CYTOKINE RESPONSE MODIFIER-A (CRMA) INHIBITS CERAMIDE FORMATION IN RESPONSE TO TUMOR-NECROSIS-FACTOR (TNF)-ALPHA - CRMA AND BCL-2 TARGET DISTINCT COMPONENTS IN THE APOPTOTIC PATHWAY

Proteases are now firmly established as major regulators of the ''exec ution'' phase of apoptosis. Here, we examine the role of proteases and their relationship to ceramide, a proposed mediator of apoptosis, in the tumor necrosis factor-alpha (TNF-alpha)-induced pathway of cell de ath. Ceramide induced activation of prICE, the protease that cleaves t he death substrate poly(ADP-ribose) polymerase. Bcl-2 inhibited cerami de-induced death, but not ceramide generation. In contrast, Cytokine r esponse modifier A (CrmA), a potent inhibitor of Interleukin-1 beta co nverting enzyme and related proteases, inhibited ceramide generation a nd prevented TNF-alpha-induced death. Exogenous ceramide could overcom e the CrmA block to cell death, but not the Bcl-2 block. CrmA, however , did not inhibit the activation of nuclear factor (NF)-kappa B by TNF -alpha, demonstrating that other signaling functions of TNF-alpha rema in intact and that ceramide does not play a role in the activation of NF-kappa B. These studies support a distinct role for proteases in the signaling/activation phase of apoptosis acting upstream of ceramide f ormation.