Crucial Involvement of CREB-Regulated Transcription Coactivator (CRTC2) and Calcineurin in FSH and TGFβ1 Upregulation of Cyp19 Expression.

Abstract

The steroid hormone estrogen governs female reproductive competence and whole-body physiology. Imbalance of estrogen often exacerbates diseases including cancer and cardiovascular risks. Estrogen is produced mainly in ovarian granulosa cells, where CYP19 (aromatase) catalyzes the conversion of theca cell-derived androgen to estrogen. The pituitary follicle-stimulating hormone (FSH) acts in concert with ovarian local factor tansforming growth factor (TGFβ1) to stimulate granulosa cell steroidogenesis, and this includes upregulation of Cyp19. However, molecular mechanisms underlying such regulation awaits further exploration. The goal of this study was to investigate FSH and TGFβ1 temporal regulation of Cyp19 expression, and the associated molecular mechanism. Primary granulosa cell culture from equine chorionic gonadotropin-primed immature rats was used. We first characterized FSH and TGFβ1 regulation of Cyp19 expression and activity. Under short-term treatment (4 h and 6 h), FSH±TGFβ1 increased Cyp19 mRNA and protein levels, while TGFβ1 alone had no effect. Under long-term treatment (24 h and 48 h) however, TGFβ1 further potentiated FSH-induced Cyp19 expression and activity. CRTC2 is a Ca2+- and cAMP-regulated CREB coactivator. Ca2+ activation of calcineurin and cAMP inhibition of salt-inducible kinase (SIK) induce dephosphorylation of CRTC2, which in turn translocates from the cytoplasm to the nucleus where it coactivates CREB. Recently, we demonstrated calcineurin and CRTC2 as crucial mediators in FSH and TGFβ1 upregulation of steroidogenic proteins StAR, P450scc and 3β-HSD enzymes. Here, we further explored the potential involvement of calcineurin/CRTC2 pathway in FSH and TGFβ1 regulation of Cyp19 expression in granulosa cells. Calcineurin autoinhibitory peptide (CNI) blocked the short-term FSH and long-term FSH+TGFβ1 effect. Moreover, chromatin immunoprecipitation analysis revealed that FSH and TGFβ1 increased the binding of CRTC2 and CREB to Cyp19 promoter at relevant post-treatment times. In all, the present study demonstrates that calcineurin and CRTC2 are crucial mediators in FSH and TGFβ1 upregulation of Cyp19 expression in ovarian granulosa cells. This study was supported by National Science Council of Taiwan. WAL was supported by an award from National Health Research Institutes, Taiwan.