Recent observations using multiphoton intravital microscopy (MP-IVM) have uncovered an unexpectedly high lymphocyte motility within peripheral lymph nodes (PLNs). Lymphocyte-expressed intracellular signaling molecules governing interstitial movement remain largely unknown. Here, we used MP-IVM of murine PLNs to examine interstitial motility of lymphocytes lacking the Rac guanine exchange factor DOCK2 and phosphoinositide-3-kinase (PI3K)gamma, signaling molecules that act downstream of G protein-coupled receptors, including chemokine receptors (CKRs). T and B cells lacking DOCK2 alone or DOCK2 and PI3Kgamma displayed markedly reduced motility inside T cell area and B cell follicle, respectively. Lack of PI3Kgamma alone had no effect on migration velocity but resulted in increased turning angles of T cells. As lymphocyte egress from PLNs requires the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor <em>1</em>, a G(alphai) protein-coupled receptor similar to CKR, we further analyzed whether DOCK2 and PI3Kgamma contributed to S<em>1</em>P-triggered signaling events. S<em>1</em>P-induced cell migration was significantly reduced in T and B cells lacking DOCK2, whereas T cell-expressed PI3Kgamma contributed to F-actin polymerization and protein kinase B phosphorylation but not migration. These findings correlated with delayed lymphocyte egress from PLNs in the absence of DOCK2 but not PI3Kgamma, and a markedly reduced cell motility of DOCK2-deficient T cells in close proximity to efferent lymphatic vessels. In summary, our data support a central role for DOCK2, and to a lesser extent T cell-expressed PI3Kgamma, for signal transduction during interstitial lymphocyte migration and S<em>1</em>P-mediated egress.

Endothelial cell invasion is a key step in angiogenic blood vessel formation. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) has been previously reported to play a role in endothelial cell proliferation, survival, migration, and angiogenesis. Here, we examine the ability of S<em>1</em>P to regulate human endothelial cell invasion into three-dimensional collagen or fibrin matrices. We show that S<em>1</em>P potently stimulated human endothelial cell invasion, lumen formation, and branching morphogenesis in collagen, and fibrin matrices, (5- and <em>1</em>5-fold increases in invasion were observed, respectively). The S<em>1</em>P-induced invasion response was pertussis-toxin sensitive and completely dependent on integrins. Addition of integrin blocking reagents revealed that the alpha2beta<em>1</em> integrin regulated invasion in collagen matrices, while a combination of alphavbeta3 and alpha5beta<em>1</em> integrins regulated invasion in fibrin. Additionally, the S<em>1</em>P-induced invasion response was dependent on matrix metalloproteinases (MMPs). Tissue inhibitor of metalloproteinase-3 (TIMP-3) was the only physiologic inhibitor of metalloproteinases that completely inhibited the potent stimulation of invasion induced by S<em>1</em>P. In contrast, TIMP-<em>1</em> had no blocking effect on invasion or morphogenesis, while TIMP-2 and TIMP-4 partially reduced invasion but completely blocked lumen formation events. Collectively, these data reveal a marked ability of S<em>1</em>P to induce metalloproteinase- and integrin-dependent human endothelial cell invasion and morphogenesis in both collagen and fibrin three-dimensional matrices, the two most physiologically relevant matrices for angiogenesis.

Intercellular coupling between ventricular myocytes and myofibroblasts was studied by co-culturing adult rabbit ventricular myocytes with previously prepared layers of cardiac myofibroblasts. Intercellular coupling was examined by: (i) tracking the movement of the fluorescent dye calcein; (ii) immunostaining for connexin 43 (Cx43); and (iii) measurement of intracellular [Ca2+] ([Ca2+]i). The effects of stimulating ventricular myocytes on the underlying myofibroblasts was examined by confocal measurements of [Ca2+]i using fluo-3. When ventricular myocytes were preloaded with calcein and co-cultured with myofibroblasts for 24 h, calcein fluorescence was detected in 52+/-4% (n=8 co-cultures) of surrounding myofibroblasts. Treatment with the gap junction uncoupler heptanol significantly reduced the movement of calcein (<em>1</em>2+/-3%, n=6 co-cultures). Immunostaining showed expression of Cx43 in co-cultured myofibroblasts and myocytes. Field stimulation of ventricular myocytes co-cultured with myofibroblasts increased myofibroblast [Ca2+]i, no response was observed after treatment with heptanol or stimulation of fibroblasts in the absence of ventricular myocytes. Action potential parameters of ventricular myocytes in co-culture were similar to control values. However, application of the hormone <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S-<em>1</em>-P) to the co-culture caused a depolarization of ventricular myocytes to approximately -20 mV. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> had no effect on ventricular myocytes alone. Voltage-clamp measurements of isolated myofibroblasts indicated that S-<em>1</em>-P activated a significant quasi-linear current with a reversal potential of approximately -40 mV. In conclusion, this study shows that stimulation of the ventricular myocyte influences the intracellular Ca2+ of the linked myofibroblast via connexons. These intercellular links also allow the myofibroblasts to influence the electrical activity of the myocyte. This work indicates the nature of the gap junction-mediated bi-directional interactions that occur between ventricular myocyte and myofibroblast.

Phylogenetic analysis of transmembrane regions of GPCRs using PHYLIP indicated that the orphan receptor P2Y(<em>1</em>0) receptor was classified into the cluster consisting nucleotide and lipid receptors. Based on the results, we studied the abilities of nucleotides and lipids to activate the P2Y(<em>1</em>0) receptors. As a result, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and lysophosphatidic acid (LPA) evoked intracellular Ca(2+) increases in the CHO cells stably expressing the P2Y(<em>1</em>0) fused with a G(<em>1</em>6alpha) protein. These Ca(2+) responses were inhibited by S<em>1</em>P receptor and LPA receptor antagonists. The introduction of siRNA designed for P2Y(<em>1</em>0) receptor into the P2Y(<em>1</em>0)-CHO cells effectively blocked both S<em>1</em>P- and LPA-induced Ca(2+) increases. RT-PCR analysis showed that the mouse P2Y(<em>1</em>0) was expressed in reproductive organs, brain, lung and skeletal muscle, suggesting the receptor plays physiological roles throughout the whole body. In conclusion, the P2Y(<em>1</em>0) receptor is the first receptor identified as a dual lysophospholipid receptor.

High density lipoproteins (HDL) possess a number of physiological activities. The most studied and, perhaps, better understood is the ability of HDL to promote excess cholesterol efflux from peripheral tissues and transport to the liver for excretion, a mechanism believed to confer protection against atherosclerotic cardiovascular disease. The ability of HDL to modulate cholesterol bioavailability in the lipid rafts, membrane microdomains enriched in glycosphingolipids and cholesterol, is evolutionary conserved and affects the properties of cells involved in the innate and adaptive immune response, tuning inflammatory response and antigen presentation functions in macrophages as well as B and T cell activation. Also <em>sphingosine</em>-<em>1</em> <em>phosphate</em> (S<em>1</em>P), a major active sphingolipid carried by HDL, is of relevance in the pathogenesis of several immuno-inflammatory disorders through the modulation of macrophage and lymphocyte functions. Furthermore, HDL influence the humoral innate immunity by modulating the activation of the complement system and the expression of pentraxin 3 (PTX3). Finally, in humans, HDL levels and functions are altered in several immune-mediated disorders, such as rheumatoid arthritis, systemic lupus eritematosus, Crohn's disease and multiple sclerosis as well as during inflammatory responses. Altogether these observations suggest that the effects of HDL in immunity could be related, to either the ability of HDL to modulate cholesterol content in immune cell lipid rafts and to their role as reservoir for several biologically active substances that may impact the immune system.

Defeating pancreatic cancer resistance to the chemotherapeutic drug gemcitabine remains a challenge to treat this deadly cancer. Targeting the sphingolipid metabolism for improving tumor chemosensitivity has recently emerged as a promising strategy. The fine balance between intracellular levels of the prosurvival <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and the proapoptotic ceramide sphingolipids determines cell fate. Among enzymes that control this metabolism, <em>sphingosine</em> kinase-<em>1</em> (SphK<em>1</em>), a tumor-associated protein overexpressed in many cancers, favors survival through S<em>1</em>P production, and inhibitors of SphK<em>1</em> are used in ongoing clinical trials to sensitize epithelial ovarian and prostate cancer cells to various chemotherapeutic drugs. We here report that the cellular ceramide/S<em>1</em>P ratio is a critical biosensor for predicting pancreatic cancer cell sensitivity to gemcitabine. A low level of the ceramide/S<em>1</em>P ratio, associated with a high SphK<em>1</em> activity, correlates with a robust intrinsic pancreatic cancer cell chemoresistance toward gemcitabine. Strikingly, increasing the ceramide/S<em>1</em>P ratio, by using pharmacologic (SphK<em>1</em> inhibitor or ceramide analogue) or small interfering RNA-based approaches to up-regulate intracellular ceramide levels or reduce SphK<em>1</em> activity, sensitized pancreatic cancer cells to gemcitabine. Conversely, decreasing the ceramide/S<em>1</em>P ratio, by up-regulating SphK<em>1</em> activity, promoted gemcitabine resistance in these cells. Development of novel pharmacologic strategies targeting the sphingolipid metabolism might therefore represent an interesting promising approach, when combined with gemcitabine, to defeat pancreatic cancer chemoresistance to this drug.

<em>Sphingosine</em>-<em>1</em> <em>phosphate</em> (S<em>1</em>P) and thrombin are agents with profound but divergent effects on vascular endothelial cell (EC) barrier properties. We have previously reported that S<em>1</em>P-induced focal adhesion (FA) remodeling involves interactions between focal adhesion kinase (FAK), paxillin, and G-protein-coupled receptor kinase-interacting proteins GIT<em>1</em> and GIT2 and suggested a critical involvement of focal adhesions in the EC barrier regulation. In this study, we examined redistribution of FA proteins (FAK, paxillin, GIT<em>1</em>, and GIT2) and site-specific FAK tyrosine phosphorylation in human pulmonary artery endothelial cells stimulated with thrombin. In contrast to S<em>1</em>P, which we have shown to induce peripheral translocation of FA proteins associated with cortical actin ring formation, thrombin caused the redistribution of FA proteins to the ends of the newly formed massive stress fibers. S<em>1</em>P and thrombin induced distinct patterns of FAK site-specific phosphorylation with the FAK Y576 phosphorylation site targeted by SIP challenge and phosphorylation of three FAK sites (Y397, Y576, and Y925) in response to thrombin stimulation. Pharmacological inhibition of Src with Src-specific inhibitor PP2 abolished S<em>1</em>P-induced translocation of FA proteins, cortical actin ring formation, and FAK [Y576] phosphorylation. However, PP2 failed to alter thrombin-induced morphological changes and exhibited only partial inhibition of FAK site-specific tyrosine phosphorylation. These observations highlight the differential mechanisms of focal adhesion protein complex remodeling and FAK activation by S<em>1</em>P and thrombin and link differential FA remodeling to EC barrier regulation.

The Mst<em>1</em> kinase is an important regulator of murine T cell adhesion, migration, proliferation, and apoptosis. In this study, we analyze mice lacking both Mst<em>1</em> and Mst2 in hematopoietic cells. Compared with wild-type mice, these double knockout (DKO) mice exhibit a severe reduction in the number of mature T cells in the circulation and in secondary lymphoid organs (SLOs). CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes in DKO mice resemble mature T cells of wild-type mice but undergo excessive apoptosis, and their egress from the thymus is reduced by >90%. Even when placed directly in the circulation, DKO SP thymocytes failed to enter SLOs. In SP thymocytes, deficiency of Mst<em>1</em> and Mst2 abolished <em>sphingosine</em>-<em>1</em> <em>phosphate</em>- and CCL2<em>1</em>-induced Mob<em>1</em> phosphorylation, Rac<em>1</em> and RhoA GTP charging, and subsequent cell migration. When phosphorylated by Mst<em>1</em> or Mst2, Mob<em>1</em> binds and activates the Rac<em>1</em> guanyl nucleotide exchanger Dock8, which is abundant in the thymus. Thus, the Mst<em>1</em> and Mst2 kinases control Rho GTPase activation and the migratory responses of SP thymocytes.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em>, a lipid mediator produced by <em>sphingosine</em> kinases, regulates diverse cellular processes, ranging from cell growth and survival to effector functions, such as proinflammatory mediator synthesis. Using human A549 epithelial lung carcinoma cells as a model system, we observed transient upregulation of <em>sphingosine</em> kinase type <em>1</em> (SPHK<em>1</em>) enzyme activity upon stimulation with both TNF-alpha or IL-<em>1</em>beta. This transient activation of SPHK<em>1</em> was found to be required for cytokine-induced COX-2 transcription and PGE2 production, since not only specific siRNA (abolishing both basal and induced SPHK<em>1</em> enzyme activity), but also a dominant-negative SPHK<em>1</em> mutant (suppressing induced SPHK<em>1</em> activity only) both reduced COX-2 and PGE2. Furthermore, TNF-alpha- or IL-<em>1</em>beta-induced transcription of selected cytokines, chemokines, and adhesion molecules (IL-6, RANTES, MCP-<em>1</em>, and VCAM-<em>1</em>) was found to require SPHK<em>1</em> activation. Suppression of SPHK<em>1</em> activation led to reduction of cytokine-induced IkappaBalpha phosphorylation and consequently diminished NFkappaB activity due to reduced nuclear translocation of RelA (p65), explaining the dependence of inflammatory mediator production on SPHK<em>1</em> activation. Inhibition of basal SPHK<em>1</em> activity by N,N-dimethyl<em>sphingosine</em> or by downregulation of its expression using siRNA induced spontaneous apoptosis in A549 cells, an effect that can be explained through interference with constitutive NFkappaB activity in this cell type. In contrast, expression of the dominant-negative mutant did not induce apoptosis. Taken together, these findings demonstrate a role of SPHK<em>1</em> activation in proinflammatory signalling and of SPHK<em>1</em> basal activity in survival of A549 lung carcinoma cells.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a sphingolipid metabolite that functions as a bioactive lipid molecule. S<em>1</em>P is degraded either by S<em>1</em>P lyase or by S<em>1</em>P phosphohydrolase. The gene encoding mammalian S<em>1</em>P lyase, SPL, has been identified. Here, we characterize the SPL protein in its expression, localization, and topology. The expression levels of the SPL protein correlated well with the dihydrosphingosine-<em>1</em>-<em>phosphate</em> (DHS<em>1</em>P) lyase activity in most tissues. However, liver and heart exhibited high DHS<em>1</em>P lyase activities compared to their SPL protein levels. The SPL mRNA expression was temporally regulated during mouse embryonal development. Immunofluorescence microscopy demonstrated that SPL is localized at the endoplasmic reticulum. Proteinase K digestion studies revealed that the large hydrophilic domain, containing the active site, faces the cytosol. This active site orientation is opposite to that of S<em>1</em>P phosphohydrolase, indicating that the degradation of S<em>1</em>P by two S<em>1</em>P-degrading enzymes occurs in spatially separated sides of the endoplasmic reticulum.

Two lysophospholipids (LPs), lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), are known to affect various cellular events. Their actions are mediated by binding to at least ten bona fide high-affinity G protein-coupled receptors referred to as LPA<em>1</em>-5 and S<em>1</em>P<em>1</em>-5. These LPs are expressed throughout the body and are involved in a range of biological activities including normal development, as well as functioning in most organ systems. A growing number of biological functions have been uncovered in vivo using single- or multiple-null mice for each LP receptor. This review will focus on findings from in vivo as well as in vitro studies using genetic null mice for the LP receptors, LPA<em>1</em>,2,3 and S<em>1</em>P<em>1</em>,2,3,5, and for the LP producing enzymes, autotaxin and <em>sphingosine</em> kinase <em>1</em>/2.

The synthesis of N-arylamide phosphonates and related arylether and arylamine analogues provided potent, subtype-selective agonists and antagonists of the five known <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors (S<em>1</em>P(<em>1</em>-5)). To this end, the syntheses of phosphoserine mimetics-selectively protected and optically active phosphonoserines-are described. In vitro binding assays showed that the implementation of phosphonates as <em>phosphate</em> mimetics provided compounds with similar receptor binding affinities as compared to their <em>phosphate</em> precursors. meta-substituted arylamide phosphonates were discovered to be antagonists of the S<em>1</em>P(<em>1</em>) and S<em>1</em>P(3) receptors. When administered to mice, an antagonist blocked the lymphopenia evoked by a S<em>1</em>P receptor agonist and caused capillary leakage in both lung and kidney.

<em>Sphingosine</em> kinase (SphK) <em>1</em> and 2 phosphorylate <em>sphingosine</em> to generate <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a pluripotent lipophilic mediator implicated in a variety of cellular events. Here we show that the activity of β-site APP cleaving enzyme-<em>1</em> (BACE<em>1</em>), the rate-limiting enzyme for amyloid-β peptide (Aβ) production, is modulated by S<em>1</em>P in mouse neurons. Treatment by SphK inhibitor, RNA interference knockdown of SphK, or overexpression of S<em>1</em>P degrading enzymes decreased BACE<em>1</em> activity, which reduced Aβ production. S<em>1</em>P specifically bound to full-length BACE<em>1</em> and increased its proteolytic activity, suggesting that cellular S<em>1</em>P directly modulates BACE<em>1</em> activity. Notably, the relative activity of SphK2 was upregulated in the brains of patients with Alzheimer's disease. The unique modulatory effect of cellular S<em>1</em>P on BACE<em>1</em> activity is a novel potential therapeutic target for Alzheimer's disease.

The induction of persistent intraepithelial CD8+ T cell responses may be key to the development of vaccines against mucosally transmitted pathogens, particularly for sexually transmitted diseases. Here we investigated CD8+ T cell responses in the female mouse cervicovaginal mucosa after intravaginal immunization with human papillomavirus vectors (HPV pseudoviruses) that transiently expressed a model antigen, respiratory syncytial virus (RSV) M/M2, in cervicovaginal keratinocytes. An HPV intravaginal prime/boost with different HPV serotypes induced <em>1</em>0-fold more cervicovaginal antigen-specific CD8+ T cells than priming alone. Antigen-specific T cell numbers decreased only 2-fold after 6 months. Most genital antigen-specific CD8+ T cells were intra- or subepithelial, expressed αE-integrin CD<em>1</em>03, produced IFN-γ and TNF-α, and displayed in vivo cytotoxicity. Using a <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> analog (FTY720), we found that the primed CD8+ T cells proliferated in the cervicovaginal mucosa upon HPV intravaginal boost. Intravaginal HPV prime/boost reduced cervicovaginal viral titers <em>1</em>,000-fold after intravaginal challenge with vaccinia virus expressing the CD8 epitope M2. In contrast, intramuscular prime/boost with an adenovirus type 5 vector induced a higher level of systemic CD8+ T cells but failed to induce intraepithelial CD<em>1</em>03+CD8+ T cells or protect against recombinant vaccinia vaginal challenge. Thus, HPV vectors are attractive gene-delivery platforms for inducing durable intraepithelial cervicovaginal CD8+ T cell responses by promoting local proliferation and retention of primed antigen-specific CD8+ T cells.

<em>Sphingosine</em> kinase (SPHK) <em>1</em> is implicated in the regulation of cell proliferation and anti-apoptotic processes by catalyzing the formation of an important bioactive messenger, <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. Unlike the proliferative action of SPHK<em>1</em>, another isozyme, SPHK2, has been shown to possess anti-proliferative or pro-apoptotic action. Molecular mechanisms of SPHK2 action, however, are largely unknown. The present studies were undertaken to characterize the N-terminal-extended form of SPHK2 (SPHK2-L) by comparing it with the originally reported form, SPHK2-S. Real-time quantitative PCR analysis revealed that SPHK2-L mRNA is the major form in several human cell lines and tissues. From sequence analyses it was concluded that SPHK2-L is a species-specific isoform that is expressed in human but not in mouse. At the protein level it has been demonstrated by immunoprecipitation studies that SPHK2-L is the major isoform in human hepatoma HepG2 cells. SPHK2-L, when expressed in human embryonic kidney (HEK) 293 cells, did not show any inhibition of DNA synthesis in the presence of serum, whereas it showed marked inhibition in the absence of serum. Moreover, serum deprivation resulted in the translocation of SPHK2-L into the nuclei. In addition, serum deprivation induced SPHK2-L expression in HEK293 cells. Furthermore, suppression of SPHK2 by small interfering RNA treatment prevented serum deprivation- or drug-induced apoptosis in HEK293 cells. Taken together, these results indicate that a major form of SPHK2 splice variant, SPHK2-L, in human cells does not inhibit DNA synthesis under normal conditions and that SPHK2-L accumulation in the nucleus induced by serum deprivation may be involved in the cessation of cell proliferation or apoptosis depending on the cell type.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a key endogenous regulator of the response to lung injury, maintaining endothelial barrier integrity through interaction with one of its receptors, S<em>1</em>P(<em>1</em>). The short-term administration of S<em>1</em>P or S<em>1</em>P(<em>1</em>) receptor agonists enhances endothelial monolayer barrier function in vitro, and attenuates injury-induced vascular leak in the lung and other organ systems in vivo. Although S<em>1</em>P(<em>1</em>) agonists bind to and activate S<em>1</em>P(<em>1</em>), several of these agents also induce receptor internalization and degradation, and may therefore act as functional antagonists of S<em>1</em>P(<em>1</em>) after extended exposure. Here we report on the effects of prolonged exposure to these agents in bleomycin-induced lung injury. We demonstrate that repeated administration of S<em>1</em>P(<em>1</em>) agonists dramatically worsened lung injury after bleomycin challenge, as manifested by increased vascular leak and mortality. Consistent with these results, prolonged exposure to S<em>1</em>P(<em>1</em>) agonists in vitro eliminated the ability of endothelial cell monolayers to respond appropriately to the barrier-protective effects of S<em>1</em>P, indicating a loss of normal S<em>1</em>P-S<em>1</em>P(<em>1</em>) signaling. As bleomycin-induced lung injury progressed, continued exposure to S<em>1</em>P(<em>1</em>) agonists also resulted in increased pulmonary fibrosis. These data indicate that S<em>1</em>P(<em>1</em>) agonists can act as functional antagonists of S<em>1</em>P(<em>1</em>) on endothelial cells in vivo, which should be considered in developing these agents as therapies for vascular leak syndromes. Our findings also support the hypothesis that vascular leak is an important component of the fibrogenic response to lung injury, and suggest that targeting the S<em>1</em>P-S<em>1</em>P(<em>1</em>) pathway may also be an effective therapeutic strategy for fibrotic lung diseases.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is now emerging as a potent lipid mediator produced by mast cells that contributes to inflammatory and allergic responses. In contrast to its weak effect on degranulation of murine mast cells, S<em>1</em>P potently induced degranulation of the human LAD2 mast-cell line and cord blood-derived human mast cells (hMCs). S<em>1</em>P also stimulated production and secretion of cytokines, TNF-alpha and IL-6, and markedly enhanced secretion of a chemokine, CCL2/MCP-<em>1</em>, important modulators of inflammation. S<em>1</em>P is produced in mast cells by the 2 <em>sphingosine</em> kinases, SphK<em>1</em> and SphK2. SphK<em>1</em> but not SphK2 plays a critical role in IgE/Ag-induced degranulation, migration toward antigen, and CCL2 secretion from hMCs, as determined by specifically down-regulating their expression. However, both isoenzymes were required for efficient TNF-alpha secretion. Taken together, our data suggest that differential formation of S<em>1</em>P by SphK<em>1</em> and SphK2 has distinct and important actions in hMCs.

OBJECTIVE

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (Sph-<em>1</em>-P), a bioactive lipid derived from activated platelets, may play an important role in coronary artery spasm and hence the pathogenesis of ischemic heart diseases, since we reported that a decrease in coronary blood flow was induced by this lysophospholipid in an in vivo canine heart model [Cardiovasc. Res. 46 (2000) <em>1</em><em>1</em>9]. In this study, metabolism related to and cellular responses elicited by Sph-<em>1</em>-P were examined in human coronary artery smooth muscle cells (CASMCs).

RESULTS

[3H]<em>Sphingosine</em> (Sph), incorporated into CASMCs, was converted to [3H]Sph-<em>1</em>-P intracellularly, but its stimulation-dependent formation and extracellular release were not observed. Furthermore, the cell surface Sph-<em>1</em>-P receptors of S<em>1</em>P family (previously called EDG) were found to be expressed in CASMCs. Accordingly, Sph-<em>1</em>-P seems to act as an extracellular mediator in CASMCs. Consistent with Sph-<em>1</em>-P-elicited coronary vasoconstriction in vivo, Sph-<em>1</em>-P strongly induced CASMC contraction, which was inhibited by JTE-0<em>1</em>3, a newly-developed specific antagonist of S<em>1</em>P(2) (EDG-5). Furthermore, C3 exoenzyme or Y-27632 inhibited the CASMC contraction induced by Sph-<em>1</em>-P, indicating Rho involvement. Finally, exogenously-added [3H]Sph-<em>1</em>-P underwent a rapid degradation. Since lipid <em>phosphate</em> phosphatases, ectoenzymes capable of dephosphorylating Sph-<em>1</em>-P, were expressed in CASMCs, Sph-<em>1</em>-P may be dephosphorylated by the ectophosphatases.

CONCLUSIONS

Sph-<em>1</em>-P, derived from platelets and dephosphorylated on the cell surface, may induce the contraction of coronary artery smooth muscle cells through the S<em>1</em>P(2)/Rho signaling.

<em>Sphingosine</em> kinase <em>1</em> (SphK<em>1</em>) is a lipid kinase that catalyses the phosphorylation of <em>sphingosine</em> to <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. There is strong evidence from cellular or animal systems that SphK<em>1</em> is involved in the major mechanisms underpinning oncogenesis, namely, the promotion of cellular survival, proliferation and transformation, the prevention of apoptosis and the stimulation of angiogenesis. Furthermore there is also good evidence from clinical samples that SphK<em>1</em> is overexpressed in many, if not most tumor types examined and that many inhibitors of SphK<em>1</em> render tumors sensitive to chemotherapeutic agents. A major question that remains concerns the exact mechanism of action of SphK<em>1</em> in cancer. The tools available to probe SphK<em>1</em> function perturb a set of cellular functions, and it is possible that several of these are involved in driving its oncogenic role. Furthermore, the importance of SphK<em>1</em> functions in normal physiology and the lack of mutations of SphK<em>1</em> in cancer, suggest that the mechanism in cancer might be an over reliance on this system of cellular signaling; an example of non-oncogene addiction.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (SPP) is a sphingolipid metabolite which has novel dual actions acting as both an intracellular second messenger and a ligand for a family of G protein-coupled receptors. This paper describes a rapid enzymatic method to quantify mass levels of SPP in serum, mammalian tissues, and cultured cells. The assay utilizes an alkaline lipid extraction to selectively separate SPP from other phospholipids and sphingolipids, including <em>sphingosine</em>. Extracted SPP is efficiently converted to <em>sphingosine</em> by alkaline phosphatase treatment. <em>Sphingosine</em> thus formed is then quantitatively phosphorylated to [(32)P]SPP using recombinant <em>sphingosine</em> kinase and [gamma-(32)P]ATP. With this procedure we were able to obtain reproducible measurements of SPP over a broad range from 0.25 pmol to 2.5 nmol. In various rat tissues, levels of SPP varied between 0. 5 and 6 pmol/mg wet wt. The lowest levels were found in heart and testes, while brain contained the highest levels. The method was adapted easily to measure minute amounts of SPP present in various cultured cell types. The amount of SPP in cell extracts was proportional to the cell number and varied between 0.04 and 2 pmol/<em>1</em>0(6) cells. Concurrent measurements of <em>sphingosine</em> levels revealed that its concentration was significantly higher than SPP in most cells and tissues. Furthermore, with this assay we were able to measure increases in intracellular SPP levels in rat pheochromocytoma PC<em>1</em>2 cells after treatment with exogenous <em>sphingosine</em> or with nerve growth factor which stimulates <em>sphingosine</em> kinase activity.

The lipid backbones of sphingolipids and their metabolites are highly bioactive compounds that affect diverse cellular functions. The metabolites that have been most extensively studied with respect to their effects on cell behavior are ceramides, <em>sphingosine</em> (and other sphingoid bases), and <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. Additionally, there is interest in other naturally occurring species such as lysosphingolipids (<em>sphingosine</em>, phosphorylcholine, and psychosines) and N-methyl (di- and tri-methyl)-<em>sphingosines</em>. In many cases, studies of cell signaling mediated by these compounds have focused on a single category (such as ceramides or <em>sphingosine</em> <em>1</em>-<em>phosphate</em>) because of the technical difficulty of more comprehensive analyses. One obstacle in such studies is that most of these compounds are metabolically interconvertable, so it is difficult to assign a conclusive relationship. In this article, we describe the analytical methods for extraction, identification, and quantitation of sphingolipids using state-of-the-art tandem mass spectrometry (MS/MS). Precursor ion scans are used to distinguish various species of sphingolipids in crude extracts by their unique molecular decomposition products. Specific headgroup, sphingoid base, and fatty acid chain combinations can be readily determined. Quantitation is achieved by multiple reaction monitoring (MRM) in conjunction with high-pressure liquid chromatography (HPLC). Compared to precursor ion scans alone, MRM experiments yield greater sensitivity and lower limits of detection by monitoring a specific precursor and product ion pair. This sensitivity facilitates detection of dozens of individual molecular species under optimal ion formation and decomposition conditions for each species, eliminating any ambiguity that may arise from differences in the kinetics of dissociation.

BACKGROUND

The oral immunomodulator fingolimod (FTY720) has recently been shown to be highly effective in relapsing-remitting multiple sclerosis (MS). Fingolimod is a functional antagonist of the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> and thereby inhibits <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-dependent lymphocyte egress from secondary lymphoid tissues, resulting in a pronounced lymphopenia in the peripheral blood. The effects of fingolimod treatment on the CSF of patients with MS have not been studied so far.

METHODS

We analyzed the leukocyte count, albumin quotient, immunoglobulin G (IgG) index, and oligoclonal bands in the CSF of fingolimod-treated patients with MS. Moreover, we performed immunophenotyping of CSF and peripheral blood leukocytes by flow cytometry. The results were compared to those from treatment-naive or natalizumab-treated patients with MS and patients with other inflammatory and noninflammatory neurologic diseases.

RESULTS

Fingolimod therapy significantly decreased CSF leukocyte counts, but had little impact on the extent of intrathecal IgG synthesis and presence of oligoclonal bands in the CSF. Fingolimod decreased the proportion of CSF CD4+ T cells but to a lesser extent than in the peripheral blood. While fingolimod strongly reduced B cells in the periphery, it had little impact on B cells in the CSF. The percentage of CSF CD8+ T cells, NK cells, and monocytes increased compared to treatment-naive patients. The CD4+/CD8+ T-cell ratio in CSF reversed in most of the patients.

CONCLUSIONS

Fingolimod treatment has a profound impact on CSF, which to some extent differs from the peripheral effects of the drug.

The lysosphingolipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a component of HDL. Findings from a growing number of studies indicate that S<em>1</em>P is a mediator of many of the cardiovascular effects of HDL, including the ability to promote vasodilation, vasoconstriction, and angiogenesis, protect against ischemia/reperfusion injury, and inhibit/reverse atherosclerosis. These latter cardioprotective effects are being shown to involve the S<em>1</em>P-mediated suppression of inflammatory processes, including reduction of the endothelial expression of monocyte and lymphocyte adhesion molecules, decreased recruitment of polymorphonuclear cells to sites of infarction, and blocking of cardiomyocyte apoptosis after myocardial infarction. This review article summarizes the evidence that S<em>1</em>P as a component of HDL serves to regulate vascular cell and lymphocyte behaviors associated with cardiovascular (patho)physiology.

Activation of the ER stress response is associated with malignant progression of B cell chronic lymphocytic leukemia (CLL). We developed a murine CLL model that lacks the ER stress-associated transcription factor XBP-<em>1</em> in B cells and found that XBP-<em>1</em> deficiency decelerates malignant progression of CLL-associated disease. XBP-<em>1</em> deficiency resulted in acquisition of phenotypes that are disadvantageous for leukemic cell survival, including compromised BCR signaling capability and increased surface expression of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> (S<em>1</em>P<em>1</em>). Because XBP-<em>1</em> expression requires the RNase activity of the ER transmembrane receptor IRE-<em>1</em>, we developed a potent IRE-<em>1</em> RNase inhibitor through chemical synthesis and modified the structure to facilitate entry into cells to target the IRE-<em>1</em>/XBP-<em>1</em> pathway. Treatment of CLL cells with this inhibitor (B-I09) mimicked XBP-<em>1</em> deficiency, including upregulation of IRE-<em>1</em> expression and compromised BCR signaling. Moreover, B-I09 treatment did not affect the transport of secretory and integral membrane-bound proteins. Administration of B-I09 to CLL tumor-bearing mice suppressed leukemic progression by inducing apoptosis and did not cause systemic toxicity. Additionally, B-I09 and ibrutinib, an FDA-approved BTK inhibitor, synergized to induce apoptosis in B cell leukemia, lymphoma, and multiple myeloma. These data indicate that targeting XBP-<em>1</em> has potential as a treatment strategy, not only for multiple myeloma, but also for mature B cell leukemia and lymphoma.