Mechanisms of insulin-like growth factor I augmentation of follicle-stimulating hormone-induced porcine steroidogenic acute regulatory protein gene promoter activity in granulosa cells

Insulin-like growth factor I(IGF-I) and the gonadotropin, FSH, can synergiz e to stimulate progesterone production in primary cultures of maturing huma n, rat, and pig granulosa cells. These trophic hormones act by increasing t he activity and production of proteins and their gene transcripts essential to sterol uptake, delivery, and utilization in steroidogenesis. We previou sly observed that FSH and IGF-I interact synergistically to promote the acc umulation of steroidogenic acute regulatory protein (StAR) messenger RNA an d protein in granulosa cells. Here we investigate potential mechanisms of I GF-I synergy with FSH and the protein kinase A (PKA) pathway in activating the porcine StAR gene promoter. To this end, we first cloned 1423 bp of the porcine StAR promoter upstream of the transcriptional start site using PCR and created 5'-deletional constructs coupled to a cytoplasmically targeted firefly luciferase reporter gene. FSH, 8-bromo-cAMP, and transient transfe ction of the protein kinase A (PKA) catalytic subunit (driven by the Rous s arcoma virus promoter) were used to activate the PKA effector pathway. All three agonists alone stimulated StAR promoter-driven luciferase activity in primary cultures of granulosa cells after 4-h treatment. IGF-I significant ly augmented PRE pathway agonist activation of the the StAR promoter, where as IGF-I had no effect alone. Binding experiments with I-125-labeled ovine FSH-20 in IGF-I(100 ng/ml)-treated granulosa cells showed that FSH binding affinity and receptor number were unchanged by IGF-I treatment. However, IG F-I augmented FSH-stimulated, but not forskolin-stimulated, cAMP accumulati on. Analysis of 5'-deletion constructs of the StAR promoter revealed three regions of stimulatory activity within the - 139-bp fragment upstream of th e transcriptional start site as well as another potentially inhibitory regi on upstream(-1115 to -905). Elimination of the putative SF-l site (-48 to - 41)virtually abolished StAR promoter responsiveness. In summary, our data i ndicate that IGF-I can act via two post FSH-binding mechanisms to augment F SH/PKA pathway-mediated StAR gene promoter transactivation: at the level of cAMP accumulation and distal to cAMP production and PKA activation.