Catalytic activity-independent pathway is involved in phospholipase A2-induced apoptotic death of human leukemia U937 cells via Ca2+-mediated p38 MAPK activation and mitochondrial depolarization

In view of the controversial role of catalytic activity on the cytotoxicity of phospholipase A2 (PLA2), the present study is conducted to explore whether PLA2 induces apoptotic process of human leukemia U937 cells through catalytic activity-independent pathway. Modification of His-48 (according to the sequence alignment with porcine pancreatic PLA2) with p-bromophenacyl bromide (BPB) caused over 99.9% drop in enzymatic activity Naja naja atra PLA2. It was found that BPB–PLA2-induced apoptotic death of U937 cells was associated with mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Upon exposure to BPB–PLA2, elevation of intracellular Ca2+ levels and p38 MAPK activation were observed in U937 cells. Pretreatment with BAPTA-AM (Ca2+ chelator) and nifedipine (L-type Ca2+ channel blocker) abrogated Ca2+ increase and p38 MAPK activation, and rescued viability of BPB–PLA2-treated U937 cells. BPB–PLA2-induced dissipation of mitochondrial membrane potential and down-regulation of Bcl-2 were suppressed by SB202190 (p38MAPK inhibitor). Although PLA2 mutants in which His-48 and Asp-49 were substituted by Ala and Lys, respectively, did not display detectable PLA2 activity, they induced death of U937 cells. The signaling pathway of PLA2 mutants in inducing cell death was indistinguishable from that of BPB–PLA2. Taken together, our data indicate that catalytic activity-independent pathway is involved in PLA2-induced apoptotic death of human leukemia U937 cells via mitochondria-mediated death pathway triggering by Ca2+-mediated p38 MAPK activation.