Activation of Ras up-regulates pro-apoptotic BNIP3 in nitric oxide-induced cell death.
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.
An
Hyun-Jung
HJ
Department of Biology, School of Biosciences and Biotechnology, Chungnam National University, Daejeon, Korea.
Maeng
Oky
O
Kang
Kyoung-Hee
KH
Lee
Jie-Oh
JO
Kim
Young-Sang
YS
Paik
Sang-Gi
SG
Lee
Hayyoung
H
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*
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