NO restores HIF-1α hydroxylation during hypoxia: Role of reactive oxygen species

Abstract

The activity of hypoxia-inducible factor 1 (HIF-1) is primarily determined by stability regulation of its α subunit, which is stabilized under hypoxia but degraded during normoxia. Hydroxylation of HIF-1α by prolyl hydroxylases (PHDs) recruits the von Hippel–Lindau (pVHL) E3 ubiquitin ligase complex to initiate proteolytic destruction of the α subunit. Hypoxic stabilization of HIF-1α has been reported to be antagonized by nitric oxide (NO). By using a HIF-1α–pVHL binding assay, we show that NO released from DETA-NO restored prolyl hydroxylase activity under hypoxia. Destabilization of HIF-1α by DETA-NO was reversed by free radical scavengers such as NAC and Tiron, thus pointing to the involvement of reactive oxygen species (ROS). Therefore, we examined the effects of ROS on HIF-1α stabilization. Treatment of cells under hypoxia with low concentrations of the superoxide generator 2,3-dimethoxy-1,4-naphthoquinone lowered HIF-1α protein stabilization. In vitro HIF-1α–pVHL interaction assays demonstrated that low-level ROS formation increased prolyl hydroxylase activity, an effect antagonized by ROS scavengers. While determining intracellular ROS formation we noticed that reduced ROS production under hypoxia was restored by the addition of DETA-NO. We propose that an increase in ROS formation contributes to HIF-1α destabilization by NO donors under hypoxia via modulation of PHD activity.