sphingosine-1-phosphate and Glioma

S1P (sphingosine 1-phosphate) is the ligand for a family of specific G-protein-coupled receptors that regulate a wide variety of important cellular functions, including vascular maturation, angiogenesis, cell growth, survival, cytoskeletal rearrangements and cell motility. However, S1P also may have intracellular functions. In this review, we discuss two examples that clearly indicate that intracellularly generated and exogenous S1P can regulate biological processes by divergent pathways.

Topics: Biopterins; Cell Division; Cell Line, Tumor; Cell Survival; Glioma; Humans; Lysophospholipids; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Sphingosine; Tumor Necrosis Factor-alpha

Topics: Animals; Antineoplastic Agents; Azo Compounds; Blood-Brain Barrier; Brain; Brain Neoplasms; Caveolin 1; Drug Compounding; Drug Liberation; Glioblastoma; Glioma; Glutathione Transferase; Humans; Liposomes; Lysophospholipids; Mice; Neoplasms, Experimental; Nitric Oxide; Piperazines; Prodrugs; Sphingosine; Sphingosine-1-Phosphate Receptors; Ultrasonography

The clarification of mechanisms that negatively regulate the invasive behavior of human glioma cells is of great importance in order to find new methods of treatment. In this study, we have focused on the negative regulation of lysophosphatidic acid (LPA)-induced migration in glioma cells. Using small interference RNA and dominant-negative gene strategies in addition to pharmacological tools, we found that isoproterenol (ISO) and sphingosine-1-phosphate (S1P) negatively but differently regulate the LPA-induced migration. ISO-induced suppression of the migration of glioma cells occurs via beta(2)-adrenergic receptor/cAMP/Epac/Rap1B/inhibition of Rac, whereas S1P has been shown to suppress the migration of the cells through S1P(2) receptor/Rho-mediated down-regulation of Rac1. The expression of tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is required for the inhibitory ISO-induced and Rap1B-mediated actions on the migration, Rac1 activation, and Akt activation in response to LPA. Thus, the PTEN-mediated down-regulation of phosphatidylinositol 3-kinase activity may be involved in the regulation of Rap1B-dependent inhibition of Rac1 activity. These findings suggest that there are at least two distinct inhibitory pathways, which are mediated by the S1P(2) receptor and beta(2)-adrenergic receptor, to control the migratory, hence invasive, behavior of glioma cells.

Topics: Cell Movement; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Glioma; Humans; Isoproterenol; Lysophospholipids; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; rac1 GTP-Binding Protein; rap GTP-Binding Proteins; Sphingosine

Sphingosine 1-phosphate (S1P) induced the inhibition of glioma cell migration. Here, we characterized the signaling mechanisms involved in the inhibitory action by S1P. In human GNS-3314 glioblastoma cells, the S1P-induced inhibition of cell migration was associated with activation of RhoA and suppression of Rac1. The inhibitory action of S1P was recovered by a small interference RNA specific to S1P(2) receptor, a carboxyl-terminal region of Galpha12 or Galpha13, an RGS domain of p115RhoGEF, and a dominant-negative mutant of RhoA. The inhibitory action of S1P through S1P(2) receptors was also observed in both U87MG glioblastoma and 1321N1 astrocytoma cells, which have no protein expression of a phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These results suggest that S1P(2) receptors/G(12/13)-proteins/Rho signaling pathways mediate S1P-induced inhibition of glioma cell migration. However, PTEN, recently postulated as an indispensable molecule for the inhibition of cell migration, may not be critical for the S1P(2) receptor-mediated action in glioma cells.

Topics: Cell Line, Tumor; Cell Movement; Glioma; GTP-Binding Protein alpha Subunits, G12-G13; Humans; Lysophospholipids; PTEN Phosphohydrolase; rac1 GTP-Binding Protein; Receptors, Lysosphingolipid; rhoA GTP-Binding Protein; Signal Transduction; Sphingosine

Treatment with isoprenaline led to a change in the cell morphology of rat C6 glioma cells. This morphological change was reverted by the addition of sphingosine 1-phosphate (S1P). Using this morphological change as a response marker we determined that DS-SG-44 ((2S,3R)-2-amino-3-hydroxy-4-(4-octylphenyl)butyl phosphoric acid) was an agonist of S1P receptors. The DS-SG-44-induced morphological reversion was not observed with such structurally related molecules as DS-SG-45 ((2S,3R)-2-amino-3-hydroxy-4-(3-octylphenyl)butyl phosphoric acid) and DS-SG-12 ((2S,3R)-2-amino-4-(4-octylphenyl)butane-1,3-diol). The S1P- and DS-SG-44-induced shape changes were neither reproduced with the S1P1/S1P3 receptor agonist VPC24191 nor inhibited by the S1P1/S1P3 receptor antagonist, VPC23019. Transfection with small interfering RNA (siRNA) for the S1P2 receptor greatly inhibited the DS-SG-44-induced shape change, and in part an S1P-induced response. In the presence of VPC23019, siRNA transfection for the S1P2 receptor almost completely blocked the S1P- and DS-SG-44-induced shape changes. Our results suggested that DS-SG-44, a newly-synthesized S1P analogue, acted as an S1P receptor agonist and that the S1P-induced shape change in rat C6 glioma cells was mediated mainly through the S1P2 receptor, and cooperatively through the S1P1/S1P3 receptors.

Topics: Analysis of Variance; Animals; Cell Shape; Glioma; Lysophospholipids; Phosphoric Acids; Rats; Receptors, Lysosphingolipid; Sphingosine; Tumor Cells, Cultured

Sphingosine-1-phosphate (S1P) caused dose-dependent and time-dependent increases in c-fos mRNA. Pretreatment with pertussis toxin (PTX; 100 ng/mlx24 h) reduced c-fos activation by S1P (100 microM-187+/-6% vs. 411+/-27%) and lysophosphatidic acid (LPA; 100 microM-90+/-34% vs. 188+/-41%), but not by sphingosylphosphorylcholine (SPC; 100 microM-390+/-47% vs. 420+/-44%). RT-PCR analysis and sequencing demonstrated the presence of previously unidentified LPA-responsive Endothelial Differentiation Gene (EDG) receptor mRNAs in C6 cells: EDG-2 and EDG-4.

Topics: Animals; Blotting, Northern; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Interactions; Estrenes; Gene Expression; Genes, fos; Glioma; Lysophospholipids; Mice; Pertussis Toxin; Phosphodiesterase Inhibitors; Pyrrolidinones; Receptors, Lysophosphatidic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sphingosine; Time Factors

A potential role for 1-oleoyl-sn-glycero-3-phosphate or lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) in the regulation of malignant diseases has been widely considered. In this study, we found that in transformed astroglial cells, the expression profile of lysophospholipid receptor mRNA and the action modes of LPA and S1P on cell motility were changed: there was a change in the acquisition of the ability of LPA to stimulate cell migration and a change in the migratory response to S1P from stimulation through S1P(1) to inhibition through S1P(2). LPA-induced cell migration was almost completely inhibited by either pertussis toxin, LPA(1) receptor antagonists including Ki16425 (3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfonyl)propanoic acid) or an inhibitor of phosphatidylinositol 3-kinase (PI3K) wortmannin. The LPA-induced action was also suppressed, although incompletely, by several specific inhibitors for intracellular signaling pathways including Rac1, Cdc42, p38 mitogen-activated protein kinase (p38MAPK) and c-Jun terminal kinase (JNK), but not extracellular signal-regulated kinase. Nearly complete inhibition of migration response to LPA, however, required simultaneous inhibition of both the p38MAPK and JNK pathways. Inhibition of Rac1 suppressed JNK but not p38MAPK, while the activity of p38MAPK was abolished by a dominant-negative form of Cdc42. These findings suggest that, in glioma cells, the PI3K/Cdc42/p38MAPK and PI3K/Rac1/JNK pathways are equally important for LPA(1) receptor-mediated migration.

Topics: Animals; Cell Line, Tumor; Cell Movement; Glioma; JNK Mitogen-Activated Protein Kinases; Lysophospholipids; p38 Mitogen-Activated Protein Kinases; Pertussis Toxin; Rats; Sphingosine

Sphingolipids participate in membrane structure and signaling in neuronal cells, and an emerging strategy for control of gliomas is to inhibit growth and/or induce apoptosis using ceramide and ceramide analogs. Nonetheless, some sphingolipids (ceramides and sphingosine) induce and others (sphingosine 1-phosphate) inhibit apoptosis; therefore, when testing putative anti-cancer agents, it is critical to obtain precise knowledge of the types and quantities of not only the test compounds, but also their effects on endogenous species. Combination of liquid chromatography and tandem mass spectrometry affords a "metabolomic" profile of all of the intermediates of ceramide biosynthesis (3-ketosphinganine, sphinganine and dihydroceramides) and the direct products of ceramide metabolism (sphingomyelins and monohexosylceramides as well as sphingosine and sphingosine 1-phosphate). This method has been applied to four human glioma cell lines (LN18, LN229, LN319 and T98G), and differences in the amounts and types of sphingolipids were found. For example, LN229 and LN319 have approximately twice the sphingosine 1-phosphate of LN18 and T98G; LN229 and LN319 have more monohexosylceramides than lactosylceramides, whereas the opposite is the case for LN18 and T98G; and the fatty acyl chain distributions of the sphingolipids differ among the cell lines. The ability to obtain this type of "metabolomic" profile allows studies of how anti-cancer agents (especially sphingolipids and sphingolipid analogs) affect the amounts of these bioactive species, and may lead to a better understanding of the abnormal phenotypes of gliomas.

Topics: Astrocytoma; Cell Line, Tumor; Ceramides; Chromatography, High Pressure Liquid; Fatty Acids; Galactosylceramides; Glioblastoma; Glioma; Glucosylceramides; Humans; Lactosylceramides; Lysophospholipids; Molecular Structure; Spectrometry, Mass, Electrospray Ionization; Sphingolipids; Sphingomyelins; Sphingosine

The regulation of glioma cell proliferation by sphingosine-1-phosphate (S1P) was studied using the human glioblastoma cell line U-373 MG. U-373 MG cells responded mitogenically to nanomolar concentrations of S1P, and express mRNA encoding the S1P receptors S1P1/endothelial differentiation gene (EDG)-1, S1P3/EDG-3 and S1P2/EDG-5. S1P-induced proliferation required extracellular signal-regulated kinase activation and was partially sensitive to pertussis toxin and wortmannin, indicating involvement of a Gi-coupled receptor and phosphatidylinositol 3-kinase. Moreover, S1P1, S1P3 and S1P2 receptors are expressed in the majority of human glioblastomas as determined by reverse transcriptase-polymerase chain reaction analysis. Thus, S1P signaling through EDG receptors may contribute to glioblastoma growth in vivo.

Topics: Androstadienes; Cell Division; DNA; Glioma; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Lysophospholipids; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; Sphingosine; Tumor Cells, Cultured; Wortmannin

Several cDNA encoding G-protein-coupled receptors, i.e. Edg-1,-3,-5,-6 and -8, have recently been identified as sphingosine 1-phosphate (S1P) receptors. However, the role of the respective receptor subtype has not been well defined. In C6 glioma cells, exogenous S1P induced expression of fibroblast growth factor-2 (FGF-2), a potent neurotrophic factor, which was associated with the stimulation of extracellular signal-regulated kinase (ERK) and the expression of early growth response-1 (Egr-1). S1P also stimulated phospholipase C (PLC)/Ca(2+) system and phospholipase D (PLD). In this study, we sought to identify S1P receptors responsible for these S1P-induced actions. Of five S1P receptor subtypes, Edg-1 and Edg-5 are expressed in the glioma cells, as evidenced by Northern blotting. We therefore prepared the cells overexpressing these S1P receptor subtypes and compared the intrinsic activities to stimulate these signaling pathways and their sensitivity to pertussis toxin (PTX). The potency of S1P and dihydrosphingosine 1-phosphate (DHS1P), another S1P receptor agonist, to stimulate the Edg-1 and Edg-5 receptors was also examined. We found that the intrinsic activity that stimulated ERK/Egr-1/FGF-2 system was much higher in Edg-1 than in Edg-5. Furthermore, DHS1P was as potent as S1P in activating ERK in control C6 cells, a pattern also observed in cells overexpressing Edg-1. On the other hand, the stimulation of the PLC/Ca(2+) system and PLD induced by S1P was PTX-insensitive, and the potency of S1P in activating PLD was roughly one order higher than that of DHS1P in control C6 cells; similar responsiveness to such pharmacological tools were observed in Edg-5-overexpressing cells. Taken together, these results suggest that Edg-1 may be the main receptor mediating the stimulation of ERK/Egr-1/FGF-2 system but that Edg-5 may be responsible for the stimulation of PLC-Ca(2+) system and PLD in native C6 glioma cells.

Topics: Animals; Calcium; Fibroblast Growth Factor 2; Gene Expression; Glioma; GTP-Binding Proteins; Immediate-Early Proteins; Lysophospholipids; MAP Kinase Signaling System; Neurons; Pertussis Toxin; Rats; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid; RNA, Messenger; Sphingosine; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

The early signaling mechanism of sphingosine 1-phosphate (S1P) on extracellular signal-regulated kinase (ERK) activation was investigated in C6 glioma cells. S1P activated the enzyme in association with a shift in the mobility on electrophoresis reflecting phosphorylation of both ERK1/ERK2 at as low as 10 nM. The lipid-induced ERK1/2 activation was partially inhibited by treatment of the cells with either phorbol 12-myristate 13-acetate (a long-term treatment to desensitize protein kinase C) or pertussis toxin (PTX) and was completely inhibited by a simultaneous treatment with both agents. Similarly, either calphostin C, an inhibitor of protein kinase C, or U73122, an inhibitor of phospholipase C, partially inhibited the S1Pinduced ERK1/2 activation in the nontreated cells with PTX and completely in the toxin-treated cells. On the other hand, the S1P-induced ERK activation was hardly affected by ethanol, which switched the product of phospholipase D from phosphatidic acid to metabolism-resistant phosphatidylethanol. S1P was able to activate ERK1/2 without a detectable increase in the intracellular content of the lipid, but sphingosine, a substrate of sphingosine kinase, which is an enzyme for S1P generation in the cells, hardly affected the ERK1/2 activation in spite of a marked elevation of intracellular S1P accumulation. This indicates that intracellular increase in S1P is not necessary for the S1P-induced ERK activation, and hence suggests the extracellular action mechanism of S1P. Supporting this idea, mRNAs of recently identified S1P specific receptors, Edg-1 and AGR16/H218, were expressed in C6 cells. Taken together, these results suggested that S1P acts on C6 cells extracellularly possibly through S1P receptors which are linked to at least two signaling pathways, i.e., the PTX-sensitive Gi/Go protein pathway and the toxin-insensitive Gq/G11-phospholipase C-PKC pathway, resulting in the activation of ERK.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; CHO Cells; Cricetinae; Estrenes; Glioma; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Pertussis Toxin; Phospholipase D; Protein Kinase C; Pyrrolidinones; Rats; Receptors, Cell Surface; Sphingosine; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

In the present study we investigate the effect of exogenous sphingosine, sphingosine 1-phosphate and sphingosylphosphorylcholine on phospholipase D (PLD) activity in glioma C6 cells. The cells were prelabeled with [1-14C]palmitic acid and PLD-mediated synthesis of [14C]phosphatidylethanol was measured. Sphingosine 1-phosphate and sphingosylphosphorylcholine did not stimulate [14C]phosphatidylethanol formation either at low (0.1-10 microM) or high (25-100 microM) concentrations. On the other hand, sphingosine at concentrations of 100-250 microM strongly stimulated PLD activity as compared to the effect of phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), known as a PLD activator. The effect of TPA on PLD is linked to the activation of protein kinase C. The present study also shows that sphingosine additively enhances TPA-mediated PLD activity. This is in contrast to the postulated role of sphingosine as a protein kinase C inhibitor. These results demonstrate that in glioma C6 cells sphingosine not only affects PLD independently of its effect on protein kinase C, but also is unable to block TPA-mediated PLD activity.

Topics: Brain Neoplasms; Enzyme Activation; Glioma; Lysophospholipids; Phospholipase D; Phosphorylcholine; Sphingosine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The effect of sphingosine, sphingosylphosphorylcholine and sphingosine 1-phosphate on L-[U-14C]serine incorporation into phosphatidylserine and phosphatidylserine-derived phosphatidylethanolamine was investigated in intact glioma C6 cells. Sphingosine, sphingosylphosphorylcholine and sphingosine 1-phosphate are potent signalling molecules which, due to their physicochemical features, may function as amphiphilic compounds. It has been found that sphingosine and sphingosylphosphorylcholine (amphiphilic cations) significantly increase [14C]phosphatidylserine synthesis and decrease the amount of 14C-labeled phosphatidylethanolamine. Sphingosine 1-phosphate (an amphiphilic anion) was without effect on phosphatidylserine synthesis but, similarly as sphingosine and sphingosylphosphorylcholine, reduced the conversion of phosphatidylserine to phosphatidylethanolamine. These results strongly suggest that sphingosine, sphingosylphosphorylcholine and sphingosine 1-phosphate can modulate cellular phospholipid homeostasis by stimulation of phosphatidylserine synthesis and an interference with phosphatidylserine decarboxylase.

Topics: Brain Neoplasms; Glioma; Homeostasis; Lysophospholipids; Phosphorylcholine; Sphingosine; Tumor Cells, Cultured

In rat type I astrocytes and C6 glioma cells, sphingosine 1-phosphate (S1P) clearly induced the expression of fibroblast growth factor-2 (FGF-2) mRNA to an extent comparable to that achieved by platelet-derived growth factor (PDGF) and endothelin. In C6 cells, Western blotting showed that S1P also induced expression of early growth response-1 (Egr-1), one of the immediate early gene products and an essential transcriptional factor for FGF-2 expression. On the other hand, sphingosine, a substrate for sphingosine kinase which forms intracellular S1P, was a very weak activator for the expression of either FGF-2 or Egr-1. The S1P-induced Egr-1 expression was partially inhibited by treatment of the cells with either calphostin C, an inhibitor of protein kinase C (PKC), or pertussis toxin (PTX), and completely inhibited by the combination of these agents. Essentially, the same inhibitory pattern by these agents has been observed for S1P-induced extracellular signal-regulated kinase (ERK) activation. The S1P-induced expression of Egr-1 was also completely inhibited in association with complete inhibition of ERK by PD 98059, an ERK kinase inhibitor. Thus, the S1P-induced activation of the Egr-1/FGF-2 system may be mediated through ERK activation, which may involve at least two signaling pathways, i.e., a PTX-sensitive G-protein-dependent pathway and a PKC-dependent pathway.

Topics: Animals; Animals, Newborn; Astrocytes; Cells, Cultured; DNA-Binding Proteins; Early Growth Response Protein 1; Endothelins; Fibroblast Growth Factor 2; Flavonoids; Gene Expression Regulation; Glioma; GTP-Binding Proteins; Immediate-Early Proteins; Lysophospholipids; Mitogen-Activated Protein Kinases; Naphthalenes; Pertussis Toxin; Platelet-Derived Growth Factor; Protein Kinase C; Rats; RNA, Messenger; Sphingosine; Tetradecanoylphorbol Acetate; Transcription Factors; Tumor Cells, Cultured; Virulence Factors, Bordetella

Treatment of rat glioma C6 cells with the beta-adrenergic agonist L-isoproterenol leads to a rise in cAMP level and a subsequent change in cell morphology from an epithelial to an astrocytic type of appearance. This morphological response is reverted by the addition of sphingosine-1-phosphate (S1P) with an EC50 of 10 nM. In rat glioma C6 cells loaded with the Ca2+ indicator Fura-2, S1P evoked Ca2+ release from internal stores and Ca2+ influx from the external medium. Half-maximal stimulation of the Ca2+ increase was 10-20 nM. A similar Ca2+ signal was observed in primary rat astrocytes loaded with the Ca2+ indicator fluo-3. Pretreatment of the C6 cells with PMA (162 nM) prevented both the S1P-induced Ca2+ increase and the morphological reversion. Ca2+ ions therefore seem essential for the morphological reversion by S1P. Pretreatment of the cells with the Clostridium botulinum C3 exoenzyme did not affect the reversion of the morphological response by S1P, indicating that the small GTP-binding protein Rho is not involved in the S1P-induced reversion.

Topics: Adrenergic beta-Agonists; Animals; Astrocytes; Calcium; Cell Size; Cells, Cultured; Glioma; Lysophospholipids; Mice; Neuroblastoma; Rats; Signal Transduction; Sphingosine; Thrombin; Tumor Cells, Cultured