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

Studies of adrenal steroidogenesis have been facilitated by the availability of immortalized mouse adrenocortical Y-1 cells. We sought to make new, alternative mouse steroidogenic cell lines by genetically targeted tumorigenesis. Transgenic mice were constructed expressing both the SV40 T-antigen and a bacterial neomycin-resistance gene under the control of the promoter for the human P450 cholesterol side-chain cleavage (P450scc) gene, which encodes the first and rate-limiting enzyme in steroidogenesis. Two female transgenic mice expressed T-antigen in various nonsteroidogenic tissues but generated tumors only in the adrenals, suggesting adrenal tumor formation was an early event. Ovarian tissues, which, unlike the adrenal, do not make steroids in fetal or early postnatal life, did not develop tumors. Cell lines derived from the adrenal tumors were resistant to the neomycin analog G418. Clonal sublines are stable, growing easily in monolayers with a doubling time of 24-60 h. The cell lines secrete progesterone and 11-deoxycorticosterone, indicating these cells express the P450scc system, 3 beta-hydroxysteroid dehydrogenase, and 21-hydroxylase activity. However the 21-hydroxylase activity was not mediated by P450c21, as the cells lacked P450c21 mRNA. The cells did not secrete any 11-hydroxylated steroids, although they contained P450c11 beta mRNA. Both the secretion of progesterone and the abundance of P450scc mRNA increase in response to 8-bromo-cAMP, but not to ACTH or angiotensin II. In addition to expression of steroidogenic enzyme mRNAs, one cell line also expresses mouse renin-1 mRNA, making these cells useful for studies of the role of adrenal renin in regulating adrenal steroidogenesis. These findings represent an approach in transgenic mice to develop highly differentiated adrenal cell lines.
Mol Endocrinol 1994 Jan
PMID:Steroidogenic adrenocortical cell lines produced by genetically targeted tumorigenesis in transgenic mice. 815 34

Production and secretion of steroid hormones throughout the ovarian cycle occurs in a highly episodic and coordinated fashion that requires precise and finely tuned regulatory mechanisms. The regulation of ovarian steroidogenesis by the gonadotropin follicle stimulating hormone (FSH) and luteinizing hormone (LH) as well as by other factors occurs, at least in part, at the level of expression of the genes encoding steroidogenic enzymes. The present study is aimed at the elucidation of regulatory mechanisms by which cyclic adenosine monophosphate (cAMP) and protein kinase C regulate cytochrome P450scc (CYP11A) gene expression in bovine granulosa cells in primary culture. As a first step we characterized the bovine granulosa cell cultures with regard to regulation of P450scc activity and mRNA levels upon treatment with forskolin and/or the phorbol ester TPA. Forskolin, a potent stimulator of cAMP generation, increased both progesterone secretion and P450scc mRNA levels. In contrast, treatment with TPA alone decreased both basal progesterone production and P450scc mRNA accumulation. Co-treatment with forskolin and TPA decreased progesterone and P450scc mRNA levels as compared to forskolin treatment alone. The possibility that TPA interfered with the forskolin-stimulated cAMP production could be excluded because simultaneous treatment of granulosa cells with TPA and forskolin potentiated the formation of cAMP. In order to identify regulatory sequences within the 5' flanking region of the bovine CYP11A gene, chimeric DNA constructs comprizing regions of the CYP11A gene fused to a beta-globin-derived reporter gene were transfected into granulosa cells in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1993 Aug
PMID:Regulation of CYP11A gene expression in bovine ovarian granulosa cells in primary culture by cAMP and phorbol esters is conferred by a common cis-acting element. 822 26

Rat adrenal mitochondria contain approximately equal levels of P450scc and P45011 beta, each reduced by NADPH through adrenodoxin reductase (ADX-reductase) and adrenodoxin (ADX). Constitutive cholesterol side-chain cleavage (SCC) can be increased over 20-fold through a combination of hormonal activation and inhibition of cholesterol metabolism in vivo prior to isolation of the mitochondria. This stimulation, which results from accumulated reactive cholesterol, does not significantly affect either the dependence of activities on the concentration of isocitrate (IC) and succinate (SU) or the ratio of maximum activities [3:1] supported by these reductants. Thus, the rate of cholesterol SCC is determined independently by electron transfer and the amount of reactive cholesterol. Hydroxylation of deoxycorticosterone (11 beta and 18 positions) required much higher levels of each reductant, indicating less effective reductant transfer to P45011 beta. Reactions at P450scc and P45011 beta, mediated by IC, are enhanced by low concentrations of various dicarboxylates anions (fumarate, SU). The actions of SU dehydrogenase inhibitors and the activity of fumarate, a poor direct reductant, suggest that higher production of NADPH results from malate-enhanced uptake of isocitrate. Only synergistic combinations of reductants are sufficient to sustain maximum rates of 11-deoxycorticosterone (DOC) metabolism, whereas IC is fully effective for P450scc. Increased reaction at P450scc (cholesterol loading or addition of 20 alpha-hydroxycholesterol) decreased simultaneous DOC metabolism at P45011 beta in inverse proportion to the estimated intramitochondrial generation of NADPH (1 mM or 10 mM SU > 1 mM IC > 10 mM IC). These decreases were reversed by inhibition of P450scc. Crossover inhibition caused by maximum DOC metabolism was less pronounced. EGTA/albumin treatment, which enhanced activities at both P450scc and P45011 beta, presumably via increased NADPH, diminished this cross-competition. The differential dependence on reductants and the characteristics of crossover competition are consistent with a roughly three-fold more favorable partitioning of electron transfer to P450scc, possibly caused by preferential interaction of reduced adrenodoxin with P450scc.
Mol Cell Endocrinol 1993 Sep
PMID:Competition for electron transfer between cytochromes P450scc and P45011 beta in rat adrenal mitochondria. 824 98

NCI-H295 is a recently described human adrenocortical carcinoma cell line that makes a variety of steroid hormones. We sought to determine if steroidogenesis in these cells employs the same enzymes as those used in normal adrenal steroidogenesis, and if the genes encoding those enzymes exhibit characteristic responsiveness to activators of the protein kinase-A and -C pathways of intracellular second messengers. Northern blots show that NCI-H295 cells contain abundant mRNAs for three key steroidogenic enzymes, cytochrome P450scc, cytochrome P450c17, and cytochrome P450c21. These mRNAs accumulated in a time- and dose-dependent fashion in response to 8-bromo-cAMP (8Br-cAMP), forskolin, cholera toxin, and 3-isobutyl-1-methylxanthine, all activators of the protein kinase-A pathway. Nuclear run-on assays and actinomycin-D transcriptional inhibition experiments show that cAMP regulates the expression of all three genes primarily at the transcriptional level. Inhibition of protein synthesis with cycloheximide did not prevent the cAMP-induced accumulation of P450scc or P450c17 mRNAs, but did inhibit accumulation of P450c21 mRNA, suggesting that cAMP is acting through a mechanism dependent on protein synthesis to promote accumulation of P450c21 mRNA. Stimulation of the protein kinase-C pathway with phorbol ester decreased P450scc and P450c17 mRNAs, but stimulated the accumulation of P450c21 mRNA. RNase protection experiments, Northern blot hybridizations, and reverse transcription-polymerase chain reaction show that NCI-H295 cells express both the 11 beta-hydroxylase (P450c11 beta) encoded by the P450c11B1 gene and the aldosterone synthetase (P450c11AS) encoded by the P450c11B2 gene. 8Br-cAMP increased the abundance of both of these mRNAs with similar kinetics, with maximal accumulation of both after about 24 h. NCI-H295 cells also contain the mRNAs for aromatase and insulin-like growth factor-II. 8Br-cAMP increased the abundance of aromatase mRNA and decreased the abundance of IGF-II mRNA. These studies show that NCI-H295 cells express most of the enzymes needed for human adrenal steroidogenesis, and that the genes encoding these enzymes respond to stimulation of second messenger pathways in a manner similar to that of human adrenals. NCI-H295 cells appear to be a good model for studying the molecular regulation of human adrenal steroidogenesis.
Mol Endocrinol 1993 Mar
PMID:Regulation of steroidogenesis in NCI-H295 cells: a cellular model of the human fetal adrenal. 838 59

The study of regulation of steroidogenesis in primary cultures of rat granulosa cells is difficult because the cells do not undergo more than one cell doubling in culture. Furthermore, there is size and steroidogenic heterogeneity in granulosa cells and it is difficult to obtain pure, functionally defined populations. Hence, it is advantageous to develop a homogeneous population of granulosa cells. In this report we describe the characterization of one such cell line (Rao-gcl-29) developed from diethylstilbestrol treated immature rat granulosa cells by transformation with SV40 T antigen. In this cell line cyclic AMP analogs induce high levels of progesterone biosynthesis, though there was no effect on estradiol biosynthesis. Also, FSH and hCG have no effect on progesterone biosynthesis. In the presence of FBS medium (20% fetal bovine serum in DMEM/F-12) and enriched medium (10% fetal bovine serum, 10% horse serum and 2% UltraSer G in DMEM/F-12 medium), 1 mM cAMP analogs induce high levels of progesterone biosynthesis up to 96 h. Ultrastructural features of the cell line resemble those of primary granulosa cells, in addition to forming gap junctions. Cyclic AMP analogs also induced cytochrome P450scc mRNA in this cell line by 48 h, and this effect is apparent by 24 h. Thus, this cell line could be useful in understanding the molecular mechanisms of regulation of cytochrome P450scc gene regulation.
Mol Cell Endocrinol 1993 Jul
PMID:Characterization of progesterone biosynthesis in a transformed granulosa cell line. 839 19

In the most severe form of congenital adrenal hyperplasia (CAH), termed lipoid CAH, both the adrenals and gonads fail to convert cholesterol to pregnenolone, so that no steroid hormones are made. Newborns have female external genitalia irrespective of karyotype, and suffer a severe salt-losing form of CAH. Previous studies have shown that adrenal or gonadal mitochondria from these patients also fail to convert cholesterol to pregnenolone in vitro, implicating a lesion in the single gene for P450scc, which is the sole enzyme converting cholesterol to pregnenolone. Two patients with XY karyotypes had female genitalia and unmeasurable steroids after stimulation with ACTH and hCG. ACTH stimulation tests of parents, obligate heterozygotes, showed normal stimulation of all precursor steroids. Southern blotting patterns of the P450scc gene were normal. Oligonucleotide-initiated enzymatic amplification (PCR) of all P450scc exons showed normal sequences on multiple amplifications and sequencing reactions, indicating normal P450scc genes. Northern blots of testicular RNA from a 6-month-old patient and from a control fetus showed normal P450scc mRNA, indicating a normal P450scc promoter. Reprobing of the blot with our cloned human cDNAs for adrenodoxin reductase and adrenodoxin showed that these electron transport cofactors used by P450scc were also normal. Similarly, probing with cDNAs for all three known factors involved in cholesterol transport to the mitochondria-sterol carrier protein 2, endozepine, and steroidogenesis activator peptide were also normal. These results suggest that the lesion in lipoid CAH is not in the P450scc system or in any known step upstream from P450scc.
J Steroid Biochem Mol Biol 1993 Apr
PMID:Congenital lipoid adrenal hyperplasia--genes for P450scc, side chain cleavage enzyme, are normal. 848 56

We have previously reported the co-localization [Cherradi et al., Endocrinology 134 (1994) 1358-1364] of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) and cytochrome P450scc (cyt. P450scc) in the inner membrane and in the intermembrane contact sites of adrenocortical mitochondria. This observation raises the question of a possible functional association between the two proteins. Isolated bovine adrenocortical mitochondria are able to convert cholesterol to progesterone without the need of exogenous cofactors. An association of 3 beta-HSD and cyt. P450scc is observed during the purification of 3 beta-HSD from mitochondria. The behaviour of 3 beta-HSD on a column of Heparin-Sepharose is modified by the presence of cyt. P450scc. Immunoprecipitations from mitochondria with either anti-cyt. P450scc or anti 3 beta-HSD antibodies result in a co-precipitation of the two proteins. Both proteins engaged in these immunocomplexes are catalytically active. The interaction was further demonstrated by the surface plasmon resonance method using purified components. An affinity demonstrated by the surface plasmon resonance method using purified components. An affinity constant of 0.12 microM between 3 beta-HSD and P450scc was obtained. These observations suggest that P450scc and 3 beta-HSD may associate into a molecular complex in the mitochondrial compartment and may constitute a functional steroidogenic unit, thus opening new possibilities in the regulation of the production of progesterone and its flow in the adrenocortical cell.
J Steroid Biochem Mol Biol 1995 Dec
PMID:Organization of 3 beta-hydroxysteroid dehydrogenase/isomerase and cytochrome P450scc into a catalytically active molecular complex in bovine adrenocortical mitochondria. 854 75

Much of the cholesterol used in steroid synthesis is stored in lipid droplets in the cytoplasm of steroid-forming cells. The cholesterol ester in these droplets is transported to the inner mitochondrial membrane where it enters the pathway to steroid hormones as free cholesterol--the substrate for the first enzyme, namely P450scc. It has been shown that this transport process governs the rate of steroid synthesis and is specifically stimulated by ACTH and its second messenger. The stimulating influence of ACTH on cholesterol transport is inhibited by cytochalasins, by monospecific anti-actin and by DNase I demonstrating that the steroidogenic cell must possess a pool of monomeric actin available for polymerization to F actin if it is to respond to ACTH and cyclic AMP. It has been shown that the two structures involved in cholesterol transport (droplets and mitochondria) are both bound to vimentin intermediate filaments in adrenal and Leydig cells. In addition these filaments are closely associated with the circumferential actomyosin ring in which they are crosslinked by actin microfilaments. In permeabilized adrenal cells Ca2+/calmodulin phosphorylates vimentin and this change is known to disrupt intermediate filaments and to cause contraction of actomyosin by phosphorylating myosin light chain kinase. Ca2+/calmodulin stimulated cholesterol transport and steroid synthesis and causes rounding of the responding cells by contraction of the actomyosin, if ATP is also added at the same time. Other agents that disrupt intermediate filaments include anti-vimentin plus ATP in permeabilized cells which also results in rounding of the cell. Acrylamide exerts a similar effect in intact adrenal cells and in addition causes rounding of the cells and increase in steroid synthesis without increase in cyclic AMP. It is also known that if adrenal cells are grown on surfaces treated with poly(HEMA), the cells grow in rounded form and steroid synthesis is increased in proportion to the degree of rounding (r = 0.92). This response does not involve increase in cellular levels of cyclic AMP. It is proposed that in vivo where the cell is always round and cannot show more than strictly limited change in shape, ACTH activates Ca2+/calmodulin possibly by redistributing cellular Ca2+. Ca2+/calmodulin in turn promotes phosphorylation of vimentin and myosin light chain. The first of these phosphorylations shortens intermediate filaments and the second promotes contraction of the actomyosin ring with internal shortening and approximation of lipid droplets and mitochondria. Details of the earlier events (activation of Ca2+/calmodulin) and later changes (transfer of cholesterol to the inner membrane) remain to be elucidated. It is clear however that the action of ACTH requires increase in cellular cyclic AMP. These experimental responses bypass this step in the response to ACTH.
J Steroid Biochem Mol Biol 1995 Dec
PMID:The roles of microfilaments and intermediate filaments in the regulation of steroid synthesis. 854 87

Studies in human beings, animals, and cell systems show that the rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone. In the adrenals and gonads, this step is subject to both acute and chronic regulation. Chronic regulation is primarily, but not exclusively at the level of gene transcription, leading to the production of more steroidogenic machinery and thus increasing the cellular capacity for steroidogenesis. Chronic regulation can be inhibited by inhibiting protein synthesis with cycloheximide, but this response varies among various cell types and species. Although the P450scc enzyme system that converts cholesterol to pregnenolone is inherently very slow, the principal site of acute regulation is at the delivery of free cholesterol to mitochondria, rather than at the delivery of reducing equivalents to P450scc. Even when the Vmax of the P450scc system is increased 6-fold by genetic engineering, delivery of cholesterol to the enzyme remains rate-limiting. Targeting of a genetically engineered fusion of the P450scc system to either mitochondria or to the endoplasmic reticulum of non-steroidogenic cells demonstrates that the mitochondrial environment is absolutely required for the conversion of cholesterol to pregnenolone, and that this absolute requirement is not based on either the nature of the available electron donors for P450scc or the availability of substrate. Various factors have been proposed as the essential mediator for the transport of cholesterol into mitochondria to initiate steroidogenesis. A recently identified protein termed Steroidogenic Acute Regulatory protein (StAR) has the necessary properties of enhancing steroidogenesis, rapid cAMP inducibility and rapid cycloheximide sensitivity that characterize the long-sought acute regulator of steroidogenesis. StAR is expressed in steroidogenic tissues exhibiting an acute response but not in steroidogenesis. StAR is expressed in steroidogenic tissues exhibiting an acute response but not in steroidogenic tissues (placenta, brain) that do not exhibit this response. Mutations in StAR are now shown to cause Congenital Lipoid Adrenal Hyperplasia, the last unsolved form of CAH. The actions of StAR can be circumvented by the use of hydroxycholesterols that can freely diffuse into mitochondria, proving that StAR functions as an acute regulator of cholesterol access to mitochondria.
J Steroid Biochem Mol Biol 1995 Dec
PMID:Mitochondrial specificity of the early steps in steroidogenesis. 854 88

Regulation of steroidogenesis in classic endocrine tissues is mediated by transcriptional regulation of the P450scc gene, which encodes the first and rate-limiting cholesterol side-chain cleavage enzyme. We previously showed that P450scc messenger RNA is regionally expressed in the adult rat brain, primary glial cultures, and C6 glioma cells. Expression of P450scc in the brain results in the de novo synthesis of neurosteroids, a class of steroid hormones that are active at gamma-aminobutyric acidA and N-methyl-D-aspartate receptors. We determined whether P450scc expression is transcriptionally regulated in neural cells, using the same DNA sequences and nuclear proteins as classic steroidogenic adrenal and Leydig cells. The transcriptional activity of deletional mutants of 2.5 kilobases of the 5'-flanking regulatory region of the rat P450scc gene cloned into a luciferase reporter gene was assessed in mouse adrenocortical Y-1, mouse Leydig MA-10, rat C6 glioma, rat GC somatotrope, and mouse GT1-7 neurosecretory cell lines. P450scc was transcriptionally regulated in Y-1, MA-10, and C6 glioma cells, but not in GC or GT1-7 cells. In one region (-94/-35), putative steroidogenic factor-1-binding sites appeared to be critical for the basal transcriptional activity and cAMP responsiveness in steroidogenic Y-1 and MA-10 cells, but had no function in rat C6 cells. DNA sequences between -94/-130 mediated both basal and cAMP-inducible transcriptional activity in C6 cells. Gel mobility shift assays showed that one nuclear protein binding to DNA sequences between -54 and -35 was abundant in MA-10 and Y-1 cells, but was absent from C6 cells, whereas another nuclear protein, binding to DNA sequences between -94 and -130 was abundant in C6 cells, but was rare in MA-10 cells and absent from Y-1 and other adrenocortical cells. Although the DNA sequence between -94 and -130 contains an Sp1 site, Sp1 did not bind to this site. Nevertheless, this GC-rich region was critical for nuclear protein binding and for basal and cAMP-induced transcriptional regulation in both C6 and MA-10 cells. These observations demonstrate that the rat P450scc gene is transcriptionally regulated in glioma cells, but its regulation in glial cells involves a DNA element different from those used in classic steroidogenic tissues. The results further suggest that steroidogenic factor-1 is not involved in regulating neurosteroidogenesis.
Mol Endocrinol 1995 Nov
PMID:Transcriptional regulation of P450scc gene expression in neural and steroidogenic cells: implications for regulation of neurosteroidogenesis. 858 34


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