INVOLVEMENT OF DE-NOVO CERAMIDE BIOSYNTHESIS IN TUMOR-NECROSIS-FACTOR-ALPHA CYCLOHEXIMIDE-INDUCED CEREBRAL ENDOTHELIAL-CELL DEATH

Cytokines, including tumor necrosis factor-alpha (TNF-alpha), may elic it cytotoxic response through the sphingomyelin-ceramide signal transd uction pathway by activation of sphingomyelinases and the subsequent r elease of ceramide: the universal lipid second messenger. Treatment of bovine cerebral endothelial cells (BCECs) with TNF-alpha for 16 h fol lowed by cycloheximide (CHX) for 6 h resulted in an increase in cerami de accumulation, DNA fragmentation, and cell death. Application of a c ell permeable ceramide analogue C-2 ceramide, but not the biologically inactive C-2 dihydroceramide, also induced DNA laddering and BCEC dea th in a concentration- and time-dependent manner, TNF-alpha/CHX-mediat ed ceramide production apparently is not a result of sphingomyelin hyd rolysis because sphingomyelin content does not decrease in this death paradigm, In addition, an acidic sphingomyelinase inhibitor, desiprami ne, had no effect on TNF-alpha/CHX-induced cell. death, However, addit ion of fumonisin B1, a selective ceramide synthase inhibitor, attenuat ed TNF-alpha/CHX-induced intracellular ceramide elevation and BCEC dea th. Together, these findings suggest that ceramide plays at least a pa rtial role in this paradigm of BCEC death. Our results show, for the f irst time, that ceramide derived from de novo synthesis is an alternat ive mechanism to sphingomyelin hydrolysis in the BCEC death process in itiated by TNF-alpha/CHX.