LysoPS Receptor Research Articles

Understanding the modulations of glycero-lysophospholipids in an elastase-induced murine emphysema model

BackgroundIncreasing evidence indicates that bioactive lipid mediators are involved in chronic obstructive pulmonary disease (COPD) pathogenesis. Recently, glycero-lysophospholipids, such as lysophosphatidic acid (LysoPA) and lysophosphatidylserine (LysoPS), have been recognized as significant inflammation-related lipid mediators. However, their association with COPD remains unclear. MethodsWe used an elastase-induced murine emphysema model to analyze the levels of lysophospholipids and diacyl-phospholipids in the lungs. Additionally, we assessed the expression of LysoPS-related genes and published data on smokers. ResultsIn the early phase of an elastase-induced murine emphysema model, the levels of LysoPS and its precursor (phosphatidylserine [PS]) were significantly reduced, without significant modulations in other glycero-lysophospholipids. Additionally, there was an upregulation in the expression of lysoPS receptors, specifically GPR34, observed in the lungs of a cigarette smoke-exposed mouse model and the alveolar macrophages of human smokers. Elastase stimulation induces GPR34 expression in a human macrophage cell line in vitro. ConclusionsElastase-induced lung emphysema affects the LysoPS/PS-GPR34 axis, and cigarette smoking or elastase upregulates GPR34 expression in alveolar macrophages. This novel association may serve as a potential pharmacological target for COPD treatment.

Structural basis for ligand recognition and signaling of the lysophosphatidylserine receptors GPR34 and GPR174.

Lysophosphatidylserine (LysoPS) is a naturally occurring lipid mediator involved in various physiological and pathological processes especially those related to the immune system. GPR34, GPR174, and P2Y10 have been identified as the receptors for LysoPS, and its analogues have been developed as agonists or antagonists for these receptors. However, the lack of structural information hinders the drug development with novel characteristics, such as nonlipid ligands and allosteric modulators. Here, we determined the structures of human GPR34 and GPR174 in complex with LysoPS and G protein by cryo-EM. Combined with structural analysis and functional studies, we elucidated the lipid-binding modes of these receptors. By structural comparison, we identified the structural features of GPR34 and GPR174 in active state. Taken together, our findings provide insights into ligand recognition and signaling of LysoPS receptors and will facilitate the development of novel therapeutics for related inflammatory diseases and autoimmune diseases.

Phospholipase A1 Member A Activates Fibroblast-like Synoviocytes through the Autotaxin-Lysophosphatidic Acid Receptor Axis.

Lysophosphatidylserine (lysoPS) is known to regulate immune cell functions. Phospholipase A1 member A (PLA1A) can generate this bioactive lipid through hydrolysis of sn-1 fatty acids on phosphatidylserine (PS). PLA1A has been associated with cancer metastasis, asthma, as well as acute coronary syndrome. However, the functions of PLA1A in the development of systemic autoimmune rheumatic diseases remain elusive. To investigate the possible implication of PLA1A during rheumatic diseases, we monitored PLA1A in synovial fluids from patients with rheumatoid arthritis and plasma of early-diagnosed arthritis (EA) patients and clinically stable systemic lupus erythematosus (SLE) patients. We used human primary fibroblast-like synoviocytes (FLSs) to evaluate the PLA1A-induced biological responses. Our results highlighted that the plasma concentrations of PLA1A in EA and SLE patients were elevated compared to healthy donors. High concentrations of PLA1A were also detected in synovial fluids from rheumatoid arthritis patients compared to those from osteoarthritis (OA) and gout patients. The origin of PLA1A in FLSs and the arthritic joints remained unknown, as healthy human primary FLSs does not express the PLA1A transcript. Besides, the addition of recombinant PLA1A stimulated cultured human primary FLSs to secrete IL-8. Preincubation with heparin, autotaxin (ATX) inhibitor HA130 or lysophosphatidic acid (LPA) receptor antagonist Ki16425 reduced PLA1A-induced-secretion of IL-8. Our data suggested that FLS-associated PLA1A cleaves membrane-exposed PS into lysoPS, which is subsequently converted to LPA by ATX. Since primary FLSs do not express any lysoPS receptors, the data suggested PLA1A-mediated pro-inflammatory responses through the ATX-LPA receptor signaling axis.

GPR34-mediated sensing of lysophosphatidylserine released by apoptotic neutrophils activates type 3 innate lymphoid cells to mediate tissue repair

Neutrophils migrate rapidly to damaged tissue and play critical roles in host defense and tissue homeostasis. Here we investigated the mechanisms whereby neutrophils participate in tissue repair. In an intestinal epithelia injury model, neutrophil depletion exacerbated colitis and associated with reduced interleukin (IL)-22 and limited activation of type 3 innate lymphoid cells (ILC3s). Co-culture with neutrophils activated ILC3s in a manner dependent on neutrophil apoptosis. Metabolomic analyses revealed that lysophosphatidylserine (LysoPS) from apoptotic neutrophils directly stimulated ILC3 activation. ILC3-specific deletion of Gpr34, encoding the LysoPS receptor GPR34, or inhibition of downstream PI3K-AKT or ERK suppressed IL-22 production in response to apoptotic neutrophils. Gpr34-/- mice exhibited compromised ILC3 activation and tissue repair during colon injury, and neutrophil depletion abrogated these defects. GPR34 deficiency in ILC3s limited IL-22 production and tissue repair invivo in settings of colon and skin injury. Thus, GPR34 is an ILC3-expressed damage-sensing receptor that triggers tissue repair upon recognition of dying neutrophils.

The Lysophosphatidylserines-An Emerging Class of Signalling Lysophospholipids.

Lysophospholipids are potent hormone-like signalling biological lipids that regulate many important biological processes in mammals (including humans). Lysophosphatidic acid and sphingosine-1-phosphate represent the best studied examples for this lipid class, and their metabolic enzymes and/or cognate receptors are currently under clinical investigation for treatment ofvarious neurological and autoimmune diseases in humans. Over the past two decades, the lysophsophatidylserines (lyso-PSs) have emerged as yet another biologically important lysophospholipid, and deregulation in its metabolism has been linked to various human pathophysiological conditions. Despite its recent emergence, an exhaustive review summarizing recent advances on lyso-PSs and the biological pathways that this bioactive lysophospholipid regulates has been lacking. To address this, here, we summarize studies that led to the discovery of lyso-PS as a potent signalling biomolecule, and discuss the structure, its detection in biological systems, and the biodistribution of this lysophospholipid in various mammalian systems. Further, we describe in detail the enzymatic pathways that are involved in the biosynthesis and degradation of this lipid and the putative lyso-PS receptors reported in the literature. Finally, we discuss the various biological pathways directly regulated by lyso-PSs in mammals and prospect new questions for this still emerging biomedically important signalling lysophospholipid.

Non-naturally Occurring Regio Isomer of Lysophosphatidylserine Exhibits Potent Agonistic Activity toward G Protein-Coupled Receptors.

Lysophosphatidylserine (LysoPS), an endogenous ligand of G protein-coupled receptors, consists of l-serine, glycerol, and fatty acid moieties connected by phosphodiester and ester linkages, respectively. An ester linkage of phosphatidylserine can be hydrolyzed at the 1-position or at the 2-position to give 2-acyl lysophospholipid or 1-acyl lysophospholipid, respectively. 2-Acyl lysophospholipid is in nonenzymatic equilibrium with 1-acyl lysophospholipid in vivo. On the other hand, 3-acyl lysophospholipid is not found, at least in mammals, raising the question of whether the reason for this might be that the 3-acyl isomer lacks the biological activities of the other isomers. Here, to test this idea, we designed and synthesized a series of new 3-acyl lysophospholipids. Structure-activity relationship studies of more than 100 "glycol surrogate" derivatives led to the identification of potent and selective agonists for LysoPS receptors GPR34 and P2Y10. Thus, the non-natural 3-acyl compounds are indeed active and appear to be biologically orthogonal with respect to the physiologically relevant 1- and 2-acyl lysophospholipids.

Lysophosphatidylserine induces eosinophil extracellular trap formation and degranulation: Implications in severe asthma.

Recent evidence demonstrates that activated eosinophils undergo a distinct form of lytic cell death, accompanied by formation of DNA-based eosinophil extracellular trap (EET) and degranulation, enhancing inflammatory immune responses in asthmatic airways. We previously showed that human blood eosinophils undergo degranulation in response to lysophosphatidylserine (LysoPS), an inflammatory lipid mediator, and strongly express P2Y10, a LysoPS receptor. We evaluated EET, degranulation, and cell death of eosinophils in response to various concentrations of LysoPS. We also compared responsiveness to LysoPS between eosinophils from severe and nonsevere asthmatics. Extensive EET formation was elicited from a substantial fraction of stimulated eosinophils in response to 50μmol/L LysoPS. Analyses for LDH and eosinophil-derived neurotoxin release showed that both lytic cell death and degranulation accompanied EET formation in response to LysoPS. Cytological analyses demonstrated that citrullinated histone 3 was present in the extracellular, filamentous DNA structure embedded with eosinophil granules. The LysoPS-induced EET was independent of ROS production and irrelevant to several signaling pathways examined, but dependent on protein arginine deiminase 4. A low concentration of LysoPS (5μmol/L) did not induce EET or degranulation, but significantly increased platelet-activating factor-induced degranulation. Eosinophils from severe asthmatics exhibited greater degranulation, but not EET formation, in response to LysoPS (50μmol/L), than those from nonsevere asthmatics, along with great expression of surface P2Y10. We identified a novel function of LysoPS, namely induction of EET in association with cytolysis and degranulation. LysoPS-dependent EET or degranulation plays a potential role in eosinophilic inflammation of severe asthma.

Possible involvement of PS-PLA1 and lysophosphatidylserine receptor (LPS1) in hepatocellular carcinoma

Lysophosphatidylserine (LysoPS) is a lysophospholipid, its generating enzyme, phosphatidylserine-specific phospholipase A1 (PS-PLA1), reportedly plays roles in stomach and colon cancers. Here, we examined the potential roles of LysoPS in hepatocellular carcinoma (HCC). The ninety-seven HCC patients who underwent surgical treatment were enrolled in this study and approved by the institutional review board. Among LysoPS-related enzymes and receptors, increased PS-PLA1 or LysoPS receptor 1 (LPS1) mRNA was observed in HCC tissues compared to non-HCC tissues. PS-PLA1 mRNA in HCC was associated with no clinical parameters, while LPS1 mRNA in HCC was correlated inversely with tumor differentiation. Furthermore, higher serum PS-PLA1 was observed in HCC patients compared to healthy control and correlated with PS-PLA1 mRNA in non-HCC tissues and with serum AST or ALT. Additionally, serum levels of PS-PLA1 were higher in HCC patients with HCV-related liver injury than in those with HBV or non-HBV-, non-HCV-related liver diseases. In conclusion, among LysoPS-related enzymes and receptors, PS-PLA1 and LPS1 mRNA were increased in HCC. Based on the correlation between the serum PS-PLA1 and the mRNA level of PS-PLA1 in non-HCC tissues, the liver may be the main source of serum PS-PLA1, and serum PS-PLA1 levels may be a useful marker for liver injury.

Lysophosphatidylserine receptor P2Y10: A G protein-coupled receptor that mediates eosinophil degranulation.

P2Y10, along with GPR34 and GPR174, is a G protein-coupled receptor that is activated by an endogenous lipid mediator lysophosphatidylserine (LysoPS). Its expression pattern and its function are completely unknown. We have previously shown that P2Y10 is one of the highly up-regulated genes at the late differentiation stage during invitro eosinophilopoiesis. We explored the expression and functions of P2Y10 in human cord blood (CB)-derived and peripheral blood (PB) eosinophils. Real-time PCR, FACS, Western blot, ELISA, and chemotaxis assays were performed to determine the expression and function of P2Y10. As CB cells differentiated towards eosinophils, P2Y10 mRNA and protein were abundantly expressed. P2Y10 was the most highly expressed in the granulocytes from PB, to a lesser extent in monocytes, and least in lymphocytes. Further fractionation of granulocytes revealed that eosinophils express P2Y10 much more strongly than do neutrophils. PB eosinophils solely expressed P2Y10 among the three LysoPS receptors, while PB neutrophils expressed the three at comparable levels. LysoPS activated both CB and PB eosinophils to induce a robust ERK phosphorylation. Importantly, LysoPS was capable of triggering degranulation of ECP in PB eosinophils. This response was significantly reduced by pharmacological inhibitors of TNF-alpha-converting enzyme (TACE), epidermal growth factor receptor (EGFR), and ERK1/2, which were known to be required in P2Y10-mediated signalling pathways. However, LysoPS had no effect on chemotaxis, differentiation, or eosinophil survival. LysoPS provokes eosinophil degranulation through P2Y10. Therefore, P2Y10 is a potential therapeutic target to control eosinophil-associated diseases.

Probing the Hydrophobic Binding Pocket of G-Protein-Coupled Lysophosphatidylserine Receptor GPR34/LPS1 by Docking-Aided Structure-Activity Analysis.

The ligands of certain G-protein-coupled receptors (GPCRs) have been identified as endogenous lipids, such as lysophosphatidylserine (LysoPS). Here, we analyzed the molecular basis of the structure-activity relationship of ligands of GPR34, one of the LysoPS receptor subtypes, focusing on recognition of the long-chain fatty acid moiety by the hydrophobic pocket. By introducing benzene ring(s) into the fatty acid moiety of 2-deoxy-LysoPS, we explored the binding site's preference for the hydrophobic shape. A tribenzene-containing fatty acid surrogate with modifications of the terminal aromatic moiety showed potent agonistic activity toward GPR34. Computational docking of these derivatives with a homology modeling/molecular dynamics-based virtual binding site of GPR34 indicated that a kink in the benzene-based lipid surrogates matches the L-shaped hydrophobic pocket of GPR34. A tetrabenzene-based lipid analogue bearing a bulky tert-butyl group at the 4-position of the terminal benzene ring exhibited potent GPR34 agonistic activity, validating the present hydrophobic binding pocket model.

Structure-activity relationships of lysophosphatidylserine analogs as agonists of G-protein-coupled receptors GPR34, P2Y10, and GPR174.

Lysophosphatidylserine (LysoPS) is an endogenous lipid mediator generated by hydrolysis of membrane phospholipid phosphatidylserine. Recent ligand screening of orphan G-protein-coupled receptors (GPCRs) identified two LysoPS-specific human GPCRs, namely, P2Y10 (LPS2) and GPR174 (LPS3), which, together with previously reported GPR34 (LPS1), comprise a LysoPS receptor family. Herein, we examined the structure-activity relationships of a series of synthetic LysoPS analogues toward these recently deorphanized LysoPS receptors, based on the idea that LysoPS can be regarded as consisting of distinct modules (fatty acid, glycerol, and l-serine) connected by phosphodiester and ester linkages. Starting from the endogenous ligand (1-oleoyl-LysoPS, 1), we optimized the structure of each module and the ester linkage. Accordingly, we identified some structural requirements of each module for potency and for receptor subtype selectivity. Further assembly of individually structure-optimized modules yielded a series of potent and LysoPS receptor subtype-selective agonists, particularly for P2Y10 and GPR174.

Lysophosphatidylserine has Bilateral Effects on Macrophages in the Pathogenesis of Atherosclerosis.

Lysophospholipids, particularly sphingosine 1-phosphate and lysophosphatidic acid, are known to be involved in the pathogenesis of atherosclerosis; however, the role of lysophosphatidylserine (LysoPS) in the onset of atherosclerotic diseases remains uncertain. We investigated the effects of LysoPS on the uptake of oxidized low-density lipoprotein (oxLDL) and the modulation of inflammatory mediators and ER stress utilizing RAW264.7 cells and mouse peritoneal macrophages (MPMs). We found that LysoPS augmented cholesterol accumulation in both models. Consistent with these findings, LysoPS increased the expression of scavenger receptors (CD36, MSR1, LOX1 and TLR4). Regarding the involvement of these lipids in inflammation, LysoPS significantly decreased the expression of inflammatory mediators in lipopolysaccharide (LPS)-treated RAW264.7 cells and MPMs. LysoPS also attenuated ER stress in LPS-untreated RAW264.7 cells. The expression patterns of LysoPS receptors differed considerably among the LPS-untreated RAW264.7 cells, LPS-treated RAW264.7 cells and MPMs. LysoPS may have proatherosclerotic properties in the setting of foam cell formation as well as antiatherosclerotic effects on inflammation in macrophages.

Lysophosphatidylserine analogues differentially activate three LysoPS receptors.

Lysophosphatidylserine (1-oleoyl-2 R-lysophosphatidylserine, LysoPS) has been shown to have lipid mediator-like actions such as stimulation of mast cell degranulation and suppression of T lymphocyte proliferation, although the mechanisms of LysoPS actions have been elusive. Recently, three G protein-coupled receptors (LPS1/GPR34, LPS2/P2Y10 and LPS3/GPR174) were found to react specifically with LysoPS, raising the possibility that LysoPS serves as a lipid mediator that exerts its role through these receptors. Previously, we chemically synthesized a number of LysoPS analogues and evaluated them as agonists for mast-cell degranulation. Here, we used a transforming growth factor-α (TGFα) shedding assay to see if these LysoPS analogues activated the three LysoPS receptors. Modification of the serine moiety significantly reduced the ability of the analogues to activate the three LysoPS receptors, whereas modification of other parts resulted in loss of activity in receptor-specific manner. We found that introduction of methyl group to serine moiety (1-oleoyl-lysophosphatidylallothreonine) and removal of sn-2 hydroxyl group (1-oleoyl-2-deoxy-LysoPS) resulted in reduction of reactivity with LPS1 and LPS3, respectively. Accordingly, we synthesized a LysoPS analogue with the two modifications (1-oleoyl-2-deoxy-lysophosphatidylallothreonine) and found it to be an LPS2-selective agonist. These pharmacological tools will definitely help to identify the biological roles of these LysoPS receptors.

GPR34 as a lysophosphatidylserine receptor

GPR34, a P2Y receptor family member, was identified as a candidate lysophosphatidylserine (LysoPS) receptor in 2006. However, it was controversial whether LysoPS is a real ligand for GPR34. Kitamura et al. (GPR34 is a receptor for lysophosphatidylserine with a fatty acid at the sn-2 position. J. Biochem. 2012;151:511-518), using several methods for detecting activation of G protein-coupled receptor (GPCR) and chemically synthesized LysoPS analogues, concluded that GPR34 is a cellular receptor for LysoPS, especially with a fatty acid at the sn-2 position. Furthermore, three additional GPCRs belonging to the P2Y family were recently identified as GPCRs for LysoPS, supporting the idea that GPR34 is a LysoPS receptor.

Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP

We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell–cell adhesion with localization of actin filaments to cell membrane, and revealed significant delay of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of Gαs protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with Gαs, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.

Synthesis and Evaluation of Lysophosphatidylserine Analogues as Inducers of Mast Cell Degranulation. Potent Activities of Lysophosphatidylthreonine and Its 2-Deoxy Derivative

In response to various exogenous stimuli, mast cells (MCs) release a wide variety of inflammatory mediators stored in their cytoplasmic granules and this release initiates subsequent allergic reactions. Lysophosphatidylserine (lysoPS) has been known as an exogenous inducer to potentiate histamine release from MCs, though even at submicromolar concentrations. In this study, through SAR studies on lysoPS against MC degranulation, we identified lysoPT, a threonine-containing lysophospholipid and its 2-deoxy derivative as novel strong agonists. LysoPT and its 2-deoxy derivative induced histamine release from MCs both in vitro and in vivo at a concentration less than one-tenth that of lysoPS. Notably, lysoPT did not activate a recently proposed lysoPS receptor on MCs, GPR34, demonstrating the presence of another undefined receptor reactive to both lysoPS and lysoPT that is involved in MC degranulation. Thus, the present strong agonists, lysoPT and its 2-deoxy derivative, will be useful tools to understand the mechanisms of lysoPS-induced activation of degranulation of MCs.

Identification of a lysophosphatidylserine receptor on mast cells

Lysophosphatidyl- l-serine (lysoPS) is thought to be an immunological regulator because it dramatically augments the degranulation of rat peritoneal mast cells (RPMCs). This stimulatory effect may be mediated by a lysoPS receptor, but its molecule has not been identified yet. During a ligand fishing study for the orphan G-protein-coupled receptor 34 (GPR34), we found that lysoPS caused a dose-dependent inhibition of forskolin-stimulated cAMP accumulation in human GPR34-expressing Chinese hamster ovary (CHO/hGPR34) cells. The CHO/hGPR34 cells were unresponsive to other structurally related phospholipids examined. Quantitative real-time-PCR demonstrated that mRNAs of GPR34 are particularly abundant in mast cells. The effective lysoPS concentration for RPMC degranulation was similar to that required for GPR34 activation, and the structural requirement of lysoPS for RPMC degranulation was in good agreement with that observed in CHO/hGPR34 cells. These results suggest that GPR34 is the functional mast cell lysoPS receptor.

Identification of alterations in gene expression in rat recipients with long-term–surviving cardiac grafts

Various human tissues and cells express phospholipase A1 member A (PLA1A), including the liver, lung, prostate gland, and immune cells. The enzyme belongs to the pancreatic lipase family. PLA1A specifically hydrolyzes sn-1 fatty acid of phosphatidylserine (PS) or 1-acyl-lysophosphatidylserine (1-acyl-lysoPS). PS externalized by activated cells or apoptotic cells or extracellular vesicles is a potential source of substrate for the production of unsaturated lysoPS species by PLA1A. Maturation and functions of many immune cells, such as T cells, dendritic cells, macrophages, and mast cells, can be regulated by PLA1A and lysoPS. Several lysoPS receptors, including GPR34, GPR174 and P2Y10, have been identified. High serum levels and high PLA1A expression are associated with autoimmune disorders such as Graves' disease and systemic lupus erythematosus. Increased expression of PLA1A is associated with metastatic melanomas. PLA1A may contribute to cardiometabolic disorders through mediating cholesterol transportation and producing lysoPS. Furthermore, PLA1A is necessary for hepatitis C virus assembly and can play a role in the antivirus innate immune response. This review summarizes recent findings on PLA1A expression, lysoPS and lysoPS receptors in autoimmune disorders, cancers, cardiometabolic disorders, antivirus immune responses, as well as regulations of immune cells.