Insulin-Like Growth Factor-I Activates Extracellularly Regulated Kinase to Regulate the P450 Side-Chain Cleavage Insulin-Like Response Element in Granulosa Cells

IGF regulates steroidogenesis in granulosa cells through expression of the cytochrome P450 side-chain cleavage enzyme (P450scc) (CYP11A1), the rate-limiting enzyme in this biosynthetic process. We showed previously that the polypyrimidine tract-binding protein-associated splicing factor (PSF) acts as a repressor, whereas Sp1 is an activator, of P450 gene expression. The aim of the present study was to investigate IGF-stimulated ERK signaling regulating P450scc gene expression in the immortalized porcine granulosa cell line JC-410. We used a reporter gene under control of the IGF response element from the P450scc promoter. Inhibition of ERK phosphorylation with U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene] blocked IGF-I induction of IGF response element reporter gene activity. Western blotting revealed that IGF-I treatment resulted in phosphorylation of ERK that was specifically inhibited by U0126. ERK activation led to phosphorylation of T739 (an ERK site) on Sp1 that was diminished by U0126 or overexpression of PSF. Coimmunoprecipitation and Western blotting of nuclear extracts showed that phosphorylated ERK (pERK) bound PSF under basal conditions. IGF-I caused dissociation of pERK from PSF. Finally, chromatin immunoprecipitation analysis showed that PSF and Sp1 constitutively occupy the P450scc promoter independent of IGF-I treatment. These events provide a potential molecular mechanism for release of PSF repression of P450scc expression by dissociation of pERK and subsequent pERK-mediated phosphorylation of Sp1 to drive transcriptional induction of the P450scc gene in the absence of altered binding of PSF or Sp1 to the promoter. Understanding IGF-I regulation of these critical ovarian signaling pathways is the first step to delineating ovarian hyperstimulation syndromes such as polycystic ovarian syndrome.