Soluble and hydrophobic lipid breakdown products have a variety of important signaling roles in cells. Here sphingoid bases derived in cells from sphingolipid breakdown are shown to have a potent and direct effect in mediating calcium release from intracellular stores. <em>Sphingosine</em> must be enzymically converted within the cell to a product believed to be <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, which thereafter effects calcium release from a pool including the inositol <em>1</em>,4,5-tris<em>phosphate</em>-sensitive calcium pool. The sensitivity, molecular specificity, and reversibility of the effect on calcium movements closely parallel sphingoid base-mediated inhibition of protein kinase C. Generation of sphingoid bases in cells may activate a dual signaling pathway involving regulation of calcium and protein kinase C, comparable perhaps to the phosphatidylinositol and calcium signaling pathway.

Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined. Work in mice showed that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor-2 (S<em>1</em>PR2), a Gα<em>1</em>2 and Gα<em>1</em>3 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA<em>1</em>3 (encoding Gα<em>1</em>3) and S<em>1</em>PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S<em>1</em>PR2 frequently disrupt the receptor's Akt and migration inhibitory functions. Gα<em>1</em>3-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S<em>1</em>P, and Gα<em>1</em>3-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Gα<em>1</em>3, but not S<em>1</em>PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Gα<em>1</em>3 effector ARHGEF<em>1</em>, and Arhgef<em>1</em> deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Gα<em>1</em>3- and S<em>1</em>PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt's lymphoma, also represses germinal centre B-cell growth and promotes confinement via Gα<em>1</em>3. These findings identify a Gα<em>1</em>3-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma.

Lysophospholipids (LPs), including lysophosphatidic acid and <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, produce many cellular effects. However, the prolonged absence of any cloned and identified LP receptor has left open the question of how these lipids actually bring about these effects. The cloning and functional identification of the first LP receptor, lp(A<em>1</em>)/vzg-<em>1</em>, has led rapidly to the identification and classification of multiple orphan receptors/expression sequence tags known by many names (e.g. edg, mrec<em>1</em>.3, gpcr26, H2<em>1</em>8, AGR<em>1</em>6, nrg-<em>1</em>) as members of a common cognate G protein-coupled receptor family. We review features of the LP receptor family, including molecular characteristics, genomics, signaling properties, and gene expression. A major question for which only partial answers are available concerns the biological significance of receptor-mediated LP signaling. Recent studies that demonstrate the role of receptor-mediated LP signaling in the nervous system, cardiovascular system, and other organ systems indicate the importance of this signaling in development, function, and pathophysiology and portend an exciting time ahead for this growing field.

Follicular dendritic cells (FDCs) retain and display opsonized antigens in primary follicles and germinal centers (GCs). However, their roles beyond antigen presentation have been incompletely defined. In this study, we tested the impact of selective FDC ablation on short-term follicle and GC function. Within 2 d of FDC ablation, primary follicles lost their homogeneity and became disorganized bands of cells around T zones. These B cell areas retained CXCL<em>1</em>3-expressing stromal cells but often exhibited inappropriate ER-TR7 and CCL2<em>1</em> expression. Ablation of GC FDCs led to the disappearance of GCs. When B cell death was prevented using a Bcl2 transgene, FDC ablation led to splenic GC B cell dispersal. Mesenteric lymph node GCs were more resistant but became dispersed when <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor-2 was also removed. These experiments indicate that FDCs help maintain primary follicles as a B cell exclusive niche and define a critical role for FDCs in cell retention within GCs.

Lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), two of the best-studied lysophospholipids, are known to influence diverse biological events, including organismal development as well as function and pathogenesis within multiple organ systems. These functional roles are due to a family of at least <em>1</em><em>1</em> G protein-coupled receptors (GPCRs), named LPA(<em>1</em>-6) and S<em>1</em>P(<em>1</em>-5), which are widely distributed throughout the body and that activate multiple effector pathways initiated by a range of heterotrimeric G proteins including G(i/o), G(<em>1</em>2/<em>1</em>3), G(q) and G(s), with actual activation dependent on receptor subtypes. In the central nervous system (CNS), a major locus for these signaling pathways, LPA and S<em>1</em>P have been shown to influence myriad responses in neurons and glial cell types through their cognate receptors. These receptor-mediated activities can contribute to disease pathogenesis and have therapeutic relevance to human CNS disorders as demonstrated for multiple sclerosis (MS) and possibly others that include congenital hydrocephalus, ischemic stroke, neurotrauma, neuropsychiatric disorders, developmental disorders, seizures, hearing loss, and Sandhoff disease, based upon the experimental literature. In particular, FTY720 (fingolimod, Gilenya, Novartis Pharma, AG) that becomes an analog of S<em>1</em>P upon phosphorylation, was approved by the FDA in 20<em>1</em>0 as a first oral treatment for MS, validating this class of receptors as medicinal targets. This review will provide an overview and update on the biological functions of LPA and S<em>1</em>P signaling in the CNS, with a focus on results from studies using genetic null mutants for LPA and S<em>1</em>P receptors. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

FTY720 is the first of a new immunomodulator class: <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor agonist. In <em>1</em>994, an immunosuppressive natural product, ISP-I (myriocin), was isolated from the culture broth of Isaria sinclairii, a type of vegetative wasp. The chemical modification of ISP-I yielded a new compound, FTY720, which has more potent immunosuppressive activity and less toxicity than ISP-I does. FTY720 has been shown to be highly effective in experimental allotransplantation models and autoimmune disease models. A striking feature of FTY720 is the induction of a marked decrease in peripheral blood T- and B-cells at doses that show immunosuppressive activity in these models. Reportedly, FTY720 is rapidly converted to FTY720-<em>phosphate</em> (FTY720-P) by <em>sphingosine</em> kinase 2 in vivo, and FTY720-P acts as a potent agonist at S<em>1</em>P receptors. Recently, it has been suggested that FTY720-P internalizes S<em>1</em>P<em>1</em> on lymphocytes and thereby inhibits the migration of lymphocytes toward S<em>1</em>P. Thus, it is likely that the reduction of circulating lymphocytes by FTY720 is due to the inhibition of S<em>1</em>P/S<em>1</em>P<em>1</em>-dependent lymphocyte egress from secondary lymphoid tissues and thymus. Because FTY720 displays a novel mechanism of action that has not been observed with other immunosuppressive agents and shows a synergism with cyclosporin A (CsA) and tacrolimus, it is presumed that FTY720 provides a useful tool for the prevention of transplant rejection and a new therapeutic approach for autoimmune diseases including multiple sclerosis and rheumatoid arthritis.

<em>Sphingosine</em> kinases (SphKs) catalyze the phosphorylation of <em>sphingosine</em> to <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). Together with other sphingolipid metabolizing enzymes, SphKs regulate the balance of the lipid mediators, ceramide, <em>sphingosine</em>, and S<em>1</em>P. The ubiquitous mediator S<em>1</em>P regulates cellular functions such as proliferation and survival, cytoskeleton architecture and Ca(2+) homoeostasis, migration, and adhesion by activating specific high-affinity G-protein-coupled receptors or by acting intracellularly. In mammals, two isoforms of SphK have been identified. They are activated by G-protein-coupled receptors, receptor tyrosine kinases, immunoglobulin receptors, cytokines, and other stimuli. The molecular mechanisms by which SphK<em>1</em> and SphK2 are specifically regulated are complex and only partially understood. Although SphK<em>1</em> and SphK2 appear to have opposing roles, promoting cell growth and apoptosis, respectively, they can obviously also substitute for each other, as mice deficient in either SphK<em>1</em> or SphK2 had no obvious abnormalities, whereas double-knockout animals were embryonic lethal. In this review, our understanding of structure, regulation, and functional roles of SphKs is updated and discussed with regard to their implication in pathophysiological and disease states.

PTEN, a tumor suppressor phosphatase, is important in the regulation of cell migration and invasion. Physiological regulation of PTEN (phosphatase and tensin homolog deleted on chromosome <em>1</em>0) by cell surface receptors has not been described. Here, we show that the bioactive lipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), which acts through the S<em>1</em>P2 receptor (S<em>1</em>P2R) G protein-coupled receptor (GPCR) to inhibit cell migration, utilizes PTEN as a signaling intermediate. S<em>1</em>P2R inhibition of cell migration is abrogated by dominant-negative PTEN expression. S<em>1</em>P was unable to efficiently inhibit the migration of Pten(DeltaloxP/DeltaloxP) mouse embryonic fibroblasts; however, the antimigratory effect was restored upon the expression of PTEN. S<em>1</em>P2R activation of Rho GTPase is not affected in Pten(DeltaloxP/DeltaloxP) cells, and dominant-negative Rho GTPase reversed S<em>1</em>P inhibition of cell migration in WT cells but not in Pten(DeltaloxP/DeltaloxP) cells, suggesting that PTEN acts downstream of the Rho GTPase. Ligand activation of the S<em>1</em>P2R receptor stimulated the coimmunoprecipitation of S<em>1</em>P2R and PTEN. Interestingly, S<em>1</em>P2R signaling increased PTEN phosphatase activity in membrane fractions. Furthermore, tyrosine phosphorylation of PTEN was stimulated by S<em>1</em>P2R signaling. These data suggest that the S<em>1</em>P2R receptor actively regulates the PTEN phosphatase by a Rho GTPase-dependent pathway to inhibit cell migration. GPCR regulation of PTEN maybe a general mechanism in signaling events of cell migration and invasion.

In vitro and in vivo evidence indicates that circulating platelets affect both vascular integrity and hemostasis. How platelets enhance the permeability barrier of the vascular endothelium is not well understood. We measured the effect of isolated human platelets on human pulmonary artery endothelial cell (EC) barrier integrity by monitoring transmonolayer electrical resistance. EC barrier function was significantly increased by the addition of platelets ( approximately 40% maximum, 2.5 x <em>1</em>06 platelets/ml). Platelet supernatants, derived from 2.5 x <em>1</em>06 platelets/ml, reproduced the barrier enhancement and reversed the barrier dysfunction produced by the edemagenic agonist thrombin, which implicates a soluble barrier-promoting factor. The barrier-enhancing effect of platelet supernatants was heat stable but was attenuated by either charcoal delipidation (suggesting a vasoactive lipid mediator) or pertussis toxin, implying involvement of a Gialpha-coupled receptor signal transduction pathway. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a sphingolipid that is released from activated platelets, is known to ligate G protein-coupled EC differentiation gene (EDG) receptors, increase EC electrical resistance, and reorganize the actin cytoskeleton (Garcia JG, Liu F, Verin AD, Birukova A, Dechert MA, Gerthoffer WT, Bamberg JR, and English D. J Clin Invest <em>1</em>08: 689-70<em>1</em>, 200<em>1</em>). Infection of EC with an adenoviral vector expressing an antisense oligonucleotide directed against EDG-<em>1</em> but not infection with control vector attenuated the barrier-enhancing effect of both platelet supernatants and S<em>1</em>P. These results indicate that a major physiologically relevant vascular barrier-protective mediator produced by human platelets is S<em>1</em>P.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a lipid mediator formed by the metabolism of sphingomyelin. In vertebrates, S<em>1</em>P is secreted into the extracellular environment and signals via G protein-coupled S<em>1</em>P receptors to regulate cell-cell and cell-matrix adhesion, and thereby influence cell migration, differentiation and survival. The expression and localization of S<em>1</em>P receptors is dynamically regulated and controls vascular development, vessel stability and immune cell trafficking. In addition, crucial events during embryogenesis, such as angiogenesis, cardiogenesis, limb development and neurogenesis, are regulated by S<em>1</em>P signalling. Here, and in the accompanying poster, we provide an overview of S<em>1</em>P signalling in development and in disease.

Macular oedema typically results from blood-retinal barrier disruption. It has recently been reported that patients with multiple sclerosis treated with FTY-720 (fingolimod) may exhibit macular oedema. Multiple sclerosis is not otherwise thought to be associated with macular oedema except in the context of comorbid clinical uveitis. Despite a lack of myelin, the retina is a site of inflammation and microglial activation in multiple sclerosis and demonstrates significant neuronal and axonal loss. We unexpectedly observed microcystic macular oedema using spectral domain optical coherence tomography in patients with multiple sclerosis who did not have another reason for macular oedema. We therefore evaluated spectral domain optical coherence tomography images in consecutive patients with multiple sclerosis for microcystic macular oedema and examined correlations between macular oedema and visual and ambulatory disability in a cross-sectional analysis. Participants were excluded if there was a comorbidity that could account for the presence of macular oedema, such as uveitis, diabetes or other retinal disease. A microcystic pattern of macular oedema was observed on optical coherence tomography in <em>1</em>5 of 3<em>1</em>8 (4.7%) patients with multiple sclerosis. No macular oedema was identified in 52 healthy controls assessed over the same period. The microcystic oedema predominantly involved the inner nuclear layer of the retina and tended to occur in small, discrete patches. Patients with multiple sclerosis with microcystic macular oedema had significantly worse disability [median Expanded Disability Score Scale 4 (interquartile range 3-6)] than patients without macular oedema [median Expanded Disability Score Scale 2 (interquartile range <em>1</em>.5-3.5)], P = 0.0002. Patients with multiple sclerosis with microcystic macular oedema also had higher Multiple Sclerosis Severity Scores, a measure of disease progression, than those without oedema [median of 6.47 (interquartile range 4.96-7.98) versus 3.65 (interquartile range <em>1</em>.92-5.87), P = 0.0009]. Microcystic macular oedema occurred more commonly in eyes with prior optic neuritis than eyes without prior optic neuritis (50 versus 27%) and was associated with lower visual acuity (median logMAR acuity of 0.<em>1</em>7 versus -0.<em>1</em>) and a thinner retinal nerve fibre layer. The presence of microcystic macular oedema in multiple sclerosis suggests that there may be breakdown of the blood-retinal barrier and tight junction integrity in a part of the nervous system that lacks myelin. Microcystic macular oedema may also contribute to visual dysfunction beyond that explained by nerve fibre layer loss. Microcystic changes need to be assessed, and potentially adjusted for, in clinical trials that evaluate macular volume as a marker of retinal ganglion cell survival. These findings also have implications for clinical monitoring in patients with multiple sclerosis on <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor modulating agents.

Disruption of vascular barrier integrity markedly increases permeability to fluid and solute and is the central pathophysiologic mechanism of many inflammatory disease processes, including sepsis and acute lung injury (ALI). Dynamic control of the endothelial barrier involves complex signaling to the endothelial cytoskeleton and to adhesion complexes between neighboring cells and between cells and the underlying matrix. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a biologically active lipid generated by hydrolysis of membrane lipids in activated platelets, organizes actin into a strong cortical ring and strengthens both intercellular and cell-matrix adherence. The mechanisms by which S<em>1</em>P increases endothelial barrier integrity remain the focus of intense basic research. The downstream structural changes induced by S<em>1</em>P interact to decrease vascular permeability to fluid and solute, which translates into a reduction lung edema formation in animal models of ALI, thus suggesting a potentially life-saving therapeutic role for vascular barrier modulation in critically ill patients.

The endothelium can evoke relaxations (dilatations) of the underlying vascular smooth muscle, by releasing vasodilator substances. The best characterized endothelium-derived relaxing factor is nitric oxide (NO), which is synthesized by the endothelial isoform of nitric oxide synthase (eNOS). Endothelium-dependent relaxations involve both pertussis-toxin-sensitive G(i) (e.g., responses to serotonin, <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, alpha(2)-adrenergic agonists, and thrombin) and pertussis-toxin-insensitive G(q) (e.g., adenosine di<em>phosphate</em> and bradykinin) coupling proteins. eNOS undergoes a complex pattern of intracellular regulation, including post-translational modifications involving enzyme acylation and phosphorylation. eNOS is reversibly targeted to signal-transducing plasmalemmal caveolae where the enzyme interacts with a number of regulatory proteins, many of which are modified in cardiovascular disease states. The release of nitric oxide by the endothelial cell can be up- (e.g., by estrogens, exercise, and dietary factors) and down-regulated (e.g. oxidative stress, smoking, and oxidized low-density lipoproteins). It is reduced in the course of vascular disease (e.g., diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively lose the pertussis-toxin-sensitive pathway for NO release which favors vasospasm, thrombosis, penetration of macrophages, cellular growth, and the inflammatory reaction leading to atherosclerosis. The unraveling of the complex interaction of the pathways regulating the presence and the activity of eNOS will enhance the understanding of the perturbations in endothelium-dependent signaling that are seen in cardiovascular disease states, and may lead to the identification of novel targets for therapeutic intervention.

Humoral immune responses depend on B cells encountering antigen, interacting with helper T cells, proliferating and differentiating into low-affinity plasma cells or, after organizing into a germinal center (GC), high-affinity plasma cells and memory B cells. Remarkably, each of these events occurs in association with distinct stromal cells in separate subcompartments of the lymphoid tissue. B cells must migrate from niche to niche in a rapid and highly regulated manner to successfully mount a response. The chemokine, CXCL<em>1</em>3, plays a central role in guiding B cells to follicles whereas T-zone chemokines guide activated B cells to the T zone. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) promotes cell egress from the tissue, as well as marginal-zone B-cell positioning in the spleen. Recent studies have identified a role for the orphan receptor, EBV-induced molecule 2 (EBI2; GPR<em>1</em>83), in guiding activated B cells to inter and outer follicular niche(s) and down-regulation of this receptor is essential for organizing cells into GCs. In this review, we discuss current understanding of the roles played by chemokines, S<em>1</em>P and EBI2 in the migration events that underlie humoral immune responses.

Innate lymphoid cells (ILCs) are innate counterparts of adaptive T lymphocytes, contributing to host defense, tissue repair, metabolic homeostasis, and inflammatory diseases. ILCs have been considered to be tissue-resident cells, but whether ILCs move between tissue sites during infection has been unclear. We show here that interleukin-25- or helminth-induced inflammatory ILC2s are circulating cells that arise from resting ILC2s residing in intestinal lamina propria. They migrate to diverse tissues based on <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P)-mediated chemotaxis that promotes lymphatic entry, blood circulation, and accumulation in peripheral sites, including the lung, where they contribute to anti-helminth defense and tissue repair. This ILC2 expansion and migration is a behavioral parallel to the antigen-driven proliferation and migration of adaptive lymphocytes to effector sites and indicates that ILCs complement adaptive immunity by providing both local and distant tissue protection during infection.

Human umbilical vein endothelial cells (HUVEC), like most normal cells, are resistant to tumor necrosis factor-alpha (TNF)-induced apoptosis in spite of TNF activating sphingomyelinase and generating ceramide, a known inducer of apoptosis. Here we report that TNF activates another key enzyme, <em>sphingosine</em> kinase (SphK), in the sphingomyelin metabolic pathway resulting in production of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and that S<em>1</em>P is a potent antagonist of TNF-mediated apoptosis. The TNF-induced SphK activation is independent of sphingomyelinase and ceramidase activities, suggesting that TNF affects this enzyme directly other than through a mass effect on sphingomyelin degradation. In contrast to normal HUVEC, in a spontaneously transformed endothelial cell line (C<em>1</em><em>1</em>) TNF stimulation failed to activate SphK and induced apoptosis as characterized by morphological and biochemical criteria. Addition of exogenous S<em>1</em>P or increasing endogenous S<em>1</em>P by phorbol ester markedly protected C<em>1</em><em>1</em> cell line from TNF-induced apoptosis. Conversely, N, N-dimethyl<em>sphingosine</em>, an inhibitor of SphK, profoundly sensitized normal HUVEC to killing by TNF. Thus, we demonstrate that the activation of SphK by TNF is an important signaling for protection from the apoptotic effect of TNF in endothelial cells.

Autotaxin (ATX) or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is an NPP family member that promotes tumor cell motility, experimental metastasis, and angiogenesis. ATX primarily functions as a lysophospholipase D, generating the lipid mediator lysophosphatidic acid (LPA) from lysophosphatidylcholine. ATX uses a single catalytic site for the hydrolysis of both lipid and non-lipid phosphodiesters, but its regulation is not well understood. Using a new fluorescence resonance energy transfer-based phosphodiesterase sensor that reports ATX activity with high sensitivity, we show here that ATX is potently and specifically inhibited by LPA and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) in a mixed-type manner (Ki approximately <em>1</em>0(-7) M). The homologous ecto-phosphodiesterase NPP<em>1</em>, which lacks lysophospholipase D activity, is insensitive to LPA and S<em>1</em>P. Our results suggest that, by repressing ATX activity, LPA can regulate its own biosynthesis in the extracellular environment, and they reveal a novel role for S<em>1</em>P as an inhibitor of ATX, in addition to its well established role as a receptor ligand.

Lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (Sph<em>1</em>P) production was examined in vitro under conditions that simulated blood clotting. Several approaches were utilized to elucidate the metabolic pathways. <em>1</em>) Platelet phospholipids were labeled using [32P]ortho<em>phosphate</em>, and the production of [32P]Sph<em>1</em>P and LPA was examined. Thrombin stimulation of platelets resulted in rapid secretion of Sph<em>1</em>P stored within the platelet. In contrast, LPA was neither stored within nor secreted from platelets. Nonetheless, extracellular levels of LPA gradually increased following stimulation. 2) Stable-isotope dilution mass spectrometry was used to quantify the molecular species of LPA generated from platelets in vitro. Only <em>1</em>0% of the LPA generated following thrombin stimulation was associated with platelets, the remaining 90% was contained within the extracellular medium. The acyl composition of LPA produced by platelets differed depending on the presence or absence of plasma in the incubation. 3) The fate of exogenously added fluorescent phospholipid analogs was determined. Incubation of [(7-nitro-2-<em>1</em>,3-benzoxadiazol-4-yl)amino]dodecanoyl-(NBD)-labeled phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine with the supernatant fractions from thrombin-stimulated platelets yielded no LPA production. However, these lipids were converted to the corresponding lysolipids by released PLA<em>1</em> and PLA2 activities. When incubated with plasma or serum the NBD-labeled lysophospholipids were readily converted to LPA. Inhibitors of lysophospholipase D and the biological activity of LPA were detected in plasma. These results suggest that the bulk of LPA produced through platelet activation results from the sequential cleavage of phospholipids to lysophospholipids by released phospholipases A<em>1</em> and A2 and then to LPA by plasma lysophospholipase D.

The endothelial-derived G-protein-coupled receptor EDG-<em>1</em> is a high-affinity receptor for the bioactive lipid mediator <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP). In the present study, we constructed the EDG-<em>1</em>-green fluorescent protein (GFP) chimera to examine the dynamics and subcellular localization of SPP-EDG-<em>1</em> interaction. SPP binds to EDG-<em>1</em>-GFP and transduces intracellular signals in a manner indistinguishable from that seen with the wild-type receptor. Human embryonic kidney 293 cells stably transfected with the EDG-<em>1</em>-GFP cDNA expressed the receptor primarily on the plasma membrane. Exogenous SPP treatment, in a dose-dependent manner, induced receptor translocation to perinuclear vesicles with a tau<em>1</em>/2 of approximately <em>1</em>5 min. The EDG-<em>1</em>-GFP-containing vesicles are distinct from mitochondria but colocalize in part with endocytic vesicles and lysosomes. Neither the low-affinity agonist lysophosphatidic acid nor other sphingolipids, ceramide, ceramide-<em>1</em>-<em>phosphate</em>, or sphingosylphosphorylcholine, influenced receptor trafficking. Receptor internalization was completely inhibited by truncation of the C terminus. After SPP washout, EDG-<em>1</em>-GFP recycles back to the plasma membrane with a tau<em>1</em>/2 of approximately 30 min. We conclude that the high-affinity ligand SPP specifically induces the reversible trafficking of EDG-<em>1</em> via the endosomal pathway and that the C-terminal intracellular domain of the receptor is critical for this process.

We have previously shown that glycosaminoglycan (GAG) storage in animal models of the mucopolysaccharidoses (MPS) leads to inflammation and apoptosis within cartilage. We have now extended these findings to synovial tissue and further explored the mechanism underlying GAG-mediated disease. Analysis of MPS rats, cats, and/or dogs revealed that MPS synovial fibroblasts and fluid displayed elevated expression of numerous inflammatory molecules, including several proteins important for lipopolysaccharide signaling (eg, Toll-like receptor 4 and lipoprotein-binding protein). The expression of tumor necrosis factor, in particular, was elevated up to 50-fold, leading to up-regulation of the osteoclast survival factor, receptor activator of nuclear factor-kappaB ligand, and the appearance of multinucleated osteoclast-like cells in the MPS bone marrow. Treatment of normal synovial fibroblasts with GAGs also led to production of the prosurvival lipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, resulting in enhanced cell proliferation, consistent with the hyperplastic synovial tissue observed in MPS patients. In contrast, GAG treatment of normal chondrocytes led to production of the proapoptotic lipid ceramide, confirming the enhanced cell death we had previously observed in MPS cartilage. These findings have important implications for the pathogenesis and treatment of MPS and have further defined the mechanism of GAG-stimulated disease.

Multiple sclerosis (MS) is a chronic autoimmune disorder affecting the central nervous system (CNS) through demyelination and neurodegeneration. Until recently, major therapeutic treatments have relied on agents requiring injection delivery. In September 20<em>1</em>0, fingolimod/FTY720 (Gilenya, Novartis) was approved by the FDA as the first oral treatment for relapsing forms of MS. Fingolimod is a novel compound produced by chemical modification of a fungal precursor. Its active metabolite, formed by in vivo phosphorylation, modulates <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors that are a subset of a larger family of cell-surface, G protein-coupled receptors (GPCRs) mediating the effects of bioactive lipids known as lysophospholipids. Fingolimod's mechanism of action in MS is not completely understood; however, its relevant biology indicates a fundamentally different mechanism compared to all previously approved MS therapies, with evolving research supporting both immunological and nervous system activities. This duality may herald a paradigm shift in the treatment of MS and other neurological disorders.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) regulates diverse cellular functions through extracellular ligation to S<em>1</em>P receptors, and it also functions as an intracellular second messenger. Human pulmonary artery endothelial cells (HPAECs) effectively utilized exogenous S<em>1</em>P to generate intracellular S<em>1</em>P. We, therefore, examined the role of lipid <em>phosphate</em> phosphatase (LPP)-<em>1</em> and <em>sphingosine</em> kinase<em>1</em> (SphK<em>1</em>) in converting exogenous S<em>1</em>P to intracellular S<em>1</em>P. Exposure of (32)P-labeled HPAECs to S<em>1</em>P or <em>sphingosine</em> (Sph) increased the intracellular accumulation of [(32)P]S<em>1</em>P in a dose- and time-dependent manner. The S<em>1</em>P formed in the cells was not released into the medium. The exogenously added S<em>1</em>P did not stimulate the sphingomyelinase pathway; however, added [(3)H]S<em>1</em>P was hydrolyzed to [(3)H]Sph in HPAECs, and this was blocked by XY-<em>1</em>4, an inhibitor of LPPs. HPAECs expressed LPP<em>1</em>-3, and overexpression of LPP-<em>1</em> enhanced the hydrolysis of exogenous [(3)H]S<em>1</em>P to [(3)H]Sph and increased intracellular S<em>1</em>P production by 2-3-fold compared with vector control cells. Down-regulation of LPP-<em>1</em> by siRNA decreased intracellular S<em>1</em>P production from extracellular S<em>1</em>P but had no effect on the phosphorylation of Sph to S<em>1</em>P. Knockdown of SphK<em>1</em>, but not SphK2, by siRNA attenuated the intracellular generation of S<em>1</em>P. Overexpression of wild type SphK<em>1</em>, but not SphK2 wild type, increased the accumulation of intracellular S<em>1</em>P after exposure to extracellular S<em>1</em>P. These studies provide the first direct evidence for a novel pathway of intracellular S<em>1</em>P generation. This involves the conversion of extracellular S<em>1</em>P to Sph by LPP-<em>1</em>, which facilitates Sph uptake, followed by the intracellular conversion of Sph to S<em>1</em>P by SphK<em>1</em>.

FTY720 is a prodrug for FTY-<em>phosphate</em>, an agonist at four of the five known receptors for <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). We show that administration of either FTY720 or FTY-P to SJL mice with established relapsing-remitting experimental autoimmune encephalitis (EAE) results in a rapid and sustained improvement in their clinical status, and a reversal of changes in expression of mRNAs encoding some myelin proteins and inflammatory mediators. EAE produced by adoptively transferring lymph node cells from immunized mice to naïve hosts is similarly ameliorated by FTY-P. Treatment with FTY-P is accompanied by a dose-responsive peripheral lymphopoenia.

Sphingolipids elicit a wide variety of eukaryotic cellular responses, most involving regulation of cell growth, differentiation, and apoptosis. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em>, a sphingolipid catabolite, is mitogenic in fibroblasts and inhibits the chemotactic mobility and invasiveness of human tumor cells. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> degradation requires cleavage at the C2-3 carbon bond by <em>sphingosine</em> <em>phosphate</em> lyase. A yeast genetic approach was used to clone the first <em>sphingosine</em> <em>phosphate</em> lyase gene, BST<em>1</em>. BST<em>1</em> overexpression conferred resistance to <em>sphingosine</em> in yeast. BST<em>1</em> deletion produced sensitivity to exogenous D-erythro-<em>sphingosine</em> and phyto<em>sphingosine</em> and intracellular accumulation of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> upon exposure to exogenous <em>sphingosine</em>. This study confirms that sphingoid base metabolism is similar in all eukaryotes and suggests that yeast genetics may be useful in the isolation and identification of other genes involved in sphingolipid signaling and metabolism.