Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1950946 | Biochimica et Biophysica Acta (BBA) - Molecular Cell Research | 2010 | 14 Pages |
Induction of endoplasmic reticulum (ER) stress by the complement membrane attack complex is enhanced by activation of cytosolic phospholipase A2-α (cPLA2). To address mechanisms by which cPLA2 may modulate ER stress, we produced a mutant cPLA2, containing an ER targeting domain (cPLA2-ERmut). After transfection and fractionation of COS-1 cells, cPLA2-ERmut was present mainly in the membrane fraction, whereas wild type (wt) cPLA2 was principally in the cytosol. By fluorescence microscopy, cPLA2-ERmut was enriched in a perinuclear distribution under basal conditions, colocalizing with the ER protein, calnexin, while cPLA2-wt was mainly cytosolic. Both forms of cPLA2 transiently expressed in COS cells showed basal phosphorylation at serine505, which correlates with catalytic activity. Expression of cPLA2-wt was ∼ 5-fold greater, compared with cPLA2-ERmut, but both enzymes produced comparable increases in free arachidonic acid, implying that cPLA2-ERmut effectively hydrolyzed ER membrane phospholipids. Although transfection of cPLA2-ERmut or wt did not induce ER stress independently, cPLA2-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. cPLA2-ERmut enhanced production of superoxide. Induction of ER stress in tunicamycin-treated cells expressing cPLA2-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 cPLA2-ERmut exacerbated tunicamycin-induced apoptosis. Thus, induction of ER stress is facilitated by the activation of cPLA2 at the ER. The mechanism involves ER membrane phospholipid hydrolysis, and accumulation of reactive oxygen species.