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

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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-1α and HIF-1β 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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  1. Cardeza Foundation for Hematologic Research, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, PA, 19107, USA

    Alexander Minchenko & Jaime Caro

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  1. Alexander Minchenko
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Minchenko, A., Caro, J. Regulation of endothelin-1 gene expression in human microvascular endothelial cells by hypoxia and cobalt: Role of hypoxia responsive element. Mol Cell Biochem 208, 53–62 (2000). https://doi.org/10.1023/A:1007042729486

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