Long-term regulation of mammalian steroid hormone synthesis occurs principally by transcriptional regulation of the gene for the rate-limiting cholesterol side-chain cleavage enzyme P450scc. Adrenal steroidogenesis is regulated primarily by two hormones: adrenocorticotropin, which works via cyclic AMP (cAMP) and protein kinase A, and angiotensin H, which works via Ca2+ and protein kinase C. Forskolin and 8-bromo-cAMP stimulated, while prolonged treatment with a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA]) and a calcium ionophore (A23187) additively suppressed accumulation of endogenous P450scc mRNA in transformed murine adrenal Yl cells. In Yl cells transfected with 2,327 base pairs of the human P450scc promoter fused to the bacterial gene for chloramphenicol acetyltransferase (CAT), forskolin increased CAT activity 900% while combined TPA plus A23187 reduced CAT activity to 15% of the control level. Forskolin induced the P450scc promoter as rapidly as a promoter containing two cAMP-responsive elements fused to a simian virus 40 promoter, a system known to respond directly to cAMP. Basal expression was increased by sequences between -89 and -152 and was increased further by sequences between -605 and -2327. This upstream region also conferred inducibility by cAMP. TPA plus A23187 transiently increased CAT activity before repressing it, reflecting the complex actions of angiotensin II in vivo. Repression by prolonged treatment with TPA plus A23187 was mediated by multiple elements between -89 and -343. Induction of CAT activity by forskolin was not diminished by treatment with TPA plus A23187, nor were the regions of the promoter responsible for regulation by the two pathways coisolated. Thus, the human gene for P450scc is repressed by TPA plus A23187 by mechanisms and sequences independent of those that mediate induction by cAMP.Steroid hormones, which work by controlling transcription of specific genes, are critical regulators of physiological processes (5). The first, rate-limiting, and hormonally regulated step in the generation of steroid hormones is conversion of cholesterol to pregnenolone by the cholesterol sidechain cleavage enzyme P450scc. This mitochondrial cytochrome P450 enzyme receives electrons from NADPH via two protein intermediates, a flavoprotein, adrenodoxin reductase, and an iron-sulfur protein, adrenodoxin. By using these electrons, P450scc catalyzes three sequential reactions-22 hydroxylation, 20 hydroxylation, and C20,22 bond cleavage-apparently at a single active site (reviewed in reference 39). As side-chain cleavage is the rate-limiting step in steroidogenesis (51), it is important to understand both the hormonal regulation and tissue-specific expression of this gene.The developmental patterns of expression and hormonal regulation of P450scc are specific to each steroidogenic tissue. Increased steroidogenesis and accumulation of P450scc mRNA are stimulated by adrenocorticotropin in the human adrenal zonae fasciculata and reticularis, by luteinizing hormone and follicle-s...