title: Nitric oxide produced endogenously is responsible for hypoxia-induced HIF-1α stabilization in colon carcinoma cells.
abstract: Hypoxia-inducible factor-1α (HIF-1α) is a critical regulator of cellular responses to hypoxia. Under normoxic conditions, the cellular HIF-1α 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-1α; thus, NO is suggested to mimic hypoxia. However, the role of low levels of endogenously produced NO generated during hypoxia in HIF-1α 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-1α accumulation in hypoxia. The antioxidant N-acetyl-L-cysteine (NAC) and NO synthase inhibitor N(G)-monomethyl L-arginine (L-NMMA) effectively reduced HIF-1α stabilization and decreased HIF-1α 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-1α stabilization in hypoxic cells, while dinitrochlorobenzene, which stabilizes S-nitrosothiols, favored its accumulation. This suggested that ROS- and NO-mediated HIF-1α stabilization involved S-nitrosation, which was confirmed by demonstrating increased S-nitrosation of PHD2 during hypoxia. Our results support a regulatory mechanism of HIF-1α during hypoxia in which endogenously generated NO and ROS promote inhibition of PHD2 activity, probably by its S-nitrosation.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Rajdeep
authors.initials: R
authors.lastname: Chowdhury
authors.forename: Luiz C
authors.initials: LC
authors.lastname: Godoy
authors.forename: Apinya
authors.initials: A
authors.lastname: Thiantanawat
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Trudel
authors.forename: William M
authors.initials: WM
authors.lastname: Deen
authors.forename: Gerald N
authors.initials: GN
authors.lastname: Wogan
mesh_terms: D015687:Cell Hypoxia; D003106:Colon / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D045325:HCT116 Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000032:analysis / Q000378:metabolism*; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D009569:Nitric Oxide / Q000378:metabolism*; D015538:Nitrosation; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases