Export and functions of sphingosine-1-phosphate

The sphingolipid metabolite, sphingosine-1-phosphate (S1P), has emerged as a critical player in a number of fundamental biological processes and is important in cancer, angiogenesis, wound healing, cardiovascular function, atherosclerosis, immunity and asthma, among others. Activation of sphingosine kinases, enzymes that catalyze the phosphorylation of sphingosine to S1P, by a variety of agonists, including growth factors, cytokines, hormones, and antigen, increases intracellular S1P. Many of the biological effects of S1P are mediated by its binding to five specific G protein-coupled receptors located on the cell surface in an autocrine and/or paracrine manner. Therefore, understanding the mechanism by which intracellularly generated S1P is released out of cells is both interesting and important. In this review, we will discuss how S1P is formed and released. We will focus particularly on the current knowledge of how the S1P gradient between tissues and blood is maintained, and the role of ABC transporters in S1P release.     

Silencing of the MT1-MMP/ G6PT axis suppresses calcium mobilization by sphingosine-1-phosphate in glioblastoma cells

The contributions of membrane type-1 matrix metalloproteinase (MT1-MMP) and of the glucose-6-phosphate transporter (G6PT) in sphingosine-1-phosphate (S1P)-mediated Ca(2+) mobilization were assessed in glioblastoma cells. We show that gene silencing of MT1-MMP or G6PT decreased the extent of S1P-induced Ca(2+) mobilization, chemotaxis, and extracellular signal-related kinase phosphorylation. Chlorogenic acid and (-)-epigallocatechin-3-gallate, two diet-derived inhibitors of G6PT and of MT1-MMP, respectively, reduced S1P-mediated Ca(2+) mobilization. An intact MT1-MMP/G6PT signaling axis is thus required for efficient Ca(2+) mobilization in response to bioactive lipids such as S1P. Targeted inhibition of either MT1-MMP or G6PT may lead to reduced infiltrative and invasive properties of brain tumor cells.     

Sphingosine 1-phosphate induces Mcl-1 upregulation and protects multiple myeloma cells against apoptosis

Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. However, neither the distribution of S1P receptors nor the effects of S1P on multiple myeloma (MM) cells are fully understood. Here, we show that MM cells express the S1P receptors, S1P1, S1P2, and S1P3. Furthermore, S1P protects MM cells against Dex-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time- and concentration-dependent manner in human MM cell lines. Treatment of MM cells with pertussis toxin (PTX), a pan-S1P receptor inhibitor, results in blockage of S1P-induced upregulation of Mcl-1. These data demonstrate that S1P upregulates the expression of Mcl-1 and protects MM cells from Dex-induced apoptosis, providing the preclinical framework for novel therapeutics targeting at both Mcl-1 and/or S1P to improve the patient outcome in MM.     

Implication of sphingosine-1-phosphate signaling in diseases: molecular mechanism and therapeutic strategies

Sphingosine-1-phosphate signaling is emerging as a critical regulator of cellular processes that is initiated by the intracellular production of bioactive lipid molecule, sphingosine-1-phosphate. Binding of sphingosine-1-phosphate to its extracellular receptors activates diverse downstream signaling that play a critical role in governing physiological processes. Increasing evidence suggests that this signaling pathway often gets impaired during pathophysiological and diseased conditions and hence manipulation of this signaling pathway may be beneficial in providing treatment. In this review, we summarized the recent findings of S1P signaling pathway and the versatile role of the participating candidates in context with several disease conditions. Finally, we discussed its possible role as a novel drug target in different diseases.     

Sphingosine-1-phosphate signaling regulates lamellipodia localization of cortactin complexes in endothelial cells

Sphingosine-1-phosphate (S1P), a serum-borne lipid mediator, was demonstrated to be a potent chemoattractant of endothelial cells. It was recently shown that the colocalization of cortactin and actin related protein 2/3 (Arp2/3) in the lamellipodia is critical to S1P-induced endothelial chemotaxis. In this report, we describe that S1P-stimulated cortactin translocation to the cell periphery to form lamellipodia is specifically mediated by the endothelial S1P1 G-protein coupled receptor, and is regulated by G_i-mediated Akt-dependent S1P1 receptor phosphorylation and Cdc42/Rac activation pathways. In contrast to Src-dependent fibroblast growth factor-induced cortactin translocation, tyrosine phosphorylation cascades are not required for S1P-mediated lamellipodia formation and chemotaxis. Furthermore, we also demonstrate that S1P signaling, via the G_i/Akt/S1P1 phosphorylation/Rac pathway, regulates the cortactin–Arp2/3 complex formation, which ultimately results in membrane ruffling, formation of the lamellipodia and endothelial migration.J.F. Lee and H. Ozaki contributed equally to this work     

Isoforms of protein kinase C involved in phorbol ester-induced sphingosine 1-phosphate receptor 1 phosphorylation and desensitization

The role of protein kinase C (PKC) isozymes in phorbol myristate acetate (PMA)-induced sphingosine 1-phosphate (S1P) receptor 1 (S1P₁) phosphorylation was studied. Activation of S1P₁ receptors induced an immediate increase in intracellular calcium, which was blocked by preincubation with PMA. Both S1P and PMA were able to increase S1P₁ phosphorylation in a concentration- and time-dependent fashion. Down-regulation of PKC (overnight incubation with PMA) blocked the subsequent effect of the phorbol ester on S1P₁ phosphorylation, without decreasing that of the natural agonist. Pharmacological inhibition of PKC α prevented the effects of PMA on S1P-triggered intracellular calcium increase and on S1P₁ phosphorylation; no such effect was observed on the effects of the sphingolipid agonist. The presence of PKC α and β isoforms in S1P₁ immunoprecipitates was evidenced by Western blotting. Additionally, expression of dominant-negative mutants of PKC α or β and knockdown of these isozymes using short hairpin RNA, markedly attenuated PMA-induced S1P₁ phosphorylation. Our results indicate that the classical isoforms, mainly PKC α, mediate PMA-induced phosphorylation and desensitization of S1P₁.     

Identification of the orphan GPCR, P2Y(10) receptor as the sphingosine-1-phosphate and lysophosphatidic acid receptor

Phylogenetic analysis of transmembrane regions of GPCRs using PHYLIP indicated that the orphan receptor P2Y₁₀ 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 P2Y₁₀ receptors. As a result, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) evoked intracellular Ca²⁺ increases in the CHO cells stably expressing the P2Y₁₀ fused with a G_16α protein. These Ca²⁺ responses were inhibited by S1P receptor and LPA receptor antagonists. The introduction of siRNA designed for P2Y₁₀ receptor into the P2Y₁₀-CHO cells effectively blocked both S1P- and LPA-induced Ca²⁺ increases. RT-PCR analysis showed that the mouse P2Y₁₀ was expressed in reproductive organs, brain, lung and skeletal muscle, suggesting the receptor plays physiological roles throughout the whole body. In conclusion, the P2Y₁₀ receptor is the first receptor identified as a dual lysophospholipid receptor.     

Lentiviral siRNA silencing of sphingosine-1-phosphate receptors S1P1 and S1P2 in smooth muscle

Sphingosine-1-phosphate regulates diverse biological processes through five receptor types, S1P(1-5). Two or more S1P receptors are usually co-expressed on target cells. We have previously shown that smooth muscle cells of the gut co-express S1P(1) and S1P(2) receptors that could mediate distinct functions. In the absence of selective agonists and antagonists, we developed siRNA constructs to silence each receptor separately. The constructs were based on identical sequences in several mammalian species. A lentiviral vector-based system was used to deliver siRNA into HEK293T cells and smooth muscle cells. One S1P(1) and two S1P(2) siRNA constructs specifically inhibited ectopic expression of S1P(1) and S1P(2) receptors, respectively, as determined by immunocytochemistry and Western blot, and endogenous expression of S1P(1) and S1P(2) receptors in smooth muscle cells, as determined by RT-PCR. Measurement of PLC-beta and Rho kinase activities, which mediate initial and sustained muscle contraction, confirmed receptor silencing and showed that contraction is mediated exclusively by S1P(2) receptors.     

The role of glycerol-3-phosphate dehydrogenase 1 in the progression of fatty liver after acute ethanol administration in mice

Acute ethanol consumption leads to the accumulation of triglycerides (TGs) in hepatocytes. The increase in lipogenesis and reduction of fatty acid oxidation are implicated as the mechanisms underlying ethanol-induced hepatic TG accumulation. Although glycerol-3-phosphate (Gro3P), formed by glycerol kinase (GYK) or glycerol-3-phosphate dehydrogenase 1 (GPD1), is also required for TG synthesis, the roles of GYK and GPD1 have been the subject of some debate. In this study, we examine (1) the expression of genes involved in Gro3P production in the liver of C57BL/6J mice in the context of hepatic TG accumulation after acute ethanol intake, and (2) the role of GPD1 in the progression of ethanol-induced fatty liver using GPD1 null mice. As a result, in C57BL/6J mice, ethanol-induced hepatic TG accumulation began within 2 h and was 1.7-fold greater than that observed in the control group after 6 h. The up-regulation of GPD1 began 2 h after administering ethanol, and significantly increased 6 h later with the concomitant escalation in the glycolytic gene expression. The incorporation of ¹⁴C-labelled glucose into TG glycerol moieties increased during the same period. On the other hand, in GPD1 null mice carrying normal GYK activity, no significant increase in hepatic TG level was observed after acute ethanol intake. In conclusion, GPD1 and glycolytic gene expression is up-regulated by ethanol, and GPD1-mediated incorporation of glucose into TG glycerol moieties together with increased lipogenesis, is suggested to play an important role in ethanol-induced hepatic TG accumulation.     

Differential involvement of ezrin/radixin/moesin proteins in sphingosine 1-phosphate-induced human pulmonary endothelial cell barrier enhancement

Endothelial cell (EC) barrier dysfunction induced by inflammatory agonists is a frequent pathophysiologic event in multiple diseases. The platelet-derived phospholipid sphingosine-1 phosphate (S1P) reverses this dysfunction by potently enhancing the EC barrier through a process involving Rac GTPase-dependent cortical actin rearrangement as an integral step. In this study we explored the role of the ezrin, radixin, and moesin (ERM) family of actin-binding linker protein in modulating S1P-induced human pulmonary EC barrier enhancement. S1P induces ERM translocation to the EC periphery and promotes ERM phosphorylation on a critical threonine residue (Ezrin-567, Radixin-564, Moesin-558). This phosphorylation is dependent on activation of PKC isoforms and Rac1. The majority of ERM phosphorylation on these critical threonine residues after S1P occurs in moesin and ezrin. Baseline radixin phosphorylation is higher than in the other two ERM proteins but does not increase after S1P. S1P-induced moesin and ezrin threonine phosphorylation is not mediated by the barrier enhancing receptor S1PR1 because siRNA downregulation of S1PR1 fails to inhibit these phosphorylation events, while stimulation of EC with the S1PR1-specific agonist SEW2871 fails to induce these phosphorylation events. Silencing of either all ERM proteins or radixin alone (but not moesin alone) reduced S1P-induced Rac1 activation and phosphorylation of the downstream Rac1 effector PAK1. Radixin siRNA alone, or combined siRNA for all three ERM proteins, dramatically attenuates S1P-induced EC barrier enhancement (measured by transendothelial electrical resistance (TER), peripheral accumulation of di-phospho-MLC, and cortical cytoskeletal rearrangement. In contrast, moesin depletion has the opposite effects on these parameters. Ezrin silencing partially attenuates S1P-induced EC barrier enhancement and cytoskeletal changes. Thus, despite structural similarities and reported functional redundancy, the ERM proteins differentially modulate S1P-induced alterations in lung EC cytoskeleton and permeability. These results suggest that ERM activation is an important regulatory event in EC barrier responses to S1P.     

The Na(+)/H(+) exchanger regulatory factor 2 mediates phosphorylation of serum- and glucocorticoid-induced protein kinase 1 by 3-phosphoinositide-dependent protein kinase 1

The Na(+)/H(+) exchanger regulatory factor 2 (NHERF2/TKA-1/E3KARP) contains two PSD-95/Dlg/ZO-1 (PDZ) domains which interact with the PDZ docking motif (X-(S/T)-X-(V/L)) of proteins to mediate the assembly of transmembrane and cytosolic proteins into functional signal transduction complexes. One of the PDZ domains of NHERF2 interacts specifically with the DSLL, DSFL, and DTRL motifs present at the carboxy-termini of the 2-adrenergic receptor, the platelet-derived growth factor receptor, and the cystic fibrosis transmembrane conductance regulator, respectively. Serum- and glucocorticoid-induced protein kinase 1 (SGK1) also carries a putative PDZ-binding motif (D-S-F-L) at its carboxy tail, implicated in the specific interaction with NHERF2. There is a 3-phosphoinositide-dependent protein kinase 1 (PDK1) interacting fragment (PIF) in the tail of NHERF2. Using pull-down assays and co-transfection experiments, we demonstrated that the DSFL tail of SGK1 interacts with the first PDZ domain of NHERF2 and the PIF of NHERF2 binds to the PIF-binding pocket of PDK1 to form an SGK1-NHERF2-PDK1 complex. Formation of the protein complex promoted the phosphorylation and activation of SGK1 by PDK1. Thus, it was suggested that NHERF2 mediates the activation and phosphorylation of SGK1 by PDK1 through its first PDZ domain and PIF motif, as a novel SGK1 activation mechanism.     

The role of agrin in synaptic development,plasticity and signaling in the central nervous system

Development of the neuromuscular junction (NMJ) requires secretion of specific isoforms of the proteoglycan agrin by motor neurons. Secreted agrin is widely expressed in the basal lamina of various tissues, whereas a transmembrane form is highly expressed in the brain. Expression in the brain is greatest during the period of synaptogenesis, but remains high in regions of the adult brain that show extensive synaptic plasticity. The well-established role of agrin in NMJ development and its presence in the brain elicited investigations of its possible role in synaptogenesis in the brain. Initial studies on the embryonic brain and neuronal cultures of agrin-null mice did not reveal any defects in synaptogenesis. However, subsequent studies in culture demonstrated inhibition of synaptogenesis by agrin antisense oligonucleotides or agrin siRNA. More recently, a substantial loss of excitatory synapses was found in the brains of transgenic adult mice that lacked agrin expression everywhere but in motor neurons. The mechanisms by which agrin influences synapse formation, maintenance and plasticity may include enhancement of excitatory synaptic signaling, activation of the “muscle-specific” receptor tyrosine kinase (MuSK) and positive regulation of dendritic filopodia. In this article I will review the evidence that agrin regulates synapse development, plasticity and signaling in the brain and discuss the evidence for the proposed mechanisms.     

Sustained intracellular acidosis activates the myocardial Na/H exchanger independent of amino acid Ser and p90

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the myocardium and other tissues. It is an important mediator of the myocardial damage that occurs after ischemia-reperfusion injury and is implicated in heart hypertrophy. Regulation of NHE1 has been proposed as a therapeutic target for cardioprotection. We therefore examined mechanisms of control of NHE1 in the myocardium. Several different amino acids have been implicated as a being critical to NHE1 regulation in a number of tissues including Ser⁷⁰³, Ser⁷⁷⁰, and Ser⁷⁷¹. In the myocardium, NHE1 is activated in response to a variety of stimuli including activation by an ERK-dependent sustained intracellular acidosis. In this study, we determined whether Ser⁷⁰³ and p90^rsk activity are critical in activation of NHE1 by sustained intracellular acidosis. In vitro phosphorylation of NHE1 C-terminal fusion proteins determined that ERK-dependent phosphorylation of the cytoplasmic region was not dependent on Ser⁷⁰³; however, phosphorylation by p90^rsk required Ser⁷⁰³. A Ser703Ala mutation decreased basal NHE1 activity in CHO cells but not in cardiomyocytes. NHE1 with a Ser703Ala mutation was activated in response to sustained intracellular acidosis in CHO cells. In addition, sustained intracellular acidosis also activated the Ser703Ala mutant protein in isolated cardiomyocytes and phosphorylation levels were also increased by acidosis. The presence of a dominant-negative p90^rsk kinase also did not prevent activation and phosphorylation of NHE1 by sustained intracellular acidosis in isolated cardiomyocytes. We conclude that Ser⁷⁰³ and p90^rsk are not required for activation by sustained intracellular acidosis and that p90^rsk phosphorylation of Ser⁷⁰³ is independent of this type of activation.     

A bidirectional crosstalk between glioblastoma and brain endothelial cells potentiates the angiogenic and proliferative signaling of sphingosine-1-phosphate in the glioblastoma microenvironment

Glioblastoma is one of the most malignant, angiogenic, and incurable tumors in humans. The aberrant communication between glioblastoma cells and tumor microenvironment represents one of the major factors regulating glioblastoma malignancy and angiogenic properties. Emerging evidence implicates sphingosine-1-phosphate signaling in the pathobiology of glioblastoma and angiogenesis, but its role in glioblastoma-endothelial crosstalk remains largely unknown. In this study, we sought to determine whether the crosstalk between glioblastoma cells and brain endothelial cells regulates sphingosine-1-phosphate signaling in the tumor microenvironment. Using human glioblastoma and brain endothelial cell lines, as well as primary brain endothelial cells derived from human glioblastoma, we report that glioblastoma-co-culture promotes the expression, activity, and plasma membrane enrichment of sphingosine kinase 2 in brain endothelial cells, leading to increased cellular level of sphingosine-1-phosphate, and significant potentiation of its secretion. In turn, extracellular sphingosine-1-phosphate stimulates glioblastoma cell proliferation, and brain endothelial cells migration and angiogenesis. We also show that, after co-culture, glioblastoma cells exhibit enhanced expression of S1P₁ and S1P₃, the sphingosine-1-phosphate receptors that are of paramount importance for cell growth and invasivity. Collectively, our results envision glioblastoma-endothelial crosstalk as a multi-compartmental strategy to enforce pro-tumoral sphingosine-1-phosphate signaling in the glioblastoma microenvironment.     

Sphingosine-1-phosphate induces the association of membrane-type 1 matrix metalloproteinase with p130Cas in endothelial cells

Membrane-type 1 matrix metalloproteinase (MT1-MMP) plays an important role in sphingosine-1-phosphate(S1P)-dependent migration of endothelial cells but the underlying mechanisms remain largely unknown. Herein, we show that S1P promotes the relocalization of MT1-MMP to peripheral actin-rich membrane ruffles that is coincident with its association with the adaptor protein p130Cas at the leading edge of migrating cells. Immunoprecipitation and confocal microscopy analyses suggest that this interaction required the tyrosine phosphorylation of p130Cas and also involves S1P-dependent phosphorylation of MT1-MMP within its cytoplasmic sequence. The interaction of MT1-MMP with p130Cas at the cell periphery suggests the existence of a close interplay between pericellular proteolysis and signaling pathways involved in EC migration.     

The role of hERG1 K channels and a functional link between hERG1 K channels and SDF-1 in acute leukemic cell migration

Stromal cell-derived factor-1 (SDF-1) and its unique receptor, CXCR4, regulate stem/progenitor cell migration and retention in the bone marrow and are required for hematopoiesis. Recent studies found that hERG1 K⁺ channels were important regulators of tumor cell migration. In this study, we investigated whether SDF-1 induced acute leukemic cell migration associated with hERG1 K⁺ channels. Our results showed that E-4031, a specific hERG1 K⁺ channels inhibitor, significantly blocked SDF-1-induced migration of leukemic cell lines, primary acute leukemic cells, leukemic stem cells and HEK293T cells transfected with herg-pEGFP. The migration of phenotypically recognizable subsets gave the indication that lymphoblastic leukemic cells were inhibited more than myeloid cells while in the presence of E-4031 which maybe associated with herg expression. SDF-1 increased hERG1 K⁺ current expressed in oocytes and HEK293T cells transfected with herg-pEGFP. There were no significant changes of CXCR4 expression on both HL-60 cells and primary leukemic cells regardless if untreated or treated with E-4031 for 24 h (P > 0.05). The hERG1 K⁺ current increased by SDF-1 might contribute to the mechanism of SDF-1-induced leukemic cell migration. The data suggested that hERG1 K⁺ channels functionally linked to cell migration induced by SDF-1.     

Sphingosine-1-phosphate induces a PDGFR-dependent cell detachment via inhibiting beta1 integrin in HEK293 cells

Several different types of interactions between sphingosine-1-phosphate (S1P) receptors and platelet-derived growth factor receptor (PDGFR) have been revealed recently. In this work, we used HEK293 cells to further investigate the potential crosstalk. Interestingly, we observed that S1P specifically induced a PDGFR-dependent cell detachment in HEK293 cells, which could be inhibited by AG1296, a specific inhibitor for PDGFR. EGFR on the other hand, did not have any effect on cell detachment. The detachment was extracellular matrix (ECM) protein specific, suggesting the involvement of specific integrin molecules. When beta(1) integrin was engaged into an active state, S1P-induced cell detachment was blocked, suggesting that S1P induced an inside-out inhibitory effect on beta(1) integrin. G(i) protein and ERK activation were required for the cell detachment induced by S1P, suggesting an endogenous receptor for S1P is likely to be involved.     

New insights into the role of sphingosine 1-phosphate and lysophosphatidic acid in the regulation of skeletal muscle cell biology

Lysophospholipids are bioactive molecules that are implicated in the control of fundamental biological processes such as proliferation, differentiation, survival and motility in different cell types. Here we review the role of sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) in the regulation of skeletal muscle biology. Indeed, a wealth of experimental data indicate that these molecules are crucial players in the skeletal muscle regeneration process, acting by controllers of activation, proliferation and differentiation not only of muscle-resident satellite cells but also of mesenchymal progenitors that originate outside the skeletal muscle. Moreover, S1P and LPA are clearly involved in the regulation of skeletal muscle metabolism, muscle adaptation to different physiological needs and resistance to muscle fatigue. Notably, studies accomplished so far, have highlighted the complexity of S1P and LPA signaling in skeletal muscle cells that appears to be further complicated by their close dependence on functional cross-talks with growth factors, hormones and cytokines. Our increasing understanding of bioactive lipid signaling can individuate novel molecular targets aimed at enhancing skeletal muscle regeneration and reducing the fibrotic process that impairs full functional recovery of the tissue during aging, after a trauma or skeletal muscle diseases. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.