Human Glucocorticoid Receptor cDNA Contains Sequences Sufficient for Receptor Down-regulation*

Glucocorticoid receptors are ligand-dependent transcription factors that are subject to down-regulation by their cognate ligand; however, the mechanisms mediating this physiological response are not completely understood. Since analysis of the human glucocorticoid receptor (hGR) cDNA sequence revealed the presence of sequences with homology to both positive and negative glucocorticoid regulatory elements, we have examined the potential of hGR to bind to the hGR cDNA by Southwestern blot analysis. The data revealed that glucocorticoid receptors exhibited specific binding to their own cDNA. To determine whether this binding was of functional significance in the down-regulation of glucocorticoid receptors, we analyzed the effect of glucocorticoids on hGR protein levels from COS 1 cells transfected with an hGR cDNA expression vector. These transfected cells produced intact hGR that were capable of ligand-dependent regulation of a co-transfected glucocorticoid-responsive reporter gene. Glucocorticoid treatment of hGR-transfected cells resulted in down-regulation of hGR (assayed by both glucocorticoid binding capacity and hGR protein levels) within 24 h of steroid administration. To determine if the glucocorticoid-induced down-regulation of transfected hGR was compatible with effects at the levels of receptor gene expression and RNA stability, we examined hGR mRNA steady state levels. Reductions from 2- to 6-fold were observed in hGR mRNA levels following glucocorticoid treatment of transfected COS 1 cells. This down-regulation of transfected hGR mRNA could not be attributed to either the Rous sarcoma virus promoter, which drives hGR expression, or to other sequences present in the vector plasmid since transcription of a related plasmid containing a chloramphenicol acetyltransferase gene in place of the hGR cDNA was not regulated by glucocorticoids. Down-regulation of hGR mRNA by glucocorticoids in transfected cells occurred in a time- and dose-dependent manner that is consistent with a glucocorticoid receptor-mediated process. Glucocorticoid-induced down-regulation of hGR mRNa steady state levels was not observed in COS 1 cells transfected with cDNAs encoding mutant hGR (defective in either steroid or DNA binding), which indicates that functional steroid and DNA binding domains of the expressed hGR were required for down-regulation. Interestingly, treatment of transfected COS 1 cells with the glucocorticoid antagonist RU486 also resulted in down-regulation of transfected hGR mRNA. Deletion analysis revealed that the region of the hGR cDNA that was responsible in part for the observed down-regulation in response to glucocorticoid was contained within a 1-kilobase restriction fragment (from base pair +527 to +1526).(ABSTRACT TRUNCATED AT 400 WORDS)

Both positive and negative modulation of gene transcription in response to glucocorticoids has been well established (for review see Refs. l-3). The action of glucocorticoid hormones is mediated through specific high affinity binding to glucocorticoid receptors (GR).' These proteins, together with the other steroid receptors, belong to a family of ligand-dependent transcription factors (4). Following steroid binding, the receptor hormone complex interacts with DNA and ultimately results in alteration of the transcription of certain genes. Although the mechanism of steroid hormone action has not been completely elucidated, it is known that the GR complex interacts with specific target DNAs called glucocorticoid regulatory elements (GREs) that are associated with genes whose transcription is elevated in response to hormone. Much less is known about how GR negatively regulates gene expression; however, sequences that may serve as negative GREs (nGREs) have been recently identified for a few genes whose transcription is inhibited by glucocorticoids (5). The biologic effects of glucocorticoids are dependent on the presence of functional receptors (6). One determinant of cellular responsiveness to steroid treatment is the concentration of GR (7,8). Thus, to understand the mechanism of steroid hormone action it is of particular importance to understand how cellular GR levels are regulated. Glucocorticoids and other steroid hormones down-regulate the levels of their cognate receptors in a number of target tissues and in many different cell lines (9)(10)(11) buffer was added to filters and incubated for 30 min; filters were washed and exposed to x-ray film.

Glucocorticoid
Receptors Bind to the Human Glucocorticoid Receptor cDNA-Computer analysis of the hGR cDNA has revealed the presence of multiple sequences with homology to the GRE octamer that was originally identified by Payvar et al. (35)" and to a putative negative GRE associated with the pro-opiomelanocortin gene (36). Based on these observations, we wanted to determine whether these GRE-like elements, which reside within the glucocorticoid receptor gene, allow specific binding of receptor to its own cDNA. Such a result might suggest that these elements function in the regulation of GR gene expression by its cognate ligand. The ability of GR from HeLa cell cytosolic fractions to bind various fragments of DNA was analyzed by Southwestern blotting. This technique has been shown to permit analysis of specific GR protein-DNA interactions using cytosolic proteins immobilized on nitrocellulose (30). Only a minimal level of nonspecific protein-DNA interactions are seen with this procedure due to the inclusion of nonfat dry milk as a blocking agent during hybridization.
The identity of GR on Southwestern blots can be verified by the capacity of the receptor to saturably bind to the affinity ligand dexamethasone mesylate. The pRShGR KpnI-DraI fragment that contains the entire hGR cDNA coding sequence but lacks most of the -2.3-kb untranslated region at the 3' end (Fig. lA, hGR) was bound in preference to two other DNA fragments, which consist either of vector sequences from pRShGR (Fig. lA, RSV) or a pBR322 fragment (Fig. lA, PBR) that is devoid of GREs (33). Some binding of GR to the vector sequences in pRShGR occurs and is most likely due to a sequence that matches the GRE consensus located in the pBR322 portion of the expression vector (bp 4044-4051 of pBR322) (33). Fig. 1B  (13, 14, 17), we wanted to determine if hGR that had been introduced into cells by transfection was also susceptible to down-regulation.
We first determined that hGR from transfected cells resembled native GR both structurally and functionally. The cytosolic fraction isolated from COS cells transfected with the hGR cDNA expression vector, pRShGR, was incubated with ["Hldexamethasone and subjected to sucrose density gradient centrifugation.
This physical analysis confirmed that intact steroid binding hGR (9-10 S) was synthesized in these transfected cells (Ref. 37 and data not shown). To analyze for the function of hGR from transfected cells, a plasmid containing the glucocorticoid-sensitive mouse mammary tumor virus-LTR promoter linked to CAT was cotransfected with pRShGR into COS cells. As expected based on the work of others (24), glucocorticoid-inducible CAT activity was seen in these cells indicating that the transfected hGR was able to activate transcription from a glucocorticoidsensitive promoter (data not shown). We then analyzed the effect of dexamethasone on hGR levels in COS cells transfected with pRShGR by two criteria: 1) immunoreactivity with anti-hGR antibodies and 2) binding to the radiolabeled affinity ligand ["Hldexamethasone mesylate. To demonstrate that GR protein levels were reduced in dexamethasonetreated transfected cells, cytosolic protein extracts from washed cells were analyzed by Western blotting.
For this analysis we used antiserum generated against a peptide that is located in the immunogenic domain (NH2 terminus) of the hGR.* Immune complexes were visualized following incubation of the immobilized proteins with '251-staphylococcal protein A. The level of transfected immunoreactive hGR from cells grown in the absence of dexamethasone (Con) was greater than that from cells grown in the presence of dexamethasone (Dex) (Fig. 2A). To confirm that GR levels were reduced in dexamethasone-treated transfected cells, GR binding to ["Hldexamethasone mesylate was examined. Transfected cells were washed extensively to remove unlabeled dexamethasone (see "Materials and Methods") prior to incubation with ["Hldexamethasone mesylate. This wash procedure has been shown to remove greater than 99% of the unlabeled steroid and allows efficient labeling of GR from cytosols (9,32 is smaller than the reported size of native GR transcripts due to removal from the hGR cDNA of most of the 3'-untranslated region, which contains the GR binding sites studied by Okret et al. (13). Fifteen independent experiments showed a mean decrease of 4-fold +-0.5 SE. in hGR mRNA levels following 12-17-h treatment with 2 x 10e7 M dexamethasone.
The two blots in Fig. 3 are representative of the range of down-regulation that we observe. The blots were also hybridized with ,'"P-labeled @-actin cDNA to confirm that equivalent amounts of RNA were present in control and dexamethasone-treated sample lanes. The differential signal intensities obtained in control and dexamethasone-treated samples were unaffected by the type of hybridization probe used, either "P-labeled hGR cDNA or cRNA.
Examination of hGR mRNA Levels from Transfected Cells-To determine whether down-regulation of hGR protein was reflected at the level of mRNA we analyzed steady state hGR mRNA levels in response to hormone treatment.
The hGR cDNA was transfected into COS cells, and total cellular RNA or poly(A+) RNA was isolated from control and dexamethasone-treated cells (36-40 h post-transfection). As shown in Fig. 3, Northern blot analysis revealed that hGR mRNA levels from transfected cells were markedly decreased in response to dexamethasone treatment.
Only the transfected hGR mRNA (-3.3 kb) encoded by pRShGR was present in these Northern blots, which is consistent with the observation that endogenous GR is not detectable in COS cells (24, 37). This Negatively Acting Sequences Are Located within the hGR cDNA-Expression of the hGR cDNA in pRShGR is driven by the RSV-LTR promoter contained on a 524-bp restriction fragment (24). Other investigators have shown that the RSV-LTR promoter is not regulated by dexamethasone (38,39). To verify that the RSV promoter is not sensitive to dexamethasone and to rule out the possibility that other vector sequences in pRShGR influenced regulation by glucocorticoids, we examined CAT activity in cells cotransfected with the plasmids pRSVCAT/ori and pRShGR. Plasmid RSVCAT/ori is identical to pRShGR except the hGR cDNA has been replaced by the cDNA encoding CAT. If sequences that are located outside the hGR cDNA were responsible for down-regulation of hGR mRNA levels, then CAT activity would be expected to decrease in response to dexamethasone treatment in cells that were transfected with pRSVCAT/ori. It was necessary to cotransfect cells with hGR since, as mentioned above, COS cells contain negligible levels of endogenous GR. As given in Table I, no difference in CAT activity in cell extracts prepared from control or dexamethasone-treated transfected cells was detected. This result demonstrates that the effects of dexamethasone on hGR mRNA levels observed in pRShGR-transfected cells were specific for the hGR cDNA and were not due to the RSV promoter or to sequences present in the vector plasmid. As expected, transfection of pRSVCAT/ori alone (no cotransfected hGR) similarly resulted in no difference in CAT activity in response to dexamethasone (data not shown). Down-regulation of Transfected hGR mRNA Is Time-and Dose-dependent-To determine if the glucocorticoid-induced decrease in hGR mRNA levels in transfected cells was a glucocorticoid receptor-mediated event we examined hGR mRNA levels following treatment with varying dexamethasone concentrations.
The kinetics of this response were also examined. Treatment of hGR-transfected COS cells for 15 h with dexamethasone concentrations ranging from 1 X lo-'" to 1 x 10m6 M resulted in a dose-dependent down-regulation of hGR mRNA (Fig. 4). Data from two independent experi-  (9), which suggests that down-regulation is mediated by GR. The time course of hGR mRNA down-regulation was examined at a dexamethasone concentration of 2 X lo-' M to determine the earliest time of mRNA response. Northern blot analysis of total RNA from COS cells transfected with an hGR cDNA showed a decrease in hGR mRNA steady state levels within 1 h of dexamethasone addition (Fig. 5). Minimum levels of hGR mRNA were established between 4 and 6 h and remained decreased for at least 24 h. Due to the transient nature of hGR expression in transfected cells, hGR mRNA was not measured at times greater than 24 h after dexamethasone treatment (corresponding to 36-40 h posttransfection).
The kinetics of down-regulation resemble that seen for other receptor-mediated events such as induction of mouse mammary tumor virus RNA by glucocorticoids (40). Together these data are consistent with the hypothesis that the glucocorticoid effects on hGR mRNA levels observed in transfected cells were mediated by GR.
Functional hGR Is Required for Down-regulation of hGR mRNA-GR, like other members of the steroid receptor superfamily, contain discrete domains that are responsible for DNA binding, ligand binding, and transactivation functions (4). Data presented above suggest that down-regulation is receptor-mediated; to directly test this hypothesis we analyzed the ability of two GR mutants to down-regulate levels of hGR mRNA in response to dexamethasone.
Transfections were performed using two hGR cDNAs mutants: one that encodes a non-DNA binding hGR and one that specifies a non-steroid binding variant. Both mutants were generated by insertion of linkers, which did not disrupt the protein reading frame (24). Fig. 6 shows that mRNA levels from either of these mutant receptors were not reduced in response to dexamethasone treatment. This result contrasts sharply with the results obtained for the wild type receptor and suggests that DNA-and steroid-binding functions of the GR receptors are required for down-regulation to occur and provides direct evidence that down-regulation is GR-mediated. Down-regulation of hGR mRNA by the Glucocorticoid Antagonist RU486-RU486 binds with high affinity to glucocorticoid receptors; however, these RU486-GR complexes do not mediate transcriptional activation (41,42). Based on these properties, we wished to determine whether down-regulation of hGR mRNA could proceed in transfected cells following RU486 treatment.
This approach should help to reveal whether the transcriptionally active form of the receptor was required for down-regulation. Fig. 7 shows that hGR mRNA levels were reduced in response to RU486 (2 X lo-' M). Concentrations between low9 and lo-' M RU486 were sufficient to evoke a small (-25-30%) decrease in the levels of hGR mRNA as compared with untreated controls (data not shown). Treatment of transfected cells with steroids that interact with GR such as cortisol and progesterone caused down-regulation of hGR mRNA in contrast to estradiol (2 x lo-' M), which had no effect on transfected hGR mRNA levels (data not shown).
Down-regulation of an hGR Deletion Mutant Is Impaired-To begin to localize sequences within the hGR cDNA that are responsible for down-regulation of transfected hGR mRNA, we evaluated the regulation of a deleted hGR cDNA. When an hGR cDNA that lacks sequences from bp +527 to +1526 (an AccI-ClaI fragment) was cotransfected with the intact hGR cDNA, levels of the truncated hGR transcript were partially decreased compared with the intact receptor mRNA (Fig. 8) that was derived from GREs from a number of glucocorticoidregulated genes (4). Results of three independent experiments revealed that the levels of the truncated Act-Cla hGR mRNA were reduced to 48 + 5% whereas intact hGR mRNA levels were decreased to 26 * 3% of control levels. This result suggests that the region between nucleotides +527 and +1526 of the hGR cDNA contains at least some of the signals that are necessary for down-regulation.
Southwestern blotting revealed that hGR from HeLa cytosols bound to the 844-bp hGR AccI-ClaI restriction fragment (contained within the 1000-bp AccI-ClaI fragment) in preference to an equivalently sized non-GRE-containing fragment of pBR322 (Fig. 9A). tivity seen in Fig. 9A most likely represents a nonspecific protein-DNA interaction since binding to both fragments (Act-Cla and pBR) was comparable. The protein responsible for this DNA binding activity appears to be unrelated to GR as this protein did not bind to ["Hldexamethasone mesylate ( Fig. 9B) or to anti-GR antibodies (data not shown). Thus the observation that hGR specifically interacts with the Acc-Cla fragment of the hGR cDNA supports the theory that the region from +527 to +1526 may contain autoregulatory signals. DISCUSSION We have examined steady state hGR mRNA and protein levels from COS 1 cells following transfection with an hGR expression vector. Down-regulation of hGR mRNA and protein levels was observed in transfected cells in response to glucocorticoid treatment. Non-hGR sequences located in the vector were not responsible for the glucocorticoid-mediated alteration in receptor levels; thus, it appears that the hGR cDNA contains sequences sufficient to evoke the glucocorticoid-induced down-regulation of hGR mRNA and protein seen in transfected cells. This effect required the DNA-and steroid-binding functions of the receptor as demonstrated by the failure of mRNA from either a DNA-or a steroid-binding deficient mutant to be down-regulated. Moreover, the time and dose dependence of down-regulation by dexamethasone in transfected cells provides further evidence that this effect is receptor-mediated.
There was some variability observed in the extent of downregulation of hGR mRNA in response to dexamethasone between the different experiments.
We attribute this variability to differences in transfection efficiencies and, perhaps to a lesser extent, to endogenous steroid in the cell culture media and to slight differences in the times of exposure to dexamethasone.
Nevertheless, down-regulation was consistently observed regardless of variations in the initial levels of hGR mRNA.
We do not know if down-regulation of mRNA levels in transfected COS 1 cells is due to transcriptional or posttranscriptional effects. It has been reported that the rate of endogenous GR transcription is decreased in dexamethasonetreated human IM9 lymphocytes (17) and in rat liver (15). In addition, Rosewicz et al. (17) and Dong et al. (15) failed to see any dexamethasone effects on GR mRNA half-life in cultured cells. The kinetics of down-regulation that we observed for the transfected hGR mRNA resembled that observed for the endogenous GR mRNA (17). Also, transfected hGR mRNA displayed a sensitivity to varying dexamethasone concentra-