1451
Follicle-stimulating hormone converges with canonical WNT signaling to enhance Cyp19a1 promoter activity in granulosa cells

Wednesday, July 23, 2014
Exhibit Hall AB (Kansas City Convention Center)
Belinda I Gomez , Oklahoma State University, Stillwater, OK
Juliet O. E. , Oklahoma State University, Stillwater, OK
Craig A. Gifford , Oklahoma State University, Stillwater, OK
Dennis M. Hallford , New Mexico State University, Las Cruces, NM
Jennifer Hernandez Gifford , Oklahoma State University, Stillwater, OK
Abstract Text:

Biosynthesis of estradiol in the adult ovary requires activation of the tissue specific cytochrome P450 aromatase (Cyp19a1) type II promoter (PII) by FSH. Canonical wingless-type mammary tumor virus integration-site (WNT) signaling has been recognized to contribute to ovarian regulation of steroidogenesis by increasing the transcriptional co-factor, β-catenin. Recent data suggest WNT3A is inhibitory on FSH mediated mRNA induction of key steroidogenic enzymes and steroid biosynthesis; however, the mechanism by which WNT3A negatively regulates FSH remains to be determined. Therefore, the objective of this study was to investigate the inhibitory effects of WNT3A on FSH-mediated Cyp19a1 activity.  Immunofluorescence was performed on primary rat granulosa cells treated with WNT3A (500 ng/mL) in the presence or absence of FSH (100 ng/mL) for 24 h (n = 4) to determine if FSH prevents WNT3A translocation of β-catenin. Treatment with WNT3A and WNT3A+FSH resulted in nuclear accumulation of β-catenin, while FSH treated cells resembled control groups with the majority of β-catenin remaining at cell membrane. To identify if WNT3A+FSH prevents β-catenin ability to bind the Cyp19a1 PII, a 516 bp fragment of the Cyp19a1 PII (516-Cyp19a1 PII) was transfected into primary cultures of rat granulosa cells, treated with vehicle or WNT3A (500 ng/mL) for 24 h, then co-cultured with or without FSH (100 ng/mL) for an additional 24 h (n = 4).  Promoter activity was measured by the luciferase reporter assay and statistical differences for treatment interaction were quantified using one-way ANOVA procedure of SAS.  Activity of Cyp19a1 PII with WNT3A alone was similar to controls, while FSH treatment increased (P = 0.01) Cyp19a1 PII activity 6.65 fold when compared to controls.  Co-incubation of FSH and WNT3A was synergistic resulting in a 16.09 fold increase in Cyp19a1 PII activity (P = 0.01; n = 4). To evaluate if regions upstream of the 516 bp Cyp19a1 PII fragment are responsible for the inhibition of estradiol biosynthesis, the full length Cyp19a1 PII (full-Cyp19a1 PII) and  2,000 bp (2,000-Cyp19a1 Promoter) upstream of the ATG site on Cyp19a1 was cloned into a luciferase reporter.  Preliminary data suggest full-Cyp19a1 PII and 2,000-Cyp19a1 promoteractivity is not synergistic (n = 2) with co-incubation of WNT3A+FSH.  Future studies are needed to determine the regions on the promoter responsible for WNT3A inhibition on FSH signaling.

Keywords:

Cyp19a1, granulosa cells, WNT