<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a platelet-derived ligand for the EDG-<em>1</em> family of G protein-coupled receptors (GPCRs), has recently emerged as a regulator of vascular development. Although S<em>1</em>P has potent effects on endothelial cells and vascular smooth muscle cells (VSMCs), the functions of the specific S<em>1</em>P receptors in the latter cell type are not known. Here we show that pup-intimal VSMCs express higher levels of EDG-<em>1</em> mRNA than adult-medial VSMCs. Stable transfection of EDG-<em>1</em> into adult-medial VSMCs enhanced their proliferative response to S<em>1</em>P, concomitant with induction of p70 S6 kinase activity and expression of cyclin D<em>1</em>. Pertussis toxin treatment inhibited S<em>1</em>P-induced p70 S6 kinase activation, cyclin D<em>1</em> expression and proliferation, suggesting that EDG-<em>1</em>-coupling to the G(i) pathway is critical. Furthermore, blocking p70 S6 kinase phosphorylation with rapamycin inhibited cyclin D<em>1</em> expression and proliferation, suggesting that activation of p70 S6 kinase is critical in EDG-<em>1</em>/G(i)-mediated cell proliferation. EDG-<em>1</em> expression also profoundly enhanced the migratory response of adult-medial VSMCs to S<em>1</em>P. S<em>1</em>P-induced migration of adult-medial VSMCs expressing exogenous EDG-<em>1</em> required G(i) activation but not p70 S6 kinase. These results suggest that enhanced expression of EDG-<em>1</em> in VSMCs dramatically stimulates both the proliferative and migratory responses to S<em>1</em>P. Since EDG-<em>1</em> is expressed in the pup-intimal phenotype of VSMCs, S<em>1</em>P signaling via EDG-<em>1</em> may play a role in vascular diseases in which the proliferation and migration of VSMCs are dysregulated.

Mechanisms by which cancer cells communicate with the host organism to regulate lung colonization/metastasis are unclear. We show that this communication occurs via <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) generated systemically by <em>sphingosine</em> kinase <em>1</em> (SK<em>1</em>), rather than via tumour-derived S<em>1</em>P. Modulation of systemic, but not tumour SK<em>1</em>, prevented S<em>1</em>P elevation, and inhibited TRAMP-induced prostate cancer growth in TRAMP(+/+) SK<em>1</em>(-/-) mice, or lung metastasis of multiple cancer cells in SK<em>1</em>(-/-) animals. Genetic loss of SK<em>1</em> activated a master metastasis suppressor, Brms<em>1</em> (breast carcinoma metastasis suppressor <em>1</em>), via modulation of S<em>1</em>P receptor 2 (S<em>1</em>PR2) in cancer cells. Alterations of S<em>1</em>PR2 using pharmacologic and genetic tools enhanced Brms<em>1</em>. Moreover, Brms<em>1</em> in S<em>1</em>PR2(-/-) MEFs was modulated by serum S<em>1</em>P alterations. Accordingly, ectopic Brms<em>1</em> in MB49 bladder cancer cells suppressed lung metastasis, and stable knockdown of Brms<em>1</em> prevented this process. Importantly, inhibition of systemic S<em>1</em>P signalling using a novel anti-S<em>1</em>P monoclonal antibody (mAb), Sphingomab, attenuated lung metastasis, which was prevented by Brms<em>1</em> knockdown in MB49 cells. Thus, these data suggest that systemic SK<em>1</em>/S<em>1</em>P regulates metastatic potential via regulation of tumour S<em>1</em>PR2/Brms<em>1</em> axis.

A wealth of indirect data suggest that the H2<em>1</em>8/AGR<em>1</em>6/Edg-5/LP(B2) <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor plays important roles in development. In vitro, it activates several forms of development-related signal transduction and regulates cellular proliferation, differentiation and survival. It is expressed during embryogenesis, and mutation of an H2<em>1</em>8-like gene in zebrafish leads to profound defects in embryonic development. Nevertheless, the in vivo functions served by H2<em>1</em>8 signalling have not been directly investigated. We report here that mice in which the H2<em>1</em>8 gene has been disrupted are unexpectedly born with no apparent anatomical or physiological defects. In addition, no abnormalities were observed in general neurological development, peripheral axon growth or brain structure. However, between 3 and 7 weeks of age, H2<em>1</em>8(-/-) mice have seizures which are spontaneous, sporadic and occasionally lethal. Electroencephalographic abnormalities were identified both during and between the seizures. At a cellular level, whole-cell patch-clamp recordings revealed that the loss of H2<em>1</em>8 leads to a large increase in the excitability of neocortical pyramidal neurons. Therefore, H2<em>1</em>8 plays an essential, unanticipated and functionally important role in the proper development and/or mediation of neuronal excitability.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and lysophosphatidic acid (LPA) are blood-borne lysophospholipids with a wide spectrum of biological activities, which include stimulation of cell growth, prevention of apoptosis, regulation of actin cytoskeleton, and modulation of cell shape, cell migration, and invasion. Activated platelets appear to be a major source of both S<em>1</em>P and LPA in blood. Despite the diversity of their biosynthetic origins, they are considered to share substantial structural similarity. Indeed, recent investigation has revealed that S<em>1</em>P and LPA act via a single family of G protein-coupled receptors designated as Edg. Thus, the Edg isoforms, Edg<em>1</em> (also called S<em>1</em>P(<em>1</em>)), Edg5 (S<em>1</em>P(2)), Edg3 (S<em>1</em>P(3)), Edg6 (S<em>1</em>P(4)), and Edg8 (S<em>1</em>P(5)), are specific receptors for S<em>1</em>P (and SPC with a lower affinity), whereas Edg2 (LPA(<em>1</em>)), Edg4 (LPA(2)), and Edg7 (LPA(3)) serve as receptors specific for LPA. Each receptor isoform displays a unique tissue expression pattern and coupling to a distinct set of heterotrimeric G proteins, leading to the activation of an isoform-specific panel of multiple intracellular signaling pathways. Recent studies on knockout mice have unveiled non-redundant Edg receptor functions that are essential for normal development and vascular maturation. In addition, the Edg lysophospholipid signaling system may play a role in modulating cell motility under such pathological conditions as inflammation, tumor cell dissemination and vascular remodeling.

The sphingolipids ceramide, <em>sphingosine</em>, and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) regulate cell signaling, proliferation, apoptosis, and autophagy. <em>Sphingosine</em> kinase-<em>1</em> and -2 (SK<em>1</em> and SK2) phosphorylate <em>sphingosine</em> to form S<em>1</em>P, shifting the balanced activity of these lipids toward cell proliferation. We have previously reported that pharmacological inhibition of SK activity delays tumor growth in vivo. The present studies demonstrate that the SK2-selective inhibitor 3-(4-chlorophenyl)-adamantane-<em>1</em>-carboxylic acid (pyridin-4-ylmethyl)amide (ABC294640) induces nonapoptotic cell death that is preceded by microtubule-associated protein light chain 3 cleavage, morphological changes in lysosomes, formation of autophagosomes, and increases in acidic vesicles in A-498 kidney carcinoma cells. ABC294640 caused similar autophagic responses in PC-3 prostate and MDA-MB-23<em>1</em> breast adenocarcinoma cells. Simultaneous exposure of A-498 cells to ABC294640 and 3-methyladenine, an inhibitor of autophagy, switched the mechanism of toxicity to apoptosis, but decreased the potency of the SK2 inhibitor, indicating that autophagy is a major mechanism for tumor cell killing by this compound. Induction of the unfolded protein response by the proteasome inhibitor N-(benzyloxycarbonyl)leucinylleucinylleucinal Z-Leu-Leu-Leu-al (MG-<em>1</em>32) or the heat shock protein 90 inhibitor geldanamycin synergistically increased the cytotoxicity of ABC294640 in vitro. In severe combined immunodeficient mice bearing A-498 xenografts, daily administration of ABC294640 delayed tumor growth and elevated autophagy markers, but did not increase terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells in the tumors. These data suggest that ABC294640 promotes tumor cell autophagy, which ultimately results in nonapoptotic cell death and a delay of tumor growth in vivo. Consequently, ABC294640 may effectively complement anticancer drugs that induce tumor cell apoptosis.

Systemic exacerbation of allergic responses, in which mast cells play a critical role, results in life-threatening anaphylactic shock. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a ligand for a family of G protein-coupled receptors, is a new addition to the repertoire of bioactive lipids secreted by activated mast cells. Yet little is known of its role in human mast cell functions and in anaphylaxis. We show that S<em>1</em>P(2) receptors play a critical role in regulating human mast cell functions, including degranulation and cytokine and chemokine release. Immunoglobulin E-triggered anaphylactic responses, including elevation of circulating histamine and associated pulmonary edema in mice, were significantly attenuated by the S<em>1</em>P(2) antagonist JTE-0<em>1</em>3 and in S<em>1</em>P(2)-deficient mice, in contrast to anaphylaxis induced by administration of histamine or platelet-activating factor. Hence, S<em>1</em>P and S<em>1</em>P(2) on mast cells are determinants of systemic anaphylaxis and associated pulmonary edema and might be beneficial targets for anaphylaxis attenuation and prophylaxis.

Fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] is cytotoxic in many cancer cell types. Studies have shown that elevation of ceramide species plays a role in 4-HPR cytotoxicity. To determine 4-HPR activity in a multidrug-resistant cancer cell line as well as to study ceramide metabolism, MCF-7/AdrR cells (redesignated NCI/ADR-RES) were treated with 4-HPR and sphingolipids were analyzed. TLC analysis of cells radiolabeled with [3H]palmitic acid showed that 4-HPR elicited a dose-responsive increase in radioactivity migrating in the ceramide region of the chromatogram and a decrease in cell viability. Results from liquid chromatography/electrospray tandem mass spectrometry revealed large elevations in dihydroceramides (N-acylsphinganines), but not desaturated ceramides, and large increases in complex dihydrosphingolipids (dihydrosphingomyelins, monohexosyldihydroceramides), sphinganine, and sphinganine <em>1</em>-<em>phosphate</em>. To test the hypothesis that elevation of sphinganine participates in the cytotoxicity of 4-HPR, cells were treated with the <em>sphingosine</em> kinase inhibitor d-erythro-N,N-dimethyl<em>sphingosine</em> (DMS), with and without 4-HPR. After 24 h, the 4-HPR/DMS combination caused a 9-fold increase in sphinganine that was sustained through +48 hours, decreased sphinganine <em>1</em>-<em>phosphate</em>, and increased cytotoxicity. Increased dihydrosphingolipids and sphinganine were also found in HL-60 leukemia cells and HT-29 colon cancer cells treated with 4-HPR. The 4-HPR/DMS combination elicited increased apoptosis in all three cell lines. We propose that a mechanism of 4-HPR-induced cytotoxicity involves increases in dihydrosphingolipids, and that the synergy between 4-HPR and DMS is associated with large increases in cellular sphinganine. These studies suggest that enhanced clinical efficacy of 4-HPR may be realized through regimens containing agents that modulate sphingoid base metabolism.

Millions of platelets are produced each hour by bone marrow (BM) megakaryocytes (MKs). MKs extend transendothelial proplatelet (PP) extensions into BM sinusoids and shed new platelets into the blood. The mechanisms that control platelet generation remain incompletely understood. Using conditional mutants and intravital multiphoton microscopy, we show here that the lipid mediator <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) serves as a critical directional cue guiding the elongation of megakaryocytic PP extensions from the interstitium into BM sinusoids and triggering the subsequent shedding of PPs into the blood. Correspondingly, mice lacking the S<em>1</em>P receptor S<em>1</em>pr<em>1</em> develop severe thrombocytopenia caused by both formation of aberrant extravascular PPs and defective intravascular PP shedding. In contrast, activation of S<em>1</em>pr<em>1</em> signaling leads to the prompt release of new platelets into the circulating blood. Collectively, our findings uncover a novel function of the S<em>1</em>P-S<em>1</em>pr<em>1</em> axis as master regulator of efficient thrombopoiesis and might raise new therapeutic options for patients with thrombocytopenia.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a sphingolipid metabolite, promotes cell proliferation and survival whereas its precursor, <em>sphingosine</em>, has the opposite effects. However, much remains unknown about their regulation. Here we identify a novel human ceramidase (haCER2) that regulates the levels of both <em>sphingosine</em> and S<em>1</em>P by controlling the hydrolysis of ceramides. haCER2 is localized to the Golgi complex and is highly expressed in the placenta. High ectopic expression of haCER2 caused fragmentation of the Golgi complex and growth arrest in HeLa cells due to <em>sphingosine</em> accumulation. Low ectopic expression of haCER2 increased S<em>1</em>P without <em>sphingosine</em> accumulation, promoting cell proliferation in serum-free medium. This proliferative effect was suppressed by dimethyl<em>sphingosine</em>, an inhibitor of the S<em>1</em>P formation, or by the RNA interference (RNAi) -mediated inhibition of S<em>1</em>P(<em>1</em>,) a G-protein-coupled receptor for S<em>1</em>P. The RNAi-mediated down-regulation of haCER2 enhanced the serum deprivation-induced growth arrest and apoptosis of HeLa cells, which was inhibited by addition of exogenous S<em>1</em>P. Serum deprivation up-regulated both haCER2 mRNA and activity in HeLa cells. haCER2 mRNA is also up-regulated in some tumors. Taken together, these results suggest that haCER2 is important for the generation of S<em>1</em>P and S<em>1</em>P-mediated cell proliferation and survival, but that its overexpression may cause cell growth arrest due to an accumulation of <em>sphingosine</em>.

To determine whether activation of the neutral sphingomyelinase pathway was responsible for the immediate (<30 min) negative inotropic effects of tumor necrosis factor-alpha (TNF-alpha), we examined <em>sphingosine</em> levels in diluent and TNF-alpha-stimulated cardiac myocytes. TNF-alpha stimulation of adult feline cardiac myocytes provoked a rapid ((<em>1</em>5 min) increase in the hydrolysis of [<em>1</em>4C]sphingomyelin in cell-free extracts, as well as an increase in ceramide mass, consistent with cytokine-induced activation of the neutral sphingomyelinase pathway. High performance liquid chromatographic analysis of lipid extracts from TNF-alpha-stimulated cardiac myocytes showed that TNF-alpha stimulation produced a rapid (<30 min) increase in free <em>sphingosine</em> levels. Moreover, exogenous D-<em>sphingosine</em> mimicked the effects of TNF-alpha on intracellular calcium homeostasis, as well as the negative inotropic effects of TNF-alpha in isolated contracting myocytes; time course studies showed that exogenous D-<em>sphingosine</em> produced abnormalities in cell shortening that were maximal at 5 min. Finally, blocking <em>sphingosine</em> production using an inhibitor of ceramidase, n-oleoylethanolamine, completely abrogated the negative inotropic effects of TNF-alpha in isolated contracting cardiac myocytes. Additional studies employing biologically active ceramide analogs and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> suggested that neither the immediate precursor of <em>sphingosine</em> nor the immediate metabolite of <em>sphingosine</em>, respectively, were likely to be responsible for the immediate negative inotropic effects of TNF-alpha. Thus, these studies suggest that <em>sphingosine</em> mediates the immediate negative inotropic effects of TNF-alpha in isolated cardiac myocytes.

Upon various stimuli, cells metabolize sphingomyelin from the cellular plasma membrane to form sphingosylphosphorylcholine (SPC) or ceramide. The latter can be further metabolized to <em>sphingosine</em> and then <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). Apart from local formation, S<em>1</em>P and SPC are major constituents of blood plasma. All four sphingomyelin metabolites (SMM) can act upon intracellular targets, and at least S<em>1</em>P and probably also SPC can additionally act upon G-protein-coupled receptors. While the molecular identity of the SPC receptors remains unclear, several subtypes of S<em>1</em>P receptors have been cloned and their distribution in cardiovascular tissues is described. In the heart SMM can alter intracellular Ca(2+) release, particularly via the ryanodine receptor, and conductance of various ion channels in the plasma membrane, particularly I(K(Ach)). While the various SMM differ somewhat in their effects, the above alterations of ion homeostasis result in reduced cardiac function in most cases, and ceramide and/or <em>sphingosine</em> may be the mediators of the negative inotropic effects of tumour necrosis factor. In the vasculature, SMM mainly act as acute vasoconstrictors in most vessels, but ceramide can be a vasodilator. SMM-induced vasoconstriction involves mobilization of Ca(2+) from intracellular stores, influx of extracellular Ca(2+) via L-type channels and activation of a rho-kinase. Extended exposure to SMM, particularly S<em>1</em>P, promotes several stages of the angiogenic process like endothelial cell activation, migration, proliferation, tube formation and vascular maturation. We propose that SMM are an important class of endogenous modulators of cardiovascular function.

OBJECTIVE

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) selectively and potently constricts isolated cerebral arteries, but this response has not been pharmacologically characterized.

METHODS

The receptor subtype(s) involved in S<em>1</em>P-induced cerebrovascular constriction were characterized using genetic (S<em>1</em>P(2) and S<em>1</em>P(3) receptor null mice) and pharmacological tools (phospho-FTY720, a S<em>1</em>P(<em>1</em>/3/4/5) receptor agonist; SEW287<em>1</em>, a S<em>1</em>P(<em>1</em>) receptor agonist, JTE-0<em>1</em>3, a S<em>1</em>P(2) receptor antagonist, VPC230<em>1</em>9, a S<em>1</em>P(<em>1</em>/3) receptor antagonist). Isolated basilar or peripheral (femoral, mesenteric resistance) arteries, from either rat or mouse, were studied in a wire myograph.

RESULTS

S<em>1</em>P concentration-dependently constricted basilar artery in rat, wild-type (WT) and S<em>1</em>P(2) null mice, but barely affected vascular tone in S<em>1</em>P(3) null mice. Vasoconstriction to U466<em>1</em>9 (a thromboxane analogue) or to endothelin-<em>1</em> did not differ between WT, S<em>1</em>P(2) and S<em>1</em>P(3) null mice. JTE-0<em>1</em>3 inhibited not only S<em>1</em>P-induced vasoconstriction, but also KCl-, U466<em>1</em>9- and endothelin-<em>1</em>-induced constriction. This effect was observed in WT as well as in S<em>1</em>P(2) null mice. VPC230<em>1</em>9 increased the concentration-dependent vasoconstriction to S<em>1</em>P in both rat and mouse basilar arteries with intact endothelium, but not in rat basilar artery without endothelium. Phospho-FTY720 concentration-dependently constricted rat basilar arteries, but not femoral or mesenteric resistance arteries, while SEW287<em>1</em> did not induce any response in the same arteries.

CONCLUSIONS

S<em>1</em>P constricts cerebral arteries through S<em>1</em>P(3) receptors. The purported S<em>1</em>P(2) receptor antagonist JTE-0<em>1</em>3 does not appear to be selective, at least in rodents. Enhancement of S<em>1</em>P-induced contraction by VPC230<em>1</em>9 might be related to blockade of S<em>1</em>P(<em>1</em>) receptors and NO generation.

Lysophosphatidic acid (LPA) exhibits a wide variety of biological functions as a bio-active lysophospholipid through G-protein-coupled receptors specific to LPA. Currently at least six LPA receptors are identified, named LPA(<em>1</em>) to LPA(6), while the existence of other LPA receptors has been suggested. From studies on knockout mice and hereditary diseases of these LPA receptors, it is now clear that LPA is involved in various biological processes including brain development and embryo implantation, as well as patho-physiological conditions including neuropathic pain and pulmonary and renal fibrosis. Unlike <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, a structurally similar bio-active lysophospholipid to LPA and produced intracellularly, LPA is produced by multiple extracellular degradative routes. A plasma enzyme called autotaxin (ATX) is responsible for the most of LPA production in our bodies. ATX converts lysophospholipids such as lysophosphatidylcholine to LPA by its lysophospholipase D activity. Recent studies on ATX have revealed new aspects of LPA. In this review, we highlight recent advances in our understanding of LPA functions and several aspects of ATX, including its activity, expression, structure, biochemical properties, the mechanism by which it stimulates cell motility and its pahto-physiological function through LPA production.

Multiple sclerosis (MS) is an autoimmune, neurological disability with unknown etiology. The current therapies available for MS work by an immunomodulatory action, preventing T-cell- and macrophage-mediated destruction of brain-resident oligodendrocytes and axonal loss. Recently, FTY720 (fingolimod) was shown to significantly reduce relapse rates in MS patients and is currently in Phase III clinical trials. This drug attenuates trafficking of harmful T cells entering the brain by regulating <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors. Here, we outline the direct roles that S<em>1</em>P receptors play in the central nervous system (CNS) and discuss additional modalities by which FTY720 may provide direct neuroprotection in MS.

OBJECTIVE

Exosomes are small membrane vesicles involved in intercellular communication. Hepatocytes are known to release exosomes, but little is known about their biological function. We sought to determine if exosomes derived from hepatocytes contribute to liver repair and regeneration after injury.

METHODS

Exosomes derived from primary murine hepatocytes were isolated and characterized biochemically and biophysically. Using cultures of primary hepatocytes, we tested whether hepatocyte exosomes induced proliferation of hepatocytes in vitro. Using models of ischemia/reperfusion injury and partial hepatectomy, we evaluated whether hepatocyte exosomes promote hepatocyte proliferation and liver regeneration in vivo.

RESULTS

Hepatocyte exosomes, but not exosomes from other liver cell types, induce dose-dependent hepatocyte proliferation in vitro and in vivo. Mechanistically, hepatocyte exosomes directly fuse with target hepatocytes and transfer neutral ceramidase and <em>sphingosine</em> kinase 2 (SK2) causing increased synthesis of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) within target hepatocytes. Ablation of exosomal SK prevents the proliferative effect of exosomes. After ischemia/reperfusion injury, the number of circulating exosomes with proliferative effects increases.

CONCLUSIONS

Our data shows that hepatocyte-derived exosomes deliver the synthetic machinery to form S<em>1</em>P in target hepatocytes resulting in cell proliferation and liver regeneration after ischemia/reperfusion injury or partial hepatectomy. These findings represent a potentially novel new contributing mechanism of liver regeneration and have important implications for new therapeutic approaches to acute and chronic liver disease.

FTY720 is a potent immunomodulator drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. FTY720 is phosphorylated in vivo by <em>sphingosine</em> kinase 2 to FTY720-<em>phosphate</em>, which acts as a potent <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor agonist. However, in contrast to S<em>1</em>P, FTY720 has no effect on mast-cell degranulation, yet significantly reduces antigen-induced secretion of PGD2 and cysteinyl-leukotriene. Unexpectedly, this effect of FTY720 was independent of its phosphorylation and S<em>1</em>P receptor functions. The rate-limiting step in the biosynthesis of all eicosanoids is the phospholipase A2 (PLA2)-mediated release of arachidonic acid from glycerol phospholipids. Although FTY720 also reduced arachidonic acid release in response to antigen, it had no effect on translocation of cPLA2 or ERK<em>1</em>/2 activation, suggesting that it does not interfere with FcepsilonRI-mediated events leading to cPLA2 activation. Remarkably, however, FTY720 drastically inhibited recombinant cPLA2alpha activity, whereas FTY720-<em>phosphate</em>, <em>sphingosine</em>, or S<em>1</em>P had no effect. This study has uncovered a unique action of FTY720 as an inhibitor of cPLA2alpha and hence on production of all eicosanoids. Our results have important implications for the potential therapeutic mechanism of action of FTY720 in eicosanoid-driven inflammatory disorders such as asthma and multiple sclerosis.

The <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor agonist FTY720 is a novel immunomodulator that sequesters lymphocytes in secondary lymphoid organs and thereby prevents their migration to sites of inflammation. However, there is currently no information available on whether this drug affects Th<em>1</em> or Th2 cell-mediated lung-inflammatory responses. The effect of FTY720 was therefore investigated in a murine airway inflammation model using OVA-specific, in vitro differentiated, and adoptively transferred Th<em>1</em> and Th2 cells. Both Th<em>1</em> and Th2 cells express a similar pattern of FTY720-targeted <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptors. The OVA-induced Th<em>1</em>-mediated airway inflammation characterized by increased numbers of lymphocytes and neutrophils in bronchoalveolar lavage fluid was significantly inhibited by oral FTY720 treatment. Similarly, FTY720 suppressed the Th2 cell-induced bronchoalveolar lavage fluid eosinophilia and the infiltration of T lymphocytes and eosinophils into the bronchial tissue. Moreover, the Ag-induced bronchial hyperresponsiveness to inhaled metacholine was almost completely blocked. The inhibitory effect of FTY720 on airway inflammation, induction of bronchial hyperresponsiveness, and goblet cell hyperplasia could be confirmed in an actively Ag-sensitized murine asthma model, clearly indicating that Th2 cell-driven allergic diseases such as asthma could benefit from such treatment.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a lysophospholipid mediator that signals through G protein-coupled receptors, regulates a wide plethora of biological responses such as angiogenesis and immune cell trafficking. Detection and quantification of S<em>1</em>P in biological samples is challenging due to its unique physicochemical nature and occurrence in trace quantities. In this report, we describe a new method to selectively enrich S<em>1</em>P and dihydro-S<em>1</em>P from biological samples by the Fe(3+) gel immobilized metal affinity chromatography (IMAC). The eluted S<em>1</em>P from IMAC was dephosphorylated, derivatized with o-phthalaldehyde (OPA), and detected by high-performance liquid chromatography (HPLC) coupled to a fluorescence detector. IMAC purification of S<em>1</em>P was linear for a wide range of S<em>1</em>P concentration. Using this assay, secretion of endogenous S<em>1</em>P from endothelial cells, fibroblasts and colon cancer cells was demonstrated. We also show that dihydro-S<em>1</em>P was the major sphingoid base <em>phosphate</em> secreted from HUVEC over expressed with Sphk<em>1</em> cDNA. Pharmcological antagonists of ABC transporters, glyburide and MK-57<em>1</em> attenuated endogenous S<em>1</em>P release. This assay was also used to demonstrate that plasma S<em>1</em>P levels were not altered in mice deficient for ABC transporters, Abca<em>1</em>, Abca7 and Abcc<em>1</em>/Mrp<em>1</em>. IMAC-based affinity-enrichment coupled with a HPLC-based separation and detection system is a rapid and sensitive method to accurately quantify S<em>1</em>P.

OBJECTIVE

<em>Sphingosine</em> kinase <em>1</em> (SPHK<em>1</em>), its product <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), and S<em>1</em>P receptor subtypes have been suggested to play protective roles for cardiomyocytes in animal models of ischaemic preconditioning and cardiac ischaemia/reperfusion injury. To get more insight into roles for SPHK<em>1</em> in vivo, we have generated SPHK<em>1</em>-transgenic (TG) mice and analysed the cardiac phenotype.

RESULTS

SPHK<em>1</em>-TG mice overexpressed SPHK<em>1</em> in diverse tissues, with a nearly 20-fold increase in enzymatic activity. The TG mice grew normally with normal blood chemistry, cell counts, heart rate, and blood pressure. Unexpectedly, TG mice with high but not low expression levels of SPHK<em>1</em> developed progressive myocardial degeneration and fibrosis, with upregulation of embryonic genes, elevated RhoA and Rac<em>1</em> activity, stimulation of Smad3 phosphorylation, and increased levels of oxidative stress markers. Treatment of juvenile TG mice with pitavastatin, an established inhibitor of the Rho family G proteins, or deletion of S<em>1</em>P3, a major myocardial S<em>1</em>P receptor subtype that couples to Rho GTPases and transactivates Smad signalling, both inhibited cardiac fibrosis with concomitant inhibition of SPHK<em>1</em>-dependent Smad-3 phosphorylation. In addition, the anti-oxidant N-2-mercaptopropyonylglycine, which reduces reactive oxygen species (ROS), also inhibited cardiac fibrosis. In in vivo ischaemia/reperfusion injury, the size of myocardial infarct was 30% decreased in SPHK<em>1</em>-TG mice compared with wild-type mice.

CONCLUSIONS

These results suggest that chronic activation of SPHK<em>1</em>-S<em>1</em>P signalling results in both pathological cardiac remodelling through ROS mediated by S<em>1</em>P3 and favourable cardioprotective effects.

The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N<em>1</em>E-<em>1</em><em>1</em>5 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, or thrombin acting on their cognate G protein-coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (<em>1</em>90 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a <em>1</em><em>1</em>6-kD protein, p<em>1</em><em>1</em>6(Rip), that interacts with both the GDP- and GTP-bound forms of RhoA in N<em>1</em>E-<em>1</em><em>1</em>5 cells. Overexpression of p<em>1</em><em>1</em>6(Rip) stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p<em>1</em><em>1</em>6(Rip) fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein-coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p<em>1</em><em>1</em>6(Rip) inhibits RhoA-stimulated contractility and promotes neurite outgrowth.

The role of the common neurotrophin receptor p75 (p75NTR) in neuronal survival and cell death remains controversial. On the one hand, p75NTR provides a positive modulatory influence on nerve growth factor (NGF) signaling through the high affinity neurotrophin receptor TrkA, and hence increases NGF survival signaling. However, p75NTR may also signal independently of TrkA, causing cell death or cell survival, depending on the cell type and stage of development. Here we demonstrate that TrkA is expressed in primary cultures of hippocampal neurons and is activated by NGF within <em>1</em>0 min of exposure. In primary hippocampal cultures neuroprotection by NGF against glutamate toxicity was mediated by NF-kappaB and accompanied by an increased expression of neuroprotective NF-kappaB target genes Bcl-2 and Bcl-xl. In mouse hippocampal cells lacking p75NTR (p75NTR-/-) activation of TrkA by NGF was not detectable. Moreover, neuroprotection by NGF against glutamate toxicity was abolished in p75NTR-/- neurons, and the expression of bcl-2 and bcl-xl was markedly reduced as compared to wildtype cells. NGF increased TrkA phosphorylation in hippocampal neurons and provided protection that required phosphoinositol-3-<em>phosphate</em> (PI3)-kinase activity and Akt phosphorylation, whereas the mitogen-activated protein kinases (MAPK), extracellular-regulated kinases (Erk) <em>1</em>/2, were not involved. P75NTR signaling independent of TrkA, such as increased neutral sphingomyelinase (NSMase) activity causing enhanced levels of ceramide, were not detected after exposure of hippocampal neurons to NGF. Interestingly, inhibition of <em>sphingosine</em>-kinase blocked the neuroprotective effect of NGF, suggesting that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> was also involved in NGF-mediated survival in our cultured hippocampal neurons. Overall, our results indicate an essential role for p75NTR in supporting NGF-triggered TrkA signaling pathways mediating neuronal survival in hippocampal neurons.

FTY720 (fingolimod), an FDA-approved drug for treatment of multiple sclerosis, has beneficial effects in the CNS that are not yet well understood, independent of its effects on immune cell trafficking. We show that FTY720 enters the nucleus, where it is phosphorylated by <em>sphingosine</em> kinase 2 (SphK2), and that nuclear FTY720-P binds and inhibits class I histone deacetylases (HDACs), enhancing specific histone acetylations. FTY720 is also phosphorylated in mice and accumulates in the brain, including the hippocampus, inhibits HDACs and enhances histone acetylation and gene expression programs associated with memory and learning, and rescues memory deficits independently of its immunosuppressive actions. Sphk2(-/-) mice have lower levels of hippocampal <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, an endogenous HDAC inhibitor, and reduced histone acetylation, and display deficits in spatial memory and impaired contextual fear extinction. Thus, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> and SphK2 play specific roles in memory functions and FTY720 may be a useful adjuvant therapy to facilitate extinction of aversive memories.

On the basis of our previous studies on RhoA signaling in smooth muscle cells (SMC), we hypothesized that RhoA-mediated nuclear translocalization of the myocardin-related transcription factors (MRTFs) was important for regulating SMC phenotype. MRTF-A protein and MRTF-B message were detected in aortic SMC and in many adult mouse organs that contain a large SMC component. Both MRTFs upregulated SMC-specific promoter activity as well as endogenous SM22alpha expression in multipotential <em>1</em>0T<em>1</em>/2 cells, although to a lesser extent than myocardin. We used enhanced green fluorescent protein (EGFP) fusion proteins to demonstrate that the myocardin factors have dramatically different localization patterns and that the stimulation of SMC-specific transcription by certain RhoA-dependent agonists was likely mediated by increased nuclear translocation of the MRTFs. Importantly, a dominant-negative form of MRTF-A (DeltaB<em>1</em>/B2) that traps endogenous MRTFs in the cytoplasm inhibited the SM alpha-actin, SM22alpha, and SM myosin heavy chain promoters in SMC and attenuated the effects of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> and transforming growth factor (TGF)-beta on SMC-specific transcription. Our data confirmed the importance of the NH(2)-terminal RPEL domains for regulating MRTF localization, but our analysis of MRTF-A/myocardin chimeras and myocardin RPEL2 mutations indicated that the myocardin B<em>1</em>/B2 region can override this signal. Gel shift assays demonstrated that myocardin factor activity correlated well with ternary complex formation at the SM alpha-actin CArGs and that MRTF-serum response factor interactions were partially dependent on CArG sequence. Taken together, our results indicate that the MRTFs regulate SMC-specific gene expression in at least some SMC subtypes and that regulation of MRTF nuclear localization may be important for the effects of selected agonists on SMC phenotype.

Fingolimod, a <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor (S<em>1</em>PR) modulator, was the first oral disease-modifying therapy approved for relapsing forms of multiple sclerosis; it reduces autoreactive lymphocytes' egress from lymphoid tissues by down-regulating S<em>1</em>PRs. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> signaling is implicated in a range of physiologic functions, and S<em>1</em>PRs are expressed differentially in various tissues, including the cardiovascular system. Modulation of S<em>1</em>PRs on cardiac cells provides an explanation for the transient effects of fingolimod on heart rate and atrioventricular conduction at initiation of fingolimod therapy, and for the mild but more persistent effects on blood pressure observed in some patients on long-term treatment. This review describes the nontherapeutic actions of fingolimod in the context of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> signaling in the cardiovascular system, as well as providing a summary of the associated clinical implications useful to physicians considering initiation of fingolimod therapy in patients. A transient reduction in heart rate (mean decrease of 8 beats per minute) and, less commonly, a temporary delay in atrioventricular conduction observed in some patients when initiating fingolimod therapy are both due to activation of S<em>1</em>PR subtype <em>1</em> on cardiac myocytes. These effects are a reflection of fingolimod first acting as a full S<em>1</em>PR agonist and thereafter functioning as an S<em>1</em>PR antagonist after down-regulation of S<em>1</em>PR subtype <em>1</em> at the cell surface. For most individuals, first-dose effects of fingolimod are asymptomatic, but all patients need to be monitored for at least 6 hours after the first dose, in accordance with the label recommendations.