Differential Activity of the Cytochrome P450 17 -Hydroxylase and Steroidogenic Acute Regulatory Protein Gene Promoters in Normal and Polycystic Ovary Syndrome Theca Cells

Abstract

17alpha-Hydroxylase (CYP17) expression in propagated theca cells isolated from the ovaries of women with polycystic ovary syndrome (PCOS) is persistently elevated, compared with theca cells isolated from normal ovaries. To investigate the mechanism for increased CYP17 messenger RNA accumulation in PCOS theca cells, we examined CYP17 and steroidogenic acute regulatory protein (StAR) promoter activities in normal and PCOS theca cells. Conditions were established to transiently transfect human theca cells with reporter gene constructs containing 5' truncations of the human CYP17 and StAR promoters. Cotransfection of a steroidogenic factor-1 expression plasmid was found to be required for detection of basal and forskolin-stimulated CYP17 promoter activity but not for StAR promoter activity. However, cotransfection with a steroidogenic factor-1 expression plasmid augmented both basal and forskolin-stimulated StAR promoter activity. CYP17 reporter activity was compared in theca cells isolated from normal and PCOS patients. Basal and forskolin-stimulated CYP17 promoter activity was 4-fold greater in PCOS cells than in theca cells isolated from normal ovaries. In contrast, StAR promoter activity, and the activity of a reporter construct containing three copies of a cAMP response element (3xCRE), were similar in normal and PCOS theca cells. The results of these studies document: 1) that basal and cAMP-dependent CYP17 gene transcription is increased in PCOS theca cells; 2) that there is differential regulation of promoters of genes required for steroidogenesis in PCOS theca cells; and 3) that passaged normal and PCOS theca cells provide a model system for studying tissue-specific regulation of genes encoding steroidogenic enzymes and identifying the molecular mechanisms involved in increased androgen production in PCOS.

Overexpression of a DENND1A isoform produces a polycystic ovary syndrome theca phenotype

“…The results of the above studies showed that DENND1A.V2 significantly increased −770 CYP17A1/LUC promoter function in normal theca cells, converting the cells to a PCOS phenotype. Similar results were observed with a reporter construct containing −235/+44 of the 5′ flanking sequence of the CYP17A1 gene (−235 CYP17A1/ LUC), which also confers increased transcriptional regulation in PCOS theca cells (17,36).…”

“…This increased androgen and progesterone biosynthesis in PCOS theca cells have been attributed, in part, to increased CYP17A1 and CYP11A1 gene transcription and RNA stability (17,20,36). Our molecular characterization of normal and PCOS theca cells from multiple individuals by microarray analysis and quantitative PCR (qRT-PCR) also established that normal and PCOS cells have distinctive molecular signatures (16,37,38).…”

“…To examine the effects of DENND1A.V2 on CYP17A1 transcription, normal theca cells were transfected with pGl3 luciferase reporter plasmid containing −770/+44 of the 5′-flanking regions of the human CYP17A1 gene (−770 CYP17A1/LUC) using the calcium phosphate method (17,36). Following transfection the cells were infected with the DENND1A.V2 adenovirus or control adenovirus for 1 h, and treated with serum-free medium with and without 20 μM forskolin, an activator of adenylate cyclase.…”

“…6C shows that 3 pfu per cell DENND1A.V2 adenovirus infection increases both basal and forskolin-stimulated −770 CYP17A1/LUC promoter activity compared with control adenovirus. The −770 CYP17A1/LUC was used based on our prior reports demonstrating that the 5′ −770/+44 bp flanking sequence of the CYP17A1 gene confers increased transcriptional regulation in PCOS theca cells (17,36). The results of the above studies showed that DENND1A.V2 significantly increased −770 CYP17A1/LUC promoter function in normal theca cells, converting the cells to a PCOS phenotype.…”

“…Studies on freshly isolated thecal tissue or cultures of theca cells derived from normal and PCOS women have demonstrated that PCOS theca secretes greater amounts of androgen than theca tissue or cells from regularly ovulating women (12,(14)(15)(16)(17)(18)(19). Increased androgen biosynthesis in PCOS theca cells results from increased expression of the key enzymes involved in androgen biosynthesis, steroid-17-α-hydroxylase/17,20 lyase (encoded by the CYP17A1 gene) and cholesterol side-chain cleavage enzyme, mitochondrial (encoded by the CYP11A1 gene) (15)(16)(17)20).…”