CrmA inhibits TNFa-induced apoptosis by a mechanism upstream and distinct from Bcl-2

The sphingomyelin breakdown product, ceramide, is emerging as a novel lipid second messenger mediating apoptosis and cell cycle arrest. Sustained elevation of intracellular ceramide induced by serum starvation or Fas receptor ligation has been shown to precede cell cycle arrest and apoptosis. Here we show that the products of two antiapoptotic genes, the cowpox virus crmA gene and the cellular gene bcl-2, target the TNFa-induced ceramide pathway of apoptosis. CrmA targets a proximal step in the pathway resulting in the inhibition of ceramide generation in response to TNFa; thus protecting cells from TNFa-induced, but not ceramide-induced apoptosis. Bcl-2, in contrast, does not interfere with ceramide generation in response to TNFa. Instead, it targets the pathway at a more distal step, blocking apoptosis induced by both TNFa and exogenous ceramide. Evidence for distinct mechanisms of action of Bcl-2 and CrmA was further found in studies utilizing the alkylating agent mitomycin C. Mitomycin C caused death and poly(ADP-ribose) polymerase (PARP) cleavage without ceramide generation. Bcl-2 inhibited the effects of Mitomycin C on death and PARP cleavage, whereas CrmA was without effect. These results describe a novel mechanism for inhibition of apoptosis, the inhibition of ceramide generation by CrmA. Also, these studies distinguish the mechanism of action of CrmA from that of Bcl-2.