In HEK293 cells, exposure to various NAD(P)H oxidants, including phenazine methosulfate (PMS), that non-enzymatically oxidize intracellular NAD(P)H to NAD(P), decreased hypoxia-induced hypoxia-inducible factor 1 (HIF-1) accumulation. RT-PCR and cycloheximide inhibition experiments indicated that PMSinduced HIF-1 decrease is involved in post-translational degradation during hypoxia. The decrease in HIF-1 caused by PMS was not eliminated by proteasome inhibitor MG132. Moreover, the increase in

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... -1 induced by exposure to MG132 alone in normoxia was diminished by PMS. In contrast, calpastatin peptide, a calpain inhibitor, fully prevented PMS-induced reduction in HIF-1 in hypoxic cells. These data suggest that the decreased stability of HIF-1 induced by PMS is due to the activation by PMS of a protein degradation system that is independent of the ubiquitin-proteasome pathway. Oxygen is essential for the maintenance of life as the final electron acceptor of the mitochondrial respiratory chain in multicellular organisms. Hypoxia leads to oxygen deprivation in tissues and promotes a series of metabolic adaptations avoiding tissue damage. Regulation of the genes that respond to hypoxia is important in maintaining homeostasis. Hypoxia-inducible factor 1 (HIF-1) regulates the transcription of hypoxia-responsive genes by a unique, well-defined mechanism. 1) HIF-1 is composed of two subunits: HIF-1 and HIF-1/ ARNT (aryl hydrocarbon receptor nuclear translocator). 2) These subunits are members of the helix-loophelix/PAS family of transcription factors, and are constitutively expressed in normoxic and hypoxic cells. 3,4) Under normoxic conditions, HIF-1 is hydroxylated by oxygen-dependent prolyl hydroxylase and undergoes ubiquitination, resulting in rapid degradation by the proteasome. 5,6) When cells are transferred from hypoxia to normoxia, the degradation of HIF-1 is very rapid and is completed in several minutes. Deferoxamine and CoCl 2 , which inhibit prolyl hydroxylase, inhibit HIF-1 degradation under conditions of normoxia, leading to increased HIF-1. 7-9) The repres-sion of HIF-1 degradation by these inhibitors is similar to that observed under hypoxic conditions. Various biochemical changes are known to be induced by hypoxia in cells. 10) These appear to result from inhibition of oxidative phosphorylation, which is coupled with the electron transport system in the mitochondria, resulting in an increase in the cellular NADH/NAD ratio. 11-13) The present study investigated whether the stability of HIF-1 is affected by NAD(P)H oxidants that decrease the NAD(P)H/NAD(P) ratio. Our results indicate that NAD(P)H oxidants stimulate the degradation of HIF-1 during exposure of cells to hypoxia, and that degradation occurs via a protein degradation system other than the ubiquitin-proteasome pathway. Materials and Methods Chemicals. Phenazine methosulfate (PMS), methylene blue (MB), and cycloheximide (CHX) were purchased from Wako (Tokyo, Japan). 1-Methoxy-5-methylphenazinium methylsulfate (MPMS) and 8-dimethylamino-2,3-benzophenoxazine hemi (zinc chloride) salt (Meldola's blue) (MELB) were from Dojindo (Kumamoto, Japan). HEPESbuffered Dulbecco's Modified Eagle's Medium (DMEM) was from Invitrogen (Carlsbad, CA). MG132, PD 150606, calpastatin peptide, and CA-074 ME were purchased from Calbiochem (La Jolla, CA). Deferoxamine (DFX) and 2,6-dichloroindophenol (DCIP) were from Sigma (St. Louis, MO). Cell culture. Human embryonic kidney 293 (HEK293) cells and human MCF7 breast adenocarcinoma cells were cultured in 95% air and 5% CO 2 at 37 C in 30-mm culture dishes containing 2 mL of HEPES-buffered DMEM supplemented with penicillin (100 units/ mL), streptomycin (0.1 mg/mL), and 10% heat-inactivated fetal bovine serum. In all the experiments, the cells were grown to 70-80% confluence. The culture medium was changed 1 h prior to experiments on hypoxia and normoxia. For exposure of the cells to hypoxia, cultures were incubated in a hermetic chamber flushed with a gas mixture of 1% O 2 , 5% CO 2 , and 94% N 2 (Taiyo Nippon Sanso, Tokyo, Japan) at 37 C. After 30 min of hypoxia, various reagent solutions equilibrated previously with the gas mixture were added to the cultures, which were then incubated for the indicated durations.