Nitric oxide produced endogenously is responsible for hypoxia-induced HIF-1α stabilization in colon carcinoma cells.

Hypoxia-inducible factor-la (HIF-1 alpha) is a critical regulator of cellular responses to hypoxia. Under normoxic conditions, the cellular HIF-1 alpha level is regulated by hydroxylation by prolyl hydroxylases (PHDs), ubiquitylation, and proteasomal degradation. During hypoxia, degradation decreases, and its intracellular level is increased. Exogenously administered nitric oxide (NO)-donor drugs stabilize HIF-1 alpha; thus, NO is suggested to mimic hypoxia. However, the role of low levels of endogenously produced NO generated during hypoxia in HIF-1 alpha stabilization has not been defined. Here, we demonstrate that NO and reactive oxygen species (ROS) produced endogenously by human colon carcinoma HCT116 cells are responsible for HIF-1 alpha accumulation in hypoxia. The antioxidant N-acetyl-L-cysteine (NAC) and NO synthase inhibitor N-G-monomethyl L-arginine (L-NMMA) effectively reduced HIF-1 alpha stabilization and decreased HIF-1 alpha hydroxylation. These effects suggested that endogenous NO and ROS impaired PHD activity, which was confirmed by reversal of L-NMMA- and NAC-mediated effects in the presence of dimethyloxaloylglycine, a PHD inhibitor. Thiol reduction with dithiothreitol decreased HIF-1 alpha stabilization in hypoxic cells, while dinitrochlorobenzene, which stabilizes S-nitrosothiols, favored its accumulation. This suggested that ROS- and NO-mediated HIF-1 alpha stabilization involved S-nitrosation, which was confirmed by demonstrating increased S-nitrosation of PHD2 during hypwda. Our results support a regulatory mechanism of HIF-1 alpha during hypoxia in which endogenously generated NO and ROS promote inhibition of PHD2 activity, probably by its S-nitrosation.