Activation of hypoxia-induced transcription in normoxia.
Hypoxia-inducible factor-1 (HIF-1), the master regulator of transcriptional responses to reduced oxygen tension (hypoxia) in mammalian cells, consists of one HIF-1alpha and one HIF-1beta subunit. In normoxia, HIF-1alpha subunits are hydroxylated on specific proline residues; modifications that signal ubiquitination and degradation of HIF-1alpha by the proteasome. To test the effect of saturating HIF-1alpha degradation, we generated a construct, denoted the saturating domain (SD), based on a region surrounding proline 564 (Pro564) in HIF-1alpha. Expression of the SD led to accumulation of endogenous HIF-1alpha proteins in nuclei of normoxic cells. The induced HIF-1alpha was functional as it activated expression from a hypoxia-regulated reporter gene and from the endogenous vascular endothelial growth facor-a (Vegf-a) and carbonic anhydrase 9 (Ca9) genes. The effect of the SD was dependent on Pro564 since a mutated SD, in which Pro564 had been replaced by a glycine residue, failed to bind the von Hippel-Lindau protein (pVHL) and to stabilise HIF-1alpha. Treatment of cells with the prolylhydroxylase inhibitor dimethyloxalylglycine, or the proteasome inhibitor MG-132, mimicked the effect of the SD. In conclusion, we show that blocking HIF-1alpha degradation, either by saturation, or inhibition of prolyl hydroxylases or proteosomal degradation, leads to nuclear localisation of active HIF-1alpha proteins.
Hägg
Maria
M
Department of Genetics and Pathology, Rudbeck Laboratory, Dag Hammarskjölds väg 20, S-751 85 Uppsala, Sweden.
Wennström
Stefan
S
D000600:Amino Acids, Dicarboxylic / Q000494:pharmacology; D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics; D019556:COS Cells; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000235:genetics; D015687:Cell Hypoxia / Q000235:genetics; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007976:Leupeptins / Q000494:pharmacology; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011485:Protein Binding / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D000600:Amino Acids, Dicarboxylic; D000951:Antigens, Neoplasm; D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007976:Leupeptins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D061988:Proteasome Inhibitors; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D011393:Procollagen-Proline Dioxygenase; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D046988:Proteasome Endopeptidase Complex; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases; C497446:VHL protein, human; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D010100:Oxygen; C040947:oxalylglycine