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. 2008 Oct 1;150(2):946–956. doi: 10.1210/en.2008-0573

Regulation of P450c17 Expression in the Early Embryo Depends on GATA Factors

Yimin Shi 1, Marcus D Schonemann 1, Synthia H Mellon 1
PMCID: PMC2646537  PMID: 18832096

Abstract

The enzyme P450c17 is required for glucocorticoid, sex steroid, and some neurosteroid biosynthesis. Defective human P450c17 causes sexual infantilism and 46,XY sex reversal but is compatible with life, whereas ablation of the corresponding mouse gene causes embryonic lethality at around E7. Normal mouse embryos express P450c17 protein and activity in the embryonic endoderm at E7. Adult adrenal and gonadal steroidogenesis requires steroidogenic factor-1 (SF-1), but SF-1 is not expressed in the early mouse embryo. We show that P450c17 is expressed in differentiated mouse parietal and visceral endoderm lineages, in cultured mouse F9 embryonic carcinoma stem cells, in mouse embryonic stem cells, and in cultured mouse P19 stem cells. Bases −110 to −55 (which contain an SF-1 site and two potential GATA sites) of the rat cyp17 gene confer promoter activity in F9 cells. Overexpression of SF-1 has no effect, whereas overexpression of GATA4 in F9 cells increases transcription from −110/−55 fused to a reporter and increases endogenous P450c17 mRNA. Chromatin immunoprecipitation assays show that GATA4 binds to −215/+55 of mouse cyp17. Stimulating F9 cells with retinoic acid and cAMP differentiates them into visceral and parietal endoderm. Commensurate with cell differentiation, quantitative PCR showed increased GATA4 and GATA6 mRNAs, temporally followed by increased P450c17 mRNA. Small interfering RNA inhibition of GATA4 or GATA6 in undifferentiated or differentiated F9 cells diminished endogenous cyp17 expression. Thus, P450c17 is expressed in mouse embryonic stem cells, its expression increases upon differentiation to an early embryonic endoderm lineage, and GATA4/6 are responsible for activation of P450c17 gene expression at this early stage of embryonic development.

The unanticipated expression of P450c17 very early in embryonic development is dependent not upon SF-1, as it is in steroidogenic tissues, but rather upon the expression of GATA factors.

A single enzyme, P450c17, catalyzes the 17α-hydroxylation of c21 steroids (17α-hydroxylase activity) and also cleaves c21 steroids to c19 precursors of sex steroids (17,20 lyase activity) on a single active site (1,2). The 17α-hydroxylase activity is robust and is effective with both Δ5 steroids (pregnenolone) and Δ4 steroids (progesterone) (3). By contrast, the 17,20 lyase activity is less efficient; human P450c17 catalyzes 17,20 lyase preferentially with Δ5 substrates (3), whereas the rodent enzyme prefers Δ4 substrates (4). P450c17 also catalyzes 17,20 lyase activity with the 3α5α-reduced derivative of progesterone (allopregnanolone), leading to the so-called backdoor pathway for androgen formation (5).

P450c17 is expressed in a tissue-specific, species-specific fashion. It is expressed in the human adrenal and gonad but not placenta (6,7,8,9), and it is expressed in the rodent gonad and placenta but not adrenal (10,11,12). In addition to its expression in these steroidogenic tissues, P450c17 is also expressed in the developing rodent (13,14,15,16), bird (17,18,19,20,21), frog (22), fish (23,24), dog (25), and adult human (26,27,28,29,30,31,32) brains.

P450c17 gene expression is regulated by a variety of tissue-specific and species-specific transcription factors. P450c17 from all species is regulated by the orphan nuclear receptor steroidogenic factor-1 (SF-1) in tissues that express SF-1 (33,34,35,36,37,38,39,40,41,42,43,44,45,46,47). In the rodent placenta, which lacks SF-1 expression, regulation of P450c17 transcription is cAMP independent (48), and may be regulated by Ku autoimmune antigen (49). Similarly, in the rodent brain, which lacks SF-1, P450c17 expression may be regulated by SET and nerve growth factor IB, nur77 (14,35,43,50).

Mutations in human P450c17 result in relatively mild disorders of adrenal steroid production, sexual infantilism, and 46,XY sex reversal but is otherwise compatible with life (51,52,53). We ablated exons 5 and 6 of the P450c17 gene in mice and found that the knockout mice fail to survive past embryonic d 7.5 (E7.5) (54). At E7 of normal mice, P450c17 was expressed in the embryonic endoderm, and P450c17 enzyme activity was detectable in E7 embryos (54). The mechanism for regulating expression of P450c17 at this early stage of embryogenesis is unknown. We now show that P450c17 is not only expressed at E7 but is also expressed in mouse embryonic stem cells, in cultured mouse F9 embryonic carcinoma stem cells, and in cultured P19 stem cells. We have determined the region of the rat promoter that is active in these cells and show that this gene is regulated by GATA factors in all these cell lines, that GATA4 overexpression results in increases in P450c17 expression, and silencing GATA4 expression decreases P450c17 expression.

Materials and Methods

Plasmids

The rat P450c17 promoter reporter constructs have been described previously (50,55,56). Human GATA4-expressing vector was a gift from Dr. Walter L. Miller (University of California, San Francisco) (38).

Cell culture, transfection, and luciferase assays

Mouse embryonal carcinoma F9 cells (57,58) were grown in DMEM with 10% fetal bovine serum as described (59,60). F9 cells were differentiated into endoderm lineages by growth in medium containing 1 μm retinoic acid (primitive endoderm), 1 μm retinoic acid plus 250 μm cAMP (parietal endoderm), or 50 nm retinoic acid (visceral endoderm).

Mouse Leydig MA-10 cells (61) were grown in Waymouth’s MB752/1 medium without insulin but containing 15% horse serum, 20 mm HEPES, and gentamicin. Cells were plated at 1 × 105 cells per well in a 12-well plate on the day before transfections and were 80% confluent on the day of transfection. Transfections were performed using a lipofection reagent (FuGene6; Roche Applied Sciences, Indianapolis, IN) according to the manufacturer’s protocol, using 1 μg promoter/reporter gene construct DNA per well. When vectors expressing GATA4 were cotransfected with luciferase reporter constructs, DNA concentrations were equalized by the addition of luciferase cloning vectors.

Mouse P19 cells (62,63) were grown in DMEM supplemented with 10% fetal bovine serum. Mouse embryonic stem cell line ES14, obtained from Dr. Nigel Killeen at University of California, San Francisco, were grown on irradiated mouse feeder fibroblasts in Glasgow MEM (Sigma-Aldrich, St. Louis, MO) that contained 15% fetal bovine serum, 0.055 mm 2-mercaptoethanol, 2 mm glutamine, 0.1 mm nonessential amino acids, and 1000 U/ml human recombinant leukemia inhibitory factor (Millipore, Bedford, MA). Cells were passaged onto 0.1% gelatin-coated (gelatin from Sigma-Aldrich) dishes and expanded in feeder-free conditions.

Luciferase assays were carried out using a luciferase assay system (Promega, Madison, WI) and a Monolight 1500 luminometer (Analytical Luminescence Laboratory, San Diego, CA). Data represent the mean ± sem of three or more independent experiments, each performed in triplicate.

Short hairpin (shRNA) transfection

Plasmids expressing shRNAs for mouse GATA4 and GATA6 or for a control sequence were obtained from Dr. Yoshitaka Hayashi, Aichi Medical University, and used as described (64). Plasmids were transfected using FuGene6 (Roche), and RNA was prepared 24, 48, and 72 h after transfection.

EMSAs

Whole-cell extracts were prepared from MA-10 and F9 cells as described (65) or by using a commercial nuclear extraction reagent (NE-PER; Pierce Biotechnology, Rockford, IL). Protein concentrations of the extracts were determined using a bicinchoninic acid reagent (Bio-Rad, Richmond, CA). The cytoplasmic proteins were used to perform Western blots, and the nuclear proteins were used in EMSAs.

EMSAs were performed as described (55,56). Nuclear protein (5 μg) was incubated at room temperature with 25,000 cpm 32P-labeled double-stranded oligonucleotide probe, with or without cold competitor oligonucleotide. Incubation solutions included 100 μg/ml salmon sperm DNA, 1 mg/ml poly-deoxyinosine-deoxycytosine, 10 mg/ml BSA, and binding buffer [20 mm HEPES (pH 7.9), 60 mm KCl, 4 mm Tris-HCl (pH 8.0), 0.6 mm EDTA, 0.6 mm EGTA, and 0.6 mm dithiothreitol]. Samples were then electrophoresed on 6% polyacrylamide nondenaturing gels (in 89 mm Tris, 89 mm boric acid, and 2 mm EDTA) at 250 V for 30–45 min at room temperature. Autoradiographs of the dried gels were exposed for 12 h at −70 C. For competitive binding studies, 10–100 ng unlabeled specific and nonspecific oligonucleotides were premixed with the nuclear extract and the reaction buffer for 10 min before adding the probe. Antibody supershift assays used goat polyclonal antibodies to mouse GATA4 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), or nonimmune rabbit serum, added at the same time as the probe.

Western blots

Cytoplasmic proteins separated on 10% SDS-polyacrylamide gels were transferred to nitrocellulose membrane (Immun-Blot PVDF Membrane; Bio-Rad Laboratories, Hercules, CA). Blots were subjected to Western analysis using a polyclonal antibody raised against human P450c17 (66), that has been characterized and used in rodents previously (13,14).

RT-PCR

Total RNA was isolated from MA-10 and F9 cells using a commercial extraction system (QIAGEN, Valencia, CA). Total RNA (0.25 μg) was used to prepare cDNA and to amplify the cDNA by RT-PCR. cDNA was prepared with SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) or ISCRIPT (Bio-Rad) according to manufacturer’s protocols. cDNA from each sample was amplified by real-time PCR using TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA). Primers for GATAs 1-6, P450c17, SF-1, and GAPDH are listed in supplemental Table 1 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). RNA quantities were normalized using GAPDH mRNA as a reference. PCR products were identified by agarose gel electrophoresis or were quantitated using an Icycler (Bio-Rad, Richmond, CA). Control RT-PCR contained water in place of RNA or contained no reverse transcriptase in the cDNA synthesis reaction.

Site-directed mutagenesis

Site-directed mutagenesis was performed using a site-directed mutagenesis kit (QuikChange; Stratagene, La Jolla, CA) according to the manufacturer’s protocol. The primers used for mutagenesis are listed in supplemental Table 1.

Chromatin immunoprecipitation (ChIP)

F9 and MA-10 cells were grown to confluence on 10-cm plates, and DNA-protein complexes were cross-linked by treating the cells with 1% formaldehyde for 15 min at room temperature. The cross-linking reaction was stopped by adding 125 mm glycine for 5 min, and the cells were lysed in 1% SDS and 20 mm Tris-HCl (pH 8.1), scraped off the plates, and collected by centrifugation. The pellet was resuspended in 0.25% SDS and 20 mm Tris-HCl (pH 8.1), and 0.5% complete protease inhibitor cocktail (Roche) was added. After 10 min incubation on ice, the samples were sonicated for 10-sec intervals at 50% output to obtain DNA fragments of 0.5–1.5 kb. After centrifugation at 1000 × g for 15 min, the supernatant was diluted in modified radioimmunoprecipitation buffer [50 mm Tris-HCl (pH 7.4), 1% Nonidet P-40 (NP-40), 0.25% Na-deoxycholate, 150 mm NaCl, 1 mm EDTA], and 0.5% protease inhibitor cocktail, 2–3 μg luciferase plasmid DNA, and 1 mg BSA were added. For immunoprecipitation, 1 ml of this mixture was gently agitated overnight at 4 C with 5 μg antibody to GATA-4, GATA-6, or control goat IgG. Protein A agarose beads (Invitrogen Life Technologies, Carlsbad, CA) were diluted to 50% in radioimmunoprecipitation buffer, and 50 μl was added to each of the samples, which were then shaken gently for another 2 h. The beads were washed once with 1 ml 0.1% SDS, 1% NP-40, 2 mm EDTA, 20 mm Tris-HCl (pH 8.1), and 150 mm NaCl; once with the same buffer containing 500 mm NaCl, once with 0.25 m LiCl, 1% NP-40, 1% Na-deoxycholate, 1 mm EDTA, and 10 mm Tris-HCl (pH 8.1); and three times with 10 mm Tris-HCl (pH 8.1) and 1 mm EDTA. Beads were collected by centrifugation at 1000 × g for 2 min at 4 C after each washing step. Immunocomplexes were eluted from the beads with 1% SDS, 0.1 m NaHCO3, 0.2 m NaCl, and 0.1 m Tris-HCl (pH 8.8) at 75 C. Samples were treated with Proteinase K (Roche) at 50 C for 1 h, and cross-links were reversed by heating to 65 C for 6 h. After adding 1 μg poly-deoxyinosine-deoxycytosine to each sample, DNA was extracted using QIAquick columns (QIAGEN), and 35 cycles of PCR were performed on each template using primers as listed in supplemental Table 1.

Results

Identification of transcriptionally active region of the P450c17 promoter in embryonic F9 cells

P450c17 is expressed in rodent gonads, placenta, and brain and also in the embryonic endoderm early in embryogenesis at E7 (54). To understand the mechanism for induction of P450c17 expression in the early embryo, we used the mouse F9 cell line (57,58), which can be differentiated into early embryonic primitive, visceral, or parietal endodermal cell types by stimulation with cAMP and/or retinoic acid (59,60). To determine whether P450c17 was expressed in any of these three populations of cells, we used RT-PCR to analyze undifferentiated F9 cells and F9 cells differentiated with retinoic acid (primitive endoderm), with retinoic acid plus cAMP (parietal endoderm), or with retinoic acid and plating on a bacterial plate (visceral endoderm) (Fig. 1). We identified a 325-bp P450c17 DNA fragment obtained by RT-PCR of undifferentiated F9 cells and of cells differentiated with all three treatments, suggesting P450c17 mRNA is expressed in primitive, visceral, and parietal endoderm. Expression of P450c17 may be greater in F9 cells differentiated into parietal endoderm than it was in the parental F9 cells, because amplified P450c17 DNA fragments could be detected with fewer amplification cycles.

Figure 1.

Figure 1

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Expression of P450c17 in F9 cells. cDNA was amplified for the indicated number of cycles (20–35) using RNA from parental F9 cells or F9 cells differentiated into primitive, visceral, or parietal endoderm. The abundance of P450c17 mRNA is greater in parietal endoderm, as evidenced by detection of P450c17 cDNA after only 25 cycles. Bottom, Restriction enzyme analysis of RT-PCR-amplified product. cDNA from F9 cells amplified with P450c17 primers (325-bp fragment) was digested with KpnI or PvuII, yielding expected fragments of 239/86 bp (KpnI) or 268/57 bp (PvuII). C1, PCR in the absence of RT; C2, RT-PCR with water. Amplified DNA fragments were sequenced for further verification. M, 100-bp molecular weight markers.

To ensure the 325-bp amplified DNA fragment was P450c17, we digested this DNA with KpnI or with PvuII and obtained DNA fragments of 239 and 86 bp (KpnI) and 268 and 57 bp (PvuII) (Fig. 1, bottom gel). The 325-bp fragment was also sequenced and was shown to be a fragment of mouse P450c17. These data demonstrate that P450c17 is expressed in early embryonic F9 cells before differentiation and that differentiation into a parietal endoderm lineage may increase its expression.

To determine how P450c17 is regulated in the F9 cells, we compared the transcriptional activation of rat P450c17 in F9 cells to its established transcriptional activation in mouse Leydig MA-10 cells (50,55,56). We ligated 1560 bp of 5′-flanking DNA from the rat P450c17 gene to a luciferase reporter gene and built constructs containing deletions from the 5′ end, which we have characterized extensively in mouse Leydig MA-10 cells (14,42,43,49,50,55,56,67). We transfected F9 and MA-10 cells with these deletional promoter luciferase constructs and assayed luciferase activity (Fig. 2). In MA-10 cells, luciferase activity was greatest with the −1560-Luc construct; in F9 cells, luciferase activity was greatest with the −375-Luc construct. This −375-Luc construct had greatly reduced luciferase activity in MA-10 cells. In F9 cells, greatly reduced luciferase activity was seen in the −290-Luc construct, suggesting that there may be activating sequences between −290/−375. These data indicate that MA-10 and F9 cells regulate P450c17 transcription using (different) factors that bind to different cis-activating sequences. We previously showed detailed transcriptional activity in the −447/−419 region of the rat P450c17 gene (14,35,43,50,67). In both F9 and MA-10 cells, the minimal sequences necessary for luciferase activity were found in 110 bp of 5′-flanking DNA. This region contains an SF-1 site at −63/−55 that regulates P450c17 transcription in MA-10 cells (42,55,56).

Figure 2.

Figure 2

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Transcriptional activation of P450c17 promoters in F9 and MA-10 cells. Mouse F9 and MA-10 cells were transfected with 1 μg of the −1561/+1 P450c17-Luc construct or its serial deletion mutants, and luciferase activity was determined 24 h after transfection. Data, reported as relative luciferase units, are means ± sem of three experiments, each done in triplicate. Data were analyzed by one-way ANOVA and post hoc Bonferroni’s multiple comparison test. Luciferase activity from all constructs were significantly different from the ΔLuc vector, with P < 0.001, relative to ΔLuc vector.

Characterization of the −110/−55 region of the P450c17 gene

In addition to the SF-1 site at −63/−55, the −110/−55 region of the P450c17 gene contains potential GATA sites at −98/−92 and −77/−71 (Fig. 3). SF-1 expression is tightly restricted to the gonad, adrenal, hypothalamus, and pituitary, and previous studies suggested that SF-1 is not expressed in mouse embryonic stem cells (68). We determined whether SF-1 was found in F9 cells. Surprisingly, we found that we could amplify an SF-1 DNA fragment in undifferentiated F9 cells (Fig. 3A). We also found that the expression of SF-1 was not changed in cells differentiated to a primitive, visceral, or parietal endoderm lineage (Fig. 3A). We therefore determined whether SF-1 could stimulate P450c17 gene transcription in F9 cells (Fig. 3B). Our data demonstrate that SF-1 could stimulate P450c17 transcription slightly (∼1.3-fold) from the −1561 promoter but that it could not stimulate P450c17 transcription from the −110 promoter in F9 cells. Thus, although SF-1 may be expressed in F9 cells, it is likely not to contribute significantly to P450c17 transcriptional activation.

Figure 3.

Figure 3

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Analysis of P450c17 promoter and regulation by GATA4. The −110/−55 region of the rP450c17 promoter that is active in F9 cells is indicated, showing the location of two GATA sites and the SF-1 site. A, Expression of SF-1 mRNA in F9 cells. cDNA from parental F9 cells or F9 cells differentiated into primitive, visceral, or parietal endoderm. C1, PCR in the absence of RT; C2, RT-PCR with water; M, 100-bp molecular weight markers. B, Effect of SF-1 on transcriptional activation of P450c17. Cells were transfected with 1 μg P450c17 promoter-reporter constructs −1561-Luc or −110-Luc in the absence or presence of 0.125 μg of an SF-1-expression vector, and luciferase activity was determined 24 h after transfection. C, Expression of GATA mRNAs in F9 cells. cDNA from F9 cells was amplified using specific GATA primers. C1, PCR in the absence of RT; C2, RT-PCR with water; M, 100-bp molecular weight markers. D and E, Effect of GATA4 on P450c17 transcriptional activation in mouse F9 (D) and MA-10 (E) cells. Cells were transfected with 1 μg P450c17 promoter-reporter construct −1561/+1-Luc or its serial deletion mutants in the absence (gray bars) or presence (black bars) of 0.125 μg GATA4 expression vector, and luciferase activity was determined 24 h after transfection. The data are mean relative luciferase units ± sem of three experiments, each done in triplicate. Data were analyzed by one-way ANOVA (B, D, and E) and two-way ANOVA (F) and post hoc Bonferroni’s multiple comparison test: ***, P < 0.001; **, P < 0.01; *, P < 0.05; in F: a, comparison between GATA4 or SF-1 transfection and control; b, comparison between GATA4 transfection and SF-1 plus GATA4 transfection; NS, not significant.

Because SF-1 likely does not contribute to P450c17 transcriptional regulation in F9 cells, we therefore determined whether any members of the GATA family are expressed in F9 cells and whether they play a role in the transcriptional activation of P450c17. RT-PCR of RNA from F9 cells showed that GATA1 through GATA6 are expressed in F9 cells, indicating that they may play a role in the transcriptional activation of P450c17 (Fig. 3C).

GATA1-3 are primarily involved in hematological development, but GATA4 and -6 have been implicated in human P450c17 expression (38,69,70). Therefore, we determined whether GATA4 could activate transcription of P450c17 in F9 cells, by cotransfecting deletional promoter luciferase constructs into F9 cells, in the absence or presence of a GATA4 expression vector (Fig. 3D). We also determined whether GATA4 could similarly activate these P450c17 constructs in MA-10 cells (Fig. 3E). The data demonstrate that luciferase activity from all constructs, including the −110/+1 construct, could be activated by cotransfection of the GATA4 expression vector. The GATA4-stimulated increase in luciferase activity was greater in MA-10 cells than it was in F9 cells, but P450c17 luciferase constructs could nevertheless be stimulated by GATA4 in both cell types. Furthermore, in both F9 and in MA-10 cells, the −110/+1 P450c17 luciferase construct was stimulated by GATA4. These data suggest that GATA sites located within −110/+1 of the rat P450c17 promoter may be activated by GATA4.

Finally, although SF-1 appeared to have little or no effect by itself in F9 cells, we determined whether SF-1 could augment GATA-stimulated transcription (Fig. 3F). We transfected F9 cells with the −1561-Luc or −110-Luc constructs, with GATA4, SF-1, or both GATA4 and SF-1. Our data indicate that SF-1 inhibits GATA4-stimulated transcription from the −1561-Luc or −110-Luc vectors but that the inhibitory effect was much greater with the −1561-Luc vector. Thus, SF-1 may inhibit the activating effect of GATA4 in F9 cells.

GATA binding site in the promoter is essential for GATA4-induced transactivation of rat P450c17 promoter

GATA4 is necessary for gonadal development (71), and members of the GATA family (GATA4 and GATA6) have been shown to regulate transcription of the human P450c17 gene (38,69,70). However, GATA regulation of human P450c17 is not through GATA sites but rather through Sp sites located at −196/−188 (38).

The −110/−55 region of the rat P450c17 gene does not contain an Sp site but contains two potential GATA sites: site 1 at −98/−92 GATA and site 2 at −77/−71 GATA (Fig. 4). To determine whether these GATA sites are important for transactivation of P450c17 by GATA4, we mutated each site individually (M1 and M2) and together (M4) in the minimally active proximal promoter (−110/+1) of P450c17, using site-directed mutagenesis of the −110/+1 P450c17 luciferase construct. We also mutated the SF-1 site (M3), which is known to be active in MA-10 cells (35,42,50,55,56,67,72). These constructs were then transfected into F9 or MA-10 cells, in the absence or presence of a GATA4 expression vector (Fig. 4).

Figure 4.

Figure 4

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GATA binding sites in the P450c17 proximal promoter are required for transcriptional activation by GATA4. F9 (A) and MA-10 (B) cells were transfected with 1 μg wild-type −110/−1-Luc construct or with 1 μg constructs containing mutations in GATA binding site 1 (M1), GATA binding site 2 (M2), both sites 1 and 2 (M4), or a mutant SF-1 binding site (M3) in the absence (gray bars) or presence (black bars) of a GATA4-expressing vector. Luciferase activities were determined 24 h after transfection. The data are mean relative luciferase units ± sem from three experiments, each done in triplicate. Data were analyzed by one-way ANOVA and post hoc Bonferroni’s multiple comparison test: ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS, not significant.

In comparison with the wild-type −110-Luc construct, mutation of the GATA4 binding site 1 (−110/M1) decreased basal luciferase activity, mutation at the GATA4 binding site 2 (−110/M2) had no effect on basal luciferase activity, and mutation of both GATA sites (−110/M4) reduced basal luciferase activity to background (empty vector) levels (Fig. 4A). Cotransfection of these mutant promoter/report constructs with a GATA4 expression vector into F9 cells showed that the wild-type −110-Luc vector was stimulated about 6-fold (Fig. 4, A and B). Mutation of the GATA binding site 1 (−110/M1) had less effect on GATA4-stimulated luciferase activity and gave results similar to those of the wild-type −110-Luc construct (Fig. 4, A and B). Mutation of the GATA binding site 2 alone (−110/M2) reduced GATA4-stimulated luciferase activity; mutation of both GATA binding sites (−110/M4), however, eliminated GATA4-stimulated luciferase activity. Thus, the −110-Luc construct is stimulated by GATA4. The GATA4 binding site 2 located at −77/−71 appears to be the major site for GATA4-induced transactivation of P450c17 promoter in F9 cells, and the GATA4 binding site 1 located at −98/−92 appears to be more important for basal transcriptional activation of P450c17. Hence, both sites are necessary for maximal transcriptional activation by GATA4.

To determine whether the SF-1 site at −63/−55 plays a role in transcriptional activation of P450c17 in F9 cells as it does in MA-10 cells, we mutated the SF-1 site (−110/M3), and a luciferase vector containing this mutant oligonucleotide was transfected into F9 cells in the absence or presence of GATA4 (Fig. 4A). Although mutation of the SF-1 site reduced basal transcriptional activity, this construct could still be stimulated by GATA4. Thus, the SF-1 site was not critical to the GATA-stimulated transcription.

We determined whether these potential GATA sites were regulated by GATA in a similar fashion in MA-10 cells (Fig. 4 B), because previous studies focused on the role of the SF-1 site (42,55,56). As in F9 cells, the −110-Luc construct was stimulated by cotransfection with a GATA4 expression vector. Mutation of either GATA binding site resulted in a decrease in basal luciferase activity and reduced GATA4 stimulation of luciferase activity. Mutation of both sites (−110/M4) reduced GATA4 stimulation, but mutation of the SF-1 site (−110/M3) had no effect on GATA4 stimulation. Thus, in MA-10 cells, both GATA sites appear to regulate basal and GATA-stimulated transcription.

GATA-4 transactivates the P450c17 promoter

To determine whether additional regions of the rat P450c17 promoter could be transactivated by GATA4 in F9 cells, we inserted the GATA site mutations at −98/−92 (M1), −77/−71 (M2), both GATA mutations (M4), or the SF-1 mutation at −63/−55 (M3) into the full-length (−1561/+1) P450c17 promoter. These constructs were cotransfected with a GATA4 expression plasmid into F9 and MA-10 cells (Fig. 5). Cotransfecting the P450c17-luciferase reporter constructs with a GATA4 expression vector activated P450c17 promoter reporter constructs ∼3-fold in F9 cells (Fig. 5A). Mutation of the GATA site at −98/−92 (M1) reduced basal and GATA4-stimulated transcription, whereas mutations at −77/−71 (M2) reduced GATA4-stimulated transcription. Mutation of the SF-1 site had no effect on GATA4-stimulated transcription. These results are similar to those obtained using the smaller region of the P450c17 promoter (−110/+1, Fig. 4), suggesting that the principal sites of GATA4 action lie within the −110/−55 region.

Figure 5.

Figure 5

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The GATA binding sites in the proximal promoter of P450c17 regulate GATA-stimulated transcription from the −1561 P450c17 promoter. F9 cells (A) and MA-10 cells (B) were transfected with 1 μg wild-type −1561/+1-P450c17Luc reporter construct or with site-directed mutants within GATA binding site 1 (M1), binding site 2 (M2), both sites 1 and 2 (M4), or the SF-1 site (M4) in the absence (gray bars) or presence (black bars) of a GATA4 expression vector, and luciferase activities were determined 24 h after transfection. The data shown are mean relative luciferase units ± sem from three experiments, each done in triplicate. Data were analyzed by one-way ANOVA and post hoc Bonferroni’s multiple comparison test: ***, P < 0.001; **, P < 0.01; *, P < 0.05.

To determine whether the P450c17 gene was regulated by GATA4 in a similar fashion in mouse Leydig MA-10 cells, we performed the same transfections and assayed luciferase activity in MA-10 cells. GATA4 could stimulate luciferase activity when site 1 (−98/92; −1561/M1) was mutated but stimulated luciferase activity much less when site 2 (−77/−71; −1561/M2) was mutated, and mutation of both GATA 4 sites (M4) eliminated GATA4-induced P450c17 transcription. These data suggest that in MA-10 cells, like in F9 cells, the principal sites of GATA action are located between −110/−55 (Fig. 5B).

GATA4 factor binds GATA binding site in rat P450c17 promoter

Because GATA binding site 2 in the proximal promoter of P450c17 is essential for GATA4-induced transactivation of both the full-length and proximal P450c17 promoter, we performed gel shift assays using oligonucleotides that contain GATA binding sites 1 and 2 as a probe to verify whether GATA4 can bind to these GATA binding sites. Nuclear extracts from F9 cells formed protein-DNA complexes with the −110/−68 oligonucleotide probe that lacks the SF-1 site (Fig. 6). Formation of this complex could be competed by excess unlabeled −110/−68 oligonucleotide probe, by a −110/−82 oligonucleotide containing only GATA site 1, or by a −91/−63 oligonucleotide containing GATA binding site 2. Similarly, F9 cell extracts incubated with a −91/−63 probe (site 2) formed a protein-DNA complex that could be competed by −110/−68 (sites 1 and 2) or −110/−82 (site 1). Protein-DNA complexes formed with the −110/−68 or with the −91/−63 probes could be supershifted by an antibody against GATA4. Thus, GATA4 binds to both GATA binding sites 1 and 2 in F9 cells.

Figure 6.

Figure 6

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Endogenous GATA4 binds to the proximal promoter of rat P450c17. Gel shift assays were performed by incubation of nuclear protein from F9 cells with 32P-end-labeled double-stranded oligonucleotides (supplemental Table 1) in the presence or absence of 100-fold excess of unlabeled oligonucleotide. Antibody supershift assays used goat polyclonal antibody to mouse GATA4. Samples were separated on 6% polyacrylamide nondenaturing gels, dried, and subjected to autoradiography. The GATA4-DNA complex (GATA4) and supershifted GATA4-DNA complex (GATA4′) are indicated.

Differentiation of F9 cells, increased GATA expression, and increased P450c17 expression

Differentiation of F9 cells with retinoic acid plus cAMP increased P450c17 mRNA, as assessed by RT-PCR (Fig. 1). We determined the time course of differentiation of F9 cells, relative to the expression of GATA4 and GATA6 and to the expression of P450c17. F9 cells were stimulated with 8-Br-cAMP and retinoic acid for 24–96 h, and cells were harvested daily and analyzed for specific mRNAs by quantitative RT-PCR (Fig. 7A). The abundance of both GATA4 and GATA6 mRNAs began to increase between 24 and 48 h of stimulation; maximal induction was seen at 72 h of stimulation, and this induction was maintained at 96 h. By contrast, P450c17 mRNA abundance did not increase until 72 h and continued to increase at 96 h. Thus, the increase in GATA4 and GATA6 mRNAs precede increases in P450c17 mRNA.

Figure 7.

Figure 7

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Regulation of endogenous GATA4 and GATA6 in F9 cells regulates P450c17 mRNA expression. A, Differentiation of F9 cells by treatment with retinoic acid and cAMP. F9 cells were harvested daily and analyzed for specific mRNA abundance by quantitative RT-PCR. The expression of both GATA4 and GATA6 mRNAs increased after 24 h stimulation with retinoic acid plus 8-Br-cAMP, and the maximal induction was achieved after 72–96 h stimulation. P450c17 mRNA abundance increased only after 72 h of 8-Br-cAMP and retinoic acid stimulation and continued to increase at 96 h. B, Reduction of endogenous GATA4 and GATA6 mRNAs by siRNA. F9 cells were transfected with plasmids expressing GATA4 and/or GATA6 siRNAs (pH1GATA4 and pH1GATA6, respectively) or control vector (pH1S) and were differentiated using retinoic acid/cAMP. Ninety-six hours after transfection and cell differentiation, RNA was isolated and analyzed by quantitative RT-PCR. In both panels, results are the means ± sem from three experiments. Data were analyzed by one-way ANOVA and post hoc Newman-Keul’s multiple comparison test: **, P < 0.01; *, P < 0.05; a, comparison between control and RA/cAMP stimulation; b, comparison between RA/cAMP stimulation and RA/cAMP stimulation plus siRNA transfection.

To determine whether the increased GATA4/GATA6 mRNA expression was required for induction of P450c17 mRNA, F9 cells were transfected with plasmids expressing shRNAs for GATA4 and/or GATA6 and were differentiated using 8-Br-cAMP and retinoic acid. Ninety-six hours after transfection and cell differentiation, RNA was isolated and analyzed by quantitative RT-PCR (Fig. 7B). Plasmids expressing small interfering RNAs (siRNAs) for GATA4 (pH1GATA4) or GATA6 (pH1GATA6) specifically reduced expression of their mRNAs and proteins (our data, not shown, and Ref. 64). Reduction of either GATA4 or GATA6 mRNA decreased P450c17 mRNA expression, although this effect was greater when GATA4 mRNA was reduced. Reduction of both GATA4 and GATA6 decreased P450c17 mRNA to the same extent as when GATA4 alone was reduced. Thus, stimulation of F9 cells with 8-Br-cAMP and retinoic acid increases expression of GATA4, GATA6, and P450c17 mRNAs, and reduction of expression of either GATA4 or GATA6 mRNAs decreases expression of P450c17. These data suggest that the differentiation of F9 cells into an endoderm phenotype involves increases in the expression of GATA transcription factors and subsequently involves increased expression of P450c17 mRNA.

The P450c17 gene is bound by GATA factors in vivo

To determine whether the P450c17 gene is regulated by GATA factors in vivo, we performed chromatin immunoprecipitation (ChIP) assays. Chromatin prepared from F9 cells stimulated with 8-Br-cAMP and retinoic acid was cross-linked with formaldehyde, sonicated, and immunoprecipitated with GATA 4 or GATA6 antibodies. Immunoprecipitated chromatin was amplified with P450c17-specific primers corresponding to −215/−196 and +36/+55. PCR amplification demonstrates that a 270-bp P450c17 DNA fragment is amplified from chromatin immunoprecipitated with either a GATA4 or a GATA6 antibody but is not amplified when a nonspecific goat IgG is used (Fig. 8). These data indicate that in differentiated F9 cells, GATA4 and GATA6 are bound to the P450c17 gene. These data further indicate that these GATA transcription factors regulate the expression of the P450c17 gene in differentiated F9 cells.

Figure 8.

Figure 8

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GATA factors bind the P450c17 promoter in vivo. Chromatin was prepared from F9 cells stimulated with 8-Br-cAMP and retinoic acid, cross-linked with formaldehyde, sonicated, and immunoprecipitated with GATA4 or GATA6 antibodies. Immunoprecipitated chromatin was amplified with P450c17-specific primers corresponding to nucleotides −215/−196 and +36/+55 of proximal promoter of P450c17.

Induction of P450c17 expression in mouse embryonic stem cells

Because P450c17 was expressed in undifferentiated and differentiated mouse embryonic carcinoma F9 cells, we determined whether it was expressed in other mouse embryonic stem cell lines as well as in primary cultures of mouse embryonic stem cells. RNA was prepared from mouse P19 cells and primary mouse embryonic stem cells and was amplified with two sets of primers encompassing the entire P450c17 open reading frame. Gel electrophoresis of amplified DNA fragments indicates that mouse P450c17 mRNA is expressed not only in mouse F9 cells but is also expressed in mouse P19 cells as well as in mouse embryonic stem cells (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo. endojournals.org). The level of expression of P450c17 mRNA appears to be comparable in all three cell types. Thus, P450c17 mRNA is expressed in several embryonic stem/carcinoma cell lines as well as in primary cultures of mouse embryonic stem cells.

Discussion

The expression of P450c17 in tissues not classically involved in steroid hormone production has been well documented in many mammalian and nonmammalian species (13,17,18,20,22,73,74,75,76,77). Our creation of a transgenic mouse lacking expression of P450c17 further demonstrated important expression of P450c17 early in embryogenesis in embryonic endoderm (54). Our current study suggests that P450c17 mRNA is expressed in mouse embryonic stem cells and in mouse embryonic carcinoma stem cells before differentiation to an endoderm lineage, and its expression is increased upon differentiation to a parietal endoderm lineage.

The regulation of P450c17 gene transcription in embryonic stem cells relies on GATA factors. This regulation is also seen in mouse Leydig MA-10 cells, where SF-1 also plays a major role in transcriptional activation. GATAs 1-6 are found in low abundance in mouse embryonic carcinoma F9 cells. The abundance of GATA4 and GATA6 increase with differentiation of these cells with retinoic acid and cAMP, factors that drive this cell line toward an endoderm lineage (58,64,78,79,80). During early rodent development, the parietal endoderm produces basement membrane components such as laminin-1 and collagen IV. Development of the parietal endoderm has been shown to depend upon Sox7, which stimulates GATA4 and GATA6 (64). Overexpression of GATA4 and GATA6 in F9 cells in which Sox7 was silenced resulted in a restoration of differentiation of the F9 cells into parietal endoderm (64), suggesting that Sox7 is upstream of GATA4 and GATA6, which play a crucial role in the differentiation of F9 cells into this lineage. GATA factors are the determining factors in the formation of extraembryonic endoderm in vivo and endoderm lineage commitment in vitro (81,82,83,84). GATA6 may regulate GATA4 expression (83,84), and GATA6 increases expression of Dab2 (Disabled 2) in the extraembryonic endoderm and absence of GATA6 suppresses expression of laminin and collagen IV (85). Recent studies have suggested that GATA4 and GATA6 may play different roles in early extraembryonic development; in vitro models of embryonic stem cell differentiation suggest that GATA4 may be responsible for cell aggregation, whereas GATA6 may be responsible for responding to retinoic acid signals (86). It is unknown whether expression of P450c17 contributes to this differentiation and, if so, where it plays a role downstream of GATA expression.

Alignment of the human, bovine, murine, and rat P450c17 gene sequences indicates that two regions between −98 and −92 and between −77 and −71 are highly conserved (supplemental Fig. 2). However, this region of the human P450c17 gene is not regulated by GATA factors. Rather, another region between −196 and −188 in the human gene responds to GATA. This effect is not direct but is through its interaction with Sp1 (38). Unlike the human P450c17 gene, this −96/−188 site does not appear to be regulated by GATA in the rat P450c17 gene. We compared the GATA4 stimulation of transcription of constructs containing this region (−267-Luc) to that obtained using the −110-Luc construct. Basal transcription from the −267-Luc construct was greater than from the −110-Luc construct (Fig. 3D); however, stimulation by GATA4 was about 2-fold with the −267-Luc construct and 6-fold with the −110-Luc construct (Fig. 3, D and E), suggesting that deletion of the potential Sp site had no effect on GATA4-stimulated transcription. These data therefore suggest that the GATA4-regulated region of the rat P450c17 gene is different from the GATA4-regulated region of the human P450c17 gene, even though the DNA sequences of these two species are similar in the −110/−55 region.

The function of P450c17 in embryonic stem cells or in differentiated endoderm is unknown. Extraembryonic tissues play an important role in embryonic growth and differentiation. The primitive endoderm arises from pluripotent cells of the inner cell mass around E4, shortly before implantation. It differentiates into the parietal endoderm and the visceral endoderm (59,78,79). Both the parietal and visceral endoderms are major components of the yolk sac. Parietal endoderm cells are specialized for synthesizing and secreting the thick basement membrane, Reichert’s membrane, which is between the parietal endoderm and the trophectoderm and which separates the yolk cavity from the maternal tissues (87,88,89). Parietal endoderm cells synthesize large components of basement membrane components including laminin, entactin,and type IV procollagen (90,91,92). The visceral endoderm acts as an early organizer before formation of the primitive streak (93,94). It is also important both for uptake of substances from the maternal circulation that have filtered through Reichert’s membrane and for secretion of serum components and other proteins such as α-fetoprotein, transferrin, and high- and low-density apolipoproteins. The visceral endoderm, which appears to be lost in P450c17-deficient embryos that we created (54), is also believed to provide signals responsible for cavitation of the embryo and could provide clues as to a function for P450c17 at this time in development.

Thus, our data demonstrate that P450c17 is expressed in undifferentiated F9 cells and that its expression increases upon differentiation to an extraembryonic endoderm lineage. This increase in P450c17 expression is dependent upon increased expression of GATA factors, which precedes increased P450c17 expression. Our data further demonstrate that there are differences in the transcriptional activation of P450c17 in mouse Leydig MA-10 vs. mouse embryonic carcinoma F9 cells, that SF-1 plays a prominent role in transcriptional activation of P450c17 in MA-10 (and other steroidogenic) cells but not in F9 cells, and that GATA factors are major regulators of P450c17 expression in early embryo development.

Supplementary Material

[Supplemental Data]
en.2008-0573_index.html (1.2KB, html)

Footnotes

This work was supported by grants from the National Institutes of Health (NIH) (HD27970) and from the National Science Foundation (0090907) to S.H.M. M.D.S. was supported by a fellowship from NIH Training Grant DK07161.

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 1, 2008

Abbreviations: E7.5, Embryonic d 7.5; NP-40, Nonidet P-40; SF-1, steroidogenic factor-1; siRNA, small interfering RNA.

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