Repression of DAX-1 and Induction of SF-1 Expression
2002; Elsevier BV; Volume: 277; Issue: 43 Linguagem: Inglês
10.1074/jbc.m206595200
ISSN1083-351X
AutoresHanan Osman‐Ponchet, Claire Murigande, Anne Nadakal, Alessandro M. Capponi,
Tópico(s)Estrogen and related hormone effects
ResumoAngiotensin II (Ang II) and adrenocorticotropic hormone stimulate aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex through induction of the expression of the steroidogenic acute regulatory (StAR) protein, which promotes intramitochondrial cholesterol transfer. To understand the mechanism of this induction of the StAR protein, we have examined the effect of Ang II and forskolin, a mimicker of adrenocorticotropic hormone action, on two transcription factors known to modulate StARgene expression in opposite ways, DAX-1 and SF-1, in bovine adrenal glomerulosa cells in primary culture. Ang II markedly inhibited DAX-1 protein expression in a time- and concentration-dependent manner (to 38.7 ± 12.9% of controls at 3 nm after 6 h, p < 0.01), an effect that required de novo protein synthesis and ERK2/1 activation. This effect was associated with a concomitant decrease in DAX-1 mRNA and an increase in mitochondrial StAR protein levels. Similarly, forskolin dramatically repressed DAX-1 protein and mRNA expression (to 19.6 ± 1.8 and 50.3 ± 4.7% of controls, respectively,p < 0.01). Neither Ang II nor forskolin affected DAX-1 protein and mRNA stability. The aldosterone response to Ang II was markedly reduced (to 59 ± 4% of controls,p < 0.01) in transiently transfected cells overexpressing DAX-1. Whereas Ang II was without effect on SF-1 expression, forskolin significantly increased SF-1 protein and mRNA levels in a cycloheximide-sensitive manner (to 167.4 ± 16.6 and 173.1 ± 25.1% of controls after 6 h, respectively,p < 0.01). These results demonstrate that the balance between repressor and inducer function of DAX-1 and SF-1 are of critical importance in the regulation of adrenal aldosterone biosynthesis. Angiotensin II (Ang II) and adrenocorticotropic hormone stimulate aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex through induction of the expression of the steroidogenic acute regulatory (StAR) protein, which promotes intramitochondrial cholesterol transfer. To understand the mechanism of this induction of the StAR protein, we have examined the effect of Ang II and forskolin, a mimicker of adrenocorticotropic hormone action, on two transcription factors known to modulate StARgene expression in opposite ways, DAX-1 and SF-1, in bovine adrenal glomerulosa cells in primary culture. Ang II markedly inhibited DAX-1 protein expression in a time- and concentration-dependent manner (to 38.7 ± 12.9% of controls at 3 nm after 6 h, p < 0.01), an effect that required de novo protein synthesis and ERK2/1 activation. This effect was associated with a concomitant decrease in DAX-1 mRNA and an increase in mitochondrial StAR protein levels. Similarly, forskolin dramatically repressed DAX-1 protein and mRNA expression (to 19.6 ± 1.8 and 50.3 ± 4.7% of controls, respectively,p < 0.01). Neither Ang II nor forskolin affected DAX-1 protein and mRNA stability. The aldosterone response to Ang II was markedly reduced (to 59 ± 4% of controls,p < 0.01) in transiently transfected cells overexpressing DAX-1. Whereas Ang II was without effect on SF-1 expression, forskolin significantly increased SF-1 protein and mRNA levels in a cycloheximide-sensitive manner (to 167.4 ± 16.6 and 173.1 ± 25.1% of controls after 6 h, respectively,p < 0.01). These results demonstrate that the balance between repressor and inducer function of DAX-1 and SF-1 are of critical importance in the regulation of adrenal aldosterone biosynthesis. The biosynthesis of aldosterone, the main mineralocorticoid hormone, in the zona glomerulosa cells of the adrenal cortex is placed under the control of three principal physiological factors, the octapeptide hormone, angiotensin II (Ang II), 1The abbreviations used are: Ang II, angiotensin II; ACTH, adrenocorticotropic hormone; DAX-1, dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X-chromosome, gene 1; IOD, integrated optical density; P450scc, cholesterol side chain cleavage cytochrome P450; SF-1, steroidogenic factor-1; StAR protein, steroidogenic acute regulatory protein; RT, reverse transcriptase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 1The abbreviations used are: Ang II, angiotensin II; ACTH, adrenocorticotropic hormone; DAX-1, dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X-chromosome, gene 1; IOD, integrated optical density; P450scc, cholesterol side chain cleavage cytochrome P450; SF-1, steroidogenic factor-1; StAR protein, steroidogenic acute regulatory protein; RT, reverse transcriptase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. extracellular potassium (K+), and adrenocorticotropic hormone (ACTH) (1Müller J. Regulation of Aldosterone Biosynthesis. Springer-Verlag, Berlin1988Google Scholar). Whereas Ang II and K+ exert their effect by recruiting the calcium messenger system in glomerulosa cells (2Capponi A.M. Rossier M.F. Davies E. Vallotton M.B. J. Biol. Chem. 1988; 263: 16113-16117Google Scholar, 3Capponi A.M. Rossier M.F. Curr. Opin. Endocrinol. Diabetes. 1996; 3: 248-257Google Scholar), ACTH acts mainly by activating adenylyl cyclase and generating cAMP as an intracellular messenger (4Baukal A.J. Hunyady L. Catt K.J. Balla T. J. Biol. Chem. 1994; 269: 24546-24549Google Scholar). In the cascade of subsequent events leading to the final production of aldosterone, the regulated, rate-limiting step is the transfer of cholesterol from the relatively cholesterol-rich outer mitochondrial membrane to the cholesterol-poor inner mitochondrial membrane (5Jefcoate C.R. McNamara B.C. Artemenko I. Yamazaki T. J. Steroid Biochem. Mol. Biol. 1992; 43: 751-767Google Scholar). Cholesterol is then converted to pregnenolone by the cytochrome P450 side chain cleavage enzyme (P450scc). In all steroidogenic tissues, intramitochondrial cholesterol transfer is facilitated by the steroidogenic acute regulatory (StAR) protein (6Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-244Google Scholar). In the adrenal zona glomerulosa cell, the expression of the StAR protein is rapidly increased by factors that activate mineralocorticoid biosynthesis. Indeed, Ang II and ACTH have been shown to stimulate StAR mRNA and StAR protein expression and to concomitantly increase aldosterone production in bovine zona glomerulosa cells (7Cherradi N. Rossier M.F. Vallotton M.B Timberg R. Friedberg I. Orly J. Wang X.J. Stocco D.M. Capponi A.M. J. Biol. Chem. 1997; 272: 7899-7907Google Scholar, 8Cherradi N. Brandenburger Y. Rossier M.F. Vallotton M.B. Stocco D.M. Capponi A.M. Mol. Endocrinol. 1998; 12: 962-972Google Scholar). Moreover, in human H295R adrenocortical carcinoma cells, which bear only very few ACTH receptors, challenge with cAMP analogs, used to mimic adenylyl cyclase activation, Ang II, or K+, leads to StAR mRNA and protein expression (9Clark B.J. Pezzi V. Stocco D.M. Rainey W.E. Mol. Cell. Endocrinol. 1995; 115: 215-219Google Scholar, 10Clark B, J. Combs R. Endocrinology. 1999; 140: 4390-4398Google Scholar). Whereas the initial signal transduction mechanisms mediating the steroidogenic action of these activators of aldosterone biosynthesis are well characterized (3Capponi A.M. Rossier M.F. Curr. Opin. Endocrinol. Diabetes. 1996; 3: 248-257Google Scholar), the events occurring downstream of Ca2+ or cAMP signal generation and leading to the induction of StAR protein expression are poorly understood. No consensus cAMP response element has been found in the ∼3.6-kilobase 5′-flanking region of the mouse, rat, human, porcine, and bovine StAR gene (11Christenson L.K. Strauss J.F. Biochim. Biophys. Acta. 2000; 1529: 175-187Google Scholar). In contrast, in all species, several putative binding sites for steroidogenic factor-1 (SF-1), also called Ad4BP, are present in the StAR gene promoter (10Clark B, J. Combs R. Endocrinology. 1999; 140: 4390-4398Google Scholar,12Sugawara T. Kiriakidou M. Mcallister J.M. Holt J.A. Arakane F. Strauss J.F. Steroids. 1997; 62: 5-9Google Scholar, 13Sugawara T. Kiriakidou M. Mcallister J.M. Kallen C.B. Strauss J.F. Biochemistry. 1997; 36: 7249-7255Google Scholar, 14Caron K.M. Ikeda Y. Soo S.C. Stocco D.M. Parker K.L. Clark B.J. Mol. Endocrinol. 1997; 11: 138-147Google Scholar, 15Sandhoff T.W. Hales D.B. Hales K.H. Mclean M.P. Endocrinology. 1998; 139: 4820-4831Google Scholar, 16Rust W. Stedronsky K. Tillmann G. Morley S. Walther N. Ivell R. J. Mol. Endocrinol. 1998; 21: 189-200Google Scholar). The number and localization of these binding sites vary from one species to the other. SF-1 is a nuclear transcription factor that was first identified in adrenal cortical cells (17Morohash K. Zanger U.M. Honda S. Hara M. Waterman M.R. Omura T. Mol. Endocrinol. 1993; 7: 1196-1204Google Scholar). The orphan nuclear receptor SF-1 plays a critical role in adrenal and gonadal differentiation, development, and function (18Parker K.L. Schimmer B.P. Endocr. Rev. 1997; 18: 361-377Google Scholar). Furthermore, SF-1 has also been shown to regulate the expression of genes encoding cytochrome P450 hydroxylases and to efficiently transactivate the StARgene in transient transfection assays in various cell types (13Sugawara T. Kiriakidou M. Mcallister J.M. Kallen C.B. Strauss J.F. Biochemistry. 1997; 36: 7249-7255Google Scholar, 15Sandhoff T.W. Hales D.B. Hales K.H. Mclean M.P. Endocrinology. 1998; 139: 4820-4831Google Scholar,16Rust W. Stedronsky K. Tillmann G. Morley S. Walther N. Ivell R. J. Mol. Endocrinol. 1998; 21: 189-200Google Scholar, 19Wooton-Kee C.R. Clark B.J. Endocrinology. 2000; 141: 1345-1355Google Scholar). Although the extent of SF-1 involvement in the regulation ofStAR gene expression may present species- and cell type-dependent differences (20Silverman E. Eimerl S. Orly J. J. Biol. Chem. 1999; 274: 17987-17996Google Scholar), it appears that activation of the cAMP-signaling pathway leads to increased phosphorylation (21Gyles S.L. Burns C.J. Whitehouse B.J. Sugden D. Marsh P.J. Persaud S.J. Jones P.M. J. Biol. Chem. 2001; 276: 34888-34895Google Scholar) and/or expression (22Aesoy R. Mellgren G. Morohashi K. Lund J. Endocrinology. 2002; 144: 295-303Google Scholar) of SF-1 protein. In addition to response elements for SF-1, the StAR gene promoter also bears a binding site for another orphan member of the nuclear receptor superfamily, DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1) (23Yu R.N. Achermann J.C. Ito M. Jameson J.L. Trends Endocrinol. Metab. 1998; 5: 169-175Google Scholar, 24Lalli E. Sassone-Corsi P. Curr. Opin. Endocrinol. Diabetes. 1999; 6: 185-190Google Scholar). DAX-1 has been shown to act as a powerful repressor of StARgene expression. Indeed, overexpression of DAX-1 in Y-1 mouse adrenal tumor cells inhibits steroid synthesis, and DAX-1 represses both basal and cAMP-induced StAR promoter activity by binding to DNA hairpin secondary structures on the StAR gene promoter or to the SF-1 protein itself (25Zazopoulos E. Lalli E. Stocco D. Sassone-Corsi P. Nature. 1997; 390: 311-315Google Scholar). Furthermore, overexpression of DAX-1 in Y-1 adrenocortical cells impairs basal and cAMP-stimulated steroid production (26Tamai K.T. Monaco L. Alastalo T.P. Lalli E. Parvinen M. Sassone-Corsi P. Mol. Endocrinol. 1996; 10: 1561-1569Google Scholar). Conversely, cAMP down-regulates DAX-1 expression in cultured rat Sertoli cells (26Tamai K.T. Monaco L. Alastalo T.P. Lalli E. Parvinen M. Sassone-Corsi P. Mol. Endocrinol. 1996; 10: 1561-1569Google Scholar). Finally, SF-1 and DAX-1 are co-localized in various endocrine and steroidogenic tissues, suggesting that these two nuclear proteins may be linked in function. Because Ang II and ACTH stimulate StAR protein expression, the present study was undertaken to investigate whether the mechanism of this response involves a modulation of DAX-1 and SF-1 expression. We report here that both Ang II, a calcium mobilizing hormone, and forskolin, used as a mimicker of ACTH action to generate a pure cAMP signal, markedly inhibit DAX-1 expression at the mRNA and protein level. We also show that forskolin significantly increases SF-1 expression. This study provides evidence that the removal of the suppressor effect of DAX-1 on StAR expression is an important mechanism through which activators of aldosterone biosynthesis increase StAR expression. Bovine adrenal glands were obtained from a local slaughterhouse. Zona glomerulosa cells were prepared by enzymatic dispersion with dispase and purified on Percoll density gradients as previously reported (27Python C.P. Rossier M.F. Vallotton M.B. Capponi A.M. Endocrinology. 1993; 132: 1489-1496Google Scholar). Primary cultures of purified glomerulosa cells were maintained in Dulbecco's modified Eagle's medium as described in detail elsewhere (27Python C.P. Rossier M.F. Vallotton M.B. Capponi A.M. Endocrinology. 1993; 132: 1489-1496Google Scholar). The cells were grown on 6-well tissue culture plates (3 × 106 cells/well) and kept in serum-free medium for 24 h before experiments, which were performed on the third day of culture. Cells were then washed and incubated at 37 °C in serum-free medium containing various agents for varying periods of time as appropriate. At the end of the incubation period, the media were collected, and cells were processed for protein or total RNA extraction as described hereafter. Aldosterone content in incubation media was measured by direct radioimmunoassay using a commercially available kit (Diagnostic Systems Laboratories, Webster, TX). Aldosterone production was normalized and expressed per milligram of cellular protein. For the determination of protein expression levels, bovine glomerulosa cells were washed twice in ice-cold phosphate saline buffer (PBS) and lysed in PBS containing 1% (v/v) Triton X-100, 0.1% (w/v) sodium dodecyl sulfate, 0.5% (w/v) deoxycholate, 1 mm phenylmethylsulfonyl fluoride, and 1 μg/ml leupeptine. The cell debris were removed by centrifugation at 20,000 × g at 4 °C for 15 min, and supernatants were used as cell lysates. Proteins were quantified using a protein microassay (Bio-Rad). Equal amounts of protein (20 μg) were resolved by 12% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Western blot analysis of DAX-1 and SF-1 proteins was carried out with a mouse monoclonal antiserum directed against DAX-1 (kindly provided by Dr. Enzo Lalli, Strasbourg, France) or a rabbit polyclonal antiserum to Ad4BP/SF-1 (kindly supplied by Dr. Ken Morohashi, University of Tsukuba, Japan). Mitochondrial StAR protein levels were determined by Western blot as previously described (8Cherradi N. Brandenburger Y. Rossier M.F. Vallotton M.B. Stocco D.M. Capponi A.M. Mol. Endocrinol. 1998; 12: 962-972Google Scholar). Immunoreactive proteins were visualized by the enhanced chemiluminescence method (ECL, Amersham Biosciences). Band intensity was quantified with a Molecular Dynamics Computing Densitometer. For transfection experiments, 800,000 bovine glomerulosa cells were seeded in 60-mm Petri dishes. Cells were transfected using Effectene transfection reagent (Qiagen, Basel, Switzerland) according to the manufacturer's instructions. One μg of pBKCMV-hDAX-1 encoding the human DAX-1 protein was introduced into cells at a DNA:Effectene ratio of 1:50. The same conditions were used with the empty vector pBKCMV for mock-transfected cells. Cells were incubated with the transfection complexes for 8 h then washed twice with phosphate saline buffer and incubated for 48 h in fresh medium supplemented with serum. In separate experiments with pBKCMV-GFP encoding the green fluorescent protein we observed that the transfection efficacy amounted to 35–40% under the above conditions (data not shown). Cells were then challenged with Ang II, and DAX-1 protein levels were determined as described. Total cellular RNA was isolated from bovine glomerulosa cells using the SV Total RNA Isolation system (Promega, Zurich, Switzerland) according to the manufacturer's instructions. Total RNA (500 ng) was reverse-transcribed and amplified using the Access RT-PCR system (Promega). A fragment of bovine DAX-1 cDNA (214 base pairs) was amplified using human DAX-1 primers (5′-AGGGGACCGTGCTCTTTAAC-3′ forward and 5′-ATGATGGGCCTGAAG AACAG-3′ reverse). Primers corresponded to positions +1145–1164 and 1339–1358 of the human DAX-1 (NCBI/GenBankTM accession number NM_000457). The PCR product was purified from a 1% agarose gel, cloned into pGEM®-T Easy vector (Promega), and amplified in JM 109 competent cells. The plasmid insert was sequenced by automatic sequencing using the DyEnamics Terminator sequencing kit (Amersham Biosciences) and Applied Biosystem 3100 sequencer. RT-PCR was used to evaluate DAX-1 and SF-1 mRNA abundance in response to various treatments. RT-PCR measurements of mRNA were performed using Promega reagents and 500 ng of total RNA per reaction. Reverse transcription with avian myeloblastosis virus RT (5 units/reaction, 48 °C for 30 min) primed with specific primers was followed by PCR using Tfl DNA polymerase (5 units/reaction) with the following cycling parameters: denaturation for 5 min at 95 °C followed by 30 and 25 cycles for DAX-1 and SF-1, respectively, of 95 °C for 1 min, 56 °C for 1 min, and 68 °C for 30 s and a final extension at 68 °C for 10 min. The primers were bovine SF-1 (5′-GCAGAAGAAGGCACAGATTC-3′ (forward) and 5′-TGGGTACTCAGACTTGATGG-3′ (reverse). Primers corresponded to positions +439–459 and 693–713 of the bovine Ad4BP mRNA (NCBI/GenBankTM accession numberD13569). DAX-1 primers were as described above. To correct for potential variations in RT-PCR efficiency, an internal control, a fragment of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was co-amplified in each sample using as the sense primer 5′-ATGGTGAAGGTCGGAGTG-3′ and as the antisense primer 5′-TGCAGAGATGATGACCCTC-3′. Primers corresponded to positions +82–99 and 426–444 of the human GAPDH (NCBI/GenBankTM accession number NM_002046). The combination of GAPDH and SF-1 primers yielded two products corresponding to 362 and 274 bp for GAPDH and SF-1, respectively, and that of GAPDH and DAX-1 yielded two products corresponding to 362 and 214 bp for GAPDH and DAX-1, respectively. Ten microliters of the PCR product were separated by electrophoresis on ethidium bromide-stained 1.3% agarose gels. The intensity of each band was normalized to the intensity of the corresponding GAPDH band. Bands were quantified by image analysis using the NIH Image 1.23 software (rsb.info.nih.gov/nih-image/download.html). All amplified sequences were confirmed by automatic sequencing. Plots of product amountversus cycle number were linear, confirming that all reactions were run in the exponential part of the progress curve. The partial sequence of bovine DAX-1 has been deposited in the GenBankTM data base (Bethesda, MD) under accession number AF421373. The nucleic acid and derived amino acid sequences of DAX-1 cDNA were compared with known sequences provided by the National Center for Biotechnology Information (Bethesda, MD) using Blast searches. Comparison of nucleotide and deduced amino acid sequences were performed using ClustalW. The data presented are the mean ± S.E. The statistical significance of differences between treatments was determined by one-way analysis of variance and Fisher's protected least significant difference test. Values of p < 0.05 or less were considered statistically significant. DAX-1 protein levels were high in unstimulated bovine adrenal glomerulosa cells. When cells were treated with Ang II for 6 h, a significant, concentration-dependent inhibition of DAX-1 protein expression was observed between 0.1 and 10 nm Ang II, as determined by Western blot analysis. Maximal inhibition (to 38.7 ± 12.9% of controls, n = 3–9, p < 0.01) was achieved with 3 nm Ang II (Fig.1, A and B). Concomitantly, Ang II powerfully stimulated intramitochondrial StAR protein accumulation (Fig. 1 C) and aldosterone production (Fig. 1 D) over the same range of concentrations. At 10 nm Ang II, aldosterone production into the medium was increased by 71-fold (127.2 ± 19.4 pmol/mg of proteinversus 1.8 ± 0.26 pmol/mg in control cells,n = 3, p < 0.01). To determine the kinetics of the inhibition of DAX-1 protein expression induced by Ang II, we stimulated glomerulosa cells with 10 nm Ang II for 0–6 h. As shown in Fig.2, A and B, the inhibitory effect of Ang II was time-dependent, with DAX-1 protein levels reaching 61.5 ± 4.6% (n = 5,p < 0.01) and 40 ± 3.4% of controls (n = 5, p < 0.01) after 4 and 6 h, respectively. No further decrease in DAX-1 protein expression was observed thereafter (data not shown). The progressive inhibition of DAX-1 protein expression was accompanied with a time-dependent increase in aldosterone production from Ang II-treated adrenal glomerulosa cells (Fig. 2 C). The inhibition of DAX-1 protein expression by Ang II depended upon de novo protein synthesis. Indeed, although glomerulosa cells stimulated with 10 nm Ang II for 6 h showed a significant decrease in DAX-1 protein levels (to 40.8 ± 2.6% of controls, n = 3,p < 0.01), simultaneous treatment with cycloheximide, a protein translation inhibitor, abolished the Ang II-induced inhibition of DAX-1 protein expression (Fig.3, A and B). Cycloheximide also completely abolished the aldosterone response to Ang II, as previously reported by others (28Elliott M.E. Goodfriend T.L. Biochem. Pharmacol. 1984; 33: 1519-1524Google Scholar) (Fig. 3 C). Ang II is known to activate ERK2/1 in bovine adrenal glomerulosa cells (29Tian Y. Smith R.D. Balla T. Catt K.J. Endocrinology. 1998; 139: 1801-1809Google Scholar). To determine whether inhibition of DAX-1 protein expression by Ang II is mediated by the mitogen-activated protein kinase pathway, we stimulated cells with Ang II alone or in combination with U0126 (Biomol, Plymouth Meeting, PA), an inhibitor of MEK-1, the kinase that phosphorylates and activates ERK2/1. As shown in Fig. 4, A and B, the inhibitory effect of Ang II on DAX-1 expression was completely abolished in the presence of U0126. In contrast, the p38 mitogen-activated protein kinase inhibitor, SB203580, did not affect Ang II action (data not shown). To determine whether Ang II exerts its inhibitory effect directly on DAX-1 mRNA expression, we stimulated glomerulosa cells for 6 h with 10 nm Ang II alone or in combination with 10 μm U0126, and DAX-1 mRNA levels were then determined by semi-quantitative RT-PCR. The results shown in Fig. 4, Cand D, indicate that Ang II treatment led to a significant decrease in bovine DAX-1 mRNA levels (to 68.8 ± 4.3% of controls, n = 4, p < 0.01). This down-regulation of DAX-1 mRNA by Ang II was completely prevented by the MEK-1 inhibitor U0126. The involvement of ERK2/1 in the functional response to Ang II was also observed at the level of steroid production. Indeed, as shown in Fig.4 E, two structurally unrelated MEK-1 inhibitors, U0126 and PD98059 (Alexis Biochemicals, Läufelfingen, Switzerland), significantly reduced aldosterone production elicited by Ang II. We next examined whether another inducer of StAR expression and aldosterone production, forskolin, used as a mimicker of ACTH, which mobilizes the cAMP messenger system, also affects DAX-1 expression. As shown in Fig. 5, A andB, treatment of adrenal glomerulosa cells with 25 μm forskolin for 6 h dramatically inhibited DAX-1 protein expression (to 19.6 ± 1.82% of controls,n = 3, p < 0.001). As for Ang II, inhibition of DAX-1 protein expression by forskolin was concentration- and time-dependent (data not shown). Furthermore, the inhibition of DAX-1 protein expression by forskolin was completely prevented by cycloheximide. In contrast, the MEK-1 inhibitor, U0126, had no effect on forskolin action. The inhibition of DAX-1 protein expression provoked by forskolin was associated with a 56-fold increase in aldosterone production (Fig. 5 E). The inhibition of DAX-1 expression by forskolin was also observed at the transcriptional level, as demonstrated in Fig. 5, C andD. Indeed, in glomerulosa cells stimulated with forskolin, DAX-1 mRNA levels were decreased to 50.3 ± 4.7% of control untreated cells (n = 3, p < 0.0.01). This effect was not reversed by the MEK-1 inhibitor, U0126. Moreover, actinomycin D did not affect the reduction of DAX-1 mRNA expression elicited by forskolin (data not shown). To determine whether Ang II and forskolin decreased DAX-1 protein levels by accelerating its catabolism, we incubated bovine glomerulosa cells for 6 h in the absence or presence of either agonist and then followed DAX-1 protein levels under conditions of protein synthesis blockade. As shown in Fig.6 A, DAX-1 protein levels were quite stable over time under basal conditions and in the absence of cycloheximide. In contrast, when de novo protein synthesis was prevented, the levels of DAX-1 protein decreased markedly with time, with a half-life of ∼6 h. Neither Ang II nor forskolin accelerated this process (Fig. 6 A). The inhibition of DAX-1 mRNA expression could result from changes in transcription rate and/or in mRNA turnover. We therefore examined whether Ang II affected DAX-1 mRNA stability. Glomerulosa cells were treated for 6 h in the absence or presence of Ang II (10 nm), and then actinomycin D was added. After 6 h, DAX-1 mRNA levels decayed to 45.2% of the zero time value in cells treated with actinomycin D alone and to a similar value (53.4 ± 4.9%, n = 3, p < 0.01) in cells that had been pretreated with Ang II (Fig. 6 B). Similar results were obtained with forskolin (data not shown). The role of DAX-1 on the aldosterone response to Ang II was confirmed in bovine glomerulosa cells that had been transiently transfected with DAX-1 cDNA. In cells transfected with the empty pBKCMV vector, Ang II induced a robust aldosterone production (Fig.7, Mock). Although the absolute aldosterone values were somewhat lower than in non-transfected cells, possibly because of the transfection procedure itself, the fold increase over basal was not significantly different (see, for example, Figs. 1 D, 3 C, or 4 C). This aldosterone response was associated with the expected repression of DAX-1. In contrast, in cells transiently transfected with pBKCMV-hDAX-1, which expressed 20–30-fold higher levels of the DAX-1 protein, the aldosterone response to Ang II was significantly reduced to 59.4 ± 4% of the response measured in mock-transfected cells (n = 3, p < 0.01). Because the transcription factor SF-1 is known to regulate StAR protein expression, we examined whether the induction of StAR expression induced by both Ang II and forskolin involves changes in SF-1 protein expression in adrenal glomerulosa cells. As shown in Fig. 8, Aand B, a significant increase in SF-1 protein expression was observed in cells stimulated for 6 h with forskolin (167.4 ± 16.6% of controls, n = 7, p < 0.001), whereas Ang II had no effect. The forskolin-induced increase in SF-1 protein expression was blocked by cycloheximide. We next examined whether forskolin and Ang II affected SF-1 mRNA expression. Bovine glomerulosa cells were stimulated for 6 h with 10 nm Ang II or 25 μm forskolin, and SF-1 mRNA levels were then determined by semi-quantitative RT-PCR. The results shown in Fig. 8, C and D, indicate that forskolin treatment led to a significant increase in bovine SF-1 mRNA levels (to 173.1 ± 25.1% of controls, n= 5, p < 0.05). In contrast, Ang II had no significant effect on SF-1 mRNA expression. The forskolin-induced increase in SF-1 mRNA expression was almost completely abolished by actinomycin D (Fig. 8, C and D), suggesting a direct action of forskolin on SF-1 mRNA at the transcriptional level. The present study was undertaken in an attempt to investigate the mechanism of the known induction of StAR protein expression in adrenal glomerulosa cells in response to two physiological activators of aldosterone biosynthesis, the octapeptide hormone angiotensin II and adrenocorticotropic hormone, ACTH (10Clark B, J. Combs R. Endocrinology. 1999; 140: 4390-4398Google Scholar, 30Cherradi N. Capponi A.M. Trends Endocrinol. Metab. 1998; 9: 412-418Google Scholar). We have focused our attention on two orphan members of the nuclear receptor family of transcription factors, DAX-1 and SF-1. Indeed, on one hand, DAX-1 has been shown to repress StAR gene expression by binding to a hairpin structure located in the StAR gene promoter and to block steroidogenesis (25Zazopoulos E. Lalli E. Stocco D. Sassone-Corsi P. Nature. 1997; 390: 311-315Google Scholar). On the other hand, multiple binding elements for SF-1 have been reported in the 5′-flanking region of theStAR gene and are required for maximal promoter activity (4Baukal A.J. Hunyady L. Catt K.J. Balla T. J. Biol. Chem. 1994; 269: 24546-24549Google Scholar). Two main conclusions can be drawn from the present work. 1) Ang II, which recruits the calcium messenger signaling system (31Cherradi N. Brandenburger Y. Capponi A.M. Eur. J. Endocrinol. 1998; 139: 249-256Google Scholar), and forskolin, used as a mimicker of ACTH action via cAMP generation, both markedly repressed DAX-1 expression, and 2) forskolin significantly increased the expression of SF-1. It is worth stressing that these observations were obtained in bovine glomerulosa cells in primary culture, expressing normal levels of only endogenous DAX-1 and SF-1, rather than in artificial overexpression systems. The inhibition of DAX-1 exerted by Ang II and forskolin was manifest at both the protein and mRNA levels. In fact, although most studies have focused on the developmental and tissular distribution of DAX-1 expression in various species (
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