Lysophosphatidic Acid Receptor Research Articles

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.

Lipid phosphate phosphatase 3 in smooth muscle cells regulates angiotensin II-induced abdominal aortic aneurysm formation

Genetic variants that regulate lipid phosphate phosphatase 3 (LPP3) expression are risk factors for the development of atherosclerotic cardiovascular disease. LPP3 is dynamically upregulated in the context of vascular inflammation with particularly heightened expression in smooth muscle cells (SMC), however, the impact of LPP3 on vascular pathology is not fully understood. We investigated the role of LPP3 and lysophospholipid signaling in a well-defined model of pathologic aortic injury and observed Angiotensin II (Ang II) increases expression of PLPP3 in SMCs through nuclear factor kappa B (NF-κB) signaling Plpp3 global reduction (Plpp3+/−) or SMC-specific deletion (SM22-Δ) protects hyperlipidemic mice from AngII-mediated aneurysm formation. LPP3 expression regulates SMC differentiation state and lowering LPP3 levels promotes a fibroblast-like phenotype. Decreased inactivation of bioactive lysophosphatidic acid (LPA) in settings of LPP3 deficiency may underlie these phenotypes because deletion of LPA receptor 4 in mice promotes early aortic dilation and rupture in response to AngII. LPP3 expression and LPA signaling influence SMC and vessel wall responses that are important for aortic dissection and aneurysm formation. These findings could have important implications for therapeutics targeting LPA metabolism and signaling in ongoing clinical trials.

Activation of the ATX/LPA/LPARs axis induces a fibrotic response in skeletal muscle

Several common chronic diseases, muscular dystrophies (MDs), and aging lead to progressive fibrous connective tissue (fibrosis) accumulation in skeletal muscle. Cumulative past evidence points to the role of signaling lipids such as lysophosphatidic acid (LPA) and its receptors (LPARs) in different models of fibrosis. However, the potential contribution of these molecules to the fibrotic process in skeletal muscle has not been explored. Here, we show the expression of ATX/LPA/LPARs axis components in skeletal muscle, which suggests their potential relevance for the biology of this tissue. We investigated if the skeletal muscle responds to the stimulus of intramuscular (IM) LPA injections, finding an early induction of the pro-fibrotic factor connective tissue growth factor/Cellular Communication Network factor 2 (CCN2) and extracellular matrix (ECM) proteins. Also, we found that LPA induces an increase in the number of fibro/adipogenic progenitors (FAPs), which are the primary cellular source of myofibroblasts. These effects were for the most part prevented by the inhibitor Ki16425, which inhibits the LPA receptors LPA1 and LPA3, as well as in the LPA1-KO mice. We also evaluated the in vivo activation of extracellular signal-regulated kinases (ERK 1/2), AKT, c-Jun N-terminal kinase (JNK), and Yes-asocciated protein 1 (YAP) in response to LPA. Our results show that LPA induces ERK 1/2 phosphorylation in WT muscle, but not in LPA1-KO mice. Treatment with the ERK 1/2 inhibitor U0126 prevented the induction of fibronectin in response to LPA, suggesting that this pathway is involved in LPA-induced fibrosis. Altogether, these results demonstrate that ATX/LPA/LPARs constitute a pro-fibrotic axis and suggest a possible role in muscular diseases.

Targeting lysophosphatidic acid receptor with Ki16425 impedes T cell lymphoma progression through apoptosis induction, glycolysis inhibition, and activation of antitumor immune response.

Lysophosphatidic acid (LPA) is a small phospholipid that acts as an extracellular lipid mediator. It promotes cancer progression by altering a wide array of cellular processes, including apoptosis, survival, angiogenesis, invasion, and migration through binding with its cognate receptors. Intriguingly, our previous study showed that in vitro treatment of LPA induced survival of T lymphoma cells. Hence, the present investigation was designed to investigate the antitumor potential of Ki16425, an antagonist of LPA receptors, against T cell lymphoma. Our in vitro results showed inhibition of LPA-mediated survival and metabolic activity of T lymphoma cells by Ki16425. Further, in vivo experimental findings indicated the tumor retarding potential of Ki16425 against T cell lymphoma through apoptosis induction, glycolysis inhibition, and immunoactivation. The administration of Ki16425 triggered apoptosis by down-regulating the expression of Bcl2 and up-regulating p53, Bax, cleaved caspase-3, and Cyt c expression. Further, Ki16425 suppressed glycolytic activity with concomitantly decreased expression of GLUT3 and MCT1. Moreover, we also noticed an elevated level of NO and iNOS in tumor cells after Ki16425 administration which might also be responsible for apoptosis induction and suppressed glycolysis. Additionally, we observed an increased population of total leukocytes, lymphocytes, and monocytes along with increased thymocytes count and IL-2 and IFN-γ levels. Besides, we observed amelioration of tumor-induced kidney and liver damages by Ki16425. Taken together, this is the first study that demonstrates that LPA receptors could be potential future therapeutic targets for designing promising therapeutic strategies against T cell lymphoma.

Influence of the autotaxin-lysophosphatidic acid axis on cellular function and cytokine expression in different breast cancer cell lines

Previous studies provide high evidence that autotaxin (ATX)-lysophosphatidic acid (LPA) signaling through LPA receptors (LPAR) plays an important role in breast cancer initiation, progression, and invasion. However, its specific role in different breast cancer cell lines remains to be fully elucidated to offer improvements in targeted therapies. Within this study, we analyzed in vitro the effect of LPA 18:1 and the LPAR1, LPAR3 (and LPAR2) inhibitor Ki16425 on cellular functions of different human breast cancer cell lines (MDA-MB-231, MDA-MB-468, MCF-7, BT-474, SKBR-3) and the human breast epithelial cell line MCF-10A, as well as Interleukin 8 (IL-8), Interleukin 6 (IL-6) and tumor necrosis factor (TNF)-alpha cytokine secretion after LPA-incubation. ATX-LPA signaling showed a dose-dependent stimulatory effect especially on cellular functions of triple-negative and luminal A breast cancer cell lines. Ki16425 inhibited the LPA-induced stimulation of triple-negative breast cancer and luminal A cell lines in variable intensity depending on the functional assay, indicating the interplay of different LPAR in those assays. IL-8, IL-6 and TNF-alpha secretion was induced by LPA in MDA-MB-468 cells. This study provides further evidence about the role of the ATX-LPA axis in different breast cancer cell lines and might contribute to identify subtypes suitable for a future targeted therapy of the ATX-LPA axis.

Lysophosphatidic Acid Receptor 5 (LPA5) Knockout Ameliorates the Neuroinflammatory Response In Vivo and Modifies the Inflammatory and Metabolic Landscape of Primary Microglia In Vitro.

Systemic inflammation induces alterations in the finely tuned micromilieu of the brain that is continuously monitored by microglia. In the CNS, these changes include increased synthesis of the bioactive lipid lysophosphatidic acid (LPA), a ligand for the six members of the LPA receptor family (LPA1-6). In mouse and human microglia, LPA5 belongs to a set of receptors that cooperatively detect danger signals in the brain. Engagement of LPA5 by LPA polarizes microglia toward a pro-inflammatory phenotype. Therefore, we studied the consequences of global LPA5 knockout (-/-) on neuroinflammatory parameters in a mouse endotoxemia model and in primary microglia exposed to LPA in vitro. A single endotoxin injection (5 mg/kg body weight) resulted in lower circulating concentrations of TNFα and IL-1β and significantly reduced gene expression of IL-6 and CXCL2 in the brain of LPS-injected LPA5-/- mice. LPA5 deficiency improved sickness behavior and energy deficits produced by low-dose (1.4 mg LPS/kg body weight) chronic LPS treatment. LPA5-/- microglia secreted lower concentrations of pro-inflammatory cyto-/chemokines in response to LPA and showed higher maximal mitochondrial respiration under basal and LPA-activated conditions, further accompanied by lower lactate release, decreased NADPH and GSH synthesis, and inhibited NO production. Collectively, our data suggest that LPA5 promotes neuroinflammation by transmiting pro-inflammatory signals during endotoxemia through microglial activation induced by LPA.

Autotaxin/Lysophosphatidic Acid Axis: From Bone Biology to Bone Disorders.

Lysophosphatidic acid (LPA) is a natural bioactive phospholipid with pleiotropic activities affecting multiple tissues, including bone. LPA exerts its biological functions by binding to G-protein coupled LPA receptors (LPA1-6) to stimulate cell migration, proliferation, and survival. It is largely produced by autotaxin (ATX), a secreted enzyme with lysophospholipase D activity that converts lysophosphatidylcholine (LPC) into active LPA. Beyond its enzymatic activity, ATX serves as a docking molecule facilitating the efficient delivery of LPA to its specific cell surface receptors. Thus, LPA effects are the result of local production by ATX in a given tissue or cell type. As a consequence, the ATX/LPA axis should be considered as an entity to better understand their roles in physiology and pathophysiology and to propose novel therapeutic strategies. Herein, we provide not only an extensive overview of the relevance of the ATX/LPA axis in bone cell commitment and differentiation, skeletal development, and bone disorders, but also discuss new working hypotheses emerging from the interplay of ATX/LPA with well-established signaling pathways regulating bone mass.

Single-cell RNA sequencing identifies a migratory keratinocyte subpopulation expressing THBS1 in epidermal wound healing

SummaryKeratinocyte differentiation is an intricate process that is regulated by multiple mediators. Using cultured human keratinocytes, we found that lysophosphatidic acid (LPA) induced the differentiation of a previously unsuspected keratinocyte subpopulation expressing the extracellular matrix protein, thrombospondin-1 (THBS1). This action of LPA was mediated by the RHO/ROCK-SRF signaling downstream of LPA1 and LPA5 receptors and required ERK activity. Suppression of THBS1 in vitro suggested a migratory role of THBS1+ keratinocytes. Moreover, we analyzed publicly deposited single-cell RNA sequencing dataset and identified Thbs1-expressing keratinocytes in the mouse wound skin. Immunohistochemistry analysis revealed that Thbs1+ keratinocytes were apparently differentiated from basal keratinocytes upon wounding, subsequently polarized and migrated suprabasally toward the wound front, and eventually underwent terminal differentiation in the neo-epidermis. Importantly, inhibition of Erk activity suppressed Thbs1+ keratinocyte differentiation in wound healing. Based on these findings, we suggest that THBS1+ keratinocyte is a migratory keratinocyte subpopulation that facilitates epidermal wound healing.

LPA1 antagonist BMS-986020 changes collagen dynamics and exerts antifibrotic effects in vitro and in patients with idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a debilitating lung disease with limited treatment options. A phase 2 trial (NCT01766817) showed that twice-daily treatment with BMS-986020, a lysophosphatidic acid receptor 1 (LPA1) antagonist, significantly decreased the slope of forced vital capacity (FVC) decline over 26 weeks compared with placebo in patients with IPF. This analysis aimed to better understand the impact of LPA1 antagonism on extracellular matrix (ECM)-neoepitope biomarkers and lung function through a post hoc analysis of the phase 2 study, along with an in vitro fibrogenesis model.MethodsSerum levels of nine ECM-neoepitope biomarkers were measured in patients with IPF. The association of biomarkers with baseline and change from baseline FVC and quantitative lung fibrosis as measured with high-resolution computed tomography, and differences between treatment arms using linear mixed models, were assessed. The Scar-in-a-Jar in vitro fibrogenesis model was used to further elucidate the antifibrotic mechanism of BMS-986020.ResultsIn 140 patients with IPF, baseline ECM-neoepitope biomarker levels did not predict FVC progression but was significantly correlated with baseline FVC and lung fibrosis measurements. Most serum ECM-neoepitope biomarker levels were significantly reduced following BMS-986020 treatment compared with placebo, and several of the reductions correlated with FVC and/or lung fibrosis improvement. In the Scar-in-a-Jar in vitro model, BMS-986020 potently inhibited LPA1-induced fibrogenesis.ConclusionsBMS-986020 reduced serum ECM-neoepitope biomarkers, which were previously associated with IPF prognosis. In vitro, LPA promoted fibrogenesis, which was LPA1 dependent and inhibited by BMS-986020. Together these data elucidate a novel antifibrotic mechanism of action for pharmacological LPA1 blockade.Trial registration ClinicalTrials.gov identifier: NCT01766817; First posted: January 11, 2013; https://clinicaltrials.gov/ct2/show/NCT01766817.

Effects of lysophosphatidic acid (LPA) signaling via LPA receptors on cellular functions associated with ATP reduction in osteosarcoma cells treated with ethidium bromide.

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA1 to LPA6) exhibits a variety of malignant properties in cancer cells. Intracellular ATP depletion leads to the development of necrosis and apoptosis. The present study aimed to evaluate the effects of LPA receptor-mediated signaling on the regulation of cancer cell functions associated with ATP reduction. Long-term ethidium bromide (EtBr) treated (MG63-EtBr) cells were established from osteosarcoma MG-63 cells. The intracellular ATP levels of MG63-EtBr cells were significantly lower than that of MG-63 cells. LPAR2, LPAR3, LPAR4 and LPAR6 gene expressions were elevated in MG63-EtBr cells. The cell motile and invasive activities of MG63-EtBr cells were markedly higher than those of MG-63 cells. The cell motile activity of MG-63 cells was increased by LPA4 and LPA6 knockdowns. In cell survival assay, cells were treated with cisplatin (CDDP) every 24h for 3 days. The cell survival to CDDP of MG63-EtBr cells was lower than that of MG-63 cells. LPA2 knockdown decreased the cell survival to CDDP of MG-63 cells. The cell survival to CDDP of MG-63 cells was inhibited by (2S)-OMPT (LPA3 agonist). Moreover, the cell survival to CDDP of MG-63 cells was enhanced by LPA4 and LPA6 knockdowns. These results indicate that LPA signaling via LPA receptors is involved in the regulation of cellular functions associated with ATP reduction in MG-63 cells treated with EtBr.