Sp1 and SF-1 Interact and Cooperate in the Regulation of Human Steroidogenic Acute Regulatory Protein Gene Expression*

Steroidogenic acute regulatory (StAR) protein plays a critical role in the movement of cholesterol from the outer to the inner mitochondrial membrane. Steroidogenic factor 1 (SF-1) controls basal and cAMP-stimulated transcription of the StAR gene. The 1.3-kb StAR promoter has three SF-1 binding sites, and two consensus transcription factor Spl binding sequences near the two most distal SF-1 binding sites. Spl mediates cAMP-dependent transcription of steroidogenic P450 enzyme genes, raising the possibility of Sp1 involvement in cAMP regulation of the StAR gene. However, the mechanism of Spl-mediated, cAMP-stimulated responsiveness is not known. In this study, we elucidated the roles of Sp1 and SF-1 in the regulation of the human StAR gene promoter. We found that there was negligible promoter activity in a pGL2 StAR construct (-235 to +39) in which Spl and SF-1 binding sites were mutated in Y-1 adrenal tumor cells. An Sp1 binding site mutation (pGL2Sp1M) did not support promoter activity, suggesting that Spl cooperates with SF-1 in regulating StAR promoter function. In gel shift assays, the SF-1 binding site formed a complex with an SF-1-GST fusion protein and Spl. Coimmunoprecipitation cross-linking experiments indicated that SF-1 physically interacts with Sp1 in vitro. Finally, a mammalian two-hybrid system was employed to demonstrate that Spl and SF-1 associate in vivo. In conclusion, our data indicate that Spl and SF-1 physically interact and cooperate in the regulation of human StAR promoter activity.