The concentration of sphingosine 1-phosphate (S1P) in plasma or serum is much higher than the half-maximal concentration of the sphingolipid needed to stimulate its receptors. Nevertheless, the inositol phosphate response to plasma or serum mediated by Edg-3, one of the S1P receptors, which was overexpressed in Chinese hamster ovary cells, was much smaller than the response expected from the total amount of S1P in these samples. The inositol phosphate response to exogenous S1P was markedly attenuated in the presence of charcoal-treated low-S1P serum. The inhibitory effect was lost by boiling but not by dialysis of the serum. The inhibitory action of the serum was specific to S1P and was associated with the trapping of exogenous S1P; the inositol phosphate response to P(2)-purinergic agonists was somewhat enhanced by the charcoal-treated serum. Among the components of plasma or serum, lipoproteins such as low-density and high-density lipoproteins showed a stronger activity for trapping S1P than lipoprotein-deficient serum. Consistent with this observation, we detected a 15-100-fold higher amount of S1P per unit amount of protein in lipoproteins than in the lipoprotein-deficient serum. Thus even though the protein content of the lipoprotein fraction contributes to only 4% of the total protein content of plasma or serum, more than 60% of S1P is distributed in this fraction. These results suggest that the tight binding of S1P to the components of serum or plasma, including lipoproteins, may interfere with the S1P binding to its receptors and thereby attenuate the lipid-receptor-mediated actions in the cells.

T cell death-associated gene 8 (TDAG8) has been reported to be a receptor for psychosine. Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4, Gprotein-coupled receptors (GPCRs) closely related to TDAG8, however, have recently been identified as protonsensing or extracellular pH-responsive GPCRs that stimulate inositol phosphate and cAMP production, respectively. In the present study, we examined whether TDAG8 senses extracellular pH change. In the several cell types that were transfected with TDAG8 cDNA, cAMP was markedly accumulated in response to neutral to acidic extracellular pH, with a peak response at approximately pH 7.0 -6.5. The pH effect was inhibited by copper ions and was reduced or lost in cells expressing mutated TDAG8 in which histidine residues were changed to phenylalanine. In the membrane fractions prepared from TDAG8-transfected cells, guanosine 5-O-(3-thiotriphosphate) binding activity and adenylyl cyclase activity were remarkably stimulated in response to neutral and acidic pH. The concentration-dependent effect of extracellular protons on cAMP accumulation was shifted to the right in the presence of psychosine. The inhibitory psychosine effect was also observed for pH-dependent actions in OGR1-and GPR4-expressing cells but not for prostaglandin E 2 -and sphingosine 1-phosphate-induced actions in any pH in native and sphingosine 1-phosphate receptor-expressing cells. Glucosylsphingosine and sphingosylphosphorylcholine similarly inhibited the pHdependent action, although to a lesser extent. Psychosinesensitive and pH-dependent cAMP accumulation was also observed in mouse thymocytes. We concluded that TDAG8 is one of the proton-sensing GPCRs coupling to adenylyl cyclase and psychosine, and its related lysosphingolipids behave as if they were antagonists against proteinsensing receptors, including TDAG8, GPR4, and OGR1. TDAG81 was initially cloned as an orphan GPCR, which is up-regulated during the programmed cell death of T lymphocytes (1-3). This gene product has recently been reported (4) to be a receptor for psychosine, a lysosphingolipid, which induces the formation of multinuclear cells. OGR1, which shares 41% identical amino acids with TDAG8, was initially reported (5) to be a receptor for sphingosylphosphorylcholine (SPC). GPR4 also shares homology with TDAG8 and was identified as a receptor for lysolipids, including lysophosphatidylcholine (LPC) and SPC (6). It has recently been reported (7), however, that OGR1 and GPR4 sense extracellular protons through histidine residues of receptors and are coupled to G-proteins to stimulate intracellular signaling pathways. Thus, OGR1 stimulation causes inositol phosphate production, and the subsequent mobilization of intracellular calcium and GPR4 stimulation induces cAMP accumulation, probably reflecting the activation of adenylyl cyclase in response to an extracellular pH change (7). These results raise the possibility that TDAG8 may also respond to extracellular pH change and stimulate intracellular signaling pathways.If TDAG8 is prov...

We examined the actions of sphingosine 1-phosphate (S1P) on signaling pathways in Chinese hamster ovary cells transfected with putative S1P receptor subtypes, i.e. Edg-1, AGR16/H218 (Edg-5), and Edg-3. Among these receptor-transfected cells, there was no significant difference in the expressing numbers of the S1P receptors and their affinities to S1P, which were estimated by [ 3 H]S1P binding to the cells. In vector-transfected cells, S1P slightly increased cytosolic Ca 2؉ concentration ([Ca 2؉ ] i ) in association with inositol phosphate production, reflecting phospholipase C activation; the S1P-induced actions were markedly enhanced in the Edg-3-transfected cells and moderately so in the AGR16-transfected cells. In comparison with vector-transfected cells, the S1P-induced [Ca 2؉ ] i increase was also slightly enhanced in the Edg-1-transfected cells. In all cases, the inositol phosphate and Ca 2؉ responses to S1P were partially inhibited by pertussis toxin (PTX). S1P also significantly increased cAMP content in a PTX-insensitive manner in all the transfected cells; the rank order of their intrinsic activity of S1P receptor subtypes was AGR16 > Edg-3 > Edg-1. In the presence of forskolin, however, S1P significantly inhibited cAMP accumulation at a lower concentration (1-100 nM) of S1P in a manner sensitive to PTX in the Edg-1-transfected cells but not in either the Edg-3 or AGR16-transfected cells. As for cell migration activity evaluated by cell number across the filter of blind Boyden chamber, Edg-1 and Edg-3 were equally potent, but AGR16 was ineffective. Thus, S1P receptors may couple to both PTX-sensitive and -insensitive G-proteins, resulting in the selective regulation of the phospholipase C-Ca 2؉ system, adenylyl cyclase-cAMP system, and cell migration activity, according to the receptor subtype.Sphingosine 1-phosphate (S1P), 1 one of the sphingolipid metabolites, has recently been suggested to affect a variety of cellular processes (1, 2). These cellular responses elicited by S1P have first been ascribed to the intracellular action of the lipid, because S1P accumulated in the cells in response to some kinds of cytokines, and moreover, S1P induced Ca 2ϩ mobilization in a cell-free system (3-5). On the other hand, these S1P-induced responses are also accompanied by the stimulation of several early signaling events that are usually regulated by cell-surface receptors. These signaling events include activation of PLC (6 -9), an increase in [Ca 2ϩ ] i (10 -12), regulation of adenylyl cyclase (6, 9, 10, 13), and Rho activation (14, 15). The presence of the latter mechanism has been supported by the recent identification of several cDNAs encoding G-protein-coupled receptors for S1P, i.e. Edg-1, AGR16/H218, and .The transfection experiments of these S1P receptor subtypes demonstrated that these putative S1P receptors can actually couple to multiple signaling pathways. Thus, the previous transfection experiments suggest the involvement of these putative S1P receptor subtypes in the regulation of multiple signal...

We characterized the molecular mechanisms by which high density lipoprotein (HDL) inhibits the expression of adhesion molecules, including vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, induced by sphingosine 1-phosphate (S1P) and tumor necrosis factor (TNF) ␣ in endothelial cells. HDL inhibited S1P-induced nuclear factor B activation and adhesion molecule expression in human umbilical vein endothelial cells. The inhibitory HDL actions were associated with nitric-oxide synthase (NOS) activation and were reversed by inhibitors for phosphatidylinositol 3-kinase and NOS. The HDL-induced inhibitory actions were also attenuated by the down-regulation of scavenger receptor class B type I (SR-BI) and its associated protein PDZK1. When TNF␣ was used as a stimulant, the HDL-induced NOS activation and the inhibitory action on adhesion molecule expression were, in part, attenuated by the down-regulation of the expression of S1P receptors, especially S1P 1 , in addition to SR-BI. Reconstituted HDL composed mainly of apolipoprotein A-I and phosphatidylcholine mimicked the SR-BI-sensitive part of HDL-induced actions. Down-regulation of S1P 3 receptors severely suppressed the stimulatory actions of S1P. Although G i/o proteins may play roles in either stimulatory or inhibitory S1P actions, as judged from pertussis toxin sensitivity, the coupling of S1P 3 receptors to G 12/13 proteins may be critical to distinguish the stimulatory pathways from the inhibitory ones. In conclusion, even though S1P alone stimulates adhesion molecule expression, HDL overcomes S1P 3 receptor-mediated stimulatory actions through SR-BI/PDZK1-mediated signaling pathways involving phosphatidylinositol 3-kinase and NOS. In addition, the S1P component of HDL plays a role in the inhibition of TNF␣-induced actions through S1P receptors, especially S1P 1 .The plasma level of HDL 2 has been shown to be inversely correlated with the risk of atherosclerosis and associated cardiovascular disease (1, 2). HDL can remove excess cholesterol from arterial and nonliver cells, transport it to the liver, and excrete it as bile acids. The so-called reverse cholesterol transport is thought to be an important anti-atherogenic action of HDL (1, 2). In recent studies, however, HDL has been shown to exert a variety of actions that are independent of cholesterol metabolism. For example, HDL inhibits LDL oxidation, smooth muscle cell migration, platelet aggregation, and endothelial dysfunction (3, 4). The inhibition of endothelial dysfunction may be achieved by several responses to HDL, including the stimulation of proliferation, cell survival, migration, and NO synthesis, or the inhibition of apoptosis and of the expression of adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) (3-5). An increase in the expression of the adhesion molecules stimulates monocyte interaction with endothelial cells and cell penetration into subendothelial space or the intima of arterial walls. Thus, the expr...

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.

Objective-Plasma high-density lipoprotein (HDL) level is inversely correlated with the risk of atherosclerosis. However, the cellular mechanism by which HDL exerts antiatherogenic actions is not well understood. In this study, we focus on the lipid components of HDL as mediators of the lipoprotein-induced antiatherogenic actions. Methods and Results-HDL and sphingosine 1-phosphate (S1P) stimulated the migration and survival of human umbilical vein endothelial cells. These responses to HDL and S1P were almost completely inhibited by pertussis toxin and other specific inhibitors for intracellular signaling pathways, although the inhibition profiles of migration and survival were different. The HDL-stimulated migration and survival of the cells were markedly inhibited by antisense oligonucleotides against the S1P receptors EDG-1/S1P 1 and EDG-3/S1P 3 . Cell migration was sensitive to both receptors, but cell survival was exclusively sensitive to S1P 1 . The S1P-rich fraction and chromatographically purified S1P from HDL stimulated cell migration, but the rest of the fraction did not, as was the case of the cell survival. Key Words: high-density lipoprotein Ⅲ sphingosine 1-phosphate Ⅲ migration Ⅲ EDG Ⅲ endothelial cell P lasma lipoproteins are responsible for the transport of cholesterol to cells and the control of cholesterol synthesis. 1-3 Low-density lipoprotein (LDL) provides cholesterol to cells through LDL receptors, whereas high-density lipoprotein (HDL) has been shown to remove excess cholesterol from the cells. The so-called reverse transport of cholesterol is thought to be an important mechanism for the antiatherogenic actions of HDL. 1,2 Recent studies have shown that HDL induces cytoprotective actions, 3,4 proliferation, 5 and migration in endothelial cells, 2,6 activities presumably independent of cholesterol metabolism, 2,3 although the mechanism by which HDL induces these antiatherogenic actions has not been well characterized. It has been reported recently that HDL activates endothelial nitric oxide (NO) production through the scavenger receptor-BI (SR-BI). 7 NO production has been shown to be involved in the cytoprotective action of endothelial cells. 8 In endothelial cells, sphingosine 1-phosphate (S1P) has been shown to regulate a wide range of cellular activities associated with angiogenesis, wound healing, apoptosis, and atherosclerosis. 3,4,8 -18 S1P promotes cell migration, 10 -13,16 -18 DNA synthesis, 10 cell survival, 4,9 cell barrier integrity, 15 NO production, 8,14,16,17 and the expression of several cell adhesion molecules. 3 We recently reported that S1P accumulates in the lipoprotein fraction, especially the HDL fraction, and that HDL-associated S1P mediates the cytoprotective actions of HDL in human umbilical vein endothelial cells (HUVECs). 4,19 Nofer et al 20 reported that sphingosylphosphorylcholine (SPC) and lysosulfatide (LSF) were major components of HDL responsible for these cytoprotective actions. Thus, lipoproteinassociated lipids may also be involved in some HDL-induced...

Cytokines and growth factors in malignant ascites are thought to modulate a variety of cellular activities of cancer cells and normal host cells. The motility of cancer cells is an especially important activity for invasion and metastasis. Here, we examined the components in ascites, which are responsible for cell motility, from patients and cancer cell-injected mice. Ascites remarkably stimulated the migration of pancreatic cancer cells. This response was inhibited or abolished by pertussis toxin, monoglyceride lipase, an enzyme hydrolyzing lysophosphatidic acid (LPA), and Ki16425 and VPC12249, antagonists for LPA receptors (LPA 1 and LPA 3 ), but not by an LPA 3 -selective antagonist. These agents also inhibited the response to LPA but not to the epidermal growth factor. In malignant ascites, LPA is present at a high level, which can explain the migration activity, and the fractionation study of ascites by lipid extraction and subsequent thin-layer chromatography indicated LPA as an active component. A significant level of LPA 1 receptor mRNA is expressed in pancreatic cancer cells with high migration activity to ascites but not in cells with low migration activity. Small interfering RNA against LPA 1 receptors specifically inhibited the receptor mRNA expression and abolished the migration response to ascites. These results suggest that LPA is a critical component of ascites for the motility of pancreatic cancer cells and LPA 1 receptors may mediate this activity. LPA receptor antagonists including Ki16425 are potential therapeutic drugs against the migration and invasion of cancer cells.

Sphingosine 1-phosphate (S1P) is accumulated in lipoproteins, especially high-density lipoprotein (HDL), in plasma. However, it remains uncharacterized how extracellular S1P is produced in the CNS. The treatment of rat astrocytes with retinoic acid and dibutyryl cAMP, which induce apolipoprotein E (apoE) synthesis and HDL-like lipoprotein formation, stimulated extracellular S1P accumulation in the presence of its precursor sphingosine. The released S1P was present together with apoE particles in the HDL fraction. S1P release from astrocytes was inhibited by the treatment of the cells with glybenclamide or small interfering RNAs specific to ATPbinding cassette transporter A1 (ABCA1). Astrocytes from Abca1)/) mice also showed impairment of retinoic acid/dibutyryl cAMP-induced S1P release in association with the blockage of HDL-like lipoprotein formation. However, the formation of either apoE or lipoprotein itself was not sufficient, and additional up-regulation of ABCA1 was requisite to stimulate S1P release. We conclude that the S1P release from astrocytes is coupled with lipoprotein formation through ABCA1. Keywords: apolipoprotein E, astrocyte, ATP-binding cassette transporter A1, high-density lipoprotein, sphingosine 1-phosphate. Sphingosine 1-phosphate (S1P), a sphingolipid metabolite, is a pleiotropic lipid mediator involved in a variety of cell activities, including morphology, motility, and growth. Five subtypes of S1P-specific receptors, S1P 1-5 , have been isolated so far (Ishii et al. 2004). In neural cells, these receptors are expressed in a cell-specific manner (Van Brocklyn et al. 1999;Sato et al. 2000) and involved in the regulation of neural cell functions; for example, a rapid process retraction through S1P 5 in oligodendrocytes (Jaillard et al. 2005), rounding of the cell body in PC12 and N1E115 cells (Postma et al. 1996;Sato et al. 1997) possibly through S1P 2 (Van Brocklyn et al. 1999), and regulation of motility in astrocytes and glioma cells . In addition, S1P induces the proliferation in astroglial cells, which was associated with the activation of extracellular signal-regulated kinase (ERK), activation of the phospholipase C, and Ca 2+ mobilization (Tas and Koschel 1998;Sato et al. 1999;Pebay et al. 2001). S1P 1 is more important than S1P 2 for the stimulation of ERK, while S1P 2 may be responsible for the activation of phospholipase C and Ca 2+ mobilization (Sato et al. 2000;Malchinkhuu et al. 2003). Thus, S1P and its receptors may play important roles in maintaining the functions of the CNS.We have recently demonstrated that S1P is concentrated in lipoproteins, such as high-density lipoprotein (HDL), in circulating blood (Murata et al. 2000b), and that the lipid mediates lipoprotein-induced anti-atherogenic actions, including cell survival, cell migration, and inhibition of adhesion molecule expression through S1P receptors in endothelial cells Okajima 2002). Thus, lipoproteins seem to serve as carriers for extracellular S1P in circulating blood. In astrocytes as well, plasma HDL ...