Cholesterol Side-Chain Cleavage Gene Expression in Theca Cells: Augmented Transcriptional Regulation and mRNA Stability in Polycystic Ovary Syndrome

Abstract

Hyperandrogenism is characteristic of women with polycystic ovary syndrome (PCOS). Ovarian theca cells isolated from PCOS follicles and maintained in long-term culture produce elevated levels of progestins and androgens compared to normal theca cells. Augmented steroid production in PCOS theca cells is associated with changes in the expression of genes for several steroidogenic enzymes, including CYP11A1, which encodes cytochrome P450 cholesterol side-chain cleavage. Here, we further examined CYP11A1 gene expression, at both the transcriptional and post-transcriptional level in normal and PCOS theca cells propagated in long-term culture utilizing quantitative RT-PCR, functional promoter analyses, and mRNA degradation studies. The minimal element(s) that conferred increased basal and cAMP-dependent CYP11A1 promoter function were determined. CYP11A1 mRNA half-life in normal and PCOS theca cells was compared. Results of these cumulative studies showed that basal and forskolin stimulated steady state CYP11A1 mRNA abundance and CYP11A1 promoter activity were increased in PCOS theca cells. Deletion analysis of the CYP11A1 promoter demonstrated that augmented promoter function in PCOS theca cells results from increased basal regulation conferred by a minimal sequence between −160 and −90 bp of the transcriptional start site. The transcription factor, nuclear factor 1C2, was observed to regulate basal activity of this minimal CYP11A1 element. Examination of mRNA stability in normal and PCOS theca cells demonstrated that CYP11A1 mRNA half-life increased >2-fold, from approximately 9.22+/−1.62 h in normal cells, to 22.38+/−0.92 h in PCOS cells. Forskolin treatment did not prolong CYP11A1 mRNA stability in either normal or PCOS theca cells. The 5′-UTR of CYP11A1 mRNA confers increased basal mRNA stability in PCOS cells. In conclusion, these studies show that elevated steady state CYP11A1 mRNA abundance in PCOS cells results from increased transactivation of the CYP11A1 promoter and increased CYP11A1 mRNA stability.

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

“…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).…”

“…As a standard of care, oophorectomies were performed during the luteal phase of the cycle. Theca cells from normal cycling and PCOS follicles were isolated and grown as we have as previously reported in detail (20,46,47). The theca cell preparations used in these studies have been described and characterized previously (15-17, 20, 35-38, 47).…”

“…DENND1A.V2 data were verified with the second confirmatory DENND1A.V2 primer probe set provided. The gene-specific one-step PCR was carried out in duplicate for each mRNA sample and for a series of dilutions in an Mx3000p Thermocycler system (Stratagene) according to the manufacturer's instructions for this instrument, as previously described (20). The same process was carried out for TBP to use TBP values for normalization of each reaction.…”

“…We used a CYP11A1 pGl3 reporter construct containing the −160/ −90 bp element of the proximal CYP11A1 promoter (−160/−90 CYP11A1/LUC), which confers increased CYP11A1 expression in PCOS theca cells (20) to examine the effects of DENND1A.V2 on CYP11A1 transcription in theca cells. Normal theca cells were transfected with the −160/−90 CYP11A1/LUC plasmid with a DENND1A.V2/pCMV-XL4 or empty pCMV-XL4.…”

“…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).…”