Communication Vol. 6, OF 2581-2584,1988 CHEMISTRY 1988 …Communication Vol. 263, No. 6, Issue of February 25, pp. 2581-2584,1988 THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The

Communication Vol. 263, No. 6, Issue of February 25, pp. 2581-2584,1988 THE JOURNAL OF BIOLOGICAL CHEMISTRY

0 1988 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U. S. A.

Mechanism of Glucocorticoid Receptor Down-regulation by Glucocorticoids*

(Received for publication, October 26, 1987) Stefan Rosewiczz, Alex R. McDonald, Betty A. Maddux, Ira D. Goldfine, Roger L. Miesfelds, and Craig D. Logsdonl From the Cell Biology Laboratory and Department of Medicine, Mount Zion Hospital and Medical Center, Sun Francisco, California 94120 and the Departments of Biochemistry, Medicine, and Physiology, University of California, San Francisco, San Francisco, California 94143

The effect of glucocorticoids on the regulation of glucocorticoid receptor mRNA was studied in two dif- ferent cell lines, human IM-9 lymphocytes and rat pancreatic acinar AR42J cells. Using a glucocorticoid receptor cDNA probe, glucocorticoid receptor mRNA was examined by Northern blot hybridization and quantitated by slot-blot hybridization. In IM-9 and AR42J cells, dexamethasone decreased steady-state glucocorticoid receptor mRNA levels to approximately 50% of control. This decrease occurred with a one-half time of 3 h for IM-9 cells and 6 h for AR42J cells. Dexamethasone was the most potent steroid tested with a one-half maximal effect occurring at 10 nM and a maximal effect occurring at 100 nM. Glucocorticoid receptor mRNA half-life and gene transcription were then studied to determine the mechanism of decreased mRNA levels. The glucocorticoid mRNA half-life was approximately 120 min in IM-9 cells and 240 min in AR42J cells; these rates were not affected by dexa- methasone treatment. In contrast, the rate of glucocor- ticoid gene transcription as measured by run-on assays in IM-9 cells was decreased to 50 f 6% of control by dexamethasone. These results indicate therefore that glucocorticoids regulate glucocorticoid receptor mRNA levels by influencing gene transcription.

Glucocorticoids are important regulators of differentiation, development, and growth (1-3). The effects of glucocorticoids are mediated by the glucocorticoid receptor, an intracellular protein which is found in nearly all mammalian cells. The receptor has been purified to near homogeneity (4, 5), the cDNAs for rat, human, and mouse receptors have been cloned

* This work was supported by National Institutes of Health Grants DK35912, DK 26667, DK 32994, the Elise Stern Haas Research Fund, and the Mount Zion Hospital and Medical Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adver- tisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Supported by Deutsche Forschungsgemeinschaft Grant Ro 674/1-1. Current address: Dept. of Physiology, University of Michigan School of Medicine, Ann Arbor, MI 48109.

5 Supported by a special fellowship of the Leukemia Society of America.

11 To whom correspondence should be addressed Dept. of Physi- ology, University of Michigan School of Medicine, Ann Arbor, MI 48109.

25

(6-8), and functional domains within the receptor have been elucidated (9). The general mechanism of action of glucocor- ticoid receptors has also been characterized. Steroid hor- mones, which appear to enter cells by simple diffusion, bind to and activate the glucocorticoid receptor in a process re- ferred to as transformation (10); the transformed hormone- receptor complex then binds with increased avidity to specific DNA sequences termed glucocorticoid response elements (GREs)’ (for review see Ref. 10). Such interactions then regulate the transcription of selected genes (10-12). It is also known that the protein products of glucocorticoid responsive genes may themselves regulate the expression of other genes such that glucocorticoids may indirectly activate the expres- sion of gene networks (10).

In general, hormone receptors are regulated both by their own ligand (homologous regulation) and by other regulatory molecules (heterologous regulation). Glucocorticoids are in- volved in the heterologous up-regulation of several hormone receptors, including those for insulin (13) and cholecystokinin (14). In the case of the insulin receptor, glucocorticoids act at the level of gene transcription (15).

Glucocorticoids are involved in the homologous down-reg- ulation of glucocorticoid receptors (16). Measurements of glucocorticoid receptor mRNA have suggested that this reg- ulation takes place at the level of glucocorticoid receptor gene expression (16, 17). However, the mechanism of this down- regulation is unknown. In the present study we have investi- gated the down-regulation of the glucocorticoid receptor by glucocorticoids in two different cell lines; the human lym- phoma IM-9 and the rat pancreatic acinar AR42J cell line. We now report the glucocorticoids decrease glucocorticoid receptor steady-state mRNA levels by decreasing receptor gene transcription.

MATERIALS AND METHODS

Cell Culture-IM-9 lymphocytes (13) and AR42J cells (14) were grown as previously described. Routinely IM-9 cells were maintained at a density of 5 X lo5 cells/ml and during experiments the cell concentration was increased to 1 X lo6 cells/ml. AR42J cells were maintained as subconfluent monolayers.

RNA Isolation and Quuntitation-RNA was prepared from IM-9 cells using the proteinase K method in the presence of sodium dodecyl sulfate as previously reported (13). RNA was isolated from AR42J cells by a modification of the method of Chirgwin et al. (18) as previously reported (14). Qualitative analysis of poly(A)’ RNA was performed using Northern transfers exactly as described (19). An RNA ladder (Bethesda Research Laboratories) was used for size determination.

For quantitative comparisons, mRNA was measured by slot-blot analysis. RNA samples (4, 2, 1, and 0.5 fig of each sample) were denatured, immobilized to nitrocellulose, and prehybridized as pre- viously described (14).

Hybridization was carried out with the following plasmids: pRdN93, which contains a 2.6-kb glucocorticoid receptor cDNA in- sert, including the entire glucocorticoid receptor cDNA beginning 24 nucleotides upstream of the AUG start codon and continuing to 360 nucleotides downstream of the translational termination codon (the polyglutamine repeat sequence was removed to reduce nonspecific hybridization to non-receptor RNA (20)); pDF15, an 8.7-kilobase plasmid containing the 5’ portion of the rat 18 S ribosomal RNA gene (21) (a gift from Dr. A. P. Bollon (Wadley Institutes of Molecular Medicine, Dallas, TX)); two human insulin receptor cDNA probes,

The abbreviations used are: GREs, glucocorticoid response ele- ments; kb, kilobase.

81

2582 Glucocorticoid Receptor Down-regulation by Glucocorticoids

18.2 and 13.1 (1 kilobase and 4.2 kilobases, respectively) (a gift from Dr. G. I. Bell (University of Chicago)) and spanning the entire open reading frame of the receptor and extending into the 3"untranslated region (22); and pHF 0-actin-BUT, containing a cDNA insert probe for p-actin (23). These cDNA probes were labeled, either using a nick-translation kit (Bethesda Research Laboratories) or by the random primer labeling method (Amersham Corp.) with [a-"PJCTP (Du Pont-New England Nuclear), to specific activities of approxi- mately 1 X lo9 cpm/pg. Filters were blotted dry and exposed to x-ray film, and radioactivity was determined by scanning the autoradi- ograph with a densitometer equipped with an area integrator (Hoeffer Scientific Instruments, San Francisco, CA).

Nuclear Transcription Assay-Nuclei were isolated from cells in- cubated in the absence and presence of 100 nM dexamethasone for 24 h as described previously (15). In each reaction 5 X lo7 nuclei were employed. RNA products were purified using DNase, proteinase K in the presence of sodium dodecyl sulfate, and salt precipitation accord- ing to the procedure of Groudine and co-workers (24). 5 pg of either the control plasmid pBR327, the plasmid containing cDNA to p-actin (23), the plasmid containing cDNA to the glucocorticoid receptor (20), or the plasmid containing the 5' end of the insulin receptor (22) were denatured by heating to 65 "C for 1 h in 0.2 M NaOH, neutralized with an equal volume of cold 3 M sodium acetate, and applied to nitrocellulose using a slot-blot manifold. The slots were washed with 500 pl of 10 X SSC and the filters were baked under vacuum at 80 "C for 2 h. The filters were then prehybridized and hybridized in 2 ml of buffer as described (24) except that hybridizations were performed in 6 X 5-ml tissue culture plates. Following hybridization, filters were washed sequentially in 100-ml volumes of 2 X SSC (20 X SSC = 3 M NaC1, 0.3 M sodium citrate) a t 65 "C for 1 h, 2 X SSC with 10 pg/ml ribonuclease A a t 37 "C for 30 min, and 0.5 X SSC with 0.1% sodium dodecyl sulfate for 2 h a t 65 "C.

RESULTS

Glucocorticoid Regulation of Glucocorticoid Receptor mRNA-Poly(A)' mRNA was extracted from IM-9 lympho- cytes and AR42J cells, electrophoresed on 1% agarose gels, transferred to nitrocellulose, and hybridized with the "P- labeled glucocorticoid receptor probe (Fig. 1). In both tissues this cDNA probe hybridized to two distinct bands of 5.2 and 7.2 kb, respectively. These two different mRNA species have been reported previously (17) and they may be due to alter- native polyadenylation sites. In both cell types preincubation with the potent synthetic glucocorticoid dexamethasone (100 nM) decreased glucocorticoid receptor mRNA levels, but in contrast had no effect on @-actin mRNA levels (Fig. 1).

In AR42J cells we have previously shown that the potency of various steroid hormones to mediate biological effects are related to their relative potencies as glucocorticoids (25). In AR42J cells, down-regulation of glucocorticoid receptor mRNA was one-half maximal at 1 nM dexamethasone and maximal a t 100 nM (Fig. 2). A similar sensitivity to this glucocorticoid was seen in IM-9 cells (data not shown). Of the natural steroids tested, their relative potency values were

Glucocorticoid Receptor Beto-Actin

Kb AR42J IM-9 Kb AR42J IM-9

7 . 2 L

5.2/ 2.0 -

Dex - + - + - + - + FIG. 1. Northern blot analysis of glucocorticoid receptor

mRNA from AR42J and IM-9 cells. Both cell lines were incubated with 100 nM dexamethasone (Der) for 12 h. Poly(A)' mRNA (20 pgllane AR42J and 3 pg/lane IM-9) from each cell line was separated on a 1% agarose-formaldehyde gel, transferred onto nitrocellulose filters, and hybridized with glucocorticoid receptor cDNA and p-actin cDNA. Bars indicate molecular size in kilobases as determined by a parallel RNA ladder.

60

40 v .- .E % ,,+ $ 0 4 4 , 2 0 -10 -9 -8 -7 -6 m

Dexamethasone (log M)

B Dexamethasone

(log M)

0 - -10 - -9 - -a - -7 -

FIG. 2. Dose-dependent effects of dexamethasone on gluco- corticoid receptor mRNA. AR42J cells were incubated for 12 h with dexamethasone and mRNA levels were determined by slot-blot analysis. The mean of three experiments is shown. B shows an autoradiograph of a representative slot-blot experiment.

c 4 100. 0 0 80:L AR42J

60

40 IM-9

2o t 04 I

0 4 8 12 16 20 24 Hours

FIG. 3. Time course of dexamethasone-induced glucocorti- coid receptor mRNA down-regulation. AR42J and IM-9 cells were incubated with dexamethasone (100 nM) for the time periods indicated. Total cellular RNA was extracted, and slot blots were quantitated and normalized to p-actin. The values obtained were then expressed as percent of untreated controls. The data shown represent the mean of four experiments.

corticosterone > aldosterone > progesterone. The same order of potency values was reported for AR42J cells when other biological functions were studied (25).

In IM-9 cells glucocorticoid receptor mRNA levels were maximally decreased to 43% of control after 6 h of incubation with dexamethasone (100 nM), whereas in AR42J cells mRNA levels were maximally decreased to 50% of control after 12 h (Fig. 3). Incubation with dexamethasone for up to 72 h caused no further down-regulation (data not shown).

Effects of Dexamethasone on Glucocorticoid Receptor mRNA Stability and Transcription-To investigate whether gluco- corticoids down-regulate glucocorticoid receptor mRNA steady-state levels by decreasing mRNA half-life, studies us- ing the RNA polymerase inhibitor actinomycin D were carried out (Fig. 4). IM-9 cells and AR42J cells were preincubated with dexamethasone (100 nM) for 12 h prior to the addition of actinomycin D. The receptor half-life was 240 min in

Glucocorticoid Receptor Down-regulation by Glucocorticoids 2583

2 o l .- 6 i 1 0 4 g o 120 240 360 480

FIG. 4. Half-life studies of glucocorticoid receptor mRNA levels. AR42J and IM-9 cells were incubated with dexamethasone (100 nM) for 6 and 12 h, respectively. Actinomycin D was added at 10 pg/ml and untreated and treated cells were harvested after the indicated times. Next, mRNA was extracted and quantitated by slot- blot analysis. Results are expressed as percent initial mRNA and represent the mean of four experiments. Open symbols represent control values and closed symbols represent dexamethasone-treated samples.

Minutes

GF

IF

Beta-Actil

pBr32;

Dex - + + alpha-Amanitin - - +

FIG. 5. Effect of dexamethasone (Dex) on glucocorticoid receptor gene transcription. IM-9 cells were pretreated with 100 nM dexamethasone for 24 h, nuclei were isolated, and nuclear run-on assays were performed. ["PJUTP-labeled RNA was hybridized to cDNAs for the glucocorticoid receptor (GR), insulin receptor (ZR), B- actin, and pBR322 (5 pg/probe). In one set of nuclei, a-amanitin was added a t 2 pg/ml. A representative of three experiments is shown.

AR42J cells, whereas in the IM-9 cells the half-life was approximately 120 min. In both cells dexamethasone treat- ment had no effect on the glucocorticoid receptor mRNA half- life when compared to control cells.

Since dexamethasone had no effect on the glucocorticoid receptor mRNA stability, we investigated the effects of dex- amethasone on the transcription of the glucocorticoid receptor by means of nuclear run-on assays. Receptor transcription was measured in nuclei isolated from dexamethasone-treated IM-9 cells and allowed to elongate in the presence of ["'PI UTP (Fig. 5). Dexamethasone decreased glucocorticoid recep- tor gene transcription by 50 f 6% (mean f S.E., n = 3). In contrast, insulin receptor gene transcription was increased and P-actin was not significantly influenced. The elongation of all tested mRNAs was markedly inhibited by a-amanitin, indicating that the studied transcription occurred via RNA polymerase I1 (Fig. 5). For technical reasons it was not pos- sible to accurately measure glucocorticoid receptor gene tran- scription in AR42J cells.

DISCUSSION

The regulation of hormone receptors is an important aspect of physiologic control since the sensitivity of target cells to a hormone signal is directly related to receptor concentration (26). Previously, the down-regulation of the glucocorticoid receptor by its ligand was suggested by ligand binding studies (27). However, quantification of steroid receptors by binding studies is complicated due to the difficulty of assuring a total exchange of receptor-bound unlabled steroids for labeled hor-

mone and the binding of the labeled glucocorticoid ligand to heterologous receptors. Recently the glucocorticoid receptor gene has been cloned and this advance has made possible the investigation of glucocorticoid receptor gene expression (8). In the current study we have utilized a cDNA to the gluco- corticoid receptor both to investigate the levels of glucocorti- coid receptor mRNA and to elucidate the possible mechanism of action of glucocorticoids on glucocorticoid receptor gene expression.

With two cultured cell types, we found that glucocorticoid treatment led to an approximately 50% decrease in steady- state glucocorticoid receptor mRNA levels. This decrease is similar to the level of decreased glucocorticoid receptor mRNA previously reported by Kalinyak et al. (17) in a variety of rat tissues after glucocorticoid treatment in vivo. Okret et al. (16) have reported a 60-95% maximal decrease in gluco- corticoid receptor mRNA in cultured rat hepatoma cells 24- 48 h after dexamethasone treatment, but also described an initial 2-fold increase in glucocorticoid receptor mRNA 6 h after treatment and a spontaneous return to control levels after 72 h. In the present study we found that down-regulation of glucocorticoid receptor mRNA continued for up to 72 h. One explanation for the differences in these studies is the differences in the cell types and culture conditions employed.

Dexamethasone had no effect on the turnover of glucocor- ticoid receptor mRNA. Thus the regulation of the receptor mRNA did not appear to occur post-transcriptionally. Of interest was the fact the receptor mRNA has a relatively short half-life of 120 min in IM-9 cells and 240 min in AR42J cells. The glucocorticoid receptor half-life a t 120 min in IM-9 cells is similar to the value reported for insulin receptor mRNA in this cell line (15).

In contrast to the negative regulatory effects of glucocorti- coids on glucocorticoid receptor gene transcription, epidermal growth factor (28) and interleukin 2 (29) have been shown to increase the level of mRNA for their own receptors. The effects of epidermal growth factor on its receptor have been shown to be due to post-transcriptional mechanisms (28), while interleukin 2 augments the transcription of its receptor gene (29).

GREs have been shown to be important for both glucocor- ticoid-induced increases as well as decreases in the transcrip- tion of a number of genes (11, 15, 30, 31). Furthermore, they themselves can confer glucocorticoid responsiveness to re- porter genes (32). Okret et al. (16) have described the existence of several potential GREs in the exon that codes for the distal 3' nontranslated end of the glucocorticoid receptor mRNA. With immunoprecipitation assays and nuclease protection experiments, Okret and co-workers demonstrated that these elements interact with the glucocorticoid receptor. Whether either these or other GREs are involved in the down-regula- tion of glucocorticoid receptor gene expression is unknown, and thus further investigation is required.

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