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Molecular Endocrinology 13 (5): 729-741
Copyright © 1999 by The Endocrine Society

SF-1 (Steroidogenic Factor-1) and C/EBPß (CCAAT/Enhancer Binding Protein-ß) Cooperate to Regulate the Murine StAR (Steroidogenic Acute Regulatory) Promoter

Adam J. Reinhart, Simon C. Williams, Barbara J. Clark and Douglas M. Stocco

Department of Cell Biology and Biochemistry (A.J.R., S.C.W., D.M.S.) and Southwest Cancer Center (S.C.W.) Texas Tech University Health Science Center Lubbock, Texas 79430
Department of Biochemistry University of Louisville School of Medicine (B.J.C) Louisville, Kentucky 40292

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 promoter for putative transcription factor-binding sites that may regulate transcription in a developmental and/or hormone-induced 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/EBPß 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)2cAMP-stimulated StAR promoter activity in MA-10 Leydig tumor cells, without affecting the fold activation [(Bu)2cAMP-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/EBPß is involved in the transcriptional regulation of the StAR gene and may play an important role in developmental and hormone-responsive regulation of steroidogenesis.




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