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. 2010 Elsevier B.V. All rights reserved.