title: Vitamin C is dispensable for oxygen sensing in vivo.
abstract: Prolyl-4-hydroxylation is necessary for proper structural assembly of collagens and oxygen-dependent protein stability of hypoxia-inducible transcription factors (HIFs). In vitro function of HIF prolyl-4-hydroxylase domain (PHD) enzymes requires oxygen and 2-oxoglutarate as cosubstrates with iron(II) and vitamin C serving as cofactors. Although vitamin C deficiency is known to cause the collagen-disassembly disease scurvy, it is unclear whether cellular oxygen sensing is similarly affected. Here, we report that vitamin C-deprived Gulo(-/-) knockout mice show normal HIF-dependent gene expression. The systemic response of Gulo(-/-) animals to inspiratory hypoxia, as measured by plasma erythropoietin levels, was similar to that of animals supplemented with vitamin C. Hypoxic HIF induction was also essentially normal under serum- and vitamin C-free cell-culture conditions, suggesting that vitamin C is not required for oxygen sensing in vivo. Glutathione was found to fully substitute for vitamin C requirement of all 3 PHD isoforms in vitro. Consistently, glutathione also reduced HIF-1α protein levels, transactivation activity, and endogenous target gene expression in cells exposed to CoCl(2). A Cys201Ser mutation in PHD2 increased basal hydroxylation rates and conferred resistance to oxidative damage in vitro, suggesting that this surface-accessible PHD2 cysteine residue is a target of antioxidative protection by vitamin C and glutathione.
authors.affiliation: Institute of Physiology and Zürich Center for Integrative Human Physiology, University of Zürich, Zürich, Switzerland.
authors.forename: Katarzyna J
authors.initials: KJ
authors.lastname: Nytko
authors.forename: Nobuyo
authors.initials: N
authors.lastname: Maeda
authors.forename: Philipp
authors.initials: P
authors.lastname: Schläfli
authors.forename: Patrick
authors.initials: P
authors.lastname: Spielmann
authors.forename: Roland H
authors.initials: RH
authors.lastname: Wenger
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Stiehl
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism*; D001206:Ascorbic Acid Deficiency / Q000378:metabolism; D015687:Cell Hypoxia; D002460:Cell Line; D003035:Cobalt / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D050544:L-Gulonolactone Oxidase / Q000172:deficiency / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000378:metabolism
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D050505:Mutant Proteins; D003035:Cobalt; D050544:L-Gulonolactone Oxidase; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; C490189:Egln1 protein, mouse; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; C018021:cobaltous chloride; D005978:Glutathione; D001205:Ascorbic Acid; D010100:Oxygen