title: Activation of Ras up-regulates pro-apoptotic BNIP3 in nitric oxide-induced cell death.
abstract: Nitric oxide (NO) produced by NO synthases causes nitration and nitrosylation of cellular factors. We have shown previously that endogenously produced or exogenously added NO induces expression of BNIP3 (Bcl-2/adenovirus E1B 19 kDa-interacting protein 3), leading to death of macrophages (Yook, Y.-H., Kang, K.-H., Maeng, O., Kim, T.-R., Lee, J.-O., Kang, K.-i., Kim, Y.-S., Paik, S.-G., and Lee, H. (2004) Biochem. Biophys. Res. Commun. 321, 298-305). We now provide evidence that Ras mediates NO-induced BNIP3 expression via the MEK/ERK/hypoxia-inducible factor (HIF)-1 pathway. (a) ras-Q61L, a constitutively active form of Ras, up-regulated BNIP3 protein expression by enhancing Bnip3 promoter activity, and ras-S17N, a dominant-negative form, and ras-C118S, an S-nitrosylation mutant, blocked NO-induced BNIP3 expression, suggesting that Ras acts downstream of NO and that NO activates Ras by nitrosylation. (b) U0126, a specific MEK inhibitor, completely abolished BNIP3 expression and the stimulation of promoter activity by NO and Ras, whereas 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, SB203580, and wortmannin, specific inhibitors of soluble guanylyl cyclase, p38 MAPK, and phosphatidylinositol 3-kinase, respectively, had no effect. Ras, MEK1/2, and ERK1/2 were sequentially activated by NO treatment of macrophages. (c) Mutation of the HIF-1-binding site (hypoxia-response element) in the Bnip3 promoter abolished BNIP3 induction, and HIF-1alpha was strongly induced by NO. (d) Transient expression of activated Ras promoted macrophage death, as did NO, and this Ras-mediated cell death was inhibited by silencing BNIP3 expression. These results suggest that NO-induced death of macrophages is mediated, at least in part, by BNIP3 induction.
authors.affiliation: Department of Biology, School of Biosciences and Biotechnology, Chungnam National University, Daejeon, Korea.
authors.forename: Hyun-Jung
authors.initials: HJ
authors.lastname: An
authors.forename: Oky
authors.initials: O
authors.lastname: Maeng
authors.forename: Kyoung-Hee
authors.initials: KH
authors.lastname: Kang
authors.forename: Jie-Oh
authors.initials: JO
authors.lastname: Lee
authors.forename: Young-Sang
authors.initials: YS
authors.lastname: Kim
authors.forename: Sang-Gi
authors.initials: SG
authors.lastname: Paik
authors.forename: Hayyoung
authors.initials: H
authors.lastname: Lee
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases; D005786:Gene Expression Regulation; D006162:Guanylate Cyclase / Q000378:metabolism; D006801:Humans; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008156:Luciferases; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000633:toxicity*; D009603:Nitroso Compounds / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D015854:Up-Regulation; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C090121:BNIP3 protein, human; C508686:BNip3 protein, mouse; D004791:Enzyme Inhibitors; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D009603:Nitroso Compounds; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D009569:Nitric Oxide; D008156:Luciferases; D019869:Phosphatidylinositol 3-Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases; D018631:ras Proteins; D006162:Guanylate Cyclase