Regulation of endothelin-1 gene expression in human microvascular endothelial cells by hypoxia and cobalt: role of hypoxia responsive element.
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.
Minchenko
A
A
Cardeza Foundation for Hematologic Research, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, PA 19107, USA.
Caro
J
J
D015687:Cell Hypoxia; D002460:Cell Line; D003035:Cobalt / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019332:Endothelin-1 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D019332:Endothelin-1; D004791:Enzyme Inhibitors; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D011498:Protein Precursors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D003035:Cobalt; D019833:Genistein; D008156:Luciferases