Cytosolic phospholipase A(2)-alpha enhances induction of endoplasmic reticulum stress

Induction of endoplasmic reticulum (ER) stress by the complement membrane attack complex is enhanced by activation of cytosolic phospholipase A(2)-alpha (cPLA(2)). To address mechanisms by which cPLA(2) may modulate ER stress, we produced a mutant cPLA(2), containing an ER targeting domain (cPLA(2)-ERmut). After transfection and fractionation of COS-1 cells, cPLA(2)-ERmut was present mainly in the membrane fraction, whereas wild type (wt) cPLA(2) was principally in the cytosol. By fluorescence microscopy, cPLA(2)-ERmut was enriched in a perinuclear distribution under basal conditions, colocalizing with the ER protein, calnexin, while cPLA(2)-wt was mainly cytosolic. Both forms of cPLA(2) transiently expressed in COS cells showed basal phosphorylation at serine(505), which correlates with catalytic activity. Expression of cPLA(2)-wt was approximately 5-fold greater, compared with cPLA(2)-ERmut, but both enzymes produced comparable increases in free arachidonic acid, implying that cPLA(2)-ERmut effectively hydrolyzed ER membrane phospholipids. Although transfection of cPLA(2)-ERmut or wt did not induce ER stress independently, cPLA(2)-ERmut and wt enhanced the induction of ER stress by tunicamycin, dithiothreitol and ionomycin (monitored by induction of grp94 and C/EBP homologous protein-10), and the effect was dependent on the catalytic activity. cPLA(2)-ERmut enhanced production of superoxide. Induction of ER stress in tunicamycin-treated cells expressing cPLA(2)-ERmut was attenuated in the presence of the antioxidant, N-acetyl cysteine, and reduced glutathione, and was exacerbated by dl-buthionine-(S,R)-sulfoximine (which depletes glutathione). Expression of cPLA(2)-ERmut exacerbated tunicamycin-induced apoptosis. Thus, induction of ER stress is facilitated by the activation of cPLA(2) at the ER. The mechanism involves ER membrane phospholipid hydrolysis, and accumulation of reactive oxygen species.