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 fa ctor that is critical for hypoxic induction of a number of physiologic ally important genes, We present evidence that regulation of HIF-1 act ivity is primarily determined by the stability of the HLF-1 alpha prot ein, Both HIF-1 alpha and HIF-1 beta mRNAs were constitutively express ed in HeLa and Hep3B cells with no significant induction by hypoxia, H owever, the HIF-1 alpha protein was barely detectable in normoxic cell s, even when HLF-1 alpha was overexpressed, but was highly induced in hypoxic cells, whereas HIF-1 beta protein levels remained constant, re gardless of pO(2). Hypoxia-induced HIF-1 binding as well as the HIF-1 alpha protein were rapidly and drastically decreased in vivo following an abrupt increase to normal oxygen tension, Moreover, short pre-expo sure of cells to hydrogen peroxide selectively prevented hypoxia-induc ed HIF-1 binding via blocking accumulation of HIF-1 alpha protein, whe reas 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 destabilization of the RIF-la protei n, In hypoxic cell extracts, HIF-1 DNA binding was reversibly abolishe d by sulfhydryl oxidation, Furthermore, the addition of reduced thiore doxin to cell extracts enhanced HIF-1 DNA binding, Consistent with the se results, overexpression of thioredoxin and Ref-1 significantly pote ntiated hypoxia-induced expression of a reporter construct containing the wild-type HIF-1 binding site, These experiments indicate that acti vation of HIF-1 involves redox-dependent stabilization of HIF-1 alpha protein.