Addition of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> induces proliferation of quiescent Swiss 3T3 fibroblasts by unknown mechanisms. To identify the pathways involved, the ability of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> to activate mitogen-activated protein (MAP) kinase was studied. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> rapidly activated the Raf/MAP kinase kinase (MKK)/MAP kinase pathway, and the concentration dependence for MAP kinase activation correlated with that for induction of DNA synthesis. Both MKK<em>1</em> and MKK2 were activated by <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, assessed by specific immune complex kinase assays. Prior treatment of the Swiss 3T3 cells with pertussis toxin inhibited 70-80% of the <em>sphingosine</em> <em>1</em>-<em>phosphate</em>-stimulated MAP kinase activity. Thus, one of the direct or indirect targets of exogenous <em>sphingosine</em> <em>1</em>-<em>phosphate</em> appears to be a G(i)/G(o) protein.

B lymphocyte egress from secondary lymphoid organs requires <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and S<em>1</em>P receptor-<em>1</em> (S<em>1</em>P<em>1</em>). However, whether S<em>1</em>P contributes to immature-B cell egress from the bone marrow (BM) has not been fully assessed. Here we report that in S<em>1</em>P- and S<em>1</em>P<em>1</em>-conditionally deficient mice, the number of immature-B cells in the BM parenchyma increased, while it decreased in the blood. Moreover, a slower rate of bromodeoxyuridine incorporation suggested immature-B cells spent longer in the BM of mice in which S<em>1</em>P<em>1</em>-S<em>1</em>P signaling was genetically or pharmacologically impaired. Transgenic expression of S<em>1</em>P<em>1</em> in developing B cells was sufficient to mobilize pro- and pre-B cells from the BM. We conclude that the S<em>1</em>P<em>1</em>-S<em>1</em>P pathway contributes to egress of immature-B cells from BM, and that this mechanism is partially redundant with other undefined pathways.

There is growing evidence that <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) plays an important role in regulating the development, morphology, and function of the cardiovascular system. There is little data, however, regarding the relative contribution of endogenous S<em>1</em>P and its cognate receptors (referred to as S<em>1</em>P(<em>1</em>-5)) to cardiovascular homeostasis. We used S<em>1</em>P(2) receptor knockout mice (S<em>1</em>P(2)(-/-)) to evaluate the role of S<em>1</em>P(2) in heart and vascular function. There were no significant differences in blood pressure between wild-type and S<em>1</em>P(2)(-/-) mice, measured in awake mice. Cardiac function, evaluated in situ by using a Millar catheter, was also not different in S<em>1</em>P(2)(-/-) mice under baseline or stimulated conditions. In vivo analysis of vascular function by flowmetry revealed decreases in mesenteric and renal resistance in S<em>1</em>P(2)(-/-) mice, especially during vasoconstriction with phenylephrine. In intact aortic rings, the concentration-force relations for both KCl and phenylephrine were right shifted in S<em>1</em>P(2)(-/-) mice, whereas the maximal isometric forces were not different. By contrast, in deendothelialized rings the concentration-force relations were not different but the maximal force was significantly greater in S<em>1</em>P(2)(-/-) aorta. Histologically, there were no apparent differences in vascular morphology. These data suggest that the S<em>1</em>P(2) receptor plays an important role in the function of the vasculature and is an important mediator of normal hemodynamics. This is mediated, at least in part, through an effect on the endothelium, but direct effects on vascular smooth muscle cannot be ruled out and require further investigation.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is now recognized as a lipid mediator that acts via G-protein-coupled receptors. S<em>1</em>P receptors couple to various heterotrimeric G-proteins and regulate downstream targets and ultimately cell behaviour. The prototypical S<em>1</em>P<em>1</em> receptor is known to couple to Gi and regulates angiogenesis, vascular development, and immune cell trafficking. In this review, we focus our attention on the S<em>1</em>P2 receptor, which has a unique G-protein-coupling property in that it preferentially activates the G(<em>1</em>2/<em>1</em>3) pathway. Recent studies indicate that the S<em>1</em>P2 receptor regulates critical intracellular signalling pathways, such as Rho GTPase, the phosphatase PTEN, and VE-cadherin-based adherens junctions. Analysis of mutant mice has revealed the critical role of this receptor in inner ear physiology, heart and vascular development, vascular remodelling, and vascular tone, permeability, and angiogenesis in vertebrates. These studies suggest that selective modulation of S<em>1</em>P2 receptor function by pharmacological tools may be useful in a variety of pathological conditions.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a phosphorylated product of <em>sphingosine</em>, the core structure of the class of lipids termed sphingolipids. S<em>1</em>P is a naturally occurring lipid metabolite, and usually is present at a concentration of a few <em>1</em>00 nanomolar in human sera. S<em>1</em>P has been found to exert a diverse set of physiological and pathophysiological responses in mammalian tissues through the activation of heterotrimeric G-proteins that in turn modulate the activity of various downstream effecter molecules. In blood vessels, vascular endothelial cells and smooth muscle cells express specific receptors for S<em>1</em>P that modulate vascular tone. This article will provide a brief overview of S<em>1</em>P metabolism in the vasculature and will discuss some of the pathways whereby S<em>1</em>P regulates intracellular signal transduction pathways in endothelial and smooth muscle cells, leading to the activation of both vasorelaxation and vasoconstriction responses.

The clinical efficacy of opiates for pain control is severely limited by analgesic tolerance and hyperalgesia. Herein we show that chronic morphine upregulates both the sphingolipid ceramide in spinal astrocytes and microglia, but not neurons, and spinal <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), the end-product of ceramide metabolism. Coadministering morphine with intrathecal administration of pharmacological inhibitors of ceramide and S<em>1</em>P blocked formation of spinal S<em>1</em>P and development of hyperalgesia and tolerance in rats. Our results show that spinally formed S<em>1</em>P signals at least in part by (<em>1</em>) modulating glial function because inhibiting S<em>1</em>P formation blocked increased formation of glial-related proinflammatory cytokines, in particular tumor necrosis factor-α, interleukin-<em>1</em>βα, and interleukin-6, which are known modulators of neuronal excitability, and (2) peroxynitrite-mediated posttranslational nitration and inactivation of glial-related enzymes (glutamine synthetase and the glutamate transporter) known to play critical roles in glutamate neurotransmission. Inhibitors of the ceramide metabolic pathway may have therapeutic potential as adjuncts to opiates in relieving suffering from chronic pain.

The plasma lysophospholipid mediator <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is produced exclusively by <em>sphingosine</em> kinase (SPHK) <em>1</em> and SPHK2 in vivo, and plays diverse biological and pathophysiological roles by acting largely through three members of the G protein-coupled S<em>1</em>P receptors, S<em>1</em>P<em>1</em>, S<em>1</em>P2 and S<em>1</em>P3. S<em>1</em>P<em>1</em> expressed on endothelial cells mediates embryonic vascular maturation and maintains vascular integrity by contributing to eNOS activation, inhibiting vascular permeability and inducing endothelial cell chemotaxis via Gi-coupled mechanisms. By contrast, S<em>1</em>P2, is expressed in high levels on vascular smooth muscle cells (VSMCs) and certain types of tumor cells, inhibiting Rac and cell migration via a G(<em>1</em>2/<em>1</em>3)-and Rho-dependent mechanism. In rat neointimal VSMCs, S<em>1</em>P<em>1</em> is upregulated to mediate local production of platelet-derived growth factor, which is a key player in vascular remodeling. S<em>1</em>P3 expressed on endothelial cells also mediates chemotaxis toward S<em>1</em>P and vasorelaxation via NO production in certain vascular bed, playing protective roles for vascular integrity. S<em>1</em>P3 expressed on VSMCs and cardiac sinoatrial node cells mediates vasopressor and negative chronotropic effect, respectively. In addition, S<em>1</em>P3, together with S<em>1</em>P2 and SPHK<em>1</em>, is suggested to play a protective role against acute myocardial ischemia. However, our recent work indicates that overexpressed SPHK<em>1</em> is involved in cardiomyocyte degeneration and fibrosis in vivo, in part through S<em>1</em>P activation of the S<em>1</em>P3 signaling. We also demonstrated that exogenously administered S<em>1</em>P accelerates neovascularization and blood flow recovery in ischemic limbs, suggesting its usefulness for angiogenic therapy. These results provide evidence for S<em>1</em>P receptor subtype-specific pharmacological intervention as a novel therapeutic approach to cardiovascular diseases and cancer.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lipid generated during vascular injury that regulates cell growth, differentiation, survival, and motility via endothelial differentiation gene (EDG) family G protein-coupled receptors. Although several G protein-coupled receptor ligands transactivate receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), S<em>1</em>P-stimulated receptor tyrosine kinase transactivation has not been well studied. We show that platelet-derived growth factor beta receptor (PDGFbetaR) and EGFR are tyrosine phosphorylated in response to S<em>1</em>P in rat aortic vascular smooth muscle cells (VSMCs). S<em>1</em>P-stimulated transactivation of PDGFbetaR and EGFR was mediated via Gi-coupled EDG receptors. S<em>1</em>P-stimulated transactivation of EGFR and PDGFbetaR was dependent on Src, reactive oxygen species, and cholesterol-rich membranes. A phosphoinositide 3-kinase-Akt pathway was activated by S<em>1</em>P and blocked by AG<em>1</em>296 and AG<em>1</em>478. Activation of extracellular signal-regulated kinase (ERK) <em>1</em> and ERK2 pathway by S<em>1</em>P was blocked only by AG<em>1</em>478. In Chinese hamster ovary cells that expressed exogenous EDG-<em>1</em>, activation of Akt and ERK<em>1</em>/2 in response to S<em>1</em>P was observed and was enhanced by coexpression of PDGFbetaR or EGFR. S<em>1</em>P-mediated VSMC proliferation was shown to be secondary to transactivation, because it was suppressed by AG<em>1</em>296 and AG<em>1</em>478. These data establish S<em>1</em>P as an important stimulus for EGFR and PDGFbetaR activation in VSMCs that may have important implications for the vessel response to injury.

Memory T cells specific for donor Ags present a unique challenge in transplantation. In addition to expressing robust immune responses to a transplanted organ, memory T cells may be resistant to the effects of immunosuppressive therapies used to prolong graft survival. In this study, we explore the possibility of controlling deleterious donor-reactive memory CD4 T cells through lymphoid sequestration. We showed that <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor-<em>1</em> agonist FTY720 induces relocation of circulating memory CD4 T cells into secondary lymphoid organs. Lymphoid sequestration of these donor-reactive memory CD4 T cells prolonged survival of murine heterotopic cardiac allografts and synergizes with conventional costimulatory blockade to further increase graft survival. Despite limited trafficking, memory CD4 T cells remain capable of providing help for the induction of anti-donor CD8 T cell and alloantibody responses. Elimination of antidonor humoral immunity resulted in indefinite allograft survival proving the pathogenicity of alloantibody under these conditions. Overall, this is the first demonstration that FTY720 influences memory CD4 T cell trafficking and attenuates their contribution to allograft rejection. The data have important implications for guiding FTY720 therapy and for designing combinatorial strategies aimed at prolonging allograft survival in sensitized transplant patients with donor-specific memory T cells.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is released by immune cells and is thought to play a key role in chemotaxis and the onset of the inflammatory response. The question remains whether this lipid mediator also contributes to the enhanced sensitivity of nociceptive neurons that is associated with inflammation. Therefore we examined whether S<em>1</em>P alters the excitability of small diameter, capsaicin-sensitive sensory neurons by measuring action potential (AP) firing and two of the membrane currents critical in regulating the properties of the AP. External application of S<em>1</em>P augments the number of APs evoked by a depolarizing current ramp. The enhanced firing is associated with a decrease in the rheobase and an increase in the resistance at firing threshold although neither the firing threshold nor the resting membrane potential are changed. Treatment with S<em>1</em>P enhanced the tetrodotoxin-resistant sodium current and decreased the total outward potassium current (IK). When sensory neurons were internally perfused with GDP-beta-S, a blocker of G protein activation, the S<em>1</em>P-induced increase in APs was completely blocked and suggests the excitatory actions of S<em>1</em>P are mediated through G-protein-coupled receptors called endothelial differentiation gene or S<em>1</em>PR. In contrast, internal perfusion with GDP-beta-S and S<em>1</em>P increased the number of APs evoked by the current ramp. These results and our finding that the mRNAs for S<em>1</em>PRs are expressed in both the intact dorsal root ganglion and cultures of adult sensory neurons supports the notion that S<em>1</em>P acts on S<em>1</em>PRs linked to G proteins. Together these findings demonstrate that S<em>1</em>P can regulate the excitability of small diameter sensory neurons by acting as an external paracrine-type ligand through activation of G-protein-coupled receptors and thus may contribute to the hypersensitivity during inflammation.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S-<em>1</em>-P) has been shown to participate in the proliferative signal transduction pathways of mammalian cells. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase (SPL) catalyzes the breakdown of S-<em>1</em>-P. Using the C. elegans SPL nucleotide sequence, we identified a mouse EST as a putative candidate for the homologous gene encoding this enzyme. Sequencing of the mouse EST revealed an open reading frame of <em>1</em>707 nucleotides. This putative mouse SPL gene is 62% similar and 39% identical to the C. elegans SPL gene and 59% homologous and 39.6% identical to the yeast SPL gene. Expression of the mouse SPL gene in a yeast strain-delta bst<em>1</em>, which carries a deletion of the SPL gene and is hypersensitive to <em>sphingosine</em>, restored a <em>sphingosine</em>-resistant phenotype, suggesting this mouse gene can functionally complement the yeast defect when expressed. In vitro enzyme assay using extracts from these <em>sphingosine</em>-resistant transformants confirmed the SPL activities encoded by this mouse cDNA clone. Northern analysis indicated the mouse SPL gene is expressed at various levels in different tissues. Chromosomal localization mapped this SPL gene to Chromosome <em>1</em>0 at 32 cM. Here, we report the identification of the first mammalian <em>sphingosine</em> <em>phosphate</em> lyase gene.

Sphingolipids have been implicated in the regulation of cell growth, differentiation, and programmed cell death. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (SPP) has recently emerged as an important lipid messenger and a ligand for the endothelial differentiation gene receptor family of proteins through which it mediates its biologic effects. Recent studies in Saccharomyces cerevisiae in our laboratory implicated the yeast oligomycin resistance gene (YOR<em>1</em>), a member of the ATP binding cassette family of proteins, in the transport of SPP. The cystic fibrosis transmembrane regulator is a unique member of the ATP binding cassette transporter family and has high homology with YOR<em>1</em>. We therefore set out to investigate if this member of the family can regulate SPP transport. We demonstrate that C<em>1</em>27/cystic fibrosis transmembrane regulator (CFTR) cells, expressing wild type CFTR, exhibited significantly higher uptake of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> than either cells expressing a mutant CFTR C<em>1</em>27/DeltaF508 or C<em>1</em>27/mock-transfected cells. This effect was specific, dose-dependent, and competed off by dihydro<em>sphingosine</em> <em>1</em>-<em>phosphate</em> and lysophosphatidic acid. There was no difference in uptake of <em>sphingosine</em>, C(<em>1</em>6)-ceramide, sphingomyelin, lysophingomyelin, phosphatidylcholine, lysophosphatidylcholine, or phosphatidic acid among the different cell lines. Pretreatment with forskolin or isobutylmethylxanthine to stimulate cAMP did not affect the uptake in any of the cell lines. Moreover, we found that mitogen-activated protein kinase activation by SPP was less responsive in C<em>1</em>27/CFTR as compared with C<em>1</em>27/mock-transfected cells, suggesting that uptake of SPP by CFTR may divert it from interacting with its cell surface receptors and attenuate signaling functions. Taken together, these data implicate CFTR in uptake of SPP and the related phosphorylated lipids dihydro<em>sphingosine</em> <em>1</em>-<em>phosphate</em> and lysophosphatidic acid. This uptake influences the availability of SPP to modulate biologic activity via endothelial differentiation gene receptors. These studies may have important implications to cystic fibrosis.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a lipid mediator that exerts multiple cellular functions through activation of a subfamily of G-protein-coupled receptors. Although there is evidence that S<em>1</em>P plays a role in the developing and adult CNS, little is known about the ability of brain parenchyma to synthesize this lipid. We have therefore analyzed the brain distribution of the enzymatic activity of the S<em>1</em>P synthesizing enzyme, <em>sphingosine</em> kinase (SPHK) [EC:2.7.<em>1</em>.9<em>1</em>], as well as mRNA distribution for one of the two isoforms of this enzyme, <em>sphingosine</em> kinase 2. SPHK activity, measured by the conversion of [(3)H]<em>sphingosine</em> to [(3)H]S<em>1</em>P, is highest in cerebellum, followed by cortex and brainstem. Lowest activities were found in striatum and hippocampus. Sensitivity to 0.<em>1</em>% Triton-X suggests that this activity is accounted for by SPHK2. RT-PCR and in situ hybridization studies show that mRNA for this isoform has a distribution similar to that of SPHK activity. In vivo and in vitro ischemia increase SPHK activity and SPHK2 mRNA levels. These results indicate that SPHK2 is the predominant S<em>1</em>P-synthesizing isoform in normal brain parenchyma. Its heterogeneous distribution, in particular laminar distribution in cortex, suggests a neuronal localization and a possible role in cortical and cerebellar functions, in normal as well as ischemic brain.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a potent biomediator that can act as either an intracellular or an intercellular messenger. In the nervous system it exerts a wide range of actions, and specific membrane receptors for it have been identified in various regions. However, the physiological origin of extracellular S<em>1</em>P in the nervous system is largely unknown. We investigated cerebellar granule cells at different stages of differentiation and astrocytes in primary cultures as possible origins of extracellular S<em>1</em>P. Although these cells show marked differences in S<em>1</em>P metabolism, we found that they can all release S<em>1</em>P and express mRNAs for S<em>1</em>P specific receptors. Extracellular S<em>1</em>P derives from the export of newly synthesized intracellular S<em>1</em>P, and not from the action of a released <em>sphingosine</em> kinase. S<em>1</em>P release is rapid, efficient, and can be regulated by exogenous stimuli. Phorbol ester treatment resulted in an increase in <em>sphingosine</em> kinase <em>1</em> activity in the membranes, accompanied by a significant increase in extracellular S<em>1</em>P. S<em>1</em>P release in cells from the cerebellum emerges as a regulated mechanism, possibly related to a specific pool of newly synthesized S<em>1</em>P. To our knowledge, this is the first evidence of the extracellular release of S<em>1</em>P by primary cells from the CNS, which supports a role of S<em>1</em>P as autocrine/paracrine physiological messenger in the cerebellum.

The mitogenic role of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and its involvement in basic fibroblast growth factor (bFGF)-induced proliferation were examined in primary cultures of cerebellar astrocytes. Exposure to bFGF resulted in a rapid increase of extracellular S<em>1</em>P formation, bFGF inducing astrocytes to release S<em>1</em>P, but not <em>sphingosine</em> kinase, in the extracellular milieu. The SK inhibitor N,N-dimethyl<em>sphingosine</em> inhibited S<em>1</em>P release as well as bFGF-induced growth stimulation. S<em>1</em>P application in quiescent astrocytes caused a dose-dependent increase in DNA synthesis. This gliotrophic effect was induced by a brief exposure to low nanomolar S<em>1</em>P, mimicked by the S<em>1</em>P receptor agonist dihydro-S<em>1</em>P, and inhibited by pertussis toxin (PTX), an inactivator of G(i)/G(o)-proteins. S<em>1</em>P also induced activation of extracellular signal-regulated kinase that was inhibited again by PTX. Moreover, the S<em>1</em>P lyase inhibitor 4-deoxypyridoxine induced the cellular accumulation of S<em>1</em>P but did not affect DNA synthesis. These results support the view that S<em>1</em>P exerted a mitogenic effect on cerebellar astrocytes extracellularly, most likely through cell surface S<em>1</em>P receptors. In agreement, mRNAs for S<em>1</em>P<em>1</em>, S<em>1</em>P2, and S<em>1</em>P3 receptors are expressed in cerebellar astrocytes (Anelli et al., 2005. J Neurochem 92:<em>1</em>204-<em>1</em>2<em>1</em>5). Ceramide, a negative regulator of astrocyte proliferation and down-regulated by bFGF (Riboni et al., 2002. Cerebellum <em>1</em>:<em>1</em>29-<em>1</em>35), efficiently inhibited S<em>1</em>P-induced proliferation. The S<em>1</em>P action appears to be part of an autocrine/paracrine cascade stimulated by bFGF and, together with ceramide down-regulation, essential for astrocytes to respond to bFGF. The results suggest that S<em>1</em>P and bFGF/S<em>1</em>P may play an important role in physiopathological glial proliferation, such as brain development, reactive gliosis and brain tumor formation.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a lipid-signaling molecule produced by <em>sphingosine</em> kinase in response to a wide number of stimuli. By acting through a family of widely expressed G protein-coupled receptors, S<em>1</em>P regulates diverse physiological processes. Here we examined the role of S<em>1</em>P signaling in neurodegeneration using a mouse model of Sandhoff disease, a prototypical neuronopathic lysosomal storage disorder. When <em>sphingosine</em> kinase <em>1</em> (Sphk<em>1</em>) was deleted in Sandhoff disease mice, a milder disease course occurred, with decreased proliferation of glial cells and less-pronounced astrogliosis. A similar result of milder disease course and reduced astroglial proliferation was obtained by deletion of the gene for the S<em>1</em>P(3) receptor, a G protein-coupled receptor enriched in astrocytes. Our studies demonstrate a functional role of S<em>1</em>P synthesis and receptor expression in astrocyte proliferation leading to astrogliosis during the terminal stages of neurodegeneration in Sandhoff disease mice. Because astrocyte responses are involved in many types of neurodegeneration, the Sphk<em>1</em>/S<em>1</em>P receptor signaling axis may be generally important during the pathogenesis of neurodegenerative diseases.

Although the hippocampus is a brain region involved in short-term memory, the molecular mechanisms underlying memory formation are not completely understood. Here we show that <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) plays a pivotal role in the formation of memory. Addition of S<em>1</em>P to rat hippocampal slices increased the rate of AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) recorded from the CA3 region of the hippocampus. In addition long-term potentiation (LTP) observed in the CA3 region was potently inhibited by a <em>sphingosine</em> kinase (SphK) inhibitor and this inhibition was fully reversed by S<em>1</em>P. LTP was impaired in hippocampal slices specifically in the CA3 region obtained from SphK<em>1</em>-knockout mice, which correlates well with the poor performance of these animals in the Morris water maze test. These results strongly suggest that SphK/S<em>1</em>P receptor signaling plays an important role in excitatory synaptic transmission in the CA3 region of hippocampus and has profound effects on hippocampal function such as spatial learning.

OBJECTIVE

We examined effects of isoflurane, volatile anesthetics, on blood-brain barrier disruption in the endovascular perforation model of subarachnoid hemorrhage (SAH) in mice.

METHODS

Animals were assigned to sham-operated, SAH+vehicle-air, SAH+<em>1</em>%, or 2% isoflurane groups. Neurobehavioral function, brain water content, Evans blue dye extravasation, and Western blotting for <em>sphingosine</em> kinases, occludin, claudin-5, junctional adhesion molecule, and vascular endothelial cadherin were evaluated at 24 hours post-SAH. Effects of <em>sphingosine</em> kinase (N,N-dimethyl<em>sphingosine</em>) or <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor-<em>1</em>/3 (S<em>1</em>P<em>1</em>/3) inhibitors (VPC230<em>1</em>9) on isoflurane's action were also examined.

RESULTS

SAH aggravated neurological scores, brain edema, and blood-brain barrier permeability, which were prevented by 2% but not <em>1</em>% isoflurane posttreatment. Two percent isoflurane increased <em>sphingosine</em> kinase-<em>1</em> expression and prevented a post-SAH decrease in expressions of the blood-brain barrier-related proteins. Both N,N-dimethyl<em>sphingosine</em> and VPC230<em>1</em>9 abolished the beneficial effects of isoflurane.

CONCLUSIONS

Two percent isoflurane can suppress post-SAH blood-brain barrier disruption, which may be mediated by <em>sphingosine</em> kinase <em>1</em> expression and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor-<em>1</em>/3 activation.

RGS5 is a potent GTPase-activating protein for G(ialpha) and G(qalpha) that is expressed strongly in pericytes and is present in vascular smooth muscle cells. To study the role of RGS5 in blood vessel physiology, we generated Rgs5-deficient mice. The Rgs5(-/-) mice developed normally, without obvious defects in cardiovascular development or function. Surprisingly, Rgs5(-/-) mice had persistently low blood pressure, lower in female mice than in male mice, without concomitant cardiac dysfunction, and a lean body habitus. The examination of the major blood vessels revealed that the aortas of Rgs5(-/-) mice were dilated compared to those of control mice, without altered wall thickness. Isolated aortic smooth muscle cells from the Rgs5(-/-) mice exhibited exaggerated levels of phosphorylation of vasodilator-stimulated phosphoprotein and extracellular signal-regulated kinase in response to stimulation with either sodium nitroprusside or <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. The results of this study, along with those of previous studies demonstrating that RGS5 stability is under the control of nitric oxide via the N-end rule pathway, suggest that RGS5 may balance vascular tone by attenuating vasodilatory signaling in vivo in opposition to RGS2, another RGS (regulator of G protein signaling) family member known to inhibit G protein-coupled receptor-mediated vasoconstrictor signaling. Blocking the function or the expression of RGS5 may provide an alternative approach to treat hypertension.

Current treatments for patients with Crohn's disease (CD) are based on recent advances in elucidating the pathophysiology of the disease. A satisfactory therapeutic strategy has not been well established. A new <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor agonist, 2-amino-2-propanediol hydrochloride (KRP-203), has been developed for immunomodulation in autoimmune diseases and organ transplantation. We aimed to evaluate the efficacy and potency of KRP-203 on the treatment of chronic colitis in an interleukin (IL)-<em>1</em>0 gene-deficient (IL-<em>1</em>0(-/-)) mouse model. KRP-203 agonistic activity on S<em>1</em>P receptor was assessed in vitro. KRP-203 was administered for <em>1</em> or 4 weeks to IL-<em>1</em>0(-/-) mice with clinical signs of colitis. The histological appearance of the colon and the numbers, phenotype, and cytokine production of lymphocytes were compared with a control group. KRP-203 treatment was effective in preventing body weight loss in the IL-<em>1</em>0(-/-) colitis model. One-week administration resulted in the sequestration of circulating lymphocytes within the secondary lymphoid tissues. After 4 weeks of treatment, highly significant reductions were observed in number of CD4(+) T cell and B220(+) B cell subpopulations in the lamina propria of the colon and peripheral blood. KRP-203 obviously inhibited the production of interferon-gamma, IL-<em>1</em>2, and tumor necrosis factor-alpha by the colonic lymphocytes, but had no influence on IL-4 production. KRP-203 significantly inhibits ongoing IL-<em>1</em>0(-/-) colitis in part through decreasing the infiltration of lymphocytes at inflammatory sites and by blocking T-helper <em>1</em> cytokine production in the colonic mucosa. Therefore, the possibility arises that KRP-203 plays a potential role in control of chronic colitis.

A challenging task of the immune system is to fight cancer cells. However, a variety of human cancers educate immune cells to become tumor supportive. This is exemplified for tumor-associated macrophages (TAMs), which are polarized towards an anti-inflammatory and cancer promoting phenotype. Mechanistic explanations, how cancer cells influence the macrophage phenotype are urgently needed to address potential anti-cancer strategies along this line. One potential immune modulating compound, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), was recently highlighted in both tumor growth and immune modulation. Using a xenograft model in nude mice, we demonstrate a supportive role of <em>sphingosine</em> kinase 2 (SphK2), one of the S<em>1</em>P-producing enzymes for tumor progression. The growth of SphK2-deficient MCF-7 breast tumor xenografts was markedly delayed when compared with controls. Infiltration of macrophages in SphK2-deficient and control tumors was comparable. However, TAMs from SphK2-deficient tumors displayed a pronounced anti-tumor phenotype, showing an increased expression of pro-inflammatory markers/mediators such as NO, TNF-alpha, IL-<em>1</em>2 and MHCII and a low expression of anti-inflammatory IL-<em>1</em>0 and CD206. These data suggest a role for S<em>1</em>P, generated by SphK2, in early tumor development by affecting macrophage polarization.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) has diverse effects on T cells that are mediated by the predominant S<em>1</em>P<em>1</em> and S<em>1</em>P4 G protein-coupled receptors (GPCRs). S<em>1</em>P4 is expressed principally by leukocytes, but little is known of its T cell effects in immunity. Two approaches were used to investigate S<em>1</em>P4 signals in T cells. First, S<em>1</em>P4 was introduced into D<em>1</em>0G4.<em>1</em> mouse Th2 cells and EL4.IL-2 mouse T cells lacking endogenous S<em>1</em>P GPCRs. Second, mouse splenic CD4 T cells were treated with FTY720 to suppress S<em>1</em>P<em>1</em> and leave S<em>1</em>P4 GPCRs as the only functionally relevant S<em>1</em>P receptor. Unlike S<em>1</em>P<em>1</em>, S<em>1</em>P4 failed to transduce chemotactic responses of any of the S<em>1</em>P4-only T cells to S<em>1</em>P or the phyto-S<em>1</em>P ligand selective for S<em>1</em>P4, or to suppress their chemotactic responses to chemokines. The S<em>1</em>P-S<em>1</em>P4 axis significantly inhibited T cell proliferation in each of the S<em>1</em>P4-only T cells activated by anti-CD3 and anti-CD28 MoAbs. Secretion of IL-4 by S<em>1</em>P4-D<em>1</em>0G4.<em>1</em> cells, IL-2 by S<em>1</em>P4-EL4.IL-2, and IFN-gamma by FTY720-treated CD4 T cells were significantly inhibited by S<em>1</em>P. In contrast, S<em>1</em>P enhanced secretion of IL-<em>1</em>0 by stimulated S<em>1</em>P4-D<em>1</em>0G4.<em>1</em> T cells. Thus, S<em>1</em>P4 mediates immunosuppressive effects of S<em>1</em>P by inhibiting proliferation and secretion of effector cytokines, while enhancing secretion of the suppressive cytokine IL-<em>1</em>0.

Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment ('host', which includes stromal cells and the immune system). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth ('colonization') being critical in determining metastatic outcome. Here we show the results of screening 8<em>1</em>0 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with <em>1</em>9 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S<em>1</em>P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive sphingolipid metabolite involved in many critical cell processes. It is produced by the phosphorylation of <em>sphingosine</em> by <em>sphingosine</em> kinases (SphKs) and exported out of cells via transporters such as spinster homolog 2 (Spns2). S<em>1</em>P regulates diverse physiological processes by binding to specific G protein-binding receptors, S<em>1</em>P receptors (S<em>1</em>PRs) <em>1</em>-5, through a process coined as "inside-out signaling." The S<em>1</em>P concentration gradient between various tissues promotes S<em>1</em>PR<em>1</em>-dependent migration of T cells from secondary lymphoid organs into the lymphatic and blood circulation. S<em>1</em>P suppresses T cell egress from and promotes retention in inflamed peripheral tissues. S<em>1</em>PR<em>1</em> in T and B cells as well as Spns2 in endothelial cells contributes to lymphocyte trafficking. FTY720 (Fingolimod) is a functional antagonist of S<em>1</em>PRs that induces systemic lymphopenia by suppression of lymphocyte egress from lymphoid organs. In this review, we summarize previous findings and new discoveries about the importance of S<em>1</em>P and S<em>1</em>PR signaling in the recruitment of immune cells and lymphocyte retention in inflamed tissues. We also discuss the role of S<em>1</em>P-S<em>1</em>PR<em>1</em> axis in inflammatory diseases and wound healing.