STEROIDOGENIC FACTOR 1-DEPENDENT PROMOTER ACTIVITY OF THE HUMAN STEROIDOGENIC ACUTE REGULATORY PROTEIN (STAR) GENE

Steroidogenic acute regulatory protein (StAR) is required for efficien t adrenal cortical and gonadal but not trophoblast steroid hormone syn thesis. StAR gene expression in gonadal cells is stimulated by tropic hormones acting through the intermediacy of cAMP. DNA sequence analysi s of the human StAR gene promoter revealed two motifs resembling bindi ng sites for steroidogenic factor 1 (SF-1), a member of the orphan nuc lear receptor transcription factor family that controls expression of steroidogenic hydroxylases. The 5'-most sequence (distal site) is a co nsensus SF-1 binding site. The proximal site is a consensus estrogen r eceptor binding half-site. The StAR gene promoter is not active in BeW o choriocarcinoma cells, COS-1 cells, HeLa cells, or SK-OV-3 ovarian a denocarcinoma cells, all of which do not express significant levels of SF-1 mRNA. Introduction of SF-1 into these cells stimulated StAR prom oter activity, particularly in response to cAMP, Two orphan nuclear tr anscription factors that bind to sequences similar to SF-1 sites, NGFI -B/Nur77 and RNR-1, did not support cAMP-stimulated StAR promoter acti vity in BeWo cells. Mutation of the distal putative SF-1 binding site reduced basal and cAMP-stimulated promoter activity in BeWo cells by 8 2% and 71%, respectively, Mutation of the proximal putative SF-1 bindi ng site reduced basal and cAMP-stimulated promoter activity by 89% and 96%, respectively. Mutations in both sites reduced basal promoter act ivity to 7% of wild type promoter activity and cAMP-stimulated promote r activity to less than 5% of the wild type. Deletion analyses of prom oter activity were consistent with the mutation studies. Electrophoret ic mobility shift assays (EMSAs) demonstrated that the distal site bin ds to SF-1 expressed in COS-1 cells and to an SF-1-GST fusion protein with high affinity, but that the mutated distal sequence does not, An anti-SF-l antibody ablated the characteristic SF-1-DNA complex with th e distal sequence. The proximal site formed a number of protein-DNA co mplexes with COS-1 cell extracts, but appeared to have at best only ve ry modest affinity for SF-1. Collectively, our findings demonstrate th at SF-1 plays a key role in controlling the basal and cAMP-stimulated expression of the StAR gene. SF-1 can function at two distinct sites i n the human StAR gene promoter, apparently by two different types of i nteraction, to control transcription.