The mechanism by which <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor-<em>1</em> (S<em>1</em>P<em>1</em>) acts to promote lymphocyte egress from lymphoid organs is not defined. Here, we showed that CCR7-deficient T cells left lymph nodes more rapidly than wild-type cells did, whereas CCR7-overexpressing cells were retained for longer. After treatment with FTY720, an agonist that causes downmodulation of lymphocyte S<em>1</em>P<em>1</em>, CCR7-deficient T cells were less effectively retained than wild-type T cells. Moreover, treatment with pertussis toxin to inactivate signaling via G alpha i-protein-coupled receptors restored egress competence to S<em>1</em>P<em>1</em>-deficient lymphocytes. We also found that T cell accumulation in lymph node cortical sinusoids required intrinsic S<em>1</em>P<em>1</em> expression and was antagonized by CCR7. These findings suggest a model where S<em>1</em>P<em>1</em> acts in the lymphocyte to promote lymph node egress by overcoming retention signals mediated by CCR7 and additional G alpha i-coupled receptors. Furthermore, by simultaneously upregulating S<em>1</em>P<em>1</em> and downregulating CCR7, T cells that have divided multiple times switch to a state favoring egress over retention.

Mast cells play a pivotal role in inflammatory and immediate-type allergic reactions by secreting a variety of potent inflammatory mediators, including <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). However, it is not known how S<em>1</em>P is released from cells. Here, we report that S<em>1</em>P is exported from mast cells independently of their degranulation and demonstrate that it is mediated by ATP binding cassette (ABC) transporters. Constitutive and antigen-stimulated S<em>1</em>P release was inhibited by MK57<em>1</em>, an inhibitor of ABCC<em>1</em> (MRP<em>1</em>), but not by inhibitors of ABCB<em>1</em> (MDR-<em>1</em>, P-glycoprotein). Moreover, down-regulation of ABCC<em>1</em> with small interfering RNA, which decreased its cell surface expression, markedly reduced S<em>1</em>P export from both rat RBL-2H3 and human LAD2 mast cells. Transport of S<em>1</em>P by ABCC<em>1</em> influenced migration of mast cells toward antigen but not degranulation. These findings have important implications for S<em>1</em>P functions in mast cell-mediated immune responses.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-<em>1</em>. Based on peptide sequences obtained from purified rat kidney <em>sphingosine</em> kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian <em>sphingosine</em> kinases (murine SPHK<em>1</em>a and SPHK<em>1</em>b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison with Saccharomyces cerevisiae and Caenorhabditis elegans <em>sphingosine</em> kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either <em>sphingosine</em> kinase expression vectors had marked increases (more than <em>1</em>00-fold) in <em>sphingosine</em> kinase activity. The enzyme specifically phosphorylated D-erythro-<em>sphingosine</em> and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, D,L-threo-dihydro<em>sphingosine</em> or N, N-dimethyl<em>sphingosine</em>. The latter two sphingolipids were competitive inhibitors of <em>sphingosine</em> kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of <em>sphingosine</em> and, to a lesser extent, in ceramide levels. Our data suggest that <em>sphingosine</em> kinase is a prototypical member of a new class of lipid kinases. Cloning of <em>sphingosine</em> kinase is an important step in corroborating the intracellular role of SPP as a second messenger.

The p<em>1</em><em>1</em>0 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p<em>1</em><em>1</em>0alpha, p<em>1</em><em>1</em>0beta, and p<em>1</em><em>1</em>0delta) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p<em>1</em><em>1</em>0alpha and p<em>1</em><em>1</em>0delta to Tyr kinase signaling pathways. The p84/p<em>1</em>0<em>1</em> regulatory subunits of the p<em>1</em><em>1</em>0gamma class IB PI3K lack SH2 domains and instead couple p<em>1</em><em>1</em>0gamma to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p<em>1</em><em>1</em>0beta and cells derived from a p<em>1</em><em>1</em>0beta-deficient mouse line, that p<em>1</em><em>1</em>0beta is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p<em>1</em><em>1</em>0beta and p<em>1</em><em>1</em>0gamma contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-<em>1</em>. In fibroblasts, which express p<em>1</em><em>1</em>0beta but not p<em>1</em><em>1</em>0gamma, p<em>1</em><em>1</em>0beta mediated Akt activation by the GPCR ligands stromal cell-derived factor, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor <em>1</em>. Introduction of p<em>1</em><em>1</em>0gamma in these cells reduced the contribution of p<em>1</em><em>1</em>0beta to GPCR signaling. Taken together, these data show that p<em>1</em><em>1</em>0beta and p<em>1</em><em>1</em>0gamma can couple redundantly to the same GPCR agonists. p<em>1</em><em>1</em>0beta, which shows a much broader tissue distribution than the leukocyte-restricted p<em>1</em><em>1</em>0gamma, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p<em>1</em><em>1</em>0gamma expression is low or absent.

Prostaglandins, leukotrienes, platelet-activating factor, lysophosphatidic acid, <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, and endocannabinoids, collectively referred to as lipid mediators, play pivotal roles in immune regulation and self-defense, and in the maintenance of homeostasis in living systems. They are produced by multistep enzymatic pathways, which are initiated by the de-esterification of membrane phospholipids by phospholipase A2s or sphingo-myelinase. Lipid mediators exert their biological effects by binding to cognate receptors, which are members of the G protein-coupled receptor superfamily. The synthesis of the lipid mediators and subsequent induction of receptor activity is tightly regulated under normal physiological conditions, and enzyme and/or receptor dysfunction can lead to a variety of disease conditions. Thus, the manipulation of lipid mediator signaling, through either enzyme inhibitors or receptor antagonists and agonists, has great potential as a therapeutic approach to disease. In this review, I summarize our current state of knowledge of the synthesis of lipid mediators and the function of their cognate receptors, and discuss the effects of genetic or pharmacological ablation of enzyme or receptor function on various pathophysiological processes.

Maintenance of vascular integrity is critical for homeostasis, and temporally and spatially regulated vascular leak is a central feature of inflammation. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) can regulate endothelial barrier function, but the sources of the S<em>1</em>P that provide this activity in vivo and its importance in modulating different inflammatory responses are unknown. We report here that mutant mice engineered to selectively lack S<em>1</em>P in plasma displayed increased vascular leak and impaired survival after anaphylaxis, administration of platelet-activating factor (PAF) or histamine, and exposure to related inflammatory challenges. Increased leak was associated with increased interendothelial cell gaps in venules and was reversed by transfusion with wild-type erythrocytes (which restored plasma S<em>1</em>P levels) and by acute treatment with an agonist for the S<em>1</em>P receptor <em>1</em> (S<em>1</em>pr<em>1</em>). S<em>1</em>pr<em>1</em> agonist did not protect wild-type mice from PAF-induced leak, consistent with plasma S<em>1</em>P levels being sufficient for S<em>1</em>pr<em>1</em> activation in wild-type mice. However, an agonist for another endothelial cell Gi-coupled receptor, Par2, did protect wild-type mice from PAF-induced vascular leak, and systemic treatment with pertussis toxin prevented rescue by Par2 agonist and sensitized wild-type mice to leak-inducing stimuli in a manner that resembled the loss of plasma S<em>1</em>P. Our results suggest that the blood communicates with blood vessels via plasma S<em>1</em>P to maintain vascular integrity and regulate vascular leak. This pathway prevents lethal responses to leak-inducing mediators in mouse models.

Protection of the endothelium is provided by circulating <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), which maintains vascular integrity. We show that HDL-associated S<em>1</em>P is bound specifically to both human and murine apolipoprotein M (apoM). Thus, isolated human ApoM(+) HDL contained S<em>1</em>P, whereas ApoM(-) HDL did not. Moreover, HDL in Apom(-/-) mice contains no S<em>1</em>P, whereas HDL in transgenic mice overexpressing human apoM has an increased S<em>1</em>P content. The <em>1</em>.7-Å structure of the S<em>1</em>P-human apoM complex reveals that S<em>1</em>P interacts specifically with an amphiphilic pocket in the lipocalin fold of apoM. Human ApoM(+) HDL induced S<em>1</em>P(<em>1</em>) receptor internalization, downstream MAPK and Akt activation, endothelial cell migration, and formation of endothelial adherens junctions, whereas apoM(-) HDL did not. Importantly, lack of S<em>1</em>P in the HDL fraction of Apom(-/-) mice decreased basal endothelial barrier function in lung tissue. Our results demonstrate that apoM, by delivering S<em>1</em>P to the S<em>1</em>P(<em>1</em>) receptor on endothelial cells, is a vasculoprotective constituent of HDL.

Regulatory T cells (T(reg) cells) are critically involved in maintaining immunological tolerance, but this potent suppression must be 'quenched' to allow the generation of adaptive immune responses. Here we report that <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor type <em>1</em> (S<em>1</em>P<em>1</em>) delivers an intrinsic negative signal to restrain the thymic generation, peripheral maintenance and suppressive activity of T(reg) cells. Combining loss- and gain-of-function genetic approaches, we found that S<em>1</em>P<em>1</em> blocked the differentiation of thymic T(reg) precursors and function of mature T(reg) cells and affected T(reg) cell-mediated immune tolerance. S<em>1</em>P<em>1</em> induced selective activation of the Akt-mTOR kinase pathway to impede the development and function of T(reg) cells. Dynamic regulation of S<em>1</em>P<em>1</em> contributed to lymphocyte priming and immune homeostasis. Thus, by antagonizing T(reg) cell-mediated immune suppression, the lipid-activated S<em>1</em>P<em>1</em>-Akt-mTOR pathway orchestrates adaptive immune responses.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) elicits diverse cellular responses through a family of G-protein-coupled receptors. We have shown previously that genetic disruption of the S<em>1</em>P(<em>1</em>) receptor, the most widely expressed of the family, results in embryonic lethality because of its key role within endothelial cells in regulating the coverage of blood vessels by vascular smooth muscle cells. To understand the physiologic functions of the two other widely expressed S<em>1</em>P receptors, we generated S<em>1</em>P(2) and S<em>1</em>P(3) null mice. Neither the S<em>1</em>P(2) null mice nor the S<em>1</em>P(3) null mice exhibited significant embryonic lethality or obvious phenotypic abnormalities. To unmask possible overlapping or collaborative functions between the S<em>1</em>P(<em>1</em>), S<em>1</em>P(2), and S<em>1</em>P(3) receptors, we examined embryos with multiple S<em>1</em>P receptor mutations. We found that S<em>1</em>P(<em>1</em>) S<em>1</em>P(2) double null and S<em>1</em>P(<em>1</em>) S<em>1</em>P(2) S<em>1</em>P(3) triple null embryos displayed a substantially more severe vascular phenotype than did embryos with only S<em>1</em>P(<em>1</em>) deleted. We also found partial embryonic lethality and vascular abnormalities in S<em>1</em>P(2) S<em>1</em>P(3) double null embryos. Our results indicate that the S<em>1</em>P(<em>1</em>), S<em>1</em>P(2) and S<em>1</em>P(3) receptors have redundant or cooperative functions for the development of a stable and mature vascular system during embryonic development.

S<em>1</em>P(<em>1</em>) is a widely distributed G protein-coupled receptor whose ligand, <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, is present in high concentrations in the blood. The <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor-signaling pathway is believed to have potent effects on cell trafficking in the immune system. To determine the precise role of the S<em>1</em>P(<em>1</em>) receptor on T-cells, we established a T-cell-specific S<em>1</em>P(<em>1</em>) knock-out mouse. The mutant mice showed a block in the egress of mature T-cells into the periphery. The expression of the S<em>1</em>P(<em>1</em>) receptor was up-regulated in mature thymocytes, and its deletion altered the chemotactic responses of thymocytes to <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. The results indicated that the expression of the S<em>1</em>P(<em>1</em>) receptor on T-cells controls their exit from the thymus and entry into the blood and, thus, has a central role in regulating the numbers of peripheral T-cells.

Interleukin-6 (IL-6)-Janus kinase (JAK) signaling is viewed as crucial for persistent signal transducer and activator of transcription-3 (STAT3) activation in cancer. However, IL-6-induced STAT3 activation is normally transient. Here we identify a key mechanism for persistent STAT3 activation in tumor cells and the tumor microenvironment. We show that expression of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor-<em>1</em> (S<em>1</em>PR<em>1</em>), a G protein-coupled receptor for the lysophospholipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), is elevated in STAT3-positive tumors. STAT3 is a transcription factor for the S<em>1</em>pr<em>1</em> gene. Reciprocally, enhanced S<em>1</em>pr<em>1</em> expression activates STAT3 and upregulates Il6 gene expression, thereby accelerating tumor growth and metastasis in a STAT3-dependent manner. Silencing S<em>1</em>pr<em>1</em> in tumor cells or immune cells inhibits tumor STAT3 activity, tumor growth and metastasis. S<em>1</em>P-S<em>1</em>PR<em>1</em>-induced STAT3 activation is persistent, in contrast to transient STAT3 activation by IL-6. S<em>1</em>PR<em>1</em> activates STAT3 in part by upregulating JAK2 tyrosine kinase activity. We show that STAT3-induced S<em>1</em>PR<em>1</em> expression, as well as the S<em>1</em>P-S<em>1</em>PR<em>1</em> pathway reciprocal regulation of STAT3 activity, is a major positive feedback loop for persistent STAT3 activation in cancer cells and the tumor microenvironment and for malignant progression.

<em>Sphingosine</em> kinase (SphK) is a highly conserved lipid kinase that phosphorylates <em>sphingosine</em> to form <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). S<em>1</em>P/SphK has been implicated as a signalling pathway to regulate diverse cellular functions [<em>1</em>-3], including cell growth, proliferation and survival [4-8]. We report that cells overexpressing SphK have increased enzymatic activity and acquire the transformed phenotype, as determined by focus formation, colony growth in soft agar and the ability to form tumours in NOD/SCID mice. This is the first demonstration that a wild-type lipid kinase gene acts as an oncogene. Using a chemical inhibitor of SphK, or an SphK mutant that inhibits enzyme activation, we found that SphK activity is involved in oncogenic H-Ras-mediated transformation, suggesting a novel signalling pathway for Ras activation. The findings not only point to a new signalling pathway in transformation but also to the potential of SphK inhibitors in cancer therapy.

Under most conditions, resorbed bone is nearly precisely replaced in location and amount by new bone. Thus, it has long been recognized that bone loss through osteoclast-mediated bone resorption and bone replacement through osteoblast-mediated bone formation are tightly coupled processes. Abundant data conclusively demonstrate that osteoblasts direct osteoclast differentiation. Key questions remain, however, as to how osteoblasts are recruited to the resorption site and how the amount of bone produced is so precisely controlled. We hypothesized that osteoclasts play a crucial role in the promotion of bone formation. We found that osteoclast conditioned medium stimulates human mesenchymal stem (hMS) cell migration and differentiation toward the osteoblast lineage as measured by mineralized nodule formation in vitro. We identified candidate osteoclast-derived coupling factors using the Affymetrix microarray. We observed significant induction of <em>sphingosine</em> kinase <em>1</em> (SPHK<em>1</em>), which catalyzes the phosphorylation of <em>sphingosine</em> to form <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), in mature multinucleated osteoclasts as compared with preosteoclasts. S<em>1</em>P induces osteoblast precursor recruitment and promotes mature cell survival. Wnt<em>1</em>0b and BMP6 also were significantly increased in mature osteoclasts, whereas sclerostin levels decreased during differentiation. Stimulation of hMS cell nodule formation by osteoclast conditioned media was attenuated by the Wnt antagonist Dkk<em>1</em>, a BMP6-neutralizing antibody, and by a S<em>1</em>P antagonist. BMP6 antibodies and the S<em>1</em>P antagonist, but not Dkk<em>1</em>, reduced osteoclast conditioned media-induced hMS chemokinesis. In summary, our findings indicate that osteoclasts may recruit osteoprogenitors to the site of bone remodeling through SIP and BMP6 and stimulate bone formation through increased activation of Wnt/BMP pathways.

The blood constituent <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a specific ligand for five G-protein-coupled receptors designated S<em>1</em>P(<em>1</em>-5). Expression of the S<em>1</em>P<em>1</em> receptor on lymphocytes is required for their exit from secondary lymphoid organs, suggesting that S<em>1</em>P serves as a stimulus for maintaining lymphocyte circulation in blood. Despite its potential role in immune surveillance, the regulatory system that controls blood S<em>1</em>P levels is not well understood. This report reveals that erythrocytes constitute a buffer system for S<em>1</em>P in blood. They efficiently incorporated and stored S<em>1</em>P, and protected it from cellular degradation. They also released S<em>1</em>P into plasma, but not into other serum-free media, indicating that S<em>1</em>P release was controlled by a plasma factor. Erythrocytes did not generate S<em>1</em>P since an increase in plasma S<em>1</em>P levels was always accompanied by a decrease in cellular S<em>1</em>P levels. Thrombocytes that were reported to generate and release S<em>1</em>P after activation did not contribute to the observed S<em>1</em>P release in blood. The amount of erythrocytes as well as the proportion of plasma in the medium determined the magnitude of S<em>1</em>P release. Adoptively transferred S<em>1</em>P-loaded and unloaded mouse erythrocytes displayed a normal life span and similar S<em>1</em>P levels 24 h after recovery, indicating that S<em>1</em>P incorporation and release are dynamically regulated in vivo.

Coupling between bone formation and bone resorption refers to the process within basic multicellular units in which resorption by osteoclasts is met by the generation of osteoblasts from precursors, and their bone-forming activity, which needs to be sufficient to replace the bone lost. There are many sources of activities that contribute to coupling at remodeling sites, including growth factors released from the matrix, soluble and membrane products of osteoclasts and their precursors, signals from osteocytes and from immune cells and signaling taking place within the osteoblast lineage. Coupling is therefore a process that involves the interaction of a wide range of cell types and control mechanisms. As bone remodeling occurs at many sites asynchronously throughout the skeleton, locally generated activities comprise very important control mechanisms. In this review, we explore the potential roles of a number of these factors, including <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, semaphorins, ephrins, interleukin-6 (IL-6) family cytokines and marrow-derived factors. Their interactions achieve the essential tight control of coupling within individual remodeling units that is required for control of skeletal mass.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a secreted lipid mediator that functions in vascular development; however, it remains unclear how S<em>1</em>P secretion is regulated during embryogenesis. We identified a zebrafish mutant, ko<em>1</em>57, that displays cardia bifida (two hearts) resembling that in the S<em>1</em>P receptor-2 mutant. A migration defect of myocardial precursors in the ko<em>1</em>57 mutant is due to a mutation in a multipass transmembrane protein, Spns2, and can be rescued by S<em>1</em>P injection. We show that the export of S<em>1</em>P from cells requires Spns2. spns2 is expressed in the extraembryonic tissue yolk syncytial layer (YSL), and the introduction of spns2 mRNA in the YSL restored the cardiac defect in the ko<em>1</em>57 mutant. Thus, Spns2 in the YSL functions as a S<em>1</em>P transporter in S<em>1</em>P secretion, thereby regulating myocardial precursor migration.

FTY720, a potent immunosuppressive agent, is phosphorylated in vivo into FTY720-P, a high affinity agonist for <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors. The effects of FTY720 on vascular cells, a major target of S<em>1</em>P action, have not been addressed. We now report the metabolic activation of FTY720 by <em>sphingosine</em> kinase-2 and potent activation of vascular endothelial cell functions in vitro and in vivo by phosphorylated FTY720 (FTY720-P). Incubation of endothelial cells with FTY720 resulted in phosphorylation by <em>sphingosine</em> kinase activity and formation of FTY720-P. <em>Sphingosine</em> kinase-2 effectively phosphorylated FTY720 in the human embryonic kidney 293T heterologous expression system. FTY720-P treatment of endothelial cells stimulated extracellular signal-activated kinase and Akt phosphorylation and adherens junction assembly and promoted cell survival. The effects of FTY720-P were inhibited by pertussis toxin, suggesting the requirement for Gi-coupled S<em>1</em>P receptors. Indeed, transmonolayer permeability induced by vascular endothelial cell growth factor was potently reversed by FTY720-P. Furthermore, oral FTY720 administration in mice potently blocked VEGF-induced vascular permeability in vivo. These findings suggest that FTY720 or its analogs may find utility in the therapeutic regulation of vascular permeability, an important process in angiogenesis, inflammation, and pathological conditions such as sepsis, hypoxia, and solid tumor growth.

Ceramide is a sphingolipid that acts as a second messenger in ubiquitous, evolutionarily conserved, signaling systems. Emerging data suggest that radiation acts directly on the plasma membrane of several cell types, activating acid sphingomyelinase, which generates ceramide by enzymatic hydrolysis of sphingomyelin. Ceramide then acts as a second messenger in initiating an apoptotic response via the mitochondrial system. Radiation-induced DNA damage can also initiate ceramide generation by activation of mitochondrial ceramide synthase and de novo synthesis of ceramide. In some cells and tissues, BAX is activated downstream of ceramide, regulating commitment to the apoptotic process via release of mitochondrial cytochrome c. Genetic and pharmacologic studies in vivo showed that radiation targets the acid sphingomyelinase apoptotic system of microvascular endothelial cells in the lungs, intestines and brain, as well as in oocytes, to initiate the pathogenesis of tissue damage. Regulated ceramide metabolism may produce metabolites, such as <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, shown to signal antiapoptosis, thus controlling the intensity of the apoptotic response and constituting a mechanism for radiation sensitivity or resistance. An improved understanding of this signaling system may offer new opportunities for the modulation of radiation effects in the treatment of cancer.

Abnormal sphingolipid metabolism has been previously reported in Alzheimer's disease (AD). To extend these findings, several sphingolipids and sphingolipid hydrolases were analyzed in brain samples from AD patients and age-matched normal individuals. We found a pattern of elevated acid sphingomyelinase (ASM) and acid ceramidase (AC) expression in AD, leading to a reduction in sphingomyelin and elevation of ceramide. More <em>sphingosine</em> also was found in the AD brains, although <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) levels were reduced. Notably, significant correlations were observed between the brain ASM and S<em>1</em>P levels and the levels of amyloid beta (Abeta) peptide and hyperphosphorylated tau protein. Based on these findings, neuronal cell cultures were treated with Abeta oligomers, which were found to activate ASM, increase ceramide, and induce apoptosis. Pre-treatment of the neurons with purified, recombinant AC prevented the cells from undergoing Abeta-induced apoptosis. We propose that ASM activation is an important pathological event leading to AD, perhaps due to Abeta deposition. The downstream consequences of ASM activation are elevated ceramide, activation of ceramidases, and production of <em>sphingosine</em>. The reduced levels of S<em>1</em>P in the AD brain, together with elevated ceramide, likely contribute to the disease pathogenesis.

The lysophospholipids, lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), are now recognized as important extracellular signaling molecules. These lipid mediators are pleiotropic; among the most common cellular responses are mitogenesis, cell survival (anti-apoptosis), inhibition of adenylyl cyclase and calcium mobilization. Physiologic events associated with these mediators include platelet aggregation, vasopressor activity, wound healing, immune modulation, and angiogenesis. Many of the actions of LPA and S<em>1</em>P are mediated through a set of eight G protein-coupled receptors. Five of these are S<em>1</em>P-prefering while the remaining three are LPA receptors. These receptors are expressed widely and in aggregate signal through a variety of heterotrimeric G proteins. The lysophospholipid receptor family is referred to commonly as the "Edg" group (e.g., Edg-<em>1</em>, Edg-2, etc.). Herein, the molecular pharmacology of the lysophospholipid receptors is reviewed briefly, and a rational nomenclature for LPA and S<em>1</em>P receptors that is consistent with the International Union of Pharmacology guidelines is proposed.

<em>Sphingosine</em> kinase-<em>1</em> (SPHK<em>1</em>) is a key enzyme catalyzing the formation of an important bioactive lipid messenger, <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, and is implicated in the regulation of cell proliferation and antiapoptotic processes. Biological features of another isozyme SPHK2, however, remain unclear. The present studies were undertaken to characterize SPHK2 by comparison with SPHK<em>1</em>. When SPHK2 was transiently expressed in various cell lines, it was localized in the nuclei as well as in the cytosol, whereas SPHK<em>1</em> was distributed in the cytosol but not in the nucleus. We have mapped a functional nuclear localization signal (NLS) to the N-terminal region of SPHK2. We have observed that the expression of SPHK2 in various cell types causes inhibition of DNA synthesis, resulting in the cell cycle arrest at G<em>1</em>/S phase. We have also demonstrated that an NLS mutant of SPHK2, SPHK2R93E/R94E, failed to enter the nucleus and to inhibit DNA synthesis. Moreover, a fusion protein, NLS-SPHK<em>1</em>, where SPHK<em>1</em> was fused to the NLS sequence of SPHK2 acquired the ability to enter nuclei and inhibited DNA synthesis. These results indicate that SPHK2 localizes in the nuclei and causes inhibition of DNA synthesis, and this may affect subsequent cellular events.

The sphingolipid metabolites ceramide (Cer), <em>sphingosine</em> (Sph), and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) play an important role in the regulation of cell proliferation, survival, and cell death. Cer and Sph usually inhibit proliferation and promote apoptosis, while the further metabolite S<em>1</em>P stimulates growth and suppresses apoptosis. Because these metabolites are interconvertible, it has been proposed that it is not the absolute amounts of these metabolites but rather their relative levels that determines cell fate. The relevance of this "sphingolipid rheostat" and its role in regulating cell fate has been borne out by work in many labs using many different cell types and experimental manipulations. A central finding of these studies is that Sph kinase (SphK), the enzyme that phosphorylates Sph to form S<em>1</em>P, is a critical regulator of the sphingolipid rheostat, as it not only produces the pro-growth, anti-apoptotic messenger S<em>1</em>P, but also decreases levels of pro-apoptotic Cer and Sph. Given the role of the sphingolipid rheostat in regulating growth and apoptosis, it is not surprising that sphingolipid metabolism is often found to be disregulated in cancer, a disease characterized by enhanced cell growth, diminished cell death, or both. Anticancer therapeutics targeting SphK are potentially clinically relevant. Indeed, inhibition of SphK has been shown to suppress gastric tumor growth [Cancer Res. 5<em>1</em> (<em>1</em>99<em>1</em>) <em>1</em>6<em>1</em>3] and conversely, overexpression of SphK increases tumorigenicity [Curr. Biol. <em>1</em>0 (2000) <em>1</em>527]. Moreover, S<em>1</em>P has also been shown to regulate angiogenesis, or new blood vessel formation [Cell 99 (<em>1</em>999) 30<em>1</em>], which is critical for tumor progression. Furthermore, there is intriguing new evidence that S<em>1</em>P can act in an autocrine and/or paracrine fashion [Science 29<em>1</em> (200<em>1</em>) <em>1</em>800] to regulate blood vessel formation [J. Clin. Invest. <em>1</em>06 (2000) 95<em>1</em>]. Thus, SphK may not only protect tumors from apoptosis, it may also increase their vascularization, further enhancing growth. The cytoprotective effects of SphK/S<em>1</em>P may also be important for clinical benefit, as S<em>1</em>P has been shown to protect oocytes from radiation-induced cell death in vivo [Nat. Med. 6 (2000) <em>1</em><em>1</em>09]. Here we review the growing literature on the regulation of SphK and the role of SphK and its product, S<em>1</em>P, in apoptosis.

The <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor signaling system is a productive model system. A hydrophobic zwitterionic lysophospholipid ligand with difficult physical properties interacts with five high-affinity G protein-coupled receptors to generate multiple downstream signals. These signals modulate homeostasis and pathology on a steep agonist concentration-response curve. Ligand presence is essential for vascular development and endothelial integrity, while acute increases in ligand concentrations result in cardiac death. Understanding this integrated biochemical system has exemplified the impact of both genetics and chemistry. Developing specific tools with defined biochemical properties for the reversible modulation of signals in real time has been essential to complement insights gained from genetic approaches that may be irreversible and compensated. Despite its knife-edge between life and death, this system, based in part on receptor subtype-selectivity and in part on differential attenuation of deleterious signals, now appears to be on the cusp of meaningful therapy for multiple sclerosis.