Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit.
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that is critical for hypoxic induction of a number of physiologically important genes. We present evidence that regulation of HIF-1 activity is primarily determined by the stability of the HIF-1alpha protein. Both HIF-1alpha and HIF-1beta mRNAs were constitutively expressed in HeLa and Hep3B cells with no significant induction by hypoxia. However, the HIF-1alpha protein was barely detectable in normoxic cells, even when HIF-1alpha was overexpressed, but was highly induced in hypoxic cells, whereas HIF-1beta protein levels remained constant, regardless of pO2. Hypoxia-induced HIF-1 binding as well as the HIF-1alpha protein were rapidly and drastically decreased in vivo following an abrupt increase to normal oxygen tension. Moreover, short pre-exposure of cells to hydrogen peroxide selectively prevented hypoxia-induced HIF-1 binding via blocking accumulation of HIF-1alpha protein, whereas treatment of hypoxic cell extracts with H2O2 had no effect on HIF-1 binding. These observations suggest that an intact redox-dependent signaling pathway is required for destablization of the HIF-1alpha protein. In hypoxic cell extracts, HIF-1 DNA binding was reversibly abolished by sulfhydryl oxidation. Furthermore, the addition of reduced thioredoxin to cell extracts enhanced HIF-1 DNA binding. Consistent with these results, overexpression of thioredoxin and Ref-1 significantly potentiated hypoxia-induced expression of a reporter construct containing the wild-type HIF-1 binding site. These experiments indicate that activation of HIF-1 involves redox-dependent stabilization of HIF-1alpha protein.
Huang
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LE
Hematology-Oncology Division, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA. Bunn@calvin.bwh.harvard.edu
Arany
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Z
Livingston
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Bunn
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HF
D015153:Blotting, Western; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*
D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D013439:Sulfhydryl Reagents; D014157:Transcription Factors; D004921:Erythropoietin; D004247:DNA; D006861:Hydrogen Peroxide