Localization of Group V Phospholipase A2 in Caveolin-enriched Granules in Activated P388D1 Macrophage-like Cells

Abstract

In murine P388D 1 macrophages, the generation of prostaglandin E 2 in response to long term stimulation by lipopolysaccharide involves the action of Group V secreted phospholipase A 2 (PLA 2 ), Group IV cytosolic PLA 2 (cPLA 2 ), and cyclooxygenase-2 (COX-2). There is an initial activation of cPLA 2 that induces expression of Group V PLA 2 , which in turn induces both the expression of COX-2 and most of the arachidonic acid substrate for COX-2-dependent prostaglandin E 2 generation. Because Group V PLA 2 is a secreted enzyme, it has been assumed that after cellular stimulation, it must be released to the extracellular medium and re-associates with the outer membrane to release arachidonic acid from phospholipids. In the present study, confocal laser scanning microscopy experiments utilizing both immunofluorescence and green fluorescent protein-labeled Group V PLA 2 shows that chronic exposure of the macrophages to lipopolysaccharide results in Group V PLA 2 being associated with caveolin-2-containing granules close to the perinuclear region. Heparin, a cellimpermeable complex carbohydrate with high affinity for Group V PLA 2 , blocks that association, suggesting that the granules are formed by internalization of the Group V sPLA 2 previously associated with the outer cellular surface. Localization of Group V PLA 2 in perinuclear granules is not observed if the cells are treated with the Group IV PLA 2 inhibitor methyl arachidonyl fluorophosphonate, confirming the important role for Group IV PLA 2 in the activation process. Cellular staining with antibodies against COX-2 reveals the presence of COX-2-rich granules in close proximity to those containing Group V PLA 2 . Collectively, these results suggest that encapsulation of Group V PLA 2 into granules brings the enzyme to the perinuclear envelope during cell activation where it may be closer to Group IV PLA 2 and COX-2 for efficient prostaglandin synthesis.

Arachidonic Acid Release from Mammalian Cells Transfected with Human Groups IIA and X Secreted Phospholipase A2 Occurs Predominantly during the Secretory Process and with the Involvement of Cytosolic Phospholipase A2-α

“…Some caveolin-2 is found in caveolae, in association with caveolin-1, but even in these cases, most of the caveolin-2 detected by immunofluorescence resides in the Golgi complex (45)(46)(47). A recent study showed co-localization of human group V sPLA 2 with caveolin-2 in punctate domains in the macrophage-like cell line p388D1 (49). Given the above concerns, the localization of human group V sPLA 2 in caveolae is called into question.…”

“…Given the above concerns, the localization of human group V sPLA 2 in caveolae is called into question. Treatment of cells with extracellular heparin reduces the amount of immunofluorescence staining of hGIIA and group V sPLA 2 in punctate domains (21,49), and this is cited as evidence that the punctate localization of sPLA 2 derives from re-entry of extracellular enzyme. It may be noted that heparin treatment did not eliminate punctate staining (49).…”

“…Treatment of cells with extracellular heparin reduces the amount of immunofluorescence staining of hGIIA and group V sPLA 2 in punctate domains (21,49), and this is cited as evidence that the punctate localization of sPLA 2 derives from re-entry of extracellular enzyme. It may be noted that heparin treatment did not eliminate punctate staining (49). Furthermore, because heparin forms high molecular mass, multi-protein/polymer aggregates with basic sPLA 2 s (50), it is possible that the effect of heparin is to prevent the transfer of extracellular sPLA 2 into detergentpermeabilized cells, given that formaldehyde fixation is typically not a complete process.…”

Liberating Chiral Lipid Mediators, Inflammatory Enzymes, and LIPID MAPS from Biological Grease

“…This led to characterizing the eicosanoids produced downstream of PLA 2 and their autocrine and paracrine function in lipid signaling in the cellular milieu (99 -101). This all became considerably more complex as we realized that the activation of eicosanoid synthesis could involve activation of multiple PLA 2 s (102)(103)(104)(105)(106) stimulated by the products of other PLA 2 s from distinct subcellular locations (107,108).…”

Group V Phospholipase A2 in Bone Marrow-derived Myeloid Cells and Bronchial Epithelial Cells Promotes Bacterial Clearance after Escherichia coli Pneumonia

“…The observations that GV sPLA 2 can hydrolyze Gram-negative bacterial phospholipids (6) and that phagocytosis by peritoneal macrophages is modulated by GV sPLA 2 (7) suggests that GV sPLA 2 participates in the killing of bacteria by leukocytes (5). GV sPLA 2 can be expressed by macrophages (7,8), mast cells (9,10), airway smooth muscle cells (11), and bronchial epithelial cells (12) in mice and by alveolar macrophages, bronchial epithelial cells, and interstitial fibroblasts in patients with pneumonia, where expression of GV sPLA 2 correlates with the severity of inflammation (13). Overexpression of GV sPLA 2 leads to increased PGE 2 biosynthesis by cultured bronchial epithelial cells (13) and to surfactant degradation, respiratory distress, and neonatal lethality in transgenic mice that constitutively express the enzyme under an actin promotor (14).…”