ACIDIC SPHINGOMYELINASE-GENERATED CERAMIDE IS NEEDED BUT NOT SUFFICIENT FOR TNF-INDUCED APOPTOSIS AND NUCLEAR FACTOR-KAPPA-B ACTIVATION

Previously we have shown that treatment of ML-1a cells with TNF in the presence of cycloheximide triggers apoptosis within 90 min. In the pr esent report we used this system to investigate the role of ceramide i n TNF action. We found that while maximum DNA fragmentation response w as induced by 1 nM TNF, the synthetic membrane-permeable C-2, C-6, and C-8 ceramides had no effect even up to 40 mu M concentration. To inve stigate the roles of ceramide in TNF action, we used nethyl)amino]prop yloxy)benzenesulfonyl))indolizine (SR33557), a potent inhibitor of aci dic but not neutral sphingomyelinase (SMase). Even though ceramides by themselves did not mimic TNF, we found that SR33557 inhibited TNF-ind uced apoptosis and the addition of ceramide reversed this effect, indi cating that ceramide generated by acidic SMase is involved in TNF acti on. The addition of SR33557 to cells at 0 or at 30 min after TNF treat ment showed inhibition, but the addition 60 min later had no effect, i ndicating that an SR33557-sensitive step is located between 30 and 60 min of signal transduction. Since ceramide has been shown to play a ro le in TNF-mediated activation of nuclear factor-kappa B (NF-kappa B), we examined the effect of SR33557 on this early cellular response of T NF. Surprisingly, this inhibitor of ceramide production was found to h ave no effect on TNF-mediated NF-kappa B activation, thus suggesting t hat SR33557-sensitive acidic SMase is not involved in this process. Fr om these results, we concluded that ceramide is needed for certain TNF -mediated cellular responses, but it alone may not be sufficient.