Cytosolic phospholipase A 2-α enhances induction of endoplasmic reticulum stress

Abstract

Induction of endoplasmic reticulum (ER) stress by the complement membrane attack complex is enhanced by activation of cytosolic phospholipase A 2-α (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 ∼ 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.