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A functional role for eicosanoid-lysophospholipids in activating monocyte signaling.

ABSTRACT: Recently, eicosanoid-lysophospholipids were identified as novel metabolites generated from the direct cyclooxygenase- or lipoxygenase-catalyzed oxidation of 2-arachidonoyl-lysophospholipids produced from either phospholipase A₁-mediated hydrolysis of diacyl arachidonoyl-phospholipids or through the cytochrome c-catalyzed oxidative hydrolysis of the vinyl ether linkage of arachidonoyl-plasmalogens. Although the metabolic pathways generating eicosanoid-lysophospholipids have been increasingly appreciated, the signaling functions of eicosanoid-lysophospholipids remain largely unknown. Herein, we demonstrate that 2-12(S)-HETE-lysophospholipids as well as nonesterified 12(S)-HETE are potent lipid mediators that activate THP-1 human monocytic cells to generate tumor necrosis factor α (TNFα) and interleukin 8 (IL8). Remarkably, low nanomolar concentrations of 12(S)-HETE-lysophospholipids, but not other oxidized signaling lipids examined activated THP-1 cells resulting in the production of large amounts of TNFα. Moreover, TNFα release induced by 12(S)-HETE-lysophospholipids was inhibited by the TNFα converting enzyme inhibitor TAPI-0 indicating normal processing of TNFα in THP-1 cells stimulated with these agonists. Western blotting analyses revealed that 12(S)-HETE-lysophospholipids activated the phosphorylation of NFκB p65, suggesting activation of the canonical NFκB signaling pathway. Importantly, activation of THP-1 cells to release TNFα was stereoselective with 12(S)-HETE favored over 12(R)-HETE. Furthermore, the EC₅₀ of 2-12(S)-HETE-lysophosphatidylcholine in activating THP-1 cells was 2.1 nm, whereas the EC₅₀ of free 12(S)-HETE was 23 nm Additionally, lipid extracts of activated platelets were separated by RP-HPLC demonstrating the coelution of 12(S)-HETE with fractions initiating TNFα release. Collectively, these results demonstrate the potent signaling properties of 2-12(S)-HETE-lysophospholipids and 12(S)-HETE by their ability to release TNFα and activate NFκB signaling thereby revealing a previously unknown role of 2-12(S)-HETE-lysophospholipids in mediating inflammatory responses.

SUBMITTER: Liu GY

PROVIDER: S-EPMC7443508 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Synovial fluid (SF) from human knee joints with osteoarthritis (OA) has elevated levels of lysophosphatidylcholine (LPC) species, but their functional role is not well understood. This in vitro study was designed to test the hypothesis that various LPCs found elevated in OA SF and their metabolites, lysophosphatidic acids (LPAs), modulate the abundance of proteins and phospholipids (PLs) in human fibroblast-like synoviocytes (FLSs), with even minute chemical variations in lysophospholipids determining the extent of regulation. Cultured FLSs (n = 5-7) were treated with one of the LPC species, LPA species, IL-1β, or a vehicle. Tandem mass tag peptide labeling coupled with LC-MS/MS/MS was performed to quantify proteins. The expression of mRNA from regulated proteins was analyzed using RT-PCR. PL synthesis was determined via ESI-MS/MS, and the release of radiolabeled PLs was determined by means of liquid scintillation counting. In total, 3960 proteins were quantified using multiplexed MS, of which 119, 8, and 3 were significantly and reproducibly regulated by IL-1β, LPC 16:0, and LPC 18:0, respectively. LPC 16:0 significantly inhibited the release of PLs and the synthesis of phosphatidylcholine, LPC, and sphingomyelin. Neither LPC metabolite-LPA 16:0 nor LPA 18:0-had any reproducible effect on the levels of each protein. In conclusion, small chemical variations in LPC species can result in the significantly altered expression and secretion of proteins and PLs from FLSs. IL-1β influenced all proteins that were reproducibly regulated by LPC 16:0. LPC species are likely to modulate FLS protein expression only in more advanced OA stages with low IL-1β levels. None of the eight proteins being significantly regulated by LPC 16:0 have been previously reported in OA. However, our in vitro findings show that the CD81 antigen, calumenin, and B4E2C1 are promising candidates for further study, focusing in particular on their potential ability to modulate inflammatory and catabolic mechanisms.

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A functional role for eicosanoid-lysophospholipids in activating monocyte signaling.

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