CERAMIDE IS NOT A SIGNAL FOR TUMOR NECROSIS FACTOR-INDUCED GENE-EXPRESSION BUT DOES CAUSE PROGRAMMED CELL-DEATH IN HUMAN VASCULAR ENDOTHELIAL-CELLS

Tumor necrosis factor (TNF) activates transcription of endothelial leu kocyte adhesion molecule-1 (CD62E) in endothelial cells (ECs) through the binding to the gene promoter of the p50/p65 heterodimeric form of nuclear factor-kappa B (NF-kappa B) and of the N-terminal phosphorylat ed form of the ATF2/c-Jun transcription factor, which is phosphorylate d by Jun N-terminal kinase (JNK). However, the intracellular signaling pathways that activate endothelial NF-kappa B and JNK in TNF-induced responses are unknown. In this study we have examined the role of a re cently described TNF signaling pathway involving sphingomyelin activat ion to generate ceramide, a potential intracellular mediator. We find that concentrations of TNF that strongly activate NF-kappa B and JNK w ithin 15 minutes do not produce either a measurable decline in sphingo myelin or a measurable generation of ceramide in cultured human umbili cal vein ECs at any rime examined. Stimulation of ECs with purified sp hingomyelinase (SMase) enzyme causes a rapid 60% to 80% decrease in ce llular sphingomyelin content and a large increase in ceramide. However , SMase treatment only minimally activates NF-kappa B, achieving level s that are insufficient to initiate gene transcription. Extracellular SMase does not have access to intracellular sphingomyelin, but treatme nt of ECs with membrane-permeant ceramide analogues still completely f ails to activate NF-kappa B and only activates JNK at late times. Neit her SMase nor ceramide analogues induce gene transcription or surface expression of endothelial leukocyte adhesion molecules that are readil y induced by TNF. Strikingly, low concentrations of membrane-permeant ceramide cause programmed cell death in ECs, a finding not observed al any concentrations of TNF tested. We conclude that ceramide is not an important second messenger for TNF signaling of gene transcription in ECs but may be a second messenger for cell death in response to as-ye t-unidentified signals.