Selective Involvement of Ceramide in Cytokine-induced Apoptosis

Among its diverse biologic effects, the cytokine tumor necrosis factor α causes the rapid nuclear translocation of the transcription factor, nuclear factor κB (NF-κB). The p55 tumor necrosis factor (TNF) receptor shares with the related APO-1/Fas antigen the ability to initiate apoptosis. We investigated the role of the sphingolipid mediator ceramide in the cytokine-induced signaling mechanisms leading to NF-κB activation and cell death. Several lines of evidence presented here suggest that ceramide generated in response to TNFα or Fas activation is not involved in NF-κB activation. (i) Cell-permeable ceramides and exogenous sphingomyelinase failed to induce either nuclear translocation of NF-κB or degradation of its cytosolic inhibitor, I-κB, in Jurkat T cells. (ii) Ceramide treatment of cells inhibited phorbol ester-induced activation of NF-κB. (iii) TNFα potently activated NF-κB in a cell line deficient in acid sphingomyelinase. (iv) TNFα activated NF-κB within minutes without altering ceramide levels. (v) Treatment of Jurkat cells with cross-linking antibodies to APO-1/Fas induced large scale increases in ceramide and apoptosis without affecting NF-κB. (vi) Ceramide generation in response to Fas activation was inhibited byN-acetyltyrosinylvalinylalanylaspartyl chloromethyl ketone, a peptide inhibitor of interleukin-1β-converting enzyme-like proteases, whereas TNFα-induced NF-κB activation was unaffected by the inhibitor. These results show that ceramide accumulation belongs selectively to the apoptotic pathway(s) induced by cytokines, and, if anything, ceramide may participate in negative feedback regulation of NF-κB.