CCAAT enhancer-binding protein beta and GATA-4 binding regions within the promoter of the steroidogenic acute regulatory protein (StAR) gene are required for transcription in rat ovarian cells

Steroidogenic acute regulatory protein (StAR) is a vital accessory protein required for biosynthesis of steroid hormones from cholesterol. The present study shows that in primary granulosa cells from prepubertal rat ovary, St AR transcript and protein are acutely induced by gonadotropin (FSH). To det ermine the sequence elements required for hormone inducibility of the StAR promoter, truncated regions of the -1002/+6 sequence of the mouse gene were ligated to pCAT-Basic plasmid and transfected by electroporation to freshl y prepared cells. FSH inducibility determined over a 6-h incubation was 10- 40-fold above basal levels of chloramphenicol acetyltransferase activity. T hese functional studies, supported by electrophoretic mobility shift assays indicated that two sites were sufficient for transcription of the StAR pro moter constructs: a non-consensus binding sequence (-81/-72) for CCAAT enha ncer-binding protein beta (C/EBP beta) and a consensus motif for GATA-4 bin ding (-61/-66). Western analyses showed that GATA-4 is constitutively expre ssed in the granulosa cells, while all isoforms of C/EBP beta were markedly inducible by FSH. Site-directed mutations of both binding sequences practi cally ablated both basal and hormone-driven chloramphenicol acetyltransfera se activities to less than 5% of the parental -96/+6 construct. Unlike earl ier notions, elimination of potential binding sites for steroidogenic facto r-1, a well known tissue-specific transcription factor, did not impair StAR transcription. Consequently, we propose that C/EBP beta and GATA-4 represe nt a novel combination of transcription factors capable of conferring an ac ute response to hormones upon their concomitant binding to the StAR promote r.