Actions Of Sphingosine-1-phosphate Research Articles

Sphingosine 1-phosphate evokes calcium signals in C2C12 myoblasts via Edg3 and Edg5 receptors

Sphingosine 1-phosphate (SPP) is a bioactive lipid that exerts multiple biological effects in a large variety of cell types, acting as either an intracellular messenger or an extracellular ligand coupled to Edg-family receptors (where Edg stands for endothelial differentiation gene). Here we report that in C2C12 myoblasts SPP elicited significant Ca2+ mobilization. Analysis of the process using a confocal laser-scanning microscope showed that the Ca2+ response occurred in a high percentage of cells, despite variations in amplitude and kinetics. Quantitative analysis of SPP-induced Ca2+ transients performed with a spectrophotofluorimeter showed that the rise in Ca2+ was strictly dependent on availability of extracellular Ca2+. Cell treatment with pertussis toxin partially prevented the Ca2+ response induced by SPP, indicating that Gi-coupled-receptors were involved. Indeed, SPP action was shown to be mediated by agonist-specific Edg receptors. In particular, suramin, an antagonist of the SPP-specific receptor Edg3, as well as down-regulation of Edg3 by cell transfection with antisense oligodeoxyribonucleotides (ODN), significantly reduced agonist-mediated Ca2+ mobilization. Moreover, an antisense ODN designed to inhibit Edg5 expression also decreased the SPP-induced rise in Ca2+, although to a lesser extent than that observed by inhibiting Edg3. On the contrary, the SPP response was unaffected in myoblasts loaded with antisense ODN specific for Edg1. Remarkably, the concomitant inhibition of Edg3 and Edg5 receptors abolished the SPP-induced Ca2+ increase, supporting the notion that Ca2+ mobilization in C2C12 cells induced by SPP is a receptor-mediated process that involves Edg3 and Edg5, but not Edg1.

Sphingosine 1-phosphate evokes calcium signals in C2C12 myoblasts via Edg3 and Edg5 receptors.

Sphingosine 1-phosphate (SPP) is a bioactive lipid that exerts multiple biological effects in a large variety of cell types, acting as either an intracellular messenger or an extracellular ligand coupled to Edg-family receptors (where Edg stands for endothelial differentiation gene). Here we report that in C(2)C(12) myoblasts SPP elicited significant Ca(2+) mobilization. Analysis of the process using a confocal laser-scanning microscope showed that the Ca(2+) response occurred in a high percentage of cells, despite variations in amplitude and kinetics. Quantitative analysis of SPP-induced Ca(2+) transients performed with a spectrophotofluorimeter showed that the rise in Ca(2+) was strictly dependent on availability of extracellular Ca(2+). Cell treatment with pertussis toxin partially prevented the Ca(2+) response induced by SPP, indicating that G(i)-coupled-receptors were involved. Indeed, SPP action was shown to be mediated by agonist-specific Edg receptors. In particular, suramin, an antagonist of the SPP-specific receptor Edg3, as well as down-regulation of Edg3 by cell transfection with antisense oligodeoxyribonucleotides (ODN), significantly reduced agonist-mediated Ca(2+) mobilization. Moreover, an antisense ODN designed to inhibit Edg5 expression also decreased the SPP-induced rise in Ca(2+), although to a lesser extent than that observed by inhibiting Edg3. On the contrary, the SPP response was unaffected in myoblasts loaded with antisense ODN specific for Edg1. Remarkably, the concomitant inhibition of Edg3 and Edg5 receptors abolished the SPP-induced Ca(2+) increase, supporting the notion that Ca(2+) mobilization in C(2)C(12) cells induced by SPP is a receptor-mediated process that involves Edg3 and Edg5, but not Edg1.

Sphingosine-1-Phosphate, a Platelet-Derived Lysophospholipid Mediator, Negatively Regulates Cellular Rac Activity and Cell Migration in Vascular Smooth Muscle Cells

Previous studies demonstrated that sphingosine-1-phosphate (S1P) induced migration of human umbilical vein endothelial cells (HUVECs) whereas it inhibited that of vascular smooth muscle cells (SMCs). This study explored the molecular mechanisms underlying the contrasting S1P actions on vascular cell motility. In rat and human aortic SMCs, the chemoattractant platelet-derived growth factor B-chain (PDGF) induced rapid 5- to 6-fold increases in the cellular amount of GTP-bound, active form of Rac. S1P did not affect PDGF-stimulated tyrosine phosphorylation of PDGF-beta receptor, but strongly inhibited PDGF-induced Rac activation, with a dose-response relationship similar to that for inhibition of PDGF-elicited chemotaxis. Dihydrosphingosine-1-phosphate, which is a weaker agonist for the S1P receptors, but not an inactive ligand sphingosine, also inhibited PDGF-stimulated chemotaxis and Rac activation although to lesser extents compared with S1P, suggesting that negative regulation by S1P of both chemotaxis and Rac was a receptor-mediated process. In contrast, S1P by itself stimulated Rac activity in HUVECs. Among the five S1P receptor isoforms, SMCs prominently expressed Edg-5 mRNA, whereas HUVECs expressed abundant Edg-1 mRNA but lacked detectable expression of Edg-5 mRNA. Adenovirus-mediated expression of a dominant-negative form of either Rac or Cdc42, but not RhoA, markedly attenuated chemotaxis of SMCs and HUVECs toward PDGF and S1P, respectively. Overexpression of Edg-1 in SMCs and Edg-5 in HUVECs reduced S1P-induced inhibition and stimulation, respectively, of Rac activity and migration. These results together indicate that Edg isoform-specific, negative or positive regulation of cellular Rac activity is critically involved in S1P-mediated bimodal regulation of cell motility in SMCs and HUVECs.

Sphingosine 1-phosphate induces chemotaxis of immature and modulates cytokine-release in mature human dendritic cells for emergence of Th2 immune responses.

Sphingosine 1-phosphate (S1P) is a potent extracellular lysolipid phosphoric acid mediator that is released after IgE-stimulation of mast cells. Here we investigated the biological activity and intracellular signaling of S1P on human dendritic cells (DC), which are specialized antigen presenting cells with the ability to migrate into peripheral tissues and lymph nodes, as well as control the activation of naive T cells. We show that immature and mature DC express the mRNA for different S1P receptors, such as endothelial differentiation gene (EDG)-1, EDG-3, EDG-5, and EDG-6. In immature DC, S1P stimulated pertussis toxin-sensitive Ca2+ increase actin-polymerization and chemotaxis. These responses were lost by DC matured with lipopolysaccharide. In maturing DC, however, S1P inhibited the secretion of tumor necrosis factor alpha and interleukin (IL)-12, whereas it enhanced secretion of IL-10. As a consequence, mature DC exposed to S1P showed a reduced and increased capacity to generate allogeneic Th1 and Th2 responses, respectively. In summary, our study implicates that S1P might regulate the trafficking of DC and ultimately favor Th2 lymphocyte-dominated immunity.

Extracellular Export of Sphingosine Kinase-1 Enzyme: SPHINGOSINE 1-PHOSPHATE GENERATION AND THE INDUCTION OF ANGIOGENIC VASCULAR MATURATION

The enzyme sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a bioactive lipid that acts extracellularly on G protein-coupled receptors of the S1P(1)/EDG-1 subfamily. Although S1P is formed in the cytosol of various cells, S1P release is not understood and is controversial because this lipid mediator is also regarded as a second messenger. In this report, we describe the existence of an extracellular S1P-generating system in vascular endothelial cells. Endothelial cells release SK constitutively and form S1P in the range of receptor stimulation. Levels of sphingosine but not ATP in the extracellular environment are rate-limiting. Treatment of endothelial cells with small interfering RNA for SK-1 transcript specifically inhibited SK export, and SK-1-transfected human embryonic kidney 293 cells exhibited enhanced release of SK-1. The export of SK-1 is constitutive and is inhibited by cytochalasin D and treatment at 4 degrees C but not by brefeldin A or nocodazole, suggesting that a nonclassical secretory pathway that requires the actin cytoskeleton dynamics is involved. Because S1P regulates angiogenesis and vascular maturation, we overexpressed SK-1 using an adenoviral vector in vivo in the Matrigel system of angiogenesis. Overexpression of SK-1 resulted in enhanced release of SK activity and induced angiogenesis and vascular maturation. These findings suggest that S1P is made in the extracellular milieu and that extracellular export of SK contributes to the action of S1P in the vascular system.

EDG1 Receptor Stimulation Leads to Cardiac Hypertrophy in Rat Neonatal Myocytes

Sphingosine 1 phosphate (S1P), an aminophospholipid, acts extracellularly as a ligand via the specific G protein-coupled receptors of the endothelial differentiation gene (EDG) 1, 3, 5, 6 and 8 receptors family and intracellularly as a second messenger in various cellular types. The aim of this work was to investigate biological activity of S1P in cardiomyocytes with respect to related sphingolipids. S1P was applied for 48 h on rat neonatal cardiomyocytes at 10 n m, 100 n m and 1 μ m. S1P induced a concentration-dependent cellular hypertrophy evidenced by an increase in cell size, [3H]-phenylalanine incorporation, protein content and Brain Natriuretic Peptide (BNP) secretion. Among the lipids tested S1P exhibits the lower EC50 (67 n m) followed by dihydro-S1P (107 n m) and sphingosylphosphorylcholine (1.6 μ m). The effect of S1P could be related to a stimulation of the EDG1 receptor since we showed that the EDG1 receptor is predominantly expressed at the mRNA and protein levels in rat cardiomyocytes and that specific anti-EDG1 antibodies inhibited the hypertrophic effect induced by S1P. Furthermore the expression level of most other EDG receptors for S1P appeared very low in cardiac myocytes. S1P (100 n m) increased the phosphorylation of p42/44MAPK, p38MAPK, JNK, Akt and p70S6K, this effect being reversed by inhibitors of their respective phosphorylation which also rescue the hypertrophic phenotype. Finally, S1P stimulated actin stress fibre formation reverted by the Rho inhibitor, the C3 exoenzyme. Altogether, our results show that S1P induces cardiomyocyte hypertrophy mainly via the EDG1 receptor and subsequently via Gi through ERKs, p38 MAPK, JNK, PI3K and via Rho pathway.

Sphingosine 1-Phosphate May Be a Major Component of Plasma Lipoproteins Responsible for the Cytoprotective Actions in Human Umbilical Vein Endothelial Cells

Sphingosine 1-phosphate (S1P), a novel lipid mediator, is concentrated in the fraction of lipoproteins that include high density lipoprotein (HDL) and low density lipoprotein (LDL) in human plasma. Here, we show that oxidation of LDL resulted in a marked reduction in the S1P level in association with a marked accumulation of lysophosphatidylcholine (LPC). We therefore investigated the role of the lipoprotein-associated lipids especially S1P in the lipoprotein-induced cytoprotective or cytotoxic actions in human umbilical vein endothelial cells. The viability of the cells gradually decreased in the absence of serum or growth factors in the culture medium. The addition of oxidized LDL (ox-LDL) accelerated the decrease in the cell viability. LPC and 7-ketocholesterol mimicked ox-LDL actions. On the other hand, HDL and LDL almost completely reversed the serum deprivation- or ox-LDL-induced cytotoxicity. Exogenous S1P mimicked cytoprotective actions. Moreover, the S1P-rich fraction and chromatographically purified S1P from HDL exerted cytoprotective actions, but the rest of the fractions did not. The cytoprotective actions of HDL and S1P were associated with extracellular signal-regulated kinase (ERK) activation and were almost completely inhibited by pertussis toxin and PD98059, an ERK kinase inhibitor. The HDL-induced action was specifically desensitized in the S1P-pretreated cells. Taken together, these results indicate that the lipoprotein-associated S1P and the lipid receptor-mediated signal pathways may be responsible for the lipoprotein-induced cytoprotective actions. Furthermore, the decrease in the S1P content, in addition to the accumulation of cytotoxic substances such as LPC, may be important for the acquisition of the cytotoxic property to ox-LDL.

Pleiotypic mechanisms of cellular responses to biologically active lysophospholipids

The activities of cell-derived lysophospholipid (LPL) growth factors on cellular proliferation and a range of proliferation-independent functions are regulated at multiple levels. This section focuses first on the capacity of the actin-severing protein gelsolin to bind lysophosphatidic acid (LPA), but not sphingosine 1-phosphate (S1P), and either sequester LPA or present it to responsive cells. Expression of members of the family of endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs) for LPLs is controlled developmentally and by cell-activating stimuli. Edg R transduction of cellular effects of LPLs involves both direct actions on target cells and induction of generation of proteins with relevant actions capable of amplifying or diminishing primary direct effects of LPLs. These general mechanisms are evident in Edg R mediation of proliferation, cytokine secretion and suppression of apoptosis. The availability of functionally-active anti-Edg R antibodies and Edg R-specific pharmacological probes, establishment of Edg R transgenes and gene knockouts, and identification of natural genetic anomalies of LPL metabolism and recognition by Edg Rs will permit elucidation of the in vivo activities of LPA and S1P normally and in disease states.

Sphingosine 1-Phosphate and Activation of Endothelial Nitric-oxide Synthase: DIFFERENTIAL REGULATION OF Akt AND MAP KINASE PATHWAYS BY EDG AND BRADYKININ RECEPTORS IN VASCULAR ENDOTHELIAL CELLS

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elicits numerous biological responses in endothelial cells mediated by a family of G protein-coupled EDG receptors. Stimulation of EDG receptors by S1P has been shown to activate the endothelial isoform of nitric-oxide synthase (eNOS) in heterologous expression systems (Igarashi, J., and Michel, T. (2000) J. Biol. Chem. 275, 32363-32370). However, the signaling pathways that modulate eNOS regulation by S1P/EDG in vascular endothelial cells remain less well understood. We now report that S1P treatment of bovine aortic endothelial cells (BAEC) acutely increases eNOS enzyme activity; the EC(50) for S1P activation of eNOS is approximately 10 nm. The magnitude of eNOS activation by S1P in BAEC is equivalent to that elicited by the agonist bradykinin. S1P treatment activates Akt, a protein kinase implicated in phosphorylation of eNOS. S1P treatment of BAEC leads to eNOS phosphorylation at Ser(1179), a residue phosphorylated by Akt; an eNOS mutant in which this Akt phosphorylation site is inactivated shows attenuated S1P-induced eNOS activation. S1P-induced activation both of Akt and of eNOS is inhibited by pertussis toxin, by the phosphoinositide 3-kinase inhibitor wortmannin, and by the intracellular calcium chelator BAPTA (1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). By contrast to S1P, activation of G protein-coupled bradykinin B2 receptors neither activates kinase Akt nor promotes Ser(1179) eNOS phosphorylation despite robustly activating eNOS enzyme activity. Understanding the differential regulation of protein kinase pathways by S1P and bradykinin may lead to the identification of new points for eNOS regulation in vascular endothelial cells.

Extracellular mechanism through the Edg family of receptors might be responsible for sphingosine-1-phosphate-induced regulation of DNA synthesis and migration of rat aortic smooth-muscle cells

Exogenous sphingosine 1-phosphate (S1P) increased cytosolic Ca(2+) concentration, stimulated thymidine incorporation (DNA synthesis) and inhibited cell migration in rat aortic smooth-muscle cells (AoSMCs). Although exogenous sphingosine, a substrate of sphingosine kinase or a precursor of S1P, markedly induced the intracellular accumulation of S1P, the lipid failed to mimic the S1P-induced actions. In contrast, dihydrosphingosine 1-phosphate (DHS1P), an S1P receptor agonist, duplicated these S1P actions even though DHS1P was approx. 20-50-fold less potent than S1P. The pharmacological properties of DHS1P for the S1P receptor subtypes Edg-1, Edg-3, Edg-5 and Edg-6 were compared in Chinese hamster ovary (CHO) cells that were overexpressing the respective receptor. In these S1P-receptor-overexpressing cells, DHS1P was approx. 20-30-fold less potent than S1P for the displacement of [(3)H]S1P binding and inositol phosphate response in Edg-5-expressing CHO cells, as was the case for AoSMCs. However, it was slightly (not more than 3-fold) less potent than S1P in cells expressing Edg-1, Edg-3 or Edg-6. Of the above-mentioned four types of S1P receptor, Edg-5 was abundantly expressed in AoSMCs, as demonstrated by Northern blotting. These results suggest that the intracellular accumulation of S1P is not necessary for the S1P-induced Ca(2+) response, for the stimulation of DNA synthesis or for the inhibition of cell migration. Thus these S1P-induced actions might be mediated through extracellular (or cell-surface) S1P receptors in AoSMCs: Edg-5 might be a most important receptor subtype.

Differential Coupling of the Sphingosine 1-Phosphate Receptors Edg-1, Edg-3, and H218/Edg-5 to the Gi, Gq, and G12 Families of Heterotrimeric G Proteins

Sphingosine 1-phosphate (S1P) is one of several bioactive phospholipids that exert profound mitogenic and morphogenic actions. Originally characterized as a second messenger, S1P is now recognized to achieve many of its effects through cell surface, G protein-coupled receptors. We used a subunit-selective [(35)S]GTPgammaS binding assay to investigate whether the variety of actions exerted through Edg-1, a recently identified receptor for S1P, might be achieved through multiple G proteins. We found, employing both Sf9 and HEK293 cells, that Edg-1 activates only members of the G(i) family, and not G(s), G(q), G(12), or G(13). We additionally established that Edg-1 activates G(i) in response not only to S1P but also sphingosylphosphorylcholine; no effects of lysophosphatidic acid through Edg-1 were evident. Our assays further revealed a receptor(s) for S1P endogenous to HEK293 cells that mediates activation of G(13) as well as G(i). Because several of the biological actions of S1P are assumed to proceed through the G(12/13) family, we tested whether Edg-3 and H218/Edg-5, two other receptors for S1P, might have a broader coupling profile than Edg-1. Indeed, Edg-3 and H218/Edg-5 communicate not only with G(i) but also with G(q) and G(13). These studies represent the first characterization of S1P receptor activity through G proteins directly and establish fundamental differences in coupling.

Assessment of the Extracellular and Intracellular Actions of Sphingosine 1-phosphate by Using the p42/p44 Mitogen-Activated Protein Kinase Cascade as a Model

We have investigated the extracellular and intracellular actions of sphingosine 1-phosphate (S1P) by using cultured airway smooth muscle cells. We have demonstrated that exogenous S1P elicited an activation of mitogen-activated protein kinase (p42/p44 MAPK) that was abolished by pertussis toxin (0.1 μg/mL, 24 h), which was used to inactivate G i. The effect of exogenous S1P might therefore be attributed to an action at a putative G i-coupled receptor. The regulation of the p42/p44 MAPK cascade by S1P was also shown to include a protein kinase C (PKC)-dependent intermediate step. Platelet-derived growth factor (PDGF) stimulates intracellular S1P formation and was therefore used to evaluate the intracellular action of S1P. This has previously been investigated by others using the sphingosine kinase inhibitors d, l- threo-dihydrosphingosine and N,N-dimethylsphingosine. We have demonstrated here that both inhibitors block the PDGF-dependent activation of p42/p44 MAPK. However, both are also PKC inhibitors, which might account for their effect because PDGF utilises PKC as an intermediate in the regulation of the p42/p44 MAPK cascade. Significantly, sphingosine, which is the substrate of sphingosine kinase and a PKC inhibitor, blocked the activation of p42/p44 MAPK by PDGF with an almost identical concentration dependence compared with d, l- threo-dihydrosphingosine and N,N-dimethylsphingosine. Therefore, the use of so-called sphingosine kinase inhibitors might lead to misleading interpretations because of their additional effect on PKC. Other approaches, such as oligodeoxynucleotide anti-sense against sphingosine kinase, are required to address the intracellular role of S1P.

Sphingosine 1-phosphate stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle

We report here that cultured airway smooth muscle cells contain transcripts of endothelial differentiation gene 1 (EDG-1), a prototypical orphan Gi-coupled receptor whose natural ligand is sphingosine 1-phosphate (S1P). This is consistent with data that showed that S1P activated both c-Src and p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) in a pertussis toxin (PTX)-sensitive manner in these cells. An essential role for c-Src was confirmed by using the c-Src inhibitor, PP1, which markedly decreased p42/p44 MAPK activation. We have also shown that phosphoinositide 3-kinase (PI-3K) inhibitors (wortmannin and LY294002) decreased p42/p44 MAPK activation. An essential role for PI-3K was supported by experiments that showed that PI-3K activity was increased in Grb-2 immunoprecipitates from S1P-stimulated cells. Significantly, Grb-2 associated PI-3K activity was decreased by pretreatment of cells with PTX. Finally, we have shown that the co-stimulation of cells with platelet-derived growth factor (PDGF) and S1P (which failed to stimulate DNA synthesis) elicited a larger p42/p44 MAPK activation over a 30 min stimulation compared with each agonist alone. This was associated with a S1P-dependent increase in PDGF-stimulated DNA synthesis. These results demonstrate that S1P activates c-Src and Grb-2-PI-3K (intermediates in the p42/p44 MAPK cascade) via a PTX-sensitive mechanism. This action of S1P is consistent with the stimulation of EDG-1 receptors. S1P might also function as a co-mitogen with PDGF, producing a more robust activation of a common permissive signal transduction pathway linked to DNA synthesis.

Sphingosine 1-phosphate stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle

We report here that cultured airway smooth muscle cells contain transcripts of endothelial differentiation gene 1 (EDG-1), a prototypical orphan Gi-coupled receptor whose natural ligand is sphingosine 1-phosphate (S1P). This is consistent with data that showed that S1P activated both c-Src and p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) in a pertussis toxin (PTX)-sensitive manner in these cells. An essential role for c-Src was confirmed by using the c-Src inhibitor, PP1, which markedly decreased p42/p44 MAPK activation. We have also shown that phosphoinositide 3-kinase (PI-3K) inhibitors (wortmannin and LY294002) decreased p42/p44 MAPK activation. An essential role for PI-3K was supported by experiments that showed that PI-3K activity was increased in Grb-2 immunoprecipitates from S1P-stimulated cells. Significantly, Grb-2 associated PI-3K activity was decreased by pretreatment of cells with PTX. Finally, we have shown that the co-stimulation of cells with platelet-derived growth factor (PDGF) and S1P (which failed to stimulate DNA synthesis) elicited a larger p42/p44 MAPK activation over a 30 min stimulation compared with each agonist alone. This was associated with a S1P-dependent increase in PDGF-stimulated DNA synthesis. These results demonstrate that S1P activates c-Src and Grb-2–PI-3K (intermediates in the p42/p44 MAPK cascade) via a PTX-sensitive mechanism. This action of S1P is consistent with the stimulation of EDG-1 receptors. S1P might also function as a co-mitogen with PDGF, producing a more robust activation of a common permissive signal transduction pathway linked to DNA synthesis.

Sphingosine 1-phosphate signalling through the G-protein-coupled receptor Edg-1.

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are structurally related lipid mediators that act on distinct G-protein-coupled receptors to evoke similar responses, including Ca2+ mobilization, adenylate cyclase inhibition, and mitogen-activated protein (MAP) kinase activation. However, little is still known about the respective receptors. A recently cloned putative LPA receptor (Vzg-1/Edg-2) is similar to an orphan Gi-coupled receptor termed Edg-1. Here we show that expression of Edg-1 in Sf9 and COS-7 cells results in inhibition of adenylate cyclase and activation of MAP kinase (Gi-mediated), but not Ca2+ mobilization, in response to S1P. These responses are specific in that (i) S1P action is not mimicked by LPA, and (ii) Vzg-1/Edg-2 cannot substitute for Edg-1. Thus the Edg-1 receptor is capable of mediating a subset of the cellular responses to S1P.

Signaling mechanisms and molecular characteristics of G protein-coupled receptors for lysophosphatidic acid and sphingosine 1-phosphate.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are potent phospholipid mediators with diverse biological activities. Their appearance and functional properties suggest possible roles in development, wound healing, and tissue regeneration. The growth-stimulating and other complex biological activities of LPA and S1P are attributable in part to the activation of multiple G protein-mediated intracellular signaling pathways. Several heterotrimeric G proteins, as well as Ras- and Rho-dependent pathways play central roles in the cellular responses to LPA and S1P. Recently, several G protein-coupled receptors encoded by a family of endothelial differentiation genes (edg) have been shown to bind LPA or S1P and transduce responses of cAMP, Ca2+ , MAP kinases, Rho, and gene transcription. This review summarizes our current understanding of signaling pathways critical for cellular responses to LPA and S1P and of recent progress in the molecular biological analyses of the Edg receptors. J. Cell. Biochem. Suppls. 30/31:147-157, 1998. © 1998 Wiley-Liss, Inc.

Phosphocholine and sphingosine-1-phosphate synergistically stimulate DNA synthesis by a MAP kinase-dependent mechanism

We have previously shown that in NIH 3T3 fibroblasts phosphocholine (PCho) potentiates sphingosine-1-phosphate (S1P)-induced mitogenesis. Here we report that PCho and S1P also synergistically stimulate DNA synthesis in mouse Swiss 3T3 fibroblasts and in mouse JB6 epidermal cells. The combined actions of PCho and S1P on DNA synthesis were associated with synergistic activation of the p42/p44 mitogen-activated protein (MAP) kinases. Ethanolamine (50–100 μM) further enhanced the synergistic effects of PCho and S1P on DNA synthesis but not on MAP kinase activity. The results indicate that the synergistic mitogenic effects of PCho and S1P (i) are not restricted to NIH 3T3 fibroblasts, (ii) are predominantly mediated by the MAP kinase-dependent signal transduction pathway, and (iii) are enhanced by ethanolamine via a MAP kinase-independent mechanism.

Sphingosine 1-phosphate stimulates hydrogen peroxide generation through activation of phospholipase C-Ca2+ system in FRTL-5 thyroid cells: possible involvement of guanosine triphosphate-binding proteins in the lipid signaling.

Exogenous sphingosine 1-phosphate (S1P) stimulated hydrogen peroxide (H2O2) generation in association with an increase in intracellular Ca2+ concentration in FRTL-5 thyroid cells. S1P also induced inositol phosphate production, reflecting activation of phospholipase C (PLC) in the cells. These three S1P-induced events were inhibited partially by pertussis toxin (PTX) and markedly by U73122, a PLC inhibitor, and were conversely potentiated by N6-(L-2-phenylisopropyl)adenosine, an A1-adenosine receptor agonist. In FRTL-5 cell membranes, S1P also activated PLC in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), but not in its absence. Guanosine 5'-O-(2-thiodiphosphate) inhibited the S1P-induced GTP gamma S-dependent activation of the enzyme. To characterize the signaling pathways, especially receptors and G proteins involved in the S1P-induced responses, cross-desensitization experiments were performed. Under the conditions where homologous desensitization occurred in S1P-, lysophosphatidic acid (LPA)-, and bradykinin-induced induction of Ca2+ mobilization, no detectable cross-desensitization of S1P and bradykinin was observed. This suggests that the primary action of S1P in its activation of the PLC-Ca2+ system was not the activation of G proteins common to S1P and bradykinin, but the activation of a putative S1P receptor. On the other hand, there was a significant cross-desensitization of S1P and LPA; however, a still significant response to S1P (50-80% of the response in the nontreated control cells) was observed depending on the lipid dose employed after a prior LPA challenge. S1P also inhibited cAMP accumulation in a PTX-sensitive manner. We conclude that S1P stimulates H2O2 generation through a PLC-Ca2+ system and also inhibits adenylyl cyclase in FRTL-5 thyroid cells. The S1P-induced responses may be mediated partly through a putative lipid receptor that is coupled to both PTX-sensitive and insensitive G proteins.

Sphingosine 1-Phosphate Stimulates Hydrogen Peroxide Generation through Activation of Phospholipase C-Ca2+ System in FRTL-5 Thyroid Cells: Possible Involvement of Guanosine Triphosphate-Binding Proteins in the Lipid Signaling

Exogenous sphingosine 1-phosphate (S1P) stimulated hydrogen peroxide (H2O2) generation in association with an increase in intracellular Ca2+ concentration in FRTL-5 thyroid cells. S1P also induced inositol phosphate production, reflecting activation of phospholipase C (PLC) in the cells. These three S1P-induced events were inhibited partially by pertussis toxin (PTX) and markedly by U73122, a PLC inhibitor, and were conversely potentiated by N6-(l-2-phenylisopropyl)adenosine, an A1-adenosine receptor agonist. In FRTL-5 cell membranes, S1P also activated PLC in the presence of guanosine 5′-O-(3-thiotriphosphate) (GTPγS), but not in its absence. Guanosine 5′-O-(2-thiodiphosphate) inhibited the S1P-induced GTPγS-dependent activation of the enzyme. To characterize the signaling pathways, especially receptors and G proteins involved in the S1P-induced responses, cross-desensitization experiments were performed. Under the conditions where homologous desensitization occurred in S1P-, lysophosphatidic acid (LPA)-, and bradykinin-induced induction of Ca2+ mobilization, no detectable cross-desensitization of S1P and bradykinin was observed. This suggests that the primary action of S1P in its activation of the PLC-Ca2+ system was not the activation of G proteins common to S1P and bradykinin, but the activation of a putative S1P receptor. On the other hand, there was a significant cross-desensitization of S1P and LPA; however, a still significant response to S1P (50–80% of the response in the nontreated control cells) was observed depending on the lipid dose employed after a prior LPA challenge. S1P also inhibited cAMP accumulation in a PTX-sensitive manner. We conclude that S1P stimulates H2O2 generation through a PLC-Ca2+ system and also inhibits adenylyl cyclase in FRTL-5 thyroid cells. The S1P-induced responses may be mediated partly through a putative lipid receptor that is coupled to both PTX-sensitive and insensitive G proteins.

Sphingosine-1-phosphate rapidly induces Rho-dependent neurite retraction: action through a specific cell surface receptor.

Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid implicated in mitogenesis and cytoskeletal remodelling, but its mechanism of action is poorly understood. We report here that in N1E-115 neuronal cells, S1P mimics the G protein-coupled receptor agonist lysophosphatidic acid (LPA) in rapidly inducing neurite retraction and soma rounding, a process driven by Rho-dependent contraction of the actin cytoskeleton. S1P is approximately 100-fold more potent than LPA in evoking these shape changes, with an EC50 as low as 1.5 nM. Microinjection of S1P has no effect, neither has addition of sphingosine or ceramide. As with LPA, S1P action is inhibited by suramin and subject to homologous desensitization; however, the responses to S1P and LPA do not show cross-desensitization. We conclude that S1P activates its own high affinity receptor to trigger Rho-regutated cytoskeletal events. Thus, S1P and LPA may belong to an emerging family of bioactive lysophospholipids that act through distinct G protein-coupled receptors to mediate similar actions.