Redox factor-1 mediates NF-kappaB nuclear translocation for LPS-induced iNOS expression in murine macrophage cell line RAW 264.7.
Redox-sensitive transcriptional regulator redox factor-1 (Ref-1) is induced by oxidative stress and protects cells against it. However, the function of Ref-1 in regulating nitric oxide (NO) synthesis in macrophages has not been defined. We investigated the role of Ref-1 related to the regulation of NO synthesis in lipopolysaccharide (LPS)-stimulated macrophage RAW 264.7 cells. LPS stimulates the up-regulation and nuclear translocation of Ref-1 in macrophages. Importantly, Ref-1-deficient macrophages using a small interfering RNA did not stimulate inducible NO synthase (iNOS) expression as well as nuclear factor-kappaB nuclear translocation by stimulation with LPS. When the cells were pretreated with diphenyleneiodonium or p47(phox) small interfering RNA for inhibition of NADPH oxidase activity, LPS did not stimulate the nuclear translocation of Ref-1. We next asked whether reactive oxygen species are sufficient for the nuclear translocation of Ref-1 in macrophages. The direct use of H2O2 stimulated the translocation to the nucleus of nuclear factor-kappaB, but not Ref-1 and antioxidant N-acetyl cysteine did not inhibit the LPS-stimulated nuclear translocation of Ref-1. These data suggest that Ref-1 nuclear translocation in LPS-stimulated macrophages requires the activation of other signalling molecules aside from reactive oxygen species followed by the activation of NADPH oxidase.
Song
Ju Dong
JD
Department of Microbiology & Immunology, Medical Research Institute, Pusan National University School of Medicine, Busan, Korea.
Lee
Sang Kwon
SK
Kim
Kang Mi
KM
Kim
Jong Won
JW
Kim
Jong Min
JM
Yoo
Young Hyun
YH
Park
Young Chul
YC
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000502:physiology*; D008070:Lipopolysaccharides / Q000276:immunology; D008262:Macrophage Activation / Q000502:physiology; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D019255:NADPH Oxidases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis*; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D014178:Translocation, Genetic; D015854:Up-Regulation
D008070:Lipopolysaccharides; D016328:NF-kappa B; D034741:RNA, Small Interfering; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D019255:NADPH Oxidases; C486216:Apex1 protein, mouse; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase