The bioactive sphingolipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) that is increased in airways of asthmatic subjects markedly induced contraction of human airway smooth muscle (HASM) cells embedded in collagen matrices in a Gi-independent manner. Dihydro-S<em>1</em>P, which binds to S<em>1</em>P receptors, also stimulated contractility. S<em>1</em>P induced formation of stress fibers, contraction of individual HASM cells, and stimulated myosin light chain phosphorylation, which was inhibited by the Rho-associated kinase inhibitor Y-27632. S<em>1</em>P-stimulated HASM cell contractility was independent of the ERK<em>1</em>/2 and PKC signaling pathways, important regulators of airway smooth muscle contraction. However, removal of extracellular calcium completely blocked S<em>1</em>P-mediated contraction and Y-27632 reduced it. S<em>1</em>P also induced calcium mobilization that was not desensitized by repeated additions. Pretreatment with thapsigargin to deplete InsP3-sensitive calcium stores partially blocked increases in [Ca2+]i induced by S<em>1</em>P, yet did not inhibit S<em>1</em>P-stimulated contraction. In sharp contrast, the L-type calcium channel blocker verapamil markedly decreased S<em>1</em>P-induced HASM cell contraction, supporting a role for calcium influx from extracellular sources. Collectively, our results suggest that S<em>1</em>P may regulate HASM contractility, important in the pathobiology of asthma.

The lymphatic system plays pivotal roles in mediating tissue fluid homeostasis and immunity, and excessive lymphatic vessel formation is implicated in many pathological conditions, which include inflammation and tumor metastasis. However, the molecular mechanisms that regulate lymphatic vessel formation remain poorly characterized. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a potent bioactive lipid that is implicated in a variety of biologic processes such as inflammatory responses and angiogenesis. Here, we first report that S<em>1</em>P acts as a lymphangiogenic mediator. S<em>1</em>P induced migration, capillary-like tube formation, and intracellular Ca(2+) mobilization, but not proliferation, in human lymphatic endothelial cells (HLECs) in vitro. Moreover, a Matrigel plug assay demonstrated that S<em>1</em>P promoted the outgrowth of new lymphatic vessels in vivo. HLECs expressed S<em>1</em>P<em>1</em> and S<em>1</em>P3, and both RNA interference-mediated down-regulation of S<em>1</em>P<em>1</em> and an S<em>1</em>P<em>1</em> antagonist significantly blocked S<em>1</em>P-mediated lymphangiogenesis. Furthermore, pertussis toxin, U73<em>1</em>22, and BAPTA-AM efficiently blocked S<em>1</em>P-induced in vitro lymphangiogenesis and intracellular Ca(2+) mobilization of HLECs, indicating that S<em>1</em>P promotes lymphangiogenesis by stimulating S<em>1</em>P<em>1</em>/G(i)/phospholipase C/Ca(2+) signaling pathways. Our results suggest that S<em>1</em>P is the first lymphangiogenic bioactive lipid to be identified, and that S<em>1</em>P and its receptors might serve as new therapeutic targets against inflammatory diseases and lymphatic metastasis in tumors.

SK<em>1</em> (<em>sphingosine</em> kinase <em>1</em>) plays an important role in many aspects of cellular regulation. Most notably, elevated cellular SK<em>1</em> activity leads to increased cell proliferation, protection from apoptosis, and induction of neoplastic transformation. We have previously shown that translocation of SK<em>1</em> from the cytoplasm to the plasma membrane is integral for oncogenesis mediated by this enzyme. The molecular mechanism mediating this translocation of SK<em>1</em> has remained undefined. Here, we demonstrate a direct role for CIB<em>1</em> (calcium and integrin-binding protein <em>1</em>) in this process. We show that CIB<em>1</em> interacts with SK<em>1</em> in a Ca(2+)-dependent manner at the previously identified "calmodulin-binding site" of SK<em>1</em>. We also demonstrate that CIB<em>1</em> functions as a Ca(2+)-myristoyl switch, providing a mechanism whereby it translocates SK<em>1</em> to the plasma membrane. Both small interfering RNA knockdown of CIB<em>1</em> and the use of a dominant-negative CIB<em>1</em> we have generated prevent the agonist-dependent translocation of SK<em>1</em>. Furthermore, we demonstrate the requirement of CIB<em>1</em>-mediated translocation of SK<em>1</em> in controlling cellular <em>sphingosine</em> <em>1</em>-<em>phosphate</em> generation and associated anti-apoptotic signaling.

Ample evidence indicates that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP) can serve as an intracellular second messenger regulating calcium mobilization, and cell growth and survival. Moreover, the dynamic balance between levels of the sphingolipids metabolites, ceramide and SPP, and consequent regulation of opposing signaling pathways, is an important factor that determines whether a cell survives or dies. SPP has also recently been shown to be the ligand for the EDG-<em>1</em> family of G protein-coupled receptors, which now includes EDG-<em>1</em>, -3, -5, -6, and -8. SPP is thus a lipid mediator that has novel dual actions signaling inside and outside of the cell. This review is focussed on <em>sphingosine</em> kinase, the enzyme that regulates levels of SPP and thus plays a critical role in diverse biological processes.

Recent studies reveal that metabolites of sphingomyelin are critically important for initiation and maintenance of diverse aspects of immune cell activation and function. The conversion of sphingomyelin to ceramide, <em>sphingosine</em>, or <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) provides interconvertible metabolites with distinct biological activities. Whereas ceramide and <em>sphingosine</em> function to induce apoptosis and to dampen mast cell responsiveness, S<em>1</em>P functions as a chemoattractant and can up-regulate some effector responses. Many of the S<em>1</em>P effects are mediated through S<em>1</em>P receptor family members (S<em>1</em>P(<em>1</em>-5)). S<em>1</em>P(<em>1</em>), which is required for thymocyte emigration and lymphocyte recirculation, is also essential for Ag-induced mast cell chemotaxis, whereas S<em>1</em>P(2) is important for mast cell degranulation. S<em>1</em>P is released to the extracellular milieu by Ag-stimulated mast cells, enhancing inflammatory cell functions. Modulation of S<em>1</em>P receptor expression profiles, and of enzymes involved in sphingolipid metabolism, particularly <em>sphingosine</em> kinases, are key in balancing mast cell and immune cell responses. Current efforts are unraveling the complex underlying mechanisms regulating the sphingolipid pathway. Pharmacological intervention of these key processes may hold promise for controlling unwanted immune responses.

While most of the pharmacological therapies for melanoma utilize the apoptotic machinery of the cells, the available therapeutic options are limited due to the ability of melanoma cells to resist programmed cell death. Human melanoma cell lines A-375 and M<em>1</em>86 are sensitive to ceramide- and Fas-induced cell death, while Mel-2a and M22<em>1</em> are resistant. We have now found that Mel-2a and M22<em>1</em> cells have a significantly higher ceramide/<em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) ratio than A-375 and M<em>1</em>86 cells. As <em>sphingosine</em> kinase (SphK) type <em>1</em> plays a critical role in determining the dynamic balance between the proapoptotic sphingolipid metabolite ceramide and the prosurvival S<em>1</em>P, we examined its role in apoptosis of melanoma cells. Increasing SphK<em>1</em> expression reduced the sensitivity of A-375 melanoma cells to Fas- and ceramide-mediated apoptosis. Conversely, downregulation of SphK<em>1</em> with small interfering RNA decreased the resistance of Mel-2a cells to apoptosis. Importantly, overexpression of the prosurvival protein Bcl-2 in A-375 cells markedly stimulated SphK<em>1</em> expression and activity, while downregulation of Bcl-2 reduced SphK<em>1</em> expression. This link between Bcl-2 and SphK<em>1</em> might be an additional clue to chemotherapy resistance of malignant melanoma.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors are widely expressed in the central nervous system where they are thought to regulate glia cell function. The phosphorylated version of fingolimod/FTY720 (FTY720P) is active on a broad spectrum of S<em>1</em>P receptors and the parent compound is currently in phase III clinical trials for the treatment of multiple sclerosis. Here, we aimed to identify which cell type(s) and S<em>1</em>P receptor(s) of the central nervous system are targeted by FTY720P. Using calcium imaging in mixed cultures from embryonic rat cortex we show that astrocytes are the major cell type responsive to FTY720P in this assay. In enriched astrocyte cultures, we detect expression of S<em>1</em>P<em>1</em> and S<em>1</em>P3 receptors and demonstrate that FTY720P activates Gi protein-mediated signaling cascades. We also show that FTY720P as well as the S<em>1</em>P<em>1</em>-selective agonist SEW287<em>1</em> stimulate astrocyte migration. The data indicate that FTY720P exerts its effects on astrocytes predominantly via the activation of S<em>1</em>P<em>1</em> receptors, whereas S<em>1</em>P signals through both S<em>1</em>P<em>1</em> and S<em>1</em>P3 receptors. We suggest that this distinct pharmacological profile of FTY720P, compared with S<em>1</em>P, could play a role in the therapeutic effects of FTY720 in multiple sclerosis.

Missing in metastasis (MIM) gene encodes an actin binding protein that is expressed at low levels in a subset of malignant cell lines. MIM protein tagged by green fluorescent protein (GFP) colocalizes with cortactin, an Arp2/3 complex activator, and interacts directly with the SH3 domain of cortactin. Recombinant full-length MIM promotes markedly cortactin and Arp2/3 complex-mediated actin polymerization in an SH3 dependent manner. In contrast, MIM-CT, a short splicing variant of MIM, binds poorly to cortactin in vitro and is unable to enhance actin polymerization. Full-length MIM binds to G-actin with a similar affinity as N-WASP-VCA, a constitutively active form of N-WASP, and inhibits N-WASP-VCA-mediated actin polymerization as analysed in vitro. The significance of the association of MIM with cortactin and G-actin was evaluated in NIH3T3 cells expressing several MIM constructs. Overexpression of full-length wild-type MIM-GFP inhibited markedly the motility of NIH3T3 cells induced by PDGF and that of human vein umbilical endothelial cells induced by <em>sphingosine</em> <em>1</em> <em>phosphate</em>. However, an MIM mutant with deletion of the WH2 domain, which is responsible for G-actin binding, enhanced cell motility. The motility inhibition imposed by MIM was compromised in the cells overexpressing N-WASP. In contrast, deletion of an MIM proline-rich domain, which is required for an optimal binding to cortactin, substantiated the MIM-mediated inhibition of cell motility. These data imply that MIM regulates cell motility by modulating different Arp2/3 activators in a distinguished manner.

The aim of this study was to characterize endogenous nitroproteins, and those proteins that interact with nitroproteins, in a human pituitary nonfunctional adenoma so as to clarify the role of protein nitration in adenomas. A nitrotyrosine affinity column (NTAC) was used to preferentially enrich and isolate endogenous nitroproteins and nitroprotein-protein complexes from a tissue homogenate that was prepared from a human pituitary nonfunctional pituitary adenoma. The preferentially enriched endogenous nitroproteins and nitroprotein-protein complexes were subjected to trypsin digestion, desalination, and tandem mass spectrometry analysis. Nine nitroproteins (Rho-GTPase-activing protein 5, leukocyte immunoglobulin-like receptor subfamily A member 4 precursor, zinc finger protein 432, cAMP-dependent protein kinase type I-beta regulatory subunit, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase <em>1</em>, centaurin beta <em>1</em>, proteasome subunit alpha type 2, interleukin <em>1</em> family member 6, and rhophilin 2) and three proteins (interleukin <em>1</em> receptor-associated kinase-like 2, glutamate receptor-interacting protein 2, and ubiquitin) that interacted with nitroproteins were discovered. The nitration site of each nitroprotein was located onto the functional domain where nitration occurred, and each nitroprotein was related to a corresponding functional system. Those data indicate that protein nitration might be an important molecular event in the formation of a human pituitary nonfunctional adenoma.

The ability of pro-inflammatory cytokines such as interleukin-<em>1</em>beta (IL-<em>1</em>beta) to induce the major inflammatory mediator prostaglandin (PG) E(2) depends on the activation of two rate-limiting enzymes, phospholipase A(2) (PLA(2)) and cyclooxygenase 2 (COX-2). PLA(2) acts to generate arachidonic acid, which serves as the precursor substrate for COX-2 in the metabolic pathway leading to PGE(2) production. However, less is known about the mechanisms that coordinate the regulation of these two enzymes. We have provided prior evidence that <em>sphingosine</em> kinase <em>1</em> and its bioactive lipid product <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) mediate the effects of cytokines on COX-2 induction, whereas ceramide kinase and its distinct product, ceramide-<em>1</em>-<em>phosphate</em> (C<em>1</em>P), are required for the activation and translocation of cPLA(2) (FASEB J <em>1</em>7:<em>1</em>4<em>1</em><em>1</em>-<em>1</em>42<em>1</em>. 2003; J Biol Chem 278:38206-382<em>1</em>3, 2003; J Biol Chem 279:<em>1</em><em>1</em>320-<em>1</em><em>1</em>326, 2004). Herein, we show that these two pathways are independent but coordinated, resulting in synergistic induction of PGE(2). Moreover, the combination of both S<em>1</em>P and C<em>1</em>P recapitulates the temporal and spatial activation of cPLA(2) and with COX-2 seen IL-<em>1</em>beta. Taken together, the results provide, for the first time, a mechanism that assures the coordinate expression and activation in time and space of COX-2 and cPLA(2), assuring maximal production of PGE(2).

Phosphatidic acid (PA), lysophosphatidic acid, ceramide <em>1</em>-<em>phosphate</em> (C<em>1</em>P), and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) are lipid mediators generated by phospholipases, sphingomyelinases, and lipid kinases. The major pathway for degradation of these lipids is dephosphorylation catalyzed by members of two classes (types <em>1</em> and 2) of phosphohydrolase activities (PAPs). cDNAs encoding two type 2 PAPs, PAP-2a and -2b, have been expressed by transient transfection and shown to catalyze hydrolysis of PA, C<em>1</em>P, and S<em>1</em>P (Kai, M., Wada, I., Imai, S., Sakane, F. and Kanoh, H. (<em>1</em>997) J. Biol. Chem. 272, 24572-24578). We report the cloning and expression of a third type 2 PAP enzyme (288 amino acids, predicted molecular mass of 32.6 kDa), PAP-2c, which exhibits 54 and 43% sequence homology to PAPs 2a and 2b. Expression of HA epitope-tagged PAP-2a, -2b, and 2c in HEK293 cells produced immunoreactive proteins and increased membrane-associated PAP activity. Sf9 insect cells contain very low endogenous PAP activity. Recombinant expression of the three PAP enzymes using baculovirus vectors produces dramatic increases in membrane-associated Mg2+-independent, N-ethylmaleimide-insensitive PAP activity. Expression of PAP-2a but not PAP-2b or -2c resulted in high levels of cell surface PAP activity in intact insect cells. Kinetic analysis of PAP-2a, -2b, and -2c activity against PA, lysophosphatidic acid, C<em>1</em>P, and S<em>1</em>P presented in mixed micelles of Triton X-<em>1</em>00 revealed differences in substrate specificity and susceptibility to inhibition by <em>sphingosine</em>, Zn2+, and propranol.

Ovarian transplantation is one of the key approaches to restoring fertility in women who became menopausal as a result of cancer treatments. A major limitation of human ovarian transplants is massive follicular loss during revascularization. Here we investigated whether <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> or its receptor agonists could enhance neoangiogenesis and follicle survival in ovarian transplants in a xenograft model. Human ovarian tissue xenografts in severe-combined-immunodeficient mice were treated with <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, its analogs, or vehicle for <em>1</em>-<em>1</em>0 days. We found that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> treatment increased vascular density in ovarian transplants significantly whereas FTY720 and SEW287<em>1</em> had the opposite effect. In addition, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> accelerated the angiogenic process compared to vehicle-treated controls. Furthermore, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> treatment was associated with a significant proliferation of ovarian stromal cell as well as reduced necrosis and tissue hypoxia compared to the vehicle-treated controls. This resulted in a significantly lower percentage of apoptotic follicles in <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-treated transplants. We conclude that while <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> promotes neoangiogenesis in ovarian transplants and reduces ischemic reperfusion injury, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor agonists appear to functionally antagonize this process. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> holds great promise to clinically enhance the survival and longevity of human autologous ovarian transplants.

The MST<em>1</em> kinase was recently identified as playing an essential role in the promotion of lymphocyte polarization and adhesion stimulated by chemokines and TCR signaling. However, the physiological relevance of the Mst<em>1</em> pathway in thymocyte development is not completely understood. In this study, we analyzed the effect of Mst<em>1</em> disruption on thymocyte development and migration. Mst<em>1</em>-deficient (Mst<em>1</em>(-/-)) mice displayed an accumulation of mature thymocytes in the thymus, a dramatic reduction of lymphocytes in blood and peripheral lymphoid tissues, and a decrease of homing ability to peripheral lymph nodes. Mst<em>1</em>(-/-) thymocytes were impaired in chemotactic response to chemokines, such as CCL<em>1</em>9, but not to <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. Further analyses of Mst<em>1</em>(-/-) mice revealed a severe impairment in the egress of mature T cells from the thymus. T lineage-specific knockout of the Mst<em>1</em> gene demonstrates a cell-intrinsic role for Mst<em>1</em> in regulating T cell development. Our study indicates that Mst<em>1</em> is crucial in controlling lymphocyte chemotaxis and thymocyte emigration.

Circulation of mature lymphocytes between blood and secondary lymphoid tissues plays a central role in the immune system. Homing of lymphocytes from blood into secondary lymphoid tissues beyond high endothelial venules is highly dependent on the interaction between the chemokines CCL<em>1</em>9, CCL2<em>1</em>, CXCL<em>1</em>2, and CXCL<em>1</em>3, and their receptors CCR7, CXCR4 and CXCR5. However, the molecular mechanism(s) of lymphocyte egress from secondary lymphoid tissues to lymph remained unclear. We have found a new class of immunomodulator, FTY720 by chemical modification of vegetative wasp-derived natural product, ISP-I (myriocin). FTY720 has been shown to be highly effective in experimental allograft and autoimmune disease models. A striking feature of FTY720 is the induction of a marked decrease in peripheral blood lymphocytes at doses that show immunomodulating activity in these models. The reduction of circulating lymphocytes by FTY720 is caused by sequestration of lymphocytes into secondary lymphoid tissues and thymus. FTY720 is rapidly converted to (S)-enantiomer of FTY720-<em>phosphate</em> [(S)-FTY720-P] by <em>sphingosine</em> kinase 2 in vivo. (S)-FTY720-P acting as a potent agonist of S<em>1</em>P receptor type <em>1</em> (S<em>1</em>P<em>1</em>), induces long-term down-regulation of S<em>1</em>P<em>1</em> on lymphocytes, and thereby inhibits the migration of lymphocytes toward S<em>1</em>P. Thus, it is presumed that FTY720-induced lymphocyte sequestration is due to the inhibition of S<em>1</em>P/S<em>1</em>P<em>1</em>-dependent lymphocyte egress from secondary lymphoid tissues and thymus by its active metabolite (S)-FTY720-P. Throughout the analysis of the mechanism of action of FTY720, it is clarified that S<em>1</em>P/S<em>1</em>P<em>1</em> interaction plays an important role for lymphocyte egress from secondary lymphoid tissues and thymus.

BACKGROUND

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), which is produced by 2 <em>sphingosine</em> kinase (SphK) isoenzymes, SphK<em>1</em> and SphK2, has been implicated in IgE-mediated mast cell responses. However, studies of allergic inflammation in isotype-specific SphK knockout mice have not clarified their contribution, and the role that S<em>1</em>P plays in vivo in a mast cell- and IgE-dependent murine model of allergic asthma has not yet been examined.

OBJECTIVE

We used an isoenzyme-specific SphK<em>1</em> inhibitor, SK<em>1</em>-I, to investigate the contributions of S<em>1</em>P and SphK<em>1</em> to mast cell-dependent airway hyperresponsiveness (AHR) and airway inflammation in mice.

METHODS

Allergic airway inflammation and AHR were examined in a mast cell-dependent murine model of ovalbumin (OVA)-induced asthma. C57BL/6 mice received intranasal delivery of SK<em>1</em>-I before sensitization and challenge with OVA or only before challenge.

RESULTS

SK<em>1</em>-I inhibited antigen-dependent activation of human and murine mast cells and suppressed activation of nuclear factor κB (NF-κB), a master transcription factor that regulates the expression of proinflammatory cytokines. SK<em>1</em>-I treatment of mice sensitized to OVA in the absence of adjuvant, in which mast cell-dependent allergic inflammation develops, significantly reduced OVA-induced AHR to methacholine; decreased numbers of eosinophils and levels of the cytokines IL-4, IL-5, IL-6, IL-<em>1</em>3, IFN-γ, and TNF-α and the chemokines eotaxin and CCL2 in bronchoalveolar lavage fluid; and decreased pulmonary inflammation, as well as activation of NF-κB in the lungs.

CONCLUSIONS

S<em>1</em>P and SphK<em>1</em> play important roles in mast cell-dependent, OVA-induced allergic inflammation and AHR, in part by regulating the NF-κB pathway.

<em>Sphingosine</em> kinase <em>1</em> (SK<em>1</em>) is an enzyme that catalyzes the phosphorylation of <em>sphingosine</em> to produce the bioactive lipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P). We demonstrate here that the SK<em>1</em> inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole) induces the proteasomal degradation of SK<em>1</em> in human pulmonary artery smooth muscle cells, androgen-sensitive LNCaP prostate cancer cells, MCF-7 and MCF-7 HER2 breast cancer cells and that this is likely mediated by ceramide as a consequence of catalytic inhibition of SK<em>1</em> by SKi. Moreover, SK<em>1</em> is polyubiquitinated under basal conditions, and SKi appears to increase the degradation of SK<em>1</em> by activating the proteasome. In addition, the proteasomal degradation of SK<em>1</em>a and SK<em>1</em>b in androgen-sensitive LNCaP cells is associated with the induction of apoptosis. However, SK<em>1</em>b in LNCaP-AI cells (androgen-independent) is less sensitive to SKi-induced proteasomal degradation and these cells are resistant to SKi-induced apoptosis, thereby implicating the ubiquitin-proteasomal degradation of SK<em>1</em> as an important mechanism controlling cell survival.

We recently showed that mucus from patients with ulcerative colitis, a chronic inflammatory disorder of the colon, is characterized by a low level of phosphatidylcholine (PC) while clinical studies reveal that therapeutic addition of PC using slow release preparations is beneficial. The positive role of PC in this disease is still elusive. Here we tested the hypothesis that exogenous application of PC has anti-inflammatory properties using three model systems. First, human Caco-2 cells were treated with tumor necrosis factor-alpha (TNF-alpha) to induce a pro-inflammatory response via activation of NF-kappaB. Second, latex bead phagosomes were analyzed for their ability to assemble actin in vitro, a process linked to pro-inflammatory signaling and correlating with the growth versus killing of mycobacteria in macrophages. The third system used was the rapid assembly of plasma membrane actin in macrophages in response to <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. TNF-alpha induced a pro-inflammatory response in Caco-2 cells, including <em>1</em>) assembly of plasma membrane actin; 2) activation of both MAPKs ERK and p38; 3) transport of NF-kappaB subunits to the nucleus; and 4) subsequent up-regulation of the synthesis of pro-inflammatory gene products. Exogenous addition of most PCs tested significantly inhibited these processes. Other phospholipids like sphingomyelin or phosphatidylethanolamine showed no effects in these assays. PC also inhibited latex bead phagosome actin assembly, the killing of Mycobacterium tuberculosis in macrophages, and the <em>sphingosine</em> <em>1</em>-<em>phosphate</em>-induced actin assembly in macrophages. TNF-alpha induces the activation of signaling molecules and the reorganization of the actin cytoskeleton in human intestinal cells. Exogenous application of PC blocks pro-inflammatory signaling in Caco-2 cells, in phagosomes in vitro and facilitates intracellular survival of mycobacteria. We provide further evidence that actin assembly by membranes is part of the pro-inflammatory response. Collectively, these results provide a molecular foundation for the clinical studies showing a beneficial effect of PC therapy in ulcerative colitis.

The process of neurite extension after activation of the TrkA tyrosine kinase receptor by nerve growth factor (NGF) involves complex signaling pathways. Stimulation of <em>sphingosine</em> kinase <em>1</em> (SphK<em>1</em>), the enzyme that phosphorylates <em>sphingosine</em> to form <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), is part of the functional TrkA signaling repertoire. In this paper, we report that in PC<em>1</em>2 cells and dorsal root ganglion neurons, NGF translocates SphK<em>1</em> to the plasma membrane and differentially activates the S<em>1</em>P receptors S<em>1</em>P<em>1</em> and S<em>1</em>P2 in a SphK<em>1</em>-dependent manner, as determined with specific inhibitors and small interfering RNA targeted to SphK<em>1</em>. NGF-induced neurite extension was suppressed by down-regulation of S<em>1</em>P<em>1</em> expression with antisense RNA. Conversely, when overexpressed in PC<em>1</em>2 cells, transactivation of S<em>1</em>P<em>1</em> by NGF markedly enhanced neurite extension and stimulation of the small GTPase Rac, important for the cytoskeletal changes required for neurite extension. Concomitantly, differentiation down-regulated expression of S<em>1</em>P2 whose activation would stimulate Rho and inhibit neurite extension. Thus, differential transactivation of S<em>1</em>P receptors by NGF regulates antagonistic signaling pathways that modulate neurite extension.

Complex sphingolipids are abundant as eukaryotic cell membrane components, whereas their metabolites, in particular ceramide, <em>sphingosine</em>, and <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, are involved in diverse cell signaling processes. In mammals, degradation of ceramide by ceramidase yields <em>sphingosine</em>, which is phosphorylated by the action of <em>sphingosine</em> kinase to generate <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. Therefore, ceramidases are key enzymes in the regulation of the cellular levels of ceramide, <em>sphingosine</em>, and <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. To explore the physiological functions of a neutral ceramidase with diverse cellular locations, we disrupted the Asah2 gene in mice. Asah2 null mice have a normal life span and do not show obvious abnormalities or major alterations in total ceramide levels in tissues. The Asah2-encoded neutral ceramidase is highly expressed in the small intestine along the brush border, suggesting that the neutral ceramidase may be involved in a pathway for the digestion of dietary sphingolipids. Indeed, Asah2 null mice were deficient in the intestinal degradation of ceramide. Thus, the results indicate that the Asah2-encoded neutral ceramidase is a key enzyme for the catabolism of dietary sphingolipids and regulates the levels of bioactive sphingolipid metabolites in the intestinal tract.

Glioblastoma multiforme (GBM) is an aggressively invasive brain neoplasm with poor patient prognosis. We have previously shown that the bioactive lipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) stimulates in vitro invasiveness of GBM cells and that high expression levels of the enzyme that forms S<em>1</em>P, <em>sphingosine</em> kinase-<em>1</em> (SphK<em>1</em>), correlate with shorter survival time of GBM patients. We also recently showed that S<em>1</em>P induces expression of CCN<em>1</em> (also known as Cyr6<em>1</em>), a matricellular protein known to correlate with poor patient prognosis, in GBM cells. In this study, we further explored the role of CCN<em>1</em> as well as the urokinase plasminogen activator (uPA), a protein known to stimulate GBM cell invasiveness, in S<em>1</em>P-induced invasion using a spheroid invasion assay. We also investigated the roles of various S<em>1</em>P receptors in stimulating invasiveness through these pathways. S<em>1</em>P induced expression of uPA and its receptor, uPAR, in GBM cells. Whereas S<em>1</em>P(<em>1</em>), S<em>1</em>P(2), and S<em>1</em>P(3) receptors all contribute, at least partially, S<em>1</em>P(<em>1</em>) overexpression led to the most dramatic induction of the uPA system and of spheroid invasion, even in the absence of added S<em>1</em>P. Furthermore, neutralizing antibodies directed against uPA or CCN<em>1</em> significantly decreased both basal and S<em>1</em>P-stimulated GBM cell invasiveness. Inhibition of SphK blocked basal expression of uPA and uPAR, as well as glioma cell invasion; however, overexpression of SphK did not augment S<em>1</em>P receptor-mediated enhancement of uPA activity or invasion. Thus, SphK is necessary for basal activity of the uPA system and glioma cell invasion, whereas S<em>1</em>P receptor signaling enhances invasion, partially through uPA and CCN<em>1</em>.

The anticoagulant factor protein S (PS) has direct cellular activities. Lack of PS in mice causes lethal coagulopathy, ischemic/thrombotic injuries, vascular dysgenesis, and blood-brain barrier (BBB) disruption with intracerebral hemorrhages. Thus, we hypothesized that PS maintains and/or enhances the BBB integrity. Using a BBB model with human brain endothelial cells, we show PS inhibits time- and dose-dependently (half maximal effective concentration [EC(50)] = 27 +/- 3 nM) oxygen/glucose deprivation-induced BBB breakdown, as demonstrated by measurements of the transmonolayer electrical resistance, permeability of endothelial monolayers to dextran (40 kDa), and rearrangement of F-actin toward the cortical cytoskeletal ring. Using Tyro-3, Axl, and Mer (TAM) receptor, tyrosine kinase silencing through RNA interference, specific N-terminus-blocking antibodies, Tyro3 phosphorylation, and Tyro3-, Axl- and Mer-deficient mouse brain endothelial cells, we show that Tyro3 mediates PS vasculoprotection. After Tyro3 ligation, PS activated <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor (S<em>1</em>P(<em>1</em>)), resulting in Rac<em>1</em>-dependent BBB protection. Using 2-photon in vivo imaging, we show that PS blocks postischemic BBB disruption in Tyro3(+/+), Axl(-/-), and Mer(-/-) mice, but not in Tyro3(-/-) mice or Tyro3(+/+) mice receiving low-dose W<em>1</em>46, a S<em>1</em>P(<em>1</em>)-specific antagonist. Our findings indicate that PS protects the BBB integrity via Tyro3 and S<em>1</em>P(<em>1</em>), suggesting potentially novel treatments for neurovascular dysfunction resulting from hypoxic/ischemic BBB damage.

Alterations in lipid metabolism may contribute to diabetic complications. Sphingolipids are essential components of cell membranes and have essential roles in homeostasis and in the initiation and progression of disease. However, the role of sphingolipids in type <em>1</em> diabetes remains largely unexplored. Therefore, we sought to quantify sphingolipid metabolites by LC-MS/MS from two animal models of type <em>1</em> diabetes (streptozotocin-induced diabetic rats and Ins2(Akita) diabetic mice) to identify putative therapeutic targets and biomarkers. The results reveal that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (So<em>1</em>P) is elevated in both diabetic models in comparison to respective control animals. In addition, diabetic animals demonstrated reductions in plasma levels of omega-9 24:<em>1</em> (nervonic acid)-containing ceramide, sphingomyelin, and cerebrosides. Reduction of 24:<em>1</em>-esterfied sphingolipids was also observed in liver and heart. Nutritional stress via a high-fat diet also reduced 24:<em>1</em> content in the plasma and liver of mice, exacerbating the decrease in some cases where diabetes was also present. Subcutaneous insulin corrected both circulating So<em>1</em>P and 24:<em>1</em> levels in the murine diabetic model. Thus, changes in circulating sphingolipids, as evidenced by an increase in bioactive So<em>1</em>P and a reduction in cardio- and neuro-protective omega-9 esterified sphingolipids, may serve as biomarkers for type <em>1</em> diabetes and represent novel therapeutic targets.

S<em>1</em>P(2) <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor signaling can regulate proliferation, survival, morphology, and migration in many cell types in vitro. Here, we report that S<em>1</em>P(2)(-/-) mice develop clonal B-cell lymphomas with age, such that approximately half of the animals display this neoplasm by <em>1</em>.5 to 2 years of age. Histologic, immunophenotypic, and molecular analyses revealed a uniform tumor phenotype with features of germinal center (GC)-derived diffuse large B-cell lymphoma (DLBCL). Tumor formation was preceded by increases in GC B cells and CD69(+) T cells, as well as an increased formation of spontaneous GCs, suggesting that S<em>1</em>P(2) loss may promote lymphomagenesis in part by disrupting GC B-cells homeostasis. With the sole exception of rare lung tumors, the effect of S<em>1</em>P(2) gene disruption is remarkably restricted to DLBCL. In humans, 28 of <em>1</em>06 (26%) DLBCL samples were found to harbor multiple somatic mutations in the 5' sequences of the S<em>1</em>P(2) gene. Mutations displayed features resembling those generated by the IgV-associated somatic hypermutation mechanism, but were not detected at significant levels in normal GC B cells, indicating a tumor-associated aberrant function. Collectively, our data suggest that S<em>1</em>P(2) signaling may play a critical role in suppressing DLBCL formation in vivo. The high incidence of DLBCL in S<em>1</em>P(2)(-/-) mice, its onset at old age, and the relative lack of other neoplasms identify these mice as a novel, and potentially valuable, model for this highly prevalent and aggressive human malignancy.