title: NO restores HIF-1alpha 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 alpha subunit, which is stabilized under hypoxia but degraded during normoxia. Hydroxylation of HIF-1alpha by prolyl hydroxylases (PHDs) recruits the von Hippel-Lindau (pVHL) E3 ubiquitin ligase complex to initiate proteolytic destruction of the alpha subunit. Hypoxic stabilization of HIF-1alpha has been reported to be antagonized by nitric oxide (NO). By using a HIF-1alpha-pVHL binding assay, we show that NO released from DETA-NO restored prolyl hydroxylase activity under hypoxia. Destabilization of HIF-1alpha 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-1alpha stabilization. Treatment of cells under hypoxia with low concentrations of the superoxide generator 2,3-dimethoxy-1,4-naphthoquinone lowered HIF-1alpha protein stabilization. In vitro HIF-1alpha-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-1alpha destabilization by NO donors under hypoxia via modulation of PHD activity.
authors.affiliation: Institute of Biochemistry I, Faculty of Medicine, Johann Wolfgang Goethe-University, 60590 Frankfurt, Germany.
authors.forename: Melvin
authors.initials: M
authors.lastname: Callapina
authors.forename: Jie
authors.initials: J
authors.lastname: Zhou
authors.forename: Tobias
authors.initials: T
authors.lastname: Schmid
authors.forename: Roman
authors.initials: R
authors.lastname: Köhl
authors.forename: Bernhard
authors.initials: B
authors.lastname: Brüne
mesh_terms: D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt / Q000494:pharmacology; D000111:Acetylcysteine / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D006801:Humans; D006900:Hydroxylation; D000860:Hypoxia / Q000503:physiopathology*; D009285:Naphthoquinones / Q000494:pharmacology; D009569:Nitric Oxide / Q000494:pharmacology / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014226:Triazenes / Q000037:antagonists & inhibitors / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000378:metabolism
chemical_list: C105351:1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene; D009285:Naphthoquinones; D020030:Nitric Oxide Donors; D017382:Reactive Oxygen Species; D014226:Triazenes; D009569:Nitric Oxide; D014013:1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; C063002:2,3-dimethoxy-1,4-naphthoquinone; D011393:Procollagen-Proline Dioxygenase; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human; D000111:Acetylcysteine