Mechanism of glucocorticoid receptor down-regulation by ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Communication

Vol. 263, No. 6 , Issue of February 25, pp. 2581-2584,1988 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

(6-8), and functional domains within the receptor have been elucidated (9). The general mechanism of action of glucocorticoid receptors has also been characterized. Steroid hormones, which appear to enter cells by simple diffusion, bind to and activate the glucocorticoid receptor in a process re(Received for publication, October 26, 1987) ferred to as transformation (10); the transformed hormoneStefan Rosewiczz, Alex R. McDonald, receptor complex then binds with increased avidity to specific Betty A. Maddux, Ira D. Goldfine, DNA sequences termed glucocorticoid response elements Roger L. Miesfelds, and CraigD. Logsdonl (GREs)’ (for reviewseeRef. 10). Such interactionsthen From the Cell Biology Laboratory and Department of regulate the transcription of selected genes (10-12). It is also Medicine, Mount Zion Hospital and Medical Center, Sun known that theprotein products of glucocorticoid responsive Francisco, California 94120 and the Departments of genes may themselves regulate the expression of other genes Biochemistry, Medicine, and Physiology, University of such that glucocorticoids may indirectly activate the expresCalifornia, San Francisco, San Francisco, California 94143 sion of gene networks (10). In general, hormone receptors are regulated both by their The effect of glucocorticoids on the regulation of own ligand (homologous regulation) and by other regulatory glucocorticoid receptor mRNA was studied in two differentcelllines, human IM-9 lymphocytes and rat molecules (heterologous regulation). Glucocorticoids are inpancreatic acinar AR42J cells. Usinga glucocorticoid volved in the heterologous up-regulation of several hormone receptor cDNA probe, glucocorticoid receptor mRNA receptors, including those for insulin (13) and cholecystokinin was examined by Northern blot hybridization and (14). Inthe case of the insulin receptor, glucocorticoids act at quantitated by slot-blot hybridization. In IM-9 and the level of gene transcription (15). Glucocorticoids are involved in the homologous down-regAR42Jcells, dexamethasone decreased steady-state glucocorticoid receptor mRNA levels to approximately ulation of glucocorticoid receptors (16). Measurements of 50% of control. This decrease occurred with a one-half glucocorticoid receptor mRNA have suggested that this regtime of 3 h for IM-9 cells and 6 h for AR42J cells. ulation takes place at thelevel of glucocorticoid receptor gene Dexamethasone was themost potent steroid tested with expression (16, 17). However, the mechanism of this downa one-half maximal effect occurring at 10 nM and a regulation is unknown. In the present study we have investimaximal effect occurring at 100 nM. Glucocorticoid gated the down-regulation of the glucocorticoid receptor by receptor mRNA half-life and gene transcription were glucocorticoids in two different cell lines; the human lymthen studied to determine the mechanism of decreased phoma IM-9 and the rat pancreatic acinar AR42J cell line. mRNA levels. The glucocorticoid mRNA half-life was We now report the glucocorticoids decrease glucocorticoid approximately 120 min in IM-9 cells and 240 min in receptor steady-state mRNAlevels by decreasing receptor AR42J cells; these rates were not affected by dexamethasone treatment. In contrast, the rate of glucocor- gene transcription. ticoid gene transcription as measuredby run-on assays MATERIALS ANDMETHODS in IM-9 cells was decreased to 50 f 6%of control by Cell Culture-IM-9 lymphocytes (13) and AR42J cells (14) were dexamethasone. These results indicate therefore that glucocorticoids regulate glucocorticoid receptor grown as previously described. Routinely IM-9 cells were maintained at a density of 5 X lo5 cells/ml and during experiments the cell mRNA levels by influencing gene transcription. concentration was increased to 1 X lo6 cells/ml. AR42J cells were

Mechanism of Glucocorticoid Receptor Down-regulation by Glucocorticoids*

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, andmouse receptors have been cloned

* This work wassupported by National Institutes of Health Grants DK35912, DK 26667, DK 32994, the Elise Stern HaasResearch 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 “advertisement’’ in accordance with 18U.S.C. Section 1734 solelyto 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 Physiology, University of Michigan School of Medicine, Ann Arbor, MI 48109.

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 forsize 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 previously described (14). Hybridization was carried out with the following plasmids: pRdN93, which contains a 2.6-kb glucocorticoid receptor cDNA insert, 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 wasremoved 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 elements; kb, kilobase.

2581

2582

by Glucocorticoids

Glucocorticoid Receptor Down-regulation

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 entireopen 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-translationkit(Bethesda ResearchLaboratories) or by the random primer labeling method (Amersham Corp.) with [a-"PJCTP (Du Pont-New England Nuclear), to specific activities of approximately 1 X lo9 cpm/pg. Filters were blotted dry andexposed to x-ray film, and radioactivity was determined by scanning the autoradiograph with adensitometer equipped withan area integrator(Hoeffer Scientific Instruments, San Francisco, CA). Nuclear Transcription Assay-Nuclei were isolated from cells incubated in the absence and presence of 100 nM dexamethasone for 24 h as described previously (15). In each reaction 5 X lo7nuclei were employed. RNA products were purified using DNase,proteinase K in the presence of sodium dodecyl sulfate, and salt precipitationaccording to the procedure of Groudine and co-workers (24). 5 pg of either the control plasmidpBR327, the plasmid containing cDNA to p-actin (23), the plasmid containing cDNA to the glucocorticoid receptor (20), or theplasmid containing the 5' end of the insulin receptor (22) were denatured by heating to 65 "C for 1h 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 slotswere washed with 500 pl of 10 X SSC and the filterswere baked under vacuum at 80"C for 2 h. The filters were then prehybridized and hybridized in 2 ml of buffer as described (24) exceptthat 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 lymphocytes and AR42J cells, electrophoresed on 1% agarose gels, transferred to nitrocellulose, and hybridized with the "Plabeled glucocorticoid receptor probe (Fig. 1).In both tissues this cDNA probehybridized 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 alternative polyadenylation sites. In bothcell types preincubation with the potent syntheticglucocorticoid dexamethasone (100 nM) decreased glucocorticoid receptor mRNA levels, but in contrast had noeffect on @-actin mRNAlevels (Fig. 1). In AR42J cells we have previously shown that thepotency of various steroid hormones to mediatebiological 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 similarsensitivity to this Of the glucocorticoid wasseen inIM-9 cells (data not shown). naturalsteroidstested,theirrelativepotency valueswere Glucocorticoid Beto-Actin Receptor Kb AR42J IM-9

Kb AR42J

IM-9

7.2L 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 v .-

.E%

$

40

,,+ 0

2 m

4

-10

0

4 -9

-8

,

-7

-6

Dexamethasone (log M)

B

Dexamethasone

(log M)

--

0 -10 -9

-a -7

-

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

4 0 0

100.

80 t AR42J

60 40

IM-9

2o

04

0

I

20 4

16 8

12

24

Hours

FIG. 3. Time course of dexamethasone-induced glucocorticoid 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 slotblots were quantitated andnormalized to p-actin. The values obtained were then expressed as percent of untreated controls. The datashown 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 after6 h of incubation with dexamethasone(100 nM), whereas inAR42J cellsmRNA levels were maximally decreased to 50% of control after 12 h (Fig. 3). Incubation with dexamethasonefor up to72 h caused no further down-regulation (data not shown). Effects of Dexamethasone on GlucocorticoidReceptor mRNA Stability and Transcription-To investigate whether glucocorticoids down-regulate glucocorticoid receptor mRNA steady-state levels by decreasing mRNA half-life, studies using the RNA polymerase inhibitor actinomycinD 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. Thereceptor half-lifewas 240 minin

Glucocorticoid Receptor Down-regulation

by Glucocorticoids

2583

mone and the bindingof 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 glucocorticoid receptor both to investigate the levels of glucocorticoid receptor mRNA and to elucidate possible the mechanism .of action of glucocorticoids on glucocorticoid receptor gene 6 expression. i 104 g o 120 240 360 480 With two cultured cell types, we found that glucocorticoid Minutes treatment led to an approximately 50% decrease in steadyFIG. 4. Half-life studies of glucocorticoid receptor mRNA state glucocorticoid receptor mRNA levels. This decrease is levels. AR42J and IM-9 cells were incubated with dexamethasone level of decreased glucocorticoid receptor (100 nM) for 6 and 12 h, respectively. Actinomycin D was added at similar tothe mRNA previously reported by Kalinyak et al. (17) in a variety 10 pg/ml and untreated and treated cells were harvested after the indicated times. Next, mRNA was extracted and quantitatedby slot- of rat tissues after glucocorticoid treatment in vivo. Okret et blot analysis. Results are expressed as percent initial mRNA and al. (16) have reported a 60-95% maximal decrease in glucorepresent the mean of fourexperiments. Opensymbols represent corticoid receptor mRNA in cultured rat hepatomacells 24control values and closed symbols represent dexamethasone-treated 48 h after dexamethasone treatment, but also described an samples. 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 thatdown-regulation GF of glucocorticoid receptor mRNA continued for up to 72 h. IF One explanation for the differences in these studies is the differences in the cell types and culture conditions employed. Beta-Actil Dexamethasone had no effect on the turnoverof glucocorticoid receptor mRNA. Thus the regulation of the receptor pBr32; mRNA did notappearto occur post-transcriptionally. Of Dex - + + relatively short interest was the fact the receptor mRNA a has half-life of 120 min in IM-9 cells and 240 min in AR42J cells. alpha-Amanitin - - + The glucocorticoid receptor half-life a t 120 min in IM-9cells FIG. 5. Effect of dexamethasone (Dex) on glucocorticoid is similar to the value reported for insulin receptor mRNAin receptor gene transcription. IM-9 cells were pretreated with 100 nM dexamethasone for 24 h, nuclei were isolated, and nuclear run-on this cell line (15). In contrast to thenegative regulatory effects of glucocortiassays were performed.["PJUTP-labeledRNA was hybridized to B- coids on glucocorticoid receptor gene transcription, epidermal cDNAs for the glucocorticoid receptor (GR),insulin receptor (ZR), actin, and pBR322 (5 pg/probe). In one set of nuclei, a-amanitin was growth factor (28) and interleukin2 (29) have been shown to added a t 2 pg/ml. A representative of three experiments is shown. increase the level of mRNA for their own receptors. The effects of epidermal growth factor on its receptor have been AR42J cells, whereasintheIM-9 cells the half-lifewas (28), shown to be due to post-transcriptional mechanisms approximately 120 min. In both cells dexamethasone treat- while interleukin 2 augments the transcriptionof its receptor ment had no effect on theglucocorticoid receptor mRNA half- gene (29). life when compared to control cells. GREs have been shown to be important for bothglucocorSince dexamethasone had no effect on the glucocorticoid ticoid-induced increases as well as decreases in the transcripreceptor mRNA stability, we investigated the effects of dex- tion of a number of genes (11, 15, 30, 31). Furthermore, they amethasone on the transcription of the glucocorticoid receptor themselves can confer glucocorticoid responsiveness to reby means of nuclear run-on assays. Receptor transcription porter genes (32).Okret et al. (16) havedescribed the existence was measured in nuclei isolated from dexamethasone-treated of several potential GREs in the exon that codes for the distal IM-9 cells and allowed to elongate in the presence of ["'PI 3' nontranslated end of the glucocorticoid receptor mRNA. U T P (Fig. 5). Dexamethasone decreasedglucocorticoid recep- Withimmunoprecipitationassaysand nuclease protection tor gene transcription by 50 f 6% (mean f S.E., n = 3). In experiments, Okret and co-workers demonstrated that these contrast, insulin receptor gene transcription was increased elements interact with the glucocorticoid receptor. Whether and P-actin was not significantly influenced. The elongation either these or other GREs areinvolved in the down-regulaof all tested mRNAs was markedly inhibited by a-amanitin, tion of glucocorticoid receptor gene expression is unknown, indicating that the studied transcription occurred via RNA and thus further investigation required. is polymerase I1 (Fig. 5). For technical reasons it was not posREFERENCES sible to accurately measureglucocorticoid receptor gene transcription in AR42Jcells. 1. Ballard, P. L. (1979) in Glucocorticoid Hormone Action, Mono-

2ol

graphs in Endocrinology (Baxter, J. D., and Roussear, G. G., eds) Vol. 12, pp. 493-497, Springer-Verlag, Berlin 2. Cidlowski, J. A., and Cidlowski, N. B. (1982) Endocrinology 110, 1653-1662 The regulationof hormone receptors isan important aspect 3. Chang, W. C., and Roth, G. S. (1979) J. Steroid Biochem. 11, of physiologic control since the sensitivityof target cells to a 889-892 hormone signal is directly related to receptor concentration 4. Wrange, O., Carlstedt-Duke, J., and Gustafsson, J.-A. (1979) J. (26).Previously, the down-regulation of the glucocorticoid Biol. C h m . 254,9284-9290 receptor by its ligand was suggested by ligandbinding studies 5. Wrange, O., Okret, S., Radajcic, M., Carlstedt-Duke, J., and (27). However, quantification of steroid receptors by binding Gustaffson, J.-A. (1984) J. Biol. Chem. 259, 4534-4541 studies is complicated due to the difficulty of assuring a total 6. Miesfeld, R., Okret, S., Wikstrom, A.-C., Wrange, O., Gustafsson, J.-A., and Yamamoto, K. R. (1984) Nature 3 1 2 , 779-781 exchange of receptor-bound unlabled steroids for labeled horDISCUSSION

2584

Glucocorticoid Receptor Dou .regulation by Glucocorticoids

7. Miesfeld, R., Rusconi, S., Godowski, P. J., Maler, B.A., Okret, S., Wikstrom, A.-C., Gustafsson, J.-A., and Yamamoto, K. R. (1986) Cell 4 6 , 389-399 8. Hollenberg, S. M., Weinberger, C., Ong, E. S., Cerelli, G., Oro, A., Lebo, R., Thompson, E. B., Rosenfeld, M. G., and Evans, R. M. (1985) Nature 318,635-641 9. Gustaffson, J.-A., Carlstedt-Duke, J., Poellinger, L., Okret, S., Wikstrom, A.-C., Broennegard, M., Gillner, M., Dong, Y.,Fuxe, K., Cintra, A., Haerfstrand, A., and Agnati, L. (1987) Endocr. Reu. 8 , 185-234 10. Yamamoto, K. R. (1985) Annu. Reu. Genet. 1 9 , 209-252 11. Ringold, G. (1985) Annu. Reu. Phurmacol. Toxicol. 2 5 , 529-566 12. Yamamoto, K. R., and Alberts, B. M. (1976) Annu. Reu. Biochem. 45,721-746 13. McDonald, A. R., Maddux, B. A., Okabayashi, Y.,Wong, K. Y., Hawley,D.M., Logsdon, C.D., and Goldhe, I. D. (1987) Diabetes 36,779-781 14. Logsdon, C. D. (1986) J. Biol. Chem. 2 6 1 , 2096-2101 15. McDonald, A.R., and Goldfine, I. D. (1987) J. Clin. Znuest., in press 16. Okret, S., Poellinger, L., Dong, Y., and Gustaffsson, J.-A. (1986) Proc. Natl. Acad. Sci. U. S. A . 83, 5899-5903 17. Kalinyak, J . E., Dorin, R. I., Hoffman, A. R., and Perlman, A. J. (1987) J. Biol. Chem. 262,10441-10445 18. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18,5294-5299 19. Maniatis, R., Fritsch, R. F., and Sambrook, J . K. (1982) Molecular

20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Miesfeld, R., Godowski, P. J., Maler, B. A., and Yamamoto, K. R. (1987) Science 236,423-427 Fluke, M., Dennis, K. J., and Bush, H. (1981) Mol. Gen. Genet. 182,25-30 Shaw, D. J., and Bell, G. I. (1985) Nucleic Acids Res. 13, 86598661 Cleveland, D. W., Lopata, M.A., MacDonald, R. A., Cowan, N. J., Rutter, W. J., and Kirschner, M. W. (1980) Cell 2 0 , 95-105 Thompson, C. B., Challoner, P. E., Neiman, P. E., and Groudine, M. (1986) Nature 3 1 9 , 374-380 Logsdon, C. D., Moessner, J., Williams, J. A., and Goldfhe, I. D. (1985) J. Cell Biol. 100, 1200-1208 Danielse, M., and Stallcup, M. R. (1984) Mol. Cell. B i o l 4 , 449453 Svec, F., and Rudis, M. (1981) J. Bwl. Chem. 256, 5984-5987 Clark, A. J. L., Ishii, S., Richert, N., Merlino, G. T., and Pastan I. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,8374-8378 Depper, J. M., Leonard, W. J., Drogula, C., Kroenke, M., Waldmann, T. A., and Greene, W. C. (1985) Proc. Natl. Acad. Sci. U. S. A . 82,4230-4234 Birnberg, N.C., Lisstzky, J.-C., Hinman, M., and Herbert, E. (1983) Proc. Natl. Acad. Sei. U. S. A. 80, 6982-6986 Potter, E., Nicholaisen, A.,Ong,E., Evans, R., and Rosenfeld, M. (1981) Proc. Natl. Acad. Sci. U. S. A . 78,6662-6666 Chandler, V. L., Maler, B. A., and Yamamoto, K. R. (1983) Cell 33,489-499