Depletion of intracellular ascorbate by the carcinogenic metals nickel and cobalt results in the induction of hypoxic stress.
Exposure of cells to carcinogenic compounds of nickel(II) and cobalt(II) causes activation of the HIF-1 transcription factor and up-regulates a battery of hypoxia-inducible genes. However, the mechanism of HIF-1 activation by these metals is not known. It was shown recently that hydroxylation of prolines in the HIFalpha subunit of HIF-1 is required for its binding with the von Hippel-Lindau tumor suppressor protein and the subsequent proteasomal destruction. Here we show that responsible prolyl hydroxylases are targets for both nickel(II) and cobalt(II) because degradation of a reporter protein containing the oxygen-dependent degradation domain (Pro-402/564) of HIFalpha was abolished in a von Hippel-Lindau-dependent manner in cells exposed to nickel(II) or cobalt(II). The enzymatic activity of prolyl hydroxylases depends on iron as the activating metal, 2-oxoglutarate as a co-substrate, and ascorbic acid as a cofactor. Hydroxylase activity can be impaired by the depletion of any of these factors. We found that exposure of cells to nickel(II) or cobalt(II) did not affect the level of intracellular iron. Instead, nickel(II) or cobalt(II) exposure greatly depleted intracellular ascorbate. Co-exposure of cells to metals and ascorbate resulted in the increase of intracellular ascorbate and reversed both metal-induced stabilization of HIF-1alpha and HIF-1-dependent gene transcription. Because ascorbate is essential for maintaining iron in prolyl hydroxylases in the active iron(II) state, we suggest that the observed depletion of ascorbate by nickel(II) or cobalt(II) favors iron oxidation and thus inactivation of the enzyme.
NCI-Frederick, National Institutes of Health, Frederick, Maryland 21702, USA. salnikow@ncifcrf.gov
Konstantin
K
Salnikow
Steven P
SP
Donald
Richard K
RK
Bruick
Anatoly
A
Zhitkovich
James M
JM
Phang
Kazimierz S
KS
Kasprzak
D001205:Ascorbic Acid / Q000378:metabolism*; D015153:Blotting, Western; D002273:Carcinogens; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003035:Cobalt / Q000737:chemistry*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D006801:Humans; D000860:Hypoxia; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007501:Iron / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000378:metabolism; D009154:Mutation; D009532:Nickel / Q000737:chemistry*; D009687:Nuclear Proteins / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014157:Transcription Factors; D014158:Transcription, Genetic; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D015854:Up-Regulation; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D002273:Carcinogens; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007656:Ketoglutaric Acids; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D003035:Cobalt; D009532:Nickel; D011392:Proline; D007501:Iron; D011393:Procollagen-Proline Dioxygenase; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C497446:VHL protein, human; D001205:Ascorbic Acid; D010100:Oxygen