It has become increasingly difficult to find an area of cell biology in which lipids do not have important, if not key, roles as signalling and regulatory molecules. The rapidly expanding field of bioactive lipids is exemplified by many sphingolipids, such as ceramide, <em>sphingosine</em>, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), ceramide-<em>1</em>-<em>phosphate</em> and lyso-sphingomyelin, which have roles in the regulation of cell growth, death, senescence, adhesion, migration, inflammation, angiogenesis and intracellular trafficking. Deciphering the mechanisms of these varied cell functions necessitates an understanding of the complex pathways of sphingolipid metabolism and the mechanisms that regulate lipid generation and lipid action.

Adaptive immunity depends on T-cell exit from the thymus and T and B cells travelling between secondary lymphoid organs to survey for antigens. After activation in lymphoid organs, T cells must again return to circulation to reach sites of infection; however, the mechanisms regulating lymphoid organ exit are unknown. An immunosuppressant drug, FTY720, inhibits lymphocyte emigration from lymphoid organs, and phosphorylated FTY720 binds and activates four of the five known <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors. However, the role of S<em>1</em>P receptors in normal immune cell trafficking is unclear. Here we show that in mice whose haematopoietic cells lack a single S<em>1</em>P receptor (S<em>1</em>P<em>1</em>; also known as Edg<em>1</em>) there are no T cells in the periphery because mature T cells are unable to exit the thymus. Although B cells are present in peripheral lymphoid organs, they are severely deficient in blood and lymph. Adoptive cell transfer experiments establish an intrinsic requirement for S<em>1</em>P<em>1</em> in T and B cells for lymphoid organ egress. Furthermore, S<em>1</em>P<em>1</em>-dependent chemotactic responsiveness is strongly upregulated in T-cell development before exit from the thymus, whereas S<em>1</em>P<em>1</em> is downregulated during peripheral lymphocyte activation, and this is associated with retention in lymphoid organs. We find that FTY720 treatment downregulates S<em>1</em>P<em>1</em>, creating a temporary pharmacological S<em>1</em>P<em>1</em>-null state in lymphocytes, providing an explanation for the mechanism of FTY720-induced lymphocyte sequestration. These findings establish that S<em>1</em>P<em>1</em> is essential for lymphocyte recirculation and that it regulates egress from both thymus and peripheral lymphoid organs.

BACKGROUND

Oral fingolimod, a <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-receptor modulator that prevents the egress of lymphocytes from lymph nodes, significantly improved relapse rates and end points measured on magnetic resonance imaging (MRI), as compared with either placebo or intramuscular interferon beta-<em>1</em>a, in phase 2 and 3 studies of multiple sclerosis.

METHODS

In our 24-month, double-blind, randomized study, we enrolled patients who had relapsing-remitting multiple sclerosis, were <em>1</em>8 to 55 years of age, had a score of 0 to 5.5 on the Expanded Disability Status Scale (which ranges from 0 to <em>1</em>0, with higher scores indicating greater disability), and had had one or more relapses in the previous year or two or more in the previous 2 years. Patients received oral fingolimod at a dose of 0.5 mg or <em>1</em>.25 mg daily or placebo. End points included the annualized relapse rate (the primary end point) and the time to disability progression (a secondary end point).

RESULTS

A total of <em>1</em>033 of the <em>1</em>272 patients (8<em>1</em>.2%) completed the study. The annualized relapse rate was 0.<em>1</em>8 with 0.5 mg of fingolimod, 0.<em>1</em>6 with <em>1</em>.25 mg of fingolimod, and 0.40 with placebo (P<0.00<em>1</em> for either dose vs. placebo). Fingolimod at doses of 0.5 mg and <em>1</em>.25 mg significantly reduced the risk of disability progression over the 24-month period (hazard ratio, 0.70 and 0.68, respectively; P=0.02 vs. placebo, for both comparisons). The cumulative probability of disability progression (confirmed after 3 months) was <em>1</em>7.7% with 0.5 mg of fingolimod, <em>1</em>6.6% with <em>1</em>.25 mg of fingolimod, and 24.<em>1</em>% with placebo. Both fingolimod doses were superior to placebo with regard to MRI-related measures (number of new or enlarged lesions on T(2)-weighted images, gadolinium-enhancing lesions, and brain-volume loss; P<0.00<em>1</em> for all comparisons at 24 months). Causes of study discontinuation and adverse events related to fingolimod included bradycardia and atrioventricular conduction block at the time of fingolimod initiation, macular edema, elevated liver-enzyme levels, and mild hypertension.

CONCLUSIONS

As compared with placebo, both doses of oral fingolimod improved the relapse rate, the risk of disability progression, and end points on MRI. These benefits will need to be weighed against possible long-term risks. (ClinicalTrials.gov number, NCT00289978.)

The evolutionarily conserved actions of the sphingolipid metabolite, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), in yeast, plants and mammals have shown that it has important functions. In higher eukaryotes, S<em>1</em>P is the ligand for a family of five G-protein-coupled receptors. These S<em>1</em>P receptors are differentially expressed, coupled to various G proteins, and regulate angiogenesis, vascular maturation, cardiac development and immunity, and are important for directed cell movement.

BACKGROUND

Fingolimod (FTY720), a <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-receptor modulator that prevents lymphocyte egress from lymph nodes, showed clinical efficacy and improvement on imaging in a phase 2 study involving patients with multiple sclerosis.

METHODS

In this <em>1</em>2-month, double-blind, double-dummy study, we randomly assigned <em>1</em>292 patients with relapsing-remitting multiple sclerosis who had a recent history of at least one relapse to receive either oral fingolimod at a daily dose of either <em>1</em>.25 or 0.5 mg or intramuscular interferon beta-<em>1</em>a (an established therapy for multiple sclerosis) at a weekly dose of 30 microg. The primary end point was the annualized relapse rate. Key secondary end points were the number of new or enlarged lesions on T(2)-weighted magnetic resonance imaging (MRI) scans at <em>1</em>2 months and progression of disability that was sustained for at least 3 months.

RESULTS

A total of <em>1</em><em>1</em>53 patients (89%) completed the study. The annualized relapse rate was significantly lower in both groups receiving fingolimod--0.20 (95% confidence interval [CI], 0.<em>1</em>6 to 0.26) in the <em>1</em>.25-mg group and 0.<em>1</em>6 (95% CI, 0.<em>1</em>2 to 0.2<em>1</em>) in the 0.5-mg group--than in the interferon group (0.33; 95% CI, 0.26 to 0.42; P<0.00<em>1</em> for both comparisons). MRI findings supported the primary results. No significant differences were seen among the study groups with respect to progression of disability. Two fatal infections occurred in the group that received the <em>1</em>.25-mg dose of fingolimod: disseminated primary varicella zoster and herpes simplex encephalitis. Other adverse events among patients receiving fingolimod were nonfatal herpesvirus infections, bradycardia and atrioventricular block, hypertension, macular edema, skin cancer, and elevated liver-enzyme levels.

CONCLUSIONS

This trial showed the superior efficacy of oral fingolimod with respect to relapse rates and MRI outcomes in patients with multiple sclerosis, as compared with intramuscular interferon beta-<em>1</em>a. Longer studies are needed to assess the safety and efficacy of treatment beyond <em>1</em> year. (ClinicalTrials.gov number, NCT00340834.)

Blood lymphocyte numbers, essential for the development of efficient immune responses, are maintained by recirculation through secondary lymphoid organs. We show that lymphocyte trafficking is altered by the lysophospholipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and by a phosphoryl metabolite of the immunosuppressive agent FTY720. Both species were high-affinity agonists of at least four of the five S<em>1</em>P receptors. These agonists produce lymphopenia in blood and thoracic duct lymph by sequestration of lymphocytes in lymph nodes, but not spleen. S<em>1</em>P receptor agonists induced emptying of lymphoid sinuses by retention of lymphocytes on the abluminal side of sinus-lining endothelium and inhibition of egress into lymph. Inhibition of lymphocyte recirculation by activation of S<em>1</em>P receptors may result in therapeutically useful immunosuppression.

Immunosuppressant drugs such as cyclosporin have allowed widespread organ transplantation, but their utility remains limited by toxicities, and they are ineffective in chronic management of autoimmune diseases such as multiple sclerosis. In contrast, the immune modulating drug FTY720 is efficacious in a variety of transplant and autoimmune models without inducing a generalized immunosuppressed state and is effective in human kidney transplantation. FTY720 elicits a lymphopenia resulting from a reversible redistribution of lymphocytes from circulation to secondary lymphoid tissues by unknown mechanisms. Using FTY720 and several analogs, we show now that FTY720 is phosphorylated by <em>sphingosine</em> kinase; the phosphorylated compound is a potent agonist at four <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptors and represents the therapeutic principle in a rodent model of multiple sclerosis. Our results suggest that FTY720, after phosphorylation, acts through <em>sphingosine</em> <em>1</em>-<em>phosphate</em> signaling pathways to modulate chemotactic responses and lymphocyte trafficking.

Naive lymphocytes continually enter and exit lymphoid organs in a recirculation process that is essential for immune surveillance. During immune responses, the egress process can be shut down transiently. When this occurs locally it increases lymphocyte numbers in the responding lymphoid organ; when it occurs systemically it can lead to immunosuppression as a result of the depletion of recirculating lymphocytes. Several mediators of the innate immune system are known to cause shutdown, including interferon alpha/beta (IFN-alpha/beta) and tumour necrosis factor, but the mechanism has been unclear. Here we show that treatment with the IFN-alpha/beta inducer polyinosine polycytidylic acid (hereafter 'poly(I:C)') inhibited egress by a mechanism that was partly lymphocyte-intrinsic. The transmembrane C-type lectin CD69 was rapidly induced and CD69-/- cells were poorly retained in lymphoid tissues after treatment with poly(I:C) or infection with lymphocytic choriomeningitis virus. Lymphocyte egress requires <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor-<em>1</em> (S<em>1</em>P<em>1</em>), and IFN-alpha/beta was found to inhibit lymphocyte responsiveness to S<em>1</em>P. By contrast, CD69-/- cells retained S<em>1</em>P<em>1</em> function after exposure to IFN-alpha/beta. In coexpression experiments, CD69 inhibited S<em>1</em>P<em>1</em> chemotactic function and led to downmodulation of S<em>1</em>P<em>1</em>. In a reporter assay, S<em>1</em>P<em>1</em> crosslinking led to co-crosslinking and activation of a CD69-CD3zeta chimaera. CD69 co-immunoprecipitated with S<em>1</em>P<em>1</em> but not the related receptor, S<em>1</em>P3. These observations indicate that CD69 forms a complex with and negatively regulates S<em>1</em>P<em>1</em> and that it functions downstream of IFN-alpha/beta, and possibly other activating stimuli, to promote lymphocyte retention in lymphoid organs.

The discovery of fingolimod (FTY720/Gilenya; Novartis), an orally active immunomodulatory drug, has opened up new approaches to the treatment of multiple sclerosis, the most common inflammatory disorder of the central nervous system. Elucidation of the effects of fingolimod--mediated by the modulation of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors--has indicated that its therapeutic activity could be due to regulation of the migration of selected lymphocyte subsets into the central nervous system and direct effects on neural cells, particularly astrocytes. An improved understanding of the biology of S<em>1</em>P receptors has also been gained. This article describes the discovery and development of fingolimod, which was approved by the US Food and Drug Administration in September 20<em>1</em>0 as a first-line treatment for relapsing forms of multiple sclerosis, thereby becoming the first oral disease-modifying therapy to be approved for multiple sclerosis in the United States.

Ceramide is an important regulatory participant of programmed cell death (apoptosis) induced by tumour-necrosis factor (TNF)-alpha and Fas ligand, members of the TNF superfamily. Conversely, <em>sphingosine</em> and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, which are metabolites of ceramide, induce mitogenesis and have been implicated as second messengers in cellular proliferation induced by platelet-derived growth factor and serum. Here we report that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> prevents the appearance of the key features of apoptosis, namely intranucleosomal DNA fragmentation and morphological changes, which result from increased concentrations of ceramide. Furthermore, inhibition of ceramide-mediated apoptosis by activation of protein kinase C results from stimulation of <em>sphingosine</em> kinase and the concomitant increase in intracellular <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. Finally <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> not only stimulates the extracellular signal-regulated kinase (ERK) pathway, it counteracts the ceramide-induced activation of stress-activated protein kinase (SAPK/JNK). Thus, the balance between the intracellular levels of ceramide and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> and their regulatory effects on different family members of mitogen-activated protein kinases determines the fate of the cell.

Sphingolipid signaling pathways have been implicated in many critical cellular events. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP), a sphingolipid metabolite found in high concentrations in platelets and blood, stimulates members of the endothelial differentiation gene (Edg) family of G protein-coupled receptors and triggers diverse effects, including cell growth, survival, migration, and morphogenesis. To determine the in vivo functions of the SPP/Edg signaling pathway, we disrupted the Edg<em>1</em> gene in mice. Edg<em>1</em>(-/-) mice exhibited embryonic hemorrhage leading to intrauterine death between E<em>1</em>2.5 and E<em>1</em>4.5. Vasculogenesis and angiogenesis appeared normal in the mutant embryos. However, vascular maturation was incomplete due to a deficiency of vascular smooth muscle cells/pericytes. We also show that Edg-<em>1</em> mediates an SPP-induced migration response that is defective in mutant cells due to an inability to activate the small GTPase, Rac. Our data reveal Edg-<em>1</em> to be the first G protein-coupled receptor required for blood vessel formation and show that sphingolipid signaling is essential during mammalian development.

Lymphocytes require <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor-<em>1</em> to exit lymphoid organs, but the source(s) of extracellular S<em>1</em>P and whether S<em>1</em>P directly promotes egress are unknown. By using mice in which the two kinases that generate S<em>1</em>P were conditionally ablated, we find that plasma S<em>1</em>P is mainly hematopoietic in origin, with erythrocytes a major contributor, whereas lymph S<em>1</em>P is from a distinct radiation-resistant source. Lymphocyte egress from thymus and secondary lymphoid organs was markedly reduced in kinase-deficient mice. Restoration of S<em>1</em>P to plasma rescued egress to blood but not lymph, and the rescue required lymphocyte expression of S<em>1</em>P-receptor-<em>1</em>. Thus, separate sources provide S<em>1</em>P to plasma and lymph to help lymphocytes exit the low-S<em>1</em>P environment of lymphoid organs. Disruption of compartmentalized S<em>1</em>P signaling is a plausible mechanism by which S<em>1</em>P-receptor-<em>1</em> agonists function as immunosuppressives.

Substances released by platelets during blood clotting are essential participants in events that link hemostasis and angiogenesis and ensure adequate wound healing and tissue injury repair. We assessed the participation of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (Sph-<em>1</em>-P), a biologically active phosphorylated lipid growth factor released from activated platelets, in the regulation of endothelial monolayer barrier integrity, which is key to both angiogenesis and vascular homeostasis. Sph-<em>1</em>-P produced rapid, sustained, and dose-dependent increases in transmonolayer electrical resistance (TER) across both human and bovine pulmonary artery and lung microvascular endothelial cells. This substance also reversed barrier dysfunction elicited by the edemagenic agent thrombin. Sph-<em>1</em>-P-mediated barrier enhancement was dependent upon G(ialpha)-receptor coupling to specific members of the endothelial differentiation gene (Edg) family of receptors (Edg-<em>1</em> and Edg-3), Rho kinase and tyrosine kinase-dependent activation, and actin filament rearrangement. Sph-<em>1</em>-P-enhanced TER occurred in conjunction with Rac GTPase- and p2<em>1</em>-associated kinase-dependent endothelial cortical actin assembly with recruitment of the actin filament regulatory protein, cofilin. Platelet-released Sph-<em>1</em>-P, linked to Rac- and Rho-dependent cytoskeletal rearrangement, may act late in angiogenesis to stabilize newly formed vessels, which often display abnormally increased vascular permeability.

The adipocyte-derived secretory factor adiponectin promotes insulin sensitivity, decreases inflammation and promotes cell survival. No unifying mechanism has yet explained how adiponectin can exert such a variety of beneficial systemic effects. Here, we show that adiponectin potently stimulates a ceramidase activity associated with its two receptors, AdipoR<em>1</em> and AdipoR2, and enhances ceramide catabolism and formation of its antiapoptotic metabolite--<em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P)--independently of AMP-dependent kinase (AMPK). Using models of inducible apoptosis in pancreatic beta cells and cardiomyocytes, we show that transgenic overproduction of adiponectin decreases caspase-8-mediated death, whereas genetic ablation of adiponectin enhances apoptosis in vivo through a sphingolipid-mediated pathway. Ceramidase activity is impaired in cells lacking both adiponectin receptor isoforms, leading to elevated ceramide levels and enhanced susceptibility to palmitate-induced cell death. Combined, our observations suggest a unifying mechanism of action for the beneficial systemic effects exerted by adiponectin, with sphingolipid metabolism as its core upstream signaling component.

Lymphocyte egress from the thymus and from peripheral lymphoid organs depends on <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor-<em>1</em> and is thought to occur in response to circulatory S<em>1</em>P. However, the existence of an S<em>1</em>P gradient between lymphoid organs and blood or lymph has not been established. To further define egress requirements, we addressed why treatment with the food colorant 2-acetyl-4-tetrahydroxybutylimidazole (THI) induces lymphopenia. We found that S<em>1</em>P abundance in lymphoid tissues of mice is normally low but increases more than <em>1</em>00-fold after THI treatment and that this treatment inhibits the S<em>1</em>P-degrading enzyme S<em>1</em>P lyase. We conclude that lymphocyte egress is mediated by S<em>1</em>P gradients that are established by S<em>1</em>P lyase activity and that the lyase may represent a novel immunosuppressant drug target.

Constitutive egress of bone marrow (BM)-resident hematopoietic stem and progenitor cells (HSPCs) into the blood is a well-established phenomenon, but the ultimate fate and functional relevance of circulating HSPCs is largely unknown. We show that mouse thoracic duct (TD) lymph contains HSPCs that possess short- and long-term multilineage reconstitution capacity. TD-derived HSPCs originate in the BM, enter the blood, and traffic to multiple peripheral organs, where they reside for at least 36 hr before entering draining lymphatics to return to the blood and, eventually, the BM. HSPC egress from extramedullary tissues into lymph depends on <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptors. Migratory HSPCs proliferate within extramedullary tissues and give rise to tissue-resident myeloid cells, preferentially dendritic cells. HSPC differentiation is amplified upon exposure to Toll-like receptor agonists. Thus, HSPCs can survey peripheral organs and can foster the local production of tissue-resident innate immune cells under both steady-state conditions and in response to inflammatory signals.

There is substantial evidence that <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is involved in cancer. S<em>1</em>P regulates processes such as inflammation, which can drive tumorigenesis; neovascularization, which provides cancer cells with nutrients and oxygen; and cell growth and survival. This occurs at multiple levels and involves S<em>1</em>P receptors, <em>sphingosine</em> kinases, S<em>1</em>P phosphatases and S<em>1</em>P lyase. This Review summarizes current research findings and examines the potential for new therapeutics designed to alter S<em>1</em>P signalling and function in cancer.

Vascular endothelial cells undergo morphogenesis into capillary networks in response to angiogenic factors. We show here that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP), a platelet-derived bioactive lipid, activates the EDG-<em>1</em> and -3 subtypes of G protein-coupled receptors on endothelial cells to regulate angiogenesis. SPP induces the Gi/mitogen-activated protein kinase/cell survival pathway and the small GTPase Rho- and Raccoupled adherens junction assembly. Both EDG-<em>1</em>-and EDG-3-regulated signaling pathways are required for endothelial cell morphogenesis into capillary-like networks. Indeed, SPP synergized with polypeptide angiogenic growth factors in the formation of mature neovessels in vivo. These data define SPP as a novel regulator of angiogenesis.

The bioactive sphingolipid metabolite <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is now recognized as a critical regulator of many physiological and pathophysiological processes, including cancer, atherosclerosis, diabetes and osteoporosis. S<em>1</em>P is produced in cells by two <em>sphingosine</em> kinase isoenzymes, SphK<em>1</em> and SphK2. Many cells secrete S<em>1</em>P, which can then act in an autocrine or paracrine manner. Most of the known actions of S<em>1</em>P are mediated by a family of five specific G protein-coupled receptors. More recently, it was shown that S<em>1</em>P also has important intracellular targets involved in inflammation, cancer and Alzheimer's disease. This suggests that S<em>1</em>P actions are much more complex than previously thought, with important ramifications for development of therapeutics. This review highlights recent advances in our understanding of the mechanisms of action of S<em>1</em>P and its roles in disease.

Secondary lymphoid organs serve as hubs for the adaptive immune system, bringing together antigen, antigen-presenting cells, and lymphocytes. Two families of G protein-coupled receptors play essential roles in lymphocyte migration through these organs: chemokine receptors and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors. Chemokines expressed by lymphoid stromal cells guide lymphocyte and dendritic cell movements during antigen surveillance and the initiation of adaptive immune responses. S<em>1</em>P receptor-<em>1</em> is required for lymphocyte egress from thymus and secondary lymphoid organs and is downregulated by the immunosuppressive drug FTY720. Here, we review the steps associated with the initiation of adaptive immune responses in secondary lymphoid organs, highlighting the roles of chemokines and S<em>1</em>P.

Tumour-necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is a key component in NF-kappaB signalling triggered by TNF-alpha. Genetic evidence indicates that TRAF2 is necessary for the polyubiquitination of receptor interacting protein <em>1</em> (RIP<em>1</em>) that then serves as a platform for recruitment and stimulation of IkappaB kinase, leading to activation of the transcription factor NF-kappaB. Although TRAF2 is a RING domain ubiquitin ligase, direct evidence that TRAF2 catalyses the ubiquitination of RIP<em>1</em> is lacking. TRAF2 binds to <em>sphingosine</em> kinase <em>1</em> (SphK<em>1</em>), one of the isoenzymes that generates the pro-survival lipid mediator <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) inside cells. Here we show that SphK<em>1</em> and the production of S<em>1</em>P is necessary for lysine-63-linked polyubiquitination of RIP<em>1</em>, phosphorylation of IkappaB kinase and IkappaBalpha, and IkappaBalpha degradation, leading to NF-kappaB activation. These responses were mediated by intracellular S<em>1</em>P independently of its cell surface G-protein-coupled receptors. S<em>1</em>P specifically binds to TRAF2 at the amino-terminal RING domain and stimulates its E3 ligase activity. S<em>1</em>P, but not dihydro-S<em>1</em>P, markedly increased recombinant TRAF2-catalysed lysine-63-linked, but not lysine-48-linked, polyubiquitination of RIP<em>1</em> in vitro in the presence of the ubiquitin conjugating enzymes (E2) UbcH<em>1</em>3 or UbcH5a. Our data show that TRAF2 is a novel intracellular target of S<em>1</em>P, and that S<em>1</em>P is the missing cofactor for TRAF2 E3 ubiquitin ligase activity, indicating a new paradigm for the regulation of lysine-63-linked polyubiquitination. These results also highlight the key role of SphK<em>1</em> and its product S<em>1</em>P in TNF-alpha signalling and the canonical NF-kappaB activation pathway important in inflammatory, antiapoptotic and immune processes.

Mural cells are essential components of blood vessels and are necessary for normal development, homeostasis, and organ function. Alterations in mural cell density or the stable attachment of mural cells to the endothelium is associated with several human diseases such as diabetic retinopathy, venous malformation, and hereditary stroke. In addition mural cells are implicated in regulating tumor growth and have thus been suggested as potential antiangiogenic targets in tumor therapy. In recent years our knowledge of mural cell function and endothelial-mural cell signaling has increased dramatically, and we now begin to understand the mechanistic basis of the key signaling pathways involved. This is mainly thanks to sophisticated in vivo experiments using a broad repertoire of genetic technologies. In this review, we summarize the five currently best understood signaling pathways implicated in mural cell biology. We discuss PDGFB/PDGFRbeta- dependent pericyte recruitment, as well as the role of angiopoietins and Tie receptors in vascular maturation. In addition, we highlight the effects of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> signaling on adherens junction assembly and vascular stability, as well as the role of TGF-beta-signaling in mural cell differentiation. We further reflect recent data suggesting an important function for Notch3 signaling in mural cell maturation.

Much has been learned about how cells enter lymphoid tissues. But how do they leave? <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) has emerged over the past decade as a central mediator of lymphocyte egress. In this review, we summarize the current understanding of how S<em>1</em>P promotes exit from the secondary lymphoid organs and thymus. We review what is known about additional requirements for emigration and summarize the mostly distinct requirements for exit from the bone marrow. Egress from lymphoid organs is limited during immune responses, and we examine how this regulation works. There is accumulating evidence for roles of S<em>1</em>P in directing immune cell behavior within lymphoid tissues. How such actions can fit together with the egress-promoting role of S<em>1</em>P is discussed. Finally, we examine current understanding of how FTY720, a drug that targets S<em>1</em>P receptors and is approved for the treatment of multiple sclerosis, causes immune suppression.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), an important sphingolipid metabolite, regulates diverse cellular processes, including cell survival, growth, and differentiation. Here we show that S<em>1</em>P signaling is critical for neural and vascular development. <em>Sphingosine</em> kinase-null mice exhibited a deficiency of S<em>1</em>P which severely disturbed neurogenesis, including neural tube closure, and angiogenesis and caused embryonic lethality. A dramatic increase in apoptosis and a decrease in mitosis were seen in the developing nervous system. S<em>1</em>P(<em>1</em>) receptor-null mice also showed severe defects in neurogenesis, indicating that the mechanism by which S<em>1</em>P promotes neurogenesis is, in part, signaling from the S<em>1</em>P(<em>1</em>) receptor. Thus, S<em>1</em>P joins a growing list of signaling molecules, such as vascular endothelial growth factor, which regulate the functionally intertwined pathways of angiogenesis and neurogenesis. Our findings also suggest that exploitation of this potent neuronal survival pathway could lead to the development of novel therapeutic approaches for neurological diseases.