The down side of glucocorticoid receptor regulation

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Moleculur und Cellular Endocrinology, 83 (1992) Cl -C8 0 1992 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/92/$05.00

MOLCEL

02705

At the Cutting

Edge

The down side of glucocorticoid receptor regulation Kerry L. Burnstein

and John A. Cidlowski

Lineherger Cornprehensil,e Cancer Research Center und Department of Physiology. Unirersif): of North Curolinu ut Chapel Hill, Chupel Hill, NC 27599, USA (Accepted

Key words: Steroid

receptor;

Down-regulation;

Autoregulation;

Glucocorticoid receptors are ligand-dependent transcription factors that belong to a large family of related proteins, which includes receptors for steroid and thyroid hormones as well as the morphogen retinoic acid (Evans, 1988). It is well known that glucocorticoid action is mediated through high affinity binding of steroid to the glucocorticoid receptor (GR). Glucocorticoid action is initiated when the hormone-occupied receptor binds to discrete target DNA sequences and, through mechanisms that are not completely understood, stimulates or represses the transcription of specific genes (Yamamoto, 1985; Beato, 1989; Burnstein and Cidlowski, 1989). The role of the receptor is to ‘transduce’ hormonal signals by altering the expression of specific genes or gene networks ultimately resulting in a change in cellular phenotype (Yamamoto, 1985). The pivotal role that receptor plays in hormone action is underscored by observations that the development of a steroid resistant state is virtually always accompanied by either mutation of the receptor or decreased receptor content (Yamamoto et al., 1974; Chrousos et al., 1982; Iida et al., 1985; Lipsett et al., 1986). A direct correlation between receptor number and cellular responsiveness to hormone has been demonstrated in a variety of cell lines (Bourgeois and Newby, 1974; Gehring et al., 1984;

Correspondence to: Kerry L. Burnstein, Department of Physiology, C.B. No. 7545, 460 Med. Sci. Res. Bldg., University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

23 October

1991)

Desensitization

Vanderbilt et al., 1987) and in intact animals (Smith and Shuster, 1984; Yi Li et al., 1989) as well as in humans (Pui et al., 1984; Iida et al., 1985). The existence of such a relationship between hormone sensitivity and receptor concentration in target cells and tissues necessitates a thorough understanding of the mechanisms responsible for maintaining receptor levels. In this review, we discuss the development of the ideas that have led to the concept of glucocorticoid receptor autoregulation. A unifying model that reflects the current state of knowledge of glucocorticoid receptor down-regulation is presented. The concentration of GR varies between different tissues and even within a given tissue; receptor levels may fluctuate with changes in the cell cycle (Cidlowski and Michaels, 19771, during aging (Chang and Roth, 1979) and in response to hormone exposure (Cidlowski and Cidlowski, 1981; Svec and Rudis, 1981; Sapolsky et al., 1984). Several years ago we and others demonstrated that glucocorticoid treatment of cells results in decreased levels of GR as determined by measurement of the capacity of receptors to bind glucocorticoid. This phenomenon, termed downregulation, is also well documented for a variety of different cell surface receptors including neurotransmitter and peptide receptors (Lefkowitz, 19811. As might be predicted based on the relationship between receptor number and responsiveness, down-regulation is accompanied by a decrease in the biological effects of hormone administration. However, this desensitization to

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hormone is not permanent and upon removal of ligand, steroid binding capacity and responsiveness is eventually regained (Seigler and Svec, 1984; McIntyre and Samuels, 198.5). Down-regulation of GR following glucocorticoid administration has been demonstrated in a variety of cell lines (Cidlowski and Cidlowski, 1981; Svec and Rudis, 19811, intact rats (Tornello et al., 1982) and peripheral blood cells from human volunteers (Schlccte et al., 1982; Shipman et al., 1983). The role of glucocorticoids in the regulation of GR was first demonstrated in 1981 in two established cell culture lines. Exposure of AtT-20 (mouse pituitary tumor) (Svec and Rudis, 1981) and HeLa S, (h uman cervical carcinoma) (Cidlowski and Cidlowski, 1981) to glucocorticoids resulted in an approximately 75% reduction in glucocorticoid receptors determined by whole cell binding assays using tritiated Iigand. In these experiments residual unlabeled glucocorticoid was removed by extensive washing and dilution of the cells in hormone-free media, which enabled examination of the glucocorticoid binding capacity of the hormone-pretreated cells. Cells that were pretreated with glucocorticoids for 1-4 days exhibited a decrease in the number of steroid binding sites. The steroid dissociation constant and the physical properties of the residual receptors in the hormone-treated cells were unchanged relative to untreated cells, suggesting that these receptors retained function. Only glucocorticoids and not other steroid hormones were able to elicit down-regulation of glucocorticoid receptors (Cidlowski and Cidlowski, 1981). In addition, ongoing protein synthesis was not required for glucocorticoid-induced down-regulation of GR (Svec and Rudis, 1981), which is consistent with the idea that receptor plays a direct role in its own regulation. However, these experiments, which measured receptor by steroid binding capacity, did not exclude the possibility that receptor protein levels may have stayed constant. It remained plausible that some receptors were not detected by steroid binding assays but were present in hormone-treated cells in a form that was not competent for Iigand binding. Alternatively, receptors may have been sequestered in a cellular compartment that was not susceptible to detection.

Availability of antisera against the GR protein (Gametchu and Harrison, 1984; Westphal et al., 1984) has permitted comparative analysis of receptor protein levels in hormone-treated and untreated cells. Decreased GR protein levels (4080%) were seen by Western blot analysis of extracts from a variety of cell lines and rat liver following 24 h of glucocorticoid treatment (Dong et al., 1988; Hoeck et al., 1989; Burnstein et al., 1990, 1991). To gain insight into the mechanism responsible for this reduction in receptor protein, a number of investigators have examined the effect of glucocorticoids on the half-life of the receptor. Most data are consistent with some role of glucocorticoid in moduIating the stability of the receptor protein. McIntyre and Samucls (1985) were the first to examine this issue. Using dense amino acid labeling studies of the rat pituitary cell line GHl. they found glucocorticoid receptor half-life to be reduced by Z-fold following exposure to agonist ~half-life of approximateIy 10 h) compared to receptor half-life in the absence of Iigand (half-life of approximately 20 h). No difference in the synthetic rate of receptor protein was found in the presence or absence of hormone. Dong et al. (19881 reported a similar finding in HTC (hepatoma) ceils treated with or without hormone and exposed to the protein synthesis inhibitor cycloheximide. Receptor half-life in these HTC cells was also reduced by 2-fold in the hormone-treated ceils. In NIH3T3 cells, the half-life of GR was significantly shorter than in HTC or GHl cells as determined by pulse chase analysis. Despite possible differences in receptor turnover in NIH3T3 cells compared to GHl or rat liver cells, a comparable reduction in the receptor half-life of approximately 2-fold was seen following glucocorticoid administration (Hoeck et al., 1989). In contrast to these findings, Distelhorst and Howard (1989) reported that GR protein half-life was unaffected by glucocorticoids in S49 mouse iymphoma cells. In this report, steady-state levels of GR protein were not examined in response to glucocorticoid and it remains formally possible that in these cells, in contrast to the majority of GR-containing cell lines and tissues that have been examined, down-regulation does not occur. AItered receptor half-life therefore appears to be

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one of the mechanisms by which down-regulation is achieved; however, this effect may not occur in all cell types and may not be sufficient to account completely for receptor down-regulation. Cloning of cDNAs that encode GR (Meisfeld et al., 1984; Hollenberg et al., 1985) has provided investigators a tool with which to examine GR mRNA following GR down-regulation. These analyses have shown that GR mRNA levels are decreased significantly (50-95%) in all cell lines and tissues that undergo glucocorticoid-mediated down-regulation of GR protein (Okret et al., 1986; Kalinyak et al., 1987; Dong et al., 1988; Rosewicz et al., 1988; Hoeck et al., 1989; Burnstein et al., 1990, 1991). Based on these findings it appears that an important element in the control of GR down-regulation is exerted at the levels of GR mRNA synthesis or stability. Additional studies have been performed to determine whether transcriptional or post-transcriptional mechanisms are responsible for hormone-induced reductions in steady-state levels of GR mRNA. Examination of transcription in isolated nuclei from hormone-treated or untreated IM9 (human lymphocytes) (Rosewicz et al., 1988) and rat liver cells (Dong et al., 1988) has revealed that glucocorticoids repress the transcription rate of the GR gene. In studies designed to determine the effect of glucocorticoids on GR mRNA stability, hormone treatment was found to have no effect on GR mRNA half-life in IM9 (Rosewicz et al., 1988), HTC (hepatoma) or AR42J (rat pancreatic acinar) cells (Dong et al., 1988). However, in each of these studies, cells were pretreated with glucocorticoids for sufficient lengths of time to permit maximal down-regulation of receptor mRNA prior to determination of GR mRNA half-life. Therefore, the effects of hormone on GR mRNA stability were determined after new steady-state levels of GR mRNA were established. Preliminary work by Vedeckis (1989) demonstrated that GR mRNA half-life was decreased in AtT-20 cells exposed to hormone. In these experiments RNA stability was analyzed during glucocorticoid-induced downregulation instead of after down-regulated levels of GR were achieved. Collectively these studies indicate that GR down-regulation is quite complex and may occur at multiple levels (transcrip-

tional, post-transcriptional, post-translational) with different cell types utilizing a combination of general and/or tissue-specific mechanisms of receptor regulation. Since receptor numbers vary widely among different cell types (Ballard et al., 19741, it is perhaps not surprising that a variety of mechanisms might be available for maintaining receptor levels. In view of the fact that GR interacts with target genes to modulate gene transcription, it is possible that receptor may directly influence its own transcription by binding to the GR gene. Okret et al. (1986) demonstrated that GR protein specifically binds to sequences in the 3’ untranslated region of the rat GR cDNA. We have found that the human GR cDNA in which most of the 3’ untranslated region has been removed (including the aforementioned GR binding sites) is still bound preferentially by GR (Burnstein et al., 1990). Based on the specific interaction between the GR protein and GR cDNA we predicted that the human GR cDNA may contain requisite sequences for ligand-induced down-regulation of GR. Our laboratory has tested this theory in cells that were either transiently (Burnstein et al., 1990) or stably transfected with the human GR cDNA (Burnstein et al., 1991; Bellingham et al., submitted). COS 1 (monkey kidney) cells were used for transient expression and CHO (Chinese hamster ovary) cells were used for stable expression of the human GR cDNA from the plasmid, pRShGR (obtained from Dr. R. Evans). Both cell lines produced intact, functional GR. Following glucocorticoid treatment of COS cells and CHO cells containing transfected GR, we found that levels of GR protein and mRNA were significantly reduced (50-80%X This glucocorticoid-mediated down-regulation was not due to the Rous sarcoma virus (RSV) promoter, which drives expression of the hGR cDNA in pRShGR. This promoter is unable to regulate the expression of a reporter gene encoding chloramphenicol acetyltransferase in response to glucocorticoid, confirming that the RSV promoter is insensitive to glucocorticoids (Majors and Varmus, 1983; Camper et al., 1985). These experiments suggest that the GR cDNA itself contains signals involved in down-regulation. Further characterization of the kinetics, dose-dependency and steroid

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specificity of transfected GR mRNA regulation showed that this process very closely resembles glucocorticoid-mediated down-regulation seen in cells that contain native, non-transfected GR (Burnstein et al., 1990; Bellingham et al., submitted). Although it is likely that sequences present in the promoter of the endogenous receptor gene play a role in GR autoregulation, it is also clear that sequences found in the receptor cDNA provide sufficient information to permit down-regulation. The significance of the location of such intragenic GR regulatory sequences is not known, perhaps they allow for more specific or stringent control of receptor mRNA synthesis or receptor mRNA half-life. A number of genes have been shown to contain regulatory sequences located downstream from the site of transcription initiation in both introns and exons. For instance, the first intron of the human growth hormone gene contains a glucocorticoid response element @later et al., 1985) and the thyrotropin P-subunit gene contains thyroid hormone regulatory sequences within the coding region (Wondisford et al., 1989). The nature of the intragenic GR sequences and how they function in down-regulation is not known. A provocative model for down-regulation invokes the possibility that GR binding to sequences within its own gene affects transcription directly, either by repressing transcription initiation or by blocking elongation. Alternatively a post-transcriptional role of these regulatory sequences can be envisioned in which GR binding to GR mRNA alters the stability of the receptor mRNA. Since down-regulation occurs in the presence of cycloheximide it is unlikely that glucocorticoids induce a protein that is responsible for down-regulation. The potential for GR interation with mRNA has not been explored; however, several years ago Ah and Vedeckis (1987) found that cytoplasmic GR is bound to tRNA. Receptor binding to tRNA may be indicative of receptor interaction with mRNA as well. Thus, a combination of transcriptional and post-transcriptional mechanisms may be utilized to achieve precise control of receptor levels. A ‘whimsical’ model of GR down-regulation that is consistent with the experimental data from a variety of different cell types is shown in Fig. 1.

The first three steps are common to current models that depict the mechanism of glucocorticoid action. Prior to steroid binding (step l), GR exist in inactive, heteromeric complexes which consist of the glucocorticoid binding receptor polypeptide and ancillary proteins that have been identified as heat shock proteins. In particular, GR is bound by a 90 kDa heat shock protein (‘hsp’) (for review see Pratt, 1990). This ubiquitous heat shock protein is thought to maintain the receptor in a’ conformation that permits steroid binding (Picard ct al., 1990). Binding of steroid to the heteromeric receptor triggers release of the 90 kDa heat shock protein and may result in a conformational change in receptor structure (step 2) (Pratt, 1990). The hormone-receptor complex migrates to the nucleus of a given target cell (step 3). Two receptor regions are necessary for this process to occur, although how these domains function in nuclear translocation is not well understood (Picard and Yamamoto, 1987). Glucocorticoid receptor binding to regulatory sequences in chromatin results in alterations in the expression of specific target genes either by enhancing or repressing basal transcription rates. Glucocorticoid effects on transcription appear to involve the concerted action of GR and other transcription factors (for review see Beato, 1991) and in fact, the glucocorticoid receptor gene is itself a target for glucocorticoid action as discussed above. There is evidence that autoregulation of GR gene expression may occur at the transcriptional, post-transcriptional or post-translational level. Down-regulation of receptor mRNA in response to glucocorticoid treatment may involve the direct interaction of GR with its own gene. GR, which binds DNA as a dimer (Tsai et al., 1988), is shown (step 4a) binding to the GR gene and repressing transcription. The ensuing decrease in the transcription rate of the GR gene following receptor binding could result from either decreased initiation of transcription or blockage of RNA chain elongation. Alternatively or perhaps in addition to transcriptional influences, decreased steady-state levels of GR mRNA may be the result of effects on receptor mRNA processing, transport or stability. In step 4b, receptor is shown binding to nascent receptor mRNA resulting in enhanced GR mRNA degra-

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dation. Receptor effects on receptor mRNA stability could potentially occur in the nucleus or the cytoplasm. Examination of glucocorticoid effects on nuclear and cytoplasmic receptor mRNA levels should reveal if effects on GR mRNA stability occur in the nucleus or cytoplasm. A key question in glucocorticoid hormone action is: What triggers cessation of the hormonal response? In our model (step 51, receptor ‘falls off the DNA and is recycled back to the cytoplasm. These events probably involve alteration of the receptor protein (indicated as cross-hatching). Movement of the receptor back to the cytoplasm may involve post-translational modifications such as a change in the phospho~lation state of the receptor (Orti et al., 1989). It is not known whether post-translational modification of GR affects GR regulation. The contribution of phosphorylation in GR down-regulation was ad-

Fig. 1. whimsical

dressed by Hoeck et al. (1989) who demonstrated down-regulation of GR mRNA and protein in NIH3T3 cells following treatment with the antiglucocorticoid RU486. RU486, unlike giucocorticoid agonists, did not promote phosphotylation of GR which led to the conclusion that phosphorylation is not involved in GR down-regulation. However, this study examined only net phospho~lation of GR and it is possible that RU486 affected the phospho~lation/ dephosphorylation of specific GR residues without changing the overall phosphorylation state of GR. Clearly, this issue requires further study. As discussed earlier, decreased receptor protein half-life has been observed following glucocorticoid treatment and this is shown in step 6 of the model. It is not known whether ‘recycled’ and ‘naive’ receptors are equally susceptible to hormone-induced degradation and so our model en-

model of GR down-regulation.

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compasses both of these possibilities. Yet another component that may be involved in down-regulation is recycling of GR. For example, down-regulation might occur when the capacity of cells to recycle receptors is exceeded. Receptor recycling may also account for the inability to see complete down-regulation of GR in response to hormone. The model that we have proposed illustrates that glucocorticoids have the potential to influence receptor concentration at a number of discrete steps, which suggests that the capacity of glucocorticoids to modulate GR levels is critical for maintaining cellular homeostasis. In contrast to the prevailing notion that glucocorticoid receptors are down-regulated by glucocorticoid treatment, CEM C7 cells exhibit hormone-mediated up-regulation of GR. Eisen et al. (1988) have shown that glucocorticoids promote an increase in GR protein and mRNA in this human leukemic T cell line. These cells display another hormonal response that is observed only in certain cells of lymphoid origin, namely, growth arrest in the Gl stage of the cell cycle followed by cell death. Since GR levels are demonstrably higher in late Gl and early S phase of HeLa cells (Cidlowski and Cidlowski, 19821, increased levels of GR in CEM C7 cells in response to glucocorticoid treatment may be explained by the synchronization of these cells in Gl. Alternatively, upregulation of GR in CEM C7 cells may reflect the use of novel mechanisms for receptor regulation. The modulation of GR expression by glucocorticoids raises a number of questions concerning the clinical use of chronic glucocorticoid therapy. Does GR down-regulation occur in humans and, if so, does down-regulation result in desensitization to hormone therapy? A number of investigators have attempted to determine if high levels of serum cortisol down-regulate GR content in humans. These studies are somewhat hampered by the necessity of using peripheral blood cells as a source of GR, since there is considerable variation in the GR content of the different types of white blood cells. For instance, polymorphs and monocytes contain more GR than T and B lymphocytes (Lippman and Barr, 1977); therefore, it is critical to use homogeneous populations of white blood cell for determination of GR numbers. In addition, glucocorticoid treatment pro-

motes redistribution of leukocytes from circulation to the bone marrow (Cupps and Fauci, 19821, again underscoring the importance of monitoring the homogeneity of the lymphocyte cell preparation. Nonetheless, studies of GR levels in circulating lymphocytes from normal human volunteers after glucocorticoid administration displayed hormone-mediated down-regulation of GR (Schlecte et al., 1982; Shipman et al., 1983). Although these studies did not examine any other aspects of glucocorticoid responsiveness before and after hormone administration, down-regulation of GR by glucocorticoids was clearly established in healthy humans. GR levels measured as a function of changes in endogenous cortisol levels, either as a result of normal circadian variation or due to increased production in diseased states (e.g. Cushing’s syndrome or major depressive disorder) have not been reliably correlated (Kontula et al., 1980; Junker, 1983; Doe et al., 1986; Whalley et al., 1986; Lefebvre et al., 1989; Pardes et al., 1989; Wassef et al., 1990; Weiss et al., 1990). These discrepancies may be due to the high degree of variability in GR content measured in human lymphocytes or may be the result of limitations in the techniques used to quantitate GR levels. Virtually all studies that examine GR in humans utilize a ligand binding assay. Accurate estimation of GR using these assays depends on removal of unlabeled glucocorticoid and quantitative receptor binding to tritiated ligand. Moreover, it is not known whether GR levels in lymphocytes accurately reflect GR levels present in the brain and pituitary, which is where circulating levels of glucocorticoid are controlled. Since the clinical use of glucocorticoids is extensive and frequently chronic, the issue of GR down-regulation during glucocorticoid therapy warrants further examination using newer, more sensitive techniques that monitor GR protein and mRNA levels. The process of glucocorticoid-induced glucocorticoid receptor down-regulation is an important influence on the capacity of target cells and tissues to respond to hormonal stimuli. The phenomenon of GR down-regulation may be a protective mechanism against the deleterious effects of prolonged hormone exposure by tempering

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biological responses. Whether such a mechanism is utilized by healthy animals following periods of stress when glucocorticoid levels are elevated is not known. Given that high levels of glucocorticoids are seen under a variety of clinical situations, an understanding of receptor regulation may be vital in the treatment of certain endocrine disorders and in the development of appropriate steroid hormone therapies. Acknowledgements

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