Transcriptional Regulation of the 17alpha-Hydroxylase Gene in Normal and Polycystic Ovary Syndrome Ovarian Theca Cells
Open Access
- Author:
- Wickenheisser, Jessica K
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 07, 2002
- Committee Members:
- Janette Marie Mcallister, Committee Chair/Co-Chair
Patrick Quinn, Committee Member
Gary Alan Clawson, Committee Member
Richard Scott Legro, Committee Member
Robert G Levenson, Committee Member - Keywords:
- theca cells
ovary
transcription
CYP17
polycystic ovary syndrome - Abstract:
- Polycystic ovary syndrome (PCOS) is an endocrine disorder affecting approximately 5-10% of women of reproductive age, and is characterized by infertility, hirsutism, abnormal follicular development, and increased ovarian androgen production. The phenotype of elevated androgen production persists in theca cells isolated from PCOS women and maintained in long-term culture. Increased androgen biosynthesis in PCOS theca cells is correlated with increased enzyme activity and gene expression of several steroidogenic enzymes responsible for androgen biosynthesis including cytochrome P450 17a-hydroxylase (CYP17) and cytochrome P450 cholesterol side chain cleavage (CYP11A). The overall objective of this thesis research was to test the hypothesis that altered transcriptional activation of the CYP17 promoter contributes to increased CYP17 gene expression in PCOS. Specifically, the mechanism(s) involved in increased CYP17 expression was examined by I) comparison of CYP17 promoter activities in normal and PCOS theca cells, II) examination of minimal region(s) of the CYP17 promoter required for increased CYP17 gene expression, and III) identification of putative transcriptional regulators involved in CYP17 gene expression in normal and PCOS theca cells. Studies were also initiated to examine whether the transcriptions factors involved in increased CYP17 transcription in PCOS theca cells coordinately increase CYP11A gene expression. The comparison of CYP17 promoter function in normal and PCOS theca cells indicated that CYP17 transcription was increased in PCOS theca cells. Sequences within the general boundaries of -235 to -109 bp of the CYP17 promoter were required for increased promoter function in PCOS theca cells. Additional deletion and mutational analysis of the CYP17 promoter was performed to characterize basal and cAMP-response elements and identify the minimal sequences involved in increased CYP17 promoter function in PCOS. Results of these analyses demonstrated that sequences between -188 and -147 bp of the CYP17 promoter were required for significant basal promoter function, whereas, cAMP-dependent regulation was conferred by two separate regions located within -172/-140 bp and -109/-61 bp. Sequences between -188 and -140 bp were both necessary and sufficient for increased promoter function in PCOS theca cells. Scanning mutation analysis of this region demonstrated that a 16 bp sequence, between -174 and -158 bp, was required for increased promoter function in PCOS theca cells. Examination of this sequence revealed a nuclear factor-1 (NF-1) binding site within -174/-162 bp of the promoter. A specific NF-1 family member, NF-1C, was found to bind this sequence and repress CYP17 promoter function in theca cells. Mutation of this NF-1 site ablated NF-1C binding and NF-1C-dependent CYP17 repression. Comparison of NF-1C binding in normal and PCOS nuclear extracts demonstrated that the level of NF-1C binding was reduced in PCOS. CYP11A promoter function was also repressed by cotransfection of NF-1C in theca cells. The C-terminal domain of NF-1C was found to be required for NF-1C-dependent repression of CYP17 and CYP11A transcription. These studies suggest that the diminished NF-1C-dependent repression in PCOS theca cells may be associated with increased CYP17 and CYP11A promoter activity in PCOS. The identification of NF-1C as an inhibitor of both CYP17 and CYP11A promoter function provides a possible common mechanism by which the expression of multiple steroidogenic enzymes are dysregulated in PCOS. Future studies are required to identify how NF-1C and additional transcription factors coordinately regulate CYP17 and CYP11A transcription in PCOS theca cells.