Lysophosphatidic Acid Receptor Research Articles

Abstract 2636: Targeting Autotaxin to suppress stromal signaling in the tumor microenvironment to improve outcome to therapy in fibrotic tumor types

Abstract Recent findings indicate that in cancer, host immune suppression and resistance to therapy is orchestrated by the presence and activity of stromal cells in the tumor microenvironment. Therefore novel approaches to overcome stromal mediated resistance to therapy represent a potential strategy to improve the outcome to chemotherapy and immune checkpoint blockade, particularly in tumors that have a fibrotic microenviroment. Autotaxin is an enzyme that converts Lysophosphatidylcholine (LPC) to Lysophosphatidic Acid (LPA), a key pathway that is aberrantly activated in fibrosis. LPA has also been described to act directly on tumor cells, promoting their growth and proliferation via G protein-coupled LPA Receptors (LPARs). IOA-289 is a novel, potent, selective and orally bioavailable inhibitor of Autotaxin, a target with known clinical application in fibrotic diseases and with a strong rationale for exploration in cancer. In preclinical models, IOA-289 modulated levels of circulating LPA in a dose dependent manner. At doses achieving at least 50% reduction of circulating LPA, IOA-289 showed monotherapeutic efficacy in in vivo mouse models of cancer. Notably the effects were most prominent in orthotopic models that recapitulate the stromal components of the microenvironment. In vitro studies investigated the mechanism of action of IOA-289, and it was shown to be driven by: (i)Direct anti-tumor activity on cancer cell lines in vitro (ii)Modulation of the secretory profile of fibroblasts(iii)Increased T cell infiltration in vitro and in vivo In a healthy volunteer study following a single oral ascending dose, IOA-289 showed a dose dependent increase in plasma and a corresponding decrease in circulating LPA. We also demonstrated that ATX was elevated in plasma from pancreatic cancer patients compared to samples from healthy volunteers and are correlated with soluble CA19-9. Based on our preclinical data we postulate that inhibition of the ATX/LPA pathway with IOA-289 may be a beneficial therapeutic strategy for cancer patients with fibrotic tumor microenvironments. The predicted biologically effective dose of IOA-289 has been calculated using PK/PD modelling combining data from preclinical studies and from the phase Ia healthy volunteer study. IOA-289 is scheduled to enter a phase Ib clinical trial in pancreatic cancer. Citation Format: Zoe Johnson, Marcel Deken, Ragini Medhi, Lauren Maggs, Alan Carruthers, Anne Cheasty, Alessia Tagliavini, Andrea Nizzardo, Marco Pergher, Karolina Niewola, Amy Fraser, Lars van der Veen, Pritom Shah, Luigi ZIviani, Stefano Milleri, Michael Lahn. Targeting Autotaxin to suppress stromal signaling in the tumor microenvironment to improve outcome to therapy in fibrotic tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2636.

Lysophosphatidic acid receptor type-1 mediates brain activation in micro-positron emission tomography analysis in a fibromyalgia-like mouse model.

The intermittent cold stress-induced generalized pain response mimics the pathophysiological and pharmacotherapeutic features reported for fibromyalgia patients, including the presence of chronic generalized pain and female dominance. In addition, the intermittent cold stress-induced generalized pain is abolished in lysophosphatidic acid receptor type-1 knockout mice, as reported in many cases of neuropathic pain models. This study aimed to identify the brain loci involved in the intermittent cold stress generalized pain response and test their dependence on the lysophosphatidic acid receptor type-1. Positron emission tomography analyses using 2-deoxy-2-[18 F]fluoro-d-glucose in the presence of a pain stimulus showed that intermittent cold stress causes a significant increase in uptake in the ipsilateral regions, including the salience networking-related anterior cingulate cortex and insular cortex and the cognition-related hippocampus. A significant decrease was observed in the default mode network-related posterior cingulate cortex. Almost these intermittent cold stress-induced changes were abolished in lysophosphatidic acid receptor type-1 knockout mice. There results suggest that the intermittent cold stress-induced generalized pain response is mediated by the lysophosphatidic acid receptor type-1 in specific brain loci related to salience networking and cognition, which may lead to further developments in the treatment of fibromyalgia.

Lysophosphatidic acid stimulates pericyte migration via LPA receptor 1

Lysophosphatidic acid (LPA) is a bioactive compound known to regulate various vascular functions. However, despite the fact that many vascular functions are regulated by peri-vascular cells such as pericytes, the effect of LPA on brain pericytes has not been fully evaluated. Thus, we designed this study to evaluate the effects of LPA on brain pericytes. These experiments revealed that while LPA receptors (LPARs) are expressed in cultured pericytes from mouse brains, LPA treatment does not influence the proliferation of these cells but does have a profound impact on their migration, which is regulated via the expression of LPAR1. LPAR1 expression was also detected in human pericyte culture and LPA treatment of these cells also induced migration. Taken together these findings imply that LPA-LPAR1 signaling is one of the key mechanisms modulating pericyte migration, which may help to control vascular function during development and repair processes.

Sesamol protects against liver fibrosis induced in rats by modulating lysophosphatidic acid receptor expression and TGF-β/Smad3 signaling pathway

The present study aimed to investigate the hepatoprotective effect of sesamol (SML), a nutritional phenolic compound obtained from sesame seeds, in liver fibrosis induced by thioacetamide (TAA) in rats and to explore the underlying mechanisms. Thirty-two male Sprague–Dawley rats were equally divided into four groups: control, TAA, TAA + SML 50 mg/kg, and TAA + SML 100 mg/kg groups. Liver functions and hepatic contents of glutathione (GSH) and malondialdehyde (MDA) were measured colorimetrically. Gene expressions of lysophosphatidic acid receptor (LPAR)-1 and -3, connective tissue growth factor (CTGF), transforming growth factor (TGF)-β1, small mothers against decapentaplegic (Smad)-3 and -7, α-smooth muscle actin (α-SMA), and cytokeratin 19 (CK19) were analyzed by qRT-PCR. Moreover, phosphorylated Smad3 (pSmad3) was quantified by ELISA. Additionally, TGF-β1, α-SMA, CK19, and vascular endothelial growth factor (VEGF) protein concentrations were semi-quantitatively analyzed by immunostaining of liver sections. SML treatment markedly improved liver index and liver functions. Moreover, SML protected against liver fibrosis in a dose-dependent manner as indicated by down-regulation of LPAR1, LPAR3, CTGF, TGF-β1/Smad3, and α-SMA expressions and a decrease in pSmad3 level, as well as an up-regulation of Smad7 expression. In addition, SML suppressed ductular reaction hinted by the decrease in CK19 expression. These results reveal the anti-fibrotic effect of SML against liver fibrosis that might be attributed to down-regulation of LPAR1/3 expressions, inhibition of TGF-β1/Smad3 pathway, and ductular reaction.

Lysophosphatidic acid supports the development of vitrified ovarian follicles by decreasing the incidence of cell death: An experimental study

BackgroundLysophosphatidic acid (LPA) contributes to follicular activation, oocyte maturation, in vitro fertilization, and embryo implantation.ObjectiveThis study was designed to evaluate the effects of LPA to improve the development of isolated follicles derived from whole mouse cultured vitrified ovaries.Materials and Methods In this experimental study, first, the 1-wk-old mouse ovaries in the non-vitrified and vitrified groups were cultured in the presence of 20 µM of LPA for 1 wk. Then, their isolated preantral follicles were cultured individually for 12 days in the presence or absence of 40 µM of LPA. The following evaluations were done for the cultured follicles: a viability test using Calcein AM staining, flow cytometry using annexin V/Pi, and analysis of the expression of genes by real-time reverse transcription polymerase chain reaction. The maturation rates of the oocytes were compared among groups and some of the released metaphase II oocytes were subjected to in vitro fertilization.ResultsIn all LPA treated groups, the rates of survival and follicular development were higher, and the incidence of cell death and expression of pro-apoptotic genes were lower, than in the non-LPA supplemented groups (p = 0.035). There was no significant difference between the vitrified and non-vitrified groups regarding follicular or oocyte development, but the expression of Bad and LPA receptors genes was significantly altered in the vitrified LPA supplemented group in comparison with the non-vitrified LPA supplemented group (p = 0.028).ConclusionLPA improved the survival and developmental potential of the isolated follicles. Despite some alterations in the expression of apoptosis-related genes in the vitrified ovaries, LPA had positive effects on the survival and development of these follicles.

Effects of a lysophosphatidic acid receptor 1 antagonist on hypertensive renal injury in Dahl-Iwai salt-sensitive rats

Lysophosphatidic acid (LPA) is a biologically active lysophospholipid, and acts on six types of LPA receptors (LPA1-LPA6). LPA-LPA1 signaling has been suggested as a therapeutic target for inflammatory and fibrotic disorders, including renal fibrosis.In this study, we investigated the effects of AM095, an LPA1 selective antagonist, on hypertensive renal injury in Dahl-Iwai salt-sensitive (DS) rats. We evaluated the preventive as well as therapeutic efficacy of AM095 in reducing proteinuria, and improving impaired renal function and renal fibrosis in the hypertensive DS rat.Preventive administration of AM095 suppressed proteinuria, renal function impairment and renal fibrosis in the hypertensive DS rats. In addition, therapeutic administration of AM095 reduced the levels of proximal tubular injury markers and suppressed renal fibrosis. Furthermore, combined administration of AM095 with an angiotensin-converting enzyme (ACE) inhibitor reduced the levels of proximal tubular injury markers and kidney weight increase, and suppressed renal fibrosis more effectively than administration of either agent alone, independent of the antihypertensive effect of the ACE inhibitor.These results provide the first evidence of the potential efficacy of LPA1 antagonist in suppressing renal injury in hypertensive DS rats, suggesting the promise of LPA1 antagonists as a novel therapeutic option for hypertensive renal injury.

LPAR2 receptor stimulates progression of gastric cancer through β‐catenin signaling pathway

IntroductionLysophosphatidic acid (LPA) is a simple lipid that has been implicated in cellular homeostasis and pathology of different cancer by binding with G‐protein coupled receptors (LPAR1‐6). The aberrant Wnt signaling pathway has been reported to facilitate a crucial role in tumorigenesis and therapy responses. Although dysregulated β‐catenin signaling pathway has been established as a critical mediator of gastric cancer, whether LPA and LPAR2 play a role in the β ‐catenin signaling pathway during gastric cancer progression remains elusive.ObjectiveOur present study explored an unknown role of LPA and LPAR2 in LPA‐induced activation of β‐catenin and its downstream signaling mechanism in β‐catenin mediated gastric cancer.MethodWe analyzed the mRNA levels of different LPA receptors in human gastric cancer by ULCAN database using TCGA dataset. LPA ELISA, Western blotting, and immunohistochemical analysis were done in the human normal stomach and cancerous stomach to measure the LPA level and check the expression of different LPA receptors. To investigate the functional role of LPAR2 in LPA‐mediated gastric cancer progression, we performed ECIS proliferation, migration assay, scratch assay, and transwell invasion assay in LPA or LPA with LPAR2 antagonist treated cells. To elucidate the molecular mechanism of LPA‐mediated gastric cancer progression, we performed Western blotting, Real‐time PCR, Luciferase assay, Immunocytochemistry, and nuclear fractionation in gastric cancer cells treated with LPA or LPA with LPAR2 antagonist.ResultWe confirmed a robust increase in LPA level and significantly increased expression of LPAR2 receptor in human gastric cancer tissue samples (P<0.001) by LPA ELISA, western blotting analysis, and immunohistochemistry, suggesting a possible role of LPA in gastric cancer through LPAR2 receptor. In cultured AGS and NCI‐N87 gastric cancer cell line, LPA treatment increased proliferation, migration, and invasion activity, whereas LPAR2 receptor antagonist abrogated the LPA induced effect. Further, when the cells were treated with LPA, it increased the β‐catenin activity, nuclear localization of β ‐catenin, and the expression of the downstream target genes of the β‐catenin signaling pathway. In addition, we confirmed LPA‐induced activation of the β‐catenin signaling pathway through the LPAR2 receptor by using LPAR2 antagonist and by knockdown of LPAR2 where LPAR2 antagonist treatment or LPAR2 knockdown rescued LPA induced activation of the β ‐catenin signaling pathway in both cell lines.Conclusionwe established a novel LPA‐LPAR2‐β‐catenin signaling pathway in the progression of gastric cancer. All our results support that LPAR2 is a potential marker and target for developing novel therapeutic strategies in the treatment of gastric cancer.

Novel GPR87/SDC‐1 Complex Modulates Lacritin Rescue of Homeostasis

IntroductionOcular tears refract 80% of light and are essential for epithelial homeostasis. 7% of the world’s population suffer from tear insufficiency associated with compromised visual acuity and chronic ocular surface inflammation. Lacritin, a prosecretory mitogenic tear and plasma glycoprotein, restores homeostasis in a mouse dry eye model, and in human corneal epithelial cells (HCET) subjected to dry eye‐like IFNG and TNF stress. It does so by transiently accelerating epithelial autophagy (FOXO1/ATG7; FOXO3/ATG101) thereby restoring oxidative phosphorylation, as validated in a recent 204 patient phase 2 clinical trial of human Sjögren's Syndrome Dry Eye. Lacritin also promotes basal tearing by increasing the sensitivity of ion channel TRPM8 on corneal sensory nerves to tear drying. Here we report the identification of G protein‐coupled receptor 87 (GPR87) as the lacritin signaling receptor out of a genome‐wide CRISPR/Cas9 death screen using the C‐terminal lacritin synthetic peptide N‐94 as probe. GPR87 is expressed in the cornea and has a critical role in cellular proliferation and survival. Years ago, GPR87 was thought to be an LPA receptor, later disproven by Arfelt et al, ′17. Finally, we also report that GPR87 interacts with syndecan‐1 (SDC1), a cell surface co‐receptor for lacritin. We observed a direct binding of recombinant lacritin to GPR87, corroborating with the hypothesis that GPR87 is a critical receptor for lacritin homeostatic signaling.MethodsValidation studies of GPR87 were performed by generation of GPR87 knockdown cell lines by shRNA depletion by transducing HCE‐T cells with lentiviral GPR87 shRNA and non‐target controls. Depletion of GPR87 protein level was confirmed by western blot. Complementation assays were performed by transducing depleted HCE‐T’s with lentiviral GPR87 mouse cDNA. Lacritin affinity binding to GPR87 was first studied out of recombinant lacritin‐ or lacritin C‐25‐intein pulldowns. Truncation mutant ‘C‐25’, lacks lacritin's ‘N‐94’ bioactive domain. Lacritin pulldowns were challenged using each of eleven synthetic peptides corresponding to or together spanning all eight extracellular or intracellular strands or loops of GPR87 ‐ each at increasing concentrations (20, 100, 400 µM). To explore whether GPR87 can couple with SDC1, we blotted for GPR87 out of SDC‐1 pulldowns from HEK2936E cells co‐transfected with wild type or mutated GPR87 and SDC1 cDNA’s.ResultsLacritin, but not C‐25, binds GPR87 via GPR87's outer three loops with outer loop 3 peptide most inhibitory. GPR87 complexes with SDC1 in the absence of N‐94, but not with SDC1 lacking the PDZ binding domain nor with GPR87 lacking the G‐protein binding site “DRY” motif. SDC‐1 lacking N‐terminal domain heparan sulfate at S15 or membrane proximal chondroitin sulfate at S184 and S185 failed to bind GPR87. SDC‐1 also failed to complex with GPR87 in cells treated with chlorate to suppress sulfation.ConclusionsLacritin appears to bind directly to the outer loop domains of GPR87 as part of a preformed SDC1‐GPR87 complex ‐ the latter linked in part by glycosaminoglycans and apparently by cytoplasmic SDC1 and GPR87 domains to regulate ocular surface health.

Lipid phosphate phosphatase‐3 dysfunction exaggerates neointimal hyperplasia following diabetic vascular injury

IntroductionDiabetes mellitus is a major risk factor for cardiovascular diseases and promotes neointimal hyperplasia characterized by occlusive disorders such as atherosclerosis and stenosis, caused by excessive proliferation and migration of vascular smooth muscle cells (VSMCs) following endovascular interventions such as balloon angioplasty and stent placement. Lipid phosphate phosphatase‐3 (LPP3) is an enzyme that regulates the availability of lysophosphatidic acid (LPA) and has been reported to play a significant role in vascular genesis, cellular permeability, tumor progression, vascular remodeling, and myocardial infarction. However, the functional role and mechanism of LPP3 in diabetic neointimal hyperplasia is unclear. LPP3 dysfunction may lead to LPA accumulation at the vascular injury site, leading to vascular inflammation, proliferation, and migration, causing restenosis. Recently, the Receptor for Advanced Glycation End product (RAGE) has been presented as a potential receptor for LPA leading to downstream signaling in vascular smooth muscle cells (VSMCs).MethodsIn this study, we used a streptozocin‐induced diabetes mice model. We made SM‐22 specific LPP3 knock‐out mice and performed endothelial denudation surgery in the carotid artery to induce neointima hyperplasia to tease out the role of LPP3 in restenosis. We isolated aortic VSMCs from LPP3 floxed and LPP3 knock‐out mice and cultured them under normal or high glucose media. The immunoblotting was performed and probed with antibodies for total extracellular signal‐regulated kinase (ERK) and phosphorylated‐ERK. The cell proliferation and migration assay was performed using the Electric Cell substrate Impedance Sensing (ECIS) with or without LPP3 in VSMC under normal or high glucose conditions. Data are mean ± SEM; P‐values were determined by Student t‐test or one‐way ANOVA with Tukey's post‐hoc tests.ResultWe observed a significant increase in proliferation (P<0.01) and exaggerated migration (p<0.01) of VSMC that lack LPP3 under the hyperglycemic condition in real‐time using ECIS. These effects are at least partially a consequence of decreased lysophosphatidic acid hydrolysis due to lack of LPP3 activity. Furthermore, VSMC that lack LPP3 showed increased phospho‐ERK expression (P<0.01) under hyperglycemic conditions. The diabetic LPP3 knock‐out mice exhibited exaggerated neo‐intimal areas after the carotid injury compared to LPP3 floxed mice.ConclusionThese findings implicate that LPP3 plays a vital role as a negative regulator of smooth muscle phenotypic modulation. We speculate that changes in LPP3 expression control the development of neo‐intima by limiting smooth muscle cell proliferation, migration, and vascular inflammation in diabetic restenosis.

Activation of Lysophosphatidic Acid Receptor 3 Promotes Mitochondrial Homeostasis in aged cells

Activation of Lysophosphatidic Acid Receptor 3 Promotes Mitochondrial Homeostasis in aged cells

Cerebral Ischemic Reperfusion Increases Autotaxin Expression Elevating Brain Endothelial Permeability

IntroductionIschemic reperfusion during stroke treatment increases the pathological progression of stroke. Reperfusion with r‐TPA and mechanical thrombectomy applied to stroke patients increases the risk of intracerebral hemorrhage. In addition, various molecular signals initiated during ischemic reperfusion exacerbate the disease due to increased endothelial permeability. However, a detailed molecular therapeutic regimen with r‐TPA and mechanical thrombectomy could limit the detrimental effects and increase the survival rate. Lysophosphatidic acid (LPA) is a bioactive phospholipid regulated in physiological and pathological conditions. LPA is produced by the enzyme autotaxin (ATX). ATX and LPA have been observed to deteriorate physiological status in various diseases such as myocardial infarction, Alzheimer’s disease, liver fibrosis, neuropathic pain etc. In cerebral ischemic‐reperfusion, the ATX‐LPA axis could initiate the endothelium disruption.HypothesisIncreased ATX expression in endothelium disrupts the endothelial barrier by increasing LPA during ischemic‐reperfusion.MethodsTransient middle cerebral artery occlusion with 90 minutes of ischemia and 24 hours of reperfusion (I/R) was used as a stroke model in mice. LPA was analyzed with mass spectrometry and immunohistochemistry. AR‐2 probe fluorescence assay was used to measure ATX activity. Evans Blue Permeability in vivo was measured with an infrared imager. For the in‐vitro study, oxygen and glucose deprivation and reperfusion (OGDR) were carried out in mouse brain microvascular endothelial cells (MBMEC).ResultsATX enzymatic activity is prominently raised (P<0.01) in I/R mice compared to sham mice. Increased ATX activity coheres with the Evans Blue permeability in the mice brain. Simultaneously LPA levels are elevated (P<0.01) in I/R mice. LPA was increased in brain tissue along the vasculature, indicating an unfavorable role of ATX in maintaining endothelial integrity. LPA receptors expression was regulated following stroke with upregulation of LPAR1, LPAR2, LPAR5, and LPAR6 following I/R. Permeability measured in MBMEC showed that LPA treatment increased the permeability in a time and concentration‐dependent manner. ATX expression was increased (P<0.05) in MBMEC with OGDR suggesting ATX regulation in endothelial cells following ischemic‐reperfusion. LPA produced during the process can disrupt the blood‐brain barrier, increasing cerebral permeability.ConclusionAfter a stroke, enhanced LPA production by ATX can disrupt the blood‐brain barrier, leading to brain edema and neural cell damage followed by irreversible brain injury. Inhibition of the ATX‐LPA axis prospects to be a better therapeutic avenue for cerebral ischemic reperfusion.

Roles for CCN proteins in Lysophosphatidic Acid and Sphingosine 1‐Phosphate Signaling in Prostate Cancer Cells

IntroductionCysteine rich angiogenic factor (CCN1/Cyr61) and connective tissue growth factor (CCN2/CTGF) are members of the CCN family of matricellular proteins. These secreted proteins interact with extracellular matrix proteins and integrins to modulate cell signaling. CCN proteins have been implicated in survival and other mitogenic responses in various cancers. CCN1 and CCN2 are the two early inducible genes in this family. Lysophosphatidic acid (LPA) and sphingosine‐1‐phosphate (S1P) are lipid growth factors known to contribute to tumor progression in prostate cancer. In previous work, our group showed that LPA stimulates proliferation and migration in prostate cancer cells via the G protein‐coupled receptor LPAR1. Free fatty acid receptor 4 (FFAR4) agonists, inhibit LPA‐induced responses in prostate cancer cells. We also demonstrated that CCN1 expression is induced by LPA in human prostate cancer cells. However, the roles of CCN1 and CCN2 in cancer cell signaling have not been fully elucidated.HypothesisWe hypothesize that CCN proteins contribute to LPA‐ and S1P‐induced adhesion and mitogenic signaling in prostate cancer cells.Methods and ResultsPC‐3 human prostate cancer cells were used in the current study. For all experiments, cells were serum starved for 24 hours and then treated with and without 10 µM LPA or S1P. Immunoblotting showed that CCN1 and CCN2 were induced by LPA after 2‐5 hours in PC‐3 cells. The response was inhibited by the LPA receptor antagonist Ki1625. TUG‐891, an FFAR4 agonist, inhibited LPA‐induced CCN1 expression in PC‐3 cells. S1P also induced CCN1 expression in PC‐3 cells. In related experiments, whole cell extracts, and extracellular matrix were subjected to immunoblotting for CCN1, and immunofluorescence microscopy was used to localize the induced CCN1. CCN1 protein levels in extracellular matrix were increased 2‐5 hours after LPA treatment, as compared to untreated controls, and CCN1 was detected outside the cells by confocal microscopy. In cell adhesion assays, LPA‐treated PC‐3 cells exhibited an increase in adhesion to fibronectin‐coated 96‐well plates that was detected by 2 hours. Erk MAPK activation in response to LPA was biphasic, with the later phase occurring after CCN1/2 induction.ConclusionThe results of this study suggest that CCN proteins may play roles in LPA‐ and S1P‐induced signaling in prostate cancer cells and are thus potential therapeutic targets.

Rho5 regulates lysophosphatidic acid induced macropinocytosis in Entamoeba histolytica

Entamoeba histolytica is a unicellular enteric protozoan responsible for variety of gastrointestinal pathologies. The pathogenicity of the parasite is attributed to its dynamic cytoskeleton. Rho family GTPases, a major class of molecular switches, play an indispensable role in controlling cytoskeleton remodelling. Rho GTPases are known to be activated by specific Guanine nucleotide Exchange Factors (GEF) upon stimulation by various extracellular stimuli to regulate signalling.We have established that EhRho5 is important for LPA induced micropinocytosis in the amoebic trophozoites. EhRho5 translocated from cytosol to plasma membrane and endomembranous compartments when stimulated with LPA, a bioactive phospholipid known to activate Rho subfamily members (1,2). Our findings were strengthened when FRAP (Fluorescence recovery after photobleaching) study showed that LPA treated cells show 30% faster recovery of Rho5 when compared to serum starved cells. The translocation is accompanied by activation of the GTPase as revealed by effector pull down assay(2). LPA stimulation enhanced the macropinocytic efficiency of trophozoites in a Rho5 dependent manner. We then identified the GEF for Rho5 which too, shows translocation upon stimulation with LPA. Also, depletion of the GEF leads to decreased pinocytic efficiency of the trophozoites. Using inhibitor based studies, we further demonstrated that PI3K acts as an intermediate cue for EhRho5 activation during LPA induced macropinocytosis.Moreoever, Rho5 expression showed contrary relationship with random migration of the trophozoites suggesting a pivotal role of the GTPase in mutally antagonistic cellular processes(3) in the amoeba. Reference: Fleming IN, Elliott CM, Exton JH. Differential Translocation of Rho Family GTPases by Lysophosphatidic Acid, Endothelin‐1, and Platelet‐derived Growth Factor, J. Biol. Chem. 1996, 271: 33067–33073 Van Leeuwen FN, Olivo C, Grivell S, Giepmans BNG, Collard JG, Moolenaar WH. Rac Activation by Lysophosphatidic Acid LPA1 Receptors through the Guanine Nucleotide Exchange Factor Tiam1, J. Biol. Chem. 2003, 278:400–406 Veltman DM, Lemieux MG, Knecht DA, Insall RH. PIP3‐dependent macropinocytosis is incompatible with chemotaxis. Journal of Cell Biology. 2014 Feb 17;204(4):497‐505.

Construction of endogenous RNA regulatory network for colorectal cancer based on bioinformatics.

The high morbidity and mortality of colorectal cancer (CRC) have posed great threats to human health. Circular RNA (circRNA) and microRNA (miRNA), acting as competing endogenous RNAs (ceRNAs), have been found to play vital roles in carcinogenesis. This paper aims to construct a circRNA/miRNA/mRNA regulatory network so as to explore the molecular mechanism of CRC. The sequencing data of circRNA from CRC were obtained from Gene Expression Omnibus (GEO). The differential circRNA was screened and its structure was identified by Cancer-specific CircRNA Database (CSCD); the sequencing data of miRNA and messenger RNA (mRNAs) were downloaded from The Cancer Genome Atlas (TCGA) database and the differentially expressed genes were screened; the corresponding miRNA of differential circRNAs were predicted by CircInteractome database; DIANA, Miranda, PicTar, and TargetScan databases were used to predict the target genes of different miRNAs; the target genes from Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were enriched by R language; String database combined with Cytoscape 3.7.2 software was used to construct protein-protein interaction (PPI) network and hub genes were screened; the expressions of mRNAs in the Top10 hub genes were verified in CRC. The network diagrams of circRNAs/miRNAs/mRNAs and circRNAs/miRNAs/Top10 hub mRNAs were constructed by Cytoscape3.7.2. Real-time PCR was used to examine the expression levels of hsa_circRNA_0065173, hsa-mir-450b, hsa-mir-582, adenylate cyclase 5 (ADCY5), muscarinic acetylcholine receptor M2 (CHRM2), cannabinoid receptor 1 (CNR1), and lysophosphatidic acid receptor 1 (LPAR1) in the CRC tissues and the adjacent normal tissues. A total of 14 differential circRNAs were identified, and 8 were found in CSCD; 34 miRNAs targeted by circRNAs were obtained. The PPI network was constructed, and the Top10 hub genes were identified, which were CHRM2, melanin concentrating hormone receptor 2 (MCHR2), G-protein gamma 3 subunit (GNG3), neuropeptide Y receptor Y1 (NPY1R), CNR1, LPAR1, ADCY5, adenylate cyclase 2 (ADCY2), gamma 7 (GNG7) and chemokine 12 (CXCL12), respectively. The expressions of Top 10 hub genes were also verified, and the results showed that the Top 10 hub genes were down-regulated in CRC; the constructed network diagram showed that hsa_circRNA_0065173 may regulate ADCY5, CHRM2, and Hsa-mir-450b by modulating hsa-mir-450b and hsa-mir-582. CNR1 and LPAR1 genes might serve as potentially relevant targets for the treatment of CRC. Real-time PCR results showed that the expression levels of hsa_circRNA_0065173, ADCY5, CHRM2, CNR1 and LPAR1 in the CRC tissues were significantly reduced compared with the adjacent normal tissues (all P<0.05); the expression levels of hsa-mir-450b and hsa-miR-582 were significantly increased (both P<0.05). In this study, a potential circRNAs/miRNAs/mRNAs network is successfully constructed, which provides a new insight for CRC development mechanism through ceRNA mediated by circRNAs.

An Update on Sphingosine-1-Phosphate and Lysophosphatidic Acid Receptor Transcripts in Rodent Olfactory Mucosa.

Olfactory neurons connect the external environment and the brain, allowing the translocation of materials from the nasal cavity into the brain. The olfactory system is involved in SARS-CoV-2 infections; early in the pandemic declared in 2020, a loss of the sense of smell was found in many infected patients. Attention has also been focused on the role that the olfactory epithelium appears to play in the entry of the SARS-CoV-2 virus into the brain. Specifically, SARS-CoV-2 enters cells via the angiotensin-converting enzyme 2 protein (ACE2), which is found on supporting cells in the olfactory epithelium. The intranasal administration of sphingosine has been proposed to prevent the binding of SARS-CoV-2 to ACE2. Further, sphingosine-1-phosphate (S1P) receptors appear to facilitate the entry of SARS-CoV-2 into the brain. The goal of these studies was to characterize S1P receptor expression status in rodent olfactory mucosa. The expression of receptors for a related sphingolipid, lysophosphatidic acid (LPA), was also assessed. The results confirm previous reports of S1P1 and S1P3 receptor expression, as well as LPA receptor 1, in mouse olfactory mucosa; moreover, they extend the previous findings to identify additional S1P and LPA receptor transcripts in rat and mouse olfactory mucosa, as well as in cultured olfactory neurons. These findings may enhance the utility of rodent models in identifying agonists and/or antagonists of S1P and LPA receptors that may block the entry of SARS-CoV-2 and other viruses into nasal epithelial cells, and prevent transmission from the nasal cavity into the brain.

Association between the Expression Levels of MicroRNA-101, -103, and -29a with Autotaxin and Lysophosphatidic Acid Receptor 2 Expression in Gastric Cancer Patients

Background Gastric cancer (GC) is regarded as the most prevalent malignancy with the high mortality rate, worldwide. However, gastroscopy, a biopsy of suspected sample, and detecting CEA, CA19-9, and CA72-4 are presently used, but these diagnostic approaches have several limitations. Recently, microRNAs as the most important member of noncoding RNAs (ncRNAs) have received attention; recent evidence demonstrates that they can be used as the promising candidate biomarkers for GC diagnosis. We aimed to investigate the association between the microRNA-29a, -101, and -103 expression and autotaxin (ATX) and lysophosphatidic acid receptor 2 (LPA2) expression in GC patients. Material and Methods. The present study was conducted on 40 paired samples of primary GC tissue and adjacent noncancerous tissue. The gene expression levels of miR-101, -103, -29, ATX, and LPA2 were analyzed by quantitative reverse-transcription PCR (qRT-PCR). Besides, the protein levels of ATX and LPA2 were evaluated using western blot. Results The expression levels of miR-29 and miR-101 were significantly lower (p value < 0.0001), but the miR-103 and LPA2 were significantly higher in gastric tumor samples compared to the corresponding nontumor tissues (p value < 0.0001). Moreover, the diagnostic accuracy of miRs to discrimine the GC patients from noncancerous controls was reliable (miR-101, sensitivity: 82.5% and specificity: 85%; miR-103, sensitivity: 72.5% and specificity: 90%; miR-29, sensitivity: 77.5% and specificity: 70%). Conclusion It seems that determining the expression level of miR-101, -103, and -29, as the novel diagnostic biomarkers, has diagnostic value to distinguish GC patients from healthy individuals.

Glucagon-like Peptide-1 Secretion Is Inhibited by Lysophosphatidic Acid.

Glucagon-like peptide-1 (GLP-1) potentiates glucose-stimulated insulin secretion (GSIS). While dozens of compounds stimulate GLP-1 secretion, few inhibit. Reduced GLP-1 secretion and impaired GSIS occur in chronic inflammation. Lysophosphatidic acids (LPAs) are bioactive phospholipids elevated in inflammation. The aim of this study was to test whether LPA inhibits GLP-1 secretion in vitro and in vivo. GLUTag L-cells were treated with various LPA species, with or without LPA receptor (LPAR) antagonists, and media GLP-1 levels, cellular cyclic AMP and calcium ion concentrations, and DPP4 activity levels were analyzed. Mice were injected with LPA, with or without LPAR antagonists, and serum GLP-1 and DPP4 activity were measured. GLUTag GLP-1 secretion was decreased ~70–90% by various LPAs. GLUTag expression of Lpar1, 2, and 3 was orders of magnitude higher than Lpar4, 5, and 6, implicating the former group in this effect. In agreement, inhibition of GLP-1 secretion was reversed by the LPAR1/3 antagonist Ki16425, the LPAR1 antagonists AM095 and AM966, or the LPAR2 antagonist LPA2-antagonist 1. We hypothesized involvement of Gαi-mediated LPAR activity, and found that intracellular cyclic AMP and calcium ion concentrations were decreased by LPA, but restored by Ki16425. Mouse LPA injection caused an ~50% fall in circulating GLP-1, although only LPAR1 or LPAR1/3 antagonists, but not LPAR2 antagonism, prevented this. GLUTag L-cell and mouse serum DPP4 activity was unchanged by LPA or LPAR antagonists. LPA therefore impairs GLP-1 secretion in vitro and in vivo through Gαi-coupled LPAR1/3 signaling, providing a new mechanism linking inflammation with impaired GSIS.

The ATX-LPA Axis Regulates Vascular Permeability during Cerebral Ischemic-Reperfusion.

Endothelial permeability is a major complication that must be addressed during stroke treatment. Study of the mechanisms underlying blood–brain barrier (BBB) disruption and management of the hypoxic stress-induced permeability of the endothelium following reperfusion are both urgently needed for stroke management. Lysophosphatidic acid (LPA), a bioactive lipid essential for basic cellular functions, causes unfavorable outcomes during stroke progression. LPA-producing enzyme autotaxin (ATX) is regulated in ischemic stroke. We used an electrical cell-substrate impedance sensor (ECIS) to measure endothelial permeability. Mitochondrial bioenergetics were obtained using a Seahorse analyzer. AR-2 probe fluorescence assay was used to measure ATX activity. LPA increased endothelial permeability and reduced junctional protein expression in mouse brain microvascular endothelial cells (MBMEC). LPA receptor inhibitors Ki16425 and AM095 attenuated the LPA-induced changes in the endothelial permeability and junctional proteins. LPA significantly diminished mitochondrial function in MBMEC. ATX was upregulated (p < 0.05) in brain microvascular endothelial cells under hypoxic reperfusion. ATX activity and permeability were attenuated with the use of an ATX inhibitor in a mouse stroke model. The upregulation of ATX with hypoxic reperfusion leads to LPA production in brain endothelial cells favoring permeability. Inhibition of the ATX–LPA–LPAR axis could be therapeutically targeted in stroke to achieve better outcomes.

Lysophosphatidic Acid Receptor 3 Suppress Neutrophil Extracellular Traps Production and Thrombosis During Sepsis.

Sepsis consists of life-threatening organ dysfunction resulting from a dysregulated response to infection. Recent studies have found that excessive neutrophil extracellular traps (NETs) contribute to the pathogenesis of sepsis, thereby increasing morbidity and mortality. Lysophosphatidic acid (LPA) is a small glycerophospholipid molecule that exerts multiple functions by binding to its receptors. Although LPA has been functionally identified to induce NETs, whether and how LPA receptors, especially lysophosphatidic acid receptor 3 (LPA3), play a role in the development of sepsis has never been explored. A comprehensive understanding of the impact of LPA3 on sepsis is essential for the development of medical therapy. After intraperitoneal injection of lipopolysaccharide (LPS), Lpar3 -/-mice showed a substantially higher mortality, more severe injury, and more fibrinogen content in the lungs than wild-type (WT) mice. The values of blood coagulation markers, plasma prothrombin time (PT) and fibrinogen (FIB), indicated that the Lpar3 -/- mice underwent a severe coagulation process, which resulted in increased thrombosis. The levels of NETs in Lpar3 -/- mice were higher than those in WT mice after LPS injection. The mortality rate and degree of lung damage in Lpar3 -/- mice with sepsis were significantly reduced after the destruction of NETs by DNaseI treatment. Furthermore, in vitro experiments with co-cultured monocytes and neutrophils demonstrated that monocytes from Lpar3 -/- mice promoted the formation of NETs, suggesting that LPA3 acting on monocytes inhibits the formation of NETs and plays a protective role in sepsis. Mechanistically, we found that the amount of CD14, an LPS co-receptor, expressed by monocytes in Lpar3 -/-mice was significantly elevated after LPS administration, and the MyD88-p65-NFκB signaling axis, downstream of toll-like receptor 4 signaling, in monocytes was overactivated. Finally, after an injection of the LPA3 agonist (2S)-1-oleoyl-2-methylglycero-3-phosphothionate (OMPT), the survival rate of mice with sepsis was improved, organ damage was reduced, and the production of NETs was decreased. This suggested the possible translational value and application prospects of (2S)-OMPT in the treatment of sepsis. Our study confirms an important protective role of LPA3 in curbing the development of sepsis by suppressing NETs production and thrombosis and provides new ideas for sepsis treatment strategies.

Autotaxin signaling facilitates β cell dedifferentiation and dysfunction induced by Sirtuin 3 deficiency

Objectiveβ cell dedifferentiation may underlie the reversible reduction in pancreatic β cell mass and function in type 2 diabetes (T2D). We previously reported that β cell-specific Sirt3 knockout (Sirt3f/f;Cre/+) mice developed impaired glucose tolerance and glucose-stimulated insulin secretion after feeding with high fat diet (HFD). RNA sequencing showed that Sirt3-deficient islets had enhanced expression of Enpp2 (Autotaxin, or ATX), a secreted lysophospholipase which produces lysophosphatidic acid (LPA). Here, we hypothesized that activation of the ATX/LPA pathway contributed to pancreatic β cell dedifferentiation in Sirt3-deficient β cells. MethodsWe applied LPA, or lysophosphatidylcoline (LPC), the substrate of ATX for producing LPA, to MIN6 cell line and mouse islets with altered Sirt3 expression to investigate the effect of LPA on β cell dedifferentiation and its underlying mechanisms. To examine the pathological effects of ATX/LPA pathway, we injected the β cell selective adeno-associated virus (AAV-Atx-shRNA) or negative control AAV-scramble in Sirt3f/f and Sirt3f/f;Cre/+ mice followed by 6-week of HFD feeding. ResultsIn Sirt3f/f;Cre/+ mouse islets and Sirt3 knockdown MIN6 cells, ATX upregulation led to increased LPC with increased production of LPA. The latter not only induced reversible dedifferentiation in MIN6 cells and mouse islets, but also reduced glucose-stimulated insulin secretion from islets. In MIN6 cells, LPA induced phosphorylation of JNK/p38 MAPK which was accompanied by β cell dedifferentiation. The latter was suppressed by inhibitors of LPA receptor, JNK, and p38 MAPK. Importantly, inhibiting ATX in vivo improved insulin secretion and reduced β cell dedifferentiation in HFD-fed Sirt3f/f;Cre/+ mice. ConclusionsSirt3 prevents β cell dedifferentiation by inhibiting ATX expression and upregulation of LPA. These findings support a long-range signaling effect of Sirt3 which modulates the ATX-LPA pathway to reverse β cell dysfunction associated with glucolipotoxicity.