Biochem. J. (1986) 233, 513-518 (Printed in Great Britain) 513 Glucocorticoids antagonize induction of prolactin-gene expression by calcitriol in rat pituitary tumour cells John D. WARK and Volker GURTLER University of Melbourne, Department of Medicine, The Royal Melbourne Hospital, Melbourne, Vic. 3050, Australia Clonal strains of rat pituitary tumour (GH4C1) cells are known to possess specific intracellular binding sites for calcitriol (1,25-dihydroxycholecalciferol, 1,25-dihydroxyvitamin D3). GH4C1 cells respond to calcitriol by a selective increase in prolactin(PRL)-gene expression. The interaction between calcitriol and glucocorti- coids was studied by using this cultured-cell model. It was found that cortisol potently antagonized the induction of PRL mRNA and PRL production by calcitriol. The effects were concentration-dependent and were evident at glucocorticoid concentrations that did not alter basal PRL production. Inhibition was half-maximal at 3.2 nM-cortisol and 0.4 nM-dexamethasone. Calcitriol-induced PRL mRNA fell by more than 50% at 25 h and reached the control level 50 h after treatment with cortisol. The inhibition by cortisol of calcitriolinduction ofPRLproduction was selective whencomparedwitheffects on otherinducersofPRL-gene expression [thyroliberin, epidermal growth factor and phorbol myristate acetate (' 12-O-tetradecanoylphorbol 13-acetate')]. Potent antagonism by glucocorticoids ofvitamin D action on specific gene expression has been demonstrated. Further studies with this cultured-cell model may help to explain the mechanism of this hormonal interaction, which assumes particular importance at major sites of vitamin D action such as the intestine. INTRODUCTION D metabolites and analogues were similar to those which have been observed for other vitamin D-responsive An imposing body of evidence indicates that calcitriol processes in vitro (Corradino, 1979; Franceschi & (1,25-dihydroxycholecalciferol, 1,25-dihydroxyvitamin DeLuca, 1981). The effect of calcitriol on PRL D3) acts in target cells by a nuclear mechanism analogous production and PRL mRNA was delayed in onset to that ofthe 'classical' steroid hormones (Norman et al., (requiring at least 24 h to become evident), and depended 1982). Thus the presence ofa specific intracellular binding on extracellular Ca2+ for its full expression. macromolecule, the calcitriol 'receptor', has come to be Although studies of this type could not prove a regarded as a marker of putative sites of action of physiological role for vitamin D in the regulation of calcitriol. After its discovery in the rat pituitary gland pituitary-cell function, they did support that possibility. (Haussler et al., 1980), the calcitriol receptor was also Moreover, the findings clearly demonstrated the useful- detected in clonal strains of rat pituitary tumour (GH) ness ofGH4C1 cells as a model in which to investigate the cells (Murdoch & Rosenfeld, 1981; Haussler et al., 1981). cellular and molecular mechanisms involved in calcitriol Subsequently, the effects of calciol (cholecalciferol, action. These studies have now been extended. In the vitamin D3) metabolites on hormone production by GH experiments reported here, glucocorticoids were found to cells have been examined in several laboratories antagonize potently and selectively calcitriol induction of (Murdoch & Rosenfeld, 1981; Wark & Tashjian, 1982; PRL-gene expression. Further studies with the present Haug et al., 1982; Wark & Tashjian, 1983). Because experimental model may lead to an increased under- serum-supplemented culture media contain potentially standing of the cellular and molecular mechanisms by confounding factors such as vitamin D metabolites, their D serum binding protein and a complex mixture of which glucocorticoids antagonize vitamin action. hormones and other humoral agents, it seemed appro- priate to study the effects ofvitamin D metabolites in cells MATERIALS AND METHODS incubated in serum-free, chemically defined medium. When such conditions were used, calcitriol potently and Materials selectively increased PRL production and PRL mRNA [a-32P]dCTP (2000-3000 Ci/mmol) and nick-trans- in GH cells of the GH4C1 strain (Wark & Tashjian, 1982, lation reagents were obtained from Amersham Australia 1983). Half-maximal effects were observed at 0. 1-0.2 nM- Pty. Ltd. Recombinant plasmid pPRL-1 (Gubbins et al., calcitriol. Effects on growth-hormone production were 1979) was a gift from Dr. D. K. Biswas, Harvard School minimal, attesting to the selectivity of the effect on of Dental Medicine, Boston, MA, U.S.A. Zetapor PRL-gene expression. The relative potencies of vitamin membrane (0.45 ,um pore size) was obtained from Abbreviations used: CS, corticosterone (11,8,21-dihydroxypregn-4-ene-3,20-dione); DXM, dexamethasone (9a-fluoro-1 1,B,17,21-trihydroxy- 16a-methylpregna-1,4-diene-3,20-dione); EGF, fl-epidermal growth factor; F10+, Ham's FIO medium supplemented with 15% (v/v) foetal bovine serum; IC50, concentration causing 50% inhibition; MEM, Minimum Essential Medium; PRL, prolactin; 15 x SSC, 2.25 M-NaCI/0.225 M-trisodium citrate; 20 x SSC, 3.0 M-NaCI/0.3M-trisodium citrate; PMA, phorbol myristate acetate ('TPA'); TRH, thyroliberin ('thyrotropin-releasing hormone'). Vol. 233 514 J. D. Wark and V. Gurtler AMF-Cuno, Meriden, CT, U.S.A. Radioimmunoassay- 10.0 r kit reagents for measuring rat PRL were gifts from the National Hormone and Pituitary Program, Baltimore, 9.0 . MD, U.S.A. Calcitriol was a gift from Dr. M. Uskokovic, Hoffmann-La Roche, Nutley, NJ, U.S.A.; other steroid Nl I hormones, PMA and Nonidet P40 were obtained from 8.0 F Sigma Chemical Co., St. Louis, MO, U.S.A.; TRH was ._ obtained from Peninsula Laboratories, Belmont, CA, 0 7.0 F U.S.A.; ,-EGF from mouse salivary glands was kindly provided by the Ludwig Institute for Cancer Research, E 6.0 F The Royal Melbourne Hospital, Melbourne, Vic., Q 0. Australia. 5.0 I- -o..0 Cell culture 00. C: 4.0 F GH4C1 cells were a gift from Dr. A. H. Tashjian, Jr., L- Harvard School of Public Health, Boston, MA, U.S.A. 0w 3.0 - -Z- Media and medium components for cell culture generally L -9- -1 . , were obtained from Gibco Laboratories, Grand Island, NY, U.S.A. The cells were plated [(2.5-5.0) x 105/60 mm 2.0 L dish] and grown for 4-5 days in Fl0+. Before an experiment, F10+ was replaced by chemically defined 1.0 medium after gently washing the cultures with the same solution. This synthetic medium was constituted as 0 previously described (Wark & Tashjian, 1982), or was based on Ham's F12 medium to which supplementary 0 10-9 10-8 10-7 amino acids and 10% serum substitute (Bauer et al., 1976) [Cortisol] (M) were added. In either case, the medium was hormone-free; Fig. 1. Effect of cortisol and cortisol-plus-calcitriol on PRL the total Ca2+ concentration was adjusted to 0.4 mm by production by GH4C1 cells addition of CaCl2. Steroid hormones in concentrated Cell cultures were prepared as described in the Materials ethanolic solutions were diluted in medium before and methods section. After equilibration for 24 h in addition to cultures. The final concentration of ethanol serum-free Eagle's MEM-based medium 96 h treatment did not exceed 0.06% (v/v) and was the same in all dishes. was commenced with 1 nM-calcitriol (@) or ethanol- Peptides were added in 155 mM-NaCl. PMA was stored containing vehicle (0), plus various concentrations of at a concentration of 6.17 mg/ml (10 mM) in dimethyl cortisol or vehicle. The cultures received fresh medium and sulphoxide and diluted with phosphate-buffered saline treatment after 48 h. The results shown are the rates of (137 mM-NaCl/2.7 mM-KCl/8.9 mM-NaHPO4, 7H20/ PRL accumulation in the medium in the final 48 h of 1.5 mM-KH2PO4, pH 7.2) before use. treatment. Medium PRL was measured by radio- immunoassay. Each point represents the mean (±S.E.M.) Measurement of specific mRNA value for six replicate dishes. The mean final cell protein PRL mRNA was measured by cytoplasmic dot hyb- content per culture was 1.2 mg and did not vary with ridization (White & Bancroft, 1982), with minor mod- treatment. ifications as previously decribed (Wark & Tashjian, 1983). The cells were mobilized mechanically, washed radiography was performed for 24 h at -70 °C using twice in Hanks balanced salt solution, suspended in 45 ,1 of ice-cold 10 mM-Tris/HCl (pH 7.4)/I mM-EDTA and X-Omat XAR-5 film (Eastman Kodak Co.) and two lysed by the addition of 5 #1 of 5% (v/v) Nonidet P40 image-intensifying screens (Du Pont Chronex Lightning twice over 7.5 min during gentle mixing on ice. After Plus). After autoradiography, the filters were cut into centrifugation of the samples (15000g, 4 °C, 2.5 min) in small squares and hybridization quantified by liquid- an Eppendorf model 5414S centrifuge, 50 ,1 of super- scintillation counting. natant were added to 210 ,1 of 20 x SSC and 65 ,ul of PRL production formaldehyde solution (37.0-40.0%, w/v). Samples were incubated at 60 °C for 15 min, rapidly cooled and stored PRL accumulation in the medium was determined by at -70 'C. Denatured cytoplasmic samples were serially rPRL radioimmunoassay and was used as a measure of diluted in 15 x SSC and applied to Zetapor membrane PRL synthesis, since GH cells store only a minimal using a Minifold filtration manifold (Schleicher and amount of PRL and degradation of PRL by the cells is Schuell, Keene, NH, U.S.A.). The filters were baked in negligible (Haug et al., 1982). Total protein was measured vacuo at 80 'C for90 min. Prehybridization, hybridization by using the Coomassie Brilliant Blue dye method and washing conditions were as described by Wark & (Bradford, 1976). Tashjian (1983). Plasmid pPRL-1 was prepared by established methods (Clewell & Helinski, 1969) and RESULTS labelled with [a-32P]dCTP by nick translation (Benz et al., As previously reported (Wark & Tashjian, 1982), 1979) to a specific radioactivity of approximately 10 nM-calcitriol caused highly significant stimulation of 1.0 x 108 d.p.m./ug.