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Sample records for glucocorticoid receptor down-regulation

  1. 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.

  2. 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.

  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. 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.

  5. 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.

  6. 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

  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

    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.

  8. Desensitization and Down Regulation of Muscarinic Acetylcholine Receptors

    DTIC Science & Technology

    1988-06-22

    decrease in the relative densities ofNMlImuscarinic receptors which have a high affinity for the selective antagonist pirenzepine . Activation of...which is readily susceptible to regu- lation by receptor agonists, while the other is resistant in this regard. In control cells, both pirenzepine and... pirenzepine and carbamylcholine with [3H]N-methylsco- polamine showed that the receptor sites remaining upon desensitization are composed mainly of pirenzepine

  9. 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

  10. Optimal experimental design in an epidermal growth factor receptor signalling and down-regulation model.

    PubMed

    Casey, F P; Baird, D; Feng, Q; Gutenkunst, R N; Waterfall, J J; Myers, C R; Brown, K S; Cerione, R A; Sethna, J P

    2007-05-01

    We apply the methods of optimal experimental design to a differential equation model for epidermal growth factor receptor signalling, trafficking and down-regulation. The model incorporates the role of a recently discovered protein complex made up of the E3 ubiquitin ligase, Cbl, the guanine exchange factor (GEF), Cool-1 (beta -Pix) and the Rho family G protein Cdc42. The complex has been suggested to be important in disrupting receptor down-regulation. We demonstrate that the model interactions can accurately reproduce the experimental observations, that they can be used to make predictions with accompanying uncertainties, and that we can apply ideas of optimal experimental design to suggest new experiments that reduce the uncertainty on unmeasurable components of the system.

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

    SciTech Connect

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

    1987-08-01

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

  12. 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.

  13. Prolonged administration of pyridostigmine impairs neuromuscular function with and without down-regulation of acetylcholine receptors.

    PubMed

    Richtsfeld, Martina; Yasuhara, Shingo; Fink, Heidrun; Blobner, Manfred; Martyn, J A Jeevendra

    2013-08-01

    The acetylcholinesterase inhibitor, pyridostigmine, is prophylactically administered to mitigate the toxic effects of nerve gas poisoning. The authors tested the hypothesis that prolonged pyridostigmine administration can lead to neuromuscular dysfunction and even down-regulation of acetylcholine receptors. Pyridostigmine (5 or 25 mg·kg·day) or saline was continuously administered via osmotic pumps to rats, and infused for either 14 or 28 days until the day of neuromuscular assessment (at day 14 or 28), or discontinued 24 h before neuromuscular assessment. Neurotransmission and muscle function were examined by single-twitch, train-of-four stimulation and 100-Hz tetanic stimulation. Sensitivity to atracurium and acetylcholine receptor number (quantitated by I-α-bungarotoxin) provided additional measures of neuromuscular integrity. Specific tetanic tensions (Newton [N]/muscle weight [g]) were significantly (P < 0.05) decreased at 14 (10.3 N/g) and 28 (11.1 N/g) days of 25 mg·kg·day pyridostigmine compared with controls (13.1-13.6 N/g). Decreased effective dose (0.81-1.05 vs. 0.16-0.45 mg/kg; P < 0.05) and decreased plasma concentration (3.02-3.27 vs. 0.45-1.37 μg/ml; P < 0.05) of atracurium for 50% paralysis (controls vs. 25 mg·kg·day pyridostigmine, respectively), irrespective of discontinuation of pyridostigmine, confirmed the pyridostigmine-induced altered neurotransmission. Pyridostigmine (25 mg·kg·day) down-regulated acetylcholine receptors at 28 days. Prolonged administration of pyridostigmine (25 mg·kg·day) leads to neuromuscular impairment, which can persist even when pyridostigmine is discontinued 24 h before assessment of neuromuscular function. Pyridostigmine has the potential to down-regulate acetylcholine receptors, but induces neuromuscular dysfunction even in the absence of receptor changes.

  14. Down-regulation of pancreatic transcription factors and incretin receptors in type 2 diabetes

    PubMed Central

    Kaneto, Hideaki; Matsuoka, Taka-aki

    2013-01-01

    Type 2 diabetes is one of the most prevalent and serious metabolic diseases. Under diabetic conditions, chronic hyperglycemia and subsequent induction of oxidative stress deteriorate pancreatic β-cell function, which leads to the aggravation of type 2 diabetes. Although such phenomena are well known as glucose toxicity, its molecular mechanism remains unclear. In this review article, we describe the possible molecular mechanism for β-cell dysfunction found in type 2 diabetes, focusing on (1) oxidative stress, (2) pancreatic transcription factors (PDX-1 and MafA) and (3) incretin receptors (GLP-1 and GIP receptors). Under such conditions, nuclear expression levels of PDX-1 and MafA are decreased, which leads to suppression of insulin biosynthesis and secretion. In addition, expression levels of GLP-1 and GIP receptors are decreased, which likely contributes to the impaired incretin effects found in diabetes. Taken together, it is likely that down-regulation of pancreatic transcription factors (PDX-1 and MafA) and down-regulation of incretin receptors (GLP-1 and GIP receptors) explain, at least in part, the molecular mechanism for β-cell dysfunction found in type 2 diabetes. PMID:24379916

  15. Down-Regulation of the Met Receptor Tyrosine Kinase by Presenilin-dependent Regulated Intramembrane Proteolysis

    PubMed Central

    Foveau, Bénédicte; Ancot, Frédéric; Leroy, Catherine; Petrelli, Annalisa; Reiss, Karina; Vingtdeux, Valérie; Giordano, Silvia; Fafeur, Véronique

    2009-01-01

    Hepatocyte growth factor/scatter factor (HGF/SF) acts through the membrane-anchored Met receptor tyrosine kinase to induce invasive growth. Deregulation of this signaling is associated with tumorigenesis and involves, in most cases, overexpression of the receptor. We demonstrate that Met is processed in epithelial cells by presenilin-dependent regulated intramembrane proteolysis (PS-RIP) independently of ligand stimulation. The proteolytic process involves sequential cleavage by metalloproteases and the γ-secretase complex, leading to generation of labile fragments. In normal epithelial cells, although expression of cleavable Met by PS-RIP is down-regulated, uncleavable Met displayed membrane accumulation and induced ligand-independent motility and morphogenesis. Inversely, in transformed cells, the Met inhibitory antibody DN30 is able to promote Met PS-RIP, resulting in down-regulation of the receptor and inhibition of the Met-dependent invasive growth. This demonstrates the original involvement of a proteolytic process in degradation of the Met receptor implicated in negative regulation of invasive growth. PMID:19297528

  16. 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

  17. Down-regulation of tumor necrosis factor receptors by blockade of mitochondrial respiration.

    PubMed

    Sánchez-Alcázar, J A; Hernández, I; De la Torre, M P; García, I; Santiago, E; Muñoz-Yagüe, M T; Solís-Herruzo, J A

    1995-10-13

    We have studied the effect of blockade of mitochondrial respiration on the binding of human 125I-TNF alpha to L929 cell receptors. Specific TNF alpha binding was decreased to about 20-40% of controls by blocking mitochondrial respiration. This effect was dose- and time-related and was observed independently of the level at which the respiration was blocked (respiratory chain, proton backflow, ATPase, anaerobiosis). This blockade had no effect on the half-life of the specific TNF alpha binding, the internalization or degradation of TNF alpha-receptor complexes, or the number of TNF alpha-binding sites. Scatchard analysis of TNF alpha binding data indicated a 2-4-fold decrease in the affinity of these binding sites. These effects did not appear to be related to the protein kinase C activity or to reactive oxygen radicals, since they were not antagonized by pretreatment of cells with oxygen radical scavengers, deferoxamine, or inhibitors of protein kinase C. Decrease in TNF alpha binding capacity correlated significantly with cellular ATP content (r = 0.94; p < 0.01) and with the cytocidal activity of TNF alpha against L929 cells. These findings suggest that blockade of mitochondrial respiration down-regulates the binding of TNF alpha to cells, most likely by changing the affinity of receptors for this cytokine. This down-regulation may increase the resistance of cells to TNF alpha cytotoxicity.

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

    SciTech Connect

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

    2014-10-01

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

  19. Remote ischaemic preconditioning down-regulates kinin receptor expression in neutrophils of patients undergoing heart surgery

    PubMed Central

    Saxena, Pankaj; Aggarwal, Shashi; Misso, Neil L.; Passage, Jurgen; Newman, Mark A. J.; Thompson, Philip J.; d'Udekem, Yves; Praporski, Slavica; Konstantinov, Igor E.

    2013-01-01

    OBJECTIVES Remote ischaemic preconditioning (RIPC) may protect distant organs against ischaemia-reperfusion injury. We investigated the impact of RIPC on kinin receptor expression in neutrophils following RIPC in patients undergoing coronary artery bypass grafting (CABG). METHODS Patients undergoing elective CABG with cardiopulmonary bypass (CPB) were randomized to RIPC (n = 15) or control (n = 15) groups. The study group underwent RIPC by inflation of a blood pressure cuff on the arm. Expression of kinin receptors, plasma concentrations of IL-6, IL-8, IL-10, TNF-α and neutrophil elastase were determined at baseline (before RIPC/sham), immediately before surgery (after RIPC/sham) and 30 min and 24 h after surgery. Plasma bradykinin levels were assessed before and after RIPC/sham, and at 30 min, 6, 12 and 24 h after surgery. Serum creatine kinase (CK), troponin I, C-reactive protein (CRP) and lactate levels were measured immediately prior to surgery and 30 min, 6, 12, 24 and 48 h after surgery. RESULTS Kinin B2 receptor expression did not differ between the groups at baseline (pre-RIPC), but was significantly lower in the RIPC group than in the control group after RIPC/sham (P < 0.05). Expressions of both kinin B1 and B2 receptors were significantly down-regulated in the RIPC group, and this persisted to 24 h after surgery (P < 0.001). Neutrophil elastase levels were significantly increased after surgery. There were no differences in CK, CRP, cytokine, lactate or troponin I levels between the groups. CONCLUSIONS RIPC down-regulated the expression of kinin B1 and B2 receptors in neutrophils of patients undergoing CABG. PMID:23814135

  20. Surfactant prevents quartz induced down-regulation of complement receptor 1 in human granulocytes.

    PubMed

    Zetterberg, G; Lundahl, J; Curstedt, T; Eklund, A

    1997-02-01

    Quartz is known to induce an inflammatory response in the alveolar space by recruitment of different effector cells. We investigated the interaction between granulocytes and quartz with respect to expression of complement receptor type 1 (CR1) and CR3, with and without the presence of surfactant. Granulocytes from hemolyzed blood were stimulated by N-formyl-methionyl-leucyl-phenylalanine (fMLP), which mobilize the intracellular pool of CR1 to the surface, and the mean fluorescence intensity (MFI) measured by cytofluorometry was 47.4 (46-63.6) (median; interquartile range). Quartz exposure reduced the CR1 expression to 23.2 (22.8-30.6) MFI units (P < 0.01), a porcine surfactant preparation added during quartz exposure abolished the down-regulation completely, 47.7 (43.2-62.3) MFI units (P < 0.001). Similar results were obtained after preincubation of the cells with surfactant followed by quartz exposure. No significant influence on CR1 expression was found by a synthetic lipid mixture, nor was the CR3 expression affected. In conclusion, this study demonstrates that the presence of surfactant inhibits quartz induced down-regulation of CR1 on activated granulocytes.

  1. Dysregulation of Ack1 inhibits down-regulation of the EGF receptor

    SciTech Connect

    Grovdal, Lene Melsaether; Johannessen, Lene E.; Rodland, Marianne Skeie; Madshus, Inger Helene; Stang, Espen

    2008-04-01

    The protein tyrosine kinase Ack1 has been linked to cancer when over-expressed. Ack1 has also been suggested to function in clathrin-mediated endocytosis and in down-regulation of the epidermal growth factor (EGF) receptor (EGFR). We have studied the intracellular localization of over-expressed Ack1 and found that Ack1 co-localizes with the EGFR upon EGF-induced endocytosis in cells with moderate over-expression of Ack. This co-localization is mainly observed in early endosomes. Furthermore, we found that over-expression of Ack1 retained the EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. Down-regulation of Ack1 in HeLa cells resulted in reduced rate of {sup 125}I-EGF internalization, whereas internalization of {sup 125}I-transferrin was not affected. In cells where Ack1 had been knocked down by siRNA, recycling of internalized {sup 125}I-EGF was increased, while degradation of {sup 125}I-EGF was inhibited. Together, these data suggest that Ack1 is involved in an early step of EGFR desensitization.

  2. 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

  3. Hydrogen peroxide down-regulates inositol 1,4,5-trisphosphate receptor content through proteasome activation.

    PubMed

    Martín-Garrido, A; Boyano-Adánez, M C; Alique, M; Calleros, L; Serrano, I; Griera, M; Rodríguez-Puyol, D; Griendling, K K; Rodríguez-Puyol, M

    2009-11-15

    Hydrogen peroxide (H(2)O(2)) is implicated in the regulation of signaling pathways leading to changes in vascular smooth muscle function. Contractile effects produced by H(2)O(2) are due to the phosphorylation of myosin light chain kinase triggered by increases in intracellular calcium (Ca(2+)) from intracellular stores or influx of extracellular Ca(2+). One mechanism for mobilizing such stores involves the phosphoinositide pathway. Inositol 1,4,5-trisphosphate (IP(3)) mobilizes intracellular Ca(2+) by binding to a family of receptors (IP(3)Rs) on the endoplasmic-sarcoplasmic reticulum that act as ligand-gated Ca(2+) channels. IP(3)Rs can be rapidly ubiquitinated and degraded by the proteasome, causing a decrease in cellular IP(3)R content. In this study we show that IP(3)R(1) and IP(3)R(3) are down-regulated when vascular smooth muscle cells (VSMC) are stimulated by H(2)O(2), through an increase in proteasome activity. Moreover, we demonstrate that the decrease in IP(3)R by H(2)O(2) is accompanied by a reduction in calcium efflux induced by IP(3) in VSMC. Also, we observed that angiotensin II (ANGII) induces a decrease in IP(3)R by activation of NADPH oxidase and that preincubation with H(2)O(2) decreases ANGII-mediated calcium efflux and planar cell surface area in VSMC. The decreased IP(3) receptor content observed in cells was also found in aortic rings, which exhibited a decreased ANGII-dependent contraction after treatment with H(2)O(2). Altogether, these results suggest that H(2)O(2) mediates IP(3)R down-regulation via proteasome activity.

  4. 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.

  5. 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.

  6. Insulin receptor activation and down-regulation by cationic lipid transfection reagents.

    PubMed

    Pramfalk, Camilla; Lanner, Johanna; Andersson, Monica; Danielsson, Eva; Kaiser, Christina; Renström, Ing-Marie; Warolén, Malin; James, Stephen R

    2004-01-26

    Transfection agents comprised of cationic lipid preparations are widely used to transfect cell lines in culture with specific recombinant complementary DNA molecules. We have found that cells in culture are often resistant to stimulation with insulin subsequent to treatment with transfection agents such as LipofectAMINE 2000 and FuGENE-6. This is seen with a variety of different readouts, including insulin receptor signalling, glucose uptake into muscle cells, phosphorylation of protein kinase B and reporter gene activity in a variety of different cell types We now show that this is due in part to the fact that cationic lipid agents activate the insulin receptor fully during typical transfection experiments, which is then down-regulated. In attempts to circumvent this problem, we investigated the effects of increasing concentrations of LipofectAMINE 2000 on insulin receptor phosphorylation in Chinese hamster ovary cells expressing the human insulin receptor. In addition, the efficiency of transfection that is supported by the same concentrations of transfection reagent was studied by using a green fluorescent protein construct. Our data indicate that considerably lower concentrations of LipofectAMINE 2000 can be used than are recommended by the manufacturers. This is without sacrificing transfection efficiency markedly and avoids the problem of reducing insulin receptor expression in the cells. Widely-used cationic lipid transfection reagents cause a state of insulin unresponsiveness in cells in culture due to fully activating and subsequently reducing the expression of the receptor in cells. This phenomenon can be avoided by reducing the concentration of reagent used in the transfection process.

  7. 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.

  8. 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

  9. Dystroglycan down-regulation links EGF receptor signaling and anterior–posterior polarity formation in the Drosophila oocyte

    PubMed Central

    Poulton, John S.; Deng, Wu-Min

    2006-01-01

    Anterior–posterior axis formation in the Drosophila oocyte requires activation of the EGF receptor (EGFR) pathway in the posterior follicle cells (PFC), where it also redirects them from the default anterior to the posterior cell fate. The relationship between EGFR activity in the PFC and oocyte polarity is unclear, because no EGFR-induced changes in the PFC have been observed that subsequently affect oocyte polarity. Here, we show that an extracellular matrix receptor, Dystroglycan, is down-regulated in the PFC by EGFR signaling, and this down-regulation is necessary for proper localization of posterior polarity determinants in the oocyte. Failure to down-regulate Dystroglycan disrupts apicobasal polarity in the PFC, which includes mislocalization of the extracellular matrix component Laminin. Our data indicate that Dystroglycan links EGFR-induced repression of the anterior follicle cell fate and anterior–posterior polarity formation in the oocyte. PMID:16908845

  10. Capsaicin protects cortical neurons against ischemia/reperfusion injury via down-regulating NMDA receptors.

    PubMed

    Huang, Ming; Cheng, Gen; Tan, Han; Qin, Rui; Zou, Yimin; Wang, Yun; Zhang, Ying

    2017-09-01

    Capsaicin, the ingredient responsible for the pungent taste of hot chili peppers, is widely used in the study and management of pain. Recently, its neuroprotective effect has been described in multiple studies. Herein, we investigated the underlying mechanisms for the neuroprotective effect of capsaicin. Direct injection of capsaicin (1 or 3nmol) into the peri-infarct area reduced the infarct volume and improved neurological behavioral scoring and motor coordination function in the middle cerebral artery occlusion (MCAO)/reperfusion model in rats. The time window of the protective effect of capsaicin was within 1h after reperfusion, when excitotoxicity is the main reason of cell death. In cultured cortical neurons, administration of capsaicin attenuated glutamate-induced excitotoxic injury. With respect to the mechanisms of the neuroprotective effect of capsaicin, reduced calcium influx after glutamate stimulation was observed following capsaicin pretreatment in cortical neurons. Trpv1 knock-out abolished the inhibitory effect of capsaicin on glutamate-induced calcium influx and subsequent neuronal death. Reduced expression of GluN1 and GluN2B, subunits of NMDA receptor, was examined after capsaicin treatment in cortical neurons. In summary, our studies reveal that the neuroprotective effect of capsaicin in cortical neurons is TRPV1-dependent and down-regulation of the expression and function of NMDA receptors contributes to the protection afforded by capsaicin. Copyright © 2017. Published by Elsevier Inc.

  11. 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

  12. Dehydroepiandrosterone down-regulates the expression of peroxisome proliferator-activated receptor gamma in adipocytes.

    PubMed

    Kajita, Kazuo; Ishizuka, Tatsuo; Mune, Tomoatsu; Miura, Atsushi; Ishizawa, Masayoshi; Kanoh, Yoshinori; Kawai, Yasunori; Natsume, Yoshiyuki; Yasuda, Keigo

    2003-01-01

    Dehydroepiandrosterone (DHEA) is expected to have a weight-reducing effect. In this study, we evaluated the effect of DHEA on genetically obese Otsuka Long Evans Fatty rats (OLETF) compared with Long-Evans Tokushima rats (LETO) as control. Feeding with 0.4% DHEA-containing food for 2 wk reduced the weight of sc, epididymal, and perirenal adipose tissue in association with decreased plasma leptin levels in OLETF. Adipose tissue from OLETF showed increased expression of peroxisome proliferator-activated receptor gamma (PPARgamma) protein, which was prevented by DHEA treatment. Further, we examined the effect of DHEA on PPARgamma in primary cultured adipocytes and monolayer adipocytes differentiated from rat preadipocytes. PPARgamma protein level was decreased in a time- and concentration-dependent manner, and DHEA significantly reduced mRNA levels of PPARgamma, adipocyte lipid-binding protein, and sterol regulatory element-binding protein, but not CCAAT/enhancer binding protein alpha. DHEA-sulfate also reduced the PPARgamma protein, but dexamethasone, testosterone, or androstenedione did not alter its expression. In addition, treatment with DHEA for 5 d reduced the triglyceride content in monolayer adipocytes. These results suggest that DHEA down-regulates adiposity through the reduction of PPARgamma in adipocytes.

  13. 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

  14. Investigation of (E)-3-[4-(2-Oxo-3-aryl-chromen-4-yl)oxyphenyl]acrylic Acids as Oral Selective Estrogen Receptor Down-Regulators.

    PubMed

    Degorce, Sébastien L; Bailey, Andrew; Callis, Rowena; De Savi, Chris; Ducray, Richard; Lamont, Gillian; MacFaul, Philip; Maudet, Mickael; Martin, Scott; Morgentin, Rémy; Norman, Richard A; Peru, Aurélien; Pink, Jennifer H; Plé, Patrick A; Roberts, Bryan; Scott, James S

    2015-04-23

    A novel estrogen receptor down-regulator, 7-hydroxycoumarin (5, SS5020), has been reported with antitumor effects against chemically induced mammary tumors. Here, we report on our own investigation of 7-hydroxycoumarins as potential selective estrogen receptor down-regulators, which led us to the discovery of potent down-regulating antagonists, such as 33. Subsequent optimization and removal of the 7-hydroxy group led to coumarin 59, which had increased potency and improved rat bioavailability relative to SS5020.

  15. Prostaglandin E2 mediates growth arrest in NFS-60 cells by down-regulating interleukin-6 receptor expression.

    PubMed Central

    de Silva, Kumudika I; Daud, Asif N; Deng, JiangPing; Jones, Stephen B; Gamelli, Richard L; Shankar, Ravi

    2003-01-01

    Interleukin-6 (IL-6), a potent myeloid mitogen, and the immunosuppressive prostanoid prostaglandin E2 (PGE2) are elevated following thermal injury and sepsis. We have previously demonstrated that bone marrow myeloid commitment shifts toward monocytopoiesis and away from granulocytopoiesis during thermal injury and sepsis and that PGE2 plays a central role in this alteration. Here we investigated whether PGE2 can modulate IL-6-stimulated growth in the promyelocytic cell line, NFS-60, by down-regulating IL-6 receptor (IL-6r) expression. Exposure of NFS-60 cells to PGE2 suppressed IL-6-stimulated proliferation as well as IL-6r expression. Receptor down-regulation is functionally significant since IL-6-induced signal transduction through activators of transcription (STAT)-3 is also decreased. Down-regulation of IL-6r correlated with the ability of PGE2 to arrest cells in the G0/G1 phase of the cell cycle. PGE2 appears to signal through EP2 receptors. Butaprost (EP2 agonist) but not sulprostone (EP3 agonist) inhibited IL-6-stimulated proliferation. In addition, an EP2 antagonist (AH6809) alleviated the anti-proliferative effects of PGE2. NFS-60 cells express predominantly EP2 and EP4 receptors. While PGE2 down-regulated both the IL-6r protein and mRNA expression, it had no influence on EP2 or EP4 mRNA expression. The present study demonstrates that PGE2 is a potent down-regulator of IL-6r expression and thus may provide a mechanistic explanation for the granulocytopenia seen in thermal injury and sepsis. PMID:12429018

  16. Reversal of islet GIP receptor down-regulation and resistance to GIP by reducing hyperglycemia in the Zucker rat

    SciTech Connect

    Piteau, Shalea; Olver, Amy; Kim, Su-Jin; Winter, Kyle; Pospisilik, John Andrew; Lynn, Francis; Manhart, Susanne; Demuth, Hans-Ulrich; Speck, Madeleine; Pederson, Raymond A.; McIntosh, Christopher H.S.

    2007-11-03

    In type 2 diabetes (T2DM) {beta}-cell responsiveness to glucose-dependent insulinotropic polypeptide (GIP) is reduced. In a model of T2DM, the VDF Zucker rat, GIP receptor mRNA and protein levels were shown to be down-regulated. Possible restoration of responsiveness to GIP in Zucker rats by reducing hyperglycemia has been examined. ZDF rats with extreme hyperglycemia demonstrated greater islet GIP receptor mRNA down-regulation (94.3 {+-} 3.8%) than ZF rats (48.8 {+-} 22.8%). GIP receptor mRNA levels in ZDF rats returned to 83.0 {+-} 17.9% of lean following normalization of hyperglycemia by phlorizin treatment and pancreas perfusions demonstrated markedly improved GIP responsiveness. Treatment of VDF rats with a DP IV inhibitor (P32/98) resulted in improved glucose tolerance and restored sensitivity to GIP in isolated pancreata. These findings support the proposal that GIP receptor down-regulation in rodent T2DM is secondary to chronic hyperglycemia and that normalization of glycemia can restore GIP sensitivity.

  17. Homologous down-regulation of growth hormone-releasing hormone receptor messenger ribonucleic acid levels.

    PubMed

    Aleppo, G; Moskal, S F; De Grandis, P A; Kineman, R D; Frohman, L A

    1997-03-01

    Repeated stimulation of pituitary cell cultures with GH-releasing hormone (GHRH) results in diminished responsiveness, a phenomenon referred to as homologous desensitization. One component of GHRH-induced desensitization is a reduction in GHRH-binding sites, which is reflected by the decreased ability of GHRH to stimulate a rise in intracellular cAMP. In the present study, we sought to determine if homologous down-regulation of GHRH receptor number is due to a decrease in GHRH receptor synthesis. To this end, we developed and validated a quantitative RT-PCR assay system that was capable of assessing differences in GHRH-R messenger RNA (mRNA) levels in total RNA samples obtained from rat pituitary cell cultures. Treatment of pituitary cells with GHRH, for as little as 4 h, resulted in a dose-dependent decrease in GHRH-R mRNA levels. The maximum effect was observed with 0.1 and 1 nM GHRH, which reduced GHRH-R mRNA levels to 49 +/- 4% (mean +/- SEM) and 54 +/- 11% of control values, respectively (n = three separate experiments; P < 0.05). Accompanying the decline in GHRH-R mRNA levels was a rise in GH release; reaching 320 +/- 31% of control values (P < 0.01). Because of the possibility that the rise in medium GH level is the primary regulator of GHRH-R mRNA, we pretreated pituitary cultures for 4 h with GH to achieve a concentration comparable with that induced by a maximal stimulation with GHRH (8 micrograms GH/ml medium). Following pretreatment, cultures were stimulated for 15 min with GHRH and intracellular cAMP accumulation was measured by RIA. GH pretreatment did not impair the ability of GHRH to induce a rise in cAMP concentrations. However, as anticipated, GHRH pretreatment (10 nM) significantly reduced subsequent GHRH-stimulated cAMP to 46% of untreated controls. These data suggest that GHRH, but not GH, directly reduces GHRH-R mRNA levels. To determine whether this effect was mediated through cAMP, cultures were treated with forskolin, a direct stimulator of

  18. 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

  19. [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.

  20. Fulvestrant, a selective estrogen receptor down-regulator, sensitizes estrogen receptor negative breast tumors to chemotherapy.

    PubMed

    Jiang, Donghai; Huang, Yuan; Han, Ning; Xu, Mingjie; Xu, Liang; Zhou, Lin; Wang, Shu; Fan, Weimin

    2014-05-01

    Drug resistance frequently results in poor prognosis and high 5-year recurrence rate in estrogen receptor-negative (ER-) breast cancer patients. Herein, we examined the reversal effects of fulvestrant on multidrug resistance (MDR) in ER- breast cancer cells. Co-administration of fulvestrant significantly sensitized ER- MDR tumors to paclitaxel both in vitro and in vivo. Further analyses indicated that fulvestrant did not affect P-gp expression, but could inhibit P-gp function and subsequently reverse P-gp mediated drug resistance in ER- breast cancer cells. These results showed that combination of fulvestrant and chemotherapeutic agents might provide an effective treatment for ER- MDR breast cancers.

  1. Involvement of tyrosine residues located in the carboxyl tail of the human beta 2-adrenergic receptor in agonist-induced down-regulation of the receptor.

    PubMed Central

    Valiquette, M; Bonin, H; Hnatowich, M; Caron, M G; Lefkowitz, R J; Bouvier, M

    1990-01-01

    Chronic exposure of various cell types to adrenergic agonists leads to a decrease in cell surface beta 2-adrenergic receptor (beta 2AR) number. Sequestration of the receptor away from the cell surface as well as a down-regulation of the total number of cellular receptors are believed to contribute to this agonist-mediated regulation of receptor number. However, the molecular mechanisms underlying these phenomena are not well characterized. Recently, tyrosine residues located in the cytoplasmic tails of several membrane receptors, such as the low density lipoprotein and mannose-6-phosphate receptors, have been suggested as playing an important role in the agonist-induced internalization of these receptors. Accordingly, we assessed the potential role of two tyrosine residues in the carboxyl tail of the human beta 2AR in agonist-induced sequestration and down-regulation of the receptor. Tyr-350 and Tyr-354 of the human beta 2AR were replaced with alanine residues by site-directed mutagenesis and both wild-type and mutant beta 2AR were stably expressed in transformed Chinese hamster fibroblasts. The mutation dramatically decreased the ability of the beta 2AR to undergo isoproterenol-induced down-regulation. However, the substitution of Tyr-350 and Tyr-354 did not affect agonist-induced sequestration of the receptor. These results suggest that tyrosine residues in the cytoplasmic tail of human beta 2AR are crucial determinants involved in its down-regulation. PMID:2164220

  2. Internalization and down-regulation of human muscarinic acetylcholine receptor m2 subtypes. Role of third intracellular m2 loop and G protein-coupled receptor kinase 2.

    PubMed

    Tsuga, H; Kameyama, K; Haga, T; Honma, T; Lameh, J; Sadée, W

    1998-02-27

    Internalization and down-regulation of human muscarinic acetylcholine m2 receptors (hm2 receptors) and a hm2 receptor mutant lacking a central part of the third intracellular loop (I3-del m2 receptor) were examined in Chinese hamster ovary (CHO-K1) cells stably expressing these receptors and G protein-coupled receptor kinase 2 (GRK2). Agonist-induced internalization of up to 80-90% of hm2 receptors was demonstrated by measuring loss of [3H]N-methylscopolamine binding sites from the cell surface, and transfer of [3H]quinuclidinyl benzilate binding sites from the plasma membrane into the light-vesicle fractions separated by sucrose density gradient centrifugation. Additionally, translocation of hm2 receptors with endocytic vesicles were visualized by immunofluorescence confocal microscopy. Agonist-induced down-regulation of up to 60-70% of hm2 receptors was demonstrated by determining the loss of [3H]quinuclidinyl benzilate binding sites in the cells. The half-time (t1/2) of internalization and down-regulation in the presence of 10(-4) M carbamylcholine was estimated to be 9.5 min and 2.3 h, respectively. The rates of both internalization and down-regulation of hm2 receptors in the presence of 10(-6) M or lower concentrations of carbamylcholine were markedly increased by coexpression of GRK2. Agonist-induced internalization of I3-del m2 receptors was barely detectable upon incubation of cells for 1 h, but agonist-induced down-regulation of up to 40-50% of I3-del m2 receptors occurred upon incubation with 10(-4) M carbamylcholine for 16 h. However, the rate of down-regulation was lower compared with wild type receptors (t1/2 = 9.9 versus 2.3 h). These results indicate that rapid internalization of hm2 receptors is facilitated by their phosphorylation with GRK2 and does not occur in the absence of the third intracellular loop, but down-regulation of hm2 receptors may occur through both GRK2-facilitating pathway and third intracellular loop-independent pathways.

  3. Fasting Induces CART Down-Regulation in the Zebrafish Nervous System in a Cannabinoid Receptor 1-Dependent Manner

    PubMed Central

    Nishio, Shin-Ichi; Gibert, Yann; Berekelya, Liubov; Bernard, Laure; Brunet, Frédéric; Guillot, Etienne; Le Bail, Jean-Christophe; Sánchez, Juan Antonio; Galzin, Anne Marie; Triqueneaux, Gerard

    2012-01-01

    Central and peripheral mechanisms modulate food intake and energy balance in mammals and the precise role of the type 1 cannabinoid receptor (CB1) in these processes is still being explored. Using the zebrafish, Danio rerio, we show that rimonabant, a CB1-specific antagonist with an EC50 of 5.15 × 10−8 m, decreases embryonic yolk sac reserve use. We reveal a developmental overlap between CART genes and CB1 expression in the hypothalamus and medulla oblongata, two brain structures that play crucial roles in appetite regulation in mammals. We show that morpholino knockdown of CB1 or fasting decreases cocaine- and amphetamine-related transcript (CART)-3 expression. Strikingly, this down-regulation occurs only in regions coexpressing CB1 and CART3, reinforcing the link between CB1, CART, and appetite regulation. We show that rimonabant treatment impairs the fasting-induced down-regulation of CART expression in specific brain regions, whereas vehicle alone-treated embryos do not display this rescue of CART expression. Our data reveal that CB1 lies upstream of CART and signals the appetite through the down-regulation of CART expression. Thus, our results establish the zebrafish as a promising system to study appetite regulation. PMID:22700585

  4. Fasting induces CART down-regulation in the zebrafish nervous system in a cannabinoid receptor 1-dependent manner.

    PubMed

    Nishio, Shin-Ichi; Gibert, Yann; Berekelya, Liubov; Bernard, Laure; Brunet, Frédéric; Guillot, Etienne; Le Bail, Jean-Christophe; Sánchez, Juan Antonio; Galzin, Anne Marie; Triqueneaux, Gerard; Laudet, Vincent

    2012-08-01

    Central and peripheral mechanisms modulate food intake and energy balance in mammals and the precise role of the type 1 cannabinoid receptor (CB1) in these processes is still being explored. Using the zebrafish, Danio rerio, we show that rimonabant, a CB1-specific antagonist with an EC(50) of 5.15 × 10(-8) m, decreases embryonic yolk sac reserve use. We reveal a developmental overlap between CART genes and CB1 expression in the hypothalamus and medulla oblongata, two brain structures that play crucial roles in appetite regulation in mammals. We show that morpholino knockdown of CB1 or fasting decreases cocaine- and amphetamine-related transcript (CART)-3 expression. Strikingly, this down-regulation occurs only in regions coexpressing CB1 and CART3, reinforcing the link between CB1, CART, and appetite regulation. We show that rimonabant treatment impairs the fasting-induced down-regulation of CART expression in specific brain regions, whereas vehicle alone-treated embryos do not display this rescue of CART expression. Our data reveal that CB1 lies upstream of CART and signals the appetite through the down-regulation of CART expression. Thus, our results establish the zebrafish as a promising system to study appetite regulation.

  5. The herpes simplex virus receptor nectin-1 is down-regulated after trans-interaction with glycoprotein D

    SciTech Connect

    Stiles, Katie M.; Milne, Richard S.B.; Cohen, Gary H.; Eisenberg, Roselyn J.; Krummenacher, Claude

    2008-03-30

    During herpes simplex virus (HSV) entry, membrane fusion occurs either on the cell surface or after virus endocytosis. In both cases, binding of glycoprotein D (gD) to a receptor such as nectin-1 or HVEM is required. In this study, we co-cultured cells expressing gD with nectin-1 expressing cells to investigate the effects of gD on nectin-1 at cell contacts. After overnight co-cultures with gD expressing cells, there was a down-regulation of nectin-1 in B78H1-C10, SY5Y, A431 and HeLa cells, which HSV enters by endocytosis. In contrast, on Vero cells, which HSV enters at the plasma membrane, nectin-1 was not down-regulated. Further analysis of B78H1-derived cells showed that nectin-1 down-regulation corresponds to the ability of gD to bind nectin-1 and is achieved by internalization and low-pH-dependent degradation of nectin-1. Moreover, gD is necessary for virion internalization in B78H1 cells expressing nectin-1. These data suggest that the determinants of gD-mediated internalization of nectin-1 may direct HSV to an endocytic pathway during entry.

  6. Distribution of beta-adrenergic receptors in failing human myocardium. Implications for mechanisms of down-regulation

    SciTech Connect

    Murphree, S.S.; Saffitz, J.E.

    1989-06-01

    The density of beta-adrenergic receptors is reduced in crude membranes prepared from failing human myocardium. We used quantitative autoradiography of radioligand binding sites in intact tissue slices to determine whether the total tissue content of receptors is reduced and to characterize the transmural distribution of receptors in cardiac myocytes and the coronary vasculature in hearts obtained from nine cardiac transplant patients with severe congestive failure. Binding of (125Iodo)cyanopindolol to transmural slices of human myocardium was rapid, saturable, stereoselective, and displaceable by agonists and antagonists with an appropriate rank order of potency. Binding isotherms in four normal and nine failing ventricles showed a significant reduction in the total tissue content of beta-receptors in failing myocardium (38.3 +/- 2.0 fmol/mg protein) compared with normal tissue (52.4 +/- 1.7 fmol/mg protein, p = 0.038). In the normal ventricles, the greatest receptor density was observed autoradiographically in myocytic regions of the subendocardium. Receptor density of the coronary arterioles was approximately 70% of that in adjacent myocytic regions. The density of binding sites in both myocytic regions and arterioles was diminished in all regions of the failing ventricles, but down-regulation was due primarily to a selective reduction of beta-receptors of subendocardial myocytes (63 +/- 5% of subepicardial receptor density vs. 115 +/- 6% in controls, p less than 0.0001). These observations indicate that down-regulation occurs nonuniformly in the transmural distribution and thus is likely not related simply to elevated circulating catecholamine levels.

  7. Expression of thyroid hormone receptor isoforms down-regulated by thyroid hormone in human medulloblastoma cells.

    PubMed

    Monden, Tsuyoshi; Nakajima, Yasuyo; Hashida, Tetsu; Ishii, Sumiyasu; Tomaru, Takuya; Shibusawa, Nobuyuki; Hashimoto, Koshi; Satoh, Teturou; Yamada, Masanobu; Mori, Masatomo; Kasai, Kikuo

    2006-04-01

    The role of thyroid hormone (T3) in the regulation of growth and development of the central nervous system including the cerebellum has been well established. However, the effects of thyroid hormone on malignant tumors derived from the cerebellum remain poorly understood. Our analysis mainly focused on expression levels of TR isoforms and the effects of thyroid hormone in human medulloblastoma HTB-185 cells. Northern blot analysis revealed TRalpha2 mRNA but not TRalpha1, beta1 or beta2 mRNA in the cell. The TRalpha1 and TRbeta1 mRNAs were detected only by RT-PCR method and TRbeta2 was not expressed. Incubation of T3 for 24 h decreased TRalpha1, TRalpha2 and TRbeta1 mRNA. Addition of actinomycin D caused an acute increase in the basal TR mRNA levels and the rate of decrease of all kinds of TR isoform mRNA was accelerated in the T3-treated groups compared to controls, indicating that the stability of TR mRNA was affected by T3. Incubation with cycloheximide also blocked a decrease in TR mRNA levels in the T3-treated HTB-185 cells suggesting that down-regulation of TR mRNA required the synthesis of new protein. Our data provide novel evidence for the expression of TRs down-regulated by T3 in HTB-185 cells, suggesting that TR expression is post-transcriptionally regulated by T3 at the level of RNA stability.

  8. MicroRNA-122 down-regulation is involved in phenobarbital-mediated activation of the constitutive androstane receptor.

    PubMed

    Shizu, Ryota; Shindo, Sawako; Yoshida, Takemi; Numazawa, Satoshi

    2012-01-01

    Constitutive androstane receptor (CAR) is a nuclear receptor that regulates the transcription of target genes, including CYP2B and 3A. Phenobarbital activates CAR, at least in part, in an AMP-activated protein kinase (AMPK)-dependent manner. However, the precise mechanisms underlying phenobarbital activation of AMPK are still unclear. In the present study, it was demonstrated that phenobarbital administration to mice decreases hepatic miR-122, a liver-enriched microRNA involved in both hepatic differentiation and function. The time-course change in the phenobarbital-mediated down-regulation of miR-122 was inversely correlated with AMPK activation. Phenobarbital decreased primary miR-122 to approximately 25% of the basal level as early as 1 h and suppressed transactivity of mir-122 promoter in HuH-7 cells, suggesting that the down-regulation occurred at the transcriptional level. AMPK activation by metformin or 5-aminoimidazole-4-carboxamide 1-β-D-ribonucleoside had no evident effect on miR-122 levels. An inhibitory RNA specific for miR-122 increased activated AMPK and CAR-mediated trancactivation of the phenobarbital-responsive enhancer module in HepG2 cells. Conversely, the reporter activity induced by the ectopic CAR was almost completely suppressed by co-transfection with the miR-122 mimic RNA. GFP-tagged CAR was expressed in the cytoplasm in addition to the nucleus in the majority of HuH-7 cells in which miR-122 was highly expressed. Co-transfection of the mimic or the inhibitor RNA for miR-122 further increased or decreased, respectively, the number of cells that expressed GFP-CAR in the cytoplasm. Taken together, these results suggest that phenobarbital-mediated down-regulation of miR-122 is an early and important event in the AMPK-dependent CAR activation and transactivation of its target genes.

  9. Down-regulation of the A3 adenosine receptor in human mast cells upregulates mediators of angiogenesis and remodeling.

    PubMed

    Rudich, Noam; Dekel, Ornit; Sagi-Eisenberg, Ronit

    2015-05-01

    Adenosine activated mast cells have been long implicated in allergic asthma and studies in rodent mast cells have assigned the A3 adenosine receptor (A3R) a primary role in mediating adenosine responses. Here we analyzed the functional impact of A3R activation on genes that are implicated in tissue remodeling in severe asthma in the human mast cell line HMC-1 that shares similarities with lung derived human mast cells. Quantitative real time PCR demonstrated upregulation of IL6, IL8, VEGF, amphiregulin and osteopontin. Moreover, further upregulation of these genes was noted upon the addition of dexamethasone. Unexpectedly, activated A3R down regulated its own expression and knockdown of the receptor replicated the pattern of agonist induced gene upregulation. This study therefore identifies the human mast cell A3R as regulator of tissue remodeling gene expression in human mast cells and demonstrates a heretofore-unrecognized mode of feedback regulation that is exerted by this receptor.

  10. 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.

  11. Synthesis and evaluation of tamoxifen derivatives with a long alkyl side chain as selective estrogen receptor down-regulators.

    PubMed

    Shoda, Takuji; Kato, Masashi; Harada, Rintaro; Fujisato, Takuma; Okuhira, Keiichiro; Demizu, Yosuke; Inoue, Hideshi; Naito, Mikihiko; Kurihara, Masaaki

    2015-07-01

    Estrogen receptors (ERs) play a major role in the growth of human breast cancer cells. An antagonist that acts as not only an inhibitor of ligand binding but also an inducer of the down-regulation of ER would be useful for the treatment for ER-positive breast cancer. We previously reported the design and synthesis of a selective estrogen receptor down-regulator (SERD), (E/Z)-4-(1-{4-[2-(dodecylamino)ethoxy]phenyl}-2-phenylbut-1-en-1-yl)phenol (C12), which is a tamoxifen derivative having a long alkyl chain on the amine moiety. This compound induced degradation of ERα via a proteasome-dependent pathway and showed an antagonistic effect in MCF-7 cells. With the aim of increasing the potency of SERDs, we designed and synthesized various tamoxifen derivatives that have various lengths and terminal groups of the long alkyl side chain. During the course of our investigation, C10F having a 10-fluorodecyl group on the amine moiety of 4-OHT was shown to be the most potent compound among the tamoxifen derivatives. Moreover, computational docking analysis suggested that the long alkyl chain interacted with the hydrophobic region on the surface of the ER, which is a binding site of helix 12 and coactivator. These results provide useful information to develop promising candidates as SERDs.

  12. Berberine Inhibits Proliferation and Down-Regulates Epidermal Growth Factor Receptor through Activation of Cbl in Colon Tumor Cells

    PubMed Central

    Wang, Lihong; Cao, Hailong; Lu, Ning; Liu, Liping; Wang, Bangmao; Hu, Tianhui; Israel, Dawn A.; Peek, Richard M.; Polk, D. Brent; Yan, Fang

    2013-01-01

    Berberine, an isoquinoline alkaloid, is an active component of Ranunculaceae and Papaveraceae plant families. Berberine has been found to suppress growth of several tumor cell lines in vitro through the cell-type-dependent mechanism. Expression and activation of epidermal growth factor receptor (EGFR) is increased in colonic precancerous lesions and tumours, thus EGFR is considered a tumour promoter. The aim of this study was to investigate the effects and mechanisms of berberine on regulation of EGFR activity and proliferation in colonic tumor cell lines and in vivo. We reported that berberine significantly inhibited basal level and EGF-stimulated EGFR activation and proliferation in the immorto Min mouse colonic epithelial (IMCE) cells carrying the APCmin mutation and human colonic carcinoma cell line, HT-29 cells. Berberine acted to inhibit proliferation through inducing G1/S and G2/M cell cycle arrest, which correlated with regulation of the checkpoint protein expression. In this study, we also showed that berberine stimulated ubiquitin ligase Cbl activation and Cbl's interaction with EGFR, and EGFR ubiquitinylation and down-regulation in these two cell lines in the presence or absence of EGF treatment. Knock-down Cbl expression blocked the effects of berberine on down-regulation of EGFR and inhibition of proliferation. Furthermore, berberine suppressed tumor growth in the HT-29 cell xenograft model. Cell proliferation and EGFR expression level was decreased by berberine treatment in this xenograft model and in colon epithelial cells of APCmin/+ mice. Taken together, these data indicate that berberine enhances Cbl activity, resulting in down-regulation of EGFR expression and inhibition of proliferation in colon tumor cells. PMID:23457600

  13. Amphiphilic Nanoparticles Repress Macrophage Atherogenesis: Novel Core/Shell Designs for Scavenger Receptor Targeting and Down-Regulation

    PubMed Central

    2015-01-01

    Atherosclerosis, an inflammatory lipid-rich plaque disease is perpetuated by the unregulated scavenger-receptor-mediated uptake of oxidized lipoproteins (oxLDL) in macrophages. Current treatments lack the ability to directly inhibit oxLDL accumulation and foam cell conversion within diseased arteries. In this work, we harness nanotechnology to design and fabricate a new class of nanoparticles (NPs) based on hydrophobic mucic acid cores and amphiphilic shells with the ability to inhibit the uncontrolled uptake of modified lipids in human macrophages. Our results indicate that tailored NP core and shell formulations repress oxLDL internalization via dual complementary mechanisms. Specifically, the most atheroprotective molecules in the NP cores competitively reduced NP-mediated uptake to scavenger receptor A (SRA) and also down-regulated the surface expression of SRA and CD36. Thus, nanoparticles can be designed to switch activated, lipid-scavenging macrophages to antiatherogenic phenotypes, which could be the basis for future antiatherosclerotic therapeutics. PMID:24972372

  14. Morphine-induced desensitization and down-regulation at mu-receptors in 7315C pituitary tumor cells

    SciTech Connect

    Puttfarcken, P.S.; Cox, B.M. )

    1989-01-01

    Pituitary 7315c tumor cells maintained in culture were treated with varying concentrations of morphine from 10 nM to 300 {mu}M, for periods of five or forty-eight hours. The ability of the mu-opioid receptor agonist, DAMGO, to inhibit forskolin-stimulated adenylyl cyclase in washed membrane preparations from the treated cells was compared with its activity in membranes from cells incubated in the absence of added morphine. In the same membrane preparations, the number and affinity of mu-opioid receptors was estimated by measurements of ({sup 3}H)diprenorphine binding. After 5 hr of treatment with morphine concentrations of 100 nM or higher, a significant reduction in inhibition of adenylyl cyclase by DAMGO was observed. Little further loss of agonist activity was observed when the incubations were extended to 48 hr. After 5 hr of morphine treatment, there was no change in either the number of receptors, or their affinity for ({sup 3}H)diprenorphine. However after 48 hr of morphine treatment, greater than 25% reductions in receptor number were apparent with morphine pretreatment concentrations of 10 {mu}M or higher. These results suggest that opioid tolerance in this system is primarily associated with a reduced ability of agonist-occupied receptor to activate the effector system. Receptor down-regulation was not necessary for loss of agonist response, although a reduction in receptor number occurred after exposure to high concentrations of morphine for periods longer than 5hr.

  15. 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.

  16. Phospholipase C-epsilon augments epidermal growth factor-dependent cell growth by inhibiting epidermal growth factor receptor down-regulation.

    PubMed

    Yun, Sanguk; Hong, Won-Pyo; Choi, Jang Hyun; Yi, Kye Sook; Chae, Suhn-Kee; Ryu, Sung Ho; Suh, Pann-Ghill

    2008-01-04

    The down-regulation of the epidermal growth factor (EGF) receptor is critical for the termination of EGF-dependent signaling, and the dysregulation of this process can lead to oncogenesis. In the present study, we suggest a novel mechanism for the regulation of EGF receptor down-regulation by phospholipase C-epsilon. The overexpression of PLC-epsilon led to an increase in receptor recycling and decreased the down-regulation of the EGF receptor in COS-7 cells. Adaptor protein complex 2 (AP2) was identified as a novel binding protein that associates with the PLC-epsilon RA2 domain independently of Ras. The interaction of PLC-epsilon with AP2 was responsible for the suppression of EGF receptor down-regulation, since a perturbation in this interaction abolished this effect. Enhanced EGF receptor stability by PLC-epsilon led to the potentiation of EGF-dependent growth in COS-7 cells. Finally, the knockdown of PLC-epsilon in mouse embryo fibroblast cells elicited a severe defect in EGF-dependent growth. Our results indicated that PLC-epsilon could promote EGF-dependent cell growth by suppressing receptor down-regulation.

  17. The atypical antipsychotics clozapine and olanzapine promote down-regulation and display functional selectivity at human 5-HT7 receptors

    PubMed Central

    Andressen, K W; Manfra, O; Brevik, C H; Ulsund, A H; Vanhoenacker, P; Levy, F O; Krobert, K A

    2015-01-01

    Background and Purpose Classically, ligands of GPCRs have been classified primarily upon their affinity and efficacy to activate a signal transduction pathway. Recent reports indicate that the efficacy of a particular ligand can vary depending on the receptor-mediated response measured (e.g. activating G proteins, other downstream responses, internalization). Previously, we reported that inverse agonists induce both homo- and heterologous desensitization, similar to agonist stimulation, at the Gs-coupled 5-HT7 receptor. The primary objective of this study was to determine whether different inverse agonists at the 5-HT7 receptor also induce internalization and/or degradation of 5-HT7 receptors. Experimental Approach HEK293 cells expressing 5-HT7(a, b or d) receptors were pre-incubated with 5-HT, clozapine, olanzapine, mesulergine or SB269970 and their effects upon receptor density, AC activity, internalization, recruitment of β-arrestins and lysosomal trafficking were measured. Key Results The agonist 5-HT and three out of four inverse agonists tested increased internalization independently of β-arrestin recruitment. Among these, only the atypical antipsychotics clozapine and olanzapine promoted lysosomal sorting and reduced 5-HT7 receptor density (∼60% reduction within 24 h). Inhibition of lysosomal degradation with chloroquine blocked the clozapine- and olanzapine-induced down-regulation of 5-HT7 receptors. Incubation with SB269970 decreased both 5-HT7(b) constitutive internalization and receptor density but increased 5-HT7(d) receptor density, indicating differential ligand regulation among the 5-HT7 splice variants. Conclusions and Implications Taken together, we found that various ligands differentially activate regulatory processes governing receptor internalization and degradation in addition to signal transduction. Thus, these data extend our understanding of functional selectivity at the 5-HT7 receptor. PMID:25884989

  18. Down regulation of the muscarinic cholinergic receptor of the rat prostate following castration

    SciTech Connect

    Shapiro, E.; Miller, A.R.; Lepor, H.

    1985-07-01

    Prostatic secretion is dependent upon the integrity of the endocrine and autonomic nervous systems and is dramatically influenced by muscarinic cholinergic analogs. In this study, the authors have used radioligand receptor binding methods on whole tissue homogenates and slide mounted tissue sections of rat prostate to determine whether androgens regulate the density of muscarinic cholinergic receptors in the prostate. The muscarinic cholinergic receptor binding affinities (Kd) of (/sup 3/H) N-methylscopolamine in prostatic homogenates obtained from intact, castrate, and castrate rats receiving testosterone replacement (castrate + T) were similar (0.07 to 0.10 nM). The muscarinic cholinergic receptor binding capacity decreased 73 per cent following castration. Testosterone administration restored the density of muscarinic cholinergic receptors in castrate rats to intact levels. In order to ensure that the loss of receptor density was not due to a decrease in the epithelial: stromal cell ratio, the number of muscarinic cholinergic receptors per unit area of epithelium was determined in the 3 treatment groups using autoradiography on slide mounted tissue sections. The density of muscarinic cholinergic receptors in a unit area of epithelium was decreased 91 per cent following castration. Testosterone administration restored the density of muscarinic cholinergic receptors in the castrate rats to intact levels. The modulation of neurotransmitter receptors by steroid hormones may be a mechanism by which sex steroids regulate biological responsiveness of target tissues.

  19. Dopamine D2-Receptor Antagonists Down-Regulate CYP1A1/2 and CYP1B1 in the Rat Liver

    PubMed Central

    Harkitis, P.; Lang, M. A.; Marselos, M.; Fotopoulos, A.; Albucharali, G.; Konstandi, M.

    2015-01-01

    Dopaminergic systems regulate the release of several hormones including growth hormone (GH), thyroid hormones, insulin, glucocorticoids and prolactin (PRL) that play significant roles in the regulation of various Cytochrome P450 (CYP) enzymes. The present study investigated the role of dopamine D2-receptor-linked pathways in the regulation of CYP1A1, CYP1A2 and CYP1B1 that belong to a battery of genes controlled by the Aryl Hydrocarbon Receptor (AhR) and play a crucial role in the metabolism and toxicity of numerous environmental toxicants. Inhibition of dopamine D2-receptors with sulpiride (SULP) significantly repressed the constitutive and benzo[a]pyrene (B[a]P)-induced CYP1A1, CYP1A2 and CYP1B expression in the rat liver. The expression of AhR, heat shock protein 90 (HSP90) and AhR nuclear translocator (ARNT) was suppressed by SULP in B[a]P-treated livers, whereas the AhRR expression was increased by the drug suggesting that the SULP-mediated repression of the CYP1 inducibility is due to inactivation of the AhR regulatory system. At signal transduction level, the D2-mediated down-regulation of constitutive CYP1A1/2 and CYP1B1 expression appears to be mediated by activation of the insulin/PI3K/AKT pathway. PRL-linked pathways exerting a negative control on various CYPs, and inactivation of the glucocorticoid-linked pathways that positively control the AhR-regulated CYP1 genes, may also participate in the SULP-mediated repression of both, the constitutive and induced CYP1 expression. The present findings indicate that drugs acting as D2-dopamine receptor antagonists can modify several hormone systems that regulate the expression of CYP1A1, CYP1A2 and CYP1B1, and may affect the toxicity and carcinogenicity outcome of numerous toxicants and pre-carcinogenic substances. Therefore, these drugs could be considered as a part of the strategy to reduce the risk of exposure to environmental pollutants and pre-carcinogens. PMID:26466350

  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. Down-regulation of chicken interleukin-17 receptor A in Eimeria infection

    USDA-ARS?s Scientific Manuscript database

    Both IL-17A and IL-17F are proinflammatory cytokines, which play an important role in intestinal homeostasis through their receptor signaling. In chickens, these two cytokines have been recently characterized, but to date, very little is known about their receptors and their functional activity. Th...

  3. First Pharmacophore-Based Identification of Androgen Receptor Down-regulating Agents: Discovery of Potent Anti-Prostate Cancer Agents

    PubMed Central

    Purushottamachar, Puranik; Khandelwal, Aakanksha; Chopra, Pankaj; Maheshwari, Neha; Gediya, Lalji K; Vasaitis, Tadas S.; Bruno, Robert; Clement, Omoshile O.; Njar, Vincent C. O.

    2007-01-01

    A qualitative 3D pharmacophore model (a common feature based model or Catalyst HipHop algorithm) was developed for well known natural product androgen receptor down-regulating agents (ARDAs). The four common chemical features identified included: one hydrophobic group, one ring aromatic group and two hydrogen bond acceptors. This model served as a template in virtual screening of the Maybridge and NCI databases that resulted in identification of 6 new ARDAs (EC50 values 17.5 – 212 μM). Five of these molecules strongly inhibited the growth of human prostate LNCaP cells. These novel compounds may be used as leads to develop other novel anti-prostate cancer agents. PMID:17383188

  4. 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.

  5. 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

  6. CRM 1-mediated degradation and agonist-induced down-regulation of beta-adrenergic receptor mRNAs.

    PubMed

    Bai, Ying; Lu, Huafei; Machida, Curtis A

    2006-10-01

    The beta1-adrenergic receptor (beta1-AR) mRNAs are post-transcriptionally regulated at the level of mRNA stability and undergo accelerated agonist-mediated degradation via interaction of its 3' untranslated region (UTR) with RNA binding proteins, including the HuR nuclear protein. In a previous report [Kirigiti et al. (2001). Mol. Pharmacol. 60:1308-1324], we examined the agonist-mediated down-regulation of the rat beta1-AR mRNAs, endogenously expressed in the rat C6 cell line and ectopically expressed in transfectant hamster DDT1MF2 and rat L6 cells. In this report, we determined that isoproterenol treatment of neonatal rat cortical neurons, an important cell type expressing beta1-ARs in the brain, results in significant decreases in beta1-AR mRNA stability, while treatment with leptomycin B, an inhibitor of the nuclear export receptor CRM 1, results in significant increases in beta1-AR mRNA stability and nuclear retention. UV-crosslinking/immunoprecipitation and glycerol gradient fractionation analyses indicate that the beta1-AR 3' UTR recognize complexes composed of HuR and multiple proteins, including CRM 1. Cell-permeable peptides containing the leucine-rich nuclear export signal (NES) were used as inhibitors of CRM 1-mediated nuclear export. When DDT1MF2 transfectants were treated with isoproterenol and peptide inhibitors, only the co-addition of the NES inhibitor reversed the isoproterenol-induced reduction of beta1-AR mRNA levels. Our results suggest that CRM 1-dependent NES-mediated mechanisms influence the degradation and agonist-mediated down-regulation of the beta1-AR mRNAs.

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

    PubMed

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

    2005-04-01

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

  8. 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

  9. Interferon-alpha down-regulates the interleukin-6 receptor in a human multiple myeloma cell line, U266.

    PubMed Central

    Anthes, J C; Zhan, Z; Gilchrest, H; Egan, R W; Siegel, M I; Billah, M M

    1995-01-01

    The effects of interferon-alpha (IFN-alpha) on the interleukin-6 (IL-6) receptor in a multiple myeloma cell line, U266, have been examined. IFN-alpha inhibits [3H]thymidine incorporation in U266 cells in a time- and dose-dependent manner. Furthermore, IFN-alpha inhibits the ability of IL-6 to induce increases in [3H]thymidine incorporation. While IFN-alpha suppresses the ability of 125I-IL-6 to bind to the IL-6 receptor on U266 cells, this effect is not due to competition of IFN-alpha with IL-6 for the IL-6 receptor. Although IFN-alpha induces IL-6 synthesis in the U266 cell, inhibition of IL-6 binding occurs when IL-6 synthesis is minimal. Furthermore, after pretreatment of U266 cells with neutralizing anti-IL-6 antibodies, IFN-alpha still inhibits 125I-IL-6 binding. These data suggest that IFN-alpha inhibition of 125I-IL-6 binding does not involve IL-6 synthesis. IFN-alpha reduces 125I-IL-6 binding without affecting its affinity, suggesting that IFN-alpha inhibits IL-6 receptor expression. Although pretreatment with cycloheximide inhibits 125I-IL-6 binding, IFN-alpha does not cause a selective decrease in the levels of gp130 or IL-6 receptor mRNA at times when 125I-IL-6 binding is inhibited. These observations indicate that IFN-alpha lowers IL-6 receptor density on U266 cells by mechanisms other than competitive binding or lowering IL-6 receptor mRNA production. Receptor down-regulation may be a mechanism of IFN-alpha-induced inhibition of growth in U266 cells. Images Figure 9 PMID:7619053

  10. 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.

  11. Internalization and Down-Regulation of the ALK Receptor in Neuroblastoma Cell Lines upon Monoclonal Antibodies Treatment

    PubMed Central

    Mazot, Pierre; Cazes, Alex; Dingli, Florent; Degoutin, Joffrey; Irinopoulou, Théano; Boutterin, Marie-Claude; Lombard, Bérangère; Loew, Damarys; Hallberg, Bengt; Palmer, Ruth Helen; Delattre, Olivier

    2012-01-01

    Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALKWT), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALKWT and ALKF1174L receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALKWT whereas both ALKWT and ALKF1174L were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALKWT. We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization. PMID:22479414

  12. Polyubiquitination of Insulin-like Growth Factor I Receptor (IGF-IR) Activation Loop Promotes Antibody-induced Receptor Internalization and Down-regulation*

    PubMed Central

    Mao, Yifan; Shang, Yonglei; Pham, Victoria C.; Ernst, James A.; Lill, Jennie R.; Scales, Suzie J.; Zha, Jiping

    2011-01-01

    Ubiquitination has been implicated in negatively regulating insulin-like growth factor I receptor (IGF-IR) activity. Because of the relative stability of IGF-IR in the presence of ligand stimulation, IGF-IR ubiquitination sites have yet to be mapped and characterized, thus preventing a direct demonstration of how the receptor ubiquitination contributes to downstream molecular cascades. We took advantage of an anti-IGF-IR antibody (h10H5) that induces more efficient receptor down-regulation to show that IGF-IR is promptly and robustly ubiquitinated. The ubiquitination sites were mapped to the two lysine residues in the IGF-IR activation loop (Lys-1138 and Lys-1141) and consisted of polyubiquitin chains formed through both Lys-48 and Lys-29 linkages. Mutation of these ubiquitinated lysine residues resulted in decreased h10H5-induced IGF-IR internalization and down-regulation as well as a reduced cellular response to h10H5 treatment. We have therefore demonstrated that IGF-IR ubiquitination contributes critically to the down-regulating and antiproliferative activity of h10H5. This finding is physiologically relevant because insulin-like growth factor I appears to mediate ubiquitination of the same major sites as h10H5 (albeit to a lesser extent), and ubiquitination is facilitated by pre-existing phosphorylation of the receptor in both cases. Furthermore, identification of a breast cancer cell line with a defect in IGF-IR ubiquitination suggests that this could be an important tumor resistance mechanism to evade down-regulation-mediated negative regulation of IGF-IR activity in cancer. PMID:21994939

  13. 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.

  14. Rapamycin down-regulates LDL-receptor expression independently of SREBP-2

    SciTech Connect

    Sharpe, Laura J.; Brown, Andrew J.

    2008-09-05

    As a key regulator of cholesterol homeostasis, sterol-regulatory element binding protein-2 (SREBP-2) up-regulates expression of genes involved in cholesterol synthesis (e.g., 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) Reductase) and uptake (the low density lipoprotein (LDL)-receptor). Previously, we showed that Akt, a critical kinase in cell growth and proliferation, contributes to SREBP-2 activation. However, the specific Akt target involved is unknown. A potential candidate is the mammalian target of rapamycin, mTOR. Rapamycin can cause hyperlipidaemia clinically, and we hypothesised that this may be mediated via an effect of mTOR on SREBP-2. Herein, we found that SREBP-2 activation and HMG-CoA Reductase gene expression were unaffected by rapamycin treatment. However, LDL-receptor gene expression was decreased by rapamycin, suggesting that this may contribute to the hyperlipidaemia observed in rapamycin-treated patients. Rapamycin did not affect mRNA stability, so the decrease in LDL-receptor gene expression is likely to be occurring at the transcriptional level, although independently of SREBP-2.

  15. Berberine suppresses migration of MCF-7 breast cancer cells through down-regulation of chemokine receptors

    PubMed Central

    Ahmadiankia, Naghmeh; Moghaddam, Hamid Kalalian; Mishan, Mohammad Amir; Bahrami, Ahmad Reza; Naderi-Meshkin, Hojjat; Bidkhori, Hamid Reza; Moghaddam, Maryam; Mirfeyzi, Seyed Jamal Aldin

    2016-01-01

    Objective(s): Berberine is one of the main alkaloids and it has been proven to have different pharmacological effects including inhibition of cell cycle and progression of apoptosis in various cancerous cells; however, its effects on cancer metastasis are not well known. Cancer cells obtain the ability to change their chemokine system and convert into metastatic cells. In this study, we examined the effect of berberine on breast cancer cell migration and its probable interaction with the chemokine system in cancer cells. Materials and Methods: The MCF-7 breast cancer cell line was cultured, and then, treated with berberine (10, 20, 40 and 80 μg/ml) for 24 hr. MTT assay was used in order to determine the cytotoxic effect of berberine on MCF-7 breast cancer cells. Wound healing assay was applied to determine the inhibitory effect of berberine on cell migration. Moreover, real-time quantitative PCR analysis of selected chemokine receptors was performed to determine the probable molecular mechanism underlying the effect of berberine on breast cancer cell migration. Results: The results of wound healing assay revealed that berberine decreases cell migration. Moreover, we found that the mRNA levels of some chemokine receptors were reduced after berberine treatment, and this may be the underlying mechanism for decreased cell migration. Conclusion: Our results indicate that berberine might be a potential preventive biofactor for human breast cancer metastasis by targeting chemokine receptor genes. PMID:27081456

  16. Cobalt chloride-induced estrogen receptor alpha down-regulation involves hypoxia-inducible factor-1alpha in MCF-7 human breast cancer cells.

    PubMed

    Cho, Jungyoon; Kim, Dukkyung; Lee, SeungKi; Lee, YoungJoo

    2005-05-01

    The estrogen receptor (ER) is down-regulated under hypoxia via a proteasome-dependent pathway. We studied the mechanism of ERalpha degradation under hypoxic mimetic conditions. Cobalt chloride-induced ERalpha down-regulation was dependent on the expression of newly synthesized protein(s), one possibility of which was hypoxia-inducible factor-1alpha (HIF-1alpha). To examine the role of HIF-1alpha expression in ERalpha down-regulation under hypoxic-mimetic conditions, we used a constitutively active form of HIF-1alpha, HIF-1alpha/herpes simplex viral protein 16 (VP16), constructed by replacing the transactivation domain of HIF-1alpha with that of VP16. Western blot analysis revealed that HIF-1alpha/VP16 down-regulated ERalpha in a dose-dependent manner via a proteasome-dependent pathway. The kinase pathway inhibitors PD98059, U0126, wortmannin, and SB203580 did not affect the down-regulation. A mammalian two-hybrid screen and immunoprecipitation assays indicated that ERalpha interacted with HIF-1alpha physically. These results suggest that ERalpha down-regulation under hypoxia involves protein-protein interactions between the ERalpha and HIF-1alpha.

  17. Leptin down-regulates insulin action through phosphorylation of serine-318 in insulin receptor substrate 1.

    PubMed

    Hennige, Anita M; Stefan, Norbert; Kapp, Katja; Lehmann, Rainer; Weigert, Cora; Beck, Alexander; Moeschel, Klaus; Mushack, Joanne; Schleicher, Erwin; Häring, Hans-Ulrich

    2006-06-01

    Insulin resistance in skeletal muscle is found in obesity and type 2 diabetes. A mechanism for impaired insulin signaling in peripheral tissues is the inhibition of insulin action through serine phosphorylation of insulin receptor substrate (Irs) proteins that abolish the coupling of Irs proteins to the activated insulin receptor. Recently, we described serine-318 as a protein kinase C (PKC)-dependent phosphorylation site in Irs1 (Ser-318) activated by hyperinsulinemia. Here we show in various cell models that the adipose hormone leptin, a putative mediator in obesity-related insulin resistance, promotes phosphorylation of Ser-318 in Irs1 by a janus kinase 2, Irs2, and PKC-dependent pathway. Mutation of Ser-318 to alanine abrogates the inhibitory effect of leptin on insulin-induced Irs1 tyrosine phosphorylation and glucose uptake in L6 myoblasts. In C57Bl/6 mice, Ser-318 phosphorylation levels in muscle tissue were enhanced by leptin and insulin administration in lean animals while in diet-induced obesity Ser-318 phosphorylation levels were already up-regulated in the basal state, and further stimulation was diminished. In analogy, in lymphocytes of obese hyperleptinemic human subjects basal Ser-318 phosphorylation levels were increased compared to lean individuals. During a hyperinsulinemic euglycemic clamp, the increment in Ser-318 phosphorylation observed in lean individuals was absent in obese. In summary, these data suggest that phosphorylation of Ser-318 in Irs1 mediates the inhibitory signal of leptin on the insulin-signaling cascade in obese subjects.

  18. Internalization and down-regulation of mu opioid receptors by endomorphins and morphine in SH-SY5Y human neuroblastoma cells.

    PubMed

    Horner, Kristen A; Zadina, James E

    2004-12-03

    The human neuroblastoma cell line, SH-SY5Y, was used to examine the effects of morphine and the endogenous opioid peptides, endomorphin-1 (EM-1) and endomorphin-2 (EM-2), on mu opioid receptor (MOR) internalization and down-regulation. Treatment for 24 h with EM-1, EM-2 or morphine at 100 nM, 1 microM and 10 microM resulted in a dose-dependent down-regulation of mu receptors. Exposure of cells to 10 microM EM-1 for 2.5, 5 and 24 h resulted in a time-dependent down-regulation of mu receptors. Down-regulation of mu receptors by morphine and EM-1 was blocked by treatment with hypertonic sucrose, consistent with an endocytosis-dependent mechanism. Sensitive cell-surface binding studies with a radiolabeled mu antagonist revealed that morphine was able to induce internalization of mu receptors naturally expressed in SH-SY5Y cells. EM-1 produced a more rapid internalization of mu receptors than morphine, but hypertonic sucrose blocked the internalization induced by each of these agonists. This study demonstrates that, like morphine, the endomorphins down-regulate mu opioid receptors in a dose- and time-dependent manner. This study also demonstrates that morphine, as well as EM-1, can induce rapid, endocytosis-dependent internalization of mu opioid receptors in SH-SY5Y cells. These results may help elucidate the ability of mu agonists to regulate the number and responsiveness of their receptors.

  19. Soluble Nogo Receptor Down-regulates Expression of Neuronal Nogo-A to Enhance Axonal Regeneration*

    PubMed Central

    Peng, Xiangmin; Zhou, Zhigang; Hu, Jian; Fink, David J.; Mata, Marina

    2010-01-01

    Nogo-A, a member of the reticulon family, is present in neurons and oligodendrocytes. Nogo-A in central nervous system (CNS) myelin prevents axonal regeneration through interaction with Nogo receptor 1, but the function of Nogo-A in neurons is less known. We found that after axonal injury, Nogo-A is increased in dorsal root ganglion (DRG) neurons unable to regenerate following a dorsal root injury or a sciatic nerve ligation-cut injury and that exposure in vitro to CNS myelin dramatically enhanced neuronal Nogo-A mRNA and protein through activation of RhoA while inhibiting neurite growth. Knocking down neuronal Nogo-A by small interfering RNA results in a marked increase of neurite outgrowth. We constructed a nonreplicating herpes simplex virus vector (QHNgSR) to express a truncated soluble fragment of Nogo receptor 1 (NgSR). NgSR released from QHNgSR prevented myelin inhibition of neurite extension by hippocampal and DRG neurons in vitro. NgSR prevents RhoA activation by myelin and decreases neuronal Nogo-A. Subcutaneous inoculation of QHNgSR to transduce DRG neurons resulted in improved regeneration of myelinated fibers in both the dorsal root and the spinal dorsal root entry zone, with concomitant improvement in sensory behavior. The results indicate that neuronal Nogo-A is an important intermediate in neurite growth dynamics and its expression is regulated by signals related to axonal injury and regeneration, that CNS myelin appears to activate signaling events that mimic axonal injury, and that NgSR released from QHNgSR may be used to improve recovery after injury. PMID:19901030

  20. Down-regulation of endothelin receptors in the ventrolateral medulla of spontaneously hypertensive rats

    SciTech Connect

    Gulati, A.; Rebello, S. )

    1991-01-01

    The binding of ({sup 125}I) sarafotoxin 6b (SRT 6b) and ({sup 125}I) endothelin-1 (ET-1) to endothelin (ET) receptors of neuronal membranes prepared from cerebral cortex and ventrolateral medulla of 8 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. ({sup 125}I) SRT 6b bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of ({sup 125}I) SRT 6b in the cerebral cortex was found to be similar in SHR and WKY rats. However, in the ventrolateral medulla ({sup 125}I) SRT 6b binding was found to be significantly lower in SHR as compared to WKY rats. The decreased binding was due to decrease (48%) in the B{sub max} values in SHR rats as compared to WKY rats. The K{sub d} values were similar in SHR and WKY rats. ({sup 125}I) ET-1 also bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of ({sup 125}I) ET-1 in the cerebral cortex was found to be similar in SHR and WKY rats. However, in the ventrolateral medulla ({sup 125}I) ET-1 binding was found to be significantly lower in SHR as compared to WKY rats. The decreased binding was due to 36% decrease in the B{sub max} values in SHR rats as compared to WKY rats. The K{sub d} values were similar in SHR and WKY rats. It is concluded that the population of ET receptors is less in the ventrolateral medulla of SHR rats and may be contributing to the regulation of blood pressure.

  1. Down-regulation of types I, II and III inositol 1,4,5-trisphosphate receptors is mediated by the ubiquitin/proteasome pathway.

    PubMed Central

    Oberdorf, J; Webster, J M; Zhu, C C; Luo, S G; Wojcikiewicz, R J

    1999-01-01

    Activation of certain phosphoinositidase-C-linked cell-surface receptors is known to cause an acceleration of the proteolysis of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptors and, thus, lead to Ins(1,4,5)P3-receptor down-regulation. In the current study we have sought to determine whether the ubiquitin/proteasome pathway is involved in this adaptive response. The data presented show (i) that activation of phosphoinositidase-C-linked receptors causes Ins(1,4,5)P3-receptor ubiquitination in a range of cell types (AR4-2J cells, INS-1 cells and rat cerebellar granule cells), (ii) that the Ins(1,4,5)P3-receptor down-regulation induced by activation of these receptors is blocked by proteasome inhibitors, (iii) that all known Ins(1,4,5)P3 receptors (types I, II and III) are substrates for ubiquitination, (iv) that ubiquitination occurs while Ins(1,4,5)P3 receptors are membrane-bound, (v) that Ins(1,4, 5)P3-receptor ubiquitination and down-regulation are stimulated only by those agonists that elevate Ins(1,4,5)P3 concentration persistently, and (vi) that a portion of cellular Ins(1,4,5)P3 receptors (those that are not type-I-receptor-associated) can be resistant to ubiquitination and degradation. In total these data indicate that the ubiquitin/proteasome pathway mediates Ins(1,4, 5)P3-receptor down-regulation and suggest that ubiquitination is stimulated by the binding of Ins(1,4,5)P3 to its receptor. PMID:10191279

  2. Manganese induces oligomerization to promote down-regulation of the intracellular trafficking receptor used by Shiga toxin

    PubMed Central

    Tewari, Ritika; Jarvela, Timothy; Linstedt, Adam D.

    2014-01-01

    Manganese (Mn) protects cells against lethal doses of purified Shiga toxin by causing the degradation of the cycling transmembrane protein GPP130, which the toxin uses as a trafficking receptor. Mn-induced GPP130 down-regulation, in addition to being a potential therapeutic approach against Shiga toxicosis, is a model for the study of metal-regulated protein sorting. Significantly, however, the mechanism by which Mn regulates GPP130 trafficking is unknown. Here we show that a transferable trafficking determinant within GPP130 bound Mn and that Mn binding induced GPP130 oligomerization in the Golgi. Alanine substitutions blocking Mn binding abrogated both oligomerization of GPP130 and GPP130 sorting from the Golgi to lysosomes. Further, oligomerization was sufficient because forced aggregation, using a drug-controlled polymerization domain, redirected GPP130 to lysosomes in the absence of Mn. These experiments reveal metal-induced oligomerization as a Golgi sorting mechanism for a medically relevant receptor for Shiga toxin. PMID:25079690

  3. 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

  4. IL-20 receptor 2 signaling down-regulates antigen-specific T cell responses.

    PubMed

    Wahl, Christian; Müller, Werner; Leithäuser, Frank; Adler, Guido; Oswald, Franz; Reimann, Jörg; Schirmbeck, Reinhold; Seier, Anne; Weiss, Johannes Martin; Prochnow, Blair; Wegenka, Ursula Maria

    2009-01-15

    The recently described cytokines IL-19, IL-20, and IL-24 share structural homology with IL-10 and are therefore classified as members of the IL-10 family of cytokines. Although it has long been speculated that signaling by their heterodimeric receptor complexes (IL-20R1/IL-20R2 and IL-22R/IL-20R2) influences immunological processes, the target cells for this group of cytokines are still unclear. By generating a knockout mouse strain deficient for the common IL-20R beta-chain (IL-20R2), we show that IFN-gamma and IL-2 secretion is significantly elevated after stimulation of IL-20R2-/--deficient CD8 and CD4 T cells with Con A or anti-CD3/CD28 in vitro. IL-10 secretion by activated IL-20R2-/- CD4 cells was diminished. Consistent with our in vitro results, significantly more Ag-specific CD8 IFN-gamma+ and CD4 IFN-gamma+ T cells developed to locally applied DNA vaccines in IL-20R2-deficient mice. In a T cell-dependent model of contact hypersensitivity, IL-20R2 knockout mice were more sensitive to the contact allergen trinitro-chloro-benzene. Thus, IL-20R2 signaling directly regulates CD8 and CD4 T cell answers in vitro and in vivo. For the first time, we provide evidence that IL-19, IL-20, and IL-24 are part of a signaling network that normally down-modulates T cell responses in mice.

  5. Down regulation of cerebellar serotonergic receptors in streptozotocin induced diabetic rats: Effect of pyridoxine and Aegle marmelose.

    PubMed

    Abraham, Pretty Mary; Paul, Jes; Paulose, C S

    2010-04-29

    Oxidative stress plays an important role in cerebellar damage caused by diabetes, leading to deterioration in glucose homeostasis causing metabolic disorders. The present study was carried out to find the effects of Aegle marmelose leaf extract and insulin alone and in combination with pyridoxine on the cerebellar 5-HT through 5-HT(2A) receptor subtype, gene expression studies on the status of antioxidants-superoxide dismutase (SOD), glutathione peroxidase (GPx), 5-HT(2A) and 5-HT transporter (5-HTT) and immunohistochemical studies in streptozotocin induced diabetic rats. 5-HT and 5-HT(2A) receptor binding parameters, B(max) and K(d), showed a significant decrease (p<0.001) in the cerebellum of diabetic rats compared to control. Gene expression studies of SOD, GPx, 5-HT(2A) and 5-HTT in cerebellum showed a significant down regulation (p<0.001) in diabetic rats compared to control. Pyridoxine treated alone and in combination with insulin, A. marmelose to diabetic rats reversed the B(max), K(d) of 5-HT, 5-HT(2A) and the gene expression of SOD, GPx, 5-HT(2A) and 5-HTT in cerebellum to near control. The gene expression of 5-HT(2A) and 5-HTT were confirmed by immunohistochemical studies. Also, the Rotarod test confirms the motor dysfunction and recovery by treatment. These data suggest the antioxidant and neuroprotective role of pyridoxine and A. marmelose through the up regulation of 5-HT through 5-HT(2A) receptor in diabetic rats. Our results suggest that pyridoxine treated alone and in combination with insulin and A. marmelose has a role in the regulation of insulin synthesis and release, normalizing diabetic related oxidative stress and neurodegeneration affecting the motor ability of an individual by serotonergic receptors through 5-HT(2A) function. This has clinical significance in the management of diabetes.

  6. The mouse dendritic cell marker CD11c is down-regulated upon cell activation through Toll-like receptor triggering.

    PubMed

    Singh-Jasuja, Harpreet; Thiolat, Allan; Ribon, Matthieu; Boissier, Marie-Christophe; Bessis, Natacha; Rammensee, Hans-Georg; Decker, Patrice

    2013-01-01

    Dendritic cells (DC) play a key role in regulating immune responses and are the best professional antigen-presenting cells. Two major DC populations are defined in part according to cell surface CD11c expression levels. Unexpectedly, we observed that mouse DC strongly down-regulate the typical DC marker CD11c upon activation. To better characterize DC responses, we have analyzed CD11c expression on mouse and human myeloid DC after Toll-like receptor (TLR) triggering. Here we show that mouse bone marrow-derived DC (BMDC) as well as spleen DC down-regulate cell surface CD11c upon activation by TLR3/4/9 agonists. In all cases, full DC activation was reached, as determined by cytokine secretion, cell stimulation in mixed leukocyte reactions (MLR), and CD40/CD86/major histocompatibility complex (MHC) up-regulation. Interestingly, membrane CD11c down-regulation correlated with increased cytoplasmic pools of CD11c. In contrast to the up-regulation of CD40 and MHC class II molecules, lipopolysaccharide (LPS)-induced CD11c down-regulation was MyD88-dependent. Polyinosinic-polycytidylic acid (poly I:C), which does not signal through MyD88, also induced cell surface CD11c down-regulation. Notably, CD11c down-regulation was not observed upon activation of human DC, either through TLR-dependent or -independent cell activation. Thus, activated mouse DC may be transiently CD11c-negative in vivo, hampering the identification of those cells. On the other hand, cell surface CD11c down-regulation may serve as a new activation marker for mouse DC. Copyright © 2012 Elsevier GmbH. All rights reserved.

  7. X-linked Ectodermal Dysplasia Receptor (XEDAR) is Down-regulated in Breast Cancer via Promoter Methylation

    PubMed Central

    Punj, Vasu; Matta, Hittu; Chaudhary, Preet M.

    2009-01-01

    Purpose The X-linked ectodermal dysplasia receptor (XEDAR) is a novel receptor of the Tumor Necrosis Factor Receptor Family that binds to ectodysplasin-A2 (EDA-A2) and induces cell death. The purpose of this study was to determine the tumor-suppressive potential of XEDAR in the development of breast cancer. Experimental Design We analyzed the expression of XEDAR in breast cancer cell lines and tumor samples using quantitative real-time RT-PCR analysis and immunoblotting. We analyzed the human XEDAR gene promoter for the presence of any CpG island and examined its methylation status using methylation-specific real-time PCR. We examined the effect of 5-aza-2′-deoxycytidine (5-Aza-dC) on the expression of XEDAR and sensitivity to EDA-A2-induced apoptosis in breast cancer cell lines. Results Expression of XEDAR, but not EDA-A2, was downregulated in most tumorigenic breast cancer cell lines and tumor samples. Loss of XEDAR expression correlated with the hypermethylation of its promoter. Ectopic expression of XEDAR in MDA-MB-231 cells resulted in significant induction of apoptosis and reduction in colony formation. Treatment with 5-Aza-dC restored XEDAR expression in breast cancer cell lines with methylated XEDAR promoter and sensitized them to EDA-A2-induced cell death. Conclusions Our results suggest that XEDAR expression is down-regulated in most breast cancers via promoter methylation, which may contribute to accelerated tumor development by blocking EDA-A2-induced cell death. XEDAR may represent a novel breast tumor suppressor gene and restoration of its expression by treatment with DNA demethylating agents may represent an attractive approach for the treatment of breast cancer. PMID:20145163

  8. Down-Regulation of 11β-Hydroxysteroid Dehydrogenase Type 2 by Bortezomib Sensitizes Jurkat Leukemia T Cells against Glucocorticoid-Induced Apoptosis

    PubMed Central

    Wu, Xiaosong; Wang, Hongmei; Yang, Guang; Zhan, Fenghuang; Shi, Jumei

    2013-01-01

    11β-hydroxysteroid dehydrogenases type 2 (11β-HSD2), a key regulator for pre-receptor metabolism of glucocorticoids (GCs) by converting active GC, cortisol, to inactive cortisone, has been shown to be present in a variety of tumors. But its expression and roles have rarely been discussed in hematological malignancies. Proteasome inhibitor bortezomib has been shown to not only possess antitumor effects but also potentiate the activity of other chemotherapeutics. In this study, we demonstrated that 11β-HSD2 was highly expressed in two GC-resistant T-cell leukemic cell lines Jurkat and Molt4. In contrast, no 11β-HSD2 expression was found in two GC-sensitive non-hodgkin lymphoma cell lines Daudi and Raji as well as normal peripheral blood T cells. Inhibition of 11β-HSD2 by 11β-HSD inhibitor 18β-glycyrrhetinic acid or 11β-HSD2 shRNA significantly increased cortisol-induced apoptosis in Jurkat cells. Additionally, pretreatment of Jurkat cells with low-dose bortezomib resulted in increased cellular sensitivity to GC as shown by elevated induction of apoptosis, more cells arrested at G1 stage and up-regulation of GC-induced leucine zipper which is an important mediator of GC action. Furthermore, we clarified that bortezomib could dose-dependently inhibit 11β-HSD2 messenger RNA and protein levels as well as activity (cortisol-cortisone conversion) through p38 mitogen-activated protein kinase signaling pathway. Therefore, we suggest 11β-HSD2 is, at least partially if not all, responsible for impaired GC suppression in Jurkat cells and also indicate a novel mechanism by which proteasome inhibitor bortezomib may influence GC action. PMID:23826195

  9. Down-regulation of 11β-hydroxysteroid dehydrogenase type 2 by bortezomib sensitizes Jurkat leukemia T cells against glucocorticoid-induced apoptosis.

    PubMed

    Tao, Yi; Gao, Lu; Wu, Xiaosong; Wang, Hongmei; Yang, Guang; Zhan, Fenghuang; Shi, Jumei

    2013-01-01

    11β-Hydroxysteroid dehydrogenases type 2 (11β-HSD2), a key regulator for pre-receptor metabolism of glucocorticoids (GCs) by converting active GC, cortisol, to inactive cortisone, has been shown to be present in a variety of tumors. But its expression and roles have rarely been discussed in hematological malignancies. Proteasome inhibitor bortezomib has been shown to not only possess antitumor effects but also potentiate the activity of other chemotherapeutics. In this study, we demonstrated that 11β-HSD2 was highly expressed in two GC-resistant T-cell leukemic cell lines Jurkat and Molt4. In contrast, no 11β-HSD2 expression was found in two GC-sensitive non-hodgkin lymphoma cell lines Daudi and Raji as well as normal peripheral blood T cells. Inhibition of 11β-HSD2 by 11β-HSD inhibitor 18β-glycyrrhetinic acid or 11β-HSD2 shRNA significantly increased cortisol-induced apoptosis in Jurkat cells. Additionally, pretreatment of Jurkat cells with low-dose bortezomib resulted in increased cellular sensitivity to GC as shown by elevated induction of apoptosis, more cells arrested at G1 stage and up-regulation of GC-induced leucine zipper which is an important mediator of GC action. Furthermore, we clarified that bortezomib could dose-dependently inhibit 11β-HSD2 messenger RNA and protein levels as well as activity (cortisol-cortisone conversion) through p38 mitogen-activated protein kinase signaling pathway. Therefore, we suggest 11β-HSD2 is, at least partially if not all, responsible for impaired GC suppression in Jurkat cells and also indicate a novel mechanism by which proteasome inhibitor bortezomib may influence GC action.

  10. Androgen Receptor and MicroRNA-21 axis down-regulates transforming growth factor beta receptor II (TGFBR2) expression in Prostate Cancer

    PubMed Central

    Mishra, Sweta; Deng, Janice J.; Gowda, Pramod S.; Rao, Manjeet K.; Lin, Chun-Lin; Chen, Chun Liang; Huang, Tim; Sun, Lu-Zhe

    2014-01-01

    Prostate cancer cells escape growth inhibition from TGFβ by down-regulating TGFβ receptors. However, the mechanism by which cancer cells down-regulate TGFβ receptors in prostate is not clear. Here, we showed that coordinated action of miR-21 and androgen receptor (AR) signaling played a critical role in inhibiting TGFβ receptor II (TGFBR2) expression in prostate cancer cells. Our results revealed that miR-21 suppresses TGFBR2 levels by binding to its 3'UTR and AR signaling further potentiates this effect in both untransformed and transformed human prostate epithelial cells as well as in human prostate cancers. Analysis of primary prostate cancers showed that increased miR-21/AR expression parallel a significantly reduced expression of TGFBR2. Manipulation of androgen signaling or the expression levels of AR or miR-21 negatively altered TGFBR2 expression in untransformed and transformed human prostate epithelial cells, human prostate cancer xenografts, and mouse prostate glands. Importantly, we demonstrated that miR-21 and AR regulated each other's expression resulting in a positive feedback loop. Our results indicated that miR-21/AR mediate its tumor promoting function by attenuating TGFβ-mediated Smad2/3 activation, cell growth inhibition, cell migration, and apoptosis. Together, these results suggest that the AR and miR-21 axis exerts its oncogenic effects in prostate tumors by down-regulating TGFBR2, hence inhibiting the tumor suppressive activity of TGFβ pathway. Targeting miR-21 alone or in combination with AR may restore the tumor inhibitory activity of TGFβ in prostate cancer. PMID:24037531

  11. The association of down-regulated toll-like receptor 4 expression with airflow limitation and emphysema in smokers

    PubMed Central

    2012-01-01

    Background An association between innate immunity including Toll-like receptors (TLRs) and COPD is reported recently; TLR4 deficiency in lung can cause emphysema in animals, which is not evident in humans. We analyzed the association of TLR4 expression, airflow limitation and emphysema in smokers. Methods We enrolled patients of ≥40years old with smoking histories of ≥10 pack-years and who had undergone lung resection. We measured TLR4 expression in lung lysates. The severity of emphysema was evaluated on computed tomography. TLR4 expression was also evaluated immunohistochemically. Results In total, 53 patients were enrolled. Forced expiratory volume in one second per forced vital capacity (FEV1/FVC) increased (P=0.03) and emphysema score decreased (P=0.01) as TLR4 expression increased. These were still significant, in multiple regression analysis including sex, age, tuberculosis history, smoking history and inhaled corticosteroid (ICS) usage. We also classified patients as high, intermediate, and low expressers according to TLR4 expression. Although no differences in age, gender, tuberculosis, or smoking history were observed among the groups, emphysema severity increased significantly (P = 0.02) and FEV1/FVC decreased significantly (P = 0.006) in TLR4 low expresser. The difference in TLR4 expression based on immunohistochemistry was most prominent in bronchial and alveolar epithelial cells. Conclusion Down-regulated TLR4 expression in lung was associated with emphysema and airflow limitation in smokers. PMID:23170858

  12. Ro52/SSA sensitizes cells to death receptor-induced apoptosis by down-regulating c-FLIP(L).

    PubMed

    Zhang, Jing; Fang, Lei; Zhu, Xuguo; Qiao, Yiting; Yu, Mei; Wang, Lu; Chen, Yuan; Yin, Wu; Hua, Zi-Chun

    2012-05-01

    Ro52/SSA is an autoantigen that presents in patients with SS (Sjögren's syndrome) and SLE (systemic lupus erythematosus). It increases cell death and redistributes itself to apoptotic blebs, but its pro-apoptotic function has not been completely identified. Overexpression of Ro52/SSA promoted cell apoptosis induced by DR (death receptor) in caspase-8-dependent manner. Ro52/SSA expression down-regulated c-FLIP(L) [cellular (Fas-associated death domain)-like interleukin 1β-converting enzyme-inhibitory protein long form] expression, and Ro52/SSA siRNAs (small interfering RNAs) increased c-FLIP(L) production, indicating that Ro52/SSA plays a role in c-FLIP(L) regulation. Ro52/SSA negatively regulated c-FLIP(L) transcriptional level probably by suppressing NF-κB (nuclear factor κB) signalling. The data suggest that Ro52/SSA is involved in DR-mediated apoptosis by regulating c-FLIP(L).

  13. Engagement of the T-cell receptor during positive selection in the thymus down-regulates RAG-1 expression.

    PubMed Central

    Brändle, D; Müller, C; Rülicke, T; Hengartner, H; Pircher, H

    1992-01-01

    We have examined the expression of the recombination activating gene RAG-1 by in situ hybridization to thymi from mice bearing transgenes for the T-cell receptor (TCR) alpha chain, TCR beta chain, or both TCR alpha and beta chains. RAG-1 transcription was found in the thymic cortex of transgenic mice carrying a single TCR alpha- or TCR beta-chain transgene, comparable to normal mice. However, RAG-1 transcription was strikingly reduced in the thymic cortex from transgenic mice carrying both TCR alpha- and beta-chain genes and expressing major histocompatibility complex (MHC) class I (H-2b) molecules necessary for positive selection of the transgenic TCR. In contrast, thymi of transgenic mice also carrying both TCR alpha- and beta-chain genes but expressing MHC molecules (H-2d) that did not positively select the transgenic TCR displayed high levels of RAG-1 transcription. The low thymic RAG-1 expression coincided with high transgenic TCR alpha-chain surface expression and with inhibition of endogenous TCR alpha-chain rearrangement. Our findings suggest that binding of the TCR to self MHC molecules during positive selection down-regulates RAG-1 transcription in cortical thymocytes and thereby prevents further TCR alpha-chain rearrangements. Images PMID:1329099

  14. 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.

  15. 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.

  16. Interleukin-10 receptor expression and signalling were down-regulated in CD4+ T cells of lupus nephritis patients

    PubMed Central

    Cui, H D; Qi, Z M; Yang, L L; Qi, L; Zhang, N; Zhang, X L; Du, S Y; Jiang, Y

    2011-01-01

    Studies have indicated that interleukin (IL)-10 has a pathogenic role in systemic lupus erythematosus (SLE); however, a protective effect of IL-10 in SLE was also observed. Because the exact mechanism of IL-10 signalling in the pathogenesis of SLE is unclear, this study sought to assess the expression and signalling of interleukin-10 receptor (IL-10R) in peripheral leucocytes from patients with SLE. We used flow cytometry to examine the expression of IL-10R1 on different peripheral leucocytes from 28 SLE patients, of whom 14 had lupus nephritis (LN) and 14 were healthy controls. We also examined the effects of IL-10 on phosphorylation of signal transducer and activator of transcription (STAT)-3 and STAT-1 in peripheral blood mononuclear cells (PBMCs) obtained from 13 SLE patients and seven healthy controls. Plasma cytokines were detected by flow cytometric bead array (CBA) techniques. Although IL-10R1 expression levels on each peripheral leucocyte subset from 28 SLE patients and 14 healthy controls were similar, the expression levels on CD4+ T cells from LN patients were significantly lower than on CD4+ T cells from controls and SLE patients without nephritis (P < 0·01). IL-10R1 expression levels on CD4+ and CD8+ T cells were correlated negatively with the SLE disease activity index (P < 0·01). Additionally, the phosphorylation of STAT-3 was delayed and reduced in PBMCs from LN patients and active SLE patients. Plasma IL-10 levels were significantly higher in LN patients than controls. IL-10R1 expression on CD4+ T cells and signalling in PBMCs were down-regulated in LN patients, indicating that IL-10 and its receptor may have a special role in LN pathogenesis. PMID:21635228

  17. Molecular mechanisms of benzodiazepine-induced down-regulation of GABAA receptor alpha 1 subunit protein in rat cerebellar granule cells.

    PubMed Central

    Brown, M. J.; Bristow, D. R.

    1996-01-01

    1. Chronic benzodiazepine treatment of rat cerebellar granule cells induced a transient down-regulation of the gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit protein, that was dose-dependent (1 nM-1 microM) and prevented by the benzodiazepine antagonist flumazenil (1 microM). After 2 days of treatment with 1 microM flunitrazepam the alpha 1 subunit protein was reduced by 41% compared to untreated cells, which returned to, and remained at, control cell levels from 4-12 days of treatment. Chronic flunitrazepam treatment did not significantly alter the GABAA receptor alpha 6 subunit protein over the 2-12 day period. 2. GABA treatment for 2 days down-regulates the alpha 1 subunit protein in a dose-dependent (10 microM-1 mM) manner that was prevented by the selective GABAA receptor antagonist bicuculline (10 microM). At 10 microM and 1 mM GABA the reduction in alpha 1 subunit expression compared to controls was 31% and 66%, respectively. 3. The flunitrazepam-induced decrease in alpha 1 subunit protein is independent of GABA, which suggests that it involves a mechanism distinct from the GABA-dependent action of benzodiazepines on GABAA receptor channel activity. 4. Simultaneous treatment with flunitrazepam and GABA did not produce an additive down-regulation of alpha 1 subunit protein, but produced an effect of the same magnitude as that of flunitrazepam alone. This down-regulation induced by the combination of flunitrazepam and GABA was inhibited by flumazenil (78%), but unaffected by bicuculline. 5. The flunitrazepam-induced down-regulation of alpha 1 subunit protein at 2 days was completely reversed by the protein kinase inhibitor staurosporine (0.3 microM). 6. This study has shown that both flunitrazepam and GABA treatment, via their respective binding sites, caused a reduction in the expression of the GABAA receptor alpha 1 subunit protein; an effect mediated through the same neurochemical mechanism. The results also imply that the benzodiazepine effect

  18. Fungal pattern receptors down-regulate the inflammatory response by a cross-inhibitory mechanism independent of interleukin-10 production.

    PubMed

    Rodríguez, Mario; Márquez, Saioa; de la Rosa, Juan Vladimir; Alonso, Sara; Castrillo, Antonio; Sánchez Crespo, Mariano; Fernández, Nieves

    2017-02-01

    Cyclic AMP regulatory element binding protein and signal transducer and activator of transcription 3 (STAT3) may control inflammation by several mechanisms, one of the best characterized is the induction of the expression of the anti-inflammatory cytokine interleukin-10 (IL-10). STAT3 also down-regulates the production of pro-inflammatory cytokines induced by immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, a mechanism termed cross-inhibition. Because signalling via ITAM-dependent mechanisms is a hallmark of fungal pattern receptors, STAT3 activation might be involved in the cross-inhibition associated with invasive fungal infections. The fungal surrogate zymosan produced the phosphorylation of Y705-STAT3 and the expression of Ifnb1 and Socs3, but did not induce the interferon (IFN)-signature cytokines Cxcl9 and Cxcl10 in bone marrow-derived dendritic cells. Unlike lipopolysaccharide (LPS), zymosan induced IL-10 and phosphorylated Y705-STAT3 to a similar extent in Irf3 and Ifnar1 knockout and wild-type mice. Human dendritic cells showed similar results, although the induction of IFNB1 was less prominent. These results indicate that LPS and zymosan activate STAT3 through different routes. Whereas type I IFN is the main effector of LPS effect, the mechanism involved in Y705-STAT3 phosphorylation by zymosan is more complex, cannot be associated with type I IFN, IL-6 or granulocyte-macrophage colony-stimulating factor, and seems dependent on several factors given that it was partially inhibited by the platelet-activating factor antagonist WEB2086 and high concentrations of COX inhibitors, p38 mitogen-activate protein kinase inhibitors, and blockade of tumour necrosis factor-α function. Altogether, these results indicate that fungal pattern receptors share with other ITAM-coupled receptors the capacity to produce cross-inhibition through a mechanism involving STAT3 and induction of SOCS3 and IL-10, but that cannot be explained through type I IFN

  19. 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.

  20. HIV-1 gp120Bal down-regulates phosphorylated NMDA receptor subunit 1 in cortical neurons via activation of glutamate and chemokine receptors

    PubMed Central

    Ru, Wenjuan; Tang, Shao-Jun

    2015-01-01

    HIV-1 envelope glycoprotein gp120 (gp120) is a major virulence protein implicated in the pathogenesis of HIV-associated neurocognitive disorders (HAND). Although gp120 has been suggested to cause synaptic and neuronal injuries by disrupting NMDA receptor (NMDAR) function, the underlying mechanism is unclear. Here, we show that gp120Bal down-regulates the phosphorylation of the NMDAR subunit 1 NR1 (at Ser896 and Ser897), which is essential for NMDAR function. This effect of gp120Bal is blocked by specific antagonists of both NMDA and AMPA receptors, indicating a critical role of synaptic activation. Furthermore, AMD3100 and maraviroc, antagonists of CCR5 and CXCR4 chemokine receptors, respectively, inhibit the effect of gp120Bal on NR1, suggesting that CXCR4 and CCR5 activation are involved. These findings may provide mechanistic insights into the synaptopathogenesis caused by HIV-1 infection. PMID:26582091

  1. Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

    SciTech Connect

    Berger, Christian; Madshus, Inger Helene; Stang, Espen

    2012-12-10

    The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: Black-Right-Pointing-Pointer Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. Black-Right-Pointing-Pointer Antibody combination causes internalization of EGFR by macropinocytosis. Black-Right-Pointing-Pointer Antibody-induced internalization of EGFR is independent of EGFR kinase activity. Black-Right-Pointing-Pointer Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

  2. A model for migratory B cell oscillations from receptor down-regulation induced by external chemokine fields.

    PubMed

    Chan, Cliburn; Billard, Matthew; Ramirez, Samuel A; Schmidl, Harald; Monson, Eric; Kepler, Thomas B

    2013-01-01

    receptor down-regulation induced by external chemokine fields can give rise to spontaneous interzonal and intrazonal oscillations in the absence of any extrinsic regulation. While the extent to which such simple feedback mechanisms contributes to B cell migration patterns in the germinal center is unknown, the model provides an alternative hypothesis for how complex B cell migration patterns might arise from very simple mechanisms.

  3. Endothelin Receptor Down-Regulation Mediated Ligand Regulation Mechanisms Protect Against Cellular Hypoxia Injury in Rat Vascular Endothelial Cells.

    PubMed

    Li, Long; Hu, Mushuang; Zheng, Long; Zhang, Chao; Li, Jiawei; Rong, Ruiming; Zhu, Tongyu; Jia, Yichen

    2016-01-01

    than the ETaR siRNA group. PKG and sGC expression levels significantly descended after mild oxygen deprivation. While such levels were higher in the ETaR siRNA group, compared with the hypoxia and negative siRNA groups, the L-NAME group had lower levels of PKG and sGC compared with the ETaR siRNA group. ETaR siRNA is capable of down-regulating the expression of inflammatory and transcription factors among endothelial cells treated with hypoxia. Down-regulation of ET-1 is triggered by altered nucleus transcription factor activity through the sGC/PKG signal pathway, and results in enhanced eNOS activity through the PI3K/Akt signal pathway. We suspect this to be the mechanism of the protective effect of ETaR siRNA. © 2016 The Author(s) Published by S. Karger AG, Basel.

  4. The ACAT inhibitor VULM1457 significantly reduced production and secretion of adrenomedullin (AM) and down-regulated AM receptors on human hepatoblastic cells.

    PubMed

    Drímal, J; Fáberová, V; Schmidtová, L; Bednáriková, M; Drímal, J; Drímal, D

    2005-12-01

    Acyl-CoA:cholesterol acyltransferase (ACAT) is an important enzyme in the pathways of cholesterol esterification. It has been shown that new ACAT inhibitor 1-(2,6-diisopropyl-phenyl)-3-[4-(4'-nitrophenylthio)phenyl] urea (VULM1457) significantly reduced atherogenic activity in animal experimental atherosclerosis. Proliferative hormone adrenomedullin (AM) has been shown to be released in response to hypoxia, however, its role in cellular protection has remained elusive. The effect of increased local production of AM in cells and resultant down-regulation of AM receptors has not been investigated yet. We hypothesized that increased expression of AM in hypoxic cells was the result of excessive AM production with resultant AM receptor down-regulation, surface-membrane protein degradation and that the new specific ACAT inhibitor would reduce AM induction in hypoxia and thus proliferation of cells. In order to investigate specific cellular AM signaling and protection induced by VULM1457, we characterized specific surface-membrane [125I]AM receptors expressed on cells, evaluated AM secretion (RIA assays), AM mRNA expression in cultured cells (RT-PCR analysis) and proliferation (incorporation of [3H]thymidine) in control, hypoxic and metabolically stressed human hepatoblastoma cell lines exposed to gradually increasing concentrations of VULM1457. The new ACAT inhibitor VULM1457 in concentration 0.03 and 0.1 micromol/l significantly down-regulated specific AM receptors on HepG2 cells, reduced AM secretion of HepG2 cells exposed to hypoxia. These results suggest that VULM1457, as new member of ACAT family of inhibitors could negatively regulate cell proliferation induced by AM, which may correlate with down-regulation of membrane-bound AM receptors on HepG2 cells, and moreover, with the induction and expression of AM in hypoxia.

  5. p120-Catenin Down-Regulation and Epidermal Growth Factor Receptor Overexpression Results in a Transformed Epithelium That Mimics Esophageal Squamous Cell Carcinoma

    PubMed Central

    Lehman, Heather L.; Yang, Xuebin; Welsh, Patricia A.; Stairs, Douglas B.

    2016-01-01

    Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy with a poor prognosis due to its highly invasive and metastatic potential. The molecular pathogenesis underlying the invasive mechanism of ESCC is not well known because of the lack of existing models to study this disease. p120-Catenin (p120ctn) and the epidermal growth factor receptor (EGFR) have each been implicated in several cancers, including ESCC. p120ctn is down-regulated in 60% of ESCC tumors, whereas EGFR is the most commonly overexpressed oncogene in ESCC. For these reasons, we investigated the cooperation between p120ctn and EGFR and its effect on ESCC invasion. We show that p120ctn down-regulation is commonly associated with EGFR overexpression. By using a three-dimensional culture system, we demonstrate that the inverse relationship between p120ctn and EGFR has biological implications. Specifically, p120ctn down-regulation coupled with EGFR overexpression in human esophageal keratinocytes (EPC1-PE) was required to promote invasion. Morphological comparison of EPC1-PE cells grown in three-dimensional culture and human ESCC revealed identical features, including significantly increased cellularity, nuclear grade, and proliferation. Molecular characteristics were measured by keratin expression patterns, which were nearly identical between EPC1-PE cells in three-dimensional culture and ESCC samples. Altogether, our analyses have demonstrated that p120ctn down-regulation and EGFR overexpression are able to mimic human ESCC in a relevant three-dimensional culture model. PMID:25529795

  6. 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

  7. 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

  8. 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.

  9. 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

  10. IFN-gamma and IFN-alpha posttranscriptionally down-regulate the IL-4-induced IL-4 receptor gene expression.

    PubMed

    So, E Y; Park, H H; Lee, C E

    2000-11-15

    As Th1 and Th2 cytokines, IFN-gamma/alpha and IL-4 counterregulate diverse immune functions. In particular, IFN-gamma and IFN-alpha have been reported to markedly suppress the IL-4-induced IgE production and type II IgE receptor (FcepsilonRII/CD23) expression. Because modulation of IL-4R may be an important mechanism in the regulation of IL-4 response, we have investigated the effect of IFN-gamma/alpha on IL-4R expression and signal transduction mechanisms involved in this process. In human mononuclear cells and B cells isolated from tonsil or peripheral blood, IL-4 up-regulates IL-4R(alpha) expression at surface protein and mRNA levels, and the IL-4-induced IL-4R(alpha) is significantly down-regulated by both IFN-gamma and IFN-alpha to a similar extent. The inhibitory effects of IFN-gamma/alpha on the IL-4R mRNA expression require a lag period of about 8 h, and are sensitive to cycloheximide treatment, which suggests that the suppressive effect of IFNs on IL-4R gene expression is a secondary response requiring de novo synthesis of IFN-induced factors. Under such conditions that the inhibitory effects of IFNs are observed, IFNs do not affect the IL-4-induced STAT6 activation and IL-4R transcription, as analyzed by EMSA and nuclear run-on assays, respectively. Subsequently, mRNA stability studies have indicated that the action of IFN-gamma/alpha is primarily mediated by an accelerated decay of IL-4-induced IL-4R mRNA. Thus, it appears that, as already shown in the case of the IL-4-induced FcepsilonRII regulation, posttranscriptional inhibition of IL-4-inducible genes by mRNA destabilization is a common mechanism by which type I and II IFNs antagonize the IL-4 response in human immune cells.

  11. Expression of Vascular Endothelial Growth Factor A During Ligand-Induced Down-Regulation of Luteinizing Hormone Receptor in the Ovary☆

    PubMed Central

    Harada, M.; Peegel, H.; Menon, K. M. J.

    2010-01-01

    Vascular endothelial growth factor A (VEGF-A) is one of the most important regulators of ovarian angiogenesis. In this study, we examined the temporal relationship between VEGF-A and luteinizing hormone receptor (LHR) mRNA expression during ligand-induced down-regulation of LHR. Immature female rats were treated with pregnant mare’s serum gonadotropin followed by 25 IU hCG 56h later (day 0). On day 5, treatment with hCG (50 IU) to down-regulate LHR showed a temporal decrease in VEGF-A mRNA and protein levels in parallel with decreasing LHR mRNA. This effect was specific since the expression of CYP11A1 mRNA showed no decline. Examination of VEGF-A mRNA expression, using in situ hybridization histochemistry with 35S-labeled antisense VEGF-A mRNA probe, showed intense signal in the corpora lutea on day 5. Treatment with 50 IU hCG to down-regulate LHR mRNA showed a decline in the intensity of VEGF-A mRNA in the corpora lutea. VEGF-A mRNA expression returned to control level 53 hours later when the expression of LHR mRNA also recovered. These results show that the transient down-regulation of VEGF-A mRNA and protein closely parallels the ligand-induced down-regulation of LHR mRNA. The present study establishes a close association between VEGF-A and LHR mRNA expression, suggesting the possibility that VEGF-A-induced vascularization of the ovary is dictated by the expression of LHR and this might play a regulatory role in ovarian physiology. PMID:20619315

  12. Apigenin suppresses migration and invasion of transformed cells through down-regulation of C-X-C chemokine receptor 4 expression

    SciTech Connect

    Wang, Lei; Kuang, Lisha; Hitron, John Andrew; Son, Young-Ok; Wang, Xin; Budhraja, Amit; Lee, Jeong-Chae; Pratheeshkumar, Poyil; Chen, Gang; Zhang, Zhuo; Luo, Jia; Shi, Xianglin

    2013-10-01

    Environmental exposure to arsenic is known to cause various cancers. There are some potential relationships between cell malignant transformation and C-X-C chemokine receptor type 4 (CXCR4) expressions. Metastasis, one of the major characteristics of malignantly transformed cells, contributes to the high mortality of cells. CXCR4 and its natural chemokine ligand C-X-C motif ligand 12 (CXCL12) play a critical role in metastasis. Therefore, identification of nutritional factors which are able to inhibit CXCR4 is important for protection from environmental arsenic-induced carcinogenesis and for abolishing metastasis of malignantly transformed cells. The present study demonstrates that apigenin (4′,5,7-trihydroxyflavone), a natural dietary flavonoid, suppressed CXCR4 expression in arsenic-transformed Beas-2B cells (B-AsT) and several other types of transformed/cancer cells in a dose- and time-dependent manner. Neither proteasome nor lysosome inhibitor had any effect in reducing the apigenin-induced down-regulation of CXCR4, indicating that apigenin-induced down-regulation of CXCR4 is not due to proteolytic degradation. The down-regulation of CXCR4 is mainly due to the inhibition of nuclear factor κB (NF-κB) transcriptional activity. Apigenin also abolished migration and invasion of transformed cells induced by CXCL12. In a xenograft mouse model, apigenin down-regulated CXCR4 expression and suppressed tumor growth. Taken together, our results show that apigenin is a novel inhibitor of CXCR4 expression. This dietary flavonoid has the potential to suppress migration and invasion of transformed cells and prevent environmental arsenic-induced carcinogenesis. - Highlights: • Apigenin has a potential in preventing environmental arsenic induced carcinogenesis. • Apigenin suppresses CXCR4 in malignant transformed cells in vitro and in vivo. • The down-regulation of CXCR4 is mainly due to inhibition of NF-κB activity.

  13. 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

  14. Down-Regulation of GABAA Receptor via Promiscuity with the Vasoactive Peptide Urotensin II Receptor. Potential Involvement in Astrocyte Plasticity

    PubMed Central

    Lecointre, Céline; Schouft, Marie-Thérèse; Leprince, Jérôme; Compère, Vincent; Morin, Fabrice; Proust, François; Gandolfo, Pierrick; Tonon, Marie-Christine; Castel, Hélène

    2012-01-01

    GABAA receptor (GABAAR) expression level is inversely correlated with the proliferation rate of astrocytes after stroke or during malignancy of astrocytoma, leading to the hypothesis that GABAAR expression/activation may work as a cell proliferation repressor. A number of vasoactive peptides exhibit the potential to modulate astrocyte proliferation, and the question whether these mechanisms may imply alteration in GABAAR-mediated functions and/or plasma membrane densities is open. The peptide urotensin II (UII) activates a G protein-coupled receptor named UT, and mediates potent vasoconstriction or vasodilation in mammalian vasculature. We have previously demonstrated that UII activates a PLC/PIPs/Ca2+ transduction pathway, via both Gq and Gi/o proteins and stimulates astrocyte proliferation in culture. It was also shown that UT/Gq/IP3 coupling is regulated by the GABAAR in rat cultured astrocytes. Here we report that UT and GABAAR are co-expressed in cerebellar glial cells from rat brain slices, in human native astrocytes and in glioma cell line, and that UII inhibited the GABAergic activity in rat cultured astrocytes. In CHO cell line co-expressing human UT and combinations of GABAAR subunits, UII markedly depressed the GABA current (β3γ2>α2β3γ2>α2β1γ2). This effect, characterized by a fast short-term inhibition followed by drastic and irreversible run-down, is not relayed by G proteins. The run-down partially involves Ca2+ and phosphorylation processes, requires dynamin, and results from GABAAR internalization. Thus, activation of the vasoactive G protein-coupled receptor UT triggers functional inhibition and endocytosis of GABAAR in CHO and human astrocytes, via its receptor C-terminus. This UII-induced disappearance of the repressor activity of GABAAR, may play a key role in the initiation of astrocyte proliferation. PMID:22563490

  15. Down-regulation of cerebellar 5-HT(2C) receptors in pilocarpine-induced epilepsy in rats: therapeutic role of Bacopa monnieri extract.

    PubMed

    Krishnakumar, Amee; Abraham, Pretty Mary; Paul, Jes; Paulose, C S

    2009-09-15

    Epilepsy is a syndrome of episodic brain dysfunction characterized by recurrent unpredictable, spontaneous seizures. Cerebellar dysfunction is a recognized complication of temporal lobe epilepsy and it is associated with seizure generation, motor deficits and memory impairment. Serotonin is known to exert a modulatory action on cerebellar function through 5HT(2C) receptors. 5-HT(2C) receptors are novel targets for developing anti-convulsant drugs. In the present study, we investigated the changes in the 5-HT(2C) receptors binding and gene expression in the cerebellum of control, epileptic and Bacopa monnieri treated epileptic rats. There was a significant down regulation of the 5-HT content (p<0.001), 5-HT(2C) gene expression (p<0.001) and 5-HT(2C) receptor binding (p<0.001) with an increased affinity (p<0.001). Carbamazepine and B. monnieri treatments to epileptic rats reversed the down regulated 5-HT content (p<0.01), 5-HT(2C) receptor binding (p<0.001) and gene expression (p<0.01) to near control level. Also, the Rotarod test confirms the motor dysfunction and recovery by B. monnieri treatment. These data suggest the neuroprotective role of B. monnieri through the upregulation of 5-HT(2C) receptor in epileptic rats. This has clinical significance in the management of epilepsy.

  16. Binding and cross-linking of recombinant mouse interferon-. gamma. to receptors in mouse leukemic L1210 cells; interferon-. gamma. internalization and receptor down-regulation

    SciTech Connect

    Wietzerbin, J.; Gaudelet, C.; Aguet, M.; Falcoff, E.

    1986-04-01

    Recombinant E. coli-derived murine IFN-..gamma.. (Mu-rIFN-..gamma..; 5 x 10/sup 7/ U/mg) was radiolabeled with /sup 125/I by the chloramine-T method without loss of its antiviral activity. The /sup 125/I-Mu-rIFN-..gamma.. showed specific binding to L1210 cells. Scatchard analysis indicates about 4000 binding sites per cell and an apparent Kd of 5 x 10/sup -10/ M. Binding of /sup 125/I-Mu-rIFn-..gamma.. to cells inhibited by both natural (glycosylated) and rIFN-..gamma.., but not by IFN-..gamma../..beta... Receptor-bound /sup 125/I-Mu-rIFN-..gamma.. was rapidly internalized when incubation temperature was raised from 4/sup 0/C to 37/sup 0/C. On internalization, almost no IFN-..gamma.. degradation was observed during 16 hr incubation. /sup 125/I-Mu-rIFN-..gamma.. binding capacity decreased in cells preincubated with low doses of unlabeled Mu-rIFN-..gamma.., but not with IFN-..cap alpha../..beta... This receptor down-regulation was dose-dependent: 90% reduction of /sup 125/I-Mu-rIFN-..gamma.. binding was observed after preincubation with 100 U/ml. After removal of IFN-..gamma.. from the culture medium, the binding capacity increased with time. However, reappearance of receptor was completely blocked by cycloheximide or tunicamycin, suggesting that re-expression of receptors is not due to recycling but to the synthesis of new receptors, and that the receptor is probably a glycoprotein. Cross-linking of /sup 125/I-Mu-rIFN-..gamma.. to surface L1210 cell proteins by using bifunctional agents yielded a predominant complex of m.w. 110,000 +/- 5000. Thus, assuming a bimolecular complex, the m.w. of the receptor or receptor subunit would be close to 95,000 +/- 5000.

  17. 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.

  18. Ligation of human Fc receptor like-2 by monoclonal antibodies down-regulates B-cell receptor-mediated signalling

    PubMed Central

    Shabani, Mahdi; Bayat, Ali Ahmad; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Hojjat-Farsangi, Mohammad; Ulivieri, Cristina; Amirghofran, Zahra; Baldari, Cosima Tatiana; Shokri, Fazel

    2014-01-01

    B-cell antigen receptor (BCR) signalling and its regulation through negative and positive regulators are critical for balancing B-cell response and function. Human Fc receptor like-2 (FCRL2), a member of the newly identified FCRL family, could influence B-cell signalling due to possession of both immunoreceptor tyrosine-based activation and inhibitory motifs (ITAM and ITIM). Since the natural ligand of FCRL2 has not been identified, we generated FCRL2-specific monoclonal antibodies (mAbs) and employed them to investigate the influence of FCRL2 stimulation on BCR signalling in an FCRL2-expressing B-cell line. Two anti-FCRL2 mAb-producing hybridoma clones (5A7-E7 and 3D8-G8) were selected. None of the mAbs displayed any cross-reactivity with the other members of the FCRL family including recombinant FCRL1, -3, -4 and -5, as tested by FACS and ELISA techniques. Engagement of the FCRL2 by these mAbs resulted in significant inhibition of BCR signalling mediators such as calcium mobilization and phosphorylation of the mitogen-activated protein kinases Erk, p38 and Jnk. These findings indicate that the FCRL2 ITIM motifs are functional and the anti-FCRL2 mAbs may mimic the natural ligand of FCRL2 by induction of inhibitory signals in B cells. PMID:24797767

  19. 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.

  20. The down-regulation of the mitogenic fibrinogen receptor (MFR) in serum-containing medium does not occur in defined medium.

    PubMed

    Levesque, J P; Hatzfeld, A; Domart, I; Hatzfeld, J

    1990-02-01

    Normal human hemopoietic cells such as early bone marrow progenitors, or lymphoma-derived cell lines such as Raji or JM cells, possess a low-affinity receptor specific for fibrinogen. This receptor triggers a mitogenic effect. It differs from the glycoprotein IIb-IIIa which is involved in fibrinogen-induced platelet aggregation. We demonstrate here that this mitogenic fibrinogen receptor (MFR) can be internalized or reexpressed, depending on culture conditions. Internalization was temperature-dependent. At 37 degrees C in the presence of cycloheximide or actinomycin D, the half-life of cell surface MFRs was 2 h, independent of receptor occupancy. Binding of fibrinogen to the MFR resulted in a down-regulation which was fibrinogen dose-dependent. This occurred in serum-supplemented medium but not in defined medium supplemented with fatty acids. Reexpression of MFRs could be induced in 28 to 42 h by serum removal. The down-regulation of mitogenic receptors in plasma or serum could explain why normal cells do not proliferate in the peripheral blood.

  1. 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.

  2. The effects of the β-agonist isoproterenol on the down-regulation, functional responsiveness, and trafficking of β2-adrenergic receptors with amino-terminal polymorphisms

    PubMed Central

    Koryakina, Yulia; Jones, Stacie M.; Cornett, Lawrence E.; Seely, Kathryn; Brents, Lisa; Prather, Paul L.; Kofman, Alexander; Kurten, Richard C.

    2014-01-01

    The β2-adrenergic receptor (β2AR) is an important target for respiratory and cardiovascular disease medications. Clinical studies suggest that amino-terminal polymorphisms of the β2AR may act as disease modifiers. We hypothesized that polymorphisms at amino acids 16 and 27 result in differential trafficking and down-regulation of β2AR variants following β-agonist exposure. The functional consequences of the four possible combinations of these polymorphisms in the human β2AR (designated β2AR-RE, -GE, -RQ and -GQ) were studied using site-directed mutagenesis and recombinant expression in HEK 293 cells. Ligand binding assays demonstrated that after 24 h exposure to 1 μM isoproterenol, isoforms with Arg16 (β2AR-RE and β2AR-RQ) underwent increased down-regulation compared to isoforms with Gly16 (β2AR-GE and β2AR-GQ). Consistent with these differences in down-regulation between isoforms, prolonged isoproterenol treatment resulted in diminished cyclic AMP response to subsequent isoproterenol challenge in β2AR-RE relative to β2AR-GE. Confocal microscopy revealed that the receptor isoforms had similar co-localization with the early endosomal marker EEA1 following isoproterenol treatment, suggesting that they had similar patterns of internalization. None of the isoforms exhibited significant co-localization with the recycling endosome marker Rab11 in response to isoproterenol treatment. Furthermore, we found that prolonged isoproterenol treatment led to a higher degree of co-localization of β2AR-RE with the lysosomal marker Lamp1 compared to that of β2AR-GE. Taken together, these results indicate that a mechanism responsible for differential responses of these receptor isoforms to β-agonist involves differences in the efficiency with which agonist-activated receptors are trafficked to lysosomes for degradation, or differences in degradation in the lysosomes. PMID:22938397

  3. 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

  4. miR-129-2 mediates down-regulation of progesterone receptor in response to progesterone in breast cancer cells.

    PubMed

    Godbole, Mukul; Chandrani, Pratik; Gardi, Nilesh; Dhamne, Hemant; Patel, Kuldeep; Yadav, Neelima; Gupta, Sudeep; Badwe, Rajendra; Dutt, Amit

    2017-09-06

    Hormonal therapy is an important component of first line of treatment for breast cancer. Response to hormonal therapy is influenced by the progesterone receptor (PR)-status of breast cancer patients. However as an early effect, exposure to progesterone decreases expression of PR in breast cancer cells. An understanding of the mechanism underlying down-regulation of PR could help improve response to hormonal therapy. We performed small RNA sequencing of breast cancer cells for identification of microRNAs targeting PR in response to progesterone treatment. Biochemical approaches were used to validate the findings in breast cancer cells. Analysis of small RNA sequencing of four breast cancer cell lines treated with progesterone revealed an up-regulation of miR-129-2 independent of the PR status of the cells. We show that miR-129-2 targets 3'UTR of PR to down-regulate its expression. Furthermore, inhibition of miR-129-2 expression rescues the down-regulation of PR in breast cancer cells. Also, the expression levels of miR-129-2 was observed to be elevated in patients with low expression of PR in the TCGA cohort (n = 359). miR-129-2 mediates down-regulation of PR in breast cancer cells in response to progesterone, while anti-miR-129-2 could potentiate PR expression levels among patients with inadequate PR levels. Thus, modulation of activity of miR-129-2 could stabilize PR expression and potentially improve response to hormonal therapy under adjuvant or neo-adjuvant settings.

  5. Apigenin suppresses migration and invasion of transformed cells through down-regulation of C-X-C chemokine receptor 4 expression

    PubMed Central

    Wang, Lei; Kuang, Lisha; Hitron, John Andrew; Son, Young-Ok; Wang, Xin; Budhraja, Amit; Lee, Jeong-Chae; Poyil, Pratheeshkumar; Chen, Gang; Zhang, Zhuo; Luo, Jia; Shi, Xianglin

    2013-01-01

    Environmental exposure to arsenic is known to cause various cancers. There are some potential relationships between cell malignant transformation and C-X-C chemokine receptor type 4 (CXCR4) expressions. Metastasis, one of the major characteristics of malignantly transformed cells, contributes to the high mortality of cells. CXCR4 and its natural chemokine ligand C-X-C motif ligand 12 (CXCL12) play a critical role in metastasis. Therefore, identification of nutritional factors which are able to inhibit CXCR4 is important for protection from environmental arsenic-induced carcinogenesis and for abolishing metastasis of malignantly transformed cells. The present study demonstrates that apigenin (4′, 5, 7-trihydroxyflavone), a natural dietary flavonoid, suppressed CXCR4 expression in arsenic-transformed Beas-2B cells (B-AsT) and several other type of transformed/cancer cells in a dose- and time-dependent manner. Neither proteasome nor lysosome inhibitor had any effect in reducing the apigenin-induced down-regulation of CXCR4, indicating that apigenin-induced down-regulation of CXCR4 is not due to proteolytic degradation. The down-regulation of CXCR4 is mainly due to the inhibition of nuclear factor κB (NF-κB) transcriptional activity. Apigenin also abolished migration and invasion of transformed cells induced by CXCL12. In a xenograft mouse model, apigenin down-regulated CXCR4 expression and suppressed tumor growth. Taken together, our results show that apigenin is a novel inhibitor of CXCR4 expression. This dietary flavonoid has the potential to suppress migration and invasion of transformed cells and prevent environmental arsenic-induced carcinogenesis. PMID:23743303

  6. Vasoactive Intestinal Peptide Down-Regulates Pro- While Up-Regulating Anti-Inflammatory Toll-like Receptors in the Infected Cornea

    PubMed Central

    Jiang, Xiaoyu; McClellan, Sharon A.; Barrett, Ronald P.; Zhang, Yunfan; Hazlett, Linda D.

    2012-01-01

    Toll-like receptors (TLRs) recognize microbial pathogens and trigger an immune response, but their regulation by neuropeptides such as vasoactive intestinal peptide (VIP) during Pseudomonas aeruginosa corneal infection remains unexplored. Therefore, C57BL/6 (B6) mice were injected intraperitoneally with VIP and mRNA, protein and immunostaining assays done. After VIP treatment PCR array and real-time RT-PCR demonstrated that pro-inflammatory TLRs (Chuk, IRAK1, TLR1, 4, 6, 8, 9 and TRAF6) were down-regulated, while anti-inflammatory TLRs (SIGIRR and ST2) were up-regulated. ELISA showed that VIP modestly down-regulated phosphorylated IKKα, but up-regulated ST2 almost 2 fold. SIGIRR also was up-regulated, while TLR4 immunostaining was reduced in cornea; all confirming the mRNA data. To determine if VIP effects were cAMP dependent, mice were injected with siRNA for type 7 adenylate cyclase (AC7) with or without VIP treatment. After silencing AC7, changes in mRNA levels of TLR1, TRAF6 and ST2 were seen and unchanged with addition of VIP, indicating their regulation was cAMP dependent. In contrast, changes were seen in mRNA levels of Chuk, IRAK1, 2, TLR4, 9 and SIGIRR following AC7 silencing alone, which were modified by VIP addition, indicating their cAMP independence. In vitro studies tested the effects of VIP on TLR regulation in macrophages and Langerhans cells. VIP down-regulated mRNA expression of pro-inflammatory, while up-regulating anti-inflammatory TLRs in both cell types. Collectively, the data provide evidence that VIP down-regulates pro- and up-regulates anti-inflammatory TLRs, that this regulation is both cAMP dependent and independent, and involves immune cell types found in the infected cornea. PMID:22661083

  7. 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

  8. 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.

  9. 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

  10. Repeated treatment with electroconvulsive seizures induces HDAC2 expression and down-regulation of NMDA receptor-related genes through histone deacetylation in the rat frontal cortex.

    PubMed

    Park, Hong Geun; Yu, Hyun Sook; Park, Soyoung; Ahn, Yong Min; Kim, Yong Sik; Kim, Se Hyun

    2014-09-01

    The enzymatic activity of histone deacetylases (HDACs) leads to a histone deacetylation-mediated condensed chromatic structure, resulting in transcriptional repression, which has been implicated in the modifications of neural circuits and behaviors. Repeated treatment with electroconvulsive seizure (ECS) induces changes in histone acetylation, expression of various genes, and intrabrain cellular changes, including neurogenesis. In this study, we examined the effects of repeated ECS on the expression of class I HDACs and related changes in histone modifications and gene expression in the rat frontal cortex. Ten days of repeated ECS treatments (E10X) up-regulated HDAC2 expression at the mRNA and protein levels in the rat frontal cortex compared with sham-treated controls; this was evident in the nuclei of neuronal cells in the prefrontal, cingulate, orbital, and insular cortices. Among the known HDAC2 target genes, mRNA expression of N-methyl-d-aspartate (NMDA) receptor signaling-related genes, including early growth response-1 (Egr1), c-Fos, glutamate receptor, ionotropic, N-methyl d-aspartate 2A (Nr2a), Nr2b, neuritin1 (Nrn1), and calcium/calmodulin-dependent protein kinase II alpha (Camk2α), were decreased, and the histone acetylation of H3 and/or H4 proteins was also reduced by E10X. Chromatin immunoprecipitation analysis revealed that HDAC2 occupancy in the promoters of down-regulated genes was increased significantly. Moreover, administration of sodium butyrate, a HDAC inhibitor, during the course of E10X ameliorated the ECS-induced down-regulation of genes in the rat frontal cortex. These findings suggest that induction of HDAC2 by repeated ECS treatment could play an important role in the down-regulation of NMDA receptor signaling-related genes in the rat frontal cortex through histone modification.

  11. 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.

  12. The Liver X Receptor Ligand T0901317 Down-regulates APOA5 GeneExpression through Activation of SREBP-1c

    SciTech Connect

    Jakel, Heidelinde; Nowak, Maxime; Moitrot, Emanuelle; Dehondt, Helene; Hum, Dean W.; Pennacchio, Len A.; Fruchart-Najib, Jamila; Fruchart,Jean-Charles

    2004-07-23

    Alterations in the expression of the recently discovered apolipoprotein A5 gene strongly affect plasma triglyceride levels. In this study, we investigated the contribution of APOA5 to the liver X-receptor (LXR) ligand mediated effect on plasma triglyceride levels.Following treatment with the LXR ligand T0901317, we found that APOA5mRNA levels were decreased in hepatoma cell lines. The observation that no down-regulation of APOA5 promoter activity was obtained by LXR-retinoid X receptor (RXR) co-transfection prompted us to explore the possible involvement of the known LXR target gene SREBP-1c (sterol regulatory element-binding protein 1c). In fact, we found that co-transfection with the active form of SREBP-1c down-regulated APOA5promoter activity in a dose-dependent manner. We then scanned the human APOA5 promoter sequence and identified two putative E-box elements that were able to bind specifically SREBP-1c in gel-shift assays and were shown to be functional by mutation analysis. Subsequent suppression of SREBP-1 mRNA through small interfering RNA interference abolished the decrease of APOA5 mRNA in response to T0901317. Finally, administration of T0901317 to hAPOA5 transgenic mice revealed a significant decrease OF APOA5 mRNA in liver tissue and circulating apolipoprotein AV protein in plasma, confirming that the described down-regulation also occurs in vivo. Taken together, our results demonstrate that APOA5 gene expression is regulated by the LXR ligand T0901317 in a negative manner through SREBP-1c. These findings may provide a new mechanism responsible for the elevation of plasma triglyceride levels by LXR ligands and support the development of selective LXR agonists, not affecting SREBP-1c, as beneficial modulators of lipid metabolism.

  13. [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.

  14. Down-regulation of transcobalamin receptor TCblR/CD320 by siRNA inhibits cobalamin uptake and proliferation of cells in culture

    SciTech Connect

    Lai, Shao-Chiang; Nakayama, Yasumi; Sequeira, Jeffrey M.; Quadros, Edward V.

    2011-07-01

    The clinical phenotype of cobalamin (Cbl) deficiency is dictated by the essential role of this vitamin in two key enzymatic reactions. Multiple proteins and receptors participate in the absorption, transport and delivery of this vitamin to tissue cells. Cellular uptake of Cbl is mediated by transcobalamin (TC), a plasma protein and a transmembrane receptor (TCblR) with high affinity for TC saturated with Cbl. Knockdown of TCblR with siRNA results in decreased TC-Cbl uptake. The ensuing Cbl deficiency leads to an increase in doubling time and decreased proliferation of these cells. The study confirms the seminal role of this receptor in the cellular uptake of Cbl and its down-regulation as a potential strategy to inhibit proliferation of cancer cells.

  15. 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.

  16. Selective down-regulation of α4β2 neuronal nicotinic acetylcholine receptors in the brain of uremic rats with cognitive impairment.

    PubMed

    Ballesta, Juan J; del Pozo, Carlos; Castelló-Banyuls, Juan; Faura, Clara C

    2012-07-01

    Cognitive impairment is common in patients with chronic kidney disease. Brain nicotinic acetylcholine receptors modulate cognitive functions, such as learning and memory. Pharmacological cholinergic enhancement is useful in patients with cognitive dysfunction. The major nicotinic acetylcholine receptor subtypes in the brain are heteromeric α4β2 and homomeric α7 receptors. To study the involvement of neuronal acetylcholine receptors in cognitive impairment in uremic rats, bilateral nephrectomy was performed. 24 weeks after nephrectomy, memory was assessed using the one trial step-down inhibitory avoidance test. Neuronal nicotinic acetylcholine receptors in the brain were studied by radioligand binding, immunoprecipitation, Western blot and sucrose gradient experiments. We demonstrated that rats with severe renal failure show disorders of short term memory. Long term memory was not altered in these rats. The number of functional α4β2 heteromeric neuronal nicotinic receptors was decreased in the brains of rats with severe renal failure. There was a significant correlation between the degree of renal impairment and the number of heteromeric nicotinic acetylcholine receptors in the brain. The down-regulation of functional α4β2 receptors in the brains of rats with severe renal failure was not due to a reduction of α4 or β2 subunit proteins. The number of α7 homomeric neuronal nicotinic acetylcholine receptors was not altered. These findings may have important clinical significance for the management of cognitive impairment in patients with chronic kidney disease. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. 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.

  18. 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.

  19. 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.

  20. Down-regulation of the tumor suppressor gene retinoic acid receptor beta2 through the phosphoinositide 3-kinase/Akt signaling pathway.

    PubMed

    Lefebvre, Bruno; Brand, Céline; Flajollet, Sébastien; Lefebvre, Philippe

    2006-09-01

    The retinoic acid receptor beta2 (RARbeta2) is a potent, retinoid-inducible tumor suppressor gene, which is a critical molecular relay for retinoid actions in cells. Its down-regulation, or loss of expression, leads to resistance of cancer cells to retinoid treatment. Up to now, no primary mechanism underlying the repression of the RARbeta2 gene expression, hence affecting cellular retinoid sensitivity, has been identified. Here, we demonstrate that the phosphoinositide 3-kinase/Akt signaling pathway affects cellular retinoid sensitivity, by regulating corepressor recruitment to the RARbeta2 promoter. Through direct phosphorylation of the corepressor silencing mediator for retinoic and thyroid hormone receptors (SMRT), Akt stabilized RAR/SMRT interaction, leading to an increased tethering of SMRT to the RARbeta2 promoter, decreased histone acetylation, down-regulation of the RARbeta2 expression, and impaired cellular differentiation in response to retinoid. The phosphoinositide 3-kinase/Akt signaling pathway, an important modulator of cellular survival, has thus a direct impact on cellular retinoid sensitivity, and its deregulation may be the triggering event in retinoid resistance of cancer cells.

  1. Clobetasol down-regulates SLPI expression in U937 monocytoid cells.

    PubMed

    Okumura, Naoko; Yoshida, Hitomi; Kitagishi, Yasuko; Nishimura, Yuri; Matsuda, Satoru

    2012-02-01

    In order to investigate how glucocorticoids affect the expression of secretory leukocyte peptidase inhibitor (SLPI), which is overexpressed in a variety of cancers, clobetasol was added to cell culture medium of U937 cells and the SLPI mRNA levels were examined. The in vitro effect of the treatment on SLPI expression was detected by reverse transcriptase-polymerase chain reaction. Clobetasol treatment of U937 cells induced an up- and down-regulation of SLPI expression in a dose-dependent manner. Western blotting confirmed the down-regulation of SLPI protein expression. We hypothesized a loop formation in the SLPI genome domain, in which the glucocorticoid receptor regulates bi-directional transcriptional activity.

  2. 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.

  3. Ischemic postconditioning and pinacidil suppress calcium overload in anoxia-reoxygenation cardiomyocytes via down-regulation of the calcium-sensing receptor

    PubMed Central

    Deng, Shengli; Yao, Gang

    2016-01-01

    Ischemic postconditioning (IPC) and ATP sensitive potassium channel (KATP) agonists (e.g. pinacidil and diazoxide) postconditioning are effective methods to defeat myocardial ischemia-reperfusion (I/R) injury, but their specific mechanisms of reducing I/R injury are not fully understood. We observed an intracellular free calcium ([Ca2+]i) overload in Anoxia/reoxygenation (A/R) cardiomyocytes, which can be reversed by KATP agonists diazoxide or pinacidil. The calcium-sensing receptor (CaSR) regulates intracellular calcium homeostasis. CaSR was reported to be involved in the I/R-induced apoptosis in rat cardiomyocytes. We therefore hypothesize that IPC and pinacidil postconditioning (PPC) reduce calcium overload in I/R cardiomyocytes by the down-regulation of CaSR. A/R model was established with adult rat caridomyocyte. mRNA and protein expression of CaSR were detected, IPC, PPC and KATP’s effects on [Ca2+]i concentration was assayed too. IPC and PPC ameliorated A/R insult induced [Ca2+]i overload in cardiomyocytes. In addition, they down-regulated the mRNA and protein level of CaSR as we expected. CaSR agonist spermine and KATP blocker glibenclamide offset IPC’s effects on CaSR expression and [Ca2+]i modulation. Our data indicate that CaSR down-regulation contributes to the mitigation of calcium overload in A/R cardiomyocytes, which may partially represents IPC and KATP’s myocardial protective mechanism under I/R circumstances. PMID:27833799

  4. 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.

  5. Prostaglandin E₂ down-regulates the expression of CD25 on bovine T cells, and this effect is mediated through the EP4 receptor.

    PubMed

    Maślanka, Tomasz; Spodniewska, Anna; Barski, Dariusz; Jasiecka, Agnieszka; Zuśka-Prot, Monika; Ziółkowski, Hubert; Markiewicz, Włodzimierz; Jaroszewski, Jerzy Jan

    2014-08-15

    A crucial event in the initiation of an immune response is the activation of T cells, which requires IL-2 binding to its high-affinity IL-2 receptor for optimal signaling. The IL-2 receptor α-chain (CD25) is needed for the high affinity binding of IL-2 to effector cells and is potently induced after T cell activation. The aim of this research has been to determine whether prostaglandin E2 (PGE2) affects the CD25 expression on bovine T cells, and if it does, then which of the PGE2 receptor (EP) subtype(s) mediate(s) this effect. Herein, we report that exposure of peripheral blood mononuclear cells (PBMC) to PGE2 considerably reduces the percentage and absolute counts of CD25(+)CD4(+), CD25(+)CD8(+) and CD25(+)WC1(+) T cells, significantly increases the value of these parameters with respect of CD25(-)CD4(+), CD25(-)CD8(+) and CD25(-)WC1(+) T cells, and does not affect counts of the total populations of CD4(+), CD8(+) and WC1(+) T cells. These results indicate that PGE2 down-regulates the CD25 expression on bovine T cells. Moreover, we show that the selective blockade of EP4 receptor, but not EP1 and EP3 receptors, prevents this effect. Interestingly, the exposure of PBMC to a selective EP2 receptor agonist leads to a substantial increase in the percentage and absolute number of CD25(+)CD4(+), CD25(+)CD8(+) and CD25(+)WC1(+) T cells. In conclusions, the PGE2-induced down-regulation of CD25 expression on bovine CD4(+), CD8(+) and WC1(+) T cells should be considered as immunosuppressive and anti-inflammatory action, because these lymphocytes primarily represent effector cells and adequate CD25 expression is essential for their correct functioning. The PGE2-mediated down-regulation of the CD25 expression on bovine T cells is mediated via the EP4 receptor, although selective activation of the EP2 receptor up-regulates the CD25 expression on these cells. Thus, with respect to the effect of PGE2 on the CD25 expression on bovine T cells, EP4 receptor serves as an

  6. Pigment epithelium-derived factor 34-mer peptide prevents liver fibrosis and hepatic stellate cell activation through down-regulation of the PDGF receptor.

    PubMed

    Tsai, Tung-Han; Shih, Shou-Chuan; Ho, Tsung-Chuan; Ma, Hsin-I; Liu, Ming-Ying; Chen, Show-Li; Tsao, Yeou-Ping

    2014-01-01

    Pigment epithelium-derived factor (PEDF) has been shown previously to prevent liver fibrosis and hepatic stellate cell (HSC) activation. By investigating the functional domains in PEDF, we identified a 34-mer peptide (residues Asp44-Asn77) that harbors the same function as the full-length PEDF protein. Not only did the 34-mer suppress the development of fibrosis in carbon tetrachloride (CCl4)-treated mouse liver but it also upregulated peroxisome proliferator-activated receptor-gamma (PPARγ) expression in HSCs in vivo. Platelet-derived growth factor (PDGF) plays a crucial role on the process of HSC activation in response to liver damage. The 34-mer suppressed PDGF-induced cell proliferation and expression of myofibroblastic marker proteins in primary rat HSC culture, increased the levels of PPARγ mRNA and protein in a dose-dependent manner and markedly reduced the level of active β-catenin protein, an HSC activating factor, in HSC-T6 cells. Similarly, IWR-1, an inhibitor of the Wnt response, displayed the same effect as the 34-mer in preventing HSC-T6 activation. The Wnt signaling-mediated PPARγ suppression was abolished by both the IWR-1 inhibitor and a small interfering RNA (siRNA) targeting β-catenin and the Wnt coreceptor, LRP6. Both PEDF and the 34-mer down-regulated PDGF receptor-α/β expression and blocked the PDGF-induced phosphorylation of Akt and ERK. Moreover, the inhibitory effect on PDGF receptor expression was abolished by PPARγ antagonists and PPARγ siRNA. Our observations indicate that the PEDF-derived 34-mer peptide can mimic PEDF in attenuating HSC activation. Investigation of this 34-mer peptide led to the identification of a signaling mechanism involving PPARγ induction, suppression of Wnt/β-catenin signaling and down-regulation of the PDGF receptor-α/β.

  7. 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.

  8. MicroRNA-Mediated Down-Regulation of M-CSF Receptor Contributes to Maturation of Mouse Monocyte-Derived Dendritic Cells

    PubMed Central

    Riepsaame, Joey; van Oudenaren, Adri; den Broeder, Berlinda J. H.; van IJcken, Wilfred F. J.; Pothof, Joris; Leenen, Pieter J. M.

    2013-01-01

    Dendritic cell (DC) maturation is a tightly regulated process that requires coordinated and timed developmental cues. Here we investigate whether microRNAs are involved in this process. We identify microRNAs in mouse GM-CSF-generated, monocyte-related DC (GM-DC) that are differentially expressed during both spontaneous and LPS-induced maturation and characterize M-CSF receptor (M-CSFR), encoded by the Csf1r gene, as a key target for microRNA-mediated regulation in the final step toward mature DC. MicroRNA-22, -34a, and -155 are up-regulated in mature MHCIIhi CD86hi DC and mediate Csf1r mRNA and protein down-regulation. Experimental inhibition of Csf1r-targeting microRNAs in vitro results not only in sustained high level M-CSFR protein expression but also in impaired DC maturation upon stimulation by LPS. Accordingly, over-expression of Csf1r in GM-DC inhibits terminal differentiation. Taken together, these results show that developmentally regulated microRNAs control Csf1r expression, supplementing previously identified mechanisms that regulate its transcription and protein surface expression. Furthermore, our data indicate a novel function for Csf1r in mouse monocyte-derived DC, showing that down-regulation of M-CSFR expression is essential for final DC maturation. PMID:24198819

  9. MicroRNA-Mediated Down-Regulation of M-CSF Receptor Contributes to Maturation of Mouse Monocyte-Derived Dendritic Cells.

    PubMed

    Riepsaame, Joey; van Oudenaren, Adri; den Broeder, Berlinda J H; van Ijcken, Wilfred F J; Pothof, Joris; Leenen, Pieter J M

    2013-01-01

    Dendritic cell (DC) maturation is a tightly regulated process that requires coordinated and timed developmental cues. Here we investigate whether microRNAs are involved in this process. We identify microRNAs in mouse GM-CSF-generated, monocyte-related DC (GM-DC) that are differentially expressed during both spontaneous and LPS-induced maturation and characterize M-CSF receptor (M-CSFR), encoded by the Csf1r gene, as a key target for microRNA-mediated regulation in the final step toward mature DC. MicroRNA-22, -34a, and -155 are up-regulated in mature MHCII(hi) CD86(hi) DC and mediate Csf1r mRNA and protein down-regulation. Experimental inhibition of Csf1r-targeting microRNAs in vitro results not only in sustained high level M-CSFR protein expression but also in impaired DC maturation upon stimulation by LPS. Accordingly, over-expression of Csf1r in GM-DC inhibits terminal differentiation. Taken together, these results show that developmentally regulated microRNAs control Csf1r expression, supplementing previously identified mechanisms that regulate its transcription and protein surface expression. Furthermore, our data indicate a novel function for Csf1r in mouse monocyte-derived DC, showing that down-regulation of M-CSFR expression is essential for final DC maturation.

  10. Calcitriol May Down-Regulate mRNA Over-Expression of Toll-Like Receptor-2 and -4, LL-37 and Proinflammatory Cytokines in Cultured Human Keratinocytes

    PubMed Central

    Jeong, Mi Sook; Kim, Ji-Yun; Lee, He In

    2014-01-01

    Background Although vitamin D analogs have been used in the topical treatment of psoriasis, their mechanisms of action are not well understand. Calcitriol, the hormonally active vitamin D3 metabolite, has been demonstrated to exert immunomodulatory effects in the skin by down-regulating the expression of Toll-like receptors (TLRs) and proinflammatory cytokines. Objective We investigated the effects of calcitriol on the expression of TLR2, TLR4, antimicrobial peptide LL-37, and proinflammatory cytokines in cultured human keratinocytes. Methods The mRNA expression levels of TLR2, TLR4, tumor necrosis factor α (TNF-α), interleukin (IL)-1β and LL-37 in cultured human keratinocytes were measured by real-time polymerase chain reaction (PCR) and reverse transcription (RT). Furthermore, we measured supernatant TNF-α levels by an enzyme-linked immunosorbent assay (ELISA) to confirm the effects of calcitriol on TLR2 and TLR4. Results As measured by RT-PCR and real-time PCR, calcitriol was found to suppress the lipopolysaccharide- and ultraviolet B radiation-mediated induction of expression of TLRs, LL-37 and proinflammatory cytokines such as TNF-α and IL-1β in normal human keratinocytes. The supernatant TNF-α levels measured by ELISA were also suppressed after treatment with calcitriol. Conclusion Calcitriol may down-regulate inflammatory stated over-expression of LL-37 and proinflammatory cytokines. PMID:24966627

  11. Lentivirus-mediated RNAi knockdown of insulin-like growth factor-1 receptor inhibits the growth and invasion of hepatocellular carcinoma via down-regulating midkine expression

    PubMed Central

    Huang, Qiu Yan; Tang, Hui Jun; Wang, Min; Cao, Guo Li; Yi, Ting Zhuang; Wu, Sheng Lan; Xu, Wei Jie; Tang, Shao Hui

    2016-01-01

    The insulin-like growth factor-1 receptor (IGF-1R) overexpression contributes to the development of a variety of cancers. The present study explored the role of IGF-1R in the development and progression of hepatocellular carcinoma (HCC) and the possibility of IGF-1R silencing by lentivirus-mediated RNA interference (RNAi) as a therapeutic target for HCC. We showed that IGF-1R mRNA was up-regulated in Huh7 and Hep3B cells and human HCC tissues, and that IGF-1R knockdown by RNAi led to decreased proliferation, apoptosis induction, and decreased migration and invasion of Huh7 and Hep3B cells. Further, the in vivo study indicated that IGF-1R knockdown markedly diminished the tumorigenesis and metastasis of Huh7 xenograft. Moreover, the intratumoral administration of lentivirus-IGF-1R siRNA led to significant tumor growth inhibition in an established Huh7 xenograft model. Mechanistic investigations showed that midkine was found to be the most significantly down-regulated protein in Huh7 cells with IGF-1R knockdown, and ectopic overexpression of midkine significantly rescued inhibition of Huh7 cell proliferation, migration, and invasion caused by IGF-1R suppression. Collectively, these data suggest that IGF-1R inhibition by RNAi can significantly suppress HCC growth and invasion at least partially through down-regulating midkine expression, and IGF-1R is a potential target for HCC gene therapy. PMID:27813495

  12. 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.

  13. 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

  14. 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.

  15. Down-regulation of muscarinic acetylcholine receptor M2 adversely affects the expression of Alzheimer's disease-relevant genes and proteins.

    PubMed

    Zuchner, Thole; Schliebs, Reinhard; Perez-Polo, J Regino

    2005-10-01

    Beta-amyloid peptides play a major role in the pathogenesis of Alzheimer's disease (AD). Therefore, preventing beta-amyloid formation by inhibition of the beta site amyloid precursor protein-cleaving enzyme (BACE) 1 is considered as a potential strategy to treat AD. Cholinergic mechanisms have been shown to control amyloid precursor protein processing and the number of muscarinic M2-acetylcholine receptors is decreased in brain regions of patients with AD enriched with senile plaques. Therefore, the present study investigates the effect of this M2 muscarinic receptor down-regulation by siRNA on total gene expression and on regulation of BACE1 in particular in SK-SH-SY5Y cells. This model system was used for microarray analysis after carbachol stimulation of siRNA-treated cells compared with carbachol stimulated, non-siRNA-treated cells. The same model system was used to elucidate changes at the protein level by using two-dimensional gels followed by Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF) analysis. Taken together, the results indicate that the M2 acetylcholine receptor down-regulation in brains of patients with AD has important effects on the expression of several genes and proteins with major functions in the pathology of AD. This includes beta-secretase BACE1 as well as several modulators of the tau protein and other AD-relevant genes and proteins. Moreover, most of these genes and proteins are adversely affected against the background of AD.

  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. 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

  18. 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.

  19. Desensitization of pigment granule aggregation in Xenopus leavis melanophores: melatonin degradation rather than receptor down-regulation is responsible.

    PubMed

    Teh, Muy-Teck; Sugden, David

    2002-05-01

    Xenopus laevis melanophores express a high density (B(max) 1224 fmol/mg protein) of high-affinity (K(d) 37 pm) cell membrane melatonin receptors. Treatment of melanophores with melatonin resulted in a loss of membrane melatonin receptors reaching a maximum (approximately 60%) by 6 h. In addition to receptor loss, a decline in the potency of melatonin to produce pigment aggregation was observed on prolonged treatment. However, the loss of potency (3.8-fold in 24 h and 162-fold in 96 h) was much slower than loss of receptors, and was completely prevented by inclusion of eserine (100 microm), an inhibitor of melatonin deacetylation in the culture medium. Incubation of melanophores with [(3)H]-melatonin showed that eserine prevented metabolism of melatonin to 5-methoxytryptamine. These results indicate that although receptor density does decline on prolonged treatment, this is not responsible for the diminishing melatonin potency, which is entirely due to degradation of melatonin by deacetylation and subsequent deamination in melanophores.

  20. Angiotensin II receptor blockade promotes repair of skeletal muscle through down-regulation of aging-promoting C1q expression

    PubMed Central

    Yabumoto, Chizuru; Akazawa, Hiroshi; Yamamoto, Rie; Yano, Masamichi; Kudo-Sakamoto, Yoko; Sumida, Tomokazu; Kamo, Takehiro; Yagi, Hiroki; Shimizu, Yu; Saga-Kamo, Akiko; Naito, Atsuhiko T.; Oka, Toru; Lee, Jong-Kook; Suzuki, Jun-ichi; Sakata, Yasushi; Uejima, Etsuko; Komuro, Issei

    2015-01-01

    Disruption of angiotensin II type 1 (AT1) receptor prolonged life span in mice. Since aging-related decline in skeletal muscle function was retarded in Atgr1a−/− mice, we examined the role of AT1 receptor in muscle regeneration after injury. Administration of AT1 receptor blocker irbesartan increased the size of regenerating myofibers, decreased fibrosis, and enhanced functional muscle recovery after cryoinjury. We recently reported that complement C1q, secreted by macrophages, activated Wnt/β-catenin signaling and promoted aging-related decline in regenerative capacity of skeletal muscle. Notably, irbesartan induced M2 polarization of macrophages, but reduced C1q expression in cryoinjured muscles and in cultured macrophage cells. Irbesartan inhibited up-regulation of Axin2, a downstream gene of Wnt/β-catenin pathway, in cryoinjured muscles. In addition, topical administration of C1q reversed beneficial effects of irbesartan on skeletal muscle regeneration after injury. These results suggest that AT1 receptor blockade improves muscle repair and regeneration through down-regulation of the aging-promoting C1q-Wnt/β-catenin signaling pathway. PMID:26571361

  1. Bryostatin-5 blocks stromal cell-derived factor-1 induced chemotaxis via desensitization and down-regulation of cell surface CXCR4 receptors.

    PubMed

    He, Xing; Fang, Liyan; Wang, Jue; Yi, Yanghua; Zhang, Shuyu; Xie, Xin

    2008-11-01

    The chemokine receptor CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1), play important roles in hematopoiesis regulation, lymphocyte activation, and trafficking, as well as in developmental processes, including organogenesis, vascularization, and embryogenesis. The receptor is also involved in HIV infection and tumor growth and metastasis. Antagonists of CXCR4 have been widely evaluated for drugs against HIV and tumors. In an effort to identify novel CXCR4 antagonists, we screened a small library of compounds derived from marine organisms and found bryostatin-5, which potently inhibits chemotaxis induced by SDF-1 in Jurkat cells. Bryostatin-5 is a member of the macrolactones, and its analogue bryostatin-1 is currently being evaluated in clinical trials for its chemotherapeutic potential. The involvement of bryostatins in the SDF-1/CXCR4 signaling process has never been reported. In this study, we found that bryostatin-5 potently inhibits SDF-1-induced chemotaxis but does not affect serum-induced chemotaxis. Further studies indicate that this inhibitory effect is not due to receptor antagonism but rather to bryostatin-5-induced receptor desensitization and down-regulation of cell surface CXCR4. We also show that these effects are mediated by the activation of conventional protein kinase C.

  2. 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

  3. 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.

  4. Estrogen Receptor α Mediates Proliferation of Osteoblastic Cells Stimulated by Estrogen and Mechanical Strain, but Their Acute Down-regulation of the Wnt Antagonist Sost Is Mediated by Estrogen Receptor β*

    PubMed Central

    Galea, Gabriel L.; Meakin, Lee B.; Sugiyama, Toshihiro; Zebda, Noureddine; Sunters, Andrew; Taipaleenmaki, Hanna; Stein, Gary S.; van Wijnen, Andre J.; Lanyon, Lance E.; Price, Joanna S.

    2013-01-01

    Mechanical strain and estrogens both stimulate osteoblast proliferation through estrogen receptor (ER)-mediated effects, and both down-regulate the Wnt antagonist Sost/sclerostin. Here, we investigate the differential effects of ERα and -β in these processes in mouse long bone-derived osteoblastic cells and human Saos-2 cells. Recruitment to the cell cycle following strain or 17β-estradiol occurs within 30 min, as determined by Ki-67 staining, and is prevented by the ERα antagonist 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride. ERβ inhibition with 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-β]pyrimidin-3-yl] phenol (PTHPP) increases basal proliferation similarly to strain or estradiol. Both strain and estradiol down-regulate Sost expression, as does in vitro inhibition or in vivo deletion of ERα. The ERβ agonists 2,3-bis(4-hydroxyphenyl)-propionitrile and ERB041 also down-regulated Sost expression in vitro, whereas the ERα agonist 4,4′,4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl]tris-phenol or the ERβ antagonist PTHPP has no effect. Tamoxifen, a nongenomic ERβ agonist, down-regulates Sost expression in vitro and in bones in vivo. Inhibition of both ERs with fulvestrant or selective antagonism of ERβ, but not ERα, prevents Sost down-regulation by strain or estradiol. Sost down-regulation by strain or ERβ activation is prevented by MEK/ERK blockade. Exogenous sclerostin has no effect on estradiol-induced proliferation but prevents that following strain. Thus, in osteoblastic cells the acute proliferative effects of both estradiol and strain are ERα-mediated. Basal Sost down-regulation follows decreased activity of ERα and increased activity of ERβ. Sost down-regulation by strain or increased estrogens is mediated by ERβ, not ERα. ER-targeting therapy may facilitate structurally appropriate bone formation by enhancing the distinct ligand-independent, strain-related contributions to proliferation

  5. 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.

  6. Dienogest, a synthetic progestin, down-regulates expression of CYP19A1 and inflammatory and neuroangiogenesis factors through progesterone receptor isoforms A and B in endometriotic cells.

    PubMed

    Ichioka, Masayuki; Mita, Shizuka; Shimizu, Yutaka; Imada, Kazunori; Kiyono, Tohru; Bono, Yukiko; Kyo, Satoru

    2015-03-01

    Dienogest (DNG) is a selective progesterone receptor (PR) agonist and oral administration of DNG is used for the treatment of endometriosis. DNG is considered to act on PR to down-regulate pathophysiological factors associated with endometriosis. PR exists as two major isoforms, PR-A and PR-B, and their physiological functions are mostly distinct. It was suggested that PR isoform expression patterns are altered in endometriosis, but it is unknown whether the pharmacological effects of DNG are exerted through PR-A, PR-B or both. In the present study, we investigated the pharmacological effects of DNG through these PR isoforms on the expression of CYP19A1 which encodes aromatase and inflammatory and neuroangiogenesis factors associated with the pain and progression of endometriosis. We used immortalized human endometriotic epithelial cell lines that specifically express PR-A or PR-B in a spheroid cell culture system, and treated them with DNG. We evaluated messenger RNA (mRNA) expression of CYP19A1, prostaglandin (PG)E2 synthase (cyclooxygenase (COX)-2 and microsomal PGE2 synthase (mPGES)-1), inflammatory cytokines (interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1) and neuroangiogenesis factors (vascular endothelial growth factor (VEGF) and nerve growth factor (NGF)) using real-time polymerase chain reaction. In addition, PGE2 production was measured by enzyme immunoassay. We found that DNG down-regulated mRNA expression of CYP19A1, COX-2, mPGES-1, IL-6, IL-8, MCP-1, NGF and VEGF, and PGE2 production in human endometriotic epithelial cell lines that specifically express either PR-A or PR-B. These results demonstrate that DNG activates both PR-A and PR-B and down-regulates the expression of pathophysiological factors associated with pain and progression of endometriosis. Our results suggest that DNG exerts therapeutic efficacy against the pain and progression of endometriosis regardless of PR isoform expression patterns.

  7. 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.

  8. Cytisine confers neuronal protection against excitotoxic injury by down-regulating GluN2B-containing NMDA receptors.

    PubMed

    Li, Yu-Jiao; Yang, Qi; Zhang, Kun; Guo, Yan-Yan; Li, Xu-Bo; Yang, Le; Zhao, Ming-Gao; Wu, Yu-Mei

    2013-01-01

    Cytisine (CYT), one of the principal bioactive components derived from the seeds of Cytisus laborinum L, has been widely used for central nervous system (CNS) diseases treatment. The present study investigated the protective effect of CYT on cultured cortical neural injury induced by N-methyl-d-aspartate (NMDA). Our data showed that CYT conferred protective effect against loss of cellular viability induced by brief exposure to 200 μM NMDA in a concentration-dependent manner. CYT significantly inhibited the neuronal apoptosis induced by NMDA exposure by reversing intracellular Ca(2+) overload and balancing Bcl-2 and Bax expression levels. Furthermore, CYT significantly reversed the up-regulation of GluN2B-containing NMDA receptors by exposure to NMDA, but it did not affect the level of GluN2A-containing NMDA receptors. These findings suggest that CYT protects cortical neurons, at least partially, by inhibiting the level of GluN2B-containing NMDA receptors and regulating Bcl-2 family.

  9. Reduction of fibroblast size/mechanical force down-regulates TGF-β type II receptor: implications for human skin aging.

    PubMed

    Fisher, Gary J; Shao, Yuan; He, Tianyuan; Qin, Zhaoping; Perry, Daniel; Voorhees, John J; Quan, Taihao

    2016-02-01

    The structural integrity of human skin is largely dependent on the quality of the dermal extracellular matrix (ECM), which is produced, organized, and maintained by dermal fibroblasts. Normally, fibroblasts attach to the ECM and thereby achieve stretched, elongated morphology. A prominent characteristic of dermal fibroblasts in aged skin is reduced size, with decreased elongation and a more rounded, collapsed morphology. Here, we show that reduced size of fibroblasts in mechanically unrestrained three-dimensional collagen lattices coincides with reduced mechanical force, measured by atomic force microscopy. Reduced size/mechanical force specifically down-regulates TGF-β type II receptor (TβRII) and thus impairs TGF-β/Smad signaling pathway. Both TβRII mRNA and protein were decreased, resulting in 90% loss of TGF-β binding to fibroblasts. Down-regulation of TβRII was associated with significantly decreased phosphorylation, DNA-binding, and transcriptional activity of its key downstream effector Smad3 and reduced expression of Smad3-regulated essential ECM components type I collagen, fibronectin, and connective tissue growth factor (CTGF/CCN2). Restoration of TβRII significantly increased TGF-β induction of Smad3 phosphorylation and stimulated expression of ECM components. Reduced expression of TβRII and ECM components in response to reduced fibroblast size/mechanical force was fully reversed by restoring size/mechanical force. Reduced fibroblast size was associated with reduced expression of TβRII and diminished ECM production, in aged human skin. Taken together, these data reveal a novel mechanism that provides a molecular basis for loss of dermal ECM, with concomitant increased fragility, which is a prominent feature of human skin aging.

  10. The vitamin D analogue paricalcitol attenuates hepatic ischemia/reperfusion injury through down-regulation of Toll-like receptor 4 signaling in rats

    PubMed Central

    Kim, Min Sung; Lee, Soyoung; Jung, Namhee; Lee, Kiho; Choi, Jinwoo; Kim, Sang-Hoon; Jun, Jinhyun; Lee, Won-Mee; Chang, Yeonsoo

    2016-01-01

    Introduction Recent studies have revealed that vitamin D and its synthetic analogues have a protective effect on experimental ischemia/reperfusion (I/R) models in several organs, but little is known about its effect on the liver. The aim of this study was to evaluate the beneficial effects of vitamin D in a model of liver I/R in rats, focusing on Toll-like receptor (TLR) 4 signaling, which has been shown to be involved in I/R injury. Material and methods Twenty-four male Wistar rats were randomized into four groups: Saline + Sham, Saline + I/R, Paricalcitol + Sham, and Paricalcitol + I/R. A synthetic vitamin D2 analogue, paricalcitol, was intraperitoneally injected 24 h prior to surgery. The animals were subjected to 60 min of partial warm ischemia (70%), followed by reperfusion for 6 h on the same day. The ischemic lobe of the liver and blood were collected for molecular biochemical analyses. Results Liver damage following I/R was diminished by pretreatment with paricalcitol. Pretreatment with paricalcitol decreased the levels of pro-inflammatory mediators, such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and macrophage migration inhibitory factor (MIF), in both plasma and liver tissue. In addition, pretreatment with paricalcitol markedly down-regulated the expression of TLR4, HMGB1, TNF-α and NF-κB. Conclusions The vitamin D analogue paricalcitol attenuates hepatic I/R injury through down-regulation of the TLR4 signaling pathway and might be considered to be a potential nutritional therapeutic agent against I/R injury in the liver. PMID:28261302

  11. Down-regulation of E-cadherin in human bronchial epithelial cells leads to epidermal growth factor receptor-dependent Th2 cell-promoting activity.

    PubMed

    Heijink, Irene H; Kies, P Marcel; Kauffman, Henk F; Postma, Dirkje S; van Oosterhout, Antoon J M; Vellenga, Edo

    2007-06-15

    Airway epithelial cells are well-known producers of thymus- and activation-regulated chemokine (TARC), a Th2 cell-attracting chemokine that may play an important role in the development of allergic airway inflammation. However, the mechanism responsible for up-regulation of TARC in allergy is still unknown. In the asthmatic airways, loss of expression of the cell-cell contact molecule E-cadherin and reduced epithelial barrier function has been observed, which may be the result of an inadequate repair response. Because E-cadherin also suppressed multiple signaling pathways, we studied whether disruption of E-cadherin-mediated cell contact may contribute to increased proallergic activity of epithelial cells, e.g., production of the chemokine TARC. We down-regulated E-cadherin in bronchial epithelial cells by small interference RNA and studied effects on electrical resistance, signaling pathways, and TARC expression (by electric cell-substrate impedance sensing, immunodetection, immunofluorescent staining, and real-time PCR). Small interference RNA silencing of E-cadherin resulted in loss of E-cadherin-mediated junctions, enhanced phosphorylation of epidermal growth factor receptor (EGFR), and the downstream targets MEK/ERK-1/2 and p38 MAPK, finally resulting in up-regulation of TARC as well as thymic stromal lymphopoietin expression. The use of specific inhibitors revealed that the effect on TARC is mediated by EGFR-dependent activation of the MAPK pathways. In contrast to TARC, expression of the Th1/Treg cell-attracting chemokine RANTES was unaffected by E-cadherin down-regulation. In summary, we show that loss of E-cadherin-mediated epithelial cell-cell contact by damaging stimuli, e.g., allergens, may result in reduced suppression of EGFR-dependent signaling pathways and subsequent induction of Th2 cell-attracting molecule TARC. Thus, disruption of intercellular epithelial contacts may specifically promote Th2 cell recruitment in allergic asthma.

  12. AB283. SPR-10 Down-regulation of ryanodine receptor gene expression in murine urinary bladder smooth muscle following partial bladder outlet obstruction

    PubMed Central

    Boopathi, Ettickan; Javed, Elham; Addya, Shankar; Fortina, Paolo; Zderic, Stephen; Wein, Alan; Chacko, Samuel

    2016-01-01

    Objective Urinary bladder smooth muscle (UBSM) displays spontaneous action potentials and this potential is related to the phasic nature of spontaneous contractions in this tissue. The amplitude of a phasic contraction depends on the increase in Ca2+ entry caused by membrane depolarization. Ryanodine receptors (RyRs) in UBSM decreases the force production by decreasing the frequency of phasic contractions through interactions with large-conductance Ca2+-activated K+ (BK) and small-conductance Ca2+-activated K+ (SK) channels. Microarray and network analysis were employed to determine the changes in mRNA in 14-day obstructed murine bladders. We found that obstruction significantly down-regulated the RyRs in bladder smooth muscle (BSM). Methods Male C57Bl/6 mice were surgically obstructed and kept for 14 days. Sham-operated mice served as a control. Bladders were excised; urothelium scraped off with a scalpel, and the serosa was removed. BSM obtained from PBOO and sham control animals were used for microarray and western blotting Results Pathway-based analysis of these gene signatures showed significant number of under-expressed genes in obstructed bladder and they were mapped to proteins involved in calcium signaling. We focused our work on RyR protein expression in BSM. There was a four-fold reduction of RyR3 in BSM in 14-day obstructed groups as shown by microarray and immunoblotting compared to that of sham-operated animals. Conclusions These results confirm that the RyR gene expression is down-regulated in obstructed murine bladder smooth muscle. Funding Source(s) None

  13. Styrene Trimer May Increase Thyroid Hormone Levels via Down-Regulation of the Aryl Hydrocarbon Receptor (AhR) Target Gene UDP-Glucuronosyltransferase

    PubMed Central

    Yanagiba, Yukie; Ito, Yuki; Yamanoshita, Osamu; Zhang, Shu-Yun; Watanabe, Gen; Taya, Kazuyoshi; Li, Chun Mei; Inotsume, Yuko; Kamijima, Michihiro; Gonzalez, Frank J.; Nakajima, Tamie

    2008-01-01

    Background Styrene trimers (STs) are polystyrene-container–eluted materials that are sometimes detected in packaged foods. Although the possible endocrine-disrupting effects of STs, such as estrogenic activities, have been reported, their potential thyroid toxicity, such as that caused by the related endocrine disruptor 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has not been studied in detail. Objective Using wild-type and aryl hydrocarbon receptor (Ahr)–null mice, we investigated whether 2,4,6-triphenyl-1-hexene (ST-1), an isomer of STs, influences thyroxin (T4) levels in the same manner as TCDD, which induces UDP-glucuronosyltransferase (UGT) via the AhR, resulting in a decrease in T4 levels in the plasma of mice. Methods Both wild-type and Ahr-null mice (five mice per group) were treated for 4 days by gavage with ST-1 (0, 32, or 64 μmol/kg). Results High-dose (64 μmol/kg) ST-1 decreased the expression of AhR, cytochrome P450 (CYP) 1A1/2, UGT1A1/A6, and CYP2B10 mRNAs and the enzyme activity for CYP1A and UGT1A only in the wild-type mice. This dose decreased AhR DNA binding, but paradoxically increased AhR translocation to the nucleus. In contrast, a high dose of ST-1 increased T4 levels in the plasma in wild-type mice but did not influence T4 levels in AhR-null mice. Conclusions Although ST-1 treatment might cause an increase in AhR levels in the nucleus by inhibiting AhR export, this chemical down-regulated AhR mRNA, thus leading to down-regulation of AhR target genes and an increase in plasma T4 levels. PMID:18560529

  14. Inhibition of mTOR down-regulates scavenger receptor, class B, type I (SR-BI) expression, reduces endothelial cell migration and impairs nitric oxide production.

    PubMed

    Fruhwürth, Stefanie; Krieger, Sigurd; Winter, Katharina; Rosner, Margit; Mikula, Mario; Weichhart, Thomas; Bittman, Robert; Hengstschläger, Markus; Stangl, Herbert

    2014-07-01

    The mammalian target of rapamycin (mTOR) inhibiting drug rapamycin (Sirolimus) has severe side effects in patients including hyperlipidemia, an established risk factor for atherosclerosis. Recently, it was shown that rapamycin decreases hepatic LDL receptor (LDL-R) expression, which likely contributes to hypercholesterolemia. Scavenger receptor, class B, type I (SR-BI) is the major HDL receptor and consequently regulating HDL-cholesterol levels and the athero-protective effects of HDL. By using the mTOR inhibitor rapamycin, we show that SR-BI is down-regulated in human umbilical vein endothelial cells (HUVECs). This reduction of SR-BI protein as well as mRNA levels by about 50% did not alter HDL particle uptake or HDL-derived lipid transfer. However, rapamycin reduced HDL-induced activation of eNOS and stimulation of endothelial cell migration. The effects on cell migration could be counteracted by SR-BI overexpression, indicating that decreased SR-BI expression is in part responsible for the rapamycin-induced effects. We demonstrate that inhibition of mTOR leads to endothelial cell dysfunction and decreased SR-BI expression, which may contribute to atherogenesis during rapamycin treatment.

  15. 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.

  16. Prolonged nicotine exposure down-regulates presynaptic NMDA receptors in dopaminergic terminals of the rat nucleus accumbens.

    PubMed

    Salamone, Alessia; Zappettini, Stefania; Grilli, Massimo; Olivero, Guendalina; Agostinho, Paula; Tomé, Angelo R; Chen, Jiayang; Pittaluga, Anna; Cunha, Rodrigo A; Marchi, Mario

    2014-04-01

    The presynaptic control of dopamine release in the nucleus accumbens (NAc) by glutamate and acetylcholine has a profound impact on reward signaling. Here we provide immunocytochemical and neurochemical evidence supporting the co-localization and functional interaction between nicotinic acetylcholine receptors (nAChRs) and N-methyl-D-aspartic acid (NMDA) receptors in dopaminergic terminals of the NAc. Most NAc dopaminergic terminals possessed the nAChR α4 subunit and the pre-exposure of synaptosomes to nicotine (30 μM) or to the α4β2-containing nAChR agonist 5IA85380 (10 nM) selectively inhibited the NMDA (100 μM)-evoked, but not the 4-aminopyridine (10 μM)-evoked, [(3)H] dopamine outflow; this inhibition was blunted by mecamylamine (10 μM). Nicotine and 5IA85380 pretreatment also inhibited the NMDA (100 μM)-evoked increase of calcium levels in single nerve terminals, an effect prevented by dihydro-β-erythroidine (1 μM). This supports a functional interaction between α4β2-containing nAChR and NMDA receptors within the same terminal, as supported by the immunocytochemical co-localization of α4 and GluN1 subunits in individual NAc dopaminergic terminals. The NMDA-evoked [(3)H]dopamine outflow was blocked by MK801 (1 μM) and inhibited by the selective GluN2B-selective antagonists ifenprodil (1 μM) and RO 25-6981 (1 μM), but not by the GluN2A-preferring antagonists CPP-19755 (1 μM) and ZnCl2 (1 nM). Notably, nicotine pretreatment significantly decreased the density of biotin-tagged GluN2B proteins in NAc synaptosomes. These results show that nAChRs dynamically and negatively regulate NMDA receptors in NAc dopaminergic terminals through the internalization of GluN2B receptors.

  17. 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.

  18. 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.

  19. Muscarinic receptor plasticity in the brain of senescent rats: down-regulation after repeated administration of diisopropyl fluorophosphate

    SciTech Connect

    Pintor, A.; Fortuna, S.; Volpe, M.T.; Michalek, H.

    1988-01-01

    Potential age-related differences in the response of Fischer 344 rats to subchronic treatment with diisopropylfluorophosphate (DFP) were evaluated in terms of brain cholinesterase (ChE) inhibition and muscarinic receptor sites. Male 3- and 24-month old rats were sc injected with sublethal doses of DFP for 2 weeks and killed 48 hrs after the last treatment. In the cerebral cortex, hippocampus and striatum of control rats a significant age-related reduction of ChE and of maximum number of /sup 3/H-QNB binding sites (Bmax) was observed. The administration of DFP to senescent rats resulted in more pronounced and longer lasting syndrome of cholinergic stimulation, with marked body weight loss and 60% mortality. The percentage inhibition of brain ChE induced by DFP did not differ between young and senescent rats. As expected, in young rats DFP caused a significant decrease of Bmax, which in the cerebral cortex reached about 40%. In the surviving senescent rats, the percentage decrease of Bmax due to DFP with respect to age-matched controls was very similar to that of young animals, especially in the cerebral cortex. Thus there is great variability in the response of aged rats to DFP treatment, from total failure of adaptive mechanisms resulting in death to considerable muscarinic receptor plasticity.

  20. Insulin-like growth factor type-1 receptor down-regulation associated with dwarfism in Holstein calves.

    PubMed

    Blum, J W; Elsasser, T H; Greger, D L; Wittenberg, S; de Vries, F; Distl, O

    2007-10-01

    Perturbations in endocrine functions can impact normal growth. Endocrine traits were studied in three dwarf calves exhibiting retarded but proportionate growth and four phenotypically normal half-siblings, sired by the same bull, and four unrelated control calves. Plasma 3,5,3'-triiodothyronine and thyroxine concentrations in dwarfs and half-siblings were in the physiological range and responded normally to injected thyroid-releasing hormone. Plasma glucagon concentrations were different (dwarfs, controls>half-siblings; P<0.05). Plasma growth hormone (GH), insulin-like growth factor-1 (IGF-1) and insulin concentrations in the three groups during an 8-h period were similar, but integrated GH concentrations (areas under concentration curves) were different (dwarfs>controls, P<0.02; half-siblings>controls, P=0.08). Responses of GH to xylazine and to a GH-releasing-factor analogue were similar in dwarfs and half-siblings. Relative gene expression of IGF-1, IGF-2, GH receptor (GHR), insulin receptor, IGF-1 type-1 and -2 receptors (IGF-1R, IGF-2R), and IGF binding proteins were measured in liver and anconeus muscle. GHR mRNA levels were different in liver (dwarfs

  1. 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

  2. Leucine Stimulates Insulin Secretion via Down-regulation of Surface Expression of Adrenergic α2A Receptor through the mTOR (Mammalian Target of Rapamycin) Pathway

    PubMed Central

    Yang, Jun; Dolinger, Michael; Ritaccio, Gabrielle; Mazurkiewicz, Joseph; Conti, David; Zhu, Xinjun; Huang, Yunfei

    2012-01-01

    The amino acid leucine is a potent secretagogue, capable of inducing insulin secretion. It also plays an important role in the regulation of mTOR activity, therefore, providing impetus to investigate if a leucine-sensing mechanism in the mTOR pathway is involved in insulin secretion. We found that leucine-induced insulin secretion was inhibited by both the mTOR inhibitor rapamycin as well as the adrenergic α2 receptor agonist clonidine. We also demonstrated that leucine down-regulated the surface expression of adrenergic α2A receptor via activation of the mTOR pathway. The leucine stimulatory effect on insulin secretion was attenuated in diabetic Goto-Kakizaki rats that overexpress adrenergic α2A receptors, confirming the role of leucine in insulin secretion. Thus, our data demonstrate that leucine regulates insulin secretion by modulating adrenergic α2 receptors through the mTOR pathway. The role of the mTOR pathway in metabolic homeostasis led us to a second important finding in this study; retrospective analysis of clinical data showed that co-administration of rapamycin and clonidine was associated with an increased incidence of new-onset diabetes in renal transplantation patients over those receiving rapamycin alone. We believe that inhibition of mTOR by rapamycin along with activation of adrenergic α2 receptors by clonidine represents a double-hit to pancreatic islets that synergistically disturbs glucose homeostasis. This new insight may have important implications for the clinical management of renal transplant patients. PMID:22645144

  3. Long-term nicotine treatment down-regulates α6β2* nicotinic receptor expression and function in nucleus accumbens.

    PubMed

    Perez, Xiomara A; McIntosh, J Michael; Quik, Maryka

    2013-12-01

    Long-term nicotine exposure induces alterations in dopamine transmission in nucleus accumbens that sustain the reinforcing effects of smoking. One approach to understand the adaptive changes that arise involves measurement of endogenous dopamine release using voltammetry. We therefore treated rats for 2-3 months with nicotine and examined alterations in nAChR subtype expression and electrically evoked dopamine release in rat nucleus accumbens shell, a region key in addiction. Long-term nicotine treatment selectively decreased stimulated α6β2* nAChR-mediated dopamine release compared with vehicle-treated rats. It also reduced α6β2* nAChRs, suggesting the receptor decline may contribute to the functional loss. This decreased response in release after chronic nicotine treatment was still partially sensitive to the agonist nicotine. Studies with an acetylcholinesterase inhibitor demonstrated that the response was also sensitive to increased endogenous acetylcholine. However, unlike the agonists, nAChR antagonists decreased dopamine release only in vehicle- but not nicotine-treated rats. As antagonists function by blocking the action of acetylcholine, their ineffectiveness suggests that reduced acetylcholine levels partly underlie the dampened α6β2* nAChR-mediated function in nicotine-treated rats. As long-term nicotine modifies dopamine release by decreasing α6β2* nAChRs and their function, these data suggest that interventions that target this subtype may be useful for treating nicotine dependence. Long-term nicotine treatment decreases dopamine (DA) transmission in the mesolimbic dopaminergic system. Our data suggest this may involve a decrease in α6β2* nicotinic receptor expression and function. These changes may play a key role in nicotine reward and dependence.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. 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

  9. 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

  10. Ciliary neurotrophic factor induces down-regulation of its receptor and desensitization of signal transduction pathways in vivo: non-equivalence with pharmacological activity.

    PubMed

    DiStefano, P S; Boulton, T G; Stark, J L; Zhu, Y; Adryan, K M; Ryan, T E; Lindsay, R M

    1996-09-13

    Despite the widespread use of polypeptide growth factors as pharmacological agents, little is known about the extent to which these molecules regulate their cognate cell surface receptors and signal transduction pathways in vivo. We have addressed this issue with respect to the neurotrophic molecule ciliary neurotrophic factor (CNTF). Administration of CNTF in vivo resulted in modest decreases in levels of CNTFRalpha mRNA and protein in skeletal muscle. CNTF causes the rapid tyrosine phosphorylation of LIFRbeta and gp130 and the induction of the immediate-early gene, tis11; injection of CNTF 3-7 h after an initial exposure failed to re-stimulate these immediate-early responses, suggesting a biochemical desensitization to CNTF not accounted for by decreased receptor protein. To determine whether the desensitization of immediate-early responses caused by CNTF resulted in a functional desensitization, we compared the efficacy of multiple daily injections versus a single daily dose of CNTF in preventing the denervation-induced atrophy of skeletal muscle. Surprisingly, injections of CNTF every 6 h, which falls within the putative refractory period for biochemical responses, resulted in efficacy equal to or greater than injections once daily. These results suggest that although much of the CNTF signal transduction machinery is down-regulated with frequent CNTF dosing, biological signals continue to be recognized and interpreted by the cell.

  11. Angiotensin-converting Enzyme Inhibition Down-regulates the Pro-atherogenic Chemokine Receptor 9 (CCR9)-Chemokine Ligand 25 (CCL25) Axis*

    PubMed Central

    Abd Alla, Joshua; Langer, Andreas; Elzahwy, Sherif S.; Arman-Kalcek, Gökhan; Streichert, Thomas; Quitterer, Ursula

    2010-01-01

    Many experimental and clinical studies suggest a relationship between enhanced angiotensin II release by the angiotensin-converting enzyme (ACE) and the pathophysiology of atherosclerosis. The atherosclerosis-enhancing effects of angiotensin II are complex and incompletely understood. To identify anti-atherogenic target genes, we performed microarray gene expression profiling of the aorta during atherosclerosis prevention with the ACE inhibitor, captopril. Atherosclerosis-prone apolipoprotein E (apoE)-deficient mice were used as a model to decipher susceptible genes regulated during atherosclerosis prevention with captopril. Microarray gene expression profiling and immunohistology revealed that captopril treatment for 7 months strongly decreased the recruitment of pro-atherogenic immune cells into the aorta. Captopril-mediated inhibition of plaque-infiltrating immune cells involved down-regulation of the C-C chemokine receptor 9 (CCR9). Reduced cell migration correlated with decreased numbers of aorta-resident cells expressing the CCR9-specific chemoattractant factor, chemokine ligand 25 (CCL25). The CCL25-CCR9 axis was pro-atherogenic, because inhibition of CCR9 by RNA interference in hematopoietic progenitors of apoE-deficient mice significantly retarded the development of atherosclerosis. Analysis of coronary artery biopsy specimens of patients with coronary artery atherosclerosis undergoing bypass surgery also showed strong infiltrates of CCR9-positive cells in atherosclerotic lesions. Thus, the C-C chemokine receptor, CCR9, exerts a significant role in atherosclerosis. PMID:20504763

  12. Down-regulation of endothelial protein C receptor shedding by persicarin and isorhamnetin-3-O-galactoside.

    PubMed

    Ku, Sae-Kwang; Han, Min-Su; Bae, Jong-Sup

    2013-07-01

    Increasing evidence has shown that beyond its role in coagulation, endothelial protein C receptor (EPCR) plays an important role in the cytoprotective pathway. Previous reports have shown that EPCR can be shed from the cell surface, and that this is mediated by tumor necrosis factor-α converting enzyme (TACE) and that sEPCR levels are increased in patients with systemic inflammatory diseases. Persicarin and isorhamnetin-3-O-galactoside (I3G) are active compounds from Oenanthe javanica, which has been widely studied for its neuroprotective, antioxidant, and barrier protective activities. However, little is known of the effects of persicarin on EPCR shedding. Here, we investigated this issue by monitoring the effects of persicarin and I3G on phorbol-12-myristate 13-acetate (PMA) and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. According to the results, persicarin and I3G induced potent inhibition of PMA and CLP-induced EPCR shedding by suppressing expression of TACE. In addition, persicarin and I3G reduced PMA-stimulated phosphorylation of p38MAPK, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). Given these results, persicarin and I3G could be used as a candidate therapeutic for treatment of severe vascular inflammatory diseases.

  13. Inhibition of mouse mammary ductal morphogenesis and down-regulation of the EGF receptor by epidermal growth factor.

    PubMed

    Coleman, S; Daniel, C W

    1990-02-01

    EGF, initially demonstrated to be a potent mitogen for a variety of cell types, has more recently been shown to inhibit proliferation of several cell lines. Few studies, however, have addressed the effects of EGF on growth and morphogenesis of tissues in vivo, particularly with regard to EGF as a possible inhibitor. We now demonstrate that EGF treatment of vigorously growing mammary ducts, administered directly to the glands by slow release plastic implants, inhibited normal ductal growth. Inhibition was restricted to the region around the implant and untreated glands in the same animal were normal, indicating direct effects of EGF. EGF-treated end buds were smaller and demonstrated reduced levels of DNA synthesis, although remnants of a stem (cap) cell layer persisted. Full inhibition of growth occurred within 3 days of implantation and required extended exposure to EGF, since treatment of 5 hr or less had no effect on ductal growth. At the lower inhibitory doses tested, growth resumed within 8 days, indicating reversibility of inhibition. No lobuloalveolar or hyperplastic response was seen. 125I-EGF autoradiography revealed that ductal growth inhibition was preceded by the disappearance of EGF receptors located in the cap cell layer of the end bud epithelium and in stromal cells adjacent to the buds. These results, in conjunction with our previous evidence demonstrating the growth-stimulatory effect by EGF on nonproliferating mammary ducts, suggest a growth regulatory role for EGF in mouse mammary ductal morphogenesis.

  14. 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.

  15. Statins decrease vascular epithelial growth factor expression via down-regulation of receptor for advanced glycation end-products.

    PubMed

    Tsujinaka, Hiroki; Itaya-Hironaka, Asako; Yamauchi, Akiyo; Sakuramoto-Tsuchida, Sumiyo; Shobatake, Ryogo; Makino, Mai; Masuda, Naonori; Hirai, Hiromasa; Takasawa, Shin; Ogata, Nahoko

    2017-09-01

    Statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, possess pleiotropic effects that have been extended to modulation of various cellular behaviors. This study aimed to examine whether statins modulate vascular endothelial growth factor A (VEGF-A) expression in human retinal pigment epithelium (RPE) cells. Human RPE cells (h1RPE7), damaged by hydroquinone (HQ) + advanced glycation endproducts (AGE) in an in vitro AMD model, were treated with atorvastatin or lovastatin for 24 h. The expression of VEGF-A and receptor for AGE (RAGE) was evaluated by real-time RT-PCR. VEGF-A secretion was measured by ELISA. To investigate the impact of RAGE on VEGF-A expression, small interfering RNA (siRNA) for RAGE (siRAGE) was introduced into h1RPE7 cells and VEGF-A expression was measured by real-time RT-PCR. Deletions of VEGF-A and RAGE promoters were performed and transcriptional activities were measured after the addition of statins to HQ + AGE-damaged RPE cells. The mRNA levels of VEGF-A and RAGE and the levels of VEGF-A in the culture medium were increased by HQ + AGE. Both atorvastatin and lovastatin attenuated HQ + AGE-induced VEGF-A and RAGE expression. These statins also decreased VEGF-A levels in the culture medium. RNA interference of RAGE attenuated the up-regulation of VEGF-A in the HQ + AGE treated cells. The deletion analysis demonstrated that these statins attenuated RAGE promoter activation in HQ + AGE-damaged RPE cells. Statins attenuated HQ + AGE-induced VEGF expression by decreasing RAGE expression. As VEGF is an important factor in developing wet AMD, statins could decrease the risk of wet-type AMD and be used as preventive medicines.

  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. 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

  19. 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.

  20. Thyroid hormone receptors are down-regulated in skeletal muscle of patients with non-thyroidal illness syndrome secondary to non-septic shock.

    PubMed

    Lado-Abeal, J; Romero, A; Castro-Piedras, I; Rodriguez-Perez, A; Alvarez-Escudero, J

    2010-11-01

    Non-thyroidal illness syndrome (NTIS) is related to changes in thyroid hormone (TH) physiology. Skeletal muscle (SM) plays a major role in metabolism, and TH regulates SM phenotype and metabolism. We aimed to characterize the SM of non-septic shock NTIS patients in terms of: i) expression of genes and proteins involved in TH metabolism and actions; and ii) NFKB's pathway activation, a responsible factor for some of the phenotypic changes in NTIS. We also investigated whether the patient's serum can induce in vitro the effects observed in vivo. Serum samples and SM biopsies from 14 patients with non-septic shock NTIS and 11 controls. Gene and protein expression and NFKB1 activation were analyzed by quantitative PCR and immunoblotting. Human SM cell (HSkMC) cultures to investigate the effects of patient's serum on TH action mediators. Patients with non-septic shock NTIS showed higher levels of pro-inflammatory cytokines than controls. Expression of TRβ (THRB), TRα1 (THRA), and retinoid X receptor γ (RXRG) was decreased in NTIS patients. RXRA gene expression was higher, but its protein was lower in NTIS than controls, suggesting the existence of a post-transcriptional mechanism that down-regulates protein levels. NFKB1 pathway activation was not different between NTIS and control patients. HSkMC incubated with patient's serum increased TH receptor and RXRG gene expression after 48  h. Patients with non-septic shock NTIS showed decreased expression of TH receptors and RXRs, which were not related to increased activation of the NFKB1 pathway. These findings could not be replicated in cultures of HSkMCs incubated in the patient's serum.

  1. LOT1 is a growth suppressor gene down-regulated by the epidermal growth factor receptor ligands and encodes a nuclear zinc-finger protein.

    PubMed

    Abdollahi, A; Bao, R; Hamilton, T C

    1999-11-11

    We previously reported cloning the rLot1 gene, and its human homolog (hLOT1), through analysis of differential gene expression in normal and malignant rat ovarian surface epithelial cells. Both human and rat ovarian carcinoma cell lines exhibited lost or decreased expression of this gene. Interestingly, the LOT1 gene localized at band q25 of human chromosome 6 which is a frequent site for LOH in many solid tumors including ovarian cancer. In this report we have further characterized the potential role of LOT1 in malignant transformation and developed evidence that the gene is a novel target of growth factor signaling pathway. Assays using transient transfections showed that LOT1 is a nuclear protein and may act as a transcription factor. In vitro and in vivo studies involving ovarian cancer cell lines revealed that expression of LOT1 is directly associated with inhibition of cellular proliferation and induction of morphological transformations. Additionally, we show that in normal rat ovarian surface epithelial cells Lot1 gene expression is responsive to growth factor stimulation. Its mRNA is strongly down-regulated by epidermal growth factor receptor (EGFR) ligands, namely EGF and TGF-alpha. Blocking the ligand-activated EGFR signal transduction pathway by the specific EGF receptor inhibitor, tyrphostin AG1478, and the MEK inhibitor, PD098059, restores the normal level of Lot1 gene expression. It appears that the regulation of Lot1 gene is unique to these ligands, as well as the growth promoting agent TPA, since other factors either did not affect Lot1 expression, or the effect was modest and transient. Altogether, the results suggest that Lot1 expression is primarily mediated via EGF receptor or a related pathway and it may regulate the growth promoting signals as a zinc-finger motif containing nuclear transcription factor.

  2. 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.

  3. Protein tyrosine phosphatase non-receptor 22 and C-Src tyrosine kinase genes are down-regulated in patients with rheumatoid arthritis.

    PubMed

    Remuzgo-Martínez, Sara; Genre, Fernanda; Castañeda, Santos; Corrales, Alfonso; Moreno-Fresneda, Pablo; Ubilla, Begoña; Mijares, Verónica; Portilla, Virginia; González-Vela, Jesús; Pina, Trinitario; Ocejo-Vinyals, Gonzalo; Irure-Ventura, Juan; Blanco, Ricardo; Martín, Javier; Llorca, Javier; López-Mejías, Raquel; González-Gay, Miguel A

    2017-09-05

    Several protein tyrosine phosphatase non-receptor 22 (PTPN22) single-nucleotide polymorphisms (SNPs) have been significantly related with rheumatoid arthritis (RA) susceptibility. Nevertheless, its potential influence on PTPN22 expression in RA has not been completely elucidated. Furthermore, PTPN22 binds to C-Src tyrosine kinase (CSK) forming a key complex in autoimmunity. However, the information of CSK gene in RA is scarce. In this study, we analyzed the relative PTPN22 and CSK expression in peripheral blood from 89 RA patients and 43 controls to determine if the most relevant PTPN22 (rs2488457, rs2476601 and rs33996649) and CSK (rs34933034 and rs1378942) polymorphisms may influence on PTPN22 and CSK expression in RA. The association between PTPN22 and CSK expression in RA patients and their clinical characteristics was also evaluated. Our study shows for the first time a marked down-regulation of PTPN22 expression in RA patients carrying the risk alleles of PTPN22 rs2488457 and rs2476601 compared to controls (p = 0.004 and p = 0.007, respectively). Furthermore, CSK expression was significantly lower in RA patients than in controls (p < 0.0001). Interestingly, a reduced PTPN22 expression was disclosed in RA patients with ischemic heart disease (p = 0.009). The transcriptional suppression of this PTPN22/CSK complex may have a noteworthy clinical relevance in RA patients.

  4. Down-Regulation of Ca2+-Activated K+ Channel KCa1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists

    PubMed Central

    Khatun, Anowara; Fujimoto, Mayu; Kito, Hiroaki; Niwa, Satomi; Suzuki, Takayoshi; Ohya, Susumu

    2016-01-01

    Vitamin D (VD) reduces the risk of breast cancer and improves disease prognoses. Potential VD analogs are being developed as therapeutic agents for breast cancer treatments. The large-conductance Ca2+-activated K+ channel KCa1.1 regulates intracellular Ca2+ signaling pathways and is associated with high grade tumors and poor prognoses. In the present study, we examined the effects of treatments with VD receptor (VDR) agonists on the expression and activity of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, flow cytometry, and voltage-sensitive dye imaging. Treatments with VDR agonists for 72 h markedly decreased the expression levels of KCa1.1 transcripts and proteins in MDA-MB-453 cells, resulting in the significant inhibition of depolarization responses induced by paxilline, a specific KCa1.1 blocker. The specific proteasome inhibitor MG132 suppressed VDR agonist-induced decreases in KCa1.1 protein expression. These results suggest that KCa1.1 is a new downstream target of VDR signaling and the down-regulation of KCa1.1 through the transcriptional repression of KCa1.1 and enhancement of KCa1.1 protein degradation contribute, at least partly, to the antiproliferative effects of VDR agonists in breast cancer cells. PMID:27973439

  5. Down-regulation of vitamin D receptor in mammospheres: implications for vitamin D resistance in breast cancer and potential for combination therapy.

    PubMed

    Pervin, Shehla; Hewison, Martin; Braga, Melissa; Tran, Lac; Chun, Rene; Karam, Amer; Chaudhuri, Gautam; Norris, Keith; Singh, Rajan

    2013-01-01

    Vitamin D signaling in mammary cancer stem cells (MCSCs), which are implicated in the initiation and progression of breast cancer, is poorly understood. In this study, we examined vitamin D signaling in mammospheres which are enriched in MCSCs from established breast cancer cell lines. Breast cancer cells positive for aldehyde dehydrogenase (ALDH(+)) had increased ability to form mammospheres compared to ALDH(-) cells. These mammospheres expressed MCSC-specific markers and generated transplantable xenografts in nude mice. Vitamin D receptor (VDR) was significantly down-regulated in mammospheres, as well as in ALDH(+) breast cancer cells. TN aggressive human breast tumors as well as transplantable xenografts obtained from SKBR3 expressed significantly lower levels of VDR but higher levels of CD44 expression. Snail was up-regulated in mammospheres isolated from breast cancer cells. Inhibition of VDR expression by siRNA led to a significant change in key EMT-specific transcription factors and increased the ability of these cells to form mammospheres. On the other hand, over-expression of VDR led to a down-regulation of Snail but increased expression of E-cad and significantly compromised the ability of cells to form mammospheres. Mammospheres were relatively insensitive to treatment with 1,25-dihydroxyvitamin D (1,25D), the active form of vitamin D, compared to more differentiated cancer cells grown in presence of serum. Treatment of H-Ras transformed HMLE(HRas) cells with DETA NONOate, a nitric oxide (NO)-donor led to induction of MAP-kinase phosphatase -1 (MKP-1) and dephosphorylation of ERK1/2 in the mammospheres. Combined treatment of these cells with 1,25D and a low-concentration of DETA NONOate led to a significant decrease in the overall size of mammospheres and reduced tumor volume in nude mice. Our findings therefore, suggest that combination therapy using 1,25D with drugs specifically targeting key survival pathways in MCSCs warrant testing in prospective

  6. Down-Regulation of Vitamin D Receptor in Mammospheres: Implications for Vitamin D Resistance in Breast Cancer and Potential for Combination Therapy

    PubMed Central

    Pervin, Shehla; Hewison, Martin; Braga, Melissa; Tran, Lac; Chun, Rene; Karam, Amer; Chaudhuri, Gautam; Norris, Keith; Singh, Rajan

    2013-01-01

    Vitamin D signaling in mammary cancer stem cells (MCSCs), which are implicated in the initiation and progression of breast cancer, is poorly understood. In this study, we examined vitamin D signaling in mammospheres which are enriched in MCSCs from established breast cancer cell lines. Breast cancer cells positive for aldehyde dehydrogenase (ALDH+) had increased ability to form mammospheres compared to ALDH− cells. These mammospheres expressed MCSC-specific markers and generated transplantable xenografts in nude mice. Vitamin D receptor (VDR) was significantly down-regulated in mammospheres, as well as in ALDH+ breast cancer cells. TN aggressive human breast tumors as well as transplantable xenografts obtained from SKBR3 expressed significantly lower levels of VDR but higher levels of CD44 expression. Snail was up-regulated in mammospheres isolated from breast cancer cells. Inhibition of VDR expression by siRNA led to a significant change in key EMT-specific transcription factors and increased the ability of these cells to form mammospheres. On the other hand, over-expression of VDR led to a down-regulation of Snail but increased expression of E-cad and significantly compromised the ability of cells to form mammospheres. Mammospheres were relatively insensitive to treatment with 1,25-dihydroxyvitamin D (1,25D), the active form of vitamin D, compared to more differentiated cancer cells grown in presence of serum. Treatment of H-Ras transformed HMLEHRas cells with DETA NONOate, a nitric oxide (NO)-donor led to induction of MAP-kinase phosphatase -1 (MKP-1) and dephosphorylation of ERK1/2 in the mammospheres. Combined treatment of these cells with 1,25D and a low-concentration of DETA NONOate led to a significant decrease in the overall size of mammospheres and reduced tumor volume in nude mice. Our findings therefore, suggest that combination therapy using 1,25D with drugs specifically targeting key survival pathways in MCSCs warrant testing in prospective clinical

  7. Down-regulation of P2U-purinergic nucleotide receptor messenger RNA expression during in vitro differentiation of human myeloid leukocytes by phorbol esters or inflammatory activators.

    PubMed

    Martin, K A; Kertesy, S B; Dubyak, G R

    1997-01-01

    HL-60 human promyelocytic leukocytes express G protein-coupled P2U-purinergic nucleotide receptors (P2UR or P2Y2R) that activate inositol phospholipid hydrolysis and Ca24 mobilization in response to ATP or UTP. We examined the expression of functional P2UR and P2UR mRNA levels during in vitro differentiation of HL-60 cells by dibutyryl-cAMP (Bt2cAMP), which induces a granulocyte/neutrophil phenotype, or by phorbol-12-myristate-13-acetate (PMA), which induces a monocyte/macrophage phenotype. Both P2UR function and P2UR mRNA levels were only modestly attenuated during granulocytic differentiation by Bt2cAMP. In contrast, P2UR function, as assayed by either Ca2+ mobilization or inositol trisphosphate generation, was greatly reduced in PMA-differentiated cells. This inhibition of P2UR function was strongly correlated with PMA-induced decreases in P2UR mRNA levels, as assayed by Northern blot analysis or reverse transcription-polymerase chain reaction-based quantification. Although PMA induced an early, transient up-regulation of P2UR mRNA, this was rapidly followed by a sustained decrease in P2UR mRNA to a level 5-10-fold lower than that in undifferentiated HL-60 cells. The half-life of the P2UR transcript in HL-60 cells was approximately 60 min, and this was not affected by acute exposure (< or = 4 hr) to Bt2cAMP or PMA. PMA down-regulated P2UR mRNA in THP-1 monocytes and HL-60 granulocytes but not in A431 human epithelial cells or human keratinocytes. P2UR mRNA was also down-regulated in THP-1 monocytes differentiated into inflammatory macrophages by gamma-interferon and endotoxin. These data indicate that myeloid leukocytes possess tissue-specific mechanisms for the rapid modulation of P2UR expression and function during differentiation and inflammatory activation.

  8. miR-223 contributes to the AGE-promoted apoptosis via down-regulating insulin-like growth factor 1 receptor in osteoblasts

    PubMed Central

    Qin, Yi; Ye, Jichao; Wang, Peng; Gao, Liangbin; Wang, Suwei; Shen, Huiyong

    2016-01-01

    Advanced glycation end products (AGEs) have been confirmed to induce bone quality deterioration in diabetes mellitus (DM), and to associate with abnormal expression of miRNAs in DM patients or in vitro. Recently, miRNAs have been recognized to mediate the onset or progression of DM. In the present study, we investigated the regulation on miR-223 level by AGE-BSA treatment in osteoblast-like MC3T3-E1 cells, with real-time quantitative PCR assay. And then we examined the inhibition of insulin-like growth factor 1 receptor (IGF-1R) expression by miR-223, via targeting of the 3′ UTR of IGF-1R with real-time quantitative PCR, western blotting and luciferase reporter assay. Then we explored the regulation of miR-223 and IGF-1R levels, via the lentivirus-mediated miR-223 inhibition and IGF-1R overexpression in the AGE-BSA-induced apoptosis in MC3T3-E1 cells. It was demonstrated that AGE-BSA treatment with more than 100 μg/ml significantly up-regulated miR-223 level, whereas down-regulated IGF-1R level in MC3T3-E1 cells. And the up-regulated miR-223 down-regulated IGF-1R expression in both mRNA and protein levels, via targeting the 3′ UTR of IGF-1R. Moreover, though the AGE-BSA treatment promoted apoptosis in MC3T3-E1 cells, the IGF-1R overexpression or the miR-223 inhibition significantly attenuated the AGE-BSA-promoted apoptosis in MC3T3-E1 cells. In summary, our study recognized the promotion of miR-223 level by AGE-BSA treatment in osteoblast-like MC3T3-E1 cells. The promoted miR-223 targeted IGF-1R and mediated the AGE-BSA-induced apoptosis in MC3T3-E1 cells. It implies that miR-223 might be an effective therapeutic target to antagonize the AGE-induced damage to osteoblasts in DM. PMID:26893485

  9. 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

  10. Endothelin-1 down-regulates matrix metalloproteinase 14 and 15 expression in human first trimester trophoblasts via endothelin receptor type B

    PubMed Central

    Majali-Martinez, Alejandro; Velicky, Philipp; Pollheimer, Jürgen; Knöfler, Martin; Yung, Hong wa; Burton, Graham J.; Tabrizi-Wizsy, Nassim Ghaffari; Lang, Uwe; Hiden, Ursula; Desoye, Gernot; Dieber-Rotheneder, Martina

    2017-01-01

    STUDY QUESTION Does endothelin-1 (ET-1) regulate matrix metalloproteinase (MMP) 14 and 15 production and invasion of human first trimester trophoblasts? SUMMARY ANSWER ET-1 in pathophysiological concentrations down-regulates MMP14 and MMP15 expression via endothelin receptor (ETR) type B and decreases trophoblast migration and invasion. WHAT IS KNOWN ALREADY MMP14 and MMP15 are involved in trophoblast invasion. Impairment of invasion has been linked to pregnancy complications such as pre-eclampsia (PE). ET-1 is up-regulated in PE. STUDY DESIGN, SIZE, DURATION In vitro study using primary human trophoblasts from 50 first trimester placentas (gestational week 7–12). PARTICIPANTS/MATERIALS, SETTING, METHODS Trophoblasts were cultured in the absence or presence of 10–100 nM ET-1. MMP14 and MMP15 mRNA and protein were quantified by RT-qPCR and Western blotting, respectively. Selective antagonists for ETRA (BQ-123) or ETRB (BQ-788) were used to identify ETR subtypes involved. Functional ET-1 effects were tested in first trimester chorionic villous explants and transwell invasion assays. The roles of tumor necrosis factor (TNF)-α (25 ng/ml) and oxygen (1%) in ET-1 regulation of MMP14 and 15 expression were assessed by Western blotting. MAIN RESULTS AND THE ROLE OF CHANCE ET-1 down-regulated MMP14 and MMP15 mRNA (−21% and −26%, respectively, P < 0.05) and protein levels (–18% and –22%, respectively, P < 0.05). This effect was mediated via ETRB. ET-1 decreased trophoblast outgrowth in placental explants (−24%, P < 0.05) and trophoblast invasion (−26%, P ≤ 0.01). TNF-α enhanced ET-1 mediated MMP15 down-regulation (by 10%, P < 0.05), whereas hypoxia abolished the effect of ET-1 on both MMPs. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Only primary trophoblasts were used in this study. Since trophoblast yield from first trimester placental material is limited, further aspects of MMP14 and 15 regulation could not be characterized. Other anti

  11. 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

  12. 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).

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. Four-and-a-half-LIM protein 1 down-regulates estrogen receptor α activity through repression of AKT phosphorylation in human breast cancer cell.

    PubMed

    Zhang, Fan; Feng, Fan; Yang, Pingxun; Li, Zijian; You, Junhao; Xie, Wenxiu; Gao, Xudong; Yang, Junlan

    2012-02-01

    The Four-and-a-half LIM protein 1 (FHL-1) is a member of LIM-only protein family. It plays important roles in proliferation and apoptosis regulation of certain hepatocellular carcinoma and human breast cancer. Estrogen receptor α (ERα) is involved in the development and progression of human breast cancer. IGF/PI3K/AKT signaling pathway also plays certain roles in the program and regulation of human breast cancer and ovary cancer. However, the biological function of FHL-1 in regulation of human breast cancer and in the cross-talk of estrogen and IGF signaling pathway remains largely unknown. In this paper, we show that FHL-1 protein interacts with ERα and AKT. FHL-1 represses the translation and transcription of estrogen receptor-responsive genes through down-regulating AKT activation. In addition, FHL-1 is not only an ERα-interacting co-regulation protein, but also decreases the phosphorylation of AKT and ERα. Depression of endogenous FHL-1 by FHL-1 targeted small interfering RNA enhances the expression of these proteins and phosphorylation of AKT and ERα. These data suggest that FHL-1 may regulate ER signaling function through regulation of AKT activation besides the physical and functional interaction with ERα. By establishing a linkage role of the FHL-1 between the estrogen ERα signaling pathway and IGF/PI3K/AKT signaling pathway, this study identifies that FHL-1 proteins may be a useful molecular target for human breast cancer therapy. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. 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).

  1. 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.

  2. 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

  3. MicroRNA-mediated GABA Aα-1 receptor subunit down-regulation in adult spinal cord following neonatal cystitis-induced chronic visceral pain in rats.

    PubMed

    Sengupta, Jyoti N; Pochiraju, Soumya; Pochiraju, Soumiya; Kannampalli, Pradeep; Bruckert, Mitchell; Addya, Sankar; Yadav, Priyanka; Miranda, Adrian; Shaker, Reza; Banerjee, Banani

    2013-01-01

    The nociceptive transmission under pathological chronic pain conditions involves transcriptional and/or translational alteration in spinal neurotransmitters, receptor expressions, and modification of neuronal functions. Studies indicate the involvement of microRNA (miRNA) - mediated transcriptional deregulation in the pathophysiology of acute and chronic pain. In the present study, we tested the hypothesis that long-term cross-organ colonic hypersensitivity in neonatal zymosan-induced cystitis is due to miRNA-mediated posttranscriptional suppression of the developing spinal GABAergic system. Cystitis was produced by intravesicular injection of zymosan (1% in saline) into the bladder during postnatal (P) days P14 through P16 and spinal dorsal horns (L6-S1) were collected either on P60 (unchallenged groups) or on P30 after a zymosan re-challenge on P29 (re-challenged groups). miRNA arrays and real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed significant, but differential, up-regulation of mature miR-181a in the L6-S1 spinal dorsal horns from zymosan-treated rats compared with saline-treated controls in both the unchallenged and re-challenged groups. The target gene analysis demonstrated multiple complementary binding sites in miR-181a for GABA(A) receptor subunit GABA(Aα-1) gene with a miRSVR score of -1.83. An increase in miR-181a concomitantly resulted in significant down-regulation of GABA(Aα-1) receptor subunit gene and protein expression in adult spinal cords from rats with neonatal cystitis. Intrathecal administration of the GABA(A) receptor agonist muscimol failed to attenuate the viscero-motor response (VMR) to colon distension in rats with neonatal cystitis, whereas in adult zymosan-treated rats the drug produced significant decrease in VMR. These results support an integral role for miRNA-mediated transcriptional deregulation of the GABAergic system in neonatal cystitis-induced chronic pelvic pain. Copyright © 2012 International

  4. Down-regulation of muscarinic receptors and the m3 subtype in white-footed mice by dietary exposure to parathion

    USGS Publications Warehouse

    Jett, D.A.; Hill, E.F.; Fernando, J.C.; Eldefrawi, M.E.; Eldefrawi, A.T.

    1993-01-01

    The effect of ad libitum dietary exposure (as occurs in the field) to parathion for 14 d was investigated on the muscarinic acetylcholine receptor (mAChR) in brains and submaxillary glands of adults of a field species, the white-footed mouse Peromyscus leucopus. Immunoprecipitation using subtype selective antibodies revealed that the relative ratios of the m1-m5 mAChR subtypes in Peromyscus brain were similar to those in rat brain. There was little variability in acetylcholinesterase (AChE) activity in control mice brains but large variability in 39 exposed mice, resulting from differences in food ingestion and parathion metabolism. Accordingly, data on radioligand binding to mAChRs in each mouse brain were correlated with brain AChE activity in the same mouse, and AChE inhibition served as a biomarker of exposure reflecting in situ paraoxon concentrations. Exposure to parathion for 14 d reduced maximal binding (Bmax) of [3H]quinuclidinyl benzilate ([3H]QNB), [3H]-N-methylscopolamine ([3H]NMS), and [3H]-4-diphenylacetoxy-N-methylpiperidine methiodide ([3H]-4-DAMP) by up to approximately 58% without affecting receptor affinities for these ligands. Maximal reduction in Bmax of [3H]QNB and [3H]-4-DAMP binding occurred in mice with highest AChE inhibition, while equivalent maximal reduction in Bmax of [3H]NMS occurred in mice with only approximately 10% AChE inhibition, without further change at higher parathion doses. This is believed to be due to the hydrophilicity of [3H]NMS, which limits its accessibility to internalized desensitized receptors. In submaxillary glands (mAChRs are predominantly m3 subtype), there were significant dose-dependent reductions in [3H]QNB binding and m3 mRNA levels in exposed mice, revealed by Northern blot analyses. The reduction in m3 receptors is suggested to result mostly from reduced synthesis at the transcription level, rather than from translational or posttranslational events. The data suggest that down-regulation of mAChRs occurs

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. Impaired recovery of brain muscarinic receptor sites following an adaptive down-regulation induced by repeated administration of diisopropyl fluorophosphate in aged rats

    SciTech Connect

    Pintor, A.; Fortuna, S.; De Angelis, S.; Michalek, H. )

    1990-01-01

    Potential age-related differences in the recovery rate of brain cholinesterase activity (ChE) and muscarinic acetylcholine receptor binding sites (mAChRs) following reduction induced by repeated treatment with diisopropyl fluorophosphate (DFP) were evaluated in Sprague-Dawley rats. Male 3- and 24-month old rats were s.c. injected with DFP on alternate days for 2 weeks and killed 48 hr and 7, 14, 21, 28 and 35 days after the last treatment. In the hippocampus and striatum, but not in the cerebral cortex, of control rats there as a significant age-related decline of ChE activity and maximal density of 3H-QNB binding sites (Bmax). The repeated administration of DFP during the first week caused a syndrome of cholinergic stimulation both in aged and young rats. The syndrome was more pronounced, in terms of intensity and duration in aged than in young animals resulting in 40 and 12% mortality, respectively; during the second week the syndrome attenuated in the two age-groups. The percentage inhibition of brain ChE at the end of DFP treatment did not differ between young and surviving aged rats. The down-regulation of mACRs was present in the three brain regions of both young and age rats (from 20 to 40%). Factorial analysis of variance showed significant differences for age, recovery rate, and significant interaction between age and recovery rate, both for ChE and mAChRs in young rats the three brain areas.

  10. Estrogen regulates Snail and Slug in the down-regulation of E-cadherin and induces metastatic potential of ovarian cancer cells through estrogen receptor alpha.

    PubMed

    Park, Se-Hyung; Cheung, Lydia W T; Wong, Alice S T; Leung, Peter C K

    2008-09-01

    Tumorigenesis is a multistep process involving dysregulated cell growth and metastasis. Considerable evidence implicates a mitogenic action of estrogen in early ovarian carcinogenesis. In contrast, its influence in the metastatic cascade of ovarian tumor cells remains obscure. In the present study, we showed that 17beta-estradiol (E2) increased the metastatic potential of human epithelial ovarian cancer cell lines. E2 treatment led to clear morphological changes characteristic of epithelial-mesenchymal transition (EMT) and an enhanced cell migratory propensity. These morphological and functional alterations were associated with changes in the abundance of EMT-related genes. Upon E2 stimulation, expression and promoter activity of the epithelial marker E-cadherin were strikingly suppressed, whereas EMT-associated transcription factors, Snail and Slug, were significantly up-regulated. This up-regulation was attributed to the increase in gene transcription activated by E2. Depletion of endogenous Snail or Slug using small interfering RNA (siRNA) attenuated E2-mediated decrease in E-cadherin. In addition, E2-induced cell migration was also neutralized by the siRNAs, suggesting that both transcription factors are indispensable for the prometastatic actions of E2. More importantly, by using selective estrogen receptor (ER) agonists, forced expression, and siRNA approaches, we identified that E2 triggered the metastatic behaviors exclusively through an ERalpha-dependent pathway. We also showed that ERbeta had an opposing action on ERalpha because the presence of ERbeta completely inhibited the EMT and down-regulation of E-cadherin induced by ERalpha. Collectively, this study provides a compelling argument that estrogen can potentiate tumor progression by EMT induction and highlights the crucial role of ERalpha in ovarian tumorigenesis.

  11. The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces apoptosis, down-regulates the CXCR4 chemokine receptor and impairs migration of chronic lymphocytic leukemia cells

    PubMed Central

    Stamatopoulos, Basile; Meuleman, Nathalie; De Bruyn, Cécile; Delforge, Alain; Bron, Dominique; Lagneaux, Laurence

    2010-01-01

    Background Chronic lymphocytic leukemia is a neoplastic disorder that arises largely as a result of defective apoptosis leading to chemoresistance. Stromal cell-derived factor-1 and its receptor, CXCR4, have been shown to play an important role in chronic lymphocytic leukemia cell trafficking and survival. Design and Methods Since histone acetylation is involved in the modulation of gene expression, we evaluated the effects of suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, on chronic lymphocytic leukemia cells and in particular on cell survival, CXCR4 expression, migration, and drug sensitization. Results Here, we showed that treatment with suberoylanilide hydroxamic acid (20 μM) for 48 hours induced a decrease in chronic lymphocytic leukemia cell viability via apoptosis (n=20, P=0.0032). Using specific caspase inhibitors, we demonstrated the participation of caspases-3, -6 and -8, suggesting an activation of the extrinsic pathway. Additionally, suberoylanilide hydroxamic acid significantly decreased CXCR4 mRNA (n=10, P=0.0010) and protein expression (n=40, P<0.0001). As a result, chronic lymphocytic leukemia cell migration in response to stromal cell-derived factor-1 (n=23, P<0.0001) or through bone marrow stromal cells was dramatically impaired. Consequently, suberoylanilide hydroxamic acid reduced the protective effect of the microenvironment and thus sensitized chronic lymphocytic leukemia cells to chemotherapy such as fludarabine. Conclusions In conclusion, suberoylanilide hydroxamic acid induces apoptosis in chronic lymphocytic leukemia cells via the extrinsic pathway and down-regulates CXCR4 expression leading to decreased cell migration. Suberoylanilide hydroxamic acid in combination with other drugs represents a promising therapeutic approach to inhibiting migration, chronic lymphocytic leukemia cell survival and potentially overcoming drug resistance. PMID:20145270

  12. Dietary sodium deprivation evokes activation of brain regional neurons and down-regulation of angiotensin II type 1 receptor and angiotensin-convertion enzyme mRNA expression.

    PubMed

    Lu, B; Yang, X J; Chen, K; Yang, D J; Yan, J Q

    2009-12-15

    Previous studies have indicated that the renin-angiotensin-aldosterone system (RAAS) is implicated in the induction of sodium appetite in rats and that different dietary sodium intakes influence the mRNA expression of central and peripheral RAAS components. To determine whether dietary sodium deprivation activates regional brain neurons related to sodium appetite, and changes their gene expression of RAAS components of rats, the present study examined the c-Fos expression after chronic exposure to low sodium diet, and determined the relationship between plasma and brain angiotensin I (ANG I), angiotensin II (ANG II) and aldosterone (ALD) levels and the sodium ingestive behavior variations, as well as the effects of prolonged dietary sodium deprivation on ANG II type 1 (AT1) and ANG II type 2 (AT2) receptors and angiotensin-convertion enzyme (ACE) mRNA levels in the involved brain regions using the method of real-time polymerase chain reaction (PCR). Results showed that the Fos immunoreactivity (Fos-ir) expression in forebrain areas such as subfornical organ (SFO), paraventricular hypothalamic nuclei (PVN), supraoptic nucleus (SON) and organum vasculosum laminae terminalis (OVLT) all increased significantly and that the levels of ANG I, ANG II and ALD also increased in plasma and forebrain in rats fed with low sodium diet. In contrast, AT1, ACE mRNA in PVN, SON and OVLT decreased significantly in dietary sodium depleted rats, while AT2 mRNA expression did not change in the examined areas. These results suggest that many brain areas are activated by increased levels of plasma and/or brain ANG II and ALD, which underlies the elevated preference for hypertonic salt solution after prolonged exposure to low sodium diet, and that the regional AT1 and ACE mRNA are down-regulated after dietary sodium deprivation, which may be mediated by increased ANG II in plasma and/or brain tissue.

  13. The Neuropeptide Y Y1 Receptor: A Diagnostic Marker? Expression in MCF-7 Breast Cancer Cells Is Down-Regulated by Antiestrogens In Vitro and in Xenografts

    PubMed Central

    Memminger, Martin; Keller, Max; Lopuch, Miroslaw; Pop, Nathalie; Bernhardt, Günther; von Angerer, Erwin; Buschauer, Armin

    2012-01-01

    The neuropeptide Y (NPY) Y1 receptor (Y1R) has been suggested as a tumor marker for in vivo imaging and as a therapeutic target. In view of the assumed link between estrogen receptor (ER) and Y1R in mammary carcinoma and with respect to the development of new diagnostic tools, we investigated the Y1R protein expression in human MCF-7 cell variants differing in ER content and sensitivity against antiestrogens. ER and Y1R expression were quantified by radioligand binding using [3H]-17β-estradiol and the Y1R selective antagonist [3H]-UR-MK114, respectively. The latter was used for cellular binding studies and for autoradiography of MCF-7 xenografts. The fluorescent ligands Cy5-pNPY (universal Y1R, Y2R and Y5R agonist) and UR-MK22 (selective Y1R antagonist), as well as the selective antagonists BIBP3226 (Y1R), BIIE0246 (Y2R) and CGP71683 (Y5R) were used to identify the NPY receptor subtype(s) by confocal microscopy. Y1R functionality was determined by mobilization of intracellular Ca2+. Sensitivity of MCF-7 cells against antiestrogen 4-hydroxytamoxifen correlated directly with the ER content. The exclusive expression of Y1Rs was confirmed by confocal microscopy. The Y1R protein was up-regulated (100%) by 17β-estradiol (EC50 20 pM) and the predominant role of ERα was demonstrated by using the ERα-selective agonist “propylpyrazole triol”. 17β-Estradiol-induced over-expression of functional Y1R protein was reverted by the antiestrogen fulvestrant (IC50 5 nM) in vitro. Furthermore, tamoxifen treatment of nude mice resulted in an almost total loss of Y1Rs in MCF-7 xenografts. In conclusion, the value of the Y1R as a target for therapy and imaging in breast cancer patients may be compromised due to Y1R down-regulation induced by hormonal (antiestrogen) treatment. PMID:23236424

  14. 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.

  15. 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.

  16. 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.

  17. Neuroprotective Effect of Scutellarin on Ischemic Cerebral Injury by Down-Regulating the Expression of Angiotensin-Converting Enzyme and AT1 Receptor

    PubMed Central

    Han, Jichun; Zhou, Mingjie; Ren, Huanhuan; Pan, Qunwen; Zheng, Chunli; Zheng, Qiusheng

    2016-01-01

    Background and Purpose Previous studies have demonstrated that angiotensin-converting enzyme (ACE) is involved in brain ischemic injury. In the present study, we investigated whether Scutellarin (Scu) exerts neuroprotective effects by down-regulating the Expression of Angiotensin-Converting Enzyme and AT1 receptor in a rat model of permanent focal cerebral ischemia. Methods Adult Sprague–Dawley rats were administrated with different dosages of Scu by oral gavage for 7 days and underwent permanent middle cerebral artery occlusion (pMCAO). Blood pressure was measured 7 days after Scu administration and 24 h after pMCAO surgery by using a noninvasive tail cuff method. Cerebral blood flow (CBF) was determined by Laser Doppler perfusion monitor and the neuronal dysfunction was evaluated by analysis of neurological deficits before being sacrificed at 24 h after pMCAO. Histopathological change, cell apoptosis and infarct area were respectively determined by hematoxylin–eosin staining, terminal deoxynucleotidyl transfer-mediated dUTP nick end labeling (TUNEL) analysis and 2,3,5-triphenyltetrazolium chloride staining. Tissue angiotensin II (Ang II) and ACE activity were detected by enzyme-linked immunosorbent assays. The expression levels of ACE, Ang II type 1 receptor (AT1R), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were measured by Western blot and real-time PCR. ACE inhibitory activity of Scu in vitro was detected by the photometric determination. Results Scu treatment dose-dependently decreased neurological deficit score, infarct area, cell apoptosis and morphological changes induced by pMCAO, which were associated with reductions of ACE and AT1R expression and the levels of Ang II, TNF-α, IL-6, and IL-1β in ischemic brains. Scu has a potent ACE inhibiting activity. Conclusion Scu protects brain from acute ischemic injury probably through its inhibitory effect on the ACE/Ang II/AT1 axis, CBF preservation and

  18. Lentiviral vector-mediated down-regulation of IL-17A receptor in hepatic stellate cells results in decreased secretion of IL-6

    PubMed Central

    Zhang, Sheng-Chu; Zheng, Yi-Hu; Yu, Pan-Pan; Min, Tan Hooi; Yu, Fu-Xiang; Ye, Chao; Xie, Yuan-Kang; Zhang, Qi-Yu

    2012-01-01

    AIM: To investigate the mechanism of interleukin (IL)-6 secretion through blocking the IL-17A/IL-17A receptor (IL-17RA) signaling pathway with a short hairpin RNA (shRNA) in hepatic stellate cells (HSCs) in vitro. METHODS: HSCs were derived from the livers of adult male Sprague-Dawley rats. IL-6 expression was evaluated using real-time quantitative polymerase chain reaction and enzyme linked immunosorbent assay. The phosphorylation activity of p38 mitogen activated protein kinases (MAPK) and extracellular regulated protein kinases (ERK) 1/2 upon induction by IL-17A and suppression by IL-17RA shRNA were examined using Western blotting. RESULTS: IL-6 expression induced by IL-17A was significantly increased compared to control in HSCs (P < 0.01 in a dose-dependent manner). Suppression of IL-17RA using lentiviral-mediated shRNA inhibited IL-6 expression induced by IL-17A compared to group with only IL-17A treatment (1.44 ± 0.17 vs 4.07 ± 0.43, P < 0.01). IL-17A induced rapid phosphorylation of p38 MAPK and ERK1/2 after 5 min exposure, and showed the strongest levels of phosphorylation of p38 MAPK and ERK1/2 at 15 min in IL-17A-treated HSCs. IL-6 mRNA expression induced by IL-17A (100 ng/mL) for 3 h exposure was inhibited by preincubation with specific inhibitors of p38 MAPK (SB-203580) and ERK1/2 (PD-98059) compared to groups without inhibitors preincubation (1.67 ± 0.24, 2.01 ± 0.10 vs 4.08 ± 0.59, P < 0.01). Moreover, Lentiviral-mediated IL-17RA shRNA 1 inhibited IL-17A-induced IL-6 mRNA expression compared to random shRNA in HSCs (1.44 ± 0.17 vs 3.98 ± 0.68, P < 0.01). Lentiviral-mediated IL-17RA shRNA 1 inhibited phosphorylation of p38 MAPK and ERK1/2 induced by 15 min IL-17A (100 ng/mL) exposure. CONCLUSION: Down-regulation of the IL-17RA receptor by shRNA decreased IL-6 expression induced by IL-17A via p38 MAPK and ERK1/2 phosphorylation in HSCs. Suppression of IL-17RA expression may be a strategy to reduce the inflammatory response induced by IL-17A in

  19. K20E, an oxidative-coupling compound of methyl caffeate, exhibits anti-angiogenic activities through down-regulations of VEGF and VEGF receptor-2

    SciTech Connect

    Pan, Chun-Hsu; Lin, Wen-Hsin; Chien, Yi-Chung; Liu, Fon-Chang; Sheu, Ming-Jyh; Kuo, Yueh-Hsiung; Wu, Chieh-Hsi

    2015-01-15

    Anti-angiogenesis is one of the most popular clinical interventions for cancer chemotherapy. A series of synthesized derivative of methyl caffeate were used to evaluate the anti-angiogenic activity and to investigate possible pharmacological mechanisms in the present study. The most potent anti-angiogenic compound was evaluated in the experiments of murine allograft tumor model and Matrigel plug assay as well as cell models in the human umbilical vascular endothelial cells (HUVECs) and the LLC1 lung cancer cells. Our results suggested that K20E suppressed the tumor growth in the allograft tumor model and exhibited anti-angiogenic activity in Matrigel plug assay. Besides, HUVEC viability was found to be significantly reduced by arresting cell cycle at G{sub 2}/M phase and apoptosis. Cell migration, invasion, and tube formation of the HUVECs were also markedly suppressed by K20E treatment. K20E largely down-regulated the intracellular and secreted vascular endothelial growth factor (VEGF) in the LLC1 cancer cells. Besides, VEGF receptor-2 (VEGFR-2) and its downstream signaling cascades (AKT-mTOR and MEK1/2-ERK1/2) as well as gelatinases were all evidently reduced in the HUVECs treated with K20E. Inversely, K20E can up-regulate the expression levels of p53 and p21 proteins in the HUVECs. Based on these results, our study suggested that K20E possessed inhibiting angiogenesis through regulation of VEGF/VEGFR-2 and its downstream signaling cascades in the vascular endothelial cells (VECs). - Highlights: • K20E is an oxidative-coupling compound of methyl caffeate. • K20E exhibits anti-tumor and anti-angiogenesis effects. • K20E suppresses the expressions of VEGF and VEGF receptor-2 (VEGFR-2) proteins. • K20E deactivates VEGFR-2-mediated downstream signaling pathways to inhibit angiogenesis. • K20E up-regulates p53-p21 pathway to induce apoptosis and cell arrest at G2/M phase.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. D1-like receptors inhibit insulin-induced vascular smooth muscle cell proliferation via down-regulation of insulin receptor expression

    PubMed Central

    Zeng, Chunyu; Han, Yu; Huang, Hefei; Yu, Changqing; Ren, Hongmei; Shi, Weibin; He, Duofen; Huang, Lan; Yang, Chengming; Wang, Xukai; Zhou, Lin; Jose, Pedro A.

    2013-01-01

    Objective Vascular smooth muscle cell (VSMC) proliferation is central to the development of vascular diseases, including hypertension, which is regulated by numerous hormones and humoral factors. Our previous study showed that the stimulatory effect of norepinephrine on VSMC proliferation is inhibited by D1-like receptors and the D3 dopamine receptor, a member of the D2-like receptor family. Insulin is a proliferative hormone but it is not known if there is any interaction between insulin and D1-like receptors. We hypothesized that Dl-like receptors may have an inhibitory effect on the insulin-induced VSMC proliferation; aberrant insulin and Dl-like receptor functions could be involved in the pathogenesis of essential hypertension. Methods VSMC proliferation was determined by [3H]-thymidine incorporation; insulin receptor mRNA and protein expressions were determined by RT-PCR, immunoblotting, and immunohistochemistry. Results Insulin increased VSMC proliferation in immortalized aortic A10 cells, determined by [3H]-thymidine incorporation. Although the D1-like receptor, by itself, had no effect on VSMC proliferation, stimulation with fenoldopam, a D1-like receptor agonist, inhibited the stimulatory effect of insulin. The inhibitory effect of fenoldopam on insulin-mediated VSMC proliferation was receptor specific, because its effect could be blocked by SCH23390, a D1-like receptor antagonist. Fenoldopam also inhibited insulin receptor mRNA and protein expression, which was time dependent and concentration dependent. A PKC or MAP kinase inhibitor blocked the inhibitory effect of fenoldopam on insulin receptor expression, indicating that PKC and MAP kinase were involved in the signaling pathway. Conclusion The inhibitory effect of D1-like receptors on insulin-mediated VSMC proliferation may play an important role in the regulation of blood pressure. PMID:19293728

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. Lysophosphatidic acid inhibits adipocyte differentiation via lysophosphatidic acid 1 receptor-dependent down-regulation of peroxisome proliferator-activated receptor gamma2.

    PubMed

    Simon, Marie Françoise; Daviaud, Danièle; Pradère, Jean Philippe; Grès, Sandra; Guigné, Charlotte; Wabitsch, Martin; Chun, Jerold; Valet, Philippe; Saulnier-Blache, Jean Sébastien

    2005-04-15

    Lysophosphatidic acid (LPA) is a bioactive phospholipid acting via specific G protein-coupled receptors that is synthesized at the extracellular face of adipocytes by a secreted lysophospholipase D (autotaxin). Preadipocytes mainly express the LPA(1) receptor subtype, and LPA increases their proliferation. In monocytes and CV1 cells LPA was recently reported to bind and activate peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor also known to play a pivotal role in adipogenesis. Here we show that, unlike the PPARgamma agonist rosiglitazone, LPA was unable to increase transcription of PPARgamma-sensitive genes (PEPCK and ALBP) in the mouse preadipose cell line 3T3F442A. In contrast, treatment with LPA decreased PPARgamma2 expression, impaired the response of PPARgamma-sensitive genes to rosiglitazone, reduced triglyceride accumulation, and reduced the expression of adipocyte mRNA markers. The anti-adipogenic activity of LPA was also observed in the human SGBS (Simpson-Golabi-Behmel syndrome) preadipocyte cell line, as well as in primary preadipocytes isolated from wild type mice. Conversely, the anti-adipogenic activity of LPA was not observed in primary preadipocytes from LPA(1) receptor knock-out mice, which, in parallel, exhibited a higher adiposity than wild type mice. In conclusion, LPA does not behave as a potent PPARgamma agonist in adipocytes but, conversely, inhibits PPARgamma expression and adipogenesis via LPA(1) receptor activation. The local production of LPA may exert a tonic inhibitory effect on the development of adipose tissue.

  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. Xenoestrogens down-regulate aryl-hydrocarbon receptor nuclear translocator 2 mRNA expression in human breast cancer cells via an estrogen receptor alpha-dependent mechanism.

    PubMed

    Qin, Xian-Yang; Zaha, Hiroko; Nagano, Reiko; Yoshinaga, Jun; Yonemoto, Junzo; Sone, Hideko

    2011-10-10

    Environmental chemicals with estrogenic activity, known as xenoestrogens, may cause impaired reproductive development and endocrine-related cancers in humans by disrupting endocrine functions. Aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) is believed to play important roles in a variety of physiological processes, including estrogen signaling pathways, that may be involved in the pathogenesis and therapeutic responses of endocrine-related cancers. However, much of the underlying mechanism remains unknown. In this study, we investigated whether ARNT2 expression is regulated by a range of representative xenoestrogens in human cancer cell lines. Bisphenol A (BPA), benzyl butyl phthalate (BBP), and 1,1,1-trichloro-2,2-bis(2-chlorophenyl-4-chlorophenyl)ethane (o,p'-DDT) were found to be estrogenic toward BG1Luc4E2 cells by an E-CALUX bioassay. ARNT2 expression was downregulated by BPA, BBP, and o,p'-DDT in a dose-dependent manner in estrogen receptor 1 (ESR1)-positive MCF-7 and BG1Luc4E2 cells, but not in estrogen receptor-negative LNCaP cells. The reduction in ARNT2 expression in cells treated with the xenoestrogens was fully recovered by the addition of a specific ESR1 antagonist, MPP. In conclusion, we have shown for the first time that ARNT2 expression is modulated by xenoestrogens by an ESR1-dependent mechanism in MCF-7 breast cancer cells. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  7. 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.

  8. Cell-based Fluorescence Complementation Reveals a Role for HIV-1 Nef Protein Dimerization in AP-2 Adaptor Recruitment and CD4 Co-receptor Down-regulation.

    PubMed

    Shu, Sherry T; Emert-Sedlak, Lori A; Smithgall, Thomas E

    2017-02-17

    The HIV-1 Nef accessory factor enhances viral infectivity, immune evasion, and AIDS progression. Nef triggers rapid down-regulation of CD4 via the endocytic adaptor protein 2 (AP-2) complex, a process linked to enhanced viral infectivity and immune escape. Here, we describe a bimolecular fluorescence complementation (BiFC) assay to visualize the interaction of Nef with AP-2 and CD4 in living cells. Interacting protein pairs were fused to complementary non-fluorescent fragments of YFP and co-expressed in 293T cells. Nef interactions with both CD4 and AP-2 resulted in complementation of YFP and a bright fluorescent signal by confocal microcopy that localized to the cell periphery. Co-expression of the AP-2 α subunit enhanced the Nef·AP-2 σ2 subunit BiFC signal and vice versa, suggesting that the AP-2 α-σ2 hemicomplex interacts cooperatively with Nef. Mutagenesis of Nef amino acids Arg-134, Glu-174, and Asp-175, which stabilize Nef for AP-2 α-σ2 binding in a recent co-crystal structure, substantially reduced AP-2 interaction without affecting CD4 binding. A dimerization-defective mutant of Nef failed to interact with either CD4 or AP-2 in the BiFC assay, indicating that Nef quaternary structure is required for CD4 and AP-2 recruitment as well as CD4 down-regulation. A small molecule previously shown to bind the Nef dimerization interface also reduced Nef interactions with AP-2 and CD4 and restored CD4 expression to the surface of HIV-infected cells. Our findings provide a mechanistic explanation for previous observations that dimerization-defective Nef mutants fail to down-regulate CD4 and validate the Nef dimerization interface as a target site for antiretroviral drug development.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. The p.Leu167del Mutation in APOE Gene Causes Autosomal Dominant Hypercholesterolemia by Down-regulation of LDL Receptor Expression in Hepatocytes.

    PubMed

    Cenarro, Ana; Etxebarria, Aitor; de Castro-Orós, Isabel; Stef, Marianne; Bea, Ana M; Palacios, Lourdes; Mateo-Gallego, Rocío; Benito-Vicente, Asier; Ostolaza, Helena; Tejedor, Teresa; Martín, César; Civeira, Fernando

    2016-05-01

    The p.Leu167del mutation in the APOE gene has been associated with hyperlipidemia. Our objective was to determine the frequency of p.Leu167del mutation in APOE gene in subjects with autosomal dominant hypercholesterolemia (ADH) in whom LDLR, APOB, and PCSK9 mutations had been excluded and to identify the mechanisms by which this mutant apo E causes hypercholesterolemia. The APOE gene was analyzed in a case-control study. The study was conducted at a University Hospital Lipid Clinic. Two groups (ADH, 288 patients; control, 220 normolipidemic subjects) were included. We performed sequencing of APOE gene and proteomic and cellular experiments. To determine the frequency of the p.Leu167del mutation and the mechanism by which it causes hypercholesterolemia. In the ADH group, nine subjects (3.1%) were carriers of the APOE c.500_502delTCC, p.Leu167del mutation, cosegregating with hypercholesterolemia in studied families. Proteomic quantification of wild-type and mutant apo E in very low-density lipoprotein (VLDL) from carrier subjects revealed that apo E3 is almost a 5-fold increase compared to mutant apo E. Cultured cell studies revealed that VLDL from mutation carriers had a significantly higher uptake by HepG2 and THP-1 cells compared to VLDL from subjects with E3/E3 or E2/E2 genotypes. Transcriptional down-regulation of LDLR was also confirmed. p.Leu167del mutation in APOE gene is the cause of hypercholesterolemia in the 3.1% of our ADH subjects without LDLR, APOB, and PCSK9 mutations. The mechanism by which this mutation is associated to ADH is that VLDL carrying the mutant apo E produces LDLR down-regulation, thereby raising plasma low-density lipoprotein cholesterol levels.

  18. 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.

  19. 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.

  20. 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.

  1. Induction of GST-P-positive proliferative lesions facilitating lipid peroxidation with possible involvement of transferrin receptor up-regulation and ceruloplasmin down-regulation from the early stage of liver tumor promotion in rats.

    PubMed

    Mizukami, Sayaka; Ichimura, Ryohei; Kemmochi, Sayaka; Taniai, Eriko; Shimamoto, Keisuke; Ohishi, Takumi; Takahashi, Miwa; Mitsumori, Kunitoshi; Shibutani, Makoto

    2010-04-01

    To elucidate the role of metal-related molecules in hepatocarcinogenesis, we examined immunolocalization of transferrin receptor (Tfrc), ceruloplasmin (Cp) and metallothionein (MT)-1/2 in relation to liver cell foci positive for glutathione-S-transferase placental form (GST-P) in the early stage of tumor promotion by fenbendazole (FB), phenobarbital, piperonyl butoxide or thioacetamide in a rat two-stage hepatocarcinogenesis model. To estimate the involvement of oxidative stress responses to the promotion, immunolocalization of 4-hydroxy-2-nonenal, malondialdehyde and acrolein was similarly examined. Our findings showed that MT-1/2 immunoreactivity was not associated with the cellular distribution of GST-P and proliferating cell nuclear antigen, suggesting no role of MT-1/2 in hepatocarcinogenesis. We also found enhanced expression of Tfrc after treatment with strong tumor-promoting chemicals. With regard to Cp, the population showing down-regulation was increased in the GST-P-positive foci in relation to tumor promotion. Up-regulation of Tfrc and down-regulation of Cp was maintained in GST-P-positive neoplastic lesions induced after long-term promotion with FB, suggesting the expression changes occurring downstream of the signaling pathway involved in the formation of GST-P-positive lesions. Furthermore, enhanced accumulation of lipid peroxidation end products was observed in the GST-P-positive foci by promotion. Post-initiation treatment with peroxisome proliferator-activated receptor alpha agonists did not enhance any such distribution changes in GST-P-negative foci. The results thus suggest that facilitation of lipid peroxidation is involved in the induction of GST-P-positive lesions by tumor promotion from an early stage, and up-regulation of Tfrc and down-regulation of Cp may be a signature of enhanced oxidative cellular stress in these lesions.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. The adenovirus E3 10.4K and 14.5K proteins, which function to prevent cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor, are localized in the plasma membrane.

    PubMed Central

    Stewart, A R; Tollefson, A E; Krajcsi, P; Yei, S P; Wold, W S

    1995-01-01

    The adenovirus type 2 and 5 E3 10,400- and 14,500-molecular-weight (10.4K and 14.5K) proteins are both required to protect some cell lines from lysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor. We have shown previously that both 10.4K and 14.5K are integral membrane proteins and that 14.5K is phosphorylated and O glycosylated. The 10.4K protein coimmunoprecipitates with 14.5K, indicating that the two proteins function as a complex. Here we show, using immunofluorescence and two different cell surface-labeling techniques, that both proteins are localized in the plasma membrane. In addition, we show that trafficking of each protein to the plasma membrane depends on concomitant expression of the other protein. Finally, neither protein could be immunoprecipitated from conditioned media, indicating that neither is secreted. Taken together, these results suggest that the plasma membrane is the site at which 10.4K and 14.5K function to inhibit cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor. PMID:7983708

  9. 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.

  10. 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

  11. Angiotensin II down-regulates natriuretic peptide receptor-A expression and guanylyl cyclase activity in H9c2 (2-1) cardiac myoblast cells: Role of ROS and NF-κB.

    PubMed

    Gopi, Venkatachalam; Subramanian, Vimala; Manivasagam, Senthamizharasi; Vellaichamy, Elangovan

    2015-11-01

    Atrial natriuretic peptide (ANP)/natriuretic peptide receptor-A (NPR-A) system is suggested as an endogenous anti-hypertrophic protective mechanism of the heart. We have shown previously that Angiotensin II (ANG II), an effector molecule of renin-angiotensin-aldosterone system, down-regulates NPR-A expression and its activity in vivo rat heart. However, the underlying mechanism by which ANG II down-regulates NPR-A expression in the heart is not well understood. Hence, the present investigation was aimed to determine whether ANG II-stimulated reactive oxygen species (ROS) and NF-κB are involved in the down-regulation of NPR-A activity in H9c2 (2-1) cardiac myoblast cells. The H9c2 (2-1) cardiac myoblast cells were exposed to ANG II (10(-7) M for 20 h) with/or without blocker treatment (losartan-10 µM, N-acetyl cysteine (NAC)-10 mM and pyrrolidine dithiocarbamate (PDTC)-100 µM). On exposure, ANG II induced a significant decrease (P < 0.001) in the expression of Npr1 (coding for NPR-A) gene and NPR-A receptor-dependent guanylyl cyclase (GC) activity. The level of expression of proto-oncogenes (c-fos, c-myc, and c-jun) and natriuretic peptides (ANP and BNP) was increased in ANG II-treated cells when compared with control cells. Interestingly, ANG II-dependent repression of Npr1 gene expression and guanylyl cyclase (GC) activity was completely restored on treatment with losartan, while only a partial reversal was observed in NAC- and PDTC-co-treated cells. In conclusion, the results of this study suggest that ROS-mediated NF-κB activation mechanism is critically involved in the ANG II-mediated down-regulation of NPR-A expression and its GC activity.

  12. Oxygenized low density lipoprotein down-regulates the TRPV4 protein expression of macrophage through activation of peroxisome proliferator-activated receptor γ.

    PubMed

    Xue, Chao; Gong, Jun; Guo, Yuming; Yin, Jun; He, Xiaohua; Huang, Hua; Zhou, Xuefeng; Zhao, Jinping

    2017-02-01

    TRPV4, a non-selective cation channel, is involved in lipometabolism and atherosclerosis. However, whether TRPV4 participates in oxygenized low density lipoprotein (oxLDL)-induced foam cell formation remains unknown. The present study investigates the effect of oxLDL on the expression of TRPV4 in macrophages and its underlying mechanisms. The expression of TRPV4 in RAW264.7 and phorbol-12-myristate-13-acetate (PMA) induced U937, THP-1 cells was detected by immunofluorescence, and western blot was used to detect the TRPV4 expression before and after PMA induction. Each cell line was divided into three groups, including control group, native low-density lipoprotein (nLDL) (100 μg/mL) group and oxLDL (100μg/mL) group; the expression of TRPV4 in each group was measured using immunohistochemistry and western blot. TRPV4 protein expression was detected by western blot after RAW 264.7 cells were treated with 0, 0.01 μM, 0.1 μM and 1 μM T0070907 or preincubated with 0.1 μM T0070709 for 1 h before incubation with oxLDL for 24 h. In all macrophage cell lines, TRPV4 was widely expressed. PMA increased TRPV4 expression in U937 and THP-1 cells. There was no significant difference in TRPV4 expression in the nLDL group compared to that in the control group; however a significant reduction in TRPV4 expression was detected in the oxLDL group compared to that in the control and nLDL groups using measurements obtained from both immunohistochemistry and western blot. The PPARγ inhibitor T0070907 enhanced the basal expression of TRPV4 and protected RAW264.7 cells from oxLDL-induced TRPV4 down-regulation. This study revealed that TRPV4 was widely expressed in macrophages and that oxLDL could induce the down-regulation of TRPV4 expression through its actions on PPARγ. This study may serve as an important first step for further investigation into the roles of TRPV4 in macrophage-derived foam cell formation in atherosclerosis.

  13. 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.

  14. Peroxisome proliferator-activated receptor γ down-regulation mediates the inhibitory effect of d-δ-tocotrienol on the differentiation of murine 3T3-F442A preadipocytes.

    PubMed

    Torabi, Sheida; Yeganehjoo, Hoda; Shen, Chwan-Li; Mo, Huanbiao

    2016-12-01

    Tocotrienols accelerate the degradation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase that catalyzes the biosynthesis of mevalonate; the latter is essential for preadipocyte differentiation. Tocotrienols also down-regulate peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation. We hypothesized that mevalonate deprivation and PPARγ down-regulation mediate d-δ-tocotrienol-induced inhibition of adipocyte differentiation. The objectives of this study were to determine the effect of d-δ-tocotrienol on 3T3-F442A preadipocyte differentiation and the involvement of PPARγ and mevalonate. Murine 3T3-F442A preadipocytes were incubated with d-δ-tocotrienol (2.5-10 μmol/L) for 8 days. AdipoRed assay and Oil Red O staining showed that d-δ-tocotrienol dose-dependently reduced the intracellular triglyceride content. Concomitantly, d-δ-tocotrienol dose-dependently inhibited glucose uptake by 3T3-F442A cells and the expression of GLUT4, HMG CoA reductase, and p-Akt proteins. The effects of d-δ-tocotrienol on intracellular triglyceride content and glucose uptake were attenuated by rosiglitazone, an agonist of PPARγ, but not supplemental mevalonate (100 μmol/L). In contrast, mevalonate, but not rosiglitazone, reversed the effects of lovastatin, a competitive inhibitor of HMG CoA reductase shown to inhibit adipocyte differentiation via mevalonate deprivation. Trypan blue staining revealed no changes in cell viability after a 48-hour incubation of 3T3-F442A cells with d-δ-tocotrienol (0-80 μmol/L), suggesting that the adipogenesis-suppressive activity of d-δ-tocotrienol was independent of cytotoxicity. In conclusion, these findings demonstrate the antiadipogenic effect of d-δ-tocotrienol via PPARγ down-regulation.

  15. 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

  16. 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

  17. Resveratrol regulates the cell viability promoted by 17β-estradiol or bisphenol A via down-regulation of the cross-talk between estrogen receptor α and insulin growth factor-1 receptor in BG-1 ovarian cancer cells.

    PubMed

    Kang, Nam-Hee; Hwang, Kyung-A; Lee, Hye-Rim; Choi, Dal-Woong; Choi, Kyung-Chul

    2013-09-01

    Endocrine disrupting chemicals (EDCs) and estrogens appear to promote development of estrogen-dependent cancers, including breast and ovarian carcinomas. In this study, we evaluated the cell viability effect of BPA on BG-1 human ovarian cancer cells, along with the growth inhibitory effect of resveratrol (trans-3,4,5-trihydroxystilbene; RES), a naturally occurring phytoestrogen. In addition, we investigated the underlying mechanism(s) of BPA and RES in regulating the interaction between estrogen receptor alpha (ERα) and insulin-like growth factor-1 receptor (IGF-1R) signals, a non- genomic pathway induced by 17β-estradiol (E2). BPA induced a significant increase in BG-1 cell growth and up-regulated mRNA levels of ERα and IGF-1R. In parallel with its mRNA level, the protein expression of ERα was induced, and phosphorylated insulin receptor substrate-1 (p-IRS-1), phosphorylated Akt1/2/3, and cyclin D1 were increased by BPA or E2. However, RES effectively reversed the BG-1 cell proliferation induced by E2 or BPA by inversely down-regulating the expressions of ERα, IGF-1R, p-IRS-1, and p-Akt1/2/3, and cyclin D1 at both transcriptional and translational levels. Taken together, these results suggest that RES is a novel candidate for prevention of tumor progression caused by EDCs, including BPA via effective inhibition of the cross-talk of ERα and IGF-1R signaling pathways. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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).

  3. 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

  4. 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.

  5. Down-regulation of peroxisome proliferator activated receptor γ coactivator 1α induces oxidative stress and toxicity of 1-(4-Chlorophenyl)-benzo-2,5-quinone in HaCaT human keratinocytes

    PubMed Central

    Xiao, Wusheng; Goswami, Prabhat C.

    2015-01-01

    Peroxisome proliferator activated receptor γ coactivator 1α (PGC-1α) is a transcriptional coactivator that is known to regulate oxidative stress response by enhancing the expression of antioxidant genes. We have shown previously that 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ), a quinone-metabolite of 4-monochlorobiphenyl (PCB3) induces oxidative stress and toxicity in human skin keratinocytes, and breast and prostate epithelial cells. In this study, we investigate whether PGC-1α regulates oxidative stress and toxicity in 4-ClBQ treated HaCaT human keratinocytes. Results showed significant down-regulation in the expression of PGC-1α and catalase in 4-ClBQ treated HaCaT cells. Down-regulation of PGC-1α expression was associated with 4-ClBQ induced increase in the steady-state levels of cellular reactive oxygen species (ROS) and toxicity. Overexpression of pgc-1α enhanced the expression of catalase and suppressed 4-ClBQ induced increase in cellular ROS levels and toxicity. These results suggest that pgc-1α mediates 4-ClBQ induced oxidative stress and toxicity in HaCaT cells presumably by regulating catalase expression. PMID:26004620

  6. Expression of Caveolin-1 reduces cellular responses to TGF-{beta}1 through down-regulating the expression of TGF-{beta} type II receptor gene in NIH3T3 fibroblast cells

    SciTech Connect

    Lee, Eun Kyung; Lee, Youn Sook; Han, In-Oc; Park, Seok Hee . E-mail: parks@skku.edu

    2007-07-27

    Transcriptional repression of Transforming Growth Factor-{beta} type II receptor (T{beta}RII) gene has been proposed to be one of the major mechanisms leading to TGF-{beta} resistance. In this study, we demonstrate that expression of Caveolin-1 (Cav-1) gene in NIH3T3 fibroblast cells down-regulates the expression of T{beta}RII gene in the transcriptional level, eventually resulting in the decreased responses to TGF-{beta}. The reduced expression of T{beta}RII gene by Cav-1 appeared to be due to the changes of the sequence-specific DNA binding proteins to either Positive Regulatory Element 1 (PRE1) or PRE2 of the T{beta}RII promoter. In addition, Cav-1 expression inhibited TGF-{beta}-mediated cellular proliferation and Plasminogen Activator Inhibitor (PAI)-1 gene expression as well as TGF-{beta}-induced luciferase activity. Furthermore, the inhibition of endogeneous Cav-1 by small interfering RNA increased the expression of T{beta}RII gene. These findings strongly suggest that expression of Cav-1 leads to the decreased cellular responsiveness to TGF-{beta} through down-regulating T{beta}RII gene expression.

  7. 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.

  8. 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

  9. Down-regulation of miR-135b in colon adenocarcinoma induced by a TGF-β receptor I kinase inhibitor (SD-208)

    PubMed Central

    Akbari, Abolfazl; Ghahremani, Mohammad Hossein; Mobini, Gholam Reza; Abastabar, Mahdi; Akhtari, Javad; Bolhassani, Manzar; Heidari, Mansour

    2015-01-01

    Objective(s): Transforming growth factor-β (TGF-β) is involved in colorectal cancer (CRC). The SD-208 acts as an anti-cancer agent in different malignancies via TGF-β signaling. This work aims to show the effect of manipulation of TGF-β signaling on some miRNAs implicated in CRC. Materials and Methods: We investigated the effects of SD-208 on SW-48, a colon adenocarcinoma cell line. The cell line was treated with 0.5, 1 and 2 μM concentrations of SD-208. Then, the xenograft model of colon cancer was established by subcutaneous inoculation of SW-48 cell line into the nude mice. The animals were treated with SD-208 for three weeks. A quantitative real-time PCR was carried out for expression level analysis of selected oncogenic (miR-21, 31, 20a and 135b) and suppressor-miRNAs (let7-g, miR-133b, 145 and 200c). Data were analyzed using the 2-∆∆CT method through student’s t-test via the GraphPad Prism software. Results: Our results revealed that SD-208 could significantly down-regulate the expression of one key onco-miRNA, miR-135b, in either SW-48 colon cells (P=0.006) or tumors orthotopically implanted in nude mice (P=0.018). Our in silico study also predicted that SD-208 could modulate the expression of potential downstream tumor suppressor targets of the miR135b. Conclusion: Our data provide novel evidence that anticancer effects of SD-208 (and likely other TGF-β inhibitors) may be owing to their ability to regulate miRNAs expression. PMID:26523217

  10. Down-regulation of miR-135b in colon adenocarcinoma induced by a TGF-β receptor I kinase inhibitor (SD-208).

    PubMed

    Akbari, Abolfazl; Ghahremani, Mohammad Hossein; Mobini, Gholam Reza; Abastabar, Mahdi; Akhtari, Javad; Bolhassani, Manzar; Heidari, Mansour

    2015-09-01

    Transforming growth factor-β (TGF-β) is involved in colorectal cancer (CRC). The SD-208 acts as an anti-cancer agent in different malignancies via TGF-β signaling. This work aims to show the effect of manipulation of TGF-β signaling on some miRNAs implicated in CRC. We investigated the effects of SD-208 on SW-48, a colon adenocarcinoma cell line. The cell line was treated with 0.5, 1 and 2 μM concentrations of SD-208. Then, the xenograft model of colon cancer was established by subcutaneous inoculation of SW-48 cell line into the nude mice. The animals were treated with SD-208 for three weeks. A quantitative real-time PCR was carried out for expression level analysis of selected oncogenic (miR-21, 31, 20a and 135b) and suppressor-miRNAs (let7-g, miR-133b, 145 and 200c). Data were analyzed using the 2-∆∆CT method through student's t-test via the GraphPad Prism software. Our results revealed that SD-208 could significantly down-regulate the expression of one key onco-miRNA, miR-135b, in either SW-48 colon cells (P=0.006) or tumors orthotopically implanted in nude mice (P=0.018). Our in silico study also predicted that SD-208 could modulate the expression of potential downstream tumor suppressor targets of the miR135b. Our data provide novel evidence that anticancer effects of SD-208 (and likely other TGF-β inhibitors) may be owing to their ability to regulate miRNAs expression.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. microRNA-34a-Mediated Down-Regulation of the Microglial-Enriched Triggering Receptor and Phagocytosis-Sensor TREM2 in Age-Related Macular Degeneration

    PubMed Central

    Dua, Prerna; Rogaev, Evgeny I.; Lukiw, Walter J.

    2016-01-01

    The aggregation of Aβ42-peptides and the formation of drusen in age-related macular degeneration (AMD) are due in part to the inability of homeostatic phagocytic mechanisms to clear self-aggregating Aβ42-peptides from the extracellular space. The triggering receptor expressed in myeloid/microglial cells-2 (TREM2), a trans-membrane-spanning, sensor-receptor of the immune-globulin/lectin-like gene superfamily is a critical component of Aβ42-peptide clearance. Here we report a significant deficit in TREM2 in AMD retina and in cytokine- or oxidatively-stressed microglial (MG) cells. RT-PCR, miRNA-array, LED-Northern and Western blot studies indicated up-regulation of a microglial-enriched NF-кB-sensitive miRNA-34a coupled to a down-regulation of TREM2 in the same samples. Bioinformatics/transfection-luciferase reporter assays indicated that miRNA-34a targets the 299 nucleotide TREM2-mRNA-3’UTR, resulting in TREM2 down-regulation. C8B4-microglial cells challenged with Aβ42 were able to phagocytose these peptides, while miRNA-34a down-regulated both TREM2 and the ability of microglial-cells to phagocytose. Treatment of TNFα-stressed MG cells with phenyl-butyl nitrone (PBN), caffeic-acid phenethyl ester (CAPE), the NF-B-inhibitor/resveratrol analog CAY10512 or curcumin abrogated these responses. Incubation of anti-miRNA-34a (AM-34a) normalized miRNA-34a abundance and restored TREM2 back to homeostatic levels. These data support five novel observations: (i) that a ROS- and NF-B-sensitive, miRNA-34a-mediated modulation of TREM2 may in part regulate the phagocytic response; (ii) that gene products encoded on two different chromosomes (miRNA-34a at chr1q36.22 and TREM2 at chr6p21.1) orchestrate a phagocytic-Aβ42-peptide clearance-system; (iii) that this NF-kB-mediated-miRNA-34a-TREM2 mechanism is inducible from outside of the cell; (iv) that when operating normally, this pathway can clear Aβ42 peptide monomers from the extracellular medium; and (v) that anti

  16. 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

  17. 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

  18. Dopamine D1 and D2 receptor functional down regulation in the cerebellum of hypoxic neonatal rats: neuroprotective role of glucose and oxygen, epinephrine resuscitation.

    PubMed

    Joseph, Binoy; Nandhu, M S; Paulose, C S

    2010-02-01

    Brain damage due to an episode of hypoxia remains a major problem in infants causing deficit in motor and sensory function. Molecular processes regulating the dopamine receptors play a very important role in motor and cognitive functions. Disturbances in the development of the dopaminergic system lead to dyskinesia, dystonia, tics and abnormal eye movements. The present study is to understand the hypoxic damage to the dopamine content and dopamine D(1), dopamine D(2) receptors in cerebellum and the neuroprotective effect of glucose supplementation prior to the current sequence of resuscitation-oxygen and epinephrine supplementation in neonatal rats. Dopamine content in the cerebellum showed a significant decrease in hypoxic neonatal rats when compared to control. Dopamine D(1) and dopamine D(2) receptors showed a decrease in B(max) during hypoxia. The cerebellar dopamine, dopamine D(1) and dopamine D(2) receptors showed significant decrease on supplementation of 100% oxygen alone to hypoxic rats when compared to control rats. Dopamine D(1) and dopamine D(2) receptors mRNA showed significant decrease during epinephrine supplementation prior to resuscitation. These dopaminergic receptor alterations were reversed to near control by glucose supplementation. Thus our results suggest that glucose acts as a neuroprotective agent in dopaminergic receptors function. This has immense clinical significance to correct the resuscitation sequence in neonatal care.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. Peroxisome proliferator-activated receptor alpha (PPARalpha) agonists down-regulate alpha2-macroglobulin expression by a PPARalpha-dependent mechanism.

    EPA Science Inventory

    Peroxisome proliferator-activated receptor alpha (PPARα) regulates transcription of genes involved both in lipid and glucose metabolism as well as inflammation. Fibrates are PPARα ligands used to normalize lipid and glucose parameters and exert anti-inflammatory effects. Fibrates...

  4. Peroxisome proliferator-activated receptor alpha (PPARalpha) agonists down-regulate alpha2-macroglobulin expression by a PPARalpha-dependent mechanism.

    EPA Science Inventory

    Peroxisome proliferator-activated receptor alpha (PPARα) regulates transcription of genes involved both in lipid and glucose metabolism as well as inflammation. Fibrates are PPARα ligands used to normalize lipid and glucose parameters and exert anti-inflammatory effects. Fibrates...

  5. Interleukin-18 Down-Regulates Multidrug Resistance-Associated Protein 2 Expression through Farnesoid X Receptor Associated with Nuclear Factor Kappa B and Yin Yang 1 in Human Hepatoma HepG2 Cells.

    PubMed

    Liu, Xiao-cong; Lian, Wei; Zhang, Liang-jun; Feng, Xin-chan; Gao, Yu; Li, Shao-xue; Liu, Chang; Cheng, Ying; Yang, Long; Wang, Xiao-Juan; Chen, Lei; Wang, Rong-quan; Chai, Jin; Chen, Wen-sheng

    2015-01-01

    Multidrug resistance-associated protein 2 (MRP2) plays an important role in bile acid metabolism by transporting toxic organic anion conjugates, including conjugated bilirubin, glutathione, sulfate, and multifarious drugs. MRP2 expression is reduced in cholestatic patients and rodents. However, the molecular mechanism of MRP2 down-regulation remains elusive. In this report, we treated human hepatoma HepG2 cells with interleukin-18 (IL-18) and measured the expression of MRP2, nuclear factor kappa B (NF-κB), farnesoid X receptor (FXR), and the transcription factor Yin Yang 1 (YY1) by quantitative real-time quantitative polymerase chain reaction (PCR) and western blotting. We found that expression of MRP2 was repressed by IL-18 at both the mRNA and protein levels in a dose- and time-dependent manner. Furthermore, the activated NF-κB pathway increased YY1 and reduced FXR. These changes were all attenuated in HepG2 cells with knockdown of the NF-κB subunit, p65. The reduced expression of FXR and MRP2 in HepG2 cells that had been caused by IL-18 treatment was also attenuated by YY1 knockdown. We further observed significantly elevated IL-18, NF-κB, and YY1 expression and decreased FXR and MRP2 expression in bile duct-ligated Sprague Dawley rat livers. Chromatin immunoprecipitation assays also showed that FXR bound to the promoter region in MRP2 was less abundant in liver extracts from bile duct-ligated rats than sham-operated rats. Our findings indicate that IL-18 down-regulates MRP2 expression through the nuclear receptor FXR in HepG2 cells, and may be mediated by NF-κB and YY1.

  6. 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

  7. Chronic morphine induces up-regulation of the pro-apoptotic Fas receptor and down-regulation of the anti-apoptotic Bcl-2 oncoprotein in rat brain

    PubMed Central

    Boronat, M Assumpció; García-Fuster, M Julia; García-Sevilla, Jesús A

    2001-01-01

    This study was designed to assess the influence of activation and blockade of the endogenous opioid system in the brain on two key proteins involved in the regulation of programmed cell death: the pro-apoptotic Fas receptor and the anti-apoptotic Bcl-2 oncoprotein. The acute treatment of rats with the μ-opioid receptor agonist morphine (3 – 30 mg kg−1, i.p., 2 h) did not modify the immunodensity of Fas or Bcl-2 proteins in the cerebral cortex. Similarly, the acute treatment with low and high doses of the antagonist naloxone (1 and 100 mg kg−1, i.p., 2 h) did not alter Fas or Bcl-2 protein expression in brain cortex. These results discounted a tonic regulation through opioid receptors on Fas and Bcl-2 proteins in rat brain. Chronic morphine (10 – 100 mg kg−1, 5 days, and 10 mg kg−1, 13 days) induced marked increases (47 – 123%) in the immunodensity of Fas receptor in the cerebral cortex. In contrast, chronic morphine (5 and 13 days) decreased the immunodensity of Bcl-2 protein (15 – 30%) in brain cortex. Chronic naloxone (10 mg kg−1, 13 days) did not alter the immunodensities of Fas and Bcl-2 proteins in the cerebral cortex. The concurrent chronic treatment (13 days) of naloxone (10 mg kg−1) and morphine (10 mg kg−1) completely prevented the morphine-induced increase in Fas receptor and decrease in Bcl-2 protein immunoreactivities in the cerebral cortex. The results indicate that morphine, through the sustained activation of opioid receptors, can promote abnormal programmed cell death by enhancing the expression of pro-apoptotic Fas receptor protein and damping the expression of anti-apoptotic Bcl-2 oncoprotein. PMID:11704646

  8. 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

  9. 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

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

    PubMed

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

    2015-05-01

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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. Down-Regulation of Type I Runx2 Mediated by Dexamethasone Is Required for 3T3-L1 Adipogenesis

    PubMed Central

    Zhang, You-you; Li, Xi; Qian, Shu-wen; Guo, Liang; Huang, Hai-yan; He, Qun; Liu, Yuan; Ma, Chun-gu

    2012-01-01

    Runx2, a runt-related transcriptional factor family member, is involved in the regulation of osteoblast differentiation. Interestingly, it is abundant in growth-arrested 3T3-L1 preadipocytes and was dramatically down-regulated during adipocyte differentiation. Knockdown of Runx2 expression promoted 3T3-L1 adipocyte differentiation, whereas overexpression inhibited adipocyte differentiation and promoted the trans-differentiation of 3T3-L1 preadipocytes to bone cells. Runx2 was down-regulated specifically by dexamethasone (DEX). Only type I Runx2 was expressed in 3T3-L1 preadipocytes. Using luciferase assay and chromatin immunoprecipitation-quantitative PCR analysis, it was found that DEX repressed this type of Runx2 at the transcriptional level through direct binding of the glucocorticoid receptor (GR) to a GR-binding element in the Runx2 P2 promoter. Further studies indicated that GR recruited histone deacetylase 1 to the Runx2 P2 promoter which then mediated the deacetylation of histone H4 and down-regulated Runx2 expression. Runx2 might play its repressive role through the induction of p27 expression, which blocked 3T3-L1 adipocyte differentiation by inhibiting mitotic clonal expansion. Taken together, we identified Runx2 as a new downstream target of DEX and explored a new pathway between DEX, Runx2, and p27 which contributed to the mechanism of the 3T3-L1 adipocyte differentiation. PMID:22422618

  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. The 10,400- and 14,500-dalton proteins encoded by region E3 of adenovirus form a complex and function together to down-regulate the epidermal growth factor receptor.

    PubMed Central

    Tollefson, A E; Stewart, A R; Yei, S P; Saha, S K; Wold, W S

    1991-01-01

    In adenovirus-infected cells, the epidermal growth factor receptor (EGF-R) is internalized from the cell surface via endosomes and is degraded, and the E3 10,400-dalton protein (10.4K protein) is required for this effect (C. R. Carlin, A. E. Tollefson, H. A. Brady, B. L. Hoffman, and W. S. M. Wold, Cell 57:135-144, 1989). We now report that both the E3 10.4K and E3 14.5K proteins are required for this down-regulation of EGF-R in adenovirus-infected cells. Down-regulation of cell surface EGF-R was demonstrated by results from several methods, namely the absence of EGF-R autophosphorylation in an immune complex kinase assay, the inability to iodinate EGF-R on the cell surface, the formation of endosomes containing EGF-R as detected by immunofluorescence, and the degradation of the metabolically [35S]Met-labeled fully processed 170K species of EGF-R. No effect on the initial synthesis of EGF-R was observed. This down-regulation was ascribed to the 10.4K and 14.5K proteins through the analysis of cells infected with rec700 (wild-type), dl748 (10.4K-, 14.5K+), or dl764 (10.4K+, 14.5K-) or coinfected with dl748 plus dl764. Further evidence that the 10.4K and 14.5K proteins function in concert was obtained by demonstrating that the 10.4K protein was coimmunoprecipitated with the 14.5K protein by using three different antisera to the 14.5K protein, strongly implying that the 10.4K and 14.5K proteins exist as a complex. Together, these results indicate that the 10.4K and 14.5K proteins function as a complex to stimulate endosome-mediated internalization and degradation of EGF-R in adenovirus-infected cells. Images PMID:1851870

  2. Galphas-coupled receptor signaling actively down-regulates α4β1-integrin affinity: A possible mechanism for cell de-adhesion

    PubMed Central

    Chigaev, Alexandre; Waller, Anna; Amit, Or; Sklar, Larry A

    2008-01-01

    Background Activation of integrins in response to inside-out signaling serves as a basis for leukocyte arrest on endothelium, and migration of immune cells. Integrin-dependent adhesion is controlled by the conformational state of the molecule (i.e. change in the affinity for the ligand and molecular unbending (extension)), which is regulated by seven-transmembrane Guanine nucleotide binding Protein-Coupled Receptors (GPCRs). α4β1-integrin (CD49d/CD29, Very Late Antigen-4, VLA-4) is expressed on leukocytes, hematopoietic stem cells, hematopoietic cancer cells, and others. Affinity and extension of VLA-4 are both rapidly up-regulated by inside-out signaling through several Gαi-coupled GPCRs. The goal of the current report was to study the effect of Gαs-coupled GPCRs upon integrin activation. Results Using real-time fluorescent ligand binding to assess affinity and a FRET based assay to probe α4β1-integrin unbending, we show that two Gαs-coupled GPCRs (H2-histamine receptor and β2-adrenergic receptor) as well as several cAMP agonists can rapidly down modulate the affinity of VLA-4 activated through two Gαi-coupled receptors (CXCR4 and FPR) in U937 cells and primary human peripheral blood monocytes. This down-modulation can be blocked by receptor-specific antagonists. The Gαs-induced responses were not associated with changes in the expression level of the Gαi-coupled receptors. In contrast, the molecular unbending of VLA-4 was not significantly affected by Gαs-coupled GPCR signaling. In a VLA-4/VCAM-1-specific myeloid cell adhesion system, inhibition of the VLA-4 affinity change by Gαs-coupled GPCR had a statistically significant effect upon cell aggregation. Conclusion We conclude that Gαs-coupled GPCRs can rapidly down modulate the affinity state of VLA-4 binding pocket through a cAMP dependent pathway. This plays an essential role in the regulation of cell adhesion. We discuss several possible implications of this described phenomenon. PMID:18534032

  3. 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.

  4. 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

  5. Fenofibrate down-regulates the expressions of androgen receptor (AR) and AR target genes and induces oxidative stress in the prostate cancer cell line LNCaP

    SciTech Connect

    Zhao, Hu; Zhu, Chen; Qin, Chao; Tao, Tao; Li, Jie; Cheng, Gong; Li, Pu; Cao, Qiang; Meng, Xiaoxin; Ju, Xiaobing; Shao, Pengfei; Hua, Lixin; Gu, Min; Yin, Changjun

    2013-03-08

    Highlights: ► Fenofibrate induces cell cycle arrest in G1 phase and apoptosis in LNCaP cells. ► Fenofibrate reduces the expressions of androgen receptor in LNCaP cells. ► Fenofibrate induces oxidative stress in the prostate cancer cell line LNCaP. -- Abstract: Fenofibrate, a peroxisome proliferator-androgen receptor-alpha agonist, is widely used in treating different forms of hyperlipidemia and hypercholesterolemia. Recent reports have indicated that fenofibrate exerts anti-proliferative and pro-apoptotic properties. This study aims to investigate the effects of fenofibrate on the prostate cancer (PCa) cell line LNCaP. The effects of fenofibrate on LNCaP cells were evaluated by flow cytometry, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assays, Western blot analysis, and dual-luciferase reporter assay. Fenofibrate induces cell cycle arrest in G1 phase and apoptosis in LNCaP cells, reduces the expressions of androgen receptor (AR) and AR target genes (prostate-specific antigen and TMPRSS2), and inhibits Akt phosphorylation. Fenofibrate can induce the accumulation of intracellular reactive oxygen species and malondialdehyde, and decrease the activities of total anti-oxidant and superoxide dismutase in LNCaP cells. Fenofibrate exerts an anti-proliferative property by inhibiting the expression of AR and induces apoptosis by causing oxidative stress. Therefore, our data suggest fenofibrate may have beneficial effects in fenofibrate users by preventing prostate cancer growth through inhibition of androgen activation and expression.

  6. Activation of autophagic flux by epigallocatechin gallate mitigates TRAIL-induced tumor cell apoptosis via down-regulation of death receptors

    PubMed Central

    Park, Sang-Youel

    2016-01-01

    Epigallocatechin gallate (EGCG) is a major polyphenol in green tea. Recent studies have reported that EGCG can inhibit TRAIL-induced apoptosis and activate autophagic flux in cancer cells. However, the mechanism behind these processes is unclear. The present study found that EGCG prevents tumor cell death by antagonizing the TRAIL pathway and activating autophagy flux. Our results indicate that EGCG dose-dependently inhibits TRAIL-induced apoptosis and decreases the binding of death receptor 4 and 5 (DR4 and 5) to TRAIL. In addition, EGCG activates autophagy flux, which is involved in the inhibition of TRAIL cell death. We confirmed that the protective effect of EGCG can be reversed using genetic and pharmacological tools through re-sensitization to TRAIL. The inhibition of autophagy flux affects not only the re-sensitization of tumor cells to TRAIL, but also the restoration of death receptor proteins. This study demonstrates that EGCG inhibits TRAIL-induced apoptosis through the manipulation of autophagic flux and subsequent decrease in number of death receptors. On the basis of these results, we suggest further consideration of the use of autophagy activators such as EGCG in combination anti-tumor therapy with TRAIL. PMID:27582540

  7. Dietary bitter melon seed increases peroxisome proliferator-activated receptor-γ gene expression in adipose tissue, down-regulates the nuclear factor-κB expression, and alleviates the symptoms associated with metabolic syndrome.

    PubMed

    Gadang, Vidya; Gilbert, William; Hettiararchchy, Navam; Horax, Ronny; Katwa, Laxmansa; Devareddy, Latha

    2011-01-01

    The objective of this study was to examine the extent to which bitter melon seed (BMS) alleviates the symptoms associated with metabolic syndrome and elucidate the mechanism by which BMS exerts beneficial effects. Three-month-old female Zucker rats were assigned to following groups: lean control (L-Ctrl), obese control (O-Ctrl), and obese + BMS (O-BMS). The control groups were fed AIN-93M purified rodent diet, and the O-BMS group was fed AIN-93M diet modified to contain 3.0% (wt/wt) ground BMS for 100 days. After 100 days of treatment, BMS supplementation in the obese rats lowered the total serum cholesterol by 38% and low-density lipoprotein-cholesterol levels by about 52% and increased the ratio of serum high-density lipoprotein-cholesterol to total cholesterol compared to the O-Ctrl group. The percentage of total liver lipids was about 32% lower and serum triglyceride levels were 71% higher in the O-BMS group compared to the O-Ctrl group. Serum glucose levels were significantly lowered partly because of the increase in the serum insulin levels in the BMS-based diet groups. BMS supplementation increased the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) in the white adipose tissue of the obese rats significantly (P < .05) and down-regulated the expression of PPAR-γ, nuclear factor-κB (NF-κB), and interferon-γ mRNA in heart tissue of the obese rats. The findings of this study suggest that BMS improves the serum and liver lipid profiles and serum glucose levels by modulating PPAR-γ gene expression. To our knowledge, this study for the first time shows that BMS exerts cardioprotective effects by down-regulating the NF-κB inflammatory pathway.

  8. Neuromodulatory propensity of Bacopa monniera against scopolamine-induced cytotoxicity in PC12 cells via down-regulation of AChE and up-regulation of BDNF and muscarnic-1 receptor expression.

    PubMed

    Pandareesh, M D; Anand, T

    2013-10-01

    Scopolamine is a competitive antagonist of muscarinic acetylcholine receptors, and thus classified as an anti-muscarinic and anti-cholinergic drug. PC12 cell lines possess muscarinic receptors and mimic the neuronal cells. These cells were treated with different concentrations of scopolamine for 24 h and were protected from the cellular damage by pretreatment with Bacopa monniera extract (BME). In current study, we have explored the molecular mechanism of neuromodulatory and antioxidant propensity of (BME) to attenuate scopolamine-induced cytotoxicity using PC12 cells. Our results elucidate that pretreatment of PC12 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by 3 μg/ml scopolamine to 54.83 and 30.30 % as evidenced by MTT and lactate dehydrogenase assays respectively. BME (100 μg/ml) ameliorated scopolamine effect by down-regulating acetylcholine esterase and up-regulating brain-derived neurotropic factor and muscarinic muscarinic-1 receptor expression. BME pretreated cells also showed significant protection against scopolamine-induced toxicity by restoring the levels of antioxidant enzymes and lipid peroxidation. This result indicates that the scopolamine-induced cytotoxicity and neuromodulatory changes were restored with the pretreatment of BME.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. Down-regulation of G protein-coupled receptor 137 by RNA interference inhibits cell growth of two hepatoma cell lines.

    PubMed

    Shao, Xin; Liu, Yong; Huang, Hai; Zhuang, Linyuan; Luo, Tianping; Huang, Huping; Ge, Xinguo

    2015-04-01

    G protein-coupled receptors (GPCRs) are important signal transduction mediators and pharmacological therapeutic targets. G protein-coupled receptor 137 (GPR137) was initially reported as a novel orphan GPCR around 10 years ago. Some orphan GPCRs have been implicated in cancer cell proliferation and migration. The aim of this study is to investigate the role of GPR137 in hepatocellular carcinoma (HCC). GPR137 is widely expressed in several human HCC cell lines, as determined by real-time PCR. We then applied lentivirus mediated RNA interference (RNAi) to knock down GPR137 expression in two HCC cell lines HepG2 and Bel7404. Depletion of GPR137 remarkably inhibited cell proliferation and colony formation capacity. Knockdown of GPR137 in HepG2 cells led to cell cycle arrest at G0/G1 phase and G2/M phase, and induced cell apoptosis, as determined by flow cytometry analysis, which contributed to cell growth inhibition. Our findings suggested that GPR137 could facilitate HCC cell proliferation and thus promote hepatocarcinogenesis.

  14. 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

  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. 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.

  17. BDE-47 causes developmental retardation with down-regulated expression profiles of ecdysteroid signaling pathway-involved nuclear receptor (NR) genes in the copepod Tigriopus japonicus.

    PubMed

    Hwang, Dae-Sik; Han, Jeonghoon; Won, Eun-Ji; Kim, Duck-Hyun; Jeong, Chang-Bum; Hwang, Un-Ki; Zhou, Bingsheng; Choe, Joonho; Lee, Jae-Seong

    2016-08-01

    2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is a persistent organic pollutant (POP) in marine environments. Despite its adverse effects (e.g. developmental retardation) in ecdysozoa, the effects of BDE-47 on transcription of ecdysteroid signaling pathway-involved-nuclear receptor (NR) genes and metamorphosis-related genes have not been examined in copepods. To examine the deleterious effect of BDE-47 on copepod molting and metamorphosis, BDE-47 was exposed to the harpacticoid copepod Tigriopus japonicus, followed by monitoring developmental retardation and transcriptional alteration of NR genes. The developmental rate was significantly inhibited (P<0.05) in response to BDE-47 and the agricultural insecticide gamma-hexachlorocyclohexane. Conversely, the ecdysteroid agonist ponasterone A (PoA) led to decreased molting and metamorphosis time (P<0.05) from the nauplius stage to the adult stage. In particular, expression profiles of all NR genes were the highest at naupliar stages 5-6 except for SVP, FTZ-F1, and HR96 genes. Nuclear receptor USP, HR96, and FTZ-F1 genes also showed significant sex differences (P<0.05) in gene expression levels over different developmental stages, indicating that these genes may be involved in vitellogenesis. NR gene expression patterns showed significant decreases (P<0.05) in response to BDE-47 exposure, implying that molting and metamorphosis retardation is likely associated with NR gene expression. In summary, BDE-47 leads to molting and metamorphosis retardation and suppresses transcription of NR genes. This information will be helpful in understanding the molting and metamorphosis delay mechanism in response to BDE-47 exposure.

  18. 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

  19. MicroRNA 339 down-regulates μ-opioid receptor at the post-transcriptional level in response to opioid treatment

    PubMed Central

    Wu, Qifang; Hwang, Cheol Kyu; Zheng, Hui; Wagley, Yadav; Lin, Hong-Yiou; Kim, Do Kyung; Law, Ping-Yee; Loh, Horace H.; Wei, Li-Na

    2013-01-01

    μ-Opioid receptor (MOR) level is directly related to the function of opioid drugs, such as morphine and fentanyl. Although agonist treatment generally does not affect transcription of mor, previous studies suggest that morphine can affect the translation efficiency of MOR transcript via microRNAs (miRNAs). On the basis of miRNA microarray analyses of the hippocampal total RNA isolated from mice chronically treated with μ-opioid agonists, we found a miRNA (miR-339-3p) that was consistently and specifically increased by morphine (2-fold) and by fentanyl (3.8-fold). miR-339-3p bound to the MOR 3′-UTR and specifically suppressed reporter activity. Suppression was blunted by adding miR-339-3p inhibitor or mutating the miR-339-3p target site. In cells endogenously expressing MOR, miR-339-3p inhibited the production of MOR protein by destabilizing MOR mRNA. Up-regulation of miR-339-3p by fentanyl (EC50=0.75 nM) resulted from an increase in primary miRNA transcript. Mapping of the miR-339-3p primary RNA and its promoter revealed that the primary miR-339-3p was embedded in a noncoding 3′-UTR region of an unknown host gene and was coregulated by the host promoter. The identified promoter was activated by opioid agonist treatment (10 nM fentanyl or 10 μM morphine), a specific effect blocked by the opioid antagonist naloxone (10 μM). Taken together, these results suggest that miR-339-3p may serve as a negative feedback modulator of MOR signals by regulating intracellular MOR biosynthesis.—Wu, Q., Hwang, C. K., Zheng, H., Wagley, Y., Lin, H.-Y., Kim, D. K., Law, P.-Y., Loh, H. H., Wei, L.-N. MicroRNA 339 downregulates mu opioid receptor at the post-transcriptional level in response to opioid treatment. PMID:23085997

  20. CB1 receptors down-regulate a cAMP/Epac2/PLC pathway to silence the nerve terminals of cerebellar granule cells.

    PubMed

    Alonso, Beatris; Bartolomé-Martín, David; Ferrero, José Javier; Ramírez-Franco, Jorge; Torres, Magdalena; Sánchez-Prieto, José

    2017-08-01

    Cannabinoid receptors mediate short-term retrograde inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at excitatory synapses. The responses of individual nerve terminals in VGLUT1-pHluorin transfected cerebellar granule cells to cannabinoids have shown that prolonged activation of cannabinoid type 1 receptors (CB1Rs) silences a subpopulation of previously active synaptic boutons. Adopting a combined pharmacological and genetic approach to study the molecular mechanisms of CB1R-induced silencing, we found that adenylyl cyclase inhibition decreases cAMP levels while it increases the number of silent synaptic boutons and occludes the induction of further silencing by the cannabinoid agonist HU-210. Guanine nucleotide exchange proteins directly activated by cAMP (Epac proteins) mediate some of the presynaptic effects of cAMP in the potentiation of synaptic transmission. ESI05, a selective Epac2 inhibitor, and U-73122, the specific inhibitor of phospholipase C (PLC), both augment the number of silent synaptic boutons. Moreover, they abolish the capacity of the Epac activator, 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate, to prevent HU-210-induced silencing consistent with PLC signaling lying downstream of Epac2 proteins. Furthermore, Rab3-interacting molecule (RIM)1α KO cells have many more basally silent synaptic boutons (12.9 ± 3.5%) than wild-type cells (1.1 ± 0.5%). HU-210 induced further silencing in these mutant cells, although 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate only awoke the HU-210-induced silence and not the basally silent synaptic boutons. This behavior can be rescued by expressing RIM1α in RIM1α KO cells, these cells behaving very much like wild-type cells. These findings support the hypothesis that a cAMP/Epac/PLC signaling pathway targeting the release machinery appears to mediate cannabinoid

  1. [Efficacy and Safety of the Selective Estrogen Receptor Down-Regulator "Fulvestrant" in Japanese Patients with Advanced, Recurrent, ER-Positive Postmenopausal Breast Cancer].

    PubMed

    Egawa, Chiyomi; Okishiro, Masatsugu; Takatsuka, Yuichi

    2015-07-01

    Fulvestrant is a novel endocrine therapy for breast cancer that exerts both anti-estrogenic and down-regulatory effects by binding to and degrading estrogen receptors (ERs). In the present study, the safety and effectiveness of 500 mg fulvestrant in 69 patients with advanced, recurrent, ER-positive postmenopausal breast cancer were investigated retrospectively. Outcomes were favorable for fulvestrant. The objective response rate was 24.6%, the clinical benefit rate was 49.2%, the median progression-free survival (PFS) was 203 days, and the median overall survival was 794 days. PFS tended to be longer in patients without a history of previous treatment or visceral metastasis. The main adverse events included injection site reactions and hot flushes; however, the majority of these events were mild to moderate. The present findings suggest that, among Japanese patients with advanced, recurrent, ER-positive postmenopausal breast cancer, 500 mg fulvestrant is effective and safe in those without metastasis and a minimal history of receipt of previous treatment regimens.

  2. Down-regulation of tryptamine binding sites following chronic molindone administration. A comparison with responses of dopamine and 5-hydroxytryptamine receptors.

    PubMed

    Nguyen, T V; Juorio, A V

    1989-10-01

    The present study assessed changes of tryptamine, dopamine D2, 5-HT1 and 5-HT2 binding sites in rat brain following chronic treatment with low (5 mg/kg/day) and high (40 mg/kg/day) doses of molindone, a clinically effective psychotropic drug. The high-dose molindone treatment produced a decrease in the number of tryptamine binding sites while both high and low doses caused an increase in the number of dopamine D2 binding sites in the striatum. No significant changes were observed in either 5-HT1 or 5-HT2 binding sites in the cerebral cortex. Competition binding experiments showed that molindone was a potent inhibitor at dopamine D2 but less effective at tryptamine, 5-HT1 and 5-HT2 binding sites. The inhibition activity of molindone towards type A monoamine oxidase produced a significant increase in endogenous tryptamine accumulation rate which was much higher than that of dopamine and 5-HT. These findings suggest that the reduction in the number of tryptamine binding sites produced by chronic molindone administration is related to monoamine oxidase inhibition and that the increase in the number of dopamine D2 binding sites is correlated to receptor blocking activity of the drug.

  3. Simvastatin inhibits the proliferation of human prostate cancer PC-3 cells via down-regulation of the insulin-like growth factor 1 receptor

    SciTech Connect

    Sekine, Yoshitaka Furuya, Yosuke; Nishii, Masahiro; Koike, Hidekazu; Matsui, Hiroshi; Suzuki, Kazuhiro

    2008-07-25

    Recently, statins have been being studied for their proapoptic and antimetastatic effects. However, the exact mechanisms of their anticancer action are still unclear. Dolichyl phosphate is a nonsterol isoprenoid derivative in the mevalonate pathway that affects the expression of the Insulin-like growth factor 1 receptor (IGF-1R). IGF-1R activation is required for prostate cell proliferation; therefore, IGF-1R inhibitory agents may be of preventive and/or therapeutic value. In this study, the effects of simvastatin on IGF-1R signaling in prostate cancer PC-3 cells were examined. Simvastatin suppressed proliferation and induced apoptosis of PC-3, and the expression of IGF-1R was suppressed by simvastatin. Knockdown of IGF-1R by siRNA led to inhibition of proliferation of PC-3. Simvastatin also inhibited IGF-1-induced activation of both ERK and Akt signaling and IGF-1-induced PC-3 cell proliferation. Our results suggest statins are potent inhibitors of the IGF-1/IGF-1R system in prostate cancer cells and may be beneficial in prostate cancer treatment.

  4. Distal-less homeobox 5 inhibits adipogenic differentiation through the down-regulation of peroxisome proliferator-activated receptor γ expression.

    PubMed

    Lee, Hye-Lim; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa

    2013-01-01

    Distal-less homeobox 5 (Dlx5) is a positive regulator of osteoblast differentiation that contains a homeobox domain. Because there are possible reciprocal relationships between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (MSCs), we examined the regulatory role of Dlx5 in adipogenic differentiation in this study. Adipogenic stimuli suppressed the expression levels of Dlx5 mRNA in mouse bone marrow stromal cells. Over-expression of Dlx5 inhibited adipogenic differentiation in human bone marrow MSCs and 3T3-L1 preadipocytic cells whereas knockdown of Dlx5 enhanced adipogenic differentiation in 3T3-L1 cells. Over-expression of Dlx5 suppressed the expression of adipogenic marker genes, including CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ). Dlx5-mediated suppression of adipogenic differentiation was overcome by over-expression of PPARγ but not by that of cAMP response element binding protein (CREB) or C/EBPα. Dlx5 decreased the transcriptional activity of CREB and C/EBPα in a dose-dependent manner. Dlx5 directly bound to CREB and C/EBPα and prevented them from binding to and subsequently transactivating the PPARγ promoter. These results suggest that Dlx5 plays an important regulatory role in fate determination of bone marrow MSCs toward the osteoblast lineage through the inhibition of adipocyte differentiation as well as the direct stimulation of osteoblast differentiation.

  5. 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.

  6. 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…

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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…

  12. 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

  13. Down-regulation of adenosine A1 and A2A receptors in peripheral cells from idiopathic normal-pressure hydrocephalus patients.

    PubMed

    Casati, Martina; Arosio, Beatrice; Gussago, Cristina; Ferri, Evelyn; Magni, Lorenzo; Assolari, Lara; Scortichini, Valeria; Nani, Carolina; Rossi, Paolo Dionigi; Mari, Daniela

    2016-02-15

    Idiopathic normal-pressure hydrocephalus (iNPH) is a neurological disease that usually develops in the elderly. Natural history of iNPH is still unknown. It has been hypothesized that cerebrovascular diseases could have a role in etiology of chronic hydrocephalus and studies show an increased prevalence of cardiovascular diseases in iNPH patients. Moreover, evidences show a possible alteration of immune system in iNPH patients. Adenosine (Ado) is a metabolite produced in response to metabolic stress and injury. Adenosine and its receptors play an important role in vascular protection and in the modulation of inflammatory reactions and neuroinflammation. Our aim is to evaluate gene and protein expression of A1R and A2AR in the peripheral blood mononuclear cells (PBMCs) from iNPH patients compared to control subjects. We investigate if Ado system, that plays an important role in central nervous system, in vascular system, and also in inflammation, is involved in pathophysiology of iNPH disease. Our analysis showed that A1R mRNA levels and A1R density in PBMCs from iNPH patients were significantly lower than CT subjects (0.84 ± 0.12 and 2.42 ± 0.42, p<0.001 and 0.31 ± 0.02 and 0.42 ± 0.04, p=0.043; respectively). About A2AR, the gene expression in PBMCs was significantly lower in iNPH than CT (0.65 ± 0.09 and 1.5 ± 0.14, p<0.001) as well as there was a trend in protein expression: iNPH and CT (0.51 ± 0.05 and 0.62 ± 0.03; p=0.172). This preliminary study underlines the involvement of Ado system in iNPH disease whose pathophysiology is still unclear.

  14. Down-regulation of intestinal scavenger receptor class B, type I (SR-BI) expression in rodents under conditions of deficient bile delivery to the intestine.

    PubMed Central

    Voshol, P J; Schwarz, M; Rigotti, A; Krieger, M; Groen, A K; Kuipers, F

    2001-01-01

    Scavenger receptor class B, type I (SR-BI) is expressed in the intestines of rodents and has been suggested to be involved in the absorption of dietary cholesterol. The aim of this study was to determine whether intestinal SR-BI expression is affected in animal models with altered bile delivery to the intestine and impaired cholesterol absorption. SR-BI protein and mRNA levels were determined in proximal and distal small intestine from control, bile-duct-ligated and bile-diverted rats and from control and bile-duct-ligated mice. Two genetically altered mouse models were studied: multidrug resistance-2 P-glycoprotein-deficient [Mdr2((-/-))] mice that produce phospholipid/cholesterol-free bile, and cholesterol 7alpha-hydroxylase-deficient [Cyp7a((-/-))] mice, which exhibit qualitative and quantitative changes in the bile-salt pool. Cholesterol-absorption efficiency was quantified using a dual-isotope ratio method. SR-BI was present at the apical membrane of enterocytes in control rats and mice and was more abundant in proximal than in distal segments of the intestine. In bile-duct-ligated animals, levels of SR-BI protein were virtually absent and mRNA levels were decreased by approximately 50%. Bile-diverted rats, Mdr2((-/-)) mice and Cyp7a((-/-)) mice showed decreased levels of intestinal SR-BI protein while mRNA levels were unaffected. Cholesterol absorption was reduced by >90% in bile-duct-ligated and bile-diverted animals and in Cyp7a((-/-)) mice, whereas Mdr2((-/-)) mice showed an approximately 50% reduction. This study shows that SR-BI is expressed at the apical membrane of enterocytes of rats and mice, mainly in the upper intestine where cholesterol absorption is greatest, and indicates that bile components play a role in post-transcriptional regulation of SR-BI expression. Factors associated with cholestasis appear to be involved in transcriptional control of intestinal SR-BI expression. The role of SR-BI in the cholesterol-absorption process remains to be

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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

  3. The coupling of epidermal growth factor receptor down regulation by 1alpha,25-dihydroxyvitamin D3 to the hormone-induced cell cycle arrest at the G1-S checkpoint in ovarian cancer cells.

    PubMed

    Shen, Zheng; Zhang, Xiaohui; Tang, Jinfu; Kasiappan, Ravi; Jinwal, Umesh; Li, Pengfei; Hann, Shan; Nicosia, Santo V; Wu, Jie; Zhang, Xiaohong; Bai, Wenlong

    2011-05-16

    1alpha,25-dihydroxyvitamin D3, 1,25(OH)(2)D(3), regulates gene expression through the vitamin D receptor. The present studies identify the epidermal growth factor receptor, EGFR, as a target gene suppressed by 1,25(OH)(2)D(3) in human ovarian cancer cells. The suppression was detected at both mRNA and protein levels in vitamin D-sensitive human ovarian cancer cells. A novel vitamin D response element was identified in intron 1 of the EGFR genome, a known hotspot for its transcriptional regulation. Chromatin immunoprecipitations and reporter gene analyses showed that the intronic DNA element bound to vitamin D receptor and a co-repressor and was functional in mediating transcriptional suppression of EGFR promoter by 1,25(OH)(2)D(3) under stable transfection conditions. Consistent with the EGFR down regulation, 1,25(OH)(2)D(3) suppressed activation of the external signal regulated kinase by epidermal growth factors. Over expression of an active EGFR in vitamin D sensitive ovarian cancer cells caused resistance to 1,25(OH)(2)D(3)-induced growth suppression and diminished the hormonal regulation of cyclin D1, cyclin E, Skp2 and p27, a group of cell cycle regulators that mediate 1,25(OH)(2)D(3)-induced cell cycle arrest at G1-S checkpoint. Taken together, our studies demonstrate that 1,25(OH)(2)D(3) suppresses the response of human ovarian cancer cells to mitogenic growth factors and couple the suppression to the cell cycle arrest at G1-S checkpoint by the hormone. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  4. 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.

  5. 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.

  6. DT-13, a saponin of dwarf lilyturf tuber, exhibits anti-cancer activity by down-regulating C-C chemokine receptor type 5 and vascular endothelial growth factor in MDA-MB-435 cells.

    PubMed

    Ren-Ping, Zhao; Sen-Sen, Lin; Yuan, Sheng-Tao; Yu, Bo-Yang; Bai, Xian-Shu; Sun, Li; Zhang, Lu-Yong

    2014-01-01

    To investigate the anticancer activity of DT-13 under normoxia and determine the underlying mechanisms of action. MDA-MB-435 cell proliferation, migration, and adhesion were performed to assess the anticancer activity of DT-13, a saponin from Ophiopogon japonicus, in vitro. In addition, the effects of DT-13 on tumor growth and metastasis in vivo were evaluated by orthotopic implantation of MDA-MB-435 cells into nude mice; mRNA levels of vascular endothelial growth factor (VEGF), C-C chemokine receptor type 5 (CCR5) and hypoxia-inducible factor 1α (HIF-1α) were evaluated by real-time quantitative PCR; and CCR5 protein levels were detected by Western blot assay. At 0.01 to 1 μmol·L(-1), DT-13 inhibited MDA-MB-435 cell proliferation, migration, and adhesion significantly in vitro. DT-13 reduced VEGF and CCR5 mRNAs, and decreased CCR5 protein expression by down-regulating HIF-1α. In addition, DT-13 inhibited MDA-MB-435 cell lung metastasis, and restricted tumor growth slightly in vivo. DT-13 inhibited MDA-MB-435 cell proliferation, adhesion, and migration in vitro, and lung metastasis in vivo by reducing VEGF, CCR5, and HIF-1α expression. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  7. Ferulic acid attenuated acetaminophen-induced hepatotoxicity though down-regulating the cytochrome P 2E1 and inhibiting toll-like receptor 4 signaling-mediated inflammation in mice

    PubMed Central

    Yuan, Junhui; Ge, Kuang; Mu, Junhuan; Rong, Jiang; Zhang, Li; Wang, Bin; Wan, Jingyuan; Xia, Gong

    2016-01-01

    Ferulic acid (FA), a phenolic acid which is abundant in vegetables and fruits, has been reported to exert anti-oxidative and anti-inflammatory activities. In the present study, the pharmacological effects and the underlying mechanisms of FA in mice with acetaminophen-induced hepatotoxicity were investigated. Our results revealed that FA pretreatment inhibited the augments of serum aminotransferases in a dose-dependent manner and attenuated the hepatic histopathological abnormalities and hepatocellular apoptosis in acetaminophen (APAP) exposed mice. Moreover, FA inhibited the expression of cytochrome P450 2E1 (CYP2E1), enhanced the activities of superoxide dismutase (SOD) and catalase (CAT) as well as the contents of glutathione (GSH). Furthermore, FA markedly attenuated acetaminophen-induced serum tumor necrosis factor (TNF)-α and interleukin (IL)-1β production, suppressed Toll-like receptor (TLR) 4 expression and dampened p38 mitogen-activated (MAPK) and nuclear factor kappa (NF-κB) activation. These data suggested that FA could effectively protect against APAP-induced liver injury by down-regulated expression of CYP 2E1 and the suppression of TLR4-mediated inflammatory responses. PMID:27830004

  8. The down-regulation of IL-6-stimulated fibrinogen steady state mRNA and protein levels by human recombinant IL-1 is not PGE2-dependent: effects of IL-1 receptor antagonist (IL-1RA).

    PubMed

    Conti, P; Bartle, L; Barbacane, R C; Reale, M; Sipe, J D

    1995-01-26

    Infections, trauma and inflammatory processes induce a host response with increases in a large group of structurally and functionally diverse plasma proteins. Parental administration of foreign proteins also induce an increase in plasma fibrinogen. Interleukin-6 (IL-6) is a monocyte-derived mediator and has regulatory effects on acute phase protein genes which result in the induction of fibrinogen synthesis in primary hepatocytes, while the addition of interleukin-1 (IL-1) exerts a negative modulating influence on the IL-6-stimulated fibrinogen. In order to understand the mechanisms by which IL-1 inhibits IL-6-stimulated fibrinogen transcription and translation, and since IL-1 is believed to act through PGE2 stimulation, we have studied the influence of PGE2 in IL-6 or IL-1, alone and in combination, on Fg mRNA expression (by Northern blot analysis) and the influence of PGE2, indomethacin, and arachidonic acid on Fg secretion. Moreover, since human recombinant interleukin-1 receptor antagonist (hrIL-1ra) is a strong inhibitor of IL-1 induced IL-1 transcription and translation and has an inhibitory effect on PGE2, we have studied the effects of IL-1ra on the down-regulation of IL-6 stimulated fibrinogen by IL-1, using an Fg ELISA method.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Mitogenicity and down-regulation of high-affinity interleukin 2 receptor by YTA-1 and YTA-2, monoclonal antibodies that recognize 75-kDa molecules on human large granular lymphocytes.

    PubMed Central

    Nakamura, Y; Inamoto, T; Sugie, K; Masutani, H; Shindo, T; Tagaya, Y; Yamauchi, A; Ozawa, K; Yodoi, J

    1989-01-01

    A large number of interleukin 2 receptors lacking the Tac epitope (IL-2R/p75) were found to be constitutively expressed on the human large granular lymphocyte/natural killer cell line YT, which bears inducible IL-2R/p55 associated with Tac antigen. Two anti-YT IgG1 monoclonal antibodies, YTA-1 and YTA-2, recognizing different epitopes of the same 75- to 80-kDa molecule, were established. The 75-kDa antigen recognized by these monoclonal antibodies was strongly expressed on the large granular lymphocytes of normal peripheral blood mononuclear cells and on various lymphoid cell lines bearing IL-2R/p75. The YTA-1 and YTA-2 antibodies were mitogenic and were different from other mitogenic monoclonal antibodies such as anti-T3 (CD3), anti-T11 (CD2), and KOLT-2 (CD28). Further, they down-regulated the high-affinity IL-2R of peripheral blood mononuclear cells within 24 hr in culture. The relationship between the YTA-1/2 antigen and the IL-2R system is discussed. Images PMID:2465549

  10. Persimmon tannin represses 3T3-L1 preadipocyte differentiation via up-regulating expression of miR-27 and down-regulating expression of peroxisome proliferator-activated receptor-γ in the early phase of adipogenesis.

    PubMed

    Zou, Bo; Ge, Zhenzhen; Zhu, Wei; Xu, Ze; Li, Chunmei

    2015-12-01

    Currently, obesity has become a worldwide health problem. Adipocyte differentiation is closely associated with the onset of obesity. Our previous studies suggested that persimmon tannin might be a potent anti-adipogenic dietary bioactive compound. However, the mechanism of persimmon tannin on adipocyte differentiation is still unknown. The purpose of this study was to investigate the effect of persimmon tannin on adipogenic differentiation in 3T3-L1 preadipocytes and the underlying mechanisms. Adipogenic differentiation was induced by cocktail in the presence or absence of persimmon tannin. Intracellular lipid accumulation was determined by Oil red O staining and enzymatic colorimetric methods. Gene expression and protein levels were measured by real time RT-PCR and Western blot. Persimmon tannin inhibited intracellular lipid accumulation markedly, and the inhibitory effect was largely limited to the early stage of adipocyte differentiation. Persimmon tannin suppressed the expression of C/EBPα and peroxisome proliferator-activated receptor-γ (PPARγ), significantly. Furthermore, genes related to lipogenesis, such as sterol regulatory element-binding protein 1, were down-regulated by persimmon tannin. In addition, adipocyte fatty acid binding protein (aP2), which is a target gene of PPARγ, was suppressed by persimmon tannin notably. Correspondingly, the expression of miR-27a and miR-27b were up-regulated by persimmon tannin from Day 2 to Day 8 significantly. Persimmon tannin inhibited adipocyte differentiation through regulation of PPARγ, C/EBPα and miR-27 in early stage of adipogenesis.

  11. Down-Regulation of Ca(2+)-Activated K⁺ Channel KCa1.1 in Human Breast Cancer MDA-MB-453 Cells Treated with Vitamin D Receptor Agonists.

    PubMed

    Khatun, Anowara; Fujimoto, Mayu; Kito, Hiroaki; Niwa, Satomi; Suzuki, Takayoshi; Ohya, Susumu

    2016-12-11

    Vitamin D (VD) reduces the risk of breast cancer and improves disease prognoses. Potential VD analogs are being developed as therapeutic agents for breast cancer treatments. The large-conductance Ca(2+)-activated K⁺ channel KCa1.1 regulates intracellular Ca(2+) signaling pathways and is associated with high grade tumors and poor prognoses. In the present study, we examined the effects of treatments with VD receptor (VDR) agonists on the expression and activity of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, flow cytometry, and voltage-sensitive dye imaging. Treatments with VDR agonists for 72 h markedly decreased the expression levels of KCa1.1 transcripts and proteins in MDA-MB-453 cells, resulting in the significant inhibition of depolarization responses induced by paxilline, a specific KCa1.1 blocker. The specific proteasome inhibitor MG132 suppressed VDR agonist-induced decreases in KCa1.1 protein expression. These results suggest that KCa1.1 is a new downstream target of VDR signaling and the down-regulation of KCa1.1 through the transcriptional repression of KCa1.1 and enhancement of KCa1.1 protein degradation contribute, at least partly, to the antiproliferative effects of VDR agonists in breast cancer cells.

  12. 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.

  13. 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

  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. AZD3514, an oral selective androgen receptor down-regulator in patients with castration-resistant prostate cancer - results of two parallel first-in-human phase I studies.

    PubMed

    Omlin, A; Jones, R J; van der Noll, R; Satoh, T; Niwakawa, M; Smith, S A; Graham, J; Ong, M; Finkelman, R D; Schellens, J H M; Zivi, A; Crespo, M; Riisnaes, R; Nava-Rodrigues, D; Malone, M D; Dive, C; Sloane, R; Moore, D; Alumkal, J J; Dymond, A; Dickinson, P A; Ranson, M; Clack, G; de Bono, J; Elliott, T

    2015-06-01

    AZD3514 is a first-in-class, orally bio-available, androgen-dependent and -independent androgen receptor inhibitor and selective androgen-receptor down-regulator (SARD). In study 1 and 2, castration-resistant prostate cancer (CRPC) patients (pts) were initially recruited into a once daily (QD) oral schedule (A). In study 1, pharmacokinetic assessments led to twice daily (BID) dosing (schedule B) to increase exposure. Study 2 explored a once daily schedule. In study 1, 49 pts were treated with escalating doses of AZD3514 (A 35 pts, B 14 pts). Starting doses were 100 mg (A) and 1000 mg (B). The AZD3514 formulation was switched from capsules to tablets at 1000 mg QD. 2000 mg BID was considered non-tolerable due to grade (G) 2 toxicities (nausea [N], vomiting [V]). No adverse events (AEs) met the dose-limiting toxicity (DLT) definition. Thirteen pts received AZD3514 in study 2, with starting doses of 250 mg QD. The most frequent drug-related AEs were N: G1/2 in 55/70 pts (79 %); G3 in 1 pt (1.4 %); & V: G1/2 in 34/70 pts (49 %) & G3 in 1 pt (1.4 %). PSA declines (≥50 %) were documented in 9/70 patients (13 %). Objective soft tissue responses per RECIST1.1 were observed in 4/24 (17 %) pts in study 1. AZD3514 has moderate anti-tumour activity in pts with advanced CRPC but with significant levels of nausea and vomiting. However, anti-tumour activity as judged by significant PSA declines, objective responses and durable disease stabilisations, provides the rationale for future development of SARD compounds.

  18. NFκB pathway is down-regulated by 1α,25(OH)(2)-vitamin D(3) in endothelial cells transformed by Kaposi sarcoma-associated herpes virus G protein coupled receptor.

    PubMed

    Gonzalez-Pardo, Verónica; D'Elia, Noelia; Verstuyf, Annemieke; Boland, Ricardo; Russo de Boland, Ana

    2012-09-01

    We have previously demonstrated that 1α,25 dihydroxy-vitamin D(3) (1α,25(OH)(2)D(3)) has antiproliferative effects on the growth of endothelial cells transformed by the viral G protein-coupled receptor associated to Kaposi sarcoma (vGPCR). In this work, we have investigated whether 1α,25(OH)(2)D(3) exerts its growth inhibitory effects by inhibiting the Nuclear Factor κ B (NFκB) pathway which is highly activated by vGPCR. Cell proliferation studies demonstrated that 1α,25(OH)(2)D(3), similarly to bortezomib, a proteosome inhibitor that suppresses the activation of NFκB, reduced the proliferation of endothelial cells transformed by vGPCR (SVEC-vGPCR). The activity of NFκB in these cells decreased by 70% upon 1α,25(OH)(2)D(3) treatment. Furthermore, time and dose response studies showed that the hormone significantly decreased NFκB and increased IκBα mRNA and protein levels in SVEC-vGPCR cells, whereas in SVEC only IκBα increased significantly. Moreover, NFκB translocation to the nucleus was inhibited and occurred by a mechanism independent of NFκB association with vitamin D(3) receptor (VDR). 1α,25(OH)(2)D(3)-induced increase in IκBα required de novo protein synthesis, and was independent of MAPK and PI3K/Akt pathways. Altogether, these results suggest that down-regulation of the NFκB pathway is part of the mechanism involved in the antiproliferative effects of 1α,25(OH)(2)D(3) on endothelial cells transformed by vGPCR. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Heat shock protein 70 down-regulates the production of toll-like receptor-induced pro-inflammatory cytokines by a heat shock factor-1/constitutive heat shock element-binding factor-dependent mechanism.

    PubMed

    Ferat-Osorio, Eduardo; Sánchez-Anaya, Aldair; Gutiérrez-Mendoza, Mireille; Boscó-Gárate, Ilka; Wong-Baeza, Isabel; Pastelin-Palacios, Rodolfo; Pedraza-Alva, Gustavo; Bonifaz, Laura C; Cortés-Reynosa, Pedro; Pérez-Salazar, Eduardo; Arriaga-Pizano, Lourdes; López-Macías, Constantino; Rosenstein, Yvonne; Isibasi, Armando

    2014-01-01

    Heat shock protein 70 (Hsp70) is an intracellular chaperone protein with regulatory and cytoprotective functions. Hsp70 can also be found in the extracellular milieu, as a result of active secretion or passive release from damaged cells. The role of extracellular Hsp70 is not fully understood. Some studies report that it activates monocytes, macrophages and dendritic cells through innate immune receptors (such as Toll-like receptors, TLRs), while others report that Hsp70 is a negative regulator of the inflammatory response. In order to address this apparent inconsistency, in this study we evaluated the response of human monocytes to a highly purified recombinant Hsp70. Human peripheral blood monocytes were stimulated with Hsp70, alone or in combination with TLR agonists. Cytokines were quantified in culture supernatants, their mRNAs were measured by RT-PCR, and the binding of transcription factors was evaluated by electrophoretic mobility shift assay (EMSA). Kruskal-Wallis test or one-way or two-way ANOVA were used to analyze the data. The addition of Hsp70 to TLR-activated monocytes down-regulated TNF-α as well as IL-6 levels. This effect was independent of a physical interaction between Hsp70 and TLR agonists; instead it resulted of changes at the TNF-α gene expression level. The decrease in TNF-α expression correlated with the binding of HSF-1 (heat shock transcription factor 1, a transcription factor activated in response to Hsp70) and CHBF (constitutive HSE-binding factor) to the TNF-α gene promoter. Extracellular Hsp70 negatively regulates the production of pro-inflammatory cytokines of monocytes exposed to TLR agonists and contributes to dampen the inflammatory response.

  20. Heat shock protein 70 down-regulates the production of toll-like receptor-induced pro-inflammatory cytokines by a heat shock factor-1/constitutive heat shock element-binding factor-dependent mechanism

    PubMed Central

    2014-01-01

    Background Heat shock protein 70 (Hsp70) is an intracellular chaperone protein with regulatory and cytoprotective functions. Hsp70 can also be found in the extracellular milieu, as a result of active secretion or passive release from damaged cells. The role of extracellular Hsp70 is not fully understood. Some studies report that it activates monocytes, macrophages and dendritic cells through innate immune receptors (such as Toll-like receptors, TLRs), while others report that Hsp70 is a negative regulator of the inflammatory response. In order to address this apparent inconsistency, in this study we evaluated the response of human monocytes to a highly purified recombinant Hsp70. Methods Human peripheral blood monocytes were stimulated with Hsp70, alone or in combination with TLR agonists. Cytokines were quantified in culture supernatants, their mRNAs were measured by RT-PCR, and the binding of transcription factors was evaluated by electrophoretic mobility shift assay (EMSA). Kruskal-Wallis test or one-way or two-way ANOVA were used to analyze the data. Results The addition of Hsp70 to TLR-activated monocytes down-regulated TNF-α as well as IL-6 levels. This effect was independent of a physical interaction between Hsp70 and TLR agonists; instead it resulted of changes at the TNF-α gene expression level. The decrease in TNF-α expression correlated with the binding of HSF-1 (heat shock transcription factor 1, a transcription factor activated in response to Hsp70) and CHBF (constitutive HSE-binding factor) to the TNF-α gene promoter. Conclusion Extracellular Hsp70 negatively regulates the production of pro-inflammatory cytokines of monocytes exposed to TLR agonists and contributes to dampen the inflammatory response. PMID:25053922

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

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

    PubMed Central

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

    2013-01-01

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

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. The E3-14.5K integral membrane protein of adenovirus that is required for down-regulation of the EGF receptor and for prevention of TNF cytolysis is O-glycosylated but not N-glycosylated.

    PubMed

    Krajcsi, P; Tollefson, A E; Wold, W S

    1992-06-01

    The adenovirus E3-14.5K protein is a cytoplasmic integral membrane protein that functions in concert with the E3-10.4K protein to down-regulate the epidermal growth factor receptor and to prevent tumor necrosis factor cytolysis in adenovirus-infected cells. The 14.5K protein migrates as multiple bands in SDS-PAGE, indicating that it undergoes post-translational modification. The 14.5K protein is known to be phosphorylated on serine. We show here that 14.5K can be metabolically labeled with [3H]glucosamine, that the label is labile to alkali, and that the SDS-PAGE band pattern is simplified in a cell line that is defective in O-glycosylation. Thus, 14.5K is O-glycosylated, probably at a single site in the NH2-terminal lumenal domain. The protein was not metabolically labeled with [3H]mannose, and its SDS-PAGE band pattern was not affected by tunicamycin treatment in vivo or endo F treatment in vitro; thus, 14.5K is not N-glycosylated. There was no evidence that the 10.4K protein is glycosylated, and the 10.4K protein was not required for glycosylation of 14.5K. Virtually all 14.5K molecules appear to contain the core disaccharide Gal beta 1-3GalNAc alpha 1-Ser/Thr which is commonly found on mucin-type O-glycoproteins, and neuraminidase digestion experiments indicated that this disaccharide contains terminal sialic acid.

  16. The adenosine A3 receptor agonist Cl-IB-MECA induces cell death through Ca²⁺/ROS-dependent down regulation of ERK and Akt in A172 human glioma cells.

    PubMed

    Kim, Thae Hyun; Kim, Yong Keun; Woo, Jae Suk

    2012-12-01

    Adenosine A(3) receptor (A3AR) is coupled to G proteins that are involved in a variety of intracellular signaling pathways and physiological functions. 2-Chloro-N(6)-(3-iodobenzyl) adenosine-5'-N-methylcarboxamide (Cl-IB-MECA), an agonist of A3AR, has been reported to induce cell death in various cancer cells. However, the effect of CI-IB-MECA on glioma cell growth is not clear. This study was undertaken to examine the effect of CI-IB-MECA on glioma cell viability and to determine its molecular mechanism. CI-IB-MECA inhibited cell proliferation and induced cell death in a dose- and time-dependent manner. Treatment of CI-IB-MECA resulted in an increase in intracellular Ca(2+) followed by enhanced reactive oxygen species (ROS) generation. EGTA and N-acetylcysteine (NAC) blocked the cell death induced by CI-IB-MECA, suggesting that Ca(2+) and ROS are involved in the Cl-IB-MECA-induced cell death. Western blot analysis showed that CI-IB-MECA induced the down-regulation of extracellular signal-regulated kinases (ERK) and Akt, which was prevented by EGTA, NAC, and the A3AR antagonist MRS1191. Transfection of constitutively active forms of MEK, the upstream kinase of ERK, and Akt prevented the cell death. CI-IB-MECA induced caspase-3 activation and the CI-IB-MECA-induced cell death was blocked by the caspase inhibitors DEVD-CHO and z-VAD-FMK. In addition, expression of XIAP and Survivin were decreased in cells treated with Cl-IB-MECA. Collectively, these findings demonstrate that CI-IB-MECA induce a caspase-dependent cell death through suppression of ERK and Akt mediated by an increase in intracellular Ca(2+) and ROS generation in human glioma cells. These suggest that A3AR agonists may be a potential therapeutic agent for induction of apoptosis in human glioma cells.

  17. Presence of a non-neuronal cholinergic system and occurrence of up- and down-regulation in expression of M2 muscarinic acetylcholine receptors: new aspects of importance regarding Achilles tendon tendinosis (tendinopathy).

    PubMed

    Bjur, Dennis; Danielson, Patrik; Alfredson, Håkan; Forsgren, Sture

    2008-02-01

    Limited information is available concerning the existence of a cholinergic system in the human Achilles tendon. We have studied pain-free normal Achilles tendons and chronically painful Achilles tendinosis tendons with regard to immunohistochemical expression patterns of the M(2) muscarinic acetylcholine receptor (M(2)R), choline acetyltransferase (ChAT), and vesicular acetylcholine transporter (VAChT). M(2)R immunoreactivity was detected in the walls of blood vessels. As evidenced via parallel staining for CD31 and alpha-smooth muscle actin, most M(2)R immunoreactivity was present in the endothelium. M(2)R immunoreactivity also occured in tenocytes, which regularly immunoreact for vimentin. The degree of M(2)R immunoreactivity was highly variable, tendinosis tendons that exhibit hypercellularity and hypervascularity showing the highest levels of immunostaining. Immunoreaction for ChAT and VAChT was detected in tenocytes in tendinosis specimens, particularly in aberrant cells. In situ hybridization revealed that mRNA for ChAT is present in tenocytes in tendinosis specimens. Our results suggest that autocrine/paracrine effects occur concerning the tenocytes in tendinosis. Up-regulation/down-regulation in the levels of M(2)R immunoreactivity possibly take place in tenocytes and blood vessel cells during the various stages of tendinosis. The presumed local production of acetylcholine (ACh), as evidenced by immunoreactivity for ChAT and VAChT and the detection of ChAT mRNA, appears to evolve in response to tendinosis. These observations are of importance because of the well-known vasoactive, trophic, and pain-modulating effects that ACh is known to have and do unexpectedly establish the presence of a non-neuronal cholinergic system in the Achilles tendon.

  18. 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.

  19. 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.

  20. 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.

  1. 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

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

    PubMed

    Hochhaus, G; Gonzalez-Rothi, R J; L