Inhibition of ceramide pathway does not affect ability of TNF-alpha to activate nuclear factor-kappa B.
TNF-alpha is a multifunctional cytokine that has been shown to activate a number of intracellular second messenger pathways. Recent studies demonstrate that the sphingomyelinase-ceramide pathway plays a potential role in the activation of nuclear factor-kappa B (NF-kappa B) by TNF-alpha. The following experiments both confirm that the addition of ceramide to cells can activate NF-kappa B and demonstrate that a 48-h pretreatment with phorbol 12-myristate (PMA) results in the loss of the ceramide-induced NF-kappa B response. In parallel experiments, in which SW480 cells were pretreated with PMA, TNF-alpha provided a signal resulting in the nuclear translocation of NF-kappa B that was similar to untreated cells. These data combined suggest that additional pathways exist that TNF-alpha can use for the activation of NF-kappa B. Supplementary data demonstrates that TNF-alpha, ceramide, and PMA activate a human cytomegalovirus-(HCMV) beta gal construct (promoter is responsive to NF-kappa B) that was stably transfected into the TNF receptor-bearing tumor cell line, SW480. PMA pretreatment of these cells resulted in a significant decrease in both the PMA and ceramide generated responses, 6% and 0% of controls, respectively. However, the response generated by TNF-alpha was not inhibited significantly (96% of control cells). This data suggests that although ceramide and 1,2-diacylglycerol (DAG) pathways may contribute to TNF-alpha activation of NF-kappa B, impedance of these pathways does not block TNF-alpha from activating NF-kappa B nor induction of the functional activation of the NF-kappa B responsive reporter construct, HCMV.
Johns
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LD
Institute for Inflammation and Experimental Medicine, Miles Research Center, West Haven, CT 06516.
Sarr
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Ranges
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism*; D016384:Consensus Sequence; D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA / Q000235:genetics; D004075:Diglycerides / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D002518:Ceramides; D004075:Diglycerides; D016328:NF-kappa B; D016755:Proto-Oncogene Proteins c-jun; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; D011493:Protein Kinase C; D013108:Sphingomyelin Phosphodiesterase; D013755:Tetradecanoylphorbol Acetate