Catalytic activity-independent pathway is involved in phospholipase A(2)-induced apoptotic death of human leukemia U937 cells via Ca(2+)-mediated p38 MAPK activation and mitochondrial depolarization.
In view of the controversial role of catalytic activity on the cytotoxicity of phospholipase A(2) (PLA(2)), the present study is conducted to explore whether PLA(2) 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 PLA(2)) with p-bromophenacyl bromide (BPB) caused over 99.9% drop in enzymatic activity Naja naja atra PLA(2). It was found that BPB-PLA(2)-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-PLA(2), elevation of intracellular Ca(2+) levels and p38 MAPK activation were observed in U937 cells. Pretreatment with BAPTA-AM (Ca(2+) chelator) and nifedipine (L-type Ca(2+) channel blocker) abrogated Ca(2+) increase and p38 MAPK activation, and rescued viability of BPB-PLA(2)-treated U937 cells. BPB-PLA(2)-induced dissipation of mitochondrial membrane potential and down-regulation of Bcl-2 were suppressed by SB202190 (p38MAPK inhibitor). Although PLA(2) mutants in which His-48 and Asp-49 were substituted by Ala and Lys, respectively, did not display detectable PLA(2) activity, they induced death of U937 cells. The signaling pathway of PLA(2) mutants in inducing cell death was indistinguishable from that of BPB-PLA(2). Taken together, our data indicate that catalytic activity-independent pathway is involved in PLA(2)-induced apoptotic death of human leukemia U937 cells via mitochondria-mediated death pathway triggering by Ca(2+)-mediated p38 MAPK activation.
Liu
Wen-Hsin
WH
Institute of Biomedical Sciences, National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, National Sun Yat-Sen University, Kaohsiung 804, Taiwan.
Kao
Pei-Hsiu
PH
Chiou
Yi-Ling
YL
Lin
Shinne-Ren
SR
Wu
Ming-Jung
MJ
Chang
Long-Sen
LS
D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002384:Catalysis; D002470:Cell Survival / Q000187:drug effects; D002942:Circular Dichroism; D004546:Elapid Venoms / Q000201:enzymology; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D054467:Phospholipases A2 / Q000302:isolation & purification / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D020298:U937 Cells; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D000098:Acetophenones; D004546:Elapid Venoms; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D048051:p38 Mitogen-Activated Protein Kinases; D054467:Phospholipases A2; D053148:Caspase 3; D053453:Caspase 9; C006821:4-bromophenacyl bromide; D002118:Calcium