Attenuated Free Cholesterol Loading-induced Apoptosis but Preserved Phospholipid Composition of Peritoneal Macrophages from Mice That Do Not Express Group VIA Phospholipase A2

Abstract

Mouse macrophages undergo ER stress and apoptosis upon free cholesterol loading (FCL). We recently generated iPLA 2 ␤-null mice, and here we demonstrate that iPLA 2 ␤-null macrophages have reduced sensitivity to FCL-induced apoptosis, although they and wild-type (WT) cells exhibit similar increases in the transcriptional regulator CHOP. iPLA 2 ␤-null macrophages are also less sensitive to apoptosis induced by the sarcoplasmic reticulum Ca 2؉ -ATPase inhibitor thapsigargin and the scavenger receptor A ligand fucoidan, and restoring iPLA 2 2 ␤ modulates mitochondrial cytochrome c release, and we find that thapsigargin and fucoidan induce mitochondrial phospholipid loss and cytochrome c release into WT macrophage cytosol and that these events are blunted in iPLA 2 ␤-null cells. Immunoblotting studies indicate that iPLA 2 ␤ associates with mitochondria in macrophages subjected to ER stress. AA incorporation into glycerophosphocholine lipids is unimpaired in iPLA 2 ␤-null macrophages upon electrospray ionization-tandem mass spectrometry analyses, and their complex lipid composition is similar to WT cells. These findings suggest that iPLA 2 ␤ participates in ER stress-induced macrophage apoptosis caused by FCL or thapsigargin but that deletion of iPLA 2 ␤ does not impair macrophage arachidonate incorporation or phospholipid composition.

Group VIA Phospholipase A2 Mitigates Palmitate-induced β-Cell Mitochondrial Injury and Apoptosis

“…Materials-Most materials were obtained from sources specified previously (21)(22)(23)(24). Rainbow molecular mass standards, PVDF membranes, and Triton X-100 were obtained from Bio-Rad.…”

“…Immunoblotting Analyses-Cells were harvested and sonicated (15-30 s at 1-s intervals) in appropriate buffer, and an aliquot (30 g) of lysate protein was analyzed by SDS-PAGE (4 -20% Tris-glycine gel, Invitrogen), transferred onto Immobilon-P polyvinylidene difluoride membranes (Bio-Rad), and processed for immunoblotting analyses as described (21)(22)(23)(24). Targeted proteins and primary antibody concentrations were as follows: iPLA 2 ␤ (1:2000 dilution of T-14 antibody from Santa Cruz Biotechnology, Santa Cruz, CA, sc-14463), caspase-3 (1:1000 dilution of H-277 antibody from Santa Cruz Biotechnology, sc7148), cytochrome c oxidase complex IV (COX IV) (1:1000 dilution of antibody 4844 from Cell Signaling Technology, Beverly, MA), anti-FLAG (1:1000; Sigma, F1804), and antipolyhistidine (1:1000; Sigma, H1029).…”

Effects of Synthetic Sphingosine-1-Phosphate Analogs on Cytosolic Phospholipase A2α–Independent Release of Arachidonic Acid and Cell Toxicity in L929 Fibrosarcoma Cells: the Structure–Activity Relationship

“…Since treatment with 10 μM BEL, which does not inhibit secretory PLA 2 or cPLA 2 α (12), inhibited the S1P analog-induced responses, the analogs may stimulate Ca 2+ -independent PLA 2 , resulting in the release of AA. It is reported that free cholesterol loading, which might be attributable to induction of endoplasmic reticulum stress, released AA and caused apoptosis of mouse macrophages in a β-isoform Ca 2+ -independent PLA 2 -mediated pathway (34). The effective S1P analogs for the release of AA such as DMB-mC11S (GS13), which are lipophilic compounds with larger log P values, may interact with intracellular organelles, resulting in the activation of secretory and Ca 2+ -independent PLA 2 s. Further identification of the molecular target(s) of the effective S1P analogs including the subtypes of secretory and Ca 2+ -independent PLA 2 s remains to be conducted.…”

“…In L929 cells, AA is reported to increase ceramide levels via the activation of sphingomyelinase and resulting cell death (35). Ca 2+ -independent PLA 2 (β-isofrom) is reported to participate in endoplasmic reticulum stress-induced apoptosis in insulinoma cells (36) and in macrophages (34). Secretory PLA 2 s play important roles in cell damage under pathological conditions including inflammation and atherogeneis (14,16,20).…”

“…Thus, the S1P analog-induced release of AA appears to be linked with cell death in L929 cells. AA has been known to regulate the activity and function of many proteins and cell growth, differentiation, and survival in direct and indirect manners (16,34). In L929 cells, AA is reported to increase ceramide levels via the activation of sphingomyelinase and resulting cell death (35).…”