Abstract
An important regulator involved in oxygen-dependent gene expression is the transcription factor HIF (hypoxia-inducible factor), which is composed of an oxygen-sensitive alpha-subunit (HIF-1alpha or HIF-2alpha) and a constitutively expressed beta-subunit. In normoxia, HIF-1alpha is destabilized by post-translational hydroxylation of Pro-564 and Pro-402 by a family of oxygen-sensitive dioxygenases. The three HIF-modifying human enzymes have been termed prolyl hydroxylase domain containing proteins (PHD1, PHD2 and PHD3). Prolyl hydroxylation leads to pVHL (von-Hippel-Lindau protein)-dependent ubiquitination and rapid proteasomal degradation of HIF-1alpha. In the present study, we report that human PHD2 and PHD3 are induced by hypoxia in primary and transformed cell lines. In the human osteosarcoma cell line, U2OS, selective suppression of HIF-1alpha expression by RNA interference resulted in a complete loss of hypoxic induction of PHD2 and PHD3. Induction of PHD2 by hypoxia was lost in pVHL-deficient RCC4 cells. These results suggest that hypoxic induction of PHD2 and PHD3 is critically dependent on HIF-alpha. Using a VHL capture assay, we demonstrate that HIF-alpha prolyl-4-hydroxylase capacity of cytoplasmic and nuclear protein extracts was enhanced by prolonged exposure to hypoxia. Degradation of HIF-1alpha after reoxygenation was accelerated, which demonstrates functional relevance of the present results. We propose a direct, negative regulatory mechanism, which limits accumulation of HIF-1alpha in hypoxia and leads to accelerated degradation on reoxygenation after long-term hypoxia.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenocarcinoma, Clear Cell / enzymology
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Adenocarcinoma, Clear Cell / metabolism
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Adenocarcinoma, Clear Cell / pathology
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Bone Neoplasms / enzymology
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Bone Neoplasms / metabolism
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Bone Neoplasms / pathology
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Carcinoma, Hepatocellular / enzymology
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Carcinoma, Hepatocellular / metabolism
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Carcinoma, Hepatocellular / pathology
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Cell Line
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Cell Line, Tumor
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DNA-Binding Proteins / biosynthesis
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DNA-Binding Proteins / metabolism
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DNA-Binding Proteins / physiology*
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Dioxygenases
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Enzyme Induction / physiology
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Epithelial Cells / enzymology
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Epithelial Cells / metabolism
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Gene Expression Regulation, Enzymologic / physiology
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Gene Expression Regulation, Neoplastic / physiology
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Half-Life
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Humans
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Hydroxylation
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Hypoxia / enzymology*
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Hypoxia-Inducible Factor 1
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Hypoxia-Inducible Factor 1, alpha Subunit
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Hypoxia-Inducible Factor-Proline Dioxygenases
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Immediate-Early Proteins / biosynthesis
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Kidney Neoplasms / enzymology
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Kidney Neoplasms / metabolism
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Kidney Neoplasms / pathology
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Kidney Tubules, Proximal / cytology
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Kidney Tubules, Proximal / enzymology
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Kidney Tubules, Proximal / metabolism
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Liver Neoplasms / enzymology
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Liver Neoplasms / metabolism
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Liver Neoplasms / pathology
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Nuclear Proteins / metabolism
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Nuclear Proteins / physiology*
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Osteosarcoma / enzymology
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Osteosarcoma / metabolism
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Osteosarcoma / pathology
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Oxygen / metabolism
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Procollagen-Proline Dioxygenase / biosynthesis*
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Procollagen-Proline Dioxygenase / metabolism
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RNA, Messenger / biosynthesis
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Transcription Factors / metabolism
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Transcription Factors / physiology*
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Tumor Suppressor Proteins / physiology
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Ubiquitin-Protein Ligases / physiology
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Von Hippel-Lindau Tumor Suppressor Protein
Substances
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DNA-Binding Proteins
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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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Immediate-Early Proteins
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Nuclear Proteins
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RNA, Messenger
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Transcription Factors
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Tumor Suppressor Proteins
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Dioxygenases
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EGLN1 protein, human
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Procollagen-Proline Dioxygenase
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EGLN3 protein, human
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Hypoxia-Inducible Factor-Proline Dioxygenases
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Ubiquitin-Protein Ligases
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Von Hippel-Lindau Tumor Suppressor Protein
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VHL protein, human
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Oxygen