OBJECTIVE

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lipid, which influences migration and proliferation of endothelial cells through activation of S<em>1</em>P receptors and has been shown to support SDF-<em>1</em> induced migration and bone marrow homing of CD34+ progenitors.

RESULTS

Here, we show that incubation of patient-derived endothelial progenitor cells (EPCs) with S<em>1</em>P or its synthetic analog FTY720 improved blood flow recovery in ischemic hind limbs. Likewise, recovery of blood flow was dramatically reduced after induction of hindlimb ischemia in mice deficient for the S<em>1</em>P receptor 3 (S<em>1</em>P3). S<em>1</em>P3-/- bone marrow-derived mononuclear cells (BMCs) failed to augment neovascularization after hind limb ischemia. Of note, treatment of BMCs derived from S<em>1</em>P3-/- mice with S<em>1</em>P did not rescue blood flow recovery. Mechanistically, S<em>1</em>P and FTY720 induced phosphorylation of CXCR4, activated the Src kinase, and stimulated phosphorylation of JAK2. The contribution of CXCR4 for S<em>1</em>P-mediated effects was further supported by the findings that S<em>1</em>P preincubation failed to stimulate invasion capacity and in vivo blood flow recovery of BMCs from CXCR4+/- mice. The activation of CXCR4 was dependent on the Src kinase family as demonstrated by preincubation with the Src inhibitor PP2. The activation of the CXCR4 signaling by S<em>1</em>P is mediated via the S<em>1</em>P3 receptor, since S<em>1</em>P-induced Src phosphorylation was abrogated in EPC from S<em>1</em>P3-/- mice.

CONCLUSIONS

S<em>1</em>P agonists might serve as sensitizers of CXCR4-mediated signaling and may be applied in clinical progenitor cell therapy to improve EPC or BMC function in patients with coronary artery disease.

Recent investigations have identified a signal-transduction system involving sphingomyelin and derivatives. In this paradigm, sphingomyelin hydrolysis by a sphingomyelinase generates ceramide, which may be converted to the protein kinase C inhibitor <em>sphingosine</em> or to ceramide <em>1</em>-<em>phosphate</em>. Ceramide may have second-messenger function because it induces epidermal growth factor receptor phosphorylation, presumably on Thr-669 in A-43<em>1</em> cells. The present studies describe a kinase that may mediate ceramide action. With a <em>1</em>9-amino acid epidermal growth factor receptor peptide containing Thr-669, a membrane-bound activity that phosphorylated the peptide was detected in A-43<em>1</em> cells. Activity was linearly related to ATP (0.3-300 microM) and peptide concentration (0.02-<em>1</em> mg/ml), possessed a physiologic pH optimum (pH 7.0-7.4), and was Mg(2+)-dependent. Other cations--Ca2+, Mn2+, and Zn(2+)--were ineffective. Natural and synthetic ceramide induced time- and concentration-dependent enhancement of kinase activity. Ceramide (0.5 microM) increased kinase activity 2-fold by 30 s, and activity remained elevated for at least <em>1</em>5 min. As little as 0.00<em>1</em> microM ceramide was effective, and <em>1</em> microM ceramide induced maximal phosphorylation. <em>Sphingosine</em> was similarly effective. Because tumor necrosis factor (TNF) alpha rapidly induces sphingomyelin hydrolysis to ceramide during monocytic differentiation of HL-60 cells, its effects on kinase activity were assessed. Kinase activity was increased <em>1</em>.5-fold at 5 min and 2-fold at 2 hr in membranes derived from TNF-stimulated cells. The effective concentration range was 3 pM-30 nM TNF. Exogenous ceramide induced a similar effect. In sum, these studies demonstrate the existence of an unusual Mg(2+)-dependent ceramide-activated protein kinase that may mediate some aspects of TNF-alpha function.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (SPP) binds to members of the endothelial differentiation gene family (EDG) of receptors and leads to diverse signaling events including cell survival, growth, migration and differentiation. However, the mechanisms of how SPP activates these proangiogenic pathways are poorly understood. Here we show that SPP signals through the EDG-<em>1</em> receptor to the heterotrimeric G protein G(i), leading to activation of the serine/threonine kinase Akt and phosphorylation of the Akt substrate, endothelial nitric-oxide synthase (eNOS). Inhibition of G(i) signaling, and phosphoinositide 3-kinase (PI 3-kinase) activity resulted in a decrease in SPP-induced endothelial cell chemotaxis. SPP also stimulates eNOS phosphorylation and NO release and these effects are also attenuated by inhibition of G(i) signaling, PI 3-kinase, and Akt. However, inhibition of NO production did not influence SPP-induced chemotaxis but effectively blocked the chemotactic actions of vascular endothelial growth factor. Thus, SPP signals through G(i) and PI 3-kinase leading to Akt activation and eNOS phosphorylation.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive sphingolipid that has recently been shown to bind cell surface S<em>1</em>P receptors (previously called endothelial differentiation gene (Edg) receptors), which are members of the G-protein-coupled family of receptors. Signaling via S<em>1</em>P is a complex process, as cells usually express a number of these receptors on their surfaces. Many of the S<em>1</em>P receptors share common G-proteins, invoking the question of how these receptors are specific in their actions. This review describes the coupling pathways of S<em>1</em>P receptors, and highlights the in vitro and in vivo evidence for the "uniqueness" of each receptor in activating downstream signaling pathways, taking the effect of S<em>1</em>P on migration as an example.

Exposure of renal mesangial cells to <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) leads to a rapid and transient activation of the mitogen- and stress-activated protein kinases but also the protein kinase B. Here, we show that S<em>1</em>P also induces phosphorylation of Smad proteins, which are members of the transforming growth factor-beta (TGF-beta) signaling device. However, Smad phosphorylation occurred more slowly with a maximal effect after 20-30 min of S<em>1</em>P stimulation when compared with the rapid activation of the MAPKs. Interestingly, Smad phosphorylation is increased by pertussis toxin, which is in contrast to the complete inhibition of S<em>1</em>P-induced MAPK phosphorylation by pertussis toxin. TGF-beta is a potent anti-inflammatory cytokine, which in mesangial cells attenuates the expression of (i) inducible nitricoxide synthase (iNOS) caused by interleukin (IL)-<em>1</em>beta, (ii) secreted phospholipase A(2) (sPLA(2)), and (iii) matrix metalloproteinase-9 (MMP-9). These gene products are also down-regulated by S<em>1</em>P in a concentration-dependent manner. Furthermore, the expression of connective tissue growth factor is enhanced by both TGF-beta(2) and S<em>1</em>P. These effects of S<em>1</em>P are not mediated by the MAPK cascade as neither pertussis toxin nor the MAPK cascade inhibitor U0<em>1</em>26 are able to reverse this inhibition. Overexpression of the inhibitory Smad-7 or down-regulation of co-Smad-4 lead to a reversal of the blocking effect of S<em>1</em>P on IL-<em>1</em>beta-induced NO release. Moreover, down-regulating the TGF-beta receptor type II by the siRNA technique or antagonizing the S<em>1</em>P(3) receptor subtype with suramin abrogates S<em>1</em>P-stimulated Smad phosphorylation. In summary, our data show that S<em>1</em>P trans-activates the TGF-beta receptor and triggers activation of Smads followed by activation of connective tissue growth factor gene transcription and inhibition of IL-<em>1</em>beta-induced expression of iNOS, sPLA(2), and MMP-9.

After induction in secondary lymphoid organs, a subset of antibody-secreting cells (ASCs) homes to the bone marrow (BM) and contributes to long-term antibody production. The factors determining secondary lymphoid organ residence versus BM tropism have been unclear. Here we demonstrate that in mice treated with FTY720 or that lack <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor-<em>1</em> (S<em>1</em>P<em>1</em>) in B cells, IgG ASCs are induced and localize normally in secondary lymphoid organs but they are reduced in numbers in blood and BM. Many IgG ASCs home to BM on day 3 of the secondary response and day 3 splenic ASCs exhibit S<em>1</em>P responsiveness, whereas the cells remaining at day 5 are unable to respond. S<em>1</em>P<em>1</em> mRNA abundance is higher in ASCs isolated from blood compared to spleen, whereas CXCR4 expression is lower. Blood ASCs also express higher amounts of Kruppel-like factor (KLF)2, a regulator of S<em>1</em>P<em>1</em> gene expression. These findings establish an essential role for S<em>1</em>P<em>1</em> in IgG plasma cell homing and they suggest that differential regulation of S<em>1</em>P<em>1</em> expression in differentiating plasma cells may determine whether they remain in secondary lymphoid organs or home to BM.

In this report, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a serum-borne bioactive lipid, is shown to activate tight-junction-associated protein Zonula Occludens-<em>1</em> (ZO-<em>1</em>), which in turn plays a critical role in regulating endothelial chemotaxis and barrier integrity. After S<em>1</em>P stimulation, ZO-<em>1</em> was redistributed to the lamellipodia and cell-cell junctions via the S<em>1</em>P<em>1</em>/G(i)/Akt/Rac pathway. Similarly, both endothelial barrier integrity and cell motility were significantly enhanced in S<em>1</em>P-treated cells through the G(i)/Akt/Rac pathway. Importantly, S<em>1</em>P-enhanced barrier integrity and cell migration were abrogated in ZO-<em>1</em> knockdown cells, indicating ZO-<em>1</em> is functionally indispensable for these processes. To investigate the underlying mechanisms, we demonstrated that cortactin plays a critical role in S<em>1</em>P-induced ZO-<em>1</em> redistribution to the lamellipodia. In addition, S<em>1</em>P significantly induced the formation of endothelial tight junctions. ZO-<em>1</em> and alpha-catenin polypeptides were colocalized in S<em>1</em>P-induced junctional structures; whereas, cortactin was not observed in these regions. Together, these results suggest that S<em>1</em>P induces the formation of two distinct ZO-<em>1</em> complexes to regulate two different endothelial functions: ZO-<em>1</em>/cortactin complexes to regulate chemotactic response and ZO-<em>1</em>/alpha-catenin complexes to regulate endothelial barrier integrity. The concerted operation of these two ZO-<em>1</em> complexes may coordinate two important S<em>1</em>P-mediated functions, i.e. migration and barrier integrity, in vascular endothelial cells.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is accumulated in platelets and released on stimulation by thrombin or Ca(2+). Thrombin-stimulated S<em>1</em>P release was inhibited by staurosporin, whereas Ca(2+)-stimulated release was not. When the platelet plasma membrane was permeabilized with streptolysin O (SLO), S<em>1</em>P leaked out with cytosol markers, whereas granular markers remained in the platelets. The SLO-induced S<em>1</em>P leakage required BSA, probably for solubilization of S<em>1</em>P in the medium. These results indicate that S<em>1</em>P is localized in the inner leaflet of the plasma membrane and that its release is a carrier-mediated process. We also used alpha-toxin (ATX), which makes smaller pores in the plasma membrane than SLO and depletes cytosolic ATP without BSA-dependent S<em>1</em>P leakage. The addition of ATP drove S<em>1</em>P release from ATX platelets. The ATP-driven S<em>1</em>P release from ATX platelets was greatly enhanced by thrombin. An ATP binding cassette transporter inhibitor, glyburide, prevents ATP- and thrombin-induced S<em>1</em>P release from platelets. Ca(2+) also stimulated S<em>1</em>P release from ATX platelets without ATP, whereas the Ca(2+)-induced release was not inhibited by glyburide. Our results indicate that two independent S<em>1</em>P release systems might exist in the platelet plasma membrane, an ATP-dependent system stimulated by thrombin and an ATP-independent system stimulated by Ca(2+).

The therapeutic options for ameliorating the profound vascular permeability, alveolar flooding, and organ dysfunction that accompanies acute inflammatory lung injury (ALI) remain limited. Extending our previous finding that the intravenous administration of the sphingolipid angiogenic factor, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), attenuates inflammatory lung injury and vascular permeability via ligation of S<em>1</em>PR(<em>1</em>), we determine that a direct intratracheal or intravenous administration of S<em>1</em>P, or a selective S<em>1</em>P receptor (S<em>1</em>PR(<em>1</em>)) agonist (SEW-287<em>1</em>), produces highly concentration-dependent barrier-regulatory responses in the murine lung. The intratracheal or intravenous administration of S<em>1</em>P or SEW-287<em>1</em> at < 0.3 mg/kg was protective against LPS-induced murine lung inflammation and permeability. However, intratracheal delivery of S<em>1</em>P at 0.5 mg/kg (for 2 h) resulted in significant alveolar-capillary barrier disruption (with a 42% increase in bronchoalveolar lavage protein), and produced rapid lethality when delivered at 2 mg/kg. Despite the greater selectivity for S<em>1</em>PR(<em>1</em>), intratracheally delivered SEW-287<em>1</em> at 0.5 mg/kg also resulted in significant alveolar-capillary barrier disruption, but was not lethal at 2 mg/kg. Consistent with the S<em>1</em>PR(<em>1</em>) regulation of alveolar/vascular barrier function, wild-type mice pretreated with the S<em>1</em>PR(<em>1</em>) inverse agonist, SB-649<em>1</em>46, or S<em>1</em>PR(<em>1</em>)(+/-) mice exhibited reduced S<em>1</em>P/SEW-287<em>1</em>-mediated barrier protection after challenge with LPS. In contrast, S<em>1</em>PR(2)(-/-) knockout mice as well as mice with reduced S<em>1</em>PR(3) expression (via silencing S<em>1</em>PR3-containing nanocarriers) were protected against LPS-induced barrier disruption compared with control mice. These studies underscore the potential therapeutic effects of highly selective S<em>1</em>PR(<em>1</em>) receptor agonists in reducing inflammatory lung injury, and highlight the critical role of the S<em>1</em>P delivery route, S<em>1</em>PR(<em>1</em>) agonist concentration, and S<em>1</em>PR(<em>1</em>) expression in target tissues.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) stimulates thymidine incorporation (DNA synthesis), cell growth and cell migration in human aortic endothelial cells (HAECs). The extent of the S<em>1</em>P-induced responses are comparable to those stimulated by vascular endothelial growth factor, one of the most potent stimulators of angiogenesis. These responses to S<em>1</em>P were mimicked by dihydrosphingosine <em>1</em>-<em>phosphate</em>, an S<em>1</em>P receptor agonist, and inhibited by pertussis toxin (PTX), an inactivator of G(i)/G(o)-proteins. S<em>1</em>P also induced activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAP kinase). The activation of these enzymes was inhibited again by PTX and also by suramin, a non-selective receptor antagonist. S<em>1</em>P-induced DNA synthesis and ERK activation were inhibited by PD98059, an ERK kinase inhibitor, but not by SB203580, a p38 MAP kinase inhibitor. In contrast, cell migration and p38 MAP kinase activation, in response to S<em>1</em>P, were inhibited by SB203580 but not by PD98059. In HAECs, high-affinity S<em>1</em>P binding activity and expression of Edg-<em>1</em> and Edg-3 mRNA were detected. These results suggest that S<em>1</em>P might be a novel angiogenesis factor and that the lipid-induced proliferation and migration of endothelial cells are possibly mediated through cell-surface S<em>1</em>P receptors, Edg-<em>1</em> and Edg-3, which are linked to signalling pathways.

The <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor <em>1</em> (S<em>1</em>P(<em>1</em>)) promotes lymphocyte egress from lymphoid organs. Previous work showed that agonist-induced internalization of this G protein-coupled receptor correlates with inhibition of lymphocyte egress and results in lymphopenia. However, it is unclear if S<em>1</em>P(<em>1</em>) internalization is necessary for this effect. We characterize a knockin mouse (S<em>1</em>p<em>1</em>r(S5A/S5A)) in which the C-terminal serine-rich S<em>1</em>P(<em>1</em>) motif, which is important for S<em>1</em>P(<em>1</em>) internalization but dispensable for S<em>1</em>P(<em>1</em>) signaling, is mutated. T cells expressing the mutant S<em>1</em>P(<em>1</em>) showed delayed S<em>1</em>P(<em>1</em>) internalization and defective desensitization after agonist stimulation. Mutant mice exhibited significantly delayed lymphopenia after S<em>1</em>P(<em>1</em>) agonist administration or disruption of the vascular S<em>1</em>P gradient. Adoptive transfer experiments demonstrated that mutant S<em>1</em>P(<em>1</em>) expression in lymphocytes, rather than endothelial cells, facilitated this delay in lymphopenia. Thus, cell-surface residency of S<em>1</em>P(<em>1</em>) on T cells is a primary determinant of lymphocyte egress kinetics in vivo.

The cleavage of sphingoid base <em>phosphates</em> by <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) lyase to produce phosphoethanolamine and a fatty aldehyde is the final degradative step in the sphingolipid metabolic pathway. We have studied mice with an inactive S<em>1</em>P lyase gene and have found that, in addition to the expected increase of sphingoid base <em>phosphates</em>, other sphingolipids (including <em>sphingosine</em>, ceramide, and sphingomyelin) were substantially elevated in the serum and/or liver of these mice. This latter increase is consistent with a reutilization of the <em>sphingosine</em> backbone for sphingolipid synthesis due to its inability to exit the sphingolipid metabolic pathway. Furthermore, the S<em>1</em>P lyase deficiency resulted in changes in the levels of serum and liver lipids not directly within the sphingolipid pathway, including phospholipids, triacyglycerol, diacylglycerol, and cholesterol. Even though lipids in serum and lipid storage were elevated in liver, adiposity was reduced in the S<em>1</em>P lyase-deficient mice. Microarray analysis of lipid metabolism genes in liver showed that the S<em>1</em>P lyase deficiency caused widespread changes in their expression pattern, with a significant increase in the expression of PPARgamma, a master transcriptional regulator of lipid metabolism. However, the mRNA expression of the genes encoding the <em>sphingosine</em> kinases and S<em>1</em>P phosphatases, which directly control the levels of S<em>1</em>P, were not significantly changed in liver of the S<em>1</em>P lyase-deficient mice. These results demonstrate that S<em>1</em>P lyase is a key regulator of the levels of multiple sphingolipid substrates and reveal functional links between the sphingolipid metabolic pathway and other lipid metabolic pathways that may be mediated by shared lipid substrates and changes in gene expression programs. The disturbance of lipid homeostasis by altered sphingolipid levels may be relevant to metabolic diseases.

Acid sphingomyelinase (ASM) is an important lipid-metabolizing enzyme cleaving sphingomyelin to ceramide, mainly within lysosomes. Acid ceramidase (AC) further degrades ceramide to <em>sphingosine</em> which can then be phosphorylated to <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. Ceramide and its metabolite <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> have been shown to antagonistically regulate apoptosis, cellular differentiation, proliferation and cell migration. Inhibitors of ASM or AC therefore hold promise for a number of new clinical therapies, e.g. for Alzheimer's disease and major depression on the one hand and cancer on the other. Inhibitors of ASM have been known for a long time. Cationic amphiphilic substances induce the detachment of ASM protein from inner lysosomal membranes with its consecutive inactivation, thereby working as functional inhibitors of ASM. We recently experimentally identified a large number of hitherto unknown functional inhibitors of ASM and determined specific physicochemical properties of such cationic amphiphilic substances that functionally inhibit ASM. We propose the acronym "FIASMA" (Functional Inhibitor of Acid SphingoMyelinAse) for members of this large group of compounds with a broad range of new clinical indications. FIASMAs differ markedly with respect to molecular structure and current clinical indication. Most of the available FIASMAs are licensed for medical use in humans, are minimally toxic and may therefore be applied for disease states associated with increased activity of ASM.

The sphingolipid metabolites ceramide and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> are second messengers with opposing roles in mammalian cell growth arrest and survival; their relative cellular level has been proposed to be a rheostat that determines the fate of cells. This report demonstrates that this rheostat is an evolutionarily conserved stress-regulatory mechanism that influences growth and survival of yeast. Although the role of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> in yeast was not previously examined, accumulation of ceramide has been shown to induce G<em>1</em> arrest and cell death. We now have identified a gene in Saccharomyces cerevisiae, LBP<em>1</em>, that regulates the levels of phosphorylated sphingoid bases and ceramide. LBP<em>1</em> was cloned from a yeast mutant that accumulated phosphorylated long-chain sphingoid bases and diverted sphingoid base intermediates from sphingolipid pathways to glycerophospholipid biosynthesis. LBP<em>1</em> and its homolog, LBP2, encode very hydrophobic proteins that contain a novel-conserved sequence motif for lipid phosphatases, and both have long-chain sphingoid base <em>phosphate</em> phosphatase activity. In vitro characterization of Lbp<em>1</em>p shows that this phosphatase is Mg2+-independent with high specificity for phosphorylated long-chain bases, phyto<em>sphingosine</em> and <em>sphingosine</em>. The deletion of LBP<em>1</em> results in the accumulation of phosphorylated long-chain sphingoid bases and reduced ceramide levels. Moreover, deletion of LBP<em>1</em> and LBP2 results in dramatically enhanced survival upon severe heat shock. Thus, these phosphatases play a previously unappreciated role in regulating ceramide and phosphorylated sphingoid base levels in yeast, and they modulate stress responses through sphingolipid metabolites in a manner that is reminiscent of their effects on mammalian cells.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (SPP), a lipid second messenger formed by the action of <em>sphingosine</em> kinase, has been implicated in regulating diverse biological processes, including growth, survival, and differentiation. N,N-Dimethyl<em>sphingosine</em> (DMS) inhibits <em>sphingosine</em> kinase and has been used to investigate the biological roles of SPP; however, little is known of the mechanism of inhibition of <em>sphingosine</em> kinase by DMS. In addition, DMS has been shown to inhibit protein kinase C in vitro. Here we report that DMS is a competitive inhibitor of <em>sphingosine</em> kinase from U937 monoblastic leukemia cells, Swiss 3T3 fibroblasts, and PC<em>1</em>2 pheochromocytoma cells. DMS decreases basal levels of SPP and prevents increases in SPP in response to physiological stimuli known to activate <em>sphingosine</em> kinase. DMS also effectively increases cellular levels of ceramide in a variety of cell types, and resetting of the ceramide/SPP rheostat may account for the pro-apoptotic effects of DMS. Moreover, DMS, at concentrations which effectively inhibit <em>sphingosine</em> kinase, has no effect on protein kinase C activity or its membrane translocation. Thus, DMS acts as a specific competitive inhibitor of <em>sphingosine</em> kinase in diverse cell types and is a useful tool to elucidate the role of SPP as an intracellular second messenger.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive sphingolipid metabolite that regulates diverse biological processes by binding to a family of G protein-coupled receptors or as an intracellular second messenger. Mammalian S<em>1</em>P phosphatase (SPP-<em>1</em>), which degrades S<em>1</em>P to terminate its actions, was recently cloned based on homology to a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast. Confocal microscopy surprisingly revealed that epitope-tagged SPP-<em>1</em> is intracellular and colocalized with the ER marker calnexin. Moreover, SPP-<em>1</em> activity and protein appeared to be mainly enriched in the intracellular membranes with lower expression in the plasma membrane. Treatment of SPP-<em>1</em> transfectants with S<em>1</em>P markedly increased ceramide levels, predominantly in the intracellular membranes, diminished survival, and enhanced apoptosis. Remarkably, dihydro-S<em>1</em>P, although a good substrate for SPP-<em>1</em> in situ, did not cause significant ceramide accumulation or increase apoptosis. Ceramide accumulation induced by S<em>1</em>P was completely blocked by fumonisin B<em>1</em>, an inhibitor of ceramide synthase, but only partially reduced by myriocin, an inhibitor of serine palmitoyltransferase, the first committed step in de novo synthesis of ceramide. Furthermore, S<em>1</em>P, but not dihydro-S<em>1</em>P, stimulated incorporation of [3H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C<em>1</em>6-ceramide. Collectively, our results suggest that SPP-<em>1</em> functions in an unprecedented manner to regulate sphingolipid biosynthesis and is poised to influence cell fate.

Fingolimod <em>phosphate</em> (FTY720) is a <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor agonist that is being used as a new oral drug for multiple sclerosis. FTY720 prevents lymphocytes from moving out of the lymphoid organs and inhibits autoreactive lymphocytes from infiltrating the central nervous system. Whether FTY720 directly affects microglia-the innate immune cells of the central nervous system-is unclear. Here we show that FTY720 binds S<em>1</em>P<em>1</em> receptors to downregulate activated microglial production of such pro-inflammatory cytokines as tumor necrosis factor-α, interleukin-<em>1</em>β, and interleukin-6. FTY720 also upregulates microglial production of brain-derived neurotrophic factor and glial cell-derived neurotrophic factor. These results suggested that FTY720 directly promotes the neuroprotective effects of microglia. Therefore, FTY720 may be a potent therapeutic agent for not only multiple sclerosis but also other neurologic diseases associated with microglial activation.

Sphingolipids play a very important role in cell membrane formation, signal transduction, and plasma lipoprotein metabolism, and all these functions may have an impact on atherosclerotic development. Serine palmitoyl-CoA transferase (SPT) is the key enzyme in sphingolipid biosynthesis. To evaluate in vivo SPT activity and its role in sphingolipid metabolism, we applied homologous recombination to embryonic stem cells, producing mice with long chain base <em>1</em> (Sptlc<em>1</em>) and long chain base 2 (Sptlc2), two subunits of SPT, gene deficiency. Homozygous Sptlc<em>1</em><em>1</em> and Sptlc2 mice are embryonic lethal, whereas heterozygous versions of both animals (Sptlc<em>1</em>(+/-), Sptlc2(+/-)) are healthy. Analysis showed that, compared with WT mice, Sptlc<em>1</em>(+/-) and Sptlc2(+/-) mice had: (<em>1</em>) decreased liver Sptlc<em>1</em> and Sptlc2 mRNA by 44% and 57% (P<0.0<em>1</em> and P<0.000<em>1</em>, respectively); (2) decreased liver Sptlc<em>1</em> mass by 50% and Sptlc2 mass by 70% (P<0.0<em>1</em> and P<0.0<em>1</em>, respectively), moreover, Sptlc<em>1</em> mass decreased by 70% in Sptlc2(+/-) mouse liver, while Sptlc2 mass decreased by 53% in Sptlc<em>1</em>(+/-) mouse liver (P<0.00<em>1</em> and P<0.0<em>1</em>, respectively); (3) decreased liver SPT activity by 45% and 60% (P<0.0<em>1</em>, respectively); (4) decreased liver ceramide (22% and 39%, P<0.05 and P<0.0<em>1</em>, respectively) and <em>sphingosine</em> levels (22% and 3<em>1</em>%, P<0.05 and P<0.0<em>1</em>, respectively); (5) decreased plasma ceramide (45% and 39%, P<0.0<em>1</em>, respectively), <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (3<em>1</em>% and 32%, P<0.0<em>1</em>, respectively) and <em>sphingosine</em> levels (22.5% and 25%, P<0.0<em>1</em>, respectively); (6) dramatically decreased plasma lysosphingomyelin (<em>1</em>7-fold and <em>1</em>6-fold, P<0.000<em>1</em>, respectively); and (7) no change of plasma sphingomyelin, triglyceride, total cholesterol, phospholipids, and liver sphingomyelin levels. These results indicated that both Sptlc<em>1</em> and Sptlc2 interactions are necessary for SPT activity in vivo, and that SPT activity directly influences plasma sphingolipid levels. Furthermore, manipulation of SPT activity might well influence the course of such diseases as atherosclerosis.

Previous studies demonstrated that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) 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 S<em>1</em>P 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. S<em>1</em>P 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. Dihydro<em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, which is a weaker agonist for the S<em>1</em>P receptors, but not an inactive ligand <em>sphingosine</em>, also inhibited PDGF-stimulated chemotaxis and Rac activation although to lesser extents compared with S<em>1</em>P, suggesting that negative regulation by S<em>1</em>P of both chemotaxis and Rac was a receptor-mediated process. In contrast, S<em>1</em>P by itself stimulated Rac activity in HUVECs. Among the five S<em>1</em>P receptor isoforms, SMCs prominently expressed Edg-5 mRNA, whereas HUVECs expressed abundant Edg-<em>1</em> 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 S<em>1</em>P, respectively. Overexpression of Edg-<em>1</em> in SMCs and Edg-5 in HUVECs reduced S<em>1</em>P-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 S<em>1</em>P-mediated bimodal regulation of cell motility in SMCs and HUVECs.

The lysophospholipids, lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), regulate various signaling pathways within cells by binding to multiple G protein-coupled receptors. Receptor-mediated LPA and S<em>1</em>P signaling induces diverse cellular responses including proliferation, adhesion, migration, morphogenesis, differentiation and survival. This review will focus on major components of lysophospholipid signaling: metabolism, identification and expression of LPA and S<em>1</em>P receptors, general signaling pathways and specific signaling mechanisms in mouse embryonic fibroblasts. Finally, in vivo effects of LP receptor gene deletion in mice will be discussed.

OBJECTIVE

Isoflurane, administered before or during cerebral ischemia, has been shown to exhibit neuroprotection in animal models of ischemic stroke. However, the underlying mechanism remains to be elucidated. In the present study, we determined whether isoflurane posttreatment provides neuroprotection after neonatal hypoxia-ischemia (HI) in rats and evaluated the role of the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>/phosphatidylinositol-3-kinase/Akt pathway in this volatile anesthetic-mediated neuroprotection.

METHODS

HI was induced in postnatal day <em>1</em>0 (P<em>1</em>0) rat pups by unilateral carotid ligation and 2 hours of hypoxia. For treatment, 2% isoflurane was administered immediately after HI for <em>1</em> hour. As pharmacological interventions, the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> antagonist VPC230<em>1</em>9, phosphatidylinositol-3-kinase inhibitor wortmannin, or opioid antagonist naloxone was administered before HI. Isoflurane posttreatment was evaluated for effects on infarct volume at 48 hours after HI and brain atrophy and neurological outcomes at 4 weeks after HI. The expression of phosphorylated Akt and cleaved caspase-3 was determined by Western blotting and immunofluorescence analysis.

RESULTS

Isoflurane posttreatment significantly reduced infarct volume at 48 hours after HI. VPC230<em>1</em>9 or wortmannin abrogated the neuroprotective effect of isoflurane, whereas naloxone did not inhibit the isoflurane-induced neuroprotection. Isoflurane posttreatment significantly preserved phosphorylated Akt expression and decreased cleaved caspase-3 levels. These effects were reversed by VPC230<em>1</em>9 and wortmannin, respectively. Isoflurane also confers long-term neuroprotective effects against brain atrophy and neurological deficits at 4 weeks after HI.

CONCLUSIONS

Isoflurane posttreatment provides lasting neuroprotection against hypoxic-ischemic brain injury in neonatal rats. Activation of the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>/phosphatidylinositol-3-kinase/Akt pathway may play a key role in isoflurane posttreatment-induced neuroprotection.

Lipid <em>phosphate</em> phosphatases (LPPs) are a family of integral membrane glycoproteins that catalyze the dephosphorylation of a number of bioactive lipid mediators including lysophosphatidic acid (LPA), <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and phosphatidic acid (PA). These mediators exert complex effects on cell function through both actions at cell surface receptors and on intracellular targets. The LPP-catalyzed dephosphorylation of these substrates can both terminate their signaling actions and itself generate further molecules with biological activity. Recent advances have revealed that a family of structurally related genes is responsible for LPP activities in species from yeast to mammals. These genes exhibit distinct but overlapping expression patterns and their products appear to be heterogeneous with respect to their posttranslational modification and subcellular localizations. Here we review the structure and catalytic properties of the LPPs and consider recent developments in understanding their cellular biology and functions.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and dihydro<em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (DHS<em>1</em>P) are important signaling sphingolipids. The presence of nanomolar levels of S<em>1</em>P and DHS<em>1</em>P in tissues, cells, and biological fluids requires a highly sensitive and selective assay method for their reliable detection and quantitation. Preliminary findings employing positive ion electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated significant sample carryover from previous injections of authentic standards of S<em>1</em>P and DHS<em>1</em>P. This article details a negative ion ESI LC-MS/MS technique following modification of the zwitterionic nature of S<em>1</em>P and DHS<em>1</em>P via derivatization. A highly selective and sensitive LC-MS/MS technique capable of reliable detection of less than 50 fmol of the derivatives of S<em>1</em>P and DHS<em>1</em>P without significant sample carryover was developed. Standard curves for S<em>1</em>P and DHS<em>1</em>P are linear over wide ranges (0-300 pmol) of analyte concentrations with correlation coefficients (r2) greater than 0.995. The levels of S<em>1</em>P and DHS<em>1</em>P in human platelet poor plasma were 590.8+/-42.<em>1</em> and <em>1</em>30.7+/-20.7 pmol/ml, respectively. The levels of S<em>1</em>P and DHS<em>1</em>P in fetal bovine serum were <em>1</em>4<em>1</em>.7+/-4.6 and 0.6+/-0.2 pmol/ml, respectively. The addition of <em>sphingosine</em> (<em>1</em> microM) to human pulmonary artery endothelial cells in culture resulted in a more than 20-fold increase in the cellular level of S<em>1</em>P, whereas the level of DHS<em>1</em>P was unchanged.

Following the present concepts, the synthetic <em>sphingosine</em> analog of myriocin FTY720 alters migration and homing of lymphocytes via <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptors. However, several studies indicate that the immunosuppressive properties of FTY720 may alternatively be due to tolerogenic activities via modulation of dendritic cell differentiation or based on direct effects on CD4(+)CD25(+) regulatory T cells (Treg). As Treg play an important role for the cure of inflammatory colitis, we used the Th<em>1</em>-mediated 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis model to address the therapeutic potential of FTY720 in vivo. A rectal enema of TNBS was given to BALB/c mice. FTY720 was administered i.p. from days 0 to 3 or 3 to 5. FTY720 substantially reduced all clinical, histopathologic, macroscopic, and microscopic parameters of colitis analyzed. The therapeutic effects of FTY720 were associated with a down-regulation of IL-<em>1</em>2p70 and subsequent Th<em>1</em> cytokines. Importantly, FTY720 treatment resulted in a prominent up-regulation of FoxP3, IL-<em>1</em>0, TGFbeta, and CTLA4. Supporting the hypothesis that FTY720 directly affects functional activity of CD4(+)CD25(+) Treg, we measured a significant increase of CD25 and FoxP3 expression in isolated lamina propria CD4(+) T cells of FTY720-treated mice. The impact of FTY720 on Treg induction was further confirmed by concomitant in vivo blockade of CTLA4 or IL-<em>1</em>0R which significantly abrogated its therapeutic activity. In conclusion, our data provide clear evidence that in addition to its well-established effects on migration FTY720 leads to a specific down-regulation of proinflammatory signals while simultaneously inducing functional activity of CD4(+)CD25(+) Treg. Thus, FTY720 may offer a promising new therapeutic strategy for the treatment of IBD.