reconsolidation of other reward-related memories.

  3. Glucocorticoid receptor antagonism disrupts reconsolidation of social reward-related memories in rats

    PubMed Central

    Achterberg, E.J. Marijke; Trezza, Viviana; Vanderschuren, Louk J.M.J.

    2014-01-01

    Reconsolidation is the process whereby consolidated memories are destabilized upon retrieval and restabilized to persist for later use. Although the neurobiology of reconsolidation of both appetitive and aversive memories has been intensively investigated, reconsolidation of memories of physiologically relevant social rewards has received little attention. Social play, the most characteristic social behaviour displayed by young mammals, is highly rewarding, illustrated by the fact that it can induce conditioned place preference (CPP). Here, we investigated the role of signaling mechanisms implicated in memory processes including reconsolidation, i.e. glucocorticoid, mineralocorticoid, NMDA glutamatergic and CB1 cannabinoid receptors, in the reconsolidation of social play-induced CPP in rats. Systemic treatment with the glucocorticoid receptor antagonist mifepristone before, but not immediately after retrieval, disrupted the reconsolidation of social play-induced CPP. Mifepristone did not affect social play-induced CPP in the absence of memory retrieval. Treatment with the NMDA receptor antagonist MK-801 modestly affected reconsolidation of social play-induced CPP. However, reconsolidation of social play-induced CPP was not affected by treatment with the mineralocorticoid and CB1 cannabinoid receptor antagonists spironolactone and rimonabant, respectively. We conclude that glucocorticoid neurotransmission mediates the reconsolidation of social reward-related memories in rats. These data indicate that the neural mechanisms of the reconsolidation of social reward-related memories only partially overlap with those underlying reconsolidation of other reward-related memories. PMID:24776489

  4. 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.

  5. Glucocorticoid receptor mediated the propofol self-administration by dopamine D1 receptor in nucleus accumbens.

    PubMed

    Wu, Binbin; Liang, Yuyuan; Dong, Zhanglei; Chen, Zhichuan; Zhang, Gaolong; Lin, Wenxuan; Wang, Sicong; Wang, Benfu; Ge, Ren-Shan; Lian, Qingquan

    2016-07-22

    Propofol, a widely used anesthetic, can cause addictive behaviors in both human and experimental animals. In the present study, we examined the involvement of glucocorticoid receptor (GR) signaling in the molecular process by which propofol may cause addiction. The propofol self-administration model was established by a fixed ratio 1 (FR1) schedule of reinforced dosing over successive 14days in rats. On day 15, the rats were treated with dexamethasone, a GR agonist (10-100μg/kg), or RU486, a GR antagonist (10-100μg/kg) at 1h prior to the last training. The animal behaviors were recorded automatically by the computer. The expression of dopamine D1 receptor in the nucleus accumbens (NAc) was examined by Western blot and the concentrations of plasma corticosterone were measured by enzyme-linked immunosorbent assay (ELISA). To further examine the specificity of GR in the process, mineralocorticoid receptor (MR) antagonist, spironolactone, and dexamethasone plus MR agonist, aldosterone, were also tested. Administration of dexamethasone (100μg/kg) or RU486 (⩾10mg/kg) significantly attenuated the rate of propofol maintained active nose-poke responses and infusions, which were accompanied by reductions in both plasma corticosterone level and the expression of D1 receptor in the NAc. Neither spironolactone alone nor dexamethasone combined with aldosterone affected the propofol-maintaining self-administrative behavior, indicating GR, but not MR, modulates the propofol reward in rats. In addition, neither the food-maintaining sucrose responses under FR1 schedule nor the locomotor activity was affected by any doses of dexamethasone or RU486 tested. These findings provide evidence that GR signaling may play an important role in propofol reward. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. 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

  7. Identification of hormone-interacting amino acid residues within the steroid-binding domain of the glucocorticoid receptor in relation to other steroid hormone receptors

    SciTech Connect

    Carlstedt-Duke, J.; Stroemstedt, P.E.; Persson, B.; Cederlund, E.; Gustafsson, J.A.; Joernvall, H.

    1988-05-15

    Purified rat liver glucocorticoid receptor was covalently charged with (/sup 3/H)glucocorticoid by photoaffinity labeling (UV irradiation of (/sup 3/H)triamcinolone acetonide-glucocorticoid receptor) or affinity labeling (incubation with (/sup 3/H)dexamethasone mesylate). After labeling, separate samples of the denatured receptor were cleaved with trypsin (directly or after prior succinylation), chymotrypsin, and cyanogen bromide. Labeled residues in the peptides obtained were identified by radiosequence analysis. The peaks of radioactivity corresponded to Met-622 and Cys-754 after photoaffinity labeling with (/sup 3/H)triamcinolone acetonide and Cys-656 after affinity labeling with (/sup 3/H)dexamethasone mesylate. The labeled residues are all positioned within hydrophobic segments of the steroid-binding domain. The patterns of hydropathy and secondary structure for the glucocorticoid receptor are highly similar to those for the progestin receptor and similar but less so to those for the estrogen receptor and to those for c-erb A.

  8. Low glucocorticoid receptor (GR), high Dig2 and low Bcl-2 expression in double positive thymocytes of BALB/c mice indicates their endogenous glucocorticoid hormone exposure.

    PubMed

    Boldizsár, Ferenc; Pálinkás, László; Czömpöly, Tamás; Bartis, Domokos; Németh, Péter; Berki, Timea

    2006-01-01

    Several studies have shown that of the four major thymocyte subsets, the CD4/CD8 double positive (DP) thymocytes are the most sensitive to in vivo glucocorticoid hormone (GC)-induced apoptosis. Our aim was to analyse fine molecular differences among thymocyte subgroups that could underlie this phenomenon. Therefore, we characterised the glucocorticoid hormone receptor (GR) expression of thymocyte subgroups both at the mRNA and protein levels by real-time PCR and flow cytometry, and correlated these features to their apoptotic sensitivity. We also investigated the time-dependent effects of the GC agonist dexamethasone (DX) with or without GC antagonist (RU486) treatments on GR mRNA/protein expression. We also analysed the expression of two apoptosis-related gene products: dexamethasone-induced gene 2 (Dig2) mRNA and Bcl-2 protein. We found that DN thymocytes had the highest GR expression, followed by CD8 single positive (SP), CD4 SP and DP thymocytes in 4-week-old BALB/c mice, both at the mRNA and protein levels, respectively. In DP cells, the Dig2 expression was significantly higher, while the Bcl-2 expression was significantly lower than in DN, CD4 SP and CD8 SP thymocytes. Single high dose DX treatment caused time-dependent depletion of DP thymocytes due to their higher apoptosis rate, which could not be abolished with RU486 pretreatment. After a single high dose DX treatment, there was a transient, significant increase of the GR mRNA and protein level of unsorted thymocytes after 8 and 16 h, followed by a significant decrease at 24 h, respectively. The time-dependent GR expression changes after DX administration could not be inhibited by the GC antagonist RU486. Twenty-four hours after exposure to high dose DX the DN, CD4 SP and CD8 SP cells showed a significant decrease of GR mRNA and protein expression, whereas the DP thymocytes, showed no significant alteration of GR mRNA or protein expression. The kinetical analysis of GR expression and apoptotic marker

  9. Expression of estrogen, androgen, and glucocorticoid receptors in recent striae distensae.

    PubMed

    Cordeiro, Raquel Cristina Tancsik; Zecchin, Karina Gotardello; de Moraes, Aparecida Machado

    2010-01-01

    Stretch marks or striae distensae (SD) can be considered a common skin disorder, but their physiopathogenic mechanisms have not been totally clarified. Although it is considered an esthetic complaint, it may have serious psychosocial consequences besides the local and systemic alterations of the conjunctive tissue. This study aims at assessing and quantifying the estrogen, androgen and glucocorticoid receptors in skin samples with striae and comparing with normal skin. Skin samples for biopsy were obtained from eight patients with SD and eight patients without lesions. The samples were frozen at -80 degrees C and underwent processing to obtain proteic extract to quantify the estrogen, androgen and glucocorticoid receptors with the Western Blot method. When the estrogen receptor in the skin with SD was compared with healthy skin, it was observed to have increased twice as much (P = 0.00001). The androgen and glucocorticoid receptors in the SD skin had also increased (P = 0.00015 and P = 0.00083, respectively). These findings indicate that under certain conditions there is an increase in hormonal receptor expression, suggesting that regions that undergo greater mechanical stretching of the skin may express greater hormonal receptor activity. This activity may influence the metabolism of the extracellular matrix, causing the formation of SD. Alterations in hormone receptors occur within a well-defined time period during the formation of SD; however, there are differences in the functionality of hormone receptors during different stages in the development of the lesions. The preliminary results appear to be relevant and represent an initial step towards an understanding of the pathophysiology of SD.

  10. Knockout of the vascular endothelial glucocorticoid receptor abrogates dexamethasone-induced hypertension

    PubMed Central

    GOODWIN, Julie E.; ZHANG, Junhui; GONZALEZ, David; ALBINSSON, Sebastian; GELLER, David S.

    2012-01-01

    Glucocorticoid-mediated hypertension is incompletely understood. Recent studies have suggested the primary mechanism of this form of hypertension may be through the effects of glucocorticoids on vascular tissues and not to excess sodium and water reabsorption as traditionally believed. Objective The goal of this study was to better understand the role of the vasculature in the generation and maintenance of glucocorticoid-mediated hypertension. Methods We created a mouse model with a tissue-specific knockout of the glucocorticoid receptor in the vascular endothelium. Results We show that these mice are relatively resistant to dexamethasone-induced hypertension. After one week of dexamethasone treatment, control animals have a mean blood pressure increase of 13.1 mm Hg while knockout animals have only a 2.7 mm Hg increase (p<0.001). Interestingly, the knockout mice have slightly elevated baseline BP compared to the controls (112.2 ± 2.5 mm Hg vs. 104.6 ± 1.2 mm Hg, p = 0.04), a finding which is not entirely explained by our data. Furthermore, we demonstrate that the knockout resistance arterioles have a decreased contractile response to dexamethasone with only 6.6% contraction in knockout vessels compared to 13.4% contraction in control vessels (p=0.034). Finally, we show that in contrast to control animals, the knockout animals are able to recover a significant portion of their normal circadian blood pressure rhythm suggesting that the vascular endothelial glucocorticoid receptor may function as a peripheral circadian clock. Conclusions Our study highlights the importance of the vascular endothelial GR in several fundamental physiologic processes, namely blood pressure homeostasis and circadian rhythm. PMID:21659825

  11. Nonsteroidal 2,3-dihydroquinoline glucocorticoid receptor agonists with reduced PEPCK activation.

    PubMed

    Hudson, Andrew R; Higuchi, Robert I; Roach, Steven L; Valdez, Lino J; Adams, Mark E; Vassar, Angie; Rungta, Deepa; Syka, Peter M; Mais, Dale E; Marschke, Keith B; Zhi, Lin

    2011-03-15

    Continuing studies based on dihydroquinoline glucocorticoid receptor agonists lead to the discovery of a series of C4-oxime analogs. Representative compounds exhibited potent transrepression activity with minimal transactivation of phosphoenolpyruvate caboxykinase (PEPCK), a key protein in the gluconeogenesis pathway. These compounds represent promising leads in identifying GR agonists with high anti-inflammatory activity and attenuated potential for glucose elevation. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Discovery of nonsteroidal glucocorticoid receptor ligands based on 6-indole-1,2,3,4-tetrahydroquinolines.

    PubMed

    Roach, Steven L; Higuchi, Robert I; Adams, Mark E; Liu, Yan; Karanewsky, Donald S; Marschke, Keith B; Mais, Dale E; Miner, Jeffrey N; Zhi, Lin

    2008-06-15

    A series of nonsteroidal glucocorticoid receptor (GR) ligands based on a 6-indole-1,2,3,4-tetrahydroquinoline scaffold are reported. Structure-activity relationship (SAR) of the pendent indole group identified compound 20 exhibiting good GR binding affinity (K(i)=1.5nM) and 100- to 1000-fold selectivity over MR, PR, and AR while showing activity in an E-selectin repression assay.

  13. Cortisol Induces Reactive Oxygen Species Through a Membrane Glucocorticoid Receptor in Rainbow Trout Myotubes.

    PubMed

    Espinoza, Marlen B; Aedo, Jorge E; Zuloaga, Rodrigo; Valenzuela, Cristian; Molina, Alfredo; Valdés, Juan A

    2017-04-01

    Cortisol is an essential regulator of neuroendocrine stress responses in teleosts. Cortisol predominantly affects target tissues through the genomic pathway, which involves interacting with cytoplasmic glucocorticoid receptors, and thereby, modulating stress-response gene expressions. Cortisol also produces rapid effects via non-genomic pathways, which do not involve gene transcription. Although cortisol-mediated genomic pathways are well documented in teleosts, non-genomic pathways are not fully understood. Moreover, no studies have focused on the contribution of non-genomic cortisol pathways in compensatory stress responses in fish. In this study, rainbow trout (Oncorhynchus mykiss) skeletal myotubes were stimulated with physiological concentrations of cortisol and cortisol-BSA, a membrane-impermeable agent, resulting in an early induction of reactive oxygen species (ROS). This production was not suppressed by transcription or translation inhibitors, suggesting non-genomic pathway involvement. Moreover, myotube preincubation with RU486 and NAC completely suppressed cortisol- and cortisol-BSA-induced ROS production. Subcellular fractionation analysis revealed the presence of cell membrane glucocorticoid receptors. Finally, cortisol-BSA induced a significant increase in ERK1/2 and CREB phosphorylation, as well as in CREB-dependent transcriptional activation of the pgc1a gene expression. The obtained results strongly suggest that cortisol acts through a non-genomic glucocorticoid receptor-mediated pathway to induce ROS production and contribute to ERK/CREB/PGC1-α signaling pathway activation as stress compensation mechanisms. J. Cell. Biochem. 118: 718-725, 2017. © 2016 Wiley Periodicals, Inc.

  14. Highly inducible expression from vectors containing multiple GRE's in CHO cells overexpressing the glucocorticoid receptor.

    PubMed Central

    Israel, D I; Kaufman, R J

    1989-01-01

    A conditional glucocorticoid-responsive expression vector system is described for highly inducible expression of heterologous genes in mammalian cells. This host-vector system requires high level expression of the glucocorticoid receptor (GR) protein in the host cell and multiple copies of the receptor binding site within the expression vector. Transfection and selection of Chinese hamster ovary cells with expression vectors encoding the rat GR yielded cell lines which express functional receptor at high levels. Insertion of multiple copies of the MMTV enhancer (glucocorticoid responsive element, GRE) into an Adenovirus major late promoter (AdMLP) based expression vector yielded greater than 1000-fold inducible expression by dexamethasone (dex) in transient DNA transfection assays. The induced expression level was 7-fold greater than that obtained with an AdMLP based vector containing an SV40 enhancer, but lacking GRE's. Vectors containing the SV40 enhancer in combination with multiple GRE's exhibited elevated basal expression in the absence of dex, but retained inducibility in both transient assays and after integration and amplification in the CHO genome. This expression system should be of general utility for studying gene regulation and for expressing heterologous genes in a regulatable fashion. Images PMID:2546123

  15. Assessment of glucocorticoid lung targeting by ex-vivo receptor binding studies in rats.

    PubMed

    Hochhaus, G; Gonzalez-Rothi, R J; Lukyanov, A; Derendorf, H; Schreier, H; Dalla Costa, T

    1995-01-01

    Triamcinolone acetonide (TA, 22 micrograms) was given to rats by intravenous (i.v.) injection or intratracheal (IT) instillation. Free glucocorticoid receptors were monitored over time in liver and lung using an ex-vivo receptor binding technique. After i.v. administration of a TA solution, the reduction of free receptors over time was very similar in lung and liver (AUCLung = 280 +/- 47% h; AUCLiver = 320 +/- 76% h). Intratracheal instillation of the same solution produced time profiles which mirrored those of i.v. injection (AUCLung = 260 +/- 41% h; AUCLiver = 330 +/- 50% h). The lack of lung targeting was also reflected in the failure to show any significant difference in the pulmonary targeting factor T (AUCLung/AUCLiver) between i.v. (T = 0.84 +/- 0.18) and IT (T = 0.78 +/- 0.03) administration. In contrast, a certain degree of lung specificity was observed after IT instillation of a glucocorticoid suspension (22 micrograms; AUCLung = 160 +/- 135% h; AUCLiver = 65 +/- 91% h, T = 2.3 +/- 0.5) as indicated by significant differences in T between i.v. injection and IT instillation (p = 0.038). The method presented provides a means of simultaneously assessing pulmonary and systemic effects after different forms and routes of administration and might be of value in further studying multiple aspects of inhalation glucocorticoid therapy.

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

    PubMed

    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.

  17. Developmental Expression and Glucocorticoid Control of the Leptin Receptor in Fetal Ovine Lung.

    PubMed

    De Blasio, Miles J; Boije, Maria; Vaughan, Owen R; Bernstein, Brett S; Davies, Katie L; Plein, Alice; Kempster, Sarah L; Smith, Gordon C S; Charnock-Jones, D Stephen; Blache, Dominique; Wooding, F B Peter; Giussani, Dino A; Fowden, Abigail L; Forhead, Alison J

    2015-01-01

    The effects of endogenous and synthetic glucocorticoids on fetal lung maturation are well-established, although the role of leptin in lung development before birth is unclear. This study examined mRNA and protein levels of the signalling long-form leptin receptor (Ob-Rb) in fetal ovine lungs towards term, and after experimental manipulation of glucocorticoid levels in utero by fetal cortisol infusion or maternal dexamethasone treatment. In fetal ovine lungs, Ob-Rb protein was localised to bronchiolar epithelium, bronchial cartilage, vascular endothelium, alveolar macrophages and type II pneumocytes. Pulmonary Ob-Rb mRNA abundance increased between 100 (0.69 fractional gestational age) and 144 days (0.99) of gestation, and by 2-4-fold in response to fetal cortisol infusion and maternal dexamethasone treatment. In contrast, pulmonary Ob-Rb protein levels decreased near term and were halved by glucocorticoid treatment, without any significant change in phosphorylated signal transducer and activator of transcription-3 (pSTAT3) at Ser727, total STAT3 or the pulmonary pSTAT3:STAT3 ratio. Leptin mRNA was undetectable in fetal ovine lungs at the gestational ages studied. These findings demonstrate differential control of pulmonary Ob-Rb transcript abundance and protein translation, and/or post-translational processing, by glucocorticoids in utero. Localisation of Ob-Rb in the fetal ovine lungs, including alveolar type II pneumocytes, suggests a role for leptin signalling in the control of lung growth and maturation before birth.

  18. Research resource: modulators of glucocorticoid receptor activity identified by a new high-throughput screening assay.

    PubMed

    Blackford, John A; Brimacombe, Kyle R; Dougherty, Edward J; Pradhan, Madhumita; Shen, Min; Li, Zhuyin; Auld, Douglas S; Chow, Carson C; Austin, Christopher P; Simons, S Stoney

    2014-07-01

    Glucocorticoid steroids affect almost every type of tissue and thus are widely used to treat a variety of human pathological conditions. However, the severity of numerous side effects limits the frequency and duration of glucocorticoid treatments. Of the numerous approaches to control off-target responses to glucocorticoids, small molecules and pharmaceuticals offer several advantages. Here we describe a new, extended high-throughput screen in intact cells to identify small molecule modulators of dexamethasone-induced glucocorticoid receptor (GR) transcriptional activity. The novelty of this assay is that it monitors changes in both GR maximal activity (A(max)) and EC(50) (the position of the dexamethasone dose-response curve). Upon screening 1280 chemicals, 10 with the greatest changes in the absolute value of A(max) or EC(50) were selected for further examination. Qualitatively identical behaviors for 60% to 90% of the chemicals were observed in a completely different system, suggesting that other systems will be similarly affected by these chemicals. Additional analysis of the 10 chemicals in a recently described competition assay determined their kinetically defined mechanism and site of action. Some chemicals had similar mechanisms of action despite divergent effects on the level of the GR-induced product. These combined assays offer a straightforward method of identifying numerous new pharmaceuticals that can alter GR transactivation in ways that could be clinically useful.

  19. Reciprocal regulation of a glucocorticoid receptor-steroidogenic factor-1 transcription complex on the Dax-1 promoter by glucocorticoids and adrenocorticotropic hormone in the adrenal cortex.

    PubMed

    Gummow, Brian M; Scheys, Joshua O; Cancelli, Victoria R; Hammer, Gary D

    2006-11-01

    Numerous genes required for adrenocortical steroidogenesis are activated by the nuclear hormone receptor steroidogenic factor 1 (SF-1) (NR5A1). Dax-1 (NR0B1), another nuclear hormone receptor, represses SF-1-dependent activation. Glucocorticoid products of the adrenal cortex provide negative feedback to the production of hypothalamic CRH and pituitary ACTH. We hypothesized that glucocorticoids stimulate an intraadrenal negative feedback loop via activation of Dax-1 expression. Reporter constructs show glucocorticoid-dependent synergy between SF-1 and glucocorticoid receptor (GR) in the activation of Dax-1, which is antagonized by ACTH signaling. We map the functional glucocorticoid response element between -718 and -704 bp, required for activation by GR and synergy with SF-1. Of three SF-1 response elements, only the -128-bp SF-1 response element is required for synergy with GR. Chromatin immunoprecipitation (ChIP) assays demonstrate that dexamethasone treatment increases GR and SF-1 binding to the endogenous murine Dax-1 promoter 10- and 3.5-fold over baseline. Serial ChIP assays reveal that that GR and SF-1 are part of the same complex on the Dax-1 promoter, whereas coimmunoprecipitation assay confirms the presence of a protein complex that contains both GR and SF-1. ACTH stimulation disrupts the formation of this complex by abrogating SF-1 binding to the Dax-1 promoter, while promoting SF-1 binding to the melanocortin-2 receptor (Mc2r) and steroidogenic acute regulatory protein (StAR) promoters. Finally, dexamethasone treatment increases endogenous Dax-1 expression and concordantly decreases StAR expression. ACTH signaling antagonizes the increase in Dax-1 yet strongly activates StAR transcription. These data indicate that GR provides feedback regulation of adrenocortical steroid production through synergistic activation of Dax-1 with SF-1, which is antagonized by ACTH activation of the adrenal cortex.

  20. Chromatin immunoprecipitation scanning identifies glucocorticoid receptor binding regions in the proximal promoter of a ubiquitously expressed glucocorticoid target gene in brain.

    PubMed

    van der Laan, Siem; Sarabdjitsingh, R Angela; Van Batenburg, Marcel F; Lachize, Servane B; Li, Hualing; Dijkmans, Thomas F; Vreugdenhil, Erno; de Kloet, E Ron; Meijer, Onno C

    2008-09-01

    While the actions of glucocorticoids on brain functions have been comprehensively studied, the underlying genomic mechanisms are poorly understood. In this study, we show that glucocorticoid-induced leucine zipper (GILZ) mRNA is strongly and ubiquitously induced in rat brain. To decipher the molecular mechanisms underlying these genomic effects, it is of interest to identify the regulatory sites in the promoter region. Alignment of the rat GILZ promoter with the well-characterized human promoter resulted in poor sequence homology. Consequently, we analyzed the rat 5' flanking sequence by Matrix REDUCE and identified two high-affinity glucocorticoid response elements (GRE) located 2 kb upstream of the transcription start site. These findings were corroborated using the glucocorticoid receptor (GR) expressing Ns-1 PC12 rat cell-line. In these cells, dexamethasone treatment leads to a progressive increase of GILZ mRNA expression levels via a GR-dependent mechanism. Subsequently, using chromatin immunoprecipitation assays we show that the two high-affinity GREs are located within the GR-binding regions. Lastly, we demonstrate using multiple tissue in situ hybridization a marked increase in mRNA expression levels in spleen, thymus, heart, lung, liver, muscle, testis, kidney, colon, ileum, as well as in brain and conclude that the GILZ gene can be used to study glucocorticoid effects in many additional rodent tissues.

  1. Separate regions of glucocorticoid receptor, coactivator TIF2, and comodulator STAMP modify different parameters of glucocorticoid-mediated gene induction.

    PubMed Central

    Awasthi, Smita; Simons, S. Stoney

    2012-01-01

    Increased specificity in steroid-regulated gene expression is a long-sought goal of endocrinologists. Considerable progress has resulted from the discovery of coactivators, corepressors, and comodulators that adjust the total activity (Amax) of gene induction. Two less frequently quantitated, but equally potent, means of improving specificity are the concentration of agonist steroid required for half-maximal activity (EC50) and the residual or partial agonist activity displayed by most antisteroids (PAA). It is usually assumed that the modulatory activity of transcriptional cofactors coordinately regulates Amax, EC50, and PAA. Here we examine the hypothesis that these three parameters can be independently modified by separate protein domains. The test system involves three differently sized fragments of each of three factors (glucocorticoid receptor [GR], coactivator TIF2, and comodulator STAMP), which are shown to form a ternary complex and similarly affect the induction properties of transfected and endogenous genes. Twenty five different fragment combinations of the ternary complex are examined for their ability to modulate the Amax, EC50, and PAA of a transiently transfected synthetic reporter gene. Different combinations selectively alter one, two, or all three parameters. These results clearly demonstrate that Amax, EC50, and PAA can be independently regulated under some conditions by different pathways or molecular interactions. This new mechanistic insight suggests that selected activities of individual transcription factors are attractive targets for small molecules, which would have obvious clinical applications for increasing the specificity of steroids during endocrine therapies. PMID:22342989

  2. Separate regions of glucocorticoid receptor, coactivator TIF2, and comodulator STAMP modify different parameters of glucocorticoid-mediated gene induction.

    PubMed

    Awasthi, Smita; Simons, S Stoney

    2012-05-15

    Increased specificity in steroid-regulated gene expression is a long-sought goal of endocrinologists. Considerable progress has resulted from the discovery of coactivators, corepressors, and comodulators that adjust the total activity (A(max)) of gene induction. Two less frequently quantitated, but equally potent, means of improving specificity are the concentration of agonist steroid required for half-maximal activity (EC(50)) and the residual or partial agonist activity displayed by most antisteroids (PAA). It is usually assumed that the modulatory activity of transcriptional cofactors coordinately regulates A(max), EC(50), and PAA. Here we examine the hypothesis that these three parameters can be independently modified by separate protein domains. The test system involves three differently sized fragments of each of three factors (glucocorticoid receptor [GR], coactivator TIF2, and comodulator STAMP), which are shown to form a ternary complex and similarly affect the induction properties of transfected and endogenous genes. Twenty-five different fragment combinations of the ternary complex are examined for their ability to modulate the A(max), EC(50), and PAA of a transiently transfected synthetic reporter gene. Different combinations selectively alter one, two, or all three parameters. These results clearly demonstrate that A(max), EC(50), and PAA can be independently regulated under some conditions by different pathways or molecular interactions. This new mechanistic insight suggests that selected activities of individual transcription factors are attractive targets for small molecules, which would have obvious clinical applications for increasing the specificity of steroids during endocrine therapies.

  3. [Effect of atopy on serum glucocorticoid receptor levels in children with bronchiolitis].

    PubMed

    Yao, Huan-Yin; Liu, Wei-Rong; Zhang, Hang-Hu; Li, Hua-Jun; Wang, Xiao-Xian; Liu, Shu-Mei; Chen, Xiao-Hong

    2017-02-01

    To investigate the effect of atopy on the expression of glucocorticoid receptors in children with bronchiolitis. ELISA was used to measure the changes in the serum levels of glucocorticoid receptor α (GRα) and glucocorticoid receptor β (GRβ) in the bronchiolitis group (77 children, including 34 children with atopy) and pneumonia group (68 children). Thirty-eight children who were prepared to undergo surgeries for non-infectious diseases and had no atopy or family history of allergic diseases were enrolled as the control group. The bronchiolitis group and the pneumonia group had significant increases in the serum levels of GRα and GRβ compared with the control group (P<0.01), and the bronchiolitis group had significant increases in these levels compared with the pneumonia group (P<0.01). Compared with the control group and the pneumonia group, the bronchiolitis group had a significant increase in the GRα/GRβ ratio (P<0.01). Compared with the control group, the children with or without atopy in the bronchiolitis group had significant increases in the serum levels of GRα and GRβ (P<0.01). The non-atopic children in the bronchiolitis group had a significant increase in the serum level of GRβ compared with the atopic children (P<0.01). The atopic children in the bronchiolitis group had a significant increase in the GRα/GRβ ratio compared with the control group and non-atopic children in the bronchiolitis group (P<0.01). Children with bronchiolitis have increased serum levels of GRα and GRβ. The children with atopy have an increased GRα/GRβ ratio, suggesting that the atopic children with bronchiolitis are highly sensitive to glucocorticoids.

  4. Microarray analysis of spaceflown murine thymus tissue reveals changes in gene expression regulating stress and glucocorticoid receptors.

    PubMed

    Lebsack, Ty W; Fa, Vuna; Woods, Chris C; Gruener, Raphael; Manziello, Ann M; Pecaut, Michael J; Gridley, Daila S; Stodieck, Louis S; Ferguson, Virginia L; Deluca, Dominick

    2010-05-15

    The detrimental effects of spaceflight and simulated microgravity on the immune system have been extensively documented. We report here microarray gene expression analysis, in concert with quantitative RT-PCR, in young adult C57BL/6NTac mice at 8 weeks of age after exposure to spaceflight aboard the space shuttle (STS-118) for a period of 13 days. Upon conclusion of the mission, thymus lobes were extracted from space flown mice (FLT) as well as age- and sex-matched ground control mice similarly housed in animal enclosure modules (AEM). mRNA was extracted and an automated array analysis for gene expression was performed. Examination of the microarray data revealed 970 individual probes that had a 1.5-fold or greater change. When these data were averaged (n = 4), we identified 12 genes that were significantly up- or down-regulated by at least 1.5-fold after spaceflight (P < or = 0.05). The genes that significantly differed from the AEM controls and that were also confirmed via QRT-PCR were as follows: Rbm3 (up-regulated) and Hsph110, Hsp90aa1, Cxcl10, Stip1, Fkbp4 (down-regulated). QRT-PCR confirmed the microarray results and demonstrated additional gene expression alteration in other T cell related genes, including: Ctla-4, IFN-alpha2a (up-regulated) and CD44 (down-regulated). Together, these data demonstrate that spaceflight induces significant changes in the thymic mRNA expression of genes that regulate stress, glucocorticoid receptor metabolism, and T cell signaling activity. These data explain, in part, the reported systemic compromise of the immune system after exposure to the microgravity of space. (c) 2010 Wiley-Liss, Inc.

  5. 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.

  6. Mapping the Dynamics of the Glucocorticoid Receptor within the Nuclear Landscape.

    PubMed

    Stortz, Martin; Presman, Diego M; Bruno, Luciana; Annibale, Paolo; Dansey, Maria V; Burton, Gerardo; Gratton, Enrico; Pecci, Adali; Levi, Valeria

    2017-07-24

    The distribution of the transcription machinery among different sub-nuclear domains raises the question on how the architecture of the nucleus modulates the transcriptional response. Here, we used fluorescence fluctuation analyses to quantitatively explore the organization of the glucocorticoid receptor (GR) in the interphase nucleus of living cells. We found that this ligand-activated transcription factor diffuses within the nucleus and dynamically interacts with bodies enriched in the coregulator NCoA-2, DNA-dependent foci and chromatin targets. The distribution of the receptor among the nuclear compartments depends on NCoA-2 and the conformation of the receptor as assessed with synthetic ligands and GR mutants with impaired transcriptional abilities. Our results suggest that the partition of the receptor in different nuclear reservoirs ultimately regulates the concentration of receptor available for the interaction with specific targets, and thus has an impact on transcription regulation.

  7. Antenatal glucocorticoid treatment and polymorphisms of the glucocorticoid and mineralocorticoid receptors are associated with IQ and behavior in young adults born very preterm.

    PubMed

    van der Voorn, Bibian; Wit, Jan M; van der Pal, Sylvia M; Rotteveel, Joost; Finken, Martijn J J

    2015-02-01

    Preterm survivors exhibit neurodevelopmental impairments. Whether this association is influenced by antenatal glucocorticoid treatment and glucocorticoid sensitivity is unknown. This study aimed to study the effects of antenatal glucocorticoid treatment and glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) polymorphisms on behavior and intelligence quotient (IQ). This study was part of the 19-year follow-up of the Project On Preterm and Small-for-gestational-age birth cohort. Multicenter study. Three hundred forty-four 19-year-olds born very preterm (gestational age < 32 wk), of whom 71 had received betamethasone antenatally. Single antenatal treatment course of betamethasone. Behavior (Young Adult Self Report and Young Adult Behavior Checklist for parents) and IQ (digital Multicultural Capacity Test-intermediate level). Data were analyzed by linear regression and presented as regression coefficient (95% confidence interval [CI]). Sex ratio, GR (R23K; N363S) and MR (-2G/C; I180V) genotypes were equally distributed between treated and nontreated subjects. Independent of treatment, R23K carriers had improved IQ scores (β 9.3; 95% CI, 3.4 to 15.1) and a tendency toward more favorable total problem behavior scores (β -8.5; 95% CI, -17.3 to 0.2) ; -2G/C CC carriers had poorer IQ scores (β -6.2; 95% CI, -10.5 to -1.9); I180V carriers had more favorable internalizing behavior scores (β -2.0; 95% CI, -3.9 to -0.1). Antenatal glucocorticoid treatment was associated with more unfavorable behavior scores, especially internalizing behavior (β 2.4; 95% CI, 0.3 to 4.5). Interaction between GR and MR polymorphisms and antenatal glucocorticoid treatment was observed, with poorer IQ scores for exposed N363S carriers; poorer intellectual subdomain scores for exposed I180V-carriers; more favorable total problem behavior scores for exposed R23K carriers. Genetic variations in glucocorticoid sensitivity and antenatal glucocorticoid treatment are associated with IQ

  8. 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.

  9. Caveolin-1 regulates genomic action of the glucocorticoid receptor in neural stem cells.

    PubMed

    Peffer, Melanie E; Chandran, Uma R; Luthra, Soumya; Volonte, Daniela; Galbiati, Ferruccio; Garabedian, Michael J; Monaghan, A Paula; DeFranco, Donald B

    2014-07-01

    While glucocorticoids (GCs) are used clinically to treat many conditions, their neonatal and prenatal usage is increasingly controversial due to reports of delayed adverse outcomes, especially their effects on brain development. Such alterations may reflect the impact of GCs on neural progenitor/stem cell (NPSC) function. We previously demonstrated that the lipid raft protein caveolin-1 (Cav-1) was required for rapid GC signaling in embryonic mouse NPSCs operating through plasma membrane-bound glucocorticoid receptors (GRs). We show here that genomic GR signaling in NPSCs requires Cav-1. Loss of Cav-1 impacts the transcriptional response of many GR target genes (e.g., the serum- and glucocorticoid-regulated kinase 1 gene) that are likely to mediate the antiproliferative effects of GCs. Microarray analysis of wild-type C57 or Cav-1-deficient NPSCs identified approximately 100 genes that are differentially regulated by GC treatment. These changes in hormone responsiveness in Cav-1 knockout NPSCs are associated with the loss of GC-regulated phosphorylation of GR at serine 211 but not at serine 226. Chromatin recruitment of total GR to regulatory regions of target genes such as Fkbp-5, RhoJ, and Sgk-1, as well as p211-GR recruitment to Sgk-1, are compromised in Cav-1 knockout NPSCs. Cav-1 is therefore a multifunctional regulator of GR in NPSCs influencing both rapid and genomic action of the receptor to impact cell proliferation.

  10. 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.

  11. Differences in nuclear retention characteristics of agonist-activated glucocorticoid receptor may determine specific responses.

    PubMed

    Vicent, Guillermo P; Pecci, Adalí; Ghini, Alberto; Piwien-Pilipuk, Graciela; Galigniana, Mario D

    2002-06-10

    We studied the glucocorticoid response to the synthetic steroid pregna-1,4-diene-11beta-ol-3,20-dione (DeltaHOP) in several cell types and correlated its biological effect with the ability of the glucocorticoid receptor (GR) to be retained in the nuclear compartment. We observed that the DeltaHOP-transformed GR was diffusely distributed in the nucleus compared to the discrete structures observed for the dexamethasone (DEX)-transformed GR. Despite the fact that the receptor was entirely nuclear upon binding of each steroid and exhibited identical nuclear export rates, a greater amount of DeltaHOP-transformed GR was recovered in the cytoplasmic fraction after hypotonic cell lysis. Furthermore, accelerated nuclear export of GR was evidenced in digitonin-permeabilized cells treated with ATP and molybdate. Inasmuch as limited trypsinization of DEX-GR and DeltaHOP-GR complexes yielded different proteolytic products, we conclude that GR undergoes a differential conformational change upon binding of each ligand. We propose that these conformational differences may consequently lead to changes of stability in the interaction of the GR with chromatin. Therefore, the dynamic exchange of liganded GR with chromatin is likely to have significant consequences for the observed pleiotropic physiological responses triggered by glucocorticoid ligands, not only in different tissues but also in the same cell type. (c) 2002 Elsevier Science (USA).

  12. Transient generalized glucocorticoid hypersensitivity.

    PubMed

    Nicolaides, Nicolas C; Lamprokostopoulou, Agaristi; Polyzos, Alexandros; Kino, Tomoshige; Katsantoni, Eleni; Triantafyllou, Panagiota; Christophoridis, Athanasios; Katzos, George; Dracopoulou, Maria; Sertedaki, Amalia; Chrousos, George P; Charmandari, Evangelia

    2015-12-01

    Transient generalized glucocorticoid hypersensitivity is a rare disorder characterized by increased tissue sensitivity to glucocorticoids and compensatory hypo-activation of the hypothalamic-pituitary-adrenal axis. The condition itself and the underlying molecular mechanisms have not been elucidated. To present the clinical manifestations, endocrinologic evaluation and transcriptomic profile in a patient with transient generalized glucocorticoid hypersensitivity. A 9-year-old girl presented with an 8-month history of clinical manifestations suggestive of Cushing syndrome. Endocrinologic evaluation revealed undetectable 08:00 h ACTH (<1 pg/mL) and cortisol (0·025 μg/dL) concentrations, which remained decreased throughout the 24-h period and did not respond to stimulation with ovine CRH. The disease gradually resolved spontaneously over the ensuing 3 months. Sequencing of the human glucocorticoid receptor gene revealed no mutations or polymorphisms. Western blot analysis in peripheral blood mononuclear cells revealed equal protein expression of hGRα of the patient in the disease and postresolution phases compared with a control subject. Transcriptomic analysis in peripheral blood mononuclear cells in the disease and postresolution phases identified 903 differentially expressed genes. Of these, 106 genes were up-regulated and 797 were down-regulated in the disease compared with the resolution phase. Bioinformatics analysis on the differentially expressed gene networks revealed Nuclear Factor-κB as the predominant transcription factor influencing the expression of the majority of differentially expressed genes. Our findings indicate that a transient postreceptor defect, or a virus- or bacterium-encoded molecule, may have enhanced glucocorticoid signal transduction, leading to transient generalized glucocorticoid hypersensitivity and hypo-activation of the HPA axis. © 2015 Stichting European Society for Clinical Investigation Journal Foundation.

  13. Reversible and regionally selective downregulation of brain cannabinoid CB1 receptors in chronic daily cannabis smokers

    PubMed Central

    Hirvonen, J; Goodwin, RS; Li, C-T; Terry, GE; Zoghbi, SS; Morse, C; Pike, VW; Volkow, ND; Huestis, MA; Innis, RB

    2011-01-01

    Chronic cannabis (marijuana, hashish) smoking can result in dependence. Rodent studies show reversible downregulation of brain cannabinoid CB1 (cannabinoid receptor type 1) receptors after chronic exposure to cannabis. However, whether downregulation occurs in humans who chronically smoke cannabis is unknown. Here we show, using positron emission tomography imaging, reversible and regionally selective downregulation of brain cannabinoid CB1 receptors in human subjects who chronically smoke cannabis. Downregulation correlated with years of cannabis smoking and was selective to cortical brain regions. After ~4 weeks of continuously monitored abstinence from cannabis on a secure research unit, CB1 receptor density returned to normal levels. This is the first direct demonstration of cortical cannabinoid CB1 receptor downregulation as a neuroadaptation that may promote cannabis dependence in human brain. PMID:21747398

  14. 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.

  15. Pregnancy effects on distribution of progesterone receptors, oestrogen receptor alpha, glucocorticoid receptors, Ki-67 antigen and apoptosis in the bovine interplacentomal uterine wall and foetal membranes.

    PubMed

    Boos, A; Kohtes, J; Janssen, V; Mülling, C; Stelljes, A; Zerbe, H; Hässig, M; Thole, H H

    2006-01-01

    Until recently, studies dealing with the uterus of the pregnant cow focus primarily on the placentome or on early and late pregnancy. Thus, there is a paucity of information about many aspects of the interplacentomal uterine wall including adherent foetal membranes. Corresponding tissue specimens were collected at the slaughterhouse and in animals undergoing premature caesarean section. Two specimens per month of pregnancy were assessed immunohistochemically for progesterone receptors, oestrogen receptor alpha and glucocorticoid receptors, Ki-67 protein and TUNEL procedure was performed. The latter two methods were employed in three animals each per months 1 and 2, 3 and 4, 7 and 8 and in six animals undergoing caesarean section at days 274 and 275 post insemination or during spontaneous labour. Results indicate that proliferation and apoptosis are of minor importance for tissue homeostasis since both can histochemically be detected only sporadically. Thus, at the sites investigated here, cellular hypertrophy plays an important role for tissue growth during pregnancy. Progesterone receptors, oestrogen receptor alpha and glucocorticoid receptors, however, exhibit cell type and pregnancy stage specific distribution patterns within the tissues assessed. Progesterone receptor immunoreactive scores remained fairly unchanged during pregnancy. Oestrogen receptor alpha scores, however, generally decreased and glucocorticoid receptors increased with ongoing gestation. Progesterone receptors and oestrogen receptor alpha were present in endometrial stroma and in myometrial smooth muscle cells during whole pregnancy. Oestrogen receptor alpha was detectable during whole pregnancy also in uterine glands. Progesterone receptors were, however, present at a very low level at the latter site only during months 1-3 and 6-9. Oestrogen receptor alpha and glucocorticoid receptors may also mediate uterine blood flow since they were present in the tunica media of uterine blood vessels

  16. Downregulation of Chicken Interleukin-17 Receptor A during Eimeria Infection

    PubMed Central

    Kim, Woo H.; Jeong, Jipseol; Park, Ae R.; Yim, Dongjean; Kim, Suk; Chang, Hong H.; Yang, Seung-Hak; Kim, Dong-Hee; Lillehoj, Hyun S.

    2014-01-01

    Both interleukin-17A (IL-17A) and IL-17F are proinflammatory cytokines that have an important role in intestinal homeostasis via receptor signaling. These cytokines have been characterized in chickens, but very little is known about their receptors and their functional activity. We provide here the first description of the sequence analysis, bioactivity, and comparative expression analysis of chicken IL-17RA (chIL-17RA) in chickens infected with Salmonella and Eimeria, two major infectious agents of gastrointestinal diseases of poultry of economic importance. A full-length chIL-17RA cDNA with a 2,568-bp coding region was identified from chicken thymus cDNA. chIL-17RA shares ca. 46% identity with mammalian homologues and 29.2 to 31.5% identity with its piscine counterparts. chIL-17RA transcript expression was relatively high in the thymus and in the chicken macrophage cell line HD11. The chIL-17RA-specific small interfering RNA inhibits interleukin-6 (IL-6), IL-8, and IL-1β mRNA expression in chicken embryo fibroblast cells (but not in DF-1 cells) stimulated with chIL-17A or chIL-17F. Interaction between chIL-17RA and chIL-17A was confirmed by coimmunoprecipitation. Downregulation of chIL-17RA occurred in concanavalin A- or lipopolysaccharide-activated splenic lymphocytes but not in poly(I·C)-activated splenic lymphocytes. In Salmonella- and Eimeria-infected chickens, the expression levels of the chIL-17RA transcript were downregulated in intestinal tissues from chickens infected with two Eimeria species, E. tenella or E. maxima, that preferentially infect the cecum and jejunum, respectively. However, chIL-17RA expression was generally unchanged in Salmonella infection. These results suggest that chIL-17RA has an important role in mucosal immunity to intestinal intracellular parasite infections such as Eimeria infection. PMID:24980970

  17. Downregulation of chicken interleukin-17 receptor A during Eimeria infection.

    PubMed

    Kim, Woo H; Jeong, Jipseol; Park, Ae R; Yim, Dongjean; Kim, Suk; Chang, Hong H; Yang, Seung-Hak; Kim, Dong-Hee; Lillehoj, Hyun S; Min, Wongi

    2014-09-01

    Both interleukin-17A (IL-17A) and IL-17F are proinflammatory cytokines that have an important role in intestinal homeostasis via receptor signaling. These cytokines have been characterized in chickens, but very little is known about their receptors and their functional activity. We provide here the first description of the sequence analysis, bioactivity, and comparative expression analysis of chicken IL-17RA (chIL-17RA) in chickens infected with Salmonella and Eimeria, two major infectious agents of gastrointestinal diseases of poultry of economic importance. A full-length chIL-17RA cDNA with a 2,568-bp coding region was identified from chicken thymus cDNA. chIL-17RA shares ca. 46% identity with mammalian homologues and 29.2 to 31.5% identity with its piscine counterparts. chIL-17RA transcript expression was relatively high in the thymus and in the chicken macrophage cell line HD11. The chIL-17RA-specific small interfering RNA inhibits interleukin-6 (IL-6), IL-8, and IL-1β mRNA expression in chicken embryo fibroblast cells (but not in DF-1 cells) stimulated with chIL-17A or chIL-17F. Interaction between chIL-17RA and chIL-17A was confirmed by coimmunoprecipitation. Downregulation of chIL-17RA occurred in concanavalin A- or lipopolysaccharide-activated splenic lymphocytes but not in poly(I·C)-activated splenic lymphocytes. In Salmonella- and Eimeria-infected chickens, the expression levels of the chIL-17RA transcript were downregulated in intestinal tissues from chickens infected with two Eimeria species, E. tenella or E. maxima, that preferentially infect the cecum and jejunum, respectively. However, chIL-17RA expression was generally unchanged in Salmonella infection. These results suggest that chIL-17RA has an important role in mucosal immunity to intestinal intracellular parasite infections such as Eimeria infection.

  18. Role of TATA-element in transcription from glucocorticoid receptor-responsive model promoters.

    PubMed Central

    Wieland, S; Schatt, M D; Rusconi, S

    1990-01-01

    Transcription activation properties of the rat glucocorticoid receptor (GR) on minimal, TATA-box containing or depleted promoters have been tested. We show that a cluster of Glucocorticoid Responsive Elements (GRE), upon activation by the GR, is sufficient to mediate abundant RNA-polymerase II transcription. We find that in absence of a bona fide TATA-element transcription initiates at a distance of 45-55bp from the activated GRE cluster with a marked preference for sequences homologous to the initiator element (Inr). Analyzing defined, bi-directional transcription units we demonstrate that the apparent reduction of specific transcription in strong, TATA-depleted promoters, is mainly due to loss of short-range promoter polarization. The implications for long-range promoter/enhancer communication mechanisms are also discussed. Images PMID:2402438

  19. 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

  20. The role of glucocorticoid receptor phosphorylation in Mcl-1 and NOXA gene expression

    PubMed Central

    2010-01-01

    Background The cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK) mediated phosphorylation of glucocorticoid receptor (GR) exerts opposite effects on GR transcriptional activity and affects other posttranslational modifications within this protein. The major phosphorylation site of human GR targeted by MAPK family is the serine 226 and multiple kinase complexes phosphorylate receptor at the serine 211 residue. We hypothesize that GR posttranslational modifications are involved in the determination of the cellular fate in human lymphoblastic leukemia cells. We investigated whether UV signalling through alternative GR phosphorylation determined the cell type specificity of glucocorticoids (GCs) mediated apoptosis. Results We have identified putative Glucocorticoid Response Elements (GREs) within the promoter regulatory regions of the Bcl-2 family members NOXA and Mcl-1 indicating that they are direct GR transcriptional targets. These genes were differentially regulated in CEM-C7-14, CEM-C1-15 and A549 cells by glucocorticoids and JNK pathway. In addition, our results revealed that the S211 phosphorylation was dominant in CEM-C7-14, whereas the opposite was the case in CEM-C1-15 where prevalence of S226 GR phosphorylation was observed. Furthermore, multiple GR isoforms with cell line specific patterns were identified in CEM-C7-14 cells compared to CEM-C1-15 and A549 cell lines with the same antibodies. Conclusions GR phosphorylation status kinetics, and site specificity as well as isoform variability differ in CEM-C7-14, CEM-C1-15, and A549 cells. The positive or negative response to GCs induced apoptosis in these cell lines is a consequence of the variable equilibrium of NOXA and Mcl-1 gene expression potentially mediated by alternatively phosphorylated GR, as well as the balance of MAPK/CDK pathways controlling GR phosphorylation pattern. Our results provide molecular base and valuable knowledge for improving the GC based therapies of

  1. Selective glucocorticoid receptor modulation inhibits cytokine responses in a canine model of mild endotoxemia.

    PubMed

    Bartko, Johann; Derhaschnig, Ulla; Neels, Tania; Nabozny, Gerald H; Harcken, Christian; Leuschner, Jost; De Vries, Frerich; Jilma, Bernd

    2017-09-18

    Selective glucocorticoid receptor modulators (GRMs) promise to reduce adverse events of glucocorticoids while maintaining anti-inflammatory potency. The present study tested the anti-inflammatory activity of two novel non-steroidal GRMs (GRM1: BI 607812 BS, GRM2: BI 653048 BS*H3PO4) in comparison to prednisolone in a canine model of low dose endotoxemia. This study compared the anti-inflammatory and pharmacokinetic profile of escalating daily oral doses of GRM1 (1, 2.5, 5 and 10mg/kg) and GRM2 (0.1, 0.25 and 1mg/kg) with prednisolone (0.25 and 0.5mg/kg) and placebo after intravenous infusion of endotoxin (0.1μg/kg) to Beagle dogs. This was followed by a 14-day evaluation study of safety and pharmacokinetics. Endotoxin challenge increased TNF-α ∼2000-fold and interleukin-6 (IL-6) 100-fold. Prednisolone and both GRMs suppressed peak TNF-α and IL-6 by 71-82% as compared with placebo. The highest doses of GRM1 and GRM2 reduced the mean body temperature increase by ∼30%. The endotoxin-induced rise in plasma cortisol was strongly suppressed in all treatment groups. Pharmacokinetics of both GRMs were non-linear. Adverse effects of endotoxemia such as vomiting were mitigated by GRM2 and prednisolone, indicating an antiemetic effect. During the 14-day treatment period, the adverse event profile of both GRMs appeared to be similar to prednisolone. Both GRMs had anti-inflammatory effects comparable to prednisolone and showed good safety profiles. Compounds targeting the glucocorticoid receptor selectively may provide an alternative to traditional glucocorticoids in the treatment of inflammatory disease. Copyright © 2017. Published by Elsevier Ltd.

  2. Glucocorticoid receptor blockade inhibits brain cell addition and aggressive signaling in electric fish, Apteronotus leptorhynchus.

    PubMed

    Dunlap, Kent D; Jashari, Denisa; Pappas, Kristina M

    2011-08-01

    When animals are under stress, glucocorticoids commonly inhibit adult neurogenesis by acting through glucocorticoid receptors (GRs). However, in some cases, conditions that elevate glucocorticoids promote adult neurogenesis, and the role of glucocorticoid receptors in these circumstances is not well understood. We examined the involvement of GRs in social enhancement of brain cell addition and aggressive signaling in electric fish, Apteronotus leptorhynchus. In this species, long-term social interaction simultaneously elevates plasma cortisol, enhances brain cell addition and increases production of aggressive electrocommunication signals ("chirps"). We implanted isolated and paired fish with capsules containing nothing (controls) or the GR antagonist, RU486, recorded chirp production and locomotion for 7d, and measured the density of newborn cells in the periventricular zone. Compared to isolated controls, paired controls showed elevated chirping in two phases: much higher chirp rates in the first 5h and moderately higher nocturnal rates thereafter. Treating paired fish with RU486 reduced chirp rates in both phases to those of isolated fish, demonstrating that GR activation is crucial for socially induced chirping. Neither RU486 nor social interaction affected locomotion. RU486 treatment to paired fish had a partial effect on cell addition: paired RU486 fish had less cell addition than paired control fish but more than isolated fish. This suggests that cortisol activation of GRs contributes to social enhancement of cell addition but works in parallel with another GR-independent mechanism. RU486 also reduced cell addition in isolated fish, indicating that GRs participate in the regulation of cell addition even when cortisol levels are low.

  3. A mixed glucocorticoid/mineralocorticoid receptor modulator dampens endocrine and hippocampal stress responsivity in male rats.

    PubMed

    Nguyen, Elizabeth T; Streicher, Joshua; Berman, Sarah; Caldwell, Jody L; Ghisays, Valentina; Estrada, Christina M; Wulsin, Aynara C; Solomon, Matia B

    2017-09-01

    Aberrant glucocorticoid secretion is implicated in the pathophysiology of stress-related disorders (i.e., depression, anxiety). Glucocorticoids exert biological effects via mineralocorticoid (MR) and glucocorticoid (GR) receptors. Previous data from our laboratory indicate that GR antagonism/modulation (i.e., mifepristone, CORT 108297) regulate endocrine, behavioral, and central stress responses. Because of the dynamic interplay between MR and GR on HPA axis regulation and emotionality, compounds targeting both receptors are of interest for stress-related pathology. We investigated the effects of CORT 118335 (a dual selective GR modulator/MR antagonist) on endocrine, behavioral, and central (c-Fos) stress responses in male rats. Rats were treated for five days with CORT 118335, imipramine (positive control), or vehicle and exposed to restraint or forced swim stress (FST). CORT 118335 dampened corticosterone responses to both stressors, without a concomitant antidepressant-like effect in the FST. Imipramine decreased corticosterone responses to restraint stress; however, the antidepressant-like effect of imipramine in the FST was independent of circulating glucocorticoids. These findings indicate dissociation between endocrine and behavioral stress responses in the FST. CORT 118335 decreased c-Fos expression only in the CA1 division of the hippocampus. Imipramine decreased c-Fos expression in the basolateral amygdala and CA1 and CA3 divisions of the hippocampus. Overall, the data indicate differential effects of CORT 118335 and imipramine on stress-induced neuronal activity in various brain regions. The data also highlight a complex relationship between neuronal activation in stress and mood regulatory brain regions and the ensuing impact on endocrine and behavioral stress responses. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. 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...

  5. 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...

  6. 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.

  7. Characterization of the angiotensin (AT1b) receptor promoter and its regulation by glucocorticoids

    PubMed Central

    Bogdarina, Irina G; King, Peter J; Clark, Adrian J L

    2009-01-01

    Angiotensin II acts through two pharmacologically distinct receptors known as AT1 and AT2. Duplication of the AT1 receptor in rodents into At1a and b subtypes allows tissue-specific expression of the AT1b in adrenal and pituitary tissue. Adrenal expression of this receptor is increased in the offspring of rat mothers exposed to a low-protein diet and this is associated with the undermethylation of its promoter. This phenomenon is blocked by the inhibition of maternal glucocorticoid synthesis by metyrapone. We have mapped the transcriptional start site of the promoter and demonstrated that a 1·2 kbp fragment upsteam of this site is effective in driving luciferase expression in mouse Y1 cells. A combination of bioinformatic analysis, electrophoretic mobility shift analysis (EMSA), and mutagenesis studies demonstrates: i) the presence of a putative TATA box and CAAT box; ii) the presence of three Sp1 response elements, capable of binding SP1; mutation of any pair of these sites effectively disables this promoter; iii) the presence of four potential glucocorticoid response elements which each bind glucocorticoid receptor in EMSA, although only two confer dexamethasone inhibition on the promoter; iv) the presence of two AP1 sites. Mutagenesis of the distal AP1 site greatly diminishes promoter function but this is also associated with the loss of dexamethasone inhibition. These studies will facilitate an understanding of the mechanisms by which fetal programming leads to long term alterations in gene expression and the development of adult disease. PMID:19411305

  8. REV-ERBα influences the stability and nuclear localization of the glucocorticoid receptor

    PubMed Central

    Okabe, Takashi; Chavan, Rohit; Fonseca Costa, Sara S.; Brenna, Andrea; Ripperger, Jürgen A.

    2016-01-01

    ABSTRACT REV-ERBα (encoded by Nr1d1) is a nuclear receptor that is part of the circadian clock mechanism and regulates metabolism and inflammatory processes. The glucocorticoid receptor (GR, encoded by Nr3c1) influences similar processes, but is not part of the circadian clock, although glucocorticoid signaling affects resetting of the circadian clock in peripheral tissues. Because of their similar impact on physiological processes, we studied the interplay between these two nuclear receptors. We found that REV-ERBα binds to the C-terminal portion and GR to the N-terminal portion of HSP90α and HSP90β, a chaperone responsible for the activation of proteins to ensure survival of a cell. The presence of REV-ERBα influences the stability and nuclear localization of GR by an unknown mechanism, thereby affecting expression of GR target genes, such as IκBα (Nfkbia) and alcohol dehydrogenase 1 (Adh1). Our findings highlight an important interplay between two nuclear receptors that influence the transcriptional potential of each other. This indicates that the transcriptional landscape is strongly dependent on dynamic processes at the protein level. PMID:27686098

  9. Structural Analysis on the Pathologic Mutant Glucocorticoid Receptor Ligand-Binding Domains

    PubMed Central

    Hurt, Darrell E.; Suzuki, Shigeru; Mayama, Takafumi; Charmandari, Evangelia

    2016-01-01

    Glucocorticoid receptor (GR) gene mutations may cause familial or sporadic generalized glucocorticoid resistance syndrome. Most of the missense forms distribute in the ligand-binding domain and impair its ligand-binding activity and formation of the activation function (AF)-2 that binds LXXLL motif-containing coactivators. We performed molecular dynamics simulations to ligand-binding domain of pathologic GR mutants to reveal their structural defects. Several calculated parameters including interaction energy for dexamethasone or the LXXLL peptide indicate that destruction of ligand-binding pocket (LBP) is a primary character. Their LBP defects are driven primarily by loss/reduction of the electrostatic interaction formed by R611 and T739 of the receptor to dexamethasone and a subsequent conformational mismatch, which deacylcortivazol resolves with its large phenylpyrazole moiety and efficiently stimulates transcriptional activity of the mutant receptors with LBP defect. Reduced affinity of the LXXLL peptide to AF-2 is caused mainly by disruption of the electrostatic bonds to the noncore leucine residues of this peptide that determine the peptide's specificity to GR, as well as by reduced noncovalent interaction against core leucines and subsequent exposure of the AF-2 surface to solvent. The results reveal molecular defects of pathologic mutant receptors and provide important insights to the actions of wild-type GR. PMID:26745667

  10. Clinical and biological significance of glucocorticoid receptor (GR) expression in breast cancer.

    PubMed

    Abduljabbar, Rezvan; Negm, Ola H; Lai, Chun-Fui; Jerjees, Dena A; Al-Kaabi, Methaq; Hamed, Mohamed R; Tighe, Patrick J; Buluwela, Lakjaya; Mukherjee, Abhik; Green, Andrew R; Ali, Simak; Rakha, Emad A; Ellis, Ian O

    2015-04-01

    The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily of transcription factors, which exerts anti-proliferative and anti-apoptotic activities. The GR is expressed in a large proportion of breast cancer (BC) although levels generally decrease during cancer progression. This study aimed to determine the clinical and biological significance of GR expression using a large series of early-stage BC with long-term follow-up and BC cell lines. Immunohistochemistry was used to assess the expression of GR in 999 cases of primary invasive BC prepared as tissue microarrays. Reverse phase protein microarray was used to assess the expression of GR in MCF7 and MDA-MB-231 cell lines. Nuclear expression of GR was observed in 61.6 % of breast tumours and was associated with features of good prognosis including smaller tumour size and lower grade with less pleomorphism and low mitotic count. GR expression was positively correlated with expression of oestrogen (ER) and progesterone receptors. In ER-positive tumours, GR was associated with other features of favourable outcome including FOXA1, GATA3 and BEX1 expression, while low GR expression was associated with high Ki67, p53 and CD71 expression. GR expression is associated with features of good outcome but does not provide prognostic information independent of size, stage and grade. Understanding the receptor and its effects on BC behaviour is essential for avoiding any unwanted effects from the use of glucocorticoids in routine oncology practice.

  11. REV-ERBα influences the stability and nuclear localization of the glucocorticoid receptor.

    PubMed

    Okabe, Takashi; Chavan, Rohit; Fonseca Costa, Sara S; Brenna, Andrea; Ripperger, Jürgen A; Albrecht, Urs

    2016-11-01

    REV-ERBα (encoded by Nr1d1) is a nuclear receptor that is part of the circadian clock mechanism and regulates metabolism and inflammatory processes. The glucocorticoid receptor (GR, encoded by Nr3c1) influences similar processes, but is not part of the circadian clock, although glucocorticoid signaling affects resetting of the circadian clock in peripheral tissues. Because of their similar impact on physiological processes, we studied the interplay between these two nuclear receptors. We found that REV-ERBα binds to the C-terminal portion and GR to the N-terminal portion of HSP90α and HSP90β, a chaperone responsible for the activation of proteins to ensure survival of a cell. The presence of REV-ERBα influences the stability and nuclear localization of GR by an unknown mechanism, thereby affecting expression of GR target genes, such as IκBα (Nfkbia) and alcohol dehydrogenase 1 (Adh1). Our findings highlight an important interplay between two nuclear receptors that influence the transcriptional potential of each other. This indicates that the transcriptional landscape is strongly dependent on dynamic processes at the protein level. © 2016. Published by The Company of Biologists Ltd.

  12. Screening of bisphenol A, triclosan and paraben analogues as modulators of the glucocorticoid and androgen receptor activities.

    PubMed

    Kolšek, Katra; Gobec, Martina; Mlinarič Raščan, Irena; Sollner Dolenc, Marija

    2015-02-01

    A homeostasis of the glucocorticoid and androgen endocrine system is essential to human health. Their disturbance can lead to various diseases, for example cardiovascular, inflammatory and autoimmune diseases, infertility, cancer. Fifteen widely used industrial chemicals that disrupt endocrine activity were selected for evaluation of potential (anti)glucocorticoid and (anti)androgenic activities. The human breast carcinoma MDA-kb2 cell line was utilized for reporter gene assays, since it expresses both the androgen and the glucocorticoid-responsive reporter. Two new antiandrogens, 4,4'-sulfonylbis(2-methylphenol) (dBPS) and 4,4'-thiodiphenol (THIO), and two new antiglucocorticoids, bisphenol Z and its analog bis[4-(2-hydroxyethoxy)phenyl] sulfone (BHEPS) were identified. Moreover, four new glucocorticoid agonists (methyl paraben, ethyl paraben, propyl paraben and bisphenol F) were found. To elucidate the structure-activity relationship of bisphenols, we performed molecular docking experiments with androgen and glucocorticoid receptor. These docking experiments had shown that bulky structures such as BHEPS and bisphenol Z act as antiglucocorticoid, because they are positioned toward helix H12 in the antagonist conformation and could therefore be responsible for H12 conformational change and the switch between agonistic and antagonistic conformation of receptor. On the other hand smaller structures cannot interact with H12. The results of in vitro screening of fifteen industrial chemicals as modulators of the glucocorticoid and androgen receptor activities demand additional in vivo testing of these chemicals for formulating any relevant hazard identification to human health.

  13. Delayed secondary glucocorticoid response elements. Unusual nucleotide motifs specify glucocorticoid receptor binding to transcribed regions of alpha 2u-globulin DNA.

    PubMed

    Chan, G C; Hess, P; Meenakshi, T; Carlstedt-Duke, J; Gustafsson, J A; Payvar, F

    1991-11-25

    Glucocorticoids stimulate the transcription of rat alpha 2u-globulin (RUG) genes. Because this induction occurs after a time lag of several hours and is blocked by inhibitors of protein synthesis, it exemplifies a delayed secondary response to steroid hormones. In this report, we show that a region of RUG-transcribed DNA (approximately +1800 to +2174) contains multiple footprint sites for glucocorticoid receptor that are, apparently, organized into at least three independent binding clusters. The DNA sequences bound by the receptor and the location of binding sites were determined. A family of sequences related to half-sites of the consensus primary glucocorticoid response element (GRE) is discernible at each cluster of sites. Compared to the consensus GRE, which contains two pseudo-palindromic hexanucleotides arranged in a tail-to-tail fashion and separated by three bases, the arrangements of hexanucleotides within this segment of RUG DNA are unusual and heterogeneous. Methylation interference of a binding cluster containing three receptor footprints demonstrates that certain guanines of the GRE-like hexanucleotides are essential for efficient receptor binding. A synthetic 29-base pair (bp) RUG element, containing one receptor footprint from this cluster, selectively binds the receptor. Within this 29-bp element, six nucleotides separate two directly repeated copies of GRE-like hexanucleotides. RUG DNA fragments containing all or part of the three binding clusters, including the 29-bp element, confer a delayed secondary hormone responsiveness upon a linked heterologous promoter and reporter gene in stably transfected cell lines. We speculate that the unusual DNA sequence motifs of the receptor-binding sites are crucial for the generation of certain delayed secondary responses.

  14. Subunit dissociation and activation of wild-type and mutant glucocorticoid receptors.

    PubMed

    Gehring, U; Mugele, K; Arndt, H; Busch, W

    1987-09-01

    Apparent molecular weights of wild-type and nti ('increased nuclear transfer') mutant glucocorticoid receptors were obtained from Stokes radii and sedimentation coefficients. At low salt concentrations molecular forms of Mr 328,000 and 298,000 of the wild-type and mutant, respectively, were predominant. Increasing ionic strength resulted in receptor dissociation. Dissociated forms of Mr 130,000 and 63,000 of the wild-type and mutant, respectively, were obtained at 300 mM KCl and above. Some metal oxi-anions prevented dissociation. Receptor activation to allow DNA binding produced the dissociated forms which could be separated from non-activated receptors by filtration through DNA-cellulose or by DEAE-cellulose chromatography. Non-activated wild-type and nti receptors eluted from DEAE-cellulose under identical conditions while activated wild-type and nti receptors eluted differently. Partially proteolyzed wild-type receptors behaved identically to nti receptors. We conclude that the large forms of wild-type and nti receptors are heteromeric and contain only one hormone-building polypeptide per complex.

  15. Retinoic acid increases glucocorticoid receptor phosphorylation via cyclin-dependent kinase 5.

    PubMed

    Brossaud, Julie; Roumes, Hélène; Helbling, Jean-Christophe; Moisan, Marie-Pierre; Pallet, Véronique; Ferreira, Guillaume; Biyong, Essi-Fanny; Redonnet, Anabelle; Corcuff, Jean-Benoît

    2017-07-01

    Glucocorticoid receptor (GR) function is modulated by phosphorylation. As retinoic acid (RA) can activate some cytoplasmic kinases able to phosphorylate GR, we investigated whether RA could modulate GR phosphorylation in neuronal cells in a context of long-term glucocorticoid exposure. A 4-day treatment of dexamethasone (Dex) plus RA, showed that RA potentiated the (Dex)-induced phosphorylation on GR Serine 220 (pSer220GR) in the nucleus of a hippocampal HT22 cell line. This treatment increased the cytoplasmic ratio of p35/p25 proteins, which are major CDK5 cofactors. Roscovitine, a pharmacological CDK5 inhibitor, or a siRNA against CDK5 prevented RA potentiation of GR phosphorylation. Furthermore, roscovitine counter-acted the effect of RA on GR sensitive target proteins such as BDNF or tissue-transglutaminase. These data help understanding the interaction between RA- and glucocorticoid-signalling pathways, both of which have strong influences on the adult brain. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Non-canonical Glucocorticoid Receptor Transactivation of gilz by Alcohol Suppresses Cell Inflammatory Response

    PubMed Central

    Ng, Hang Pong; Jennings, Scott; Wang, Jack; Molina, Patricia E.; Nelson, Steve; Wang, Guoshun

    2017-01-01

    Acute alcohol exposure suppresses cell inflammatory response. The underlying mechanism has not been fully defined. Here we report that alcohol was able to activate glucocorticoid receptor (GR) signaling in the absence of glucocorticoids (GCs) and upregulated glucocorticoid-induced leucine zipper (gilz), a prominent GC-responsive gene. Such a non-canonical activation of GR was not blocked by mifepristone, a potent GC competitor. The proximal promoter of gilz, encompassing five GC-responsive elements (GREs), was incorporated and tested in a luciferase reporter system. Deletion and/or mutation of the GREs abrogated the promoter responsiveness to alcohol. Thus, the GR–GRE interaction transduced the alcohol action on gilz. Alcohol induced GR nuclear translocation, which was enhanced by the alcohol dehydrogenase inhibitor fomepizole, suggesting that it was alcohol, not its metabolites, that engendered the effect. Gel mobility shift assay showed that unliganded GR was able to bind GREs and such interaction withstood clinically relevant levels of alcohol. GR knockout via CRISPR/Cas9 gene targeting or GILZ depletion via small RNA interference diminished alcohol suppression of cell inflammatory response to LPS. Thus, a previously unrecognized, non-canonical GR activation of gilz is involved in alcohol modulation of cell immune response. PMID:28638383

  17. Ligand-dependent genomic function of glucocorticoid receptor in triple-negative breast cancer.

    PubMed

    Chen, Zhong; Lan, Xun; Wu, Dayong; Sunkel, Benjamin; Ye, Zhenqing; Huang, Jiaoti; Liu, Zhihua; Clinton, Steven K; Jin, Victor X; Wang, Qianben

    2015-09-16

    Glucocorticoids (GCs) have been widely used as coadjuvants in the treatment of solid tumours, but GC treatment may be associated with poor pharmacotherapeutic response or prognosis. The genomic action of GC in these tumours is largely unknown. Here we find that dexamethasone (Dex, a synthetic GC)-regulated genes in triple-negative breast cancer (TNBC) cells are associated with drug resistance. Importantly, these GC-regulated genes are aberrantly expressed in TNBC patients and are associated with unfavourable clinical outcomes. Interestingly, in TNBC cells, Compound A (CpdA, a selective GR modulator) only regulates a small number of genes not involved in carcinogenesis and therapy resistance. Mechanistic studies using a ChIP-exo approach reveal that Dex- but not CpdA-liganded glucocorticoid receptor (GR) binds to a single glucocorticoid response element (GRE), which drives the expression of pro-tumorigenic genes. Our data suggest that development of safe coadjuvant therapy should consider the distinct genomic function between Dex- and CpdA-liganded GR.

  18. Evaluation of glucocorticoid receptor function in COPD lung macrophages using beclomethasone-17-monopropionate.

    PubMed

    Plumb, Jonathan; Robinson, Laura; Lea, Simon; Banyard, Antonia; Blaikley, John; Ray, David; Bizzi, Andrea; Volpi, Giorgina; Facchinetti, Fabrizio; Singh, Dave

    2013-01-01

    Previous studies of glucocorticoid receptor (GR) function in COPD lung macrophages have used dexamethasone to evaluate inhibition of cytokine production. We have now used the clinically relevant corticosteroid beclomethasone-17-monopropionate (17-BMP) to assess GR function in COPD lung macrophages, and investigated the transactivation of glucocorticoid sensitive genes and GR phosphorylation in addition to cytokine production. Lung macrophages were purified from surgically acquired lung tissue, from patients with COPD, smokers, and non-smokers. The transactivation of glucocorticoid sensitive genes (FKBP51 and GILZ) by 17-BMP were analysed by polymerase chain reaction. 17-BMP suppression of LPS-induced TNFα, IL-6 and CXCL8 was measured by ELISA and GR phosphorylation was measured by immunohistochemistry and Western blot. 17-BMP reduced cytokine release in a concentration dependent manner, with >70% inhibition of all cytokines, and no difference between COPD patients and controls. Similarly, the transactivation of FKBP51 and GILZ, and GR phosphorylation was similar between COPD patients and controls. In this context, GR function in COPD lung macrophages is unaltered. 17-BMP effectively suppresses cytokine production in COPD lung macrophages.

  19. New selective glucocorticoid receptor modulators reverse amyloid-β peptide-induced hippocampus toxicity.

    PubMed

    Pineau, Fanny; Canet, Geoffrey; Desrumaux, Catherine; Hunt, Hazel; Chevallier, Nathalie; Ollivier, Matthias; Belanoff, Joseph K; Givalois, Laurent

    2016-09-01

    In Alzheimer's disease (AD), cognitive deficits and psychological symptoms are associated with an early deregulation of the hypothalamic-pituitary-adrenal axis. Here, in an acute model of AD, we investigated if antiglucocorticoid strategies with selective glucocorticoid receptor (GR) modulators (CORT108297 and CORT113176) that combine antagonistic and agonistic GR properties could offer an interesting therapeutic approach in the future. We confirm the expected properties of the nonselective GR antagonist (mifepristone) because in addition to restoring basal circulating glucocorticoids levels, mifepristone totally reverses synaptic deficits and hippocampal apoptosis processes. However, mifepristone only partially reverses cognitive deficit, effects of the hippocampal amyloidogenic pathway, and neuroinflammatory processes, suggesting limits in its efficacy. By contrast, selective GR modulators CORT108297 and CORT113176 at a dose of 20 and 10 mg/kg, respectively, reverse hippocampal amyloid-β peptide generation, neuroinflammation, and apoptotic processes, restore the hippocampal levels of synaptic markers, re-establish basal plasma levels of glucocorticoids, and improve cognitive function. In conclusion, selective GR modulators are particularly attractive and may pave the way to new strategies for AD treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. 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

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

    PubMed

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

    2016-01-05

    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.

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

    PubMed

    Jacobson, Lauren

    2014-10-02

    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. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. The role of glucocorticoids in pregnancy, parturition, lactation, and nurturing in melanocortin receptor 2-deficient mice.

    PubMed

    Chida, Dai; Miyoshi, Keiko; Sato, Tsuyoshi; Yoda, Tetsuya; Kikusui, Takefumi; Iwakura, Yoichiro

    2011-04-01

    Maternal glucocorticoids are critical for fetal development, but overexpression can be deleterious. Previously we established a mouse line deficient in melanocortin receptor 2 (MC2R). MC2R(-/-) mice have undetectable levels of corticosterone despite high levels of ACTH and defects resembling those in patients with familial glucocorticoid deficiency. Here we analyzed the role of glucocorticoids in pregnancy, parturition, lactation, and nurturing in MC2R(-/-) mice. MC2R(-/-) mice were fertile and produced normal litters when crossed with MC2R(+/+) mice. However, MC2R(-/-) females crossed with MC2R(-/-) males had no live births, and approximately 20% of the embryos at d 18.5 of pregnancy were of normal body size but were dead when born. MC2R(-/-) pregnant females crossed with MC2R(+/+) males had detectable serum corticosterone levels, suggesting the transplacental passage of corticosterone from fetus to mother. MC2R(+/-) pups delivered from MC2R(-/-) females crossed with MC2R(+/+) males mice thrived poorly with MC2R(-/-) mothers but grew to adulthood when transferred to foster mothers after birth, suggesting that MC2R(-/-) females are poor mothers or cannot nurse. MC2R(-/-) females had normal alveoli, but penetration of mammary epithelium into fat pads and expression of milk proteins were reduced. Myoepithelial cells, which force milk out of the alveoli, were fully developed and differentiated. Pup retrieval behavior was normal in MC2R(-/-) mice. Exogenous corticosterone rescued expression of milk proteins in MC2R(-/-) mothers, and the pups of treated mothers grew to adulthood. Our results reveal the importance of glucocorticoids for fetal survival late in pregnancy, mammary gland development, and milk protein gene expression.

  4. 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

  5. Cytosolic glucocorticoid receptor interaction with nuclear factor-kappa B proteins in rat liver cells.

    PubMed

    Widén, Christina; Gustafsson, Jan-Ake; Wikström, Ann-Charlotte

    2003-07-01

    The glucocorticoid receptor (GR) acts as an anti-inflammatory factor. To a large extent, this activity is exerted by the interference of pro-inflammatory nuclear factor kappa B (NF-kappa B) activity. In their respective inactive forms, both GR and NF-kappa B reside in the cytoplasm and translocate to the nucleus on relevant stimulation. Previously, p65, a component of the NF-kappa B complex, and GR have been shown to interact physically in vitro, and the interaction is assumed to take place in the nucleus of cells [McKay and Cidlowski (1999) Endocrine Rev. 20, 435-459]. We have studied the interaction between GR and NF-kappa B using in vivo -like conditions. Using immunoaffinity chromatography or immunoprecipitation, combined with Western blotting, we observed that, with endogenous protein levels in cytosolic extracts of rat liver and of H4-II-E-C3 hepatoma cells and in contrast with the current belief, p65, p50 and inhibitory kappa B alpha complex interact with GR, even in the absence of glucocorticoid or an inflammatory signal. The interaction between non-liganded/non-activated GR and p65/p50 has also been verified by both p65 and p50 co-immunoprecipitations. Intracellular localization studies, using Western blotting, revealed that glucocorticoids can decrease tumour necrosis factor alpha (TNFalpha)-induced nuclear entry of p65, whereas glucocorticoid-induced GR translocation was much less affected by TNFalpha. We were also able to demonstrate a nuclear interaction of GR and p65 and p50 using in vivo -like protein concentrations. Furthermore, nuclear GR interaction with heat-shock protein 90 was enhanced distinctly by TNFalpha treatment. In conclusion, our studies suggest a strong interconnectivity between the NF-kappa B and GR-signalling pathways where also, somewhat unexpectedly, a physical interaction in the cytosol constitutes an integral part of GR-NF-kappa B cross-talk.

  6. Neuroendocrine Function After Hypothalamic Depletion of Glucocorticoid Receptors in Male and Female Mice.

    PubMed

    Solomon, Matia B; Loftspring, Matthew; de Kloet, Annette D; Ghosal, Sriparna; Jankord, Ryan; Flak, Jonathan N; Wulsin, Aynara C; Krause, Eric G; Zhang, Rong; Rice, Taylor; McKlveen, Jessica; Myers, Brent; Tasker, Jeffrey G; Herman, James P

    2015-08-01

    Glucocorticoids act rapidly at the paraventricular nucleus (PVN) to inhibit stress-excitatory neurons and limit excessive glucocorticoid secretion. The signaling mechanism underlying rapid feedback inhibition remains to be determined. The present study was designed to test the hypothesis that the canonical glucocorticoid receptors (GRs) is required for appropriate hypothalamic-pituitary-adrenal (HPA) axis regulation. Local PVN GR knockdown (KD) was achieved by breeding homozygous floxed GR mice with Sim1-cre recombinase transgenic mice. This genetic approach created mice with a KD of GR primarily confined to hypothalamic cell groups, including the PVN, sparing GR expression in other HPA axis limbic regulatory regions, and the pituitary. There were no differences in circadian nadir and peak corticosterone concentrations between male PVN GR KD mice and male littermate controls. However, reduction of PVN GR increased ACTH and corticosterone responses to acute, but not chronic stress, indicating that PVN GR is critical for limiting neuroendocrine responses to acute stress in males. Loss of PVN GR induced an opposite neuroendocrine phenotype in females, characterized by increased circadian nadir corticosterone levels and suppressed ACTH responses to acute restraint stress, without a concomitant change in corticosterone responses under acute or chronic stress conditions. PVN GR deletion had no effect on depression-like behavior in either sex in the forced swim test. Overall, these findings reveal pronounced sex differences in the PVN GR dependence of acute stress feedback regulation of HPA axis function. In addition, these data further indicate that glucocorticoid control of HPA axis responses after chronic stress operates via a PVN-independent mechanism.

  7. Neuroendocrine Function After Hypothalamic Depletion of Glucocorticoid Receptors in Male and Female Mice

    PubMed Central

    Loftspring, Matthew; de Kloet, Annette D.; Ghosal, Sriparna; Jankord, Ryan; Flak, Jonathan N.; Wulsin, Aynara C.; Krause, Eric G.; Zhang, Rong; Rice, Taylor; McKlveen, Jessica; Myers, Brent; Tasker, Jeffrey G.; Herman, James P.

    2015-01-01

    Glucocorticoids act rapidly at the paraventricular nucleus (PVN) to inhibit stress-excitatory neurons and limit excessive glucocorticoid secretion. The signaling mechanism underlying rapid feedback inhibition remains to be determined. The present study was designed to test the hypothesis that the canonical glucocorticoid receptors (GRs) is required for appropriate hypothalamic-pituitary-adrenal (HPA) axis regulation. Local PVN GR knockdown (KD) was achieved by breeding homozygous floxed GR mice with Sim1-cre recombinase transgenic mice. This genetic approach created mice with a KD of GR primarily confined to hypothalamic cell groups, including the PVN, sparing GR expression in other HPA axis limbic regulatory regions, and the pituitary. There were no differences in circadian nadir and peak corticosterone concentrations between male PVN GR KD mice and male littermate controls. However, reduction of PVN GR increased ACTH and corticosterone responses to acute, but not chronic stress, indicating that PVN GR is critical for limiting neuroendocrine responses to acute stress in males. Loss of PVN GR induced an opposite neuroendocrine phenotype in females, characterized by increased circadian nadir corticosterone levels and suppressed ACTH responses to acute restraint stress, without a concomitant change in corticosterone responses under acute or chronic stress conditions. PVN GR deletion had no effect on depression-like behavior in either sex in the forced swim test. Overall, these findings reveal pronounced sex differences in the PVN GR dependence of acute stress feedback regulation of HPA axis function. In addition, these data further indicate that glucocorticoid control of HPA axis responses after chronic stress operates via a PVN-independent mechanism. PMID:26046806

  8. Antenatal Hypoxia Induces Epigenetic Repression of Glucocorticoid Receptor and Promotes Ischemic-Sensitive Phenotype in the Developing Heart

    PubMed Central

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

    2016-01-01

    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

  9. 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.

  10. 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

  11. Glucocorticoid receptor Antagonist and siRNA Prevent Senescence of Human Bone Marrow Mesenchymal Stromal Cells in vitro

    PubMed Central

    Wei, Na; Yu, Yang; Joshi, Vijaya; Schmidt, Thomas; Qian, Fang; Salem, Aliasger K.; Stanford, Clark; Hong, Liu

    2016-01-01

    We investigated the effects mediated by glucocorticoid (GC) receptor (GR) blockage using RU486, a GR antagonist, and GR siRNA on the proliferative and differentiation capabilities of human bone marrow mesenchymal stromal/stem cells (MSCs), as well as on their senescence and antioxidant levels during extended in vitro culture. Treatment with either RU486 or GR siRNA for a 7 day period significantly increased the proliferation of MSCs as well as their osteogenic capabilities, as reflected by an increase in alkaline phosphatase (ALP) level after differentiation. After 4 weeks of the treatments MSCs improved or maintained their proliferation rates, while the control MSCs exhibited decreased proliferation. While all MSCs exhibited reduced osteogenic potential after 4 weeks of in vitro culture, the MSCs treated with GR inhibitors showed higher ALP levels than untreated MSCs after they were subjected to osteogenic differentiation. These treatment also significantly down-regulated the adipogenic capabilities of MSCs. Telomere lengths as well as the telomerase and superoxide dismutase activities of MSCs treated with either RU486 or GR siRNA appeared to be higher than those detected in controls. These results demonstrate that blockage of the effects mediated by the GCs normally found in fetal bovine serum may postpone senescence of these cells by up-regulating their antioxidant levels. These data suggested that blocking the effects mediated by GCs could potentially extend the lifespan of endogenous MSCs in patients who have elevated GC levels as a consequence of advancing age or estrogen depletion. PMID:23963647

  12. Distinct, genome-wide, gene-specific selectivity patterns of four glucocorticoid receptor coregulators.

    PubMed

    Wu, Dai-Ying; Ou, Chen-Yin; Chodankar, Rajas; Siegmund, Kimberly D; Stallcup, Michael R

    2014-01-01

    Glucocorticoids are a class of steroid hormones that bind to and activate the glucocorticoid receptor (GR), which then positively or negatively regulates transcription of many genes that govern multiple important physiological pathways such as inflammation and metabolism of glucose, fat and bone. The remodeling of chromatin and regulated assembly or disassembly of active transcription complexes by GR and other DNA-binding transcription factors is mediated and modulated by several hundred transcriptional coregulator proteins. Previous studies focusing on single coregulators demonstrated that each coregulator is required for regulation of only a subset of all the genes regulated by a steroid hormone. We hypothesized that the gene-specific patterns of coregulators may correspond to specific physiological pathways such that different coregulators modulate the pathway-specificity of hormone action, thereby providing a mechanism for fine tuning of the hormone response. We tested this by direct comparison of multiple coregulators, using siRNA to deplete the products of four steroid hormone receptor coregulator genes (CCAR1, CCAR2, CALCOCO1 and ZNF282). Global analysis of glucocorticoid-regulated gene expression after siRNA mediated depletion of coregulators confirmed that each coregulator acted in a selective and gene-specific manner and demonstrated both positive and negative effects on glucocorticoid-regulated expression of different genes. We identified several classes of hormone-regulated genes based on the effects of coregulator depletion. Each coregulator supported hormonal regulation of some genes and opposed hormonal regulation of other genes (coregulator-modulated genes), blocked hormonal regulation of a second class of genes (coregulator-blocked genes), and had no effect on hormonal regulation of a third gene class (coregulator-independent genes). In spite of previously demonstrated physical and functional interactions among these four coregulators, the majority

  13. Expression of glucocorticoid receptor and coactivators in ependymal cells of male rats.

    PubMed

    Iwata, Kinuyo; Ozawa, Hitoshi

    2014-08-29

    Glucocorticoid receptor (GR) is a ligand-activated nuclear receptor which is widely distributed in the brain. Many types of neurons and glial cells are known to express GR, but the expression of GR in ependymal cells has yet to be identified. The present study therefore was undertaken to determine whether ependymal cells express GR and coactivators of GR, such as steroid receptor coactivator 1 (SRC-1) and p300. GR immunoreactivity was found in cells immunopositive to vimentin, a marker of ependymal cells, around the third ventricle (3V), the lateral ventricle (LV), the cerebral aqueduct and the fourth ventricle (4V), whereas the expression of GR in vimentin-immunoreactive (ir) cells was significantly reduced by adrenalectomy (ADX) in male rats. Vimentin-ir cells also expressed both SRC-1 and p300 at around 3V, LV, the cerebral aqueduct and 4V. ADX had no effect on the expression of SRC-1 or p300 in vimentin-ir cells. These results suggest that glucocorticoid may exert effects on ependymal cells through binding to GR followed by association with SRC-1 and p300 to maintain brain environment under stressful conditions.

  14. Regulation of expanded polyglutamine protein aggregation and nuclear localization by the glucocorticoid receptor.

    PubMed

    Diamond, M I; Robinson, M R; Yamamoto, K R

    2000-01-18

    Spinobulbar muscular atrophy and Huntington's disease are caused by polyglutamine expansion in the androgen receptor and huntingtin, respectively, and their pathogenesis has been associated with abnormal nuclear localization and aggregation of truncated forms of these proteins. Here we show, in diverse cell types, that glucocorticoids can up- or down-modulate aggregation and nuclear localization of expanded polyglutamine polypeptides derived from the androgen receptor and huntingtin through specific regulation of gene expression. Wild-type glucocorticoid receptor (GR), as well as C-terminal deletion derivatives, suppressed the aggregation and nuclear localization of these polypeptides, whereas mutations within the DNA binding domain and N terminus of GR abolished this activity. Surprisingly, deletion of a transcriptional regulatory domain within the GR N terminus markedly increased aggregation and nuclear localization of the expanded polyglutamine proteins. Thus, aggregation and nuclear localization of expanded polyglutamine proteins are regulated cellular processes that can be modulated by a well-characterized transcriptional regulator, the GR. Our findings suggest approaches to study the molecular pathogenesis and selective neuronal degeneration of polyglutamine expansion diseases.

  15. Regulation of expanded polyglutamine protein aggregation and nuclear localization by the glucocorticoid receptor

    PubMed Central

    Diamond, Marc I.; Robinson, Melissa R.; Yamamoto, Keith R.

    2000-01-01

    Spinobulbar muscular atrophy and Huntington's disease are caused by polyglutamine expansion in the androgen receptor and huntingtin, respectively, and their pathogenesis has been associated with abnormal nuclear localization and aggregation of truncated forms of these proteins. Here we show, in diverse cell types, that glucocorticoids can up- or down-modulate aggregation and nuclear localization of expanded polyglutamine polypeptides derived from the androgen receptor and huntingtin through specific regulation of gene expression. Wild-type glucocorticoid receptor (GR), as well as C-terminal deletion derivatives, suppressed the aggregation and nuclear localization of these polypeptides, whereas mutations within the DNA binding domain and N terminus of GR abolished this activity. Surprisingly, deletion of a transcriptional regulatory domain within the GR N terminus markedly increased aggregation and nuclear localization of the expanded polyglutamine proteins. Thus, aggregation and nuclear localization of expanded polyglutamine proteins are regulated cellular processes that can be modulated by a well-characterized transcriptional regulator, the GR. Our findings suggest approaches to study the molecular pathogenesis and selective neuronal degeneration of polyglutamine expansion diseases. PMID:10639135

  16. Endogenous hepatic glucocorticoid receptor signaling coordinates sex-biased inflammatory gene expression

    PubMed Central

    Quinn, Matthew A.; Cidlowski, John A.

    2016-01-01

    An individual’s sex affects gene expression and many inflammatory diseases present in a sex-biased manner. Glucocorticoid receptors (GRs) are regulators of inflammatory genes, but their role in sex-specific responses is unclear. Our goal was to evaluate whether GR differentially regulates inflammatory gene expression in male and female mouse liver. Twenty-five percent of the 251 genes assayed by nanostring analysis were influenced by sex. Of these baseline sexually dimorphic inflammatory genes, 82% was expressed higher in female liver. Pathway analyses defined pattern-recognition receptors as the most sexually dimorphic pathway. We next exposed male and female mice to the proinflammatory stimulus LPS. Female mice had 177 genes regulated by treatment with LPS, whereas males had 149, with only 66% of LPS-regulated genes common between the sexes. To determine the contribution of GR to sexually dimorphic inflammatory genes we performed nanostring analysis on liver-specific GR knockout (LGRKO) mice in the presence or absence of LPS. Comparing LGRKO to GRflox/flox revealed that 36 genes required GR for sexually dimorphic expression, whereas 24 genes became sexually dimorphic in LGRKO. Fifteen percent of LPS-regulated genes in GRflox/flox were not regulated in male and female LGRKO mice treated with LPS. Thus, GR action is influenced by sex to regulate inflammatory gene expression.—Quinn, M. A., Cidlowski, J. A. Endogenous hepatic glucocorticoid receptor signaling coordinates sex-biased inflammatory gene expression. PMID:26581598

  17. Structural analysis of the evolution of steroid specificity in the mineralocorticoid and glucocorticoid receptors.

    PubMed

    Baker, Michael E; Chandsawangbhuwana, Charlie; Ollikainen, Noah

    2007-02-16

    The glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) evolved from a common ancestor. Still not completely understood is how specificity for glucocorticoids (e.g. cortisol) and mineralocorticoids (e.g. aldosterone) evolved in these receptors. Our analysis of several vertebrate GRs and MRs in the context of 3D structures of human GR and MR indicates that with the exception of skate GR, a cartilaginous fish, there is a deletion in all GRs, at the position corresponding to Ser-949 in human MR. This deletion occurs in a loop before helix 12, which contains the activation function 2 (AF2) domain, which binds coactivator proteins and influences transcriptional activity of steroids. Unexpectedly, we find that His-950 in human MR, which is conserved in the MR in chimpanzee, orangutan and macaque, is glutamine in all teleost and land vertebrate MRs, including New World monkeys and prosimians. Evolution of differences in the responses of the GR and MR to corticosteroids involved deletion in the GR of a residue corresponding to Ser-949 in human MR. A mutation corresponding to His-950 in human MR may have been important in physiological changes associated with emergence of Old World monkeys from prosimians.

  18. Synthesis and characterization of nonsteroidal glucocorticoid receptor modulators for multiple myeloma.

    PubMed

    Hudson, Andrew R; Roach, Steven L; Higuchi, Robert I; Phillips, Dean P; Bissonnette, Reid P; Lamph, William W; Yen, Jean; Li, Yongkai; Adams, Mark E; Valdez, Lino J; Vassar, Angie; Cuervo, Catalina; Kallel, E Adam; Gharbaoui, Catherine J; Mais, Dale E; Miner, Jeffrey N; Marschke, Keith B; Rungta, Deepa; Negro-Vilar, Andrés; Zhi, Lin

    2007-09-20

    Structure-activity relationship studies centered around 3'-substituted (Z)-5-(2'-(thienylmethylidene))1,2-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5H-chromeno[3,4-f]quinolines are described. A series of highly potent and efficacious selective glucocorticoid receptor modulators were identified with in vitro activity comparable to dexamethasone. In vivo evaluation of these compounds utilizing a 28 day mouse tumor xenograft model demonstrated efficacy equal to dexamethasone in the reduction of tumor volume.

  19. Novel ketal ligands for the glucocorticoid receptor: in vitro and in vivo activity.

    PubMed

    Smith, Cameron J; Ali, Amjad; Balkovec, James M; Graham, Donald W; Hammond, Milton L; Patel, Gool F; Rouen, Gregory P; Smith, Scott K; Tata, James R; Einstein, Monica; Ge, Lan; Harris, Georgianna S; Kelly, Theresa M; Mazur, Paul; Thompson, Chris M; Wang, Chuanlin F; Williamson, Joanne M; Miller, Douglas K; Pandit, Shilpa; Santoro, Joseph C; Sitlani, Ayesha; Yamin, Ting-Ting D; O'Neill, Edward A; Zaller, Dennis M; Carballo-Jane, Ester; Forrest, Michael J; Luell, Silvi

    2005-06-02

    A novel series of selective ligands for the human glucocorticoid receptor is described. Structure-activity studies focused on variation of B-ring size, ketal ring size, and ketal substitution. These analogs were found to be potent and selective ligands for GR and have partial agonist profiles in functional assays for transactivation (TAT, GS) and transrepression (IL-6). Of these compounds, 27, 28, and 35 were evaluated further in a mouse LPS-induced TNF-alpha secretion model. Compound 28 had an ED(50) of 14.1 mg/kg compared with 0.5 mg/kg for prednisolone in the same assay.

  20. 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.

  1. Ligand-induced repression of the glucocorticoid receptor gene is mediated by an NCoR1 repression complex formed by long-range chromatin interactions with intragenic glucocorticoid response elements.

    PubMed

    Ramamoorthy, Sivapriya; Cidlowski, John A

    2013-05-01

    Glucocorticoids are among the most potent and effective agents for treating inflammatory diseases and hematological cancers. However, subpopulations of patients are often resistant to steroid therapy, and determining the molecular mechanisms that contribute to glucocorticoid resistance is thus critical to addressing this clinical problem affecting patients with chronic inflammatory disorders. Since the cellular level of the glucocorticoid receptor (GR) is a critical determinant of glucocorticoid sensitivity and resistance, we investigated the molecular mechanisms mediating repression of glucocorticoid receptor gene expression. We show here that glucocorticoid-induced repression of GR gene expression is mediated by inhibition of transcription initiation. This process is orchestrated by the recruitment of agonist-bound GR to exon 6, followed by the assembly of a GR-NCoR1-histone deacetylase 3-containing repression complex at the transcriptional start site of the GR gene. A functional negative glucocorticoid response element (nGRE) in exon 6 of the GR gene and a long-range interaction occurring between this intragenic response element and the transcription start site appear to be instrumental in this repression. This autoregulatory mechanism of repression implies that the GR concentration can coordinate repression with excess ligand, regardless of the combinatorial associations of tissue-specific transcription factors. Consequently, the chronic nature of inflammatory conditions involving long-term glucocorticoid administration may lead to constitutive repression of GR gene transcription and thus to glucocorticoid resistance.

  2. Targeted ablation reveals a novel role of FKBP52 in gene-specific regulation of glucocorticoid receptor transcriptional activity.

    PubMed

    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.

  3. 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

  4. Requisite Role of Basolateral Amygdala Glucocorticoid Receptor Stimulation in Drug Context-Induced Cocaine-Seeking Behavior

    PubMed Central

    Stringfield, Sierra J.; Higginbotham, Jessica A.

    2016-01-01

    Background: Exposure to cocaine-associated stimuli triggers a robust rise in circulating glucocorticoid levels. Glucocorticoid receptors are richly expressed in the basolateral amygdala, a brain region that controls the reinstatement of cocaine-seeking behavior upon exposure to a previously cocaine-paired environmental context. In the present study, we investigated whether glucocorticoid receptor stimulation in the basolateral amygdala is integral to drug context-induced motivation to seek cocaine in a rat model of drug relapse. Methods: Rats were trained to lever press for cocaine reinforcement in a distinct environmental context and were then given daily extinction training sessions in a different context. At test, the rats received bilateral glucocorticoid receptor antagonist (mifepristone; 3 or 10ng/hemisphere) or vehicle microinfusions into either the basolateral amygdala or the overlying posterior caudate-putamen (anatomical control region). Immediately thereafter, drug-seeking behavior (i.e., nonreinforced lever presses) was assessed in the previously cocaine-paired context and locomotor activity was assessed in a novel context. Results: Intra-basolateral amygdala, but not intra-posterior caudate-putamen, mifepristone dose-dependently attenuated drug context-induced cocaine-seeking behavior relative to vehicle, such that responding was similar to that observed in the extinction context. In contrast, mifepristone treatment did not alter locomotor activity. Conclusions: These findings suggest that basolateral amygdala glucocorticoid receptor stimulation is necessary for drug context-induced motivation to seek cocaine. PMID:27521756

  5. Isoform switching of steroid receptor co-activator-1 attenuates glucocorticoid-induced anxiogenic amygdala CRH expression.

    PubMed

    Zalachoras, I; Verhoeve, S L; Toonen, L J; van Weert, L T C M; van Vlodrop, A M; Mol, I M; Meelis, W; de Kloet, E R; Meijer, O C

    2016-12-01

    Maladaptive glucocorticoid effects contribute to stress-related psychopathology. The glucocorticoid receptor (GR) that mediates many of these effects uses multiple signaling pathways. We have tested the hypothesis that manipulation of downstream factors ('coregulators') can abrogate potentially maladaptive GR-mediated effects on fear-motivated behavior that are linked to corticotropin releasing hormone (CRH). For this purpose the expression ratio of two splice variants of steroid receptor coactivator-1 (SRC-1) was altered via antisense-mediated 'exon-skipping' in the central amygdala of the mouse brain. We observed that a change in splicing towards the repressive isoform SRC-1a strongly reduced glucocorticoid-induced responsiveness of Crh mRNA expression and increased methylation of the Crh promoter. The transcriptional GR target gene Fkbp5 remained responsive to glucocorticoids, indicating gene specificity of the effect. The shift of the SRC-1 splice variants altered glucocorticoid-dependent exploratory behavior and attenuated consolidation of contextual fear memory. In conclusion, our findings demonstrate that manipulation of GR signaling pathways related to the Crh gene can selectively diminish potentially maladaptive effects of glucocorticoids.

  6. Dexamethasone enhances serum deprivation-induced necrotic death of rat C6 glioma cells through activation of glucocorticoid receptors.

    PubMed

    Morita, K; Ishimura, K; Tsuruo, Y; Wong, D L

    1999-01-23

    Glucocorticoids have been shown to be neurotoxic and appear to play a role in neuronal cell loss during aging and following neuropathological insults. However, very little is known about the effects of these steroid hormones on glial cells. The effect of the synthetic glucocorticoid dexamethasone (DEX) on glial cell viability was therefore examined by measuring neutral red uptake into rat C6 glioma cells. Serum deprivation markedly reduced cell viability, and this effect was significantly enhanced by DEX. Electrophoretic analysis showed that the cell damage induced by either serum deprivation alone or in combination with DEX was not accompanied by the degradation of DNA into nucleosomic fragments. Electron microscopic studies confirmed that serum deprivation and glucocorticoid treatment caused necrotic cell death. Furthermore, the effect of DEX on cell viability could be mimicked by the glucocorticoid receptor agonist RU28362, and completely prevented by the glucocorticoid receptor antagonist RU38486. These results indicate that dexamethasone can enhance the necrotic death of glioma cells induced by serum deprivation, suggesting that glucocorticoids may be involved in the chronic alteration of brain function arising from neuropathological damage to glial cells.

  7. Glucocorticoid Receptor, C/EBP, HNF3, and Protein Kinase A Coordinately Activate the Glucocorticoid Response Unit of the Carbamoylphosphate Synthetase I Gene

    PubMed Central

    Christoffels, Vincent M.; Grange, Thierry; Kaestner, Klaus H.; Cole, Timothy J.; Darlington, Gretchen J.; Croniger, Colleen M.; Lamers, Wouter H.

    1998-01-01

    A single far-upstream enhancer is sufficient to confer hepatocyte-specific, glucocorticoid- and cyclic AMP-inducible periportal expression to the carbamoylphosphate synthetase I (CPS) gene. To identify the mechanism of hormone-dependent activation, the composition and function of the enhancer have been analyzed. DNase I protection and gel mobility shift assays revealed the presence of a cyclic AMP response element, a glucocorticoid response element (GRE), and several sites for the liver-enriched transcription factor families HNF3 and C/EBP. The in vivo relevance of the transcription factors interacting with the enhancer in the regulation of CPS expression in the liver was assessed by the analysis of knockout mice. A strong reduction of CPS mRNA levels was observed in glucocorticoid receptor- and C/EBPα-deficient mice, whereas the CPS mRNA was normally expressed in C/EBPβ knockout mice and in HNF3α and -γ double-knockout mice. (The role of HNFβ could not be assessed, because the corresponding knockout mice die at embryonic day 10). In hepatoma cells, most of the activity of the enhancer is contained within a 103-bp fragment, which depends for its activity on the simultaneous occupation of the GRE, HNF3, and C/EBP sites, thus meeting the requirement of a glucocorticoid response unit. In fibroblast-like CHO cells, on the other hand, the GRE in the CPS enhancer does not cooperate with the C/EBP and HNF3 elements in transactivation of the CPS promoter. In both hepatoma and CHO cells, stimulation of expression by cyclic AMP depends mainly on the integrity of the glucocorticoid pathway, demonstrating cross talk between this pathway and the cyclic AMP (protein kinase A) pathway. PMID:9774647

  8. 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

  9. Noncoding RNA gas5 is a growth arrest- and starvation-associated repressor of the glucocorticoid receptor.

    PubMed

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

    2010-02-02

    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 because of 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 "riborepressor" of the GR, influencing cell survival and metabolic activities during starvation by modulating the transcriptional activity of the GR.

  10. Glucocorticoid receptors take part in the apoptotic process of human lens epithelial cells, but the glucocorticoid receptor antagonist RU486 does not rescue the cells fully.

    PubMed

    Wang, Lin; Zhao, Wencheng; Leng, Fei; Ge, Jinying; Bu, Zhigao; Zhang, Yi; Liu, Ping

    2011-06-01

    To identify an agent with specific activity against human lens epithelial cells (HLECs), we confirmed the presence of glucocorticoid receptors (GRs) and GR-α genes and evaluated whether GRs have a relationship with the apoptotic process in cultured HLECs. We also determined whether the inhibitor RU486 could rescue the cells from apoptosis when the HLECs were exposed to dexamethasone (Dex), a steroid, in 4 concentrations for 4 periods, or were co-treated with the antagonist RU486. We found that Dex, which has been used as a medical agent for a long time, resulted in increased expression of GRE-luciferase, the GR-α gene and GR-protein and, in contrast, decreased the viability of HLECs. The expression of Bax protein was increased in an earlier stage in contrast to the expression of Bcl-2 protein, which was increased in a later stage. Caspase-3 activity was significantly increased under lower concentrations of Dex in the last stage. The nuclear morphology of HLECs showed an obvious apoptotic phenomenon under greater concentrations of Dex in the last stage. However, RU486, a GR antagonist, could partially inhibit GR and Bax expressions and the expression of caspase-3 was increased so that there was not a decrease in the ratio of apoptotic cells and an increase in the viability of HLECs. Our data showed that GRs had a partial relationship to the apoptotic process of HLECs when exposed to Dex and RU486 did not rescue the cells fully. Because of its toxicity, RU486 did not provide a therapeutic benefit in a glucocorticoid induced cataract (GIC) for the in vitro model, however, its activity and pathway targeting should still be studied further with appropriate drug combinations.

  11. Post-Stress Combined Administration of Beta-Receptor and Glucocorticoid Antagonists as a Novel Preventive Treatment in an Animal Model of PTSD

    DTIC Science & Technology

    2009-07-01

    glucocorticoid receptors, propranolol, mifepristone 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME...PTSD (Stam, 2007). We could then administer a combination of the β-receptor antagonist propranolol and the glucocorticoid antagonist mifepristone ...antagonist, mifepristone , immediately after each MFS session In this first test of the combined drug intervention intended to attenuate

  12. Corticosteroid receptors and glucocorticoid content in microdissected brain regions: correlative aspects.

    PubMed

    Magariños, A M; Ferrini, M; De Nicola, A F

    1989-12-01

    Stereoselective competition was used to determine (3H)-aldosterone binding to type I corticosteroid receptors, and (3H)-dexamethasone binding to type II receptors in punches obtained from 11 brain regions of short-term adrenalectomized (ADX) rats. It was observed that type I receptor binding was almost exclusive of the hippocampus (HIPPO), while type II receptor binding was more generally distributed among HIPPO, cerebral cortex, lateral septum, ventromedial and arcuate hypothalamic nuclei, with lower levels in 6 additional regions studies. We determined corticosterone (CORT) in brain punches from ADX rats, ADX rats receiving CORT for 5 days, intact rats and intact rats receiving ACTH for 5 days. We correlated (3H)-ligand binding with CORT content in punches obtained from identical brain regions and showed a significant positive correlation in the case of the ADX plus CORT group, for type II corticosteroid receptors. Similarly, a significant correlation emerged with type II sites, when binding capacity was correlated with percentage increases of CORT in brain areas of rats receiving ACTH. It is suggested that in situations where CORT levels are elevated, changes in CORT retention throughout the brain occur as a function of the type II glucocorticoid receptor, although at the level of the HIPPO, both receptors may provide appropriate control of the CNS-pituitary-adrenal axis, according to the physiological or stress levels of circulating hormone.

  13. 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

  14. 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

  15. Effect of the glucocorticoid receptor antagonist RU486 on MK-801 induced behavioural sensitisation

    PubMed Central

    Lefevre, Emilia M.; Medley, Gregory A.; Reeks, Timothy; Alexander, Suzy; Burne, Thomas H. J.; Eyles, Darryl W.

    2017-01-01

    Stress is known to modulate sensitisation to repeated psychostimulant exposure. However, there is no direct evidence linking glucocorticoids and sensitisation achieved by repeated administration of the NMDA receptor antagonist MK-801. We tested the hypothesis that co-administration of RU486, a glucocorticoid receptor (GR) antagonist, prior to repeated daily MK-801 injections would block the expression of locomotor sensitisation due to its dual effects on corticosterone and dopamine. We employed a repeated MK-801 administration locomotor sensitisation paradigm in male Sprague Dawley rats. RU486 or a dimethyl sulfoxide (DMSO) vehicle was co-administered with MK-801 or saline during the induction phase. Subsequent to withdrawal, rats were challenged with MK-801 alone to test for the expression of sensitisation. In a separate cohort of rats, plasma corticosterone levels were quantified from blood samples taken on the 1st, 4th and 7th day of induction and at expression. One day after challenge, nucleus accumbens tissue levels of dopamine and its metabolites DOPAC and HVA were measured. During the induction phase, RU486 progressively enhanced locomotor sensitisation to MK-801. RU486 and MK-801 both showed stimulatory effects on corticosterone levels and this was further augmented when given in combination. Contrary to our hypothesis, RU486 did not block the expression of locomotor sensitisation to MK-801 and actually increased levels of dopamine, DOPAC and HVA in nucleus accumbens tissue. Our results showed that RU486 has augmentative rather than inhibitory effects on MK-801-induced sensitisation. This study indicates a divergent role for glucocorticoids in sensitisation to MK-801 compared to sensitisation with other psychostimulants. PMID:28430805

  16. Glucocorticoid receptor signaling is essential for mesoderm formation and muscle development in zebrafish.

    PubMed

    Nesan, Dinushan; Kamkar, Maryam; Burrows, Jeffrey; Scott, Ian C; Marsden, Mungo; Vijayan, Mathilakath M

    2012-03-01

    Glucocorticoid receptor (GR) signaling is thought to play a key role in embryogenesis, but its specific developmental effects remain unclear. Cortisol is the primary ligand for GR activation in teleosts, and in zebrafish (Danio rerio), the prehatch embryo content of this steroid is of maternal origin. Using early zebrafish developmental stages, we tested the hypothesis that GR signaling is critical for embryo growth and hatching. In zebrafish, maternal GR mRNA is degraded quickly, followed by zygotic synthesis of the receptor. GR protein is widely expressed throughout early development, and we were able to knockdown this protein using morpholino oligonucleotides. This led to a more than 70% reduction in mRNA abundance of matrix metalloproteinase-13 (mmp13), a glucocorticoid-responsive gene. The GR morphants displayed delayed somitogenesis, defects in somite and tail morphogenesis, reduced embryo size, and rarely survived after hatch. This correlated with altered expression of myogenic markers, including myogenin, myostatin, and muscle-specific myosin heavy chain and troponin genes. A key finding was a 70-90% reduction in the mRNA abundance of bone morphogenetic proteins (BMP), including bmp2a, bmp2b, and bmp4 in GR morphants. Bioinformatics analysis confirmed multiple putative glucocorticoid response elements upstream of these BMP genes. GR morphants displayed reduced expression of BMP-modulated genes, including eve1 and pax3. Zebrafish GR mRNA injection rescued the GR morphant phenotype and reversed the disrupted expression of BMP and myogenic genes. Our results for the first time indicate that GR signaling is essential for zebrafish muscle development, and we hypothesize a role for BMP morphogens in this process.

  17. Deletion of Mesenchymal Glucocorticoid Receptor Attenuates Embryonic Lung Development and Abdominal Wall Closure

    PubMed Central

    Stoner, Shihani; Tuckermann, Jan; Seibel, Markus; Zhou, Hong

    2013-01-01

    As a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors, the glucocorticoid receptor (GR) is essential for normal embryonic development. To date, the role of mesenchymal glucocorticoid signaling during development has not been fully elucidated. In the present study, we investigated the role of the GR during embryogenesis specifically in mesenchymal tissues. To this aim, we crossed GRflox mice with Dermo1-Cre mice to generate GRDermo1 mice, where the GR gene was deleted within mesenchymal cells. Compared to their wild type littermates, GRDermo1 mice displayed severe pulmonary atelectasis, defects in abdominal wall formation resulting in intestinal herniation, abnormal extracellular matrix synthesis in connective tissues and high postnatal lethality. Lungs of GRDermo1 mice failed to progress from the canalicular to saccular stage, as evidenced by the presence of immature air sacs, thickened interstitial mesenchyme and an underdeveloped vascular network between E17.5 and E18.5. Furthermore, myofibroblasts and vascular smooth muscle cells, although present in normal numbers in GRDermo1 animals, were characterized by significantly reduced elastin synthesis, whilst epithelial lining cells of the immature saccules were poorly differentiated. A marked reduction in normal elastin and collagen deposits were also observed in connective tissues adjacent to the umbilical hernia. This study demonstrates that eliminating the GR in cells of the mesenchymal lineage results in marked effects on interstitial fibroblast function, including a significant decrease in elastin synthesis. This results in lung atelectasis and postnatal lethality, as well as additional and hitherto unrecognized developmental defects in abdominal wall formation. In addition, altered glucocorticoid signaling in the mesenchyme attenuates normal lung epithelial differentiation. PMID:23696835

  18. Aminosulfhydryl and Aminodisulfide Compounds Enhance Binding of the Glucocorticoid Receptor Complex to Deoxyribonucleic Acid-Coated Cellulose and to Chromatin

    DTIC Science & Technology

    1993-01-01

    glucocorticoid receptor [21]. Diaminosulfhydryl chloroacetic acid was obtained from the Fisher compounds are more active at enhancing GRC Scientific...phase consisting of 0. I M BASE containing 25mM KCI and 3 mM chloroacetic acid and 5mM d/-10-camphorsul- MgCI2, pH 7.6 at 0 0C) was added to each tube...Enhance Binding of the Glucocorticoid Receptor Complex to Deoxy- ribonucleic Acid -Coated Cellulose and to Chromatin 4. AUThOR(S)’ J.M. Karle, R. Olmeda and

  19. Glucocorticoid receptors in primary cultures of mouse mammary epithelial cells: characterization and modulation by prolactin and cortisol

    SciTech Connect

    Schneider, W.; Shyamala, G.

    1985-06-01

    Mammary epithelial cells isolated from midpregant mice and cultured on collagen gels contain soluble glucocorticoid receptors. The kinetics of binding of dexamethasone reveal a saturable binding site (dissociation constant (K /sub d/), approximately 1 nM), and the binding site obeys a steroid specificity characteristic of a glucocorticoid receptor. As with the receptor isolated from intact glands, the receptor from the cultured cells also requires the addition of dithiothreitol for maximal binding of dexamethasone. The receptors are maintained at in vivo levels (approximately 1.3 pmol/mg DNA) for at least a period of 10 days in culture. However, the presence of both cortisol and PRL is required for the maintenance of the receptors, and the effect of both these hormones is dose dependent.

  20. 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

  1. 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

  2. Activated glucocorticoid receptor interacts with the INHAT component Set/TAF-Ibeta 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

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

    2008-02-13

    Set/template-activating factor (TAF)-Ibeta, part of the Set-Can oncogene product found in acute undifferentiated leukemia, is a component of the inhibitor of acetyltransferases (INHAT) complex. Set/TAF-Ibeta 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-Ibeta 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-Ibeta 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-Ibeta 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.

  3. Farnesyl pyrophosphate inhibits epithelialization and wound healing through the glucocorticoid receptor.

    PubMed

    Vukelic, Sasa; Stojadinovic, Olivera; Pastar, Irena; Vouthounis, Constantinos; Krzyzanowska, Agata; Das, Sharmistha; Samuels, Herbert H; Tomic-Canic, Marjana

    2010-01-15

    Farnesyl pyrophosphate (FPP), a key intermediate in the mevalonate pathway and protein farnesylation, can act as an agonist for several nuclear hormone receptors. Here we show a novel mechanism by which FPP inhibits wound healing acting as an agonist for glucocorticoid receptor (GR). Elevation of endogenous FPP by the squalene synthetase inhibitor zaragozic acid A (ZGA) or addition of FPP to the cell culture medium results in activation and nuclear translocation of the GR, a known wound healing inhibitor. We used functional studies to evaluate the effects of FPP on wound healing. Both FPP and ZGA inhibited keratinocyte migration and epithelialization in vitro and ex vivo. These effects were independent of farnesylation and indicate that modulation of FPP levels in skin may be beneficial for wound healing. FPP inhibition of keratinocyte migration and wound healing proceeds, in part, by repression of the keratin 6 gene. Furthermore, we show that the 3-hydroxy-3-methylglutaryl-CoA-reductase inhibitor mevastatin, which blocks FPP formation, not only promotes epithelialization in acute wounds but also reverses the effect of ZGA on activation of the GR and inhibition of epithelialization. We conclude that FPP inhibits wound healing by acting as a GR agonist. Of special interest is that FPP is naturally present in cells prior to glucocorticoid synthesis and that FPP levels can be further altered by the statins. Therefore, our findings may provide a better understanding of the pleiotropic effects of statins as well as molecular mechanisms by which they may accelerate wound healing.

  4. The glucocorticoid receptor type II complex is a target of the HIV-1 vpr gene product.

    PubMed Central

    Refaeli, Y; Levy, D N; Weiner, D B

    1995-01-01

    The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a 15-kDa virion-associated protein that functions as a regulator of cellular processes linked to the HIV life cycle. We report the interaction of a 41-kDa cytosolic viral protein R interacting protein 1 (Rip-1) with Vpr in vitro. Rip-1 displays a wide tissue distribution, including relevant targets of HIV infection. Vpr protein induced nuclear translocation of Rip-1, as did glucocorticoid receptor (GR)-II-stimulating steroids. Importantly, Vpr and Rip-1 coimmunoprecipitated with the human GR as part of an activated receptor complex. Vpr complementation of a vpr mutant virus was also mimicked by GR-II-stimulating steroids. Vpr and GR-II actions were inhibited by mifepristone, a GR-II pathway inhibitor. Together these data directly link the activity of the vpr gene product to the glucocorticoid steroid pathway and provide a biochemical mechanism for the cellular and viral activity of Vpr, as well as suggest that a unique class of antivirals, which includes mifepristone (RU486), may influence HIV-1 replication. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 PMID:7724608

  5. Glucocorticoid receptor represses proinflammatory genes at distinct steps of the transcription cycle.

    PubMed

    Gupte, Rebecca; Muse, Ginger W; Chinenov, Yurii; Adelman, Karen; Rogatsky, Inez

    2013-09-03

    Widespread anti-inflammatory actions of glucocorticoid hormones are mediated by the glucocorticoid receptor (GR), a ligand-dependent transcription factor of the nuclear receptor superfamily. In conjunction with its corepressor GR-interacting protein-1 (GRIP1), GR tethers to the DNA-bound activator protein-1 and NF-κB and represses transcription of their target proinflammatory cytokine genes. However, these target genes fall into distinct classes depending on the step of the transcription cycle that is rate-limiting for their activation: Some are controlled through RNA polymerase II (PolII) recruitment and initiation, whereas others undergo signal-induced release of paused elongation complexes into productive RNA synthesis. Whether these genes are differentially regulated by GR is unknown. Here we report that, at the initiation-controlled inflammatory genes in primary macrophages, GR inhibited LPS-induced PolII occupancy. In contrast, at the elongation-controlled genes, GR did not affect PolII recruitment or transcription initiation but promoted, in a GRIP1-dependent manner, the accumulation of the pause-inducing negative elongation factor. Consistently, GR-dependent repression of elongation-controlled genes was abolished specifically in negative elongation factor-deficient macrophages. Thus, GR:GRIP1 use distinct mechanisms to repress inflammatory genes at different stages of the transcription cycle.

  6. 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.

  7. The glucocorticoid receptor dimer interface allosterically transmits sequence-specific DNA signals.

    PubMed

    Watson, Lisa C; Kuchenbecker, Kristopher M; Schiller, Benjamin J; Gross, John D; Pufall, Miles A; Yamamoto, Keith R

    2013-07-01

    Glucocorticoid receptor (GR) binds to genomic response elements and regulates gene transcription with cell and gene specificity. Within a response element, the precise sequence to which the receptor binds has been implicated in directing its structure and activity. Here, we use NMR chemical-shift difference mapping to show that nonspecific interactions with bases at particular positions in the binding sequence, such as those of the 'spacer', affect the conformation of distinct regions of the rat GR DNA-binding domain. These regions include the DNA-binding surface, the 'lever arm' and the dimerization interface, suggesting an allosteric pathway that signals between the DNA-binding sequence and the associated dimer partner. Disrupting this pathway by mutating the dimer interface alters sequence-specific conformations, DNA-binding kinetics and transcriptional activity. Our study demonstrates that GR dimer partners collaborate to read DNA shape and to direct sequence-specific gene activity.

  8. 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.

  9. 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

  10. Blockade of glucocorticoid receptors with ORG 34116 does not normalize stress-induced symptoms in male tree shrews.

    PubMed

    Van Kampen, Marja; De Kloet, E Ronald; Flügge, Gabriele; Fuchs, Eberhard

    2002-12-20

    Glucocorticoid receptors play an important role in the regulation of the activity of the hypothalamo-pituitary-adrenal axis, and are thought to be involved in the pathophysiology of depressive disorders. The present study investigated the effect of the specific glucocorticoid receptor antagonist ORG 34116 (a substituted 11,21 bisarylsteroid compound) in the tree shrew (Tupaia belangeri) chronic psychosocial stress model, an established animal model for depressive disorders. Animals were stressed for 10 days before treatment with ORG 34116 started (25 mg/kg p.o. for 28 days). Stress induced a decrease in body weight, which just failed significance, whereas ORG 34116 did not affect body weight in stress and control animals. ORG 34116 enhanced the stress-induced increase in the concentration of urinary-free cortisol, although no differences between the different experimental groups existed during the last week of treatment. In stressed animals, ORG 34116 did not affect marking behavior, but decreased locomotor activity. Post mortem analysis of 5-HT(1A) receptors revealed a decreased affinity of 3[H]-8-OH-DPAT (3[H]-8-hydroxy-2-[di-n-propylamino]tetralin) binding sites in the hippocampus of animals treated with the glucocorticoid receptor antagonist. In conclusion, under our experimental conditions, the glucocorticoid receptor antagonist ORG 34116 did not normalize the depressive-like symptoms in the psychosocial stress model of male tree shrews. This finding, however, does not exclude that specific central, neuroendocrine and behavioral features are affected by the compound.

  11. Glucocorticoids and the Cardiovascular System.

    PubMed

    Goodwin, Julie E

    2015-01-01

    Glucocorticoids affect the developing and mature cardiovascular system in profound and, at times, contradictory ways. The glucocorticoid receptor is ubiquitous in most cell types and conserved across species, highlighting its importance in development and homeostasis. Despite the fact that the glucocorticoid receptor is widely expressed, tissue-specific effects of glucocorticoids may have pronounced effects on whole organism phenotypes. Here we will review the interactions between glucocorticoids and the cardiovascular system.

  12. Association between reduced expression of hippocampal glucocorticoid receptors and cognitive dysfunction in a rat model of traumatic brain injury due to lateral head acceleration.

    PubMed

    Gao, Wei; Xu, Hongyu; Liang, Ming; Huang, Jason H; He, Xiaosheng

    2013-01-15

    Expression of hippocampal glucocorticoid receptors (GRs) and spatial learning and memory were observed in rat model of diffuse traumatic brain injury (TBI) due to lateral head acceleration with an aim at investigating the relation between GRs expression and cognitive deficits. Immunohistochemical staining, Western blotting, and RT-PCR indicated that down-regulation of GRs expression occurred in the hippocampus among TBI-rats which demonstrated reduced performance of learning and memory in Morris water maze. As the GRs expression bounced up, the cognitive function approached to normal. It is concluded that reduced expression of hippocampal GRs was closely associated with learning and memory deficits in TBI-rats. Hippocampal GRs was involved in the biochemical mechanisms of cognitive deficits after TBI. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  13. 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. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  14. 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.

  15. Distribution of glucocorticoid receptors and 11β-hydroxysteroid dehydrogenase isoforms in the human inner ear.

    PubMed

    Kumagami, Hidetaka; Terakado, Mariko; Takahashi, Haruo

    2013-01-01

    Glucocorticoids (GCs) are widely used as a therapeutic modality for the inner ear disorders including Ménière's disease (MD). The concentration of GCs in the target cells is known to be regulated by 11β-hydroxysteroid dehydrogenase (11β-HSD), an enzyme complex responsible for the conversion of hormonally active cortisol into inactive cortisone. There is no morphologic indication of glucocorticoid receptors (GRs) and 11β-HSD isoforms (11β-HSD1 and 2) in human inner ear. The objectives of this study are to determine whether GRs and the isoforms of 11β-HSD are present in human inner ear tissues and to reveal their precise distribution. This study investigated the expression of GRs and 11β-HSD isoforms (11β-HSD1 and 2) in the human inner ear. In humans, immunostaining of GRs, 11β-HSD1, and 11β-HSD2 was performed in the stria vascularis (SV) and the vestibular tissues, whereas in the cochlear tissues except for the SV, only GRs were investigated. Immunoreactivity of GRs was detected in the SV, outer hair cells, inner hair cell, spiral ligament, Reissner's membrane, vestibular hair cells, vestibular nerve, transitional cells, and dark cells of the crista ampullaris. 11β-HSD1 was observed in the SV, the apical area of the vestibular hair cells, the transitional cells, and the dark cells. However, no immunoreactivity of 11β-HSD2 was observed. Those data indicate that different local steroid regulation by GRs and the isoforms of 11β-HSD is present in various parts of the human inner ear tissues and that the tissues are a direct therapeutic target of glucocorticoids in the inner ear diseases.

  16. Shear stress causes nuclear localization of endothelial glucocorticoid receptor and expression from the GRE promoter.

    PubMed

    Ji, Julie Y; Jing, Huiyan; Diamond, Scott L

    2003-02-21

    We tested the hypothesis that steady laminar shear stress activates the glucocorticoid receptor (GR) and its transcriptional signaling pathway in an effort to investigate the potential involvement of GR in shear stress-induced antiatherosclerosis actions in the vasculature. In both bovine aortic endothelial cells (BAECs) and NIH3T3 cells expressing GFP-GR chimeric protein, wall shear stress of 10 or 25 dynes/cm2 caused a marked nuclear localization of GFP-GR within 1 hour to an extent comparable to induction with 25 micromol/L dexamethasone. The shear mediated nuclear localization of GFP-GR was significantly reduced by 25 micromol/L of the MEK1 inhibitor (PD098059) or the PI 3-kinase inhibitor (LY294002). Also, Western blots demonstrated translocation of endogenous GR into nucleus of sheared BAECs. Promoter construct studies using glucocorticoid response element (GRE)-driven expression of secreted alkaline phosphatase (SEAP) indicated that BAECs exposed to shear stress of 10 and 25 dynes/cm2 for 8 hours produced >9-fold more SEAP (n=6; P<0.005) than control cells, a level comparable to that observed with dexamethasone. Shear stress enhanced SEAP expression at 6 hours was reduced 50% (n=5; P<0.005) by MEK1/2 or PI 3-kinase inhibitors, but not by the NO inhibitor, L-NAME. Finally, in human internal mammary artery, endothelial GR is found to be highly nuclear localized. We report a new shear responsive transcriptional element, GRE. The finding that hemodynamic forces can be as potent as high dose glucocorticoid steroid in activating GR and GRE-regulated expression correlates with the atheroprotective responses of endothelial cells to unidirectional arterial shear stress.

  17. A genetically encoded indicator for assaying bioactive chemicals that induce nuclear transport of glucocorticoid receptor.

    PubMed

    Kim, Sung Bae; Ozawa, Takeaki; Umezawa, Yoshio

    2005-12-15

    Glucocorticoids, the adrenal steroid hormones secreted during stress, are essential to homeostasis and metabolism in the human body. An impaired glucocorticoid signaling due to dysfunction of the glucocorticoid receptor (GR) by synthetic chemicals can cause diseases and disruptions of the homeostasis and metabolism. Here we demonstrate the development of a method for screening endocrine-disrupting chemicals and potent risk factors of human diseases based on the nuclear trafficking of the GR. We constructed a new assay using a pair of genetic indicators with the full length of the GR, split Renilla luciferase (RLuc), and split DnaE (a protein splicing element). The GR-containing fusion protein with C-terminal halves of DnaE and RLuc is localized in cytosol due to the cytosolic character of the GR, whereas the fusion protein with N-terminal halves of DnaE and RLuc stays in the nucleus due to the cofused nucleus localization signal. On being stimulated with a ligand, the GR is translocated into the cellular nucleus. Thus, a protein splicing occurs in the nucleus by an interaction between the splicing junctions of each DnaE fragment. The enzymatic activities from the reconstituted RLuc allow the ligand-dependent luminescence intensities. The feasibility of the method was evaluated by quantifying the hormonal activities of 20 different kinds of steroids and synthetic chemicals using the NIH 3T3 cells carrying the pair of indicators. The hormonal activities of tested ligands are discussed based on the chemical structure-activity relationship. We found that androgens, testosterone, and 19-nortestosterone weakly induce the nuclear transport of the GR. The current assay allows high-throughput screening of risk chemicals and drug candidates influential to a signal transduction pathway of the GR.

  18. Expression of glucocorticoid receptors α and ß in steroid sensitive and steroid insensitive interstitial lung diseases

    PubMed Central

    Pujols, L; Xaubet, A; Ramirez, J; Mullol, J; Roca-Ferrer, J; Torrego, A; Cidlowski, J; Picado, C

    2004-01-01

    Background: Sensitivity to glucocorticoids may be related to the concentration of glucocorticoid receptors α (GRα) and ß (GRß). A study was undertaken to assess GRα and GRß expression in steroid insensitive interstitial lung disease (idiopathic pulmonary fibrosis (IPF)) and steroid sensitive interstitial lung diseases (sarcoidosis and cryptogenic organising pneumonia (COP)). Methods: Lung tissue was obtained from control subjects and from patients with IPF, sarcoidosis, and COP. Pulmonary function tests were carried out at the time of lung biopsy and every 3 months. GRα and GRß expression was evaluated by both competitive RT-PCR and immunohistochemistry. Data are presented as median and 25–75th percentile. Results: GRα mRNA expression (105 cDNA copies/µg total RNA) was higher in patients with steroid sensitive interstitial lung diseases (10.0; 7.8–14.9; n = 11) than in patients with IPF (4.4; 3.2–6.6; n = 19; p<0.001). GRß expression was at least 1000 times lower than that of GRα and did not differ between the three groups. A negative correlation was found between GRα mRNA levels and the fibrotic pathology score of the tissue (r = –0.484, p<0.01) and a positive correlation was found between GRα mRNA levels and improvement in forced vital capacity (r = 0.633; p<0.01) after treatment of patients with glucocorticoids. Immunoreactivity for GR protein was also higher in patients with sarcoidosis and COP than in those with IPF. Conclusion: The variable response of some interstitial lung diseases to steroid treatment may be the result of differences in the expression of GRα. PMID:15282390

  19. Ligand structural motifs can decouple glucocorticoid receptor transcriptional activation from target promoter occupancy.

    PubMed

    Blind, Raymond D; Pineda-Torra, Inés; Xu, Yong; Xu, H Eric; Garabedian, Michael J

    2012-04-20

    Glucocorticoid (GC) induction of the tyrosine aminotransferase (TAT) gene by the glucocorticoid receptor (GR) is a classic model used to investigate steroid-regulated gene expression. Classic studies analyzing GC-induction of the TAT gene demonstrated that despite having very high affinity for GR, some steroids cannot induce maximal TAT enzyme activity, but the molecular basis for this phenomenon is unknown. Here, we used RT-PCR and chromatin immunoprecipitation to determine TAT mRNA accumulation and GR recruitment to the TAT promoter (TAT-GRE) in rat hepatoma cells induced by seven GR ligands: dexamethasone (DEX), cortisol (CRT), corticosterone (CCS), 11-deoxycorticosterone (DOC), aldosterone (ALD), progesterone (PRG) and 17-hydroxyprogesterone (17P). As expected, DEX, CRT, CCS and ALD all induced both TAT mRNA and GR recruitment to the TAT-GRE, while PRG and 17P did not. However, while DOC could not induce significant TAT mRNA, it did induce robust GR occupancy of the TAT-GRE. DOC also induced recruitment of the histone acetyltransferase p300 to the TAT-GRE as efficiently as DEX. These DOC-induced effects recapitulated at another GR target gene (sulfonyltransferase 1A1), and DOC also failed to promote the multiple changes in gene expression required for glucocorticoid-dependent 3T3-L1 adipocyte differentiation. Structural simulations and protease sensitivity assays suggest that DOC and DEX induce different conformations in GR. Thus, although steroids that bind GR with high affinity can induce GR and p300 occupancy of target promoters, they may not induce a conformation of GR capable of activating transcription.

  20. Adenosine Receptor Stimulation Improves Glucocorticoid-Induced Osteoporosis in a Rat Model.

    PubMed

    Pizzino, Gabriele; Irrera, Natasha; Galfo, Federica; Oteri, Giacomo; Atteritano, Marco; Pallio, Giovanni; Mannino, Federica; D'Amore, Angelica; Pellegrino, Enrica; Aliquò, Federica; Anastasi, Giuseppe P; Cutroneo, Giuseppina; Squadrito, Francesco; Altavilla, Domenica; Bitto, Alessandra

    2017-01-01

    Glucocorticoid-induced osteoporosis (GIO) is a secondary cause of bone loss. Bisphosphonates approved for GIO, might induce jaw osteonecrosis; thus additional therapeutics are required. Adenosine receptor agonists are positive regulators of bone remodeling, thus the efficacy of adenosine receptor stimulation for treating GIO was tested. In a preventive study GIO was induced in Sprague-Dawley rats by methylprednisolone (MP) for 60 days. Animals were randomly assigned to receive polydeoxyribonucleotide (PDRN), an adenosine A2 receptor agonist, or PDRN and DMPX (3,7-dimethyl-1-propargylxanthine, an A2 antagonist), or vehicle (0.9% NaCl). Another set of animals was used for a treatment study, following the 60 days of MP-induction rats were randomized to receive (for additional 60 days) PDRN, or PDRN and DMPX (an adenosine A2 receptor antagonist), or zoledronate (as control for gold standard treatment), or vehicle. Control animals were administered with vehicle for either 60 or 120 days. Femurs were analyzed after treatments for histology, imaging, and breaking strength analysis. MP treatment induced severe bone loss, the concomitant use of PDRN prevented the developing of osteoporosis. In rats treated for 120 days, PDRN restored bone architecture and bone strength; increased b-ALP, osteocalcin, osteoprotegerin and stimulated the Wnt canonical and non-canonical pathway. Zoledronate reduced bone resorption and ameliorated the histological features, without significant effects on bone formation. Our results suggest that adenosine receptor stimulation might be useful for preventing and treating GIO.

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

    PubMed Central

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

    2016-01-01

    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

  2. BclI polymorphism of the glucocorticoid receptor gene is associated with increased bone resorption in patients on glucocorticoid replacement therapy.

    PubMed

    Koetz, Kathrin R; van Rossum, Elisabeth F C; Ventz, Manfred; Diederich, Sven; Quinkler, Marcus

    2013-06-01

    Patients with primary adrenal insufficiency (PAI) and patients with congenital adrenal hyperplasia (CAH) receive weight-adapted standard glucocorticoid replacement therapy. Clinically, some patients appear more sensitive to therapeutic administration of glucocorticoids than others. Glucocorticoid sensitivity is at least partially genetically determined by polymorphisms of the glucocorticoid receptor (GR) and might influence bone mineral density (BMD). To determine if bone turnover markers and BMD are associated with the GR gene polymorphism BclI in patients with PAI and CAH. A prospective, cross-sectional study including 74 PAI and 38 CAH patients. BMD was evaluated by DXA. Serum levels of bone turnover markers, minerals, vitamins and hormones, and urinary crosslinks were measured. Patients carrying the homozygous BclI polymorphism (GG) had significantly higher serum β-CrossLaps (0.37 ± 0.34 μg/l; P < 0.05) and urinary collagen crosslinks (NTX, 68.1 ± 32.4 nmol/g; P < 0.005) despite receiving the lowest average daily hydrocortisone dose of 9.9 ± 3.7 mg/m(2) (P < 0.05). The GG genotype occurred significantly more frequently in patients with increased NTX (OR=6.7, 95% CI = 1.78-25.38) than in patients with normal NTX. However, BMD was not significantly different between different allelic variants. No significant differences in associations of the genotypes with outcomes (or in clinical characteristics) were found between the sexes. Although the sample sizes were relatively small and the results should be interpreted with caution, this study suggests that the homozygous (GG) genotype may be associated with higher bone resorption in adult PAI and CAH patients. GG-carriers needed a lower hydrocortisone dose on average supporting the concept that this GR variant is associated with increased cortisol sensitivity. © 2012 John Wiley & Sons Ltd.

  3. The BclI polymorphism of the glucocorticoid receptor gene is associated with emotional memory performance in healthy individuals.

    PubMed

    Ackermann, Sandra; Heck, Angela; Rasch, Björn; Papassotiropoulos, Andreas; de Quervain, Dominique J-F

    2013-07-01

    Glucocorticoids, stress hormones released from the adrenal cortex, are important players in the regulation of emotional memory. Specifically, in animals and in humans, glucocorticoids enhance memory consolidation of emotionally arousing experiences, but impair memory retrieval. These glucocorticoid actions are partly mediated by glucocorticoid receptors in the hippocampus, amygdala and prefrontal cortex, key brain regions for emotional memory. In a recent study in patients who underwent cardiac surgery, the BclI polymorphism of the glucocorticoid receptor gene (NR3C1) was associated with traumatic memories and posttraumatic stress disorder symptoms after intensive care therapy. Based on this finding, we investigated if the BclI polymorphism is also associated with emotional memory in healthy young subjects (N=841). We used a picture-learning task consisting of learning and recalling neutral and emotional photographs on two consecutive days. The BclI variant was associated with short-delay recall of emotional pictures on both days, with GG carriers showing increased emotional memory performance as compared to GC and CC carriers. We did not detect a genotype-dependent difference in recall performance for neutral pictures. These findings suggest that the Bcll polymorphism contributes to inter-individual differences in emotional memory also in healthy humans.

  4. Glucocorticoid receptor activation lowers the threshold for NMDA-receptor-dependent homosynaptic long-term depression in the hippocampus through activation of voltage-dependent calcium channels.

    PubMed

    Coussens, C M; Kerr, D S; Abraham, W C

    1997-07-01

    The effects of the glucocorticoid receptor agonist RU-28362 on homosynaptic long-term depression (LTD) were examined in hippocampal slices obtained from adrenal-intact adult male rats. Field excitatory postsynaptic potentials were evoked by stimulation of the Schaffer collateral/commissural pathway and recorded in stratum radiatum of area CA1. Low-frequency stimulation (LFS) was delivered at LTD threshold (2 bouts of 600 pulses, 1 Hz, at baseline stimulation intensity). LFS of the Schaffer collaterals did not produce significant homosynaptic LTD in control slices. However, identical conditioning in the presence of the glucocorticoid receptor agonist RU-28362 (10 microM) produced a robust LTD, which was blocked by the selective glucocorticoid antagonist RU-38486. The LTD induced by glucocorticoid receptor activation was dependent on N-methyl-D-aspartate (NMDA) receptor activity, because the specific NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (D-AP5) blocked the facilitation. However, the facilitation of LTD was not due to a potentiation of the isolated NMDA receptor potential by RU-28362. The facilitation of LTD by RU-28362 was also blocked by coincubation of the L-type voltage-dependent calcium channel (VDCC) antagonist nimodipine. Selective activation of the L-type VDCCs by the agonist Bay K 8644 also facilitated LTD induction. Both nimodipine and D-AP5 were effective in blocking the facilitation of LTD by Bay K 8644. These results indicate that L-type VDCCs can contribute to NMDA-receptor-dependent LTD induction.

  5. Subchronic glucocorticoid receptor inhibition rescues early episodic memory and synaptic plasticity deficits in a mouse model of Alzheimer's disease.

    PubMed

    Lanté, Fabien; Chafai, Magda; Raymond, Elisabeth Fabienne; Pereira, Ana Rita Salgueiro; Mouska, Xavier; Kootar, Scherazad; Barik, Jacques; Bethus, Ingrid; Marie, Hélène

    2015-06-01

    The early phase of Alzheimer's disease (AD) is characterized by hippocampus-dependent memory deficits and impaired synaptic plasticity. Increasing evidence suggests that stress and dysregulation of the hypothalamo-pituitary-adrenal (HPA) axis, marked by the elevated circulating glucocorticoids, are risk factors for AD onset. How these changes contribute to early hippocampal dysfunction remains unclear. Using an elaborated version of the object recognition task, we carefully monitored alterations in key components of episodic memory, the first type of memory altered in AD patients, in early symptomatic Tg2576 AD mice. We also combined biochemical and ex vivo electrophysiological analyses to reveal novel cellular and molecular dysregulations underpinning the onset of the pathology. We show that HPA axis, circadian rhythm, and feedback mechanisms, as well as episodic memory, are compromised in this early symptomatic phase, reminiscent of human AD pathology. The cognitive decline could be rescued by subchronic in vivo treatment with RU486, a glucocorticoid receptor antagonist. These observed phenotypes were paralleled by a specific enhancement of N-Methyl-D-aspartic acid receptor (NMDAR)-dependent LTD in CA1 pyramidal neurons, whereas LTP and metabotropic glutamate receptor-dependent LTD remain unchanged. NMDAR transmission was also enhanced. Finally, we show that, as for the behavioral deficit, RU486 treatment rescues this abnormal synaptic phenotype. These preclinical results define glucocorticoid signaling as a contributing factor to both episodic memory loss and early synaptic failure in this AD mouse model, and suggest that glucocorticoid receptor targeting strategies could be beneficial to delay AD onset.

  6. Lose dose genistein inhibits glucocorticoid receptor and ischemic brain injury in female rats.

    PubMed

    Shi, Rengfei; Wang, Shunli; Qi, Xiang; Chen, Si; Chen, Peijie; Zhang, Quanguang

    2014-01-01

    Although acute bolus of genistein treatment has been shown to protect against neuronal damage in experimental brain injury animal models, chronic continuous low dose treatment of genistein on ischemic brain injury has not been well elucidated. In the present study, female rats were received either pure genistein (0.1mg/kg/day via osmotic minipumps) or placebo at the time of ovariectomy, and transient forebrain ischemia was induced 7days later. Results demonstrated that genistein treatment for 14days significantly improved ischemic neuronal survival in hippocampal CA1 region of ovariectomized rats. Glucocorticoid receptor (GR) is a member of the adrenal steroid hormone receptor, which is highly expressed in the rat hippocampus. Activation of the GR plays a critical role in the neuronal stress responses, including ischemic brain damage. This study therefore examined the potential mechanisms by which genistein regulates GR signaling, including the protein distribution and receptor activation in hippocampus following ischemic reperfusion (I/R). Results showed that GR expression in the ovariectomized rats was excessively increased both in neurons (I/R 6h) and activated microglial cells (I/R 7d) in hippocampal CA1 region. Genistein treatment significantly attenuated GR induction and the enhanced GR nuclear translocation and DNA-binding capacity. The effects of genistein on the GR levels was accompanied with decreased blood plasma levels of corticosterone (primary glucocorticoid in rodents) and coupled to an E3 ubiquitin ligase Mdm2 targeted proteasomal degradation of GR, because genistein treatment could enhance the GR-Mdm2 interaction and the ubiquitination level of GR protein. In addition, our results indicated that genistein markedly prevented the excessive activation of microglia in CA1 sector. These results demonstrate the neuroprotective action of chronic low dose genistein replacement against ischemic brain damage, and a potential mechanism associated with the

  7. In vitro activation of rat cardiac glucocorticoid antagonist- versus agonist-receptor complexes.

    PubMed

    Schmidt, T J; Diehl, E E

    1988-06-30

    The synthetic antiglucocorticoid RU 38486 interacts with cardiac cytoplasmic glucocorticoid receptors and competes for in vitro binding with the potent agonist triamcinolone acetonide. In addition to binding to receptors with high affinity, RU 38486 also facilitates the in vitro conformational change in the receptor which is a consequence of the physiologically relevant activation step during which the receptor is converted from a non DNA- to a DNA-binding form. This ability of RU 38486 to promote receptor activation is reflected by both the appropriate shift in the elution profile of [3H]RU 38486-receptor complexes from DEAE-cellulose as well as by an increased binding of these complexes to DNA-cellulose. Although less effective than triamcinolone acetonide, RU 38486 promotes in vitro receptor activation under a variety of experimental conditions, including incubation of labeled cardiac cytosols at 25 degrees C for 30 min or at 15 degrees C for 30 min in the presence of 5 mM pyridoxal 5'-phosphate. Once thermally activated, the cardiac [3H]triamcinolone acetonide and [3H]RU 38486-receptor complexes bind to nonspecific DNA-cellulose with the same relative affinities, as evidenced by the fact that 50% of both activated complexes are eluted at approx. 215-250 mM NaCl. Thus, this pure antiglucocorticoid does promote, at least to some extent, many of the crucial in vitro events including high-affinity binding, activation, and DNA binding which have been shown to be required to elicit a physiological response in vivo.

  8. Effects of a glucocorticoid receptor agonist, dexamethasone, on fathead minnow reproduction, growth, and development.

    PubMed

    LaLone, Carlie A; Villeneuve, Daniel L; Olmstead, Allen W; Medlock, Elizabeth K; Kahl, Michael D; Jensen, Kathleen M; Durhan, Elizabeth J; Makynen, Elizabeth A; Blanksma, Chad A; Cavallin, Jenna E; Thomas, Linnea M; Seidl, Sara M; Skolness, Sarah Y; Wehmas, Leah C; Johnson, Rodney D; Ankley, Gerald T

    2012-03-01

    Synthetic glucocorticoids are pharmaceutical compounds prescribed in human and veterinary medicine as anti-inflammatory agents and have the potential to contaminate natural watersheds via inputs from wastewater treatment facilities and confined animal-feeding operations. Despite this, few studies have examined the effects of this class of chemicals on aquatic vertebrates. To generate data to assess potential risk to the aquatic environment, we used fathead minnow 21-d reproduction and 29-d embryo-larvae assays to determine reproductive toxicity and early-life-stage effects of dexamethasone. Exposure to 500 µg dexamethasone/L in the 21-d test caused reductions in fathead minnow fecundity and female plasma estradiol concentrations and increased the occurrence of abnormally hatched fry. Female fish exposed to 500 µg dexamethasone/L also displayed a significant increase in plasma vitellogenin protein levels, possibly because of decreased spawning. A decrease in vitellogenin messenger ribonucleic acid (mRNA) expression in liver tissue from females exposed to the high dexamethasone concentration lends support to this hypothesis. Histological results indicate that a 29-d embryo-larval exposure to 500 µg dexamethasone/L caused a significant increase in deformed gill opercula. Fry exposed to 500 µg dexamethasone/L for 29 d also exhibited a significant reduction in weight and length compared with control fry. Taken together, these results indicate that nonlethal concentrations of a model glucocorticoid receptor agonist can impair fish reproduction, growth, and development.

  9. Effect of glucocorticoid receptor gene polymorphisms in Guillain-Barré syndrome.

    PubMed

    Dekker, Marieke J H J; van den Akker, Erica L T; Koper, Jan Willem; Manenschijn, Laura; Geleijns, Karin; Ruts, Liselotte; van Rijs, Wouter; Tio-Gillen, Anne P; van Doorn, Pieter A; Lamberts, Steven W J; Jacobs, Bart C

    2009-06-01

    Guillain-Barré syndrome (GBS) is a postinfectious immune-mediated polyneuroradiculopathy in which host factors influence disease susceptibility and clinical course. Single-nucleotide polymorphisms (SNPs) in the glucocorticoid receptor (GR) gene influence the sensitivity to glucocorticoids and are related to both microbial colonization and susceptibility to develop auto-immune disease. This genetic variation may therefore also influence the chance to develop GBS. In this study, we genotyped 318 GBS patients and 210 control subjects for five known SNPs in the GR gene. We could distinguish six different GR haplotypes of which two carried the BclI polymorphism: haplotype 1, which consists of the minor allele of BclI in combination with the common variant of TthIIII and haplotype 2, which carries the minor allele of BclI as well as the minor allele of TthIIII. The GR haplotypes were not related to susceptibility to develop GBS. Carriers of haplotype 2 had more frequently preceding diarrhea, serum antibodies to GM1 and GD1a, and more severe muscle weakness at entry. Haplotype 1 carriers had a significantly better prognosis. In conclusion, GR haplotypes are not a susceptibility factor for GBS. However, haplotypes carrying the minor allele of the BclI polymorphism were related to the phenotype and outcome of GBS.

  10. Arsenic alters the function of the glucocorticoid receptor as a transcription factor.

    PubMed Central

    Kaltreider, R C; Davis, A M; Lariviere, J P; Hamilton, J W

    2001-01-01

    Chronic human exposure to nonovertly toxic doses of arsenic is associated with an increased risk of cancer. Although its carcinogenic mechanism is still unknown, arsenic does not directly cause DNA damage or mutations and is therefore thought to act principally as a co-mutagen, co-carcinogen, and/or tumor promoter. Previous studies in our laboratory demonstrated that effects of low-dose arsenic (III) (arsenite) on expression of the hormone-regulated phosphoenolpyruvate carboxykinase (PEPCK) gene were strongly associated with the glucocorticoid receptor (GR)-mediated regulatory pathway. We therefore examined specifically the effects of arsenite on the biochemical function of GR in hormone-responsive H4IIE rat hepatoma cells. Completely noncytotoxic arsenite treatments (0.3-3.3 microM) significantly decreased dexamethasone-induced expression of transiently transfected luciferase constructs containing either an intact hormone-responsive promoter from the mammalian PEPCK gene or two tandem glucocorticoid response elements (GRE). Western blotting and confocal microscopy of a green fluorescent protein-tagged-GR fusion protein demonstrated that arsenite pretreatment did not block the normal dexamethasone-induced nuclear translocation of GR. These data indicate that nontoxic doses of arsenite can interact directly with GR complexes and selectively inhibit GR-mediated transcription, which is associated with altered nuclear function rather than a decrease in hormone-induced GR activation or nuclear translocation. PMID:11333185

  11. Maturation and maintenance of cholinergic medial septum neurons require glucocorticoid receptor signaling.

    PubMed

    Guijarro, Christian; Rutz, Susanne; Rothmaier, Katharina; Turiault, Marc; Zhi, Qixia; Naumann, Thomas; Frotscher, Michael; Tronche, Francois; Jackisch, Rolf; Kretz, Oliver

    2006-05-01

    Glucocorticoids have been shown to influence trophic processes in the nervous system. In particular, they seem to be important for the development of cholinergic neurons in various brain regions. Here, we applied a genetic approach to investigate the role of the glucocorticoid receptor (GR) on the maturation and maintenance of cholinergic medial septal neurons between P15 and one year of age by using a mouse model carrying a CNS-specific conditional inactivation of the GR gene (GRNesCre). The number of choline acetyltransferase and p75NTR immuno-positive neurons in the medial septum (MS) was analyzed by stereology in controls versus mutants. In addition, cholinergic fiber density, acetylcholine release and cholinergic key enzyme activity of these neurons were determined in the hippocampus. We found that in GRNesCre animals the number of medial septal cholinergic neurons was significantly reduced during development. In addition, cholinergic cell number further decreased with aging in these mutants. The functional GR gene is therefore required for the proper maturation and maintenance of medial septal cholinergic neurons. However, the loss of cholinergic neurons in the medial septum is not accompanied by a loss of functional cholinergic parameters of these neurons in their target region, the hippocampus. This pinpoints to plasticity of the septo-hippocampal system, that seems to compensate for the septal cell loss by sprouting of the remaining neurons.

  12. Anti- trachea inflammatory effects of diosgenin from Dioscorea nipponica through interactions with glucocorticoid receptor α.

    PubMed

    Junchao, Yang; Zhen, Wang; Yuan, Wang; Liying, Xu; Libin, Jiang; Yuanhong, Zhu; Wei, Zhao; Ruilin, Chen; Lu, Zhai

    2017-02-01

    Asthma is a heterogeneous disease characterized by symptoms of chronic inflammation and airway structural and functional changes. It affects about 300 million people worldwide and causes 250 000 deaths annually, but its symptoms can be greatly relieved by regular use of inhaled glucocorticoids (GCs). GCs exert their function through interacting with glucocorticoid receptors (GRs). Diosgenin is a naturally occurring steroidal saponin abundantly present in many medicinal plants, including Dioscorea nipponica, which shares a similar steroidal structure with GC. In this study, ovalbumin (OVA)-induced asthmatic mice and primary tracheal epithelial cells (TECs) were used as research models. ELISAs were applied to measure the secretion of TNF-α, IL-1β, and IL-6, while quantitative PCR and western blotting were applied to evaluate expression of GRs SLPI, TTP, GILZ, MKP-1, and NF-κB. Our data demonstrated that diosgenin suppressed the secretion of TNF-α, IL-1β, and IL-6 by enhancing the expression of GRs, SLPI, GILZ, and MKP-1, and inhibiting the expression of HSP70. These data provide some evidence on the molecular mechanism of diosgenin, which might facilitate its clinical applications.

  13. Glucocorticoid Receptor β Stimulates Akt1 Growth Pathway by Attenuation of PTEN*

    PubMed Central

    Stechschulte, Lance A.; Wuescher, Leah; Marino, Joseph S.; Hill, Jennifer W.; Eng, Charis; Hinds, Terry D.

    2014-01-01

    Glucocorticoids (GCs) are known inhibitors of proliferation and are commonly prescribed to cancer patients to inhibit tumor growth and induce apoptosis via the glucocorticoid receptor (GR). Because of alternative splicing, the GR exists as two isoforms, GRα and GRβ. The growth inhibitory actions of GCs are mediated via GRα, a hormone-induced transcription factor. The GRβ isoform, however, lacks helix 12 of the ligand-binding domain and cannot bind GCs. While we have previously shown that GRβ mRNA is responsive to insulin, the role of GRβ in insulin signaling and growth pathways is unknown. In the present study, we show that GRβ suppresses PTEN expression, leading to enhanced insulin-stimulated growth. These characteristics were independent of the inhibitory qualities that have been reported for GRβ on GRα. Additionally, we found that GRβ increased phosphorylation of Akt basally, which was further amplified following insulin treatment. In particular, GRβ specifically targets Akt1 in growth pathways. Our results demonstrate that the GRβ/Akt1 axis is a major player in insulin-stimulated growth. PMID:24817119

  14. Glucocorticoid attenuates brain-derived neurotrophic factor-dependent upregulation of glutamate receptors via the suppression of microRNA-132 expression.

    PubMed

    Kawashima, H; Numakawa, T; Kumamaru, E; Adachi, N; Mizuno, H; Ninomiya, M; Kunugi, H; Hashido, K

    2010-02-17

    Brain-specific microRNAs (miRs) may be involved in synaptic plasticity through the control of target mRNA translation. Brain-derived neurotrophic factor (BDNF) also contributes to the regulation of synaptic function. However, the possible involvement of miRs in BDNF-regulated synaptic function is poorly understood. Importantly, an increase in glucocorticoid levels and the downregulation of BDNF are supposed to be involved in the pathophysiology of depressive disorders. Previously, we reported that glucocorticoid exposure inhibited BDNF-regulated synaptic function via weakening mitogen-activated protein kinase/extracellular signal-regulated kinase1/2 (MAPK/ERK) and/or phospholipase C-gamma (PLC-gamma) intracellular signaling in cultured neurons [Kumamaru et al (2008) Mol Endocrinol 22:546-558; Numakawa et al (2009) Proc Natl Acad Sci U S A 106:647-652]. Therefore, in this study, we investigate the possible influence of glucocorticoid on BDNF/miRs-stimulated biological responses in cultured cortical neurons. Significant upregulation of miR-132 was caused by BDNF, although miR-9, -124, -128a, -128b, -134, -138, and -16 were intact. Transfection of exogenous ds-miR-132 induced marked upregulation of glutamate receptors (NR2A, NR2B, and GluR1), suggesting that miR-132 has a positive effect on the increase in postsynaptic proteins levels. Consistently, transfection of antisense RNA to inhibit miR-132 function decreased the BDNF-dependent increase in the expression of postsynaptic proteins. U0126, an inhibitor of the MAPK/ERK pathway, suppressed the BDNF-increased miR-132, suggesting that BDNF upregulates miR-132 via the MAPK/ERK1/2 pathway. Interestingly, pretreatment with glucocorticoid (dexamethasone, DEX) reduced BDNF-increased ERK1/2 activation, miR-132 expression, and postsynaptic proteins. We demonstrate that the exposure of neurons to an excess glucocorticoid results in a decrease in the BDNF-dependent neuronal function via suppressing miR-132 expression.

  15. Effects of glucocorticoid receptor antagonist, RU486, on the proliferative and differentiation capabilities of bone marrow mesenchymal stromal cells in ovariectomized rats.

    PubMed

    Wei, Na; Yu, Yang; Schmidt, Thomas; Stanford, Clark; Hong, Liu

    2013-05-01

    Glucocorticoids (GCs) potentially regulate the proliferation, differentiation, and premature senescence of bone marrow mesenchymal stem/stromal cells (MSCs). In the present study we investigated the effects mediated by endogenous GCs and the effects of an antagonist of the glucocorticoid receptor, RU486, on the proliferative and differentiation capabilities of MSCs using an ovariectomized (OVX) animal model. Following ovariectomy and a decrease in systemic estradiol levels, the serum concentration of corticosterone is significantly increased in OVX rats. Compared to sham-operated controls, the total superoxide dismutase (SOD) activity in serum of OVX rats and the proliferation of their MSCs are significantly reduced. Furthermore, the osteogenic differentiation capabilities of OVX rat MSCs are significantly decreased, while adipogenic capabilities tend to increase. Subcutaneous administration of RU486 effectively increases the population and proliferative capacity of the MSCs in OVX rats. RU486 treatment also improves osteogenic capabilities and down-regulates adipogenic capabilities of MSCs. These results strongly indicate that the elevated levels of endogenous GCs induced by estrogen depletion might accelerate the premature senescence of MSCs and reduce their proliferative and osteogenic differentiation capabilities, while the blockage of the effects of endogenous GCs may restore their capabilities. These responses could potentially be developed to protect the capabilities of MSCs from oxidative stress-induced premature senescence and extend their lifespan in patients with advancing age and estrogen depletion.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Nuclear export of the glucocorticoid receptor is accelerated by cell fusion-dependent release of calreticulin.

    PubMed

    Walther, Rhian F; Lamprecht, Claudia; Ridsdale, Andrew; Groulx, Isabelle; Lee, Stephen; Lefebvre, Yvonne A; Haché, Robert J G

    2003-09-26

    Nucleocytoplasmic exchange of nuclear hormone receptors is hypothesized to allow for rapid and direct interactions with cytoplasmic signaling factors. In addition to recycling between a naïve, chaperone-associated cytoplasmic complex and a liganded chaperone-free nuclear form, the glucocorticoid receptor (GR) has been observed to shuttle between nucleus and cytoplasm. Nuclear export of GR and other nuclear receptors has been proposed to depend on direct interactions with calreticulin, which is predominantly localized to the lumen of the endoplasmic reticulum. We show that rapid calreticulin-mediated nuclear export of GR is a specific response to transient disruption of the endoplasmic reticulum that occurs during polyethylene glycol-mediated cell fusion. Using live and digitonin-permeabilized cells we demonstrate that, in the absence of cell fusion, GR nuclear export occurs slowly over a period of many hours independent of direct interaction with calreticulin. Our findings temper expectations that nuclear receptors respond rapidly and directly to cytoplasmic signals in the absence of additional regulatory control. These results highlight the importance of verifying findings of nucleocytoplasmic trafficking using techniques in addition to heterokaryon cell fusion.

  1. Glucocorticoid--receptor interactions. Studies of the negative co-operativity induced by steroid interactions with a secondary, hydrophobic, binding site.

    PubMed Central

    Jones, T R; Bell, P A

    1980-01-01

    The effects of steroids on the binding of [1,2-3H]dexamethasone and [1,2-3H]progesterone to the glucocorticoid receptor of rat thymus cytosol were studied. Although both glucocorticoid agonists and antagonists competed with [1,2-3H]dexamethasone for binding to the receptor under equilibrium conditions, only glucocorticoid antagonists of partial agonists, at micromolar concentrations, were capable of accelerating the rate of dissociation of previously bound [1,2-3H]dexamethasone from the receptor. Antagonists or partial agonists also enhanced the rate of dissociation of [1,2-3H]progesterone from the glucocorticoid receptor, with identical specificity and concentration--response characteristics. These effects are attributed to the presence on the receptor of a secondary, low-affinity, binding site for glucocorticoid antagonists, the occupancy of which produces negatively co-operative interactions with the primary glucocorticoid-binding site. In contrast with the interactions with the primary site, the interactions of steroids with the negatively co-operative site appear to be primarily hydrophobic in nature, and the site resembles the steroid-binding site of progestin-binding proteins in its specificity, though not its affinity. The results also suggest that the initial interactions of both glucocorticoid agonists and antagonists with the receptor under equilibrium conditions are with one primary site on a receptor existing in one conformation only. PMID:7406882

  2. Glucocorticoids curtail stimuli-induced CREB phosphorylation in TRH neurons through interaction of the glucocorticoid receptor with the catalytic subunit of protein kinase A.

    PubMed

    Sotelo-Rivera, Israim; Cote-Vélez, Antonieta; Uribe, Rosa-María; Charli, Jean-Louis; Joseph-Bravo, Patricia

    2017-03-01

    Corticosterone prevents cold-induced stimulation of thyrotropin-releasing hormone (Trh) expression in rats, and the stimulatory effect of dibutyryl cyclic-adenosine monophosphate (dB-cAMP) on Trh transcription in hypothalamic cultures. We searched for the mechanism of this interference. Immunohistochemical analyses of phosphorylated cAMP-response element binding protein (pCREB) were performed in the paraventricular nucleus (PVN) of Wistar rats, and in cell cultures of 17-day old rat hypothalami, or neuroblastoma SH-SY5Y cells. Cultures were incubated 1h with dB-cAMP, dexamethasone and both drugs combined; their nuclear extracts were used for chromatin immunoprecipitation; cytosolic or nuclear extracts for coimmunoprecipitation analyses of catalytic subunit of protein kinase A (PKAc) and of glucocorticoid receptor (GR); their subcellular distribution was analyzed by immunocytochemistry. Cold exposure increased pCREB in TRH neurons of rats PVN, effect blunted by corticosterone previous injection. Dexamethasone interfered with forskolin increase in nuclear pCREB and its binding to Trh promoter; antibodies against histone deacetylase-3 precipitated chromatin from nuclear extracts of hypothalamic cells treated with tri-iodothyronine but not with dB-cAMP + dexamethasone, discarding chromatin compaction as responsible mechanism. Co-immunoprecipitation analyses of cytosolic or nuclear extracts showed protein:protein interactions between activated GR and PKAc. Immunocytochemical analyses of hypothalamic or SH-SY5Y cells revealed diminished nuclear translocation of PKAc and GR in cells incubated with forskolin + dexamethasone, compared to either forskolin or dexamethasone alone. Glucocorticoids and cAMP exert mutual inhibition of Trh transcription through interaction of activated glucocorticoid receptor with protein kinase A catalytic subunit, reducing their nuclear translocation, limiting cAMP-response element binding protein phosphorylation and its binding to Trh promoter.

  3. Glucocorticoid receptor in T cells mediates protection from autoimmunity in pregnancy

    PubMed Central

    Engler, Jan Broder; Kursawe, Nina; Solano, María Emilia; Patas, Kostas; Wehrmann, Sabine; Heckmann, Nina; Lühder, Fred; Reichardt, Holger M.; Arck, Petra Clara; Gold, Stefan M.

    2017-01-01

    Pregnancy is one of the strongest inducers of immunological tolerance. Disease activity of many autoimmune diseases including multiple sclerosis (MS) is temporarily suppressed by pregnancy, but little is known about the underlying molecular mechanisms. Here, we investigated the endocrine regulation of conventional and regulatory T cells (Tregs) during reproduction. In vitro, we found the pregnancy hormone progesterone to robustly increase Treg frequencies via promiscuous binding to the glucocorticoid receptor (GR) in T cells. In vivo, T-cell–specific GR deletion in pregnant animals undergoing experimental autoimmune encephalomyelitis (EAE), the animal model of MS, resulted in a reduced Treg increase and a selective loss of pregnancy-induced protection, whereas reproductive success was unaffected. Our data imply that steroid hormones can shift the immunological balance in favor of Tregs via differential engagement of the GR in T cells. This newly defined mechanism confers protection from autoimmunity during pregnancy and represents a potential target for future therapy. PMID:28049829

  4. The glucocorticoid receptor hormone binding domain mediates transcriptional activation in vitro in the absence of ligand.

    PubMed Central

    Schmitt, J; Stunnenberg, H G

    1993-01-01

    We show that recombinant rat glucocorticoid receptor (vvGR) expressed using vaccinia virus is indistinguishable from authentic GR with respect to DNA and hormone binding. In the absence of hormone, vvGR is mainly found in the cytoplasm in a complex with heat shock protein 90. Upon incubation with ligand, vvGR is released from this complex and translocated to the nucleus. Thus, the ligand binding domain displays the known biochemical properties. However, in vitro, transcription from a synthetic promoter and from the mouse mammary tumor virus (MMTV) promoter is enhanced by recombinant GR in a ligand independent manner. Both transactivation domains contribute to the transcriptional activity, additively on a synthetic promoter and cooperatively on the MMTV promoter. We thus provide the first evidence that in vitro the hormone binding domain has a transcriptional activity even in the absence of ligand. Images PMID:8392705

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

    PubMed

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

    1985-02-01

    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.

  6. Stress Increases Peripheral Axon Growth and Regeneration through Glucocorticoid Receptor-Dependent Transcriptional Programs

    PubMed Central

    Alexander, Jessica K.; Madalena, Kathryn M.; Motti, Dario; Quach, Tam; Zha, Alicia; Webster Marketon, Jeanette

    2017-01-01

    Abstract Stress and glucocorticoid (GC) release are common behavioral and hormonal responses to injury or disease. In the brain, stress/GCs can alter neuron structure and function leading to cognitive impairment. Stress and GCs also exacerbate pain, but whether a corresponding change occurs in structural plasticity of sensory neurons is unknown. Here, we show that in female mice (Mus musculus) basal GC receptor (Nr3c1, also known as GR) expression in dorsal root ganglion (DRG) sensory neurons is 15-fold higher than in neurons in canonical stress-responsive brain regions (M. musculus). In response to stress or GCs, adult DRG neurite growth increases through mechanisms involving GR-dependent gene transcription. In vivo, prior exposure to an acute systemic stress increases peripheral nerve regeneration. These data have broad clinical implications and highlight the importance of stress and GCs as novel behavioral and circulating modifiers of neuronal plasticity. PMID:28828403

  7. Stress Increases Peripheral Axon Growth and Regeneration through Glucocorticoid Receptor-Dependent Transcriptional Programs.

    PubMed

    Lerch, Jessica K; Alexander, Jessica K; Madalena, Kathryn M; Motti, Dario; Quach, Tam; Dhamija, Akhil; Zha, Alicia; Gensel, John C; Webster Marketon, Jeanette; Lemmon, Vance P; Bixby, John L; Popovich, Phillip G

    2017-01-01

    Stress and glucocorticoid (GC) release are common behavioral and hormonal responses to injury or disease. In the brain, stress/GCs can alter neuron structure and function leading to cognitive impairment. Stress and GCs also exacerbate pain, but whether a corresponding change occurs in structural plasticity of sensory neurons is unknown. Here, we show that in female mice (Mus musculus) basal GC receptor (Nr3c1, also known as GR) expression in dorsal root ganglion (DRG) sensory neurons is 15-fold higher than in neurons in canonical stress-responsive brain regions (M. musculus). In response to stress or GCs, adult DRG neurite growth increases through mechanisms involving GR-dependent gene transcription. In vivo, prior exposure to an acute systemic stress increases peripheral nerve regeneration. These data have broad clinical implications and highlight the importance of stress and GCs as novel behavioral and circulating modifiers of neuronal plasticity.

  8. A naturally occurring insertion of a single amino acid rewires transcriptional regulation by glucocorticoid receptor isoforms.

    PubMed

    Thomas-Chollier, Morgane; Watson, Lisa C; Cooper, Samantha B; Pufall, Miles A; Liu, Jennifer S; Borzym, Katja; Vingron, Martin; Yamamoto, Keith R; Meijsing, Sebastiaan H

    2013-10-29

    In addition to guiding proteins to defined genomic loci, DNA can act as an allosteric ligand that influences protein structure and activity. Here we compared genome-wide binding, transcriptional regulation, and, using NMR, the conformation of two glucocorticoid receptor (GR) isoforms that differ by a single amino acid insertion in the lever arm, a domain that adopts DNA sequence-specific conformations. We show that these isoforms differentially regulate gene expression levels through two mechanisms: differential DNA binding and altered communication between GR domains. Our studies suggest a versatile role for DNA in both modulating GR activity and also in directing the use of GR isoforms. We propose that the lever arm is a "fulcrum" for bidirectional allosteric signaling, conferring conformational changes in the DNA reading head that influence DNA sequence selectivity, as well as conferring changes in the dimerization domain that connect functionally with remote regulatory surfaces, thereby influencing which genes are regulated and the magnitude of their regulation.

  9. Combination of chewing and stress up-regulates hippocampal glucocorticoid receptor in contrast to the increase of mineralocorticoid receptor under stress only.

    PubMed

    Sasaguri, Ken-Ichi; Yoshikawa, Gota; Yamada, Kentaro; Miyake, Shinjiro; Kubo, Kin-Ya; Yamamoto, Toshiharu

    2012-06-21

    In general, acute immobilization stress increases plasma corticosterone levels that signal the hypothalamic-pituitary-adrenal axis. Mineralocorticoid receptors and glucocorticoid receptors in the hippocampus perform crucial roles in this feedback mechanism. In the present study, we investigated the effects of chewing under stress on the rat hippocampal mineralocorticoid and glucocorticoid receptors by immunohistochemistry. We separated rats into a control group, a 2-h immobilization stress group (stress only group), and a 2-h immobilization stress group that was allowed to chew on a wooden stick for the latter 1h (stress with chewing group). Mineralocorticoid receptor immunoreactive cells with nucleus staining in the hippocampal CA1 area were scattered in the pyramidal cell layer. The stress only group showed the densest distribution of immunoreactive cells; however, the density of the immunoreactive cells in the stress with chewing group was similar to that of the control group. Changes in immunoreactive cell density were not visible in other areas of the hippocampus, namely, the CA3 area and dentate gyrus. Image analysis indicated that the increase in the mineralocorticoid receptor immunoreactive area within a fixed area in the stress only group was statistically significant compared with those in the control group and the stress with chewing group. On the other hand, glucocorticoid receptor immunoreactive cells in the CA1 area seemed to be increased in the stress with chewing group, but not in the stress only group. Image analysis indicated that this increase was statistically significant. These results suggest that immobilization and immobilization with chewing differentially affect these two types of glucocorticoid receptors in the rat hippocampus. Considering that chewing has alleviative effects against stress, glucocorticoid receptor elevation in the hippocampal CA1 area is one of the neuronal mechanisms of coping with stress. Copyright © 2012 Elsevier

  10. Glucocorticoid receptor gene inactivation in dopamine-innervated areas selectively decreases behavioral responses to amphetamine

    PubMed Central

    Parnaudeau, Sébastien; Dongelmans, Marie-louise; Turiault, Marc; Ambroggi, Frédéric; Delbes, Anne-Sophie; Cansell, Céline; Luquet, Serge; Piazza, Pier-Vincenzo; Tronche, François; Barik, Jacques

    2014-01-01

    The meso-cortico-limbic system, via dopamine release, encodes the rewarding and reinforcing properties of natural rewards. It is also activated in response to abused substances and is believed to support drug-related behaviors. Dysfunctions of this system lead to several psychiatric conditions including feeding disorders and drug addiction. These disorders are also largely influenced by environmental factors and in particular stress exposure. Stressors activate the corticotrope axis ultimately leading to glucocorticoid hormone (GCs) release. GCs bind the glucocorticoid receptor (GR) a transcription factor ubiquitously expressed including within the meso-cortico-limbic tract. While GR within dopamine-innervated areas drives cocaine's behavioral responses, its implication in responses to other psychostimulants such as amphetamine has never been clearly established. Moreover, while extensive work has been made to uncover the role of this receptor in addicted behaviors, its contribution to the rewarding and reinforcing properties of food has yet to be investigated. Using mouse models carrying GR gene inactivation in either dopamine neurons or in dopamine-innervated areas, we found that GR in dopamine responsive neurons is essential to properly build amphetamine-induced conditioned place preference and locomotor sensitization. c-Fos quantification in the nucleus accumbens further confirmed defective neuronal activation following amphetamine injection. These diminished neuronal and behavioral responses to amphetamine may involve alterations in glutamate transmission as suggested by the decreased MK801-elicited hyperlocomotion and by the hyporeactivity to glutamate of a subpopulation of medium spiny neurons. In contrast, GR inactivation did not affect rewarding and reinforcing properties of food suggesting that responding for natural reward under basal conditions is preserved in these mice. PMID:24574986

  11. 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

  12. 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

  13. Glucocorticoid Receptor ChIP-seq Identifies PLCD1 as a KLF15 Target that Represses Airway Smooth Muscle Hypertrophy.

    PubMed

    Sasse, Sarah K; Kadiyala, Vineela; Danhorn, Thomas; Panettieri, Reynold A; Phang, Tzu L; Gerber, Anthony N

    2017-04-04

    Glucocorticoids exert important therapeutic effects on airway smooth muscle (ASM), yet few direct targets of glucocorticoid signaling in ASM have been definitively identified. Here, we show that the transcription factor, KLF15, is directly induced by glucocorticoids in primary human ASM and that KLF15 represses ASM hypertrophy. We integrated transcriptome data from KLF15 overexpression with genome-wide analysis of RNA Polymerase II (RNAPII) and glucocorticoid receptor (GR) occupancy (i.e. ChIP-seq) to identify PLCD1 as both a KLF15-regulated gene and a novel repressor of ASM hypertrophy. Our ChIP-seq data also allowed us to establish numerous direct transcriptional targets of GR in ASM. Genes with inducible GR occupancy and putative anti-inflammatory properties included IRS2, APPL2, RAMP1 and MFGE8. Surprisingly, we also observed GR occupancy in the absence of supplemental ligand, including robust GR binding peaks within the IL11 and LIF loci. Detection of antibody-GR complexes at these areas was abrogated by dexamethasone treatment in association with reduced RNAPII occupancy, suggesting that non-canonical pathways contribute to cytokine repression by glucocorticoids in ASM. Through defining GR interactions with chromatin on a genome-wide basis in ASM, our data also provide an important resource for future studies of GR in this therapeutically relevant cell type.

  14. Prenatal Dexamethasone Exposure Increases the Susceptibility to Autoimmunity in Offspring Rats by Epigenetic Programing of Glucocorticoid Receptor.

    PubMed

    Sun, Yanhong; Wan, Xiaoyan; Ouyang, Juan; Xie, Renfeng; Wang, Xueping; Chen, Peisong

    2016-01-01

    Objective. Prenatal glucocorticoids (GC) can induce long term effects on offspring health. However, reports and related studies regarding the prolonged effects of prenatal GC on the development of autoimmunity are limited. Here, we aimed to explore the immunological effects of dexamethasone (DEX) exposure on young adults and whether glucocorticoid receptor (GR) is involved in this process. Methods. Wistar rats were given DEX during pregnancy. Susceptibility to autoimmunity in offspring was assessed using experimental autoimmune encephalomyelitis (EAE) and adjuvant-induced arthritis (AIA) animal models. To reveal the possible mechanism, glucocorticoid response, GR expression, and methylation status were measured in peripheral blood mononuclear cells (PBMCs). Results. Our results showed that the DEX-treated rats had greater susceptibility to EAE (100% versus 62.5%, P < 0.05) and AIA (63.6% versus 0%, P < 0.05) than saline control group. Glucocorticoid response and GR expression were decreased in DEX rats. Significant difference was also found in the methylation levels of GR exon 1-10 to exon 1-11 region. Conclusions. Prenatal DEX administration increases the susceptibility to autoimmune diseases, which is potentially mediated by programming GR methylation status and glucocorticoid sensitivity.

  15. Prenatal Dexamethasone Exposure Increases the Susceptibility to Autoimmunity in Offspring Rats by Epigenetic Programing of Glucocorticoid Receptor

    PubMed Central

    Sun, Yanhong; Ouyang, Juan; Xie, Renfeng; Wang, Xueping

    2016-01-01

    Objective. Prenatal glucocorticoids (GC) can induce long term effects on offspring health. However, reports and related studies regarding the prolonged effects of prenatal GC on the development of autoimmunity are limited. Here, we aimed to explore the immunological effects of dexamethasone (DEX) exposure on young adults and whether glucocorticoid receptor (GR) is involved in this process. Methods. Wistar rats were given DEX during pregnancy. Susceptibility to autoimmunity in offspring was assessed using experimental autoimmune encephalomyelitis (EAE) and adjuvant-induced arthritis (AIA) animal models. To reveal the possible mechanism, glucocorticoid response, GR expression, and methylation status were measured in peripheral blood mononuclear cells (PBMCs). Results. Our results showed that the DEX-treated rats had greater susceptibility to EAE (100% versus 62.5%, P < 0.05) and AIA (63.6% versus 0%, P < 0.05) than saline control group. Glucocorticoid response and GR expression were decreased in DEX rats. Significant difference was also found in the methylation levels of GR exon 1-10 to exon 1-11 region. Conclusions. Prenatal DEX administration increases the susceptibility to autoimmune diseases, which is potentially mediated by programming GR methylation status and glucocorticoid sensitivity. PMID:28078304

  16. Familial glucocorticoid receptor haploinsufficiency by non-sense mediated mRNA decay, adrenal hyperplasia and apparent mineralocorticoid excess.

    PubMed

    Bouligand, Jérôme; Delemer, Brigitte; Hecart, Annie-Claude; Meduri, Geri; Viengchareun, Say; Amazit, Larbi; Trabado, Séverine; Fève, Bruno; Guiochon-Mantel, Anne; Young, Jacques; Lombès, Marc

    2010-10-22

    Primary glucocorticoid resistance (OMIM 138040) is a rare hereditary disease that causes a generalized partial insensitivity to glucocorticoid action, due to genetic alterations of the glucocorticoid receptor (GR). Investigation of adrenal incidentalomas led to the discovery of a family (eight affected individuals spanning three generations), prone to cortisol resistance, bilateral adrenal hyperplasia, arterial hypertension and hypokalemia. This phenotype exacerbated over time, cosegregates with the first heterozygous nonsense mutation p.R469[R,X] reported to date for the GR, replacing an arginine (CGA) by a stop (TGA) at amino-acid 469 in the second zinc finger of the DNA-binding domain of the receptor. In vitro, this mutation leads to a truncated 50-kDa GR lacking hormone and DNA binding capacity, devoid of hormone-dependent nuclear translocation and transactivation properties. In the proband's fibroblasts, we provided evidence for the lack of expression of the defective allele in vivo. The absence of detectable mutated GR mRNA was accompanied by a 50% reduction in wild type GR transcript and protein. This reduced GR expression leads to a significantly below-normal induction of glucocorticoid-induced target genes, FKBP5 in fibroblasts. We demonstrated that the molecular mechanisms of glucocorticoid signaling dysfunction involved GR haploinsufficiency due to the selective degradation of the mutated GR transcript through a nonsense-mediated mRNA Decay that was experimentally validated on emetine-treated propositus' fibroblasts. GR haploinsufficiency leads to hypertension due to illicit occupation of renal mineralocorticoid receptor by elevated cortisol rather than to increased mineralocorticoid production reported in primary glucocorticoid resistance. Indeed, apparent mineralocorticoid excess was demonstrated by a decrease in urinary tetrahydrocortisone-tetrahydrocortisol ratio in affected patients, revealing reduced glucocorticoid degradation by renal activity of

  17. 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

  18. Overexpression of pregnane X and glucocorticoid receptors and the regulation of cytochrome P450 in human epileptic brain endothelial cells.

    PubMed

    Ghosh, Chaitali; Hossain, Mohammed; Solanki, Jesal; Najm, Imad M; Marchi, Nicola; Janigro, Damir

    2017-04-01

    Recent evidence suggests a metabolic contribution of cytochrome P450 enzymes (CYPs) to the drug-resistant phenotype in human epilepsy. However, the upstream molecular regulators of CYP in the epileptic brain remain understudied. We therefore investigated the expression and function of pregnane xenobiotic (PXR) and glucocorticoid (GR) nuclear receptors in endothelial cells established from post-epilepsy surgery brain samples. PXR/GR localization was evaluated by immunohistochemistry in specimens from subjects who underwent temporal lobe resections to relieve drug-resistant seizures. We used primary cultures of endothelial cells obtained from epileptic brain tissues (EPI-ECs; n = 8), commercially available human brain microvascular endothelial cells (HBMECs; n = 8), and human hepatocytes (n = 3). PXR/GR messenger RNA (mRNA) levels in brain ECs was initially determined by complementary DNA (cDNA) microarrays. The expression of PXR/GR proteins was quantified by Western blot. PXR and GR silencing was performed in EPI-ECs (n = 4), and the impact on downstream CYP expression was determined. PXR/GR expression was detected by immunofluorescence in ECs and neurons in the human temporal lobe samples analyzed. Elevated mRNA and protein levels of PXR and GR were found in EPI-ECs versus control HBMECs. Hepatocytes, used as a positive control, displayed the highest levels of PXR/GR expression. We confirmed expression of PXR/GR in cytoplasmic-nuclear subcellular fractions, with a significant increase of PXR/GR in EPI-ECs versus controls. CYP3A4, CYP2C9, and CYP2E1 were overexpressed in EPI-ECs versus control, whereas CYP2D6 and CYP2C19 were downregulated or absent in EPI-ECs. GR silencing in EPI-ECs led to decreased CYP3A4, CYP2C9, and PXR expression. PXR silencing in EPI-ECs resulted in the specific downregulation of CYP3A4 expression. Our results indicate increased PXR and GR in primary ECs derived from human epileptic brains. PXR or GR may be responsible for a local drug brain

  19. Developmental reprogramming of rat GLUT5 requires glucocorticoid receptor translocation to the nucleus

    PubMed Central

    Douard, Véronique; Choi, Hye-In; Elshenawy, Summer; Lagunoff, David; Ferraris, Ronaldo P

    2008-01-01

    Fructose consumption has increased dramatically but little is known about mechanisms regulating the intestinal fructose transporter GLUT5 in vivo. In neonatal rats, GLUT5 can be induced only by luminal fructose and only after 14 days of age, unless the gut is primed with dexamethasone prior to fructose perfusion. To elucidate the mechanisms underlying dexamethasone modulation of GLUT5 development, we first identified the receptor mediating its effects then determined whether those effects were genomic. The glucocorticoid receptor (GR) antagonist RU486 dose-dependently prevented the dexamethasone-mediated effects on body weight, intestinal arginase2 (a known GR-regulated gene) and GLUT5. In contrast, an antagonist of the mineralocorticoid receptor as well as agonists of progesterone (PR) and pregnane-X (PXR) receptors did not block the effects of dexamethasone. These receptor antagonists and agonists had no effect on the intestinal glucose transporter SGLT1. Translocation of the GR into the enterocyte nucleus occurred only in dexamethasone-injected pups perfused with fructose, was accompanied by marked increases in brush border GLUT5 abundance, and was blocked by RU486. A priming duration of ∼24 h is optimal for induction but actinomycin D injection before dexamethasone priming prevented dexamethasone from allowing luminal fructose to induce GLUT5. Actinomycin D had no effect on dexamethasone-independent fructose-induced increases in glucose-6-phosphatase mRNA abundance, suggesting that it did not prevent fructose-induction of GLUT5, but instead prevented dexamethasone-induced synthesis of an intermediate required by fructose for GLUT5 regulation. In suckling rats < 14 days old, developmental regulation of transporters may involve cross-talk between hormonal signals modulating intestinal maturation and nutrient signals regulating specific transporters. PMID:18556366

  20. Glucocorticoid Receptor-DNA Dissociation Kinetics Measured in Vitro Reveal Exchange on the Second Time Scale.

    PubMed

    De Angelis, Rolando W; Maluf, Nasib K; Yang, Qin; Lambert, James R; Bain, David L

    2015-09-01

    The glucocorticoid receptor (GR) is a member of the steroid receptor family of ligand-activated transcription factors. Recent live cell imaging studies have revealed that interactions of GR with chromatin are highly dynamic, with average receptor residence times of only seconds. These findings were surprising because early kinetic studies found that GR-DNA interactions in vitro were much slower, having calculated residence times of minutes to hours. However, these latter analyses were conducted at a time when it was possible to work with only either partially purified holoreceptor or its purified but isolated DNA binding domain. Noting these limitations, we reexamined GR-DNA dissociation kinetics using a highly purified holoreceptor shown to be amenable to rigorous study. We first observe that GR-DNA interactions in vitro are not slow as previously thought but converge with in vivo behavior, having residence times of only seconds to tens of seconds. This rapid exchange is seen at six individual response elements and the multisite MMTV promoter used in live cell imaging. Second, GR dissociation rates are identical for all response elements. Thus, previously observed differences in receptor affinity toward these sequences are not due to differences in off rate but in on rate. Finally, dissociation kinetics are biphasic in character. A minimal kinetic model consistent with the data is that in which DNA-bound GR interconverts between states on a second time scale, with dissociation occurring via a multistep process. We speculate that receptor interconversion in this time frame can be recognized by the coregulatory proteins that interact with GR, leading to unique transcriptional responses.

  1. Endothelial nuclear lamina is not required for glucocorticoid receptor nuclear import but does affect receptor-mediated transcription activation.

    PubMed

    Nayebosadri, Arman; Ji, Julie Y

    2013-08-01

    The lamina serves to maintain the nuclear structure and stiffness while acting as a scaffold for heterochromatin and many transcriptional proteins. Its role in endothelial mechanotransduction, specifically how nuclear mechanics impact gene regulation under shear stress, is not fully understood. In this study, we successfully silenced lamin A/C in bovine aortic endothelial cells to determine its role in both glucocorticoid receptor (GR) nuclear translocation and glucocorticoid response element (GRE) transcriptional activation in response to dexamethasone and shear stress. Nuclear translocation of GR, an anti-inflammatory nuclear receptor, in response to dexamethasone or shear stress (5, 10, and 25 dyn/cm(2)) was observed via time-lapse cell imaging and quantified using a Bayesian image analysis algorithm. Transcriptional activity of the GRE promoter was assessed using a dual-luciferase reporter plasmid. We found no dependence on nuclear lamina for GR translocation from the cytoplasm into the nucleus. However, the absence of lamin A/C led to significantly increased expression of luciferase under dexamethasone and shear stress induction as well as changes in histone protein function. PCR results for NF-κB inhibitor alpha (NF-κBIA) and dual specificity phosphatase 1 (DUSP1) genes further supported our luciferase data with increased expression in the absence of lamin. Our results suggest that absence of lamin A/C does not hinder passage of GR into the nucleus, but nuclear lamina is important to properly regulate GRE transcription. Nuclear lamina, rather than histone deacetylase (HDAC), is a more significant mediator of shear stress-induced transcriptional activity, while dexamethasone-initiated transcription is more HDAC dependent. Our findings provide more insights into the molecular pathways involved in nuclear mechanotransduction.

  2. Endothelial nuclear lamina is not required for glucocorticoid receptor nuclear import but does affect receptor-mediated transcription activation

    PubMed Central

    Nayebosadri, Arman

    2013-01-01

    The lamina serves to maintain the nuclear structure and stiffness while acting as a scaffold for heterochromatin and many transcriptional proteins. Its role in endothelial mechanotransduction, specifically how nuclear mechanics impact gene regulation under shear stress, is not fully understood. In this study, we successfully silenced lamin A/C in bovine aortic endothelial cells to determine its role in both glucocorticoid receptor (GR) nuclear translocation and glucocorticoid response element (GRE) transcriptional activation in response to dexamethasone and shear stress. Nuclear translocation of GR, an anti-inflammatory nuclear receptor, in response to dexamethasone or shear stress (5, 10, and 25 dyn/cm2) was observed via time-lapse cell imaging and quantified using a Bayesian image analysis algorithm. Transcriptional activity of the GRE promoter was assessed using a dual-luciferase reporter plasmid. We found no dependence on nuclear lamina for GR translocation from the cytoplasm into the nucleus. However, the absence of lamin A/C led to significantly increased expression of luciferase under dexamethasone and shear stress induction as well as changes in histone protein function. PCR results for NF-κB inhibitor alpha (NF-κBIA) and dual specificity phosphatase 1 (DUSP1) genes further supported our luciferase data with increased expression in the absence of lamin. Our results suggest that absence of lamin A/C does not hinder passage of GR into the nucleus, but nuclear lamina is important to properly regulate GRE transcription. Nuclear lamina, rather than histone deacetylase (HDAC), is a more significant mediator of shear stress-induced transcriptional activity, while dexamethasone-initiated transcription is more HDAC dependent. Our findings provide more insights into the molecular pathways involved in nuclear mechanotransduction. PMID:23703529

  3. Identification of constitutive androstane receptor and glucocorticoid receptor binding sites in the CYP2C19 promoter.

    PubMed

    Chen, Yuping; Ferguson, Stephen S; Negishi, Masahiko; Goldstein, Joyce A

    2003-08-01

    CYP2C19 is an important human drug-metabolizing enzyme that metabolizes a number of clinically used drugs including the antiulcer drug omeprazole, the anxiolytic drug diazepam, the beta-blocker propranolol, the antimalarial drug proguanil, certain antidepressants and barbiturates, and the prototype substrate S-mephenytoin. Previous studies show that compounds such as rifampicin and dexamethasone induce CYP2C19 both in vivo in humans and in vitro in human hepatocytes. This study examines the transcriptional regulation of CYP2C19. Analysis of the CYP2C19 promoter revealed a single constitutive androstane receptor (CAR) binding site (CAR-RE; -1891/-1876 bp) and a glucocorticoid-responsive element (GRE; -1750/-1736 bp). Gel-shift assays showed that CAR-RE binds CAR and pregnane X receptor (PXR). Cotransfection with hCAR, mCAR, or hPXR in HepG2 cells up-regulated transcription of CYP2C19 promoter constructs, whereas mutation of the -1891-bp CAR-RE abolished up-regulation. Expression with hCAR also up-regulated endogenous CYP2C19 mRNA content in HepG2 cells. Androstenol repressed the mCAR-mediated constitutive activation of the CYP2C19 promoter in HepG2 cells, whereas the potent mCAR ligand 1,4-bis[2-3,5-dichloropyridyloxyl)] benzene derepressed this response. Rifampicin produced a modest increase in promoter activity in cells cotransfected with hPXR. Dexamethasone activated the -2.7-kb CYP2C19 promoter constructs in HepG2 cells only in the presence of cotransfected glucocorticoid receptor (GR), whereas the GR antagonist mifepristone inhibits this response. Mutation of the GRE abolishes dexamethasone activation. This is the first study to identify nuclear receptor binding sites (CAR/PXR and GR) in the CYP2C19 promoter and to suggest that these receptors may up-regulate CYP2C19 constitutively and possibly its response to drugs.

  4. BclI polymorphism of the glucocorticoid receptor and adrenal crisis in primary adrenal insufficiency.

    PubMed

    Zopf, Kathrin; Frey, Kathrin; Kienitz, Tina; Ventz, Manfred; Bauer, Britta; Quinkler, Marcus

    2017-09-27

    Patients with primary adrenal insufficiency (PAI) or congenital adrenal hyperplasia (CAH) are at high risk of adrenal crisis (AC). Glucocorticoid sensitivity is at least partially genetically determined by polymorphisms of the glucocorticoid receptor (GR). To determine if number of intercurrent illnesses and AC are associated with the GR gene polymorphism BclI in patients with PAI and CAH. This prospective, longitudinal study over 37.7±10.1 months included 47 PAI and 25 CAH patients. During the study period intercurrent illness episodes and AC were documented. The study period covered 223 patient years, in which 21 AC occurred (9.4 AC/100 pat years). There were no significant differences between BclI polymorphisms (CC (n=29), CG (n=34), GG (n=9)) regarding BMI, hydrocortisone equivalent daily dose, and blood pressure. We did not find a difference in number of intercurrent illnesses/patient year between BclI polymorphisms (CC (1.5±1.4/pat year), CG (1.2±1.2/pat year), GG (1.6±2.2/pat year)). The occurrence of AC was not significantly different between the homozygous (GG) genotype (32.5 AC/100 pat years), the CC (6.7 AC/100 pat years) and the CG genotype (4.9 AC/100 pat years). Concomitant hypothyroidism was highest in the GG genotype group (5/9), compared to the others (CC (11/29), CG (11/34)). Although sample sizes were relatively small and results should be interpreted with caution, this study suggests that the GR gene polymorphism BclI may not be associated with the frequency of intercurrent illnesses and of AC.

  5. [Effects of electromagnetic irradiation on glucocorticoid in serum and its receptor expression in rat hippocampus].

    PubMed

    Li, Mao-quan; Wang, Yan-yan; Zhang, Guang-bin; Yu, Zheng-ping

    2007-04-01

    To explore the role and mechanism of glucocorticoid (GC) in the harmful bio-effects of electromagnetic irradiation. Rats were exposed to 65 mW/cm(2) electromagnetic wave for 20 min. At 10 min, 30 min, 3 h, 12 h after irradiation, their learning and memory abilities were tested by Morris water maze. The levels of corticosterone (CORT) in serum were measured by radioimmunoprecipitation assay and the changes of total glucocorticoid receptor (GR) expression and GR nuclear translocation in rat hippocampus were measured by reverse transcription-polymerase chain reaction and Western blot. The rats had learning and memory deficits at 10 min, 30 min and 3 h after irradiation, but at 12 h had no difference from the normal control. The levels of corticosterone in serum increased significantly at 10 min, 30 min, decreased at 3 h and increased significantly compared with 12 h after irradiation. GR mRNA and total GR protein expression in rat hippocampus had no significant changes at 10 min, 30 min after irradiation. At 3 h, 12 h GR mRNA expression significantly decreased by 69%, 76% respectively and GR total protein decreased by 58%, 67% respectively. There were significant differences between the two groups and the corresponding controls (P<0.05). And compared with the control, the GR nuclear translocation increased significantly at 3 h and 12 h (P<0.05). GC may take part in the injury to learning and memory abilities after electromagnetic irradiation, and the non-genomic and genomic effects of GC may play a major role in the early and late stage, respectively.

  6. The prognostic value of glucocorticoid receptors for adult acute lymphoblastic leukemia

    PubMed Central

    EL-Maghraby, Shereen M.; Kandil, Noha S.; El-Bendary, Waleed R.

    2015-01-01

    Background Therapeutic protocols used in adult acute lymphoblastic leukemia (ALL) are widely variable, and glucocorticoids (GCs) are essential components in ALL treatment. Therefore, this study aimed to evaluate the distribution of prominent glucocorticoid receptor (GR) gene polymorphic variants among adult ALL patients. We also investigated the association between GR messenger ribonucleic acid (mRNA) isoform expressions and the response to chemotherapy. Methods Fifty-two newly diagnosed Philadelphia-negative adult ALL patients and 30 healthy control subjects were enrolled in this study. Genotyping was carried out using a polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis. GR mRNA isoform expressions were assayed by quantitative real-time PCR. Results ALL patients in this study had a median age of 34 years (range, 18-75). GRα expression was associated with complete remission (P=0.03), while GRγ mRNA expression was significantly higher in GC resistant patients (P=0.032) and in non-responders (P=0.019). However, there were no significant associations with GC resistance. The BclI polymorphic variant of the GR gene was the most frequent in adult ALL patients and was not associated with the GC response. Both higher GRα expression and lower GRγ expression were associated with achievement of complete remission, while higher GRγ expression was associated with GC-resistance. Conclusion Our data suggest that the level of GR isoform expression may be useful in predicting GC response, achievement of complete remission, and better event-free survival in ALL patients. However, further evaluation with a larger cohort of patients is warranted. PMID:26770951

  7. 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.

  8. Hepatic Glucocorticoid Receptor Plays a Greater Role Than Adipose GR in Metabolic Syndrome Despite Renal Compensation.

    PubMed

    Bose, Sandip K; Hutson, Irina; Harris, Charles A

    2016-12-01

    Exogenous glucocorticoid administration results in hyperglycemia, insulin resistance, hepatic dyslipidemia, and hypertension, a constellation of findings known as Cushing's syndrome. These effects are mediated by the glucocorticoid receptor (GR). Because GR activation in liver and adipose has been implicated in metabolic syndrome (MS), we wanted to determine the role of GR in these tissues in the development of MS. Because GR knockout (KO) mice (whole-body KO) exhibit perinatal lethality due to respiratory failure, we generated tissue-specific (liver or adipose) GRKO mice using cre-lox technology. Real-time PCR analysis of liver mRNA from dexamethasone-treated wildtype (WT) and liver GRKO mice indicated that hepatic GR regulates the expression of key genes involved in gluconeogenesis and glycogen metabolism. Interestingly, we have observed that liver-specific deletion of GR resulted in a significant increase in mRNA expression of key genes involved in gluconeogenesis and glycogen metabolism in kidney tissue, indicating a compensatory mechanism to maintain glucose homeostasis. We have also observed that GR plays an important role in regulating the mRNA expression of key genes involved in lipid metabolism. Liver GRKO mice demonstrated decreased fat mass and liver glycogen content compared with WT mice administered dexamethasone for 2 weeks. Adipose-specific deletion of GR did not alter glucose tolerance or insulin sensitivity of adipose GRKO mice compared with WT mice administrated dexamethasone. This indicates that liver GR might be more important in development of MS in dexamethasone-treated mice, whereas adipose GR plays a little role in these paradigms.

  9. 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

  10. Hypercortisolemia and glucocorticoid receptor-signaling insufficiency in Alzheimer's disease initiation and development.

    PubMed

    Notarianni, Elena

    2013-09-01

    The cause and mechanism of development of Alzheimer' s disease (AD) remain unexplained. Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis, denoted by adrenal cortisol hypersecretion, is a recognised feature of the condition but generally disregarded as causative, due to lack of association between AD and other hypercortisolemic states. However, a meta-analysis of published studies suggests a need for reappraisal. A specific circadian rhythm of cortisol hypersecretion pertains at mild-to-moderate AD stages, entailing increased levels at the circadian peak from a low nadir. This is in contrast to the continuously elevated levels that are characteristic of other hypercortisolemic states, e.g. Cushing' s disease or major depression. This previously overlooked detail provides a starting premise here: that equating the form of hypercortisolism in AD with that in other states is inappropriate, as phasic and chronic elevation elicit different neuroendocrine effects. Theoretical implications are discussed in this review. Given the capacity of glucocorticoids and corticotropin-releasing hormone to induce AD-associated pathologies, I suggest a role for circadian cortisol hypersecretion in the initiation of sporadic AD; and propose a temporal mechanism for AD development featuring neuroinflammation- mediated suppression of central glucocorticoid receptor (GR) signaling. This latter may represent a critical phase in AD development, where the density of functional GR is proposed to underlie the "cognitive reserve". Supporting evidence for this mechanism is drawn from the brain regional locations of AD neuropathologies, and from risk factors for AD development (aging, ApoE-4 genotype, and hypertension). Thus, it is argued that basal hypercortisolemia merits further scrutiny regarding AD causation and development.

  11. 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.

  12. Glucocorticoid receptor antagonism as a novel therapy for triple-negative breast cancer

    PubMed Central

    Skor, Maxwell N.; Wonder, Erin L.; Kocherginsky, Masha; Goyal, Anju; Hall, Ben A.; Cai, Yi; Conzen, Suzanne D.

    2013-01-01

    Purpose: Triple-negative breast cancer (TNBC) accounts for 10-20% of newly diagnosed invasive breast cancer. Finding effective targets for chemotherapy-resistant TNBC has proven difficult in part because of TNBC’s molecular heterogeneity. We have previously reported that, likely because of GR’s anti-apoptotic activity in ER-negative breast epithelial and cancer cells, high glucocorticoid receptor (GR) expression/activity in early-stage TNBC significantly correlates with chemotherapy-resistance and increased recurrence. We hypothesized that pre-treatment with mifepristone, a (GR)-antagonist, would potentiate the efficacy of chemotherapy in GR+ TNBC by inhibiting GR’s anti-apoptotic signaling pathways and increasing the cytotoxic efficiency of chemotherapy. Experimental Design: TNBC cell apoptosis was examined in the context of physiological glucocorticoid concentrations, chemotherapy, and/or pharmacologic concentrations of mifepristone. We used high-throughput live microscopy with continuous recording to measure apoptotic cells stained with a fluorescent dye, and Western analysis to detect caspase-3 and PARP cleavage. The effect of mifepristone on GR-mediated gene expression was also measured. TNBC xenograft studies were performed in female severe combined immunodeficient (SCID) mice and tumors were measured following treatment with vehicle, paclitaxel or mifepristone/paclitaxel. Results: We found that although mifepristone treatment alone had no significant effect on TNBC cell viability or clonogenicity in the absence of chemotherapy, the addition of mifepristone to dexamethasone/paclitaxel treatment significantly increased cytotoxicity and caspase-3/PARP cleavage. Mifepristone also antagonized GR-induced SGK1 and MKP1/DUSP1 gene expression, while significantly augmenting paclitaxel-induced GR+ MDA-MB-231 xenograft tumor shrinkage in vivo. Conclusions: These results suggest that mifepristone pre-treatment could be a useful strategy for increasing tumor cell

  13. Steroid hormone receptor gene expression in human breast cancer cells: inverse relationship between oestrogen and glucocorticoid receptor messenger RNA levels.

    PubMed

    Hall, R E; Lee, C S; Alexander, I E; Shine, J; Clarke, C L; Sutherland, R L

    1990-12-15

    The relative expression in human breast cancer cells of messenger ribonucleic acids (mRNA) encoding different steroid hormone receptors is unknown. Accordingly, mRNA levels in total RNA extracted from 13 human breast cancer cell lines were measured by Northern analysis employing complementary DNA probes for the human oestrogen (ER), progesterone (PR), androgen (AR), vitamin D3 (VDR) and glucocorticoid receptors (GR). The 7 ER+ lines expressed a single 6.4 kilobases (kb) ER mRNA. Interestingly, low concentrations of ER mRNA were detected in the ER- cell lines, MDA-MB-330 and BT 20. PR mRNA, predominantly a 13.5 kb species, was expressed in the 6 lines known to be ER+, PR+ by radioligand binding; however, one ER+ cell line, MDA-MB-134, failed to express PR mRNA. A 10.5 kb AR mRNA was expressed at significantly higher levels in ER+ than ER- cell lines. All cell lines expressed a single 4.6 kb mRNA for VDR and a single 7.4 kb mRNA for GR. ER and PR mRNA levels were positively correlated (p = 0.011) and each was positively correlated with androgen receptor (AR) mRNA levels (p less than or equal to 0.009). ER, PR and AR mRNAs were negatively associated with GR levels (p less than or equal to 0.012), while ER and AR mRNA levels were negatively correlated with mRNA for the epidermal growth factor receptor. In contrast, levels of VDR mRNA were unrelated to the concentration of any other steroid receptor mRNA. Our data demonstrate the coordinate expression of ER, PR and AR genes, and an inverse relationship between sex steroid hormone receptor and GR gene expression in human breast cancer cell lines.

  14. Region-specific Alterations in Glucocorticoid Receptor Expression in the Postmortem Brain of Teenage Suicide Victims

    PubMed Central

    Pandey, Ghanshyam N.; Rizavi, Hooriyah S.; Ren, Xinguo; Dwivedi, Yogesh; Palkovits, Miklós

    2013-01-01

    Introduction Abnormal function of the hypothalamic-pituitary-adrenal (HPA) axis has been implicated in the pathophysiology of depression and suicide. The purpose of this study was to test the hypothesis that the reported dysregulation of the HPA axis in suicide may be related to a disturbed feedback inhibition caused by decreased corticoid receptors in the brain. We therefore determined the protein and gene expression of glucocorticoid (GR) and mineralocorticoid receptors (MR) in the postmortem brain of teenage suicide victims and matched normal controls. Methods Protein and mRNA expression of GR (GR-α and GR-β) and MR and the mRNA expression of glucocorticoid-induced leucine zipper (GILZ), a target gene for GR were determined by immunolabeling using Western blot technique and the real-time RT-polymerase chain reaction (qPCR) technique in the prefrontal cortex (PFC), hippocampus, subiculum, and amygdala obtained from 24 teenage suicide victims and 24 teenage control subjects. Results We observed that protein and gene expression of GR-α was significantly decreased in the PFC and amygdala, but not in the hippocampus or subiculum, of teenage suicide victims compared with normal control subjects. Also, the mRNA levels of GR inducible target gene GILZ was significantly decreased in PFC and amygdaloid nuclei but not in hippocampus compared with controls. In contrast, no significant differences were observed in protein or gene expression of MR in any of the areas studied between teenage suicide victims and normal control subjects. There was no difference in the expression of GR-β in the PFC between suicide victims and normal controls. Conclusions These results suggested that the observed dysregulation of the HPA axis in suicide may be related to a decreased expression of GR-α and GR inducible genes in the PFC and amygdala of teenage suicide victims. The reason why GR receptors are not dysregulated in the hippocampus or subiculum, presumably two sites of stress action

  15. Phenylalanine-780 near the C-terminus of the mouse glucocorticoid receptor is important for ligand binding affinity and specificity.

    PubMed

    Chen, D; Kohli, K; Zhang, S; Danielsen, M; Stallcup, M R

    1994-04-01

    Site-directed mutagenesis was employed to make two single amino acid substitutions for highly conserved amino acid residues near the C-terminus of the 783-amino acid mouse glucocorticoid receptor. Substitution of leucine for histidine-781 caused little or no change in the concentration of dexamethasone required for half-maximal activation of a chloramphenicol acetyltransferase reporter gene expressed from a mouse mammary tumor virus promoter. However, when phenylalanine-780 was changed to alanine, the half-maximal concentrations of various agonists were increased as follows, compared with the wild-type glucocorticoid receptor: triamcinolone acetonide by 7-fold, dexamethasone by 25-fold, and hydrocortisone and deoxycorticosterone by more than 150-fold. Binding of labeled steroids by the mutant receptor in vitro and in vivo was also decreased. In contrast, this mutation caused a small decrease in the concentration of RU486 required for antagonist or partial agonist activity. Thus, the phenyl group of phenylalanine-780 of the mouse glucocorticoid receptor is an important determinant of ligand binding affinity and specificity.

  16. Mild Hyperthermia Downregulates Receptor-dependent Neutrophil Function

    PubMed Central

    Fröhlich, Dieter; Wittmann, Sigrid; Rothe, Gregor; Sessler, Daniel I.; Vogel, Peter; Taeger, Kai

    2005-01-01

    Mild hypothermia impairs resistance to infection and, reportedly, impairs phagocytosis and oxidative killing of un-opsonized bacteria. We evaluated various functions at 33 to 41°C in neutrophils taken from volunteers. Adhesion on endothelial cells was determined using light microscopy. Adhesion molecules expression and receptors, phagocytosis, and release of reactive oxidants were assessed using flow cytometric assays. Adhesion protein CD11b expression on resting neutrophils was temperature independent. However, upregulation of CD11b with TNF-α was increased by hypothermia and decreased with hyperthermia. Neutrophil adhesion to either resting or activated endothelial cells was not temperature dependent. Bacterial uptake was inversely related to temperature, more so with E. coli than S. aureus. Temperature dependence of phagocytosis occurred only with opsonized bacteria. Hypothermia slightly increased N-Formyl-L-methionyl-L-leucyl-phenylalanine (FMLP) receptors on neutrophils: hyperthermia decreased expression, especially with TNF-α. FMLP-induced H2O2 production was inversely related to temperature, especially in the presence of TNF-α. Conversely, phorbol-13-myristate-12-acetate, an activator of protein kinase C, induced an extreme and homogenous release of reactive oxidants that increased with temperature. In contrast to non-receptor dependent phagocytosis and oxidative killing, several crucial receptor-dependent neutrophil activities show temperature-dependent regulation, with hypothermia increasing function. The temperature dependence of neutrophil function is thus more complicated than previously appreciated. PMID:15281545

  17. Glucocorticoid-induced changes in glucocorticoid receptor mRNA and protein expression in the human placenta as a potential factor for altering fetal growth and development.

    PubMed

    Bivol, Svetlana; Owen, Suzzanne J; Rose'Meyer, Roselyn B

    2016-02-05

    Glucocorticoids (GCs) control essential metabolic processes in virtually every cell in the body and play a vital role in the development of fetal tissues and organ systems. The biological actions of GCs are mediated via glucocorticoid receptors (GRs), the cytoplasmic transcription factors that regulate the transcription of genes involved in placental and fetal growth and development. Several experimental studies have demonstrated that fetal exposure to high maternal GC levels early in gestation is associated with adverse fetal outcomes, including low birthweight, intrauterine growth restriction and anatomical and structural abnormalities that may increase the risk of cardiovascular, metabolic and neuroendocrine disorders in adulthood. The response of the fetus to GCs is dependent on gender, with female fetuses becoming hypersensitive to changes in GC levels whereas male fetuses develop GC resistance in the environment of high maternal GCs. In this paper we review GR function and the physiological and pathological effects of GCs on fetal development. We propose that GC-induced changes in the placental structure and function, including alterations in the expression of GR mRNA and protein levels, may play role in inhibiting in utero fetal growth.

  18. Endogenous hepatic glucocorticoid receptor signaling coordinates sex-biased inflammatory gene expression.

    PubMed

    Quinn, Matthew A; Cidlowski, John A

    2016-02-01

    An individual's sex affects gene expression and many inflammatory diseases present in a sex-biased manner. Glucocorticoid receptors (GRs) are regulators of inflammatory genes, but their role in sex-specific responses is unclear. Our goal was to evaluate whether GR differentially regulates inflammatory gene expression in male and female mouse liver. Twenty-five percent of the 251 genes assayed by nanostring analysis were influenced by sex. Of these baseline sexually dimorphic inflammatory genes, 82% was expressed higher in female liver. Pathway analyses defined pattern-recognition receptors as the most sexually dimorphic pathway. We next exposed male and female mice to the proinflammatory stimulus LPS. Female mice had 177 genes regulated by treatment with LPS, whereas males had 149, with only 66% of LPS-regulated genes common between the sexes. To determine the contribution of GR to sexually dimorphic inflammatory genes we performed nanostring analysis on liver-specific GR knockout (LGRKO) mice in the presence or absence of LPS. Comparing LGRKO to GR(flox/flox) revealed that 36 genes required GR for sexually dimorphic expression, whereas 24 genes became sexually dimorphic in LGRKO. Fifteen percent of LPS-regulated genes in GR(flox/flox) were not regulated in male and female LGRKO mice treated with LPS. Thus, GR action is influenced by sex to regulate inflammatory gene expression.

  19. Working memory performance is associated with common glucocorticoid receptor gene polymorphisms.

    PubMed

    Kumsta, R; Entringer, S; Koper, J W; van Rossum, E F C; Hellhammer, D H; Wüst, S

    2010-01-01

    Cortisol has a modulatory influence on cognitive functions in humans. Both impairing and enhancing effects of cortisol administration have been shown for hippocampus-dependent declarative memory, and impairing effects have been shown for prefrontal-cortex-dependent working memory function. Given the high density of glucocorticoid (GC) receptors in the prefrontal cortex, we investigated whether common polymorphisms of the GC receptor (GR) gene (ER22/23EK, N363S, BclI, 9 beta A3669G) modulate the influence of cortisol administration on working memory. Working memory performance was investigated in 169 subjects on 10 mg hydrocortisone (cortisol) and placebo using an item recognition task. No impairing effect of hydrocortisone treatment became evident. However, a sex x genotype interaction on general working memory performance was revealed (p = 0.02). While female heterozygous carriers of the 9 beta G allele displayed faster reaction times than the other genotype groups, 9 beta G heterozygous men were relatively slower. Heritability estimates for memory are roughly 50%, indicating that common genetic polymorphisms have an important impact on cognitive performance. Our results suggest that variants of the GR gene might explain some of the variance attributable to genetic factors. Furthermore, it can be speculated that they modulate the individual vulnerability for memory impairments related to stress-related psychiatric disorders. (c) 2009 S. Karger AG, Basel.

  20. 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. © 2016 by the Society for Experimental Biology and Medicine.

  1. 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.

  2. 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

  3. Trehalose induces functionally active conformation in the intrinsically disordered N-terminal domain of glucocorticoid receptor.

    PubMed

    Khan, Shagufta H; Jasuja, Ravi; Kumar, Raj

    2016-08-05

    Glucocorticoid receptor (GR) is a classic member of the nuclear receptor superfamily and plays pivotal roles in human physiology at the level of gene regulation. Various constellations of cellular cofactors are required to associate with GR to activate/repress genes. The effects of specific ligands on the AF2 structure and consequent preferential binding of co-activators or co-repressors have helped our understanding of the mechanisms involved. But the data so far fall short of fully explaining GR actions. We believe that this is because work so far has largely avoided detailed examination of the contributions of AF1 to overall GR actions. It has been shown that the GR containing only the N-terminal domain (NTD) and the DNA-binding domain (GR500) is constitutively quite active in stimulating transcription from simple promoters. However, we are only beginning to understand structure and functions of GR500 in spite of the fact that AF1 located within the NTD serves as major transactivation domain for GR. Lack of this information has hampered our complete understanding of how GR regulates its target gene(s). The major obstacle in determining GR500 structure has been due to its intrinsically disordered NTD conformation, frequently found in transcription factors. In this study, we tested whether a naturally occurring osmolyte, trehalose, can promote functionally ordered conformation in GR500. Our data show that in the presence of trehalose, GR500 is capable of formation of a native-like functionally folded conformation.

  4. Insights into negative regulation by the glucocorticoid receptor from genome-wide profiling of inflammatory cistromes.

    PubMed

    Uhlenhaut, N Henriette; Barish, Grant D; Yu, Ruth T; Downes, Michael; Karunasiri, Malith; Liddle, Christopher; Schwalie, Petra; Hübner, Norbert; Evans, Ronald M

    2013-01-10

    How the glucocorticoid receptor (GR) activates some genes while potently repressing others remains an open question. There are three current models for suppression: transrepression via GR tethering to AP-1/NF-κB sites, direct GR association with inhibitory elements (nGREs), and GR recruitment of the corepressor GRIP1. To gain insights into GR suppression, we used genomic analyses and genome-wide profiling of GR, p65, and c-Jun in LPS-stimulated macrophages. We show that GR mediates both activation and repression at tethered sites, GREs, and GRIP1-bound elements, indicating that motif classification is insufficient to predict regulatory polarity of GR binding. Interestingly, sites of GR repression utilize GRIP1's corepressor function and display reduced histone acetylation. Together, these findings suggest that while GR occupancy confers hormone responsiveness, the receptor itself may not participate in the regulatory effects. Furthermore, transcriptional outcome is not established by sequence but is influenced by epigenetic regulators, context, and other unrecognized regulatory determinants.

  5. Corticosterone targets distinct steps of synaptic transmission via concentration specific activation of mineralocorticoid and glucocorticoid receptors.

    PubMed

    Chatterjee, Sreejata; Sikdar, Sujit K

    2014-02-01

    Hippocampal neurons are affected by chronic stress and have a high density of cytoplasmic mineralocorticoid and glucocorticoid receptors (MR and GR). Detailed studies on the genomic effects of the stress hormone corticosterone at physiologically relevant concentrations on different steps in synaptic transmission are limited. In this study, we tried to delineate how activation of MR and GR by different concentrations of corticosterone affects synaptic transmission at various levels. The effect of 3-h corticosterone (25, 50, and 100 nM) treatment on depolarization-mediated calcium influx, vesicular release and properties of miniature excitatory post-synaptic currents (mEPSCs) were studied in cultured hippocampal neurons. Activation of MR with 25 nM corticosterone treatment resulted in enhanced depolarization-mediated calcium influx via a transcription-dependent process and increased frequency of mEPSCs with larger amplitude. On the other hand, activation of GR upon 100 nM corticosterone treatment resulted in increase in the rate of vesicular release via the genomic actions of GR. Furthermore, GR activation led to significant increase in the frequency of mEPSCs with larger amplitude and faster decay. Our studies indicate that differential activation of the dual receptor system of MR and GR by corticosterone targets the steps in synaptic transmission differently.

  6. Early postnatal handling alters glucocorticoid receptor concentrations in selected brain regions.

    PubMed

    Meaney, Michael J; Aitken, David H; Bodnoff, Shari R; Iny, Linda J; Tatarewicz, Joseph E; Sapolsky, Robert M

    2013-10-01

    Norway rat pups were either handled (H) or undisturbed (nonhandled, NH) in the period between birth and weaning on Day 21. Following weaning, half of the animals in each group were housed socially (Soc), and half were housed in isolation (Isol). At 120-150 days of age, all animals were sacrificed, and the following regions were dissected and frozen at -70 °C until the time of assay: frontal cortex, hippocampus, hypothalamus, amygdala, septum, and pituitary. [3H]Dexamethasone (3H Dex) binding in each region was examined by an in vitro, cytosol, receptor assay. 3H Dex binding was significantly higher in the hippocampus of both H-Soc and H-Isol than in NH groups. In the frontal cortex, 3H Dex binding was higher in the H-Soc animals than in the H-Isol and NH-Isol animals. There were no significant handling or housing effects found in the amygdala, hypothalamus, septum, or pituitary. Thus, early postnatal handling appears to influence the development of the glucocorticoid receptor system in the hippocampus and frontal cortex. These results are discussed as providing a possible mechanism for some of the previously reported effects of early handling on the development of the pituitary-adrenal response to stress. 2013 APA, all rights reserved

  7. Corticosterone Inhibits the Proliferation of C6 Glioma Cells via the Translocation of Unphosphorylated Glucocorticoid Receptor.

    PubMed

    Nakatani, Yoshihiko; Amano, Taku; Takeda, Hiroshi

    2016-01-01

    Astroglial cells have been considered to have passive brain function by helping to maintain neurons. However, recent studies have revealed that the dysfunction of such passive functions may be associated with various neuropathological diseases, such as schizophrenia, Alzheimer's disease, amyotrophic lateral sclerosis and major depression. Corticosterone (CORT), which is often referred to as the stress hormone, is a well-known regulator of peripheral immune responses and also shows anti-inflammatory properties in the brain. However, it is still obscure how CORT affects astroglial cell function. In this study, we investigated the effects of CORT on the proliferation and survival of astroglial cells using C6 glioma cells. Under treatment with CORT for 24h, the proliferation of C6 glioma cells decreased in a dose-dependent manner. Moreover, this inhibition was diminised by treatment with mifepristone, a glucocorticoid receptor (GR) antagonist, but not by spironolactone, a mineralocorticoid receptor (MR) antagonist, and was independent of GR phosphorylation and other GR-related intracellular signaling cascades. Furthermore, it was observed that the translocation of GR from the cytosol to the nucleus was promoted by the treatment with CORT. These results indicate that CORT decreases the proliferation of C6 glioma cells by modifying the transcription of a particular gene related to cell proliferation independent of GR phosphorylation.

  8. Gene and protein alterations of FKBP5 and glucocorticoid receptor in the amygdala of suicide victims.

    PubMed

    Pérez-Ortiz, José M; García-Gutiérrez, María S; Navarrete, Francisco; Giner, Salvador; Manzanares, Jorge

    2013-08-01

    Recent reports suggest that FKBP5 gene and its corresponding FKBP5 protein play a relevant role in the regulation of anxiety and depression in animal models and human stress-related disorders. In the present study, FKBP5 and glucocorticoid receptor (GR) gene and protein expression were analyzed in the amygdala (AMY) of suicide victims (n=13 males, without clinical psychiatric history and non-treated with anxiolytic or antidepressant drugs) and its corresponding controls (n=13 males) by real-time PCR and Western blotting. The results revealed that FKBP5 and GR gene expression were significantly reduced in the AMY (-38% and -48%, respectively) of suicide victims compared with controls. Interestingly, FKBP5 and GR protein expression were also significantly decreased (-41% and -42%, respectively) in the AMY of suicide victims compared with controls. These results suggest that the FKBP5 plays a relevant role in human emotional responses and suggest this receptor as a new promising target in the treatment of suicide behavior.

  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. Human glucocorticoid-induced TNF receptor ligand regulates its signaling activity through multiple oligomerization states

    PubMed Central

    Zhou, Zhaocai; Song, Xiaomin; Berezov, Alan; Zhang, Geng; Li, Yanjing; Zhang, Hongtao; Murali, Ramachandran; Li, Bin; Greene, Mark I.

    2008-01-01

    Ligation between glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand (GITRL) provides an undefined signal that renders CD4+CD25− effector T cells resistant to the inhibitory effects of CD4+CD25+ regulatory T cells. To understand the structural basis of GITRL function, we have expressed and purified the extracellular domain of human GITR ligand in Escherichia coli. Chromotography and cross-linking studies indicate that human GITRL (hGITRL) exists as dimers and trimers in solution and also can form a supercluster. To gain insight into the nature of GITRL oligomerization, we determined the crystallographic structures of hGITRL, which revealed a loosely associated open trimer with a deep cavity at the molecular center and a flexible C-terminal tail bent for trimerization. Moreover, a tetramer of trimers (i.e., supercluster) has also been observed in the crystal, consistent with the cross-linking analysis. Deletion of the C-terminal distal three residues disrupts the loosely assembled trimer and favors the formation of a dimer that has compromised receptor binding and signaling activity. Collectively, our studies identify multiple oligomeric species of hGITRL that possess distinct kinetics of ERK activation. The studies address the functional implications and structural models for a process by which hGITRL utilizes multiple oligomerization states to regulate GITR-mediated signaling during T cell costimulation. PMID:18378892

  11. Identification of the BclI polymorphism in the glucocorticoid receptor gene: association with sensitivity to glucocorticoids in vivo and body mass index.

    PubMed

    van Rossum, Elisabeth F C; Koper, Jan W; van den Beld, Annewieke W; Uitterlinden, André G; Arp, Pascal; Ester, Wietske; Janssen, Joop A M J L; Brinkmann, Albert O; de Jong, Frank H; Grobbee, Diederick E; Pols, Huibert A P; Lamberts, Steven W J

    2003-11-01

    Sensitivity to glucocorticoids differs between individuals, partially due to genetic variation in the glucocorticoid receptor (GR) gene. We studied the sequence alteration of a previously described intronic BclI polymorphism of the GR gene, and investigated whether there was an association with sensitivity to glucocorticoids and anthropometric parameters in a group of healthy elderly individuals. In study group 1, two overnight dexamethasone suppression tests (DSTs) were performed: with 1 mg dexamethasone, and 2.5 years later with 0.25 mg dexamethasone. Anthropometric parameters were measured in a larger population (study group 2), as well as in a third study group, in which we also measured body composition by dual-energy X-ray absorbtiometry (DEXA) scans. Groups 1 and 2, respectively, 191 and 1963 male and female participants of the Rotterdam study, a population-based study in Dutch elderly. Study group 3: 370 elderly males (mean age 77.8 +/- 0.2 years) from Zoetermeer, the Netherlands. We identified the BclI restriction site polymorphism as a C/G substitution in intron 2, 646 nucleotides downstream from exon 2. After both 1 mg and 0.25 mg DST, heterozygous (CG) and homozygous G-allele carriers (GG) had lower cortisol levels than CC-carriers (P = 0.01 and P = 0.02, respectively). In study group 2, we found a lower body mass index (BMI; P = 0.006) and waist-hip ratio (WHR; P = 0.02) in G-allele carriers. In study group 3, again we found a lower BMI (P = 0.05) in G-allele carriers. No differences were found in fat mass. However, lean mass tended to be lower in G-allele carriers (P = 0.07). We characterized a BclI-RFLP (restriction fragment length polymorphism) of the GR gene as a C/G polymorphism in intron 2 of which the G-allele was associated with hypersensitivity to glucocorticoids. This resulted in a lower BMI in older individuals in general, while our study in elderly males suggests that the lower BMI is probably due to a greater loss of lean mass during the

  12. Glucocorticoid receptor binding to a specific DNA sequence is required for hormone-dependent repression of pro-opiomelanocortin gene transcription.

    PubMed Central

    Drouin, J; Trifiro, M A; Plante, R K; Nemer, M; Eriksson, P; Wrange, O

    1989-01-01

    Glucocorticoids rapidly and specifically inhibit transcription of the pro-opiomelanocortin (POMC) gene in the anterior pituitary, thus offering a model for studying negative control of transcription in mammals. We have defined an element within the rat POMC gene 5'-flanking region that is required for glucocorticoid inhibition of POMC gene transcription in POMC-expressing pituitary tumor cells (AtT-20). This element contains an in vitro binding site for purified glucocorticoid receptor. Site-directed mutagenesis revealed that binding of the receptor to this site located at position base pair -63 is essential for glucocorticoid repression of transcription. Although related to the well-defined glucocorticoid response element (GRE) found in glucocorticoid-inducible genes, the DNA sequence of the POMC negative glucocorticoid response element (nGRE) differs significantly from the GRE consensus; this sequence divergence may result in different receptor-DNA interactions and may account at least in part for the opposite transcriptional properties of these elements. Hormone-dependent repression of POMC gene transcription may be due to binding of the receptor over a positive regulatory element of the promoter. Thus, repression may result from mutually exclusive binding of two DNA-binding proteins to overlapping DNA sequences. Images PMID:2586521

  13. Downregulation of sphingosine 1-phosphate (S1P) receptor 1 by dexamethasone inhibits S1P-induced mesangial cell migration.

    PubMed

    Koch, Alexander; Jäger, Manuel; Völzke, Anja; Grammatikos, Georgios; Zu Heringdorf, Dagmar Meyer; Huwiler, Andrea; Pfeilschifter, Josef

    2015-06-01

    Sphingosine 1-phosphate (S1P) is generated by sphingosine kinase (SK)-1 and -2 and acts mainly as an extracellular ligand at five specific receptors, denoted S1P1-5. After activation, S1P receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration and survival. Previously, we showed that dexamethasone enhances SK-1 activity and S1P formation, which protected mesangial cells from stress-induced apoptosis. Here we demonstrate that dexamethasone treatment lowered S1P1 mRNA and protein expression levels in rat mesangial cells. This effect was abolished in the presence of the glucocorticoid receptor antagonist RU-486. In addition, in vivo studies showed that dexamethasone downregulated S1P1 expression in glomeruli isolated from mice treated with dexamethasone (10 mg/kg body weight). Functionally, we identified S1P1 as a key player mediating S1P-induced mesangial cell migration. We show that dexamethasone treatment significantly lowered S1P-induced migration of mesangial cells, which was again reversed in the presence of RU-486. In summary, we suggest that dexamethasone inhibits S1P-induced mesangial cell migration via downregulation of S1P1. Overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells.

  14. A glucocorticoid/retinoic acid receptor chimera that displays cytoplasmic/nuclear translocation in response to retinoic acid. A real time sensing assay for nuclear receptor ligands.

    PubMed

    Mackem, S; Baumann, C T; Hager, G L

    2001-12-07

    Members of the nuclear receptor superfamily play key roles in a host of physiologic and pathologic processes from embryogenesis to cancer. Some members, including the retinoic acid receptor (RAR), are activated by ligand binding but are unaffected in their subcellular distribution, which is predominantly nuclear. In contrast, several members of the steroid receptor family, including the glucocorticoid receptor, are cytoplasmic and only translocate to the nucleus after ligand binding. We have constructed chimeras between RAR and glucocorticoid receptor that selectively respond to RAR agonists but display cytoplasmic localization in the absence of ligand. These chimeric receptors manifest both nuclear translocation and gene activation functions in response to physiological concentrations of RAR ligands. The ability to achieve regulated subcellular trafficking with a heterologous ligand binding domain has implications both for current models of receptor translocation and for structural-functional conservation of ligand binding domains broadly across the receptor superfamily. When coupled to the green fluorescent protein, chimeric receptors offer a powerful new tool to 1) study mechanisms of steroid receptor translocation, 2) detect dynamic and graded distributions of ligands in complex microenvironments such as embryos, and 3) screen for novel ligands of "orphan" receptors in vivo.

  15. Selective Androgen Receptor Down-Regulators (SARDs): A New Prostate Cancer Therapy

    DTIC Science & Technology

    2007-10-01

    PCa (9). Thus far, the techniques that have been used to down-regulate the AR include antisense oligonucleotides (10, 11), ribozyme treatments (12...Our findings suggest that ICI may present a useful treatment option for patients with AR-dependent PCa. Unlike the ribozyme , antisense, siRNA, or...Catalytic cleavage of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme . Mol Endocrinol

  16. Subchronic Glucocorticoid Receptor Inhibition Rescues Early Episodic Memory and Synaptic Plasticity Deficits in a Mouse Model of Alzheimer's Disease

    PubMed Central

    Lanté, Fabien; Chafai, Magda; Raymond, Elisabeth Fabienne; Salgueiro Pereira, Ana Rita; Mouska, Xavier; Kootar, Scherazad; Barik, Jacques; Bethus, Ingrid; Marie, Hélène

    2015-01-01

    The early phase of Alzheimer's disease (AD) is characterized by hippocampus-dependent memory deficits and impaired synaptic plasticity. Increasing evidence suggests that stress and dysregulation of the hypothalamo-pituitary-adrenal (HPA) axis, marked by the elevated circulating glucocorticoids, are risk factors for AD onset. How these changes contribute to early hippocampal dysfunction remains unclear. Using an elaborated version of the object recognition task, we carefully monitored alterations in key components of episodic memory, the first type of memory altered in AD patients, in early symptomatic Tg2576 AD mice. We also combined biochemical and ex vivo electrophysiological analyses to reveal novel cellular and molecular dysregulations underpinning the onset of the pathology. We show that HPA axis, circadian rhythm, and feedback mechanisms, as well as episodic memory, are compromised in this early symptomatic phase, reminiscent of human AD pathology. The cognitive decline could be rescued by subchronic in vivo treatment with RU486, a glucocorticoid receptor antagonist. These observed phenotypes were paralleled by a specific enhancement of N-Methyl-D-aspartic acid receptor (NMDAR)-dependent LTD in CA1 pyramidal neurons, whereas LTP and metabotropic glutamate receptor-dependent LTD remain unchanged. NMDAR transmission was also enhanced. Finally, we show that, as for the behavioral deficit, RU486 treatment rescues this abnormal synaptic phenotype. These preclinical results define glucocorticoid signaling as a contributing factor to both episodic memory loss and early synaptic failure in this AD mouse model, and suggest that glucocorticoid receptor targeting strategies could be beneficial to delay AD onset. PMID:25622751

  17. A Pharmacokinetic/Pharmacodynamic Study of the Glucocorticoid Receptor Antagonist Mifepristone Combined with Enzalutamide in Castrate-Resistant Prostate Cancer

    DTIC Science & Technology

    2014-12-01

    combination of mifepristone and enzalutamide has been well tolerated with no dose limiting toxicities . The current cohort has dose- escalated the...Pharmacodynamic Study of the Glucocorticoid Receptor Antagonist Mifepristone Combined with Enzalutamide in Castrate-Resistant Prostate Cancer...Antagonist Mifepristone Combined with Enzalutamide in Castrate-Resistant Prostate Cancer 5b. GRANT NUMBER W81XWH-14-1-0021 5c. PROGRAM ELEMENT

  18. 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

  19. 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

  20. 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

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

    PubMed

    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

    2015-10-01

    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. 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. 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. The liver GR controls systemic and liver urea cycle function by transcriptional regulation of ARG1 expression.

  2. Eosinophil as a cellular target of the ocular anti-allergic action of mapracorat, a novel selective glucocorticoid receptor agonist

    PubMed Central

    Baiula, Monica; Spartà, Antonino; Bedini, Andrea; Carbonari, Gioia; Bucolo, Claudio; Ward, Keith W.; Zhang, Jin-Zhong; Govoni, Paolo

    2011-01-01

    Purpose Glucocorticoids can either suppress gene transcription (transrepression) or activate it (transactivation). This latter process may contribute to certain side effects caused by these agents. Mapracorat (also known as BOL-303242-X or ZK 245186) is a novel selective glucocorticoid receptor agonist that maintains a beneficial anti-inflammatory activity but seems to be less effective in transactivation, resulting in a lower potential for side effects; it has been proposed for the topical treatment of inflammatory skin disorders. This study assessed the anti-allergic activity of mapracorat at the ocular level and whether eosinophils and mast cells are targets of its action. Methods With in vitro studies apoptosis was evaluated in human eosinophils by flow cytometry and western blot of caspase-3 fragments. Eosinophil migration toward platelet-activating factor was evaluated by transwell assays. Interleukin (IL)-6, IL-8, tumor necrosis factor-α (TNF-α), and the chemokine (C-C motif) ligand 5 (CCL5)/regulated upon activation normal T cell expressed, and presumably secreted (RANTES) were measured using a high-throughput multiplex luminex technology. Annexin I and the chemochine receptor C-X-C chemokine receptor 4 (CXCR4) were detected by flow cytometry. With in vivo studies, allergic conjunctivitis was induced in guinea pigs sensitized to ovalbumin by an ocular allergen challenge and evaluated by a clinical score. Conjunctival eosinophils were determined by microscopy or eosinophil peroxidase assay. Results In cultured human eosinophils, mapracorat showed the same potency as dexamethasone but displayed higher efficacy in increasing spontaneous apoptosis and in counteracting cytokine-sustained eosinophil survival. These effects were prevented by the glucocorticoid receptor antagonist mifepristone. Mapracorat inhibited eosinophil migration and IL-8 release from eosinophils or the release of IL-6, IL-8, CCL5/RANTES, and TNF-α from a human mast cell line with equal

  3. Tetrahydroisoquinoline Phenols: Selective Estrogen Receptor Downregulator Antagonists with Oral Bioavailability in Rat.

    PubMed

    Scott, James S; Bailey, Andrew; Davies, Robert D M; Degorce, Sébastien L; MacFaul, Philip A; Gingell, Helen; Moss, Thomas; Norman, Richard A; Pink, Jennifer H; Rabow, Alfred A; Roberts, Bryan; Smith, Peter D

    2016-01-14

    A series of tetrahydroisoquinoline phenols was modified to give an estrogen receptor downregulator-antagonist profile. Optimization around the core, alkyl side chain, and pendant aryl ring resulted in compounds with subnanomolar levels of potency. The phenol functionality was shown to be required to achieve highly potent compounds, but unusually this was compatible with obtaining high oral bioavailabilities in rat.