OBJECTIVE

B-cells are important for disease pathogenesis in the nonobese diabetic (NOD) mouse model of type <em>1</em> diabetes. Recent studies demonstrate that marginal-zone B-cells (MZBs), which connect innate with adaptive immune responses, are increased in NOD mice. However, beyond this, the contribution of different B-cell subsets to diabetes pathogenesis is poorly understood.

METHODS

To better understand the role of different B-cell subsets in the etiology of type <em>1</em> diabetes, we have examined the MZB compartment in NOD mice, with respect to their number, distribution, and function.

RESULTS

We demonstrate that splenic MZB numbers in female NOD mice undergo a marked, approximately threefold expansion between approximately <em>1</em>2 and <em>1</em>6 weeks of age, coincident with the onset of frank diabetes. Functionally, NOD MZBs are hyperresponsive to toll-like receptor 9 ligation and CD40 ligation, as well as <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-dependent chemotactic cues, suggesting an increased sensitivity to selective innate- and activation-induced stimuli. Intriguingly, at <em>1</em>6 weeks of age, approximately 80% of female NOD mice present with MZB-like cells in the pancreatic lymph node (PLN). These MZB-like cells express major histocompatibility complex class II and high levels of CD80 and CD86, and their presence in the PLN is associated with an increased frequency of activated Vbeta4(+) CD4(+) T-cells. Significantly, we demonstrate that purified MZBs are able to present the autoantigen insulin to diabetogenic T-cells.

CONCLUSIONS

These data are consistent with MZBs contributing to the pathogenesis of type <em>1</em> diabetes as antigen-presenting cells. By integrating innate-derived inflammatory signals with the activation of autoreactive T-cells, MZBs may help to direct T-cell responses against beta-cell self-constituents.

FTY720 is a novel immunomodulatory agent that inhibits lymphocyte trafficking and prevents allograft rejection. FTY720 is phosphorylated in vivo, and the phosphorylated drug acts as agonist for a family of G protein-coupled receptors that recognize <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. Evidence suggests that FTY720-<em>phosphate</em>-induced activation of S<em>1</em>P<em>1</em> is responsible for its mechanism of action. FTY720 was rationally designed by modification of myriocin, a naturally occurring sphingoid base analog that causes immunosuppression by interrupting sphingolipid metabolism. In this study, we examined interactions between FTY720, FTY720-<em>phosphate</em>, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase, the enzyme responsible for irreversible <em>sphingosine</em> <em>1</em>-<em>phosphate</em> degradation. FTY720-<em>phosphate</em> was stable in the presence of active <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase, demonstrating that the lyase does not contribute to FTY720 catabolism. Conversely, FTY720 inhibited <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase activity in vitro. Treatment of mice with FTY720 inhibited tissue <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase activity within <em>1</em>2 h, whereas lyase gene and protein expression were not significantly affected. Tissue <em>sphingosine</em> <em>1</em>-<em>phosphate</em> levels remained stable or increased throughout treatment. These studies raise the possibility that disruption of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> metabolism may account for some effects of FTY720 on immune function and that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase may be a potential target for immunomodulatory therapy.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) helps mediate lymphocyte egress from lymph nodes, yet many mechanistic questions remain. Here, we show the presence of B lymphocyte egress sites located in the lymph node cortex close to lymph node follicles. B cells exited lymph nodes by squeezing through apparent portals in the lymphatic endothelium of these sinusoids. Treatment with the S<em>1</em>P receptor agonist FTY720 emptied the cortical sinusoids of lymphocytes, blocked lymphatic endothelial penetration, and displaced B lymphocytes into the T cell zone. S<em>1</em>pr3(-/-) B cells, which lack chemoattractant responses to S<em>1</em>P, transited lymph nodes normally, whereas Gnai2(-/-) B cells, which have impaired responses to chemokines and S<em>1</em>P, transited more rapidly than did wild-type cells. This study identifies a major site of B lymphocyte lymph node egress, shows that FTY720 treatment blocks passage through the cortical lymphatic endothelium, and argues against a functional role for S<em>1</em>P chemotaxis in B lymphocyte egress.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP), formed by <em>sphingosine</em> kinase, is the ligand for EDG-<em>1</em>, a GPCR important for cell migration and vascular maturation. Here we show that cytoskeletal rearrangements, lamellipodia extensions, and cell motility induced by platelet-derived growth factor (PDGF) are abrogated in EDG-<em>1</em> null fibroblasts. However, EDG-<em>1</em> appears to be dispensable for mitogenicity and survival effects, even those induced by its ligand SPP and by PDGF. Furthermore, PDGF induced focal adhesion formation and activation of FAK, Src, and stress-activated protein kinase 2, p38, were dysregulated in the absence of EDG-<em>1</em>. In contrast, tyrosine phosphorylation of the PDGFR and activation of extracellular signal regulated kinase (ERK<em>1</em>/2), important for growth and survival, were unaltered. Our results suggest that EDG-<em>1</em> functions as an integrator linking the PDGFR to lamellipodia extension and cell migration. PDGF, which stimulates <em>sphingosine</em> kinase, leading to increased SPP levels in many cell types, also induces translocation of <em>sphingosine</em> kinase to membrane ruffles. Hence, recruitment of <em>sphingosine</em> kinase to the cell's leading edge and localized formation of SPP may spatially and temporally stimulate EDG-<em>1</em>, resulting in activation and integration of downstream signals important for directional movement toward chemoattractants, such as PDGF. These results may also shed light on the vital role of EDG-<em>1</em> in vascular maturation.

Sphingolipid long chain bases (LCBs) and phosphorylated derivatives, particularly <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, are putative signaling molecules. To help elucidate the physiological roles of LCB <em>phosphates</em>, we identified two Saccharomyces cerevisiae genes, LCB4 (YOR<em>1</em>7<em>1</em>c) and LCB5 (YLR260w), which encode LCB kinase activity. This conclusion is based upon the synthesis of LCB kinase activity in Escherichia coli expressing either LCB gene. LCB4 encodes most (97%) Saccharomyces LCB kinase activity, with the remainder requiring LCB5. Log phase lcb4-deleted yeast cells make no LCB <em>phosphates</em>, showing that the Lcb4 kinase synthesizes all detectable LCB <em>phosphates</em> under these growth conditions. The Lcb4 and Lcb5 proteins are paralogs with 53% amino acid identity but are not related to any known protein, thus revealing a new class of lipid kinase. Two-thirds of the Lcb4 and one-third of the Lcb5 kinase activity are in the membrane fraction of yeast cells, a puzzling finding in that neither protein contains a membrane-localization signal. Both enzymes can use phyto<em>sphingosine</em>, dihydro<em>sphingosine</em>, or <em>sphingosine</em> as substrate. LCB4 and LCB5 should be useful for probing the functions of LCB <em>phosphates</em> in S. cerevisiae. Potential mammalian cDNA homologs of the LCB kinase genes may prove useful in helping to understand the function of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> in mammals.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP), a bioactive sphingolipid metabolite, suppresses apoptosis of many types of cells, including rat pheochromocytoma PC<em>1</em>2 cells. Elucidating the molecular mechanism of action of SPP is complicated by many factors, including uptake and metabolism, as well as activation of specific G-protein-coupled SPP receptors, known as the endothelial differentiation gene-<em>1</em> (EDG-<em>1</em>) family. In this study, we overexpressed type <em>1</em> <em>sphingosine</em> kinase (SPHK<em>1</em>), the enzyme that converts <em>sphingosine</em> to SPP, in order to examine more directly the role of intracellularly generated SPP in neuronal survival. Enforced expression of SPHK<em>1</em> in PC<em>1</em>2 cells resulted in significant increases in kinase activity, with corresponding increases in intracellular SPP levels and concomitant decreases in both <em>sphingosine</em> and ceramide, and marked suppression of apoptosis induced by trophic factor withdrawal or by C(2)-ceramide. NGF, which protects PC<em>1</em>2 cells from serum withdrawal-induced apoptosis, also stimulated SPHK<em>1</em> activity. Surprisingly, overexpression of SPHK<em>1</em> had no effect on activation of two known NGF-stimulated survival pathways, extracellular signal regulated kinase ERK <em>1</em>/2 and Akt. However, trophic withdrawal-induced activation of the stress activated protein kinase, c-Jun amino terminal kinase (SAPK/JNK), and activation of the executionary caspases 2, 3 and 7, were markedly suppressed. Moreover, this abrogation of caspase activation, which was prevented by the SPHK inhibitor N,N-dimethyl<em>sphingosine</em>, was not affected by pertussis toxin treatment, indicating that the cytoprotective effect was likely not mediated by binding of SPP to cell surface G(i)-coupled SPP receptors. In agreement, there was no detectable release of SPP into the culture medium, even after substantially increasing cellular SPP levels by NGF or <em>sphingosine</em> treatment. In contrast to PC<em>1</em>2 cells, C6 astroglioma cells secreted SPP, suggesting that SPP might be one of a multitude of known neurotrophic factors produced and secreted by glial cells. Collectively, our results indicate that SPHK/SPP may play an important role in neuronal survival by regulating activation of SAPKs and caspases.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a highly bioactive lipid that exerts numerous biological effects both intracellularly as a second messenger and extracellularly by binding to its G-protein-coupled receptors of the endothelial differentiation gene family (S<em>1</em>P receptors-(<em>1</em>-5)). Intracellularly, at least two enzymes, <em>sphingosine</em> kinase and S<em>1</em>P phosphatase, regulate the activity of S<em>1</em>P by governing the phosphorylation status of S<em>1</em>P. To study the regulation of S<em>1</em>P levels, we cloned the human isoform of S<em>1</em>P phosphatase <em>1</em> (hSPPase<em>1</em>). The hSPPase<em>1</em> has 78% homology to the mouse SPPase at the amino acid level with 6-8 possible transmembrane domains. Confocal microscopy revealed green fluorescent protein-tagged hSPPase<em>1</em>, expressed in either MCF7 or HEK293 cells, co-localized to endoplasmic reticulum with calreticulin. According to Northern blot analysis, hSPPase<em>1</em> is expressed in most tissues, with the strongest levels found in the highly vascular tissues of placenta and kidney. Transient overexpression of hSPPase<em>1</em> exhibited a 2-fold increase in phosphatase activity against S<em>1</em>P and dihydro-S<em>1</em>P, indicating that the expressed protein was functional. Small interfering RNA (siRNA) knockdown of endogenous hSPPase<em>1</em> drastically reduced hSPPase<em>1</em> mRNA levels, as confirmed by reverse transcription PCR, and resulted in an overall 25% reduction of in vitro phosphatase activity in the membrane fractions. Sphingolipid mass measurements in hSPPase<em>1</em> siRNA knockdown cells revealed a 2-fold increase of S<em>1</em>P levels and concomitant decrease in <em>sphingosine</em>. In vivo labeling of hSPPase<em>1</em> siRNA-treated cells showed accumulation of S<em>1</em>P within cells, as well as significantly increased secretion of S<em>1</em>P into the media, indicating that hSPPase<em>1</em> regulates secreted S<em>1</em>P. In addition, siRNA-induced knockdown of hSPPase<em>1</em> endowed resistance to tumor necrosis factor-alpha and the chemotherapeutic agent daunorubicin. Collectively, these data suggest that regulation of hSPPase<em>1</em> with the resultant changes in cellular and secreted S<em>1</em>P could have important implications to cell proliferation, angiogenesis, and apoptosis.

CXCL<em>1</em>2 and VCAM<em>1</em> retain hematopoietic stem cells (HSCs) in the BM, but the factors mediating HSC egress from the BM to the blood are not known. The <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> (S<em>1</em>P(<em>1</em>)) is expressed on HSCs, and S<em>1</em>P facilitates the egress of committed hematopoietic progenitors from the BM into the blood. In the present study, we show that both the S<em>1</em>P gradient between the BM and the blood and the expression of S<em>1</em>P(<em>1</em>) are essential for optimal HSC mobilization by CXCR4 antagonists, including AMD3<em>1</em>00, and for the trafficking of HSCs during steady-state hematopoiesis. We also demonstrate that the S<em>1</em>P(<em>1</em>) agonist SEW287<em>1</em> increases AMD3<em>1</em>00-induced HSC and progenitor cell mobilization. These results suggest that the combination of a CXCR4 antagonist and a S<em>1</em>P(<em>1</em>) agonist may prove to be sufficient for mobilizing HSCs in normal donors for transplantation purposes, potentially providing a single mobilization procedure and eliminating the need to expose normal donors to G-CSF with its associated side effects.

We recently demonstrated that elevation of intracellular glucosylceramide (GlcCer) levels results in increased functional Ca2+ stores in cultured neurons, and suggested that this may be due to modulation of ryanodine receptors (RyaRs) by GlcCer (Korkotian, E., Schwarz, A., Pelled, D., Schwarzmann, G., Segal, M. and Futerman, A. H. (<em>1</em>999) J. Biol. Chem. 274, 2<em>1</em>673-2<em>1</em>678). We now systematically examine the effects of exogenously added GlcCer, other glycosphingolipids (GSLs) and their lyso-derivatives on Ca2+ release from rat brain microsomes. GlcCer had no direct effect on Ca2+ release, but rather augmented agonist-stimulated Ca2+ release via RyaRs, through a mechanism that may involve the redox sensor of the RyaR, but had no effect on Ca2+ release via inositol <em>1</em>,4,5-tris<em>phosphate</em> receptors. Other GSLs and sphingolipids, including galactosylceramide, lactosylceramide, ceramide, sphingomyelin, <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, sphinganine <em>1</em>-<em>phosphate</em>, and sphingosylphosphorylcholine had no effect on Ca2+ mobilization from rat brain microsomes, but both galactosyl<em>sphingosine</em> (psychosine) and glucosyl<em>sphingosine</em> stimulated Ca2+ release, although only galactosyl<em>sphingosine</em> mediated Ca2+ release via the RyaR. Finally, we demonstrated that GlcCer levels were approximately <em>1</em>0-fold higher in microsomes prepared from the temporal lobe of a type 2 Gaucher disease patient compared with a control, and Ca2+ release via the RyaR was significantly elevated, which may be of relevance for explaining the pathophysiology of neuronopathic forms of Gaucher disease.

OBJECTIVE

Tumor necrosis factor alpha (TNF-alpha), a key proinflammatory cytokine acting on the endothelium, activates endothelial nitric oxide synthase (eNOS). We have examined the signaling pathway leading to this activation and its biological role in endothelium, which are still unknown.

RESULTS

In human endothelial cells, we found that eNOS activation by TNF-alpha is time dependent and requires activation of Akt, a known eNOS activator. eNOS activation was preceded by sequential activation of neutral-sphingomyelinase-2 (N-SMase2) and <em>sphingosine</em>-kinase-<em>1</em> (SK<em>1</em>) and generation of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (Sph<em>1</em>P). Inhibition of N-SMase2 inhibited Sph<em>1</em>P formation, whereas inhibition of SK<em>1</em> did not affect N-SMase2 activation by TNF-alpha. Blockade of N-SMase2, SK<em>1</em>, or the Sph<em>1</em>P receptors S<em>1</em>P<em>1</em> and S<em>1</em>P3, either by silencing or pharmacological inhibitors, prevented eNOS activation. Thus, eNOS is activated by TNF-alpha via S<em>1</em>P receptors, activated by Sph<em>1</em>P generated through N-SMase2 and SK<em>1</em> activation. We found that nitric oxide generated through this pathway has a biological role, because it inhibits the expression of E-selectin and the adhesion of dendritic cells to the endothelium stimulated by TNF-alpha.

CONCLUSIONS

This study establishes a previously undescribed link among TNF-alpha, Sph<em>1</em>P, and eNOS in a same signaling pathway of biological relevance in the process of endothelial cell activation by TNF-alpha.

OBJECTIVE

Sphingomyelin deposition and metabolism occurs in the atherosclerotic plaque, leading to the formation of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), which activates G protein-coupled receptors to regulate vascular and immune cells. The role of S<em>1</em>P receptors in atherosclerosis has not been examined.

RESULTS

We tested the hypothesis that S<em>1</em>P receptor-2 (S<em>1</em>PR2) regulates atherosclerosis. Apoe(-/-) S<em>1</em>pr2(-/-) mice showed greatly attenuated atherosclerosis compared with the Apoe(-/-) mice. Bone marrow transplant experiments indicate that S<em>1</em>PR2 function in the hematopoietic compartment is critical. S<em>1</em>PR2 is expressed in bone marrow-derived macrophages and in macrophage-like foam cells in atherosclerotic plaques. Reduced macrophage-like foam cells were found in the atherosclerotic plaques of Apoe(-/-)S<em>1</em>pr2(-/-) mice, suggesting that S<em>1</em>PR2 retains macrophages in atherosclerotic plaques. Lipoprotein profiles, plasma lipids, and oxidized low-density lipoprotein uptake by bone marrow-derived macrophages were not altered by the S<em>1</em>pr2 genotype. In contrast, endotoxin-induced inflammatory cytokine (interleukin [IL]-<em>1</em>β, IL-<em>1</em>8) levels in the serum of S<em>1</em>PR2 knockout mice were significantly reduced. Furthermore, treatment of wild-type mice with S<em>1</em>PR2 antagonist JTE-0<em>1</em>3 suppressed IL-<em>1</em>β and IL-<em>1</em>8 levels in plasma.

CONCLUSIONS

These data suggest that S<em>1</em>PR2 signaling in the plaque macrophage regulates macrophage retention and inflammatory cytokine secretion, thereby promoting atherosclerosis.

The bioactive sphingolipids ceramide, <em>sphingosine</em> and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) are important signalling molecules that regulate a diverse array of cellular processes. Most notably, the balance of the levels of these three sphingolipids in cells, termed the 'sphingolipid rheostat', can dictate cell fate, where ceramide and <em>sphingosine</em> enhance apoptosis and S<em>1</em>P promotes cell survival and proliferation. The <em>sphingosine</em> kinases (SKs) catalyse the production of S<em>1</em>P from <em>sphingosine</em> and are therefore central regulators of the sphingolipid rheostat and attractive targets for cancer therapy. Two SKs exist in humans: SK<em>1</em> and SK2. SK<em>1</em> has been extensively studied and there is a large body of evidence to demonstrate its role in promoting cell survival, proliferation and neoplastic transformation. SK<em>1</em> is also elevated in many human cancers which appears to contribute to carcinogenesis, chemotherapeutic resistance and poor patient outcome. SK2, however, has not been as well characterized, and there are contradictions in the key physiological functions that have been proposed for this isoform. Despite this, many studies are now emerging that implicate SK2 in key roles in a variety of diseases, including the development of a range of solid tumours. Here, we review the literature examining SK2, its physiological and pathophysiological functions, the current knowledge of its regulation, and recent developments in targeting this complex enzyme.

The pleiotropic lipid mediator <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) can act intracellularly independently of its cell surface receptors through unknown mechanisms. <em>Sphingosine</em> kinase 2 (SphK2), one of the isoenzymes that generates S<em>1</em>P, was associated with histone H3 and produced S<em>1</em>P that regulated histone acetylation. S<em>1</em>P specifically bound to the histone deacetylases HDAC<em>1</em> and HDAC2 and inhibited their enzymatic activity, preventing the removal of acetyl groups from lysine residues within histone tails. SphK2 associated with HDAC<em>1</em> and HDAC2 in repressor complexes and was selectively enriched at the promoters of the genes encoding the cyclin-dependent kinase inhibitor p2<em>1</em> or the transcriptional regulator c-fos, where it enhanced local histone H3 acetylation and transcription. Thus, HDACs are direct intracellular targets of S<em>1</em>P and link nuclear S<em>1</em>P to epigenetic regulation of gene expression.

Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. However, the lipid mediators linking these two observations remain elusive. We postulated that ER stress-regulated release of pro-inflammatory extracellular vesicles (EVs) from lipotoxic hepatocytes may be this link. EVs were isolated from cell culture supernatants of hepatocytes treated with palmitate (PA) to induce lipotoxic ER stress, characterized by immunofluorescence, Western blotting, electron microscopy, and nanoparticle tracking analysis. Sphingolipids were measured by tandem mass spectrometry. EVs were employed in macrophage chemotaxis assays. PA induced significant EV release. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme <em>1</em>α (IRE<em>1</em>α)/X-box binding protein-<em>1</em>. PA-induced EVs were enriched in C<em>1</em>6:0 ceramide in an IRE<em>1</em>α-dependent manner, and activated macrophage chemotaxis via formation of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) from C<em>1</em>6:0 ceramide. This chemotaxis was blocked by <em>sphingosine</em> kinase inhibitors and S<em>1</em>P receptor inhibitors. Lastly, elevated circulating EVs in experimental and human NASH demonstrated increased C<em>1</em>6:0 ceramide. PA induces C<em>1</em>6:0 ceramide-enriched EV release in an IRE<em>1</em>α-dependent manner. The ceramide metabolite, S<em>1</em>P, activates macrophage chemotaxis, a potential mechanism for the recruitment of macrophages to the liver under lipotoxic conditions.

Fingolimod (FTY-720), a <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor modulator, is the first US Food and Drug Administration (FDA)-approved oral agent for the treatment of relapsing forms of multiple sclerosis (MS). Two recent phase III clinical studies (TRANSFORMS [Trial Assessing Injectable Interferon vs FTY720 Oral in RRMS] and FREEDOMS [FTY720 Research Evaluating Effects of Daily Oral Therapy in MS]) demonstrated a significant reduction in the annualized relapse rate in patients with relapsing-remitting MS, compared to once weekly interferon β-<em>1</em>a and placebo. Macular edema was a prominent adverse event reported in these and prior studies of fingolimod. Thirteen of 2,564 (0.5%) patients treated with fingolimod in FREEDOMS and TRANSFORMS developed macular edema. Fingolimod-associated macular edema (FAME) appears to be dose-dependent (observed in only 2 patients taking the FDA-approved 0.5 mg dose) and typically resolves upon cessation of therapy. Although a relatively common condition in ophthalmology, most neurologists have not encountered macular edema in clinical practice. The purpose of this review is to educate the neurologist on the etiology, clinical manifestations, diagnostic modalities, and treatment approaches in patients with FAME. We also discuss the use of fingolimod in patients with uveitis and diabetes mellitus, highlight the guidelines for surveillance ophthalmic examination, and outline the key distinguishing features between FAME and optic neuritis.

The sphingolipid metabolite <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and the kinases that produce it have emerged as critical regulators of numerous fundamental biological processes important for health and disease. Activation of <em>sphingosine</em> kinases (SphKs) by a variety of agonists increases intracellular S<em>1</em>P, which in turn can be secreted out of the cell and bind to and signal through S<em>1</em>P receptors (S<em>1</em>PRs) in an autocrine and/or paracrine manner. Recent studies suggest that this "inside-out" signaling by S<em>1</em>P may play a role in many human diseases. As the roles of the S<em>1</em>PRs in cell and organismal physiology are discussed elsewhere in this volume, we focus this review mainly on recent reports showing how SphKs are activated and S<em>1</em>P reaches its receptors, the role of SphKs and S<em>1</em>P in regulating sphingolipid homeostasis, and the potential importance of the SphK/S<em>1</em>P axis as a therapeutic target in human diseases.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) levels in cells and, consequently, its bioactivity as a signalling molecule are controlled by the action of enzymes responsible for its synthesis and degradation. In the present report, we examined alterations in expression patterns of enzymes involved in S<em>1</em>P-metabolism (<em>sphingosine</em> kinases including their splice variants, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> phosphatases, and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> lyase) under certain inflammatory conditions. We found that <em>sphingosine</em> kinase type <em>1</em> (SPHK<em>1</em>) mRNA could be triggered in a cell type-specific manner; individual SPHK<em>1</em> splice variants were induced with similar kinetics. Remarkably, expression and activity of S<em>1</em>P phosphatase 2 (SPP2) was found to be highly upregulated by inflammatory stimuli in a variety of cells (e.g., neutrophils, endothelial cells). Bandshift analysis using oligonucleotides spanning predicted NFkappaB sites within the SPP2 promoter and silencing of NFkappaB/RelA via RelA-directed siRNA demonstrated that SPP2 is an NFkappaB-dependent gene. Silencing of SPP2 expression in endothelial cells, in turn, led to a marked reduction of TNF-alpha-induced IL-<em>1</em>beta mRNA and protein and to a partial reduction of induced IL-8, suggesting a pro-inflammatory role of SPP2. Notably, up-regulation of SPP2 was detected in samples of lesional skin of patients with psoriasis, an inflammatory skin disease. This study provides detailed insights into the regulation of SPP2 gene expression and suggests that SPP2 might be a novel player in pro-inflammatory signalling.

<em>Sphingosine</em> and <em>sphingosine</em> derivatives induce Ca2+ release from inositol <em>1</em>,4,5-tris<em>phosphate</em> (InsP3)-sensitive Ca2+ pools in permeabilized cells (Ghosh, T. K., Bian, J., and Gill, D. L. (<em>1</em>990) Science 248, <em>1</em>653-<em>1</em>656). To further assess the mechanism of sphingoid base-mediated Ca2+ release, the effects of <em>sphingosine</em> and <em>sphingosine</em> derivatives on Ca2+ fluxes were characterized using a microsomal membrane vesicle fraction (B3) enriched in rough endoplasmic reticulum (ER) prepared from cells of the DDT<em>1</em>MF-2 cell smooth muscle line (Ghosh, T. K., Mullaney, J. M., Tarazi, F. I., and Gill, D. L. (<em>1</em>989) Nature 340, 236-239). Addition of <em>1</em>5 microM <em>sphingosine</em> to Ca2+ pump-loaded B3 vesicles induced a delayed but thereafter rapid Ca2+ release from vesicles which was dependent on the presence of ATP and was blocked by ADP. Sphingosylphosphorylcholine (SPC) released Ca2+ to the same extent (more than 80% of pumped Ca2+), but in contrast to <em>sphingosine</em>, there was no lag and the effect was independent of ATP or ADP. The EC50 for <em>sphingosine</em> and SPC in activating Ca2+ release was <em>1</em> and 3 microM, respectively. Such observations are consistent with the view that <em>sphingosine</em>, unlike SPC, must be modified by an ATP-requiring kinase activity located within the ER membrane. Sphingoid bases do not appear to release Ca2+ through InsP3 receptors since heparin had no effect on sphingoid base-mediated Ca2+ release. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (sph-<em>1</em>-P), the likely active Ca(2+)-releasing derivative of <em>sphingosine</em>, was synthesized by phospholipase D-catalyzed cleavage of SPC, purified, and tested for Ca(2+)-releasing activity. sph-<em>1</em>-P at <em>1</em>0 microM induced Ca2+ release from both B3 vesicles and permeabilized DDT<em>1</em>MF-2 cells to exactly the same extent as <em>sphingosine</em>. Unlike <em>sphingosine</em>, the effect of sph-<em>1</em>-P was immediate and not blocked by ADP. Using B3 membrane vesicles incubated with [gamma-32P]ATP and <em>sphingosine</em> under the same conditions as Ca2+ flux studies, a labeled band was detected on TLC which ran identically with authentic sph-<em>1</em>-P. Formation of this labeled product was prevented by removal of exogenous <em>sphingosine</em> and blocked by ADP. <em>Sphingosine</em>- but not SPC-mediated Ca2+ release was blocked by <em>1</em>0 mM oxalate. <em>1</em>0 mM oxalate also blocked the formation of 32P-labeled sph-<em>1</em>-P indicating that it is an inhibitor of sph-<em>1</em>-P formation. The studies establish that the ER membrane contains the necessary kinase to convert <em>sphingosine</em> to sph-<em>1</em>-P which functions as a powerful mediator of Ca2+ release through a non-InsP3 receptor-mediated mechanism in the same ER membrane, perhaps reflecting a novel Ca2+ signaling pathway.

Endothelial cells play significant roles in conditioning tissues after injury by the production and secretion of angiocrine factors. At least two distinct subsets of monocytes, CD45(+)CD<em>1</em><em>1</em>b(+)Gr<em>1</em>(+)Ly6C(+) inflammatory and CD45(+)CD<em>1</em><em>1</em>b(+)Gr<em>1</em>(-)Ly6C(-) anti-inflammatory monocytes, respond differentially to these angiocrine factors and promote pathogen/debris clearance and arteriogenesis/tissue regeneration, respectively. We demonstrate here that local <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor 3 (S<em>1</em>P3) agonism recruits anti-inflammatory monocytes to remodeling vessels. Poly(lactic-co-glycolic acid) thin films were used to deliver FTY720, an S<em>1</em>P<em>1</em>/3 agonist, to inflamed and ischemic tissues, which resulted in a reduction in proinflammatory cytokine secretion and an increase in regenerative cytokine secretion. The altered balance of cytokine secretion results in preferential recruitment of anti-inflammatory monocytes from circulation. The chemotaxis of these cells, which express more S<em>1</em>P3 than inflammatory monocytes, toward SDF-<em>1</em>α was also enhanced with FTY720 treatment, but not in S<em>1</em>P3 knockout cells. FTY720 delivery enhanced arteriolar diameter expansion and increased length density of the local vasculature. This work establishes a role for S<em>1</em>P receptor signaling in the local conditioning of tissues by angiocrine factors that preferentially recruit regenerative monocytes that can enhance healing outcomes, tissue regeneration, and biomaterial implant functionality.

Hematopoietic stem progenitor cells (HSPCs) respond robustly to α-chemokine stromal-derived factor-<em>1</em> (SDF-<em>1</em>) gradients, and blockage of CXCR4, a seven-transmembrane-spanning G(αI)-protein-coupled SDF-<em>1</em> receptor, mobilizes HSPCs into peripheral blood. Although the SDF-<em>1</em>-CXCR4 axis has an unquestionably important role in the retention of HSPCs in bone marrow (BM), new evidence shows that, in addition to SDF-<em>1</em>, the migration of HSPCs is directed by gradients of the bioactive lipids <em>sphingosine</em>-<em>1</em> <em>phosphate</em> and ceramide-<em>1</em> <em>phosphate</em>. Furthermore, the SDF-<em>1</em> gradient may be positively primed/modulated by cationic peptides (C3a anaphylatoxin and cathelicidin) and, as previously demonstrated, HSPCs respond robustly even to very low SDF-<em>1</em> gradients in the presence of priming factors. In this review, we discuss the role of bioactive lipids in stem cell trafficking and the consequences of HSPC priming by cationic peptides. Together, these phenomena support a picture in which the SDF-<em>1</em>-CXCR4 axis modulates homing, BM retention and mobilization of HSPCs in a more complex way than previously envisioned.

B-<em>1</em> and marginal zone (MZ) B cells produce natural Abs, make Ab responses to microbial pathogens, and contribute to autoimmunity. Although the delta isoform of the PI3K p<em>1</em><em>1</em>0 catalytic subunit is essential for development of these innate-like B cells, its role in the localization, activation, and function of normal B-<em>1</em> and MZ B cells is not known. Using IC87<em>1</em><em>1</em>4, a highly selective inhibitor of p<em>1</em><em>1</em>0delta enzymatic activity, we show that p<em>1</em><em>1</em>0delta is important for murine B-<em>1</em> and MZ B cells to respond to BCR clustering, the TLR ligands LPS and CpG DNA, and the chemoattractants CXCL<em>1</em>3 and <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. In these innate-like B cells, p<em>1</em><em>1</em>0delta activity mediates BCR-, TLR- and chemoattractant-induced activation of the Akt prosurvival kinase, chemoattractant-induced migration, and TLR-induced proliferation. Moreover, we found that TLR-stimulated Ab responses by B-<em>1</em> and MZ B cells, as well as the localization of MZ B cells in the spleen, depend on p<em>1</em><em>1</em>0delta activity. Finally, we show that the in vivo production of natural Abs requires p<em>1</em><em>1</em>0delta and that p<em>1</em><em>1</em>0delta inhibitors can reduce in vivo autoantibody responses. Thus, targeting p<em>1</em><em>1</em>0delta may be a novel approach for regulating innate-like B cells and for treating Ab-mediated autoimmune diseases.

Microglial activation has been implicated as one of the causative factors for neuroinflammation in various neurodegenerative diseases. The sphingolipid metabolic pathway plays an important role in inflammation, cell proliferation, survival, chemotaxis, and immunity in peripheral macrophages. In this study, we demonstrate that <em>sphingosine</em> kinase<em>1</em> (SphK<em>1</em>), a key enzyme of the sphingolipid metabolic pathway, and its receptors are expressed in the mouse BV2 microglial cells and SphK<em>1</em> alters the expression and production of proinflammatory cytokines and nitric oxide in microglia treated with lipopolysaccharide (LPS). LPS treatment increased the SphK<em>1</em> mRNA and protein expression in microglia as revealed by the RT-PCR, Western blot and immunofluorescence. Suppression of SphK<em>1</em> by its inhibitor, N, N Dimethyl<em>sphingosine</em> (DMS), or siRNA resulted in decreased mRNA expression of TNF-alpha, IL-<em>1</em>beta, and iNOS and release of TNF-alpha and nitric oxide (NO) in LPS-activated microglia. Moreover, addition of <em>sphingosine</em> <em>1</em> <em>phosphate</em> (S<em>1</em>P), a breakdown product of sphingolipid metabolism, increased the expression levels of TNF-alpha, IL-<em>1</em>beta and iNOS and production of TNF-alpha and NO in activated microglia. Hence to summarize, suppression of SphK<em>1</em> in activated microglia inhibits the production of proinflammatory cytokines and NO and the addition of exogenous S<em>1</em>P to activated microglia enhances their inflammatory responses. Since the chronic proinflammatory cytokine production by microglia has been implicated in neuroinflammation, modulation of SphK<em>1</em> and S<em>1</em>P in microglia could be looked upon as a future potential therapeutic method in the control of neuroinflammation in neurodegenerative diseases.

Neuronal activity greatly influences the formation and stabilization of synapses. Although receptors for <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a lipid mediator regulating diverse cellular processes, are abundant in the central nervous system, neuron-specific functions of S<em>1</em>P remain largely undefined. Here, we report two novel actions of S<em>1</em>P using primary hippocampal neurons as a model system: (i) as a secretagogue where S<em>1</em>P triggers glutamate secretion and (ii) as an enhancer where S<em>1</em>P potentiates depolarization-evoked glutamate secretion. <em>Sphingosine</em> kinase <em>1</em> (SK<em>1</em>), a key enzyme for S<em>1</em>P production, was enriched in functional puncta of hippocampal neurons. Silencing SK<em>1</em> expression by small interfering RNA as well as SK<em>1</em> inhibition by dimethyl<em>sphingosine</em> resulted in a strong inhibition of depolarization-evoked glutamate secretion. Fluorescence recovery after photobleaching analysis showed translocation of SK<em>1</em> from cytosol to membranes at the puncta during depolarization, which resulted in subsequent accumulation of S<em>1</em>P within cells. Fluorescent resonance energy transfer analysis demonstrated that the S<em>1</em>P(<em>1</em>) receptor at the puncta was activated during depolarization and that depolarization-induced S<em>1</em>P(<em>1</em>) receptor activation was inhibited in SK<em>1</em>-knock-down cells. Importantly, exogenously added S<em>1</em>P at a nanomolar concentration by itself elicited glutamate secretion from hippocampal cells even when the Na(+)-channel was blocked by tetrodotoxin, suggesting that S<em>1</em>P acts on presynaptic membranes. Furthermore, exogenous S<em>1</em>P at a picomolar level potentiated depolarization-evoked secretion in the neurons. These findings indicate that S<em>1</em>P, through its autocrine action, facilitates glutamate secretion in hippocampal neurons both by secretagogue and enhancer actions and may be involved in mechanisms underlying regulation of synaptic transmission.

Activation of the PDGF receptor on human arterial smooth muscle cells (SMC) induces migration and proliferation via separable signal transduction pathways. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (Sph-<em>1</em>-P) can be formed following PDGF receptor activation and therefore may be implicated in PDGF-receptor signal transduction. Here we show that Sph-<em>1</em>-P does not significantly affect PDGF-induced DNA synthesis, proliferation, or activation of mitogenic signal transduction pathways, such as the mitogen-activated protein (MAP) kinase cascade and PI 3-kinase, in human arterial SMC. On the other hand, Sph-<em>1</em>-P strongly mimics PDGF receptor-induced chemotactic signal transduction favoring actin filament disassembly. Although Sph-<em>1</em>-P mimics PDGF, exogenously added Sph-<em>1</em>-P induces more prolonged and quantitatively greater PIP2 hydrolysis compared to PDGF-BB, a markedly stronger calcium mobilization and a subsequent increase in cyclic AMP levels and activation of cAMP-dependent protein kinase. This excessive and prolonged signaling favors actin filament disassembly by Sph-<em>1</em>-P, and results in inhibition of actin nucleation, actin filament assembly and formation of focal adhesion sites. Sph-<em>1</em>-P-induced interference with the dynamics of PDGF-stimulated actin filament disassembly and assembly results in a marked inhibition of cell spreading, of extension of the leading lamellae toward PDGF, and of chemotaxis toward PDGF. The results suggest that spatial and temporal changes in phosphatidylinositol turnover, calcium mobilization and actin filament disassembly may be critical to PDGF-induced chemotaxis and suggest a possible role for endogenous Sph-<em>1</em>-P in the regulation of PDGF receptor chemotactic signal transduction.