The inhibin α-subunit gene is expressed in the ovary, testis, adrenal, and pituitary. Because this pattern of expression corresponds to that of the orphan nuclear receptor, steroidogenic factor-1 (SF-1), we hypothesized that the inhibin α promoter might be regulated by SF-1. Expression of exogenous SF-1, in an SF-1 deficient cell line, caused modest stimulation of the inhibin α promoter. However, activation of the cAMP pathway, which is known to regulate inhibin α 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 alone. Synergistic stimulation by SF-1 and the cAMP pathway was also seen in GRMO2 granulosa cells, which express endogenous SF-1. Deletion and site-directed mutagenesis localized a novel SF-1 regulatory element (TCA GGGCCA; -137 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 α fragment (-146 and -112), as it was transferable to heterologous promoters. Mutations in either the CRE or the SF-1 regulatory element completely eliminated synergistic activation by these pathways. The binding of SF-1 and CRE binding protein (CREB) to the inhibin regulatory elements was relatively weak in gel mobility shift assays, consistent 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. Expression of CREB binding protein (CBP), a co-activator that interacts with SF-1 and CREB, further enhanced transcription by these pathways. Stimulation by the SF-1 and cAMP pathways was associated with increased histone H4 acetylation, suggesting that chromatin remodeling accompanies their actions. We propose a model in which direct interactions of SF-1, CREB, and associated coactivators like CBP induce strongly cooperative transactivation by pathways that individually have relatively weak effects on transcription.
ASJC Scopus subject areas
- Molecular Biology