Ascorbate modulates the hypoxic pathway by increasing intracellular activity of the HIF hydroxylases in renal cell carcinoma cells

Purpose: Protein levels and activity of the hypoxia-inducible transcription factors HIF-1 and HIF-2 are controlled by hydroxylation of the regulatory alpha chains. Proline hydroxylases (PHDs) target the protein for degradation via the von-Hippel-Lindau (VHL)-ubiquitin-ligase complex, and asparagine hydroxylation by Factor Inhibiting HIF (FIH) leads to transcriptional inactivation. In cell-free systems, these enzymes require ascorbate as a cofactor, and this is also inferred to be an intracellular requirement for effective hydroxylation. However, how intracellular concentrations of ascorbate affect hydroxylase activity is unknown. In this study, we investigated the modulation of the regulatory hydroxylases in cancer cells by intracellular ascorbate. Materials and methods: To facilitate this investigation, we used clear cell renal carcinoma cell lines that were VHL-proficient (Caki-1), with a normal hypoxic response, or VHL-defective (Caki-2 and 786-0), with uncontrolled accumulation of HIF-alpha chains. We monitored the effect of intracellular ascorbate on the hypoxia-induced accumulation of HIF-1 alpha, HIF-2 alpha and the expression of downstream HIF targets BNIP3, cyclin D1 and GLUT1. Changes in hydroxylation of the HIF-1 alpha protein in response to ascorbate were also investigated in 786-0 cells gene-modified to express full-length HIF-1 alpha (786-HIF1). Results: In VHL-proficient cells, hypoxia induced accumulation of HIF-1 alpha and BNIP3 which was dampened in mild hypoxia by elevated intracellular ascorbate. Increased HIF-2 alpha accumulation occurred only under severe hypoxia and this was not modified by ascorbate availability. In VHL-defective cells, ascorbate supplementation induced additional accumulation of HIF under hypoxic conditions and HIF pathway proteins were unchanged by oxygen supply. In 786-HIF1 cells, levels of hydroxylated HIF-1 alpha were elevated in response to increasing intracellular ascorbate levels. Conclusion: Our data provide evidence that the hypoxic pathway can be modulated by increasing HIF hydroxylase activity via intracellular ascorbate availability. In VHL-defective cells, accumulation of HIF-alpha proteins is independent of hydroxylation and is unaffected by intracellular ascorbate levels.