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

Abstract

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.

• Authors: A Minchenko, J Caro
• Journal: Molecular and cellular biochemistry (Mol Cell Biochem) Vol. 208 Issue 1-2 Pg. 53-62 (May 2000) ISSN: 0300-8177 [Print] Netherlands
• PMID: 10939628 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• DNA-Binding Proteins
• Endothelin-1
• Enzyme Inhibitors
• HIF1A protein, human
• Hypoxia-Inducible Factor 1
• Hypoxia-Inducible Factor 1, alpha Subunit
• Nuclear Proteins
• Protein Precursors
• RNA, Messenger
• Recombinant Fusion Proteins
• Transcription Factors
• Cobalt
• Genistein
• Luciferases

Topics

• Cell Hypoxia
• Cell Line
• Cobalt (metabolism)
• DNA-Binding Proteins (genetics, metabolism)
• Electrophoresis, Polyacrylamide Gel
• Endothelin-1 (biosynthesis, genetics, metabolism)
• Endothelium, Vascular (cytology, metabolism)
• Enzyme Inhibitors (pharmacology)
• Gene Expression Regulation
• Genes, Reporter
• Genistein (pharmacology)
• Humans
• Hypoxia-Inducible Factor 1
• Hypoxia-Inducible Factor 1, alpha Subunit
• Luciferases (genetics, metabolism)
• Nuclear Proteins (genetics, metabolism)
• Plasmids (genetics, metabolism)
• Protein Precursors (metabolism)
• RNA, Messenger (genetics, metabolism)
• Recombinant Fusion Proteins (genetics, metabolism)
• Response Elements (genetics)
• Transcription Factors (genetics, metabolism)