The endothelium provides an essential and selective membrane barrier that regulates the movement of water, solutes, gases, macromolecules and the cellular elements of the blood from the tissue compartment in health and disease. Its structure and continuous function is essential for life for all vertebrate organisms. Recent evidence indicates that the endothelial surface does not have a passive role in systemic inflammatory states such as septic shock. In fact, endothelial cells are in dynamic equilibrium with a myriad of inflammatory mediators and elements of the innate immune and coagulation systems to orchestrate the host response in sepsis. The barrier function of the endothelial surface is almost uniformly impaired in septic shock, and it is likely that this contributes to adverse outcomes. In this review, we will highlight recent advances in the understanding of the signalling events that regulate endothelial function and molecular events that induce endothelial dysfunction in sepsis. Endothelial barrier repair strategies as a treatment for sepsis include modulation of C5a, high-mobility group box <em>1</em> and VEGF receptor 2; stimulation of angiopoietin-<em>1</em>, <em>sphingosine</em> <em>1</em> <em>phosphate</em> receptor <em>1</em> and Slit; and a number of other innovative approaches.

Sphingolipids are components of all eukaryotic cells that play important roles in a wide variety of biological processes. Ceramides and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) are signaling molecules that regulate cell fate decisions in a wide array of species including yeast, plants, vertebrates, and invertebrates. Ceramides favor anti-proliferative and cell death pathways such as senescence and apoptosis, whereas S<em>1</em>P stimulates cell proliferation and survival pathways. The control of cell fate by these two interconvertible lipids has been called the sphingolipid rheostat or sphingolipid biostat. <em>Sphingosine</em> kinase, the enzyme that synthesizes S<em>1</em>P, is a crucial enzyme in regulation of the balance of these sphingolipids. <em>Sphingosine</em> kinase has been shown to play dynamic roles in the responses of cells to stress, leading to modulation of cell fate through a variety of signaling pathways impinging on the processes of cell proliferation, apoptosis, autophagy and senescence. This review summarizes the roles of <em>sphingosine</em> kinase signaling in these processes and the mechanisms mediating these responses. In addition, we discuss the evidence tying <em>sphingosine</em> kinase-mediated stress responses to the process of aging.

We examined the involvement of <em>sphingosine</em> kinase-<em>1</em>, a critical regulator of the sphingolipid balance, in susceptibility to antineoplastic agents of either sensitive or multidrug-resistant acute myeloid leukemia cells. Contrary to parental HL-60 cells, doxorubicin and etoposide failed to trigger apoptosis in chemoresistant HL-60/Doxo and HL-60NP<em>1</em>6 cells overexpressing MRP<em>1</em> and MDR<em>1</em>, respectively. Chemosensitive HL-60 cells displayed <em>sphingosine</em> kinase-<em>1</em> inhibition coupled with ceramide generation. In contrast, chemoresistant HL-60/ Doxo and HL-60/VP<em>1</em>6 had sustained <em>sphingosine</em> kinase-<em>1</em> activity and did not produce ceramide during treatment. Enforced expression of <em>sphingosine</em> kinase-<em>1</em> in chemosensitive HL-60 cells resulted in marked inhibition of apoptosis that was mediated by blockade of mitochondrial cytochrome c efflux hence suggesting a control of apoptosis at the pre-mitochondrial level. Incubation with cell-permeable ceramide of chemoresistant cells led to a <em>sphingosine</em> kinase-<em>1</em> inhibition and apoptosis both prevented by <em>sphingosine</em> kinase-<em>1</em> over-expression. Furthermore, F-<em>1</em>2509a, a new <em>sphingosine</em> kinase inhibitor, led to ceramide accumulation, decrease in <em>sphingosine</em> <em>1</em>-<em>phosphate</em> content and caused apoptosis equally in chemosensitive and chemoresistant cell lines that is inhibited by adding <em>sphingosine</em> <em>1</em>-<em>phosphate</em> or overexpressing <em>sphingosine</em> kinase-<em>1</em>. F-<em>1</em>2509a induced classical apoptosis hallmarks namely nuclear fragmentation, caspase-3 cleavage as well as downregulation of antiapoptotic XIAP, and release of cytochrome c and SMAC/Diablo.

Macrophage recruitment to sites of inflammation is an essential step in host defense. However, the mechanisms preventing excessive accumulation of macrophages remain relatively unknown. The lysophospholipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) promotes T and B cell egress from lymphoid organs by acting on S<em>1</em>P receptor <em>1</em> (S<em>1</em>P(<em>1</em>)R). More recently, S<em>1</em>P(5)R was shown to regulate NK cell mobilization during inflammation, raising the possibility that S<em>1</em>P regulates the trafficking of other leukocyte lineages. In this study, we show that S<em>1</em>P(2)R inhibits macrophage migration in vitro and that S<em>1</em>P(2)R-deficient mice have enhanced macrophage recruitment during thioglycollate peritonitis. We identify the signaling mechanisms used by S<em>1</em>P(2)R in macrophages, involving the second messenger cAMP and inhibition of Akt phosphorylation. In addition, we show that the phosphoinositide phosphatase and tensin homolog deleted on chromosome <em>1</em>0, which has been suggested to mediate S<em>1</em>P(2)R effects in other cell types, does not mediate S<em>1</em>P(2)R inhibition in macrophages. Our results suggest that S<em>1</em>P serves as a negative regulator of macrophage recruitment by inhibiting migration in these cells and identify an additional facet to the regulation of leukocyte trafficking by S<em>1</em>P.

Activated protein C (APC), the only FDA-approved biotherapeutic drug for sepsis, possesses anticoagulant, antiinflammatory, and barrier-protective activities. However, the mechanisms underlying its anti-inflammatory functions are not well defined. Here, we report that the antiinflammatory activity of APC on macrophages is dependent on integrin CD<em>1</em><em>1</em>b/CD<em>1</em>8, but not on endothelial protein C receptor (EPCR). We showed that CD<em>1</em><em>1</em>b/CD<em>1</em>8 bound APC within specialized membrane microdomains/lipid rafts and facilitated APC cleavage and activation of protease-activated receptor-<em>1</em> (PAR<em>1</em>), leading to enhanced production of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and suppression of the proinflammatory response of activated macrophages. Deletion of the gamma-carboxyglutamic acid domain of APC, a region critical for its anticoagulant activity and EPCR-dependent barrier protection, had no effect on its antiinflammatory function. Genetic inactivation of CD<em>1</em><em>1</em>b, PAR<em>1</em>, or <em>sphingosine</em> kinase-<em>1</em>, but not EPCR, abolished the ability of APC to suppress the macrophage inflammatory response in vitro. Using an LPS-induced mouse model of lethal endotoxemia, we showed that APC administration reduced the mortality of wild-type mice, but not CD<em>1</em><em>1</em>b-deficient mice. These data establish what we believe to be a novel mechanism underlying the antiinflammatory activity of APC in the setting of endotoxemia and provide clear evidence that the antiinflammatory function of APC is distinct from its barrier-protective function and anticoagulant activities.

In Chinese hamster ovary (CHO) cells transiently transfected with an expression vector for EDG<em>1</em>, but not an empty vector, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (SP) at a concentration as low as <em>1</em>0(-<em>1</em>0) M caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i) as a result of mobilization of Ca2+ from both intracellular and extracellular pools. In a CHO clone stably expressing EDG<em>1</em> receptor (CHO-EDG<em>1</em> cells), SP induced increases in the production of inositol <em>phosphates</em> and the [Ca2+]i and inhibited forskolin-induced increase in the cellular cAMP content, all in a manner sensitive to pertussis toxin. SP also activated mitogen-activated protein kinase in CHO-EDG<em>1</em> cells in pertussis toxin-sensitive and Ras-dependent manners. To evaluate the spectrum of agonists for EDG<em>1</em>, we used human erythroleukemia (HEL) cells, which at naive state do not respond to SP or structurally related lipids with an increase in the [Ca2+]i. In HEL cells stably expressing EDG<em>1</em> receptor (HEL-EDG<em>1</em> cells), SP dose-dependently increased the [Ca2+]i with half-maximal and maximal concentration values of <em>1</em>0(-9) and 3 x <em>1</em>0(-7) M, respectively; sphingosylphosphorylcholine at exclusively high concentrations, but not <em>sphingosine</em> at all, also increased the [Ca2+]i. HEL-EDG<em>1</em> cells bound 32P-labeled SP, which was displaced dose dependently by unlabeled SP. These results indicate that EDG<em>1</em>, a member of the EDG family G protein-coupled receptors, is a specific, high-affinity SP receptor.

OBJECTIVE

BAF3<em>1</em>2 is a next-generation <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor modulator, selective for S<em>1</em>P(<em>1</em>) and S<em>1</em>P(5 ) receptors. S<em>1</em>P(<em>1</em>) receptors are essential for lymphocyte egress from lymph nodes and a drug target in immune-mediated diseases. Here, we have characterized the immunomodulatory potential of BAF3<em>1</em>2 and the S<em>1</em>P receptor-mediated effects on heart rate using preclinical and human data.

METHODS

BAF3<em>1</em>2 was tested in a rat experimental autoimmune encephalomyelitis (EAE) model. Electrophysiological recordings of G-protein-coupled inwardly rectifying potassium (GIRK) channels were carried out in human atrial myocytes. A Phase I multiple-dose trial studied the pharmacokinetics, pharmacodynamics and safety of BAF3<em>1</em>2 in 48 healthy subjects.

RESULTS

BAF3<em>1</em>2 effectively suppressed EAE in rats by internalizing S<em>1</em>P(<em>1</em>) receptors, rendering them insensitive to the egress signal from lymph nodes. In healthy volunteers, BAF3<em>1</em>2 caused preferential decreases in CD4(+) T cells, T(naïve) , T(central memory) and B cells within 4-6 h. Cell counts returned to normal ranges within a week after stopping treatment, in line with the elimination half-life of BAF3<em>1</em>2. Despite sparing S<em>1</em>P(3) receptors (associated with bradycardia in mice), BAF3<em>1</em>2 induced rapid, transient (day <em>1</em> only) bradycardia in humans. BAF3<em>1</em>2-mediated activation of GIRK channels in human atrial myocytes can fully explain the bradycardia.

CONCLUSIONS

This study illustrates species-specific differences in S<em>1</em>P receptor specificity for first-dose cardiac effects. Based on its profound but rapidly reversible inhibition of lymphocyte trafficking, BAF3<em>1</em>2 may have potential as a treatment for immune-mediated diseases.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a novel lipid messenger that has important roles in a wide variety of mammalian cellular processes including growth, differentiation and death. Basal levels of S<em>1</em>P in mammalian cells are generally low, but can increase rapidly and transiently when cells are exposed to mitogenic agents and other stimuli. This increase is largely due to increased activity of <em>sphingosine</em> kinase (SK), the enzyme that catalyses its formation. In the current study we have purified, cloned and characterized the first human SK to obtain a better understanding of its biochemical activity and possible activation mechanisms. The enzyme was purified to homogeneity from human placenta using ammonium sulphate precipitation, anion-exchange chromatography, calmodulin-affinity chromatography and gel-filtration chromatography. This resulted in a purification of over <em>1</em>0(6)-fold from the original placenta extract. The enzyme was cloned and expressed in active form in both HEK-293T cells and Escherichia coli, and the recombinant E. coli-derived SK purified to homogeneity. To establish whether post-translational modifications lead to activation of human SK activity we characterized both the purified placental enzyme and the purified recombinant SK produced in E. coli, where such modifications would not occur. The premise for this study was that post-translational modifications are likely to cause conformational changes in the structure of SK, which may result in detectable changes in the physico-chemical or catalytic properties of the enzyme. Thus the enzymes were characterized with respect to substrate specificity and kinetics, inhibition kinetics and various other physico-chemical properties. In all cases, both the native and recombinant SKs displayed remarkably similar properties, indicating that post-translational modifications are not required for basal activity of human SK.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is an endogenous agonist for a family of five G protein-coupled receptors (S<em>1</em>P(<em>1</em>-5)) involved in cell proliferation, cardiovascular development and lymphocyte trafficking. The sphingolipid drug FTY720 displays structural similarity to S<em>1</em>P and efficacy as an immunosuppressant in models of autoimmune disease and in solid organ transplantation. While FTY720 is well-tolerated in humans, it produces a transient reduction of heart rate (HR). As S<em>1</em>P activates the cardiac G protein-gated potassium channel I(KACh), we speculated that the FTY720-induced HR reduction reflects I(KACh) activation. We examined FTY720 effects on atrial myocytes from wild-type and I(KACh)-deficient mice. In wild-type myocytes, the active <em>phosphate</em> metabolite of FTY720 (FTY720-P) induced single channel activity with conductance, open time, GTP sensitivity and rectification identical to that of I(KACh). In whole-cell recordings, FTY720-P evoked an inwardly rectifying potassium current in approximately 90% of myocytes responding to acetylcholine. Comparable channel activity was never observed in myocytes from I(KACh)-deficient mice. In wild-type mice, acute FTY720 administration produced a dose-dependent, robust HR reduction. In contrast, the HR reduction induced by FTY720 in I(KACh)-deficient mice was blunted. We conclude that the effect of acute FTY720 administration on HR is mediated primarily by I(KACh) activation.

Programmed cell death is vital for a number of pathophysiologic settings. Apoptotic cells are rapidly engulfed by phagocytes (ie, macrophages), which in turn acquire an anti-inflammatory phenotype known as alternative activation or the M2-type. Here we show that interaction of apoptotic cells with macrophages attenuates cell death pathways in the latter. Protection of human macrophages required phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinase <em>1</em>/2 (ERK<em>1</em>/2), and Ca2+ signaling, and correlated with Bcl-X(L) and Bcl-2 up-regulation as well as Ser<em>1</em>36-Bad phosphorylation. Unexpectedly, neither phagocytosis nor binding of apoptotic debris to the phagocyte was necessary to induce protection. Surprisingly, apoptotic cells released <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), mainly derived from <em>sphingosine</em> kinase 2, as a survival messenger. This points to an active role of apoptotic cells in preventing cell destruction in their neighborhood, with implications for innate immunity and inflammation.

FTY720, also known as fingolimod, is an orally administered <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) analogue that is under investigation as a therapy for both relapsing-remitting (RR) and progressive forms of multiple sclerosis (MS). The demonstrated beneficial effect of FTY720 on disease activity in RR-MS patients and in the animal model experimental autoimmune encephalomyelitis (EAE) is largely attributed to effects on the systemic immune system. In addition, unlike other current systemic immuno-modulators used in MS, the lipophilic nature of FTY720 allows it to cross the blood-brain barrier (BBB). Since S<em>1</em>P receptors are expressed on all cell types, FTY720 has the potential to exert effects directly on the BBB and on resident cells of the CNS. The latter include cells implicated in regulating immune reactivity within the CNS (astrocytes, microglia), those that are targeted by the disease process (oligodendrocytes, neurons), and those involved in repair (oligodendrocyte progenitor cells). In vitro studies document the dose-dependent effects of FTY720 on neural cell survival, differentiation, and cytoskeletal dynamics. Animal model studies, specifically EAE, indicate an overall neuroprotective effect of FTY720 mediated at least in part by its actions within the CNS. Ongoing studies will need to define the direct and indirect (via immune-modulation) effects of FTY720 on the CNS across the broad clinical spectrum of MS.

<em>Sphingosine</em> kinase (SK) catalyzes the formation of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a lipid messenger that plays an important role in a variety of mammalian cell processes, including inhibition of apoptosis and stimulation of cell proliferation. Basal levels of S<em>1</em>P in cells are generally low but can increase rapidly when cells are exposed to various agonists through rapid and transient activation of SK activity. To date, elucidation of the exact signaling pathways affected by these elevated S<em>1</em>P levels has relied on the use of SK inhibitors that are known to have direct effects on other enzymes in the cell. Furthermore, these inhibitors block basal SK activity, which is thought to have a housekeeping function in the cell. To produce a specific inhibitor of SK activation we sought to generate a catalytically inactive, dominant-negative SK. This was accomplished by site-directed mutagenesis of Gly(82) to Asp of the human SK, a residue identified through sequence similarity to the putative catalytic domain of diacylglycerol kinase. This mutant had no detectable SK activity when expressed at high levels in HEK293T cells. Activation of endogenous SK activity by tumor necrosis factor-alpha (TNFalpha), interleukin-<em>1</em>beta, and phorbol esters in HEK293T cells was blocked by expression of this inactive <em>sphingosine</em> kinase (hSK(G82D)). Basal SK activity was unaffected by expression of hSK(G82D). Expression of hSK(G82D) had no effect on TNFalpha-induced activation of protein kinase C and sphingomyelinase activities. Thus, hSK(G82D) acts as a specific dominant-negative SK to block SK activation. This discovery provides a powerful tool for the elucidation of the exact signaling pathways affected by elevated S<em>1</em>P levels following SK activation. To this end we have employed the dominant-negative SK to demonstrate that TNFalpha activation of extracellular signal-regulated kinases <em>1</em> and 2 (ERK<em>1</em>,2) is dependent on SK activation.

Caveolin-<em>1</em> is a scaffolding/regulatory protein that interacts with diverse signaling molecules in endothelial cells. To explore the role of this protein in receptor-modulated signaling pathways, we transfected bovine aortic endothelial cells (BAEC) with small interfering RNA (siRNA) duplexes to down-regulate caveolin-<em>1</em> expression. Transfection of BAEC with duplex siRNA targeted against caveolin-<em>1</em> mRNA selectively "knocked-down" the expression of caveolin-<em>1</em> by approximately 90%, as demonstrated by immunoblot analyses of BAEC lysates. We used discontinuous sucrose gradients to purify caveolin-containing lipid rafts from siRNA-treated endothelial cells. Despite the near-total down-regulation of caveolin-<em>1</em> expression, the lipid raft targeting of diverse signaling proteins (including the endothelial isoform of nitric-oxide synthase, Src-family tyrosine kinases, Galphaq and the insulin receptor) was unchanged. We explored the consequences of caveolin-<em>1</em> knockdown on kinase pathways modulated by the agonists <em>sphingosine</em>-<em>1</em> <em>phosphate</em> (S<em>1</em>P) and vascular endothelial growth factor (VEGF). siRNA-mediated caveolin-<em>1</em> knockdown enhanced basal as well as S<em>1</em>P- and VEGF-induced phosphorylation of the protein kinase Akt and did not modify the basal or agonist-induced phosphorylation of extracellular signal-regulated kinases <em>1</em>/2. Caveolin-<em>1</em> knock-down also significantly enhanced the basal and agonist-induced activity of the small GTPase Rac. We used siRNA to down-regulate Rac expression in BAEC, and we observed that Rac knockdown significantly reduced basal, S<em>1</em>P-, and VEGF-induced Akt phosphorylation, suggesting a role for Rac activation in the caveolin siRNA-mediated increase in Akt phosphorylation. By using siRNA to knockdown caveolin-<em>1</em> and Rac expression in cultured endothelial cells, we have found that caveolin-<em>1</em> does not seem to be required for the targeting of signaling molecules to caveolae/lipid rafts and that caveolin-<em>1</em> differentially modulates specific kinase pathways in endothelial cells.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) acts as an extracellular ligand for a family of G-protein coupled receptors that are crucial in cell migration. S<em>1</em>P5 is exclusively expressed in oligodendrocytes and oligodendrocyte precursor cells (OPCs), which migrate considerable distances during brain development. The current studies suggest a physiological role for S<em>1</em>P and S<em>1</em>P5 in regulation of OPC migration. mRNA expression levels of S<em>1</em>P2 and S<em>1</em>P5 are comparable in OPCs, but S<em>1</em>P binding specifically to the S<em>1</em>P5 receptor blocked OPC migration (IC50=29 nM). Thus, knocking down S<em>1</em>P5 using siRNA prevented the S<em>1</em>P-induced decrease in OPC migration, whereas knocking down S<em>1</em>P2 did not have any effect. S<em>1</em>P-induced modulation of OPC migration was insensitive to pertussis toxin, suggesting that S<em>1</em>P5-initiated signaling is not mediated by the G alpha(i)-protein coupled pathway. Furthermore, S<em>1</em>P5 appears to engage the G alpha(<em>1</em>2/<em>1</em>3) protein coupled Rho/ROCK signaling pathway to impede OPC migration. To modulate OPC motility, extracellular S<em>1</em>P could be derived from the export of intracellular S<em>1</em>P generated in response to glutamate treatment of OPCs. These studies suggest that S<em>1</em>P could be a part of the neuron-oligodendroglial communication network regulating OPC migration and may provide directional guidance cues for migrating OPCs in the developing brain.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a biologically active lysophospholipid that serves as a key regulator of cellular differentiation and survival. Immune stimuli increase S<em>1</em>P synthesis and secretion by mast cells and platelets, implicating this molecule in tissue responses to injury and inflammation. Binding of S<em>1</em>P to G(i) protein-coupled receptors activates phosphatidylinositol 3-kinase and Akt in a variety of tissues. To elucidate the mechanisms by which S<em>1</em>P enhances adult cardiac myocyte survival during hypoxia, we used a mouse cell culture system in which S<em>1</em>P(<em>1</em>) receptors were observed to transduce signals from exogenous S<em>1</em>P, an S<em>1</em>P(<em>1</em>) receptor antibody with agonist properties, and the pharmacological agents FTY720 and SEW287<em>1</em>. S<em>1</em>P(<em>1</em>) receptor mRNA and protein were abundantly expressed by adult mouse cardiac myocytes. S<em>1</em>P-S<em>1</em>P(<em>1</em>) receptor axis enhancement of myocyte survival during hypoxia was abolished by phosphatidylinositol 3-kinase inhibition. S<em>1</em>P(<em>1</em>) receptor function was closely associated with activation of Akt, inactivation of GSK-3beta, and reduction of cytochrome c release from heart mitochondria. These observations highlight the importance of S<em>1</em>P(<em>1</em>) receptors on ventricular myocytes as mediators of inducible resistance against cellular injury during severe hypoxic stress.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lipid molecule that acts as both an extracellular signaling mediator and an intracellular second messenger. S<em>1</em>P is synthesized from <em>sphingosine</em> by <em>sphingosine</em> kinase and is degraded either by S<em>1</em>P lyase or by S<em>1</em>P phosphohydrolase. Recently, mammalian S<em>1</em>P phosphohydrolase (SPP<em>1</em>) was identified and shown to constitute a novel lipid phosphohydrolase family, the SPP family. In this study we have identified a second human S<em>1</em>P phosphohydrolase, SPP2, based on sequence homology to human SPP<em>1</em>. SPP2 exhibited high phosphohydrolase activity against S<em>1</em>P and dihydro<em>sphingosine</em> <em>1</em>-<em>phosphate</em>. The dihydro<em>sphingosine</em>-<em>1</em>-<em>phosphate</em> phosphohydrolase activity was efficiently inhibited by excess S<em>1</em>P but not by lysophosphatidic acid, phosphatidic acid, or glycerol 3-<em>phosphate</em>, indicating that SPP2 is highly specific to sphingoid base <em>1</em>-<em>phosphates</em>. Immunofluorescence microscopic analysis demonstrated that SPP2 is localized to the endoplasmic reticulum. Although the enzymatic properties and localization of SPP2 were similar to those of SPP<em>1</em>, the tissue-specific expression pattern of SPP2 was different from that of SPP<em>1</em>. Thus, SPP2 is another member of the SPP family that may play a role in attenuating intracellular S<em>1</em>P signaling.

BACKGROUND

Phosphorylation of <em>sphingosine</em> by <em>sphingosine</em> kinase (SK) is the rate-limiting step in the cellular synthesis of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P). The monoganglioside GM<em>1</em>, which stimulates SK, is cardioprotective in part through increased generation of S<em>1</em>P that protects myocytes by diverse mechanisms. Because protein kinase C (PKC)epsilon activation is necessary for myocardial ischemic preconditioning (IPC) and PKC activators increase SK activity, we tested the hypothesis that SK may be a central mediator of IPC.

RESULTS

In adult murine hearts, IPC sufficient to reduce infarct size significantly increased cardiac SK activity, induced translocation of SK protein from the cytosol to membranes, and enhanced cardiac myocyte survival. IPC did not increase SK activity in PKCepsilon-null mice. The SK antagonist N,N-dimethylsphingosine inhibited PKCepsilon activation and directly abolished the protective effects of IPC and the enhanced SK activity induced by IPC.

CONCLUSIONS

These findings demonstrate that PKCepsilon is thus recruited by IPC and induces activation of SK that then mediates IPC-induced cardioprotection in murine heart.

The structural similarity of lysosphingolipids to lysophosphatidic acid (LPA) prompted a sequence-based search for lysosphingolipid receptors using cDNA sequence of the Edg2 human LPA receptor. Two closely related G protein-coupled receptors, rat H2<em>1</em>8 and human Edg3, are highly similar to Edg2. When overexpressed in Jurkat cells, H2<em>1</em>8 and Edg3 activated serum response element-driven transcriptional reporter gene in response to <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), dihydro-S<em>1</em>P and sphingosylphosphorylcholine, but not to LPA. H2<em>1</em>8 and Edg3 expressed in Xenopus oocytes conferred responsiveness to S<em>1</em>P and dihydro-S<em>1</em>P in agonist-triggered 45Ca2+ efflux. Therefore, H2<em>1</em>8 and Edg3 are functional receptors for S<em>1</em>P and perhaps other closely related lysosphingolipids.

<em>Sphingosine</em> kinase <em>1</em> (SphK<em>1</em>) and SphK2 are ubiquitous enzymes that generate <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a ligand for a family of G protein-coupled receptors (S<em>1</em>PR<em>1</em>-S<em>1</em>PR5) with important functions in the vascular and immune systems. Here we explore the role of these kinases and receptors in recovery from anaphylaxis in mice. We found that Sphk2-/- mice had a rapid recovery from anaphylaxis. In contrast, Sphk<em>1</em>-/- mice showed poor recovery from anaphylaxis and delayed histamine clearance. Injection of S<em>1</em>P into Sphk<em>1</em>-/- mice increased histamine clearance and promoted recovery from anaphylaxis. Adoptive cell transfer experiments demonstrated that SphK<em>1</em> activity was required in both the hematopoietic and nonhematopoietic compartments for recovery from anaphylaxis. Mice lacking the S<em>1</em>P receptor S<em>1</em>PR2 also showed a delay in plasma histamine clearance and a poor recovery from anaphylaxis. However, S<em>1</em>P did not promote the recovery of S<em>1</em>pr2-/- mice from anaphylaxis, whereas S<em>1</em>pr2+/- mice showed partial recovery. Unlike Sphk2-/- mice, Sphk<em>1</em>-/- and S<em>1</em>pr2-/- mice had severe hypotension during anaphylaxis. Thus, SphK<em>1</em>-produced S<em>1</em>P regulates blood pressure, histamine clearance, and recovery from anaphylaxis in a manner that involves S<em>1</em>PR2. This suggests that specific S<em>1</em>PR2 agonists may serve to counteract the vasodilation associated with anaphylactic shock.

Various studies in cell lines have previously demonstrated that <em>sphingosine</em> kinase <em>1</em> (SK<em>1</em>) and extracellular signal-regulated kinase <em>1</em>/2 (ERK-<em>1</em>/2) interact in an estrogen receptor (ER)-dependent manner to influence both breast cancer cell growth and migration. A cohort of 304 ER-positive breast cancer patients was used to investigate the prognostic significance of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors <em>1</em>, 2, and 3 (ie, S<em>1</em>P<em>1</em>, S<em>1</em>P2, and S<em>1</em>P3), SK<em>1</em>, and ERK-<em>1</em>/2 expression levels. Expression levels of both SK<em>1</em> and ERK-<em>1</em>/2 were already available for the cohort, and S<em>1</em>P<em>1</em>, S<em>1</em>P2, and S<em>1</em>P3 levels were established by immunohistochemical analysis. High membrane S<em>1</em>P<em>1</em> expression was associated with shorter time to recurrence (P=0.008). High cytoplasmic S<em>1</em>P<em>1</em> and S<em>1</em>P3 expression levels were also associated with shorter disease-specific survival times (P=0.036 and P=0.0<em>1</em>9, respectively). Those patients with tumors that expressed high levels of both cytoplasmic SK<em>1</em> and ERK-<em>1</em>/2 had significantly shorter recurrence times than those that expressed low levels of cytoplasmic SK<em>1</em> and cytoplasmic ERK-<em>1</em>/2 (P=0.00008), with a difference in recurrence time of <em>1</em>0.5 years. Similarly, high cytoplasmic S<em>1</em>P<em>1</em> and cytoplasmic ERK-<em>1</em>/2 expression levels (P=0.004) and high cytoplasmic S<em>1</em>P3 expression and cytoplasmic ERK-<em>1</em>/2 expression levels (P=0.004) were associated with shorter recurrence times. These results support a model in which the interaction between SK<em>1</em>, S<em>1</em>P<em>1</em>, and/or S<em>1</em>P3 and ERK-<em>1</em>/2 might drive breast cancer progression, and these findings, therefore, warrant further investigation.

This paper describes a simultaneous analytical method for the measurement of sphingoid base <em>1</em>-<em>phosphates</em> and sphingoid bases from a variety of biological samples. This method consists of two steps of sample pretreatment: the enzymatic dephosphorylation of sphingoid base <em>1</em>-<em>phosphates</em> by alkaline phosphatase (APase) and the subsequent analysis of o-phthalaldehyde (OPA) derivatives of the liberated sphingoid bases by HPLC. By introducing C<em>1</em>7-<em>sphingosine</em> <em>1</em>-<em>phosphate</em> and C<em>1</em>7-<em>sphingosine</em> as internal standards, not only phyto<em>sphingosine</em> <em>1</em>-<em>phosphate</em>, <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, and sphinganine <em>1</em>-<em>phosphate</em> but also phyto<em>sphingosine</em>, <em>sphingosine</em>, and sphinganine present in a sample could be quantified in <em>1</em>2 min on a C<em>1</em>8 reversed-phase column with a simple mobile phase of acetonitrile:deionized distilled water (90:<em>1</em>0, v/v). With this HPLC method, we could reproducibly analyze the levels of sphingoid base <em>1</em>-<em>phosphates</em> over a broad range of concentrations from 0.5 to <em>1</em>00.0 pmol from various biological samples including serum, cultured cells, and rat tissue homogenates. The conversion of sphingoid base <em>1</em>-<em>phosphates</em> into sphingoid bases increased the stability of the OPA adducts. Thus, this indirect measurement of sphingoid base <em>1</em>-<em>phosphates</em> increased the sensitivity and reproducibility of the method. This HPLC method was also used to measure the changes in the levels of sphingoid base <em>1</em>-<em>phosphates</em> in cultured cells after treatment with <em>1</em>,25-(OH)2D3, a <em>sphingosine</em> kinase activator, or with fumonisin B<em>1</em>, a sphinganine N-acyltransferase inhibitor.

Novel immunomodulatory molecule FTY720 is a synthetic analog of myriocin, but unlike myriocin FTY720 does not inhibit serine palmitoyltransferase. Although many of the effects of FTY720 are ascribed to its phosphorylation and subsequent <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P)-like action through S<em>1</em>P(<em>1</em>,3-5) receptors, studies on modulation of intracellular balance of signaling sphingolipids by FTY720 are limited. In this study, we used stable isotope pulse labeling of human pulmonary artery endothelial cells with l-[U-(<em>1</em>3)C, (<em>1</em>5)N]serine as well as in vitro enzymatic assays and liquid chromatography-tandem mass spectrometry methodology to characterize FTY720 interference with sphingolipid de novo biosynthesis. In human pulmonary artery endothelial cells, FTY720 inhibited ceramide synthases, resulting in decreased cellular levels of dihydroceramides, ceramides, <em>sphingosine</em>, and S<em>1</em>P but increased levels of dihydro<em>sphingosine</em> and dihydro<em>sphingosine</em> <em>1</em>-<em>phosphate</em> (DHS<em>1</em>P). The FTY720-induced modulation of sphingolipid de novo biosynthesis was similar to that of fumonisin B<em>1</em>, a classical inhibitor of ceramide synthases, but differed in the efficiency to inhibit biosynthesis of short-chain versus long-chain ceramides. In vitro kinetic studies revealed that FTY720 is a competitive inhibitor of ceramide synthase 2 toward dihydro<em>sphingosine</em> with an apparent K(i) of 2.<em>1</em>5 microm. FTY720-induced up-regulation of DHS<em>1</em>P level was mediated by <em>sphingosine</em> kinase (SphK) <em>1</em>, but not SphK2, as confirmed by experiments using SphK<em>1</em>/2 silencing with small interfering RNA. Our data demonstrate for the first time the ability of FTY720 to inhibit ceramide synthases and modulate the intracellular balance of signaling sphingolipids. These findings open a novel direction for therapeutic applications of FTY720 that focuses on inhibition of ceramide biosynthesis, ceramide-dependent signaling, and the up-regulation of DHS<em>1</em>P generation in cells.

During late endosome maturation, cargo molecules are sorted into intralumenal vesicles (ILVs) of multivesicular endosomes (MVEs), and are either delivered to lysosomes for degradation or fused with the plasma membranes for exosome release. The mechanism underlying formation of exosomal ILVs and cargo sorting into ILVs destined for exosome release is still unclear. Here we show that inhibitory G protein (Gi)-coupled <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors regulate exosomal MVE maturation. Gi-coupled S<em>1</em>P receptors on MVEs are constitutively activated through a constant supply of S<em>1</em>P via autocrine activation within organelles. We also found that the continuous activation of Gi-coupled S<em>1</em>P receptors on MVEs is essential for cargo sorting into ILVs destined for exosome release. Our results reveal a mechanism underlying ESCRT-independent maturation of exosomal MVEs.