STEROIDOGENIC FACTOR-1 (SF-1) AND SP1 ARE REQUIRED FOR REGULATION OF BOVINE CYP11A GENE-EXPRESSION IN BOVINE LUTEAL CELLS AND ADRENAL Y1 CELLS

Cholesterol side-chain cleavage cytochrome P450 (CYP11A; P450scc) gene expression is regulated by gonadotropins via cAMP in the ovary and by ACTH via cAMP in adrenal cortical cells. Previously, we have characte rized a response element located at -118 to -101 bp in the 5'-flanking region of the bovine P450scc gene required for cAMP-stimulated transc ription in both mouse adrenocortical Y1 cells and bovine ovarian cells in primary culture. It was shown that this region contains a binding site for the transcription factor Sp1. Deletion of this sequence aboli shed cAMP-stimulated transcription in both Y1 cells and bovine ovarian luteal cells. Another sequence element located at -57 to -32 bp upstr eam from the transcription initiation site, which is highly conserved in CYP11A of other species, contains the motif TAGCCTTG, similar to th e consensus binding site of steroidogenic factor-1, SF-1 (or Ad4-BP), but in the inverted orientation. In the present study, gel shift analy sis using nuclear extracts of either Y1 cells or bovine luteal cells d emonstrated that the sequence between -57 and -32 bp bound SF-1. A mut ation of the SF-1-binding site that abolished binding of the nuclear p rotein to DNA reduced markedly the basal transcription of the reporter gene as well as the responsiveness to cAMP, when the mutated fragment s containing the region from -186 to +12 bp were cloned into a lucifer ase construct and transfected into mouse adrenal Y1 cells and bovine l uteal cells. The role of SF-1 in P450scc transcription was further con firmed by transactivation of the -186/+12Luc construct employing an SF -1 expression vector after transfection into nonsteroidogenic COS-1 ce lls. In addition, results obtained employing a double mutation of the Sp1- and SF-1-binding sites, and from a construct containing both Sp1 and SF-1 elements upstream of the CYP11A TATA box, indicated that Sp1 and SF-1 function cooperatively in the transactivation of the bovine C YP11A promoter in both bovine luteal cells and Y1 cells. Finally, a ma mmalian two-hybrid system was employed to demonstrate that Sp1 and SF- 1 can associate in vivo. These results establish that basal and cAMP-s timulated activity of the bovine P450scc promoter in both Y1 cells and bovine luteal cells requires the combined action of at least two tran scription factors, Sp1 and SF-1.