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-Hydroxylase Promoter Function in Theca Cells Isolated from Patients with Polycystic Ovary Syndrome Involves Nuclear Factor-1
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033
Address all correspondence and requests for reprints to: Jan M. McAllister, Ph.D, Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, 500 University Drive H166, Hershey, Pennsylvania 17033. E-mail: jmcallister{at}psu.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-hydroxylase (CYP17) gene expression and androgen biosynthesis are persistently elevated in theca cells isolated from ovaries of women with polycystic ovary syndrome (PCOS). We previously reported that -235 to -109 bp of the CYP17 promoter confers increased CYP17 promoter function in PCOS theca cells. In this report, additional deletion and mutational analyses of the CYP17 promoter were performed to identify the sequences that contribute to increased CYP17 promoter function in PCOS theca cells. Results of these analyses established that augmented promoter function in PCOS theca cells results from preferentially increased basal regulation conferred by sequences between -188 and -147 bp of the CYP17 promoter. Scanning mutant analysis demonstrated that mutations within a 16-bp sequence, spanning -174 to -158 bp of the promoter, ablated increased basal CYP17 promoter function in PCOS theca cells. EMSA analysis demonstrated that the NF-1 family member, NF-1C, bound this sequence. Cotransfection of several NF-1C isoforms expressed in normal and PCOS cells repressed CYP17 promoter function. NF-1C protein and DNA binding were reduced in PCOS theca cell nuclear extracts, as compared with normal. Another NF-1C site between -102 and -90 bp of the promoter was also identified. However, mutation of this site had no effect on differential promoter function in PCOS theca cells. These studies demonstrate that 1) augmented CYP17 promoter function in PCOS theca cells results from increased basal regulation, and 2) diminished NF-1C-dependent repression may be one mechanism underlying increased basal CYP17 promoter activity and altered gene expression in PCOS theca cells. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Unlike the ovary of normal cycling women, the PCOS ovary is characterized by multiple small follicles 4–7 mm in diameter, with a theca cell compartment that is often hypertrophied. Ovarian theca cells are recognized as one of the primary sources of excess androgen biosynthesis in women with PCOS (7, 8, 9, 10). In response to LH-dependent increases in adenylate cyclase, theca cells express a variety of genes encoding components of the steroidogenic pathway that are necessary for androgen and progestin biosynthesis (11). The synthesis of thecal androgens is contingent on the expression of the cytochrome P450 17- hydroxylase (CYP17) gene, which encodes a single cytochrome P450 (P450c17) with both 17-hydroxylase and C17, 20 lyase activities.
We previously reported that androgen production is persistently elevated in theca cells isolated from the ovaries of women with PCOS and propagated for successive population doublings in vitro (12, 13). Augmented expression of several steroidogenic enzymes is associated with increased androgen biosynthesis in PCOS theca cells. In addition to CYP17, these include cytochrome P450 cholesterol side chain cleavage (P450scc), encoded by the CYP11A gene, as well as 3ß-hydroxysteroid dehydrogenase type II (3ß-HSDII), encoded by the HSD3B2 gene (12, 14). A comparison of CYP17 promoter function in normal and PCOS theca cells has demonstrated that increased CYP17 expression in PCOS results, in part, from increased transactivation of the CYP17 gene (15). In contrast, the abundance of the steroidogenic acute regulatory protein (StAR) and 17ß-hydroxysteroid dehydrogenase type V mRNAs and the transcriptional activity of the StAR promoter are not different between normal and PCOS theca cells (13, 15). Therefore, the expression and activity of only a subset of proteins that are important for androgen biosynthesis are affected in PCOS theca cells. These observations are in agreement with recent microarray analysis of differential gene expression in normal and PCOS theca cells and demonstrate that dysregulation of androgen biosynthesis involves selective differences in networks of genes involved in steroid hormone biosynthesis as well as insulin and glucose homeostasis (1, 6, 16).
In this report, we have focused and extended our studies on the regulation of CYP17 promoter function in normal and PCOS theca cells to examine the molecular mechanisms underlying dysregulated gene expression in the PCOS ovary. We previously reported that increased CYP17 gene expression in PCOS theca cells results from increased promoter function, within -235 bp to -109 bp of the start site of transcription of the CYP17 gene (15). Increased CYP17 gene expression in PCOS theca cells could result from alterations in basal promoter function, and/or changes in cAMP-dependent regulation. Here, further deletion and scanning mutational analysis of the human CYP17 promoter were performed to identify the elements involved in augmented CYP17 promoter function in PCOS theca cells. In this process, the DNA regulatory element(s) that are required for basal and cAMP- dependent CYP17 promoter regulation in normal and PCOS theca cells were also elucidated.
The data presented in this report suggest that nuclear factor-1 (NF-1) is one factor involved in increased CYP17 gene expression in the PCOS ovary. The NF-1 family of transcription factors has been shown to regulate transcriptional initiation of a variety of genes by specifically binding to the bipartite recognition sequence, (C/T)TGGC(N)6CC(N)3 (17, 18). The NF-1 family of transcription factors is comprised of four genes, NF-1A, B, C, and X, which are approximately 90% identical (19, 20, 21, 22, 23) and contain highly conserved amino terminal regions responsible for dimerization and DNA binding (24). NF-1C, also referred to as CAAT-box transcription factor (19), was the first NF-1 family member identified and has been reported to transactivate and repress the transcription of a wide variety of genes expressed in developmental and tissue-specific patterns (25, 26, 27, 28). With respect to the CYP17 promoter, recent data in adrenal H295 cells have shown that NF-1C binds the CYP17 promoter (29); however, NF-1C-dependent regulation of the CYP17 gene has not been characterized. This NF-1C binding site overlaps with putative binding sites for the orphan nuclear hormone receptor steroidogenic factor-1 (SF-1), which has also been reported to activate sequences within -235 bp of the CYP17 promoter in both ovarian and adrenal cell types (15, 30, 31).
Examination of the cellular processes involved in the dysregulation of CYP17 gene expression in PCOS theca cells is crucial for understanding the underlying mechanisms involved in excessive ovarian androgen production associated with PCOS. In this report, we show that decreased expression of NF-1C contributes to differential regulation of CYP17 promoter function in normal and PCOS theca cells. These studies provide evidence to suggest that decreased NF-1C-dependent repression may play a role in increased CYP17 gene transcription in PCOS theca cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The comparison of CYP17 promoter function in normal and PCOS demonstrated that both basal (aP < 0.01) and forskolin-stimulated (bP < 0.01) -235, -188, and -172 CYP17 promoter activities were increased in PCOS theca cells, as compared with normal theca cells (Fig. 1A
). Deletion of sequences 5' of -147 bp of the CYP17 promoter resulted in a complete ablation of increased CYP17 promoter function in PCOS theca cells, suggesting that sequences within the general boundaries of -172 to -147 bp of the CYP17 promoter are involved in augmented promoter regulation in PCOS theca cells. Further trimming of promoter sequences to -109 bp did not affect CYP17 promoter function in normal or PCOS theca cells, as compared with the activity of -147 bp of the promoter (Fig. 1B). These data suggest that augmented promoter function in PCOS theca cells primarily results from preferentially increased regulation conferred by sequences between -172 and -147 bp of the CYP17 promoter.
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and Fig. 1
). Significant forskolin-stimulated promoter function was observed with all of the CYP17 promoter constructs (i.e. -235, -188, -172, -147, and -109), with the exception of the -61 CYP17 promoter construct, in both normal and PCOS theca cells. The activity of the -61 CYP17 construct was similar to that observed with the promoterless pGL3-basic construct (data not shown). The observed reduction in forskolin-stimulated CYP17 promoter activity after removal of sequences upstream of -147 bp, as well as -61 bp, suggests the presence of two putative cAMP-regulatory elements within the general boundaries of -172 to -147 bp, and -109 to -61 bp, of the CYP17 promoter. A comparison of the fold-induction with forskolin in normal vs. PCOS theca cells (Table 1) indicates that forskolin-dependent induction is less robust in PCOS theca cells. However, the forskolin induced promoter activity, like basal activity, is significantly higher is PCOS cells. These data are consistent with quantitative real-time PCR data that also demonstrate that the fold increase in forskolin-stimulated CYP17 mRNA accumulation is blunted in PCOS theca cells, although the absolute amount is higher than in normal theca cells (data not shown).
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-188/-140) CYP17; or 2) ligated to the minimal -61 CYP17 promoter, generating (-188/-140)/-61 CYP17. Basal and forskolin-stimulated promoter function was compared with the activity of the wild-type (WT) -235 CYP17 or minimal -61 CYP17 constructs after transient transfection of normal and PCOS theca cells. As shown in Fig. 2B, both basal (aP < 0.01) and forskolin-stimulated (bP < 0.01) -235 CYP17 (WT) promoter activities were increased more than 2-fold in PCOS theca cells compared with normal theca cells. In contrast, -235(-188/-140) promoter activities were comparable in normal and PCOS theca cells under basal or forskolin-stimulated conditions. Forskolin treatment increased -235(-188/-140) CYP17 promoter activity approximately 2-fold over basal promoter activity in both normal and PCOS theca cells (Table 1). Similar results were observed after deletion of the -188- to -140-bp sequence from a promoter construct containing -750 bp of the CYP17 promoter (data not shown). These data suggest that one or more elements between -188 and -140 of the CYP17 promoter is required for increased basal regulation in PCOS theca cells.
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, -61 CYP17 promoter activity was not different in normal and PCOS theca cells, and was not induced by forskolin. Both basal (aP < 0.01) and forskolin-stimulated (-188/-140)/-61 CYP17 promoter function (bP < 0.01) was increased 2- to 3-fold in PCOS theca cells, as compared with normal theca cells. In both normal and PCOS theca cells, (-188/-140)/-61 CYP17 promoter activity was coordinately increased by 3- to 4-fold in response to forskolin treatment (Table 1). These studies suggest that sequences between -188 and -140 bp confer increased basal CYP17 promoter function in PCOS, and contribute to cAMP-dependent regulation in both normal and PCOS theca cells. The data presented in Figs. 1 and 2 indicate that increased promoter function in PCOS results from an increase in basal regulation, rather than altered cAMP-dependent regulation.
Scanning Mutant Analysis of the -188- to -140-bp Region of the CYP17 Promoter
Linker-scanning mutational analysis was performed to identify the nucleotide sequences within -188 and -140 bp of the CYP17 promoter responsible for increased promoter activity in PCOS theca cells. Mutant CYP17 promoter constructs were generated within the context of the -188 CYP17/LUC construct by introducing overlapping 5-bp mutations spanning sequences between -188 and -140 bp (Table 2 and Fig. 3). The relative basal promoter activities of the mutant CYP17 promoter constructs (i.e. M1–11) were compared with the WT -188 CYP17 promoter construct (WT) by transient transfection analysis in independent theca cell cultures isolated from five normal and five PCOS patients. In comparison to the -188 WT construct, mutation of any 5-bp sequence within -188/-140, with the exception of M5, resulted in a decrease in basal promoter activity, in both normal and PCOS theca cells. This suggests that several factors with overlapping sites contribute to regulation. The strongest reduction (90%) in promoter activity was observed with the M3 and M7 mutants. M1, M2, M3, M5, M8, M9, M10, and M11 CYP17 promoter function was significantly increased in PCOS theca cells as compared with normal theca cells. In contrast, the promoter function of mutants M4, M6, or M7 was equivalent in normal and PCOS theca cells, suggesting that one or more elements mediating differential regulation were disrupted. These data implicate nucleotide sequences from -174 to -158 bp of the CYP17 promoter (M4, M6, and M7) in differential regulation of CYP17 promoter activity in normal and PCOS theca cells. This is in agreement with results from deletion analysis, suggesting that sequences within approximately -172 bp of the CYP17 promoter are sufficient for increased CYP17 promoter function in PCOS theca cells (Fig. 1). The significant reduction in basal activity after mutation of several regions of the -188- to -140-bp element, with the exception of M5, further confirms that this region plays an important role in the basal regulation of the CYP17 promoter. Consistent with our data demonstrating that increased CYP17 promoter function in PCOS theca cells results from altered basal regulation, all of the CYP17 scanning mutants (M1-M11) retained forskolin inducibility (data not shown).
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). Lin et al. (29) have recently reported that NF-1 binds to the CYP17 promoter and contributes to basal regulation in adrenocortical H295 cells. To examine whether NF-1 binds to the region of the CYP17 promoter that confers increased regulation in PCOS theca cells, EMSA was performed using nuclear extracts (NEs) isolated from normal theca cells maintained under basal conditions. Incubation of theca cell NEs (3 µg) with a radioactively labeled oligonucleotide probe corresponding to sequences between -188 and -109 bp of the CYP17 promoter resulted in the formation of multiple protein-DNA complexes (Fig. 4B). Addition of NF-1 antisera that recognizes an amino-terminal region common to all NF-1 family members, resulted in a decrease in intensity of two specific protein/DNA complexes. These data demonstrate that NF-1 proteins in theca NEs form specific complexes with the region of the CYP17 promoter required for differential regulation.
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). Addition of an NF-1 consensus oligonucleotide prevented the formation of NF-1 complexes, further demonstrating that proteins present in these complexes interact with a consensus NF-1 binding site. Incubation of the Mut 4 or Mut 6 oligonucleotides did not compete for the NF-1 complexes, indicating that -174/-170 bp and -166/-162 bp of the CYP17 promoter are required for binding. In contrast, the Mut 5 oligonucleotide competed for NF-1 binding. These results are in agreement with the bipartite nature of the NF-1 binding site [i.e. (C/T)TGGC(N)6CC(N)3] because the sequences altered in mutants 4 and 6 correspond with the two halves of the NF-1 site required for NF-1 binding (Fig. 4A). The slower migrating, more intense complex was identified as the regulatory subunits of DNA-dependent protein kinase, Ku 86 and Ku70, which interact with double stranded DNA primarily in a nonspecific fashion (32, 33) because they can be competed by the addition of unlabeled probe (Fig. 4B).
To investigate whether the sequences mutated in Mut 4 (between -174/-170 bp) and Mut 6 (between -166/-162 bp) comprise a single bipartite NF-1- regulatory element, as discussed above, we generated a double mutant (Mut 4/6). The promoter activity of the Mut 4/6 construct was compared with the WT -188 construct, Mut 4, and Mut 6 constructs under basal conditions, in normal and PCOS theca cells. As shown in Fig. 4C, mutation of either half of the putative NF-1 site (Mut 4 or Mut 6) or both half sites of the bipartite recognition sequence (Mut 4/6) was observed to ablate increased promoter regulation in PCOS theca cells. Furthermore, mutation of both half sites did not further affect promoter function in PCOS theca cells.
Examination of NF-1C-Dependent Regulation in Normal and PCOS Theca Cells
The genes of NF-1 family members are subject to differential splicing, resulting in the formation of multiple isoforms of each gene (34). To examine whether the NF-1 family member, NF-1C, contributes to NF-1 binding to the CYP17 promoter, supershift analysis was performed with antisera specific to the C terminus of NF-1C. This antisera does not cross react with other NF-1 family members (35). Addition of NF-1C (8199)-specific antibody resulted in the formation of two supershifted complexes, whereas incubation with the NF-1 antibody (common to all NF-1 family members) competed for NF-1 binding (Fig. 5A). Along with the data in Fig. 4, these data demonstrate that an element within -174/-162 bp of the CYP17 promoter, that is required for increased promoter function in PCOS theca cells, specifically binds NF-1C.
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, these NF-1C isoforms result primarily from alternative splicing within the C-terminal region of NF-1C, which contains two domains involved in transactivation, the proline-rich domain (PRD) and a weaker transactivation domain (TAD) within the central region of the NF-1 protein (37). The C terminus of NF-1C has also been reported to function as a transcriptional repression domain, although, the specific domains involved in repression have not been identified (34). The DNA-binding domain (DBD) of NF-1 proteins is highly conserved and resides within the first 220 residues (34). To investigate the NF-1C isoforms expressed in normal and PCOS theca cells, RT-PCR was performed with primers specific for NF-1C isoforms. The primers used in these experiments amplify exon 3 through exon 11 of NF-1C (Fig. 5B), and therefore have the potential to amplify all known isoforms of NF-1C, with the exception of NF-1C3, which lacks exon 3. As shown in Fig. 5C, three NF-1C isoforms, of approximately 950, 800, and 700 bp in length were similarly expressed in normal and PCOS theca cells. Based on the predicted size of known NF-1C isoforms, these mRNA species were identified as NF-1C1 (946 bp), NF-1C2 (792 bp), and NF-1C5 (706 bp), respectively. RT-PCR of the bona fide NF-1C1, NF-1C2, and NF-1C5 cDNAs (see Materials and Methods) resulted in PCR products with sizes identical to those observed after RT-PCR of theca cell RNA. As a positive control, NF-1C isoforms were also amplified from RNA isolated from adrenocortical H295 cells, where the expression of NF-1C2 and NF-1C5 protein has been described (29). RT-PCR using alternative NF-1C specific primer pairs did not detect NF-1C3, NF-1C4, NF-1C6, or NF-1C7 in human theca cells (data not shown). Together, these data confirm that similar NF-1C mRNA splice variants are expressed in normal and PCOS theca cells.
To investigate whether NF-1C isoforms affect CYP17 gene expression, normal and PCOS theca cells were transfected with increasing concentrations of NF-1C1, NF-1C2, or NF-1C5 expression vectors, and -188 CYP17 promoter function was examined under basal conditions. In agreement with previous observations, -188 CYP17 promoter activity was 2- to 3-fold higher in PCOS theca cells as compared with normal theca cells. Cotransfection of each of the three NF-1C expression vectors resulted in a dose-dependent decrease in -188 CYP17 promoter function in both normal and PCOS theca cells (Fig. 5D). Cotransfection of 1.0 µg/dish of NF-1C1, NF-1C2, or NF-1C5 resulted in approximately 60–70% reduction in -188 CYP17 promoter activity in normal theca cells, and a 75–85% reduction in PCOS theca cells. Similar reductions in CYP17 promoter function by NF-1C isoforms were observed after forskolin treatment (data not shown). These studies demonstrate that each of the three NF-1C isoforms expressed in theca cells have the potential to inhibit CYP17 promoter function in normal and PCOS theca cells.
Comparison of NF-1 Binding in Normal and PCOS Theca Cells
To compare the relative levels of NF-1 binding in normal and PCOS theca cells, EMSA was performed using NEs isolated from normal and PCOS theca cells maintained under basal conditions, and a radiolabeled oligonucleotide corresponding to -180 to -150 bp of the CYP17 promoter. To better resolve DNA/protein binding to this element we used 8% polyacrylamide gels in these experiments. The observed intensities of NF-1-containing complexes were less in PCOS NEs than in normal NEs (Fig. 6A). A comparable reduction in NF-1 complexes in PCOS theca cell NEs was also observed under forskolin-stimulated conditions (data not shown). Quantitation of the relative NF-1 binding obtained from EMSA of five individual normal and five individual PCOS NEs demonstrates that NF-1 binding is reduced by approximately 70% in PCOS NEs (Fig. 6A).
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), with a trend toward increased SF-1 binding in PCOS NEs. In both normal and PCOS, SF-1 binding in individual NEs appeared to have a reciprocal relationship with respect to NF-1C binding (compare normal NEs, lanes 2 and 4, or PCOS NEs, lanes 5 and 6). Together, these data demonstrate that both NF-1 and SF-1 bind sequences within -180/-150 bp of the CYP17 promoter and suggest that NF-1 and SF-1 may compete for binding to overlapping elements.
Additional EMSA was performed utilizing normal and PCOS NEs and a probe corresponding to a shorter sequence of the promoter, from -178 to -158 bp, which retains the NF-1 site, but lacks the SF-1 site. As shown in Fig. 6B, the level of NF-1C binding to this sequence was also decreased approximately 70% in PCOS, similar to those presented for the -180/-150 bp probe (Fig. 6A). As expected, no SF-1 binding was observed. These data suggest that in the absence of SF-1 binding, NF-1 binding to sequences between -178 and -158 bp of the CYP17 promoter is reduced in PCOS theca cells and does not result from competition with SF-1.
Subsequent Western analysis using NF-1C antisera detected several NF-1C proteins (
50 kDa) in both normal and PCOS theca cell NEs, suggesting the presence of several NF-1C isoforms (Fig. 6C). The amount of immunoreactive NF-1C isoforms was reduced, to approximately the same extent as binding was reduced, in NEs isolated from PCOS theca cells as compared with normal theca cells. These data demonstrate that the observed decrease in NF-1C binding in PCOS theca cells may result from a reduction in nuclear NF-1C. Together with the finding that NF-1C represses promoter function, these studies suggest that increased CYP17 promoter activity in PCOS may result, in part, from a relief of NF-1C-dependent repression.
Comparison of SF-1 Binding in Normal and PCOS Theca Cells
To examine SF-1 binding, EMSA was performed using a probe corresponding to -163/-140 bp of the CYP17 promoter, which contains a consensus SF-1 site but lacks the NF-1 binding site, and NE isolated from normal and PCOS theca cells. As shown in Fig. 7A, addition of SF-1 antisera resulted in a reduction in binding of a specific complex, indicating that SF-1 binds this sequence. A comparison of SF-1 binding in normal and PCOS NEs indicated that the intensity of SF-1 binding was similar in normal and PCOS NEs. Because SF-1 cotransfection was required to investigate differential regulation of this element in normal and PCOS theca cells, experiments were also performed to examine whether cotransfected SF-1 modulates SF-1 and NF-1C binding. To investigate SF-1 binding alone, EMSA was performed using the -163- to -140-bp SF-1 site and NE isolated from cells transiently transfected with either empty pcDNA expression vector or pcDNA encoding human SF-1 (hSF-1) (0.1 µg/dish). As shown in Fig. 7B, SF-1 cotransfection did not appear to affect SF-1 binding. To investigate both NF-1 and SF-1 binding in transfected cells, these experiments were also performed with the -180 to -150-bp probe. As shown in Fig. 7C, DNA/protein binding was similar in NEs isolated from both transfected and nontransfected cells and both SF-1 and NF-1-containing complexes were observed. Furthermore, SF-1 cotransfection did not alter overall SF-1 and/or NF-1C binding. Together, these data demonstrate that SF-1 interacts with sequences within -163 /-140 bp of the CYP17 promoter and suggests that cotransfection of SF-1 does not appear to dramatically modify binding to this region.
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, CYP17 promoter activity was examined after cotransfection of full-length NF-1C2, or an expression vector encoding the DBD of NF-1C (NF-1C-DBD). As expected, inhibition was observed with full-length NF-1C2, whereas cotransfection with NF-1C-DBD resulted in an approximately 4-fold increase in CYP17 promoter activity (P < 0.001) in normal theca cells and an approximately 2-fold increase in CYP17 promoter activity in PCOS theca cells (P < 0.01), as compared with the empty vector (pcDNA) (Fig. 8). The observation of increased CYP17 promoter activity in the presence of cotransfected NF-1C-DBD also suggests that the DBD may compete with endogenous NF-1C proteins for DNA binding, and interfere with NF-1C-dependent repression. The reduced ability of the DBD to augment CYP17 promoter activity in PCOS theca cells may result from decreased endogenous NF-1 binding to the CYP17 promoter in PCOS theca cells. This suggests that a relief of repression model may explain the differential expression because CYP17 promoter function was similar in both normal and PCOS theca cells after cotransfection with NF-1C or NF-1C-DBD.
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, mutation of the NF-1 site did not ablate NF-1C2-dependent inhibition of CYP17 promoter activity. Cotransfection of NF-1C2 inhibited Mut 4/6 promoter activity by approximately 60% (*, P < 0.05). These data suggest that an additional NF-1 binding site may be involved in NF-1C-dependent repression of the CYP17 promoter.
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). Incubation with unlabeled WT -113/-75-bp, or NF-1 consensus oligonucleotide resulted in a reduction in the intensity of all protein-DNA complexes. In contrast, addition of 50-fold excess of the mutant -113/-75 oligonucleotide partially competed for the formation of all but two protein-DNA complexes, indicated by arrows. Supershift analysis utilizing the NF-1C specific antisera (8199) resulted in the formation of supershifted complexes, as well as the dimunition of two specific complexes. Addition of NF-1 antisera competed with the same two specific complexes. Together, these data verify that NF-1C in theca cell NEs also binds to sequences within -113 to -75 bp of the CYP17 promoter.
The Proximal NF-1 Site Is Not Required for Increased Promoter Function in PCOS
To evaluate the extent to which these NF-1 sites contribute to CYP17 promoter function, the promoter activity of constructs containing mutations in: 1) the distal site (site A) between -174/-162 bp (Mut A); 2) the more proximal site (site B) from -102/-90 (Mut B); or 3) both NF-1 sites (Mut A/B), was examined within the context of the WT -188 CYP17 promoter (WT) (Fig. 10A). To independently examine NF-1C-dependent regulation of the two NF-1 sites, we compared the promoter activities of the WT, Mut A, Mut B, and the double mutant (Mut A/B) after cotransfection with pcDNA vectors encoding NF-1C2 or DBD/NF1C in normal theca cells. As shown in Fig. 10B, NF-1C2 cotransfection inhibited WT, Mut A, and Mut B promoter activity approximately 60% (*, P < 0.05). In contrast, Mut A/B promoter activity was not significantly affected by cotransfection of NF-1C2. Following cotransfection with NF-1C-DBD expression vector, the promoter activity of the WT and Mut B construct was increased 3-fold (*, P < 0.05) in the presence of NF-1C-DBD. In contrast, NF-1C-DBD cotransfection did not significantly affect the promoter activity of Mut A or Mut A/B. The finding that the NF-1C-DBD enhances Mut B promoter function while having no effect on Mut A/B promoter function, suggests that the mechanism of NF-1 inhibition at each site may be different. Although both NF-1 sites confer NF-1-dependent repression by cotransfected NF-1C2, only repression at the distal (site A) NF-1 site appears to be affected by competition for DNA binding by cotransfected DBD.
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). Mutation of the proximal NF-1 site (Mut B) also reduced promoter function in both cell types. However, Mut B promoter activity was approximately 2-fold higher in PCOS cells (aP < 0.01), as compared with normal cells. Data obtained after mutation of both NF-1 sites (Mut A/B) was not significantly different from those observed with Mut A. In addition, Mut A/B promoter function was not different in normal and PCOS theca cells. In agreement with our deletion and mutation analyses (Figs. 1–3), these data suggest that the proximal NF-1 site (site B, located at -102/-90 bp of the promoter), may not be required for increased CYP17 promoter function in PCOS. These data indicate that the distal NF-1 site (site A), located within -174/-162 bp of the CYP17 promoter, appears to be required for increased CYP17 promoter function in PCOS theca cells. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Analysis of CYP17 promoter function in normal and PCOS theca cells has localized elements within the CYP17 promoter that are required for basal and cAMP-dependent regulation in human theca cells. Sequences between -188 and -147 bp of the promoter were observed to confer basal regulation, whereas cAMP-dependent regulation appears to involve two independent elements, within -172 to -147 bp and -109 to -61 bp of the CYP17 promoter. Although we have observed that promoter function is increased in PCOS theca cells under both basal and cAMP-stimulated conditions, the dysregulation of the CYP17 gene expression in PCOS theca cells appears to be primarily basal in nature (15). Deletion analyses have demonstrated that the increase in basal promoter function in PCOS theca cells is conferred by sequences between -172 and -147 bp of the CYP17 promoter. Scanning mutation analysis of the -188- to -140-bp region, within the context of -188 bp of the promoter, demonstrated that specific mutations within a 16-bp element, spanning -174 to -158 bp, ablated increased basal CYP17 promoter function in PCOS theca cells. This element coincides with a binding site for NF-1 (29). The bipartite nature of the NF-1 consensus binding site, TGGN7CCA, directly corresponds to sequences altered in scanning mutant constructs M4, M6, and M7, whereas the sequences altered in M5 correspond to the linker nucleotides (N7) within the bipartite NF-1 site, which are not required for NF-1 binding (38). Therefore, mutations of the CYP17 promoter that affect NF-1 binding are associated with a loss of augmented basal promoter function observed in PCOS theca cells. Utilization of a NF-1C specific antibody demonstrated that the NF-1 family member, NF-1C, is one primary component of the NF-1/DNA complexes. EMSA indicated that the level of NF-1/DNA complex formation is decreased in PCOS NEs. This reduction in NF-1 binding was associated with a decrease in nuclear levels of NF-1C protein in PCOS theca cells. Both normal and PCOS theca cells express several NF-1C mRNA species, including the full-length NF-1C1 and differentially spliced isoforms NF-1C2 and NF-1C5. Cotransfection of these NF-1C isoforms inhibited CYP17 promoter activity, demonstrating that NF-1C acts to repress CYP17 promoter function in theca cells. The finding that NF-1C cotransfection equalizes promoter function in normal and PCOS theca cells (Fig. 8), together with the observation that endogenous NF-1C expression and binding is reduced in PCOS theca cells, indicates that increased CYP17 expression in PCOS theca cells results, in part, from a reduction in NF-1C repression.
NF-1 family members have been implicated in the transcriptional repression of numerous genes, including the insulin-responsive glucose transporter GLUT4 (39, 40), liver X receptor (41), and placental GH (42). The mechanism of NF-1C-mediated repression is likely to depend not only on the promoter context, but the cell-specific expression of specific NF-1C isoforms and the relative abundance of transcription factors with adjacent and/or overlapping binding sites. NF-1C has been reported to actively repress transcription through the recruitment of corepressors (43) by repression domains within the C terminus, or passively as a result of competition for overlapping binding sites for other transactivators, such as Sp1 (44). From our studies with the full length CYP17 promoter, overall NF-1C repression does not appear to result simply from competition with other factors (i.e. SF-1) because cotransfection of DBD failed to inhibit CYP17 promoter function. Furthermore, the observation that the DBD of NF-1C stimulates rather than inhibits CYP17 promoter function, is in agreement with the finding that NF-1C functions as an active repressor.
The observation that cotransfection of NF-1C or DBD normalizes promoter activity in normal and PCOS theca cells suggests that altered NF-1C-dependent repression contributes to differential CYP17 promoter function. Based on our data, NF-1C dependent repression of CYP17 gene expression appears to involve regulation through two distinct sites. However, only one of these sites (site A) is required for increased CYP17 promoter function in PCOS theca cells, suggesting underlying differences in the overall regulation of these two sites (Fig. 10). In cotransfection experiments, each of the NF-1 sites examined conferred NF-1C-dependent repression, whereas the DBD affected only the distal site (site A). With respect to site A, the stimulation in CYP17 promoter function in response to DBD cotransfection is most consistent with direct competition or displacement of NF-1C, and relief of NF-1C repression. In contrast, the observation that the DBD had no effect on site B indicates that the DBD may compete with the binding of other factors to a site that overlaps this NF-1C site. Further investigation of the mechanisms regulating NF-1-dependent repression will be necessary to identify the underlying mechanisms involved in differential NF-1 binding and repression in PCOS.
The combined findings of NF-1C repression of CYP17 promoter function and reduced NF-1C binding in PCOS, appear to be in conflict with observations of diminished CYP17 promoter activity after mutation of NF-1 binding sites (Fig. 3). However, basal CYP17 promoter function was reduced after mutation of every 5-bp sequence within -188 to -140 bp, with the exception of Mut 5 (Fig. 3). Overall, these data suggest that basal regulation of the CYP17 promoter within this region is complex and may involve cooperative regulation by several elements and factors. Therefore, it is likely that the mutations generated within the NF-1 binding sites may alter overlapping recognition site(s) for stimulatory factor(s) that mediate basal CYP17 gene expression. The finding that SF-1 binds to this region suggests that SF-1 is one of these stimulatory factors. Furthermore, the observations that putative SF-1 sites may have been altered in these mutants, and that SF-1 cotransfection is required to examine promoter regulation (15), suggest that both activation of the promoter and differential regulation in normal and PCOS cells involves both NF-1C and SF-1. Our EMSA data suggests that there may be a reciprocal relationship between NF-1 and SF-1 binding to the CYP17 promoter (Fig. 6A). Increased NF-1 binding appears to be correlated with decreased SF-1 binding and visa versa. However, in the absence of an NF-1 binding site, SF-1 binding is not different in normal and PCOS theca cells, suggesting that SF-1 alone does not confer increased regulation in PCOS theca cells. In addition, StAR promoter function is not increased in PCOS theca cells (15), and multiple copies of an SF-1 consensus element fused to the -61 CYP17 promoter do not confer increased promoter function in PCOS theca cells (data not shown). Therefore, it is likely that SF-1 interacts with unidentified stimulatory factor(s) to increase CYP17 promoter function in PCOS theca cells. Mutations within NF-1 sites that eliminate NF-1 binding have been reported to reduce transactivation by members of the steroid hormone receptor superfamily (34). Therefore, NF-1 may also function as an accessory factor, which may modulate the binding of other stimulatory and/or inhibitory factors to the CYP17 promoter.
It is interesting to note that comparison of the mRNA expression profiles of normal and PCOS theca cells used throughout this report did not reveal differences in NF-1C mRNA (16). However, several genes previously reported to be regulated by NF-1 were differentially expressed in PCOS theca cells, including collagen 11 (45), Myc (46), IGF binding protein (47), IL-18 (48), and aldehyde dehydrogenase type 3 (49). In addition to altered CYP17 gene expression, we have also reported that increased androgen production in PCOS theca cells is associated with increased gene expression of several steroidogenic enzymes including CYP11A and 3ß-HSDII (12, 13). Preliminary analysis of the CYP11A promoter in normal and PCOS theca cells suggests that increased transcriptional activation contributes to increased CYP11A gene expression in PCOS. The presence of putative NF-1 sites within the CYP11A and 3ßHSDII promoters suggests that dysregulation of NF-1 may also contribute to increased CYP11A and HSD3B2 gene expression in PCOS (50). Further studies are necessary to examine whether NF-1-dependent repression is also involved in altered CYP11A and 3ß-HSDII promoter function in PCOS theca cells.
The ability of NF-1 family members to regulate gene expression in a wide variety of cell types also makes it a suitable candidate for involvement in PCOS phenotypes peripheral to the ovary. The observation that NF-1 mediates repression of the GLUT4 promoter in response to prolonged insulin stimulation in adipocytes is also interesting with respect to altered NF-1C-dependent regulation in PCOS theca cells (40). NF-1 has been implicated in the transcriptional regulation of a variety of promoters whose gene expression is controlled by cAMP (39), TGF-ß (51), TNF (52), vitamin D (53), and glucocorticoids (54, 55). This potential for NF-1 to regulate gene expression in response to a variety of stimuli suggests that NF-1 dysregulation in PCOS theca cells may result from signaling defects upstream of NF-1. Tissue-specific differences in signaling mechanisms regulating NF-1 expression and/or function may also explain tissue-specific differences in insulin resistance and steroidogenesis in PCOS. It will be of interest to determine whether dysregulation of NF-1C action is specific to theca cells or also occurs in other tissues in PCOS.
The identification of reduced NF-1-dependent repression of the CYP17 promoter in PCOS theca cells provides one potential model by which CYP17 and other genes required for androgen biosynthesis may be coordinately up-regulated in PCOS. The exact mechanism(s) underlying NF-1C-dependent regulation of the 16-bp basal element between -174 and -158 bp of the CYP17 promoter are unknown. The identification of additional factors that regulate this element, as well as contribute to increased basal regulation in PCOS theca cells, will further our understanding of the underlying cause of increased CYP17 gene expression and possibly other abnormalities in PCOS, e.g. insulin resistance, obesity, arrested follicular development, and endometrial cancer.
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MATERIALS AND METHODS |
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The PCOS and normal ovarian tissue came from age-matched women, 38–40 yr old. The diagnosis of PCOS was made according to established guidelines (57), including hyperandrogenemia, oligoovulation, and the exclusion of 21-hydroxylase deficiency, Cushing’s syndrome, and hyperprolactinemia. All of the PCOS theca cell preparations studied came from ovaries of women with fewer than six menses per year and elevated serum total testosterone or bioavailable testosterone levels, as previously described (1, 12). Each of the PCOS ovaries contained multiple subcortical follicles of less than 10 mm in diameter. The control (normal) theca cell preparations came from ovaries of fertile women with normal menstrual histories, menstrual cycles of 21–35 d, and no clinical signs of hyperandrogenism. Neither PCOS nor normal subjects were receiving hormonal medications at the time of surgery. Indications for surgery were dysfunctional uterine bleeding, endometrial cancer, and pelvic pain. The passage conditions and split ratios for all normal and PCOS cells were identical. Experiments comparing PCOS and normal theca were performed utilizing fourth-passage (31–38 population doublings) theca cells isolated from size-matched follicles obtained from age-matched subjects. Sera and growth factors were obtained from the following sources: FBS and DMEM/F12 were obtained from Irvine Scientific (Irvine, CA): horse serum was obtained from Life Technologies (Grand Island, NY); UltroSer G was from Reactifs IBF (Villeneuve- la-Garenne, France): other compounds were purchased from Sigma (St. Louis, MO).
Construction of CYP17 Luciferase Constructs
Reporter gene plasmids containing sequentially smaller fragments of the 5'-flanking region of the human CYP17 gene were constructed using either existing restriction sites; -750/+44 (KpnI/NaeI), -235/+44 (SacI/NaeI), -188/+44 (BspE1/NaeI), and -109/+44 (SacI/NaeI), or by synthesis of double-stranded oligonucleotides corresponding to the indicated sequence; -172 CYP17 LUC, -147 CYP17 LUC, and -61 CYP17 LUC. The oligonucleotides were subsequently subcloned into either the luciferase vector pGL3 basic (Promega Corp., Madison, WI) or -109/+44 CYP17 LUC utilizing a BglII (-147) or XbaI (-172) site at the 5' end and a SacI (-172, -147) or EcoNI (-61) site at the 3' end. The -235 (188/140) CYP17 construct was generated by subcloning oligonucleotides corresponding to sequences from -235 to -109, with sequences from -188 to -140 bp deleted, into -109 CYP17/LUC. The (-188/-140)-61 CYP17 construct was generated by subcloning oligonucleotides corresponding to the -188/-140 sequence, shown in Table 1, into -61 CYP17/LUC. The -188 CYP17/LUC scanning mutant constructs, containing 5-bp transition changes between -188 and -140 bp (-188 Mut 1 to -188 Mut 11 CYP17/LUC), were generated by synthesis of double-stranded oligonucleotides corresponding to sequences within -188/-109 bp of the CYP17 promoter with the indicated base pair changes (Table 1), and subcloned into -109 CYP17/LUC (5'-BglII/SacI-3'). The promoter sequences of all constructs were confirmed by automated DNA sequencing.
The -188 mutant constructs containing 5-bp transition changes within the consensus NF-1 site at -174/-170 bp (Mut 4), -166/-162 bp (Mut 6), and both -174/-170 bp and -166/-162 bp (Mut 4/6, Mut A) were generated by synthesis of oligonucleotides corresponding to sequences within -188/-109 bp of the CYP17 promoter with the indicated base pair changes (Table 1). These oligonucleotides were annealed and subcloned into -109 CYP17/LUC. The -188 CYP17/LUC constructs containing alterations in the NF-1 site at -102/-90 bp, Mut B and Mut A/B, were generated by PCR amplification of the -109 CYP17 construct with a mutant forward primer (5'-GAGCTCAGGCCTAACTGGGCTTTAGGAGAATCTTTCCAC-3'), which corresponds to the SacI site at -110 bp and contains the mutations indicated in bold (GG
AA and AATT) within the NF-1 site between -102 and -90 bp (site B). The reverse primer (5'-GAAAGATTCTCCTAAA GCC CAGTTAGGCCTGAG-3') corresponded to sequences within the polylinker of pGL3 basic, and overlapped the HindIII restriction site for cloning. The PCR product containing the appropriate mutations was amplified by 20 cycles of Expand Long Template PCR (Roche Diagnostics, Indianapolis, IN), and subcloned (SacI/HindIII) into either the WT -188 CYP17 construct, or the mutant -188 Mut 4/6(A) CYP17 construct. All constructs were confirmed by automated DNA sequencing.
Transient Transfection of Normal and PCOS Theca Cells
Human theca cells isolated from normal cycling women and women with PCOS were transfected as previously described (15, 58). One hour before transfection, the cells were transferred into DMEM high-glucose medium containing 20 mmol/liter HEPES and 2% heat-inactivated calf serum (Atlanta Biologicals, Atlanta, GA) and moved to a 3% CO2, 95% ambient air, 37 C incubator. The calcium phosphate precipitate contained 20 µg/dish of CYP17 promoter luciferase plasmid, 0.1 µg/dish of expression vector for hSF-1 (hSF-1pcDNA3.1) (31) and 1 µg/dish of an expression vector for ß-galactosidase (pSVß-gal, Promega). SF-1 cotransfection was performed based on previous observations that SF-1 may be a limiting factor for the CYP17 promoter under transfection conditions (15). In cotransfection experiments, empty pcDNA 3.1(+) expression vector (Invitrogen, Carlsbad, CA) and/or pcDNA 3.1 expression vector containing the cDNA for human NF-1C isoforms, NF-1C1, NF-1C2, or NF-1C5 (0.1–1 µg/100-mm dish), were added to the DNA/CaPO4 solution. The total amount of expression vector was kept constant at 1.0 µg/100-mm dish. A plasmid expressing ß-galactosidase (pSV-ßgal) was also cotransfected (1.0 µg/100-mm dish) for normalization of transfection efficiency between replicates and transfection experiments (15). Cotransfection of the expression vector encoding the DNA-binding domain of NF-1C (NF-1C-DBD) was performed with 1 µg/100-mm dish. Following transfection, the cells were rinsed with 15% glycerol/HEPES buffered salt solution and PBS, and treated in transfection media containing vehicle or 20 µM forskolin for 72 h. Cells were harvested with trypsin, pelleted, and resuspended in reporter lysis buffer (Promega). Luciferase activity was determined by utilizing the Luciferase Assay System (Promega) on a Sirius Luminometer (Zylux Corp., Maryville, TN). ß-Galactosidase activity was measured by the chemiluminescent assay Galacto-Light Plus (Tropix/PE Applied Biosystems, Bedford, MA) and used for normalization of transfection efficiency.
Transfections were performed in triplicate in theca cells isolated from at least five independent normal, and five independent PCOS patients, using plasmid obtained from several DNA preparations. The mean results from transfection analysis of theca cell cultures from individual patients were collected (n =5) and unpaired two-tailed t tests and/or ANOVA were performed using Prism 3.0c for Macintosh (GraphPad Software, San Diego, CA).
NE Preparation
Human theca cells were transferred into serum-free medium containing DMEM/F12, 1.0 mg/ml BSA, 100 µg/ml transferrin, 20 nM insulin, 20 nM selenium, 1.0 µM vitamin E and antibiotics, in the absence or presence of 20 µM forskolin. At 24 h, cells were harvested with trypsin/EDTA and NEs were prepared as previously described (59) with the exception that the buffers contained 1 µM dithiothreitol, 0.5 µM PMSF, 2 µg/ml leupeptin, 1 µM benzamidine, 1 µM sodium orthovanadate, and 20 µM sodium fluoride to inhibit protein phosphatases and proteases. Protein concentrations of NEs were determined by bicinchoninic acid protein assay (Pierce, Rockford, IL). NEs prepared from transfected cells were transfected as described above with 20 µg/dish of pGL3basic and 0.1 µg/dish of either empty pcDNA3.1+ or hSF-1pcDNA plasmid.
EMSA
Double-stranded oligonucleotides were generated corresponding to -188/-109 bp, -188/-140 bp, -180/-150 bp, -178/-158 bp, -163/-140 bp, or -113/-75 bp of the 5'-flanking sequence of the CYP17 promoter. Oligonucleotide probes were end labeled using [
-32P] ATP and T4 polynucleotide kinase, and incubated with NEs (2–5 µg) from human theca cells in the presence of 20 mM HEPES, 40 mM KCl, 0.5 mM EDTA, 0.5 mM EGTA, 50 µg/ml poly(deoxyinosine-deoxycytidine), 2.5 mM dithiothreitol, 0.5% Nonidet P-40, and 0.1 µg salmon sperm DNA for 15 min at room temperature. One half of the reaction was separated on a 6–8% nondenaturing PAGE for 2–3 h at 150 V with 1x Tris-glycine EDTA running buffer. The gel was then dried and exposed to autoradiography film.
For competition experiments, 50-fold excess of cold competitor oligonucleotides were included in the reaction with labeled oligonucleotide probe and incubated simultaneously with theca cell NEs. Mutant -188/-109 oligonucleotides used for competition analysis are presented in Table 1, and contain overlapping 5-bp mutations spanning sequences from 174 to -162 bp of the CYP17 promoter (M4, M5, M6). The mutant -113/-75 oligonucleotide used for competition analysis is presented in Fig. 7B, and contains mutations in both half-sites of the putative NF-1 site.
For supershift analyses, specific antisera (1–3 µl) were incubated with NE for 15 min at room temperature before addition of labeled probe. Two different NF-1 antibodies were used in these studies. An NF-1 antibody specific for the amino-terminal region of NF-1 was obtained from Santa Cruz Biotechnologies (Santa Cruz, CA) and is cross-reactive with all NF-1 family members. NF-1C (8199) antisera specific for the C-terminal half of NF-1C, was a generous gift from Dr. Naoko Tanese (New York University, NY). The antibody to SF-1 was obtained from Affinity BioReagents (Golden, CO).
Western Analysis
Theca cell NEs (15 µg/lane) were separated by 10% SDS-PAGE (60), transferred to polyvinylidene difluoride membrane, and incubated with antisera specific to NF-1C (8199) followed by donkey-antirabbit horseradish peroxidase- conjugated secondary antibody, and detected by ECL from Amersham Pharmacia Biotech (Piscataway, NJ). All autoradiograms were scanned and densitometric analysis was performed using Image Quant version 1.2 (Molecular Dynamics, Sunnyvale, CA). Data was normalized using antisera specific to Ku70, the 70-kDa subunit of DNA-dependent protein kinase (33), from Santa Cruz Biotechnology.
Amplification and Cloning of NF-1C Isoforms from Human Theca Cell RNA
The reverse transcription reaction was performed using 5 µg of total RNA isolated from normal theca cells maintained in serum-free media for 24 h, with Moloney murine leukemia virus reverse transcriptase (Promega, WI), and oligo-deoxythymidine 16 primer. For cloning purposes, 1 µg of the reverse transcriptase reaction was amplified by 30 cyc
