1. Vitamin C is dispensable for oxygen sensing in vivo
- Author
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Philipp Schläfli, Patrick Spielmann, Daniel P. Stiehl, Nobuyo Maeda, Katarzyna J. Nytko, and Roland H. Wenger
- Subjects
Vitamin ,Vitamin D-binding protein ,Immunology ,Procollagen-Proline Dioxygenase ,Ascorbic Acid ,Biology ,Biochemistry ,Cell Line ,Hypoxia-Inducible Factor-Proline Dioxygenases ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Transactivation ,0302 clinical medicine ,Alpha ketoglutarate ,Animals ,Humans ,030304 developmental biology ,Mice, Knockout ,0303 health sciences ,Vitamin C ,Cobalt ,Cell Biology ,Hematology ,Glutathione ,Hypoxia-Inducible Factor 1, alpha Subunit ,Ascorbic acid ,Cell Hypoxia ,Oxygen ,Amino Acid Substitution ,chemistry ,030220 oncology & carcinogenesis ,Knockout mouse ,Ascorbic Acid Deficiency ,Mutagenesis, Site-Directed ,Mutant Proteins ,L-Gulonolactone Oxidase ,HeLa Cells - 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 CoCl2. 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.
- Published
- 2011