Sphingosylphosphorylcholine-biological functions and mechanisms of action

Compared to the lysophospholipid mediators, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), little information is available regarding the molecular mechanisms of action, metabolism and physiological significance of the related sphingosylphosphorylcholine (SPC). S1P and LPA have recently been established as agonists at several G-protein-coupled receptors of the EDG family, S1P additionally serves an intracellular second messenger function. Several cellular effects of SPC can be explained by low-affinity binding to and activation of S1P-EDG receptors. However, certain cellular and subcellular actions of SPC are not shared by S1P, suggesting that SPC, which has been identified in normal blood plasma, ascites and various tissues, is a lipid mediator in its own right. This concept was corroborated by the recent discovery of specific high-affinity G-protein-coupled SPC receptors. In this article, our present knowledge on cellular actions and biological functions of SPC will be reviewed.     

Sphingosine 1-phosphate is a key metabolite linking sphingolipids to glycerophospholipids

The sphingolipid metabolite sphingosine 1-phosphate (S1P) is a well-known lipid mediator. As a lipid mediator, S1P must be present in extracellular space and bind to its cell surface receptors (S1P_1–5). However, most S1P, synthesized intracellularly, is metabolized without being released into extracellular space, in other words, without functioning as a lipid mediator in the vast majority of cells except those supplying plasma and lymph S1P such as blood cells and endothelial cells. Instead, intracellular S1P plays an important role as an intermediate of the sole sphingolipid-to-glycerophospholipid metabolic pathway. The degradation of S1P by S1P lyase is the first irreversible reaction (committed step) of this pathway. This metabolic pathway is conserved in eukaryotes from yeast to human, indicating its much older origin than the function of S1P as a lipid mediator, which is found to be present only in vertebrates and chordates. The sphingolipid-to-glycerophospholipid metabolism takes place ubiquitously in mammalian tissues, and its defect causes an aberration of several tissue functions as well as abnormal lipid metabolism. Although this metabolic pathway has been known for over four decades, only recently the precise reactions and enzymes involved in this pathway have been revealed. This review will focus on the recent advances in our understanding of the sphingolipid metabolic pathway via S1P and its physiological and pathological roles. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Regulation of ryanodine receptors by sphingosylphosphorylcholine: Involvement of both calmodulin-dependent and -independent mechanisms

Sphingosylphosphorylcholine (SPC), a lipid mediator with putative second messenger functions, has been reported to regulate ryanodine receptors (RyRs), Ca²⁺ channels of the sarco/endoplasmic reticulum. RyRs are also regulated by the ubiquitous Ca²⁺ sensor calmodulin (CaM), and we have previously shown that SPC disrupts the complex of CaM and the peptide corresponding to the CaM-binding domain of the skeletal muscle Ca²⁺ release channel (RyR1). Here we report that SPC also displaces Ca²⁺-bound CaM from the intact RyR1, which we hypothesized might lead to channel activation by relieving the negative feedback Ca²⁺CaM exerts on the channel. We could not demonstrate such channel activation as we have found that SPC has a direct, CaM-independent inhibitory effect on channel activity, confirmed by both single channel measurements and [³H]ryanodine binding assays. In the presence of Ca²⁺CaM, however, the addition of SPC did not reduce [³H]ryanodine binding, which we could explain by assuming that the direct inhibitory action of the sphingolipid was negated by the simultaneous displacement of inhibitory Ca²⁺CaM. Additional experiments revealed that RyRs are unlikely to be responsible for SPC-elicited Ca²⁺ release from brain microsomes, and that SPC does not exert detergent-like effects on sarcoplasmic reticulum vesicles. We conclude that regulation of RyRs by SPC involves both CaM-dependent and -independent mechanisms, thus, the sphingolipid might play a physiological role in RyR regulation, but channel activation previously attributed to SPC is unlikely.     

Sphingosylphosphorylcholine and lysophosphatidylcholine: G protein-coupled receptors and receptor-mediated signal transduction

In recent years, certain lysophospholipids (lyso-PLs) have been recognized as important cell signaling molecules. Among them, two phosphorylcholine-containing lyso-PLs, sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC), have been shown to be involved in many cellular processes and are produced under physiological and pathological conditions. Although signaling properties of SPC and LPC have been studied in a variety of cellular systems, specific cell membrane receptors for SPC and LPC have not been identified previously. Recently, ovarian cancer G protein-coupled receptor 1 (OGR1, also known as GPR68), G protein-coupled receptor 4 (GPR4), and G2A have been identified as receptors for SPC and LPC. The signaling and ligand-binding properties of these receptors are reviewed here. These discoveries provide an intriguing opportunity and a novel approach in studying the pathophysiological roles of SPC and LPC and their receptors.     

Dissociation of Calmodulin-Target Peptide Complexes by the Lipid Mediator Sphingosylphosphorylcholine: IMPLICATIONS IN CALCIUM SIGNALING*

Previously we have identified the lipid mediator sphingosylphosphorylcholine (SPC) as the first potentially endogenous inhibitor of the ubiquitous Ca²⁺ sensor calmodulin (CaM) (Kovacs, E., and Liliom, K. (2008) Biochem. J. 410, 427–437). Here we give mechanistic insight into CaM inhibition by SPC, based on fluorescence stopped-flow studies with the model CaM-binding domain melittin. We demonstrate that both the peptide and SPC micelles bind to CaM in a rapid and reversible manner with comparable affinities. Furthermore, we present kinetic evidence that both species compete for the same target site on CaM, and thus SPC can be considered as a competitive inhibitor of CaM-target peptide interactions. We also show that SPC disrupts the complex of CaM and the CaM-binding domain of ryanodine receptor type 1, inositol 1,4,5-trisphosphate receptor type 1, and the plasma membrane Ca²⁺ pump. By interfering with these interactions, thus inhibiting the negative feedback that CaM has on Ca²⁺ signaling, we hypothesize that SPC could lead to Ca²⁺ mobilization in vivo. Hence, we suggest that the action of the sphingolipid on CaM might explain the previously recognized phenomenon that SPC liberates Ca²⁺ from intracellular stores. Moreover, we demonstrate that unlike traditional synthetic CaM inhibitors, SPC disrupts the complex between not only the Ca²⁺-saturated but also the apo form of the protein and the target peptide, suggesting a completely novel regulation for target proteins that constitutively bind CaM, such as ryanodine receptors.     

Rapid glucocorticoid effects on immune cells

Apart from their classic genomic effects, it is well known that glucocorticoids also have rapid, nongenomically mediated effects. Three different mechanisms are currently under discussion as being responsible for these effects: (1) specific interaction with the cytosolic glucocorticoid receptor (cGCR), (2) nonspecific interactions with cellular membranes and (3) specific interactions with membrane-bound glucocorticoid receptors (mGCR). With regard to the first mechanism, there is evidence that although the binding of glucocorticoids to the cGCR-associated multi-protein complex induces the further processes of the classic path, it also leads to a rapid intracellular signalling through other components of the complex (e.g. Src). For the second mechanism, a nonspecific interactive effect with cellular membranes through the intercalation of glucocorticoid molecules is being discussed, which primarily alters cellular functions by influencing cation transport via the plasma membrane and by increasing the proton leak of the mitochondria. With regard to the third, mGCR-mediated mechanism, the first evidence has now been found to suggest a physiological expression of membrane-bound glucocorticoid receptors on human cells, whereas in humans this had previously only been demonstrated on lymphoma cells. The clinical importance and therapeutic relevance of these rapid glucocorticoid effects remains unclear at present, although effects on intracellular signalling, interferences with bioenergetically relevant cell functions and the induction of apoptosis via the mGCR are being discussed. This article gives a detailed presentation of the data available at present concerning rapid glucocorticoid effects on immune cells.     

Novel effects of sphingosylphosphorylcholine on the apoptosis of breast cancer via autophagy/AKT/p38 and JNK signaling

Sphingosylphosphorylcholine (SPC), an important lipid mediator in blood, inhibits the proliferation and migration of various cancer cells. However, its effect as a cell-specific sphingolipid in breast cancer cells is still unknown. Here, we showed that SPC promoted autophagy and apoptosis in triple-negative breast cancer MDA-MB-231 cells. Autophagy worked as a negative regulator of apoptosis-induced by SPC. Mechanistically, SPC mediated apoptosis via activating c-Jun N-terminal kinase (JNK). Meanwhile, p38MAPK (p38) and protein kinase B (PKB or AKT) signaling pathways were also activated to inhibit apoptosis, suggesting that SPC could evoke multiple signaling pathways to modulate cell apoptosis. In addition, the crosstalk between autophagy, p38, AKT and JNK is that autophagy, p38, and AKT attenuated the JNK. AKT and p38 were in the downstream of autophagy, which is autophagy/AKT/p38 signaling evoked by SPC to antagonize JNK signaling and subsequent apoptosis. Although the pathways that antagonize apoptosis were evoked, the cells eventually reached apoptosis by SPC. Therefore, the combination with pharmacological autophagy inhibitors would be a more effective therapeutic strategy for eliminating breast cancer cells by SPC.     

The bioactive lipid sphingosylphosphorylcholine induces differentiation of mouse embryonic stem cells and human promyelocytic leukaemia cells

Sphingosylphosphorylcholine (SPC) is the major component of high-density lipoproteins (HDL) in blood plasma. The bioactive lipid acts mainly via G protein coupled receptors (GPCRs). Similar to ligands of other GPCRs, SPC has multiple biological roles including the regulation of proliferation, migration, angiogenesis, wound healing and heart rate. Lysophospholipids and their receptors have also been implicated in cell differentiation. A potential role of SPC in stem cell or tumour cell differentiation has been elusive so far. Here we examined the effect of SPC on the differentiation of mouse embryonic stem (ES) cells and of human NB4 promyelocytic leukemia cells, a well established tumour differentiation model. Our data show that mouse embryonic stem cells and NB4 cells express the relevant GPCRs for SPC. We demonstrate both at the level of morphology and of gene expression that SPC induces neuronal and cardiac differentiation of mouse ES cells. Furthermore, SPC induces differentiation of NB4 cells by a mechanism which is critically dependent on the activity of the MEK-ERK cascade. Thus, the bioactive lipid SPC is a novel differentiation inducing agent both for mouse ES cells, but also of certain human tumour cells.     

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.     

Vesicular and conductive mechanisms of nucleotide release

Extracellular nucleotides and nucleosides promote a vast range of physiological responses, via activation of cell surface purinergic receptors. Virtually all tissues and cell types exhibit regulated release of ATP, which, in many cases, is accompanied by the release of uridine nucleotides. Given the relevance of extracellular nucleotide/nucleoside-evoked responses, understanding how ATP and other nucleotides are released from cells is an important physiological question. By facilitating the entry of cytosolic nucleotides into the secretory pathway, recently identified vesicular nucleotide and nucleotide-sugar transporters contribute to the exocytotic release of ATP and UDP-sugars not only from endocrine/exocrine tissues, but also from cell types in which secretory granules have not been biochemically characterized. In addition, plasma membrane connexin hemichannels, pannexin channels, and less-well molecularly defined ATP conducting anion channels have been shown to contribute to the release of ATP (and UTP) under a variety of conditions.     

Ceramide/sphingosine/sphingosine 1-phosphate metabolism on the cell surface and in the extracellular space

Sphingolipid metabolites, ceramide, sphingosine, and sphingosine 1-phosphate, have emerged as a new class of lipid biomodulators of various cell functions. These metabolites are known to function not only as intracellular second messengers, but also in the extracellular space. Sphingosine 1-phosphate especially has numerous functions as an important extracellular mediator that binds to cell surface S1P receptors. Recent studies have also shown that sphingolipid-metabolizing enzymes function not only in intracellular organelles but also in the extracellular spaces, including the outer leaflet of the plasma membrane. This review focuses on the metabolic enzymes (acid and alkaline sphingomyelinases, neutral ceramidase, and sphingosine kinase) that are involved in the production of the sphingolipid metabolites in these extracellular spaces, and on the metabolic pathway itself.     

Role of sphingomyelinase and ceramide in modulating rafts: do biophysical properties determine biologic outcome?

Recent biophysical data suggest that the properties of ceramide observed in model membranes may apply to biological systems. In particular, the ability of ceramide to form microdomains, which coalesce into larger platforms or macrodomains, appears to be important for some cellular signaling processes. Several laboratories have now demonstrated similar reorganization of plasma membrane sphingolipid rafts, via ceramide generation, into macrodomains. This event appeared necessary for signaling upon activation of a specific set of cell surface receptors. In this article, we review the properties and functions of rafts, and the role of sphingomyelinase and ceramide in the biogenesis and re-modeling of these rafts. As clustering of some cell surface receptors in these domains may be critical for signal transduction, we propose a new model for transmembrane signal transmission.     

Brave little yeast, please guide us to thebes: sphingolipid function in S. cerevisiae

Sphingolipids typically cover the exoplasmic leaflet of the plasma membrane of eukaryotic cells. They differ from the more abundant glycerophospholipids in that they contain ceramide instead of diacylglycerol as a hydrophobic anchor. Why did nature choose to invent this complex class of lipids, and why do eukaryotic cells follow elaborate remodelling pathways in order to generate dozens to hundreds of different molecular species of sphingolipid, depending on cell type? Yeast may, once again, serve as a model to dissect sphingolipid function at various levels. Almost the complete pathway for sphingolipid synthesis in yeast has been uncovered during the past two decades. More recently, key enzymes in sphingolipid degradation and signalling have been identified. Together with a wealth of genetic data obtained from the characterization of various suppressor mutants, this information now allows for an unprecedented analysis of sphingolipid function in this organism. This overview summarizes recent data on sphingolipid function in cell signalling, their role in the heat-stress response and Ca(2+) homeostasis, and addresses their function in transport of glycosylphosphatidylinositol-anchored proteins.     

Sphingosine-1-phosphate: signaling inside and out

Ample evidence indicates that sphingosine-1-phosphate (SPP) can serve as an intracellular second messenger regulating calcium mobilization, and cell growth and survival. Moreover, the dynamic balance between levels of the sphingolipid metabolites, ceramide and SPP, and consequent regulation of opposing signaling pathways, is an important factor that determines whether a cell survives or dies. SPP has recently also been shown to be the ligand for the EDG-1 family of G-protein-coupled receptors, which now includes EDG-1, -3, -5, -6 and -8. SPP is thus a lipid mediator that has novel dual actions signaling inside and outside of the cell.     

S1P-lyase deficiency uncouples ganglioside formation – Potential contribution to tumorigenic capacity

Sphingosine-1-phosphate (S1P) is not only a catabolic intermediate of all sphingolipids but also an evolutionary conserved bioactive lipid with critical functions in cell survival, differentiation, and migration as well as in immunity and angiogenesis. S1P-lyase (SGPL1) irreversibly cleaves S1P in the final step of sphingolipid catabolism. As sphingoid bases and their 1-phosphates are not only metabolic intermediates but also highly bioactive lipids that modulate a wide range of physiological processes, it would be predicted that their elevation might induce adjustments in other facets of sphingolipid metabolism and/or alter cell behavior. We actually found in a previous study that in terminally differentiated neurons SGPL1 deficiency increases sphingolipid formation via recycling at the expense of de novo synthesis. We now investigated whether and how SGPL1 deficiency affects the metabolism of (glyco)sphingolipids in mouse embryonic fibroblasts (MEFs). According to our previous experiments in neurons, we found a strong accumulation of S1P in SGPL1-deficient MEFs. Surprisingly, a completely different situation arose as we analyzed sphingolipid metabolism in this non-differentiated cell type. The production of biosynthetic precursors of complex glycosphingolipids including ceramide, glucosylceramide and also ganglioside GM3 via de novo synthesis and recycling pathway was substantially increased whereas the amount of more complex gangliosides dropped significantly.     

Plasma Lysosphingomyelin Demonstrates Great Potential as a Diagnostic Biomarker for Niemann-Pick Disease Type C in a Retrospective Study

Niemann-Pick disease type C (NP-C) is a devastating, neurovisceral lysosomal storage disorder which is characterised by variable manifestation of visceral signs, progressive neuropsychiatric deterioration and premature death, caused by mutations in the NPC1 and NPC2 genes. Due to the complexity of diagnosis and the availability of an approved therapy in the EU, improved detection of NP-C may have a huge impact on future disease management. At the cellular level dysfunction or deficiency of either the NPC1 or NPC2 protein leads to a complex intracellular endosomal/lysosomal trafficking defect, and organ specific patterns of sphingolipid accumulation. Lysosphingolipids have been shown to be excellent biomarkers of sphingolipidosis in several enzyme deficient lysosomal storage disorders. Additionally, in a recent study the lysosphingolipids, lysosphingomyelin (SPC) and glucosylsphingosine (GlcSph), appeared to be elevated in the plasma of three adult NP-C patients. In order to investigate the clinical utility of SPC and GlcSph as diagnostic markers, an in-depth fit for purpose biomarker assay validation for measurement of these biomarkers in plasma by liquid chromatography-tandem mass spectrometry was performed. Plasma SPC and GlcSph are stable and can be measured accurately, precisely and reproducibly. In a retrospective analysis of 57 NP-C patients and 70 control subjects, median plasma SPC and GlcSph were significantly elevated in NP-C by 2.8-fold and 1.4-fold respectively. For miglustat-naïve NP-C patients, aged 2–50 years, the area under the ROC curve was 0.999 for SPC and 0.776 for GlcSph. Plasma GlcSph did not correlate with SPC levels in NP-C patients. The data indicate excellent potential for the use of lysosphingomyelin in NP-C diagnosis, where it could be used to identify NP-C patients for confirmatory genetic testing.     

Sphingosylphosphorylcholine and lysosulfatide have inverse regulatory functions in monocytic cell differentiation into macrophages

Sphingolipids act as signaling mediators that regulate a diverse range of cellular events. Although numerous sphingolipid functions have been studied, little is known about the effect of sphingolipids on monocyte differentiation into macrophages. Here, we report that two lysosphingolipids, sphingosylphosphorylcholine (SPC) and lysosulfatide (LSF), inversely affect macrophagic differentiation of monocytic cell lines, U937 and THP-1. Molecular analyses revealed that SPC enhances, whereas LSF suppresses, phorbol ester-induced classical (M1-polarized) differentiation to macrophages. The expression of CD11b, a macrophage marker, was induced in accordance with the activation status of the Raf/MEK/ERK signaling pathway in which SPC and LSF had opposite effects. Pharmacological inhibition of this pathway aborted the differentiation, indicating that this signaling pathway is required. Consistently, SPC promoted, while LSF inhibited, monocyte adhesion to fibronectin, through the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. The effects of SPC on Raf/MEK/ERK and PI3K/Akt signaling were dependent on G_i/o, whereas the SPC-induced calcium influx was dependent on G_q. Thus SPC utilizes G-protein coupled receptor. In contrast, the effects of LSF were independent of G_i/o and G_q. These results suggest that SPC enhances, whereas LSF suppresses, monocyte differentiation into macrophages through regulating the Raf/MEK/ERK and PI3K/Akt signaling pathways via distinct mechanisms.     

Sphingosine 1-phosphate as a novel immune regulator of dendritic cells

Although originally described as an intracellular second messenger, sphingosine 1-phosphate (S1P) has recently been shown to be involved in several physiological and pathological functions as an extracellular mediator. S1P receptors are widely expressed and thought to regulate important functions in cell signalling. Recently, the role of S1P on the immune system has evoked great interest. In particular, several aspects of the effects on antigen-presenting cells (APCs) as dendritic cells (DC) in mice and humans have been reported. In this review, we focus on the role played by S1P on the DC system and its effects in immune-related pathological states.     

Sphingosine 1-phosphate as a novel immune regulator of dendritic cells

Although originally described as an intracellular second messenger, sphingosine 1-phosphate (S1P) has recently been shown to be involved in several physiological and pathological functions as an extracellular mediator. S1P receptors are widely expressed and thought to regulate important functions in cell signalling. Recently, the role of S1P on the immune system has evoked great interest. In particular, several aspects of the effects on antigen-presenting cells (APCs) as dendritic cells (DC) in mice and humans have been reported. In this review, we focus on the role played by S1P on the DC system and its effects in immune-related pathological states.     

Effect of human chorionic gonadotropin derivatives on leydig cell function.

BACKGROUND: Several human chorionic gonadotropin (hCG) derivatives have been detected in healthy human subjects, indicating that they may play a role in cell function. These hCG derivatives include deglycosylated hCG, proteolytic digestion products of hCG and free alpha and beta subunits of the hormone. It is well documented that testicular Leydig cells are responsive to luteinising hormone (LH) or its analogue hCG. These hormones have high affinity for LH/hCG receptors on the plasma membrane. METHODS: We designed functional and binding studies to compare the effects of native hCG and several hCG derivatives on a rat Leydig cell system. The molecular weight of the hCG derivatives was determined by SDS-PAGE and the binding affinity to LH/hCG receptors was measured by a radioligand assay. In addition, their ability to produce testosterone, cyclic AMP and arachidonic acid release was also studied. RESULTS: These hCG derivatives, with the exception of the free beta subunit, were able to bind to LH/hCG plasma membrane receptors with different affinities than that of native hCG. In addition, hCG derivatives did not increase intracellular cAMP levels or arachidonic acid release. However, they did increase testosterone production. CONCLUSION: Taken together, the results of this study lead us to suggest that these hCG derivatives may regulate the action of the native hormone in Leydig cells and are, thus, molecules of physiological relevance.