Glucocorticoid Receptor Nuclear Translocation Research Articles

Molecular mechanisms of glucocorticoid resistance in splenocytes of socially stressed male mice

Splenocytes from socially stressed male mice display functional glucocorticoid (GC) resistance, viz., the antiproliferative effects of GC on lipopolysaccharide (LPS)-stimulated splenocytes is absent. In this study, we investigated changes in the structure and function of the glucocorticoid receptor (GR) in socially stressed animals. Changes of GR at both DNA and RNA levels were excluded. Reduced GR function was restricted to macrophages (CD11b +) in association with impaired nuclear translocation of GR after GC stimulation. Consequently, GC failed to block the activation of NF-κB in these cells. Thus, impaired nuclear translocation of GR and the lack of transcriptional suppression of NF-κB by GC were identified as the molecular mechanisms responsible for the observed GC resistance in spleens of socially stressed mice.

Prevention of glucocorticoid receptor (GCR) nuclear translocation: A novel mechanism of IL-2-induced steroid resistance in T cells

Prevention of glucocorticoid receptor (GCR) nuclear translocation: A novel mechanism of IL-2-induced steroid resistance in T cells

Dexamethasone-induced thymocytes apoptosis requires glucocorticoid receptor nuclear translocation but not mitochondrial membrane potential transition

Dexamethasone (DEX), a glucocorticoid hormone (GCH) with specificity for the glucocorticoid receptor (GR) induces T lymphocyte and thymocyte apoptosis. DEX-activated thymocyte apoptosis requires a sequence of biochemical events including mRNA and protein synthesis. In particular, GCH treatment induces non-genomic mechanisms, such as for example Ca 2+ mobilization and PI-PLC activation, and genomic mechanisms. Most of these events, including protein synthesis, are required and precede caspase activation. As protein synthesis is required for caspases and apoptosis activation, DEX-induced GR nuclear translocation is necessary for apoptosis. Cell treatment with geldanamycin (GA) inhibits the GR nuclear translocation and consequently, caspases activation and apoptosis. Although DEX treatment induces loss of mitochondrial membrane potential (Δψm) and cytochrome c release, Δψm induction does not correlate with thymocyte apoptosis. In fact, while Cyclosporin-A and the caspase-9 inhibitor, Z-LEHD-FMK, inhibit DEX-induced Δψm, do not influence apoptosis. These data indicate many biochemical events and are activated by DEX treatment of thymocytes and some, but not all, are required for apoptosis.

The Phosphodiesterase Type 4 Inhibitor, Rolipram, Enhances Glucocorticoid Receptor Function

Previous studies have demonstrated that antidepressants can enhance glucocorticoid receptor (GR) translocation and function, possibly through activation of cAMP and downstream cAMP dependent protein kinases. Accordingly, we examined GR function in cells treated with rolipram, a phosphodiesterase (PDE) type 4 inhibitor that antagonizes cAMP breakdown. Compared with vehicle-treated cells, rolipram alone and in combination with dexamethasone significantly enhanced GR function as measured in both mouse L929 cells and rat C6 glioma cells stably transfected with reporter genes driven by upstream glucocorticoid response elements. Rolipram's facilitation of GR function was reversible by the GR antagonist, RU486, and was associated with reduced cytosloic GR binding, indicating rolipram enhancement of GR nuclear translocation. Finally, rolipram potently augmented GR enhancement by the antidepressant, desipramine. These findings broaden the potential pathways by which PDE type 4 inhibitors can influence cellular function and indicate that these agents may have special utility in disorders associated with impaired GR-mediated feedback inhibition.

Antagonism of glucocorticoid receptor transactivity and cell growth inhibition by transforming growth factor-β through AP-1-mediated transcriptional repression

We have examined the interaction of the glucocorticoid receptor (GR) and transforming growth factor-β (TGF-β) signal pathways because of their mutual involvement in the regulation of cell growth, development and differentiation. Most studies of this cross-talk event have focused on the effects of glucocorticoids (GCs) on TGF-β responses. In this work, we show that TGF-β can antagonize dexamethasone (Dex)-mediated growth suppression in mouse fibrosarcoma L929 cells. TGF-β also repressed GR-mediated reporter (pMMTV-CAT) gene expression in a concentration-dependent manner, with an IC 50 of 5 ng/ml of TGF-β. Maximal inhibition (76%) was observed at 10 ng/ml of TGF-β. Conversely, Dex inhibited TGF-β-mediated promoter (p3TP-Lux) activity in these same cells. As TGF-β inhibition of GR-mediated gene expression occurred after Dex-mediated nuclear translocation of GR, we conclude that TGF-β inhibition of GR signaling occurs at the level of GR-mediated transcription activity. However, TGF-β did not repress GR-mediated gene expression using the pGRE 2E1B-CAT minimal promoter construct, suggesting that TGF-β did not inhibit intrinsic GR activity but, rather, required DNA-binding factor(s) distinct from GR. As the MMTV promoter contains several putative AP-1 binding sites, we hypothesized that AP-1, a transcription factor composed of c-jun and c-fos proteins, might be involved in the TGF-β inhibition of GR functions. Curcumin, a potent inhibitor of AP-1 expression, completely abolished the inhibitory effect of TGF-β on GR-mediated gene expression without affecting GR activity in the absence of TGF-β, and this drug blocked TGF-β-induced binding of AP-1 to a response element derived from the MMTV sequence. Furthermore, curcumin abolished TGF-β inhibition of Dex-induced growth suppression. Taken as a whole, our data suggest that TGF-β can antagonize the growth inhibitory properties of GR by blocking GR transactivity at complex promoters through a mechanism involving transcriptional repression by DNA-bound AP-1.

Decrements in nuclear glucocorticoid receptor (GR) protein levels and DNA binding in aged rat hippocampus.

Hippocampal glucocorticoid receptors (GRs) are believed to play a major role in age-related cognitive decline and cellular vulnerability. It has been proposed that these receptors mediate damaging effects of elevated glucocorticoid secretion on cellular function. In the present report we present evidence that intracellular trafficking of the GR is impaired with hippocampal aging, manifest as decreased nuclear translocation and deficient DNA binding. We also present evidence that chaperone proteins responsible for GR nuclear translocation are decreased with hippocampal aging. Age-related nuclear GR decreases are not observed in hypothalamus, indicating regional specificity of trafficking deficits. Aging did not affect nuclear or cytosolic MR levels. These data suggest that GR signaling is diminished, rather than enhanced, during hippocampal aging. Diminished GR signaling capacity may attenuate the beneficial effects of glucocorticoids on hippocampal regulation of memory and stress integration.

Increased glucocorticoid receptor β expression leads to reduced nuclear translocation of glucocorticoid receptor α and impaired steroid responsiveness

Increased glucocorticoid receptor β expression leads to reduced nuclear translocation of glucocorticoid receptor α and impaired steroid responsiveness

Cross-talk between pro-inflammatory transcription factors and glucocorticoids.

Asthma is a chronic inflammatory disease of the airway that is characterized by cellular infiltration and activation. These processes are induced by overexpression of chemokines and cytokines, such as eotaxin, IL-1beta and GM-CSF. These mediators are downstream targets for the transcription factors activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB), which control the expression of most immunomodulatory genes and whose activity and expression are elevated in asthma. Glucocorticoids are the most effective anti-inflammatory drugs used in the treatment of chronic inflammatory diseases such as asthma. They act by binding to a specific glucocorticoid receptor (GR) that on activation translocates to the nucleus and either increases (transactivates) or decreases (transrepresses) the expression of responsive genes. Transrepression is the major mechanism of glucocorticoid action in inhibiting inflammatory gene expression. Thus, the ability of the transcription factors AP-1 and NF-kappaB to induce gene transcription is attenuated by GR. Although only 5-10% of asthmatic subjects are glucocorticoid-insensitive, these subjects account for over 50% of the health-care costs for asthma (> $6 billion per annum). Examining these patients also gives an insight into important aspects of glucocorticoid action in controlling inflammation and into the development of potential new drugs. Biochemical and genomic studies have indicated abnormal induction of the c-Jun N-terminal kinase (JNK) pathway in some of these patients. The ability of most patients to respond to dexamethasone with induction of histone acetylation correlated with nuclear translocation of GR. However, a subgroup of these patients had an inability to correctly interact with the basal transcription complex in spite of high levels of nuclear GR. This suggests that cross-talk between pro- and anti-inflammatory transcription factors may modulate activation of the transcriptional complex and thereby reduce steroid actions.

High level resistance to glucocorticoids, associated with a dysfunctional glucocorticoid receptor, in childhood acute lymphoblastic leukemia cells selected for methotrexate resistance.

The molecular basis for the clinical presentation of broad-range drug resistance in childhood ALL is poorly understood. In this study, high level cross-resistance to the glucocorticoid dexamethasone was encountered in a childhood ALL cell line selected for resistance to methotrexate (CEM MTX-R3). Compared with wild-type (WT) CEM cells, MTX-R3 cells had significantly fewer glucocorticoid binding sites, as well as reduced glucocorticoid receptor protein and mRNA levels. DNA sequencing and restriction fragment-length polymorphism (RFLP) analysis showed that WT cells expressed both a wild-type and a mutant (GR753F) glucocorticoid receptor allele, while MTX-R3 cells expressed only the GR753F allele. Therefore, the cross-resistance of MTX-R3 cells to dexamethasone appeared due to loss of expression of the wild-type glucocorticoid receptor allele. In an effort to gain insight into the underlying basis for the development of cross-resistance to methotrexate and glucocorticoids, glucocorticoid receptor nuclear translocation experiments were carried out. Exposure of WT cells to either dexamethasone or the cytotoxic agents cytarabine and methotrexate caused translocation of the glucocorticoid receptor from the cytoplasm into the nucleus. These data indicate that exposure of childhood ALL cells to cytotoxic agents may result in ligand-independent glucocorticoid receptor activation which, in the context of the outgrowth of drug-resistant cells, could lead to the co-selection of glucocorticoid resistance.

Arsenic Alters the Function of the Glucocorticoid Receptor as a Transcription Factor

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

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

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

Age-associated increase of basal corticosterone levels decreases ED2high, NF-κBhigh activated macrophages

The proportion of cells with a high density of ED2 (ED2high cells) in peritoneal cells from old rats was significantly lower than that from young rats. The expression of major histocompatibility complex class II (MHC class II) molecules, the antigen presentation, production of interleukin (IL)-1beta and IL-6, and nuclear factor-kappaB activity in ED2high cells were markedly higher than those in cells with a low density of ED2 (ED2low cells), although no significant difference was observed in the expression of MHC class II molecules and the antigen presentation between ED2high cells from young and old rats. Meanwhile, basal corticosterone concentration in serum and glucocorticoid (GC) receptor mRNA expression in peritoneal cells increased significantly in old rats. The proportion of ED2high cells was increased by adrenalectomy in young rats. Furthermore, nuclear translocation of GC receptor was observed in ED2low cells, whereas GC receptor was detected in cytoplasmic extracts from ED2high cells. These results suggest that the decrease in functional ED2high macrophages with age results in the age-associated decline of immune responses, which is regulated, in part, by the basal GC concentration.

Alteration of the glucocorticoid receptor subcellular localization by non steroidal compounds

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.

グルココルチコイド受容体の細胞質‐核間輸送の制御機構

Glucocorticoids are not only essential hormones to maintain homeostasis but also effective drugs for controlling inflammatory disorders. Glucocorticoids inhibit expression of proinflammatory genes such as cytokines, chemokines, and adhesion molecules. The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR) . The GR is localized in the cytoplasm in the absence of hormonal ligand. Upon binding ligand, the GR dissociates from molecular chaperones such as heat shock proteins and translocates into the nucleus. The GR interacts with other transcription factors such as NF-κB and AP-1 which activate proinflammatory genes and inhibits inflammation through negative regulation of these transcription factors and/or competition with these transcription factors for association with coactivators. Therefore, nuclear translocation of the GR is an important step for glucocorticoid action and is controlled by nuclear localization signals within the GR molecule and various cellular factors. It is suggested that cellular oxidation-reduction (redox) status also regulates nuclear translocation of GR by targeting the cysteine within the NL1. We demonstrated that various functions of GR are regulated by redox status.Taken together, it is important to understand the mechanisms of redox dependent regulation of GR function, to aid development of novel drugs and therapies for various disorders including inflammatory diseases.

Subcellular localization of glucocorticoid receptor protein in the human kidney glomerulus

The detailed mechanisms of glucocorticoid action in idiopathic nephrotic syndrome and progressive glomerulonephritides have not been clearly elucidated. The pharmacological actions of glucocorticoids are mediated by their binding to an intracellular protein, the glucocorticoid receptor (GR). The determination of GR localization in normal glomerular cells is essential to elucidate the mechanisms of glucocorticoid action in various glomerular diseases. We carried out an immunoblot examination using antihuman GR-specific antibody and homogenates of isolated normal human glomeruli and mesangial cells in culture. Immunohistochemical examinations were also performed on normal human kidney specimens at light and electron microscopic levels. The nuclear translocation of GRs elicited by ligand binding was further investigated by confocal laser-scanning microscopic inspection of freshly isolated glomeruli and mesangial cells cultured with dexamethasone. An immunoblot examination demonstrated the presence of a 94 kDa protein, a molecular weight consistent with that of GRs, in the homogenates of glomeruli and cultured mesangial cells. By light microscopic examination, GRs were strongly detected in the nucleus and moderately in the cytoplasm of all glomerular cells, parietal and visceral epithelial cells, endothelial cells, and mesangial cells. By electron microscopic examination, the nuclear GRs of all glomerular cells were found to be diffusely distributed in the euchromatin. Additionally, the immunofluorescence intensities of nuclear GRs in isolated glomeruli and mesangial cells in culture became more intense by the addition of dexamethasone. Our findings suggest that all subsets of human glomerular cells definitely express the GR protein, which potentially undergoes translocation by glucocorticoids.

Involvement of glucocorticoid receptor in the induction of differentiation by ginsenosides in F9 teratocarcinoma cells

We have previously reported that ginsenosides Rh1 and Rh2 induced the differentiation of F9 teratocarcinoma stem cells [Lee, Y. N., Lee, H. Y., Chung, H. Y., Kim, S. I., Lee, S. K., Park, B. C. and Kim, K. W., In vitro induction of differentiation by ginsenosides in F9 teratocarcinoma cells. Eur. J. Cancer 1996, 32, 1420–1428.]. Since the chemical structure of Rh1 and Rh2 is very similar to that of dexamethasone, a synthetic glucocorticoid, we investigated whether Rh1 and Rh2 act through the glucocorticoid receptor (GR). Immunocytochemistry showed that Rh1 or Rh2 increased the nuclear translocation of GR in the same manner of dexamethasone. In the gel shift assay, glucocorticoid response element (GRE) binding protein in F9 cells was increased by Rh1 or Rh2. To confirm whether the increased binding protein is GR, we performed the competition assay with unlabeled GRE as a specific competitor. Moreover, supershift assay with the GR antibody showed that the binding proteins are GR. In addition, to confirm the Rh1 or Rh2-induced transactivation of GRE promoter, we cotransfected GR expression vector and GRE-luciferase vector. In the luciferase assay, Rh1 or Rh2 potently induced luciferase activity and this induction was blocked by RU486, a potent GR antagonist. Taken together, we suggest that ginsenosides Rh1 and Rh2 may induce the differentiation of F9 cells by stimulating the nuclear translocation of GR.

Ursolic acid-induced down-regulation of MMP-9 gene is mediated through the nuclear translocation of glucocorticoid receptor in HT1080 human fibrosarcoma cells.

We have previously reported that ursolic acid, a pentacyclic triterpene acid, inhibited the invasion of HT1080 human fibrosarcoma cells by reducing the expression of matrix metalloproteinase-9. Since the chemical structure of ursolic acid is very similar to that of dexamethasone, a synthetic glucocorticoid, we investigated whether ursolic acid acts through the glucocorticoid receptor. The expression of matrix metalloproteinase-9 is thought to be regulated similarly with matrix metalloproteinase-1 and matrix metalloproteinase-3 as containing common 2-O-tetradecanoylphorbol-acetate responsible region, where AP-1 proteins can bind. Dexamethasone has been studied to repress the 2-O-tetradecanoylphorbol-acetate-induced expression of matrix metalloproteinase-1 and matrix metalloproteinase-3 through a glucocorticoid receptor-mediated manner. In Northern blot analysis, we found that ursolic acid reduced the expression of matrix metalloproteinase-1 and matrix metalloproteinase-3 induced by 2-O-tetradecanoylphorbol-acetate. Similarly, ursolic acid down-regulated 2-O-tetradecanoylphorbol-acetate-induction of matrix metalloproteinase-9 gene in the same manner of dexamethasone. RU486, a potent glucocorticoid receptor antagonist, was used for identifying that ursolic acid-induced down-regulation of matrix metalloproteinase-9 expression is mediated by its binding to glucocorticoid receptor. The effect of ursolic acid on the matrix metalloproteinase-9 expression was blocked by RU486, suggesting that ursolic acid acts via a glucocorticoid receptor in the regulation of matrix metalloproteinase-9. Western blot analysis and immunocytochemistry showed that ursolic acid increased glucocorticoid receptor fraction in the nucleus, although it decreased the synthesis of glucocorticoid receptor mRNA. In addition, ursolic acid did not decrease the expression of c-jun and DNA-binding activity of AP-1 to its cognate sequences. Taken together, we suggest that ursolic acid may induce the repression of matrix metalloproteinase-9 by stimulating the nuclear translocation of glucocorticoid receptor, and the translocated glucocorticoid receptor probably down-modulating the trans-activating function of AP-1 to 2-O-tetradecanoylphorbol-acetate responsible element of matrix metalloproteinase-9 promoter region.

The antibiotic rifampicin is a nonsteroidal ligand and activator of the human glucocorticoid receptor.

The glucocorticoid receptor (GR) belongs to a superfamily of ligand-regulated nuclear steroid hormone receptors. The steps in the signal transduction pathway leading to the biological effects of glucocorticoids (GCs) include sequentially binding of the steroid to the GR ligand binding domain (LBD), receptor transformation, nuclear translocation and either positive or negative gene transactivation. Rifampicin (RIF) is a macrocyclic antibiotic used as an antituberculosis agents. As the incidence of tuberculosis has been increasing, in part because of the AIDS epidemic, a growing number of patients are being exposed to the adverse effects of this antibiotic. Indeed, this compound, as are the GCs, is often implicated in noxious drug interactions, because of its strong ability to induce drug-metabolizing enzymes. Moreover, in humans, RIF, as are the GCs, has been described as a potential immunodepressor, associated notably with the reduction of mitogenic responsiveness of human peripheral blood lymphocytes. Here, we report that RIF activates the human glucocorticoid receptor (hGR). Transient expression of wild-type, deleted or mutated GRs; sucrose density gradient sedimentation; and the BIAcore technique strongly suggest that RIF binds to the receptor with the physiological consequence that this antibiotic acts as an immunodepressor. Given the wide use of RIF in the treatment of coinfection of tuberculosis and HIV, this report is highly relevant to current medical practice.

In vivo evidence for the generation of a glucocorticoid receptor-heat shock protein-90 complex incapable of binding hormone by the calmodulin antagonist phenoxybenzamine.

The glucocorticoid receptor (GR) is a ligand-regulated transcription factor whose ability to bind hormone is thought to be dependent on association with the 90-kDa heat shock protein (hsp90). In the present study, we have generated a novel form of the GR, in which the receptor remains complexed to hsp90 but has lost its ability to bind hormone, by treatment of intact cells with the calmodulin (CaM) antagonist phenoxybenzamine (POBA). Treatment of these cells, mouse L929 cells stably transfected with the mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter construct, with increasing concentrations of POBA resulted in a concentration-dependent inhibition of dexamethasone (Dex)-induced CAT gene expression, with 100 microM POBA resulting in approximately 80% inhibition. This inhibitory effect of POBA was markedly reduced if POBA was added after a short incubation with Dex, suggesting that the primary effect of POBA was on hormone-induced transformation of the GR. Using a subcellular fractionation technique, POBA inhibition of CAT gene expression was found to correlate with an inhibition of Dex-induced GR nuclear translocation. However, inhibition of translocation was not the primary effect of POBA on the GR signal pathway, as POBA was found to reduce GR hormone-binding capacity after treatment of intact cells. The inhibitory effect of POBA on hormone-binding function correlated closely with the inhibitory effect of this drug on CAT gene expression and was not due to an oxidation of sulfhydryl groups, a condition known to reduce GR hormone-binding capacity. Incubation of cytosols from untreated cells with POBA did not decrease GR steroid-binding capacity, demonstrating that this inhibitory effect was not the result of a competitive antagonism at the ligand-binding site. Quantitation of GR protein in the cytosols of POBA-treated cells revealed that the decrease in steroid-binding function was not due to a loss of GR protein. Surprisingly, the amount of GR-bound hsp90 was also unaltered in response to POBA. Taken together, the above observations provide evidence for a novel state of the GR within intact cells in which hsp90 interaction is but one step in the generation or maintenance of hormone-competent receptors. In addition, these results point to the potential use of POBA, and possibly other CaM inhibitors, as antagonists of steroid receptor actions.

Cyclosporin A and FK506 are potent activators of proopiomelanocortin-derived peptide secretion without affecting corticotrope glucocorticoid receptor function.

Unliganded glucocorticoid receptors (GR) are localized in the cytoplasm and are associated with heat shock protein (hsp)90, hsp70, and a member of the immunophilin family, FK506 binding protein 59 (FKBP59). Several members of the cyclophilin and FKBP families have now been shown to associate with unactivated steroid receptors, however the physiological role these immunophilins play in steroid receptor function is questionable. In the present study we have measured GR binding and nuclear translocation of activated receptor in corticotrope cells following treatment with the immunophilin ligands FK506 and cyclospcrin A (CsA). Extensive GR binding studies in AtT20 cells, a mouse corticotrope tumor cell line failed to demonstrate an effect of FK506 or CsA on either the ability of GR to bind ligand, or on nuclear translocation of the liganded receptor at either a saturating or subsaturating dose of dexamethasone (DEX). Consistent with the binding data, functionally, neither CsA nor FK506 altered the glucocorticoid induced decrease in either proopiomelanocortin (POMC) derived peptide secretion or POMC heteronuclear (hn) RNA expression. Despite the fact these drugs did not modulate the actions of glucocorticoids on corticotrope cells, both FK506 and CsA were potent stimulators of basal beta-endorphin secretion (4-6 fold) from rat anterior pituitary cultures and AtT20 cells. In addition, FK506 and CsA potentiated beta-endorphin secretion induced by corticotropin releasing factor (CRF) and phorbol ester, but had no apparent acute (60 min) effect on POMC hnRNA levels. Unlike the acute actions of these immunosuppressant drugs, chronic (24 h) treatment lead to a decrease in cytoplasmic POMC mRNA with no apparent change in the amount of secreted beta-endorphin. Taken together these data suggest that FK506 and CsA do not alter GR activation or function in corticotrope cells, however, they are potent but short lived stimulators of POMC-derived peptide secretion. The observation that CsA and FK506 stimulate POMC-derived peptide secretion, and potentiate both phorbol ester and CRF induced secretion, suggests that these immunosuppressant drugs are acting upon a common point within these intracellular pathways.