CD95(Fas/APO-1) signals ceramide generation independent of the effector stage of apoptosis

Although numerous studies document caspase-independent ceramide generation preceding apoptosis upon environmental stress, the molecular ordering of ce ramide generation during cytokine-induced apoptosis remains uncertain. Here , we show that CD95-induced ceramide elevation occurs during the initiation phase of apoptosis. We titrated down the amount of FADD transfected into H eLa and 293T cells until it was insufficient for apoptosis, although cycloh eximide (CHX) still triggered the effector phase. Even in the absence of CI M, ceramide levels increased rapidly, peaking at 2.7 +/- 0.2-fold of contro l 8 h post-transfection. Dominant negative FADD failed to confer ceramide g eneration or CHX-mediated apoptosis, Ceramide generation induced by FADD wa s initiator caspase-dependent, being blocked by crmA. Limited pro-caspase 8 overexpression also increased ceramide levels 2.7 +/- 0.2-fold, yet failed , without CIM, to initiate apoptosis, Expression of membrane-targeted oligo merized CD-8 caspase 8 induced apoptosis without GEM, yet elevated ceramide only to a level equivalent to limited pro-caspase 8 transfection. Ceramide elevations mere detected concurrently by diacyl-glycerol kinase and electr ospray tandem mass spectrometry. These investigations provide evidence that ceramide generation is initiator caspase-dependent and occurs prior to com mitment to the effector phase of apoptosis, definitively ordering ceramide as proximal in CD95 signaling.