10939628

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Endothelin-1 (Et-1) is a vasoconstrictor peptide that plays an important role in the pathophysiology of hypertension, myocardial ischemia, and other diseases. We examined the mechanism of regulation the Et-1 mRNA expression in human microvascular endothelial cells (HMEC-1) in response to hypoxia and cobalt. To determine whether the 5'-flanking region of Et-1 gene mediate transcriptional responses to cellular hypoxia, we constructed reporter plasmids in which Et-1 5'-flanking sequences of Et-1 gene were fused to luciferase coding sequences. Constructs, which contain native Et-1 sequence 5'-AACGTGCA-3', located between -118 and -125 in the opposite orientation as the transcriptional unit, mediate transcriptional response to hypoxia and cobalt. This responsiveness was inhibited by genistein, a tyrosine kinase selective inhibitor. Both hypoxia and cobalt induced binding of HIF-1 (hypoxia inducible-1 factor) to this Et-1 hypoxia responsive element in gel shift assays. Mutation in this sequence eliminated both the hypoxia-induced HIF-1 binding and luciferase expression. Using the supershift assay we have shown that this hypoxia responsive element binds HIF-1alpha and HIF-1beta proteins. Interestingly, genistein only slightly affected HIF-1 binding. These results indicate that the Et-1 gene contains HIF-1 binding hypoxia responsive elements which mediate transcriptional responses to hypoxia and cobalt in microvascular endothelial cells. Genistein appears to inhibit this response by affecting the transcriptional activity of the HIF-1 complex, without significantly affecting its DNA-binding properties.

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0300-8177

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53-62

pubmed-meshheading:10939628-Cell Hypoxia, pubmed-meshheading:10939628-Cell Line, pubmed-meshheading:10939628-Cobalt, pubmed-meshheading:10939628-DNA-Binding Proteins, pubmed-meshheading:10939628-Electrophoresis, Polyacrylamide Gel, pubmed-meshheading:10939628-Endothelin-1, pubmed-meshheading:10939628-Endothelium, Vascular, pubmed-meshheading:10939628-Enzyme Inhibitors, pubmed-meshheading:10939628-Gene Expression Regulation, pubmed-meshheading:10939628-Genes, Reporter, pubmed-meshheading:10939628-Genistein, pubmed-meshheading:10939628-Humans, pubmed-meshheading:10939628-Hypoxia-Inducible Factor 1, pubmed-meshheading:10939628-Hypoxia-Inducible Factor 1, alpha Subunit, pubmed-meshheading:10939628-Luciferases, pubmed-meshheading:10939628-Nuclear Proteins, pubmed-meshheading:10939628-Plasmids, pubmed-meshheading:10939628-Protein Precursors, pubmed-meshheading:10939628-RNA, Messenger, pubmed-meshheading:10939628-Recombinant Fusion Proteins, pubmed-meshheading:10939628-Response Elements, pubmed-meshheading:10939628-Transcription Factors

Regulation of endothelin-1 gene expression in human microvascular endothelial cells by hypoxia and cobalt: role of hypoxia responsive element.

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