REGULATION OF CYP11A GENE-EXPRESSION IN BOVINE OVARIAN GRANULOSA-CELLS IN PRIMARY CULTURE BY CAMP AND PHORBOL ESTERS IS CONFERRED BY A COMMON CIS-ACTING ELEMENT

Production and secretion of steroid hormones throughout the ovarian cy cle occurs in a highly episodic and coordinated fashion that requires precise and finely tuned regulatory mechanisms. The regulation of ovar ian steroidogenesis by the gonadotropin follicle stimulating hormone ( FSH) and luteinizing hormone (LH) as well as by other factors occurs, at least in part, at the level of expression of the-genes encoding ste roidogenic enzymes. The present study is aimed at the elucidation of r egulatory mechansims by which cyclic adenosine monophosphate (cAMP) an d protein kinase C regulate cytochrome P450scc (CYP11A) gene expressio n in bovine granulosa cells in primary culture. As a first step we cha racterized the bovine granulosa cell cultures with regard to regulatio n of P450scc activity and mRNA levels upon treatment with forskolin an d/or the phorbol ester TPA. Forskolin, a potent stimulator of cAMP gen eration, increased both progesterone secretion and P450scc mRNA levels . In contrast, treatment with TPA alone decreased both basal progester one production and P450scc mRNA accumulation. Co-treatment with forsko lin and TPA decreased progesterone and P450scc mRNA levels as compared to forskolin treatment alone. The possibility that TPA interfered wit h the forskolin-stimulated cAMP production could be excluded because s imultaneous treatment of granulosa cells with TPA and forskolin potent iated the formation of cAMP. In order to identify regulatory sequences within the 5' flanking region of the bovine CYP11A gene, chimeric DNA constructs comprizing regions of the CYP11A gene fused to a beta-glob in-derived reporter gene were transfected into granulosa cells in prim ary culture. The expression of reporter gene constructs containing - 8 96/ - 32, - 186/ - 32, - 118/ - 83 and - 118/100 bp of the 5' upstream region of the CYP11A gene was markedly stimulated upon forskolin trea tment. Expression in the absence of forskolin was essentially undetect able. Addition of TPA alone did not change the expression of the repor ter gene constructs as compared to control, however, co-treatment with forskolin and TPA significantly decreased the stimulation observed wi th forskolin treatment alone. Examination of the - 118/ - 100 bp seque nce revealed two regions exhibiting similarity to the consensus bindin g sites for AP1 and Sp1 transcription factors. It is likely therefore that differential regulation of bovine CYP11A by forskolin and phorbol esters is mediated by one or both of these sequences.