Raf-independent and MEKK1-dependent activation of NF-kappaB by hydrogen peroxide in 70Z/3 pre-B lymphocyte tumor cells.
We have previously demonstrated that hydrogen peroxide (H(2)O(2)) treatment of murine 70Z/3 pre-B lymphocytes inhibits the immune response to lipopolysaccharide by attenuating signaling through c-Jun N-terminal kinase (JNK) activation. In the present study, we further examined the signaling intermediates responsible for immunosuppression by H(2)O(2), focusing on NF-kappaB, a dimeric transcription factor whose activation is implicated in a number of immune response. Treatment of 70Z/3 pre-B cells with H(2)O(2) caused activation of NF-kappaB in the nuclei by detection of NF-kappaB specific DNA binding, concomitant with phosphorylation of IkappaBalpha. H(2)O(2) stimulation of NF-kappaB occurred within 20 min of treatment, reached maximum level at 60 min, and sustained for 2 h or more. Especially, MEK1 may contribute to H(2)O(2)-induced NF-kappaB activation as shown in the inhibition of NF-kappaB binding activity by the MEK1 inhibitor, PD 98059, and H(2)O(2)-induced MEK1 activation. However, H(2)O(2) exhibited no effect on the activity of Raf-1 kinase, which was an upstream activator of MEK1. Furthermore, B-58l and alpha-hydroxyfarnesylphosphonic acid, two inhibitors of Ras, did not block NF-kappaB activation. In addition, the transient transfection of a dominant negative Ras (RasN17) construct showed a negligible inhibitory effect on the activation of NF-kappaB by H(2)O(2). Instead, treatment of 70Z/3 cells with H(2)O(2) resulted in the activation of MAPK kinase kinase 1 (MEKK1) as well as JNK. Therefore, our data suggest that H(2)O(2) regulates the activity of NF-kappaB by MEK1 activation through MEKK1-dependent but Ras/Raf-independent mechanism.
Lee
Michael
M
Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, P.O. Box 107, Yusong, Daejeon 305-600, Republic of Korea. mikelee@kitox.re.kr
Koh
Woo Suk
WS
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology* / Q000378:metabolism; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism*; D020672:I-kappa B Proteins / Q000378:metabolism; D048728:MAP Kinase Kinase Kinase 1; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000502:physiology; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D016877:Oxidants / Q000378:metabolism*; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*
D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; D016877:Oxidants; D000072000:NF-KappaB Inhibitor alpha; D006861:Hydrogen Peroxide; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; D020930:MAP Kinase Kinase Kinases; C482578:Map3k1 protein, mouse