REGULATION OF EXPRESSION OF THE CYP11A (P450(SCC)) GENE IN BOVINE OVARIAN LUTEAL CELLS BY FORSKOLIN AND PHORBOL ESTERS

This study examines the transcriptional regulation of the bovine CYP11 A (P450scc) gene by activators of protein kinase A and protein kinase C in bovine ovarian luteal cells. Cells were transfected with reporter gene constructs containing deletion mutations of the 5'- flanking reg ion of the bovine CYP11A gene linked to the minimal beta-globin gene. A construct containing -118/-101 base pairs of CYP11A sequence retains the same degree of stimulation by forskolin and inhibition by co-trea tment with phorbol 12-myristate 13-acetate as larger constructs. This sequence contains two putative binding sites for nuclear proteins, an AP1-like sequence and an overlapping GA box element. Gel shift analysi s using nuclear extracts of bovine ovarian luteal cells demonstrated t hat both the wild-type -118/-101-base pair sequence and a consensus GC box bound Sp1 or Sp1-like proteins. Mutation of the GA box element co mpletely suppressed stimulation by forskolin. Absence of binding using the same mutated sequence correlated with the reporter gene transcrip tion results. Mutation of the AP1-like site had little effect on forsk olin induction of phorbol 12-myristate 13-acetate inhibition. These re sults indicate that both stimulation by forskolin and inhibition by ph orbol esters are mediated by the same GA box element, which binds Sp1 or an Sp1-like protein.