Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit

Abstract

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.