<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive sphingolipid metabolite involved in many critical cellular processes including proliferation, survival, and migration, as well as angiogenesis and allergic responses. S<em>1</em>P levels inside cells are tightly regulated by the balance between its synthesis by <em>sphingosine</em> kinases and degradation. S<em>1</em>P is interconvertible with ceramide, which is a critical mediator of apoptosis. It has been postulated that the ratio between S<em>1</em>P and ceramide determines cell fate. Activation of <em>sphingosine</em> kinase by a variety of agonists increases intracellular S<em>1</em>P, which in turn can function intracellularly as a second messenger or be secreted out of the cell and act extracellularly by binding to and signaling through S<em>1</em>P receptors in autocrine and/or paracrine manners. Recent studies suggest that this "inside-out" signaling by S<em>1</em>P may play a role in many human diseases, including cancer, atherosclerosis, inflammation, and autoimmune disorders such as multiple sclerosis. In this review we summarize metabolism of S<em>1</em>P, mechanisms of <em>sphingosine</em> kinase activation, and S<em>1</em>P receptors and their downstream signaling pathways and examine relationships to multiple disease processes. In particular, we describe recent preclinical and clinical trials of therapies targeting S<em>1</em>P signaling, including 2-amino-2-propane-<em>1</em>,3-diol hydrochloride (FTY720, fingolimod), S<em>1</em>P receptor agonists, <em>sphingosine</em> kinase inhibitors, and anti-S<em>1</em>P monoclonal antibody.

The egress of lymphocytes from the thymus and secondary lymphoid organs into circulatory fluids is essential for normal immune function. The discovery that a small-molecule inhibitor of lymphocyte exit, FTY720, is a ligand for <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors led to studies demonstrating that S<em>1</em>P receptor type <em>1</em> (S<em>1</em>P<em>1</em>) is needed in T cells and B cells for their egress from lymphoid organs. S<em>1</em>P exists in higher concentrations in blood and lymph than in lymphoid organs, and this differential is also required for lymphocyte exit. Transcriptional and post-translational mechanisms regulate S<em>1</em>P<em>1</em> and thus the egress of lymphocytes. In this review we discuss the body of evidence supporting a model in which lymphocyte egress is promoted by encounter with S<em>1</em>P at exit sites. We relate this model to work examining the effects of S<em>1</em>P receptor agonists on endothelium.

There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), <em>sphingosine</em> (Sph) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (Sph-<em>1</em>P) that necessitates development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring changes in SPL levels and composition from biological materials. Simultaneous ESI/MS/MS analysis of sphingoid bases (SBs), sphingoid base <em>1</em>-<em>phosphates</em> (SB-<em>1</em>Ps), Cers and sphingomyelins (SMs) is performed on a Thermo Finnigan TSQ 7000 triple quadrupole mass spectrometer operating in a multiple reaction monitoring (MRM) positive ionization mode. Biological materials (cells, tissues or physiological fluids) are fortified with internal standards (ISs), extracted into a one-phase neutral organic solvent system, and analyzed by a Surveyor/TSQ 7000 LC/MS system. Qualitative analysis of SPLs is performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of SPLs. Quantitative analysis is based on calibration curves generated by spiking an artificial matrix with known amounts of target synthetic standards and an equal amount of IS. The calibration curves are constructed by plotting the peak area ratios of analyte to the respective IS against concentration using a linear regression model. This robust analytical procedure can determine the composition of endogenous sphingolipids (ESPLs) in varied biological materials and achieve a detection limit at <em>1</em> pmol or lower level. This and related methodology are already defining unexpected specialization and specificity in the metabolism and function of distinct subspecies of individual bioactive SPLs.

The endothelium is a semi-permeable barrier that regulates the flux of liquid and solutes, including plasma proteins, between the blood and surrounding tissue. The permeability of the vascular barrier can be modified in response to specific stimuli acting on endothelial cells. Transport across the endothelium can occur via two different pathways: through the endothelial cell (transcellular) or between adjacent cells, through interendothelial junctions (paracellular). This review focuses on the regulation of the paracellular pathway. The paracellular pathway is composed of adhesive junctions between endothelial cells, both tight junctions and adherens junctions. The actin cytoskeleton is bound to each junction and controls the integrity of each through actin remodeling. These interendothelial junctions can be disassembled or assembled to either increase or decrease paracellular permeability. Mediators, such as thrombin, TNF-alpha, and LPS, stimulate their respective receptor on endothelial cells to initiate signaling that increases cytosolic Ca2+ and activates myosin light chain kinase (MLCK), as well as monomeric GTPases RhoA, Rac<em>1</em>, and Cdc42. Ca2+ activation of MLCK and RhoA disrupts junctions, whereas Rac<em>1</em> and Cdc42 promote junctional assembly. Increased endothelial permeability can be reversed with "barrier stabilizing agents," such as <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> and cyclic adenosine mono<em>phosphate</em> (cAMP). This review provides an overview of the mechanisms that regulate paracellular permeability.

The potent lipid mediator <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is produced inside cells by two closely related kinases, <em>sphingosine</em> kinase <em>1</em> (SPHK<em>1</em>) and SPHK2, and has emerged as a crucial regulator of immunity. Many of the actions of S<em>1</em>P in innate and adaptive immunity are mediated by its binding to five G protein-coupled receptors, designated S<em>1</em>PR<em>1</em>-5, but recent findings have also identified important roles for S<em>1</em>P as a second messenger during inflammation. In this Review, we discuss recent advances in our understanding of the roles of S<em>1</em>P receptors and describe the newly identified intracellular targets of S<em>1</em>P that are crucial for immune responses. Finally, we discuss the therapeutic potential of new drugs that target S<em>1</em>P signalling and functions.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a biologically active metabolite of plasma-membrane sphingolipids that is essential for immune-cell trafficking. Its concentration is increased in many inflammatory conditions, such as asthma and autoimmunity. Much of the immune function of S<em>1</em>P results from the engagement of a family of G-protein-coupled receptors (S<em>1</em>PR<em>1</em>-S<em>1</em>PR5). Recent findings on the role of S<em>1</em>P in immunosurveillance, the discovery of regulatory mechanisms in S<em>1</em>P-mediated immune-cell trafficking and new advances in understanding the mechanism by which S<em>1</em>P affects immune-cell function indicate that the alliance between S<em>1</em>P and its receptors has a fundamental role in immunity.

The lyso-phospholipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptors. Here, we present the crystal structure of the <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor <em>1</em> fused to T4-lysozyme (S<em>1</em>P(<em>1</em>)-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S<em>1</em>P(<em>1</em>), resulting in the modulation of immune and stromal cell responses.

The sphingolipid metabolite <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP) has been implicated as a second messenger in cell proliferation and survival. However, many of its biological effects are due to binding to unidentified receptors on the cell surface. SPP activated the heterotrimeric guanine nucleotide binding protein (G protein)-coupled orphan receptor EDG-<em>1</em>, originally cloned as Endothelial Differentiation Gene-<em>1</em>. EDG-<em>1</em> bound SPP with high affinity (dissociation constant = 8.<em>1</em> nM) and high specificity. Overexpression of EDG-<em>1</em> induced exaggerated cell-cell aggregation, enhanced expression of cadherins, and formation of well-developed adherens junctions in a manner dependent on SPP and the small guanine nucleotide binding protein Rho.

Lysophospholipids (LPs), such as lysophosphatidic acid and <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, are membrane-derived bioactive lipid mediators. LPs can affect fundamental cellular functions, which include proliferation, differentiation, survival, migration, adhesion, invasion, and morphogenesis. These functions influence many biological processes that include neurogenesis, angiogenesis, wound healing, immunity, and carcinogenesis. In recent years, identification of multiple cognate G protein-coupled receptors has provided a mechanistic framework for understanding how LPs play such diverse roles. Generation of LP receptor-null animals has allowed rigorous examination of receptor-mediated physiological functions in vivo and has identified new functions for LP receptor signaling. Efforts to develop LP receptor subtype-specific agonists/antagonists are in progress and raise expectations for a growing collection of chemical tools and potential therapeutic compounds. The rapidly expanding literature on the LP receptors is herein reviewed.

Mammalian Kruppel-like transcription factors are implicated in regulating terminal differentiation of several tissue types. Deficiency in Kruppel-like factor (KLF) 2 (also known as LKLF) leads to a massive loss of the peripheral T-cell pool, suggesting KLF2 regulates T-cell quiescence and survival. Here we show, however, that KLF2 is essential for T-cell trafficking. KLF2-deficient (Klf2-/-) thymocytes show impaired expression of several receptors required for thymocyte emigration and peripheral trafficking, including the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor S<em>1</em>P<em>1</em>, CD62L and beta7 integrin. Furthermore, KLF2 both binds and transactivates the promoter for S<em>1</em>P<em>1</em>--a receptor that is critical for thymocyte egress and recirculation through peripheral lymphoid organs. Our findings suggest that KLF2 serves to license mature T cells for trafficking from the thymus and recirculation through secondary lymphoid tissues.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a biologically active lysophospholipid that transmits signals through a family of G-protein-coupled receptors to control cellular differentiation and survival, as well as the vital functions of several types of immune cell. In this Review article, we discuss recent results that indicate that S<em>1</em>P and its receptors are required for the emigration of thymocytes from the thymus, the trafficking of lymphocytes in secondary lymphoid organs and the migration of B cells into splenic follicles. In an autocrine manner, through interactions with different G-protein-coupled receptors, S<em>1</em>P also enhances optimal mast-cell migration and release of pro-inflammatory mediators in allergic reactions. S<em>1</em>P-S<em>1</em>P-receptor regulatory systems might therefore be novel targets for the therapy of diverse immunological diseases.

Sphingolipids are ubiquitous building blocks of eukaryotic cell membranes. Progress in our understanding of sphingolipid metabolism, state-of-the-art sphingolipidomic approaches and animal models have generated a large body of evidence demonstrating that sphingolipid metabolites, particularly ceramide and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, are signalling molecules that regulate a diverse range of cellular processes that are important in immunity, inflammation and inflammatory disorders. Recent insights into the molecular mechanisms of action of sphingolipid metabolites and new perspectives on their roles in regulating chronic inflammation have been reported. The knowledge gained in this emerging field will aid in the development of new therapeutic options for inflammatory disorders.

Sphingolipid-metabolizing enzymes control the dynamic balance of the cellular levels of bioactive lipids, including the proapoptotic compound ceramide and the proliferative compound <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. Accumulating evidence indicates that <em>sphingosine</em> kinase (SK) plays a pivotal role in regulating tumor growth and that SK can act as an oncogene. Despite the importance of SK for cell proliferation, pharmacological inhibition of SK is an untested means of treating cancer because of the current lack of nonlipid inhibitors of this enzyme. To further assess the involvement of SK in human tumors, levels of RNA for SK in paired samples of cDNA prepared from tumors and normal adjacent tissue were analyzed. Expression of SK RNA was significantly elevated in a variety of solid tumors, compared with normal tissue from the same patient. To identify and evaluate inhibitors of SK, a medium throughput assay for recombinant human SK fused to glutathione S-transferase was developed, validated, and used to screen a library of synthetic compounds. A number of novel inhibitors of human SK were identified, and several representative compounds were characterized in detail. These compounds demonstrated activity at sub- to micromolar concentrations, making them more potent than any other reported SK inhibitor, and were selective toward SK compared with a panel of human lipid and protein kinases. Kinetic studies revealed that the compounds were not competitive inhibitors of the ATP-binding site of SK. The SK inhibitors were antiproliferative toward a panel of tumor cell lines, including lines with the multidrug resistance phenotype because of overexpression of either P-glycoprotein or multidrug resistance phenotype <em>1</em>, and were shown to inhibit endogenous human SK activity in intact cells. Furthermore, each inhibitor induced apoptosis concomitant with tumor cell cytotoxicity. Methods for the synthesis of a series of aurone inhibitors of SK were established, and a prototypical dihydroxyaurone was found to have moderate antitumor activity in vivo in the absence of overt toxicity to the mice. These compounds are the first examples of nonlipid inhibitors of SK with in vivo antitumor activity and so provide leads for additional development of inhibitors of this important molecular target.

Sphingolipids constitute a class of lipids defined by their eighteen carbon amino-alcohol backbones which are synthesized in the ER from nonsphingolipid precursors. Modification of this basic structure is what gives rise to the vast family of sphingolipids that play significant roles in membrane biology and provide many bioactive metabolites that regulate cell function. Despite the diversity of structure and function of sphingolipids, their creation and destruction are governed by common synthetic and catabolic pathways. In this regard, sphingolipid metabolism can be imagined as an array of interconnected networks that diverge from a single common entry point and converge into a single common breakdown pathway. In their simplest forms, <em>sphingosine</em>, phyto<em>sphingosine</em> and dihydro<em>sphingosine</em> serve as the backbones upon which further complexity is achieved. For example, phosphorylation of the C<em>1</em> hydroxyl group yields the final breakdown products and/or the important signaling molecules <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, phyto<em>sphingosine</em>-<em>1</em>-<em>phosphate</em> and dihydro<em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, respectively. On the other hand, acylation of <em>sphingosine</em>, phyto<em>sphingosine</em>, or dihydro<em>sphingosine</em> with one of several possible acyl CoA molecules through the action of distinct ceramide synthases produces the molecules defined as ceramide, phytoceramide, or dihydroceramide. Ceramide, due to the differing acyl CoAs that can be used to produce it, is technically a class of molecules rather than a single molecule and therefore may have different biological functions depending on the acyl chain it is composed of. At the apex of complexity is the group of lipids known as glycosphingolipids (GSL) which contain dozens of different sphingolipid species differing by both the order and type of sugar residues attached to their headgroups. Since these molecules are produced from ceramide precursors, they too may have differences in their acyl chain composition, revealing an additional layer of variation. The glycosphingolipids are divided broadly into two categories: glucosphingolipids and galactosphingolipids. The glucosphingolipids depend initially on the enzyme glucosylceramide synthase (GCS) which attaches glucose as the first residue to the C<em>1</em> hydroxyl position. Galactosphingolipids, on the other hand, are generated from galactosylceramide synthase (GalCerS), an evolutionarily dissimilar enzyme from GCS. Glycosphingolipids are further divided based upon further modification by various glycosyltransferases which increases the potential variation in lipid species by several fold. Far more abundant are the sphingomyelin species which are produced in parallel with glycosphingolipids, however they are defined by a phosphocholine headgroup rather than the addition of sugar residues. Although sphingomyelin species all share a common headgroup, they too are produced from a variety of ceramide species and therefore can have differing acyl chains attached to their C-2 amino groups. Whether or not the differing acyl chain lengths in SMs dictate unique functions or important biophysical distinctions has not yet been established. Understanding the function of all the existing glycosphingolipids and sphingomyelin species will be a major undertaking in the future since the tools to study and measure these species are only beginning to be developed (see Fig <em>1</em> for an illustrated depiction of the various sphingolipid structures). The simple sphingolipids serve both as the precursors and the breakdown products of the more complex ones. Importantly, in recent decades, these simple sphingolipids have gained attention for having significant signaling and regulatory roles within cells. In addition, many tools have emerged to measure the levels of simple sphingolipids and therefore have become the focus of even more intense study in recent years. With this thought in mind, this chapter will pay tribute to the complex sphingolipids, but focus on the regulation of simple sphingolipid metabolism.

Alveolar cell apoptosis is involved in the pathogenesis of emphysema, a prevalent disease primarily caused by cigarette smoking. We report that ceramide, a second messenger lipid, is a crucial mediator of alveolar destruction in emphysema. Inhibition of enzymes controlling de novo ceramide synthesis prevented alveolar cell apoptosis, oxidative stress and emphysema caused by blockade of the vascular endothelial growth factor (VEGF) receptors in both rats and mice. Emphysema was reproduced with intratracheal instillation of ceramide in naive mice. Excessive ceramide triggers a feed-forward mechanism mediated by activation of secretory acid sphingomyelinase, as suggested by experiments with neutralizing ceramide antibody in mice and with acid sphingomyelinase-deficient fibroblasts. Concomitant augmentation of signaling initiated by a prosurvival metabolite, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, prevented lung apoptosis, implying that a balance between ceramide and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> is required for maintenance of alveolar septal integrity. Finally, increased lung ceramides in individuals with smoking-induced emphysema suggests that ceramide upregulation may be a crucial pathogenic element and a promising target in this disease that currently lacks effective therapies.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP) has diverse biological functions acting inside cells as a second messenger to regulate proliferation and survival, and extracellularly, as a ligand for G protein-coupled receptors of the endothelial differentiation gene-<em>1</em> subfamily. Based on sequence homology to murine and human <em>sphingosine</em> kinase-<em>1</em> (SPHK<em>1</em>), which we recently cloned (Kohama, T., Oliver, A., Edsall, L. , Nagiec, M. M., Dickson, R., and Spiegel, S. (<em>1</em>998) J. Biol. Chem. 273, 23722-23728), we have now cloned a second type of mouse and human <em>sphingosine</em> kinase (mSPHK2 and hSPHK2). mSPHK2 and hSPHK2 encode proteins of 6<em>1</em>7 and 6<em>1</em>8 amino acids, respectively, both much larger than SPHK<em>1</em>, and though diverging considerably, both contain the conserved domains found in all SPHK<em>1</em>s. Northern blot analysis revealed that SPHK2 mRNA expression had a strikingly different tissue distribution from that of SPHK<em>1</em> and appeared later in embryonic development. Expression of SPHK2 in HEK 293 cells resulted in elevated SPP levels. d-erythro-dihydro<em>sphingosine</em> was a better substrate than d-erythro-<em>sphingosine</em> for SPHK2. Surprisingly, d, l-threo-dihydro<em>sphingosine</em> was also phosphorylated by SPHK2. In contrast to the inhibitory effects on SPHK<em>1</em>, high salt concentrations markedly stimulated SPHK2. Triton X-<em>1</em>00 inhibited SPHK2 and stimulated SPHK<em>1</em>, whereas phosphatidylserine stimulated both type <em>1</em> and type 2 SPHK. Thus, SPHK2 is another member of a growing class of sphingolipid kinases that may have novel functions.

Fingolimod (FTY720) is a first-in-class orally bioavailable compound that has shown efficacy in advanced clinical trials for the treatment of multiple sclerosis (MS). In vivo, fingolimod is phosphorylated to form fingolimod-<em>phosphate</em>, which resembles naturally occurring <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), an extracellular lipid mediator whose major effects are mediated by cognate G protein-coupled receptors. There are at least 5 S<em>1</em>P receptor subtypes, known as S<em>1</em>P subtypes <em>1</em>-5 (S<em>1</em>P<em>1</em>-5), 4 of which bind fingolimod-<em>phosphate</em>. These receptors are expressed on a wide range of cells that are involved in many biological processes relevant to MS. S<em>1</em>P<em>1</em> plays a key role in the immune system, regulating lymphocyte egress from lymphoid tissues into the circulation. Fingolimod-<em>phosphate</em> initially activates lymphocyte S<em>1</em>P<em>1</em> via high-affinity receptor binding yet subsequently induces S<em>1</em>P<em>1</em> down-regulation that prevents lymphocyte egress from lymphoid tissues, thereby reducing autoaggressive lymphocyte infiltration into the central nervous system (CNS). S<em>1</em>P receptors are also expressed by many CNS cell types and have been shown to influence cell proliferation, morphology, and migration. Fingolimod crosses the blood-brain barrier and may therefore have direct CNS effects, distinguishing it from immunologically targeted MS therapies. Prophylactic administration of fingolimod to animals with experimental autoimmune encephalitis (EAE), a model of MS, completely prevents development of EAE features, whereas therapeutic administration significantly reduces clinical severity of EAE. Therapeutic efficacy observed in animal studies has been substantiated in phase 2 and 3 trials involving patients with relapsing or relapsing-remitting MS.

The extensive diversity of membrane lipids is rarely appreciated by cell and molecular biologists. Although most researchers are familiar with the three main classes of lipids in animal cell membranes, few realize the enormous combinatorial structural diversity that exists within each lipid class, a diversity that enables functional specialization of lipids. In this brief review, we focus on one class of membrane lipids, the sphingolipids, which until not long ago were thought by many to be little more than structural components of biological membranes. Recent studies have placed sphingolipids-including ceramide, <em>sphingosine</em> and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-at the centre of a number of important biological processes, specifically in signal transduction pathways, in which their levels change in a highly regulated temporal and spatial manner. We outline exciting progress in the biochemistry and cell biology of sphingolipids and focus on their functional diversity. This should set the conceptual and experimental framework that will eventually lead to a fully integrated and comprehensive model of the functions of specific sphingolipids in regulating defined aspects of cell physiology.

Growth signalling networks that use glycerophospholipid metabolites as second messengers have been well characterized, but less is known of the second messengers derived from sphingolipids, another major class of membrane lipids. A tantalizing link between sphingolipids and cellular proliferation has emerged from the discovery that the sphingolipid metabolites <em>sphingosine</em> and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> stimulate growth of quiescent Swiss 3T3 fibroblasts by a pathway that is independent of protein kinase C. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> is rapidly produced from <em>sphingosine</em> and may mediate its biological effects. Furthermore, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> triggers the dual signal transduction pathways of calcium mobilization and activation of phospholipase D, prominent events in the control of cellular proliferation. Here we report that activation of <em>sphingosine</em> kinase and the formation of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> are important in the signal transduction pathways activated by the potent mitogens platelet-derived growth factor (PDGF) and fetal calf serum (FCS).

Endothelial cells normally form a dynamically regulated barrier at the blood-tissue interface, and breakdown of this barrier is a key pathogenic factor in inflammatory disorders such as sepsis. Pro-inflammatory signaling by the blood coagulation protease thrombin through protease activated receptor-<em>1</em> (PAR<em>1</em>) can disrupt endothelial barrier integrity, whereas the bioactive lipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) recently has been demonstrated to have potent barrier protective effects. Activated protein C (APC) inhibits thrombin generation and has potent anti-inflammatory effects. Here, we show that APC enhanced endothelial barrier integrity in a dual-chamber system dependent on binding to endothelial protein C receptor, activation of PAR<em>1</em>, and activity of cellular <em>sphingosine</em> kinase. Small interfering RNA that targets <em>sphingosine</em> kinase-<em>1</em> or S<em>1</em>P receptor-<em>1</em> blocked this protective signaling by APC. Incubation of cells with PAR<em>1</em> agonist peptide or low concentrations of thrombin (approximately 40 pM) had a similar barrier-enhancing effect. These results demonstrate that PAR<em>1</em> activation on endothelial cells can have opposite biologic effects, reveal a role for cross-communication between the prototypical barrier-protective S<em>1</em>P and barrier-disruptive PAR<em>1</em> pathway, and suggest that S<em>1</em>P receptor-<em>1</em> mediates protective effects of APC in systemic inflammation.

Our prior in vitro studies indicate that <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a phospholipid angiogenic factor, produces endothelial cell barrier enhancement through ligation of endothelial differentiation gene family receptors. We hypothesized that S<em>1</em>P may reduce the vascular leak associated with acute lung injury and found that S<em>1</em>P infusion produced a rapid and significant reduction in lung weight gain (more than 50%) in the isolated perfused murine lung. The effect of S<em>1</em>P was next assessed in a murine model of LPS-mediated microvascular permeability and inflammation with marked increases in parameters of lung injury at both 6 and 24 hours after intratracheal LPS. Each parameter assessed was significantly reduced by intravenous S<em>1</em>P (<em>1</em> microM final) and in selected experiments by the S<em>1</em>P analogue FTY720 (0.<em>1</em> mg/kg, intraperitoneally) delivered <em>1</em> hour after LPS. S<em>1</em>P produced an approximately 40-50% reduction in LPS-mediated extravasation of Evans blue dye albumin, bronchoalveolar lavage protein content, and lung tissue myeloperoxidase activity (reflecting phagocyte infiltration). Consistent with systemic barrier enhancement, S<em>1</em>P significantly decreased Evans blue dye albumin extravasation and myeloperoxidase content in renal tissues of LPS-treated mice. These studies indicate that S<em>1</em>P significantly decreases pulmonary/renal vascular leakage and inflammation in a murine model of LPS-mediated acute lung injury and may represent a novel therapeutic strategy for vascular barrier dysfunction.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive sphingolipid that is critically involved in the embryonic development of the cardiovascular and central nervous systems. In the adult, S<em>1</em>P can produce cytoskeletal re-arrangements in many cell types to regulate immune cell trafficking, vascular homeostasis and cell communication in the central nervous system. S<em>1</em>P is contained in body fluids and tissues at different concentrations, and excessive production of the pleiotropic mediator at inflammatory sites may participate in various pathological conditions. Gene deletion studies and reverse pharmacology (techniques aiming to identify both ligands and function of receptors) provided evidence that many effects of S<em>1</em>P are mediated via five G-protein-coupled S<em>1</em>P receptor subtypes, and novel therapeutic strategies based on interaction with these receptors are being initiated. The prototype S<em>1</em>P receptor modulator, FTY720 (fingolimod), targets four of the five S<em>1</em>P receptor subtypes and may act at several levels to modulate lymphocyte trafficking via lymphocytic and endothelial S<em>1</em>P<em>1</em> and, perhaps, other inflammatory processes through additional S<em>1</em>P receptor subtypes. A recently completed Phase II clinical trial suggested that the drug may provide an effective treatment of relapsing-remitting multiple sclerosis. FTY720 is currently being evaluated in larger-scale, longer-term, Phase III studies. This review provides an overview on S<em>1</em>P activities and S<em>1</em>P receptor function in health and disease, and summarizes the clinical experience with FTY720 in transplantation and multiple sclerosis.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a lipid signaling molecule that regulates many cellular functions, is synthesized from <em>sphingosine</em> and ATP by the action of <em>sphingosine</em> kinase. Two such kinases have been identified, SPHK<em>1</em> and SPHK2. To begin to investigate the physiological functions of <em>sphingosine</em> kinase and S<em>1</em>P signaling, we generated mice deficient in SPHK<em>1</em>. Sphk<em>1</em> null mice were viable, fertile, and without any obvious abnormalities. Total SPHK activity in most Sphk<em>1</em>-/-tissues was substantially, but not completely, reduced indicating the presence of multiple <em>sphingosine</em> kinases. S<em>1</em>P levels in most tissues from the Sphk<em>1</em>-/- mice were not markedly decreased. In serum, however, there was a significant decrease in the S<em>1</em>P level. Although S<em>1</em>P signaling regulates lymphocyte trafficking, lymphocyte distribution was unaffected in lymphoid organs of Sphk<em>1</em>-/- mice. The immunosuppressant FTY720 was phosphorylated and elicited lymphopenia in the Sphk<em>1</em> null mice showing that SPHK<em>1</em> is not required for the functional activation of this <em>sphingosine</em> analogue prodrug. The results with these Sphk<em>1</em> null mice reveal that some key physiologic processes that require S<em>1</em>P receptor signaling, such as vascular development and proper lymphocyte distribution, can occur in the absence of SPHK<em>1</em>.

Sphingolipids (SLs) are essential constituents of eukaryotic cells. Besides playing structural roles in cellular membranes, some metabolites, including ceramide, <em>sphingosine</em>, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, have drawn attention as bioactive signaling molecules involved in the regulation of cell growth, differentiation, senescence, and apoptosis. Understanding the many cell regulatory functions of SL metabolites requires an advanced knowledge of how and where in the cell they are generated, converted, or degraded. This review will provide a short overview of the metabolism, localization, and compartmentalization of SLs. Also, a discussion on bioactive members of the SL family and inducers of SL enzymes that lead to ceramide generation will be presented.