Recent studies by our group and others have disclosed the presence of ceramides in mitochondria, and the activities of ceramide synthase and reverse ceramidase in mitochondria have also been reported. Since a possible contamination with the ER (endoplasmic reticulum)-related compartment MAM (mitochondria-associated membrane) could not be ruled out in previous studies, we have re-investigated the presence of the enzymes of ceramide metabolism in mitochondria and MAM highly purified from rat liver. In the present paper, we show that purified mitochondria as well as MAM are indeed able to generate ceramide in vitro through both ceramide synthase or reverse ceramidase, whereas the latter enzyme activity is barely detectable in microsomes. Moreover, ceramide synthase activities were recovered in outer mitochondrial membranes as well as in inner mitochondrial membranes. Using radiolabelled <em>sphingosine</em> as a substrate, mitochondria could generate ceramide and phytoceramide. However, the in vitro sensitivity of ceramide synthase toward FB<em>1</em> (fumonisin B<em>1</em>) in mitochondria as well as in MAM was found to depend upon the sphingoid base: whereas dihydro<em>sphingosine</em> N-acyltransferase was inhibited by FB<em>1</em> in a concentration-dependent manner, FB<em>1</em> actually activated the ceramide synthase when using <em>sphingosine</em> as a substrate. Acylation of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> and dihydro<em>sphingosine</em> <em>1</em>-<em>phosphate</em>, generating ceramide <em>1</em>-<em>phosphate</em>, was also shown with both subcellular fractions. Moreover, the same difference in sensitivity towards FB<em>1</em> for the ceramide synthase activities was seen between the two phosphorylated sphingoid bases, raising the possibility that distinct base-specific enzymes may be involved as ceramide synthases. Collectively, these results demonstrate the involvement of mitochondria in the metabolism of ceramides through different pathways, thereby supporting the hypothesis that topology of ceramide formation could determine its function.

Apoptosis and autophagy are two evolutionarily conserved processes that maintain homeostasis during stress. Although the two pathways utilize fundamentally distinct machinery, apoptosis and autophagy are highly interconnected and share many key regulators. The crosstalk between apoptosis and autophagy is complex, as autophagy can function to promote cell survival or cell death under various cellular conditions. The molecular mechanisms of crosstalk are beginning to be elucidated and have critical implications for the treatment of various diseases, such as cancer. Sphingolipids are a class of bioactive lipids that mediate many key cellular processes, including apoptosis and autophagy. By targeting several of the shared regulators, sphingolipid metabolites differentially regulate the induction of apoptosis and autophagy. Importantly, individual sphingolipid species appear to "switch" autophagy toward cell survival (e.g., <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>) or cell death (e.g., ceramide, gangliosides). This review assesses the current understanding of sphingolipid-induced apoptosis and autophagy to address how sphingolipids mediate the "switch" between the cell survival and cell death. As sphingolipid metabolism is frequently dysregulated in cancer, sphingolipid-modulating agents, or sphingomimetics, have emerged as a novel chemotherapeutic strategy. Ultimately, a greater understanding of sphingolipid-mediated crosstalk between apoptosis and autophagy may be critical for enhancing the chemotherapeutic efficacy of these agents.

Activated macrophages acquire a proinflammatory (classic) or antiinflammatory (alternative) phenotype that influences atherosclerosis. The present study investigated whether <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), with its known antiinflammatory effects, could regulate the inflammatory phenotype of lipopolysaccharide (LPS)-stimulated mouse macrophages. Activation of macrophages by LPS significantly increases proinflammatory cytokine secretion. Pretreatment of macrophages with 500 nmol/L S<em>1</em>P markedly reduced LPS-mediated secretion of tumor necrosis factor-alpha, monocyte chemoattractant protein-<em>1</em>, and interleukin-<em>1</em>2. Such antiinflammatory actions were also evident in LPS-stimulated macrophages treated with the S<em>1</em>P<em>1</em> receptor-specific agonist SEW287<em>1</em>. Pharmacological antagonism of the S<em>1</em>P<em>1</em> receptor on macrophages using the S<em>1</em>P<em>1</em>-specific antagonist VPC44<em>1</em><em>1</em>6 also blocked proinflammatory cytokine secretion in response to LPS. Studies using bone marrow-derived macrophages from S<em>1</em>P2-deficient mice revealed that the S<em>1</em>P2 receptor did not play a pivotal role in this process. Thus, activation of the S<em>1</em>P<em>1</em> receptor in mouse macrophages limits the expression of proinflammatory cytokines. Furthermore, we demonstrated that S<em>1</em>P increased arginase I activity and inhibited LPS-induced inducible NO synthase activity in LPS-treated macrophages, again through S<em>1</em>P<em>1</em> receptor activation on macrophages. Analysis of a <em>1</em>.7-kb region of the murine inducible NO synthase promoter revealed the presence of putative nuclear factor kappaB, activator protein-<em>1</em>, and STAT-<em>1</em> response elements. Using inducible NO synthase promoter-reporter constructs, we found that S<em>1</em>P significantly reduced the nuclear factor kappaB-mediated induction of inducible NO synthase. These findings demonstrate an important role for S<em>1</em>P in the regulation of macrophage phenotypic switching. Therefore, we conclude that S<em>1</em>P promotes the production of an alternative antiinflammatory macrophage phenotype through activation of the macrophage S<em>1</em>P<em>1</em> receptor.

Ceramide-<em>1</em>-<em>phosphate</em> is a sphingolipid metabolite that has been implicated in membrane fusion of brain synaptic vesicles and neutrophil phagolysosome formation. Ceramide-<em>1</em>-<em>phosphate</em> can be produced by ATP-dependent ceramide kinase activity, although little is known of this enzyme because it has not yet been highly purified or cloned. Based on sequence homology to <em>sphingosine</em> kinase type <em>1</em>, we have now cloned a related lipid kinase, human ceramide kinase (hCERK). hCERK encodes a protein of 537 amino acids that has a catalytic region with a high degree of similarity to the diacylglycerol kinase catalytic domain. hCERK also has a putative N-myristoylation site on its NH(2) terminus followed by a pleckstrin homology domain. Membrane but not cytosolic fractions from HEK293 cells transiently transfected with a hCERK expression vector readily phosphorylated ceramide but not <em>sphingosine</em> or other sphingoid bases, diacylglycerol or phosphatidylinositol. This activity was clearly distinguished from those of bacterial or human diacylglycerol kinases. With natural ceramide as a substrate, the enzyme had a pH optimum of 6.0-7.5 and showed Michaelis-Menten kinetics, with K(m) values of <em>1</em>87 and 32 microm for ceramide and ATP, respectively. Northern blot analysis revealed that hCERK mRNA expression was high in the brain, heart, skeletal muscle, kidney, and liver. A BLAST search analysis using the hCERK sequence revealed that putative ceramide kinases (CERKs) exist widely in diverse multicellular organisms including plants, nematodes, insects, and vertebrates. Phylogenetic analysis revealed that CERKs are a new class of lipid kinases that are clearly distinct from <em>sphingosine</em> and diacylglycerol kinases. Cloning of CERK should provide new molecular tools to investigate the physiological functions of ceramide-<em>1</em>-<em>phosphate</em>.

FTY720, a <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor modulator, induces a marked decrease in the number of peripheral blood lymphocytes and exerts immunomodulating activity in various experimental allograft and autoimmune disease models. In this study, we evaluated the effect of FTY720 and its active metabolite, (S)-enantiomer of FTY720-<em>phosphate</em> [(S)-FTY720-P] on experimental autoimmune encephalomyelitis (EAE) in rats and mice. Prophylactic administration of FTY720 at 0.<em>1</em> to <em>1</em> mg/kg almost completely prevented the development of EAE, and therapeutic treatment with FTY720 significantly inhibited the progression of EAE and EAE-associated histological change in the spinal cords of LEW rats induced by immunization with myelin basic protein. Consistent with rat EAE, the development of proteolipid protein-induced EAE in SJL/J mice was almost completely prevented and infiltration of CD4(+) T cells into spinal cord was decreased by prophylactic treatment with FTY720 and (S)-FTY720-P. When FTY720 or (S)-FTY720-P was given after establishment of EAE in SJL/J mice, the relapse of EAE was markedly inhibited as compared with interferon-beta, and the area of demyelination and the infiltration of CD4(+) T cells were decreased in spinal cords of EAE mice. Similar therapeutic effect by FTY720 was obtained in myelin oligodendrocyte glycoprotein-induced EAE in C57BL/6 mice. These results indicate that FTY720 exhibits not only a prophylactic but also a therapeutic effect on EAE in rats and mice, and that the effect of FTY720 on EAE appears to be due to a reduction of the infiltration of myelin antigen-specific CD4(+) T cells into the inflammation site.

The endothelial isoform of nitric-oxide synthase (eNOS), a key determinant of vascular homeostasis, is a calcium/calmodulin-dependent phosphoprotein regulated by diverse cell surface receptors. Vascular endothelial growth factor (VEGF) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) stimulate eNOS activity through Akt/phosphoinositide 3-kinase and calcium-dependent pathways. AMP-activated protein kinase (AMPK) also activates eNOS in endothelial cells; however, the molecular mechanisms linking agonist-mediated AMPK regulation with eNOS activation remain incompletely understood. We studied the role of AMPK in VEGF- and S<em>1</em>P-mediated eNOS activation and found that both agonists led to a striking increase in AMPK phosphorylation in pathways involving the calcium/calmodulin-dependent protein kinase kinase beta. Treatment with tyrosine kinase inhibitors or the phosphoinositide 3-kinase inhibitor wortmannin demonstrated differential effects of VEGF versus S<em>1</em>P. Small interfering RNA (siRNA)-mediated knockdown of AMPKalpha<em>1</em>or Akt<em>1</em> impaired the stimulatory effects of both VEGF and S<em>1</em>P on eNOS activation. AMPKalpha<em>1</em> knockdown impaired agonist-mediated Akt phosphorylation, whereas Akt<em>1</em> knockdown did not affect AMPK activation, thus suggesting that AMPK lies upstream of Akt in the pathway leading from receptor activation to eNOS stimulation. Importantly, we found that siRNA-mediated knockdown of AMPKalpha<em>1</em> abrogates agonist-mediated activation of the small GTPase Rac<em>1</em>. Conversely, siRNA-mediated knockdown of Rac<em>1</em> decreased the agonist-mediated phosphorylation of AMPK substrates without affecting that of AMPK, implicating Rac<em>1</em> as a molecular link between AMPK and Akt in agonist-mediated eNOS activation. Finally, siRNA-mediated knockdown of caveolin-<em>1</em> significantly enhanced AMPK phosphorylation, suggesting that AMPK is negatively regulated by caveolin-<em>1</em>. Taken together, these results suggest that VEGF and S<em>1</em>P differentially regulate AMPK and establish a central role for an agonist-modulated AMPK ->> Rac<em>1</em> ->> Akt axis in the control of eNOS in endothelial cells.

OBJECTIVE

FTY720, a <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor agonist that crosses the blood-brain barrier, is a potential immuno-therapy for multiple sclerosis. Our objective was to assess the effect of FTY720 on process extension, differentiation, and survival of human oligodendrocyte progenitor cells (OPCs), and link the functional effects with S<em>1</em>P receptor expression and signaling.

METHODS

Functional assays and receptor expression studies were conducted on A2B5+ OPCs derived from the human fetal central nervous system. Cells were treated with physiologically relevant concentrations of the active phosphorylated form of FTY720. S<em>1</em>P receptor/signaling modulators were used to elucidate the basis of the FTY720-induced functional responses.

RESULTS

Short-term (<em>1</em> day) FTY720 treatment caused initial process retraction that was reversed by uncoupling S<em>1</em>P3 and 5 from their G protein using suramin, and with a Rho-kinase inhibitor H<em>1</em><em>1</em>52. Retraction was associated with RhoA-mediated cytoskeletal signaling and with inhibition of OPC differentiation into more mature phenotypes. Continued FTY720 treatment (2 days) induced process extension and enhanced cell survival associated with increased extracellular signal-regulated kinases <em>1</em> and 2 phosphorylation, mimicked with the S<em>1</em>P<em>1</em>-specific agonist SEW287<em>1</em>, but not reversed with suramin. Quantitative real-time polymerase chain reaction showed that FTY720 induced reciprocal and cyclic modulation of S<em>1</em>P<em>1</em> and S<em>1</em>P5 messenger RNA levels. The observed initial downregulation of S<em>1</em>P5 and subsequently of S<em>1</em>P<em>1</em> messenger RNA supports functional responses being mediated sequentially by S<em>1</em>P5- and later S<em>1</em>P<em>1</em>-associated signaling.

CONCLUSIONS

FTY720 induces time-dependent modulation of S<em>1</em>P receptors on human OPCs with consequent functional responses that are directly relevant for the remyelination process.

The mechanisms of hematopoietic progenitor cell egress and clinical mobilization are not fully understood. Herein, we report that in vivo desensitization of <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors by FTY720 as well as disruption of S<em>1</em>P gradient toward the blood, reduced steady state egress of immature progenitors and primitive Sca-<em>1</em>(+)/c-Kit(+)/Lin(-) (SKL) cells via inhibition of SDF-<em>1</em> release. Administration of AMD3<em>1</em>00 or G-CSF to mice with deficiencies in either S<em>1</em>P production or its receptor S<em>1</em>P(<em>1</em>), or pretreated with FTY720, also resulted in reduced stem and progenitor cell mobilization. Mice injected with AMD3<em>1</em>00 or G-CSF demonstrated transient increased S<em>1</em>P levels in the blood mediated via mTOR signaling, as well as an elevated rate of immature c-Kit(+)/Lin(-) cells expressing surface S<em>1</em>P(<em>1</em>) in the bone marrow (BM). Importantly, we found that S<em>1</em>P induced SDF-<em>1</em> secretion from BM stromal cells including Nestin(+) mesenchymal stem cells via reactive oxygen species (ROS) signaling. Moreover, elevated ROS production by hematopoietic progenitor cells is also regulated by S<em>1</em>P. Our findings reveal that the S<em>1</em>P/S<em>1</em>P(<em>1</em>) axis regulates progenitor cell egress and mobilization via activation of ROS signaling on both hematopoietic progenitors and BM stromal cells, and SDF-<em>1</em> release. The dynamic cross-talk between S<em>1</em>P and SDF-<em>1</em> integrates BM stromal cells and hematopoeitic progenitor cell motility.

The role for hyaluronan (HA) and CD44 in vascular barrier regulation is unknown. We examined high and low molecular weight HA (HMW-HA, approximately <em>1</em>,000 kDa; LMW-HA, approximately 2.5 kDa) effects on human transendothelial monolayer electrical resistance (TER). HMW-HA increased TER, whereas LMW-HA induced biphasic TER changes ultimately resulting in EC barrier disruption. HMW-HA induced the association of the CD44s isoform with, and AKT-mediated phosphorylation of, the barrier-promoting <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor (S<em>1</em>P<em>1</em>) within caveolin-enriched lipid raft microdomains, whereas LMW-HA induced brief CD44s association with S<em>1</em>P<em>1</em> followed by sustained association of the CD44v<em>1</em>0 isoform with, and Src and ROCK <em>1</em>/2-mediated phosphorylation of, the barrier-disrupting S<em>1</em>P3 receptor. HA-induced EC cytoskeletal reorganization and TER alterations were abolished by either disruption of lipid raft formation, CD44 blocking antibody or siRNA-mediated reductions in expression of CD44 isoforms. Silencing S<em>1</em>P<em>1</em>, AKT<em>1</em>, or Rac<em>1</em> blocked the barrier enhancing effects of HA whereas silencing S<em>1</em>P3, Src, ROCK<em>1</em>/2, or RhoA blocked the barrier disruption induced by LMW-HA. In summary, HA regulates EC barrier function through novel differential CD44 isoform interaction with S<em>1</em>P receptors, S<em>1</em>P receptor transactivation, and RhoA/Rac<em>1</em> signaling to the EC cytoskeleton.

Krüppel-like factor 2 (KLF2) is a member of zinc-finger transcription factors. Based on its expression in naive and memory T cells and the activated phenotype of few T cells in mice lacking KLF2 in the lymphoid lineage, KLF2 is postulated to regulate T cell homeostasis by promoting cell quiescence. In this study, we show that in reporter gene assays KLF2 directly activates the promoters of both CD62L and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> (S<em>1</em>P<em>1</em>), whose expression is critical for T cell egress from the thymus and homing to the lymph nodes. Correspondingly, exogenous KLF2 expression in primary T cells significantly up-regulates both CD62L and S<em>1</em>P<em>1</em>. Following adoptive transfer, KLF2-transduced T cells are much more efficient in homing to lymphoid organs than nontransduced T cells. These findings suggest that KLF2 regulates T cell homeostasis at least partly by controlling CD62L and S<em>1</em>P<em>1</em> expression, and therefore T cell egress from the thymus and circulation in the periphery.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a biologically active lipid growth factor, induces robust endothelial cell activation resulting in cellular locomotion, vascular maturation and angiogenesis. Recent work by our laboratory has demonstrated S<em>1</em>P to enhance the cellular barrier function of the vascular endothelium. S<em>1</em>P-induced modulation of vascular permeability is effected through profound cytoskeletal reorganization initiated by cell surface receptor-mediated G protein activation and downstream signaling via the Rho family of small GTPases. The details of the downstream signaling mechanism remain an active area of in vitro investigation. Translational investigation suggests a profound impact of S<em>1</em>P administration in the modulation of edema formation in disease state manifest as acute inflammatory lung injury in which increased vascular permeability is a hallmark feature. These data support an exciting potential therapeutic role for S<em>1</em>P in vascular barrier enhancement necessary for the treatment of critically ill patients.

T cell egress from the thymus is essential for adaptive immunity, yet the requirements for and sites of egress are incompletely understood. We have shown that transgenic expression of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor-<em>1</em> (S<em>1</em>P<em>1</em>) in immature thymocytes leads to their perivascular accumulation and premature release into circulation. Using an intravascular procedure to label emigrating cells, we found that mature thymocytes exit via blood vessels at the corticomedullary junction. By deleting <em>sphingosine</em> kinases in neural crest-derived pericytes, we provide evidence that these specialized vessel-ensheathing cells contribute to the S<em>1</em>P that promotes thymic egress. Lymphatic endothelial cell-derived S<em>1</em>P was not required. These studies identify the major thymic egress route and suggest a role for pericytes in promoting reverse transmigration of cells across blood vessel endothelium.

Lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) are potent biologically active lipid mediators that exert a wide range of cellular effects through specific G protein-coupled receptors. To date, four LPA receptors and five S<em>1</em>P receptors have been identified. These receptors are expressed in a large number of tissues and cell types, allowing for a wide variety of cellular responses to lysophospholipid signaling, including cell adhesion, cell motility, cytoskeletal changes, proliferation, angiogenesis, process retraction, and cell survival. In addition, recent studies in mice show that specific lysophospholipid receptors are required for proper cardiovascular, immune, respiratory, and reproductive system development and function. Lysophospholipid receptors may also have specific roles in cancer and other diseases. This review will cover identification and expression of the lysophospholipid receptors, as well as receptor signaling properties and function. Additionally, phenotypes of mice deficient for specific lysophospholipid receptors will be discussed to demonstrate how these animals have furthered our understanding of the role lysophospholipids play in normal biology and disease.

After a coagulation stimulus, the blood clotting cascade amplifies largely unchecked until very high levels of thrombin are generated. Natural anticoagulant mechanisms (for example, the protein C anticoagulant pathway) are amplified to prevent excessive thrombin generation. Thrombin binds to thrombomodulin (TM) and this complex and then activates protein C approximately <em>1</em>000 times faster than free thrombin. Protein C activation is enhanced approximately 20-fold further by the endothelial cell protein C receptor (EPCR). Activated protein C proteolytically inactivates factor Va (FVa) and FVIIIa, thereby blocking the amplification of the coagulation system, a process that is accelerated by protein S. TM not only accelerates protein C activation, but also decreases endothelial cell activation by blocking high-mobility group protein-B<em>1</em> inflammatory functions and suppressing both nuclear factor-kappa B nuclear translocation and the mitogen-activated protein kinase pathways. The thrombin-TM complex also activates thrombin-activatable fibrinolysis inhibitor, a procarboxypeptidase that renders fibrin resistant to clot lysis and neutralizes vasoactive molecules such as complement C5a. Activated protein C has a variety of antiinflammatory activities. It suppresses inflammatory cytokine elevation in animal models of severe sepsis, inhibits leukocyte adhesion, decreases leukocyte chemotaxis, reduces endothelial cell apoptosis, helps maintain endothelial cell barrier function through activation of the <em>sphingosine</em>-<em>1</em> <em>phosphate</em> receptor, and minimizes the decrease in blood pressure associated with severe sepsis. Most of these functions are dependent on binding to EPCR. Overall this pathway is critical to both regulation of the blood coagulation process, and control of the innate inflammatory response and some of its associated downstream pathologies.

The lysophospholipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a pleiotropic signaling lipid present constitutively in plasma, and secreted locally at elevated concentrations at sites of inflammation. S<em>1</em>P maintains essential variable homeostatic functions in addition to inducing pathophysiology through the activation of five specific high-affinity G-protein-coupled receptors. Therefore, S<em>1</em>P can function as an extracellular rheostat regulating tonic and acutely evoked functions. Although S<em>1</em>P receptors can regulate lymphoid development and lymphocyte trafficking, and different opinions exist on the roles of receptor agonism and functional antagonism in regulating lymphocyte recirculation, this personal perspective highlights the pivotal control points regulated by constitutive and induced S<em>1</em>P receptor tone at vascular endothelial and lymphatic endothelial barriers, through which S<em>1</em>P agonism impacts on both innate and adaptive immunity. We also emphasize how specific, proof-of-concept chemical tools complement genetic approaches by enabling reversible perturbation of the S<em>1</em>P-S<em>1</em>P(<em>1</em>) receptor axis and, thus, clarifying in vivo mechanisms in the absence of developmental compensations.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is one of several bioactive phospholipids that exert profound mitogenic and morphogenic actions. Originally characterized as a second messenger, S<em>1</em>P is now recognized to achieve many of its effects through cell surface, G protein-coupled receptors. We used a subunit-selective [(35)S]GTPgammaS binding assay to investigate whether the variety of actions exerted through Edg-<em>1</em>, a recently identified receptor for S<em>1</em>P, might be achieved through multiple G proteins. We found, employing both Sf9 and HEK293 cells, that Edg-<em>1</em> activates only members of the G(i) family, and not G(s), G(q), G(<em>1</em>2), or G(<em>1</em>3). We additionally established that Edg-<em>1</em> activates G(i) in response not only to S<em>1</em>P but also sphingosylphosphorylcholine; no effects of lysophosphatidic acid through Edg-<em>1</em> were evident. Our assays further revealed a receptor(s) for S<em>1</em>P endogenous to HEK293 cells that mediates activation of G(<em>1</em>3) as well as G(i). Because several of the biological actions of S<em>1</em>P are assumed to proceed through the G(<em>1</em>2/<em>1</em>3) family, we tested whether Edg-3 and H2<em>1</em>8/Edg-5, two other receptors for S<em>1</em>P, might have a broader coupling profile than Edg-<em>1</em>. Indeed, Edg-3 and H2<em>1</em>8/Edg-5 communicate not only with G(i) but also with G(q) and G(<em>1</em>3). These studies represent the first characterization of S<em>1</em>P receptor activity through G proteins directly and establish fundamental differences in coupling.

Immunotherapeutic drugs that mimic <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) disrupt lymphocyte trafficking and cause T helper and T effector cells to be retained in secondary lymphoid tissue and away from sites of inflammation. The prototypical therapeutic agent, 2-alkyl-2-amino-<em>1</em>,3-propanediol (FTY720), stimulates S<em>1</em>P signaling pathways only after it is phosphorylated by one or more unknown kinases. We generated <em>sphingosine</em> kinase 2 (SPHK2) null mice to demonstrate that this kinase is responsible for FTY720 phosphorylation and thereby its subsequent actions on the immune system. Both systemic and lymphocyte-localized sources of SPHK2 contributed to FTY720 induced lymphopenia. Although FTY720 was selectively activated in vivo by SPHK2, other S<em>1</em>P pro-drugs can be phosphorylated to cause lymphopenia through the action of additional <em>sphingosine</em> kinases. Our results emphasize the importance of SPHK2 expression in both lymphocytes and other tissues for immune modulation and drug metabolism.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and 5 specific high-affinity S<em>1</em>P receptor (S<em>1</em>PR) subtypes, S<em>1</em>P(<em>1</em>-5), have important regulatory functions in normal physiology and disease processes, particularly involving the immune, central nervous, and cardiovascular systems. Within the immune system, downmodulation of S<em>1</em>P(<em>1</em>) prevents the egress of B and T cells from lymph nodes (LN) into the lymphatic circulation. This is especially relevant in certain autoimmune diseases, including multiple sclerosis (MS), in which demyelination and brain atrophy occur due to the presence of autoreactive lymphocytes within the CNS. Accordingly, S<em>1</em>P(<em>1</em>)-directed pharmacologic interventions that aim to retain these autoreactive lymphocytes in the LN and thus prevent their recirculation and subsequent infiltration into the CNS have been investigated as a means of preventing disease progression in patients with MS. Fingolimod (FTY720), a structural analog of <em>sphingosine</em>, is phosphorylated in vivo into fingolimod <em>phosphate</em> by <em>sphingosine</em> kinase-2. Fingolimod <em>phosphate</em>, which binds to S<em>1</em>PRs, has been shown to modulate the activity of S<em>1</em>P(<em>1</em>) in patients with MS and to reduce immune cell infiltration into the CNS, consistent with its previously established effects in animal models of the disease. Preclinical studies also suggest that fingolimod has beneficial effects within the CNS that are independent of its immune cell trafficking activity. This review highlights the normal physiologic processes modulated by S<em>1</em>P and S<em>1</em>PRs, and the therapeutic effects of S<em>1</em>PR modulation in the immune, central nervous, and cardiovascular systems.

The <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>/Endothelial Differentiation Gene (S<em>1</em>P/EDG) family of G-protein-coupled receptors (GPCR) currently includes five different isoforms, which differentially regulate fundamental cellular processes such as migration, proliferation, cytoskeletal organization, adherens junction assembly and morphogenesis. Additionally, specific S<em>1</em>P/EDG isoforms can regulate important physiological processes such as blood vessel maturation, cardiac development and angiogenesis in vivo. Herein, we review the current state of knowledge of the expression patterns, signaling pathways and functional characteristics of the different S<em>1</em>P receptors. Further investigation in this field will likely improve our understanding of cardiovascular development as well as vascular diseases and may lead to novel therapeutic approaches.

In this chapter, roles of bioactive sphingolipids in the regulation of cancer pathogenesis and therapy will be reviewed. Sphingolipids have emerged as bioeffector molecules, which control various aspects of cell growth, proliferation, and anti-cancer therapeutics. Ceramide, the central molecule of sphingolipid metabolism, generally mediates anti-proliferative responses such as inhibition of cell growth, induction of apoptosis, and/or modulation of senescence. On the other hand, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) plays opposing roles, and induces transformation, cancer cell growth, or angiogenesis. A network of metabolic enzymes regulates the generation of ceramide and S<em>1</em>P, and these enzymes serve as transducers of sphingolipid-mediated responses that are coupled to various exogenous or endogenous cellular signals. Consistent with their key roles in the regulation of cancer growth and therapy, attenuation of ceramide generation and/or increased S<em>1</em>P levels are implicated in the development of resistance to drug-induced apoptosis, and escape from cell death. These data strongly suggest that advances in the molecular and biochemical understanding of sphingolipid metabolism and function will lead to the development of novel therapeutic strategies against human cancers, which may also help overcome drug resistance.

Fingolimod is the first oral disease-modifying therapy approved for relapsing forms of multiple sclerosis (MS). Following phosphorylation in vivo, the active agent, fingolimod <em>phosphate</em> (fingolimod-P), acts as a <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor modulator, binding with high affinity to four of the five known S<em>1</em>P receptors (S<em>1</em>P<em>1</em>, S<em>1</em>P3, S<em>1</em>P4 and S<em>1</em>P5). The mechanism of action of fingolimod in MS has primarily been considered as immunomodulatory, whereby fingolimod-P modulates S<em>1</em>P<em>1</em> on lymphocytes, selectively retaining autoreactive lymphocytes in lymph nodes to reduce damaging infiltration into the central nervous system (CNS). However, emerging evidence indicates that fingolimod has direct effects in the CNS in MS. For example, in the MS animal model of experimental autoimmune encephalomyelitis (EAE), fingolimod is highly efficacious in both a prophylactic and therapeutic setting, yet becomes ineffective in animals selectively deficient for S<em>1</em>P<em>1</em> on astrocytes, despite maintained normal immunologic receptor expression and functions, and S<em>1</em>P-mediated immune activities. Here we review S<em>1</em>P signaling effects relevant to MS in neural cell types expressing S<em>1</em>P receptors, including astrocytes, oligodendrocytes, neurons, microglia and dendritic cells. The direct effects of fingolimod on these CNS cells observed in preclinical studies are discussed in view of the functional consequences of reducing neurodegenerative processes and promoting myelin preservation and repair. The therapeutic implications of S<em>1</em>P modulation in the CNS are considered in terms of the clinical outcomes of MS, such as reducing MS-related brain atrophy, and other CNS disorders. Additionally, we briefly outline other existing and investigational MS therapies that may also have effects in the CNS.

Five cognate G protein-coupled receptors (S<em>1</em>P(<em>1</em>-5)) have been shown to mediate various cellular effects of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P). Here we report the generation of mice null for S<em>1</em>P(2) and for both S<em>1</em>P(2) and S<em>1</em>P(3). S<em>1</em>P(2)-null mice were viable and fertile and developed normally. The litter sizes from S<em>1</em>P(2)S<em>1</em>P(3) double-null crosses were remarkably reduced compared with controls, and double-null pups often did not survive through infancy, although double-null survivors lacked any obvious phenotype. Mouse embryonic fibroblasts (MEFs) were examined for the effects of receptor deletions on S<em>1</em>P signaling pathways. Wild-type MEFs were responsive to S<em>1</em>P in activation of Rho and phospholipase C (PLC), intracellular calcium mobilization, and inhibition of forskolin-activated adenylyl cyclase. S<em>1</em>P(2)-null MEFs showed a significant decrease in Rho activation, but no effect on PLC activation, calcium mobilization, or adenylyl cyclase inhibition. Double-null MEFs displayed a complete loss of Rho activation and a significant decrease in PLC activation and calcium mobilization, with no effect on adenylyl cyclase inhibition. These data extend our previous findings on S<em>1</em>P(3)-null mice and indicate preferential coupling of the S<em>1</em>P(2) and S<em>1</em>P(3) receptors to Rho and PLC/Ca(2+) pathways, respectively. Although either receptor subtype supports embryonic development, deletion of both produces marked perinatal lethality, demonstrating an essential role for combined S<em>1</em>P signaling by these receptors.

Lysophospholipids encompass a diverse range of small, membrane-derived phospholipids that act as extracellular signals. The signalling properties are mediated by 7-transmembrane GPCRs, constituent members of which have continued to be identified after their initial discovery in the mid-<em>1</em>990s. Here we briefly review this class of receptors, with a particular emphasis on their protein and gene nomenclatures that reflect their cognate ligands. There are six lysophospholipid receptors that interact with lysophosphatidic acid (LPA): protein names LPA<em>1</em> - LPA6 and italicized gene names LPAR<em>1</em>-LPAR6 (human) and Lpar<em>1</em>-Lpar6 (non-human). There are five <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors: protein names S<em>1</em>P<em>1</em> -S<em>1</em>P5 and italicized gene names S<em>1</em>PR<em>1</em>-S<em>1</em>PR5 (human) and S<em>1</em>pr<em>1</em>-S<em>1</em>pr5 (non-human). Recent additions to the lysophospholipid receptor family have resulted in the proposed names for a lysophosphatidyl inositol (LPI) receptor - protein name LPI<em>1</em> and gene name LPIR<em>1</em> (human) and Lpir<em>1</em> (non-human) - and three lysophosphatidyl serine receptors - protein names LyPS<em>1</em> , LyPS2 , LyPS3 and gene names LYPSR<em>1</em>-LYPSR3 (human) and Lypsr<em>1</em>-Lypsr3 (non-human) along with a variant form that does not appear to exist in humans that is provisionally named LyPS2L . This nomenclature incorporates previous recommendations from the International Union of Basic and Clinical Pharmacology, the Human Genome Organization, the Gene Nomenclature Committee, and the Mouse Genome Informatix.

Endothelial differentiation gene (Edg) proteins are G-protein-coupled receptors activated by lysophospholipid mediators: <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) or lysophosphatidic acid. We show that in the CNS, expression of Edg8/S<em>1</em>P5, a high-affinity S<em>1</em>P receptor, is restricted to oligodendrocytes and expressed throughout development from the immature stages to the mature myelin-forming cell. S<em>1</em>P activation of Edg8/S<em>1</em>P5 on O4-positive pre-oligodendrocytes induced process retraction via a Rho kinase/collapsin response-mediated protein signaling pathway, whereas no retraction was elicited by S<em>1</em>P on these cells derived from Edg8/S<em>1</em>P5-deficient mice. Edg8/S<em>1</em>P5-mediated process retraction was restricted to immature cells and was no longer observed at later developmental stages. In contrast, S<em>1</em>P activation promoted the survival of mature oligodendrocytes but not of pre-oligodendrocytes. The S<em>1</em>P-induced survival of mature oligodendrocytes was mediated through a pertussis toxin-sensitive, Akt-dependent pathway. Our data demonstrate that Edg8/S<em>1</em>P5 activation on oligodendroglial cells modulates two distinct functional pathways mediating either process retraction or cell survival and that these effects depend on the developmental stage of the cell.