Transcription of Cholesterol Side-Chain Cleavage Cytochrome P450 in the Placenta: Activating Protein-2 Assumes the Role of Steroidogenic Factor-1 by Binding to an Overlapping Promoter Element (original) (raw)
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2002
Progesterone is essential to the sustenance of pregnancy in humans and other mammals. From the second trimester on, the human placenta is the sole origin of de novo synthesized steroid hor- mones. In mice, placentation at midgestation is accompanied by a temporal rise of steroid hor- mone synthesis commencing in the giant cells of the mouse trophoblast. In doing so, the giant tro- phoblasts, as any other steroidogenic cell, express high levels of the key steroidogenic enzyme, cho- lesterol side-chain cleavage cytochrome P450 (P450scc). Because steroidogenic factor 1 (SF-1), the transcription factor required for expression of P450scc in the adrenals and the gonads, is not expressed in the placenta, we hypothesized that placenta-specific nuclear factor(s) (PNF) assumes the role of SF-1 by binding to the same promoter region that harbors the SF-1 recognition site in the P450scc gene. To address this possibility, we used SCC1, a well conserved proximal region in the P450scc genes (60/32 in the rat gene) to purify PNF from human term placenta. Sequencing of the purified PNF revealed that it is the isoform of the human activating protein-2 (AP-2). Specific anti- bodies tested in EMSA confirmed that AP-2 is the predominant isoform that binds SCC1 in the human placenta, whereas AP-2 is the only mouse placen- tal protein that binds this oligonucleotide. Func- tional studies showed that coexpression of the rat P450scc promoter (378/8 CAT) and AP-2 iso- forms ( or ) in human embryonic kidney 293 cells results in a marked activation of chloramphenicol acetyltransferase (CAT) transcription that is de- pendent on an intact AP-2 motif, GCCTTGAGC. This motif conforms with consensus sequences previously determined for binding of the AP-2 and isoforms. Mutations of the AP-2 element ablated binding of AP-2 to SCC1, as well as se- verely diminished the promoter activity in primary mouse giant trophoblasts and human choriocarci- noma JAR cells. Collectively, these studies sug- gest that expression of placental P450scc is gov- erned by AP-2 factors that bind to a cis-element that largely overlaps the sequence required for recognition of SF-1 in other steroidogenic tissues. (Molecular Endocrinology 16: 1864-1880, 2002)
Differential Regulation of the CYP11A1 (P450scc) and Ferredoxin Genes in Adrenal and Placental Cells
DNA and Cell Biology, 1993
The regulation of the genes encoding cholesterol side-chain cleavage enzyme (P450scc) and ferredoxin, two components in the first step of steroid synthetic pathways, was studied by RNA analyses of endogenous and transfected genes. cAMP rather than calcium was the major secondary messenger that stimulated expression of both P450scc and ferredoxin genes in human placental JEG-3 cells. The effect of cAMP on P450scc expression was abolished by cycloheximide in JEG-3 cells, but it was superinduced in mouse adrenal Yl cells. For ferredoxin expression, both reagents have synergistic effect in Yl and JEG-3 cells. To test the mechanism of regulation, DNA segments containing regulatory elements of the P450scc and ferredoxin genes were connected to reporter genes and analyzed in cotransfection experiments. The results showed that the proximal cAMP-responsive sequences of both P450scc and ferredoxin genes were stimulated by cAMP early in both Yl and JEG-3 cells, requiring no new protein synthesis. This indicates a common mechanism for the regulated expression of both genes. P450scc possessed an additional upstream cAMP-responsive sequence that also responded to cAMP induction in a different manner from the proximal element. The presence of additional upstream regulatory elements makes it possible for the P450scc gene to be further regulated.
Regulation of the Human P450scc Gene by Steroidogenic Factor 1 Is Mediated by CBP/p300
Journal of Biological Chemistry, 1998
Regulation of the human CYP11A gene encoding cytochrome P450scc, which catalyzes the first step of steroid synthesis, is regulated by many trans-acting transcription factors including steroidogenic factor 1 (SF-1). Transfection experiments in human adrenal NCI-H295 cells demonstrate regulation of the P450scc gene promoter region that contains several putative SF-1 binding sites. Cotransfection of SF-1 with a luciferase reporter construct containing the P450scc gene 5-flanking region from nucleotides ؊1676 to ؉49 increased promoter activity, and deletion of the nucleotide sequence from position ؊1676 to ؊1620, which removes a putative cAMP response element (CRE), did not affect the stimulatory response to SF-1. As well, further deletion of the promoter region to nucleotide ؊110, which contains only one SF-1 binding site, still retained the ability to respond to exogenous SF-1. However, mutation of the remaining site which abolished SF-1 protein/DNA interaction also abrogated any functional response to the factor. All the P450scc reporter constructs which responded to SF-1 were further stimulated by exogenous p300 and CREB-binding protein (CBP), suggesting interaction between SF-1 and p300/CBP. As well, mutation of the binding site that abrogated the response to SF-1 also abolished the response to p300 and CBP. Cotransfection of the adenovirus E1A oncoprotein, which has been shown to interact with p300/CBP and interfere with its function, decreased the stimulatory effect of SF-1 and p300/CBP. Cotransfection of a mutated E1A protein, RG2, which does not interact with p300/CBP, did not alter the stimulatory effect of SF-1 and p300/CBP on the P450scc promoter. Deletion of the region from amino acid residues 2-67 in E1A, which has been postulated to interact with p300/CBP, also abolished the inhibitory effect of E1A, whereas deletion of the region from residues 120 to 140 had no effect. Two regions of CBP from amino acids 1 to 451 and from 1460 to 1891 were demonstrated to interact with SF-1 in vitro. Coexpression of fragments of the p300 protein fused to the VP16 protein in the presence of SF-1 and the ؊110 P450scc reporter construct indicated in vivo the interaction of two regions of p300 with SF-1, thus confirming the in vitro
2011
BTGC or BNGC Binucleate trophoblast giant cells Ca 2 + BPs Binding proteins known as the calbindins Ca 2+ C a l c i u m Ca 2+ BP9k Calbindin-D9k calcitriol 1,25-dihydroxyvitamin D 3 cAMP Cyclic-AMP CEC Caruncular epithelial cells CL Corpus luteum CP Chorionic plate CRL Crown-rump length CSC Caruncular stromal cells CSH Chorionic somatomammotropin hormones CSH1 Chorionic somatomammotropin hormone 1 xvi (placental lactogen) CST3 Cystatin C CTSL Cathepsin L, cysteine proteinase CV Chorionic villi Cy3 Cyanine 3 CYP11A1 Cytochrome P450, family 11 subfamily, A polypeptide 1 CYP17A1 Cytochrome P450, family 17, subfamily A, polypeptide 1 CYP19A1 Cytochrome P450, family 19 subfamily, A
The FASEB Journal, 1996
Participation in steroid hormone biosynthesis in adrenal cortex and gonads is one of the important roles P450s play in endogenous substrate metabolism. The tissue-specific regulatory mechanisms by which genes encoding the steroid hydroxylases are expressed in these steroidogenic factories and not in other tissues have long been of interest. Recently an orphan nuclear receptor (Ad4BP/SF-1) has been discovered that is responsible for this tissue-specific pattern. Doctors Omura and Morohashi describe the tissue-specific and developmental patterns of expression of Ad4BP/SF-1, which provide insight into the important roles that this novel transcription factor plays in both steroidogenic and certain nonsteroidogenic cells.
Biochemical and Biophysical Research Communications, 1998
The nuclear receptor SF1 is an essential mediator in ventromedial hypothalamus-pituitary-gonadal development. As with other nuclear receptors, SF1 possesses a DNA-binding domain composed of two zinc fingers and a ligand-binding domain containing a ligand-dependent activation sequence termed AF2. To dissect the domain function of SF1, we examined various SF1 mutants in mouse adrenocortical Y1 cells and human placental JEG3 cells. Destruction of the AF2 structure removed 73-90% transactivation activity, suggesting that AF2 is indispensable for transactivation. Mutants carrying the DNA-binding domain but lacking the AF2 or the ligand-binding domain blocked the activity of normal SF1. Disrupting the zinc finger diminished the dominant negative effect of mutant. Cotransfection of SF1 with AP1 showed that the two transcription factors cooperated to activate gene expression. Some mutants lost the synergistic action with AP1, while some retained partial activity. These experiments delineate the functional domains of SF1.
Journal of Endocrinology, 1997
We have investigated the expression of cholesterol side-chain cleavage cytochrome P450 (P450scc) and 3hydroxysteroid dehydrogenase (3-HSD) type 1 genes during human trophoblast differentiation in culture and the modulation of their steady-state mRNA levels by steroids. During the first 24 to 48 h after plating, mononucleated cells aggregated, forming colonies. After 60 h in culture, cell diameters were increased and nuclei appeared centrally distributed within large cells, consistent with syncytiotrophoblast formation. During these striking morphological changes in culture the expression and activity levels of 3-HSD type 1 and P450scc increased significantly as isolated cytotrophoblasts progressed to a differentiated state, with P450scc and 3-HSD type 1 mRNAs activities being more abundant in cells cultured for 48 to 72 h. In the same culture, however, the amount of 3-HSD protein decreased during the first 12 to 24 h by 50% compared with freshly isolated trophoblasts but remained at these levels throughout the culture period. The specific activity of the 3-HSD as determined with pregnenolone or dehydroepiandrosterone was similar but increased with time as syncytiotrophoblast was formed in vitro. These observations provide additional evidence that the expression of these two progesterone-synthesizing enzymes is coincident and that they reach their maximum steady-state mRNA levels at a time when syncytium formation occurs in vitro. Incubation of trophoblast cells with progesterone or estradiol increased the abundance of P450scc and 3-HSD type 1 mRNAs but had no significant effect on the amount of 3-HSD protein. These observations of the regulation of 3-HSD type 1 mRNA levels by steroids suggest a complex relationship of the mechanisms regulating transcription/mRNA processing and transduction of the 3-HSD type 1 gene.
Gene regulation of steroidogenesis
The Journal of Steroid Biochemistry and Molecular Biology, 1995
The biosynthesis of various steroid hormones in animal tissues are catalyzed by six forms of cytochrome P450 and two hydroxysteroid dehydrogenases. The tissue-specific expression of these enzymes, which are under the control of the pituitary gland and mainly regulated at the transcriptional level, determines the steroidogenesis of animal tissues. Analysis of the promoter regions of the steroidogenic P450 genes revealed various cis-acting elements, including cAMP-responsive sequences (CRS), Ad4, and GC-rich sequences, which were needed for the tissue-specific and cAMP-dependent expression of the genes. Some of the nuclear protein factors binding to the cis-acting elements were isolated and characterized. A zinc-finger protein binding to Ad4, which was termed Ad4BP or SF-1, seems to be of particular importance in steroidogenesis. Ad4BP was expressed in the cells of the steroidogenic tissues, adrenal gland and gonadal tissues, in the rat fetus prior to the expression of steroidogenic P450s, and remained expressed only in steroidogenic cells in adult animals. Close investigation of the temporal and spacial expression of Ad4BP in the fetal tissues suggested its role in the differentiation of the steroidogenic tissues and the sex determination of the gonadal tissues.