Le. Huang et al., ACTIVATION OF HYPOXIA-INDUCIBLE TRANSCRIPTION FACTOR DEPENDS PRIMARILY UPON REDOX-SENSITIVE STABILIZATION OF ITS ALPHA-SUBUNIT, The Journal of biological chemistry, 271(50), 1996, pp. 32253-32259
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.