Abstract
Hypoxia-inducible factors (HIFs) locate to HIF-binding sites (HBSs) within the hypoxia-response elements (HREs) of oxygen-regulated genes. Whereas HIF-1alpha is expressed ubiquitously, HIF-2alpha is found primarily in the endothelium, similar to endothelin-1 (ET-1) and fms-like tyrosine kinase 1 (Flt-1), the expression of which is controlled by HREs. We identified an unique sequence alteration in both ET-1 and Flt-1 HBSs not found in other HIF-1 target genes, implying that these HBSs might cause binding of HIF-2 rather than HIF-1. However, electrophoretic mobility shift assays showed HIF-1 and HIF-2 DNA complex formation with the unique ET-1 HBS to be about equal. Both DNA-binding and hypoxic activation of reporter genes using the ET-1 HBS was decreased compared with transferrin and erythropoietin HBSs. The Flt-1 HBS was non-functional when assayed in isolation, suggesting that additional factors are required for hypoxic up-regulation via the reported Flt-1 HRE. Interestingly, HIF-1 activity could be restored fully by point-mutating the ET-1 (but not the Flt-1) HBS, suggesting that the wild-type ET-1 HBS attenuated the full hypoxic response known from other oxygen-regulated genes. Such a mechanism might serve to limit the expression of this potent vasoconstrictor in hypoxia.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Base Sequence / genetics
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Basic Helix-Loop-Helix Transcription Factors
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Binding Sites / genetics
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Binding, Competitive
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Cell Hypoxia / genetics
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DNA-Binding Proteins / metabolism
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DNA-Binding Proteins / physiology*
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Endothelin-1 / genetics
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Endothelin-1 / metabolism*
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Erythropoietin / metabolism
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Gene Expression
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Genes, Reporter
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HeLa Cells
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Humans
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Hypoxia-Inducible Factor 1
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Hypoxia-Inducible Factor 1, alpha Subunit
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Nuclear Proteins / metabolism
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Nuclear Proteins / physiology*
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Protein Biosynthesis
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism
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Receptor Protein-Tyrosine Kinases / genetics
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Receptor Protein-Tyrosine Kinases / metabolism
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Response Elements / physiology
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Trans-Activators / genetics
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Trans-Activators / metabolism
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Transcription Factors / genetics
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Transcription Factors / metabolism
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Tumor Cells, Cultured
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Vascular Endothelial Growth Factor Receptor-1
Substances
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Basic Helix-Loop-Helix Transcription Factors
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DNA-Binding Proteins
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Endothelin-1
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HIF1A protein, human
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Hypoxia-Inducible Factor 1
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Hypoxia-Inducible Factor 1, alpha Subunit
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Nuclear Proteins
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Proto-Oncogene Proteins
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Trans-Activators
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Transcription Factors
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Erythropoietin
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endothelial PAS domain-containing protein 1
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Receptor Protein-Tyrosine Kinases
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Vascular Endothelial Growth Factor Receptor-1