Stimulation of high-affinity GTPase activity and cholera toxin-catalysed [32P]ADP-ribosylation of Gi by lysophosphatidic acid (LPA) in wild-type and alpha 2C10 adrenoceptor-transfected Rat 1 fibroblasts.
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ABSTRACT:
SUBMITTER: Carr C
PROVIDER: S-EPMC1137967 | biostudies-other | 1994 Mar
REPOSITORIES: biostudies-other
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Project description: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.
| S-EPMC2677190 | biostudies-literature
Project description:NG108-15 neuroblastoma x glioma hybrid cells express a major 45 kDa substrate for cholera toxin and a 40 kDa substrate(s) for pertussis toxin when ADP-ribosylation is performed in the presence of GTP. In the absence of exogenous GTP, however, cholera toxin was shown to catalyse incorporation of radioactivity into a 40 kDa protein as well as into the 45 kDa polypeptide. In membranes of cells which had been pretreated in vivo with pertussis toxin, the 40 kDa band was no longer a substrate for either pertussis or cholera toxin in vitro, whereas in membranes from cholera-toxin-pretreated cells the 40 kDa band was still a substrate for fresh cholera toxin in vitro and for pertussis toxin. In this cell line, opioid peptides have been shown to inhibit adenylate cyclase exclusively by interacting with Gi (inhibitory G-protein) and with no other pertussis-toxin-sensitive G-protein. Opioid agonists, but not antagonists, promoted the cholera-toxin-catalysed ADP-ribosylation of the 40 kDa polypeptide, hence demonstrating that this cholera-toxin substrate was indeed the alpha-subunit of Gi. These results demonstrate that Gi can be a substrate for either cholera or pertussis toxin under appropriate conditions.
| S-EPMC1149154 | biostudies-other
Project description:Lysophosphatidic acid (LPA), a water-soluble phospholipid, has gained significant attention in recent years since the discovery that it acts as a potent signaling molecule with wide-ranging effects on many different target tissues. There are currently five identified G protein-coupled receptors for LPA and more are undergoing validation. The complexity of the expression pattern and signaling properties of LPA receptors results in multiple influences on developmental, physiological, and pathological processes. This review provides a summary of LPA receptor signaling and current views on the potential involvement of this pathway in human diseases that include cardiovascular, cancer, neuropathic pain, neuropsychiatric disorders, reproductive disorders, and fibrosis. The involvement of LPA signaling in these processes implicates multiple, potential drug targets including LPA receptor subtypes and LPA metabolizing enzymes. Modulation of LPA signaling may thus provide therapeutic inroads for the treatment of human disease.
| S-EPMC2845529 | biostudies-literature
Project description:BACKGROUND:In the extracellular environment, lysophosphatidic acid (LPA) species are generated via autotaxin (ATX)-mediated hydrolysis of lysophospholipid precursors. Members of the LPA family are potent lipid mediators transmitting signals via six different G protein-coupled LPA receptors (LPAR1-6). The LPA signaling axis is indispensable for brain development and function of the nervous system; however, during damage of the central nervous system, LPA levels can increase and aberrant signaling events counteract brain function. Here, we investigated regulation of the ATX/LPA/LPAR axis in response to lipopolysaccharide-induced systemic inflammation in mice and potential neurotoxic polarization programs in LPA-activated primary murine microglia. METHODS:In vivo, LPAR1-6 expression was established by qPCR in whole murine brain homogenates and in FACS-sorted microglia. ELISAs were used to quantitate LPA concentrations in the brain and cyto-/chemokine secretion from primary microglia in vitro. Transcription factor phosphorylation was analyzed by immunoblotting, and plasma membrane markers were analyzed by flow cytometry. We used MAPK inhibitors to study signal integration by the JNK, p38, and ERK1/2 branches in response to LPA-mediated activation of primary microglia. RESULTS:Under acute and chronic inflammatory conditions, we observed a significant increase in LPA concentrations and differential regulation of LPAR, ATX (encoded by ENPP2), and cytosolic phospholipase A2 (encoded by PLA2G4A) gene expression in the brain and FACS-sorted microglia. During pathway analyses in vitro, the use of specific MAPK antagonists (SP600125, SB203580, and PD98059) revealed that JNK and p38 inhibition most efficiently attenuated LPA-induced phosphorylation of proinflammatory transcription factors (STAT1 and -3, p65, and c-Jun) and secretion of IL-6 and TNF?. All three inhibitors decreased LPA-mediated secretion of IL-1?, CXCL10, CXCL2, and CCL5. The plasma membrane marker CD40 was solely inhibited by SP600125 while all three inhibitors affected expression of CD86 and CD206. All MAPK antagonists reduced intracellular COX-2 and Arg1 as well as ROS and NO formation, and neurotoxicity of microglia-conditioned media. CONCLUSION:In the present study, we show that systemic inflammation induces aberrant ATX/LPA/LPAR homeostasis in the murine brain. LPA-mediated polarization of primary microglia via MAPK-dependent pathways induces features reminiscent of a neurotoxic phenotype.
| S-EPMC7178949 | biostudies-literature
Project description:Emerging evidence has indicated that high-grade serous ovarian cancer (HGSOC) originates in the fallopian tube, where the earliest known genetic lesion is the mutation of TP53. In addition to such genetic changes, HGSOC is characterized by altered metabolism, including the production of oncogenic lipids such as lysophosphatidic acid (LPA). To understand the crosstalk between TP53 mutations and LPA signaling, we utilized primary fallopian tube epithelial cells (FTEC) engineered to overexpress mutant p53. We found that gain-of-function (GOF) p53 mutations downregulated the LPA-degrading enzyme lysophosphatidic acid phosphatase type 6 (ACP6), leading to upregulation of focal adhesion signaling in an LPA-dependent manner. Although highly expressed in normal fallopian tube epithelium, ACP6 expression was significantly reduced in ovarian cancer tumors and early in situ lesions. Downregulation of ACP6 in ovarian cancer cells was necessary and sufficient to support HGSOC proliferation, adhesion, migration, and invasion. Using mouse models of metastasis, we established that attenuation of ACP6 expression was associated with increased tumor burden. Conversely, overexpression of ACP6 suppressed invasive behavior. These data identify an involvement of oncogenic p53 mutations in LPA signaling and HGSOC progression through regulation of ACP6 expression.
| S-EPMC6435808 | biostudies-literature
Project description:A method is described for preparing a plasma-membrane fraction from hepatocytes by a rapid, gentle, Percoll fractionation procedure. Cholera toxin elicited the ribosylation of a number of proteins in these membranes, including the components of the stimulatory guanine nucleotide regulatory protein, Ns. Insulin, however, inhibited the ability of cholera toxin to ribosylate a protein of Mr 25 000. The action was decreased in membranes from cells that had been pre-treated with glucagon. Ribosylation of both the components of Ns and the Mr-25 000 species occurred in whole cells treated with cholera toxin, because membranes from such treated cells exhibited decreased labelling when incubated with [32P]NAD+ and activated cholera toxin. The labelling of proteins, including the Mr-25 000 species, with [32P]NAD+ and cholera toxin in the plasma membranes was decreased by an inhibitor of ribosylation. Azido-GTP photoaffinity labelling identified several high-affinity GTP-binding proteins, including one of Mr 25 000. Cholera toxin failed to ribosylate the Mr-25 000 protein in membranes from cells that had been pre-treated with the tumour-promoting agent 12-O-tetradecanoylphorbol 13-acetate (TPA). In membranes from such treated cells, insulin actually allowed cholera toxin to label this species. As TPA activates protein kinase C, it is possible that the Mr-25 000 protein, or a species that interacts with it, is a substrate for phosphorylation. These observations may offer an explanation for some of the perturbing effects that TPA exerts on insulin's action. It is suggested that the insulin receptor interacts with the guanine nucleotide regulatory protein system in the liver, and that the Mr-25 000 species may be a component of Nin, a specific guanine nucleotide regulatory protein that has been proposed to mediate certain of the actions of insulin on target cells [Houslay & Heyworth (1983) Trends Biochem. Sci. 8, 449-452].
| S-EPMC1145027 | biostudies-other
Project description:Statins such as simvastatin have many side effects, including muscle damage, which is known to be the most frequent undesirable side effect. Lysophosphatidic acid (LPA), a kind of biolipid, has diverse cellular activities, including cell proliferation, survival, and migration. However, whether LPA affects statin-linked muscle damage has not been reported yet. In the present study, to determine whether LPA might exert potential protective effect on statin-induced myocyotoxicity, the effect of LPA on cytotoxicity in rat L6 myoblasts exposed to simvastatin was explored. Viability and apoptosis of rat L6 myoblasts were detected via 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, respectively. Protein expression levels were detected via Western blotting. Simvastatin decreased viability of L6 cells. Such decrease in viability was recovered in the presence of LPA. Treatment with LPA suppressed simvastatin-induced apoptosis in L6 cells. In addition, treatment with LPA receptor inhibitor Ki16425, protein kinase C (PKC) inhibitor GF109203X, or intracellular calcium chelator BAPTA-AM attenuated the recovery effect of LPA on simvastatin-induced L6 cell toxicity. These findings indicate that LPA may inhibit simvastatin-induced toxicity in L6 cells probably by activating the LPA receptor-PKC pathway. Therefore, LPA might have potential as a bioactive molecule to protect muscles against simvastatin-induced myotoxicity.
| S-EPMC7180799 | biostudies-literature
Project description:The pleiotropic, bioactive lipid lysophosphatidic acid [(LPA), 1-acyl-sn-glycerol-3-phosphate] exerts critical regulatory actions in physiology and pathophysiology in many systems. It is present in normal bodily fluids, and is elevated in pathology (1). In vivo, "LPA" exists as distinct molecular species, each having a single fatty acid of varying chain length and degree of unsaturation covalently attached to the glycerol backbone via an acyl, alkyl, or alkenyl link. These species differ in affinities for the individual LPA receptors [(LPARs), LPA1-6] and coupling to G proteins (2). However, LPA 18:1 has been and continues to be the most commonly utilized species in reported studies. The actions of "LPA" remain poorly defined in oral biology and pathophysiology. Our laboratory has addressed this knowledge gap by studying in vitro the actions of the major human salivary LPA species [18:1, 18:0, and 16:0 (3)] in human oral cells (4-7). This includes gingival fibroblasts (GF), which our flow cytometry data from multiple donors found that they express LPA1-5 (6). We have also reported that these species are ten-fold elevated to pharmacologic levels in the saliva and gingival crevicular fluid obtained from patients with moderate-severe periodontitis (8). As the potential of LPA to regulate transcriptional activity had not been examined in the oral system, this study used whole human genome microarray analysis to test the hypothesis that LPA 18:1-treated human GF would show significant changes in gene transcripts relevant to their biology, wound-healing, and inflammatory responses. LPA 18:1 was found to significantly regulate a large, complex set of genes critical to GF biology in these categories and to periodontal disease. The raw data has been deposited at NCBI's GEO database as record GSE57496.
| S-EPMC4535903 | biostudies-literature
Project description:Autotaxin (ATX) is a multifunctional ecto-type phosphodiesterase that converts lysophospholipids, such as lysophosphatidylcholine, to lysophosphatidic acid (LPA) by its lysophospholipase D activity. LPA is a lipid mediator with diverse biological functions, most of which are mediated by G protein-coupled receptors specific to LPA (LPA1-6). Recent studies on ATX knock-out mice revealed that ATX has an essential role in embryonic blood vessel formation. However, the underlying molecular mechanisms remain to be solved. A data base search revealed that ATX and LPA receptors are conserved in wide range of vertebrates from fishes to mammals. Here we analyzed zebrafish ATX (zATX) and LPA receptors both biochemically and functionally. zATX, like mammalian ATX, showed lysophospholipase D activity to produce LPA. In addition, all zebrafish LPA receptors except for LPA5a and LPA5b were found to respond to LPA. Knockdown of zATX in zebrafish embryos by injecting morpholino antisense oligonucleotides (MOs) specific to zATX caused abnormal blood vessel formation, which has not been observed in other morphant embryos or mutants with vascular defects reported previously. In ATX morphant embryos, the segmental arteries sprouted normally from the dorsal aorta but stalled in midcourse, resulting in aberrant vascular connection around the horizontal myoseptum. Similar vascular defects were not observed in embryos in which each single LPA receptor was attenuated by using MOs. Interestingly, similar vascular defects were observed when both LPA1 and LPA4 functions were attenuated by using MOs and/or a selective LPA receptor antagonist, Ki16425. These results demonstrate that the ATX-LPA-LPAR axis is a critical regulator of embryonic vascular development that is conserved in vertebrates.
| S-EPMC3243515 | biostudies-literature
Project description:Envenomation by the brown recluse spider (Loxosceles reclusa) may cause local dermonecrosis and, rarely, coagulopathies, kidney failure and death. A venom phospholipase, SMaseD (sphingomyelinase D), is responsible for the pathological manifestations of envenomation. Recently, the recombinant SMaseD from Loxosceles laeta was demonstrated to hydrolyse LPC (lysophosphatidylcholine) to produce LPA (lysophosphatidic acid) and choline. Therefore activation of LPA signalling pathways may be involved in some manifestations of Loxosceles envenomation. To begin investigating this idea, we cloned a full-length cDNA encoding L. reclusa SMaseD. The 305 amino acid sequence of the L. reclusa enzyme is 87, 85 and 60% identical with those of L. arizonica, L. intermedia and L. laeta respectively. The recombinant enzyme expressed in bacteria had broad substrate specificity. The lysophospholipids LPC, LPI (18:1-1-oleyol lysophosphatidylinositol), LPS, LPG (18:1-1-oleoyl-lysophosphatidylglycerol), LBPA (18:1-1-oleoyl-lysobisphosphatidic acid) (all with various acyl chains), lyso-platelet-activating factor (C16:0), cyclic phosphatidic acid and sphingomyelin were hydrolysed, whereas sphingosylphosphorylcholine, PC (phosphatidylcholine; C22:6, C20:4 and C6:0), oxidized PCs and PAF (platelet-activating factor; C16:0) were not hydrolysed. The PAF analogue, edelfosine, inhibited enzyme activity. Recombinant enzyme plus LPC (C18:1) induced the migration of A2058 melanoma cells, and this activity was blocked by the LPA receptor antagonist, VPC32183. The recombinant spider enzyme was haemolytic, but this activity was absent from catalytically inactive H37N (His37-->Asn) and H73N mutants. Our results demonstrate that Loxosceles phospholipase D hydrolyses a wider range of lysophospholipids than previously supposed, and thus the term 'SMaseD' is too limited in describing this enzyme.
| S-EPMC1276930 | biostudies-literature