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

• Published in: The Journal of biological chemistry Vol. 295; no. 16; pp. 5307 - 5320
• Main Authors: Liu, Xinping, Sims, Harold F., Jenkins, Christopher M., Guan, Shaoping, Dilthey, Beverly G., Gross, Richard W.
• Format: Journal Article
• Language: English
• Published: 11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110, U.S.A Elsevier Inc 17-04-2020; American Society for Biochemistry and Molecular Biology
• Subjects: 2-arachidonoyl-lysophospholipids; aging; calcium; eicosanoid; iPLA2γ; Lipids; lysophospholipid; myocardium; platelet; platelet-type 12-lipoxygenase (12-LOX); polyunsaturated fatty acids (PUFAs); eicosanoid; calcium; 2-arachidonoyl-lysophospholipids; polyunsaturated fatty acids (PUFAs); platelet-type 12-lipoxygenase (12-LOX); platelet; iPLA2γ; myocardium; aging; lysophospholipid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.012296

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.