title: A dominant-negative isoform lacking exons 11 and 12 of the human hypoxia-inducible factor-1alpha gene.
abstract: Hypoxia-inducible factor-1alpha (HIF-1alpha), a member of the transcription family characterized by a basic helix-loop-helix (bHLH) domain and a PAS domain, regulates the transcription of hypoxia-inducible genes involved in erythropoiesis, vascular remodelling and glucose/energy metabolism. It contains bHLH/PAS domains in the N-terminal half, and a nuclear localization signal (NLS) and two transactivation domains (TADs) in the C-terminal half. It also has an oxygen-dependent degradation (ODD) domain, which is required to degrade HIF-1alpha protein by the ubiquitin-proteasome pathway. In this study, we identified a new alternatively spliced variant of human HIF-1alpha mRNA, which lacked both exons 11 and 12, producing a frame shift and giving a shorter form of HIF-1alpha. In the corresponding protein, a part of the ODD domain, both TADs and the C-terminal NLS motif were missing. Expression of endogenous HIF-1alpha variant protein was identified using immunoprecipitation and immunoblotting methods. The expressed HIF-1alpha variant exhibited neither the activity of transactivation nor hypoxia-induced nuclear translocation. In contrast with HIF-1alpha, the variant was strikingly stable in normoxic conditions and not up-regulated to such an extent by hypoxia, cobalt ions or desferrioxamine. It was also demonstrated that the HIF-1alpha variant competed with endogenous HIF-1alpha and suppressed HIF-1 activity, resulting in the down-regulation of mRNA expression of hypoxia-inducible genes. The association of the variant and arylhydrocarbon receptor nuclear translocator in the cytoplasm may be related to the inhibition of HIF-1 activity. It is assumed that this isoform preserves the balance between aerobic and anaerobic metabolism by counteracting the overaction of HIF-1alpha.
authors.affiliation: Department of Pharmacology, Seoul National University, College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, South Korea.
authors.forename: Yang-Sook
authors.initials: YS
authors.lastname: Chun
authors.forename: Eunjoo
authors.initials: E
authors.lastname: Choi
authors.forename: Tae-You
authors.initials: TY
authors.lastname: Kim
authors.forename: Myung-Suk
authors.initials: MS
authors.lastname: Kim
authors.forename: Jong-Wan
authors.initials: JW
authors.lastname: Park
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay; D005091:Exons; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D051379:Mice; D008856:Microscopy, Fluorescence; D011233:Precipitin Tests; D020033:Protein Isoforms / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018336:Receptors, Aryl Hydrocarbon; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism / Q000502:physiology
chemical_list: C497396:ARNT protein, human; C497397:Arnt protein, mouse; D017931:DNA Primers; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020033:Protein Isoforms; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator