Abstract
Hypoxia results in adaptive changes in the transcription of a range of genes including erythropoietin. An important mediator is hypoxia-inducible factor-1 (HIF-1), a DNA binding complex shown to contain at least two basic helix-loop-helix PAS-domain (bHLH-PAS) proteins, HIF-1alpha and aryl hydrocarbon nuclear receptor translocator (ARNT). In response to hypoxia, HIF-1alpha is activated and accumulates rapidly in the cell. Endothelial PAS domain protein 1 (EPAS-1) is a recently identified bHLH-PAS protein with 48% identity to HIF-1alpha, raising the question of its role in responses to hypoxia. We developed specific antibodies and studied expression and regulation of EPAS-1 mRNA and protein across a range of human cell lines. EPAS-1 was widely expressed, and strongly induced by hypoxia at the level of protein but not mRNA. Comparison of the effect of a range of activating and inhibitory stimuli showed striking similarities in the EPAS-1 and HIF-1alpha responses. Although major differences were observed in the abundance of EPAS-1 and HIF-1alpha in different cell types, differences in the inducible response were subtle with EPAS-1 protein being slightly more evident in normoxic and mildly hypoxic cells. Functional studies in a mutant cell line (Ka13) expressing neither HIF-1alpha nor EPAS-1 confirmed that both proteins interact with hypoxically responsive targets, but suggest target specificity with greater EPAS-1 transactivation (relative to HIF-1alpha transactivation) of the VEGF promoter than the LDH-A promoter.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Aryl Hydrocarbon Receptor Nuclear Translocator
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Basic Helix-Loop-Helix Transcription Factors
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CHO Cells
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COS Cells
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Cell Hypoxia / physiology*
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Cell Line
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Cobalt / pharmacology
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Cricetinae
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Cricetulus
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DNA-Binding Proteins / biosynthesis*
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DNA-Binding Proteins / genetics
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Deferoxamine / pharmacology
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Endothelial Growth Factors / genetics
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Gene Expression Regulation*
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HeLa Cells / drug effects
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HeLa Cells / metabolism
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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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Iron Chelating Agents / pharmacology
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L-Lactate Dehydrogenase / genetics
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Lymphokines / genetics
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Mice
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Mice, Inbred BALB C
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Nuclear Proteins / biosynthesis*
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Nuclear Proteins / genetics
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Onium Compounds / pharmacology
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Promoter Regions, Genetic
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RNA, Messenger / biosynthesis
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Receptors, Aryl Hydrocarbon*
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Recombinant Fusion Proteins / biosynthesis
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Trans-Activators / biosynthesis*
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Trans-Activators / genetics
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Transcription Factors / metabolism
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Transcriptional Activation
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Transfection
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Vascular Endothelial Growth Factor A
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Vascular Endothelial Growth Factors
Substances
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ARNT protein, human
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Arnt protein, mouse
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Basic Helix-Loop-Helix Transcription Factors
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DNA-Binding Proteins
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Endothelial Growth Factors
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HIF1A protein, human
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Hif1a protein, mouse
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Hypoxia-Inducible Factor 1
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Hypoxia-Inducible Factor 1, alpha Subunit
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Iron Chelating Agents
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Lymphokines
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Nuclear Proteins
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Onium Compounds
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RNA, Messenger
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Receptors, Aryl Hydrocarbon
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Recombinant Fusion Proteins
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Trans-Activators
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Transcription Factors
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Vascular Endothelial Growth Factor A
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Vascular Endothelial Growth Factors
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Aryl Hydrocarbon Receptor Nuclear Translocator
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endothelial PAS domain-containing protein 1
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Cobalt
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diphenyleneiodonium
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L-Lactate Dehydrogenase
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cobaltous chloride
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Deferoxamine