Sphingosine‐1‐phosphate receptors and innate immunity

Sphingosine‐1‐phosphate (S1P) is a signalling lipid that regulates many cellular processes in mammals. One well‐studied role of S1P signalling is to modulate T‐cell trafficking, which has a major impact on adaptive immunity. Compounds that target S1P signalling pathways are of interest for immune system modulation. Recent studies suggest that S1P signalling regulates many more cell types and processes than previously appreciated. This review will summarise current understanding of S1P signalling, focusing on recent novel findings in the roles of S1P receptors in innate immunity.     

Inhibition of Ca(2+) signalling by the sphingosine 1-phosphate receptor S1P(1)

The lysophospholipid, sphingosine 1-phosphate (S1P), regulates a multitude of cellular functions by activating specific G protein-coupled receptors (GPCRs) (S1P(1-5), plus three newly identified S1P receptors). The G(i)-coupled S1P(1) receptor inhibits adenylyl cyclase, stimulates mitogen-activated protein kinases (MAP kinases) and cell migration, and is required for blood vessel maturation. Here, we report that S1P(1) inhibits Ca(2+) signalling in a number of cell types. In HEK-293 cells, which endogenously express S1P(1-3), overexpression of S1P(1) reduced intracellular free Ca(2+) concentration ([Ca(2+)](i)) increases induced by various receptor agonists as well as thapsigargin. The inhibitory Ca(2+) signalling of S1P(1) was blocked by pertussis toxin (PTX) and the protein kinase C (PKC) inhibitor, G?6976, and imitated by phorbol ester and overexpression of classical PKC isoforms. Activation of S1P(1) stably expressed in RH7777 cells, which endogenously do not express S1P receptors, also inhibited Ca(2+) signalling, without mediating Ca(2+) mobilization on its own. It is concluded that the widely expressed S1P receptor S1P(1) inhibits Ca(2+) signalling, most likely via G(i) proteins and classical PKC isoforms. Co-expression of S1P(1) with S1P(3), but not S1P(2), reversed the inhibitory effect of S1P(1), furthermore suggesting a specific interplay of S1P receptor subtypes usually found within a single cell type.     

Redox regulation of PI 3-kinase signalling via inactivation of PTEN

The tumour suppressor PTEN is a PtdIns(3,4,5)P(3) phosphatase that regulates many cellular processes through direct antagonism of PI 3-kinase signalling. Here we show that oxidative stress activates PI 3-kinase-dependent signalling via the inactivation of PTEN. We use two assay systems to show that cellular PTEN phosphatase activity is inhibited by oxidative stress induced by 1 mM hydrogen peroxide. PTEN inactivation by oxidative stress also causes an increase in cellular PtdIns(3,4,5)P(3) levels and activation of the downstream PtdIns(3,4,5)P(3) target, PKB/Akt, that does not occur in cells lacking PTEN. We then show that endogenous oxidant production in RAW264.7 macrophages inactivates a fraction of the cellular PTEN, and that this is associated with an oxidant-dependent activation of downstream signalling. These results show that oxidants, including those produced by cells, can activate downstream signalling via the inactivation of PTEN. This demonstrates a novel mechanism of regulation of the activity of this important tumour suppressor and the signalling pathways it regulates. These results may have significant implications for the many cellular processes in which PtdIns(3,4,5)P(3) and oxidants are produced concurrently.     

An autocrine sphingosine-1-phosphate signaling loop enhances NF-κB-activation and survival

Background  

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates a multitude of cellular functions, including cell proliferation, survival, migration and angiogenesis. S1P mediates its effects either by signaling through G protein-coupled receptors (GPCRs) or through an intracellular mode of action. In this study, we have investigated the mechanism behind S1P-induced survival signalling.     

Sphingosine-1-phosphate: dual messenger functions

The sphingolipid metabolite sphingosine-1-phosphate (S1P) is a serum-borne lipid that regulates many vital cellular processes. S1P is the ligand of a family of five specific G protein-coupled receptors that are differentially expressed in different tissues and regulate diverse cellular actions. Much less is known of the intracellular actions of S1P. It has been suggested that S1P may also function as an intracellular second messenger to regulate calcium mobilization, cell growth and suppression of apoptosis in response to a variety of extracellular stimuli. Dissecting the dual actions and identification of intracellular targets of S1P has been challenging, but there is ample evidence to suggest that the balance between S1P and ceramide and/or sphingosine levels in cells is an important determinant of cell fate.     

Gαq-mediated plasma membrane translocation of sphingosine kinase-1 and cross-activation of S1P receptors

Sphingosine-1-phosphate (S1P), formed by sphingosine kinases (SphKs), regulates cellular proliferation and migration by acting as an agonist at specific receptors or intracellularly. Since S1P's effects are probably dependent on subcellular localization of its formation and degradation, we have studied the influence of G protein-coupled receptors on the localization of SphK1. Activation of G_q-coupled receptors induced a profound, rapid (half-life 3–5 s) and long-lasting (> 2 h) translocation of SphK1 to the plasma membrane. This was mimicked by expression of constitutively active G protein α-subunits specifically of the G_q family. Classical G_q signalling pathways, or phosphorylation at Ser²²⁵, phospholipase D and Ca²⁺/calmodulin were not involved in M₃ receptor-induced SphK1 translocation in HEK-293 cells. Translocation was associated with S1P receptor internalization, which was dependent on catalytic activity of SphK1 and S1P receptor binding and thus resulted from S1P receptor cross-activation. It is concluded that SphK1 is an important effector of G_q-coupled receptors, linking them via cross-activation of S1P receptors to G_i and G_12/13 signalling pathways.     

Modulation of cellular S1P levels with a novel, potent and specific inhibitor of sphingosine kinase-1

SphK (sphingosine kinase) is the major source of the bioactive lipid and GPCR (G-protein-coupled receptor) agonist S1P (sphingosine 1-phosphate). S1P promotes cell growth, survival and migration, and is a key regulator of lymphocyte trafficking. Inhibition of S1P signalling has been proposed as a strategy for treatment of inflammatory diseases and cancer. In the present paper we describe the discovery and characterization of PF-543, a novel cell-permeant inhibitor of SphK1. PF-543 inhibits SphK1 with a K(i) of 3.6 nM, is sphingosine-competitive and is more than 100-fold selective for SphK1 over the SphK2 isoform. In 1483 head and neck carcinoma cells, which are characterized by high levels of SphK1 expression and an unusually high rate of S1P production, PF-543 decreased the level of endogenous S1P 10-fold with a proportional increase in the level of sphingosine. In contrast with past reports that show that the growth of many cancer cell lines is SphK1-dependent, specific inhibition of SphK1 had no effect on the proliferation and survival of 1483 cells, despite a dramatic change in the cellular S1P/sphingosine ratio. PF-543 was effective as a potent inhibitor of S1P formation in whole blood, indicating that the SphK1 isoform of sphingosine kinase is the major source of S1P in human blood. PF-543 is the most potent inhibitor of SphK1 described to date and it will be useful for dissecting specific roles of SphK1-driven S1P signalling.     

Down-regulation of EDG5/S1P2 during myogenic differentiation results in the specific uncoupling of sphingosine 1-phosphate signalling to phospholipase D

The bioactive lipid sphingosine 1-phosphate (S1P) is known to exert powerful biological effects through the interaction with various members of the endothelial differentiation gene (EDG) receptor family, recently renamed S1P receptors. In the present study, evidence is provided that differentiation of C2C12 myoblasts into myotubes was accompanied by profound changes of EDG/S1P receptor expression. Indeed, in differentiated cells a significant increase of EDG3/S1P3 together with a large decrease of EDG5/S1P2 expression at mRNA as well as protein level was detected. Moreover, S1P was capable to initiate the signalling pathways downstream to cytosolic Ca(2+) increase in myotubes, similarly to that observed in myoblasts, whereas the signalling of the bioactive lipid to phospholipase D (PLD), but not that of bradykinin (BK) or lysophosphatidic acid (LPA), was found impaired in differentiated cells. Intriguingly, overexpression of EDG5/S1P2, but not EDG1/S1P1 or EDG3/S1P3, potentiated the efficacy of S1P to stimulate PLD, strongly suggesting a role for EDG5/S1P2 in the signalling to PLD. This view was also supported by the marked reduction of S1P-induced PLD activity in myoblasts loaded with antisense oligodeoxyribonucleotides (ODN) to EDG5/S1P2. Furthermore, overexpression of EDG5/S1P2 rescued the coupling of S1P signalling to PLD in C2C12 myotubes. Experimental evidence here provided supports the notion that EDG5/S1P2 plays a dominant role in the coupling of S1P to PLD in myoblasts and that the down-regulation of the receptor subtype is responsible for the specific uncoupling of S1P signalling to PLD in myotubes.     

Cell cycle control of Wnt/β-catenin signalling by conductin/axin2 through CDC20

Wnt/β-catenin signalling regulates cell proliferation by modulating the cell cycle and is negatively regulated by conductin/axin2/axil. We show that conductin levels peak at G2/M followed by a rapid decline during return to G1. In line with this, Wnt/β-catenin target genes are low at G2/M and high at G1/S, and β-catenin phosphorylation oscillates during the cell cycle in a conductin-dependent manner. Conductin is degraded by the anaphase-promoting complex/cyclosome cofactor CDC20. Knockdown of CDC20 blocks Wnt signalling through conductin. CDC20-resistant conductin inhibits Wnt signalling and attenuates colony formation of colorectal cancer cells. We propose that CDC20-mediated degradation of conductin regulates Wnt/β-catenin signalling for maximal activity during G1/S.     

Sphingosine Kinase 1 and Sphingosine-1-Phosphate in Oxidative Stress Evoked by 1-Methyl-4-Phenylpyridinium (MPP+) in Human Dopaminergic Neuronal Cells

Sphingosine kinases (Sphk1/2) are crucial enzymes in regulation of the biostat between sphingosine-1-phosphate (S1P) and ceramide and play an important role in the pathogenesis/pathomechanism of Alzheimer’s disease (AD). These enzymes synthesise S1P, which regulates neurotransmission, synaptic function and neuron cell proliferation, by activating five G protein-coupled receptors (S1P1-5). However, S1P synthesised by Sphk2 could be involved in amyloid β (Aβ) release by stimulation of Aβ precursor protein degradation. The significance of this bioactive sphingolipid in the pathogenesis of Parkinson’s disease (PD) is unknown. The aim of our study was to investigate the expression level of Sphk1 and its role in human dopaminergic neuronal cell (SH-SY5Y) viability under oxidative stress, evoked by 1-methyl-4-phenylpyridinium (MPP+). Moreover, the mechanism of S1P action on the death signalling pathway in these experimental conditions was evaluated. Our study indicated marked downregulation of Sphk1 expression in this cellular PD model. Inhibition of Sphk1 decreased SH-SY5Y cell viability and concomitantly enhanced the reactive oxygen species (ROS) level. It was found that exogenous S1P (1 μM) exerted the neuroprotective effect by activation of Sphk1 and S1P1 receptor gene expression. Moreover, S1P downregulated Bax and harakiri, death protein 5 (Hrk/DP5) expression and enhanced cell viability in MPP+-treated cells. The neuroprotective mechanism of S1P is mainly dependent on S1P1 receptor signalling, which was indicated by using specific agonists and antagonists of S1P1 receptor. The results show that S1P and S1P1 receptor agonists protected a significant population of neuronal cells against death.     

Sphingosine kinase type 1 inhibition reveals rapid turnover of circulating sphingosine 1-phosphate

S1P (sphingosine 1-phosphate) is a signalling molecule involved in a host of cellular and physiological functions, most notably cell survival and migration. S1P, which signals via a set of five G-protein-coupled receptors (S1P1-S1P5), is formed by the action of two SphKs (sphingosine kinases) from Sph (sphingosine). Interfering RNA strategies and SphK1 (sphingosine kinase type 1)-null (Sphk1-/-) mouse studies implicate SphK1 in multiple signalling cascades, yet there is a paucity of potent and selective SphK1 inhibitors necessary to evaluate the effects of rapid onset inhibition of this enzyme. We have identified a set of submicromolar amidine-based SphK1 inhibitors and report using a pair of these compounds to probe the cellular and physiological functions of SphK1. In so doing, we demonstrate that our inhibitors effectively lower S1P levels in cell-based assays, but we have been unable to correlate SphK1 inhibition with changes in cell survival. However, SphK1 inhibition did diminish EGF (epidermal growth factor)-driven increases in S1P levels and Akt (also known as protein kinase B)/ERK (extracellular-signal-regulated kinase) phosphorylation. Finally, administration of the SphK1 inhibitor to wild-type, but not Sphk1-/-, mice resulted in a rapid decrease in blood S1P levels indicating that circulating S1P is rapidly turned over.     

Regulation of IGF-1/PI3K/Akt signalling by the phosphoinositide phosphatase pharbin

Pharbin, a 5-phosphatase that induces arborization, is one of the phosphoinositide 5-phosphatases that is highly mutated in patients with Joubert syndrome. Pharbin can hydrolyse PI(4,5)P(2) and PI(3,4,5)P(3) and has the same substrate specificity as SHIP2 and SKIP, which negatively regulate PI3K signalling. Here, we investigated the role of pharbin in IGF-1/PI3K signalling. Ectopic expression of pharbin markedly suppressed the IGF-1-induced activation of Akt without affecting p42/44 MAP kinase phosphorylation. In contrast, pharbin silencing by RNA interference increased the IGF-1-induced phosphorylation of Akt, suggesting that pharbin negatively regulates PI3K/Akt signalling. Pharbin expression also inhibited the phosphorylation of p70 S6 kinase and 4E-BP1 as well as the subsequent protein synthesis in response to IGF-1 treatment. Taken together, these results indicate that pharbin is an important negative regulator of IGF-1/PI3K/Akt signalling and protein synthesis.     

Targeting the lipids LPA and S1P and their signalling pathways to inhibit tumour progression

The bioactive lipids lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), the enzymes that generate and degrade them, and the receptors that receive their signals are all potential therapeutic targets in cancer. LPA and S1P signalling pathways can modulate a range of cellular processes that contribute to tumourigenesis, such as proliferation and motility, and components of the signalling pathways often show aberrant expression and altered activity upon malignant transformation. This article reviews LPA- and S1P-mediated activities that might contribute to the aetiology of cancer, and examines the potential of the many antagonists that have been developed to inhibit LPA and S1P signalling pathways. In addition, the outcomes of various clinical trials using LPA- and S1P-associated targets in cancer and other diseases are described, and future directions are discussed.     

Sphingosine kinases, sphingosine 1-phosphate, apoptosis and diseases

Sphingolipids are ubiquitous components of cell membranes and their metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) have important physiological functions, including regulation of cell growth and survival. Cer and Sph are associated with growth arrest and apoptosis. Many stress stimuli increase levels of Cer and Sph, whereas suppression of apoptosis is associated with increased intracellular levels of S1P. In addition, extracellular/secreted S1P regulates cellular processes by binding to five specific G protein coupled-receptors (GPCRs). S1P is generated by phosphorylation of Sph catalyzed by two isoforms of sphingosine kinases (SphK), type 1 and type 2, which are critical regulators of the “sphingolipid rheostat”, producing pro-survival S1P and decreasing levels of pro-apoptotic Sph. Since sphingolipid metabolism is often dysregulated in many diseases, targeting SphKs is potentially clinically relevant. Here we review the growing recent literature on the regulation and the roles of SphKs and S1P in apoptosis and diseases.     

Sphingosine kinases, sphingosine 1-phosphate, apoptosis and diseases

Sphingolipids are ubiquitous components of cell membranes and their metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) have important physiological functions, including regulation of cell growth and survival. Cer and Sph are associated with growth arrest and apoptosis. Many stress stimuli increase levels of Cer and Sph, whereas suppression of apoptosis is associated with increased intracellular levels of S1P. In addition, extracellular/secreted S1P regulates cellular processes by binding to five specific G protein coupled-receptors (GPCRs). S1P is generated by phosphorylation of Sph catalyzed by two isoforms of sphingosine kinases (SphK), type 1 and type 2, which are critical regulators of the "sphingolipid rheostat", producing pro-survival S1P and decreasing levels of pro-apoptotic Sph. Since sphingolipid metabolism is often dysregulated in many diseases, targeting SphKs is potentially clinically relevant. Here we review the growing recent literature on the regulation and the roles of SphKs and S1P in apoptosis and diseases.     

Role of sphingosine kinases and lipid phosphate phosphatases in regulating spatial sphingosine 1-phosphate signalling in health and disease

Sphingosine 1-phosphate (S1P) is a bioactive lipid that is produced by the sphingosine kinase-catalysed phosphorylation of sphingosine. S1P is an important regulator of cell function, mediating many of its effects through a family of five closely related G protein-coupled receptors (GPCR) termed S1P(1-5) which exhibit high affinity for S1P. These receptors function to relay the effects of extracellular S1P via well-defined signal transduction networks linked to the regulation of cell proliferation, survival, migration etc. Diverse agonists (e.g. cytokines) also activate sphingosine kinase and the resulting S1P formed may bind to specific undefined intracellular targets to elicit cellular responses. The purpose of this review is to discuss some of the spatial/temporal aspects of intracellular S1P signalling and to define the function of sphingosine kinases and lipid phosphate phosphatases (which catalyse dephosphorylation of S1P) in terms of their regulation of cell function. Finally, we survey the function of S1P in relation to disease, where the major challenge is to dissect the role of intracellular versus extracellular actions of S1P in terms of association with defined diseased phenotypes.     

Sphingosine kinase signalling in immune cells: potential as novel therapeutic targets

During the last few years, it has become clear that sphingolipids are sources of important signalling molecules. Particularly, the sphingolipid metabolites, ceramide and S1P, have emerged as a new class of potent bioactive molecules, implicated in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation. Sphingomyelin (SM) is the major membrane sphingolipid and is the precursor for the bioactive products. Ceramide is formed from SM by the action of sphingomyelinases (SMase), however, ceramide can be very rapidly hydrolysed, by ceramidases to yield sphingosine, and sphingosine can be phosphorylated by sphingosine kinase (SphK) to yield S1P. In immune cells, the sphingolipid metabolism is tightly related to the main stages of immune cell development, differentiation, activation, and proliferation, transduced into physiological responses such as survival, calcium mobilization, cytoskeletal reorganization and chemotaxis. Several biological effectors have been shown to promote the synthesis of S1P, including growth factors, cytokines, and antigen and G-protein-coupled receptor agonists. Interest in S1P focused recently on two distinct cellular actions of this lipid, namely its function as an intracellular second messenger, capable of triggering calcium release from internal stores, and as an extracellular ligand activating specific G protein-coupled receptors. Inhibition of SphK stimulation strongly reduced or even prevented cellular events triggered by several proinflammatory agonists, such as receptor-stimulated DNA synthesis, Ca(2+) mobilization, degranulation, chemotaxis and cytokine production. Another very important observation is the direct role played by S1P in chemotaxis, and cellular escape from apoptosis. As an extracellular mediator, several studies have now shown that S1P binds a number of G-protein-coupled receptors (GPCR) encoded by endothelial differentiation genes (EDG), collectively known as the S1P-receptors. Binding of S1P to these receptors trigger an wide range of cellular responses including proliferation, enhanced extracellular matrix assembly, stimulation of adherent junctions, formation of actin stress fibres, and inhibition of apoptosis induced by either ceramide or growth factor withdrawal. Moreover, blocking S1P1-receptor inhibits lymphocyte egress from lymphatic organs. This review summarises the evidence linking SphK signalling pathway to immune-cell activation and based on these data discuss the potential for targeting SphKs to suppress inflammation and other pathological conditions.     

Sphingosine 1-phosphate lyase blockade elicits myogenic differentiation of murine myoblasts acting via Spns2/S1P2 receptor axis

The bioactive sphingolipid sphingosine 1-phosphate (S1P) has emerged in the last three decades as main regulator of key cellular processes including cell proliferation, survival, migration and differentiation. A crucial role for this sphingolipid has been recognized in skeletal muscle cell biology both in vitro and in vivo. S1P lyase (SPL) is responsible for the irreversible degradation of S1P and together with sphingosine kinases, the S1P producing enzymes, regulates cellular S1P levels. In this study is clearly showed that the blockade of SPL by pharmacological or RNA interference approaches induces myogenic differentiation of C2C12 myoblasts. Moreover, down-regulation of the specific S1P transporter spinster homolog 2 (Spns2) abrogates myogenic differentiation brought about by SPL inhibition or down-regulation, pointing at a role of extracellular S1P in the pro-myogenic action induced by SPL blockade. Furthermore, also S1P₂ receptor down-regulation was found to abrogate the pro-myogenic effect of SPL blockade. These results provide further proof that inside-out S1P signaling is critically implicated in skeletal muscle biology and provide support to the concept that the specific targeting of SPL could represent an exploitable strategy to treat skeletal muscle disorders.