12-LOX catalyzes the oxidation of 2-arachidonoyl-lysolipids in platelets generating eicosanoid-lysolipids that are attenuated by iPLA2γ knockout

Abstract

The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A2-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA2γ (patatin-like phospholipase domain containing 8 (PNPLA8)), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increases in 2-AA-LPC, 2-AA-LPE, and their downstream 12-LOX oxidation products, 12(S)-HETE-LPC and 12(S)-HETE-LPE, in calcium ionophore (A23187)-stimulated murine platelets. Mechanistically, genetic ablation of iPLA2γ markedly decreased the calcium-stimulated production of 2-AA-LPC, 2-AA-LPE, and 12-HETE-lysophospholipids in mouse platelets. Importantly, a potent and selective 12-LOX inhibitor, ML355, significantly inhibited the production of 12-HETE-LPC and 12-HETE-LPE in activated platelets. Furthermore, we found that aging is accompanied by significant changes in 12-HETE-LPC in murine serum that were also markedly attenuated by iPLA2γ genetic ablation. Collectively, these results identify previously unknown iPLA2γ-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-AA-LPC and 2-AA-LPE. This oxidation generates previously unrecognized eicosanoid-lysophospholipids that may serve as biomarkers for age-related diseases and could potentially be used as targets in therapeutic interventions.

Synergy between 15-lipoxygenase and secreted PLA 2 promotes inflammation by formation of TLR4 agonists from extracellular vesicles

“…Given that different lysoPLs are the source and that oxidized molecules are very unstable probably reduces the probability of detecting specific molecular species especially in the extracellular fluid. In support of our data Liu et al (27,28) detected eicosanoid-lysolipids formed by either COX-2 or 12-LO and iPLA 2 γ activity in murine hepatic tissue, murine and human myocardium, as well as in cells exposed to calcium ionophore. As increased concentrations of 12-HETE-LPC were detected in serum of old male mice they proposed them as specific biomarkers for various age-associated diseases (28).…”

“…The results underlie the differences in classical (pathogen-induced) and sterile inflammation. Eicosanoid-lysolipids, namely lysophospholipids (lysoPLs) with oxidized arachidonoyl acyl chain (e.g., HpETE-LPC, HETE-LPC) were first determined by Liu et al (27,28) in mice and human tissues and their role as possible signaling molecules was purposed, but neither their receptor nor signaling pathway had been identified. Herein we found that 15-LO oxidized 2-arachidonoyl-lysoPLs are more potent agonists for TLR4/MD-2 than oxidized PLs with two acyl chains.…”

Updating Phospholipase A2 Biology

“…Heart failure-induced activation of iPLA 2 γ leads to mitochondrial generation of HETEs that open the mitochondrial permeability transition pore, thus further amplifying myocardial damage [ 111 ]. Phospholipids bearing sn -2 AA are cleaved by the PLA 1 activity of iPLA 2 γ to give rise to 2-arachidonoyl-lysophospholipids, which are then oxygenized directly by cyclooxygenase-2 or 12-lipoxygenase for conversion to eicosanoid-esterified lysophospholipids [ 134 , 135 ]. The generation of eicosanoid-esterified lysophospholipids is attenuated by the absence of iPLA 2 γ, underscoring an iPLA 2 γ-initiated pathway generating new classes of lipid metabolites with potential signaling functions.…”

The Biosynthesis of Enzymatically Oxidized Lipids

“…The enzymatic activities of 12S-LOX are also proposed to function in the biosynthesis of hepoxilins and maresins (92,93). Recently, 12S-LOX was shown to oxygenate 2-AA-lysoPL, proposing alternate pathways for oxylipin and oxPL biosynthesis (94).…”

“…Recent studies found that COX-2, 15-LOX-2, and platelet 12S-LOX can catalyze the oxygenation of 2-AA-lysoPL released by iPLA 2 g in response to calcium ionophore stimulation (88,94). These reactions generate 2-eicosanoid-lysoPLs which are proposed to be a source of free eicosanoids as well as acting as signaling mediators themselves.…”