Intracellular ascorbate enhances hypoxia-inducible factor (HIF)-hydroxylase activity and preferentially suppresses the HIF-1 transcriptional response.
Hypoxia-inducible factor (HIF)-1 drives the transcription of hundreds of genes to support cell survival under conditions of microenvironmental and metabolic stress. HIF-1 is downregulated by iron-containing 2-oxoglutarate-dependent enzymes that require ascorbate as a cofactor. The HIF hydroxylases control both protein stability and the formation of an active transcription complex and, consequently, ascorbate could affect HIF-1α stabilization and/or gene expression, but the relative effect of ascorbate on these separate processes has not been well characterized. In this study we examined the effects of known intracellular ascorbate concentrations on both processes in response to various means of hydroxylase inhibition, including CoCl2, NiCl2, desferrioxamine, dimethyloxalylglycine, and hypoxia. Ascorbate inhibited HIF-1 activity most dramatically with all mechanisms of iron competition. In addition, HIF-1-dependent gene expression was effectively prevented by ascorbate and was inhibited even under conditions that allowed HIF-1α protein stabilization. This suggests that (1) ascorbate acts primarily to stabilize and reduce the iron atom in the hydroxylase active site and (2) the asparagine hydroxylase controlling HIF-1 transcriptional activity is particularly susceptible to fluctuations in intracellular ascorbate. These findings suggest that ascorbate plays a significant role in supporting HIF-hydroxylase function and that it could thereby modulate the cell survival response.
Centre for Free Radical Research, Department of Pathology and Biomedical Science, University of Otago at Christchurch, Christchurch 8140, New Zealand. Electronic address: caroline.kuiper@otago.ac.nz.
Caroline
C
Kuiper
Mackenzie Cancer Research Group, Department of Pathology and Biomedical Science, University of Otago at Christchurch, Christchurch 8140, New Zealand.
Gabi U
GU
Dachs
Mackenzie Cancer Research Group, Department of Pathology and Biomedical Science, University of Otago at Christchurch, Christchurch 8140, New Zealand.
Margaret J
MJ
Currie
Centre for Free Radical Research, Department of Pathology and Biomedical Science, University of Otago at Christchurch, Christchurch 8140, New Zealand.
Margreet C M
MC
Vissers
D001205:Ascorbic Acid / Q000378:metabolism*; D020134:Catalytic Domain; D015687:Cell Hypoxia / Q000235:genetics; D002470:Cell Survival / Q000235:genetics; D005786:Gene Expression Regulation; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry / Q000378:metabolism*; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry* / Q000378:metabolism; D055550:Protein Stability; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry / Q000378:metabolism
C090121:BNIP3 protein, human; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D042461:Vascular Endothelial Growth Factor A; D007501:Iron; D011393:Procollagen-Proline Dioxygenase; D001205:Ascorbic Acid
Ascorbate; BNIP3; Bcl-2/adenovirus E1B 19-kDa interacting protein 3; Cancer; Free radicals; Hydroxylase; Hypoxia-inducible factor-1; Vascular endothelial cell growth factor