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.
Highlights
We found that aging is accompanied by significant changes in 12(S)-hydroxy-eicosatetraenoic acid (12-HETE)-LPC in murine serum that were markedly attenuated by iPLA2␥ genetic ablation
Because calcium activates iPLA2␥ to produce 2-arachidonic acid (AA)-lysophospholipids from arachidonate-containing phospholipids [37], these results indicate that the production of 2–12–HETElysophospholipids in platelets is initiated by iPLA2␥–mediated PLA1 hydrolysis of AA-phospholipids to produce 2-AA-lysophospholipids that can be directly oxidized by 12-LOX to generate 2–12–HETE-lysophospholipids (Fig. 7)
We demonstrated that calcium ion induces the activation of iPLA2␥ leading to the production of 2-AA-LPC and 2-AA-LPE [37]
Summary
12-LOX, 12-lipoxygenase; iPLA2␥, calcium-independent phospholipase A2␥; cPLA2␣, cytosolic phospholipase A2␣; PPH3, triphenylphosphine; AA-LPC, arachidonoyl-lysophosphatidylcholine; AALPE, arachidonoyl-lysophosphatidylethanolamine; 12-HETE, 12-hydroxy-eicosatetraenoic acid; 12-HpETE, 12-hydroperoxy-eicosatetraenoic acid; ML355, N-2-benzothiaoly-4-[[(2-hydroxy-3-methoxyphenyl)methyl]amino]benzenesulfonamide; HRAM, high resolution high mass accuracy; CID, collision-induced dissociation; Ni-NTA, nickel-nitrilotriacetic acid; AA, arachidonic acid. The crystal structure of the 12-LOX catalytic domain possesses a U-shaped substrate-binding site that facilitates the precise positioning of the C10 hydrogens in arachidonic acid for the regioselective and stereospecific oxidization of AA resulting in the production of 12(S)-HpETE [32]. The regiospecificity of iPLA2␥ for the sn-1 position of polyunsaturated phospholipids suggested the existence of a previously unknown signaling pathway initiated by the generation of 2-arachidonoyl-lysophosphatidylcholine, which serves as a signaling node for multiple lipid 2nd messenger cascades [37, 38]. We demonstrated that aging in mice is accompanied by increased levels of 12-HETE-LPC in serum that are attenuated by genetic ablation of iPLA2␥ These results identify previously unknown iPLA2␥-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-arachidonoyl-lysophospholipids to generate previously unrecognized eicosanoid-lysophospholipid metabolites in critical cells of the cardiovascular system that could potentially serve as biomarkers of disease and inflammation during aging
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
