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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Addition of cholesterol to rat adrenal mitochondria resulted in a stimulation of pregnenolone synthesis. The slow step of the mitochondrial cholesterol side-chain cleavage reaction could be the interaction of the sterol with cytochrome P-450. The rate of cholesterol binding to this enzyme as observed spectroscopically correlated with the equilibration period (20 min) of the mitochondria and exogenous cholesterol required for maximal rates of pregnenolone synthesis. It is suggested that translocation of cholesterol between different sterol pools occurs within the mitochondria. Potential intracellular effectors that could be of importance in the movement or regulation of mitochondrial cholesterol include bivalent metallic ions, prostaglandins, cyclic nucleotides, polyamines and polylysine. Of the effectors studied, only calcium ions and polylysine markedly stimulated pregnenolone synthesis. These effectors might stimulate steroidogenesis by lateral displacement of cholesterol in the mitochondrial membrane into a compartment easily accessible to the cholesterol side-chain cleavage enzyme complex.
Mol Cell Endocrinol 1978 Apr
PMID:Regulation of cholesterol metabolism in rat adrenal mitochondria. 20 6

Long-term inductive effects of luteinizing hormone (LH) on cholesterol side-chain cleavage (CSCC) enzyme activity were studied, using cultured Leydig cells isolated from 21-day-old rats. Particular reference was given to the role of insulin-like growth factor-I (IGF-I) as an autocrine or paracrine modulator or as an essential extracellular mediator of LH action. The CSCC enzyme activity was measured using an excess of 22(R)-hydroxycholesterol as substrate to saturate the enzyme, and inhibitors of pregnenolone metabolism to concentrate all the products of the enzyme reaction in pregnenolone. The rate of sterol conversion into pregnenolone (CSCC enzyme activity) reflected the amount of cytochrome P-450scc (P-450scc), as was shown by Western blotting. In cells cultured without LH, the CSCC enzyme activity decreased to 10% on day 7 of the culture period. In the presence of various doses of LH ranging from 0.01 to 100 ng/ml, the CSCC enzyme activity also diminished during the first 3 days of culture, but during the following days, the amount of CSCC enzyme was stimulated by LH. In contrast to the absence of any LH effect on the activity of the CSCC enzyme during the first days of the culture, the endogenous steroid production (no added 22(R)-hydroxycholesterol) could be stimulated at least 10-fold by high doses of LH. When LH (1 ng/ml) was added to cells which had been cultured for 7 days without hormones, CSCC enzyme activity was elevated 8-fold after 4 days of exposure of LH. These effects of LH could be mimicked by dbcAMP (0.5 mM). No evidence could be provided that IGF-I plays any role in the LH induction of the CSCC enzyme; neither the addition of exogenous IGF-I or analogs that do not bind to IGF-I binding proteins (IGFBPs) nor the inactivation of endogenous IGF-I action (through binding to IGFBP and antibodies to IGF-I or via masking of IGF-I receptor by antibodies) could influence the LH induced CSCC enzyme activity. The present data raise the question under which conditions IGF-I is capable of modulating Leydig cell steroidogenesis.
Mol Cell Endocrinol 1992 Sep
PMID:Luteinizing hormone induction of the cholesterol side-chain cleavage enzyme in cultured immature rat Leydig cells: no role of insulin-like growth factor-I? 128 Feb 34

Adrenocorticotropin (ACTH) is known to exert an acute effect on adrenal steroidogenesis as well as long-term effects by regulation of gene expression. In order to further study the long-term action of ACTH, guinea pig fasciculata-glomerulosa (FG) cells in primary culture were treated for up to 72 h with ACTH. The effects of this treatment on steroid secretion, enzyme activity and mRNA levels for steroid enzymes were measured. While the rate of 17-deoxy C-21 steroid secretion decreased over the 72-h period of incubation with ACTH, the 17-hydroxy C-21 steroid secretion rate remained constant for the first 24 h of incubation and declined thereafter; the rate of 4-ene C-19 steroid secretion increased over the 72-h incubation period. ACTH treatment increased 17-hydroxylase and 17,20-lyase activities and the maximal stimulation was reached after 48 h. In contrast, the activity of 21-hydroxylase (P450c21) steadily declined over the 72-h incubation period. ACTH also caused an increase in mRNA levels for P450c21, 17-hydroxylase and 17,20-lyase (P450c17), 3 beta-hydroxysteroid dehydrogenase 4-ene-5-ene-isomerase (3 beta-HSD) and cholesterol side-chain cleavage enzyme (P450scc). The maximal stimulation for the four mRNAs was observed after 18 h of incubation with ACTH, decreasing afterwards except for P450c17 mRNA levels which remained elevated over the 72-h incubation period. Despite the increase in mRNA levels for 3 beta-HSD and P450c21, no increase in their respective enzyme activities was observed and 21-hydroxylase activity even declined over the 72-h incubation period with ACTH, thus suggesting that mechanism(s) other than gene expression alone regulate steroid secretion in FG cells. In conclusion ACTH caused major changes in steroid distribution due to increased 17-hydroxylase and 17,20-lyase activities and decreased 21-hydroxylase activity in FG cells in culture. Moreover, our data revealed major differences in the induction of mRNAs for steroidogenic enzymes and their activities following ACTH treatment.
J Steroid Biochem Mol Biol 1992 Jan
PMID:Effect of ACTH on steroidogenic enzymes in guinea pig fasciculata-glomerulosa cells: changes in activity and mRNA levels. 131 Apr 15

The chronic regulation of steroiodgenesis is mediated principally by transcriptional regulation of the genes encoding the various steroidogenic enzymes. The cholesterol side-chain cleavage enzyme, P450scc, is rate limiting and hormonally regulated in a tissue-specific fashion. Human placental steroidogenesis is regulated by LH and hCG through increased intracellular cAMP, and forskolin and 8-bromo-cAMP increase the abundance of human P450scc mRNA in human JEG-3 choriocarcinoma cells. We transfected JEG-3 cells with 24 promoter/reporter constructions to examine the tissue-specific and hormonally induced transcription of the human P450scc gene in these cells. A reporter construction containing only bases -79 to +49 of the human P450scc gene was expressed in JEG-3 cells. This basal expression was increased by four elements, especially by a powerful element between -152 to -142. Adding DNA sequences to -177 suppressed the basal expression seen with the -152 construction, indicating that a repressor element lies between -177 and -152. Thus, basal expression of the human P450scc gene in JEG-3 cells is mediated by the interplay of several separate cis-acting DNA elements. Forskolin induction was conferred by sequences between -108 and -89. The mechanism for cAMP induction appears to be direct, as this induction is rapid and is not blocked by inhibiting protein synthesis with cycloheximide. Gel mobility shift experiments identified six specific DNA-protein complexes. Five of these complexes correlate closely with the basal transcription activities identified by the reporter assays. The powerful basal element, the repressor element, and the cAMP element differ from those identified by similar experiments in mouse adrenal Y1 cells, suggesting that the human P450scc gene is regulated by the tissue-specific use of different regulatory elements.
Mol Endocrinol 1992 Dec
PMID:Identification of positive and negative placenta-specific basal elements and a cyclic adenosine 3',5'-monophosphate response element in the human gene for P450scc. 133 41

The regulation of steroidogenesis by luteinizing hormone (LH) was studied in granulosa cells during follicular development using a fluorescent reporter assay based on the metabolism of a fluorescent probe specific for cytochrome P-450SCC (cholesterol side-chain cleavage enzyme). Intact granulosa cells or mitochondria were obtained from the first (F1) second (F2) and third (F3) largest preovulatory follicles of the hen ovary and incubated with the fluorogenic substrate. Metabolism of this substrate by cytochrome P-450SCC generates the highly fluorescent resorufin anion (the fluorescent reporter). In both mitochondria and intact granulosa cells, incubated with the fluorescent substrate, an increase in resorufin fluorescence was observed and the increase was greater in samples derived from F1 than in samples from F2 or F3. In cells, LH added simultaneously with the P-450SCC substrate significantly increased resorufin fluorescence above control values in a time- and dose-dependent manner up to 2-3 h after the incubation was initiated. Forskolin and 8-bromo-cAMP also stimulated metabolism of the P-450SCC substrate significantly by 15 min. When granulosa cells were preincubated with LH before exposure to the P-450SCC substrate resorufin fluorescence was significantly attenuated compared to controls (not exposed to LH in the preincubation period). The decrease in resorufin fluorescence observed when cells were pretreated with LH, may be due to the release of cholesterol from endogenous pools and its competition with the exogenous fluorogenic for the substrate P-450SCC enzyme. In granulosa cells that were preloaded with the P-450SCC substrate, the stimulatory effect of LH treatment remained constant from 30 min to 2 h after hormone addition. The results show that this fluorescent probe can be used in a rapid assay for the continuous measurement of the acute effects of hormone agonists on cholesterol conversion to pregnenolone in steroidogenic cells.
J Steroid Biochem Mol Biol 1992 Nov
PMID:Hormone stimulated steroid biosynthesis in granulosa cells studied with a fluorogenic probe for cytochrome P-450SCC. 141 83

We recently showed that the production of progesterone (P4) in human placental explant culture from early gestation is enhanced by treatment with 19-nortestosterone (19-NT) or with certain androgens, namely androstenedione (A-dione), 5 alpha-androstane-3 alpha,17 beta diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta diol (3 beta-diol). This stimulation of P4 was explored further in this study. There was little metabolism of radioactive P4 when incubated for 24 h in the presence or absence of these steroids. The role of different steroids in the regulation of P450 cholesterol side-chain cleavage enzyme (P450scc) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) was evaluated by measuring the conversion of P4 derived from unlabelled 25-hydroxycholesterol and from labelled pregnenolone, respectively. The results showed that 19-NT, A-dione and 3 alpha-diol stimulated P450scc activity; however, 3 beta-diol was ineffective. While 19-NT and 3 beta-diol enhanced the bioconversion of pregnenolone to P4, A-dione and 3 alpha-diol were without effect. The initial rapid stimulation of P4 by 19-NT within 2 h of incubation was not blocked by concurrent treatment with cycloheximide (CH). However, after incubation for 24 h, 70% of the 19-NT-stimulated P4 was abolished by CH. During the same incubation period, P4 stimulation by A-dione, 3 alpha- and 3 beta-diol were completely blocked by treatment with CH. Thus our observations suggest that 19-NT-stimulated P4 accumulation is due to the combined effects on P450scc and 3 beta-HSD enzyme activities. A-dione and 3 alpha-diol increase biosynthesis of P4 by acting selectively on P450scc enzyme. However, the stimulatory action of 3 beta-diol on P4 is only at the level of 3 beta-HSD. Since CH blocks the stimulatory actions, the mechanism(s) by which androgens (A-dione, 3 alpha-diol and 3 beta-diol) and norandrogen (19-NT) augment the biosynthetic enzyme activities appears to be mediated by a process inhibited by CH. Since CH interference was absent during the initial rapid P4-stimulation by 19-NT, there may be a direct action of this steroid at the cellular level which is not dependent on new protein synthesis.
J Steroid Biochem Mol Biol 1992 Sep
PMID:Influence of 19-nortestosterone and androgens on progesterone biosynthetic enzymes in early human placental explants. 152 44

The side-chain cleavage of cholesterol by cytochrome P-450scc in mitochondria from the human placenta was studied using hydroxycholesterol substrates and intermediates of the reaction. 25-Hydroxycholesterol inhibited 3 beta-hydroxy-5-pregnen-20-one (pregnenolone) production by placental mitochondria. It was converted to pregnenolone at a maximum velocity of only 19% of that for cholesterol. Addition of 20 alpha-hydroxycholesterol or 22R-hydroxycholesterol to placental mitochondria caused a lag in pregnenolone synthesis which was concentration dependent. Measurement of the concentration of 20 alpha,22R-dihydroxycholesterol during incubation of placental mitochondria with 22R-hydroxycholesterol revealed that the lag in pregnenolone production was caused by accumulation of 20 alpha,22R-dihydroxycholesterol. This intermediate of the reaction dissociated from the active site of cytochrome P-450scc. Only after its concentration had increased, presumably to a level where it could compete with 22R-hydroxycholesterol for binding to cytochrome P-450scc, was it converted to pregnenolone. These results indicate a lack of kinetic stabilization of the cytochrome P-450scc-20 alpha,22R-dihydroxycholesterol complex with dissociation occurring more rapidly than the final hydroxylation. Similar measurements of side-chain cleavage of 22R-hydroxycholesterol by mitochondria from the bovine adrenal cortex showed that kinetic stabilization of the cytochrome P-450scc-20 alpha,22R-dihydroxycholesterol complex does not occur in that tissue either. The relative hydroxylation rates of 20 alpha-hydroxycholesterol, 22R-hydroxycholesterol and 20 alpha,22R-dihydroxycholesterol indicate that all three hydroxylations catalysed by human cytochrome P-450scc occur at approximately the same rate.
J Steroid Biochem Mol Biol 1992 Sep
PMID:Cholesterol side-chain cleavage by mitochondria from the human placenta. Studies using hydroxycholesterols as substrates. 152 48

The effects of various antimycotic reagents and some other reagents on a cytochrome P-450-linked monooxygenase system were investigated with respect to the activities of NADPH-ferricyanide reductase. NADPH-cytochrome c reductase of NADPH-adreno-ferredoxin reductase from NADPH to cytochrome c via adreno-ferredoxin, NADPH-cytochrome P-450-phenylisocyanide complex reductase, and the cholesterol side chain cleavage of the cytochrome P-450scc-linked monooxygenase system. No reagents inhibited the NADPH-ferricyanide reductase activity. Only cloconazole inhibited about 50% of NADPH-cytochrome c reductase activity. Cloconazole, econazole, clotrimazole, etomidate and ketoconazole inhibited both NADPH-cytochrome P-450-phenylisocyanide complex reductase and the side chain cleavage activity of cholesterol of the cytochrome P-450scc-linked monooxygenase system. Cloconazole, econazole, etomidate and ketoconazole behaved like non-competitive inhibitors for NADPH-cytochrome P-450-phenylisocyanide reductase activities and their Ki values were 10(-4)-10(-6) M. Cloconazole was a non-competitive inhibitor of NADPH-cytochrome c reductase and its Ki value was 8.3 x 10(-4) M. Cloconazole, clotrimazole, econazole, etomidate, ketoconazole and mitotane completely inhibited the side chain cleavage activity of cholesterol.
J Steroid Biochem Mol Biol 1992 May
PMID:Inhibition mechanism of reconstituted cytochrome P-450scc-linked monooxygenase system by antimycotic reagents and other inhibitors. 160 41

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 II, 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 Y1 cells. In Y1 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.
Mol Cell Biol 1990 Nov
PMID:Human P450scc gene transcription is induced by cyclic AMP and repressed by 12-O-tetradecanoylphorbol-13-acetate and A23187 through independent cis elements. 170 Feb 77

The adrenal cortex of the mouse coordinately expresses three cytochrome P450 enzymes that are required for the biosynthesis of corticosteroids: cholesterol side-chain cleavage enzyme (SCC), steroid 21-hydroxylase (21-OHase), and steroid 11 beta-hydroxylase (11 beta-OHase). Within their 5'-flanking regions, we previously identified six elements containing variations of an AGGTC motif that regulated expression in mouse Y1 adrenocortical cells: 21-OHase elements at -210, -140, and -65; SCC elements at -70 and -40; and an 11 beta-OHase element at -310. We demonstrate here that all six elements interact with the same, or closely related, DNA-binding protein(s). First, these elements all formed complexes of similar mobility in gel shift assays, suggesting that they interacted with protein(s) of similar size. Additional larger complexes were seen with those probes containing exact AGGTCA sequences. Second, competition experiments confirmed that the factor(s) interacting with different elements had closely related or identical recognition specificities. Finally, indistinguishable profiles of shift activities were seen upon fractionation of nuclear proteins over sequential chromatographic columns. Collectively, these results suggest that related elements interact with a shared protein to regulate three essential steroidogenic enzymes. An AGGTCA sequence motif comprises the response element for several members of the nuclear hormone receptor family. Oligonucleotide competitions and specific effects of antisera in gel shift assays implicated chicken ovalbumin upstream promoter-transcription factor in the formation of the larger complexes seen with the elements containing exact AGGTCA sequences. Therefore, this member of the nuclear hormone receptor family also may regulate the expression of the adrenal steroidogenic enzymes.
Mol Endocrinol 1991 Oct
PMID:A shared promoter element regulates the expression of three steroidogenic enzymes. 177 36


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