Localization of Group V phospholipase A2 in caveolin-enriched granules in activated P388D1 macrophage-like cells
Localization of Group V phospholipase A2 in caveolin-enriched granules in activated P388D1 macrophage-like cells
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DOI：
10.1074/jbc.m305904200
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发表时间：
2003-11-28
影响因子：
4.8
通讯作者：
Dennis, EA
中科院分区：
文献类型：
--
作者：
Balboa, MA;Shirai, Y;Dennis, EA
在鼠P388 D（1）巨噬细胞中，前列腺素E-2对脂多糖长期刺激的反应涉及V组分泌型磷脂酶A（2）（PLA（2））、IV组胞质PLA（2）（cPLA（2））和环氧合酶-2（考克斯-2）的作用。cPLA（2）的初始活化诱导V组PLA（2）的表达，V组PLA（2）又诱导考克斯-2和大多数花生四烯酸底物的表达，以产生考克斯-2依赖性前列腺素E-2。由于V族PLA（2）是一种分泌酶，因此假定在细胞刺激后，它必须释放到细胞外介质中并与外膜重新结合以从磷脂中释放花生四烯酸。在本研究中，利用免疫荧光和绿色荧光蛋白标记的V组PLA（2）的共聚焦激光扫描显微镜实验表明，巨噬细胞长期暴露于脂多糖导致V组PLA（2）与靠近核周区域的含小窝蛋白-2的颗粒相关。肝素是一种对第V族PLA（2）具有高亲和力的细胞不可渗透的复合碳水化合物，可阻断这种结合，表明颗粒是由先前与细胞外表面结合的第V族sPLA（2）内化形成的。如果用IV族PLA（2）抑制剂甲基花生四烯酸酯处理细胞，则未观察到V族PLA（2）在核周颗粒中的定位，证实了IV族PLA（2）在活化过程中的重要作用。用抗考克斯-2抗体进行细胞染色，发现富含考克斯-2的颗粒与含有V组PLA的颗粒非常接近（2）。总的来说，这些结果表明，在细胞活化过程中，将V组PLA（2）包封到颗粒中将酶带到核周包膜，在那里它可能更接近IV组PLA（2）和考克斯-2，以有效合成前列腺素。
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.