title: Hypoxia-inducible factor-1 (HIF-1) promotes its degradation by induction of HIF-alpha-prolyl-4-hydroxylases.
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.
authors.affiliation: Department of Internal Medicine I, University of Luebeck, Ratzeburger Allee 160, D-23538 Luebeck, Germany.
authors.forename: Jan H
authors.initials: JH
authors.lastname: Marxsen
authors.forename: Petra
authors.initials: P
authors.lastname: Stengel
authors.forename: Kathrin
authors.initials: K
authors.lastname: Doege
authors.forename: Pekka
authors.initials: P
authors.lastname: Heikkinen
authors.forename: Terhi
authors.initials: T
authors.lastname: Jokilehto
authors.forename: Thomas
authors.initials: T
authors.lastname: Wagner
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Jelkmann
authors.forename: Panu
authors.initials: P
authors.lastname: Jaakkola
authors.forename: Eric
authors.initials: E
authors.lastname: Metzen
mesh_terms: D018262:Adenocarcinoma, Clear Cell / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D001859:Bone Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D049308:Dioxygenases; D004790:Enzyme Induction / Q000502:physiology; D004847:Epithelial Cells / Q000201:enzymology / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D006207:Half-Life; D006801:Humans; D006900:Hydroxylation; D000860:Hypoxia / Q000201:enzymology*; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D017874:Immediate-Early Proteins / Q000096:biosynthesis; D007680:Kidney Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D008113:Liver Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology*; D012516:Osteosarcoma / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D010100:Oxygen / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000096:biosynthesis* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D014157:Transcription Factors / Q000378:metabolism / Q000502:physiology*; D025521:Tumor Suppressor Proteins / Q000502:physiology; D044767:Ubiquitin-Protein Ligases / Q000502:physiology; D051794:Von Hippel-Lindau Tumor Suppressor Protein
chemical_list: D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D049308:Dioxygenases; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; C488894:EGLN3 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human; D010100:Oxygen