Follicular helper T (Tfh) cells access the B cell follicle to promote antibody responses and are particularly important for germinal center (GC) reactions. However, the molecular mechanisms of how Tfh cells are physically associated with GCs are incompletely understood. We report that the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor 2 (S<em>1</em>PR2) gene is highly expressed in a subpopulation of Tfh cells that localizes in GCs. S<em>1</em>PR2-deficient Tfh cells exhibited reduced accumulation in GCs due to their impaired retention. T cells deficient in both S<em>1</em>PR2 and CXCR5 were ineffective in supporting GC responses compared with T cells deficient only in CXCR5. These results suggest that S<em>1</em>PR2 and CXCR5 cooperatively regulate localization of Tfh cells in GCs to support GC responses.

Lysophosphatidic acid (LPA) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) have been proposed to play a key role in oligodendrocyte maturation and myelinogenesis. In this study, we examined lysophospholipid receptor gene expression in differentiated rat oligodendrocyte cultures and signaling downstream of lysophospholipid receptor activation by LPA and S<em>1</em>P. Differentiated oligodendrocytes express mRNAs encoding lysophospholipid receptors with the relative abundance of lpa<em>1</em>)s<em>1</em>p5>s<em>1</em>p<em>1</em>=s<em>1</em>p2=lpa3>s<em>1</em>p3. LPA and S<em>1</em>P transiently increased phosphorylation of extracellular signal-regulated kinase (ERK) with EC50 values of 956 and <em>1</em>68 nM, respectively. LPA- and S<em>1</em>P-induced ERK phosphorylation was dependent on the activation of mitogen-activated protein kinase (MAPK), phospholipase C (PLC), and protein kinase C (PKC), but was insensitive to pertussis toxin (PTX). LPA increased intracellular calcium levels in oligodendrocytes and these increases were partially blocked by a PLC inhibitor but not by PTX. In contrast, S<em>1</em>P was not found to induce measurable changes of intracellular calcium. These results taken together suggest that lysophospholipid receptor activation involves receptor coupling to heterotrimeric Gq subunits with consequent activation of PLC, PKC, and MAPK pathways leading to ERK phosphorylation.

BACKGROUND

Reactive astrocytes are implicated in the development and maintenance of neuroinflammation in the demyelinating disease multiple sclerosis (MS). The <em>sphingosine</em> kinase <em>1</em> (SphK<em>1</em>)/<em>sphingosine</em><em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor signaling pathway is involved in modulation of the inflammatory response in many cell types, but the role of S<em>1</em>P receptor subtype 3 (S<em>1</em>P(3)) signaling and SphK<em>1</em> in activated rat astrocytes has not been defined.

RESULTS

Using immunohistochemistry we observed the upregulation of S<em>1</em>P(3) and SphK<em>1</em> expression on reactive astrocytes and SphK<em>1</em> on macrophages in MS lesions. Increased mRNA and protein expression of S<em>1</em>P(3) and SphK<em>1</em>, as measured by qPCR and Western blotting respectively, was observed after treatment of rat primary astrocyte cultures with the pro-inflammatory stimulus lipopolysaccharide (LPS). Activation of SphK by LPS stimulation was confirmed by SphK activity assay and was blocked by the use of the SphK inhibitor SKI (2-(p-hydroxyanilino)-4-(p-chlorphenyl) thiazole. Treatment of astrocytes with a selective S<em>1</em>P(3) agonist led to increased phosphorylation of extracellular signal-regulated kinase (ERK)-<em>1</em>/2), which was further elevated with a LPS pre-challenge, suggesting that S<em>1</em>P(3) upregulation can lead to increased functionality. Moreover, astrocyte migration in a scratch assay was induced by S<em>1</em>P and LPS and this LPS-induced migration was sensitive to inhibition of SphK<em>1</em>, and independent of cell proliferation. In addition, S<em>1</em>P induced secretion of the potentially neuroprotective chemokine CXCL<em>1</em>, which was increased when astrocytes were pre-challenged with LPS. A more prominent role of S<em>1</em>P(3) signaling compared to S<em>1</em>P(<em>1</em>) signaling was demonstrated by the use of selective S<em>1</em>P(3) or S<em>1</em>P(<em>1</em>) agonists.

CONCLUSIONS

In summary, our data demonstrate that the SphK<em>1</em>/S<em>1</em>P(3) signaling axis is upregulated when astrocytes are activated by LPS. This signaling pathway appears to play a role in the establishment and maintenance of astrocyte activation. Upregulation of the pathway in MS may be detrimental, e.g. through enhancing astrogliosis, or beneficial through increased remyelination via CXCL<em>1</em>.

Increasing evidence supports roles for lipids in the biology of immune cells. In particular, bioactive lipids such as <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) bind to cognate G protein-coupled receptors (GPCRs) and modulate leukocyte trafficking and homeostasis. Lysophosphatidic acid (LPA) represents a family of bioactive lipids, which differ in the length and degree of saturation of the fatty acyl chain. LPA is structurally related to S<em>1</em>P and exerts cellular effects by binding to five known GPCRs (LPA(<em>1</em>-5)). Its function in the immune system is less clear, although it was shown to induce chemotaxis of human dendritic cells (DCs) and activated T cells. In this study, we show that LPA can induce chemotaxis of immature but not mature mouse DCs and that only unsaturated and not saturated LPA species are efficient chemoattractants. However, both LPA species do not alter DC maturation or chemotaxis to other chemokines. The loss of DC migration capability correlated with the down-regulation of expression of the receptors LPA(3) and LPA(5), and expression of LPA(<em>1</em>), LPA(2), and LPA(4) did not change. A LPA(3) antagonist reduced immature DC migration to LPA by 70%, suggesting that LPA(3) mediates immature DC chemotaxis to unsaturated species of LPA. Furthermore, isolated, immature DCs from mice lacking LPA(3) exhibited a 50% reduction in migration to LPA. In summary, our results indicate that immature mouse DCs migrate preferentially in response to unsaturated LPA and that LPA(3) is important in this response.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is an endogenous growth factor with potent effects on many different cell types. Most of these effects are produced by activation of one or more of a family of G-protein coupled receptors. The S<em>1</em>P2 receptor can mediate S<em>1</em>P-induced proliferation, differentiation and survival in a wide variety of cells in culture. However, identifying essential in vivo functions for S<em>1</em>P2 has been hampered by its ubiquitous expression and the failure to detect any anatomical abnormalities in initial analyses of S<em>1</em>P2 knockout mice. We report here that all S<em>1</em>P2 knockout mice are profoundly deaf from postnatal day 22 and approximately half display a progressive loss of vestibular function with aging. Anatomically, both the auditory and vestibular systems appear to develop normally but then degrade. Morphological defects associated with hearing are first detected at 3 weeks postnatal as deformations of the organ of Corti/Nuel's space. By one year of age structures within the scala media are dramatically altered. The S<em>1</em>P2 knockout mice also display a loss of otoconia consistent with the vestibular impairment. The present data are the first to indicate that S<em>1</em>P signaling plays critical roles, in vivo, in auditory and vestibular functions. The data further establish that the S<em>1</em>P signaling occurs through the S<em>1</em>P2 receptor and makes an essential contribution to the structural maintenance of these systems, raising the possibility that properly targeted enhancement of this signaling may prove to be clinically beneficial.

Sphingolipids, historically described as potential reservoirs for bioactive lipids, presently define a new family of cellular mediators, joining the well-established glycerolipid-derived mediators of signal transduction such as diacylglycerol, phosphatidylinositides, and eicosanoids. Sphingolipid metabolism is clearly involved in the regulation of cell growth, differentiation, and programmed cell death. Indeed, a majority of the greater than four thousand studies conducted on sphingolipids during the past five years were investigations of the role of sphingolipids as cellular bioregulators. Studies spanning more than a decade have shown multiple interactions and intersections of the sphingolipid-mediated pathways and the eicosanoid pathway. This review will discuss the emerging mechanisms by which sphingolipids induce inflammatory responses via the eicosanoid pathway in addition to linking previous literature on sphingolipids and inflammation with newer findings of distinct roles for <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> in regulating cyclooygenase-2 and ceramide-<em>1</em>-<em>phosphate</em> in the regulation of cytosolic phospholipase A2alpha. Finally, the relationship between bioactive sphingolipids and inflammation is discussed.

The potent immunomodulator FTY720 elicits immunosuppression via acting on <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptors (S<em>1</em>PR), thereby leading to an entrapment of lymphocytes in the secondary lymphoid tissue. To elucidate the potential in vitro effects of this drug on human monocyte-derived DC, we used low nanomolar therapeutic concentrations of FTY720 and phosphorylated FTY720 (FTY720-P) and investigated their influence on DC surface marker expression, protein levels of S<em>1</em>PR and DC effector functions: antigen uptake, chemotaxis, cytokine production, allostimulatory and Th-priming capacity. We report that both FTY720 and FTY720-P reduce chemotaxis of immature and mature DC. Mature DC generated in the presence of FTY720 or FTY720-P showed an impaired immunostimmulatory capacity and reduced IL-<em>1</em>2 but increased IL-<em>1</em>0 production. T cells cultured in the presence of FTY720- or FTY720-P-treated DC showed an altered cytokine production profile indicating a shift from Th<em>1</em> toward Th2 differentiation. In treated immature and mature DC, expression levels for two S<em>1</em>PR proteins, S<em>1</em>P<em>1</em> and S<em>1</em>P4, were reduced. We conclude that in vitro treatment with FTY720 affects DC features that are essential for serving their role as antigen-presenting cells. This might represent a new aspect of the overall immunosuppressive action of FTY720 and makes DC potential targets of further sphingolipid-derived drugs.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a pleiotropic bioactive lipid thought to be dysregulated in a variety of disease conditions. In this review, we discuss the roles of S<em>1</em>P in cancer and in wet age-related macular degeneration. We also explore potential treatment strategies for these disorders, including the utility of anti-S<em>1</em>P antibodies acting as molecular sponges to neutralize dysregulated S<em>1</em>P in relevant tissues.

Recent evidence suggests that inflammation is involved in malignant progression of breast cancer. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), acting on the G-protein-coupled receptors, is known as a potent inflammatory mediator. In this study, the effect of the inflammatory lipid S<em>1</em>P on the regulation of invasive/migratory phenotypes of MCF<em>1</em>0A human breast epithelial cells was investigated to elucidate a causal relationship between inflammation and the control of invasiveness of breast cells. We show that S<em>1</em>P causes induction of matrix metalloproteinase-9 (MMP-9) in vitro and in vivo, and thus enhances invasion and migration. We also show that fos plays a crucial role in the transcriptional activation of MMP-9 by S<em>1</em>P. In addition, activation of extracellular-signal-regulated kinases <em>1</em> and 2 (ERK<em>1</em>/2), p38 and alpha serine/threonine-protein kinase (Akt) are involved in the process of S<em>1</em>P-mediated induction of MMP-9 expression and invasion. Activation of the S<em>1</em>P receptor S<em>1</em>P₃ and G(αq) are required for S<em>1</em>P-induced invasive/migratory responses, suggesting that the enhancement of S<em>1</em>P-mediated invasiveness is triggered by the specific coupling of S<em>1</em>P₃ to the heterotrimeric G(αq) subunit. Activation of phospholipase C-β₄ and intracellular Ca²⁺ release are required for S<em>1</em>P-induced MMP-9 upregulation. Taken together, this study demonstrated that S<em>1</em>P regulates MMP-9 induction and invasiveness through coupling of S<em>1</em>P₃ and G(αq) in MCF<em>1</em>0A cells, thus providing a molecular basis for the crucial role of S<em>1</em>P in promoting breast cell invasion.

The aim of this study was to test the hypothesis that plasma <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) levels are associated with the risk of cardiovascular autonomic neuropathy (CAN) in type 2 diabetes patients. This cross-sectional study included 287 individuals with type 2 diabetes. CAN was evaluated using cardiovascular reflex tests. Logistic regression analyses were conducted to assess the relationship between plasma S<em>1</em>P levels and CAN. Plasma S<em>1</em>P concentrations were significantly lower in individuals with CAN than in those without CAN. There was a significant interaction between plasma S<em>1</em>P levels and sex with respect to CAN (p for interaction = 0.003). When stratified by sex, the association between plasma S<em>1</em>P levels and CAN exhibited a sex difference; in multivariable analysis, plasma S<em>1</em>P levels were significantly associated with CAN in women (odds ratio per standard deviation increase in the log-transformed value, 0.40; 95% confidence interval, 0.23-0.70, p = 0.00<em>1</em>). However, there was no significant association between plasma S<em>1</em>P and CAN in men. Plasma S<em>1</em>P concentrations were inversely associated with CAN only in women with type 2 diabetes.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), which derives from the metabolism of sphingomyelin, is mainly synthesized, stored, and released from platelets after activation by physiological and pathophysiological events. S<em>1</em>P acts in cardiovascular tissues through cell surface G-protein-coupled receptors of the endothelial differentiation gene (EDG) family, i.e., EDG<em>1</em>, EDG3 and EDG5. The aim of the present study was to assess the precise distribution of EDG<em>1</em>, EDG3, and EDG5 receptors expressed in human cardiovascular tissues to investigate their respective physiological implication. When assessed by Northern blots, EDG<em>1</em>, EDG3, and EDG5 displayed wide expression levels in decreasing order, respectively. In particular, EDG3 was mainly detected in the aorta. Detailed analysis by in situ hybridization (ISH) and immunohistochemistry (IHC) revealed strong EDG<em>1</em> expression in cardiomyocytes and in endothelial cells of cardiac vessels. In cardiomyocytes, the EDG<em>1</em> receptor is likely to be co-expressed with EDG3 and EDG5, although EDG<em>1</em> exhibits the most prominent expression pattern. Unlike EDG3 and EDG5, which are expressed in the smooth muscle cell layer of the human aorta, no signal corresponding to EDG<em>1</em> expression could be detected in the aorta. Moreover, only EDG3 expression was also found in smooth muscle cells of cardiac vessels. The present results provide new insight into the expression pattern of S<em>1</em>P receptors in human cardiovascular tissues, indicating a differential pattern of expression for these receptors in human vessels.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lipid that promotes cell survival, proliferation and migration, platelet aggregation, mediates ischemic preconditioning, and is essential for angiogenesis and lymphocyte trafficking. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase (SPL) is the enzyme responsible for the irreversible degradation of S<em>1</em>P and is, thus, in a strategic position to regulate these same processes by removing available S<em>1</em>P signaling pools, that is, silencing the siren. In fact, recent studies have implicated SPL in the regulation of immunity, cancer surveillance and other physiological processes. Here, we summarize the current understanding of SPL function and regulation, and discuss how SPL might facilitate cancer chemoprevention and serve as a target for modulation of immune responses in transplantation settings and in the treatment of autoimmune disease.

In the present study, we determined the agonist specificity and the signalling mechanisms of a putative <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor, AGR<em>1</em>6. In CHO cells transiently transfected with an AGR<em>1</em>6 expression vector, but not in cells transfected with an empty vector, the addition of a low concentration of S<em>1</em>P (<em>1</em> nM) caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i) by mobilization of Ca2+ from both intra- and extra-cellular pools. To determine the spectrum of agonists for AGR<em>1</em>6, we employed K562 cells, which in the naive state do not respond at all to either S<em>1</em>P or structurally related lipids with an increase in [Ca2+]i. In K562 cells stably expressing AGR<em>1</em>6, S<em>1</em>P and sphingosylphosphorylcholine (SPC) dose-dependently increased [Ca2+]i with half-maximal values of 3 nM and <em>1</em>00 nM respectively. In CHO cells stably expressing AGR<em>1</em>6 (CHO-AGR<em>1</em>6), but not in parental CHO cells, we observed specific binding of [32P]S<em>1</em>P, which was displaced by unlabelled S<em>1</em>P and SPC. In CHO-AGR<em>1</em>6 cells, but not in parental CHO cells, S<em>1</em>P stimulated the production of inositol <em>phosphates</em> and Ca2+ mobilization which was only 30% inhibited by pertussis toxin (PTX), different from the case of the recently identified S<em>1</em>P receptor EDG<em>1</em>. Also in CHO-AGR<em>1</em>6 cells, but not in CHO cells, S<em>1</em>P at higher concentrations activated mitogen-activated protein kinase (MAPK) in a PTX-sensitive and Ras-dependent manner. S<em>1</em>P also induced the activation of two stress-activated MAPKs, c-Jun N-terminal kinase and p38, in a manner that was totally insensitive to PTX. In CHO-AGR<em>1</em>6 cells, S<em>1</em>P induced stress-fibre formation, with an increase in myosin light chain phosphorylation, in a PTX-insensitive and Rho-dependent manner. S<em>1</em>P also induced an increase in the cellular cAMP content in CHO-AGR<em>1</em>6 cells, which contrasts sharply with the case of EDG<em>1</em>. These results establish that the S<em>1</em>P receptor AGR<em>1</em>6 is coupled via both PTX-sensitive and -insensitive G-proteins to multiple effector pathways.

BACKGROUND

Idiopathic pulmonary fibrosis (IPF) is characterised by the aberrant epithelial to mesenchymal transition (EMT) and myofibroblast accumulation. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and <em>sphingosine</em> kinase <em>1</em> (SPHK<em>1</em>) have been implicated in lung myofibroblast transition, but their role in EMT and their expression in patients with IPF is unknown.

RESULTS

S<em>1</em>P levels were measured in serum (n=27) and bronchoalveolar lavage (BAL; n=<em>1</em>5) from patients with IPF and controls (n=30 for serum and n=<em>1</em>5 for BAL studies). SPHK<em>1</em> expression was measured in lung tissue from patients with IPF (n=<em>1</em>2) and controls (n=<em>1</em>5). Alveolar type II transformation into mesenchymal cells was studied in response to S<em>1</em>P (<em>1</em>0(-9)-<em>1</em>0(-5) M). The median (IQR) of S<em>1</em>P serum levels was increased in patients with IPF (<em>1</em>.4 (0.4) μM) versus controls (<em>1</em> (0.26) μM; p<0.000<em>1</em>). BAL S<em>1</em>P levels were increased in patients with IPF (<em>1</em>.<em>1</em>2 (0.53) μM) versus controls (0.2 (0.5); p<0.000<em>1</em>) and correlated with diffusion capacity of the lung for carbon monoxide, forced expiratory volume in <em>1</em> s and forced vital capacity (Spearman's r=-0.87, -0.72 and -0.68, respectively) in patients with IPF. SPHK<em>1</em> was upregulated in lung tissue from patients with IPF and correlated with α-smooth muscle actin, vimentin and collagen type I (Spearman's r=0.82, 0.85 and 0.72, respectively). S<em>1</em>P induced EMT in alveolar type II cells by interacting with S<em>1</em>P(2) and S<em>1</em>P(3), as well as by the activation of p-Smad3, RhoA-GTP, oxidative stress and transforming growth factor-β<em>1</em> (TGF-β<em>1</em>) release. Furthermore, TGF-β<em>1</em>-induced EMT was partially conducted by the S<em>1</em>P/SPHK<em>1</em> activation, suggesting crosstalk between TGF-β<em>1</em> and the S<em>1</em>P/SPHK<em>1</em> axis.

CONCLUSIONS

S<em>1</em>P is elevated in patients with IPF, correlates with the lung function and mediates EMT.

BACKGROUND

<em>Sphingosine</em> kinases (SphKs) <em>1</em> and 2 regulate the synthesis of the bioactive sphingolipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), an important lipid mediator that promotes cell proliferation, migration, and angiogenesis.

OBJECTIVE

We aimed to examine whether SphKs and their product, S<em>1</em>P, play a role in the development of pulmonary arterial hypertension (PAH).

METHODS

SphK<em>1</em>(-/-), SphK2(-/-), and S<em>1</em>P lyase heterozygous (Sgpl<em>1</em>(+/-)) mice, a pharmacologic SphK inhibitor (SKI2), and a S<em>1</em>P receptor 2 (S<em>1</em>PR2) antagonist (JTE0<em>1</em>3) were used in rodent models of hypoxia-mediated pulmonary hypertension (HPH). S<em>1</em>P levels in lung tissues from patients with PAH and pulmonary arteries (PAs) from rodent models of HPH were measured.

RESULTS

mRNA and protein levels of SphK<em>1</em>, but not SphK2, were significantly increased in the lungs and isolated PA smooth muscle cells (PASMCs) from patients with PAH, and in lungs of experimental rodent models of HPH. S<em>1</em>P levels were increased in lungs of patients with PAH and PAs from rodent models of HPH. Unlike SphK2(-/-) mice, SphK<em>1</em>(-/-) mice were protected against HPH, whereas Sgpl<em>1</em>(+/-) mice were more susceptible to HPH. Pharmacologic SphK<em>1</em> and S<em>1</em>PR2 inhibition prevented the development of HPH in rodent models of HPH. Overexpression of SphK<em>1</em> and stimulation with S<em>1</em>P potentially via ligation of S<em>1</em>PR2 promoted PASMC proliferation in vitro, whereas SphK<em>1</em> deficiency inhibited PASMC proliferation.

CONCLUSIONS

The SphK<em>1</em>/S<em>1</em>P axis is a novel pathway in PAH that promotes PASMC proliferation, a major contributor to pulmonary vascular remodeling. Our results suggest that this pathway is a potential therapeutic target in PAH.

It is now widely accepted that lysophospholipids (LPLs), a product of the phospholipase A reaction, function as mediators through G-protein-coupled receptors. Notably, recent studies of lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) have revealed their essential roles in vivo. On the other hand, other LPLs such as lysophosphatidylserine (LPS), lysophosphatidylthreonine (LPT), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI) and lysophosphatidylglycerol (LPG) have been reported to show lipid mediator-like responses both in vivo (LPS and LPT) and in vitro (LPS, LPT, LPE and LPG), while very little is known about their receptor, synthetic enzyme and patho-physiological roles. In this review, we summarize the actions of these LPLs as lipid mediators including LPS, LPT, LPE and LPG.

High expression of autotaxin in cancers is often associated with increased tumor progression, angiogenesis and metastasis. This is explained mainly since autotaxin produces the lipid growth factor, lysophosphatidate (LPA), which stimulates cell division, survival and migration. It has recently become evident that these signaling effects of LPA also produce resistance to chemotherapy and radiation-induced cell death. This results especially from the stimulation of LPA(2) receptors, which depletes the cell of Siva-<em>1</em>, a pro-apoptotic signaling protein and stimulates prosurvival kinase pathways through a mechanism mediated via TRIP-6. LPA signaling also increases the formation of <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, a pro-survival lipid. At the same time, LPA decreases the accumulation of ceramides, which are used in radiation therapy and by many chemotherapeutic agents to stimulate apoptosis. The signaling actions of extracellular LPA are terminated by its dephosphorylation by a family of lipid <em>phosphate</em> phosphatases (LPP) that act as ecto-enzymes. In addition, lipid <em>phosphate</em> phoshatase-<em>1</em> attenuates signaling downstream of the activation of both LPA receptors and receptor tyrosine kinases. This makes many cancer cells hypersensitive to the action of various growth factors since they often express low LPP<em>1</em>/3 activity. Increasing our understanding of the complicated signaling pathways that are used by LPA to stimulate cell survival should identify new therapeutic targets that can be exploited to increase the efficacy of chemo- and radio-therapy. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

The novel immunomodulator FTY720 is an effective immunosuppressive agent in experimental models of transplantation and autoimmunity and is currently undergoing phase III clinical trials for multiple sclerosis. Phosphorylated FTY720 is a structural analogue of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and therefore acts as a high-affinity agonist at four of the five G protein-coupled S<em>1</em>P receptors. It has been well established that there exists a crosstalk between S<em>1</em>P and transforming growth factor (TGF)-beta signaling. Because TGF-beta is the most prominent inductor of fibrosis and myofibroblasts are primarily responsible for excessive matrix protein formation, we examined whether FTY720, in analogy to TGF-beta, induces differentiation of fibroblasts into myofibroblasts. Indeed, FTY720 provoked myofibroblast differentiation comparable with that of TGF-beta. For biological efficacy, FTY720 required endogenous phosphorylation because inhibition of <em>sphingosine</em> kinase completely prevented FTY720 from inducing the differentiation process. Moreover, we identified the lysophospholipid receptor S<em>1</em>P3 as the crucial receptor subtype for FTY720-induced myofibroblast differentiation because the effect was abolished in fibroblasts isolated from S<em>1</em>P3 knockout mice. Finally, we determined that downstream of S<em>1</em>P3 signaling Smad3 activation is essential for myofibroblast differentiation in response to FTY720. Thus, FTY720 may have adverse fibrotic effects related to its activity on S<em>1</em>P3 signaling.

Vasculogenesis, angiogenesis, and maturation are three major phases of the development of blood vessels. Although many receptors required for blood vessel formation have been defined, the intracellular signal transduction pathways involved in vascular maturation remain unclear. KLF2(-/-) embryos fail to develop beyond <em>1</em>3.5 days because of a lack of blood vessel stabilization. The molecular mechanism of KLF2 function in embryonic vascular vessels is still largely unknown. Here we show a normal development pattern of endothelial cells in KLF2(-/-) embryos but a defect of smooth muscle cells at the dorsal side of the aorta. This phenotype results from arrested vascular maturation characterized by the failure of mural cells to migrate around endothelial cells. This migration defect is also observed when platelet-derived growth factor-B (PDGF) controlled migration is studied in murine embryonic fibroblast (MEF) cells from KLF2(-/-) animals. In addition, KLF2(-/-) MEFs exhibit a significant growth defect, indicating that KLF2 is required to maintain the viability of MEF cells. The PDGF signal is mediated through the Src signaling pathway, and a downstream target of KLF2 is <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor <em>1</em>. These studies demonstrate that KLF2 is required for smooth muscle cell migration and elucidate a novel mechanism involving communication between PDGF and KLF2 in vascular maturation.

The anthracycline daunorubicin is widely used in the treatment of acute nonlymphocytic leukemia. The drug has, of course, been the object of intense basic research, as well as preclinical and clinical study. As reviewed in this article, evidence stemming from this research clearly demonstrates that cell response to daunorubicin is highly regulated by multiple signaling events, including a sphingomyelinase-initiated sphingomyelin-ceramide pathway, mitogen-activated kinase and stress-activated protein/c-Jun N-terminal kinase activation, transcription factors such as nuclear factor kappa B, as well as the Fas/Fas-ligand system. These pathways are themselves influenced by a number of lipid products (diacylglycerol, <em>sphingosine</em>-<em>1</em> <em>phosphate</em>, and glucosyl ceramide), reactive oxygen species, oncogenes (such as the tumor suppressor gene p53), protein kinases (protein kinase C and phosphoinositide-3 kinase), and external stimuli (hematopoietic growth factors and the extracellular matrix). In light of the complexity and diversity of these observations, a comprehensive review has been attempted toward the understanding of their individual implication (and regulation) in daunorubicin-induced signaling. (Blood. 200<em>1</em>;98:9<em>1</em>3-924)

Ceramide and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> are related sphingolipid metabolites that can be generated through a de novo biosynthetic route or derived from the recycling of membrane sphingomyelin. Both these lipids regulate cellular responses to stress, with generally opposing effects. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> functions as a growth and survival factor, acting as a ligand for a family of G protein-coupled receptors, whereas ceramide activates intrinsic and extrinsic apoptotic pathways through receptor-independent mechanisms. A growing body of evidence has implicated ceramide, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> and the genes involved in their synthesis, catabolism and signaling in various aspects of oncogenesis, cancer progression and drug- and radiation resistance. This may be explained in part by the finding that both lipids impinge upon the PI3K/ AKT pathway, which represses apoptosis and autophagy. In addition, sphingolipids influence cell cycle progression, telomerase function, cell migration and stem cell biology. Considering the central role of ceramide in mediating physiological as well as pharmacologically stimulated apoptosis, ceramide can be considered a tumor-suppressor lipid. In contrast, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> can be considered a tumor-promoting lipid, and the enzyme responsible for its synthesis functions as an oncogene. Not surprisingly, genetic mutations that result in reduced ceramide generation, increased <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> synthesis or which reduce steady state ceramide levels and increase <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> levels have been identified as mechanisms of tumor progression and drug resistance in cancer cells. Pharmacological tools for modulating sphingolipid pathways are being developed and represent novel therapeutic strategies for the treatment of cancer.

The phospholipid growth factors <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and lysophosphatidic acid (LPA) are ligands for the related G protein-coupled receptors S<em>1</em>P(<em>1</em>)/EDG<em>1</em> and LPA(<em>1</em>)/EDG2, respectively. We have developed a model of LPA(<em>1</em>) that predicts interactions between three polar residues and LPA. One of these, glutamine <em>1</em>25, which is conserved in the LPA receptor subfamily (LPA(<em>1</em>)/EDG2, LPA(2)/EDG4, and LPA(3)/EDG7), hydrogen bonds with the LPA hydroxyl group. Our previous S<em>1</em>P(<em>1</em>) study identified that the corresponding glutamate residue, conserved in all S<em>1</em>P receptors, ion pairs with the S<em>1</em>P ammonium. These two results predict that this residue might influence ligand recognition and specificity. Characterization of glutamate/glutamine interchange point mutants of S<em>1</em>P(<em>1</em>) and LPA(<em>1</em>) validated this prediction as the presence of glutamate was required for S<em>1</em>P recognition, whereas LPA recognition was possible with either glutamine or glutamate. The most likely explanation for this dual specificity behavior is a shift in the equilibrium between the acid and conjugate base forms of glutamic acid due to other amino acids surrounding that position in LPA(<em>1</em>), producing a mixture of receptors including those having an anionic glutamate that recognize S<em>1</em>P and others with a neutral glutamic acid that recognize LPA. Thus, computational modeling of these receptors provided valid information necessary for understanding the molecular pharmacology of these receptors.

OBJECTIVE

Directed migration of hepatic myofibroblasts (hMFs) contributes to the development of liver fibrosis. However, the signals regulating the motility of these cells are incompletely understood. We have recently shown that <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and S<em>1</em>P receptors (S<em>1</em>PRs) are involved in mouse liver fibrogenesis. Here, we investigated the role of S<em>1</em>P/S<em>1</em>PRs signals in human liver fibrosis involving motility of human hMFs.

METHODS

S<em>1</em>P level in the liver was examined by high-performance liquid chromatography. Expression of S<em>1</em>PRs was characterized, in biopsy specimens of human liver and cultured hMFs, by immunofluorescence and real-time RT-PCR or Western blot analysis. Cell migration was determined in Boyden chambers, by using the selective S<em>1</em>P receptor agonist or antagonist and silencing of S<em>1</em>PRs expression with small interfering RNA.

RESULTS

S<em>1</em>P level in the human fibrotic liver was increased through up-regulation of <em>sphingosine</em> kinase (SphK), irrespective of the etiology of fibrosis. S<em>1</em>P receptors type <em>1</em>, 2, and 3 (S<em>1</em>P(<em>1</em>,2,3)) were expressed in human hMFs in vivo and in vitro. Interestingly, S<em>1</em>P(<em>1</em>,3) were strongly induced in human fibrotic samples, whereas expression of S<em>1</em>P(2) was massively decreased. S<em>1</em>P exerted a powerful migratory action on human hMFs. Furthermore, the effect of S<em>1</em>P was mimicked by SEW287<em>1</em> (an S<em>1</em>P(<em>1</em>) agonist), and blocked by suramin (an S<em>1</em>P(3) antagonist) and by silencing S<em>1</em>P(<em>1</em>,3) expression. In contrast, JTE-0<em>1</em>3 (an S<em>1</em>P(2) antagonist) and silencing of S<em>1</em>P(2) expression enhanced S<em>1</em>P-induced migration.

CONCLUSIONS

SphK/S<em>1</em>P/S<em>1</em>PRs signaling axis plays an important role in human liver fibrosis and is involved in the directed migration of human hMFs into the damaged areas.

BACKGROUND

Lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) are ubiquitous lipid messengers found in the blood and most cell types. Both lysophospholipids are ligands of G protein-coupled receptors and mediate important physiological processes. Moreover, lysophospholipids are potential biomarkers for various diseases, including atherosclerosis and cancer. Because existing methodologies are of limited value for systematic evaluations of S<em>1</em>P and LPA in clinical studies, we developed a fast and simple quantification method that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS).

METHODS

Sphingoid base <em>1</em>-<em>phosphate</em>s and LPA species were quantified in negative-ion mode with fragments of m/z 79 and <em>1</em>53, respectively. The internal standards LPA <em>1</em>7:0 and [(<em>1</em>3)C(2)D(2)]S<em>1</em>P were added before butanol extraction. Application of hydrophilic-interaction chromatography allowed coelution of analytes and internal standards with a short analysis time of 2.5 min.

RESULTS

Comparison of butanol extraction with a frequently used extraction method based on strong acidification of human plasma revealed artificial formation of LPA from lysophosphatidylcholine with the latter method. Validation according to US Food and Drug Administration guidelines showed an overall imprecision (CV) of (<em>1</em>2% and a limit of detection <6 nmol/L for all lysophospholipid species. Concentrations of S<em>1</em>P and sphinganine <em>1</em>-<em>phosphate</em> (SA<em>1</em>P) in EDTA-containing plasma were stable for 24 h at room temperature, whereas LPA concentrations increased substantially over this period.

CONCLUSIONS

Our validated LC-MS/MS methodology for quantifying LPA, S<em>1</em>P, and SA<em>1</em>P features simple sample preparation and short analysis times, therefore providing a valuable tool for diagnostic evaluation of these lysophospholipids as biomarkers.