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

• Published in: The Journal of biological chemistry 2003-11, Vol.278 (48), p.48059-48065
• Main Authors: Balboa, María A, Shirai, Yasuhito, Gaietta, Guido, Ellisman, Mark H, Balsinde, Jesús, Dennis, Edward A
• Format: Article
• Language: English
• Subjects: Animals; Arachidonic Acid - metabolism; Arachidonic Acids - pharmacology; Caveolin 1; Caveolin 2; Caveolins - metabolism; Cell Line; Cell Membrane - metabolism; Cell Nucleus - metabolism; Cyclooxygenase 2; DNA, Complementary - metabolism; Enzyme Inhibitors - pharmacology; Fibroblasts - metabolism; Green Fluorescent Proteins; Heparin - chemistry; Heparin - metabolism; Isoenzymes - metabolism; Lipopolysaccharides - chemistry; Lipopolysaccharides - metabolism; Luminescent Proteins - metabolism; Macrophages - metabolism; Mice; Microscopy, Fluorescence; Models, Biological; Organophosphonates - pharmacology; Peptides - chemistry; Phospholipases A - biosynthesis; Phospholipases A2; Plasmids - metabolism; Prostaglandin-Endoperoxide Synthases - metabolism; Prostaglandins - metabolism; Protein Binding; Time Factors; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M305904200

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 cell-impermeable 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.