TRANSCRIPTIONAL REGULATION OF THE RAT STEROIDOGENIC ACUTE REGULATORY PROTEIN GENE BY STEROIDOGENIC FACTOR-1

Steroidogenic acute regulatory (StAR) protein is synthesized in respon se to tropic hormones to facilitate cholesterol transport to the inner mitochondrial membrane-bound P450 side-chain cleavage enzyme (P450scc ), the first enzymatic step in the steroid hormone biosynthetic pathwa y. Gonadotropins activate expression of their target genes via the cAM P second messenger system. We have demonstrated that cAMP administrati on to rat luteal cells stimulates expression of both StAR messenger RN A and protein. Because cholesterol delivery is the first regulated ste p in steroidogenesis, and because StAR messenger RNA levels are increa sed in response to tropic hormone and cAMP stimulation, the mechanism by which tropic hormones/cAMP stimulate transcription needs to be eluc idated. To this end, approximately 2.7 kb of the rat StAR promoter was isolated and sequenced. Sequence analysis revealed the presence of a TATA-like element as well as multiple regulatory motifs including ster oidogenic factor 1 (SF-1) binding sites, an estrogen receptor half-sit e, and two AP-1 sites within the promoter region. 5'-RACE experiments determined the transcription start site to be located 82 bp upstream o f the ATG translation start codon. Electrophoretic mobility shift, ass ays and supershift analysis demonstrated SF-1 binding to three SF-1 bi nding sites in the rat StAR promoter with high affinity and two SF-1 b inding sites with low affinity. Transfection of mouse Y1 adrenal tumor cells and human bladder carcinoma cells (HTB9s) with the rat StAR pro moter demonstrated that SF-1 was able to activate transcription of the luciferase reporter gene and that the rat StAR promoter was responsiv e to cAMP. Nested deletions of the rat StAR promoter (1.9 kb) identifi ed a region between - 1413 and -998 that is essential for maximal acti vation of the rat StAR gene in HTB9 cells; however, deletion of this r egion does not affect responsiveness to cAMP. 5'-Deletion and site-dir ected mutagenesis experiments demonstrated that the SF-1 motifs identi fied within the rat StAR promoter (located at positions -764, -455, an d - 106) were sufficient to activate transcription as well as confer c AMP responsiveness to the rat StAR gene. Site-directed mutagenesis stu dies using the smallest promoter fragment demonstrated that the two pr oximal SF-1 binding sites are crucial for StAR gene transcription, bot h at a basal level and in response to cAMP stimulation. These studies provide novel insights into the regulation of the rat StAR gene at the transcriptional level by SF-1.