Inflammatory bowel disease is an important risk factor for colorectal cancer. We show that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) produced by upregulation of <em>sphingosine</em> kinase <em>1</em> (SphK<em>1</em>) links chronic intestinal inflammation to colitis-associated cancer (CAC) and both are exacerbated by deletion of Sphk2. S<em>1</em>P is essential for production of the multifunctional NF-κB-regulated cytokine IL-6, persistent activation of the transcription factor STAT3, and consequent upregulation of the S<em>1</em>P receptor, S<em>1</em>PR<em>1</em>. The prodrug FTY720 decreased SphK<em>1</em> and S<em>1</em>PR<em>1</em> expression and eliminated the NF-κB/IL-6/STAT3 amplification cascade and development of CAC, even in Sphk2(-/-) mice, and may be useful in treating colon cancer in individuals with ulcerative colitis. Thus, the SphK<em>1</em>/S<em>1</em>P/S<em>1</em>PR<em>1</em> axis is at the nexus between NF-κB and STAT3 and connects chronic inflammation and CAC.

Sphingolipids are a highly diverse category of compounds that serve not only as components of biologic structures but also as regulators of numerous cell functions. Because so many of the sphingolipids in a biological system are bioactive and are often closely related structurally and metabolically (for example, complex sphingolipids<->>ceramide<->><em>sphingosine</em><->><em>sphingosine</em> <em>1</em>-<em>phosphate</em>), to understand the role(s) of sphingolipids in a given context one must conduct a "sphingolipidomic" analysis-i.e., a structure-specific and quantitative measurement of all of these compounds, or at least all members of a critical subset. Liquid chromatography tandem mass spectrometry (LC MS/MS) is currently the only technology with the requisite structural specificity, sensitivity, quantitative precision, and relatively high-throughput capabilities for such analyses in small samples ( approximately <em>1</em>0(6) cells). This review describes a series of protocols that have been developed for the relatively rapid analysis of all of the molecular species from 3-ketosphinganines through sphingomyelins and some glycosphingolipids (including all the compounds that are presently regarded as sphingolipid "second messengers") using normal- and reverse-phase LC to separate isometric and isobaric species (such as glucosylceramides and galactosylceramides) in combination with triple quadrupole (for MS/MS) and hybrid quadrupole-ion trap (for MS3) mass spectrometry. Also discussed are some of the issues remaining to be resolved in the analysis of the full sphingolipidome.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) influences heart rate, coronary artery caliber, endothelial integrity, and lymphocyte recirculation through five related high affinity G-protein-coupled receptors. Inhibition of lymphocyte recirculation by non-selective S<em>1</em>P receptor agonists produces clinical immunosuppression preventing transplant rejection but is associated with transient bradycardia. Understanding the contribution of individual receptors has been limited by the embryonic lethality of the S<em>1</em>P(<em>1</em>) knock-out and the unavailability of selective agonists or antagonists. A potent, S<em>1</em>P(<em>1</em>)-receptor selective agonist structurally unrelated to S<em>1</em>P was found to activate multiple signals triggered by S<em>1</em>P, including guanosine 5'-3-O-(thio)tri<em>phosphate</em> binding, calcium flux, Akt and ERK<em>1</em>/2 phosphorylation, and stimulation of migration of S<em>1</em>P(<em>1</em>)- but not S<em>1</em>P(3)-expressing cells in vitro. The agonist also alters lymphocyte trafficking in vivo. Use of selective agonism together with deletant mice lacking S<em>1</em>P(3) receptor reveals that agonism of S<em>1</em>P(<em>1</em>) receptor alone is sufficient to control lymphocyte recirculation. Moreover, S<em>1</em>P(<em>1</em>) receptor agonist plasma levels are causally associated with induction and maintenance of lymphopenia. S<em>1</em>P(3), and not S<em>1</em>P(<em>1</em>), is directly implicated in sinus bradycardia. The sustained bradycardia induced by S<em>1</em>P receptor non-selective immunosuppressive agonists in wild-type mice is abolished in S<em>1</em>P(3)-/- mice, whereas S<em>1</em>P(<em>1</em>)-selective agonist does not produce bradycardia. Separation of receptor subtype usage for control of lymphocyte recirculation and heart rate may allow the identification of selective immunosuppressive S<em>1</em>P(<em>1</em>) receptor agonists with an enhanced therapeutic window. S<em>1</em>P(<em>1</em>)-selective agonists will be of broad utility in understanding cell functions in vitro, and vascular physiology in vivo, and the success of the chemical approach for S<em>1</em>P(<em>1</em>) suggests that selective tools for the resolution of function across this broad lipid receptor family are now possible.

<em>Sphingosine</em> kinase <em>1</em> is an agonist-activated signalling enzyme that catalyses the formation of <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, a lipid second messenger that has been implicated in a number of agonist-driven cellular responses, including stimulation of cell proliferation, inhibition of apoptosis and expression of inflammatory molecules. Although agonist-induced stimulation of <em>sphingosine</em> kinase activity is critical in a number of signalling pathways, nothing has been known of the molecular mechanism of this activation. Here we show that this activation results directly from phosphorylation of <em>sphingosine</em> kinase <em>1</em> at Ser225, and present several lines of evidence to show compellingly that the activating kinase is ERK<em>1</em>/2 or a close relative. Furthermore, we show that phosphorylation of <em>sphingosine</em> kinase <em>1</em> at Ser225 results not only in an increase in enzyme activity, but is also necessary for translocation of the enzyme from the cytosol to the plasma membrane. Thus, these studies have elucidated the mechanism of agonist-mediated <em>sphingosine</em> kinase activation, and represent a key finding in understanding the regulation of <em>sphingosine</em> kinase/<em>sphingosine</em> <em>1</em>-<em>phosphate</em>-controlled signalling pathways.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), an abundant lipid mediator in plasma, regulates vascular and immune cells by activating S<em>1</em>P receptors. In this report, we investigated the mechanisms by which high plasma S<em>1</em>P levels are maintained in mice. We found that plasma S<em>1</em>P turns over rapidly with a half-life of approximately <em>1</em>5 minutes, suggesting the existence of a high-capacity biosynthetic source(s). Transplantation of bone marrow from wild-type to Sphk<em>1</em>(-/-)Sphk2(+/-) mice restored plasma S<em>1</em>P levels, suggesting that hematopoietic cells are capable of secreting S<em>1</em>P into plasma. However, plasma S<em>1</em>P levels were not appreciably altered in mice that were thrombocytopenic, anemic, or leukopenic. Surprisingly, reconstitution of Sphk<em>1</em>(-/-)Sphk2(+/-) bone marrow cells into wild-type hosts failed to reduce plasma S<em>1</em>P, suggesting the existence of an additional, nonhematopoietic source for plasma S<em>1</em>P. Adenoviral expression of Sphk<em>1</em> in the liver of Sphk<em>1</em>(-/-) mice restored plasma S<em>1</em>P levels. In vitro, vascular endothelial cells, but not hepatocytes, secreted S<em>1</em>P in a constitutive manner. Interestingly, laminar shear stress downregulated the expression of S<em>1</em>P lyase (Sgpl) and S<em>1</em>P phosphatase-<em>1</em> (Sgpp<em>1</em>) while concomitantly stimulating S<em>1</em>P release from endothelial cells in vitro. Modulation of expression of endothelial S<em>1</em>P lyase with small interfering RNA and adenoviral expression altered S<em>1</em>P secretion, suggesting an important role played by this enzyme. These data suggest that the vascular endothelium, in addition to the hematopoietic system, is a major contributor of plasma S<em>1</em>P.

S1P has been proposed to contribute to cancer progression by regulating tumor proliferation, invasion, and angiogenesis. We developed a biospecific monoclonal antibody to S1P to investigate its role in tumorigenesis. The anti-S1P mAb substantially reduced tumor progression and in some cases eliminated measurable tumors in murine xenograft and allograft models. Tumor growth inhibition was attributed to antiangiogenic and antitumorigenic effects of the antibody. The anti-S1P mAb blocked EC migration and resulting capillary formation, inhibited blood vessel formation induced by VEGF and bFGF, and arrested tumor-associated angiogenesis. The anti-S1P mAb also neutralized S1P-induced proliferation, release of proangiogenic cytokines, and the ability of S1P to protect tumor cells from apoptosis in several tumor cell lines, validating S1P as a target for therapy.

For an epithelium to provide a protective barrier, it must maintain homeostatic cell numbers by matching the number of dividing cells with the number of dying cells. Although compensatory cell division can be triggered by dying cells, it is unknown how cell death might relieve overcrowding due to proliferation. When we trigger apoptosis in epithelia, dying cells are extruded to preserve a functional barrier. Extrusion occurs by cells destined to die signalling to surrounding epithelial cells to contract an actomyosin ring that squeezes the dying cell out. However, it is not clear what drives cell death during normal homeostasis. Here we show in human, canine and zebrafish cells that overcrowding due to proliferation and migration induces extrusion of live cells to control epithelial cell numbers. Extrusion of live cells occurs at sites where the highest crowding occurs in vivo and can be induced by experimentally overcrowding monolayers in vitro. Like apoptotic cell extrusion, live cell extrusion resulting from overcrowding also requires <em>sphingosine</em> <em>1</em>-<em>phosphate</em> signalling and Rho-kinase-dependent myosin contraction, but is distinguished by signalling through stretch-activated channels. Moreover, disruption of a stretch-activated channel, Piezo<em>1</em>, in zebrafish prevents extrusion and leads to the formation of epithelial cell masses. Our findings reveal that during homeostatic turnover, growth and division of epithelial cells on a confined substratum cause overcrowding that leads to their extrusion and consequent death owing to the loss of survival factors. These results suggest that live cell extrusion could be a tumour-suppressive mechanism that prevents the accumulation of excess epithelial cells.

The splenic marginal zone is a site of blood flow, and the specialized B cell population that inhabits this compartment has been linked to the capture and follicular delivery of blood-borne antigens. However, the mechanism of this antigen transport has remained unknown. Here we show that marginal zone B cells were not confined to the marginal zone but continuously shuttled between the marginal zone and follicular areas, such that many of the cells visited a follicle every few hours. Migration to the follicle required the chemokine receptor CXCR5, whereas return to the marginal zone was promoted by the <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptors S<em>1</em>P<em>1</em> and S<em>1</em>P3. Treatment with an S<em>1</em>P<em>1</em> antagonist caused displacement of marginal zone B cells from the marginal zone. Marginal zone-follicle shuttling of marginal zone B cells provides an efficient mechanism for systemic antigen capture and delivery to follicular dendritic cells.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a lysophospholipid, has gained relevance to multiple sclerosis through the discovery of FTY720 (fingolimod), recently approved as an oral treatment for relapsing forms of multiple sclerosis. Its mechanism of action is thought to be immunological through an active phosphorylated metabolite, FTY720-P, that resembles S<em>1</em>P and alters lymphocyte trafficking through receptor subtype S<em>1</em>P(<em>1</em>). However, previously reported expression and in vitro studies of S<em>1</em>P receptors suggested that direct CNS effects of FTY720 might theoretically occur through receptor modulation on neurons and glia. To identify CNS cells functionally contributing to FTY720 activity, genetic approaches were combined with cellular and molecular analyses. These studies relied on the functional assessment, based on clinical score, of conditional null mouse mutants lacking S<em>1</em>P(<em>1</em>) in CNS cell lineages and challenged by experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. All conditional null mutants displayed WT lymphocyte trafficking that responded normally to FTY720. In marked contrast, EAE was attenuated and FTY720 efficacy was lost in CNS mutants lacking S<em>1</em>P(<em>1</em>) on GFAP-expressing astrocytes but not on neurons. In situ hybridization studies confirmed that astrocyte loss of S<em>1</em>P(<em>1</em>) was the key alteration in functionally affected mutants. Reductions in EAE clinical scores were paralleled by reductions in demyelination, axonal loss, and astrogliosis. Receptor rescue and pharmacological experiments supported the loss of S<em>1</em>P(<em>1</em>) on astrocytes through functional antagonism by FTY720-P as a primary FTY720 mechanism. These data identify nonimmunological CNS mechanisms of FTY720 efficacy and implicate S<em>1</em>P signaling pathways within the CNS as targets for multiple sclerosis therapies.

The potent sphingolipid metabolite <em>sphingosine</em> <em>1</em>-<em>phosphate</em> is produced by phosphorylation of <em>sphingosine</em> catalyzed by <em>sphingosine</em> kinase (SphK) types <em>1</em> and 2. In contrast to pro-survival SphK<em>1</em>, the putative BH3-only protein SphK2 inhibits cell growth and enhances apoptosis. Here we show that SphK2 catalytic activity also contributes to its ability to induce apoptosis. Overexpressed SphK2 also increased cytosolic free calcium induced by serum starvation. Transfer of calcium to mitochondria was required for SphK2-induced apoptosis, as cell death and cytochrome c release was abrogated by inhibition of the mitochondrial Ca(2+) transporter. Serum starvation increased the proportion of SphK2 in the endoplasmic reticulum and targeting SphK<em>1</em> to the endoplasmic reticulum converted it from anti-apoptotic to pro-apoptotic. Overexpression of SphK2 increased incorporation of [(3)H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C<em>1</em>6-ceramide, whereas SphK<em>1</em> decreased it. Electrospray ionizationmass spectrometry/mass spectrometry also revealed an opposite effect on ceramide mass levels. Importantly, specific down-regulation of SphK2 reduced conversion of <em>sphingosine</em> to ceramide in the recycling pathway and conversely, down-regulation of SphK<em>1</em> increased it. Our results demonstrate that SphK<em>1</em> and SphK2 have opposing roles in the regulation of ceramide biosynthesis and suggest that the location of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> production dictates its functions.

FTY720 is an immunosuppressant that reduces circulating levels of naïve lymphocytes by increasing their localization and sequestration in secondary lymphoid organs. It is considered to be an agonist for <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) G protein-coupled receptors (GPCRs) after phosphorylation at micromolar concentrations. We now describe its nonagonist and noncompetitive inhibitory activity at low nanomolar concentrations for types <em>1</em> and 5 S<em>1</em>P-GPCRs and of moderate potency for type 2 S<em>1</em>P-GPCRs. FTY720 blocks S<em>1</em>P signaling through S<em>1</em>P<em>1</em>,2,5 by inducing their internalization and intracellular partial degradation without affecting S<em>1</em>P3 or S<em>1</em>P4. S<em>1</em>P-R internalization is maximal several hours after only seconds of incubation with FTY720 at 37 degrees C and washing, and continues for days before recovery of surface expression and functions. The timing and extent of S<em>1</em>P-R internalization are highly dependent on FTY720 concentration. FTY720 is therefore an S<em>1</em>P-GPCR-selective and noncompetitive inhibitor with a unique mechanism of action.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> is formed in cells in response to diverse stimuli, including growth factors, cytokines, G-protein-coupled receptor agonists, antigen, etc. Its production is catalysed by <em>sphingosine</em> kinase, while degradation is either via cleavage to produce palmitaldehyde and phosphoethanolamine or by dephosphorylation. In this review we discuss the most recent advances in our understanding of the role of the enzymes involved in metabolism of this lysolipid. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> can also bind to members of the endothelial differentiation gene (EDG) G-protein-coupled receptor family [namely EDG<em>1</em>, EDG3, EDG5 (also known as H2<em>1</em>8 or AGR<em>1</em>6), EDG6 and EDG8] to elicit biological responses. These receptors are coupled differentially via G(i), G(q), G(<em>1</em>2/<em>1</em>3) and Rho to multiple effector systems, including adenylate cyclase, phospholipases C and D, extracellular-signal-regulated kinase, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase and non-receptor tyrosine kinases. These signalling pathways are linked to transcription factor activation, cytoskeletal proteins, adhesion molecule expression, caspase activities, etc. Therefore <em>sphingosine</em> <em>1</em>-<em>phosphate</em> can affect diverse biological responses, including mitogenesis, differentiation, migration and apoptosis, via receptor-dependent mechanisms. Additionally, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> has been proposed to play an intracellular role, for example in Ca(2+) mobilization, activation of non-receptor tyrosine kinases, inhibition of caspases, etc. We review the evidence for both intracellular and extracellular actions, and extensively discuss future approaches that will ultimately resolve the question of dual action. Certainly, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> will prove to be unique if it elicits both extra- and intra-cellular actions. Finally, we review the evidence that implicates <em>sphingosine</em> <em>1</em>-<em>phosphate</em> in pathophysiological disease states, such as cancer, angiogenesis and inflammation. Thus there is a need for the development of new therapeutic compounds, such as receptor antagonists. However, identification of the most suitable targets for drug intervention requires a full understanding of the signalling and action profile of this lysosphingolipid. This article describes where the research field is in relation to achieving this aim.

Sphingolipids are ubiquitous components of cell membranes and their metabolites ceramide (Cer), <em>sphingosine</em> (Sph), and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) have important physiological functions, including regulation of cell growth and survival. Cer and Sph are associated with growth arrest and apoptosis. Many stress stimuli increase levels of Cer and Sph, whereas suppression of apoptosis is associated with increased intracellular levels of S<em>1</em>P. In addition, extracellular/secreted S<em>1</em>P regulates cellular processes by binding to five specific G protein coupled-receptors (GPCRs). S<em>1</em>P is generated by phosphorylation of Sph catalyzed by two isoforms of <em>sphingosine</em> kinases (SphK), type <em>1</em> and type 2, which are critical regulators of the "sphingolipid rheostat", producing pro-survival S<em>1</em>P and decreasing levels of pro-apoptotic Sph. Since sphingolipid metabolism is often dysregulated in many diseases, targeting SphKs is potentially clinically relevant. Here we review the growing recent literature on the regulation and the roles of SphKs and S<em>1</em>P in apoptosis and diseases.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P, <em>1</em>) regulates vascular barrier and lymphoid development, as well as lymphocyte egress from lymphoid organs, by activating high-affinity S<em>1</em>P<em>1</em> receptors. We used reversible chemical probes (i) to gain mechanistic insights into S<em>1</em>P systems organization not accessible through genetic manipulations and (ii) to investigate their potential for therapeutic modulation. Vascular (but not airway) administration of the preferred R enantiomer of an in vivo-active chiral S<em>1</em>P<em>1</em> receptor antagonist induced loss of capillary integrity in mouse skin and lung. In contrast, the antagonist did not affect the number of constitutive blood lymphocytes. Instead, alteration of lymphocyte trafficking and phenotype required supraphysiological elevation of S<em>1</em>P<em>1</em> tone and was reversed by the antagonist. In vivo two-photon imaging of lymph nodes confirmed requirements for obligate agonism, and the data were consistent with the presence of a stromal barrier mechanism for gating lymphocyte egress. Thus, chemical modulation reveals differences in S<em>1</em>P-S<em>1</em>P<em>1</em> 'set points' among tissues and highlights both mechanistic advantages (lymphocyte sequestration) and risks (pulmonary edema) of therapeutic intervention.

The <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) family of G protein-coupled receptors (GPCR) regulates essential cellular processes such as proliferation, migration, cytoskeletal organization, adherens junction assembly, and morphogenesis. S<em>1</em>P, a product from the breakdown of sphingomyelin, binds to the five members of this receptor family, S<em>1</em>P(<em>1</em>), S<em>1</em>P(2), S<em>1</em>P(3), S<em>1</em>P(4), and S<em>1</em>P(5), previously referred to as endothelial differentiation gene (EDG)-<em>1</em>, -5, -3, -6, and -8. S<em>1</em>P receptors are widely expressed in different tissues, so it is not surprising that the S<em>1</em>P receptor family regulates many physiological processes, such as vascular maturation, cardiac development, lymphocyte trafficking, and vascular permeability. FTY720, a new S<em>1</em>P receptor agonist, is undergoing clinical trials as an immunosuppressor. Understanding the physiological role of these receptors and the basics of the ligand-receptor interaction will potentially provide new therapies to control a variety of diseases.

Osteoclasts are the only somatic cells with bone-resorbing capacity and, as such, they have a critical role not only in normal bone homeostasis (called 'bone remodelling') but also in the pathogenesis of bone destructive disorders such as rheumatoid arthritis and osteoporosis. A major focus of research in the field has been on gene regulation by osteoclastogenic cytokines such as receptor activator of NF-kappaB-ligand (RANKL, also known as TNFSF<em>1</em><em>1</em>) and TNF-alpha, both of which have been well documented to contribute to osteoclast terminal differentiation. A crucial process that has been less well studied is the trafficking of osteoclast precursors to and from the bone surface, where they undergo cell fusion to form the fully differentiated multinucleated cells that mediate bone resorption. Here we report that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a lipid mediator enriched in blood, induces chemotaxis and regulates the migration of osteoclast precursors not only in culture but also in vivo, contributing to the dynamic control of bone mineral homeostasis. Cells with the properties of osteoclast precursors express functional S<em>1</em>P(<em>1</em>) receptors and exhibit positive chemotaxis along an S<em>1</em>P gradient in vitro. Intravital two-photon imaging of bone tissues showed that a potent S<em>1</em>P(<em>1</em>) agonist, SEW287<em>1</em>, stimulated motility of osteoclast precursor-containing monocytoid populations in vivo. Osteoclast/monocyte (CD<em>1</em><em>1</em>b, also known as ITGAM) lineage-specific conditional S<em>1</em>P(<em>1</em>) knockout mice showed osteoporotic changes due to increased osteoclast attachment to the bone surface. Furthermore, treatment with the S<em>1</em>P(<em>1</em>) agonist FTY720 relieved ovariectomy-induced osteoporosis in mice by reducing the number of mature osteoclasts attached to the bone surface. Together, these data provide evidence that S<em>1</em>P controls the migratory behaviour of osteoclast precursors, dynamically regulating bone mineral homeostasis, and identifies a critical control point in osteoclastogenesis that may have potential as a therapeutic target.

Cell-mediated immunity critically depends on the localization of lymphocytes at sites of infection. While some memory T cells recirculate, a distinct lineage (resident memory T cells (T(RM) cells)) are embedded in nonlymphoid tissues (NLTs) and mediate potent protective immunity. However, the defining transcriptional basis for the establishment of T(RM) cells is unknown. We found that CD8(+) T(RM) cells lacked expression of the transcription factor KLF2 and its target gene S<em>1</em>pr<em>1</em> (which encodes S<em>1</em>P<em>1</em>, a receptor for <em>sphingosine</em> <em>1</em>-<em>phosphate</em>). Forced expression of S<em>1</em>P<em>1</em> prevented the establishment of T(RM) cells. Cytokines that induced a T(RM) cell phenotype (including transforming growth factor-β (TGF-β), interleukin 33 (IL-33) and tumor-necrosis factor) elicited downregulation of KLF2 expression in a pathway dependent on phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt, which suggested environmental regulation. Hence, regulation of KLF2 and S<em>1</em>P<em>1</em> provides a switch that dictates whether CD8(+) T cells commit to recirculating or tissue-resident memory populations.

Ceramide is an important lipid signaling molecule and a key intermediate in sphingolipid biosynthesis. Recent studies have implied a previously unappreciated role for the ceramide N-acyl chain length, inasmuch as ceramides containing specific fatty acids appear to play defined roles in cell physiology. The discovery of a family of mammalian ceramide synthases (CerS), each of which utilizes a restricted subset of acyl-CoAs for ceramide synthesis, strengthens this notion. We now report the characterization of mammalian CerS2. qPCR analysis reveals that CerS2 mRNA is found at the highest level of all CerS and has the broadest tissue distribution. CerS2 has a remarkable acyl-CoA specificity, showing no activity using C<em>1</em>6:0-CoA and very low activity using C<em>1</em>8:0, rather utilizing longer acyl-chain CoAs (C20-C26) for ceramide synthesis. There is a good correlation between CerS2 mRNA levels and levels of ceramide and sphingomyelin containing long acyl chains, at least in tissues where CerS2 mRNA is expressed at high levels. Interestingly, the activity of CerS2 can be regulated by another bioactive sphingolipid, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), via interaction of S<em>1</em>P with two residues that are part of an S<em>1</em>P receptor-like motif found only in CerS2. These findings provide insight into the biochemical basis for the ceramide N-acyl chain composition of cells, and also reveal a novel and potentially important interplay between two bioactive sphingolipids that could be relevant to the regulation of sphingolipid metabolism and the opposing functions that these lipids play in signaling pathways.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the S<em>1</em>P<em>1</em> receptor. This receptor is activated by FTY720-P, a phosphorylated derivative of the immunosuppressant and vasoactive compound FTY720. However, in contrast to the natural ligand S<em>1</em>P, FTY720-P appears to act as a functional antagonist, even though the mechanisms involved are poorly understood. In this study, we investigated the fate of endogenously expressed S<em>1</em>P<em>1</em> receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S<em>1</em>P<em>1</em>. We show that FTY720-P is more potent than S<em>1</em>P at inducing receptor degradation. Pretreatment with an antagonist of S<em>1</em>P<em>1</em>, VPC 44<em>1</em><em>1</em>6, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient S<em>1</em>P<em>1</em> receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and beta-arrestins abolished FTY720-P-induced S<em>1</em>P<em>1</em> receptor degradation. These data suggest that agonist-induced phosphorylation of S<em>1</em>P<em>1</em> and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S<em>1</em>P<em>1</em> degradation is blocked by MG<em>1</em>32, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of S<em>1</em>P<em>1</em> receptor, whereas S<em>1</em>P at concentrations that induced complete internalization was not as efficient, suggesting that receptor internalization is required but not sufficient for ubiquitinylation and degradation. We propose that the ability of FTY720-P to target the S<em>1</em>P<em>1</em> receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties.

Increased endothelial cell (EC) permeability is central to the pathophysiology of inflammatory syndromes such as sepsis and acute lung injury (ALI). Activated protein C (APC), a serine protease critically involved in the regulation of coagulation and inflammatory processes, improves sepsis survival through an unknown mechanism. We hypothesized a direct effect of APC to both prevent increased EC permeability and to restore vascular integrity after edemagenic agonists. We measured changes in transendothelial electrical resistance (TER) and observed that APC produced concentration-dependent attenuation of TER reductions evoked by thrombin. We next explored known EC barrier-protective signaling pathways and observed dose-dependent APC-mediated increases in cortical myosin light chain (MLC) phosphorylation in concert with cortically distributed actin polymerization, findings highly suggestive of Rac GTPase involvement. We next determined that APC directly increases Rac<em>1</em> activity, with inhibition of Rac<em>1</em> activity significantly attenuating APC-mediated barrier protection to thrombin challenge. Finally, as these signaling events were similar to those evoked by the potent EC barrier-enhancing agonist, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), we explored potential cross-talk between endothelial protein C receptor (EPCR) and S<em>1</em>P<em>1</em>, the receptors for APC and S<em>1</em>P, respectively. EPCR-blocking antibody (RCR-252) significantly attenuated both APC-mediated barrier protection and increased MLC phosphorylation. We next observed rapid, EPCR and PI 3-kinase-dependent, APC-mediated phosphorylation of S<em>1</em>P<em>1</em> on threonine residues consistent with S<em>1</em>P<em>1</em> receptor activation. Co-immunoprecipitation studies demonstrate an interaction between EPCR and S<em>1</em>P<em>1</em> upon APC treatment. Targeted silencing of S<em>1</em>P<em>1</em> expression using siRNA significantly reduced APC-mediated barrier protection against thrombin. These data suggest that novel EPCR ligation and S<em>1</em>P<em>1</em> transactivation results in EC cytoskeletal rearrangement and barrier protection, components potentially critical to the improved survival of APC-treated patients with severe sepsis.

Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell proliferation and migration, primarily through the receptor tyrosine kinase VEGF receptor2 (Flk<em>1</em>/KDR). Reactive oxygen species (ROS) derived from NAD(P)H oxidase are critically important in many aspects of vascular cell regulation, and both the small GTPase Rac<em>1</em> and gp9<em>1</em>(phox) are critical components of the endothelial NAD(P)H oxidase complex. A role of NAD(P)H oxidase in VEGF-induced angiogenesis, however, has not been defined. In the present study, electron spin resonance spectroscopy is utilized to demonstrate that VEGF stimulates O2*- production, which is inhibited by the NAD(P)H oxidase inhibitor, diphenylene iodonium, as well as by overexpression of dominant-negative Rac<em>1</em> (N<em>1</em>7Rac<em>1</em>) and transfection of gp9<em>1</em>(phox) antisense oligonucleotides in human umbilical vein endothelial cells (ECs). Antioxidants, including N-acetylcysteine (NAC), various NAD(P)H oxidase inhibitors, and N<em>1</em>7Rac<em>1</em> significantly attenuate not only VEGF-induced KDR tyrosine phosphorylation but also proliferation and migration of ECs. Importantly, these effects of VEGF are dramatically inhibited in cells transfected with gp9<em>1</em>(phox) antisense oligonucleotides. By contrast, ROS are not involved in mediating these effects of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) on ECs. Sponge implant assays demonstrate that VEGF-, but not S<em>1</em>P-, induced angiogenesis is significantly reduced in wild-type mice treated with NAC and in gp9<em>1</em>(phox-/-) mice, suggesting that ROS derived from gp9<em>1</em>(phox)-containing NAD(P)H oxidase play an important role in angiogenesis in vivo. These studies indicate that VEGF-induced endothelial cell signaling and angiogenesis is tightly controlled by the reduction/oxidation environment at the level of VEGF receptor and provide novel insights into the NAD(P)H oxidase as a potential therapeutic target for angiogenesis-dependent diseases.

Lymphocyte egress from lymph nodes (LNs) is dependent on <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), but the cellular source of this S<em>1</em>P is not defined. We generated mice that expressed Cre from the lymphatic vessel endothelial hyaluronan receptor <em>1</em> (Lyve-<em>1</em>) locus and that showed efficient recombination of loxP-flanked genes in lymphatic endothelium. We report that mice with Lyve-<em>1</em> CRE-mediated ablation of <em>sphingosine</em> kinase (Sphk) <em>1</em> and lacking Sphk2 have a loss of S<em>1</em>P in lymph while maintaining normal plasma S<em>1</em>P. In Lyve-<em>1</em> Cre+ Sphk-deficient mice, lymphocyte egress from LNs and Peyer's patches is blocked. Treatment with pertussis toxin to overcome Galphai-mediated retention signals restores lymphocyte egress. Furthermore, in the absence of lymphatic Sphks, the initial lymphatic vessels in nonlymphoid tissues show an irregular morphology and a less organized vascular endothelial cadherin distribution at cell-cell junctions. Our data provide evidence that lymphatic endothelial cells are an in vivo source of S<em>1</em>P required for lymphocyte egress from LNs and Peyer's patches, and suggest a role for S<em>1</em>P in lymphatic vessel maturation.

HDL is a major atheroprotective factor, but the mechanisms underlying this effect are still obscure. HDL binding to scavenger receptor-BI has been shown to activate eNOS, although the responsible HDL entities and signaling pathways have remained enigmatic. Here we show that HDL stimulates NO release in human endothelial cells and induces vasodilation in isolated aortae via intracellular Ca2+ mobilization and Akt-mediated eNOS phosphorylation. The vasoactive effects of HDL could be mimicked by three lysophospholipids present in HDL: sphingosylphosphorylcholine (SPC), <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), and lysosulfatide (LSF). All three elevated intracellular Ca2+ concentration and activated Akt and eNOS, which resulted in NO release and vasodilation. Deficiency of the lysophospholipid receptor S<em>1</em>P3 (also known as LPB3 and EDG3) abolished the vasodilatory effects of SPC, S<em>1</em>P, and LSF and reduced the effect of HDL by approximately 60%. In endothelial cells from S<em>1</em>P3-deficient mice, Akt phosphorylation and Ca2+ increase in response to HDL and lysophospholipids were severely reduced. In vivo, intra-arterial administration of HDL or lysophospholipids lowered mean arterial blood pressure in rats. In conclusion, we identify HDL as a carrier of bioactive lysophospholipids that regulate vascular tone via S<em>1</em>P3-mediated NO release. This mechanism may contribute to the vasoactive effect of HDL and represent a novel aspect of its antiatherogenic function.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP) is a bioactive lipid that has recently been identified as the ligand for the EDG family of G protein-coupled cell surface receptors. However, the mitogenic and survival effects of exogenous SPP may not correlate with binding to cell-surface receptors (Van Brocklyn, J.R., M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier, D.M. Thomas, P.J.P. Coopman, S. Thangada, T. Hla, and S. Spiegel. <em>1</em>998. J. Cell Biol. <em>1</em>42:229-240). The recent cloning of <em>sphingosine</em> kinase, a unique lipid kinase responsible for the formation of SPP, has provided a new tool to investigate the role of intracellular SPP. Expression of <em>sphingosine</em> kinase markedly increased SPP levels in NIH 3T3 fibroblasts and HEK293 cells, but no detectable secretion of SPP into the medium was observed. The increased <em>sphingosine</em> kinase activity in NIH 3T3 fibroblasts was sufficient to promote growth in low- serum media, expedite the G(<em>1</em>)/S transition, and increase DNA synthesis and the proportion of cells in the S phase of the cell cycle with a concomitant increase in cell numbers. Transient or stable overexpression of <em>sphingosine</em> kinase in NIH 3T3 fibroblasts or HEK293 cells protected against apoptosis induced by serum deprivation or ceramide elevation. N,N-Dimethyl<em>sphingosine</em>, a competitive inhibitor of <em>sphingosine</em> kinase, blocked the effects of <em>sphingosine</em> kinase overexpression on cell proliferation and suppression of apoptosis. In contrast, pertussis toxin did not abrogate these biological responses. In Jurkat T cells, overexpression of <em>sphingosine</em> kinase also suppressed serum deprivation- and ceramide-induced apoptosis and, to a lesser extent, Fas-induced apoptosis, which correlated with inhibition of DEVDase activity, as well as inhibition of the executionary caspase-3. Taken together with ample evidence showing that growth and survival factors activate <em>sphingosine</em> kinase, our results indicate that SPP functions as a second messenger important for growth and survival of cells. Hence, SPP belongs to a novel class of lipid mediators that can function inside and outside cells.