Biochimie ( 199 ! ) 73, 51-59 © Soci6t6 fran~;aise de biochimie et biologic mol6culaire / Elsevier, Paris
51
Regulation of glucocorticoid receptor expression S Okret, Y Dong, M Br6nneg~rd, JA G u s t a f s s o n Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital F60. Novum. S-141 86 Huddinge, Swede:!
(Received 12 November 1990; accepted 5 December 1990)
Summary - - A limiting factor determining the sensitivity of a cell to glucocorticoid hormones is the intracellular concentration of the glucocorticoid receptor (GR) protein. By regulating the expression of GR the cell is able to adapt to both changes in its hormone environment and to the varying requirements for biological response. Studies on the regulation of GR expression have shown this to be a complex process which involves both transcriptional and posttranscriptional mechanisms. Although GR is more or less constitutively expressed in most tissues its concentration varies under different physiological conditions. GR expression is regulated by a number of different agents including factors which act through a second messenger pathway. This allows the cell in control glucocorticoid regulated gene expression through a complex but integrated hormonal network. Here we summarize our studies on GR regulation with emphasis on: i), GR autoregulation; ii), the effect of cAMP on GR expression; and iii), GR expression during fetal rat lung development.
glucocorticoid receptor / autoregulation / cAMP / development Introduction Early studies demonstrated that in most systems the magnitude of biological response elicited by GR is proportional to the number of receptor molecules expressed in the cell [1-3]. In fact, GR concentration has been suggested to be the limiting factor in !he signal transduction pathway leading to transcriptional response [4, 5]. This situation also means that small changes in GR concentration will affect cellular glucocorticoid sensitivity. It is therefore of interest to study factors and the mechanism regulating GR expression. Cellular GR levels have been shown to vary as a result of endocrine manipulations [6-9], neural influence and cell contact [10, 11] during different stages in the cell cycle [12, 13], development [14, 151 and ageing [ 16]. Initial studies utilized labeled ligands to study GR expression [17]. In many situations this required an exchange of receptor-bound steroids with a labeled tracer, which in turn required experimental manipulations that might affect receptor quantification. Furthermore, ligand binding activity of the GR is altered by ' m o d i f y i n g ' substances, eg ATP in that the receptor Abbreviations: GR, glucocorticoid receptor; MMTV, mouse mammary tumor virus
can exist in 2 states regarding its ability to bind ligand [181. In view of the availability of antibodies [191 and c D N A probes for rat GR [20, 21], we undertook studies on GR regulation during various physiological conditions. Use of the above probes enabled us to: i), overcome possible problems quantifying GR by !igand binding assays; and ii), determine regulatory, levels at the 'pre-hormone binding' level. This report summarizes our data regarding GR expression with special emphasis on GR autoregulation, GR expression during rat lung development and effects on GR levels by cAMP.
Results GR autoregulation
Previous studies using ligand binding assays have shown that GR expression is under autoregulatory control. For instance, the presence of glucocorticoids has been reported to cause a 5 0 - 7 5 % down-regulation of cellular GR in a number of tissues and cell lines [17], while adrenalectomy caused = 2-fold up-regulation of GR concentration [9, 22]. Using monoclonal anti-GR antibodies and cloned rat GR cDNA, we investigated whether autoregulation of GR expression was reflected at the protein and pretranslational levels.
52
S Okrct e t al
A
0
4
A single dose injection of the synthetic glucocorticoid dexamethasone caused down-regulation of the steady state level of both rat liver GR mRNA and GR protein (fig 1). GR mRNA levels decreased by 60-80%, already starting 4 h after hormone administration, being maximal at 18-24 h and returning to their original level at 48 h when dexamethasone concentration in the body decreased (fig I A). GR protein levels followed the same pattern, although downregulation was not as pronounced as the downregulation of GR mRNA (fig I B). Furthermore, changes in GR protein concentration were slightly delayed in comparison with GR mRNA, which could be explained by different haif-lifes for the protein and the mRNA, respectively (see below). Studies on the rat HTC cell line, H4IIE, showed a much more complex autoregulatory pattern of GR expression (fig 2). Continuous presence of dexamethasone in the media led to a transient 2-fold increase in GR mRNA levels (peak 6 h after addition of hormone) followed by -- 80% down-regulation 18--42 h later (fig 2). At 72 h, GR mRNA had almost returned to the initial value. This cyclic pattern was also reflected at the protein level [23]. The mechanism for the cyclic pattern and the initial up-regulation is not known. However, the mechanisms of GR mRNA down-regulation were investigaged. Glucocorticoid-induced down-regulation of GR mRNA did not require ongoing protein synthesis, since it still occurred in the presence of the protein synthesis inhibitor cycloheximide [24]. However, cycloheximide by itself caused a super-induction of GR mRNA level. Furthermore, down-regulation of GR by dexamethasone was dose-dependent and a maximal response was obtained with i0-20 nM, close to the concentration required for receptor saturation [23, 241. To elucidate whether down-regulation of GR expression involved transcriptional and/or posttranscriptional processes, transcriptional activity of the GR gene and GR mRNA stability were measured in the presence of absence of dexamethasone. Using a
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Fig 1. The analysis of GR mRNA and protein levels in rat liver after treatment with dexamethasone. Rats were adrenalectomized 9 d before treatment with dexamethasone-lphosphate (4 mg/kg BW). At indicated times, total RNA and protein were isolated from the livers and analyzed for GR and 6-actin mRNA by Northern blot hybridization and for GR and actin proteins by Western immunoblotting. A, Autoradiogram of RNA blots. The sizes of the signals are calculated from the position of 18S and 28S RNAs, respectively. B, Western blots. The sizes of the hybridization signals are determined from the mobility c,f slandard proteins. C, Summary of densitometric analysis of the RNA and Western blots, respectively. Values are expressed as percent of control (untreated animals). For further experimental details, see [23].
Glucocorticoid receptor regulation
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Fig 10. Correlation between cellular GR protein level and the inducibility of MMTV and tyrosine aminotransferase by dexamethasone and forskolin. Cells were exposed to 0.5 lttM dexamethasone (DEX) in the presence or absence of 25 ~M forskolin (Fors) for 18 h.. For the study of GR protein level, cellular extracts were prepared and subjected to Western immunoblot analysis using monoclonal anti-GR antibody (see [23]). The mRNA levels of MMTV were determined by RNA blot hybridization with 32p-labeled cDNA probe coding for MMTV. The enzyme activity of tyrosine aminotransferase was studied in isolated cell extract. The specific tyrosine aminotransferase activity is calculated as nmol product/min/mg protein. All data shown in this figure were obtained from 3 experiments and are presented as the mean value + SD relative to control. For further experimental details see [5].
57
Adrenalectomy of rats has shown differences in upregulation of GR mRNA in various tissues [9. 401. Finally. feedback regulation of GR expression has been shown to vary during development and may differ from tissue to tissue [15]. The mechanism(s) determining the differences in autoregulatory response is unknown. However, data presented above have shown that with regard to the negative feedback control of GR expression, at least 2 independent mechanisms are involved, namely transcriptional repression of GR gene expression and increased turnover of the GR protein. These data have been confirmed by other reports 127-281. In addition, indirect data support supplementing mechanisms, eg at the translational level. These include discrepancies between changes in GR mRNA and protein levels (see above), changes in GR mRNA which are not reflected at the protein level (see above) and the lack of changes in receptor synthesis despite changes in GR mRNA 1271. Complex regulation of receptor expression has also been demonstrated for other steroid receptors. Ree et al [41] showed that treatment of MCF-7 cells with a saturating dose of estradiol gave a transient downregulation of the estrogen receptor mRNA. while the estrogen receptor protein remained low. In addition, a positive feedback mechanism on estrogen receptor mRNA was seen with low concentration of estradiol while a negative feedback was obtained with higher concentration of the hormone. Saceda et al [42] demonstrated that down-regulation of estrogen-receptor expression by estradiol was mainly a post-transcriptional response and that a discrepancy between gene transcription and mRNA levels existed. Estrogen receptor mRNA and protein remained low despite the fact that estrogen receptor gene transcriptional activity followir:g a short transient down-regulation increased and remained high (2-fold increase). The observation that down-regulation of GR mRNA by glucocorticoids did occur at the transcriptional level and did not require ongoing protein synthesis, may indicate that the GR protein interacts with its own gene, controlling GR gene activity. In line with this, we have shown that the GR cDNA contains at least one binding site for the GR protein. althgouh its functional implications are unknown [24]. However, post-transcriptional mechanisms could be the major determinants regulating GR expression. Such mechanisms could be controlled by eg, differences in the 5' untranslated region of GR mRNA, created by different protnotor utilization and/or splicing events. Indications for heterogeneity in the 5' untranslated region of GR mRNA have recently been reported 1431.5' Untranslated regions have been shown to be important in translational control [441, as well as mRNA stability [451.
5S
s Okret et al
A~ demonstrated above, it is norteworthy that the c A M P second messenger pathway may influence GR expression and thereby biological response to glucocorticoids. Together with recently published data on the interaction between the AP-I binding factors, c-jun and c-3'os and the GR, respectively, it is clear that an interplay between membrane and intracellularly acting signals, respectively, in regulating the cellular response for either system exists 146-501. Since small changes in GR activity in many cases will be reflected in a parallel change in biological response to glucocorticoids, it is of great importance to understand how other systems or factors influence GR expression. As shown, regulation of GR expression is controlled at transcriptional as well as posttranscriptional levels, some of which will be more or less important in generating changes in the amount of active GR protein. The complex regulation may also enable a versatile control of GR activity during various physiological and environmental conditions.
10
Acknowledgments
18
This work was supported by grants from the Swedish Cancer Society and Magnus Bergvali Foundation (to 50), Swedish Medical Research Council (No 13X-2819 to JAG) and the Swedish Society of Medical Research (to YD).
I!
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References ! 2
3 4
5
6 7 8 9
Bourgeois S, Newby RF (1979) Correlation between glucocorticoid receptor and cytolytic response of murine lymphoid cell lines. Cancer Res 39, 4749--4751 Bloom F, Metulich DT, Lan NC, Higgins SJ, Simons SS, Baxter JD (1980) Nuclear binding of glucocorticoid receptors: correlation between cytosol binding, activation and the biological response. J Steroid Biochem 12, 175-184 Bloomfield CD, Smith KA, Peterson BA, Munck A (1981) Glucocorticoid receptors in adult acute lymphoblastic leukemia. Cancer Res 4 I, 4857--4860 Vanderbilt JN, Miesfeld R, Maler BA, Yamamoto KR (1987) lntraceilular receptor concentration limits glucocorticoid-dependent enhancer activity. Mol Endocrinol 1, 68-74 Dong Y, Aronsson M, Gustafsson J,&, Okret S (1989) The mechanism of cAMP-induced glucocorticoid receptor expression. Correlation to cellular glucocorticoid response. J Biol Chem 264, 13679-I 3683 Svec F, Rudis M (1981) Glucocorticoids regulate the glucocorticoid receptor in the AtT-20 cell. J Bi'ol Chem 256, 5984-5987 Cidlowski JA, Cidlowski NB (1981) Regulation of glucocorticoid receptors by glucocorticoids in cultured HeLa $3 cells. Endocrinology 109, 1975-1982 Sapolsky RM, Krey LC, McEwen BS (1984) Stress downregulates corticosterone receptors in a site-specific manner in the brain. Endocrinology 114, 287-292 Turner AB (1986) Tissue differences in the up-regulation of glucocorticoid-binding proteins in the rat. Endocrinology 118, 1211-1216
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McGinnis JF, De Vellis J (1981) Cell surface modulation of gene expression in brain cells by down-regulation. Proc N,ttl Acad Sci USA 78, 1288-1292 Antakly T, Sasuki A, Liota AS, Palkovits M, Krieger DT (1985) Induced expression of the glucocorticoid receptor in the rat intermediate pituitary lobe. Science 229, 277-279 Cidlowski JA, Michaels GA (1977) Alteration in glucocorticoid binding site number during the cell cycle in HeLa cells. Nature 266, 643-645 Crabtree Gr, Munck A, Smith KA (1980) Giucocorticoids and iympocytes. II. Cell cycle-dependent changes in glucocorticoid receptor content. J Immunol 125, 13-17 Br6nnegard M, Okret S (1988) Characterization of the glucocorticoid receptor in fetal rat lung during development: influence of proteolytic activity. J Steroid Biochem 3 I, 809-817 Kalinyak JE, Griffin CA, Hamilton RW, Bradshaw JG, Perlman AJ, Hoffman AR (1989) Developmental and hormonal regulation of glucocorticoid receptor messenger RNA in the rat. J Clin Invest 84, 1843-1848 Chang WC, Roth GS (1979) Changes in the mechanism of steroid action during ageing. J Bioi Chem 11,889-892 Svec F (1985) Glucocorticoid receptor regulation. Life Sci 36, 2359-2366 Mendel DB, Bodwell JE, Munck A (1986) Glucocorticoid receptors lacking hormone-binding activity are bound in nuclei of ATP-depleted cells. Nature 324, 478-480 Okret S, Wikstr6m AC, Wrange O, Andersson B, Gustafsson J/~ (1984) Monoclonal antibodies against the rat liver glucocorticoid receptor. Proc Natl Acad Sci USA 81, 1609-1613 Miesfeld R, Okret S, Wikstrtim AC, Wrange O, Gustafsson JA, Yamamoto KR (1984) Characterization of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature 312, 779-78 i Miesfeld R, Rusconi S, Godowski PH, Maler BA, Okret S, Wikstr6m AC, Gustafsson JA, Yamamoto KR (1986) Genetic complemer'ation of a glucocorticoid receptor deficiency by exp.,~ssion of cloned receptor cDNA. Ceil 46, J ~ - j ~ Smith K, Shuster S (1984) Effect of adrenalectomy and steroid treatment on rat skin cytosol glucocorticoid receptor. J Endocrinol 102, 16 I- 165 Dong Y, Poellinger L, Gustafsson J,~,, Okret S (1988) Regulation of glucocorticoid receptor expression: evidence for transcriptional and posttranslational mechanisms. Mol Endt)crinol 2, 1256-1264 Okret S, Poellinger L, Dong Y, Gustafsson Jt~, (1986) Down-regulation of glucocorticoid receptor mRNA by glucocorticoid hormones and recognition by the receptor of a specific binding sequence within a receptor cDNA clone. Proc Natl Acad Sci USA 83, 5899-5903 Walsh MJ, LeLeiko NS, Sterling Jr KM (1987) Regulation of type 1, IIl and IV procollagen mRNA synthesis in glucocorticoid-mediated intestinal development. J Biol Chem 262, 10814-10818 Rosewicz S, McDonald AR, Maddux BA, Goldfine JD, Miesfeld RL, Logsdon CD (1988) Mechanism of glucocorticoid receptor down-regulation by glucocorticoids. J Biol Chem 263,2581-2584 Mclntyre WR, Samuels HH (1985) Triamcinolone acetonide regulates glucocorticoid-receptor levels by decreasing the half-life of the activated nuclear-receptor form. J Biol Chem 260, 418-427
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Hoeck W, Rusconi S, Groner B (1989) Down-regulation and phosphorylation of glucocorticoid receptors in cultured cells. ,I Biol Chem 264, 14396-14402 Rajpert EJ, Lemaigre FP, Eliard PH, Place M, Lafontaine DA, Economidis IV, Belayew A, Martial JA, Rousseau GG (1987) Giucocorticoid receptors bound to the antagonist RU486 are not downregulated despite their capacity to interact in vitro with defined gene regions. J Steroid Biochem 26, 513-520 Denis M, Poellinger L, Wikstrtim AC, Gustafsson JA (1988) Requirement of hormone for thermal activation of the glucocorticoid receptor to a DNA binding state. Nature 333,686-688 Picard D, Salser SJ, Yamamoto KR (1988) A movable and regulable inactivation function within the steroid binding domain of the glucocorticoid receptor. Cell 54, ! 073-1080 Wikstrtim AC, Bakke O, Okret S, Brtinneg3.rd M, Gustafsson JA (1987) lntracellular localization of the glucocorticoid receptor: evidence for cytoplasmic and nuclear localization. Endocrinology 120, 1232-1242 Liley HG, Hawgood S, Wellenstein GA, Benson B, Tyler White R, Ballard PL (1987) Surfactant protein of molecular weight 28, 000-36,000 in cultured human fetal lung: cellular localization and effect of dexamethasone. Mol Endocrinol 1,205-215 Oikarinen J, Hamalainen L, Oikarinen A (1984) Modulation of glucocorticoid receptor activity by cyclic nucleotides and its implications on the regulation of human skin fibroblast growth and protein synthesis. Biochim Biophys Acta 779, 158-165 Gruol DJ, Faith Campbell N, Bourgeois S (1986) Cyclic AMP-dependent protein kinase promotes glucocorticoid receptor function. J Bioi Chem 261,4909-4914 Darneil J, Codish H, Baltimore D (1986) in: Molecular Cell Biology. Scientific American Books, NY Eisen LP, Elsasser MS, Harmon JM (1988) Positive regulation of the glucocorticoid receptor in human T-cells sensitive to the cytolytic effects of glucocorticoids. J Biol Chem 263, 12044-- 12048 ~UHH lvl, tViOliWi.thl ~, ~utagir: Kul:aiu Y Thomp son EB (1990) Glucocorticoid effects on myeloma cells in culture: correlation of growth inhibition with induction of glucocorticoid receptor messenger RNA. Cancer Res 50, 1873-1878 Gadson P, McCoy J, Wikstrtim AC, Gustafsson JA (1990) Suppression of protein kinase C and the stimulation of glucocorticoid receptor synthesis by dexamethasone in human fibroblasts derived from tumor tissue. J Cell Biochem 43, 185- ! 89 A,"~___:
ILJl
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....
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|I
. . . .
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49
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Kalinyak JE. Dorin RI. Hoffman AR, Perlman AJ (1987~ Tissue-specific regulation of glucoconicoid receptor mRNA by dexamethasone. J Biol C/tent 262. 1044 ! - 10,!44 Ree AH. Landmark BF. Eskild W. Levy FO. Lahooti H. Jahnsen T. Aakvaag A, Hansson V (1989) Autologous down-regulation of messenger ribonuc!eic acid and protein levels tor estrogen receptors in MCF-7 cells: an reverse correlation to progesterone receptor levels. Endocrinoh~gy 124, 2577-2583 Saceda M, Lippman ME. Chambon P, Lindsey RL, Ponglikitmongkol M. Puente M, Martin MM (19881 Regulation of the estrogen receptor in MCF-7 cells by estradiol. Mol Endocrinol 2. 1157-1162 Dieken ES, Meese EU, Miesfeld RL (1990) nti Giucocorticoid receptor transcripts lack sequences encoding the amino-terminal transcriptional modulatory domain. Mol Cell Biol I0. 4574--458 I Horiuchi T, Macon KJ. Kidd VJ. Volanskis JE (19901 Translational regulation of complement protein C2 expression by differential utilization of the 5'-untranslated region of mRNA. J Biol Chem 265,6521-6524 Rabbits PH. Forster A, Stinson MA, Rabbitts TH (19851 Truncation of exon 1 from the c-mv~' gene results in prolonged c-rove mRNa stability. EMBO J 4, 3727-3733 Diamond MI, Miner JN, Yoshinaga SK, Yamamoto KR (1990) Transcription factor interactions: selectors of positive or negative regulation from a single DNA element. Science 249, i 266-1272 Jonat C, Rahmsdorf H J, Park KK, Cato ACB. Gebei S. Ponta H, Herrlich P (1990) Antitumor promotion and antiinflammation: down-modulation of AP-I (fos/jun) activity by glucocorticoid hormone. Cell 62, 1189-1204 Yang-Yen HF, Chambard JC, Sun YL, Smeal T. Schmidt T, Drouin J, Karin M (1990) Transcriptional interference between c-jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62, 1205-1215 Schiile R, Rangaraja- P, K, ewer S, Ransone LJ, Bolado J, Yang N, Verma IM, Evans RM (1990) Functional antagoHism between oncoproieiH c-iuu and ihe glucocotaicoid receptor. Cell 62, 1217-1226 Lucibeilo FC, Siater EP, Jooss KU, Beato M. Miiller R (1990) Mutual transrepression of fi~s and the glucocorticoid receptor: involvement of a functional domain in fi>s which is absent infi~sB. I:,MBO J 9, 2827-2834 Dong Y, Cairns ~ , Okret S, Gustafsson JA (1990) A glucocorticoid-resistant rat hepatoma cell variant contains functional glucocorticoid receptor. ,I Biol Chem 265. 7525-7531
51
Regulation of glucocorticoid receptor expression S Okret, Y Dong, M Br6nneg~rd, JA G u s t a f s s o n Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital F60. Novum. S-141 86 Huddinge, Swede:!
(Received 12 November 1990; accepted 5 December 1990)
Summary - - A limiting factor determining the sensitivity of a cell to glucocorticoid hormones is the intracellular concentration of the glucocorticoid receptor (GR) protein. By regulating the expression of GR the cell is able to adapt to both changes in its hormone environment and to the varying requirements for biological response. Studies on the regulation of GR expression have shown this to be a complex process which involves both transcriptional and posttranscriptional mechanisms. Although GR is more or less constitutively expressed in most tissues its concentration varies under different physiological conditions. GR expression is regulated by a number of different agents including factors which act through a second messenger pathway. This allows the cell in control glucocorticoid regulated gene expression through a complex but integrated hormonal network. Here we summarize our studies on GR regulation with emphasis on: i), GR autoregulation; ii), the effect of cAMP on GR expression; and iii), GR expression during fetal rat lung development.
glucocorticoid receptor / autoregulation / cAMP / development Introduction Early studies demonstrated that in most systems the magnitude of biological response elicited by GR is proportional to the number of receptor molecules expressed in the cell [1-3]. In fact, GR concentration has been suggested to be the limiting factor in !he signal transduction pathway leading to transcriptional response [4, 5]. This situation also means that small changes in GR concentration will affect cellular glucocorticoid sensitivity. It is therefore of interest to study factors and the mechanism regulating GR expression. Cellular GR levels have been shown to vary as a result of endocrine manipulations [6-9], neural influence and cell contact [10, 11] during different stages in the cell cycle [12, 13], development [14, 151 and ageing [ 16]. Initial studies utilized labeled ligands to study GR expression [17]. In many situations this required an exchange of receptor-bound steroids with a labeled tracer, which in turn required experimental manipulations that might affect receptor quantification. Furthermore, ligand binding activity of the GR is altered by ' m o d i f y i n g ' substances, eg ATP in that the receptor Abbreviations: GR, glucocorticoid receptor; MMTV, mouse mammary tumor virus
can exist in 2 states regarding its ability to bind ligand [181. In view of the availability of antibodies [191 and c D N A probes for rat GR [20, 21], we undertook studies on GR regulation during various physiological conditions. Use of the above probes enabled us to: i), overcome possible problems quantifying GR by !igand binding assays; and ii), determine regulatory, levels at the 'pre-hormone binding' level. This report summarizes our data regarding GR expression with special emphasis on GR autoregulation, GR expression during rat lung development and effects on GR levels by cAMP.
Results GR autoregulation
Previous studies using ligand binding assays have shown that GR expression is under autoregulatory control. For instance, the presence of glucocorticoids has been reported to cause a 5 0 - 7 5 % down-regulation of cellular GR in a number of tissues and cell lines [17], while adrenalectomy caused = 2-fold up-regulation of GR concentration [9, 22]. Using monoclonal anti-GR antibodies and cloned rat GR cDNA, we investigated whether autoregulation of GR expression was reflected at the protein and pretranslational levels.
52
S Okrct e t al
A
0
4
A single dose injection of the synthetic glucocorticoid dexamethasone caused down-regulation of the steady state level of both rat liver GR mRNA and GR protein (fig 1). GR mRNA levels decreased by 60-80%, already starting 4 h after hormone administration, being maximal at 18-24 h and returning to their original level at 48 h when dexamethasone concentration in the body decreased (fig I A). GR protein levels followed the same pattern, although downregulation was not as pronounced as the downregulation of GR mRNA (fig I B). Furthermore, changes in GR protein concentration were slightly delayed in comparison with GR mRNA, which could be explained by different haif-lifes for the protein and the mRNA, respectively (see below). Studies on the rat HTC cell line, H4IIE, showed a much more complex autoregulatory pattern of GR expression (fig 2). Continuous presence of dexamethasone in the media led to a transient 2-fold increase in GR mRNA levels (peak 6 h after addition of hormone) followed by -- 80% down-regulation 18--42 h later (fig 2). At 72 h, GR mRNA had almost returned to the initial value. This cyclic pattern was also reflected at the protein level [23]. The mechanism for the cyclic pattern and the initial up-regulation is not known. However, the mechanisms of GR mRNA down-regulation were investigaged. Glucocorticoid-induced down-regulation of GR mRNA did not require ongoing protein synthesis, since it still occurred in the presence of the protein synthesis inhibitor cycloheximide [24]. However, cycloheximide by itself caused a super-induction of GR mRNA level. Furthermore, down-regulation of GR by dexamethasone was dose-dependent and a maximal response was obtained with i0-20 nM, close to the concentration required for receptor saturation [23, 241. To elucidate whether down-regulation of GR expression involved transcriptional and/or posttranscriptional processes, transcriptional activity of the GR gene and GR mRNA stability were measured in the presence of absence of dexamethasone. Using a
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Fig 1. The analysis of GR mRNA and protein levels in rat liver after treatment with dexamethasone. Rats were adrenalectomized 9 d before treatment with dexamethasone-lphosphate (4 mg/kg BW). At indicated times, total RNA and protein were isolated from the livers and analyzed for GR and 6-actin mRNA by Northern blot hybridization and for GR and actin proteins by Western immunoblotting. A, Autoradiogram of RNA blots. The sizes of the signals are calculated from the position of 18S and 28S RNAs, respectively. B, Western blots. The sizes of the hybridization signals are determined from the mobility c,f slandard proteins. C, Summary of densitometric analysis of the RNA and Western blots, respectively. Values are expressed as percent of control (untreated animals). For further experimental details, see [23].
Glucocorticoid receptor regulation
1.50
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Fig 10. Correlation between cellular GR protein level and the inducibility of MMTV and tyrosine aminotransferase by dexamethasone and forskolin. Cells were exposed to 0.5 lttM dexamethasone (DEX) in the presence or absence of 25 ~M forskolin (Fors) for 18 h.. For the study of GR protein level, cellular extracts were prepared and subjected to Western immunoblot analysis using monoclonal anti-GR antibody (see [23]). The mRNA levels of MMTV were determined by RNA blot hybridization with 32p-labeled cDNA probe coding for MMTV. The enzyme activity of tyrosine aminotransferase was studied in isolated cell extract. The specific tyrosine aminotransferase activity is calculated as nmol product/min/mg protein. All data shown in this figure were obtained from 3 experiments and are presented as the mean value + SD relative to control. For further experimental details see [5].
57
Adrenalectomy of rats has shown differences in upregulation of GR mRNA in various tissues [9. 401. Finally. feedback regulation of GR expression has been shown to vary during development and may differ from tissue to tissue [15]. The mechanism(s) determining the differences in autoregulatory response is unknown. However, data presented above have shown that with regard to the negative feedback control of GR expression, at least 2 independent mechanisms are involved, namely transcriptional repression of GR gene expression and increased turnover of the GR protein. These data have been confirmed by other reports 127-281. In addition, indirect data support supplementing mechanisms, eg at the translational level. These include discrepancies between changes in GR mRNA and protein levels (see above), changes in GR mRNA which are not reflected at the protein level (see above) and the lack of changes in receptor synthesis despite changes in GR mRNA 1271. Complex regulation of receptor expression has also been demonstrated for other steroid receptors. Ree et al [41] showed that treatment of MCF-7 cells with a saturating dose of estradiol gave a transient downregulation of the estrogen receptor mRNA. while the estrogen receptor protein remained low. In addition, a positive feedback mechanism on estrogen receptor mRNA was seen with low concentration of estradiol while a negative feedback was obtained with higher concentration of the hormone. Saceda et al [42] demonstrated that down-regulation of estrogen-receptor expression by estradiol was mainly a post-transcriptional response and that a discrepancy between gene transcription and mRNA levels existed. Estrogen receptor mRNA and protein remained low despite the fact that estrogen receptor gene transcriptional activity followir:g a short transient down-regulation increased and remained high (2-fold increase). The observation that down-regulation of GR mRNA by glucocorticoids did occur at the transcriptional level and did not require ongoing protein synthesis, may indicate that the GR protein interacts with its own gene, controlling GR gene activity. In line with this, we have shown that the GR cDNA contains at least one binding site for the GR protein. althgouh its functional implications are unknown [24]. However, post-transcriptional mechanisms could be the major determinants regulating GR expression. Such mechanisms could be controlled by eg, differences in the 5' untranslated region of GR mRNA, created by different protnotor utilization and/or splicing events. Indications for heterogeneity in the 5' untranslated region of GR mRNA have recently been reported 1431.5' Untranslated regions have been shown to be important in translational control [441, as well as mRNA stability [451.
5S
s Okret et al
A~ demonstrated above, it is norteworthy that the c A M P second messenger pathway may influence GR expression and thereby biological response to glucocorticoids. Together with recently published data on the interaction between the AP-I binding factors, c-jun and c-3'os and the GR, respectively, it is clear that an interplay between membrane and intracellularly acting signals, respectively, in regulating the cellular response for either system exists 146-501. Since small changes in GR activity in many cases will be reflected in a parallel change in biological response to glucocorticoids, it is of great importance to understand how other systems or factors influence GR expression. As shown, regulation of GR expression is controlled at transcriptional as well as posttranscriptional levels, some of which will be more or less important in generating changes in the amount of active GR protein. The complex regulation may also enable a versatile control of GR activity during various physiological and environmental conditions.
10
Acknowledgments
18
This work was supported by grants from the Swedish Cancer Society and Magnus Bergvali Foundation (to 50), Swedish Medical Research Council (No 13X-2819 to JAG) and the Swedish Society of Medical Research (to YD).
I!
12 13 14
15
16 17
19
20
References ! 2
3 4
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