Zhang Y, Lemasters J, Herman B
Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7762, USA.
J Biol Chem. 1999 Sep 24;274(39):27726-33. doi: 10.1074/jbc.274.39.27726.
Mammalian group IIA phospholipase A(2) (PLA(2)) is believed to play important roles in inflammation, cell injury, and tumor resistance. However, the cellular site of action has not been clearly defined as it has long been recognized that group IIA PLA(2) is both a secretory and mitochondrial protein. The purpose of this study was to determine the subcellular target of the group IIA PLA(2) and its role in apoptosis stimulated by growth factor withdrawal. Cloning of the rat liver group IIA PLA(2) demonstrated a typical secretory signal and no alternative splicing of the primary transcript. When a sequence including the signal peptide and first 8 residues in the mature enzyme or the entire PLA(2) (including the signal peptide) was fused to enhanced green fluorescent protein, the fusion protein was directed to the secretory pathway rather than mitochondria in baby hamster kidney (BHK) cells. To examine the role of group IIA PLA(2) in cell injury, wild type (wt) rat group IIA PLA(2) and a mutant group IIA PLA(2) containing a His-47 --> Gln mutation (at the catalytic center) were transfected into BHK cells and cells stably expressing these constructs were isolated. After deprivation of growth factors, both normal BHK cells and BHK cells expressing mutant PLA(2) underwent massive apoptosis, while BHK cells expressing wt PLA(2) showed considerable resistance to growth factor withdrawal-induced apoptosis. The secretory PLA(2) inhibitors 12-epi-scalaradial and aristolochic acid abrogated resistance to apoptosis in the wt PLA(2) expressing cells. These two inhibitors did not induce cell death in the presence of fetal bovine serum, suggesting that they induce cell death by blocking PLA(2) generated survival signals. This study demonstrates that group IIA PLA(2) generates anti-apoptotic survival signals in BHK cells targeting the secretory pathway, and suggests that high levels of group IIA PLA(2) accumulated at inflammatory sites may not only regulate inflammation, but also may protect cells from unnecessary death induced by pro-inflammatory agents.
哺乳动物IIA组磷脂酶A2（PLA2）被认为在炎症、细胞损伤和肿瘤抗性中发挥重要作用。然而，其细胞作用位点尚未明确界定，因为长期以来人们认识到IIA组PLA2既是一种分泌蛋白，也是一种线粒体蛋白。本研究的目的是确定IIA组PLA2的亚细胞靶点及其在生长因子撤除诱导的细胞凋亡中的作用。大鼠肝脏IIA组PLA2的克隆显示出典型的分泌信号，且初级转录本无选择性剪接。当将包含信号肽和成熟酶中前8个残基的序列或整个PLA2（包括信号肽）与增强型绿色荧光蛋白融合时，融合蛋白在幼仓鼠肾（BHK）细胞中被导向分泌途径而非线粒体。为了研究IIA组PLA2在细胞损伤中的作用，将野生型（wt）大鼠IIA组PLA2和含有His-47→Gln突变（位于催化中心）的突变型IIA组PLA2转染到BHK细胞中，并分离出稳定表达这些构建体的细胞。在撤除生长因子后，正常BHK细胞和表达突变型PLA2的BHK细胞均发生大量凋亡，而表达wt PLA2的BHK细胞对生长因子撤除诱导的凋亡表现出相当的抗性。分泌型PLA2抑制剂12-表-司卡勒拉地尔和马兜铃酸消除了表达wt PLA2的细胞对凋亡的抗性。这两种抑制剂在胎牛血清存在下不诱导细胞死亡，表明它们通过阻断PLA2产生的存活信号诱导细胞死亡。本研究表明，IIA组PLA2在靶向分泌途径的BHK细胞中产生抗凋亡存活信号，并提示在炎症部位积累的高水平IIA组PLA2不仅可能调节炎症，还可能保护细胞免受促炎剂诱导的不必要死亡。
