pyrophosphate and lysophosphatidic-acid

Lysophosphatidic acid (LPA) affects several female reproductive functions through G-protein-coupled receptors. LPA contributes to embryo implantation via the lysophospholipid LPA

Topics: Animals; Decidua; Diphosphates; Embryo Implantation; Female; Glycerol; Interleukin-10; Lysophospholipids; Neovascularization, Physiologic; Placenta; Pregnancy; Rats; Receptors, Lysophosphatidic Acid; Signal Transduction; Uterine Artery; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1

Reactive oxygen species (ROS) are known to mediate a variety of biological responses, including cell motility. Recently, we indicated that lysophosphatidic acid (LPA) receptor-3 (LPA3) increased cell motile activity stimulated by hydrogen peroxide. In the present study, we assessed the role of LPA1 in the cell motile activity mediated by ROS in mouse fibroblast 3T3 cells. 3T3 cells were treated with hydrogen peroxide and 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) at concentrations of 0.1 and 1 μM for 48 h. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3 cells treated with hydrogen peroxide and DMNQ were significantly higher than those of untreated cells. 3T3 cells treated with hydrogen peroxide and DMNQ showed elevated expression levels of the Lpar3 gene, but not the Lpar1 and Lpar2 genes. To investigate the effects of LPA1 on the cell motile activity induced by hydrogen peroxide and DMNQ, Lpar1-overexpressing (3T3-a1) cells were generated from 3T3 cells and treated with hydrogen peroxide and DMNQ. The cell motile activities stimulated by hydrogen peroxide and DMNQ were markedly suppressed in 3T3-a1 cells. These results suggest that LPA signaling via LPA1 inhibits the cell motile activities stimulated by hydrogen peroxide and DMNQ in 3T3 cells.

Topics: 3T3 Cells; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Diphosphates; Fibroblasts; Gene Expression Regulation; Glycerol; Hydrogen Peroxide; Lysophospholipids; Mice; Naphthoquinones; Neuroblastoma; Rats; Receptors, Lysophosphatidic Acid

Lysophosphatidic acid (LPA) mediates a variety of cellular responses with atleast six G protein-coupled transmembrane receptors (LPA receptor-1 (LPA(1)-LPA(6))). The interaction between LPA receptors and other cellular molecules on the biological function is not fully understood. Recently, we have reported that LPA(1) suppressed and LPA(3) stimulated cell migration of pancreatic cancer cells. In the present study, to evaluate the function of LPA(2) on motile and invasive activities of pancreatic cancer cells, we generated Lpar2 knockdown (HPD-sh2) cells from hamster pancreatic cancer cells and measured their cell migration ability. In cell motility and invasive assays with an uncoated Cell Culture Insert, HPD-sh2 cells showed significantly lower intrinsic activity than control (HPD-GFP) cells. Since K-ras mutations were frequently detected in pancreatic cancer, we next investigated whether oncogenic K-ras is involved in cell migration induced by LPA(2) using K-ras knockdown (HPD-K2) cells. The cell motile ability of HPD-K2 cells was significantly lower than that of control cells. To confirm LPA(2) increases cell migration activity, cells were pretreated with dioctylglycerol pyrophosphate (DGPP) which is the antagonist of LPA(1)/LPA(3). The cell motile and invasive abilities of DGPP -treated HPD-GFP cells were markedly higher than those of untreated cells, but DGPP did not stimulate cell migration of HPD-K2 cells. These results suggest that cell migration activity of pancreatic cancer cells stimulated by LPA(2) may be enhanced by oncogenic K-ras.

Topics: Adenocarcinoma; Animals; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Movement; Cricetinae; Diphosphates; Gene Knockdown Techniques; Genes, ras; Glycerol; Lysophospholipids; Neoplasm Invasiveness; Pancreatic Neoplasms; Receptors, Lysophosphatidic Acid; Up-Regulation

The lipidic metabolite, diacylglycerol pyrophosphate (DGPP), in its dioctanoyl form (DGPP 8:0), has been described as an antagonist for mammalian lysophosphatidic acid (LPA) receptors LPA1 and LPA3. In this study we show that DGPP 8:0 does not antagonize LPA dependent activation of ERK(1/2) MAP kinases but strongly stimulated them in various mammalian cell lines. LPA and DGPP 8:0 stimulation of ERK(1/2) occurred through different pathways. The DGPP 8:0 effect appeared to be dependent on PKC, Raf and MEK but was insensitive to pertussis toxin and did not involve G protein activation. Finally we showed that DGPP 8:0 effect on ERK(1/2) was dependent on its dephosphorylation by a phosphatase activity sharing lipid phosphate phosphatase properties. The inhibition of this phosphatase activity by VPC32183, a previously characterized LPA receptor antagonist, blocked the DGPP 8:0 effect on ERK(1/2) activation. Moreover, down-regulation of lipid phosphate phosphatase 1 (LPP1) expression by RNA interference technique also reduced DGPP 8:0-induced ERK(1/2) activation. Consistently, over expression of LPP1 in HEK293 cells increases DGPP 8:0 hydrolysis and this increased activity was inhibited by VPC32183. In conclusion, DGPP 8:0 does not exert its effect by acting on a G protein coupled receptor, but through its dephosphorylation by LPP1, generating dioctanoyl phosphatidic acid which in turn activates PKC. These results suggest that LPP1 could have a positive regulatory function on cellular signaling processes such as ERK(1/2) activation.

Topics: Blotting, Western; Cell Membrane; Cells, Cultured; Diphosphates; Glycerol; Humans; Hydrolysis; Kidney; Lysophospholipids; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Organophosphates; Phosphatidate Phosphatase; Phosphatidic Acids; Phosphorylation; Protein Kinase C; Pyridines; raf Kinases; Real-Time Polymerase Chain Reaction; Receptors, Lysophosphatidic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Lysophosphatidic acid (LPA) is a bioactive lipid with diverse physiological effects via activation of G protein-coupled receptors (GPCRs). It has been implicated as a specific dedifferentiation factors that can promote phenotypic modulation of cultured vascular smooth muscle cells (VSMCs) which is critically involved in various vascular disease. However, the role of LPA receptors and details of their signaling in LPA induced phenotypic modulation are largely unexplored. In this study we detect the expression of LPA1 and LPA3 in rat aortic smooth muscle cells (RASMCs). LPA promoted RASMCs phenotypic modulation in a dose-dependent manner and coordinated induced the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK). LPA-induced cell phenotypic modulation was significantly inhibited by specific LPA1/LPA3-receptor antagonist dioctyl-glycerol pyrophosphate (DGPP8:0) at concentration, but this inhibitive effect was lost when the antagonist was coadministered with a highly selective LPA3 agonist,1-oleoyl-2-Omethyl-rac-glycero-phosphothionate (OMPT). In addition, pertussis toxin (PTX), a Gi protein inhibitor had little affect on the LPA-induced phenotypic modulation in RASMC. These data suggest that LPA-induced phenotypic modulation is mediated through the PTX-insensitive G-protein(s), possibly Gq-coupled LPA3 receptor.

Topics: Animals; Aorta; Diphosphates; Enzyme Activation; Gene Expression Regulation; Glycerol; GTP-Binding Proteins; Lysophospholipids; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Pertussis Toxin; Phenotype; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptors, Lysophosphatidic Acid

Lysophosphatidic acid (LPA) has been shown to be a potent modulator of prostaglandin (PG) secretion during the luteal phase of the estrous cycle in the bovine endometrium in vivo. The aims of the present study were to determine the cell types of the bovine endometrium (epithelial or stromal cells) responsible for the secretion of PGs in response to LPA, the cellular, receptor, intracellular, and enzymatic mechanisms of LPA action. Cultured bovine epithelial and stromal cells were exposed to LPA (10(-5)-10(-9) M), tumor necrosis factor alpha (TNFalpha; 10 ng/mL) or oxytocin (OT; 10(-7) M) for 24 h. LPA treatment resulted in a dose-dependent increase of PGE(2) production in stromal cells, but not in epithelial cells. LPA did not influence PGF(2alpha) production in stromal or epithelial cells. To examine which type of LPA G-protein-coupled receptor (LP-GPCR; LPA1, LPA2, or LPA3) is responsible for LPA action, stromal cells were preincubated with three selected blockers of LPA receptors: NAEPA, DGPP, and Ki16425 for 0.5 h, and then stimulated with LPA. Only Ki16425 inhibited the stimulatory effect of LPA on PGE(2) production and cell proliferation in the stromal cells. LPA-induced intracellular calcium ion mobilization was also inhibited only by Ki16425. Finally, we examined whether LPA-induced PGE(2) synthesis in stromal cells is via the influence on mRNA expression for the enzymes responsible for PGE(2) synthesis-PGE(2) synthase (PGES) and PG-endoperoxide synthase 2 (PTGS2). We demonstrated that the stimulatory effect of LPA on PGE(2) production in stromal cells is via the stimulation of PTGS2 and PGES mRNA expression in the cells. The overall results indicate that LPA stimulates PGE(2) production, cell viability, and intracellular calcium ion mobilization in cultured stromal endometrial cells via Ki16425-sensitive LPA1 receptors. Moreover, LPA exerts a stimulatory effect on PGE(2) production in stromal cells via the induction of PTGS2 and PGES mRNA expression.

Topics: Animals; Calcium Signaling; Cattle; Cell Survival; Cells, Cultured; Cyclooxygenase 2; Dinoprost; Dinoprostone; Diphosphates; Endometrium; Epithelial Cells; Female; Glycerol; Intracellular Space; Intramolecular Oxidoreductases; Isoxazoles; Lysophospholipids; Propionates; Prostaglandin-E Synthases; Receptors, Lysophosphatidic Acid; RNA, Messenger; Stromal Cells

Lysophosphatidic acid (LPA), a naturally occurring glycerophospholipid, can evoke various biological responses, including cell migration, proliferation and survival, via activation of G protein-coupled receptors (GPCRs). However, the role of LPA receptors and details of LPA signaling in migration are largely unexplored. In this study we detect the expression of LPA1 and LPA3 receptors in rat aortic smooth muscle cells (RASMCs). LPA stimulated RASMCs migration in a dose-dependent manner and induced the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK). LPA-induced cell migration was significantly inhibited by specific LPA1/LPA3-receptor antagonist Dioctylglycerol pyrophosphate (8:0) (DGPP8.0) at higher concentration. Migration of cells toward LPA was partially, but significantly, reduced in the presence of SB-203580, a p38 MAPK inhibitor, but not PD98059, an ERK inhibitor. In addition, pertussis toxin (PTX), a Gi protein inhibitor, induced an inhibitory effect on p38 MAPK, ERK phosphorylation and RASMCs migration. These data suggest that LPA-induced migration is mediated through the Gi-protein-coupled LPA1 receptor involving activation of a PTX-sensitive Gi / p38MAPK pathway.

Topics: Animals; Cell Movement; Cells, Cultured; Diphosphates; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Glycerol; Imidazoles; Lysophospholipids; MAP Kinase Signaling System; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; p38 Mitogen-Activated Protein Kinases; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Lysophosphatidic Acid

Lysophosphatidic acid (LPA) is a bioactive phospholipid with diverse functions mediated via G-protein-coupled receptors (GPCRs). In view of the elevated levels of LPA in acute myocardial infarction (MI) patients we have conducted studies aimed at identifying specific LPA receptor subtypes and signaling events that may mediate its actions in hypertrophic remodeling. Experiments were carried out in cultured neonatal rat cardiomyocytes (NRCMs) exposed to LPA and in a rat MI model. In NRCMs, LPA-induced hypertrophic growth was completely abrogated by DGPP, an LPA1/LPA3 antagonist. The LPA3 agonist OMPT, but not the LPA2 agonist dodecylphosphate, promoted hypertrophy as examined by 3[H]-Leucine incorporation, ANF-luciferase expression and cell area. In in vivo experiments, LPA1, LPA2 and LPA3 mRNA levels as well as LPA1 and LPA3 protein levels increased together with left ventricular remodeling (LVRM) after MI. In addition, LPA stimulated the phosphorylation of Akt and p65 protein and activated NF-kappaB-luciferase expression. Inhibitors of PI3K (wortmannin), mTOR (rapamycin), and NF-kappaB (PDTC or SN50) effectively prevented LPA-induced 3[H]-Leucine incorporation and ANF-luciferase expression. Furthermore, ERK inhibitors (U0126 and PD98059) suppressed LPA-stimulated activation of NF-kappaB and p65 phosphorylation whereas wortmannin showed no effect on NF-kappaB activation. Our findings indicate that LPA3 and/or LPA1 mediate LPA-induced hypertrophy of NRCMs and that LPA1 and LPA3 may be involved in LVRM of MI rats. Moreover, Akt and NF-kappaB signaling pathways independently implicate in LPA-stimulated myocardial hypertrophic growth.

Topics: Animals; Animals, Newborn; Cell Survival; Cells, Cultured; Diphosphates; Enzyme Inhibitors; Female; Glycerol; Hypertrophy; Lysophospholipids; MAP Kinase Signaling System; Myocardial Infarction; Myocytes, Cardiac; NF-kappa B; Organothiophosphates; Phosphatidic Acids; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Receptors, Lysophosphatidic Acid; Signal Transduction; Ventricular Function, Left; Ventricular Remodeling

Lysophosphatidic acid (LPA), the simplest of the water-soluble phospholipids, can evoke various biological responses. The present study examined the activity of LPA to induce plasma exudation and histamine release in mice. Plasma exudation was assessed by extravasation of Evans blue. Subcutaneous administration of LPA (1 - 100 microg/site) led to increased plasma exudation in the skin. The LPA-induced plasma exudation was inhibited by ketotifen, a histamine H1-receptor antagonist, and diacylglycerol pyrophosphate (DGPP), a LPA1/LPA3-receptor antagonist. Moreover, pretreatment with pertussis toxin and DGPP inhibited the histamine release from peritoneal mast cells induced by LPA. These findings indicate that plasma exudation induced by LPA is mediated by histamine release from mast cells via LPA receptor(s), presumably LPA1 and/or LPA3, coupled to G(i/o) proteins. Moreover, these findings point to a role of LPA in the pathomechanisms of various allergic disorders.

Topics: Animals; Capillary Permeability; Diphosphates; Dose-Response Relationship, Drug; Exudates and Transudates; Glycerol; Histamine H1 Antagonists; Histamine Release; In Vitro Techniques; Injections, Subcutaneous; Ketotifen; Lysophospholipids; Male; Mast Cells; Mice; Mice, Inbred ICR; Pertussis Toxin; Plasma; Receptors, Lysophosphatidic Acid; Skin

To determine whether lysophosphatidic acid (LPA) or sphingosine-1-phosphate (S1P) affects transcellular resistance across cultured rabbit corneal epithelial and endothelial cells.. Electric cell-substrate impedance sensing (ECIS) was used to measure electrical resistance across cultured rabbit corneal epithelial and endothelial monolayers. After a 1-hour equilibration period, different concentrations of LPA or S1P were added to each well, and the effect observed for 4 hours. For cells significantly affected by LPA or S1P, pertussis toxin (PTX) or dioctyl-glycerol pyrophosphate (DGPP 8:0) was added along with LPA or S1P in separate experiments. Cells were also treated with phorbol 12-myristate 13-acetate (PMA) in the presence of LPA or S1P in different tests. The influence of LPA and S1P on epithelial and endothelial cell F-actin was determined with immunohistochemistry.. LPA significantly increased the resistance of both the epithelial and endothelial monolayers, whereas S1P increased the resistance in only the endothelial cells. PTX blocked both the LPA- and S1P-induced increases in resistance, and DGPP (8:0) inhibited LPA-induced transcellular resistance in both the epithelium and endothelium. LPA and S1P prevented PMA-induced resistance decreases across epithelial and endothelial cells. F-actin staining around cell borders was more intense in both LPA- and S1P-treated cells.. LPA increases transcellular resistance across cultured rabbit corneal epithelial and endothelial cell monolayers, and the effect is mediated through the LPA(1) receptor and signaled through Galpha(i/o). S1P-stimulated increases in endothelial resistance are also signaled through Galpha(i/o). Both LPA and S1P prevented increased transcellular permeabilities induced by PMA, and increased actin stress fiber formation in epithelial and endothelial cells.

Topics: Actins; Animals; Cell Culture Techniques; Diphosphates; Electric Impedance; Endothelium, Corneal; Epithelium, Corneal; Fluorescent Antibody Technique, Indirect; Glycerol; GTP-Binding Protein alpha Subunits, Gi-Go; Lysophospholipids; Pertussis Toxin; Rabbits; Receptors, Lysophosphatidic Acid; Sphingosine; Tetradecanoylphorbol Acetate

Lysophosphatidic acid (LPA) is a bioactive lipid mediator, which is generated by secretory type II phospholipase A(2) and is thought to play a major role in the pathogenesis of atopic diseases. In this study, the biological activity of LPA on human eosinophils was characterized. We showed by reverse transcription and PCR that human eosinophils express the mRNA of the LPA receptors endothelial differentiation gene (EDG)-2 and EDG-7. Experiments revealed that LPA has chemotactic activity toward eosinophils, stimulates the production of reactive oxygen metabolites, and induces up-regulation of the integrin CD11b. Signal pathway measurements indicated Ca(2+)-mobilization from intracellular stores and transient actin polymerization upon stimulation with LPA. Cell responses elicited by LPA were inhibited by pertussis toxin indicating that in eosinophils the LPA receptor(s), presumably EDG-2 and/or EDG-7, are coupled to G(i/o) proteins. Moreover, LPA-induced activation of eosinophils could be completely blocked by the EDG-2/EDG-7 antagonist diacylglycerol pyrophosphate. In addition, at optimal doses the changes induced by LPA were comparable to those obtained by the other well-characterized chemotaxins. These results indicate that LPA is a strong chemotaxin and activator of eosinophils. These findings point to a novel role of LPA in the pathogenesis of diseases with eosinophilic inflammation such as atopic diseases as chemotaxin as well as activator of proinflammatory effector functions.

Topics: Actins; Calcium Signaling; CD11b Antigen; Chemotactic Factors, Eosinophil; Chemotaxis, Leukocyte; Diphosphates; Eosinophils; Glycerol; GTP-Binding Proteins; Humans; Intracellular Fluid; Lysophospholipids; Pertussis Toxin; Reactive Oxygen Species; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; Respiratory Burst; RNA, Messenger; Up-Regulation

Neural stem/progenitor cells are clonogenic in vitro and produce neurospheres in serum-free medium containing epidermal growth factor (EGF) and fibroblast growth factor (FGF2). Here, we demonstrate that lysophosphatidic acid (LPA) instigated the clonal generation of neurospheres from dissociated mouse postnatal forebrain in the absence of EGF and FGF2. LPA induced proliferation of cells which co-expressed Sca-1 antigen and AC133, markers of primitive hematopoietic and neural stem/progenitor cells. Clonal expansion of these cells induced by LPA was inhibited by diacylglycerol- pyrophosphate (DGPP), an antagonist of the LPA receptor subtypes LPA1 and LPA3. Moreover, Sca-1- and AC133-positive cells of these neurospheres expressed LPA1, LPA2, and LPA3, suggesting important roles for these LPA receptors in proliferation of neural progenitors. LPA induced neurospheres to differentiate on an adherent laminin/poly-L-ornithine matrix. In differentiating neurospheres, LPA receptors co-localized with betaIII-tubulin, nestin, and CNPase, but not with glial fibrillary acidic protein (GFAP), a marker of astrocyte lineage. Our results demonstrate for the first time that lysophosphatidic acid induces clonal neurosphere development via proliferation of AC133/Sca-1-positive stem cells by a receptor-dependent mechanism. This differentiation was characterized by the initial co-localization of neural specific antigens at sites of LPA receptor expression upon their interaction with the inducing agonist.

Topics: AC133 Antigen; Animals; Antigens, CD; Astrocytes; Ataxin-1; Ataxins; Brain; Cell Differentiation; Cell Lineage; Cell Proliferation; Cells, Cultured; Culture Media, Serum-Free; Diphosphates; Epidermal Growth Factor; Fibroblast Growth Factor 2; Glial Fibrillary Acidic Protein; Glycerol; Glycoproteins; Immunohistochemistry; Lysophospholipids; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Neurons; Nuclear Proteins; Oligodendroglia; Peptides; Prosencephalon; Receptors, Lysophosphatidic Acid; Stem Cells