Ligand-dependent down-regulation of stably transfected human glucocorticoid receptors is associated with the loss of functional glucocorticoid responsiveness.

Ligand-Dependent Down-Regulation of Stably Transfected Human Glucocorticoid Receptors Is Associated with the Loss of Functional Glucocorticoid Responsiveness

Deborah L. Bellingham*, John A. Cidlowski

Madhabananda

Sar, and

Department of Biochemistry and Biophysics (D.L.B., Department of Physiology (J.A.C.) Department of Cell Biology and Anatomy (M.S.) Lineberger Cancer Cell Biology Program University of North Carolina Chapel Hill, North Carolina 27599

With prolonged exposure to dexamethasone (>12 h) a second, more gradual decline in human glucocorticoid receptor mRNA was observed. This biphasic pattern of glucocorticoid receptor gene expression was not reflected at the level of receptor protein, suggesting that both transcriptional and translational control mechanisms may be involved in ligand-dependent receptor regulation. When cells were removed from dexamethasone after up to 48 h of treatment, glucocorticoid receptor mRNA levels fully recovered within 12 h. Receptor protein recovered only partially during this same time period. Down-regulation of glucocorticoid receptor protein and mRNA levels by dexamethasone in stably transfected cells led to corresponding reductions in the hormone sensitivity of two glucocorticoid-regulated genes: a transiently transfected chloramphenicol acetyltransferase receptor gene and a stably integrated dihydrofolate reductase gene. These results demonstrate that stably transfected human glucocorticoid receptors are subject to ligand-induced down-regulation in a heterologous cell line. Moreover, glucocorticoid receptor autoregulation appears to be a highly conserved mechanism for attenuating cellular responsiveness to hormone. (Molecular Endocrinology 8: 2090-2102, 1992)

The effect of glucocorticoids on the regulation of stably transfected human glucocorticoid receptors has been examined. Exposure of a Chinese hamster ovary-derived cell line containing stably transfected human glucocorticoid receptor genes and glucocorticoid-responsive dihydrofolate reductase genes to 5 nM dexamethasone resulted in a rapid, time-dependent reduction in the level of glucocorticoid receptor protein to 50% of control levels within 5 h of steroid treatment. This decrease in receptor protein was persistent, with a maximal 70% reduction observed even after 4 weeks of dexamethasone treatment. lmmunocytochemical analysis of the influence of dexamethasone on stably transfected glucocorticoid receptors revealed efficient translocation of receptors to the nucleus within 1 h of hormone treatment. However, upon longer exposure to dexamethasone (5 h), immunoreactive glucocorticoid receptors were localized primarily to the cytoplasm. By 24 h of treatment, glucocorticoid receptors were absent from the cytoplasm and the nucleus, suggesting that the ligand-induced loss of glucocorticoid receptors may be a cytoplasmic event. The decrease in transfected glucocorticoid receptor protein was largely reflected by similar changes in steady state levels of human glucocorticoid receptor mRNA; however, the effects of hormone on receptor protein levels were more profound than on receptor mRNA. There was an initial rapid reduction in transfected glucocorticoid receptor mRNA to 50% of control levels within 2 h of dexamethasone treatment. This reduction was followed by a transient rise in mRNA expression after 12 h of hormone treatment. o&%3-8809/92/2090-2102$03.00/0 Molecular Endocrmology Copyright 0 1992 by The Endocrine

J.A.C.),

INTRODUCTION Glucocorticoid hormones elicit a variety of biological responses in many different eukaryotic cells and tissues. The primary determinant of these effects is the glucocorticoid receptor protein, which is a member of a large family of highly specialized transcription factors

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that includes the other steroid hormone receptors as well as receptors for structurally unrelated molecules such as retinoic acid and thyroid hormone (1, 2). In response to hormone binding, the glucocorticoid receptor is converted into a DNA-binding protein, which subsequently interacts with enhancer-like glucocorticoid regulatory elements (GREs) located near or within hormonally responsive genes (3, 4). Upon activation by hormone, the glucocorticoid receptor is also thought to interact with one or more components of the transcriptional machinery, ultimately resulting in positive or in some cases, negative regulation of gene expression (5). As a ligand-activated transcriptional regulator, the glucocorticoid receptor necessarily participates in multiple reactions to convert the signals that originate outside a target cell into the appropriate changes in gene expression. In this capacity, the glucocorticoid receptor does not exist as a static entity within the cell; rather, it is subject to regulation by a number of different factors. For example, glucocorticoid receptor levels have been shown to vary as a function of the cell cycle (6, 7) after mitogenic stimulation (8) during development (9) and after exposure to certain drugs (10) or hormone ligands (11, 12). Since it appears in many cases that a direct correlation exists between the number of receptors and the magnitude of a particular glucocorticoid response (13-l 5) the question arises as to whether those factors that affect the intracellular concentration of glucocorticoid receptors play a physiological role in regulating the capacity of a given cell type to respond to hormone. Among the most intensively studied modulators of glucocorticoid receptor number are the glucocorticoids themselves (reviewed in Refs. 16-18). The phenomenon of homologous down-regulation or autoregulation has been documented in various cell lines (7, 12, 19,20), intact animals (21-23) and healthy humans (24). Similar observations have been made regarding other steroid hormone receptors (25-28) as well as for membrane-bound receptors (29). These findings have led to the more general proposal that ligand-induced down-regulation of steroid hormone receptors represents a feedback mechanism to limit the duration of steroid hormone action in the cell. Despite the important clinical ramifications of this proposal, the data on glucocorticoid receptor pharmacology in normal and disease states have often been conflicting (30-32). The mechanism(s) responsible for ligand-induced down-regulation of glucocorticoid receptor levels are not well understood; however, the majority of evidence to date indicates that the receptor is an integral part of this process. Down-regulation occurs in the presence of inhibitors of protein synthesis (33,34) and is dependent on hormone concentration (11). Analysis of the effects of ligand on receptor protein and mRNA levels have suggested that at least two mechanisms may be responsible for down-regulation. Exposure to ligand causes a reduction in the transcription rate of the glucocorticoid receptor gene (33-36) and in some

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cases, it appears to decrease the half-life of the receptor protein as well (20, 34). An attractive model to explain these findings invokes interaction of the glucocorticoid receptor protein with its own gene in analogy to the mechanism by which the receptor regulates other genes. However, the promoter of the human glucocorticoid receptor gene lacks consensus GREs (35) suggesting that novel regulatory mechanisms may be involved. In this regard, we recently demonstrated that the human glucocorticoid receptor cDNA contains sequences sufficient to mediate down-regulation after transient transfection into receptor-deficient COS cells (36). These findings raise the interesting possibility that intragenic elements may be involved in autoregulation of glucocorticoid receptor gene expression. In the present study, we sought to determine whether homologous down-regulation occurs when mammalian cells are stably transfected with human glucocorticoid receptors and if so, to determine whether autoregulation plays a functional role in modulating steroid action. We report here that Chinese hamster ovary (CHO) cells stably transfected with the human glucocorticoid receptor cDNA are subject to down-regulation by cognate ligand. Furthermore, we show that this ligand-dependent reduction in receptor levels is functionally associated with the cessation of two different glucocorticoid responses.

RESULTS Stable Expression of Intact Human Glucocorticoid Receptors in CHO Ceils We have previously demonstrated that cotransfection of dihydrofolate reductase (DHFR)-deficient CHO cells with a plasmid containing a modified, glucocorticoidresponsive DHFR gene and a plasmid encoding the human glucocorticoid receptor cDNA is a useful method for obtaining stable expression of intact glucocor-ticoid receptors in mammalian cells (37). The structural features of the glucocorticoid receptors expressed in MG/ hGR (Mst/GRE + human GR) cells, which were created using this linked cotransfection strategy, are summarized in Fig. 1. Electrophoretic analysis of the cytosolic proteins covalently labeled by the affinity label [3H] dexamethasone mesylate ([3H]DM) in MG/hGR cells indicates that a saturably labeled species is present at M, of approximately 94,000 (Fig. 1A). Previous studies have shown that this hormone-binding species comigrates with [3H]DM-labeled endogenous hamster receptor (37). Several smaller, saturably labeled proteins are also visible, which probably represent proteolytic degradation products. lmmunoblot analysis of labeled cytosolic proteins in MG/hGR cells using an antipeptide antibody (number 57) directed against the amino-terminal region of the human glucocorticoid receptor (38) demonstrates the presence of a reactive species at M, of approximately 94,000 (Fig. 1B). The identity of this protein as the human glucocorticoid receptor is verified


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Fig. 1. Stable Overproduction of intact Glucocorticoid Receptors in CHO Cells MG/hGR cells were created by cotransfecting a glucocorticoid-responsive DHFR gene and the human glucocorticoid receptor gene Into DHFR-deficient CHO cells as previously described (37). A, Affinity labeling of glucocorticoid receptors with [3H]DM. MG/ hGR cells were incubated with 40 nM [3H]DM in the absence (H) or presence (H + C) of a lOOO-fold excess of unlabeled dexamethasone. Cytosolic extracts were prepared as described in Materials and Methods, subjected to SDS-PAGE, and electrophoretically transferred to nitrocellulose. Samples were normalized to equivalent protein concentrations (400 pg) before loading. After transfer, nitrocellulose was sprayed with EN[3]HANCE and exposed to x-ray film for 3-4 days at -70 C to visualize saturably labeled proteins. The posrtions of prestained molecular mass standards are indicated at the leff of the panel: phosphorylase b, 97,400 (97.4); BSA, 68,000 (68). B, Western blot analysis of the glucocorticoid receptors in MG/hGR cells. Before processing for fluorography, samples shown in A were incubated with antihuman glucocorticoid receptor antibody 59. This antibody is directed against a 1%amino acid sequence located near the DNA-binding domain of the human glucocorticoid receptor (38). Immune complexes were visualized by staining with alkaline phosphatase-conjugated secondary antibody. The arrow to the right of the figure indicates the position of the immune complex that is saturably labeled by [3H]DM in A. C, Northern blot analysis of glucocorticoid receptor mRNA in MG/hGR cells. Total RNA was isolated from MG/hGR cells. RNA (20 fig) was denatured, separated on a 1% agarose gel, and transferred to a nylon membrane. The filter was then hybridized with a 32P-labeled human glucocorticoid receptor cRNA probe and processed for autoradiography. The position of the transfected human glucocorticoid receptor transcript (hGR) is indicated to the right of the figure. Mol wt standards (in kb) are shown to the leff of the figure.

by the fact that it comigrates with the species that is affinity labeled by [3H]DM shown in Fig. IA. Northern blot analysis of total RNA produced in MG/hGR cells after hybridization with a human glucocorticoid receptor cRNA probe reveals the presence of an abundant transcript at approximately 3.5 kilobases (kb) (Fig. 1 C). This hybridizing species corresponds to the predicted size of the transfected human glucocorticoid receptor transcript (39). Under the hybridization conditions shown, endogenous hamster glucocorticoid receptor mRNA cannot be detected (37). Regulation of Glucocorticoid Receptor MG/hGR Cells By Dexamethasone

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Based on several independent criteria discussed above, the additional receptors expressed in MG/hGR cells appear to be indentical to endogenous hamster glucocorticoid receptors. These findings suggested that MG/ hGR cells would be an appropriate model system in which to examine glucocorticoid receptor regulation. Specifically we wished to determine whether stably transfected glucocorticoid receptors are subject to ligand-mediated down-regulation as are endogenous receptors (11, 12). To investigate this issue, whole cell lysates were prepared from MG/hGR cells after an extended time course of treatment with dexamethasone. A dose of 5 nM dexamethasone was initially chosen for study, as this value is on the order of the binding affinity of glucocorticoid receptors for dexa-

methasone in MG/hGR cells (dissociation constant -2 nM) (37). Cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then analyzed via immunoblotting techniques using antireceptor antibody 57 (38). As shown in Fig. 2, dexamethasone had a marked inhibitory effect on the level of glucocorticoid receptors in MG/hGR cells. This effect was very rapid with a 50% reduction in receptor protein evident by 6 h of hormone treatment. Moreover, the reduction in receptor protein was persistent. A maximal 70% reduction in receptor protein was achieved within 9 h of exposure to hormone and was maintained after 4 weeks of dexamethasone treatment. It is interesting that the inhibitory effect of dexamethasone on glucocorticoid receptor levels is not complete. There is a residual amount of receptor (25-30%) remaining after 4 weeks of chronic exposure to dexamethasone that appears to be refractory to downregulation. Affinity labeling of receptor protein in MG/ hGR cells after extended dexamethasone treatment demonstrates that this population of residual receptors is capable of binding steroid (data not shown). The incomplete nature of down-regulation in MG/hGR cells is similar to the observed effects of ligand on endogenous receptor levels (11, 12, 19). lmmunocytochemical Localization of Glucocorticoid Receptor after Treatment With Dexamethasone As demonstrated by the immunoblot analysis shown in Fig. 2, it is evident that dexamethasone treatment


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Fig. 2. lmmunoblot Analysis of Glucocor-ticoid Receptor Protein in MG/hGR Cells Treated with 5 nM Dexamethasone MG/hGR cells were treated with 5 nM dexamethasone for times ranging from 1 h to 4 weeks as indicated above the inset. Whole cell lysates were prepared from cells at each time point, resolved by SDS-PAGE, and transferred to nitrocellulose. Samples were normalized to equivalent protein concentrations (200 pg) before loading. lmmunoreactive glucocorticoid receptor protein was detected by incubating the filter with epitope-purified antihuman glucocorticoid receptor antibody 57. Antibody signals were visualized with ‘251-conjugated protein A and autoradiography (inset). Autoradiograms were scanned densitometrically and the relative absorbances plotted as a function of time of dexamethasone treatment.

causes a rapid, time-dependent reduction in the steady state levels of glucocorticoid receptor protein in MG/ hGR cells. We next wished to determine whether this effect was in fact reflected at the level of the intact cell and was not the result of receptor protein sequestration, as has been reported for other signal transducing receptors (29). To examine the intracellular distribution of glucocorticoid receptors in response to dexametha-

sone, MG/hGR cellswere treated with 5 nMdexamethasonefor times ranging from 1 to 24 h and then stained for glucocorticoid receptor immunoreactivity as previously described (38). As shown in Fig. 3, there is a specific amount of staining evident in untreated MG/ hGR cells (CONTROL), which appears to be largely cytoplasmic in nature. There is a smalldegree of heterogeneity in the overall pattern of staining in the absence of exogenous hormone, which may reflect the low level of corticosteroids in the mediumcontributed by the fetal calf serum. After 1 h of treatment with dexamethasonethere is a profound redistributionin the pattern of glucocorticoid receptor immunoreactivity (Fig. 3, +DEX, 1 h). This pattern appears to reflect a predominantly nuclear location for receptor protein in the presence of hormone. After 3 h of exposure to ligand, immunoreactiveglucocorticoidreceptors remain largely nuclear(Fig. 3, +DEX, 3 h). In contrast, by 5 h of treatment there appearsto be a substantialreduction in the level of nuclear receptors (Fig. 3, +DEX, 5 h). This decrease is accompaniedby an increasein cytoplasmic staining, which may reflect a movement of glucocorticoid receptors from the nucleus back to the cytoplasm. With longer exposure to dexamethasone

(24 h), there is a substantial reduction in the intensity of cytoplasmic glucocorticoid receptor immunoreactivity (Fig. 3, +DEX, 24 h). Indeed, the relative level of staining is comparable to the amount of background staining observed with preimmune serum (Fig. 3, PREIMMUNE). Based on these results, it appearsthat the dexamethasone-inducedloss of glucocorticoid receptors may occur in the cytoplasmof hormone-treated cells. Influence of Dexamethasone on Steady State Levels of Glucocorticoid Receptor mRNA

Based on the profound effect of dexamethasoneon glucocotticoid receptor protein levels in MG/hGR cells (Figs. 2 and 3) are these changes reflected by alterations in the steady state levels of transfected glucocorticoid receptor mRNA? To addressthis question, cells were treated with 5 nM dexamethasone,and RNA was isolatedat time points rangingfrom 30 min to 4 weeks. After hybridization of sampleswith a humanglucocorticoid receptor cRNA probe and a control P-actin cRNA probe (Fig. 4, inset), the relative amountsof transfected humanreceptor transcript were quantified by scanning densitometry. The concentrations of human glucocorticoid receptor mRNA in dexamethasone-treatedsamples expressed as a percentage of the control sample are representedgraphicallyin Fig. 4. The level of human glucocorticoid receptor mRNA was reduced to 50% of control levels within 2 h of hormonetreatment, indicating that the initial effect of dexamethasoneon receptor expressionin MG/hGR cellsis very rapid. Thisreduction


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3. lmmunocytochemical Analysis of the Effect of Dexamethasone on Glucocorticoid Receptor Levels in MG/hGR Ceils MG/hGR cells were plated in two-well chamber slides and treated with 5 nM dexamethasone for 0 h (CONTROL), 1 h [+DEX (1 h)], 3 h [+DEX (3 h)], 5 h [+DEX (5 h)], or 24 h [+DEX (24 h)]. After fixation and permeabilization steps as described in Materials and Methods, cells were incubated with epitope-purified antihuman glucocorticoid receptor antibody 57 or preimmune serum (PREIMMUNE) at a concentration of 1:7500. lmmunoreactivity was visualized by staining with avidin-biotin-peroxidase. Magnification, x650. Fig.

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4. Influence of Dexamethasone Treatment on Human Glucocorticoid Receptor mRNA Levels in MG/hGR Calls MG/hGR cells were treated with 5 nM dexamethasone for times ranging from 1 h to 4 weeks as indicated in the inset. Total RNA was isolated from cells at each time point, and poly(A)+ RNA was selected using oligo dT cellulose. Samples (5 fig) were fractionated by agarose gel electrophoresis, transferred to nylon, and hybridized with a 3’P-labeled human glucocorticoid receptor cRNA probe (inset, hGR). The membrane was then stripped and hybridized with a 32P-labeled chick P-actin cRNA probe (inset,Actin). After exposure to x-ray film, autoradiograms were scanned densitometrically. The human glucocorticoid receptor mRNA concentrations in dexamethasone-treated cells are expressed as percentages of the glucocorticoid receptor concentration in control cells after normalization of all hybridization signals to actin. The data shown are representative of three independent experiments. Fig.


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was followed by a transient rise in glucocorticoid receptor gene expression after 12 h of hormone treatment. Interestingly, this increase was not reflected at the protein level (Fig. 2) and it was followed by another, more gradual decline in human glucocorticoid receptor mRNA with longer exposure to dexamethasone (>24 h). Maximal down-regulation of receptor mRNA was achieved by 96 h of treatment with hormone, which persisted for greater than 4 weeks of exposure to dexamethasone. In addition, the inhibitory effects of glucocorticoids on transfected receptor gene expression were dose- and steroid-specific (data not shown). Together these data suggest that down-regulation of transfected glucocorticoid receptors is a rapid, receptor-mediated event. Analysis of Glucocorticoid Receptor mRNA Levels after Dexamethasone Withdrawal

Protein and Treatment and

Chronic exposure to glucocorticoids has been reported in several cases to result in the selection of new populations of cells with reduced receptor numbers (19). To address the possibility that extended treatment of MG/ hGR cells with dexamethasone could be selecting for cell populations with permanently altered glucocorticoid receptor content, we examined the reversibility of ligand-induced down-regulation. Cells were treated with 5 nM dexamethasone for 12,24, or 48 h and were then withdrawn from hormone for either 12 or 24 h. Samples were subsequently processed for comparison of glucocorticoid receptor protein levels (Fig. 5A) or RNA levels (Fig. 5B). Western blot analysis of the relative levels of glucocorticoid receptor protein after treatment for 12 h with dexamethasone [Fig. 5A, +DEX (12 h)] followed by withdrawal for either 12 or 24 h (Fig. 5A, -12 h, -24 h) resulted in virtually complete restoration of receptor levels to control values. In contrast, hormone treatment for either 24 h [Fig. 5A, +DEX (24 h)] or 48 h [+DEX (48 h)] followed by a similar withdrawal schedule (i.e. -12 or -24 h) resulted in restoration of only 40-60% of the control levels of receptor protein. These data indicate that down-regulation of dexamethasone was fully reversible with short treatment times (12 h); however, this process was only partially reversible with longer exposure to dexamethasone (24 h, 48 h). In contrast, the kinetics of the reversibility of glucocorticoid receptor mRNA down-regulation in MG/hGR cells appear to be more rapid than those of the receptor protein. Indeed, there was almost full reversibility of glucocorticoid receptor mRNA levels after reduction by treatment with dexamethasone for either 12, 24, or 48 h [Fig. 5B (+DEX)], which occurred within 12 h of hormone withdrawal (-12 h). By 24 h of hormone withdrawal (Fig. 5B, -24 h), all receptor mRNA levels were restored to control values and in some cases slightly exceeded controls (e.g. +DEX 12 h/-24 h). Down-Regulation of Glucocorticoid Receptors Associated with the Loss of Glucocorticoid Responsiveness Examination glucocorticoid

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strongly suggests that CHO cells have the capacity to regulate the expression of a heterologous glucocorticoid receptor gene. We therefore wished to determine whether the observed down-regulation of transfected receptors might be functionally significant. To address this issue, we examined the effect of dexamethasone treatment on the magnitude of trans-activation by glucocorticoid receptors. MG/hGR cells were grown in the presence of 5 nM dexamethasone for 0, 24, 48, or 72 h. Cells were then transfected with a GRE-containing chloramphenicol acetyltransferase (CAT) reporter plasmid. After transfection, groups were either maintained in 5 nM dexamethasone (defined as control conditions) or treated with 100 nM dexamethasone (induced conditions). The results of this assay are presented in Fig. 6. The magnitude of glucocorticoid-inducible CAT activity (dashed line) underwent more than a 2-fold decrease (from 47% to 20% conversion of CAT) within 24 h in cells pretreated with dexamethasone (0 vs. 24 h). This effect appears to be maximal within 24 h of pretreatment with hormone, since the reduction in the extent of CAT activity was similar after 24 and 48 h of dexamethasone pretreatment (18% and 17%, respectively). These effects cannot be attributed to differential uptake of CAT reporter plasmid since cotransfection with a Rous sarcoma virus (RSV) P-galactosidase control plasmid demonstrates that the transfection efficiencies of MG/hGR cells are unaffected by pretreatment with dexamethasone (data not shown). Based on the levels of immunoreactive glucocorticoid receptor protein shown in Fig. 2, which indicate that the amount of glucocorticoid receptor protein in MG/hGR cells is reduced to 30% of control within 12 h of dexamethasone treatment, these findings suggest that the observed 2fold decrease in CAT activity is the direct result of a comparable decrease in the amount of glucocorticoid receptor protein. As an independent measure of the influence of dexamethasone treatment on the sensitivity of MG/hGR cells to hormone, we next examined whether the stably transfected, glucocorticoid-responsive DHFR reporter gene would also be affected by extended exposure to hormone. Accordingly, we examined the transcriptional activity of the DHFR gene as a function of the length of treatment with 5 nM dexamethasone. As seen in Fig. 7 (inset), hybridization of poly(A)’ RNA with a DHFR cDNA fragment resulted in the appearance of multiple DHFR transcripts ranging in size from 1.8-1.2 kb. These transcripts, which differ in their 3’-termini, are a characteristic feature of the DHFR gene (40). Moreover, a 6-fold induction in the steady state level of DHFR mRNA was observed at early dexamethasone treatment times, which was maximal by 9 h of exposure to hormone. After longer treatment with dexamethasone, DHFR expression underwent a marked 3-fold decrease, and by 24 h DHFR mRNAs were present at a level only slightly above basal expression. The kinetics of this biphasic change in steady state levels of DHFR mRNAs coincide with the disappearance of the glucocorticoid receptor protein observed in Fig. 2. Down-regulation of receptor protein is maximal by 12 h of dexamethasone


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Fig. 5. Effect of Dexamethasone Treatment and Withdrawal on Glucccorticoid Receptor Protein and mRNA Levels in MG/hGR Cells MG/hGR cells were plated in triplicate and treated with 5 nM dexamethasone for 12, 24, or 48 h [+DEX (12 h), +DEX (24 h), +DEX (48 h)]. Dexamethasone-containing media was then removed and was replaced with media lacking exogenous hormone for 0, 12, or 24 h (0, -12 h, -24 h). A, Western blot analysis of glucocorticoid receptor protein levels in MG/hGR cells after hormone treatment and withdrawal. Whole cell extracts of cells harvested at each time point were separated by SDS-PAGE and blotted to nitrocellulose. lmmunoreactive glucocorticoid receptor protein was detected by incubating the filter with epitope-purified antihuman glucocorticoid receptor antibody 57. Antibody signals were visualized with ‘251-conjugated protein A and autoradiography. The positions of prestained molecular mass standards are indicated at the left of the panel: myosin, 200,000 (200); phosphorylase b, 97,400 (97.4): BSA, 68,000 (68). B, Northern blot analysis of steady state human glucocorticoid receptor mRNA levels after dexamethasone treatment and withdrawal. Total RNA was isolated from MG/hGR cells harvested at each time Point as described in Materials and Methods. Twenty micrograms of RNA were separated by agarose gel electrophoresis and analyzed by Northern blot analysis using a 32P-labeled human glucocotticoid receptor cRNA probe. The position of the transfected human glucocorticoid receptor transcript (hGR) is indicated to the rigighrof the figure.

treatment, which corresponds to the point at which DHFR expression begins to decline (Fig. 7, lanes 12 h vs. 24 h). This biphasic pattern of DHFR expression is consistent with a hormone response mechanism that involves an initial rise due to transcriptional activation by giucocorticoid receptors, followed by a decline in DHFR expression as a result of receptor down-regulation.

DISCUSSION

Regulation of glucocorticoid receptor gene expression by cognate ligand has been documented in a variety of cell lines, intact animals, and humans (11, 12, 19, 2124). Despite the ubiquity of this process, the functional consequences of receptor down-regulation have not yet been elucidated. Similarly, the mechanisms responsible for down-regulation are not well understood; however, the evidence to date strongly suggests that the receptor plays an integral role in modulating its own

expression (33, 41, 42). The present studies were initiated to determine whether the human glucocorticoid receptor cDNA is subject to ligand-mediated downregulation when stably incorporated within the context of a native chromatin environment. We used a CHOderived cell line (MG/hGR) that had been stably cotransfected with a glucocotticoid-responsive DHFR gene and the cDNA encoding the human glucocorticoid receptor (37). MG/hGR cells express intact, tranfected glucocorticoid receptors (Fig. 1, A-C). Moreover, these cells also contain glucocorticoid-responsive DHFR genes. Analysis of glucocorticoid receptor protein (Fig. 2) and mRNA levels (Fig. 4) in MG/hGR cells as a function of exposure to a low concentration (5 nM) of dexamethasone revealed a rapid, time-dependent reduction in receptor expression. Ligandinduced down-regulation of a stably transfected human glucocorticoid receptor gene in CHO cells confirms and extends the findings of Burnstein et al. (36) who initially demonstrated that the glucocorticoid receptor cDNA is sensitive to modulation by ligand after transient transfection into COS cells (36). Taken together, the results obtained with stable and


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0 HOURS Fig. 6. Effect of Glucocorticoid Treatment on Subsequent Glucocorticoid Responsiveness MG/hGR cells were pretreated in duplicate with 5 nrv dexamethasone for 0, 24, 48, or 72 h. All groups were then transfected with pGMCS reporter plasmid and pRSV P-gal as described in Materials and Methods. After removal of DNA, each group was grown for an additional 18 h in either 5 nM dexamethasone (0) or 100 nM dexamethasone (A). Cell extracts were prepared and assayed for analysis of CAT activity essentially as described by Gorman et al. (56). The difference in the percent total of [‘*Cl chloramphenicol converted to acetylated products after treatment with 5 nM dexamethasone or 100 nM dexamethasone is represented by dashed lines. The data shown is representative of three independent experiments performed in a similar fashion. Transfection efficiencies were determined by analysis of P-galactosidase activity from the RSV P-gal plasmid and were essentially the same for all treatment groups (57).

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Fig. 7. Influence of Dexamethasone Treatment on Glucocorticoid-Responsive DHFR Gene Expression MG/hGR cells were treated with 5 nM dexamethasone for intervals ranging from 30 min to 4 weeks as described in Fig. 4. Poly(A)+ RNA was isolated and transferred to nylon membrane. After hybridization with glucocorticoid receptor cRNA (shown in Fig. 4) the membrane was stripped and hybridized with a random primed 32P-labeled DHFR cDNA fragment (inset, DHFR). Mol wt standards (in kb) are shown to the left of the figure. Autoradiograms were scanned densitometrically and the relative absorbances plotted as a function of time of dexamethasone treatment. The lower panel shows the same blot probed with @actin cRNA after removal of the DHFR probe (inset, Actin).


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transiently transfected glucocorticoid receptor cDNAs suggest that receptor autoregulation can occur in heterologous cell lines without an absolute requirement for endogenous promoter elements. During the preparation of this article, Gustafsson and coworkers (43) confirmed our preliminary findings (44) that stably transfected human glucocorticoid receptors expressed in CHO cells undergo ligand-induced downregulation. However, our studies clearly demonstrate that down-regulation is more rapid and occurs at lower concentrations of hormone than was reported by Alksnis et al. (43). Kinetic analysis of glucocorticoid receptor levels as a function of treatment time shows that downregulation of receptor protein and mRNA by 5 nM dexamethasone (Figs. 2 and 4, respectively) is evident well before 24 h, which was the earliest time point examined by Alksnis et al. (43). Moreover, the changes observed in transfected glucocorticoid receptor levels in CHO cells after hormone treatment are reflected in the whole cell (Fig. 3). Analysis of the intracellular distribution pattern of glucocorticoid receptor protein as a function of time of exposure to ligand reveals that there is a rapid movement of glucocorticoid receptors into nuclei with short-term dexamethasone treatment (Fig. 3, +DEX, 1 h, 3 h). In contrast, by 5 h of exposure to ligand, immunoreactive glucocorticoid receptors appear to have moved out of the nucleus and back into the cytoplasm. After 24 h of dexamethasone, there is little immunoreactivity remaining in the cytoplasm of MG/hGR cells. These data suggest that down-regulation of transfected receptors occurs in the cytoplasm of hormone-treated CHO cells. Moreover, these findings demonstrate that glucocorticoid receptor protein is turned over in MG/hGR cells and is not sequestered in response to ligand (29). The changes in glucocorticoid receptor protein in MG/ hGR cells in response to dexamethasone were rapid, with a maximal reduction in protein occurring by 12 h of hormone treatment (Fig. 2). This finding contrasts with previous studies on the effects of hormone on endogenous glucocorticoid receptor protein levels, which typically required at least 24 h of exposure to glucocorticoid to reach a minimum (11, 12, 20). Indeed, in rat neural tissue, 4 days of corticosterone treatment were necessary to maximally repress glucocorticoid receptor protein levels (23). Such variation in the rate of receptor protein turnover could be the result of a number of factors, including other regulatory elements within the promoter or the introns of endogenous glucocorticoid receptor genes or cell- and tissue-specific components (9, 45). At the RNA level, down-regulation of transfected glucocorticoid receptor expression in MG/hGR cells is initially very rapid, with a 50% reduction in steady state receptor levels detected within 2 h (Fig. 4). This initial reduction in glucocorticoid receptor mRNA levels in response to dexamethasone treatment is similar in magnitude and time of onset to the effects reported by other labs. For example, Rosewicz et al. (33) reported a 50% decrease in receptor mRNA within 3 h in IM-9

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cells. Similarly, Hoeck et al. (34) found that dexamethasone reduced the level of glucocorticoid receptor mRNA to 25% of control levels within 3 h in NIH 3T3 cells. After extended exposure of MG/hGR cells to hormone (>24 h), the level of glucocorticoid receptor mRNA underwent an additional decline to about 50% of control levels and remained at this reduced level for more than 4 weeks of chronic hormone treatment. Comparison of the kinetics of glucocorticoid receptor protein turnover in the presence of hormone (Fig. 2) with receptor mRNA levels in MG/hGR cells (Fig. 4) reveals that there is about a 6-h lag between a change in receptor message and an observable effect on receptor protein. This finding is consistent with a regulatory mechanism that acts initially (~6 h) at the level of receptor transcription. With longer exposure to hormone (>12 h), there is a transient rise in receptor mRNA, which was unexpected (Fig. 4). The basis for this increase is unclear; however, there have been other reports of similar variations in endogenous glucocorticoid receptor mRNA levels in response to dexamethasone (41). Whether this rise reflects a direct transcriptional response to the loss of glucocorticoid receptor protein remains to be determined. Studies on the regulation of endogenous glucocorticoid receptors by ligand indicate that at least two mechanisms may be responsible for this process. These mechanisms include: 1) reduced transcription of the glucocorticoid receptor gene (33); and 2) accelerated turnover of hormone-bound receptor (20, 34, 41). It is clear from Figs. 2 and 4 that glucocorticoid receptor protein and mRNA are not under synchronous control in MG/hGR cells. It is especially interesting that the transient rise in human glucocorticoid receptor mRNA levels in MG/hGR cells after 12 h of dexamethasone treatment is not reflected by a similar increase in receptor protein. These findings are consistent with a mechanism in which transfected glucocorticoid receptor protein is more unstable in the continued presence of hormone. This result is in agreement with the findings of McIntyre and Samuels (20) who initially reported that glucocorticoid treatment regulates receptor levels by decreasing the half-life of the receptor protein. It is also intriguing that continued exposure of MG/ hGR cells to dexamethasone does not cause a total elimination of glucocorticoid receptor protein. Where it has been examined, down-regulation does not proceed to completion (11, 12, 19). Typically, 20-40% of total receptors remain in the continued presence of hormone. The remaining receptors are competent to respond to hormone. Based on these results, it appears that the cellular mechanisms of receptor replenishment exceed those of receptor depletion. The general reversibility of dexamethasone-induced down-regulation of glucocorticoid receptor protein and RNA (Fig. 5, A and B) further suggests that extended hormone treatment does not select for a subpopulation of cells that are receptordeficient. Rather, down-regulation in MG/hGR cells appears to be a transient process that can be reversed fairly rapidly.


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The question of the functional significance of glucocorticoid receptor autoregulation has been difficult to address in intact animals and in humans. Nonetheless, there have been several cases where down-regulation of endogenous glucocorticoid receptor levels is correlated with a loss of glucocorticoid responsiveness (46, 47). In MG/hGR cells the results obtained with a transiently transfected, glucocorticoid-responsive CAT reporter gene (Fig. 6) as well as a glucocorticoid-responsive DHFR gene (Fig. 7) suggest that a direct relation exists between stably transfected glucocorticoid receptor levels and glucocorticoid responsiveness. Pretreatment of MG/hGR cells with 5 nM dexamethasone for times sufficient to cause greater than 50% reduction in glucocorticoid receptor protein and mRNA (Figs. 2 and 4, respectively) were associated with greater than a 2fold reduction in glucocorticoid-responsive CAT activity (Fig. 6). In addition, examination of the effect of dexamethasone on the expression of the glucocorticoidresponsive DHFR gene in MG/hGR cells revealed a biphasic pattern of DHFR gene expression (Fig. 7). It is interesting to note that a similar response has been observed with a resident aromatase gene after glucocorticoid treatment of fibroblasts (46). The clinical importance of understanding ligand-induced down-regulation of glucocorticoid receptors is particularly relevant with respect to its occurrence during extended therapeutic glucocorticoid treatment, thus leading to glucocorticoid resistance (48, 49). With the identification of an apparently conserved mechanism to down-regulate glucocorticoid receptors, MG/hGR cells will be useful to examine in greater detail the mechanisms responsible for modulating steroid hormone receptor gene expression.

MATERIALS

AND

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METHODS

Materials Alpha minimal essential medium (MEM) and trypsin were obtained from GIBCO BRL (Gaithersburg, MD). Fetal calf serum was from Flow Labs (McLean, VA). [6,7-3H]Dexamethasone mesylate (49.9 Ci/mmol), [‘251]staphylococcal protein A (33.2 mCi/mg), and [‘4C]chloramphenicol (60 mCi/mmol) were obtained from Du Pont-New England Nuclear (Boston, MA). Dexamethasone was from Steraloids (Wilton, NH). Acetyl coenzyme A, diethylaminoethyl (DEAE)-dextran, and unlabeled nucleotides were obtained from Pharmacia LKB Biotechnology (Piscataway, NJ). Oligo (dT)-cellulose was obtained from Collaborative Research (Lexinqton, MA). Biotrans nylon membrane was from ICN (Irvine,?CA), and nitrocellulose was from Schleicher 8 Schuell (Keene. NH). f&“PlUTP (600 Ci/mmol) and [&‘P]dCTP (3006 Ci/mmol) used to prepare hybridization probes were obtained from ICN. Riboprobe reaction components (T3 RNA polymerase, RNAse-free DNase, reaction buffers) and T4 DNA polymerase were from GIBCO BRL. Randomprimed DNA labeling reactions were performed using a kit that was purchased from Boehringer Mannheim (Indianapolis, IN). Relative quantification of bands on autoradiographs was obtained using a GS3000 scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA). Anti-peptide antibodies 57 and 59, which are directed against amino acids 346-367 and 245-259, respectively, from the amino-terminal portion of

the human glucocorticoid receptor, were prepared as previously described (38). Reagents for SDS-PAGE and agarose were from GIBCO BRL. Restriction digests of X and $X174 DNA and prestained molecular mass standard proteins were also from BRL. Protease inhibitors (phenylmethylsulfonyl fluoride and aprotinin), dimethyl sulfoxide (DMSO), and all other reagent grade chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Methods Cell Culture MG/hGR cells were created by stably cotransfecting DHFR-deficient CHO cells with a plasmid containing a modified, glucocorticoid-responsive DHFR gene and a plasmid encoding the human glucocorticoid receptor cDNA as previously described (37). MG/hGR cells were propagated as monolayer cultures in MEM lacking nucleosides but supplemented with 8% dialyzed fetal calf serum, 2 mM glutamine, penicillin (100 U/ml), and streptomycin (75 U/ml). Cells were routinely passaged by brief trypsinization and were counted in a hemocytometer to obtain cell numbers. Affinity Labeling of Glucocorticoid Receptors To prepare affinity-labeled glucocorticoid receptors, MG/hGR cells were harvested from culture plates by incubation in PBS containing 5 mM EDTA for 5 min at 37 C, followed by scraping with a rubber policeman. Cells were washed in PBS, resuspended in unsupplemented MEM at a density of l-2 x 10’ cells/ml, and incubated for 2 h at 0 C with 40 nM [3H]DM in the absence or presence of a 1 ,OOO-fold excess of unlabeled dexamethasone. After incubation with steroid, cells were homogenized in 10 mrv Tris-HCI, pH 8.3, 1 mM EDTA, and the homogenate was centrifuged at 100,000 x g for 1 h at 4 C. The resulting supernatant (cytosol) was collected, and free steroid was removed by adding cytosol to the pellet obtained from centrifugation of an equivalent volume of a dextran-coated charcoal suspension (1% activated charcoal (wt/vol), 0.1% dextran (wt/ vol) in 1.5 mM MgCI,). The cytosol/charcoal suspension was vortexed, incubated on ice for 5 min, and centrifuged at 10,000 x g for 1 min. The supernatant was collected, concentrated using Centricon 30 filters (Amicon, Danvers, MA), and processed for SDS-PAGE and fluorography as described below. Gel Electrophoresis Protein samples were mixed with an equal volume of 2x Fairbanks sample buffer (20 mM Tris-HCI, pH 7.5, 2 mM EDTA, 2% SDS, 10% sucrose, 20 Kg pyronin Y tracking dye/ml) (50) heated at 100 C for 2 min. and stored at -70 C. Before electrophoresis, protein concentrations were determined by the method of Bradford (51). Samples (200400 pg) were resolved by electrophoresis through 7.5% SDS polyacrylamide gels and then electroblotted to nitrocellulose according to the method of Towbin et al. (52). To visualize affinity-labeled glucocorticoid receptor protein, nitrocellulose was sprayed with EN[3H]ANCE (DuPont-NEN, Boston, MA) and exposed to x-ray film for 3-4 days at -70 C. lmmunoblot Analysis Before fluorography, [3H]DM-labeled cytosols were subjected to immunoblot analysis using antiglucocorticoid receptor antibody 59 as previously described (38). To analyze immunoreactive glucocorticoid receptor protein in MG/hGR cells after extended treatments with dexamethasone, whole cell lysates were prepared. Briefly, cells were washed in incomplete MEM for 2 x 30 min, harvested by scraping in PBS containing 5 mM EDTA, and collected by centrifugation at 600 x g for 5 min. Cell pellets were resuspended in lysis buffer (10 mM sodium phosphate, pH 7.2, 150 mM NaCI, 1% sodium deoxycholate, 1% NP-40, 0.1% SDS, 0.5 mM phenylmethylsulfonyl fluoride, and 1 pg/pl aprotinin) at a density of 3 x 10’ cells/ml, essentially as described by Sambrook et al. (53). Lysates were vortexed briefly and centrifuged at 15,000 x g for 30 min at 4 C to remove insoluble debris. Supernatants were then transferred to fresh microfuge tubes, mixed with an equal volume of 2x Fairbanks sample buffer, and processed for SDS-PAGE as described above. After electrophoretic transfer of proteins, nitrocellulose was


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1992

incubated with blocking buffer (10 mM Tris-HCI, pH 7.4, 10% nonfat dry milk, 0.05% Tween-20, 0.9% NaCI) for 2-3 h at room temperature, followed by overnight incubation at 4 C with epitope-purified, antiglucocorticoid receptor antibody 57 at a dilution of 1 :lOO (38). After incubation with antibody, filters were briefly washed in blocking buffer at room temperature and incubated with 5 PCi ‘251-conjugated protein A in 10 ml blocking buffer for 2 h at room temperature. Filters were again washed in blocking buffer, followed by several washes in blocking buffer without nonfat milk, air dried, and exposed to x-ray film at -70 C. lmmunocytochemical Procedures MG/hGR cells were plated in duplicate in two-chamber glass slides in complete MEM and cultured for 3 days at 37 C. Dexamethasone (5 nM final concentration) was added at indicated times before processing. The immunocytochemical staining procedure used was essentially as described in Cidlowski et al. (38). Briefly, after ftxation in 2% paraformaldehyde, slides were washed in PBS and permeabilized in 0.2% Triton-X for 20 min. After an additional wash in PBS, cells were treated with 2.0% normal goat serum, washed in PBS, and then incubated with epitope purified antiglucocorticoid receptor antibody 57 (1:7500) for 20 h at 4 C. The cells were washed in PBS followed by incubation with biotinylated goat antirabbit immunoglobulin G (1:400) for 1 h at room temperature. lmmunoreactive proteins were visualized by incubating cells in avidin-biotin-peroxidase (1:400) for 1 h followed by treatment with a diaminobenzidinehydrogen peroxide solution for 10 min. Northern Blot Analysis Total RNA was isolated from MG/ hGR cells by lysis in 4 M guanidinium thiocyanate and centrifugation through a cushion of cesium chloride (54). Polyadenylated [poly(A)+] RNA was selected using oligo (dT)-cellulose according to standard procedures (53). Total RNA (20 pg) or poly(A)’ RNA (5 Kg) samples were denatured in glyoxal and DMSO and size fractionated on 1% agarose gels. After electrophoresis, RNA was transferred to a nylon membrane for 18-24 h and immobilized by UV crosslinking (UV Stratalinker 1800, Stratagene, La Jolla, CA). Filters were then hybridized by standard methods with the following 32P-labeled probes: human glucocorticoid receptor antisense RNA, DHFR cDNA (2500 base pairs EcoRI/Pstl fragment), or chick @actin antisense RNA. Riboprobes were generated from the dual promoter vector pT7/T3-18 containing either the human glucocorticoid receptor cDNA (39) or the chick P-actin cDNA, according to the procedure recommended by the supplier, BRL. 3*P-Labeled DHFR DNA was prepared using a random priming kit. After hybridization, membranes were extensively washed at 70 C and exposed to x-ray film. Before hybridization with a different probe, membranes were stripped according to the instruction of the commercial source, ICN. Determination of CAT Activity MG/hGR cells were propagated in MEM containing 5 nM dexamethasone for times ranging from 24-72 h. After hormone pretreatment, cells were transiently transfected with a reporter plasmid containing the mouse mammary tumor virus promoter linked to the CAT gene. This plasmid (pGMCS) was provided by Dr. Keith Yamamoto (University of California, San Francisco, CA). Transient transfections were performed by the DEAE-dextran method as modified by Lopata et a/. (5.5). Briefly, control and dexamethasone-treated cells were plated in duplicate into lo-cm dishes at a density of l-3 x lo6 cells per dish in complete MEM. The followina dav. 10 ua DGMCS reported plasmid and 5 pg RSV promote;-P-galact&ase control plasmid (pRSV$gal, kindly provided by Dr. William Rutter, University of California, San Francisco, CA) were mixed with DEAE-dextran and incubated with cells for 4 h at 37 C. After incubation with the DNA/DEAE-dextran mixture, cells were washed and exposed to 10% DMSO for 90 sec. Complete MEM containing either 5 nM dexamethasone or 100 nM dexamethasone was then added to each group, and cells were incubated for 18 h at 37 C. Cells were harvested in PBS and assayed for CAT activity essentially as described by Gorman et al. (56). Typi-

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tally, 40 pg cell extract was used per reaction and was incubated for 3 h at 37 C. f’4C1Chloramphenicol and its acetylated forms were separated by TLC in a solvent of CHCla:methanol (95:5). After autoradiography, the amount of CAT activity in each sample was assayed by excising radioactive spots from silica gel plates and counting in Scintiverse (Fisher Scientific Co., Pittsburgh, PA). Relative levels of conversion were calculated after the transfection efficiencies of all cell lines had been determined. These values were determined by calorimetric assay of P-galactosidase activity (57) produced by the control plasmid pRSV-P-gal and were essentially identical for all lines tested. Acknowledgments We thank Dr. Yoshie Itoh-Lindstrom and assistance with CAT assays.

for her helpful

suggestions

Received August 11, 1992. Revision received September 17, 1992. Accepted September 21, 1992. Address requests for reprints to: Dr. John A. Cidlowski, CB #7545 460 Medical Science Research Building, University of North Carolina, Chapel Hill, North Carolina 27599-7545. This work was supported by NIH Grant DK-32460. Portions of this work have been presented at the 72nd Annual Meeting of The Endocrine Society, Atlanta, GA, 1990 (44). ’ Current address: Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 752359050.

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Modulation of glucocorticoid induction of stably transfected tyrosine aminotransferase gene constructs involves elements up-stream of the glucocorticoid-responsive element.

Glucocorticoid receptors.

Mineralocorticoid and glucocorticoid receptors in human kidney.

Glucocorticoid receptors and glucocorticoid sensitivity of mitogen stimulated and unstimulated human lymphocytes.

Glucocorticoid receptors: evidence for a second, non-glucocorticoid binding site.

Demonstration of glucocorticoid receptors in human mammary carcinomas.

Two glucocorticoid binding sites on the human glucocorticoid receptor.

The glucocorticoid receptors regulation in Alzheimer's disease.

Loss of glucocorticoid fast feedback in depression.

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

Unraveling the functions of the membrane-bound glucocorticoid receptors: first clues on origin and functional activity.

Enhancer Turnover Is Associated with a Divergent Transcriptional Response to Glucocorticoid in Mouse and Human Macrophages.

The glucocorticoid receptor 1A3 promoter correlates with high sensitivity to glucocorticoid-induced apoptosis in human lymphocytes.

Comparing the rules of engagement of androgen and glucocorticoid receptors.

Early environments, glucocorticoid receptors, and behavioral epigenetics.

Evidence that dephosphorylation inactivates glucocorticoid receptors.

Glucocorticoid and mineralocorticoid receptors in gut mucosa.

Involvement of the Androgen and Glucocorticoid Receptors in Bladder Cancer.

Loss of the podocyte glucocorticoid receptor exacerbates proteinuria after injury.

The mechanism for glucocorticoid-resistance in a rat hepatoma cell variant that contains functional glucocorticoid receptor.

The role of cytoplasmic glucocorticoid receptors in the cytolysis of human lymphoblastoid cells by methylprednisolone [proceedings].

Structural Stereochemistry of Androstene Hormones Determines Interactions with Human Androgen, Estrogen, and Glucocorticoid Receptors.

Methylation of the Glucocorticoid Receptor (NR3C1) in Placenta Is Associated with Infant Cry Acoustics.

Implication of glucocorticoid receptors in the stimulation of human glioma cell proliferation by dexamethasone.