Induction of HIF–1α in response to hypoxia is instantaneous

Abstract

Despite the pivotal role the hypoxia‐inducible factor‐1α (HIF‐1α) plays in physiological and pathological processes, little is known regarding the timeframe and mechanisms involved in its regulation. We determined the onset, accumulation, and degradation of HIF‐1α and a number of redox‐sensitive nuclear factors over a range of pathophysiological oxygen concentrations. Experiments were carried out on nonadherent human HeLaS3 cells placed in tonometers to achieve rapid equilibration between the cell suspension and the various hypoxic/reoxygenation conditions. Exposure to hypoxia for less than 2 min already revealed nuclear HIF‐1α protein induction on Western blots and HIF‐1 DNA binding in EMSAs. One hour after anoxic/hypoxic exposure, nuclear HIF‐1α proteins reached maximal levels, which were maintained for 4 h. Reoxygenation reduced HIF‐1 DNA binding within 2 min, and nuclear HIF‐1α protein levels within 4 to 8 min, down to a level below the detection limit within 32 min. Western blot analysis of the redox sensitive nuclear factors NF‐κB, c‐Fos, c‐Jun, Ref‐1, and thioredoxin showed no alteration in their nuclear levels in response to anoxia/hypoxia, but reoxygenation rapidly caused a transient increase in nuclear NF‐κB and thioredoxin protein levels. The instant initiation of HIF‐1α accumulation shown here limits the hypoxic signaling pathway to below 2 min.