Abstract
Hypoxia-inducible factor (HIF), a transcriptional complex conserved from Caenorhabditis elegans to vertebrates, plays a pivotal role in cellular adaptation to low oxygen availability. In normoxia, the HIF-alpha subunits are targeted for destruction by prolyl hydroxylation, a specific modification that provides recognition for the E3 ubiquitin ligase complex containing the von Hippel-Lindau tumour suppressor protein (pVHL). Three HIF prolyl-hydroxylases (PHD1, 2 and 3) were identified recently in mammals and shown to hydroxylate HIF-alpha subunits. Here we show that specific 'silencing' of PHD2 with short interfering RNAs is sufficient to stabilize and activate HIF-1alpha in normoxia in all the human cells investigated. 'Silencing' of PHD1 and PHD3 has no effect on the stability of HIF-1alpha either in normoxia or upon re-oxygenation of cells briefly exposed to hypoxia. We therefore conclude that, in vivo, PHDs have distinct assigned functions, PHD2 being the critical oxygen sensor setting the low steady-state levels of HIF-1alpha in normoxia. Interestingly, PHD2 is upregulated by hypoxia, providing an HIF-1-dependent auto-regulatory mechanism driven by the oxygen tension.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Biological Transport
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Cell Hypoxia
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Cell Line
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Cell Nucleus / metabolism
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DNA-Binding Proteins / chemistry
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DNA-Binding Proteins / drug effects
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DNA-Binding Proteins / metabolism*
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Dose-Response Relationship, Drug
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Gene Silencing
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HeLa Cells
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Humans
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Hydroxylation
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Hypoxia / genetics
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Hypoxia / metabolism
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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*
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Isoenzymes / metabolism
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Nuclear Proteins / chemistry
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Nuclear Proteins / drug effects
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Nuclear Proteins / metabolism*
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Oxygen / metabolism*
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Procollagen-Proline Dioxygenase / metabolism*
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RNA, Messenger / genetics
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RNA, Messenger / metabolism
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RNA, Small Interfering / metabolism
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RNA, Small Interfering / pharmacology
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Recombinant Proteins / metabolism
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Transcription Factors / metabolism*
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Transfection
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Tumor Cells, Cultured
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Up-Regulation
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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Isoenzymes
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Nuclear Proteins
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RNA, Messenger
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RNA, Small Interfering
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Recombinant Proteins
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Transcription Factors
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EGLN1 protein, human
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Procollagen-Proline Dioxygenase
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Hypoxia-Inducible Factor-Proline Dioxygenases
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Oxygen