Synergistic activation of the inhibin alpha-promoter by steroidogenic factor-1 and cyclic adenosine 3',5'-monophosphate

The inhibin alpha-subunit gene is expressed in the ovary, testis, adrenal, and pituitary. Because this pattern of expression corresponds to that of th e orphan nuclear receptor, steroidogenic factor-1 (SF-1), we hypothesized t hat the inhibin alpha promoter might be regulated by SF-1. Expression of ex ogenous SF-1, in an SF-1 deficient cell line, caused modest stimulation of the inhibin alpha promoter. However, activation of the cAMP pathway, which is known to regulate inhibin alpha expression, greatly enhanced the actions of SF-1. Coexpression of SF-1 with the catalytic subunit of cAMP-dependent protein kinase A caused greater than 250-fold stimulation, whereas only 4- or 7-fold stimulation was seen by the SF-1 or protein kinase A pathway alo ne. Synergistic stimulation by SF-1 and the cAMP pathway was also seen in G RMO2 granulosa cells, which express endogenous SF-1. Deletion and site-dire cted mutagenesis localized a novel SF-1 regulatory element (TCA GGGCCA; -13 7 to -129) adjacent to a Variant cAMP-response element (CRE; -120 to -114). The synergistic property of SF-1 and cAMP stimulation was inherent within this composite inhibin alpha fragment (-146 and -112), as it was transferab le to heterologous promoters. Mutations in either the CRE or the SF-1 regul atory element completely eliminated synergistic activation by these pathway s. The binding of SF-1 and CRE binding protein (CREB) to the inhibin alpha regulatory elements was relatively weak in gel mobility shift assays, consi stent with their deviation from consensus binding sites. However, SF-1 was found to interact with CREB using an assay in which epitope-tagged SF-1 was expressed in cells and used to pull down in vitro translated CREB. Express ion of CREB binding protein (CBP), a coactivator that interacts with SF-1 a nd CREB, further enhanced transcription by these pathways. Stimulation by t he SF-1 and cAMP pathways was associated with increased histone H4 acetylat ion, suggesting that chromatin remodeling accompanies their actions. We pro pose a model in which direct interactions of SF-1, CREB, and associated coa ctivators like CBP induce strongly cooperative transactivation by pathways that individually have relatively weak effects on transcription.