Abstract
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.
Highlights
Mammalian group IIA phospholipase A2 (PLA2)1 catalyzes the release of sn-2 fatty acid from membrane phospholipids
This study demonstrates that group IIA PLA2 generates anti-apoptotic survival signals in baby hamster kidney (BHK) cells targeting the secretory pathway, and suggests that high levels of group IIA PLA2 accumulated at inflammatory sites may regulate inflammation, and may protect cells from unnecessary death induced by pro-inflammatory agents
Using PLA2 inhibitors and antisense oligonucleotides specific for group IIA PLA2, we found that group IIA PLA2 contributed to cell injury during chemical hypoxia in isolated rat hepatocytes [16, 17]
Summary
Mammalian group IIA phospholipase A2 (PLA2) catalyzes the release of sn-2 fatty acid from membrane phospholipids. Using PLA2 inhibitors and antisense oligonucleotides specific for group IIA PLA2, we found that group IIA PLA2 contributed to cell injury during chemical hypoxia in isolated rat hepatocytes [16, 17]. Certain reagents used to modulate group IIA PLA2 activity such as inhibitors, antisense oligonucleotides, and antibodies thought to be specific to group IIA PLA2, have been shown to act nonspecifically via group V PLA2. Given these concerns, the most direct way of elucidating group IIA PLA2 function is to study cellular phenotype changes after expression of group IIA PLA2 and its mutants. This finding sheds new light on the understanding of the function of group IIA PLA2 in inflammation and other pathological processes
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