Conditional response of the human steroidogenic acute regulatory protein gene promoter to sterol regulatory element binding protein-1a.

The steroidogenic acute regulatory protein (StAR) gene controls the rate-limiting step in the biogenesis of steroid hormones, delivery of cholesterol to the cholesterol side-chain cleavage enzyme on the inner mitochondrial membrane. We determined whether the human StAR promoter is responsive to sterol regulatory element-binding proteins (SREBPs). Expression of SREBP-1a stimulated StAR promoter activity in the context of COS-1 cells and human granulosa-lutein cells. In contrast, expression of SREBP-2 produced only a modest stimulation of StAR promoter activity. One of the SREBP-1a response elements in the StAR promoter was mapped in deletion constructs and by site-directed mutagenesis between nucleotides -81 to -70 from the transcription start site. This motif bound recombinant SREBPs in electrophoretic mobility shift assays, but with lesser affinity than a low density lipoprotein receptor SREBP-binding site. An additional binding site for the transcriptional modulator, yin yang 1 (YY1), was observed within the SREBP-binding site (nucleotides -73 to -70). Mutation of the YY1-binding site increased the responsiveness of the StAR promoter to exogenous SREBP-1a, but did not alter the affinity for SREBP-1a binding in electrophoretic mobility gel shift assays. Manipulations that altered endogenous mature SREBP-1a levels (e.g. culture in lipoprotein-deficient medium and addition of 27-hydroxycholesterol) did not affect StAR promoter function, but influenced low density lipoprotein receptor promoter activity. We conclude that 1) the human StAR promoter is conditionally responsive to SREBP-1a such that promoter activity is up-regulated in the presence of high levels of SREBP-1a, but is unaffected when mature SREBP levels are suppressed; and 2) the human StAR promoter is selectively responsive to SREBP-1a.

Dual regulation of 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, and dehydroepiandrosterone sulfotransferase by adenosine 3',5'-monophosphate and activators of protein kinase C in cultured human adrenocortical cells.

The relationship between cAMP and protein kinase C in the regulation of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), 17 alpha-hydroxylase, and sulfotransferase was examined in human fetal adrenocortical cells under defined serum-free conditions in culture. Forskolin induced 3 beta HSD and 17 alpha-hydroxylase in a dose-dependent manner, with maximal effects at 10 microM. 12-O-Tetradecanoyl phorbol 13-acetate (TPA) at 1 nM depressed the induction of 17 alpha-hydroxylase activity by forskolin by more than 95% and increased the stimulation of 3 beta HSD activity by forskolin by 4- to 5-fold. Increases were maximal at 48-72 h of incubation. Dehydroepiandrosterone sulfotransferase activity increased over 48 h when cells were transferred to serum-free defined medium. Addition of 10 microM forskolin stimulated sulfotransferase activity only when cells remained in 10% serum. TPA at 1 nM inhibited the increase in sulfotransferase activity. The concentration of TPA required for inhibition of forskolin-stimulated 17 alpha-hydroxylase and sulfotransferase activity was similar to that required for enhancement of forskolin-induced 3 beta HSD activity, suggesting that comparable levels of C kinase activation are involved in these events. Angiotensin II, carbachol, epidermal growth factor, and fibroblast growth factor had actions similar to those of TPA on one or more of these enzyme activities. TPA also had similar actions on enzyme activities when they were stimulated by cAMP analogs rather than by forskolin. These studies suggest that adrenal steroid biosynthesis is under dual regulation by cAMP and protein kinase C. cAMP induces enzymes required for synthesis of 17 alpha-hydroxylated steroids, including the adrenal androgens. Activation of protein kinase C may play a complementary role by enhancing the induction of enzymes required for non-17 alpha-hydroxylated steroid biosynthesis and inhibiting those involved in the synthesis of androgens.