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, is transcriptionally induced by FSH and cAMP during ovarian follicular development. At least one promoter element [-82/-31 base pairs (bp)] required for stimulation of the rat gene in granulosa cells binds steroidogenic factor-1, an orphan steroid receptor. In this paper, we demonstrate that an additional region, -161/-138 bp is required for cAMP regulation. This region shares homology with promoter sequences in the bovine 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-response element (CRE)-like sequence, TGCACGTCA. Deletion or mutation of this sequence reduces promoter activity of chimeric chloramphenicol acetyl transferase (CAT) reporter constructs that are transiently transfected into granulosa cells and R2C Leydig cells. Granulosa cell nuclear proteins and R2C cell nuclear proteins specifically bind the -161/-138 bp region and form three protein/DNA complexes. Recombinant CRE-binding protein (CREB) binds the CRE-like sequence and forms a single band, and a CREB antibody retards the migration of CREB and one granulosa cell protein-aromatase DNA binding complex. Using Western blot analysis, CREB was demonstrated in granulosa cell nuclear extracts from all stages of follicular development. Thus, aromatase is transcriptionally regulated by a hexameric sequence binding SF-1 and a CRE sequence binding CREB and other factors present in granulosa cells and in R2C Leydig cells. The presence of identical SF-1 and CRE-like sequences in the human ovarian aromatase promoter II suggests that the human promoter may also be regulated in a similar manner.