Lysophosphatidic acid improves development of porcine somatic cell nuclear transfer embryos.

This study was conducted to investigate whether lysophosphatidic acid (LPA) could improve the development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine SCNT-derived embryos were cultured in chemically defined polyvinyl alcohol (PVA)-based porcine zygote medium (PZM)-4 without or with LPA, and the development, cell proliferation potential, apoptosis, and expression levels of pluripotent markers were evaluated. LPA significantly increased the rates of cleavage and blastocyst formation compared to those seen in the LPA un-treatment (control) group. The expression levels of embryonic development-related genes (IGF2R, PCNA and CDH1) were higher (p < 0.05) in the LPA treatment group than in the control group. LPA significantly increased the numbers of total, inner cell mass and EdU (5-ethynyl-2'-deoxyuridine)-positive cells in porcine SCNT blastocysts compared to those seen in the control group. TUNEL assay showed that LPA significantly reduced the apoptosis rate in porcine SCNT-derived embryos; this was confirmed by decreases (p < 0.05) in the expression levels of pro-apoptotic genes, BAX and CASP3, and an increase (p < 0.05) in the expression level of the anti-apoptotic gene, BCL2L1. In addition, LPA significantly increased Oct4 expression at the gene and protein levels. Together, our data suggest that LPA improves the quality and development of porcine SCNT-derived embryos by reducing apoptosis and enhancing cell proliferation and pluripotency.

The role of lysophosphatidic acid acyltransferase 1 in reproductive growth of Arabidopsis thaliana.

Lysophosphatidic acid acyltransferase1 (LPAT1) catalyzes the second step of de novo glycerolipid biosynthesis in chloroplasts. However, the embryonic-lethal phenotype of the knockout mutant suggested an unknown role for LPAT1 in non-photosynthetic reproductive organs. Reciprocal genetic crossing of the lpat1-1 heterozygous line suggested a female gametophytic defect of the lpat1-1 knockout mutant. By suppressing LPAT1 specifically during seed development, we showed that LPAT1 suppression affected silique growth and seed production. Glycerolipid analysis of the LPAT1 knockdown lines revealed a pronounced decrease of phosphatidylcholine (PC) content in mature siliques along with an altered polyunsaturation level of the polar glycerolipids. In seeds, the acyl composition of triacylglycerol (TAG) was altered albeit not the content. These results indicate that plastidic LPAT1 plays an important role in reproductive growth and extraplastidic glycerolipid metabolism involving PC and TAG.

LPA suppresses HLA-DR expression in human melanoma cells: a potential immune escape mechanism involving LPAR1 and DR6-mediated release of IL-10.

While immune checkpoint inhibitors (ICIs) are promising in the treatment of metastatic melanoma, about half of patients do not respond well to them. Low levels of human leukocyte antigen-DR (HLA-DR) in tumors have been shown to negatively influence prognosis and response to ICIs. Lysophosphatidic acid (LPA) is produced in large amounts by melanoma and is abundantly present in the tumor microenvironment. LPA induces the release of various cytokines and chemokines from tumor cells, which affect cancer development, metastasis, and tumor immunity. In the present study, we investigated the role of LPA-induced IL-10 release in regulating HLA-DR expression and the underlying mechanisms in human melanoma cells. We showed that LPA (0.001-10 µM) dose-dependently increased DR6 transcript levels through activating LPAR1 in HEK293T cells. Knockdown of NF-κB1 abrogated the LPA-increased DR6 expression without affecting basal DR6 expression in both A2058 and A375 melanoma cell lines. LPA (10 µM) significantly increased IL-10 transcripts in A2058 and A375 melanoma cells, the effect was abolished by pharmacological inhibition of LPAR1 or knockdown of DR6. We found a statistically significant correlation between the expression of LPAR1, DR6 and IL-10 in human melanoma tissue and an association between increased expression of LPAR1 and reduced effectiveness of ICI therapy. We demonstrated that LPA (10 µM) markedly suppressed HLA-DR expression in both A375 and A2058 melanoma cells via activating the LPAR1-DR6-IL-10 pathway. These data suggest that the LPAR1-DR6-IL-10 autocrine loop could constitute a novel mechanism used by tumor cells to evade immunosurveillance by decreasing HLA-DR expression.

Lysophospholipase D activity on oral mucosa cells in whole mixed human saliva involves in production of bioactive lysophosphatidic acid from lysophosphatidylcholine.

We reported that lysophosphatidic acid (LPA) is present at 0.8 µM in mixed human saliva (MS). In this study, we examined the distribution, origin, and enzymatic generation pathways of LPA in MS. LPA was distributed in the medium and cell pellet fraction; a true level of soluble LPA in MS was about 150 nM. The soluble LPA was assumed to be generated by ecto-type lysophospholipase D on exfoliated cells in MS from LPC that originated mainly from the major salivary gland saliva. Our results with the albumin-back extraction procedures suggest that a significant pool of LPA is kept in the outer layer of the plasma membranes of detached oral mucosal cells. Such pool of LPA may contribute to wound healing in upper digestive organs including oral cavity. We obtained evidence that the choline-producing activity in MS was mainly due to Ca2+-activated lysophospholipase D activity of glycerophosphodiesterase 7.

From Classical to Alternative Pathways of 2-Arachidonoylglycerol Synthesis: AlterAGs at the Crossroad of Endocannabinoid and Lysophospholipid Signaling.

2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid (EC), acting as a full agonist at both CB1 and CB2 cannabinoid receptors. It is synthesized on demand in postsynaptic membranes through the sequential action of phosphoinositide-specific phospholipase Cß1 (PLCß1) and diacylglycerol lipase α (DAGLα), contributing to retrograde signaling upon interaction with presynaptic CB1. However, 2-AG production might also involve various combinations of PLC and DAGL isoforms, as well as additional intracellular pathways implying other enzymes and substrates. Three other alternative pathways of 2-AG synthesis rest on the extracellular cleavage of 2-arachidonoyl-lysophospholipids by three different hydrolases: glycerophosphodiesterase 3 (GDE3), lipid phosphate phosphatases (LPPs), and two members of ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP6-7). We propose the names of AlterAG-1, -2, and -3 for three pathways sharing an ectocellular localization, allowing them to convert extracellular lysophospholipid mediators into 2-AG, thus inducing typical signaling switches between various G-protein-coupled receptors (GPCRs). This implies the critical importance of the regioisomerism of both lysophospholipid (LPLs) and 2-AG, which is the object of deep analysis within this review. The precise functional roles of AlterAGs are still poorly understood and will require gene invalidation approaches, knowing that both 2-AG and its related lysophospholipids are involved in numerous aspects of physiology and pathology, including cancer, inflammation, immune defenses, obesity, bone development, neurodegeneration, or psychiatric disorders.

Lysophosphatidic Acid Receptors LPAR5 and LPAR2 Inversely Control Hydroxychloroquine-Evoked Itch and Scratching in Mice.

Lysophosphatidic acids (LPAs) evoke nociception and itch in mice and humans. In this study, we assessed the signaling paths. Hydroxychloroquine was injected intradermally to evoke itch in mice, which evoked an increase of LPAs in the skin and in the thalamus, suggesting that peripheral and central LPA receptors (LPARs) were involved in HCQ-evoked pruriception. To unravel the signaling paths, we assessed the localization of candidate genes and itching behavior in knockout models addressing LPAR5, LPAR2, autotaxin/ENPP2 and the lysophospholipid phosphatases, as well as the plasticity-related genes Prg1/LPPR4 and Prg2/LPPR3. LacZ reporter studies and RNAscope revealed LPAR5 in neurons of the dorsal root ganglia (DRGs) and in skin keratinocytes, LPAR2 in cortical and thalamic neurons, and Prg1 in neuronal structures of the dorsal horn, thalamus and SSC. HCQ-evoked scratching behavior was reduced in sensory neuron-specific Advillin-LPAR5-/- mice (peripheral) but increased in LPAR2-/- and Prg1-/- mice (central), and it was not affected by deficiency of glial autotaxin (GFAP-ENPP2-/-) or Prg2 (PRG2-/-). Heat and mechanical nociception were not affected by any of the genotypes. The behavior suggested that HCQ-mediated itch involves the activation of peripheral LPAR5, which was supported by reduced itch upon treatment with an LPAR5 antagonist and autotaxin inhibitor. Further, HCQ-evoked calcium fluxes were reduced in primary sensory neurons of Advillin-LPAR5-/- mice. The results suggest that LPA-mediated itch is primarily mediated via peripheral LPAR5, suggesting that a topical LPAR5 blocker might suppress "non-histaminergic" itch.

Mice deficient in the phosphatase activity of sEH show decreased levels of the endocannabinoid 2-AG in the olfactory bulb and depressive-like behavior.

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has epoxide hydrolase activity and phosphatase activity. Our earlier study revealed that lysophosphatidic acids are a substrate of the phosphatase activity of sEH in vitro, but its physiological function remained unknown. Herein, we used the CRISPR/Cas9 system and i-GONAD method to generate mice that are deficient in sEH phosphatase activity. In the mouse brain, sEH was highly expressed in the olfactory bulb. Deletion of the sEH phosphatase activity resulted in decreased levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG), which is a dephosphorylated form of 2-arachidonoyl-lysophosphatidic acid in the olfactory bulb. The sEH-deficient mice showed depressive-like behavior. These results indicate that sEH can regulate the production of 2-AG and brain function in vivo.

Autotaxin-Lysophosphatidate Axis: Promoter of Cancer Development and Possible Therapeutic Implications.

Autotaxin (ATX) is a member of the ectonucleotide pyrophosphate/phosphodiesterase (ENPP) family; it is encoded by the ENPP2 gene. ATX is a secreted glycoprotein and catalyzes the hydrolysis of lysophosphatidylcholine to lysophosphatidic acid (LPA). LPA is responsible for the transduction of various signal pathways through the interaction with at least six G protein-coupled receptors, LPA Receptors 1 to 6 (LPAR1-6). The ATX-LPA axis is involved in various physiological and pathological processes, such as angiogenesis, embryonic development, inflammation, fibrosis, and obesity. However, significant research also reported its connection to carcinogenesis, immune escape, metastasis, tumor microenvironment, cancer stem cells, and therapeutic resistance. Moreover, several studies suggested ATX and LPA as relevant biomarkers and/or therapeutic targets. In this review of the literature, we aimed to deepen knowledge about the role of the ATX-LPA axis as a promoter of cancer development, progression and invasion, and therapeutic resistance. Finally, we explored its potential application as a prognostic/predictive biomarker and therapeutic target for tumor treatment.

Cell type-specific and subcellular expression of phospholipid phosphatase-related proteins to modulate lyso-phosphatidic acid synaptic signaling in the developing and adult CNS.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that participates in critical processes in neural development and adult brain function and is implicated in various pathophysiological conditions. Along with its six well-characterized receptors, atypical regulators of LPA signaling have also been suggested, including phospholipid phosphatase-related proteins (PLPPRs). PLPPRs have been mostly studied in the developing brain where they control LPA-dependent axon guidance, cortical network hyperexcitability, and glutamatergic neurotransmission. PLPPR4 and PLPPR3 represent two closely related proteins reported to localize predominantly in dendrites and axons, respectively, and differ in their developmental expression patterns. Herein, we have revised the expression patterns of PLPPRs in the cerebellum, dorsal and ventral hippocampus, prefrontal cortex (PFC), nucleus accumbens, and striatum during development and in the adult using quantitative PCR. Expression patterns of Plppr2,4 and 5 were consistent with previous studies, whereas Plppr3 and Plppr1 exhibited a unique expression profile in nucleus accumbens (NAc) and striatum in later developmental and adult stages, which we verified at the protein level for PLPPR3. To investigate neuron type-specific expression at the single cell level, we developed a bioinformatic tool to analyze recent single-cell RNA-sequencing data in the cerebral cortex and hippocampus of adult mice. Our analysis revealed a widespread but also selective adult neuron-type expression with higher expression levels of Plppr3, Plppr1, and Plppr5 in GABAergic and Plppr4 and Plppr2 in glutamatergic neurons. PLPPR4 has been identified as a post-synaptic modulator of LPA levels in glutamatergic synapses operating via an uptake mechanism, to control LPA-dependent cortical network hyperexcitability. Using subcellular fractionation experiments, we found that both PLPPR4 and PLPPR3 are co-expressed in adult synaptosomal membranes. Furthermore, flow cytometry experiments in HEK293 cells showed comparable LPA uptake by PLPPR4 and PLPPR3, whereas PLPRR3, but not PLPPR4, induced also uptake of monoacylglycerol, the dephosphorylation product of LPA. We propose that synaptic LPA may be subject to both pre-synaptic and post-synaptic mechanisms of regulation by PLPPRs in addition to LPARs in developing and adult synapses.

Multi-Omics Analysis by Machine Learning Identified Lysophosphatidic Acid as a Biomarker and Therapeutic Target for Porcine Reproductive and Respiratory Syndrome.

As a significant infectious disease in livestock, porcine reproductive and respiratory syndrome (PRRS) imposes substantial economic losses on the swine industry. Identification of diagnostic markers and therapeutic targets has been a focal challenge in PPRS prevention and control. By integrating metabolomic and lipidomic serum analyses of clinical pig cohorts through a machine learning approach with in vivo and in vitro infection models, lysophosphatidic acid (LPA) is discovered as a serum metabolic biomarker for PRRS virus (PRRSV) clinical diagnosis. PRRSV promoted LPA synthesis by upregulating the autotaxin expression, which causes innate immunosuppression by dampening the retinoic acid-inducible gene I (RIG-I) and type I interferon responses, leading to enhanced virus replication. Targeting LPA demonstrated protection against virus infection and associated disease outcomes in infected pigs, indicating that LPA is a novel antiviral target against PRRSV. This study lays a foundation for clinical prevention and control of PRRSV infections.

LPA-Induced Thromboxane A2-Mediated Vasoconstriction Is Limited to Poly-Unsaturated Molecular Species in Mouse Aortas.

We have previously reported that, in aortic rings, 18:1 lysophosphatidic acid (LPA) can induce both vasodilation and vasoconstriction depending on the integrity of the endothelium. The predominant molecular species generated in blood serum are poly-unsaturated LPA species, yet the vascular effects of these species are largely unexplored. We aimed to compare the vasoactive effects of seven naturally occurring LPA species in order to elucidate their potential pathophysiological role in vasculopathies. Vascular tone was measured using myography, and thromboxane A2 (TXA2) release was detected by ELISA in C57Bl/6 mouse aortas. The Ca2+-responses to LPA-stimulated primary isolated endothelial cells were measured by Fluo-4 AM imaging. Our results indicate that saturated molecular species of LPA elicit no significant effect on the vascular tone of the aorta. In contrast, all 18 unsaturated carbon-containing (C18) LPAs (18:1, 18:2, 18:3) were effective, with 18:1 LPA being the most potent. However, following inhibition of cyclooxygenase (COX), these LPAs induced similar vasorelaxation, primarily indicating that the vasoconstrictor potency differed among these species. Indeed, C18 LPA evoked a similar Ca2+-signal in endothelial cells, whereas in endothelium-denuded aortas, the constrictor activity increased with the level of unsaturation, correlating with TXA2 release in intact aortas. COX inhibition abolished TXA2 release, and the C18 LPA induced vasoconstriction. In conclusion, polyunsaturated LPA have markedly increased TXA2-releasing and vasoconstrictor capacity, implying potential pathophysiological consequences in vasculopathies.

Phosphatidylethanolamines link ferroptosis and autophagy during appressorium formation of rice blast fungus.

A cell death pathway, ferroptosis, occurs in conidial cells and is critical for formation and function of the infection structure, the appressorium, in the rice blast fungus Magnaporthe oryzae. In this study, we identified an orthologous lysophosphatidic acid acyltransferase (Lpaat) acting at upstream of phosphatidylethanolamines (PEs) biosynthesis and which is required for such fungal ferroptosis and pathogenicity. Two PE species, DOPE and SLPE, that depend on Lpaat function for production were sufficient for induction of lipid peroxidation and the consequent ferroptosis, thus positively regulating fungal pathogenicity. On the other hand, both DOPE and SLPE positively regulated autophagy. Loss of the LPAAT gene led to a decrease in the lipidated form of the autophagy protein Atg8, which is probably responsible for the autophagy defect of the lpaatΔ mutant. GFP-Lpaat was mostly localized on the membrane of lipid droplets (LDs) that were stained by the fluorescent dye monodansylpentane (MDH), suggesting that LDs serve as a source of lipids for membrane PE biosynthesis and probably as a membrane source of autophagosome. Overall, our results reveal novel intracellular membrane-bound organelle dynamics based on Lpaat-mediated lipid metabolism, providing a temporal and spatial link of ferroptosis and autophagy.

Lysophosphatidic Acid (LPA) and Its Receptors in Mood Regulation: A Systematic Review of the Molecular Mechanisms and Therapeutic Potential.

Mood disorders affect over 300 million individuals worldwide, often characterized by their chronic and refractory nature, posing significant threats to patient life. There has been a notable increase in mood disorders among American adolescents and young adults, with a rising number of suicide attempts and fatalities, highlighting a growing association between mood disorders and suicidal outcomes. Dysregulation within the neuroimmune-endocrine system is now recognized as one of the fundamental biological mechanisms underlying mood and mood disorders. Lysophosphatidic acid (LPA), a novel mediator of mood behavior, induces anxiety-like and depression-like phenotypes through its receptors LPA1 and LPA5, regulating synaptic neurotransmission and plasticity. Consequently, LPA has garnered substantial interest in the study of mood regulation. This study aimed to elucidate the molecular mechanisms of lysophosphatidic acid and its receptors, along with LPA receptor ligands, in mood regulation and to explore their potential therapeutic efficacy in treating mood disorders. A comprehensive literature search was conducted using the PubMed and Web of Science databases, identifying 208 articles through keyword searches up to June 2024. After excluding duplicates, irrelevant publications, and those restricted by open access limitations, 21 scientific papers were included in this review. The findings indicate that LPA/LPA receptor modulation could be beneficial in treating mood disorders, suggesting that pharmacological agents or gintonin, an extract from ginseng, may serve as effective therapeutic strategies. This study opens new avenues for future research into how lysophosphatidic acid and its receptors, as well as lysophosphatidic acid receptor ligands, influence emotional behavior in animals and humans.

Neurolipid systems: A new target for the treatment of dementia.

Neurolipids comprise a diverse class of bioactive lipids that include molecules capable of activating G protein­coupled receptors, thereby inducing systemic effects that contribute to the maintenance of homeostasis. Dementia, a non­specific brain disorder characterized by a common set of signs and symptoms, usually arises subsequent to brain injuries or diseases and is often associated with the aging process. Individuals affected by dementia suffer from the disruption of several neurotransmitter and neuromodulatory systems, among which neurolipids play an important role, including the endocannabinoid, lysophosphatidic acid and sphingosine 1­phosphate systems. In this review, we present an overview of the most recent and pertinent findings regarding the involvement of these neurolipidic systems in dementia, including data from a wide range of both in vitro and in vivo experiments as well as clinical trials.

Lipid-orchestrated paracrine circuit coordinates mast cell maturation and anaphylaxis through functional interaction with fibroblasts.

Interaction of mast cells (MCs) with fibroblasts is essential for MC maturation within tissue microenvironments, although the underlying mechanism is incompletely understood. Through a phenotypic screening of >30 mouse lines deficient in lipid-related genes, we found that deletion of the lysophosphatidic acid (LPA) receptor LPA1, like that of the phospholipase PLA2G3, the prostaglandin D2 (PGD2) synthase L-PGDS, or the PGD2 receptor DP1, impairs MC maturation and thereby anaphylaxis. Mechanistically, MC-secreted PLA2G3 acts on extracellular vesicles (EVs) to supply lysophospholipids, which are converted by fibroblast-derived autotaxin (ATX) to LPA. Fibroblast LPA1 then integrates multiple pathways required for MC maturation by facilitating integrin-mediated MC-fibroblast adhesion, IL-33-ST2 signaling, L-PGDS-driven PGD2 generation, and feedforward ATX-LPA1 amplification. Defective MC maturation resulting from PLA2G3 deficiency is restored by supplementation with LPA1 agonists or PLA2G3-modified EVs. Thus, the lipid-orchestrated paracrine circuit involving PLA2G3-driven lysophospholipid, eicosanoid, integrin, and cytokine signaling fine-tunes MC-fibroblast communication, ensuring MC maturation.

Lysophosphatidic acid metabolism and signaling in heart disease.

Lysophosphatidic acid (LPA) is a bioactive lipid that is mainly produced by the secreted lysophospholipase D, autotaxin (ATX), and signals through at least six G protein-coupled receptors (LPA1-6). Extracellular LPA is degraded through lipid phosphate phosphatases (LPP1, LPP2, and LPP3) at the plasmamembrane, terminating LPA receptor signaling. The ATX-LPA-LPP3 pathway is critically involved in a wide range of physiological processes, including cell survival, migration, proliferation, angiogenesis, and organismal development. Similarly, dysregulation of this pathway has been linked to many pathological processes, including cardiovascular disease. This review summarizes and interprets current literature examining the regulation and role of the ATX-LPA-LPP3 axis in heart disease. Specifically, the contribution of altered LPA metabolism via ATX and LPP3 and resulting changes to LPA receptor signaling in obesity cardiomyopathy, cardiac mitochondrial dysfunction, myocardial infarction/ischemia-reperfusion injury, hypertrophic cardiomyopathy, and aortic valve stenosis is discussed.

Design and Characterization of a Dual-Protein Strategy for an Early-Stage Assay of Ovarian Cancer Biomarker Lysophosphatidic Acid.

The overall 5-year survival rate of ovarian cancer (OC) is generally low as the disease is often diagnosed at an advanced stage of progression. To save lives, OC must be identified in its early stages when treatment is most effective. Early-stage OC causes the upregulation of lysophosphatidic acid (LPA), making the molecule a promising biomarker for early-stage detection. An LPA assay can additionally stage the disease since LPA levels increase with OC progression. This work presents two methods that demonstrate the prospective application for detecting LPA: the electromagnetic piezoelectric acoustic sensor (EMPAS) and a chemiluminescence-based iron oxide nanoparticle (IONP) approach. Both methods incorporate the protein complex gelsolin-actin, which enables testing for detection of the biomarker as the binding of LPA to the complex results in the separation of gelsolin from actin. The EMPAS was characterized with contact angle goniometry and atomic force microscopy, while gelsolin-actin-functionalized IONPs were characterized with transmission electron microscopy and Fourier transform infrared spectroscopy. In addition to characterization, LPA detection was demonstrated as a proof-of-concept in Milli-Q water, buffer, or human serum, highlighting various LPA assays that can be developed for the early-stage detection of OC.

The Role of Lysophospholipid Metabolites LPC and LPA in the Pathogenesis of Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous lung condition characterized by persistent respiratory symptoms and airflow limitation. While there are some available treatment options, the effectiveness of treatment varies depending on individual differences and the phenotypes of the disease. Therefore, exploring or identifying potential therapeutic targets for COPD is urgently needed. In recent years, there has been growing evidence showing that lysophospholipids, namely lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA), can play a significant role in the pathogenesis of COPD. Exploring the metabolism of lysophospholipids holds promise for understanding the underlying mechanism of COPD development and developing novel strategies for COPD treatment. This review primarily concentrates on the involvement and signaling pathways of LPC and LPA in the development and progression of COPD. Furthermore, we reviewed their associations with clinical manifestations, phenotypes, and prognosis within the COPD context and discussed the potential of the pivotal signaling molecules as viable therapeutic targets for COPD treatment.

Lysophosphatidic Acid Modulates TGF-ß2-Induced Biological Phenotype in Human Conjunctival Fibroblasts.

BACKGROUND: Although lysophosphatidic acid (LPA) is known to have multiple pathophysiological roles, its contributions to ocular tissues, especially conjunctival fibrogenesis, remain to be elucidated. METHODS: To study this issue, the effects of LPA on transforming growth factor-ß2 (TGF-ß2)-induced fibrogenesis of two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblasts (HconF) were examined by the following analyses: (1) planar proliferation determined by transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran permeability measurements, (2) real-time metabolic analyses, (3) measurements of the size and stiffness of 3D spheroids, and (4) mRNA expression of extracellular matrix (ECM) molecules and their modulators. RESULTS: LPA had no effect on TGF-ß2-induced increase in the planar proliferation of HconF cells. LPA induced a more quiescent metabolic state in 2D HconF cells, but this metabolic suppression by LPA was partially blunted in the presence of TGF-ß2. In contrast, LPA caused a substantial decrease in the hardness of 3D HconF spheroids independently of TGF-ß2. In agreement with these different LPA-induced effects between 2D and 3D cultured HconF cells, mRNA expressions of ECM and their modulators were differently modulated. CONCLUSION: The findings that LPA induced the inhibition of both TGF-ß2-related and -unrelated subepithelial proliferation of HconF cells may be clinically applicable.

Lysophosphatidic Acid Stimulates Mitogenic Activity and Signaling in Human Neuroblastoma Cells through a Crosstalk with Anaplastic Lymphoma Kinase.

Lysophosphatidic acid (LPA) is a well-documented pro-oncogenic factor in different cancers, but relatively little is known on its biological activity in neuroblastoma. The LPA effects and the participation of the tyrosine kinase receptor anaplastic lymphoma kinase (ALK) in LPA mitogenic signaling were studied in human neuroblastoma cell lines. We used light microscopy and [3H]-thymidine incorporation to determine cell proliferation, Western blot to study intracellular signaling, and pharmacological and molecular tools to examine the role of ALK. We found that LPA stimulated the growth of human neuroblastoma cells, as indicated by the enhanced cell number, clonogenic activity, and DNA synthesis. These effects were curtailed by the selective ALK inhibitors NPV-TAE684 and alectinib. In a panel of human neuroblastoma cell lines harboring different ALK genomic status, the ALK inhibitors suppressed LPA-induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), which are major regulators of cell proliferation. ALK depletion by siRNA treatment attenuated LPA-induced ERK1/2 activation. LPA enhanced ALK phosphorylation and potentiated ALK activation by the ALK ligand FAM150B. LPA enhanced the inhibitory phosphorylation of the tumor suppressor FoxO3a, and this response was impaired by the ALK inhibitors. These results indicate that LPA stimulates mitogenesis of human neuroblastoma cells through a crosstalk with ALK.