INHIBITION 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 activat e a number of intracellular second messenger pathways. Recent studies demonstrate that the sphingomyelinase-ceramide pathway plays a potenti al role in the activation of nuclear factor-kappa B (NF-kappa B) by TN F-alpha. The following experiments both confirm that the addition of c eramide to cells can activate NF-kappa B and demonstrate that a 48-h p retreatment with phorbol 12-myristate (PMA) results in the loss of the ceramide-induced NF-kappa B response. In parallel experiments, in whi ch SW480 cells were pretreated with PMA, TNF-alpha provided a signal r esulting in the nuclear translocation of NF-kappa B that was similar t o untreated cells. These data combined suggest that additional pathway s exist that TNF-alpha can use for the activation of NF-kappa B. Suppl ementary data demonstrates that TNF-alpha, ceramide, and PMA activate a human cytomegalovirus-(HCMV) beta gal construct (promoter is respons ive to NF-kappa B) that was stably transfected into the TNF receptor-b earing tumor cell line, SW480. PMA pretreatment of these cells resulte d in a significant decrease in both the PMA and ceramide generated res ponses, 6% and 0% of controls, respectively. However, the response gen erated by TNF-alpha was not inhibited significantly (96% of control ce lls). 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 N F-kappa B nor induction of the functional activation of the NF-kappa B responsive reporter construct, HCMV.