DEX-induced Glucocorticoid Receptor Research Articles

Crosstalk between glucocorticoid and mineralocorticoid receptors boosts glucocorticoid-induced killing of multiple myeloma cells

The glucocorticoid receptor (GR) is a crucial drug target in multiple myeloma as its activation with glucocorticoids effectively triggers myeloma cell death. However, as high-dose glucocorticoids are also associated with deleterious side effects, novel approaches are urgently needed to improve GR action in myeloma. Here, we reveal a functional crosstalk between GR and the mineralocorticoid receptor (MR) that plays a role in improved myeloma cell killing. We show that the GR agonist dexamethasone (Dex) downregulates MR levels in a GR-dependent way in myeloma cells. Co-treatment of Dex with the MR antagonist spironolactone (Spi) enhances Dex-induced cell killing in primary, newly diagnosed GC-sensitive myeloma cells. In a relapsed GC-resistant setting, Spi alone induces distinct myeloma cell killing. On a mechanistic level, we find that a GR–MR crosstalk likely arises from an endogenous interaction between GR and MR in myeloma cells. Quantitative dimerization assays show that Spi reduces Dex-induced GR–MR heterodimerization and completely abolishes Dex-induced MR–MR homodimerization, while leaving GR–GR homodimerization intact. Unbiased transcriptomics analyses reveal that c-myc and many of its target genes are downregulated most by combined Dex-Spi treatment. Proteomics analyses further identify that several metabolic hallmarks are modulated most by this combination treatment. Finally, we identified a subset of Dex-Spi downregulated genes and proteins that may predict prognosis in the CoMMpass myeloma patient cohort. Our study demonstrates that GR–MR crosstalk is therapeutically relevant in myeloma as it provides novel strategies for glucocorticoid-based dose-reduction.

Identification of dicyclohexyl phthalate as a glucocorticoid receptor antagonist by molecular docking and multiple in vitro methods.

The potential activities of phthalate esters (PAEs) that interfere with the endocrine system have been focused recently. However, information on modulating the glucocorticoid receptor (GR) of PAEs is scarce. Our aim was to evaluate the agonistic / antagonistic properties of PAEs on human GR. Luciferase reporter gene assay revealed that the tested chemicals displayed no agonistic effects but dicyclohexyl phthalate (DCHP) exerted antagonistic activity in a dose-responsive manner for GR in HeLa cells. The effects of DCHP on dexamethasone (DEX)-induced GR nuclear translocation and gene expression of glucocorticoid-responsive gene expression (G6Pase, PEPCK, FAS, GILZ and MKP-1), as well as protein expression of G6Pase and PEPCK were further examined by RT-qPCR and western blot analysis. DCHP antagonized DEX-induced GR nuclear translocation and suppressed gene expression in both mRNA and protein levels. Furthermore, the results of molecular docking and molecular dynamics simulation showed that DCHP could bind to GR and exhibited potential regulation on this target protein. Collectively, we demonstrate that DCHP may act as a GR antagonist in vitro and is considered to exert endocrine effects via human GR.

Paeonol, an Ingredient of Kamishoyosan, Reduces Intracellular Lipid Accumulation by Inhibiting Glucocorticoid Receptor Activity in 3T3-L1 Cells.

Excessive triglyceride accumulation in lipid-metabolizing tissues is associated with an increased risk of a variety of metabolic diseases. Kamishoyosan (KSS) is a Kampo composed of 10 constituent herbs, and contains moutan cortex (MC) and paeonol (PN) as the major ingredient of MC. Here, we demonstrate the molecular mechanism underlying the effect of KSS on the differentiation of mouse preadipocytes (3T3-L1 cells). KSS inhibited the accumulation of triglycerides in a dose-dependent manner in 3T3-L1 cells that were induced to differentiate into adipocytes. We also found that MC and PN were responsible for the anti-adipogenetic effect of KSS and significantly suppressed the expression of CCAAT/enhancer-binding proteins-δ (C/EBP-δ) mRNA 3 days after the induction of differentiation. Thus, PN may contribute to the anti-adipogenetic property of MC in 3T3-L1 cells. In addition, PN inhibited dexamethasone (Dex)-induced glucocorticoid receptor (GR) promoter activity. Taken together, these results suggest that PN suppresses C/EBP-δ expression by inhibiting Dex-induced GR promoter activity at the early stage of differentiation and, consequently, delays differentiation into mature adipocytes. Our results suggest that the habitual intake of Kampo-containing PN contributes to the prevention of the onset of metabolic diseases by decreasing the excessive accumulation of triglycerides in lipid-metabolizing tissues.

Heme Oxygenase 1 Impairs Glucocorticoid Receptor Activity in Prostate Cancer.

Glucocorticoids are used during prostate cancer (PCa) treatment. However, they may also have the potential to drive castration resistant prostate cancer (CRPC) growth via the glucocorticoid receptor (GR). Given the association between inflammation and PCa, and the anti-inflammatory role of heme oxygenase 1 (HO-1), we aimed at identifying the molecular processes governed by the interaction between HO-1 and GR. PCa-derived cell lines were treated with Hemin, Dexamethasone (Dex), or both. We studied GR gene expression by RTqPCR, protein expression by Western Blot, transcriptional activity using reporter assays, and nuclear translocation by confocal microscopy. We also evaluated the expression of HO-1, FKBP51, and FKBP52 by Western Blot. Hemin pre-treatment reduced Dex-induced GR activity in PC3 cells. Protein levels of FKBP51, a cytoplasmic GR-binding immunophilin, were significantly increased in Hemin+Dex treated cells, possibly accounting for lower GR activity. We also evaluated these treatments in vivo using PC3 tumors growing as xenografts. We found non-significant differences in tumor growth among treatments. Immunohistochemistry analyses revealed strong nuclear GR staining in almost all groups. We did not observe HO-1 staining in tumor cells, but high HO-1 reactivity was detected in tumor infiltrating macrophages. Our results suggest an association and crossed modulation between HO-1 and GR pathways.

MicroRNA-9 regulates steroid-resistant airway hyperresponsiveness by reducing protein phosphatase 2A activity

Steroid-resistant asthma is a major clinical problem that is linked to activation of innate immune cells. Levels of IFN-γ and LPS are often increased in these patients. Cooperative signaling between IFN-γ/LPS induces macrophage-dependent steroid-resistant airway hyperresponsiveness (AHR) in mouse models. MicroRNAs (miRs) are small noncoding RNAs that regulate the function of innate immune cells by controlling mRNA stability and translation. Their role in regulating glucocorticoid responsiveness and AHR remains unexplored. IFN-γ and LPS synergistically increase the expression of miR-9 in macrophages and lung tissue, suggesting a role in the mechanisms of steroid resistance. Here we demonstrate the role of miR-9 in IFN-γ/LPS-induced inhibition of dexamethasone (DEX) signaling in macrophages and in induction of steroid-resistant AHR. MiRNA-9 expression was assessed by means of quantitative RT-PCR. Putative miR-9 targets were determined in silico and confirmed in luciferase reporter assays. miR-9 function was inhibited with sequence-specific antagomirs. The efficacy of DEX was assessed by quantifying glucocorticoid receptor (GR) cellular localization, protein phosphatase 2A (PP2A) activity, and AHR. Exposure of pulmonary macrophages to IFN-γ/LPS synergistically induced miR-9 expression; reduced levels of its target transcript, protein phosphatase 2 regulatory subunit B (B56) δ isoform; attenuated PP2A activity; and inhibited DEX-induced GR nuclear translocation. Inhibition of miR-9 increased both PP2A activity and GR nuclear translocation in macrophages and restored steroid sensitivity in multiple models of steroid-resistant AHR. Pharmacologic activation of PP2A restored DEX efficacy and inhibited AHR. MiR-9 expression was increased in sputum of patients with neutrophilic but not those with eosinophilic asthma. MiR-9 regulates GR signaling and steroid-resistant AHR. Targeting miR-9 function might be a novel approach for the treatment of steroid-resistant asthma.

Anti-inflammatory and corticosteroid-enhancing actions of vitamin D in monocytes of patients with steroid-resistant and those with steroid-sensitive asthma

Vitamin D is known for its anti-inflammatory effects. Vitamin D regulation of responses in patients with steroid-resistant (SR) versus steroid-sensitive (SS) asthma has not been studied. Peripheral blood cells from 11 patients with SR asthma and 8 patients with SS asthma were preincubated with 1,25-dihydroxyvitamin D (1,25[OH]2D [VitD]), followed by dexamethasone (DEX) treatment and LPS stimulation. LPS-induced phosphorylated p38 mitogen-activated protein kinase (p-p38) in monocytes was examined by means of flow cytometry. Mitogen-activated protein kinase phosphatase-1 (MKP-1) mRNA expression, which inhibits p-p38, was analyzed by means of real-time PCR. Glucocorticoid receptor (GR) binding and histone H4 acetylation in the glucocorticoid response element of the MKP-1 promoter in monocytes were analyzed by means of chromatin immunoprecipitation. DEX significantly inhibited LPS-induced p-p38 in monocytes from patients with SS asthma but not those from patients with SR asthma (P < .01). VitD inhibited LPS-induced p-p38 in monocytes from both patient groups (P < .01) but enhanced DEX suppression of LPS-induced p-p38 only in monocytes from patients with SS asthma (P < .01). VitD induced MKP-1 expression and enhanced DEX induction of MKP-1 in both patients with SS asthma and patients with SR asthma. VitD/DEX-induced MKP-1 mRNA levels remained significantly lower in monocytes from patients with SR asthma (P < .05). DEX-stimulated recruitment of GR and histone H4 acetylation at the glucocorticoid response element 4.6 kbp upstream of the MKP-1 gene were significantly lower in monocytes from patients with SR asthma compared with those from patients with SS asthma. VitD pretreatment enhanced DEX-induced GR binding and histone acetylation in monocytes from both patient groups. However, GR binding and histone H4 acetylation remained significantly lower in monocytes from patients with SR asthma. VitD demonstrated anti-inflammatory and corticosteroid-enhancing effects in monocytes of patients with SR asthma and patients with SS asthma. However, the responses to corticosteroids in patients with SR asthma remained significantly lower than those in patients with SS asthma.

Pro-Asthmatic Cytokines Regulate Unliganded and Ligand-Dependent Glucocorticoid Receptor Signaling in Airway Smooth Muscle

To elucidate the regulation of glucocorticoid receptor (GR) signaling under pro-asthmatic conditions, cultured human airway smooth muscle (HASM) cells were treated with proinflammatory cytokines or GR ligands alone and in combination, and then examined for induced changes in ligand-dependent and -independent GR activation and downstream signaling events. Ligand stimulation with either cortisone or dexamethsone (DEX) acutely elicited GR translocation to the nucleus and, comparably, ligand-independent stimulation either with the Th2 cytokine, IL-13, or the pleiotropic cytokine combination, IL-1β/TNFα, also acutely evoked GR translocation. The latter response was potentiated by combined exposure of cells to GR ligand and cytokine. Similarly, treatment with either DEX or IL-13 alone induced GR phosphorylation at its serine-211 residue (GRSer211), denoting its activated state, and combined treatment with DEX+IL-13 elicited heightened and sustained GRSer211 phosphorylation. Interestingly, the above ligand-independent GR responses to IL-13 alone were not associated with downstream GR binding to its consensus DNA sequence or GR transactivation, whereas both DEX-induced GR:DNA binding and transcriptional activity were significantly heightened in the presence of IL-13, coupled to increased recruitment of the transcriptional co-factor, MED14. The stimulated GR signaling responses to DEX were prevented in IL-13-exposed cells wherein GRSer211 phosphorylation was suppressed either by transfection with specific serine phosphorylation-deficient mutant GRs or treatment with inhibitors of the MAPKs, ERK1/2 and JNK. Collectively, these novel data highlight a heretofore-unidentified homeostatic mechanism in HASM cells that involves pro-asthmatic cytokine-driven, MAPK-mediated, non-ligand-dependent GR activation that confers heightened glucocorticoid ligand-stimulated GR signaling. These findings raise the consideration that perturbations in this homeostatic cytokine-driven GR signaling mechanism may be responsible, at least in part, for the insensirtivity to glucocorticoid therapy that is commonly seen in individuals with severe asthma.

Activation of cAMP–protein kinase A abrogates STAT5-mediated inhibition of glucocorticoid receptor signaling by interferon-alpha

IFN-alpha has been found to inhibit glucocorticoid receptor (GR) function by activating janus kinase-signal transducer and activator of transcription (JAK-STAT) inflammatory signaling pathways. In contrast, through stimulation of protein kinase A (PKA), cAMP has been shown to enhance GR function and can inhibit inflammatory signaling. We therefore examined whether increased cAMP-PKA pathway activation could reverse IFN-alpha-induced inhibition of GR function and whether decreased cAMP-PKA activity might exacerbate IFN-alpha effects on the GR. Activation of cAMP by forskolin (10 μM) reversed the inhibitory effects of mIFN-alpha (1000 U/ml) on dexamethasone (DEX)-induced MMTV-luciferase activity in hippocampal HT22 cells. Forskolin treatment also blocked both IFN-alpha-induced activation of phosphorylated STAT5 (pSTAT5) and inhibitory protein-protein interactions between pSTAT5 and GR in the nucleus of HT22 cells treated with IFN-alpha and DEX. These effects of forskolin were reversed by co-administration of the PKA inhibitor, H89. Conversely, the combination of IFN-alpha and treatment with either H89 or siRNA directed against the alpha and beta catalytic subunit isoforms of PKA led to an additive inhibitory effect on DEX-induced GR activity in HT22 cells. Taken together, these findings suggest that inhibition of GR signaling by mIFN-alpha and STAT5 can be reversed by activation of cAMP-PKA pathways, whereas decreased PKA activity increases the inhibitory effect of IFN-alpha on GR function. Given decreased PKA activity found in patients with major depression, these data suggest that depressed patients may be vulnerable to cytokine effects on GR, and cAMP-PKA agonists may serve to reverse glucocorticoid resistance in patients with depression and increased inflammation.

Characterization of a novel non-steroidal glucocorticoid receptor antagonist

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 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.

Interferon-alpha inhibits glucocorticoid receptor-mediated gene transcription via STAT5 activation in mouse HT22 cells

Interferon (IFN)-alpha is an innate immune cytokine that induces significant depressive symptoms in clinical populations. A number of mechanisms have been considered regarding the relationship between IFN-alpha and depression, including the effects of IFN-alpha on the hypothalamic–pituitary–adrenal (HPA) axis. Here, we examined the impact of mouse interferon (mIFN)-alpha and its signaling pathways on the functioning of the glucocorticoid receptor (GR), which plays a key role in HPA axis regulation. mIFN-alpha treatment (100–1000 IU/ml) of HT22 mouse hippocampal cells for 24 h was found to significantly inhibit dexamethasone (DEX)-induced GR-mediated MMTV-luciferase activity and significantly decrease DEX-induced GR-binding to its DNA response element. Of note, mIFN-alpha treatment for 24 h had no effect on DEX-induced GR translocation or GR protein expression. Inhibition of DEX-induced GR function by mIFN-alpha was significantly reversed by pharmacological inhibition of janus kinase/signal transducer and activator of transcription (Jak-STAT) signaling pathways, but not by inhibition of p38 mitogen-activated protein kinase. Moreover, pretreatment of cells with siRNA targeted to STAT5, but not STAT1 or STAT2, significantly attenuated IFN-alpha inhibition of DEX-induced MMTV-luciferase activity. Immunoprecipitation experiments revealed nuclear co-immunoprecipitation of activated STAT5 and GR following IFN-alpha plus DEX treatment. Taken together, these results indicate that negative regulation of GR function by IFN-alpha in hippocampal HT22 cells is mediated by activation of Jak/STAT signaling pathways leading to nuclear STAT5-GR protein–protein interactions. Given the role of GR in depressive disorders, IFN-alpha effects on GR function in cells of hippocampal origin may contribute to HPA axis alterations and depressive symptoms in IFN-alpha-treated patients.

Regulation of T-type Cav3.1 channels expression by synthetic glucocorticoid dexamethasone in neonatal cardiac myocytes

The effect of the dexamethasone (Dex) on the regulation of the T-type Ca(2+) channel expressions was investigated in primary cultures of neonatal rat ventricular myocytes. We found that Dex (1 microM) increases the T-type Ca(2+) current (I(CaT)) associated with an increase in Ca(v)3.1 mRNA amount. We isolated the upstream region from Ca(v)3.1 encoding gene and tested the activity of the promoter in transfected ventricular myocytes. We found a minimal Dex-responsive region that displayed putative glucocorticoid receptor (GR) and nuclear factor kappa-B (NFkappaB) targets. The GR selective antagonist, RU38486 (10 microM), nearly turned off the transcriptional activity of Ca(v)3.1 encoding gene, and an NFkappaB inhibitor, pyrrolodine dithiocarbonate (10 microM), completely abolished the Dex-induced mRNA increase. However, Dex-induced GR and NFkappaB synthesis and nuclear translocation were not timely related to Ca(v)3.1 mRNA increase. These results indicate that both GR and NFkappaB were necessary, but not sufficient, to trigger the increase in Ca(v)3.1 mRNA amount. This study showed the relationship between glucocorticoid and T-type channels up-regulation that may be involved in cardiac development and pathology.

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 proinflammatory cytokine, interleukin-1alpha, reduces glucocorticoid receptor translocation and function.

Proinflammatory cytokines have been shown to influence the expression and function of the glucocorticoid receptor (GR). Specifically, several studies have found that cytokines induce a decrease in GR function, as evidenced by reduced sensitivity to glucocorticoid effects on functional end points. To investigate the potential mechanism(s) involved, we examined the impact of the proinflammatory cytokine, interleukin-1alpha (IL-1alpha), on 1) GR translocation from cytoplasm to nucleus using GR immunostaining, 2) cytosolic radioligand GR binding, and 3) GR-mediated gene transcription in L929 cells stably transfected with the mouse mammary tumor virus-cholamphenicol acetyltransferase reporter gene. L929 cells were treated with IL-1alpha (100 and 1000 U/ml) for 24 h in the presence or absence of dexamethasone (Dex; 10 nM to 1 microM). IL-1alpha inhibited Dex-induced GR translocation and alone induced GR up-regulation. Pretreatment with IL-1alpha followed by Dex treatment for 1.5 h led to about 20% inhibition of Dex-induced GR-mediated gene transcription, whereas coincubation of IL-1alpha plus Dex for 24 h inhibited Dex-induced GR-mediated gene activity up to 42%. The latter effect was reversed by the IL-1 receptor antagonist. These results suggest that cytokines produced during an inflammatory response may induce GR resistance in relevant cell types by direct effects on the GR, thereby providing an additional pathway by which the immune system can influence the hypothalamic-pituitary-adrenal axis.

Inhibition of heat shock factor activity prevents heat shock potentiation of glucocorticoid receptor-mediated gene expression.

Using mouse L929 cells stably transfected with a glucocorticoid receptor (GR)-responsive murine mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter gene (LMCAT2 cells), we have shown that cellular stress (heat or chemical shock) can cause a dramatic increase in the levels of dexamethasone (Dex)-induced CAT gene expression. We refer to this response as the heat shock potentiation effect, or HSPE. As the cellular heat shock response also involves the activation of heat shock transcription factor (HSF), we have, in the present study, examined the role of HSF in the stress potentiation of GR by use of a flavonoid compound, quercetin, recently shown to selectively inhibit the stress response in a variety of human and murine cell lines. Analysis of the HSPE, as well as heat shock protein synthesis and activation of HSF during time-courses of recovery following heat shock, revealed a similar pattern for each response, with peak activities occurring about 16 h after stress. These data suggest a correlation between the activation of both GR and HSF in stressed cells. In L929 cells stably transfected with a CAT reporter plasmid under the control of the HSF-responsive hsp70 promoter (LHSECAT cells), pretreatment with quercetin was found to cause a dose- and time-dependent inactivation of HSF activity following heat shock, but only when added before the stress event. In LMCAT2 cells, quercetin similarly inhibited both heat and chemical shock potentiation of Dex-induced GR activity. This activity of quercetin was not the result of post-transcriptional or general cytotoxic properties, as quercetin (1) did not significantly affect GR or HSF activities when added after the stress event, (2) did not reduce CAT gene expression as controlled by the constitutive SV40 early promoter, and (3) did not alter normal (non-stress), Dex-induced MMTV-CAT expression. Thus, quercetin appears to be an effective and selective inhibitor of HSF stress-induced activation and its ability to prevent the stress potentiation of GR suggests either a direct or indirect involvement by stress-activated HSF in this process, or the existence of a regulatory step common to both the heat shock and HSPE responses.

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.