Activation of nuclear factor-kappaB in C6 rat glioma cells after transfection with glia maturation factor.
The 17-kDa endogenous brain protein glia maturation factor (GMF) was transfected into C6 rat glioma cells using a replication-defective human adenovirus vector. The cells overexpressed GMF but did not secrete the protein into the medium. Transfection with GMF led to the activation of the transcription factor nuclear factor-kappaB (NF-kappaB), as evidenced by electrophoretic mobility shift assay of the nuclear extract, using a double-stranded oligonucleotide probe containing the consensus binding sequence for NF-kappaB. The specificity of binding was demonstrated by competition with unlabeled probe and by the nonbinding of the mutant probe. Binding was detectable as early as 3 h after transfection, peaked at 6 and 12 h, and gradually declined thereafter. The observed NF-kappaB activation was reduced by cotransfection with catalase and by the presence of high concentrations of pyruvate in the medium, suggesting the involvement of H2O2. The p38 mitogen-activated protein kinase inhibitor SB-203580 also suppressed the GMF-activated NF-kappaB, suggesting the involvement of the p38 signal transduction cascade. On the other hand, the phorbol ester phorbol 12-myristate 13-acetate activated NF-kappaB whether or not GMF was overexpressed. Along with NF-kappaB activation was an enhanced expression of superoxide dismutase (SOD), which was suppressed if NF-kappaB nuclear translocation was blocked by its specific decoy DNA, implicating NF-kappaB as an upstream mediator of this antioxidant enzyme. The p38 inhibitor SB-203580 also blocked the GMF-activated SOD. As NF-kappaB and SOD are both pro-survival signals, the results suggest a cytoprotective role for endogenous GMF in glial cells.
Lim
R
R
Department of Neurology, University of Iowa College of Medicine and Veterans Affairs Medical Center, Iowa City 52242, USA. ramon-lim@uiowa.edu
Zaheer
A
A
Yorek
M A
MA
Darby
C J
CJ
Oberley
L W
LW
D000818:Animals; D002374:Catalase / Q000235:genetics / Q000502:physiology; D020904:Glia Maturation Factor / Q000235:genetics / Q000502:physiology*; D005910:Glioma / Q000378:metabolism; D006801:Humans; D016328:NF-kappa B / Q000187:drug effects / Q000502:physiology*; D019289:Pyruvic Acid / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013482:Superoxide Dismutase / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D020904:Glia Maturation Factor; D016328:NF-kappa B; D012333:RNA, Messenger; D019289:Pyruvic Acid; D002374:Catalase; D013482:Superoxide Dismutase