BACKGROUND

RhoA and Rho kinase are important modulators of microvascular tone.

RESULTS

We tested whether <em>sphingosine</em> kinase (Sphk<em>1</em>) that generates the endogenous sphingolipid mediator <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is part of a signaling cascade to activate the RhoA/Rho kinase pathway. Using a new transfection model, we report that resting tone and myogenic responses of isolated resistance arteries increased with forced expression of Sphk<em>1</em> in smooth muscle cells of these arteries. Overexpression of a dominant negative Sphk<em>1</em> mutant or coexpression of dominant negative mutants of RhoA or Rho kinase together with Sphk<em>1</em> completely inhibited development of tone and myogenic responses.

CONCLUSIONS

The tone-increasing effects of a Sphk<em>1</em> overexpression suggest that Sphk<em>1</em> may play an important role in the control of peripheral resistance.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> receptors (S<em>1</em>P<em>1</em>-5) are activated by the endogenous agonist S<em>1</em>P and are expressed in the central nervous system. In astrocytes, activation of S<em>1</em>P receptors leads to phosphorylation of extracellular-signal regulated kinase (ERK), a signaling cascade which plays intimate roles in cell proliferation. Fingolimod (FTY720) is in phase III clinical trials for the treatment of multiple sclerosis and its phosphorylated version (FTY720P) activates S<em>1</em>P receptors. We examined the effects of FTY720P on ERK phosphorylation and determined which S<em>1</em>P receptor subtype(s) mediated this signaling event. FTY720P augmented ERK phosphorylation in cortical cultures prepared from embryonic day <em>1</em>8 rat brains and was blocked by an MEK inhibitor or by pertussis toxin. Co-localisation of phosphorylated ERK occurred in glial fibrillary acidic protein (GFAP) positive astrocytes but not neurons or oligodendrocytes. Furthermore, FTY720P stimulated ERK phosphorylation in highly enriched astrocyte cultures made from postnatal day 2 rat cortices. The effects of FTY720P were mimicked by selective S<em>1</em>P<em>1</em> receptor agonists and blocked by S<em>1</em>P<em>1</em> receptor antagonists. Collectively, these results demonstrate that FTY720P mediates ERK phosphorylation in astrocytes via the activation of S<em>1</em>P<em>1</em> receptors.

The cytokine tumour necrosis factor-alpha (TNF) has been implicated in autoimmune diseases and may play an indirect role in activation of pain pathways. In this study we have investigated the possibility that TNF directly activates cultured neonatal rat dorsal root ganglion (DRG) neurones and provides a signalling pathway from cells in the immune system such as macrophages to sensory neurones. Expression of TNF receptor subtypes (TNFR<em>1</em> and TNFR2) on sensory neurones was identified using immunohistochemistry, fluorescence-activated cell sorting analysis and RT-PCR. Biochemical and immunocytochemical analysis showed that TNF activated p38 mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) but not p42/p44 MAPK. TNF treatment evoked transient Ca2+-dependent inward currents in 70% of DRG neurones. These TNF-evoked currents were significantly attenuated by ryanodine or thapsigargin or by inclusion of BAPTA in the patch pipette solution. Responses were also evoked in subpopulations of cultured DRG neurones by human mutant TNFs that cross-reacted with rat receptors and selectively activated TNFR<em>1</em> or TNFR2 subtypes. TNF-evoked transient increases in [Ca2+]i were also detected in 34% of fura-2-loaded DRG neurones. The link between TNF receptor activation and Ca2+ release from stores remains to be elucidated. However, responses to TNF were mimicked by sphingolipids, including <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, which evoked a transient rises in [Ca2+]i in a pertussis toxin-insensitive manner in fura-2-loaded DRG neurones. We conclude that distinct receptors TNFR<em>1</em> and TNFR2 are expressed on cultured DRG neurones and that they are functionally linked to intracellular Ca2+ mobilisation, a response that may involve sphingolipid signalling.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a pleiotropic bioactive lipid involved in multiple physiological processes. Importantly, dysregulated S<em>1</em>P levels are associated with several pathologies, including cardiovascular and inflammatory diseases and cancer. This report describes the successful production and characterization of a murine monoclonal antibody, LT<em>1</em>002, directed against S<em>1</em>P, using novel immunization and screening methods applied to bioactive lipids. We also report the successful generation of LT<em>1</em>009, the humanized variant of LT<em>1</em>002, for potential clinical use. Both LT<em>1</em>002 and LT<em>1</em>009 have high affinity and specificity for S<em>1</em>P and do not cross-react with structurally related lipids. Using an in vitro bioassay, LT<em>1</em>002 and LT<em>1</em>009 were effective in blocking S<em>1</em>P-mediated release of the pro-angiogenic and prometastatic cytokine, interleukin-8, from human ovarian carcinoma cells, showing that both antibodies can out-compete S<em>1</em>P receptors in binding to S<em>1</em>P. In vivo anti-angiogenic activity of all antibody variants was demonstrated using the murine choroidal neovascularization model. Importantly, intravenous administration of the antibodies showed a marked effect on lymphocyte trafficking. The resulting lead candidate, LT<em>1</em>009, has been formulated for Phase <em>1</em> clinical trials in cancer and age-related macular degeneration. The anti-S<em>1</em>P antibody shows promise as a novel, first-in-class therapeutic acting as a "molecular sponge" to selectively deplete S<em>1</em>P from blood and other compartments where pathological S<em>1</em>P levels have been implicated in disease progression or in disorders where immune modulation may be beneficial.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) in blood and lymph controls T cell traffic and proliferation through type <em>1</em> S<em>1</em>P receptor (S<em>1</em>P(<em>1</em>)) signals, but suppression of IFN-gamma generation has been the only consistently observed effect on T cell cytokines. The fact that S<em>1</em>P enhances the development of Th<em>1</em>7 cells from Ag-challenged transgenic S<em>1</em>P(<em>1</em>)-overexpressing CD4 T cells suggested that the S<em>1</em>P-S<em>1</em>P(<em>1</em>) axis may promote the expansion of Th<em>1</em>7 cells in wild-type mice. In a model of Th<em>1</em>7 cell development from CD4 T cells stimulated by anti-CD3 plus anti-CD28 Abs and a mixture of TGF-beta<em>1</em>, IL-<em>1</em>, and IL-6, S<em>1</em>P enhanced their number and IL-<em>1</em>7-generating activity the same as IL-23. As for IL-23 enhancement of Th<em>1</em>7 cell development, that by S<em>1</em>P was prevented by IL-4 plus IFN-gamma and by IL-27. The prevention of S<em>1</em>P augmentation of Th<em>1</em>7 cell development by the S<em>1</em>P receptor agonist and down-regulator FTY720 implies that FTY720 immunosuppression is attributable partially to inhibition of Th<em>1</em>7-mediated inflammation.

Here we provide evidence to show that the platelet-derived growth factor beta receptor is tethered to endogenous G-protein-coupled receptor(s) in human embryonic kidney 293 cells. The tethered receptor complex provides a platform on which receptor tyrosine kinase and G-protein-coupled receptor signals can be integrated to produce more efficient stimulation of the p42/p44 mitogen-activated protein kinase pathway. This was based on several lines of evidence. First, we have shown that pertussis toxin (which uncouples G-protein-coupled receptors from inhibitory G-proteins) reduced the platelet-derived growth factor stimulation of p42/p44 mitogen-activated protein kinase. Second, transfection of cells with inhibitory G-protein alpha subunit increased the activation of p42/p44 mitogen-activated protein kinase by platelet-derived growth factor. Third, platelet-derived growth factor stimulated the tyrosine phosphorylation of the inhibitory G-protein alpha subunit, which was blocked by the platelet-derived growth factor kinase inhibitor, tyrphostin AG <em>1</em>296. We have also shown that the platelet-derived growth factor beta receptor forms a tethered complex with Myc-tagged endothelial differentiation gene <em>1</em> (a G-protein-coupled receptor whose agonist is <em>sphingosine</em> <em>1</em>-<em>phosphate</em>) in cells co-transfected with these receptors. This facilitates platelet-derived growth factor-stimulated tyrosine phosphorylation of the inhibitory G-protein alpha subunit and increases p42/p44 mitogen-activated protein kinase activation. In addition, we found that G-protein-coupled receptor kinase 2 and beta-arrestin I can associate with the platelet-derived growth factor beta receptor. These proteins play an important role in regulating endocytosis of G-protein-coupled receptor signal complexes, which is required for activation of p42/p44 mitogen-activated protein kinase. Thus, platelet-derived growth factor beta receptor signaling may be initiated by G-protein-coupled receptor kinase 2/beta-arrestin I that has been recruited to the platelet-derived growth factor beta receptor by its tethering to a G-protein-coupled receptor(s). These results provide a model that may account for the co-mitogenic effect of certain G-protein-coupled receptor agonists with platelet-derived growth factor on DNA synthesis.

Although the requirements for T lymphocyte homing to lymph nodes (LNs) are well studied, much less is known about the requirements for T lymphocyte locomotion within LNs. Imaging of murine T lymphocyte migration in explanted LNs using two-photon laser-scanning fluorescence microscopy provides an opportunity to systematically study these requirements. We have developed a closed system for imaging an intact LN with controlled temperature, oxygenation, and perfusion rate. Naive T lymphocyte locomotion in the deep paracortex of the LN required a perfusion rate of>><em>1</em>3 microm/s and a partial pressure of O(2) (pO(2)) of >7.4%. Naive T lymphocyte locomotion in the subcapsular region was 38% slower and had higher turning angles and arrest coefficients than naive T lymphocytes in the deep paracortex. T lymphocyte activation decreased the requirement for pO(2), but also decreased the speed of locomotion in the deep paracortex. Although CCR7(-/-) naive T cells displayed a small reduction in locomotion, systemic treatment with pertussis toxin reduced naive T lymphocyte speed by 59%, indicating a contribution of Galpha(i)-mediated signaling, but involvement of other G protein-coupled receptors besides CCR7. Receptor knockouts or pharmacological inhibition in the adenosine, PG/lipoxygenase, lysophosphatidylcholine, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> pathways did not individually alter naive T cell migration. These data implicate pO(2), tissue architecture, and G-protein coupled receptor signaling in regulation of naive T lymphocyte migration in explanted LNs.

Highly up-regulated in liver cancer (HULC) is a long non-coding RNA (lncRNA). We found that HULC up-regulated <em>sphingosine</em> kinase <em>1</em> (SPHK<em>1</em>), which is involved in tumor angiogenesis. Levels of HULC were positively correlated with levels of SPHK<em>1</em> and its product, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), in patients HCC samples. HULC increased SPHK<em>1</em> in hepatoma cells. Chicken chorioallantoic membrane (CAM) assays revealed that si-SPHK<em>1</em> remarkably blocked the HULC-enhanced angiogenesis. Mechanistically, HULC activated the promoter of SPHK<em>1</em> in hepatoma cells through the transcription factor E2F<em>1</em>. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) further showed that E2F<em>1</em> was capable of binding to the E2F<em>1</em> element in the SPHK<em>1</em> promoter. HULC increased the expression of E2F<em>1</em> in hepatoma cells and levels of HULC were positively correlated with those of E2F<em>1</em> in HCC tissues. Intriguingly, HULC sequestered miR-<em>1</em>07, which targeted E2F<em>1</em> mRNA 3'UTR, by complementary base pairing. Functionally, si-SPHK<em>1</em> remarkably abolished the HULC-enhanced tumor angiogenesis in vitro and in vivo. Taken together, we conclude that HULC promotes tumor angiogenesis in liver cancer through miR-<em>1</em>07/E2F<em>1</em>/SPHK<em>1</em> signaling. Our finding provides new insights into the mechanism of tumor angiogenesis.

To maintain an intact barrier, epithelia eliminate dying cells by extrusion. During extrusion, a cell destined for apoptosis signals its neighboring cells to form and contract a ring of actin and myosin, which squeezes the dying cell out of the epithelium. Here, we demonstrate that the signal produced by dying cells to initiate this process is <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). Decreasing S<em>1</em>P synthesis by inhibiting <em>sphingosine</em> kinase activity or by blocking extracellular S<em>1</em>P access to its receptor prevented apoptotic cell extrusion. Extracellular S<em>1</em>P activates extrusion by binding the S<em>1</em>P(2) receptor in the cells neighboring a dying cell, as S<em>1</em>P(2) knockdown in these cells or its loss in a zebrafish mutant disrupted cell extrusion. Because live cells can also be extruded, we predict that this S<em>1</em>P pathway may also be important for driving delamination of stem cells during differentiation or invasion of cancer cells.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is an ubiquitously present extracellular lipid mediator that is released by several cell types, particularly by activated platelets. The effects of S<em>1</em>P are mediated by a specific family of G protein-coupled <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptors (S<em>1</em>P<em>1</em>-S<em>1</em>P5). We demonstrate that S<em>1</em>P acts on hematopoietic progenitor cells as a chemotactic factor, attracting peripheral blood CD34(+) cells in vitro. Furthermore, constant activation of S<em>1</em>P receptors augments CXCR4-mediated signal transduction induced by stromal cell-derived factor <em>1</em> (SDF-<em>1</em>). These effects are most likely mediated by the S<em>1</em>P<em>1</em> receptor consistently expressed in both primitive and committed CD34(+) hematopoietic progenitor cells (HPCs). In vivo, sustained activation of S<em>1</em>P<em>1</em> by a receptor agonist during the homing process resulted in increased engraftment. Given the fact that activated platelets represent a major source of extracellular S<em>1</em>P, SDF-<em>1</em>-mediated stem cell homing may occur at sites of tissue injury in addition to the bone marrow. This could explain the previously observed contribution of primary hematopoietic stem cells to tissue repair in myocardial infarction and other diseases.

Sphingolipid long-chain bases and their phosphorylated derivatives, for example, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> in mammals, have been implicated as signaling molecules. The possibility that Saccharomyces cerevisiae cells also use long-chain-base <em>phosphates</em> to regulate cellular processes has only recently begun to be examined. Here we present a simple and sensitive procedure for analyzing and quantifying long-chain-base <em>phosphates</em> in S. cerevisiae cells. Our data show for the first time that phyto<em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (PHS-<em>1</em>-P) is present at a low but detectable level in cells grown on a fermentable carbon source at 25 degreesC, while dihydro<em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (DHS-<em>1</em>-P) is only barely detectable. Shifting cells to 37 degreesC causes transient eight- and fivefold increases in levels of PHS-<em>1</em>-P and DHS-<em>1</em>-P, respectively, which peak after about <em>1</em>0 min. The amounts of both compounds return to the unstressed levels by 20 min after the temperature shift. These data are consistent with PHS-<em>1</em>-P and DHS-<em>1</em>-P being signaling molecules. Cells unable to break down long-chain-base <em>phosphates</em>, due to deletion of DPL<em>1</em> and LCB3, show a 500-fold increase in PHS-<em>1</em>-P and DHS-<em>1</em>-P levels, grow slowly, and survive a 44 degreesC heat stress <em>1</em>0-fold better than parental cells. These and other data for dpl<em>1</em> or lcb3 single-mutant strains suggest that DHS-<em>1</em>-P and/or PHS-<em>1</em>-P act as signals for resistance to heat stress. Our procedure should expedite experiments to determine how the synthesis and breakdown of these compounds is regulated and how the compounds mediate resistance to elevated temperature.

Safingol, the synthetic L-threo-stereoisomer of endogenous (D-erythro-) sphinganine, is an inhibitor of protein kinase C and <em>sphingosine</em> kinase in vitro, and in some cell types has been implicated in ceramide generation and induction of apoptosis. Utilizing electron microscopy, acridine orange staining, and immunoblot and fluorescent localization studies of the myosin light chain-associated protein (LC3), we determined that safingol induces cell death of an exclusively autophagic character and lacking any of the hallmarks of apoptosis. Safingol inhibited PKCbeta-I, PKC delta and PKC epsilon, and inhibited phosphorylation of critical components of the PI3k/Akt/mTOR pathway (Akt, p70S6k and rS6) and the MAPk pathway (ERK). Inhibition of PI3k with LY294002 or suppression of PKC delta and PKC epsilon with siRNA in HCT-<em>1</em><em>1</em>6 cells induced autophagy, though not to the extent caused by safingol. Conversely, activation of PKCs with phorbol <em>1</em>2,<em>1</em>3-dibutyrate (PDBu) or transient transfection of a constitutively active form of Akt each reduced safingol's autophagic induction, but not completely, indicating that Akt- and PKC-dependent pathways both contribute partially and independently to safingol-induced autophagy. Accordingly, combining siRNA depletion of PKC epsilon with LY294002 inhibition of PI3k induced autophagy to a degree comparable to safingol. Liquid chromatography, electrospray tandem mass spectrometry analysis indicated that safingol did not elevate levels of any endogenous sphingolipids previously shown to induce autophagy (ceramide, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> and dihydroceramide); therefore, these effects may be due to safingol per se or another metabolite. Thus, our studies establish that safingol induces autophagy through inhibition of PKCs and PI3k by safingol directly rather than via changes in endogenous sphingolipids.

The role of Delta4-unsaturated sphingolipid long-chain bases such as <em>sphingosine</em> was investigated in Arabidopsis (Arabidopsis thaliana). Identification and functional characterization of the sole Arabidopsis ortholog of the sphingolipid Delta4-desaturase was achieved by heterologous expression in Pichia pastoris. A P. pastoris mutant disrupted in the endogenous sphingolipid Delta4-desaturase gene was unable to synthesize glucosylceramides. Synthesis of glucosylceramides was restored by the expression of Arabidopsis gene At4g04930, and these sphingolipids were shown to contain Delta4-unsaturated long-chain bases, confirming that this open reading frame encodes the sphingolipid Delta4-desaturase. At4g04930 has a very restricted expression pattern, transcripts only being detected in pollen and floral tissues. Arabidopsis insertion mutants disrupted in the sphingolipid Delta4-desaturase At4g04930 were isolated and found to be phenotypically normal. Sphingolipidomic profiling of a T-DNA insertion mutant indicated the absence of Delta4-unsaturated sphingolipids in floral tissue, also resulting in the reduced accumulation of glucosylceramides. No difference in the response to drought or water loss was observed between wild-type plants and insertion mutants disrupted in the sphingolipid Delta4-desaturase At4g04930, nor was any difference observed in stomatal closure after treatment with abscisic acid. No differences in pollen viability between wild-type plants and insertion mutants were detected. Based on these observations, it seems unlikely that Delta4-unsaturated sphingolipids and their metabolites such as <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> play a significant role in Arabidopsis growth and development. However, Delta4-unsaturated ceramides may play a previously unrecognized role in the channeling of substrates for the synthesis of glucosylceramides.

Extracellular sphingolipid signaling has been implicated as an essential event in vascular development. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), through interactions with G protein-coupled receptors, regulates functions of endothelial and smooth muscle cells (SMCs)-the major cell types of the vasculature. The knockout of the gene encoding the S<em>1</em>P<em>1</em> receptor (formally known as Edg-<em>1</em>) in mice blocks vascular maturation, the process where SMCs and pericytes envelop nascent endothelial tubes. The question that remains is how stimulation of S<em>1</em>P receptors controls this critical event in the developmental sequence leading to the formation of functional blood vessels.

Simple bioactive sphingolipids include ceramide, <em>sphingosine</em> and their phosphorylated forms <em>sphingosine</em> <em>1</em>-<em>phosphate</em> and ceramide <em>1</em>-<em>phosphate</em>. These molecules are crucial regulators of cell functions. In particular, they play important roles in the regulation of angiogenesis, apoptosis, cell proliferation, differentiation, migration, and inflammation. Decoding the mechanisms by which these cellular functions are regulated requires detailed understanding of the signaling pathways that are implicated in these processes. Most importantly, the development of inhibitors of the enzymes involved in their metabolism may be crucial for establishing new therapeutic strategies for treatment of disease.

In yeast, the long-chain sphingoid base <em>phosphate</em> phosphohydrolase Lcb3p is required for efficient ceramide synthesis from exogenous sphingoid bases. Similarly, in this study, we found that incorporation of exogenous <em>sphingosine</em> into ceramide in mammalian cells was regulated by the homologue of Lcb3p, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> phosphohydrolase <em>1</em> (SPP-<em>1</em>), an endoplasmic reticulum resident protein. <em>Sphingosine</em> incorporation into endogenous long-chain ceramides was increased by SPP-<em>1</em> overexpression, whereas recycling of C(6)-ceramide into long-chain ceramides was not altered. The increase in ceramide was inhibited by fumonisin B(<em>1</em>), an inhibitor of ceramide synthase, but not by ISP-<em>1</em>, an inhibitor of serine palmitoyltransferase, the rate-limiting step in the de novo biosynthesis of ceramide. Mass spectrometry analysis revealed that SPP-<em>1</em> expression increased the incorporation of <em>sphingosine</em> into all ceramide acyl chain species, particularly enhancing C<em>1</em>6:0, C<em>1</em>8:0, and C20:0 long-chain ceramides. The increased recycling of <em>sphingosine</em> into ceramide was accompanied by increased hexosylceramides and, to a lesser extent, sphingomyelins. <em>Sphingosine</em> kinase 2, but not <em>sphingosine</em> kinase <em>1</em>, acted in concert with SPP-<em>1</em> to regulate recycling of <em>sphingosine</em> into ceramide. Collectively, our results suggest that an evolutionarily conserved cycle of phosphorylation-dephosphorylation regulates recycling and salvage of <em>sphingosine</em> to ceramide and more complex sphingolipids.

<em>Sphingosine</em> kinase <em>1</em> (SK<em>1</em>) catalyzes the conversion of <em>sphingosine</em> to the bioactive lipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. We have previously demonstrated that FTY720 and (S)-FTY720 vinylphosphonate are novel inhibitors of SK<em>1</em> activity. Here, we show that (S)-FTY720 vinylphosphonate binds to a putative allosteric site in SK<em>1</em> contingent on formation of the enzyme-<em>sphingosine</em> complex. We report that SK<em>1</em> is an oligomeric protein (minimally a dimer) containing noncooperative catalytic sites and that the allosteric site exerts an autoinhibition of the catalytic site. A model is proposed in which (S)-FTY720 vinylphosphonate binding to and stabilization of the allosteric site might enhance the autoinhibitory effect on SK<em>1</em> activity. Further evidence for the existence of allosteric site(s) in SK<em>1</em> was demonstrated by data showing that two new FTY720 analogues (a conjugate of <em>sphingosine</em> with a fluorophore and (S)-FTY720 regioisomer) increased SK<em>1</em> activity, suggesting relief of autoinhibition of SK<em>1</em> activity. Comparisons with the SK<em>1</em> inhibitor, SKi or siRNA knockdown of SK<em>1</em> indicated that (S)-FTY720 vinylphosphonate and FTY720 behave as typical SK<em>1</em> inhibitors in preventing <em>sphingosine</em> <em>1</em>-<em>phosphate</em>-stimulated rearrangement of actin in MCF-7 cells. These findings are discussed in relation to the anticancer properties of SK<em>1</em> inhibitors.

Sphingolipids and their synthetic enzymes are emerging as important mediators in inflammatory responses and as regulators of immune cell functions. In particular, <em>sphingosine</em> kinase (SK) and its product <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) have been extensively implicated in these processes. SK catalyzes the phosphorylation of <em>sphingosine</em> to S<em>1</em>P and exists as two isoforms, SK<em>1</em> and SK2. SK<em>1</em> has been shown to be activated by cytokines including tumor necrosis factor-alpha (TNF-alpha) and interleukin<em>1</em>-beta (IL<em>1</em>-beta). The activation of SK<em>1</em> in this pathway has been shown to be, at least in part, required for mediating TNF-alpha and IL<em>1</em>-beta inflammatory responses in cells, including induction of cyclo-oxygenase 2 (COX2). In addition to their role in inflammatory signaling, SK and S<em>1</em>P have also been implicated in various immune cell functions including, mast cell degranulation, migration of neutrophils, and migration and maturation of lymphocytes. The involvement of sphingolipids and sphingolipid metabolizing enzymes in inflammatory signaling and immune cell functions has implicated these mediators in numerous inflammatory disease states as well. The contribution of these mediators, specifically SK<em>1</em> and S<em>1</em>P, to inflammation and disease are discussed in this review.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) signaling regulates lymphocyte egress from lymphoid organs into systemic circulation. The <em>sphingosine</em> <em>phosphate</em> receptor <em>1</em> (S<em>1</em>P<em>1</em>) agonist FTY-720 (Gilenya) arrests immune trafficking and prevents multiple sclerosis (MS) relapses. However, alternative mechanisms of S<em>1</em>P-S<em>1</em>P<em>1</em> signaling have been reported. Phosphoproteomic analysis of MS brain lesions revealed S<em>1</em>P<em>1</em> phosphorylation on S35<em>1</em>, a residue crucial for receptor internalization. Mutant mice harboring an S<em>1</em>pr<em>1</em> gene encoding phosphorylation-deficient receptors (S<em>1</em>P<em>1</em>(S5A)) developed severe experimental autoimmune encephalomyelitis (EAE) due to autoimmunity mediated by interleukin <em>1</em>7 (IL-<em>1</em>7)-producing helper T cells (TH<em>1</em>7 cells) in the peripheral immune and nervous system. S<em>1</em>P<em>1</em> directly activated the Jak-STAT3 signal-transduction pathway via IL-6. Impaired S<em>1</em>P<em>1</em> phosphorylation enhances TH<em>1</em>7 polarization and exacerbates autoimmune neuroinflammation. These mechanisms may be pathogenic in MS.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) functions not only as a bioactive lipid molecule, but also as an important intermediate of the sole sphingolipid-to-glycerolipid metabolic pathway. However, the precise reactions and the enzymes involved in this pathway remain unresolved. We report here that yeast HFD<em>1</em> and the Sjögren-Larsson syndrome (SLS)-causative mammalian gene ALDH3A2 are responsible for conversion of the S<em>1</em>P degradation product hexadecenal to hexadecenoic acid. The absence of ALDH3A2 in CHO-K<em>1</em> mutant cells caused abnormal metabolism of S<em>1</em>P/hexadecenal to ether-linked glycerolipids. Moreover, we demonstrate that yeast Faa<em>1</em> and Faa4 and mammalian ACSL family members are acyl-CoA synthetases involved in the sphingolipid-to-glycerolipid metabolic pathway and that hexadecenoic acid accumulates in Δfaa<em>1</em> Δfaa4 mutant cells. These results unveil the entire S<em>1</em>P metabolic pathway: S<em>1</em>P is metabolized to glycerolipids via hexadecenal, hexadecenoic acid, hexadecenoyl-CoA, and palmitoyl-CoA. From our results we propose a possibility that accumulation of the S<em>1</em>P metabolite hexadecenal contributes to the pathogenesis of SLS.

We have previously reported that, in prostate cancer, inhibition of the oncogenic <em>sphingosine</em> kinase-<em>1</em>/<em>sphingosine</em> <em>1</em>-<em>phosphate</em> (SphK<em>1</em>/S<em>1</em>P) pathway is a key element in chemotherapy-induced apoptosis. Here, we show that selective pharmacologic inhibition of SphK<em>1</em> triggers apoptosis in LNCaP and PC-3 prostate cancer cells, an effect that is reversed by SphK<em>1</em> enforced expression. More importantly, we show for the first time that the up-regulation of the SphK<em>1</em>/S<em>1</em>P pathway plays a crucial role in the resistance of prostate cancer cells to chemotherapy. Importantly, pharmacologic SphK<em>1</em> inhibition with the B-5354c compound sensitizes LNCaP and PC-3 cells to docetaxel and camptothecin, respectively. In vivo, camptothecin and B-5354c alone display a limited effect on tumor growth in PC-3 cells, whereas in combination there is a synergy of effect on tumor size with a significant increase in the ceramide to S<em>1</em>P sphingolipid ratio. To conclude, our study highlights the notion that drugs specifically designed to inhibit SphK<em>1</em> could provide a means of enhancing the effects of conventional treatment through the prosurvival antiapoptotic SphK<em>1</em>/S<em>1</em>P pathway.

Fingolimod (Gilenya; FTY720), a synthetic compound based on the fungal secondary metabolite myriocin (ISP-I), is a potent immunosuppressant that was approved (September 20<em>1</em>0) by the U.S. FDA as a new treatment for multiple sclerosis (MS). Fingolimod was synthesized by the research group of Tetsuro Fujita at Kyoto University in <em>1</em>992 while investigating structure-activity relationships of derivatives of the fungal metabolite ISP-I, isolated from Isaria sinclairii. Fingolimod becomes active in vivo following phosphorylation by <em>sphingosine</em> kinase 2 to form fingolimod-<em>phosphate</em>, which binds to extracellular G protein-coupled receptors, <em>sphingosine</em> <em>1</em>-<em>phosphates</em>, and prevents the release of lymphocytes from lymphoid tissue. Fingolimod is orally active, which is unique among current first-line MS therapies, and it has the potential to be used in the treatment of organ transplants and cancer. This review highlights the discovery and development of fingolimod, from an isolated lead natural product, through synthetic analogues, to an approved drug.

Autotaxin (NPP2) is an extracellular protein that is upregulated in various malignancies, including breast and lung cancer. It potently stimulates cell proliferation, cell motility and angiogenesis, which is accounted for by its intrinsic lysophospholipase-D activity that generates the lipid mediators lysophosphatidic acid and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. Based on its structural similarities with the better characterized nucleotide pyrophosphatase/phosphodiesterase NPP<em>1</em>, it has always been assumed that NPP2 is also synthesized as a type-II integral membrane protein and that extracellular NPP2 is generated from this membrane precursor. We show here, however, using domain swapping and mutagenesis experiments as well as N-terminal protein sequencing, that NPP2 is actually synthesized as a pre-pro-enzyme and that the proteolytically processed protein is secreted. Following the removal of a 27-residue signal peptide by the signal peptidase, NPP2 is subsequently cleaved by proprotein convertases (PCs). The removal of an N-terminal octapeptide by PCs is associated with an enhanced activity of NPP2 as a lysophospholipase D. These novel insights in the maturation of NPP2 have also implications for the development of NPP2 inhibitors as potential anti-cancer agents.

OBJECTIVE

Although astrocytes participate in glial scar formation and tissue repair, dysregulation of the NFκB pathway and of nitric oxide (NO) production in these glia cells contributes to neuroinflammation and neurodegeneration. Here we investigated the role of the crosstalk between <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and cytokine signaling cascades in astrocyte activation and inflammation-mediated neurodegeneration, and addressed the effects of fingolimod on astrocyte-neuron interaction and NO synthesis in vivo.

METHODS

Immunohistochemistry, immunofluorescence, and confocal microscopy were used to detect S<em>1</em>P receptors, interleukin (IL) <em>1</em>R, IL<em>1</em>7RA, and nitrosative stress in multiple sclerosis (MS) plaques, experimental autoimmune encephalomyelitis (EAE) spinal cord, and the spinal cord of fingolimod-treated EAE mice. An in vitro model was established to study the effects of S<em>1</em>P, IL<em>1</em>, and IL<em>1</em>7 stimulation on NFkB translocation and NO production in astrocytes, on spinal neuron survival, and on astrocyte-neuron interaction. Furthermore, fingolimod efficacy in blocking astrocyte-mediated neurodegeneration was evaluated.

RESULTS

We found coordinated upregulation of IL<em>1</em>R, IL<em>1</em>7RA, S<em>1</em>P<em>1</em>, and S<em>1</em>P3 together with nitrosative markers in astrocytes within MS and EAE lesions. In vitro studies revealed that S<em>1</em>P, IL<em>1</em>7, and IL<em>1</em> induced NFκB translocation and NO production in astrocytes, and astrocyte conditioned media triggered neuronal death. Importantly, fingolimod blocked the 2 activation events evoked in astrocytes by either S<em>1</em>P or inflammatory cytokines, resulting in inhibition of astrocyte-mediated neurodegeneration. Finally, therapeutic administration of fingolimod to EAE mice hampered astrocyte activation and NO production.

CONCLUSIONS

A neuroprotective effect of fingolimod in vivo may result from its inhibitory action on key astrocyte activation steps.