Sphingolipid metabolism is driven by inflammatory cytokines. These cascade of events include the activation of <em>sphingosine</em> kinase (SK), and subsequent production of the mitogenic and proinflammatory lipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P). Overall, S<em>1</em>P is one of the crucial components in inflammation, making SK an excellent target for the development of new anti-inflammatory drugs. We have recently shown that SK inhibitors suppress colitis and hypothesize here that the novel SK inhibitor, ABC294640, prevents the development of colon cancer. In an azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model, there was a dose-dependent decrease in tumor incidence with SK inhibitor treatment. The tumor incidence (number of animals with tumors per group) in the vehicle, ABC294640 (20 mg/kg) and ABC294640 (50 mg/kg) groups were 80, 40 and 30%, respectively. Tumor multiplicity (number of tumors per animal) also decreased from 2.<em>1</em> ± 0.23 tumors per animal in the AOM + DSS + vehicle group to <em>1</em>.2 ± 0 tumors per animal in the AOM + DSS + ABC294640 (20 mg/kg) and to 0.8 ± 0.4 tumors per animal in the AOM + DSS + ABC294640 (50 mg/kg) group. Importantly, with ABC294640, there were no observed toxic side effects. To explore mechanisms, we isolated cells from the colon (CD45-, representing primarily colon epithelial cells) and (CD45+, representing primarily colon inflammatory cells) then measured known targets of SK that control cell survival. Results are consistent with the hypothesis that the inhibition of SK activity by our novel SK inhibitor modulates key pathways involved in cell survival and may be a viable treatment strategy for the chemoprevention colitis-driven colon cancer.

BACKGROUND

Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central nervous system which is characterized by inflammatory demyelination and neurodegeneration. Neurological symptoms include sensory disturbances, optic neuritis, limb weakness, ataxia, bladder dysfunction, cognitive deficits and fatigue.

METHODS

The inflammation process with MS is promoted by several inflammatory cytokines produced by the immune cells themselves and local resident cells like activated microglia. Consecutive damaging pathways involve the transmigration of activated B lymphocytes and plasma cells, which synthesize antibodies against the myelin sheath, boost the immune attack, and result in ultimate loss of myelin. Likewise, activated macrophages and microglia are present outside the lesions in the normal-appearing CNS tissue contributing to tissue damage. In parallel to inflammatory demyelination, axonal pathology occurs in the early phase which correlates with the number of infiltrating immune cells, and critically contributes to disease severity. The spectrum of neuronal white matter and cortical damage ranges from direct cell death to subtle neurodegenerative changes such as loss of dendritic ramification and the extent of neuronal damage is regarded as a critical factor for persisting neurological deficits. Under normal conditions, CNS microglia safeguards organ integrity by constantly scanning the tissue and responding rapidly to danger signals. The main task of microglial cells is to encapsulate dangerous foci and remove apoptotic cells and debris to protect the surrounding CNS tissue; this assists with tissue regeneration in toxin-induced demyelination. In the absence of lymphocytic inflammation and in the context of non-autoimmune, pathogen-associated triggered inflammation, microglial cells protect the neuronal compartment. These mechanisms seem to be inverted in MS and other chronic neurodegenerative disorders because activated microglia and peripherally derived macrophages are shifted towards a strongly pro-inflammatory phenotype and produce the proinflammatory cytokines TNF-α and interleukin (IL)<em>1</em>-β, as well as potentially neurotoxic substances including nitric oxide, oxygen radicals and proteolytic enzymes. Microglial silencing reduces clinical severity, demonstrating their active involvement in damage processes and in the immune attack against the CNS. In light of this, it is questionable whether microglia and monocyte-derived macrophages, the very last downstream effector cells in the immune reaction, actually have the capacity to influence their fate. It is more likely that the adaptive immune system orchestrates the attack against CNS cells and drives microglia and macrophages to attack oligodendrocytes and neurons.

UNASSIGNED

Currently, Glatiramer acetate (GA) and the interferon-β (IFN-β) variants are established as first-line disease modifying treatments that reduce the relapse rate, ameliorate relapse severity and delay the progression of disability in patients with relapsing-remitting MS. Similarily, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor agonists which influence lymphocyte migration through T cells-trapping in secondary lymphatic organs ameliorates astrogliosis and promotes remyelination by acting on S<em>1</em>P-receptors on astrocytes and oligodendrocytes. Ion channel blockers (e.g. sodium channel blockers), currently used for other indications, are now tested in neurodegenerative diseases to restore intracellular ion homeostasis in neurons. Axonal degeneration was significantly reduced and functional outcome was improved during treatment with Phenytoin, Flecainide and Lamotrigine. Although evidence for a direct protective effect on axons is still missing, additional immune-modulatory actions of sodium channel blockers on microglia and macrophages are likely available. In vitro-studies in axons subjected to anoxia in vitro or exposure to elevated levels of nitric oxide (NO) in vivo demonstrated the involvement of a direct effect on axons. As increased intracellular calcium levels contribute to axonal damage through activation of different enzymes such as proteases, blockade of voltage gated calcium channels is another promising target. For example, nitrendipin and bepridil ameliorate axonal loss and clinical symptoms in different models of chronic neurodegeneration. In addition to these exogenous neuroprotective patheways, endogenous neuroprotective mechanisms including neurotrophins, (re)myelination and, neurogenesis support restauration of neuronal integrity.

OBJECTIVE

High-density lipoprotein (HDL) levels are inversely proportional to the risk of atherosclerosis, but mechanisms of HDL atheroprotection remain unclear. Monocyte chemoatractant protein-<em>1</em> (MCP-<em>1</em>) constitutes an early component of inflammatory response in atherosclerosis. Here we investigated the influence of HDL on MCP-<em>1</em> production in vascular smooth muscle cells (VSMCs) and rat aortic explants.

RESULTS

HDL inhibited the thrombin-induced production of MCP-<em>1</em> in a concentration-dependent manner. The HDL-dependent inhibition of MCP-<em>1</em> production was accompanied by the suppression of reactive oxygen species (ROS), which regulate the MCP-<em>1</em> production in VSMCs. HDL inhibited NAD(P)H oxidase, the preponderant source of ROS in the vasculature, and prevented the activation of Rac<em>1</em>, which precedes NAD(P)H-oxidase activation. The HDL capacity to inhibit MCP-<em>1</em> production, ROS generation, and NAD(P)H-oxidase activation was emulated by <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and sphingosylphosphorylcholine (SPC), two lysosphingolipids present in HDL, but not by apolipoprotein A-I. HDL-, S<em>1</em>P-, and SPC-induced inhibition of MCP-<em>1</em> production was attenuated in VSMCs pretreated with VPC230<em>1</em>9, an antagonist of lysosphingolipid receptors S<em>1</em>P(<em>1</em>) and S<em>1</em>P(3), but not by JTE0<em>1</em>3, an antagonist of S<em>1</em>P(2). In addition, HDL, S<em>1</em>P, and SPC failed to inhibit MCP<em>1</em> production and ROS generation in aortas from S<em>1</em>P(3)- and SR-B<em>1</em>-deficient mice.

CONCLUSIONS

HDL-associated lysosphingolipids inhibit NAD(P)H oxidase-dependent ROS generation and MCP-<em>1</em> production in a process that requires coordinate signaling through S<em>1</em>P(3) and SR-B<em>1</em> receptors.

Nonmuscle myosin light chain kinase (nmMLCK), a multi-functional cytoskeletal protein critical to vascular homeostasis, is highly regulated by tyrosine phosphorylation. We identified multiple novel c-Abl-mediated nmMLCK phosphorylation sites by mass spectroscopy analysis (including Y23<em>1</em>, Y464, Y556, Y846) and examined their influence on nmMLCK function and human lung endothelial cell (EC) barrier regulation. Tyrosine phosphorylation of nmMLCK increased kinase activity, reversed nmMLCK-mediated inhibition of Arp2/3-mediated actin polymerization, and enhanced binding to the critical actin-binding phosphotyrosine protein, cortactin. EC challenge with <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a potent barrier-enhancing agonist, resulted in c-Abl and phosphorylated nmMLCK recruitment into caveolin-enriched microdomains, rapid increases in Abl kinase activity, and spatial targeting of c-Abl to barrier-promoting cortical actin structures. Conversely, reduced c-Abl expression in EC (siRNA) markedly attenuated S<em>1</em>P-mediated cortical actin formation, reduced the EC modulus of elasticity (assessed by atomic force microscopy), reduced nmMLCK and cortactin tyrosine phosphorylation, and attenuated S<em>1</em>P-mediated barrier enhancement. These studies indicate an essential role for Abl kinase in vascular barrier regulation via posttranslational modification of nmMLCK and strongly support c-Abl-cortactin-nmMLCK interaction as a novel determinant of cortical actin-based cytoskeletal rearrangement critical to S<em>1</em>P-mediated EC barrier enhancement.

There is an increasing body of evidence demonstrating a critical role for the bioactive lipid S<em>1</em>P (<em>sphingosine</em> <em>1</em>-<em>phosphate</em>) in cancer. S<em>1</em>P is synthesized and metabolized by a number of enzymes, including <em>sphingosine</em> kinase, S<em>1</em>P lyase and S<em>1</em>P phosphatases. S<em>1</em>P binds to cell-surface G-protein-coupled receptors (S<em>1</em>P<em>1</em>-S<em>1</em>P5) to elicit cell responses and can also regulate, by direct binding, a number of intracellular targets such as HDAC (histone deacetylase) <em>1</em>/2 to induce epigenetic regulation. S<em>1</em>P is involved in cancer progression including cell transformation/oncogenesis, cell survival/apoptosis, cell migration/metastasis and tumour microenvironment neovascularization. In the present paper, we describe our research findings regarding the correlation of <em>sphingosine</em> kinase <em>1</em> and S<em>1</em>P receptor expression in tumours with clinical outcome and we define some of the molecular mechanisms underlying the involvement of <em>sphingosine</em> kinase <em>1</em> and S<em>1</em>P receptors in the formation of a cancer cell migratory phenotype. The role of <em>sphingosine</em> kinase <em>1</em> in the acquisition of chemotherapeutic resistance and the interaction of S<em>1</em>P receptors with oncogenes such as HER2 is also reviewed. We also discuss novel aspects of the use of small-molecule inhibitors of <em>sphingosine</em> kinase <em>1</em> in terms of allosterism, ubiquitin-proteasomal degradation of <em>sphingosine</em> kinase <em>1</em> and anticancer activity. Finally, we describe how S<em>1</em>P receptor-modulating agents abrogate S<em>1</em>P receptor-receptor tyrosine kinase interactions, with potential to inhibit growth-factor-dependent cancer progression.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is an important vascular barrier regulatory agonist which enhances the junctional integrity of human lung endothelial cell monolayers. We have now demonstrated that S<em>1</em>P induced cortical actin ring formation and redistribution of focal adhesion kinase (FAK) and paxillin to the cell periphery suggesting the critical role of cell-cell adhesion in endothelial barrier enhancement. Co-immunoprecipitation studies revealed increased association of VE-cadherin with FAK and paxillin in S<em>1</em>P-challenged human pulmonary artery endothelial cell (HPAEC) monolayers. Furthermore, S<em>1</em>P-induced enhancement of VE-cadherin interaction with alpha-catenin and beta-catenin was associated with the increased formation of FAK-beta-catenin protein complexes. Depletion of beta-catenin (siRNA) resulted in loss of S<em>1</em>P-mediated VE-cadherin association with FAK and paxillin rearrangement. Furthermore, transendothelial electrical resistance (an index of barrier function) demonstrated that beta-catenin siRNA significantly attenuated S<em>1</em>P-induced barrier enhancement. These results demonstrate a mechanism of S<em>1</em>P-induced endothelial barrier enhancement via beta-catenin-linked adherens junction and focal adhesion interaction.

PLCepsilon (phospholipase Cepsilon) is a novel PLC that has a CDC25 guanine nucleotide exchange factor domain and two RA (Ras-association) domains of which the second (RA2) is critical for Ras activation of the enzyme. In the present studies, we examined hormonal stimulation to elucidate receptor-mediated pathways that functionally regulate PLCepsilon. We demonstrate that EGF (epidermal growth factor), a receptor tyrosine kinase agonist, and LPA (lysophosphatidic acid), S<em>1</em>P (<em>sphingosine</em> <em>1</em>-<em>phosphate</em>) and thrombin, GPCR (G-protein-coupled receptor) agonists, stimulate PLCepsilon overexpressed in COS-7 cells. EGF stimulated PLCepsilon in an RA2-dependent manner through Ras and Rap. In contrast, LPA, S<em>1</em>P and thrombin stimulated PLCepsilon by both RA2-independent and -dependent mechanisms. To determine the G-proteins that mediate the effects of these GPCR agonists, we co-expressed constitutively active G-proteins with PLCepsilon and found that G(alpha<em>1</em>2), G(alpha<em>1</em>3), Rho, Rac and Ral stimulate PLCepsilon in an RA2-independent manner; whereas TC2<em>1</em>, Rap<em>1</em>A, Rap2A and Rap2B stimulate PLCepsilon in an RA2-dependent manner similar to H-Ras. Of these G-proteins, we show that G(alpha<em>1</em>2)/G(alpha<em>1</em>3) and Rap partly mediate the effects of LPA, S<em>1</em>P and thrombin to stimulate PLCepsilon. In addition, the stimulation by LPA and S<em>1</em>P is also partly sensitive to pertussis toxin. These studies demonstrate diverse hormonal regulation of PLCepsilon by distinct and overlapping pathways.

One of the striking activities of the Edg family <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors includes receptor isotype-specific, bimodal regulatory activity on cell migration. While Edg<em>1</em> and Edg3 act as typical chemotactic receptors, Edg5 uniquely acts as a chemorepellant receptor. Consistent with this, Edg<em>1</em> and Edg3, and Edg5 regulate the activity of the Rho family GTPase Rac positively and negatively, respectively. Thus, Edg isotype-specific, differential regulatory activities on Rac seem to be important as mechanisms underlying the bimodal regulation of cell migration by S<em>1</em>P. Edg5-mediated Rac inhibition involves stimulation of Rac-GTPase-activating protein (GAP) activity, rather than inhibition of Rac-guanine nucleotide exchange factor (GEF) activity. Many cell types including vascular smooth muscle and endothelial cells express more than a single S<em>1</em>P receptor isotype. In these cells, it appears that an integration of the Edg isotype-selective, positive and negative signals on cellular Rac activity is a critical determinant for eventual direction of regulation on cell motility by S<em>1</em>P. Physiological and pathological roles for the repulsive activity of Edg5 receptor remain to be clarified.

G-protein-coupled receptors (GPCRs) are a large family of remarkably versatile membrane proteins that are attractive therapeutic targets because of their involvement in a vast range of normal physiological processes and pathological diseases. Upon activation, intracellular domains of GPCRs mediate signaling to G-proteins, but these domains have yet to be effectively exploited as drug targets. Cell-penetrating lipidated peptides called pepducins target specific intracellular loops of GPCRs and have recently emerged as effective allosteric modulators of GPCR activity. The lipid moiety facilitates translocation across the plasma membrane, where pepducins then specifically modulate signaling of their cognate receptor. To date, pepducins and related lipopeptides have been shown to specifically modulate the activity of diverse GPCRs and other membrane proteins, including protease-activated receptors (PAR<em>1</em>, PAR2, and PAR4), chemokine receptors (CXCR<em>1</em>, CXCR2, and CXCR4), <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor-3 (S<em>1</em>P3), the melanocortin-4 receptor, the Smoothened receptor, formyl peptide receptor-2 (FPR2), the relaxin receptor (LGR7), G-proteins (Gα(q/<em>1</em><em>1</em>/o/<em>1</em>3)), muscarinic acetylcholine receptor and vanilloid (TRPV<em>1</em>) channels, and the GPIIb integrin. This minireview describes recent advances made using pepducin technology in targeting diverse GPCRs and the use of pepducins in identifying potential novel drug targets.

Radiation resistance poses a major clinical challenge in cancer treatment, but little is known about how microRNA (miR) may regulate this phenomenon. In this study, we used next-generation sequencing to perform an unbiased comparison of miR expression in PC3 prostate cancer cells rendered resistant to fractionated radiation treatment. One miR candidate found to be upregulated by ionizing radiation was miR-95, the enforced expression of which promoted radiation resistance in a variety of cancer cells. miR-95 overexpression recapitulated an aggressive phenotype including increased cellular proliferation, deregulated G2-M checkpoint following ionizing radiation, and increased invasive potential. Using combined in silico prediction and microarray expression analyses, we identified and validated the sphingolipid phosphatase SGPP<em>1</em>, an antagonist of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> signaling, as a target of miR-95 that promotes radiation resistance. Consistent with this finding, cell treatment with FTY720, a clinically approved small molecule inhibitor of S<em>1</em>P signaling, sensitized miR-95 overexpressing cells to radiation treatment. In vivo assays extended the significance of these results, showing that miR-95 overexpression increased tumor growth and resistance to radiation treatment in tumor xenografts. Furthermore, reduced tumor necrosis and increased cellular proliferation were seen after radiation treatment of miR-95 overexpressing tumors compared with control tumors. Finally, miR-95 expression was increased in human prostate and breast cancer specimens compared with normal tissue. Together, our work reveals miR-95 expression as a critical determinant of radiation resistance in cancer cells.

Originating from its DNA sequence, a computational model of the Edg<em>1</em> receptor has been developed that predicts critical interactions with its ligand, <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. The basic amino acids Arg(<em>1</em>20) and Arg(292) ion pair with the <em>phosphate</em>, whereas the acidic Glu(<em>1</em>2<em>1</em>) residue ion pairs with the ammonium moiety of <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [(35)S]GTPgammaS binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg<em>1</em> receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions.

Ceramidases (CDases) play a key role in cancer therapy through enhanced conversion of ceramide into <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), but their involvement in hepatocarcinogenesis is unknown. Here, we report that daunorubicin (DNR) activated acid CDase post-transcriptionally in established human (HepG2 cells) or mouse (Hepa<em>1</em>c<em>1</em>c7) hepatoma cell lines as well as in primary cells from murine liver tumors, but not in cultured mouse hepatocytes. Acid CDase silencing by small interfering RNA (siRNA) or pharmacological inhibition with N-oleoylethanolamine (NOE) enhanced the ceramide to S<em>1</em>P balance compared to DNR alone, sensitizing hepatoma cells (HepG2, Hep-3B, SK-Hep and Hepa<em>1</em>c<em>1</em>c7) to DNR-induced cell death. DNR plus NOE or acid CDase siRNA-induced cell death was preceded by ultrastructural changes in mitochondria, stimulation of reactive oxygen species generation, release of Smac/DIABLO and cytochrome c and caspase-3 activation. In addition, in vivo siRNA treatment targeting acid CDase reduced tumor growth in liver tumor xenografts of HepG2 cells and enhanced DNR therapy. Thus, acid CDase promotes hepatocarcinogenesis and its antagonism may be a promising strategy in the treatment of liver cancer.

The pro-drug FTY720 is undergoing phase III clinical trials for prevention of allograft rejection. After phosphorylation, FTY720 targets the G protein-coupled-<em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> (S<em>1</em>PR<em>1</em>) on lymphocytes, thereby inhibiting their egress from lymphoid organs and their recirculation to inflammatory sites. Potential effects on dendritic cell (DC) trafficking have not been evaluated. Here, we demonstrate the expression of all five S<em>1</em>PR subtypes (S<em>1</em>PR<em>1</em>-5) by murine DCs. Administration of FTY720 to C57BL/<em>1</em>0 mice markedly reduced circulating T and B lymphocytes within 24 h, but not blood-borne DCs, which were enhanced significantly for up to 96 h, while DCs in lymph nodes and spleen were reduced. Numbers of adoptively transferred, fluorochrome-labeled syngeneic or allogeneic DCs in blood were increased significantly in FTY720-treated animals, while donor-derived DCs and allostimulatory activity for host naïve T cells within the spleen were reduced. Administration of the selective S<em>1</em>PR<em>1</em> agonist SEW287<em>1</em> significantly enhanced circulating DC numbers. Flow analysis revealed that CD<em>1</em><em>1</em>b, CD3<em>1</em>/PECAM-<em>1</em>, CD54/ICAM-<em>1</em> and CCR7 expression on blood-borne DCs was downregulated following FTY720 administration. Transendothelial migration of FTY720-P-treated immature DCs to the CCR7 ligand CCL<em>1</em>9 was reduced. These novel data suggest that modulation of DC trafficking by FTY720 may contribute to its immunosuppressive effects.

Membrane type <em>1</em>-matrix metalloproteinase (MT<em>1</em>-MMP) has been suggested to play an important role in angiogenesis, but the mechanisms involved remain incompletely understood. Using an in vitro model of angiogenesis in which cell migration of bovine aortic endothelial cells (BAECs) and their morphogenic differentiation into capillary-like structures on Matrigel are induced by overexpression of MT<em>1</em>-MMP, we show that the platelet-derived bioactive lipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is the predominant serum factor essential for MT<em>1</em>-MMP-dependent migration and morphogenic differentiation activities. In the presence of S<em>1</em>P, MT<em>1</em>-MMP-dependent cell migration and morphogenic differentiation were inhibited by pertussis toxin, suggesting the involvement of Gi-protein-coupled receptor-mediated signaling. Accordingly, cotransfection of BAECs with MT<em>1</em>-MMP and a constitutively active Galphai2 (Q205L) mutant increased cell migration and morphogenic differentiation, whereas treatment of BAECs overexpressing MT<em>1</em>-MMP with antisense oligonucleotides directed against S<em>1</em>P<em>1</em> and S<em>1</em>P3, the predominant S<em>1</em>P receptors, significantly inhibited both processes. These results demonstrate that MT<em>1</em>-MMP-induced migration and morphogenic differentiation involve the cooperation of the enzyme with platelet-derived bioactive lipids through S<em>1</em>P-mediated activation of Galphai-coupled S<em>1</em>P<em>1</em> and S<em>1</em>P3 receptors. Given the important contribution of platelets to tumor angiogenesis, the stimulation of endothelial MT<em>1</em>-MMP function by S<em>1</em>P may thus constitute an important molecular event linking hemostasis to angiogenesis.

Nitric oxide (NO) has been implicated as a critical signaling molecule of angiogenesis. Recently, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) has emerged as a mediator of angiogenesis, and S<em>1</em>P-induced NO synthesis in endothelial cells (ECs) has been reported. To analyze the signaling pathways involved in S<em>1</em>P-induced angiogenesis and clarify the role of NO in this process, we performed in vivo and in vitro angiogenesis assays. S<em>1</em>P activated the phosphatidylinositol-3-kinase (PI3K)/Akt/endothelial NO synthase (eNOS) pathway in ECs, since S<em>1</em>P-stimulated eNOS phosphorylation and NO production were blocked by inhibition of activities of PI3K and Akt. S<em>1</em>P increased capillary ingrowth into subcutaneously implanted Matrigel plugs in mice, and the effect of S<em>1</em>P was significantly reduced in mice that received N(G)-nitro- L-arginine methyl ester (L-NAME), an inhibitor of NOS. S<em>1</em>P stimulated EC migration and tube formation on Matrigel, which processes were significantly decreased by inhibition of activities of PI3K, Akt, or eNOS, whereas treatment with LY294002, a PI3K inhibitor, but not L-NAME, inhibited EC viability and proliferation. Thus, our results demonstrate the crucial role of NO in S<em>1</em>P-induced angiogenesis in vivo and in vitro and suggest the divergent roles of NO in the S<em>1</em>P-induced angiogenic response.

Transforming growth factor beta (TGFbeta) plays a dual role in oncogenesis, acting as both a tumor suppressor and a tumor promoter. These disparate processes of suppression and promotion are mediated primarily by Smad and non-Smad signaling, respectively. A central issue in understanding the role of TGFbeta in the progression of epithelial cancers is the elucidation of the mechanisms underlying activation of non-Smad signaling cascades. Because the potent lipid mediator <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) has been shown to transactivate the TGFbeta receptor and activate Smad3, we examined its role in TGFbeta activation of extracellular signal-regulated kinases <em>1</em> and 2 (ERK<em>1</em>/2) and promotion of migration and invasion of esophageal cancer cells. Both S<em>1</em>P and TGFbeta activate ERK<em>1</em>/2, but only TGFbeta activates Smad3. Both ligands promoted ERK<em>1</em>/2-dependent migration and invasion. Furthermore, TGFbeta rapidly increased S<em>1</em>P, which was required for TGFbeta-induced ERK<em>1</em>/2 activation, as well as migration and invasion, since downregulation of <em>sphingosine</em> kinases, the enzymes that produce S<em>1</em>P, inhibited these responses. Finally, our data demonstrate that TGFbeta activation of ERK<em>1</em>/2, as well as induction of migration and invasion, is mediated at least in part by ligation of the S<em>1</em>P receptor, S<em>1</em>PR2. Thus, these studies provide the first evidence that TGFbeta activation of <em>sphingosine</em> kinases and formation of S<em>1</em>P contribute to non-Smad signaling and could be important for progression of esophageal cancer.

Murine CD4 and CD8 T cells express predominantly types <em>1</em> and 4 <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) G protein-coupled receptors (designated S<em>1</em>P<em>1</em> and S<em>1</em>P4 or previously endothelial differentiation gene-encoded <em>1</em> and 6) for S<em>1</em>P, which has a normal plasma concentration of 0.<em>1</em>-<em>1</em> microM. S<em>1</em>P now is shown to enhance chemotaxis of CD4 T cells to CCL-2<em>1</em> and CCL-5 by up to 2.5-fold at <em>1</em>0 nM to 0.<em>1</em> microM, whereas 0.3-3 microM S<em>1</em>P inhibits this chemotaxis by up to 70%. Chemotaxis of S<em>1</em>P(<em>1</em>), but not S<em>1</em>P(4), transfectants to CXCL<em>1</em> and CXCL4 was similarly affected by S<em>1</em>P. Activation of CD4 T cells, which decreases S<em>1</em>P receptor expression, suppressed effects of S<em>1</em>P on chemotaxis. Pretreatment of labeled CD4 T cells with S<em>1</em>P before reintroduction into mice inhibited by a maximum of 75% their migration into chemokine-challenged s.c. air pouches. The S<em>1</em>P-S<em>1</em>P(<em>1</em>) receptor axis thus controls recruitment of naive T cells by maintaining their response threshold to diverse lymphotactic factors.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) rapidly increases endothelial barrier function and induces the assembly of the adherens junction proteins vascular endothelial (VE)-cadherin and catenins. Since VE-cadherin contributes to the stabilization of the endothelial barrier, we determined whether the rapid, barrier-enhancing activity of S<em>1</em>P requires VE-cadherin. Ca(2+)-dependent, homophilic VE-cadherin binding of endothelial cells, derived from human umbilical veins and grown as monolayers, was disrupted with EGTA, an antibody to the extracellular domain of VE-cadherin, or gene silencing of VE-cadherin with small interfering RNA. All three protocols caused a reduction in the immunofluorescent localization of VE-cadherin at intercellular junctions, the separation of adjacent cells, and a decrease in basal endothelial electrical resistance. In all three conditions, S<em>1</em>P rapidly increased endothelial electrical resistance. These findings demonstrate that S<em>1</em>P enhances the endothelial barrier independently of homophilic VE-cadherin binding. Junctional localization of VE-cadherin, however, was associated with the sustained activity of S<em>1</em>P. Imaging with phase-contrast and differential interference contrast optics revealed that S<em>1</em>P induced cell spreading and closure of intercellular gaps. Pretreatment with latrunculin B, an inhibitor of actin polymerization, or Y-27632, a Rho kinase inhibitor, attenuated cell spreading and the rapid increase in electrical resistance induced by S<em>1</em>P. We conclude that S<em>1</em>P rapidly closes intercellular gaps, resulting in an increased electrical resistance across endothelial cell monolayers, via cell spreading and Rho kinase and independently of VE-cadherin.

The pleiotropic cytokine transforming growth factor (TGF)-beta<em>1</em> is a key player in the onset of skeletal muscle fibrosis, which hampers tissue repair. However, the molecular mechanisms implicated in TGFbeta<em>1</em>-dependent transdifferentiation of myoblasts into myofibroblasts are presently unknown. Here, we show that TGFbeta<em>1</em> up-regulates <em>sphingosine</em> kinase (SK)-<em>1</em> in C2C<em>1</em>2 myoblasts in a Smad-dependent manner, and concomitantly modifies the expression of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors (S<em>1</em>PRs). Notably, pharmacological or short interfering RNA-mediated inhibition of SK<em>1</em> prevented the induction of fibrotic markers by TGFbeta<em>1</em>. Moreover, inhibition of S<em>1</em>P(3), which became the highest expressed S<em>1</em>PR after TGFbeta<em>1</em> challenge, strongly attenuated the profibrotic response to TGFbeta<em>1</em>. Furthermore, downstream of S<em>1</em>P(3), Rho/Rho kinase signaling was found critically implicated in the profibrotic action of TGFbeta<em>1</em>. Importantly, we demonstrate that SK/S<em>1</em>P axis, known to play a key role in myogenesis via S<em>1</em>P(2), consequently to TGFbeta<em>1</em>-dependent S<em>1</em>PR pattern remodeling, becomes responsible for transmitting a profibrotic, antidifferentiating action. This study provides new compelling information on the mechanism by which TGFbeta<em>1</em> gives rise to fibrosis in skeletal muscle, opening new perspectives for its pharmacological treatment. Moreover, it highlights the pleiotropic role of SK/S<em>1</em>P axis in skeletal myoblasts that, depending on the expressed S<em>1</em>PR pattern, seems capable of eliciting multiple, even contrasting biological responses.

BACKGROUND

Microglial activation in multiple sclerosis has been postulated to contribute to long-term neurodegeneration during disease. Fingolimod has been shown to impact on the relapsing remitting phase of disease by modulating autoreactive T-cell egress from lymph organs. In addition, it is brain penetrant and has been shown to exert multiple effects on nervous system cells.

METHODS

In this study, the impact of fingolimod and other <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor active molecules following lysophosphotidyl choline-induced demyelination was examined in the rat telencephalon reaggregate, spheroid cell culture system. The lack of immune system components allowed elucidation of the direct effects of fingolimod on CNS cell types in an organotypic situation.

RESULTS

Following demyelination, fingolimod significantly augmented expression of myelin basic protein in the remyelination phase. This increase was not associated with changes in neurofilament levels, indicating de novo myelin protein expression not associated with axonal branching. Myelin wrapping was confirmed morphologically using confocal and electron microscopy. Increased remyelination was associated with down-regulation of microglial ferritin, tumor necrosis factor alpha and interleukin <em>1</em> during demyelination when fingolimod was present. In addition, nitric oxide metabolites and apoptotic effectors caspase 3 and caspase 7 were reduced during demyelination in the presence of fingolimod. The <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> and 5 agonist BAF3<em>1</em>2 also increased myelin basic protein levels, whereas the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> agonist AUY954 failed to replicate this effect on remyelination.

CONCLUSIONS

The results presented indicate that modulation of S<em>1</em>P receptors can ameliorate pathological effectors associated with microglial activation leading to a subsequent increase in protein and morphological markers of remyelination. In addition, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor 5 is implicated in promoting remyelination in vitro. This knowledge may be of benefit for treatment of chronic microglial inflammation in multiple sclerosis.

FTY720 (Fingolimod, Gilenya) is a <em>sphingosine</em> analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V6<em>1</em>7F) oncogene. PP2A inactivation occurs in a Jak2(V6<em>1</em>7F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V6<em>1</em>7F) inactivation/downregulation and impairs clonogenic potential of Jak2(V6<em>1</em>7F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V6<em>1</em>7F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V6<em>1</em>7F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V6<em>1</em>7F) also utilizes an alternative <em>sphingosine</em> kinase-<em>1</em>-mediated pathway to inhibit PP2A and that FTY720-P, acting as a <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-receptor-<em>1</em> agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V6<em>1</em>7F) MPNs.

There is a great deal of evidence that altered sphingolipid metabolism is associated with fumonisin-induced animal diseases including increased apoptotic and oncotic necrosis, and carcinogenesis in rodent liver and kidney. The biochemical consequences of fumonisin disruption of sphingolipid metabolism most likely to alter cell regulation are increased free sphingoid bases and their <em>1</em>-<em>phosphates</em>, alterations in complex sphingolipids, and decreased ceramide (CER) biosynthesis. Because free sphingoid bases and CER can induce cell death, the fumonisin inhibition of CER synthase can inhibit cell death induced by CER but promote free sphingoid base-induced cell death. Theoretically, at any time the balance between the intracellular concentration of effectors that protect cells from apoptosis (decreased CER, increased <em>sphingosine</em> <em>1</em>-<em>phosphate</em>) and those that induce apoptosis (increased CER, free sphingoid bases, altered fatty acids) will determine the cellular response. Because the balance between the rates of apoptosis and proliferation is important in tumorigenesis, cells sensitive to the proliferative effect of decreased CER and increased <em>sphingosine</em> <em>1</em>-<em>phosphate</em> may be selected to survive and proliferate when free sphingoid base concentration is not growth inhibitory. Conversely, when the increase in free sphingoid bases exceeds a cell's ability to convert sphinganine/<em>sphingosine</em> to dihydroceramide/CER or their sphingoid base <em>1</em>-<em>phosphate</em>, then free sphingoid bases will accumulate. In this case cells that are sensitive to sphingoid base-induced growth arrest will die and insensitive cells will survive. If the cells selected to die are normal phenotypes and the cells selected to survive are abnormal, then cancer risk will increase.

The inhalational anesthetic isoflurane has been shown to protect against renal ischemia-reperfusion (IR) injury. Previous studies demonstrated that isoflurane modulates sphingolipid metabolism in renal proximal tubule cells. We sought to determine whether isoflurane stimulates <em>sphingosine</em> kinase (SK) activity and synthesis of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) in renal proximal tubule cells to mediate renal protection via the S<em>1</em>P signaling pathway. Isoflurane anesthesia reduced the degree of renal failure and necrosis in a murine model of renal IR injury. This protection with isoflurane was reversed by SK inhibitors (DMS and SKI-II) as well as an S<em>1</em>P(<em>1</em>) receptor antagonist (VPC230<em>1</em>9). In addition, mice deficient in SK<em>1</em> enzyme were not protected from IR injury with isoflurane. SK activity as well as SK<em>1</em> mRNA expression increased in both cultured human proximal tubule cells (HK-2) and mouse kidneys after exposure to isoflurane. Finally, isoflurane increased the generation of S<em>1</em>P in HK-2 cells. Taken together, our findings indicate that isoflurane activates SK in renal tubule cells and initiates S<em>1</em>P->>S<em>1</em>P(<em>1</em>) receptor signaling to mediate the renal protective effects. Our findings may help to unravel the cellular signaling pathways of volatile anesthetic-mediated renal protection and lead to new therapeutic applications of inhalational anesthetics during the perioperative period.

Among the family of lipid-derived mediators, the group of lysophospholipids including lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) have growth-related and -unrelated effects on diverse cell types including lymphocytes, macrophages, smooth muscle cells, endothelial cells, and neuronal cells. This review summarizes the known effects of lysophospholipids and their G protein-coupled receptors (GPCRs) in inflammation and immunity. Lysophospholipids have the capacity to evoke and modulate immune responses by attracting and activating T-cells, B-cells and macrophages directly and influencing their interactions with other cell types. Immune cells express multiple subsets of lysophospholipid receptors, which are critical for specific cellular responses such as proliferation and migration that are fundamental to immunity. Investigation of the expression pattern of EDG-receptors on human T-cells revealed a dynamic transcriptional regulation influenced by both developmental stages and activation states. Other lipid mediators like psychosine and other GPCRs for lipid mediators like G2A also may be involved in the development of normal immune and inflammatory reactions and diseases. These observations suggest that agonists and antagonists for lysophospholipid receptors may influence immune responses.