Stimulation of Luteinizing Hormone β Gene Promoter Activity by the Orphan Nuclear Receptor, Steroidogenic Factor-1
1996; Elsevier BV; Volume: 271; Issue: 12 Linguagem: Inglês
10.1074/jbc.271.12.6645
ISSN1083-351X
AutoresLisa M. Halvorson, Ursula B. Kaiser, William W. Chin,
Tópico(s)Sexual Differentiation and Disorders
ResumoThe orphan nuclear receptor, steroidogenic factor-1 (SF-1), is expressed in the pituitary and in the gonadotrope precursor cell line, αT3-1, where it is believed to enhance expression of the common gonadotropin α-subunit gene through transactivation of the gonadotrope-specific element (GSE). Sequence analysis of the rat luteinizing hormone β-subunit (LHβ) gene promoter revealed the presence of a consensus GSE at −127 to −119 (TGACCTTGT). We have demonstrated the ability of SF-1 to bind specifically to this putative GSE sequence by electrophoretic mobility shift assay, utilizing both αT3-1 nuclear extracts and in vitro translated SF-1. In addition, mutation of the putative LHβ-GSE (TGAAATTGT) eliminated specific DNA binding. To examine the ability of SF-1 to enhance LHβ promoter activity, CV-1 cells, which lack endogenous SF-1, were cotransfected with an SF-1-containing expression vector and an LHβ-luciferase reporter construct. When cotransfected with −209/+5 of the LHβ promoter, SF-1 increased luciferase activity by 56-fold. SF-1 responsiveness was markedly diminished with loss of the putative GSE region in deletion constructs and in the presence of a two base pair mutation, analogous to the mutation which eliminated DNA binding. Finally, the LHβ-GSE was able to confer SF-1 responsiveness on a heterologous minimal growth hormone promoter, GH50 (57-fold). We conclude that SF-1 both binds to and transactivates the rat LHβ promoter. These data suggest that SF-1 may participate in the expression of the LHβ gene by the gonadotrope. The orphan nuclear receptor, steroidogenic factor-1 (SF-1), is expressed in the pituitary and in the gonadotrope precursor cell line, αT3-1, where it is believed to enhance expression of the common gonadotropin α-subunit gene through transactivation of the gonadotrope-specific element (GSE). Sequence analysis of the rat luteinizing hormone β-subunit (LHβ) gene promoter revealed the presence of a consensus GSE at −127 to −119 (TGACCTTGT). We have demonstrated the ability of SF-1 to bind specifically to this putative GSE sequence by electrophoretic mobility shift assay, utilizing both αT3-1 nuclear extracts and in vitro translated SF-1. In addition, mutation of the putative LHβ-GSE (TGAAATTGT) eliminated specific DNA binding. To examine the ability of SF-1 to enhance LHβ promoter activity, CV-1 cells, which lack endogenous SF-1, were cotransfected with an SF-1-containing expression vector and an LHβ-luciferase reporter construct. When cotransfected with −209/+5 of the LHβ promoter, SF-1 increased luciferase activity by 56-fold. SF-1 responsiveness was markedly diminished with loss of the putative GSE region in deletion constructs and in the presence of a two base pair mutation, analogous to the mutation which eliminated DNA binding. Finally, the LHβ-GSE was able to confer SF-1 responsiveness on a heterologous minimal growth hormone promoter, GH50 (57-fold). We conclude that SF-1 both binds to and transactivates the rat LHβ promoter. These data suggest that SF-1 may participate in the expression of the LHβ gene by the gonadotrope. INTRODUCTIONThe pituitary gonadotropins, luteinizing hormone and follicle-stimulating hormone, are critical modulators of gamete maturation and gonadal steroidogenesis. These hormones are composed of a common α-subunit linked noncovalently to unique β-subunits which specify physiologic actions(1.Gharib S.D. Wierman M.E. Shupnik M.A. Chin W.W. Endocr. Rev. 1990; 11: 177-199Crossref PubMed Scopus (601) Google Scholar).Several DNA regulatory elements have been defined for the α-subunit gene promoter. GnRH 1The abbreviations used are: GnRHgonadotropin-releasing hormoneCREcAMP response elementGSEgonadotrope-specific elementSF-1steroidogenic factor-1LHβluteinizing hormone β-subunitFSHβfollicle-stimulating hormone β-subunitEMSAelectrophoretic mobility shift assayRSVRous sarcoma virus. -stimulated expression is believed to be mediated through a regulatory element located between positions −346 and −244 in the human α-subunit gene promoter, a region separate from those involved in basal and cAMP-stimulated expression(2.Kay T.W.H. Jameson J.L. Mol. Endocrinol. 1992; 6: 1767-1773PubMed Google Scholar). Activation of a cAMP response element (CRE) appears to be important for expression in both pituitary and placental cell types, while placental-specific expression occurs through the activation of a trophoblast-specific element acting in concert with the CRE(3.Delegeane A.M. Ferland L.H. Mellon P.L. Mol. Cell. Biol. 1987; 7: 3994-4002Crossref PubMed Scopus (281) Google Scholar). Pituitary-specific expression of the α-subunit gene has been attributed to the presence of both a pituitary glycoprotein basal element and a gonadotrope-specific element (GSE)(4.Horn F. Windle J.J. Barnhart K.M. Mellon P.L. Mol. Cell. Biol. 1992; 12: 2143-2153Crossref PubMed Google Scholar, 5.Schoderbek W.E. Kim K.E. Ridgway E.C. Mellon P.L. Maurer R.A. Mol. Endocrinol. 1992; 6: 893-903PubMed Google Scholar).The consensus GSE sequence (TGACCTTGT), defined in the common α-subunit by Mellon and colleagues, resembles a nuclear receptor binding half-site(4.Horn F. Windle J.J. Barnhart K.M. Mellon P.L. Mol. Cell. Biol. 1992; 12: 2143-2153Crossref PubMed Google Scholar, 6.Barnhart K.M. Mellon P.L. Mol. Endocrinol. 1994; 8: 878-885Crossref PubMed Scopus (204) Google Scholar). Variations of this sequence, alternatively called the Ad4 response element, are also present in the promoter regions of multiple genes which play a role in steroidogenesis, sexual differentiation, and adult reproductive function(7.Ingraham H.A. Lala D.S. Ikeda Y. Luo X. Shen W.-H. Nachtigal M.W. Abbud R. Nilson J.H. Parker K.L. Genes & Dev. 1994; 8: 2302-2312Crossref PubMed Scopus (510) Google Scholar). The GSE/Ad4 element has been shown to interact with the transcription factor, steroidogenic factor-1 (SF-1), in a number of genes, including the steroidogenic P450, the aromatase, and the Müllerian inhibiting substance genes(8.Rice D.A. Mouw A.R. Bogerd A.M. Parker K.L. Mol. Endocrinol. 1991; 5: 1552-1561Crossref PubMed Scopus (220) Google Scholar, 9.Lynch J.P. Lala D.S. Peluso J.J. Luo W. Parker K.L. White B.A. Mol. Endocrinol. 1993; 7: 776-786Crossref PubMed Scopus (213) Google Scholar, 10.Shen W.-H. Moore C.C.D. Ikeda Y. Parker K.L. Ingraham H.A. Cell. 1994; 77: 651-661Abstract Full Text PDF PubMed Scopus (482) Google Scholar). SF-1 is an orphan member of the nuclear hormone receptor superfamily. Best known for its selective expression in adrenal and gonadal cells, it has more recently been identified in the pituitary gland with localization to the gonadotrope(6.Barnhart K.M. Mellon P.L. Mol. Endocrinol. 1994; 8: 878-885Crossref PubMed Scopus (204) Google Scholar, 7.Ingraham H.A. Lala D.S. Ikeda Y. Luo X. Shen W.-H. Nachtigal M.W. Abbud R. Nilson J.H. Parker K.L. Genes & Dev. 1994; 8: 2302-2312Crossref PubMed Scopus (510) Google Scholar).In studies of the human α-subunit gene promoter, SF-1 has been shown to bind to the GSE region by electrophoretic gel mobility shift assay. Furthermore, reporter constructs which contain the α-subunit GSE site are expressed at higher levels in cell lines which contain endogenous SF-1 than in those cells which lack SF-1, consistent with a role for SF-1 in tissue-specific transcriptional activation of the α-subunit gene(4.Horn F. Windle J.J. Barnhart K.M. Mellon P.L. Mol. Cell. Biol. 1992; 12: 2143-2153Crossref PubMed Google Scholar, 6.Barnhart K.M. Mellon P.L. Mol. Endocrinol. 1994; 8: 878-885Crossref PubMed Scopus (204) Google Scholar).In contrast with the α-subunit, the cis-acting elements responsible for expression of either the LHβ- or FSHβ-subunit mRNAs are poorly understood. Interestingly, transgenic mice null for the gene which encodes SF-1 not only express the α-subunit in low levels, but also fail to express the β-subunits, suggesting a functional role for SF-1 in LHβ gene expression(7.Ingraham H.A. Lala D.S. Ikeda Y. Luo X. Shen W.-H. Nachtigal M.W. Abbud R. Nilson J.H. Parker K.L. Genes & Dev. 1994; 8: 2302-2312Crossref PubMed Scopus (510) Google Scholar). As previous studies of the bovine LHβ gene promoter have shown that the proximal 776 base pairs are sufficient to direct pituitary-specific expression in transgenic mice (11.Keri R.A. Wolfe M.W. Saunders T.L. Anderson I. Kendall S.K. Wagner T. Yeung J. Gorski J. Nett T.M. Camper S.A. Nilson J.H. Endocrinology. 1994; 8: 1807-1816Google Scholar), we analyzed the corresponding region of the rat LHβ promoter for the presence of an SF-1-binding site, or GSE.The rat LHβ gene promoter contains a consensus GSE at position −127 to −119 relative to the transcriptional start site (Fig. 1). Interestingly, this sequence is highly conserved across species among the LHβ genes, suggesting physiologic significance(11.Keri R.A. Wolfe M.W. Saunders T.L. Anderson I. Kendall S.K. Wagner T. Yeung J. Gorski J. Nett T.M. Camper S.A. Nilson J.H. Endocrinology. 1994; 8: 1807-1816Google Scholar, 14.Brown P. McNeilly J.R. Wallace R.M. McNeilly A.S. Clark A.J. Mol. Cell. Endocrinol. 1993; 93: 157-165Crossref PubMed Scopus (34) Google Scholar). Inasmuch as the consensus GSE sequence is present in the LHβ gene promoter, we wished to determine whether this putative GSE region has functional significance. We, therefore, investigated the ability of SF-1 to bind to and transactivate the rat LHβ gene promoter.MATERIALS AND METHODSOligonucleotides Used in Electrophoretic Mobility Shift Assay (EMSA)The nucleotide sequence of the rat LHβ gene promoter was based on Fig. 3 of Jameson et al.(12.Jameson J.L. Chin W.W. Hollenberg A.N. Chang A.S. Habener J.F. J. Biol. Chem. 1984; 259: 15474-15480Abstract Full Text PDF PubMed Google Scholar) with position −1 assigned to the nucleotide immediately 5′ to the transcriptional start site. The LHSF oligonucleotide used in EMSA corresponds to bases −134 to −113 of the rat LHβ gene (sense strand: 5′-TCCTTTCTGACCTTGTCTGTCT-3′). The LHSFM oligonucleotide sequence is identical to LHSF except for the conversion of the CC nucleotide pair at positions −124 and −123 to an AA pair (sense strand: 5′-TCCTTTCTGAAATTGTCTGTCT-3′). The Pit-1 oligonucleotide used in competition studies corresponds to −137 to −65 of the rat growth hormone promoter and contains two Pit-1/growth hormone factor-1 binding sites (sense strand: 5′-GGGAGGAGCTTCTAAATTATCCATCAGCACAAGCTGTCAGTGGCTCCAGCCATGAATAAATGTATAGGGAAA-3′) (16.Suen C.-S. Chin W.W. Mol. Cell. Biol. 1993; 13: 1719-1727Crossref PubMed Scopus (37) Google Scholar). Except for the Pit-1 oligonucleotide, all oligonucleotides used for EMSA contained 5′-BamHI and 3′-BglII restriction sites in addition to the sequences listed above.Figure 3:Mutation of the putative LHβ-GSE sequence defines nucleotides essential for binding by αT3-1 nuclear extract. Binding reactions included αT3-1 nuclear extracts and either the 32P-labeled wild-type LHβ gene oligonucleotide (LHSF, lanes 1-4) or the 32P-labeled mutated oligonucleotide (LHSFM, lanes 5-8). Competition with 500-fold molar excess of unlabeled LHSF, LHSFM, or Pit-1 oligonucleotide was performed as indicated. The specific and nonspecific binding complexes are indicated by an arrowhead and asterisk, respectively.View Large Image Figure ViewerDownload (PPT)Sense and antisense oligonucleotides were annealed and end-labeled with [γ-32P]ATP by T4 polynucleotide kinase and purified over a NICK column (Pharmacia Biotech Inc.).Nuclear Extract and in Vitro Translated ProteinsThe method of Andrews and Faller (17.Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2209) Google Scholar) was used to prepare crude nuclear extracts from a mouse gonadotrope-derived cell line (αT3-1), a monkey kidney fibroblast cell line (CV-1), and a rat somatolactotrope cell line (GH3). In vitro translated SF-1 protein was generated from a plasmid containing 2.1 kilobase pairs of the mouse SF-1 cDNA using the TNT coupled reticulocyte lysate system (Promega, Madison, WI)(18.Lala D.S. Rice D.A. Parker K.L. Mol. Endocrinol. 1992; 6: 1249-1258Crossref PubMed Scopus (514) Google Scholar). The resultant product was determined to be of appropriate size by comparison with [35S]methionine-labeled protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Electrophoretic Mobility Shift AssaysNuclear extract (5 μg) or in vitro translated protein (1, 3, or 5 μl) was incubated with 50,000 cpm of oligonucleotide probe in DNA-binding buffer (20 mM HEPES (pH 7.9), 60 mM KCl, 5 mM MgCl2, 10 mM phenylmethylsulfonyl fluoride, 10 mM dithiothreitol, 1 mg/ml bovine serum albumin, and 5% (v/v) glycerol) for 30 min on ice. For competition studies, excess unlabeled oligonucleotide was added 5 min prior to the addition of probe. Where indicated, antiserum (1 μl) was added 30 min following the addition of probe, and incubation was continued for 2 h. Protein-DNA complexes were resolved on a 5% nondenaturing polyacrylamide gel in 0.5 × Tris borate-EDTA buffer and subjected to autoradiography.Plasmids Used in Transfection StudiesThe largest LHβ reporter construct used for these studies contained 794 base pairs of the 5′-flanking sequence of the rat LHβ gene and the first 5 base pairs of the 5′-untranslated region fused to a luciferase reporter gene, pXP2(19.Nordeen S.K. BioTechniques. 1988; 6: 454-458PubMed Google Scholar). Deletions in this construct were created by subcloning polymerase chain reaction products containing the LHβ promoter sequences into the pXP2 vector using BamHI/HindIII sites which were introduced by the primers. The −209LHβ-MUT plasmid was created by introducing a two base pair mutation into the −209LHβ construct using the transformer site-directed mutagenesis kit (Clontech Laboratories, Inc., Palo Alto, California). The selection primer was located in the pXP2 polylinker and the 3′-end of the LHβ flanking sequence and converted a unique HindIII restriction site to a unique MluI site (sense strand: 5′-GGTAGGGAAGGTATCACGCGTGTCGACCCGGGTACC-3′). The mutagenic primer spanned region −147 to −104 of the LHβ promoter and eliminated a TthIII1 restriction site in addition to introducing the desired mutation (sense strand: 5′-GCTGGTCCCTGGCTTTTCTGAAATTGTCTGTCTCGCCCCCAAAG-3′). To create GSE2-GH50, an oligonucleotide was designed which contained two copies of the putative GSE region as a tandem repeat flanked by BamHI/BglII restriction sites (sense strand: 5′-GATCCTTTTCTGACCTTGTCTGTCTCGCCTCTGACCTTGTCTGTA-3′). This oligonucleotide was inserted upstream of the minimal growth hormone promoter, GH50, in the pXP1 luciferase reporter plasmid(19.Nordeen S.K. BioTechniques. 1988; 6: 454-458PubMed Google Scholar, 20.Suen C.-S. Yen P.M. Chin W.W. J. Biol. Chem. 1994; 269: 1314-1322Abstract Full Text PDF PubMed Google Scholar). All reporter constructs were confirmed by dideoxysequencing.The SF-1 expression vector contained 2.1 kilobase pairs of the mouse SF-1 cDNA driven by cytomegalovirus promoter sequences(18.Lala D.S. Rice D.A. Parker K.L. Mol. Endocrinol. 1992; 6: 1249-1258Crossref PubMed Scopus (514) Google Scholar). The Pit-1 expression vector was created by placing 915 base pairs of the rat Pit-1/growth hormone factor-1 cDNA sequence from pBluescript SK(-) (Stratagene, La Jolla, CA) into the pcDNAI vector (Invitrogen, San Diego, CA) using HindIII/NotI restriction enzyme sites(21.Bodner M. Castrillo J.-L. Theill L.E. Deerinck T. Ellisman M. Karin M. Cell. 1988; 55: 505-518Abstract Full Text PDF PubMed Scopus (629) Google Scholar).Cell Culture and AssaysMonkey kidney fibroblast (CV-1) cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells growing in 3.5-cm tissue culture wells (Flow Laboratories, McLean, VA) were transfected with expression (0.1 μg/well) and reporter (1.65 μg/well) plasmids using the calcium phosphate precipitation method(22.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Control wells received the appropriate "empty" expression vector (0.1 μg/well). Cotransfection with an RSV-β-galactosidase plasmid (1 μg/well) allowed correction for differences in transfection efficiency between wells. The cells were harvested 48 h following transfection and the cell extracts analyzed for both luciferase (23.DeWet J.R. Wood K.V. DeLuca M. Helinski D.R. Subramani S. Mol. Cell. Biol. 1987; 7: 725-737Crossref PubMed Scopus (2473) Google Scholar) and β-galactosidase (24.Edlund T. Walker M.D. Barr P.J. Rutter W.J. Science. 1985; 230: 912-916Crossref PubMed Scopus (395) Google Scholar) activities. Luciferase activity was first normalized to the level of β-galactosidase activity. Results were then calculated as fold change relative to expression in the presence of the control empty expression vector. Data are shown as the mean ± S.E. and represent a minimum of three independent experiments with each point run in triplicate in each experiment.Sources of SF-1 and Pit-1 Antibodies and PlasmidsThe rabbit SF-1 antiserum as well as the vectors containing the SF-1 cDNA were kindly provided by Dr. K. L. Parker (Duke University). The SF-1 antibody was generated against a glutathione S-transferase-SF-1 fusion protein(25.Ikeda Y. Lala D.S. Luo X. Kim E. Moisan M.-P. Parker K.L. Mol. Endocrinol. 1993; 7: 852-860Crossref PubMed Google Scholar). The Pit-1 antiserum, directed against amino acids 136-150 of rat Pit-1/growth hormone factor-1, was provided by C. Bancroft (Mt. Sinai School of Medicine) (26.Morris A.E. Jiang Y. McChesney R.E. Jackson A.E. Bancroft C. Chasin L.A. Gene (Amst.). 1990; 94: 289-294Crossref PubMed Scopus (5) Google Scholar). The Pit-1 cDNA in pBluescript SK(-) was provided by Dr. L. E. Theill (University of California, San Diego).RESULTSαT3-1 Nuclear Extract Binds to the Putative GSE Region of the LHβ Gene PromoterThe αT3-1 cell line has previously been shown to express both SF-1 mRNA and protein(6.Barnhart K.M. Mellon P.L. Mol. Endocrinol. 1994; 8: 878-885Crossref PubMed Scopus (204) Google Scholar, 7.Ingraham H.A. Lala D.S. Ikeda Y. Luo X. Shen W.-H. Nachtigal M.W. Abbud R. Nilson J.H. Parker K.L. Genes & Dev. 1994; 8: 2302-2312Crossref PubMed Scopus (510) Google Scholar). Nuclear extracts from this cell line, presumed to contain SF-1, have been shown to bind to the α-subunit promoter GSE(4.Horn F. Windle J.J. Barnhart K.M. Mellon P.L. Mol. Cell. Biol. 1992; 12: 2143-2153Crossref PubMed Google Scholar, 6.Barnhart K.M. Mellon P.L. Mol. Endocrinol. 1994; 8: 878-885Crossref PubMed Scopus (204) Google Scholar). We therefore utilized EMSA to investigate whether these extracts were able to interact with the region of the rat LHβ gene promoter that contains the putative GSE sequence (oligonucleotide LHSF). As shown in Fig. 2, the interaction of αT3-1 nuclear extracts with 32P-labeled LHSF produced a specific protein-DNA complex as demonstrated by successful competition with unlabeled LHSF (lanes 1 and 2).Figure 2:αT3-1 nuclear extracts and in vitro translated SF-1 bind the putative LHβ-GSE region with similar specificity. Binding reactions included 32P-labeled LHSF as a probe and, as indicated, either αT3-1 nuclear extract (lanes 1-4) or 1, 3, or 5 μl of in vitro translated SF-1 (lanes 5-11). Competition with 500-fold molar excess of unlabeled LHSF is shown for both the nuclear extract and in vitro translated SF-1 (lanes 2 and 8). Incubation with antiserum specific to SF-1 (lanes 3 and 9) or Pit-1 (lanes 4 and 10) was also performed using both protein preparations. Note that 3 μl of in vitro translated SF-1 were used in the cold competition and antibody studies (lanes 8-10) and therefore band intensity should be compared against lane 6. Lane 11 contains a probe and the unprogrammed reticulocyte lysate used for in vitro translation. The arrowhead indicates the specific binding complex. A nonspecific band, indicated by the asterisk, is present in unprogrammed reticulocyte lysate.View Large Image Figure ViewerDownload (PPT)In order to confirm that the complex identified in Fig. 2 contained SF-1, we investigated the effect of a SF-1-specific antibody on the formation of the αT3-1 nuclear extract-LHSF complex. This antibody has previously been shown to block the ability of SF-1 to bind to the promoter element of a number of genes, including the glycoprotein hormone α-subunit, aromatase, and 21-hydroxylase genes(6.Barnhart K.M. Mellon P.L. Mol. Endocrinol. 1994; 8: 878-885Crossref PubMed Scopus (204) Google Scholar, 9.Lynch J.P. Lala D.S. Peluso J.J. Luo W. Parker K.L. White B.A. Mol. Endocrinol. 1993; 7: 776-786Crossref PubMed Scopus (213) Google Scholar, 25.Ikeda Y. Lala D.S. Luo X. Kim E. Moisan M.-P. Parker K.L. Mol. Endocrinol. 1993; 7: 852-860Crossref PubMed Google Scholar). Treatment with this SF-1-specific antiserum substantially decreased the intensity of the protein-DNA complex while the addition of an anti-Pit-1 antiserum, used as a negative control, had no effect (Fig. 2, lanes 3 and 4). This result confirms that the GSE of the LHβ gene promoter is bound by SF-1, or an immunologically related protein, present in αT3-1 nuclear extracts.Parallel EMSA was performed using the oligonucleotide LHSF as a probe in the presence of nuclear extracts from cell lines that do not contain SF-1. No specific protein-DNA interactions were detected with the use of nuclear extracts from either monkey kidney fibroblast cells (CV-1) or rat somatolactotrope cells (GH3) (data not shown).In Vitro Translated SF-1 Binds to the LHβ Gene PromoterFurther confirmation that SF-1 binds to the LHβ-GSE promoter region was obtained by the use of in vitro translated SF-1 in EMSA. A binding reaction containing the labeled LHSF oligonucleotide and in vitro translated SF-1 resulted in a protein-DNA complex mobility similar to that obtained with the αT3-1 nuclear extracts (Fig. 2, lanes 5-10). Formation of this complex diminished in the presence of either excess unlabeled LHSF oligonucleotide or blocking antiserum directed against SF-1, but was unaffected by the Pit-1 antibody. Taken as a whole, these data clearly demonstrate that the putative GSE region of the LHβ gene is recognized by SF-1, as either an endogenous (αT3-1 nuclear extract) or an in vitro translated product.Mutation of the Putative LHβ-GSE Site Eliminates BindingIn order to localize further the SF-1 recognition site, a 2-base pair mutation was introduced in the wild-type LHβ oligonucleotide sequence (LHSF) to form LHSFM. The choice of this mutation was based on the loss of DNA binding which resulted from analogous mutations in the Müllerian inhibiting substance and glycoprotein α-subunit promoters(6.Barnhart K.M. Mellon P.L. Mol. Endocrinol. 1994; 8: 878-885Crossref PubMed Scopus (204) Google Scholar, 10.Shen W.-H. Moore C.C.D. Ikeda Y. Parker K.L. Ingraham H.A. Cell. 1994; 77: 651-661Abstract Full Text PDF PubMed Scopus (482) Google Scholar). EMSA was performed using αT3-1 nuclear extracts and either the wild-type LHSF (Fig. 3, lanes 1-4) or the mutant LHSFM (Fig. 3, lanes 5-8) as a probe. The intensity of the complex obtained with the LHSF probe was blunted by unlabeled wild-type LHSF, but not by the mutated sequence or by an unrelated oligonucleotide containing two binding sites for the pituitary transcription factor, Pit-1 (Fig. 3, lanes 2-4). As seen in lanes 5-8, the αT3-1 nuclear extract was not able to bind to LHSFM when used as a probe. These results establish that an intact LHβ-GSE sequence is required for binding by αT3-1 nuclear extract.SF-1 Specifically Increases LHβ Promoter ActivityWe next sought to determine the functional significance of the interaction between SF-1 and the LHβ gene promoter sequences. In initial investigations utilizing the gonadotrope-derived αT3-1 cell line, LHβ promoter-driven luciferase activity exceeded luciferase activity in the absence of cell extract (background activity) by less than 2-fold. At this level of expression, we were unable to evaluate reliably whether the presence or absence of the putative GSE sequence altered LHβ promoter activity in response to the endogenous SF-1 present in this cell line. Furthermore, attempts to increase LHβ gene expression through cotransfection with an SF-1 expression vector were unsuccessful.These studies were therefore performed in the monkey kidney fibroblast cell line, CV-1, a cell line which has previously been shown to support SF-1-induced transactivation of the bovine P-450 CYP11B promoter. By Northern blot analysis, this cell line lacks the mRNA which encodes the SF-1 homolog, Ad4BP(27.Honda S. Morohashi K. Nomura M. Takeya H. Kitajima M. Omura T. J. Biol. Chem. 1993; 268: 7494-7502Abstract Full Text PDF PubMed Google Scholar). As stated previously, we have also demonstrated that CV-1 nuclear extract fails to bind the LHβ-GSE region by EMSA, consistent with the absence of endogenous SF-1 (data not shown). Utilizing the CV-1 cell line, basal LHβ gene promoter activity exceeded expression of the promoterless reporter plasmid (pXP2) by an average of 15-fold.In Fig. 4A, CV-1 cells were cotransfected with region −209 to +5 of the LHβ gene promoter and cytomegalovirus-driven expression vectors containing either the SF-1 or Pit-1 cDNA. The presence of SF-1 markedly increased LHβ promoter activity (56 ± 5-fold). In contrast, the pituitary transcription factor Pit-1 did not alter luciferase levels, indicating the specificity of the SF-1 response.Figure 4:An intact putative LHβ-GSE region confers SF-1 responsiveness to both the LHβ promoter and a heterologous minimal promoter. CV-1 cells were transiently transfected with luciferase reporter constructs which contained various regions of the rat LHβ gene promoter. Cells were cotransfected with plasmids encoding either SF-1 or Pit-1 and with an RSV-β-galactosidase expression vector. Luciferase activity was normalized to β-galactosidase activity. Promoter activity was then calculated as fold change over expression in the presence of the appropriate control expression vector. Results are shown as the mean ± S.E. of at least nine samples in three independent experiments. A, comparison of LHβ promoter activity in response to SF-1 versus Pit-1. B, SF-1 stimulation of LHβ promoter activity with loss of the intact GSE sequence by sequential 5′-deletion or mutagenesis. C, SF-1 responsiveness of the growth hormone minimal promoter (GH50) (20.Suen C.-S. Yen P.M. Chin W.W. J. Biol. Chem. 1994; 269: 1314-1322Abstract Full Text PDF PubMed Google Scholar) or GH50 preceded by two copies of the putative LHβ-GSE region (GSE2-GH50).View Large Image Figure ViewerDownload (PPT)Of importance, we have recently confirmed the ability of SF-1 to increase LHβ promoter activity in the rat pituitary-derived somatolactotrope cell line, GH3. Utilizing transiently transfected GH3 cells and conditions similar to those in CV-1 cells, SF-1 increased LHβ promoter activity in the −209/+5 construct by 15 ± 1.5-fold.SF-1 Stimulation of Rat LHβ Gene Promoter Activity Is Dependent on the Presence of an Intact GSEIn order to delineate the region in the LHβ gene promoter responsible for providing SF-1 responsiveness, reporter constructs were generated which incorporated various deletions in the rat LHβ gene promoter. Of note, no systematic changes in basal expression (i.e. in the absence of SF-1) were observed in these deletion constructs.The evaluation of sequential 5′-deletion constructs revealed persistent SF-1 stimulation of LHβ promoter activity with deletion to position −134, followed by an abrupt loss of the SF-1 response with deletion to position −82 (Fig. 4B). Based on these data, loss of LHβ promoter sequences across the putative GSE region (positions −127 to −119) correlates with the loss of SF-1-stimulated promoter activity.Further definition of the SF-1-responsive cis-acting element was obtained by the introduction of a two base pair mutation into the putative GSE site of the −209LHβ luciferase reporter construct to form −209LHβ-MUT. This small change, analogous to the mutation which eliminated DNA-binding by nuclear extract (Fig. 3), substantially decreased the ability of SF-1 to increase promoter activity (6 ± 1-fold versus 56 ± 5-fold) (Fig. 4B). Thus, transactivation of the LHβ promoter by SF-1 appears to be critically dependent on the presence of an intact GSE sequence.LHβ Promoter Sequences Confer SF-1 Responsiveness to a Heterologous PromoterHaving demonstrated that the putative GSE site is necessary for SF-1 responsiveness in the context of the LHβ gene promoter, we next asked whether this sequence was sufficient to confer SF-1 responsiveness to a heterologous promoter. Two copies of the LHβ-GSE sequence were inserted upstream of the growth hormone minimal promoter, GH50. As seen in Fig. 4C, these sequences conferred a marked SF-1 response to this normally nonresponsive promoter (57 ± 10-fold versus 1.3 ± 0.2-fold).Other investigators have shown previously that the human α-subunit promoter GSE (identical to the putative rat LHβ-GSE, see Fig. 1) increases thymidine kinase minimal promoter activity in SF-1 containing cell lines, but not in cell lines which lack SF-1(6.Barnhart K.M. Mellon P.L. Mol. Endocrinol. 1994; 8: 878-885Crossref PubMed Scopus (204) Google Scholar). However, interpretation of this study was limited by the possibility that additional cell-specific factors were contributing to the observed differences in transcriptional activity. As our results were obtained in a single cell line, stimulation of promoter activity in the presence of the GSE can be attributed solely to SF-1-induced effects.DISCUSSIONOur results clearly demonstrate that SF-1 bi
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