The steroidogenic acute regulatory (StAR) protein mediates the rate-limiting step of steroidogenesis, which is the transfer of cholesterol to the inner mitochondrial membrane. In steroidogenic tissues, StAR expression is acutely regulated by trophic hormones through a cAMP second messenger pathway, leading to increased StAR mRNA levels within 30 min, reaching maximal levels after 4-6 h of stimulation. The molecular mechanisms underlying such regulation remain unknown. We have examined the StAR

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... romoter for putative transcription factor-binding sites that may regulate transcription in a developmental and/or hormoneinduced context. Through sequence analysis, deoxyribonuclease I (DNAse I) footprinting and electrophoretic mobility shift assays (EMSAs), we have identified two putative CCAAT/enhancer binding protein (C/EBP) DNA elements at ؊113 (C1) and ؊87 (C2) in the mouse StAR promoter. Characterization of these sites by EMSA indicated that C/EBP␤ bound with high affinity to C1 and C2 was a low-affinity C/EBP site. Functional analysis of these sites in the murine StAR promoter showed that mutation of one or both of these binding sites decreases both basal and (Bu) 2 cAMP-stimulated StAR promoter activity in MA-10 Leydig tumor cells, without affecting the fold activation [(Bu) 2 cAMP-stimulated/basal] of the promoter. Furthermore, we have demonstrated that these two C/EBP binding sites are required for steroidogenic factor-1 (SF-1)-dependent transactivation of the StAR promoter in a nonsteroidogenic cell line. These data indicate that in addition to SF-1, C/EBP␤ is involved in the transcriptional regulation of the StAR gene and may play an important role in developmental and hormone-responsive regulation of steroidogenesis. (Molecular Endocrinology 13: 729-741, 1999)