CCAAT Enhancer-binding Protein β and GATA-4 Binding Regions within the Promoter of the Steroidogenic Acute Regulatory Protein (StAR) Gene Are Required for Transcription in Rat Ovarian Cells
1999; Elsevier BV; Volume: 274; Issue: 25 Linguagem: Inglês
10.1074/jbc.274.25.17987
ISSN1083-351X
AutoresEran Silverman, Sarah Eimerl, Joseph Orly,
Tópico(s)interferon and immune responses
ResumoSteroidogenic acute regulatory protein (StAR) is a vital accessory protein required for biosynthesis of steroid hormones from cholesterol. The present study shows that in primary granulosa cells from prepubertal rat ovary, StAR transcript and protein are acutely induced by gonadotropin (FSH). To determine the sequence elements required for hormone inducibility of the StAR promoter, truncated regions of the −1002/+6 sequence of the mouse gene were ligated to pCAT-Basic plasmid and transfected by electroporation to freshly prepared cells. FSH inducibility determined over a 6-h incubation was 10–40-fold above basal levels of chloramphenicol acetyltransferase activity. These functional studies, supported by electrophoretic mobility shift assays indicated that two sites were sufficient for transcription of the StAR promoter constructs: a non-consensus binding sequence (−81/−72) for CCAAT enhancer-binding protein β (C/EBPβ) and a consensus motif for GATA-4 binding (−61/−66). Western analyses showed that GATA-4 is constitutively expressed in the granulosa cells, while all isoforms of C/EBPβ were markedly inducible by FSH. Site-directed mutations of both binding sequences practically ablated both basal and hormone-driven chloramphenicol acetyltransferase activities to less than 5% of the parental −96/+6 construct. Unlike earlier notions, elimination of potential binding sites for steroidogenic factor-1, a well known tissue-specific transcription factor, did not impair StAR transcription. Consequently, we propose that C/EBPβ and GATA-4 represent a novel combination of transcription factors capable of conferring an acute response to hormones upon their concomitant binding to the StAR promoter. Steroidogenic acute regulatory protein (StAR) is a vital accessory protein required for biosynthesis of steroid hormones from cholesterol. The present study shows that in primary granulosa cells from prepubertal rat ovary, StAR transcript and protein are acutely induced by gonadotropin (FSH). To determine the sequence elements required for hormone inducibility of the StAR promoter, truncated regions of the −1002/+6 sequence of the mouse gene were ligated to pCAT-Basic plasmid and transfected by electroporation to freshly prepared cells. FSH inducibility determined over a 6-h incubation was 10–40-fold above basal levels of chloramphenicol acetyltransferase activity. These functional studies, supported by electrophoretic mobility shift assays indicated that two sites were sufficient for transcription of the StAR promoter constructs: a non-consensus binding sequence (−81/−72) for CCAAT enhancer-binding protein β (C/EBPβ) and a consensus motif for GATA-4 binding (−61/−66). Western analyses showed that GATA-4 is constitutively expressed in the granulosa cells, while all isoforms of C/EBPβ were markedly inducible by FSH. Site-directed mutations of both binding sequences practically ablated both basal and hormone-driven chloramphenicol acetyltransferase activities to less than 5% of the parental −96/+6 construct. Unlike earlier notions, elimination of potential binding sites for steroidogenic factor-1, a well known tissue-specific transcription factor, did not impair StAR transcription. Consequently, we propose that C/EBPβ and GATA-4 represent a novel combination of transcription factors capable of conferring an acute response to hormones upon their concomitant binding to the StAR promoter. The first and key reaction in the enzymatic cascade of steroid hormone biosynthesis is catalyzed in the mitochondria by cholesterol side chain cleavage cytochrome P450 (P450scc) 1The abbreviations used are: P450scc, cholesterol side chain cleavage cytochrome P450; StAR, steroidogenic acute regulatory protein; SF-1, steroidogenic factor-1; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; RT, reverse transcription; EMSA, electrophoretic mobility shift assay; PEPCK, phosphoenolpyruvate carboxykinase; PAGE, polyacrylamide gel electrophoresis; hCG, human chorionic gonadotropin; PMSG, pregnant mare serum gonadotropin; FSH, follitropin. (1Hall P.F. Steroids. 1986; 48: 131-196Crossref PubMed Scopus (135) Google Scholar, 2Simpson E.R. Waterman M.R. Annu. Rev. Physiol. 1988; 50: 427-440Crossref PubMed Scopus (425) Google Scholar, 3Miller W.L. J. Steroid Biochem. Mol. Biol. 1995; 55: 607-616Crossref PubMed Scopus (105) Google Scholar). In the presence of atmospheric oxygen and reducing power provided by associated proteins, P450scc converts cholesterol substrate to the first steroid prototype molecule, pregnenolone (1Hall P.F. Steroids. 1986; 48: 131-196Crossref PubMed Scopus (135) Google Scholar). In order to do so, a supply of cholesterol is required to be transferred from cytosolic pools into the inner membranes of the mitochondrion, where P450scc resides (4Simpson E.R. McCarthy J.L. Peterson J.A. J. Biol. Chem. 1978; 253: 3135-3139Abstract Full Text PDF PubMed Google Scholar, 5Crivello J.F. Jefcoate C.R. J. Biol. Chem. 1980; 255: 8144-8151Abstract Full Text PDF PubMed Google Scholar, 6Jefcoate C.R. DiBartolomeis M.J. Williams C.A. McNamara B.C. J. Steroid Biochem. 1987; 27: 721-729Crossref PubMed Scopus (100) Google Scholar, 7Stevens V.L. Xu T. Lambeth J.D. Eur. J. Biochem. 1993; 216: 557-563Crossref PubMed Scopus (28) Google Scholar). Recently, it was found that cholesterol delivery into the mitochondria is enhanced by a novel protein (8Clark B.J. Wells J. King S.R. Stocco D.M. J. Biol. Chem. 1994; 269: 28314-28322Abstract Full Text PDF PubMed Google Scholar, 9Wang X. Liu Z. Eimerl S. Timberg R. Weiss A.M. Orly J. Stocco D.M. Endocrinology. 1998; 139: 3903-3912Crossref PubMed Scopus (0) Google Scholar) designated steroidogenic acute regulatory (StAR) protein (reviewed in Refs.10Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-244Crossref PubMed Scopus (942) Google Scholar, 11Stocco D.M. Biol. Reprod. 1997; 56: 328-336Crossref PubMed Scopus (81) Google Scholar, 12Stocco D.M. Clark B.J. Steroids. 1997; 62: 29-36Crossref PubMed Scopus (91) Google Scholar). More studies have established the fact that StAR is a vital protein essential for steroidogenesis in the adrenal cortex and the gonads (13Lin D. Sugawara T. Strauss III, J.F. Clark B.J. Stocco D.M. Saenger P. Rogol A. Miller W.L. Science. 1995; 267: 1828-1831Crossref PubMed Scopus (874) Google Scholar, 14Caron K.M. Soo S.C. Wetsel W.C. Stocco D.M. Clark B.J. Parker K.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11540-11545Crossref PubMed Scopus (387) Google Scholar). In rodents, StAR is also expressed in steroidogenic brain cells (15Furukawa A. Miyatake A. Ohnishi T. Ichikawa Y. J. Neurochem. 1998; 71: 2231-2238Crossref PubMed Scopus (205) Google Scholar) and placenta. 2Y. Arensburg and J. Orly, unpublished data. Interestingly, StAR is not expressed in human placenta, where its role is probably assumed by a less efficient StAR substitute called MLN64 (16Watari H. Arakane F. Moog L.C. Kallen C.B. Tomasetto C. Gerton G.L. Rio M.C. Baker M.E. Strauss III, J.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8462-8467Crossref PubMed Scopus (205) Google Scholar). Perhaps the most compelling evidence for the critical role of StAR in steroidogenesis was the discovery that various mutations of the StAR gene encoding a functionally impaired protein (14Caron K.M. Soo S.C. Wetsel W.C. Stocco D.M. Clark B.J. Parker K.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11540-11545Crossref PubMed Scopus (387) Google Scholar) cause a syndrome known as lipoid congenital adrenal hyperplasia (17Bose H.S. Sugawara T. Strauss III, J.F. Miller W.L. N. Engl. J. Med. 1996; 335: 1870-1878Crossref PubMed Scopus (534) Google Scholar). Affected individuals die shortly after birth in the absence of adrenal steroids, unless treated with steroid hormone replacement therapy. Similar patterns were also observed in StAR null mice (14Caron K.M. Soo S.C. Wetsel W.C. Stocco D.M. Clark B.J. Parker K.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11540-11545Crossref PubMed Scopus (387) Google Scholar). Trophic hormones, such as gonadotropins and ACTH, trigger up-regulation of StAR expression by cAMP signaling (18Pon L.A. Hartigan J.A. Orme-Johnson N.R. J. Biol. Chem. 1986; 261: 13309-13316Abstract Full Text PDF PubMed Google Scholar, 19Stocco D.M. Sodeman T.C. J. Biol. Chem. 1991; 266: 19731-19738Abstract Full Text PDF PubMed Google Scholar, 20Clark B.J. Soo S.C. Caron K.M. Ikeda Y. Parker K.L. Stocco D.M. Mol. Endocrinol. 1995; 9: 1346-1355Crossref PubMed Google Scholar). Additionally, Ca2+ changes evoke StAR expression in glomerulosa cells of the adrenal cortex (21Cherradi 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: 7899-7907Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 22Cherradi N. Brandenburger Y. Rossier M.F. Vallotton M.B. Stocco D.M. Capponi A.M. Mol. Endocrinol. 1998; 12: 962-972Crossref PubMed Scopus (46) Google Scholar). Very little is known about the factors controlling StAR expression at the transcriptional level, downstream to the signal transduction pathways. Special attention has been devoted to examine the potential involvement of the steroidogenic factor-1 (SF-1, or Ad4BP), which is a pivotal tissue-specific orphan nuclear receptor essential for regulation of many steroid hydroxylases in steroid-producing tissues (23Parker K.L. Schimmer B.P. Endocr. Rev. 1997; 18: 361-377Crossref PubMed Scopus (556) Google Scholar, 24Honda S. Morohashi K. Nomura M. Takeya H. Kitajima M. Omura T. J. Biol. Chem. 1993; 268: 7494-7502Abstract Full Text PDF PubMed Google Scholar). In light of the fact that StAR promoter includes several putative recognition sites for SF-1 binding, several attempts have be made to determine if the latter factor is involved in StAR regulation. At present, the available results are somewhat inconsistent. Using the human, mouse, and rat promoters, an apparent up-regulation of StAR transcription by SF-1 could be demonstrated upon co-transfection of SF-1 cDNA and promoter-reporter plasmids in non-steroidogenic cells (25Sugawara T. Lin D. Holt J.A. Martin K.O. Javitt N.B. Miller W.L. Strauss III, J.F. Biochemistry. 1995; 34: 12506-12512Crossref PubMed Scopus (197) Google Scholar, 26Sugawara T. Holt J.A. Kiriakidou M. Strauss III, J.F. Biochemistry. 1996; 35: 9052-9059Crossref PubMed Scopus (241) Google Scholar, 27Sugawara T. Kiriakidou M. McAllister J.M. Kallen C.B. Strauss III, J.F. Biochemistry. 1997; 36: 7249-7255Crossref PubMed Scopus (153) Google Scholar, 28Sandhoff T.W. Hales D.B. Hales K.H. McLean M.P. Endocrinology. 1998; 139: 4820-4831Crossref PubMed Scopus (141) Google Scholar, 29Christenson L.K. McAllister J.M. Martin K.O. Javitt N.B. Osborne T.F. Strauss III, J.F. J. Biol. Chem. 1998; 273: 30729-30735Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, other studies analyzing the activity of StAR promoter in SF-1-expressing cells did not support a role for SF-1 in a cAMP-inducible fashion (30Caron K.M. Ikeda Y. Soo S.C. Stocco D.M. Parker K.L. Clark B.J. Mol. Endocrinol. 1997; 11: 138-147Crossref PubMed Scopus (223) Google Scholar). These data suggested that SF-1 may not confer cAMP responsiveness in authentic steroidogenic cells and, therefore, cannot be an exclusive transcription factor controlling the acute regulation of StAR in such cells. In search for alternative regulatory elements that can mediate the acute response of StAR to hormones, we undertook to study the inducibility of the mouse promoter in ovarian granulosa cells from prepubertal rats. Earlier studies have unambiguously shown that endogenous SF-1 in these cells is critical for the induction of P450scc and P450aromatase by follicle-stimulating hormone (FSH) (31Fitzpatrick S.L. Richards J.S. Mol. Endocrinol. 1993; 7: 341-354PubMed Google Scholar, 32Clemens J.W. Lala D.S. Parker K.L. Richards J.S. Endocrinology. 1994; 134: 1499-1508Crossref PubMed Scopus (152) Google Scholar, 33Carlone D.L. Richards J.S. Mol. Endocrinol. 1997; 11: 292-304PubMed Google Scholar, 34Liu Z. Simpson E.R. Mol. Endocrinol. 1997; 11: 127-137Crossref PubMed Scopus (131) Google Scholar). In contrast, the present study suggests that SF-1 is probably not involved in hormonal activation of StAR promoter. We also demonstrate that promoter regions capable of C/EBPβ and GATA-4 binding are required for activation of StAR transcription in FSH-treated cells. Thus, StAR provides the first example of a steroidogenesis-associated protein that is transcriptionally controlled by C/EBPβ and/or GATA-4. Ovine FSH (NIDDK-oFSH-20) was kindly provided by the National Institute of Health NIAMD (Bethesda, MD.). Acetyl-CoA, poly(dI-dC), RNase A, indomethacin, proteinase K, sodium orthovanadate, aprotinin, NaF, pepstatin, phenylmethylsulfonyl fluoride, peroxidase-conjugated goat anti-rabbit and peroxidase-conjugated rabbit anti-goat sera were obtained from Sigma. Dulbecco's modified Eagle's medium and Ham's F-12 medium were from Grand Island Biological, New York. Polyclonal antisera to C/EBPβ (sc-150x), C/EBPα (sc-61x), Sp1 (sc-059x), GATA-4 (sc-1237x), GATA-6 (sc-7244x), c-Fos (sc-253x), and c-Jun/AP-1 (sc-44x) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). SF-1 antibody was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Intact, immature female Sprague-Dawley rats (21 days old) were obtained from Harlan (Jerusalem, Israel) and maintained under 16:8 light:dark schedule with food and water ad libitum. Animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All protocols had the approval of the Institutional Committee on Animal Care and Use, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem. Naive granulosa cells for CAT assays were expressed form E2-primed rats (35Orly J. Clemens J.W. Singer O. Richards J.S. Biol. Reprod. 1996; 54: 208-218Crossref PubMed Scopus (27) Google Scholar). Preovulatory PMSG-hCG-treated ovaries were prepared by administration of 15 IU of PMSG (PMSG 600, Intervet, Angers, France) to 25-day-old rats, which were further treated with 4 IU of human chorionic gonadotropin (hCG, Organon Special Chemicals, West Orange, NJ) administered (subcutaneously) 50 h later. The animals were sacrificed at 8 h after hCG, and ovaries were retrieved for protein extraction. Also, post-ovulatory ovaries enriched with corpora lutea were similarly harvested 72 h after onset of PMSG-hCG treatment. To obtain granulosa cells expressing low basal levels of CAT, we used a modification of a previously described method (35Orly J. Clemens J.W. Singer O. Richards J.S. Biol. Reprod. 1996; 54: 208-218Crossref PubMed Scopus (27) Google Scholar). Briefly, after incubation in hypertonic sucrose/EGTA-containing medium, the ovaries were incubated for additional 45 min in Dulbecco's modified Eagle's medium/F-12 medium containing 10 μm indomethacin. The same medium also served for further procedures, including needle pricking of the ovaries and post-electroporation treatments. After electroporation the cells were plated onto serum-coated wells (35Orly J. Clemens J.W. Singer O. Richards J.S. Biol. Reprod. 1996; 54: 208-218Crossref PubMed Scopus (27) Google Scholar) (24-well plated; Nunc, Copenhagen, Denmark) and incubated at 37 °C in 95% air and 5% CO2. Estradiol-primed granulosa cells (4 × 105) obtained from 3–4 ovaries were electroporated in the presence of 20 μg of DNA as described before (35Orly J. Clemens J.W. Singer O. Richards J.S. Biol. Reprod. 1996; 54: 208-218Crossref PubMed Scopus (27) Google Scholar). Cells from each cuvette were seeded into four wells and hormonal treatments were initiated after a 3-h recovery period. Following a 6-h treatment with FSH (100 ng/ml), cell lysates were prepared and CAT activity was analyzed as described previously (35Orly J. Clemens J.W. Singer O. Richards J.S. Biol. Reprod. 1996; 54: 208-218Crossref PubMed Scopus (27) Google Scholar). Quantitation of the CAT assay was performed using a Fuji Bio-Imaging analyzer (BAS-1000, Fuji Photo Film, Tokyo, Japan). Protein was determined by a modified method of Bradford (36Zor T. Selinger Z. Anal. Biochem. 1996; 236: 302-308Crossref PubMed Scopus (888) Google Scholar) using the Bio-Rad protein assay. Data are presented as percent of [14C]chloramphenicol (Amersham International, Little Chalfont, United Kingdom) converted to its acetylated products (per protein and time of assay) and the -fold induction of CAT activity over basal values measured in the absence of hormone. Data are presented as the mean ± S.E. of several independent transfections as indicated in each figure. Whole ovarian extracts for EMSA was performed as described before (37Welte T. Garimorth K. Philipp S. Doppler W. Mol. Endocrinol. 1994; 8: 1091-1102PubMed Google Scholar). Briefly, ovaries were homogenized in a Dounce homogenizer using 2–3 volumes of buffer A containing 400 mm KCl, 10 mmNaH2PO4, (pH 7.4), 10% glycerol, 1 mm EDTA, 1 mm dithiothreitol, 5 μm NaF, 1 mm sodium orthovanadate, 5 μg/ml aprotinin, 2 μm pepstatin, and 1 mmphenylmethylsulfonyl fluoride. Following homogenization, the protein slurry was freeze-thawed three times in liquid nitrogen and finally centrifuged for 2 min at 14,000 × g. After determination of the protein content, the supernatant was aliquoted and kept at −70 °C until use. Electrophoretic mobility shift assay (EMSA) was performed as described before (37Welte T. Garimorth K. Philipp S. Doppler W. Mol. Endocrinol. 1994; 8: 1091-1102PubMed Google Scholar). Briefly, whole cell extracts (3–15 μg) were incubated with 2 ng of double-stranded DNA, previously labeled by a fill-in reaction using Klenow fragment (Promega, Madison, WI) and [α-32P]-dCTP (Amersham International, Little Chalfont, United Kingdom). Incubation was performed using a final volume of 30 μl of buffer containing 100 mm KCl, 15 mm Tris-HCl (pH 7.5), 10 mm dithiothreitol, 1 mm EDTA, 5 mmMgCl2, 12% glycerol, and 4.5 μg of poly(dI-dC). After incubation for 35 min at room temperature, the binding reactions were resolved on pre-run 5% acrylamide gel as described previously for quantitative RT-PCR analyses (38Orly J. Rei Z. Greenberg N.M. Richards J.S. Endocrinology. 1994; 134: 2336-2346Crossref PubMed Scopus (124) Google Scholar). When competition experiments were conducted in the presence of molar excess of cold probe, the protein extracts were added last to the reaction mixture. When antibodies were used for supershift (or ablation) of a given protein-DNA complex, the protein extracts were preincubated for 25 min at room temperature with 2–8 μg of the antiserum, prior to the addition of the DNA-labeled probe. The following oligonucleotide probes used for EMSA included overhanging restriction site sequences: SCC1(SF-1) (32Clemens J.W. Lala D.S. Parker K.L. Richards J.S. Endocrinology. 1994; 134: 1499-1508Crossref PubMed Scopus (152) Google Scholar) (upper strand, 5′-GATCGCCCTCTCTTAGCCTTGAGCTA GTTA); consensus Sp1 (upper strand, 5′-GATCCGATCGGGGCGGGGCGAGC); −148/−127 StAR (upper strand, 5′-TGCTCCCTCCCACCTTGGCCAG); −148/−127mut2Sp1 (upper strand, 5′-TGCTCCCTCtgACCTTGGCCAG); −87/−70 StAR (upper strand, 5′-GGCCAAGCTTGCACAATGACTGATGACT); and −73/42 StAR (upper strand, 5′-GGCCAAGCTTGACTTTTTTATCTCAAGTGATGATGCACAGCC). A previously published sequence of the 5′-flanking region of the mouse StAR gene (30Caron K.M. Ikeda Y. Soo S.C. Stocco D.M. Parker K.L. Clark B.J. Mol. Endocrinol. 1997; 11: 138-147Crossref PubMed Scopus (223) Google Scholar), was used to clone most of the StAR promoter constructs by a PCR-based approach. 5′-HindIII and 3′-XbaI cloning sites were included in all forward and reverse primers, respectively. To generate the −1002/+6 DNA fragment, the oligonucleotide sequence −1002/−982 (5′-GGCCAAGCTTTTCTAAGGTTCCCTGGATCT) and −14/+6 (5′-GGCCTCTAGAAGCTGTGGCGCAGATCAAGT) were included in the PCR reaction (38Orly J. Rei Z. Greenberg N.M. Richards J.S. Endocrinology. 1994; 134: 2336-2346Crossref PubMed Scopus (124) Google Scholar) using mouse genomic DNA (prepared as described in Ref. 39Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols In Molecular Biology. 1st Ed. Greene Publishing Associates/Wiley-Interscience, New York1988Google Scholar), as template. The PCR product was digested with HindIII andXbaI (New England Biolabs) and ligated (T4-DNA ligase; Roche Molecular Biochemicals, Mannheim, Germany) into the HindIII and XbaI sites of a promoterless pCAT-Basic vector (Promega, Madison, WI). The resulting construct was designated −1002StAR. Two deletion constructs were generated from −1002StAR by StuI and AccI (New England Biolabs, Beverly, MA) to generate the −823/+6 (−823StAR) and −257/+6 (−257StAR) constructs, respectively. Further progressive deletions of the promoter constructs were prepared using the −14/+6 reverse primer and the appropriate 5′- forward primers: −152StAR (5′-GGCCAAGCTTAGTCTGCTCCCTCCCACCTTGGCCAGCACT); −123StAR (5′-GGCCAAGCTTTGCAGGATGAGGCAATCATTCCAT); −96StAR (5′-GGCCAAGCTTTGACCCTCTGCACAATGACTGA); −73StAR (the same oligonucleotide sequence used for the EMSA probe, −73/−42StAR); −51StAR (5′-GGCCAAGCTTATGCACAGCCTTCCACGG). To generate constructs with point mutations, oligonucleotides containing the point mutations of choice were used for the PCR reaction as the forward primers, using the −14/+6 as the reverse primer (unless stated otherwise): −152mutSF-1 (5′- GGCCAAGCTTAGTCTGCTCCCTCCCAtaTTGGCCAGCACT); −152mut1"Sp-1" (5′-GGCCAAGCTTAGTCTGCTCCCTCtgACCTTGGCCAGCACT); −152mut2"Sp-1" (5′-GGCCAAGCTTAGTCTGCTCCCTggCACCTTGGCCAGCACT); −123mutC/EBPβ-2 (5′-GGCCAAGCTTTGCAGGATGAGtcccaCATTCCAT); −96mutβ −2(5′-GGCCAAGCTTTGACCCTCTCtcccaGACTGAT); −96mutβ-3 (5′-GGCCAAGCTTTGACCCTCTGCACAATGAtctgTGACTT; −96mutGATA (5′-GGCCAAGCTTTGACCCTCTGCACAATGACTGATGACTTTTTTAagTCAAGTG); −73mutGATA (5′- GGCCAAGCTTGACTTTTTTAagTCAAGTGATGATGCACAGCC); The −96rStAR construct was built using the −96StAR primer as the forward primer, and the −53/+6mutStAR (5′-GGCCTCTAGATGATctcgacgtccaggacgcAAGCATTTAAGGCAGAGCACTTGATCTGCGCCACAGCT) as the reverse primer. The double mutant construct −96doublemut was generated using the −96mutβ-3 as the forward primer, the −14/+6 oligonucleotide as the reverse primer, and the construct −96mutGATA as the template. PCR reaction (total volume of 100 μl) consisted of 30 cycles at 94 °C (1 min), 60–65 °C (2 min), and 72 °C (3 min) (38Orly J. Rei Z. Greenberg N.M. Richards J.S. Endocrinology. 1994; 134: 2336-2346Crossref PubMed Scopus (124) Google Scholar). At the indicated time points, granulosa cells were extracted by lysis buffer (RIPA) and analyzed by SDS-PAGE and electro-blotting procedures as described previously (40Ronen F.T. Timberg R. King S.R. Hales K.H. Hales D.B. Stocco D.M. Orly J. Endocrinology. 1998; 139: 303-315Crossref PubMed Scopus (165) Google Scholar). After a 1-h incubation with anti-C/EBPβ (1:2000) or anti-GATA-4 (1:4000), the nitrocellulose membranes were washed and further incubated for 1 h with the appropriate peroxidase-conjugated antibodies (1:10,000 dilution). Specific signals were detected by chemiluminescence utilizing the LumiGlo substrate (New England Biolabs). Quantitation of chemiluminescence signals on x-ray films was performed as described previously (40Ronen F.T. Timberg R. King S.R. Hales K.H. Hales D.B. Stocco D.M. Orly J. Endocrinology. 1998; 139: 303-315Crossref PubMed Scopus (165) Google Scholar). Total RNA was extracted by dissolving the granulosa cells in 0.5 ml of RNAzol B (Tel-Test, Inc., Friendwood, TX) added to each culture well (16 mm). Further steps followed the manufacturer's instructions. Semiquantitative RT-PCR analysis of total RNA extracts from granulosa cells was performed exactly as described previously (40Ronen F.T. Timberg R. King S.R. Hales K.H. Hales D.B. Stocco D.M. Orly J. Endocrinology. 1998; 139: 303-315Crossref PubMed Scopus (165) Google Scholar). Student's unpaired two-tailedt test was performed using Microsoft Excel 97 statistical analysis functions. Differences between the activities of the indicated constructs were considered statistically significant atp < 0.05. Aiming to identify the regulatory elements controlling StAR expression, we have applied transient expression assays of the mouse StAR promoter by use of granulosa cells from prepubertal rat ovary. To this end, a −1002 to +6 fragment of the StAR gene was cloned by PCR and ligated to a promoterless pCAT-Basic plasmid. We reasoned that the expression of the mouse promoter in rat cells is justified by the fact that the proximal regions of the rat and mouse promoters are almost identical, in particular through the first 150 base pairs upstream to the transcription start site (Fig. 1). Testing the hormonal inducibility of the promoter constructs was performed following a 6-h incubation with FSH added shortly after transfection by electroporation. Semiquantitative RT-PCR and Western blot analyses confirmed that under similar experimental conditions the levels of StAR mRNA and protein rise acutely upon the addition of FSH (Fig. 2).Figure 2Time-dependent rise of StAR mRNA and protein induced by FSH . Freshly prepared granulosa cells were seeded to culture and 3 h later, FSH was added (100 ng/ml). At the indicated time points, duplicate wells, were harvested with either RNAzol B or lysis buffer (see "Experimental Procedures"). A, RT-PCR was performed to determine the levels of StAR mRNA as described under "Experimental Procedures." The presented autoradiogram depicts the amplified PCR signals obtained for StAR and the ribosomal protein L19 mRNAs.B, Western blot analysis was performed, and the enhanced chemiluminescence reaction depicts the mitochondrial 30-kDa StAR protein. The lower panel presents the time dependent increase of StAR transcript and protein. Quantitation of StAR mRNA and protein was performed as described under "Experimental Procedures."View Large Image Figure ViewerDownload (PPT) At large, the activity values obtained by transfecting a series of progressive deletions of the promoter showed that hormone inducibility remained high in all constructs pruned down to −96/+6 (Fig.3). The latter region exhibited the highest -fold induction by FSH (44-fold), suggesting that two potential upstream binding sites for SF-1 (−139/−132 and −102/−95), are not necessarily required for the FSH activation of the promoter. These results did not agree with earlier reports, which strongly advocated the notion that SF-1 is implicated in regulation of StAR expression (26Sugawara T. Holt J.A. Kiriakidou M. Strauss III, J.F. Biochemistry. 1996; 35: 9052-9059Crossref PubMed Scopus (241) Google Scholar, 27Sugawara T. Kiriakidou M. McAllister J.M. Kallen C.B. Strauss III, J.F. Biochemistry. 1997; 36: 7249-7255Crossref PubMed Scopus (153) Google Scholar, 28Sandhoff T.W. Hales D.B. Hales K.H. McLean M.P. Endocrinology. 1998; 139: 4820-4831Crossref PubMed Scopus (141) Google Scholar, 30Caron K.M. Ikeda Y. Soo S.C. Stocco D.M. Parker K.L. Clark B.J. Mol. Endocrinol. 1997; 11: 138-147Crossref PubMed Scopus (223) Google Scholar, 41Sugawara T. Kiriakidou M. McAllister J.M. Holt J.A. Arakane F. Strauss III, J.F. Steroids. 1997; 62: 5-9Crossref PubMed Scopus (88) Google Scholar). This inconsistency, together with the fact that deletion of the −139/−132 SF-1 site significantly reduced the basal activity of −152StAR (Fig. 3), urged us to cautiously reassess the importance of this element by site-directed mutations and EMSAs. To our surprise, SF-1 did not bind to a −148/−127 probe (Fig.4), previously shown to be capable of SF-1 binding using extracts of Y-1 adrenocortical cell line (30Caron K.M. Ikeda Y. Soo S.C. Stocco D.M. Parker K.L. Clark B.J. Mol. Endocrinol. 1997; 11: 138-147Crossref PubMed Scopus (223) Google Scholar). Instead, the rat cell extracts generated a slower migrating protein complex, which was not affected by antiserum to SF-1 (Fig.4 A, lanes 2 and 4). A closer examination of this sequence revealed a potential Sp1 site, which is overlapping the SF-1 binding element to create an "Sp1"/SF-1 motif (see Fig.5 B, probe 2). This G-rich element (−146, 5′-TGGGAGGGAG, lower strand) is nearly identical to an Sp1-like binding sequence previously reported to be involved in cAMP-dependent regulation of the bovine P450scc transcription (34Liu Z. Simpson E.R. Mol. Endocrinol. 1997; 11: 127-137Crossref PubMed Scopus (131) Google Scholar, 42Venepally P. Waterman M.R. J. Biol. Chem. 1995; 270: 25402-25410Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). In StAR promoter, this Sp1-like site, termed "Sp1," binds a protein that is antigenically cross-reactive with Sp1 antiserum (Fig. 4 B, lanes 6and 8). Moreover, molar excess of Sp1 consensus DNA can compete for the binding of "Sp1" to its site in StAR promoter (Fig.5 A, lanes 4 and 5). Finally, a site-directed mutation replacing GG with ca (Fig.5 B, lane 16) resulted in the loss of "Sp1" band shift and rendered the SF-1 site available for a typical SF-1 binding (Fig. 5 B, lane 16). Noteworthy, the "Sp1"/SF-1 element could bind both proteins, providing the extracts were prepared from the mouse MA-10 cells (Fig.5 B, lane 14), which are highly enriched with SF-1 content. These results suggest that the −148/−127 region has a dual capacity to bind both "Sp1"and SF-1, which compete with each other depending on their relative content in a given cell type.Figure 4SF-1 does not bind the −148/−127 StAR promoter region. Extracts prepared from PMSG/hCG-treated rats (see "Experimental Procedures") were used for the following electrophoretic mobility shift assays (EMSAs): A, antiserum to SF-1 (4 μg) was added to protein extracts for a 25 min preincubation period prior to addition of either a32P-labeled −148/−127 probe (lane 2), or a positive control SF-1 probe, design
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