Human P450scc gene transcription is induced by cyclic AMP and repressed by 12-O-tetradecanoylphorbol-13-acetate and A23187 through independent cis elements.

Abstract

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...

A Novel Zinc Finger Protein TReP-132 Interacts with CBP/p300 to Regulate Human CYP11A1 Gene Expression

“…Preliminary studies on the 5Ј-flanking region of the gene demonstrated the ability of a 2.5-kb 1 DNA fragment to confer basal and cAMP responsive activity when transiently transfected into mouse adrenal Y1 tumor cells (11)(12)(13). Subsequent studies in mouse Leydig MA-10 (14), I-10 (15), human placenta JEG-3 (16), and adrenal NCI-H295 (17) cells identified regions of the 5Ј-flanking DNA that conferred basal promoter activity and response to cAMP.…”

Regulatory mechanisms of cAMP‐dependent and cell‐specific expression of human steroidogenic cytochrome P450scc (CYP11A1) gene

“…The sequence of nucleotides -118 to -83 (CRS) for the bovine CYPllA gene has been shown to enhance the transcription of the gene in response to cAMP [15], while the sequence of nucleotides -325 to -34 in the mouse CYPIIA gene [13] contains several DNA elements, including the tissue-specific-factor-binding site (SFl), which were required for sufficient CAMP responsiveness. Moore and co-workers [17] reported that the nucleotides -152 to -89 were necessary for basal expression, but not for cAMP responsiveness, of the human CYPllAl gene in Y-1 cells [17], while the region composed of nucleotides -108 to -89 was needed for induction in response to forskolin treatment in human JEG-3 choriocarcinoma cells [34]. On the basis of these reports, Momoi and co-workers [ 181 discussed that regions including the bovine CRS, mouse SF-3 and the human nucleotides -152 to -89 were important for the transcription of the CYPllAl gene in these animal species [I 81.…”

“…Recently, human CYP17 and CYP21A2 genes have been demonstrated to be controlled by CREB or its related factors through binding to the DNA elements 111, 121, although they do not have a typical CRE consensus sequence. Regulatory mechanisms of CYPllAl expression have been investigated with mouse, rat, bovine, sheep and human genes [13][14][15][16][17][18] 331. In the bovine CYPllA gene [15, 181, a cis regulatory region was localized between nucleotides -118 and -83, and Spl and adrenal-specific protein Correspondence to Y. Fujii-lriyama,…”

Polar phospholipids from bovine endogenously oxidized low density lipoprotein interfere with follicular thecal function

“…Phospholipids failed to impair the forskolin-triggered increase in progesterone synthesis as opposed to LH-stimulated progesterone production, suggesting that postreceptor cAMP signalling is little affected by these lipids. 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 P450 scc (Moore et al 1990). Its expression is positively regulated via the cAMP-PKA pathway; negative regulation includes elevation of the intracellular free Ca 2+ level (Moore et al 1990).…”

“…Stimulation of follicular progesterone synthesis is brought about via numerous pathways. The preovulatory LH surge elevates the progesterone level of the follicular fluid mainly through a cAMP-protein kinase A (PKA) signalling towards nuclear steroidogenic transcription factors that stimulate expression of thecal and granulosal steroidogenic enzymes (Moore et al 1990, Mamluk et al 1998.…”