10098601

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3

The steroidogenic enzyme P450c17 (17alpha hydroxylase/C17,20 lyase) regulates a key branchpoint in steroidogenesis, as its activity directs the steroid biosynthetic pathways toward glucocorticoid or sex hormone synthesis. Expression of the P450c17 gene is transcriptionally regulated in steroidogenic tissues by cAMP. We showed that DNA between -84 and -55 in the rat P450c17 gene was bound uniquely by steroidogenic factor-1 (SF-1), which regulated both basal and cAMP-stimulated transcription in mouse adrenocortical and Leydig cells. SF-1 gene ablation experiments in mice indicate that SF-1 is not mandatory for placental steroidogenesis. We studied P450c17 gene regulation in the placenta using human placental JEG-3 trophoblast cells. Transfection of reporter luciferase gene constructs containing serial deletions of the 5' flanking region of the rat P450c17 gene showed that DNA between -98 and +13 mediated basal and cAMP-regulated transcription in placental JEG-3 cells, as it did in adrenal and Leydig cells. DNase footprints further identified a region between -88 and the TATA box that was bound by protein. Transfection of luciferase reporter constructs containing -84 to -55 of the rat P450c17 DNA ligated to the minimal promoter of the thymidine kinase gene showed that this DNA increased both basal and cAMP-simulated luciferase activity. Gel mobility shift assays identified two DNA-protein complexes with JEG-3 cell nuclear extracts that were different from complexes formed with MA-10 cell extracts and did not involve SF-1. Mutational analysis of the -84/-55 DNA showed that JEG-3 nuclear proteins bound to a site containing, but not identical to, the SF-1 sequence. One complex involved Ku autoimmune antigen, which bound to DNA sequence specifically. Overexpression of Ku antigen in MA-10 cells stimulated rat P450c17 gene transcription, thus demonstrating a biologic effect of Ku. Ku also bound to a similar region of the human P450c17 gene, and the DNA region to which Ku bound was transcriptionally active in JEG-3 cells. Ku was also found in extracts from rat placenta and bound to the -84/-55 rat P450c17 DNA. These data demonstrate a role of Ku in regulating P450c17 gene expression. These data further indicate that although human P450c17 is not normally expressed in the placenta, factors that could activate this gene are indeed present.

eng

IM

MEDLINE

1044-5498

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NLM

197-208

pubmed-meshheading:10098601-Adrenal Glands, pubmed-meshheading:10098601-Animals, pubmed-meshheading:10098601-Antigens, Nuclear, pubmed-meshheading:10098601-Base Sequence, pubmed-meshheading:10098601-Binding Sites, pubmed-meshheading:10098601-Cell Line, pubmed-meshheading:10098601-Choriocarcinoma, pubmed-meshheading:10098601-DNA, pubmed-meshheading:10098601-DNA Helicases, pubmed-meshheading:10098601-DNA-Binding Proteins, pubmed-meshheading:10098601-Female, pubmed-meshheading:10098601-Fushi Tarazu Transcription Factors, pubmed-meshheading:10098601-Gene Expression Regulation, pubmed-meshheading:10098601-Homeodomain Proteins, pubmed-meshheading:10098601-Humans, pubmed-meshheading:10098601-Leydig Cells, pubmed-meshheading:10098601-Male, pubmed-meshheading:10098601-Mice, pubmed-meshheading:10098601-Molecular Sequence Data, pubmed-meshheading:10098601-Nuclear Proteins, pubmed-meshheading:10098601-Placenta, pubmed-meshheading:10098601-Pregnancy, pubmed-meshheading:10098601-Protein Binding, pubmed-meshheading:10098601-Rats, pubmed-meshheading:10098601-Receptors, Cytoplasmic and Nuclear, pubmed-meshheading:10098601-Steroid 17-alpha-Hydroxylase, pubmed-meshheading:10098601-Steroidogenic Factor 1, pubmed-meshheading:10098601-Transcription, Genetic, pubmed-meshheading:10098601-Transcription Factors, pubmed-meshheading:10098601-Transcriptional Activation, pubmed-meshheading:10098601-Tumor Cells, Cultured

Ku autoimmune antigen is involved in placental regulation of rat P450c17 gene transcription.

Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't