vpc32183 and lysophosphatidic-acid

We have previously documented synthesis of lysophosphatidic acid (LPA) in the bovine endometrium and the increased presence of LPA receptor mRNA expression during pregnancy. Therefore, LPA could contribute to early pregnancy establishment in the cow. In the present study, we investigated the effect of intravaginally administered LPA on pregnancy rates and on the plasma levels of progesterone (P4) and prostaglandins (PGs) in heifers. Animals were inseminated and from day 15 to 18 after estrus were treated intravaginally with saline, LPA (1 mg) or LPA receptor blocker (VPC32183; 1 mg). Blood samples were collected on days 0, 6, 12, 15, 16, 17, 18 and 21 after insemination. Pregnancy was confirmed by ultrasonography and per rectum examination on days 30 and 49-50 after insemination. Intravaginal LPA administration increased the plasma P4 and PGE(2) concentrations compared with saline and VPC32183-treated heifers. In the saline and LPA-treated groups, 6 out of 8 heifers were pregnant (75%), whereas the pregnancy rate in the VPC32183-treated heifers was only 37%. We also examined the effects of LPA on PG secretion and PG synthase mRNA expression in stromal and epithelial cells of the bovine endometrium on days 16-18 of pregnancy and the estrous cycle. LPA increased PGE(2) production and PGE(2) synthase (PGES) mRNA expression in stromal cells during the estrous cycle and pregnancy. On Days 16-18 of pregnancy, LPA inhibited PGF(2alpha) production and PGFS mRNA expression in epithelial cells. The results suggest that LPA serves as a luteotropic factor during the estrous cycle and pregnancy, stimulating P4 secretion in vivo and PGE(2) secretion in vitro through activation of PGES mRNA expression in stromal cells. Moreover, during the early pregnancy, LPA decreases PGF(2alpha) synthesis and mRNA expression for PGFS in epithelial cells of the bovine endometrium.

Topics: Administration, Intravaginal; Animals; Cattle; Cells, Cultured; Dinoprostone; Embryonic Development; Endometrium; Female; Gestational Age; Intramolecular Oxidoreductases; Lysophospholipids; Organophosphates; Pregnancy; Pregnancy Rate; Pregnancy, Animal; Prostaglandin-E Synthases; Pyridines; Receptors, Lysophosphatidic Acid

Lysophosphatidylethanolamine (LPE) is a lyso-type metabolite of phosphatidylethanolamine (a plasma membrane component), and its intracellular Ca(2+) ([Ca(2+)]i) increasing actions may be mediated through G-protein-coupled receptor (GPCR). However, GPCRs for lysophosphatidic acid (LPA), a structurally similar representative lipid mediator, have not been implicated in LPE-mediated activities in SK-OV3 or OVCAR-3 ovarian cancer cells or in receptor over-expression systems. In the present study, LPE-induced [Ca(2+)]i increase was observed in MDA-MB-231 cells but not in other breast cancer cell lines. In addition, LPE- and LPA-induced responses showed homologous and heterologous desensitization. Furthermore, VPC32183 and Ki16425 (antagonists of LPA1 and LPA3) inhibited LPE-induced [Ca(2+)]i increases, and knockdown of LPA1 by transfection with LPA1 siRNA completely inhibited LPE-induced [Ca(2+)]i increases. Furthermore, the involvement of CD97 (an adhesion GPCR) in the action of LPA1 in MDA-MB-231 cells was demonstrated by siRNA transfection. Pertussis toxin (a specific inhibitor of Gi/o proteins), edelfosine (an inhibitor of phospholipase C), or 2-APB (an inhibitor of IP3 receptor) completely inhibited LPE-induced [Ca(2+)]i increases, whereas HA130, an inhibitor of autotaxin/lysophospholipase D, did not. Therefore, LPE is supposed to act on LPA1-CD97/Gi/o proteins/phospholipase C/IP3/Ca(2+) rise in MDA-MB-231 breast cancer cells.

Topics: Antigens, CD; Boron Compounds; Calcium; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Inositol 1,4,5-Trisphosphate Receptors; Isoxazoles; Lysophospholipids; Organ Specificity; Organophosphates; Pertussis Toxin; Propionates; Pyridines; Receptors, G-Protein-Coupled; Receptors, Lysophosphatidic Acid; RNA, Small Interfering; Signal Transduction; Type C Phospholipases

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 membrane-derived lysophospholipid that can induce pleomorphic effects in neural progenitor cells (NPCs) from the cerebral cortex, including alterations in ionic conductance. LPA-induced, calcium-mediated conductance changes have been reported; however, the underlying molecular mechanisms have not been determined. We show here that activation of specific cognate receptors accounts for nearly all intracellular calcium responses evoked by LPA in acutely cultured nestin-positive NPCs from the developing mouse cerebral cortex. Fast-onset changes in intracellular calcium levels required release from thapsigargin-sensitive stores by a pertussis toxin-insensitive mechanism. The influx of extracellular calcium through Cd(2+)/Ni(2+)-insensitive influx pathways, approximately one-half of which were Gd(3+) sensitive, contributed to the temporal diversity of responses. Quantitative reverse transcription-PCR revealed the presence of all five known LPA receptors in primary NPCs, with prominent expression of LPA(1), LPA(2), and LPA(4). Combined genetic and pharmacological studies indicated that NPC responses were mediated by LPA(1) (approximately 30% of the cells), LPA(2) (approximately 30%), a combination of receptors on single cells (approximately 30%), and non-LPA(1,2,3) pathways (approximately 10%). LPA responsivity was significantly reduced in more differentiated TuJ1(+) cells within cultures. Calcium transients in a large proportion of LPA-responsive NPCs were also initiated by the closely related signaling lipid S1P (sphingosine-1-phosphate). These data demonstrate for the first time the involvement of LPA receptors in mediating surprisingly diverse NPC calcium responses involving multiple receptor subtypes that function within a single cell. Compared with other known factors, lysophospholipids represent the major activator of calcium signaling identified within NPCs at this early stage in corticogenesis.

Topics: Animals; Cadmium Chloride; Calcium; Calcium Signaling; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Embryo, Mammalian; Extracellular Fluid; Female; Gene Expression Regulation, Developmental; Glutamic Acid; Intracellular Signaling Peptides and Proteins; Isoxazoles; Lysophospholipids; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nerve Tissue Proteins; Neurogenesis; Neurons; Nickel; Organophosphates; Pertussis Toxin; Pituitary Adenylate Cyclase-Activating Polypeptide; Pregnancy; Propionates; Pyridines; RNA, Messenger; Sphingosine; Thapsigargin

Lysophosphatidic acid (LPA) is produced by tumor cells and is present in the ascites fluid of ovarian cancer patients. To determine the role of endogenous LPA in the ovarian cancer cell line SKOV3, we treated cells with the LPA receptor antagonist VPC32183 and found that it inhibited cell growth and induced apoptosis. Exogenous LPA further stimulated ERK and Akt phosphorylation and NF-κB activity. To determine if reactive oxygen species (ROS), which have been implicated as second messengers in cell signaling, were also involved in LPA signaling, we treated cells with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), and antioxidants N-acetyl cysteine, EUK-134 and curcumin, and showed that all blocked LPA-dependent NF-κB activity and cell proliferation. DPI and EUK-134 also inhibited Akt and ERK phosphorylation. LPA was shown to stimulate dichlorofluorescein fluorescence, though not in the presence of DPI, apocynin (an inhibitor of NADPH oxidase), VPC32183, or PEG-catalase. Akt phosphorylation was also inhibited by PEG-catalase and apocynin. These data indicate that NADPH oxidase is a major source of ROS and H(2)O(2) is critical for LPA-mediated signaling. Thus, LPA acts as a growth factor and prevents apoptosis in SKOV3 cells by signaling through redox-dependent activation of ERK, Akt, and NF-κB-dependent signaling pathways.

Topics: Antioxidants; Apoptosis; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Female; Genes, Reporter; Humans; Lysophospholipids; NADPH Oxidases; NF-kappa B; Onium Compounds; Organophosphates; Ovarian Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyridines; Reactive Oxygen Species; Receptors, Lysophosphatidic Acid; Signal Transduction; Up-Regulation

Increased expression of autotaxin in tumors including glioblastoma, breast, renal, ovarian, lung, and thyroid cancers is associated with increased tumor aggressiveness. Autotaxin promotes metastasis as well as cell growth, survival, and migration of cancer cells. These actions could depend on the noncatalytic effects of autotaxin on cell adhesion, or the catalytic activity of autotaxin, which converts lysophosphatidylcholine into lysophosphatidate in the extracellular fluid surrounding the tumor. Both lysophosphatidylcholine (LPC) and lysophosphatidate have been reported to stimulate migration through their respective G-protein coupled receptors. The present study determines the roles of autotaxin, LPC, and lysophosphatidate in controlling the migration of two cancer cell lines: MDA-MB-231 breast cancer cells, which produce little autotaxin and MDA-MB-435 melanoma cells that secrete significant levels of autotaxin. LPC alone was unable to stimulate the migration of either cell type unless autotaxin was present. Knocking down autotaxin secretion, or inhibiting its catalytic activity, blocked cell migration by preventing lysophosphatidate production and the subsequent activation of LPA(1/3) receptors. We conclude that inhibiting autotaxin production or activity could provide a beneficial adjuvant to chemotherapy for preventing tumor growth and metastasis in patients with high autotaxin expression in their tumors.

Topics: Anilides; Blotting, Western; Breast Neoplasms; Catalysis; Cell Line; Cell Line, Tumor; Cell Movement; Enzyme Inhibitors; Humans; Kinetics; Lysophosphatidylcholines; Lysophospholipids; Melanoma; Multienzyme Complexes; Organophosphates; Organophosphonates; Phosphodiesterase I; Phosphoric Diester Hydrolases; Pyridines; Pyrophosphatases; Receptors, Lysophosphatidic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Transfection

Lysophosphatidylserine (LPS) can be generated following phosphatidylserine-specific phospholipase A2 activation. The effects of LPS on cellular activities and the identities of its target molecules, however, have not been fully elucidated. In this study, we observed that LPS stimulated intracellular calcium increased in mouse bone marrow-derived mast cells (BMMC), and rat C6 glioma and human HCT116 colon cancer cells and compared the LPS-induced Ca2+ increases with the response by lysophosphatidic acid (LPA), a structurally related bioactive lysolipid. In order to test involvement of signaling molecules in the LPS-induced Ca2+ signaling, we used pertussis toxin (PTX), U73122, and 2-APB, which are specific inhibitors for G proteins, phospholipase C (PLC), and IP3 receptors, respectively. The increases due to LPS and LPA were inhibited by PTX, U-73122 and 2-APB, suggesting that both lipids stimulate calcium signaling via G proteins (Gi/o types), PLC activation, and subsequent IP3 production, although the sensitivity to pharmacological inhibitors varied from complete inhibition to partial inhibition depending on cell type and lysolipid. Furthermore, we observed that Ki16425 completely inhibited an LPS-induced Ca2+ response in three cell types, but that the effect of VPC32183 varied from complete inhibition in BMMC and C6 glioma cells to partial inhibition in HCT116 cells. Therefore, we conclude that LPS increases [Ca2+]i through Ki16425/VPC32183-sensitive G protein-coupled receptors (GPCR), G protein, PLC, and IP3 in mouse BMMC, rat C6, and human HCT116 cells.

Topics: Animals; Bone Marrow Cells; Calcium Signaling; Colonic Neoplasms; Dose-Response Relationship, Drug; Enzyme Inhibitors; Estrenes; Glioma; HCT116 Cells; Humans; Inositol 1,4,5-Trisphosphate Receptors; Isoxazoles; Lysophospholipids; Male; Mast Cells; Mice; Mice, Inbred BALB C; Organophosphates; Pertussis Toxin; Propionates; Pyridines; Pyrrolidinones; Rats; Receptors, Lysophosphatidic Acid; Time Factors; Type C Phospholipases

Previously, we suggested that dioleoyl phosphatidic acid (PA) and lysophosphatidic acid (LPA) increased [Ca(2+)](i) through endogenous LPA receptors coupled to pertussis toxin-sensitive G proteins in rat C6 glioma cells. In the present report, we investigated morphological changes and cytotoxicity induced by PA and LPA in C6 glioma cells. Isoproterenol treatment led to changes in the cell morphology of rat C6 glioma cells, which were reverted by the addition of PA and LPA. PA-and LPA-induced morphological reversions were inhibited by treatment with Ki16425, an LPA(1)/LPA(3) receptor antagonist. VPC32183, another LPA(1)/LPA(3) receptor antagonist with a different structure, only inhibited PA-induced morphological reversion but not LPA-induced reversion. However, the reversions were not inhibited by treatment with pertussis toxin, a specific inhibitor of G(i/o) proteins. In addition, cytotoxicity was only induced by LPA but not by PA in C6 glioma cells. Our results suggest that PA may act as a partial agonist at endogenous LPA receptors, which are sensitive to Ki16425 and coupled to PTX-insensitive G proteins, to evoke morphological changes in C6 glioma cells.

Topics: Animals; Cell Line, Tumor; Cell Survival; Cytotoxins; Drug Partial Agonism; Glioma; Isoproterenol; Isoxazoles; Lysophospholipids; Organophosphates; Pertussis Toxin; Phosphatidic Acids; Propionates; Pyridines; Rats; Receptors, Lysophosphatidic Acid

Lysophosphatidic acid (LPA) is a simple lipid with many important biological functions such as the regulation of cellular proliferation, cellular migration, differentiation, and suppression of apoptosis. Although a direct angiogenic effect of LPA has not been reported to date, there are indications that LPA promotes angiogenesis. In addition, LPA is a chemoattractant for cultured endothelial cells and promotes barrier function in such cultures. To test the hypothesis that LPA is angiogenic, we used the chicken chorio-allantoic membrane (CAM) assay. Sequence analysis of the cloned, full-length chicken LPA receptor cDNAs revealed three receptor types that are orthologous to the mammalian LPA(1), LPA(2), and LPA(3) receptors. We document herein that LPA is angiogenic in the CAM system and further that synthetic LPA receptor agonists and antagonists mimic or block this response, respectively. Our results predict that LPA receptor antagonists are a possible therapeutic route to interdicting angiogenesis.

Topics: Amino Acid Sequence; Angiogenesis Inhibitors; Animals; Chick Embryo; Drug Evaluation, Preclinical; Lysophospholipids; Molecular Sequence Data; Neovascularization, Physiologic; Organophosphates; Pyridines; Receptors, Lysophosphatidic Acid; Sequence Homology, Amino Acid; Sphingosine; Substrate Specificity; Vascular Endothelial Growth Factor A