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