AP-2γ and the Homeodomain Protein Distal-less 3 Are Required for Placental-specific Expression of the Murine 3β-Hydroxysteroid Dehydrogenase VI Gene, Hsd3b6
2002; Elsevier BV; Volume: 277; Issue: 10 Linguagem: Inglês
10.1074/jbc.m106765200
ISSN1083-351X
Autores Tópico(s)Aldose Reductase and Taurine
ResumoThe enzyme 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) is essential for the biosynthesis of all active steroid hormones. It exists as multiple isoforms in humans and rodents, each the product of a distinct gene. Human 3β-HSD I in placenta is essential for placental progesterone biosynthesis and thus is essential for the maintenance of pregnancy. The murine ortholog, 3β-HSD VI, is the only isoform expressed in giant trophoblast cells during the first half of mouse pregnancy. This study was designed to identify the cis-acting element(s) and the associated transcription factors required for trophoblast-specific expression of 3β-HSD VI. Transfection studies in placental and nonplacental cells identified a novel 66-bp trophoblast-specific enhancer element located between −2896 and −2831 of the 3β-HSD VI promoter. DNase protection analysis of the enhancer element identified three trophoblast-specific binding sites, FPI, FPII, and FPIII. Electrophoretic mobility shift assays with oligonucleotides representing the protected sequences, FPI and FPIII, and nuclear extracts isolated from human JEG-3 cells and from mouse trophoblast cells, demonstrated the same binding pattern that was distinct from the binding pattern with mouse Leydig cell nuclear proteins. Further electrophoretic mobility shift assays identified AP-2γ and the homeodomain protein, Dlx 3, as the transcription factors that specifically bind to FPI and FPIII, respectively. Site-specific mutations in each of the binding sites eliminated enhancer activity indicating that AP-2γ and Dlx 3, together with an additional transcription factor(s) that are conserved between humans and mice, are required for trophoblast-specific expression of 3β-HSD VI. The enzyme 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) is essential for the biosynthesis of all active steroid hormones. It exists as multiple isoforms in humans and rodents, each the product of a distinct gene. Human 3β-HSD I in placenta is essential for placental progesterone biosynthesis and thus is essential for the maintenance of pregnancy. The murine ortholog, 3β-HSD VI, is the only isoform expressed in giant trophoblast cells during the first half of mouse pregnancy. This study was designed to identify the cis-acting element(s) and the associated transcription factors required for trophoblast-specific expression of 3β-HSD VI. Transfection studies in placental and nonplacental cells identified a novel 66-bp trophoblast-specific enhancer element located between −2896 and −2831 of the 3β-HSD VI promoter. DNase protection analysis of the enhancer element identified three trophoblast-specific binding sites, FPI, FPII, and FPIII. Electrophoretic mobility shift assays with oligonucleotides representing the protected sequences, FPI and FPIII, and nuclear extracts isolated from human JEG-3 cells and from mouse trophoblast cells, demonstrated the same binding pattern that was distinct from the binding pattern with mouse Leydig cell nuclear proteins. Further electrophoretic mobility shift assays identified AP-2γ and the homeodomain protein, Dlx 3, as the transcription factors that specifically bind to FPI and FPIII, respectively. Site-specific mutations in each of the binding sites eliminated enhancer activity indicating that AP-2γ and Dlx 3, together with an additional transcription factor(s) that are conserved between humans and mice, are required for trophoblast-specific expression of 3β-HSD VI. 3β-hydroxysteroid dehydrogenase/isomerase cholesterol side chain cleavage cytochrome P450 activator protein-2 distal-less 3 transcription enhancer factor junctional regulatory element human chorionic gonadotropin rapid amplification of cDNA ends electrophoretic mobility shift assay thymidine kinase luciferase steroidogenic factor 1 The enzyme 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD)1 is essential for the biosynthesis of all active steroid hormones: the adrenal steroid hormones, cortisol, corticosterone, and aldosterone; the testicular steroid hormone, testosterone; and the ovarian and placental hormones, progesterone and estradiol. The 3β-HSD enzyme exists as multiple isoforms in humans and rodents, each the product of a distinct gene (1Clarke T.R. Bain P.A. Burmeister M. Payne A.H. DNA Cell Biol. 1996; 15: 387-399Crossref PubMed Scopus (15) Google Scholar). To date, six isoforms have been identified in mouse and two in human. These isoforms are expressed in a tissue- and temporal-specific manner (1Clarke T.R. Bain P.A. Burmeister M. Payne A.H. DNA Cell Biol. 1996; 15: 387-399Crossref PubMed Scopus (15) Google Scholar, 2Park C.-H.J. Abbaszade I.G. Payne A.H. Mol. Cell. Endocrinol. 1996; 116: 157-164Crossref PubMed Scopus (13) Google Scholar, 3Abbaszade I.G. Arensburg J. Park C.H. Kasa-Vubu J.Z. Orly J. Payne A.H. Endocrinology. 1997; 138: 1392-1399Crossref PubMed Scopus (48) Google Scholar, 4Baker P.J. Sha J.A. McBride M.W. Peng L. Payne A.H. O'Shaughnessy P.J. Eur. J. Biochem. 1999; 260: 911-916Crossref PubMed Scopus (94) Google Scholar, 5Arensburg J. Payne A.H. Orly J. Endocrinology. 1999; 140: 5220-5232Crossref PubMed Scopus (57) Google Scholar). In the mouse, the two major isoforms involved in steroid hormone biosynthesis are 3β-HSD I and 3β-HSD VI. 3β-HSD I is the major or only isoform expressed in gonads and adrenal glands, whereas 3β-HSD VI is the only isoform expressed in giant trophoblast cells during mid-pregnancy (3Abbaszade I.G. Arensburg J. Park C.H. Kasa-Vubu J.Z. Orly J. Payne A.H. Endocrinology. 1997; 138: 1392-1399Crossref PubMed Scopus (48) Google Scholar). The orthologous isoforms in human are human 3β-HSD II, the isoform expressed in the gonads and adrenal glands (6Rheaume E. Lachance Y. Zhao H.-F. Breton N. Dumont M. de Launoit Y. Trudel C. Luu-The V. Simard J. Labrie F. Mol. Endocrinol. 1991; 5: 1147-1157Crossref PubMed Scopus (330) Google Scholar), and human 3β-HSD I (7Luu-The V. Lachance Y. Labrie C. Leblanc G. Thomas J.L. Strickler R.C. Labrie F. Mol. Endocrinol. 1989; 3: 1310-1312Crossref PubMed Scopus (296) Google Scholar, 8Guerin S.L. Leclerc S. Verreault H.L.F. Luu-The V. Mol. Endocrinol. 1995; 9: 1583-1597PubMed Google Scholar), the only isoform expressed in placenta throughout pregnancy. The expression of human 3β-HSD I in placenta is essential for placental progesterone biosynthesis and, thus, is vital for maintenance of pregnancy (9Miller W.L. Clin. Perinatol. 1998; 25: 799-817Abstract Full Text PDF PubMed Google Scholar). Consistent with the role of human 3β-HSD I in placental progesterone biosynthesis, we have shown that mouse 3β-HSD VI is required for progesterone biosynthesis in giant trophoblast cells between embryonic day (E) 9.5 and E10.5 (10Peng L. Orly J. Payne A.H. Mol. Cell. Endocrinol. 2001; (in press)PubMed Google Scholar). Progesterone biosynthesis from cholesterol requires the activity of two enzymes, cholesterol side chain cleavage cytochrome P450 (P450scc) which catalyzes the conversion of cholesterol to pregnenolone and 3β-HSD which catalyzes the conversion of pregnenolone to progesterone. This latter step is brought about by distinct tissue-specific isoforms of 3β-HSD. Previous studies designed to identify placental-specific regulatory elements in the human placental-specific 3β-HSD promoter were unsuccessful (8Guerin S.L. Leclerc S. Verreault H.L.F. Luu-The V. Mol. Endocrinol. 1995; 9: 1583-1597PubMed Google Scholar). Moreover, identity of transcription factors essential for placental-specific expression of P450scc remains to be resolved (11Huang N. Miller W.L. J. Biol. Chem. 2000; 275: 2852-2858Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Therefore, the question is whether there is a unique tissue-specific transcription factor or factors required for the expression of the trophoblast-specific isoform of 3β-HSD as well as the other enzyme required for progesterone biosynthesis, P450scc, in human placenta and mouse giant trophoblast cells. In the present study, we identify a 66-bp trophoblast-specific enhancer element located between −2896 and −2831 5′ of the transcription start site of the murine 3β-HSD VI promoter and demonstrate the requirement for two transcription factors, AP-2γ, and the homeodomain protein, Distal-less 3 (Dlx 3), in determining trophoblast-specific expression of the 3β-HSD VI gene. Dlx 3 is a member of a family comprising at least six Dlx genes sharing a homeobox sequence similar to that found in the Drosophila Distal-less gene (12Bendall A.J. Abate-Shen C. Gene (Amst.). 2000; 247: 17-31Crossref PubMed Scopus (227) Google Scholar). Dlx proteins are transcriptional activators which play an essential role during vertebrate development (12Bendall A.J. Abate-Shen C. Gene (Amst.). 2000; 247: 17-31Crossref PubMed Scopus (227) Google Scholar). AP-2γ is a member of a family of three closely related and evolutionary conserved sequence-specific DNA-binding proteins which include AP-2α, AP-2β, and AP-2γ (13Hilger-Eversheim K. Moser M. Schorle H. Buettner R. Gene (Amst.). 2000; 260: 1-12Crossref PubMed Scopus (292) Google Scholar). Dlx 3 and AP-2γ are expressed in both murine and human placental trophoblast cells and are required for the placental-specific expression of a number of genes (14Morasso M.I. Grinberg A. Robinson G. Sargent T.D. Mahon K.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 162-167Crossref PubMed Scopus (217) Google Scholar, 15Shi D. Kellems R.E. J. Biol. Chem. 1998; 273: 27331-27338Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 16LiCalsi C. Christophe S. Steger D.J. Buescher M. Fischer W. Mellon P.L. Nucleic Acids Res. 2000; 28: 1036-1043Crossref PubMed Scopus (43) Google Scholar, 17Knofler M. Saleh L. Bauer S. Vasicek R. Griesinger G. Strohmer H. Helmer H. Husslein P. Endocrinology. 2000; 141: 3737-3748Crossref PubMed Google Scholar, 18Roberson M.S. Meermann S. Morasso M.I. Mulvaney-Musa J.M. Zhang T. J. Biol. Chem. 2001; 276: 10016-10024Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Our studies demonstrate that these two transcription factors regulating the trophoblast-specific expression of the steroidogenic enzyme, 3β-HSD VI, are conserved in both murine and human trophoblast cells. This is the first report on the identification of at least two of the transcription factors required for placental-specific expression of 3β-HSD in humans and mice. A λ phage 129/SvJ mouse genomic library from Stratagene (La Jolla, CA) was screened with an 180-bp probe labeled with [α-32P]dCTP, which includes 18 bp of coding region and 167 bp of 3′-untranslated region of the 3β-HSD VI cDNA (3Abbaszade I.G. Arensburg J. Park C.H. Kasa-Vubu J.Z. Orly J. Payne A.H. Endocrinology. 1997; 138: 1392-1399Crossref PubMed Scopus (48) Google Scholar). Prehybridization and hydribization were performed as described (19Sambrook J. Fritsch F.H. Maniatis T. Molecular Cloning: A Laboratory Manual.2 Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Two clones (402 and 602) were identified to represent 3β-HSD VI. The clones were subjected to restriction enzyme and Southern blot analysis using exon-specific oligonucleotide probes (exon 2, 5′-CCAGAGGATTGTCCAGTTG-3′; exon 3, 5′-GACATCTAGGATGGTCTG-3′; exon 4, 5′-AGGAAGCTCACAGTTTCCA-3′). Restriction fragments from clone 402 were subcloned into the pBluescript-KS vector and subjected to further analysis to identify the location of exon 1 and to establish the start site of transcription. 5′-RACE was carried out using a Marathon cDNA library from CLONTECH (Palo Alto, CA) prepared from a 7-day pregnant mouse implantation site. The primary amplification was performed as described by the manufacturer using touchdown PCR techniques with adapter primer AP1 (5′-CCATCCTAATACGACTCACTATAGGGC-3′) and 3β-HSD VI-specific primer GSP1 (5′-GCCCGTACAACCGAGAATATT-3′, 628 bp 3′ of ATG in exon 4) (20Roux K.H. Hecker K.H. Methods Mol. Biol. 1997; 67: 39-45PubMed Google Scholar). Secondary amplification was performed using nested adapter primer AP2 (5′-ACTCACTATAGGGCTCGAGCGGC-3′) and 3β-HSD-specific primer GSP2 (5′-CAGACCATCCTAGATGTC-3′, 283 bp 3′ of ATG in exon 3). The secondary PCR product was subject to sequencing using primers AP2 or GSP2. Timed pregnant C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were killed by CO2 followed by cervical dislocation at E9.5 and E10.5, and the uterine horns were removed and placed in phosphate-buffered saline for further isolation of giant trophoblast cells as described (5Arensburg J. Payne A.H. Orly J. Endocrinology. 1999; 140: 5220-5232Crossref PubMed Scopus (57) Google Scholar). E9.5 and E10.5 implantation sites were separated from the myometrium and the embryos were removed. Giant trophoblast cells were gently removed by scraping the inner face of the hemi-implantation site and collected for preparation of RNA or proteins. The transcription initiation site of 3β-HSD VI was determined using the primer extension kit from Promega (Madison, MI). Messenger RNA was isolated from E10.5 mouse giant trophoblast cells and adult mouse testes using the QuickPrep Micro mRNA Purification Kit (Pharmacia, Piscataway, NJ). Primer GSP3 (5′-GGTTCTGATCTCTGCAAAGGAACCAG-3′, 132 bp 5′ of exon 1 end), which is specific for 3β-HSD VI, was end-labeled with [γ-32P]ATP using T4 polynucleotide kinase. Approximately 100 fmol of labeled primer and 3–5 μg of mRNA were hybridized at 65 °C for 20 min, followed by gradual cooling to room temperature. The reactions were extended by avian myeloblastosis virus reverse transcriptase at 42 °C for 1 h and 15 min and followed by heating at 99 °C for 5 min. The extended DNA fragments were precipitated with ethanol and subjected to electrophoresis in a 6% denaturing polyacrylamide gel. To characterize the promoter region of the mouse 3β-HSD VI gene, a series of 5′ deletions spanning between −4700 and −40 (Fig. 3) were subcloned into a promoterless luciferase reporter vector pA3LUC at theSmaI-HindIII sites (21Wood W.M. Kao M.Y. Gordon D.F. Ridgway E.C. J. Biol. Chem. 1989; 264: 14840-14847Abstract Full Text PDF PubMed Google Scholar). To make heterologous constructs, different fragments, including −3004/−1989, −3004/−2500, −3004/−2723, and −2722/−2500, were subcloned into a heterologous thymidine kinase promoter-driven luciferase reporter vector TK164LUC at the SmaI site. Further deletions within the −3004/−2723 fragment, including sequences −2896/−2725, −2830/−2725, and −2896/−2831, were generated by PCR using primers with SmaI site added at the 5′ end and subcloned into TK164LUC/SmaI. The sequences −2896/−2857 and −2867/−2831 were synthesized with a half SmaI site added in both 5′ and 3′ ends and subcloned into TK164LUC/SmaI. The PCR fidelity and orientations of inserts of each construct were verified by restriction enzyme digestion and sequencing. Mutations in the potential AP-2, TEF, or Dlx 3-binding sites in each footprint (FPI, FPII, and FPIII) (Table I) that were identified by DNase I footprinting were introduced by the ExSite PCR-based site-directed mutagenesis kit (Stratagene) following the instructions given in the manual. Positive clones were identified and verified by sequencing in the DNA Sequencing Facility at Stanford University.Table ISequences of some oligonucleotides for EMSAsOligo nameSequence (5′→3′)Position (5′→3′)FPICAGTTGGCCTGTAGGCAAGT−2891 /−2872mFPICAGTTGGCCTGggtaccAGT−2891 /−2872Ker1 (31)GTGTAGCCTGCAGGCCCACAFPIICTGGAGGGCATTCTATTGAC−2871 /−2852mFPIICTGGAGGaagctcTATTGAC−2871 /−2852FPIIITTGACTGATAATTGGTGTGA−2856 /−2837mFPIIITTGACaagcttTTGGTGTGA−2856 /−2837Nkx2–5 (34)TCGGGATCCGAGTTAATTGCGCJRE (18)GTACTTGGTGTAATTACCAT Open table in a new tab Both human choriocarcinoma cells, JEG-3 (ATCC HTB-36) and monkey kidney tumor cells, COS-7 (ATCC CRL-1651), were cultured in Dulbecco's modified Eagle's medium with 50 μg/ml gentamycin supplemented with 10% fetal bovine serum (Invitrogen, Gaithersburg, MD). MA-10 cells, a mouse Leydig tumor cell line (a gift of Dr. Mario Ascoli), were grown in Waymouth's MB752/1 medium containing 15% horse serum. JEG-3 and MA-10 cells were transfected by calcium phosphate-DNA precipitation (22Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1996Google Scholar). COS-7 cells were transfected using the FuGENE 6 reagent (Roche Molecular Biochemicals, Indianapolis, IN). Cells were plated at a density of 0.5–1 × 105 cells/20-mm well. After 20 h of culture, the testing plasmid (0.5 μg DNA) and pSV2β-Gal (23Rosenthal N. Methods Enzymol. 1987; 152: 704-720Crossref PubMed Scopus (403) Google Scholar) (0.1 μg of DNA) were co-transfected in triplicate. Following transfection (42 h), cells were lysed using the Reporter Lysis Buffer (Promega). The activities of luciferase and β-galactosidase were measured according to the manufacturer's description. The luciferase activity of each construct was normalized to the co-transfected β-galactosidase activity. Crude nuclear extracts from JEG-3 and MA-10 cells were prepared as described (24Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 192499Crossref PubMed Scopus (2214) Google Scholar). Nuclear extracts from E10.5 giant trophoblast cells and E15.5 placental tissues were isolated using the 1-h minipreparation techniques (25Deryckere F. Gannon F. BioTechniques. 1994; 16: 405PubMed Google Scholar). Total protein was quantitated by Bradford assay and normalized against extraction buffer. The extracts were aliquoted and stored at −70 °C until use for footprinting or EMSA. DNase I footprinting was carried out using Promega's core footprinting kit with modifications. To generate the probe, the 120-bp fragment (−2916/−2800) amplified from PCR was subcloned into the pBluescript-KS vector at theEcoRI-HindIII sites. The insert was sequenced for verification. A 167-bp/XbaI-XhoI fragment released from the vector was labeled with T4 polynucleotide kinase and [γ-32P]ATP. Digestion with AccI orSpeI resulted in formation of a sense or antisense probe, respectively. Increasing amounts of crude nuclear extract were incubated with the labeled probe (10,000 cpm) at room temperature for 10 min, followed by digestion with 6 units of DNase I at room temperature for 3 min. The digested products were extracted with phenol/chloroform (1:1), ethanol-precipitated, and analyzed using 5% denaturing polyacrylamide gel electrophoresis. EMSAs were performed as described previously (26Howard G. Peng L. Hyde J.F. Endocrinology. 1997; 138: 4649-4656Crossref PubMed Scopus (38) Google Scholar). The 20-μl binding reaction containing 5–10 μg of crude nuclear extracts and 0.05–0.25 ng of radiolabeled probes (50,000 cpm/reaction) was incubated for 20 min at room temperature. The sequences of the double-stranded oligonucleotides used for EMSAs are listed in TableI. For competition assays, a 50–500-fold molar excess of unlabeled oligonucleotides was added to the binding reaction mixture. For supershift assays, 2 μg of polyclonal antisera to AP-2α, AP-2γ, Nkx2-5 (Santa Cruz Biotechnology, Santa Cruz, CA), Dlx 3 (a gift of Dr. Mark Roberson), or preimmune sera were added to the mixture prior to the addition of the probe. The binding reactions were resolved on a 5% nondenaturing polyacrylamide gel. Extracts of MA-10 cells, transfected COS-7 cells, E9.5 or E10.5 giant trophoblast cells, or adrenal glands or testes from 50-day-old mice were prepared by homogenization in extraction buffer followed by centrifugation as described previously (3Abbaszade I.G. Arensburg J. Park C.H. Kasa-Vubu J.Z. Orly J. Payne A.H. Endocrinology. 1997; 138: 1392-1399Crossref PubMed Scopus (48) Google Scholar). The supernatant was subjected to SDS-PAGE and Western blot analysis. Membranes were first incubated with antiserum generated against human placental 3β-HSD and then with the horseradish peroxidase-labeled secondary antibody and exposed using the Enhanced ChemiLuminescence kit (Amersham Biosciences, Inc., Arlington Heights, IL). Cryosections (8 μm) of implantation sites from E9.5 and E10.5 were subjected to in situhybridization as described (22Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1996Google Scholar) using a 359-bp 35S-labeled sense or antisense cRNA probe representing 45 bp from the 3′ end of the coding region and 314 bp from the 3′-untranslated region of 3β-HSD VI cDNA (3Abbaszade I.G. Arensburg J. Park C.H. Kasa-Vubu J.Z. Orly J. Payne A.H. Endocrinology. 1997; 138: 1392-1399Crossref PubMed Scopus (48) Google Scholar). Slides were exposed at 4 °C and developed after 4 days. The slides were stained with hematoxylin and eosin and examined under light microscopy with bright- and dark-field illumination. To characterize the 3β-HSD VI gene, a λ phage genomic DNA library was screened with a 3β-HSD VI cDNA probe. Two phage clones, 402 and 602, were identified as encompassing the entire 3β-HSD VI gene. Restriction mapping and Southern blot analysis revealed that clone 402 contains ∼10 kb of 5′-flanking region, and exon 2 through exon 4 ending at theSacI site in the 3′-untranslated region (3Abbaszade I.G. Arensburg J. Park C.H. Kasa-Vubu J.Z. Orly J. Payne A.H. Endocrinology. 1997; 138: 1392-1399Crossref PubMed Scopus (48) Google Scholar), whereas clone 602 contains a portion of intron 2, exon 3, exon 4, and 9 kb 3′-flanking region (Fig.1 A). 5′-RACE analysis was performed to identify the exon 1 sequence. The 3′ 340-bp sequences from the 5′-RACE PCR product matched with known cDNA sequences including 206 bp of exon 2 and 134 bp of exon 3 (3Abbaszade I.G. Arensburg J. Park C.H. Kasa-Vubu J.Z. Orly J. Payne A.H. Endocrinology. 1997; 138: 1392-1399Crossref PubMed Scopus (48) Google Scholar). To confirm that the new sequence from the 5′-RACE represents the exon 1 sequence, reverse transcriptase-PCR was performed using RNA isolated from E10.5 mouse giant trophoblast cells and from mouse testicular tissues with a forward primer designed from the 5′-RACE exon 1 sequence and a backward primer designed from the known sequence of exon 2 (3Abbaszade I.G. Arensburg J. Park C.H. Kasa-Vubu J.Z. Orly J. Payne A.H. Endocrinology. 1997; 138: 1392-1399Crossref PubMed Scopus (48) Google Scholar). A fragment of the expected size was obtained. Thus, exon 1 was identified and the size of intron 1 was established to be 3.1 kb, which differs from human 3β-HSD I or mouse 3β-HSD I (1Clarke T.R. Bain P.A. Burmeister M. Payne A.H. DNA Cell Biol. 1996; 15: 387-399Crossref PubMed Scopus (15) Google Scholar). The transcription start site for 3β-HSD VI was confirmed by primer extension (Fig. 1 B). Using mRNA from adult mouse testes and from E10.5 mouse giant trophoblast cells, a single transcription start site was identified at a guanosine residue 315 bp 5′ of the translation initiation codon in exon 2 which defines the size of exon 1 as 252 bp. We previously reported the expression of 3β-HSD mRNA and protein in mouse giant trophoblast cells at E9.5 (5Arensburg J. Payne A.H. Orly J. Endocrinology. 1999; 140: 5220-5232Crossref PubMed Scopus (57) Google Scholar). However, the earlier study did not use isoform-specific probes for the identification of 3β-HSD. To determine the isoform-specific expression of 3β-HSD VI, in situ hybridization of sections of E9.5 and E10.5 implantation sites were analyzed using a 3β-HSD VI-specific antisense probe (see “Experimental Procedures”). Fig.2 A shows exclusive expression of 3β-HSD VI mRNA in giant trophoblast cells at E9.5 and E10.5 with considerably greater expression at E10.5. No expression of 3β-HSD VI mRNA was observed in decidua or embryo. The increase in 3β-HSD VI mRNA in giant trophoblast cells at E10.5 is accompanied by a parallel increase in 3β-HSD VI protein as determined by Western blot analysis (Fig. 2 B). In gonads and adrenal glands expression of steroidogenic enzymes is dependent on steroidogenic factor 1 (SF-1) (27Parker K.L. Schimmer B.P. Endocr. Rev. 1997; 18: 361-377Crossref PubMed Scopus (556) Google Scholar, 28Leers-Sucheta S. Morohashi-Ken-ichirou Mason J.I. Melner M.H. J. Biol. Chem. 1997; 272: 7960-7967Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). SF-1 null mice develop normal placental trophoblast cells that express steroidogenic enzymes (29Sadovsky Y. Crawford P.A. Woodson K.G. Polish J.A. Clements M.A. Tourtellotte L.M. Simburger K. Milbrandt J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10939-10943Crossref PubMed Scopus (395) Google Scholar) indicating that this factor is not involved in placental-specific expression of steroidogenic enzymes. Our studies (Table II) demonstrate that the transcriptional activity of the murine 3β-HSD VI proximal promoter transfected into JEG-3 cells that do not contain SF-1, is 22-fold greater than the transcriptional activity of the gonadal- and adrenal-specific 3β-HSD I promoter. Furthermore, co-transfection with an SF-1 expression vector resulted in a dose-dependent increase in 3β-HSD I transcriptional activity, but had no effect on 3β-HSD VI transcriptional activity. Thus, SF-1 is not required for expression of the 3β-HSD VI enzyme in the placenta.Table IIEffect of SF-1 on transcriptional activity of the mouse 3β-HSD I and VI promoters in JEG-3 cellsSF-1Luciferase activity2-aLuciferase activity relative to pGL3-Basic.3β-HSD I3β-HSD VIng00.715.820034004.18004.917.416005.8The sequences −359/+35 of 3β-HSD I and −443/+33 of 3β-HSD VI were subcloned into a promoterless luciferase reporter vector pGL3-Basic (Promega), respectively, and then transfected into JEG-3 cells as described under “Experimental Procedures.” Data from one experiment carried out in triplicates.2-a Luciferase activity relative to pGL3-Basic. Open table in a new tab The sequences −359/+35 of 3β-HSD I and −443/+33 of 3β-HSD VI were subcloned into a promoterless luciferase reporter vector pGL3-Basic (Promega), respectively, and then transfected into JEG-3 cells as described under “Experimental Procedures.” Data from one experiment carried out in triplicates. Because of the lack of a mouse trophoblast cell line and the difficulty in obtaining mouse primary giant trophoblast cells for transfection studies, the human placental choriocarcinoma cell line, JEG-3, was chosen for the transfection studies described herein. JEG-3 cells express human 3β-HSD I (30Tremblay Y. Beaudoin C. Mol. Endocrinol. 1993; 7: 355-364Crossref PubMed Scopus (51) Google Scholar), the tissue-specific ortholog to mouse 3β-HSD VI. To establish trophoblast-specific expression, nonplacental cell lines, both mouse Leydig tumor cells (MA-10) and monkey kidney cells (COS-7), were also used for the promoter and enhancer analyses of the mouse 3β-HSD VI gene. To characterize the promoter sequences involved in transcriptional regulation of the 3β-HSD VI gene in trophoblast cells, a series of 5′ deletions of the 3β-HSD VI gene, spanning from −4700 to −40 5′ of exon 1 (Fig. 3), were subcloned into a promoterless luciferase reporter vector, pA3LUC, and transiently transfected into JEG-3, MA-10, or COS-7 cells. As shown in Fig. 3, a marked increase in basal transcription activity was observed in all three cell lines when the promoter sequence was increased from −40 to −91, suggesting that a common positive cis-acting element is located between −40 and −91, although the luciferase activity was ∼10-fold greater in JEG-3 and MA-10 cells compared with COS-7 cells. A database analysis of this sequence shows that there are several potential binding sites for transcription factors CREB and Sp1 in the segment between −40 and −91. No further analysis was performed with this proximal sequence. As larger fragments from −91 to −4700 were included, the basal transcription activities remained essentially the same in both MA-10 and COS-7 cells. In contrast, a large increase in basal activity was observed in JEG-3 cells with fragments greater than −1989. The results suggest that the sequence between −3004 and −1989 may contain a trophoblast-specific enhancer element. To determine whether the sequence between −3004 and −1989 has the properties of an enhancer, this region was subcloned 5′ of the heterologous thymidine kinase (tk) promoter either in the sense or antisense direction and transfected into JEG-3, MA-10, and COS-7 cells. Results of the transfections are shown in Fig.4. The sequence between −3004 and −1989 increased tk promoter activity over 8-fold in either the sense or antisense directions in JEG-3 cells, but not in COS-7 or MA-10 cells. These data indicate that the sequence between −3004 and −1989 has the characteristics of a trophoblast-specific enhancer element. Because a further increase in transcriptional activity was observed between −4700 and −3004 (Fig. 3), this 1700-bp fragment was also subcloned 5′ of the tk promoter and transfected into JEG-3 cells. No increase in the promoter activity was observed with this fragment (data not shown). To further define the regulatory domains within the −3004/−1989 fragment of the 3β-HSD VI promoter, subsequent deletions were made and subcloned 5′ of the tk promoter. Fig.5 A shows the results of the deletion constructs transfected into JEG-3, MA-10, and COS-7 cells. No enhancer activity with any of the deletion constructs was observed in MA-10 or COS-7 cells. In contrast, in JEG-3 cells, deletions of the 3′ sequence from −1989 to −2500, did not change enhancer activity compared with the −3004/−1989 fragment. Further deletion from −2500 to −2723 displayed similar enhancer activity. The data suggest that the 282-bp fragment between −3004 and −2723 contains the trophoblast-specific enhancer element. To characterize the minimal enhancer region, additional 5′ deletions within −3004/−2723 were made and subcloned 5′ of the tkhet
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