IDENTIFICATION OF A CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-RESPONSE ELEMENT IN THE RAT AROMATASE PROMOTER THAT IS REQUIRED FOR TRANSCRIPTIONAL ACTIVATION IN RAT GRANULOSA-CELLS AND R2C LEYDIG-CELLS

Cytochrome P450 aromatase, which converts testosterone to estradiol, i s transcriptionally induced by FSH and cAMP during ovarian follicular development. At least one promoter element [-82/-31 base pairs (bp)] r equired for stimulation of the rat gene in granulosa cells binds stero idogenic factor-1, an orphan steroid receptor. In this paper, we demon strate that an additional region, -161/-138 bp is required for cAMP re gulation. This region shares homology with promoter sequences in the b ovine 21-hydroxylase and mouse 11 beta-hydroxylase genes that are also induced by cAMP, yet each binds different proteins in granulosa cell nuclear extracts. The aromatase -161/-138 bp region contains a cAMP-re sponse element (CRE)-like sequence, TGCACGTCA. Deletion or mutation of this sequence reduces promoter activity of chimeric chloramphenicol a cetyl transferase (CAT) reporter constructs that are transiently trans fected into granulosa cells and R2C Leydig cells. Granulosa cell nucle ar proteins and R2C cell nuclear proteins specifically bind the -161/- 138 bp region and form three protein/DNA complexes. Recombinant CRE-bi nding protein (CREB) binds the CRE-like sequence and forms a single ba nd, and a CREB antibody retards the migration of CREB and one granulos a cell protein-aromatase DNA binding complex. Using Western blot analy sis, CREB was demonstrated in granulosa cell nuclear extracts from all stages of follicular development. Thus, aromatase is transcriptionall y regulated by a hexameric sequence binding SF-1 and a CRE sequence bi nding CREB and other factors present in granulosa cells and in R2C Ley dig cells. The presence of identical SF-1 and CRE-like sequences in th e human ovarian aromatase promoter II suggests that the human promoter may also be regulated in a similar manner.