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We show that glioblastoma multiform (GBM) cells overexpressing the constitutively active form of the epidermal growth factor receptor [epidermal growth factor receptor variant III (EGFRvIII) and U87MG human GBM cell line overexpressing EGFRvIII (EGFR+) cells] possess greater invasive properties and have higher levels of extracellular sphingosine-1-phosphate (S1P) and increased sphingosine kinase-1 (SK1) activity than the empty vector-expressing cells. Notably, the inhibition of SK1 or S1P receptors decreases the invasiveness of EGFR+ cells. Moreover, EGFR and MEK1 inhibitors reduce both SK1 activation and cell invasion, suggesting that the enhanced invasiveness observed in the EGFR+ cells depends on the increased S1P secretion, downstream of the EGFRvIII-ERK-SK1-S1P pathway. Altogether, the results of the present study indicate that, in GBM cells, EGFRvIII is connected with the S1P signaling pathway to enhance cell invasiveness and tumor progression.

In MKN1 gastric cancer cells, lysophosphatidic acid (LPA) upregulates expression of sphingosine kinase 1 (SphK1) and its downregulation or inhibition suppresses LPA mediated proliferation. Although LPA activates numerous signaling pathways downstream of its receptors, including extracellular-signal-regulated kinase 1/2, p38, JNK, and Akt, and the transactivation of the epidermal growth factor receptor, pharmacological and molecular approaches demonstrated that only activation of ERK1, in addition to the CCAAT/enhancer-binding protein β transcription factor, is involved in transcriptional upregulation of SphK1 by LPA. Our data implicate ERK1 as an important mediator of LPA signaling leading to upregulation of SphK1 and point to SphK1 and sphingosine-1-phosphate production as potential therapeutic targets in gastric cancer.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) plays an important role in sphingosine-1-phosphate(S1P)-dependent migration of endothelial cells but the underlying mechanisms remain largely unknown. Herein, we show that S1P promotes the relocalization of MT1-MMP to peripheral actin-rich membrane ruffles that is coincident with its association with the adaptor protein p130Cas at the leading edge of migrating cells. Immunoprecipitation and confocal microscopy analyses suggest that this interaction required the tyrosine phosphorylation of p130Cas and also involves S1P-dependent phosphorylation of MT1-MMP within its cytoplasmic sequence. The interaction of MT1-MMP with p130Cas at the cell periphery suggests the existence of a close interplay between pericellular proteolysis and signaling pathways involved in EC migration.

Several different types of interactions between sphingosine-1-phosphate (S1P) receptors and platelet-derived growth factor receptor (PDGFR) have been revealed recently. In this work, we used HEK293 cells to further investigate the potential crosstalk. Interestingly, we observed that S1P specifically induced a PDGFR-dependent cell detachment in HEK293 cells, which could be inhibited by AG1296, a specific inhibitor for PDGFR. EGFR on the other hand, did not have any effect on cell detachment. The detachment was extracellular matrix (ECM) protein specific, suggesting the involvement of specific integrin molecules. When beta(1) integrin was engaged into an active state, S1P-induced cell detachment was blocked, suggesting that S1P induced an inside-out inhibitory effect on beta(1) integrin. G(i) protein and ERK activation were required for the cell detachment induced by S1P, suggesting an endogenous receptor for S1P is likely to be involved.

In this study, the effects of short-term diabetes (4 days) on rat renal glomerular cells proliferation and the potential involvement of sphingolipids in this process were investigated. Immunohistochemical analysis showed that streptozotocin (STZ)-induced diabetes promoted increased intra-glomerular hyperplasia, particularly marked for mesangial cells. This was associated with a concomitant increase in neutral ceramidase and sphingosine-kinase activities and the accumulation of the pro-proliferative sphingolipid sphingosine-1-phosphate, in glomeruli isolated from kidney cortex of STZ-treated rats. These results suggest a possible involvement of sphingolipid metabolites in the glomerular proliferative response during the early stages of diabetic nephropathy.

Sphingosine kinase type 1 (SphK1) and its product sphingosine-1-phosphate have been shown to promote cell growth and inhibit apoptosis of tumor cells. In an effort to further understand the regulation of SphK1, we used a yeast two-hybrid screen to find SphK1-interacting proteins. One of these was identified as aminoacylase 1 (Acy1), a metalloenzyme that removes amide-linked acyl groups from amino acids and may play a role in regulating responses to oxidative stress. Both the C-terminal fragment found in the two-hybrid screen and full-length Acy1 co-immunoprecipitate with SphK1. Though both C-terminal and full-length proteins slightly reduce SphK1 activity measured in vitro, the C-terminal fragment inhibits while full-length Acy1 potentiates the effects of SphK1 on proliferation and apoptosis. Interestingly, Acy1 induces redistribution of SphK1 as observed by immunocytochemistry and subcellular fractionation. Collectively, our data suggest that Acy1 physically interacts with SphK1 and may influence its physiological functions.

Ceramide-1-phosphate (Cer-1-P) is emerging as a novel bioactive sphingolipid. It is formed by phosphorylation of ceramide catalyzed by ceramide kinase, and has been implicated in different cellular processes. Cer-1-P is mitogenic for fibroblasts, blocks apoptosis in macrophages, controls phagocytosis in neutrophils, and mediates inflammatory responses. Only recently have we started to uncover the signaling pathways that are affected by Cer-1-P. Recent work has demonstrated that cytosolic phospholipase A2 and acid sphingomyelinase are direct intracellular targets of Cer-1-P, and that it may also induce phosphorylation of extracellular signal-regulated kinase-2 and calcium mobilization. These actions of Cer-1-P seem to be cell type-specific.

Sphingosine-1-phosphate (Sph-1-P) regulates critical cellular functions including cell proliferation, differentiation, angiogenesis, and cell survival. However, its mechanisms of action are incompletely understood. Here, we show a novel biological effect of Sph-1-P: inhibition of acidic sphingomyelinase (A-SMase) activity in apoptotic bone marrow-derived macrophages. A-SMase catalyzes the conversion of sphingomyelin to ceramides, which are pro-apoptotic. This action of Sph-1-P prevents the accumulation of ceramides and blocks apoptosis, thereby promoting survival of the macrophages.

We show here for the first time that sphingosine-1-phosphate (Sph-1-P) stimulates cortisol secretion in zona fasciculata cells of bovine adrenal glands. This effect was dependent upon protein kinase C (PKC) and extracellular Ca2+, and was inhibited by pertussis toxin. Sph-1-P activated phospholipase D (PLD) through a pertussis toxin-sensitive mechanism, also involving extracellular Ca2+ and PKC. Primary alcohols, which attenuate formation of phosphatidic acid (the product of PLD), and cell-permeable ceramides, which inhibit PLD, blocked Sph-1-P-induced cortisol secretion. In conclusion, Sph-1-P stimulates cortisol secretion through a mechanism involving Gi/o protein-coupled receptors, extracellular Ca2+, PKC and PLD.

Rho GTPases participate in various important signaling pathways and have been implicated in myogenic differentiation. Here the first evidence is provided that in C2C12 myoblasts sphingosine 1-phosphate (SPP) rapidly and transiently induced membrane association of Rho A in a pertussis toxin-insensitive manner. The bioactive lipid preferentially relocalized the GTPase to Golgi-enriched membrane. Translocation of Rho A was abolished by inhibition or down-regulation of protein kinase C (PKC). Notably, treatment with Gö6976, an inhibitor of conventional PKCs, which selectively blocked PKC alpha in these cells, prevented SPP-induced Rho A translocation. Conversely rottlerin, a selective inhibitor of PKC delta, was without effect, demonstrating that SPP signaling to Rho A involves PKC alpha but not PKC delta activation. This novel functional relationship between the two proteins may have a role in SPP-mediated regulation of downstream effectors.

Sphingosine kinase catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (SPP), a novel lipid mediator with both intra- and extracellular functions. Based on sequence identity to murine sphingosine kinase (mSPHK1a), we cloned and characterized the first human sphingosine kinase (hSPHK1). The open reading frame of hSPHK1 encodes a 384 amino acid protein with 85% identity and 92% similarity to mSPHK1a at the amino acid level. Similar to mSPHK1a, when HEK293 cells were transfected with hSPHK1, there were marked increases in sphingosine kinase activity resulting in elevated SPP levels. hSPHK1 also specifically phosphorylated D-erythro-sphingosine and to a lesser extent sphinganine, but not other lipids, such as D,L-threo-dihydrosphingosine, N,N-dimethylsphingosine, diacylglycerol, ceramide, or phosphatidylinositol. Northern analysis revealed that hSPHK1 was widely expressed with highest levels in adult liver, kidney, heart and skeletal muscle. Thus, hSPHK1 belongs to a highly conserved unique lipid kinase family that regulates diverse biological functions.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph-1-P) are known as structurally related bio-active lipids activating platelets through their respective receptors. Although the receptors for LPA and Sph-1-P have been recently identified in various cells, the identification and characterization of ones in platelets have been reported only preliminarily. In this report, we first investigated the distinct modes of LPA and Sph-1-P actions in platelet activation and found that LPA functioned as a much stronger agonist than Sph-1-P, and high concentrations of Sph-1-P specifically desensitized LPA-induced intracellular Ca2+ mobilization. In order to identify the responsible receptors underlying these observations, we analyzed the LPA and Sph-1-P receptors which might be expressed in human platelets, by RT-PCR. We found for the first time that Edg2, 4, 6 and 7 mRNA are expressed in human platelets.

Sphingosine-1-phosphate (S1P) is a bioactive lipid molecule. It stimulates the growth of some cells, but inhibits the growth of others. In this study, we describe the detection of sub-microM to microM concentrations of S1P in the ascitic fluids of patients with ovarian cancer. In ovarian cancer cells cultured in vitro, S1P exhibited a dual effect on growth and/or survival. S1P (10 microM) induced cell death when cells were in suspension but stimulated cell growth when cells were attached. The calcium-dependent induction of cell death by S1P is apparently associated with its inhibitory effect on cell attachment and cell adhesion. S1P (10-30 microM) also induced calcium-dependent cell-cell aggregation.

Sphingolipid breakdown products are now being recognized to play a dual role in cellular signalling, acting as intracellular as well as extracellular signalling molecules. Both types of action may even be found with one sphingolipid species. The recent demonstration of G protein-coupled receptors with high affinity for sphingosine 1-phosphate and sphingosylphosphorylcholine has been followed by the discovery of several novel sphingolipid actions, such as regulation of heart rate, oxidative burst, neurite retraction or platelet activation. Ligand profiles and concentration-response relationships suggest the existence of putative sphingolipid receptor subtypes. Against this background, several observations on supposed sphingolipid second messenger actions deserve a new evaluation.

Sphingosine-1-phosphate (Sph-1-P), the initial product of sphingosine (Sph) catabolism, has been reported to inhibit motility of mouse melanoma B16/F1 and other types of cells at very low concentrations (10-100 nM). Sph-1-P (100 nM-1 microM) inhibited pseudopodium formation by blocking polymerization and reorganization of actin filaments in newly formed pseudopodia, and reduced F-actin by approximately 25% in F1 cells. A pyrene-labeled actin nucleation assay revealed that Sph-1-P (100 nM) inhibits actin nucleation mediated by F1 cell plasma membranes. These results suggest that Sph-1-P interacts with molecules associated with actin nucleation to inhibit reorganization of pseudopodium formation and cell motility.

Ceramidase hydrolyzes ceramide and produces sphingosine as a substrate of sphingosine kinase (SPHK), which transforms sphingosine to sphingosine-1-phosphate. It has been reported that cytokines elicit SPHK activation in rat β-cells. As a sphingosine provider, ceramidase should also be activated. In our previous work, we showed that the increase in mRNA and protein levels in cytokine-treated INS-1 rat β-cells resulted in chronic activation of neutral ceramidase. Here we found that acid ceramidase (AC) is activated by cytokines at an early stage via tyrosine phosphorylation. In addition, basal AC activity was first detected in INS-1 cells and isolated rat islets, and cytokine-induced cell growth was significantly repressed when AC was pharmacologically inhibited.

The adipose-derived plasma protein, adiponectin (APN), has various protective effects on cardiovascular diseases. In this study, we show that endogenous APN is required for full cyclooxygenase-2 (COX-2) induction by ischemia-reperfusion injury in the heart in vivo. In rat neonatal cardiac myocytes, APN-induced COX-2 expression was reduced by treatment with a sphingosine kinase-1 (SphK-1) inhibitor or siRNA targeting SphK-1. Treatment with a sphingosine-1-phosphate (S1P) receptor antagonist also diminished COX-2 expression in response to APN stimulation. These findings suggest that APN is a physiological regulator of COX-2 signaling in the heart and that this regulation occurs in part via a SphK-1–S1P receptor dependent mechanism in cardiac myocytes.

In this study, we report that low doses of tumor necrosis factor-alpha (TNFalpha) promote myogenesis in C2C12 myoblasts. Moreover, the cytokine increased sphingosine kinase (SphK) activity and induced SphK1 translocation to membranes. The inhibition of SphK functionality by various approaches abrogated the pro-myogenic effect of TNFalpha. Moreover, silencing of S1P(2) impaired the positive action of TNFalpha on myogenesis. These results represent the first evidence that SphK/S1P(2) axis is required for the regulation of myogenesis by TNFalpha. In view of the physiological role of TNFalpha in muscle regeneration, the present finding reinforces the notion that SphK/S1P(2) signaling is critically implicated in myogenesis.

Recent studies suggest that sphingosine 1-phosphate (S1P) protects against atherosclerosis. We assessed the effects of S1P on monocyte–endothelial interaction in the presence of inflammatory mediators. Pretreatment of THP-1 cells with S1P abolished Phorbol 12 myristate 13-acetate (PMA)-induced THP-1 cell adhesion to human umbilical vein endothelial cells (HUVECs). S1P inhibited PMA-induced activation of RhoA, but not PKCs. S1P activated p190Rho GTPase activation protein (GAP) only in the presence of PMA, suggesting an inhibitory effect of S1P and PMA to suppress RhoA. In conclusion, S1P inhibited monocyte–endothelial interactions by inhibiting RhoA activity which may explain its anti-atherogenic effects.

Sphingosine-1-phosphate (S1P) is a potent lysolipid involved in a variety of biological responses important for cancer progression. Therefore, we investigated the role of sphingosine kinase type 1 (SphK1), the enzyme that makes S1P, in the motility, growth, and chemoresistance of MCF-7 breast cancer cells. Epidermal growth factor (EGF), an important growth factor for breast cancer progression, activated and translocated SphK1 to plasma membrane. SphK1 was required for EGF-directed motility. Downregulation of SphK1 in MCF-7 cells reduced EGF- and serum-stimulated growth and enhanced sensitivity to doxorubicin, a potent chemotherapeutic agent. These results suggest that SphK1 may be critical for growth, metastasis and chemoresistance of human breast cancers.

Sphingosine kinase (SK) is the enzyme that catalyzes the formation of sphingosine 1-phosphate (S1P). Although diverse biological functions have been reported for SK, its recognition site for its substrate sphingosine (Sph) is still unclear. We constructed various mutants of mouse sphingosine kinase 1a (mSK1a), carrying mutations in the C4 domain, which we had expected to encompass the Sph-binding site. We analyzed the influence of these mutations on the SK activity and substrate kinetics. One mutation, Asp177--Asn177, caused a dramatic decrease in SK activity (to approximately 6% of wild type) and an increase in the Km value for Sph (10.1--108 microM), with no change in the affinity for ATP. This result suggests that the C4 domain, especially the Asp177, is involved in the specific recognition of Sph. In this report, we are able, for the first time, to provide an account of the Sph-binding site of SK.

Sphingosine-1-phosphate (S1P), the product of sphingosine kinase, activates several widely expressed G-protein-coupled receptors (GPCR). S1P might also play a role as second messenger, but this hypothesis has been challenged by recent findings. Here we demonstrate that intracellular S1P can mobilize Ca2+ in intact cells independently of S1P-GPCR. Within seconds, S1P generated by the photolysis of caged S1P raised the intracellular free Ca2+ concentration in HEK-293, SKNMC and HepG2 cells, in which the response to extracellularly applied S1P was either blocked or absent. Ca2+ transients induced by photolysis of caged S1P were caused by Ca2+ mobilization from thapsigargin-sensitive stores. These results provide direct evidence for a true intracellular action of S1P.

The sphingolipid metabolite sphingosine-1-phosphate (S1P) is a serum-borne lipid that regulates many vital cellular processes. S1P is the ligand of a family of five specific G protein-coupled receptors that are differentially expressed in different tissues and regulate diverse cellular actions. Much less is known of the intracellular actions of S1P. It has been suggested that S1P may also function as an intracellular second messenger to regulate calcium mobilization, cell growth and suppression of apoptosis in response to a variety of extracellular stimuli. Dissecting the dual actions and identification of intracellular targets of S1P has been challenging, but there is ample evidence to suggest that the balance between S1P and ceramide and/or sphingosine levels in cells is an important determinant of cell fate.

Receptor-regulated phospholipase D (PLD) is a key signaling pathway implicated in the control of fundamental biological processes. Here evidence is presented that in addition to protein kinase C (PKC) and Rho GTPases, Ca(2+) response evoked by sphingosine 1-phosphate (S1P) also participates to the enzyme regulation. Ca(2+) was found critical for PKC(alpha)-mediated PLD activation. Moreover, S1P-induced PLD activity resulted diminished by calmodulin inhibitors such as W-7 and CGS9343B implicating its involvement in the process. A plausible candidate for Ca(2+)-dependent PLD regulation by S1P was represented by calcineurin, in view of the observed reduction of the stimulatory effect by cyclosporin A. In contrast, monomeric GTP-binding protein Ral was translocated to membranes by S1P in a Ca(2+)-independent manner, ruling out its possible role in agonist-mediated regulation of PLD.

Formation of sphingosine-1-phosphate (SPP) by sphingosine kinase serves as a signalling pathway for various membrane receptors. Here, we show that membrane depolarisation is another mechanism by which this pathway can be activated. Formation of [(3)H]SPP as well as levels of endogenous SPP were rapidly and transiently increased in PC12 pheochromocytoma cells depolarised with high KCl. Time course and maximum were similar to those induced by bradykinin. Depolarisation-induced SPP production was also observed in RINm5F insulinoma cells, dependent on extracellular Ca(2+) and fully suppressed by verapamil, thus apparently caused by Ca(2+) influx via voltage-gated Ca(2+) channels. Studies with sphingosine kinase inhibitors and overexpression of sphingosine kinase revealed a partial contribution of this pathway to depolarisation-induced noradrenaline release and Ca(2+) increase.

Subcellular fractionation revealed that a significant fraction of total sphingosine kinase, the enzyme that phosphorylates sphingosine to form the bioactive lipid metabolite sphingosine-1-phosphate, resides in the nuclei of Swiss 3T3 cells, localized to both the nuclear envelope and the nucleoplasm. Platelet-derived growth factor, in addition to rapidly stimulating cytosolic sphingosine kinase, also induced a large increase in nucleoplasm-associated activity after 12–24 h that correlated with progression of cells to the S-phase of the cell cycle and translocation of sphingosine kinase–green fluorescent protein fusion protein to the nuclear envelope. Our results add sphingosine kinase to the growing list of lipid-metabolizing enzymes associated with the nucleus, and suggest that sphingosine-1-phosphate may also play a role in signal transduction in the nucleus.

The present study showed that sphingosine 1-phosphate (SPP) induced rapid stimulation of phospholipase D (PLD) in skeletal muscle C2C12 cells. The effect was receptor-mediated since it was fully inhibited by pertussis toxin. All known SPP-specific receptors, Edg-1, Edg-3 and AGR16/H218, resulted to be expressed in C2C12 myoblasts, although at a different extent. SPP-induced PLD activation did not involve membrane translocation of PLD1 or PLD2 and appeared to be fully dependent on protein kinase C (PKC) catalytic activity. SPP increased membrane association of PKCalpha, PKCdelta and PKClambda, however, only PKCalpha and PKCdelta played a role in PLD activation since low concentrations of GF109203X and rottlerin, a selective inhibitor of PKCdelta, prevented PLD stimulation.

We recently reported that N,N-dimethylsphingosine 1-phosphate (DMS-1-P) can be formed from N,N-dimethylsphingosine (DMS) in activated platelets [Y. Yatomi et al., Biochem. Biophys. Res. Commun. 231 (1997) 848–851]. In this study, we synthesized, for the first time, DMS-1-P and examined the functional effects of DMS-1-P and its related sphingolipids on platelets. Although exogenous DMS was inactive, its phosphorylated derivative, DMS-1-P, induced platelet intracellular Ca2+ mobilization and shape change, but not aggregation or release reactions. Since sphingosine 1-phosphate (Sph-1-P) is structurally related to DMS-1-P and activates platelets more strongly than DMS-1-P, a competitive binding experiment for [3H]Sph-1-P was performed using DMS-1-P. DMS-1-P reduced the binding of [3H]Sph-1-P to platelets almost as much as unlabeled Sph-1-P did. These results suggest that DMS-1-P activates platelets via an interaction with a platelet surface receptor for Sph-1-P.

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.

Although a growing number of reports on the bioactivities of sphingosine 1-phosphate (Sph-1-P) in various systems have been appearing recently, current evidence for the involvement of Sph-1-P in signal transduction or cellular function(s) consists largely of data on cellular and biochemical effects of exogenous Sph-1-P. In this study, we measured Sph-1-P contents in various rat tissues. When the amount of this sphingoid base was measured by its N -acetylation into [ 3 H]C 2 -ceramide 1-phosphate with [ 3 H]acetic anhydride, we could detect Sph-1-P in all rat tissues examined. We also got unexpected findings that Sph-1-P exists most abundantly in testis=intestine>spleen (in the order of Sph-1-P abundance when its amounts were adjusted to the phospholipid levels). Our new data on Sph-1-P distribution in rat tissues may imply novel bioactivities for Sph-1-P. © 1997 Federation of European Biochemical Societies.

The potent immunosuppressive drug FTY720, a sphingosine analog, induces redistribution of lymphocytes from circulation to secondary lymphoid tissues. FTY720 is phosphorylated in vivo and functions as an agonist for four G-protein-coupled sphingosine-1-phosphate receptors. The identity of the kinase that phosphorylates FTY720 is still not known. Here we report that although both sphingosine kinase type 1 (SphK1) and type 2 (SphK2) can phosphorylate FTY720 with low efficiency, SphK2 is much more effective than SphK1. FTY720 inhibited phosphorylation of sphingosine catalyzed by SphK2 to a greater extent than it inhibits SphK1. Thus, SphK2 may be the relevant enzyme that is responsible for in vivo phosphorylation of FTY720.

In previous studies, we reported that sphingosine 1-phosphate (Sph-1-P) inhibits the chemotactic motility of some cancer cell lines such as mouse melanoma cells, as well as human smooth muscle cells, at a very low concentration, as demonstrated by a transwell migration assay method (Proc. Natl. Acad. Sci. USA 89, 9698, 1992; J. Cell Biol. 130, 193, 1995). In this study, we investigated the effect of Sph-1-P on the chemotactic motility and invasiveness of human neutrophils, utilizing three different assay systems: (a) a transwell migration assay where IL-8 or fLMP was added as a chemotactic factor, (b) a phagokinetic assay with gold colloids, and (c) a trans-endothelial migration assay with human umbilical vein endothelial cells (HUVECs) plated on collagen layers. We found that among various sphingosine derivatives, Sph-1-P specifically inhibited the IL-8- or fLMP-induced chemotactic migration of neutrophils at concentrations below 1 μM. Phagokinetic activity of neutrophils was also suppressed by Sph-1-P, but more moderately than by the PKC inhibitory sphingosine analog, trimethylsphingosine. Finally, Sph-1-P inhibited trans-endothelial migration and invasiveness of neutrophils into HUVEC-covered collagen layers, whereas no effect on their adhesion to HUVECs was observed. These observations strongly suggest that Sph-1-P can act as a specific and effective motility regulator of human neutrophils, raising the possibility of future applications of Sph-1-P, or its analogs, as anti-inflammatory agents regulating invasive migration of neutrophils through endothelial layers at injured vascular sites.

Sphingosine kinase (SK) catalyses the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in mediating such fundamental biological processes as cell growth and survival. Very little is currently known regarding the structure or mechanisms of catalysis and activation of SK. Here we have tested the functional importance of Gly(113), a highly conserved residue of human sphingosine kinase 1 (hSK), by site-directed mutagenesis. Surprisingly, a Gly(113)--Ala substitution generated a mutant that had 1.7-fold greater catalytic activity than wild-type hSK (hSK(WT)). Our data suggests that the Gly(113)--Ala mutation increases catalytic efficiency of hSK, probably by inducing a conformational change that increases the efficiency of phosphoryl transfer. Interestingly, hSK(G113A) activity could be stimulated in HEK293T cells by cell agonists to a comparable extent to hSK(WT).

A genomic analysis of the s1p(4)/lp(C1)/Edg6 mouse sphingosine-1-phosphate (S1P) G protein-coupled receptor gene revealed it to be located on central chromosome 10 and to consist of two exons with an intronless coding region. Surprisingly, we found the gene encoding the promiscuously coupling G(alpha15) protein (Gna15) located in tandem just upstream, an arrangement conserved in the human genome (on chromosome 19p13.3). Given that Northern blots demonstrated similar tissue distributions of the mouse s1p(4) and Gna15 transcripts, we propose that transcription of the two genes may be under control of the same enhancer elements and that their protein products may couple in vivo.

Sphingosine-1-phosphate (Sph-1-P), the initial product of Sph catabolism, inhibited chemotactic motility of a few lines of tumor cells [(1992) Proc. Natl. Acad. Sci. USA 89, 9686]. We now report that Sph-1-P even at very low concentration (10–100 nM) inhibits integrin-dependent motility of melanoma cells induced by extracellular matrix (ECM), although it did not affect integrin-dependent adhesion to ECM. Other Sph-related compounds tested (including sphinganine-1-P) were much less effective than Sph-1-P at inhibiting motility, and also had no effect on integrin-dependent adhesion of tumor cells to ECM. Our findings suggest that Sph-1-P inhibits actin filament reorganization by affecting cytoplasmic connection to integrin in ECM-stimulated motility of melanoma cells.

Sphingosine 1-phosphate (S1P) induces phospholipase C (PLC) activation and Ca2+ mobilization in many types of cells. We examined the possible involvement of Edg-3, one of the putative S1P receptors, in the phospholipase C (PLC)-Ca2+ system. S1P increased the cytoplasmic free Ca2+ concentration without detectable inositol phosphate production in vector-transfected CHO cells. In the Edg-3-transfected cells, however, the S1P-induced Ca2+ response was clearly enhanced, which was associated with a significant production of inositol phosphate. These S1P-induced responses in the Edg-3-transfected cells were inhibited by U73122, a potent PLC inhibitor. We conclude that Edg-3 may be one of the S1P receptors participating in the activation of the PLC-Ca2+ system.

FTY720 is a novel immunomodulatory drug efficacious in the treatment of multiple sclerosis. The drug is converted in vivo to the monophosphate, FTY720-P, by sphingosine kinase 2. This conversion is incomplete, suggesting opposing actions of kinase and phosphatase activities. To address which of the known lipid phosphatases might dephosphorylate FTY720-P, we overexpressed the broad specificity lipid phosphatases LPP1–3, and the specific S1P phosphatases (SPP1 and 2) in HEK293 cells, and performed in vitro assays using lysates of transfected cells. Among LPPs, only LPP3 was able to dephosphorylate FTY720-P; among SPPs, only SPP1 showed activity against FTY720-P. On intact cells, LPP3 acted as an ecto-phosphatase or FTY720-P, thus representing the major phosphatase involved in the equilibrium between FTY720 and FTY720-P observed in vivo.