Activation of nuclear factor-kappa B in C6 rat glioma cells after transfection with glia maturation factor

The 17-kDa endogenous brain protein glia maturation factor (GMF) was transf ected into C6 rat glioma cells using a replication-defective human adenovir us vector. The cells overexpressed GMF but did not secrete the protein into the medium. Transfection with GMF led to the activation of the transcripti on factor nuclear factor-kappa B (NF-kappa B), as evidenced by electrophore tic mobility shift assay of the nuclear extract, using a double-stranded ol igonucleotide probe containing the consensus binding sequence for NF-kappa B, The specificity of binding was demonstrated by competition with unlabele d probe and by the nonbinding of the mutant probe. Binding was delectable a s early as 3 h after transfection, peaked at 6 and 12 h, and gradually decl ined thereafter. The observed NF-kappa B activation was reduced by cotransf ection with catalase and by the presence of high concentrations of pyruvate in the medium, suggesting the involvement of H2O2. The p38 mitogen-activat ed protein kinase inhibitor SB-203580 also suppressed the GMF-activated NF- kappa B, suggesting the involvement of the p38 signal transduction cascade. On the other hand, the phorbol ester phorbol 12-myristate 13-acetate activ ated NF-kappa B whether or not GMF was overexpressed. Along with NF-kappa B activation was an enhanced expression of superoxide dismutase (SOD), which was suppressed if NF-kappa B nuclear translocation was blocked by its spec ific decoy DNA, implicating NF-kappa B as an upstream mediator of this anti oxidant enzyme. The p38 inhibitor SB-203580 also blocked the GMF-activated SOD, As NF-kappa B and SOD are both pro-survival signals, the results sugge st a cytoprotective role for endogenous GMF in glial cells.