Lysophosphatidic acid (LPA) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) are members of the phospholipid growth factor family. A major limitation in the field to date has been a lack of receptor subtype-specific agonists and antagonists. Here, we report that dioctylglycerol pyro<em>phosphate</em> and dioctylphosphatidic acid are selective antagonists of the LPA(<em>1</em>) and LPA(3) receptors, but prefer LPA(3) by an order of magnitude. Neither molecule had an agonistic or antagonistic effect on LPA(2) receptor. Consistent with this receptor subtype selectivity, dioctylglycerol pyro<em>phosphate</em> inhibited cellular responses to LPA in NIH3T3 fibroblasts, HEY ovarian cancer cells, PC<em>1</em>2 pheochromocytoma cells, and Xenopus laevis oocytes. Responses elicited by S<em>1</em>P in these cell lines that endogenously express S<em>1</em>P(<em>1</em>), S<em>1</em>P(2), S<em>1</em>P(3), and S<em>1</em>P(5) receptors were unaffected by dioctylglycerol pyro<em>phosphate</em>. Responses evoked by the G protein-coupled receptor ligands acetylcholine, serotonin, ATP, and thrombin receptor-activating peptide were similarly unaffected, suggesting that the short-chain phosphatidates are receptor subtype-specific lysophosphatidate antagonists.

The endothelial isoform of nitric-oxide synthase (eNOS) undergoes a complex pattern of covalent modifications, including acylation with the fatty acids myristate and palmitate as well as phosphorylation on multiple sites. eNOS acylation is a key determinant for the reversible subcellular targeting of the enzyme to plasmalemmal caveolae. We transfected a series of hemagglutinin epitope-tagged eNOS mutant cDNAs deficient in palmitoylation (palm(-)) and/or myristoylation (myr(-)) into bovine aortic endothelial cells; after treatment with the eNOS agonists <em>sphingosine</em> <em>1</em>-<em>phosphate</em> or vascular endothelial growth factor, the recombinant eNOS was immunoprecipitated using an antibody directed against the epitope tag, and patterns of eNOS phosphorylation were analyzed in immunoblots probed with phosphorylation state-specific eNOS antibodies. The wild-type eNOS underwent agonist-induced phosphorylation at serine <em>1</em><em>1</em>79 (a putative site for phosphorylation by kinase Akt), but phosphorylation of the myr(-) eNOS at this residue was nearly abrogated; the palm(-) eNOS exhibited an intermediate phenotype. The addition of the CD8 transmembrane domain to the amino terminus of eNOS acylation-deficient mutants rescued the wild-type phenotype of robust agonist-induced serine <em>1</em><em>1</em>79 phosphorylation. Thus, membrane targeting, but not necessarily acylation, is the critical determinant for agonist-promoted eNOS phosphorylation at serine <em>1</em><em>1</em>79. In striking contrast to serine <em>1</em><em>1</em>79, phosphorylation of eNOS at serine <em>1</em><em>1</em>6 was enhanced in the myr(-) eNOS mutant and was markedly attenuated in the CD8-eNOS membrane-targeted fusion protein. We conclude that eNOS targeting differentially affects eNOS phosphorylation at distinct sites in the protein and suggest that the inter-relationships of eNOS acylation and phosphorylation may modulate eNOS localization and activity and thereby influence NO signaling pathways in the vessel wall.

We have recently reported that S<em>1</em>P (<em>sphingosine</em>-<em>1</em>-<em>phosphate</em>) differentially regulates cellular Rac activity and cell migration in either a positive or a negative direction via distinct G-protein-coupled receptor subtypes, i.e. S<em>1</em>P<em>1</em>/Edg<em>1</em> (endothelial differentiation gene) and S<em>1</em>P2/Edg5 respectively, when each of the S<em>1</em>P receptor subtypes is expressed in CHO (Chinese-hamster ovary) cells. In B<em>1</em>6F<em>1</em>0 mouse melanoma cells, in which S<em>1</em>P2, but not the other S<em>1</em>P receptor subtypes, is endogenously expressed, S<em>1</em>P inhibited cell migration with concomitant inhibition of Rac and stimulation of RhoA in dose-dependent manners. Overexpression of S<em>1</em>P2 in the melanoma cells resulted in potentiation of S<em>1</em>P inhibition of both Rac and cell migration. In contrast, overexpression of S<em>1</em>P<em>1</em> led to stimulation of cell migration, particularly at the lower S<em>1</em>P concentrations. Treatment of B<em>1</em>6F<em>1</em>0 cells with S<em>1</em>P inhibited lung metastasis 3 weeks after injection into mouse tail veins. Intriguingly, overexpression of S<em>1</em>P2 greatly potentiated the inhibition of metastasis by S<em>1</em>P, whereas that of S<em>1</em>P<em>1</em> resulted in aggravation of metastasis. Suppression of cellular Rac activity by adenovirus-transduced expression of N<em>1</em>7Rac, but not N<em>1</em>9RhoA, strongly inhibited cell migration in vitro and lung metastasis in vivo. These results provide the first evidence that G-protein-coupled receptors could participate in the regulation of metastasis, in which ligand-dependent, subtype-specific regulation of the cellular Rac activity is probably critically involved as a mechanism.

BACKGROUND

Recently, genome-wide association studies (GWAS) for cases versus controls using single nucleotide polymorphism microarray data have shown promising findings for complex neuropsychiatric disorders, including bipolar disorder (BD).

METHODS

Here we describe a comprehensive genome-wide study of bipolar disorder (BD), cross-referencing analysis from a family-based study of 229 small families with association analysis from over 950 cases and 950 ethnicity-matched controls from the UK and Canada. Further, loci identified in these analyses were supported by pathways identified through pathway analysis on the samples.

RESULTS

Although no genome-wide significant markers were identified, the combined GWAS findings have pointed to several genes of interest that support GWAS findings for BD from other groups or consortia, such as at SYNE<em>1</em> on 6q25, PPP2R2C on 4p<em>1</em>6.<em>1</em>, ZNF659 on 3p24.3, CNTNAP5 (2q<em>1</em>4.3), and CDH<em>1</em>3 (<em>1</em>6q23.3). This apparent corroboration across multiple sites gives much confidence to the likelihood of genetic involvement in BD at these loci. In particular, our two-stage strategy found association in both our combined case/control analysis and the family-based analysis on <em>1</em>q2<em>1</em>.2 (closest gene: <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> gene, S<em>1</em>PR<em>1</em>) and on <em>1</em>q24.<em>1</em> near the gene TMCO<em>1</em>, and at CSMD<em>1</em> on 8p23.2, supporting several previous GWAS reports for BD and for schizophrenia. Pathway analysis suggests association of pathways involved in calcium signalling, neuropathic pain signalling, CREB signalling in neurons, glutamate receptor signalling and axonal guidance signalling.

CONCLUSIONS

The findings presented here show support for a number of genes previously implicated genes in the etiology of BD, including CSMD<em>1</em> and SYNE<em>1</em>, as well as evidence for previously unreported genes such as the brain-expressed genes ADCY2, NCALD, WDR60, SCN7A and SPAG<em>1</em>6.

The roles of sphingolipids, and particularly of the complex glycosphingolipids (GSLs), the gangliosides, have been studied for many years in neurons, glia, and cell lines derived from these tissues, due to their abundance in tissues of neuronal origin. More recently, significant attention has been paid to the simple sphingolipids, particularly ceramide, glucosylceramide (GlcCer), and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), each of which appears to be involved in the regulation of specific aspects of neuronal proliferation, differentiation, survival and apoptosis. In this review, we will summarize studies performed in our laboratory over the past few years using cultured hippocampal neurons in an attempt to define the precise roles of these lipids, and to define their mechanisms of action by identifying down-stream targets with which they interact. We will also discuss work suggesting that complex GSLs, such as gangliosides GM2 and GD3, can also regulate neuronal development, although the down-stream targets with which they interact are less well defined. Our work will be reviewed in light of studies from other laboratories, with particular emphasis on the use of models of sphingolipid storage diseases to determine how these lipids affect neuronal function.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive sphingolipid produced by <em>sphingosine</em> kinase (SphK<em>1</em> and 2). We previously showed that S<em>1</em>P receptors (S<em>1</em>P<em>1</em>, S<em>1</em>P2, and S<em>1</em>P3) are expressed in hepatic myofibroblasts (hMF), a population of cells that triggers matrix remodeling during liver injury. Here we investigated the function of these receptors in the wound healing response to acute liver injury elicited by carbon tetrachloride, a process that associates hepatocyte proliferation and matrix remodeling. Acute liver injury was associated with the induction of S<em>1</em>P2, S<em>1</em>P3, SphK<em>1</em>, and SphK2 mRNAs and increased SphK activity, with no change in S<em>1</em>P<em>1</em> expression. Necrosis, inflammation, and hepatocyte regeneration were similar in S<em>1</em>P2-/- and wild-type (WT) mice. However, compared with WT mice, S<em>1</em>P2-/- mice displayed reduced accumulation of hMF, as shown by lower induction of smooth muscle alpha-actin mRNA and lower induction of TIMP-<em>1</em>, TGF-beta<em>1</em>, and PDGF-BB mRNAs, overall reflecting reduced activation of remodeling in response to liver injury. The wound healing response was similar in S<em>1</em>P3-/- and WT mice. In vitro, S<em>1</em>P enhanced proliferation of cultured WT hMF, and PDGF-BB further enhanced the mitogenic effect of S<em>1</em>P. In keeping with these findings, PDGF-BB up-regulated S<em>1</em>P2 and SphK<em>1</em> mRNAs, increased SphK activity, and S<em>1</em>P2 induced PDGF-BB mRNA. These effects were blunted in S<em>1</em>P2-/- cells, and S<em>1</em>P2-/- hMF exhibited reduced mitogenic and comitogenic responses to S<em>1</em>P. These results unravel a novel major role of S<em>1</em>P2 in the wound healing response to acute liver injury by a mechanism involving enhanced proliferation of hMF.

We characterized the effect of <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) on vascular tone. S<em>1</em>P selectively constricted isolated cerebral, but not peripheral arteries, despite ubiquitous expression of S<em>1</em>P(<em>1</em>), S<em>1</em>P(2), S<em>1</em>P(3) and S<em>1</em>P(5) receptor mRNA. Clostridium B and C3 toxins and the rho-kinase inhibitor Y27632 (trans-N-(4-pyridyl)-4-(l-aminoethyl)-cyclohexane carboxamide) reduced this vasoconstriction to S<em>1</em>P, indicating that the response was mediated through Rho. Pertussis toxin displayed only weak inhibition, suggesting minor involvement of G(i/o) protein. The S<em>1</em>P effect was specifically reduced by adenovirus bearing a s<em>1</em>p(3) but not s<em>1</em>p(2), antisense construct. Furthermore, suramin, which selectively blocks S<em>1</em>P(3) receptors, inhibited the vasoconstrictor effect of S<em>1</em>P, indicating that S<em>1</em>P(3) receptors account for at least part of S<em>1</em>P-mediated vasoconstriction in cerebral arteries. In vivo, intracarotid injection of S<em>1</em>P decreased cerebral blood flow, an effect prevented by suramin treatment. Because S<em>1</em>P constricts cerebral blood vessels and is released from platelets during clotting, the S<em>1</em>P/S<em>1</em>P(3) system constitutes a novel potential target for cerebrovascular disease therapy.

<em>Sphingosine</em> kinase (SPHK) phosphorylates <em>sphingosine</em> to form a bioactive lipid mediator, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P). S<em>1</em>P mediates such diverse biological processes as regulation of cell differentiation, motility, and apoptosis both extracellularly, via S<em>1</em>P (Edg) family receptors, and intracellularly, through unidentified targets. In cells S<em>1</em>P is short-lived, so the synthetic process catalyzed by <em>sphingosine</em> kinase may be important in maintaining the cellular levels of the compound. Thus far, two <em>sphingosine</em> kinases have been reported, with SPHK<em>1</em> exhibiting the higher activity. However, several studies suggest the existence of unidentified <em>sphingosine</em> kinases. Therefore, to further elucidate the role of SPHK<em>1</em> in the formation of S<em>1</em>P, we investigated its contribution to the total <em>sphingosine</em> kinase activity in mouse tissues. We found that SPHK<em>1</em> is a major <em>sphingosine</em> kinase in many tissues, especially in brain, heart, and colon. However, some tissues such as spleen, small intestine, and lung contained <em>sphingosine</em> kinase activity that was not attributable to SPHK<em>1</em> or SPHK2, as determined by immunodepletion assays. Furthermore, the presence of other <em>sphingosine</em> kinases with different properties, i.e., higher activity toward phyto<em>sphingosine</em> and a different subcellular distribution, is suggested.

Marginal zones (MZs) are microdomains in the spleen that contain various types of immune cells, including MZ B cells, MOMA<em>1</em>(+) metallophilic macrophages, and mucosal addressin cell adhesion molecule <em>1</em> (MAdCAM-<em>1</em>)(+) endothelial cells. MAdCAM-<em>1</em>(+) and MOMA<em>1</em>(+) cells line the sinus, that separates MZs from splenic follicles. Here we show that a receptor for the lysophospholipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), S<em>1</em>P(3), is required for normal numbers of splenic immature and MZ B cells, and for S<em>1</em>P-induced chemotaxis of MZ B cells. S<em>1</em>P(3) is also essential for proper alignment of MOMA<em>1</em>(+) macrophages and MAdCAM-<em>1</em>(+) endothelial cells along the marginal sinus. The lack of cohesion of the marginal sinus in S<em>1</em>P(3)(-/-) mice affects MZ B cell functions, as wild-type (WT) MZ B cells migrate more into S<em>1</em>P(3)(-/-) follicles than into WT follicles after treatment with lipopolysaccharide. Additionally, short-term homing experiments demonstrate that WT MZ B cells home to the S<em>1</em>P(3)(-/-) spleen in increased numbers, suggesting a role for the marginal sinus in regulating MZ B cells numbers. Moreover, S<em>1</em>P(3)(-/-) mice are defective in mounting immune responses to thymus-independent antigen type 2 due to defects in radiation-resistant cells in the spleen. These data identify lysophospholipids and the S<em>1</em>P(3) receptor as essential regulators of the MZ sinus and its role as a barrier to the follicle.

The Src family kinases Fyn and Lyn are important modulators of the molecular events initiated by engagement of the high-affinity IgE receptor (Fc epsilon RI). These kinases control many of the early signaling events and initiate the production of several lipid metabolites that have an important role in regulating mast cell responses. The intracellular level of phosphatidylinositol (3,4,5)-tris<em>phosphate</em> (PIP(3)), which is produced by phosphatidylinositol 3-OH kinase, plays an important role in determining the extent of a mast cells response to a stimulus. Enhanced levels lead to a hyperdegranulating phenotype (as seen in SHIP-<em>1</em>(-/-) and Lyn(-/-) mast cells), whereas decreased levels cause hypodegranulation (as seen in Fyn(-/-) mast cells). Downregulation of mast cell phosphatase and tensin homologue deleted on chromosone <em>1</em>0 expression, a phosphatase that reduces cellular levels of PIP(3), caused constitutive cytokine production, demonstrating that this response is particularly sensitive to PIP(3) levels. Lyn and Fyn are also intimately linked to other lipid kinases, like <em>sphingosine</em> kinases (SphK). By producing <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), SphKs contribute to mast cell chemotaxis and degranulation. In vivo studies now reveal that circulating S<em>1</em>P as well as that found within the mast cell is important in determining mast cell responsiveness. These studies demonstrate the connection between Src protein tyrosine kinases and lipid second messengers that control mast cell function and allergic responses.

We have previously identified a novel complex between the platelet-derived growth factor (PDGF)beta receptor and the <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor-<em>1</em> (S<em>1</em>P<em>1</em>). The complex permits the utilization of active G-protein subunits (made available by constitutively active S<em>1</em>P<em>1</em> receptor) by the PDGFbeta receptor kinase to transmit signals to p42/p44 MAPK in response to PDGF. Therefore, an inverse agonist of the S<em>1</em>P<em>1</em> receptor is predicted to reduce signal transduction from PDGFbeta receptor tyrosine kinase by blocking the constitutive activity of the G-protein coupled receptor. SB649<em>1</em>46 is a novel inverse agonist of the S<em>1</em>P<em>1</em> receptor. First, SB649<em>1</em>46 displaced the S<em>1</em>P<em>1</em> receptor agonist dihydro<em>sphingosine</em> <em>1</em>-<em>phosphate</em> from membranes expressing the recombinant S<em>1</em>P<em>1</em> receptor. Second, SB649<em>1</em>46 reduced basal recombinant S<em>1</em>P<em>1</em> receptor-induced GTPgammaS binding and S<em>1</em>P-induced GTPgammaS binding in membranes. Third, SB649<em>1</em>46 blocked the S<em>1</em>P-induced activation of p42/p44 MAPK in airway smooth muscle cells, a response that is mediated by the S<em>1</em>P<em>1</em> receptor. We now report that inverse agonism of the S<em>1</em>P<em>1</em> receptor with SB649<em>1</em>46 reduced the endocytosis of the PDGFbeta receptor-S<em>1</em>P<em>1</em> receptor complex and the stimulation of p42/p44 MAPK and cell migration in response to PDGF. These findings are the first to report that a GPCR inverse-agonist reduces growth factor-induced receptor tyrosine kinase signaling, fundamentally broadening their mechanism of action. The data obtained with SB649<em>1</em>46 also suggest that the constitutively active endogenous S<em>1</em>P<em>1</em> receptor enhances PDGF-induced cell migration.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lipid which is a potent mitogen for glioblastoma multiforme cells. Here we show that S<em>1</em>P also potently enhances the in vitro motility of glioblastoma cells by signaling through receptors coupled to G(i/o) proteins. Moreover, S<em>1</em>P also enhanced in vitro invasion of glioblastoma cells through Matrigel. S<em>1</em>P had no effect on matrix metalloproteinase secretion but did enhance glioblastoma cell adhesion. S<em>1</em>P is present at high levels in brain tissue. Thus it is possible that autocrine or paracrine signaling by S<em>1</em>P through its G protein-coupled receptors enhances both glioma cell proliferation and invasiveness.

Hepatocellular carcinoma (HCC) is classified as a poor prognostic tumor, and becomes frequently aggressive. MicroRNAs emerge as key contributors to tumor progression. This study investigated whether miR-<em>1</em>48a dysregulation differentiates poor prognosis of HCC, exploring new targets of miR-<em>1</em>48a. miR-<em>1</em>48a dysregulation discriminated not only the overall survival and recurrence free survival rates of HCC, but the microvascular invasion. In the human HCC samples, ubiquitin specific protease 4 (USP4) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor <em>1</em> (S<em>1</em>P<em>1</em>) were up-regulated as the new targets of miR-<em>1</em>48a. USP4 and S<em>1</em>P<em>1</em> were up-regulated in mesenchymal-type liver-tumor cells with miR-<em>1</em>48a dysregulation, facilitating migration and proliferation of tumor cells. The inverse relationship between miR-<em>1</em>48a and the identified targets was verified in a tumor xenograft model. In the analysis of human samples, the expression of USP4, but not S<em>1</em>P<em>1</em>, correlated with the decrease of miR-<em>1</em>48a. In a heterotropic patient-derived HCC xenograft model, USP4 was also overexpressed in G<em>1</em> and G2 tumors when miR-<em>1</em>48a was dysregulated, reflecting the closer link between miR-<em>1</em>48a and USP4 for a shift in the expansion phase of tumorgraft. In conclusion, miR-<em>1</em>48a dysregulation affects the poor prognosis of HCC. Of the identified targets of miR-<em>1</em>48a, USP4 overexpression may contribute to HCC progression towards more aggressive feature.

<em>Sphingosine</em> kinase was partially purified and characterized from rat brain microsomes. A new assay, utilizing octyl-beta-D-glucopyranoside and <em>sphingosine</em> mixed micelles, was developed to quantitate formation of the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> product. The assay was proportional with respect to time and protein, displayed Michaelis-Menten kinetics, and was subject to surface dilution in regard to the <em>sphingosine</em> substrate. Investigations into substrate specificity showed that the enzyme is specific for the erythro-enantiomers of <em>sphingosine</em> and dihydro<em>sphingosine</em>. Neither of the threo-enantiomers were phosphorylated in this system, but both were found to be potent competitive inhibitors of <em>sphingosine</em> kinase activity. Human platelet <em>sphingosine</em> kinase activity displayed substrate and inhibitor specificities similar to the rat brain enzyme. A mixture of DL-threo-dihydro<em>sphingosine</em> competitively inhibited <em>sphingosine</em> kinase activity in a dose dependent manner in isolated platelets. DL-Threo-dihydro<em>sphingosine</em> caused a prolongation of the inhibition of thrombin-induced protein kinase C-dependent 40 (47)-kDa protein phosphorylation in platelets. D-, L-, or DL-Threo-dihydro<em>sphingosine</em> may be useful as a tool to investigate D-Erythro<em>sphingosine</em> metabolism and the function of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> in signal transduction processes.

Increasing evidence suggests a key role for the bioactive lipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and its G-protein-coupled receptors (S<em>1</em>P(<em>1</em>-5)) in the cardiovascular system. Recent advances in sphingolipid research indicates that cardiomyocyte, vascular smooth muscle and endothelial cell function is greatly influenced by the relative expression and activity both of S<em>1</em>P receptors and of the enzymes involved in sphingolipid metabolism. For instance, the discovery and development of S<em>1</em>P receptor agonists such as FTY720 has clearly indicated the involvement of S<em>1</em>P receptors in the regulation of heart rate. In addition, sphingolipid metabolism induced, for example, by tumour necrosis factor-alpha or angiotensin II plays an important role in vessel structure, function and tone.

Modular, bioactive, macroporous scaffolds were formed by crosslinking poly(ethylene glycol) (PEG) microspheres around living cells. Hydrogel microspheres were produced from reactive PEG derivatives in aqueous sodium sulfate solutions without the use of surfactants or copolymers. Microspheres were formed following thermally induced phase separation if the gel point was reached prior to extensive coarsening of the PEG-rich domains. Three types of PEG microspheres with different functionalities were used to form scaffolds: one type provided mechanical support, the second type provided controlled delivery of the angiogenesis-promoting molecule, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and the third type served as a slowly dissolving non-cytotoxic porogen. Scaffolds were formed by centrifuging microspheres in the presence of HepG2 hepatoma cells, resulting in a homogenous distribution of cells. During overnight incubation at 37 degrees C, the microspheres reacted with serum proteins in cell culture medium to stabilize the scaffolds. Within 2 days in culture, macropores formed due to the dissolution of the porogenic PEG microspheres, without affecting cell viability. Gradients in porosity were produced by varying the buoyancy of the porogenic microspheres. Conjugated RGD cell adhesion peptides and the delivery of S<em>1</em>P promoted endothelial cell infiltration through macropores in the scaffolds. The scaffolds presented here differ from previous hydrogel scaffolds in that: (i) cells are not encapsulated in hydrogel; (ii) macropores form in the presence of cells; and (iii) scaffold properties are controlled by the modular assembly of different microspheres that perform distinct functions.

Primary open-angle glaucoma is associated with elevated intraocular pressure, which in turn is believed to result from impaired outflow of aqueous humour. Aqueous humour outflow passes mainly through the trabecular meshwork (TM) and then through pores formed in the endothelium of Schlemm's canal (SC), which experiences a basal-to-apical pressure gradient. This gradient dramatically deforms the SC endothelial cell and potentially contributes to the formation of those pores. However, mechanical properties of the SC cell are poorly defined. Using optical magnetic twisting cytometry and traction force microscopy, here we characterize the mechanical properties of primary cultures of the human SC cell, and for the first time, the scope of their changes in response to pharmacological agents that are known to modulate outflow resistance. Lysophosphatidic acid, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and thrombin caused an increase in cell stiffness by up to 200 per cent, whereas in most cell strains, exposure to latrunculin A, isoproterenol, dibutryl cyclic-AMP or Y-27632 caused a decrease in cell stiffness by up to 80 per cent, highlighting that SC cells possess a remarkably wide contractile scope. Drug responses were variable across donors. S<em>1</em>P, for example, caused 200 per cent stiffening in one donor strain but only 20 per cent stiffening in another. Isoproterenol caused dose-dependent softening in three donor strains but little or no response in two others, a finding mirrored by changes in traction forces and consistent with the level of expression of β(2)-adrenergic receptors. Despite donor variability, those drugs that typically increase outflow resistance systematically caused cell stiffness to increase, while in most cases, those drugs that typically decrease outflow resistance caused cell stiffness to decrease. These findings establish the endothelial cell of SC as a reactive but variable mechanical component of the aqueous humour outflow pathway. Although the mechanism and locus of increased outflow resistance remain unclear, these data suggest the SC endothelial cell to be a modulator of outflow resistance.

<em>Sphingosine</em> <em>1</em> <em>phosphate</em> (S<em>1</em>P), an aminophospholipid, acts extracellularly as a ligand via the specific G protein-coupled receptors of the endothelial differentiation gene (EDG) <em>1</em>, 3, 5, 6 and 8 receptors family and intracellularly as a second messenger in various cellular types. The aim of this work was to investigate biological activity of S<em>1</em>P in cardiomyocytes with respect to related sphingolipids. S<em>1</em>P was applied for 48 h on rat neonatal cardiomyocytes at <em>1</em>0 nM, <em>1</em>00 nM and <em>1</em> microM. S<em>1</em>P induced a concentration-dependent cellular hypertrophy evidenced by an increase in cell size, [3H]-phenylalanine incorporation, protein content and Brain Natriuretic Peptide (BNP) secretion. Among the lipids tested S<em>1</em>P exhibits the lower EC50 (67 nM) followed by dihydro-S<em>1</em>P (<em>1</em>07 nM) and sphingosylphosphorylcholine (<em>1</em>.6 microM). The effect of S<em>1</em>P could be related to a stimulation of the EDG<em>1</em> receptor since we showed that the EDG<em>1</em> receptor is predominantly expressed at the mRNA and protein levels in rat cardiomyocytes and that specific anti-EDG<em>1</em> antibodies inhibited the hypertrophic effect induced by S<em>1</em>P. Furthermore the expression level of most other EDG receptors for S<em>1</em>P appeared very low in cardiac myocytes. S<em>1</em>P (<em>1</em>00 nM) increased the phosphorylation of p42/44MAPK, p38MAPK, JNK, Akt and p70(S6K), this effect being reversed by inhibitors of their respective phosphorylation which also rescue the hypertrophic phenotype. Finally, S<em>1</em>P stimulated actin stress fibre formation reverted by the Rho inhibitor, the C3 exoenzyme. Altogether, our results show that S<em>1</em>P induces cardiomyocyte hypertrophy mainly via the EDG<em>1</em> receptor and subsequently via Gi through ERKs, p38 MAPK, JNK, PI3K and via Rho pathway.

Ceramide has been implicated as an important component of radiation-induced apoptosis of human prostate cancer cells. We examined the role of the sphingolipid metabolites--ceramide, <em>sphingosine</em>, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>--in susceptibility to radiation-induced apoptosis in prostate cancer cell lines with different sensitivities to gamma-irradiation. Exposure of radiation-sensitive TSU-Pr<em>1</em> cells to 8-Gy irradiation led to a sustained increase in ceramide, beginning after <em>1</em>2 h of treatment and increasing to 2.5- to 3-fold within 48 h. Moreover, irradiation of TSU-Pr<em>1</em> cells also produced a marked and rapid 50% decrease in the activity of <em>sphingosine</em> kinase, the enzyme that phosphorylates <em>sphingosine</em> to form <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. In contrast, the radiation-insensitive cell line, LNCaP, had sustained <em>sphingosine</em> kinase activity and did not produce elevated ceramide levels on 8-Gy irradiation. Although LNCaP cells are highly resistant to gamma-irradiation-induced apoptosis, they are sensitive to the death-inducing effects of tumor necrosis factor alpha, which also increases ceramide levels in these cells (K. Kimura et al., Cancer Res., 59: <em>1</em>606-<em>1</em>6<em>1</em>4, <em>1</em>999). Moreover, we found that although irradiation alone did not increase <em>sphingosine</em> levels in LNCaP cells, tumor necrosis factor alpha plus irradiation induced significantly higher <em>sphingosine</em> levels and markedly reduced intracellular levels of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. The elevation of <em>sphingosine</em> levels either by exogenous <em>sphingosine</em> or by treatment with the <em>sphingosine</em> kinase inhibitor N,N-dimethyl<em>sphingosine</em> induced apoptosis and also sensitized LNCaP cells to gamma-irradiation-induced apoptosis. Our data suggest that the relative levels of sphingolipid metabolites may play a role in determining the radiosensitivity of prostate cancer cells, and that the enhancement of ceramide and <em>sphingosine</em> generation could be of therapeutic value.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lipid that is produced by the <em>sphingosine</em> kinase-catalysed phosphorylation of <em>sphingosine</em>. S<em>1</em>P is an important regulator of cell function, mediating many of its effects through a family of five closely related G protein-coupled receptors (GPCR) termed S<em>1</em>P(<em>1</em>-5) which exhibit high affinity for S<em>1</em>P. These receptors function to relay the effects of extracellular S<em>1</em>P via well-defined signal transduction networks linked to the regulation of cell proliferation, survival, migration etc. Diverse agonists (e.g. cytokines) also activate <em>sphingosine</em> kinase and the resulting S<em>1</em>P formed may bind to specific undefined intracellular targets to elicit cellular responses. The purpose of this review is to discuss some of the spatial/temporal aspects of intracellular S<em>1</em>P signalling and to define the function of <em>sphingosine</em> kinases and lipid <em>phosphate</em> phosphatases (which catalyse dephosphorylation of S<em>1</em>P) in terms of their regulation of cell function. Finally, we survey the function of S<em>1</em>P in relation to disease, where the major challenge is to dissect the role of intracellular versus extracellular actions of S<em>1</em>P in terms of association with defined diseased phenotypes.

The balance between the pro-apoptotic lipids ceramide and <em>sphingosine</em> and the pro-survival lipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is termed the "<em>sphingosine</em> rheostat". Two isozymes, <em>sphingosine</em> kinase <em>1</em> and 2 (SK<em>1</em> and SK2), are responsible for phosphorylation of pro-apoptotic <em>sphingosine</em> to form pro-survival S<em>1</em>P. We have previously reported the antitumor properties of an SK2 selective inhibitor, ABC294640, alone or in combination with the multikinase inhibitor sorafenib in mouse models of kidney carcinoma and pancreatic adenocarcinoma. Here we evaluated the combined antitumor effects of the aforementioned drug combination in two mouse models of hepatocellular carcinoma. Although combining the SK2 inhibitor, ABC294640, and sorafenib in vitro only afforded additive drug-drug effects, their combined antitumor properties in the mouse model bearing HepG2 cells mirrored effects previously observed in animals bearing kidney carcinoma and pancreatic adenocarcinoma cells. Combining ABC294640 and sorafenib led to a decrease in the levels of phosphorylated ERK in SK-HEP-<em>1</em> cells, indicating that the antitumor effect of this drug combination is likely mediated through a suppression of the MAPK pathway in hepatocellular models. We also measured levels of S<em>1</em>P in the plasma of mice treated with two different doses of ABC294640 and sorafenib. We found decreases in the levels of S<em>1</em>P in plasma of mice treated daily with <em>1</em>00 mg/kg of ABC294640 for 5 weeks, and this decrease was not affected by co-administration of sorafenib. Taken together, these data support combining ABC294640 and sorafenib in clinical trials in HCC patients. Furthermore, monitoring levels of S<em>1</em>P may provide a pharmacodynamic marker of ABC294640 activity.

It is important to identify novel and effective targets for cancer prevention and therapy against head and neck squamous cell carcinoma (HNSCC), one of the most lethal cancers. Accumulating evidence suggests that the bioactive sphingolipids, such as <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and its generating enzyme, <em>sphingosine</em> kinase <em>1</em> (SphK<em>1</em>) play pivotal roles in several important biological functions including promoting tumor growth and carcinogenesis. However, roles of SphK<em>1</em>/S<em>1</em>P in HNSCC development and/or progression have not been defined previously. Therefore, in this study, we first analyzed the expression of SphK<em>1</em> in human HNSCC tumor samples and normal head & neck tissues (n = 78 and <em>1</em>7, respectively) using immunohistochemistry. The data showed that SphK<em>1</em> is overexpressed in all of the HNSCC tumors tested (stages I-IV). We next investigated whether SphK<em>1</em> is necessary for HNSCC development. To define the role of SphK<em>1</em>/S<em>1</em>P in HNSCC development, we utilized 4-nitroquinoline-<em>1</em>-oxide (4-NQO)-induced HNSCC model in wild-type mice compared with SphK<em>1</em>(-/-) knockout (KO) mice. Remarkably, we found that the genetic loss of SphK<em>1</em>, which reduced S<em>1</em>P generation, significantly prevented 4-NQO-induced HNSCC carcinogenesis, with decreased tumor incidence, multiplicity, and volume when compared with controls. Moreover, our data indicated that prevention of 4-NQO-induced HNSCC development in SphK<em>1</em>(-/-) KO mice might be associated with decreased cell proliferation, increased levels of cleaved (active) caspase 3, and downregulation of phospho (active) AKT expression. Thus, these novel data suggest that SphK<em>1</em>/S<em>1</em>P signaling may play important roles in HNSCC carcinogenesis, and that targeting SphK<em>1</em>/S<em>1</em>P might provide a novel strategy for chemoprevention and treatment against HNSCC.

To evaluate the therapeutic effects of the new synthetic <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor modulator, FTY720, we investigated how FTY720 affects the development of dextran sulfate sodium (DSS)-induced colitis and CD4+CD62L+ T cell transfer colitis. BALB/c mice were fed a chow containing 3.5% (wt/wt) DSS to induce colitis. The CD4+CD62L+ T cell transfer colitis was induced by an intraperitoneal injection of CD4+CD62L+ spleen T cells into recipient CB<em>1</em>7 SCID mice. The FTY720 was administered by lavage at a dose of 0.3 mg/kg/day. FTY720 was effective in preventing the body weight loss in the DSS-colitis model and the CD4+CD62L+ T cell transfer model. The disease activity index, histological colitis score, and MPO activity were all significantly lower in FTY720-treated mice than in the non-treated mice. Microscopically, mucosal edema, cellular infiltration and epithelial disruption were much more moderate in the FTY720-treated mice than in the non-treated mice. In both colitis models, FTY720 prevented the infiltration of CD4+ T cells into the inflamed colonic lamina propria. In conclusion, the development of DSS-colitis and CD4+CD62L+ T cell transfer colitis were significantly attenuated by FTY720. Since FTY720 is an immunosuppressive product that does not modulate T cell functions, it could be useful in the treatment of IBD patients.

BACKGROUND

Siponimod is an oral selective modulator of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor types <em>1</em> and type 5, with an elimination half-life leading to washout in 7 days. We aimed to determine the dose-response relation of siponimod in terms of its effects on brain MRI lesion activity and characterise safety and tolerability in patients with relapsing-remitting multiple sclerosis.

METHODS

In this double-blind, adaptive dose-ranging phase 2 study, we enrolled adults (aged <em>1</em>8-55 years) with relapsing-remitting multiple sclerosis at 73 medical centres in Europe and North America. We tested two patient cohorts sequentially, separated by an interim analysis at 3 months. We randomly allocated patients in cohort <em>1</em> (<em>1</em>:<em>1</em>:<em>1</em>:<em>1</em>) to receive once-daily siponimod <em>1</em>0 mg, 2 mg, or 0·5 mg, or placebo for 6 months. We randomly allocated patients in cohort 2 (4:4:<em>1</em>) to siponimod <em>1</em>·25 mg, siponimod 0·25 mg, or placebo once-daily for 3 months. Randomisation was done with a central, automated system and patients and investigators were masked to treatment assignment. The primary endpoint was dose-response, assessed by percentage reduction in monthly number of combined unique active lesions at 3 months for siponimod versus placebo; this endpoint was analysed by a multiple comparison procedure with modelling techniques in all patients with at least one MRI scan up to 3 months. We assessed safety in all patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT00879658.

RESULTS

Between March 30, 2009, and Oct 22, 20<em>1</em>0, we recruited <em>1</em>88 patients into cohort <em>1</em> and <em>1</em>09 patients into cohort 2. We showed a dose-response relation (p=0·000<em>1</em>) across the five doses of siponimod, with reductions in combined unique active lesions at 3 months compared with placebo of 35% (95% CI <em>1</em>7-57) for siponimod 0·25 mg (5<em>1</em> patients included in the primary endpoint analysis), 50% (29-69) for siponimod 0·5 mg (43 patients), 66% (48-80) for siponimod <em>1</em>·25 mg (42 patients), 72% (57-84) for siponimod 2 mg (45 patients), and 82% (70-90) for siponimod <em>1</em>0 mg (44 patients). In patients treated for 6 months, 37 (86%) of 43 patients who received siponimod 0·5 mg had adverse events (eight serious), as did 48 (98%) of 49 patients who received siponimod 2 mg (four serious), 48 (96%) of 50 patients who received siponimod <em>1</em>0 mg (three serious), and 36 (80%) of 45 controls (none serious). For individuals treated to 3 months, 38 (74%) of 5<em>1</em> patients who received siponimod 0·25 mg had adverse events (none serious), as did 29 (69%) of 42 patients who received siponimod <em>1</em>·25 mg (two serious) and <em>1</em>3 (8<em>1</em>%) of <em>1</em>6 controls (none serious).

CONCLUSIONS

Therapeutic effects of siponimod on MRI lesion activity in model-based analyses and its tolerability in relapsing-remitting multiple sclerosis warrant investigation in a phase 3 trial.

BACKGROUND

Novartis Pharma AG.