Syntheses of sphingosine-1-phosphate analogues and their interaction with EDG/S1P receptors

Sphingosine-1-phosphate (S1P) is an important regulator of a wide variety of biological processes acting as an endogenous ligand to EDG/S1P receptors. In an effort to establish structure-activity relationship between EDG/S1P and ligands, we report herein homology modeling study of EDG-1/S1P(1), syntheses of S1P analogues, and cell based binding affinity study for EDG/S1P receptors.     

Structural and functional characteristics of S1P receptors

The sphingosine-1-phosphate (S1P) family of G protein-coupled receptors (GPCR) regulates essential cellular processes such as proliferation, migration, cytoskeletal organization, adherens junction assembly, and morphogenesis. S1P, a product from the breakdown of sphingomyelin, binds to the five members of this receptor family, S1P(1), S1P(2), S1P(3), S1P(4), and S1P(5), previously referred to as endothelial differentiation gene (EDG)-1, -5, -3, -6, and -8. S1P receptors are widely expressed in different tissues, so it is not surprising that the S1P receptor family regulates many physiological processes, such as vascular maturation, cardiac development, lymphocyte trafficking, and vascular permeability. FTY720, a new S1P receptor agonist, is undergoing clinical trials as an immunosuppressor. Understanding the physiological role of these receptors and the basics of the ligand-receptor interaction will potentially provide new therapies to control a variety of diseases.     

Inhibitory regulation of Rac activation, membrane ruffling, and cell migration by the G protein-coupled sphingosine-1-phosphate receptor EDG5 but not EDG1 or EDG3

Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that induces a variety of biological responses in diverse cell types. Many, if not all, of these responses are mediated by members of the EDG (endothelial differentiation gene) family G protein-coupled receptors EDG1, EDG3, and EDG5 (AGR16). Among prominent activities of S1P is the regulation of cell motility; S1P stimulates or inhibits cell motility depending on cell types. In the present study, we provide evidence for EDG subtype-specific, contrasting regulation of cell motility and cellular Rac activity. In CHO cells expressing EDG1 or EDG3 (EDG1 cells or EDG3 cells, respectively) S1P as well as insulin-like growth factor I (IGF I) induced chemotaxis and membrane ruffling in phosphoinositide (PI) 3-kinase- and Rac-dependent manners. Both S1P and IGF I induced a biphasic increase in the amount of the GTP-bound active form of Rac. In CHO cells expressing EDG5 (EDG5 cells), IGF I similarly stimulated cell migration; however, in contrast to what was found for EDG1 and EDG3 cells, S1P did not stimulate migration but totally abolished IGF I-directed chemotaxis and membrane ruffling, in a manner dependent on a concentration gradient of S1P. In EDG5 cells, S1P stimulated PI 3-kinase activity as it did in EDG1 cells but inhibited the basal Rac activity and totally abolished IGF I-induced Rac activation, which involved stimulation of Rac-GTPase-activating protein activity rather than inhibition of Rac-guanine nucleotide exchange activity. S1P induced comparable increases in the amounts of GTP-RhoA in EDG3 and EDG5 cells. Neither S1P nor IGF I increased the amount of GTP-bound Cdc42. However, expression of N(17)-Cdc42, but not N(19)-RhoA, suppressed S1P- and IGF I-directed chemotaxis, suggesting a requirement for basal Cdc42 activity for chemotaxis. Taken together, the present results demonstrate that EDG5 is the first example of a hitherto-unrecognized type of receptors that negatively regulate Rac activity, thereby inhibiting cell migration and membrane ruffling.     

EDG3 is a functional receptor specific for sphingosine 1-phosphate and sphingosylphosphorylcholine with signaling characteristics distinct from EDG1 and AGR16.

AGR16/H218/EDG5 and EDG1 are functional receptors for lysosphingolipids, whereas EDG2 and EGD4 are receptors for lysophosphatidic acid (LPA). The present study demonstrates that EDG3, the yet poorly defined member of the EDG family G protein-coupled receptors, shows identical agonist specificity, but distinct signaling characteristics, compared to AGR16 and EDG1. Overexpression of EDG3 conferred a specific [32P]S1P binding, which was displaced by S1P and sphingosylphosphorylcholine (SPC), but not by LPA or other related lipids. In cells overexpressing EDG3, S1P induced inositol phosphate production and [Ca2+]i increase in a manner only partially sensitive to pertussis toxin (PTX), which was similar to the case of AGR16, but quite different from the case of EDG1, in which the S1P-induced responses were totally abolished by PTX. EDG3 also mediated activation of mitogen-activated protein kinase (MAPK) in PTX-sensitive and Ras-dependent manners, as in the cases of EDG1 and AGR16, although EDG3 and EDG1 were more effectively coupled to activation of MAPK, compared to AGR16. Additionally, EDG3 mediated a decrease in cellular cyclic AMP content, like EDG1, but contrasting with AGR16 which mediated an increase in cyclic AMP. These and previous results establish that EDG1, AGR16 and EDG3 comprise the lysosphingolipid receptor subfamily, each showing distinct signaling characteristics.     

Syntheses of sphingosine-1-phosphate stereoisomers and analogues and their interaction with EDG receptors

Sphingosine-1-phosphate (S1P) is considered to be an important regulator of diverse biological processes acting as a natural ligand to EDG receptors. As a preliminary study to develop potent and selective agonist and antagonist for EDG receptors, we report synthesis of S1P stereoisomers and analogues and their binding affinities to EDG-1, -3, and -5.     

A single amino acid determines lysophospholipid specificity of the S1P1 (EDG1) and LPA1 (EDG2) phospholipid growth factor receptors

The phospholipid growth factors sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are ligands for the related G protein-coupled receptors S1P(1)/EDG1 and LPA(1)/EDG2, respectively. We have developed a model of LPA(1) that predicts interactions between three polar residues and LPA. One of these, glutamine 125, which is conserved in the LPA receptor subfamily (LPA(1)/EDG2, LPA(2)/EDG4, and LPA(3)/EDG7), hydrogen bonds with the LPA hydroxyl group. Our previous S1P(1) study identified that the corresponding glutamate residue, conserved in all S1P receptors, ion pairs with the S1P ammonium. These two results predict that this residue might influence ligand recognition and specificity. Characterization of glutamate/glutamine interchange point mutants of S1P(1) and LPA(1) validated this prediction as the presence of glutamate was required for S1P 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(1), producing a mixture of receptors including those having an anionic glutamate that recognize S1P 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.     

Sphingosine 1-phosphate and isoform-specific activation of phosphoinositide 3-kinase beta. Evidence for divergence and convergence of receptor-regulated endothelial nitric-oxide synthase signaling pathways

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elicits diverse biological responses, including angiogenesis, via the activation of G protein-coupled EDG receptors. S1P activates the endothelial isoform of nitric-oxide synthase (eNOS), associated with eNOS phosphorylation at Ser-1179, a site phosphorylated by protein kinase Akt. We explored the proximal signaling pathways that mediate Akt activation and eNOS regulation by S1P/EDG receptors. Akt is regulated by the lipid kinase phosphoinositide 3-kinase (PI3-K). We found that bovine aortic endothelial cells (BAEC) express both alpha and beta isoforms of PI3-K, while lacking the gamma isoform. S1P treatment led to the rapid and isoform-specific activation of PI3-Kbeta in BAEC. PI3-Kbeta can be regulated by G protein betagamma subunits (Gbetagamma). The overexpression of a peptide inhibitor of Gbetagamma attenuated S1P-induced eNOS enzyme activation, as well as S1P-induced phosphorylation of eNOS and Akt. In contrast, bradykinin, a classical eNOS agonist, neither activated any PI3-K isoform nor induced eNOS phosphorylation at Ser-1179, despite activating eNOS in BAEC. Vascular endothelial growth factor activated both PI3-Kalpha and PI3-Kbeta via tyrosine kinase pathways and promoted eNOS phosphorylation that was unaffected by Gbetagamma inhibition. These findings indicate that PI3-Kbeta (regulated by Gbetagamma) may represent a novel molecular locus for eNOS activation by EDG receptors in vascular endothelial cells. These studies also indicate that different eNOS agonists activate distinct signaling pathways that diverge proximally following receptor activation but converge distally to activate eNOS.     

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.     

EDG receptors and hepatic pathophysiology of LPA and S1P: EDG-ology of liver injury

The biological roles of phospholipid growth factors lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) have been broadly investigated. The cellular effects of LPA and S1P are mediated predominantly via endothelial differentiation gene (EDG) receptors. Yet, the biological significance of LPA, S1P and their EDG receptors in cells of the liver remains unclear. Recent data demonstrate the presence of EDG2 and EDG4 mRNA for LPA receptor in a murine hepatocyte cell line transformed with human TGF-alpha, and in primary mouse hepatocytes. EDG2 receptor protein is expressed in mouse liver, where it appears to be located in nonparenchymal cells. Moreover, we have obtained data suggesting that proliferation of small hepatocyte-progenitors and stem (oval) cells during liver injury is associated with the expression of EDG2 and EDG4 receptors. LPA, and possibly S1P, appear to be essential factors that control proliferation and motility of hepatic stellate cells (HSC) and hepatoma cells. It is proposed that LPA, S1P and their respective EDG receptors play important roles in pathophysiology of chronic liver injury and fibrogenesis. The underlying mechanisms recruited by LPA and S1P in pathogenesis of liver injury remain to be investigated.     

Cell type-specific localization of human cardiac S1P receptors.

Sphingosine 1-phosphate (S1P), which derives from the metabolism of sphingomyelin, is mainly synthesized, stored, and released from platelets after activation by physiological and pathophysiological events. S1P acts in cardiovascular tissues through cell surface G-protein-coupled receptors of the endothelial differentiation gene (EDG) family, i.e., EDG1, EDG3 and EDG5. The aim of the present study was to assess the precise distribution of EDG1, EDG3, and EDG5 receptors expressed in human cardiovascular tissues to investigate their respective physiological implication. When assessed by Northern blots, EDG1, 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 EDG1 expression in cardiomyocytes and in endothelial cells of cardiac vessels. In cardiomyocytes, the EDG1 receptor is likely to be co-expressed with EDG3 and EDG5, although EDG1 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 EDG1 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 S1P receptors in human cardiovascular tissues, indicating a differential pattern of expression for these receptors in human vessels.     

Homodimerization and heterodimerization of S1P/EDG sphingosine-1-phosphate receptors

Sphingosine-1-phosphate (S1P) binds to and signals through several members of a group of G protein-coupled receptors (GPCRs) known as the S1P/EDG family. Several of these receptors are coexpressed in various cell types and recent reports have shown that biological effects of S1P often require more than one S1P receptor subtype. Recent evidence indicates that many GPCRs exist as dimers. We show that S1P receptors form both homodimers as well as heterodimers with other members of the S1P subfamily of receptors. We also discuss the role that GPCR dimers play in receptor function and what this may mean for S1P signaling.     

Translational aspects of sphingosine 1-phosphate biology

Sphingosine 1-phosphate (S1P) is a bioactive lipid that has both physiological and pathophysiological roles. It regulates cellular processes such as proliferation, migration, survival and differentiation and affects all organ systems. S1P not only activates S1P-specific receptors to initiate cellular signalling pathways but also directly regulates specific intracellular target proteins. The therapeutic opportunities surrounding S1P signalling are numerous and exemplified by the recent approval of FTY720 (a sphingosine analogue, Gilenya?) for the treatment of relapsing multiple sclerosis. A major focus of research is to develop small-molecule antagonists/agonists/inhibitors that are specific to the different S1P receptor subtypes and the enzymes that regulate S1P levels. This review describes fundamental aspects of S1P biology with an emphasis on the translational potential of intervention therapeutics.     

Structures of signaling complexes of lipid receptors S1PR1 and S1PR5 reveal mechanisms of activation and drug recognition

Sphingosine-1-phosphate (S1P) is an important bioactive lipid molecule in cell membrane metabolism and binds to G protein-coupled S1P receptors (S1PRs) to regulate embryonic development, physiological homeostasis, and pathogenic processes in various organs. S1PRs are lipid-sensing receptors and are therapeutic targets for drug development, including potential treatment of COVID-19. Herein, we present five cryo-electron microscopy structures of S1PRs bound to diverse drug agonists and the heterotrimeric Gi protein. Our structural and functional assays demonstrate the different binding modes of chemically distinct agonists of S1PRs, reveal the mechanical switch that activates these receptors, and provide a framework for understanding ligand selectivity and G protein coupling.Subject terms: Cryoelectron microscopy, Lipid signalling     

Sphingosine 1-phosphate and activation of endothelial nitric-oxide synthase. differential regulation of Akt and MAP kinase pathways by EDG and bradykinin receptors in vascular endothelial cells

Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid that elicits numerous biological responses in endothelial cells mediated by a family of G protein-coupled EDG receptors. Stimulation of EDG receptors by S1P has been shown to activate the endothelial isoform of nitric-oxide synthase (eNOS) in heterologous expression systems (Igarashi, J., and Michel, T. (2000) J. Biol. Chem. 275, 32363-32370). However, the signaling pathways that modulate eNOS regulation by S1P/EDG in vascular endothelial cells remain less well understood. We now report that S1P treatment of bovine aortic endothelial cells (BAEC) acutely increases eNOS enzyme activity; the EC(50) for S1P activation of eNOS is approximately 10 nm. The magnitude of eNOS activation by S1P in BAEC is equivalent to that elicited by the agonist bradykinin. S1P treatment activates Akt, a protein kinase implicated in phosphorylation of eNOS. S1P treatment of BAEC leads to eNOS phosphorylation at Ser(1179), a residue phosphorylated by Akt; an eNOS mutant in which this Akt phosphorylation site is inactivated shows attenuated S1P-induced eNOS activation. S1P-induced activation both of Akt and of eNOS is inhibited by pertussis toxin, by the phosphoinositide 3-kinase inhibitor wortmannin, and by the intracellular calcium chelator BAPTA (1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). By contrast to S1P, activation of G protein-coupled bradykinin B2 receptors neither activates kinase Akt nor promotes Ser(1179) eNOS phosphorylation despite robustly activating eNOS enzyme activity. Understanding the differential regulation of protein kinase pathways by S1P and bradykinin may lead to the identification of new points for eNOS regulation in vascular endothelial cells.     

Sphingosine-1-phosphate receptor expression and signaling correlate with uterine prostaglandin-endoperoxide synthase 2 expression and angiogenesis during early pregnancy

Signaling mechanisms coordinating uterine angiogenesis and tissue remodeling during decidualization are not completely understood. Prostanoid signaling is thought to play a functionally important role in each of these events. In the present study, we demonstrate that the subfamily of G-protein-coupled receptors that binds and becomes activated by the terminal signaling lipid in the sphingolipid pathway, sphingosine-1-phosphate (S1P), were expressed during uterine decidualization. Three of the five known S1P receptors, termed endothelial differentiation genes (Edg; Edg1, Edg3, and Edg5) were upregulated in the uterine deciduum from Day of Pregnancy (DOP) 4.5 to 7.5, while Edg6 and Edg8 expression remained unchanged. Consistent with angiogenesis in general during decidualization, we believe EDG1 and EDG5 to be regulated by the embryo because no microvascular expression for these receptors was observed in oil-induced deciduomas. Observed expression of EDG1 and EDG5 showed a similar expression pattern to that previously reported for prostaglandin-endoperoxide synthase 2 (PTGS2), transitioning from the sublumenal stromal compartment in the antimesometrial pole (DOP 5) to the microvasculature of the mesometrial pole (DOP 7). Furthermore, these two receptors colocalized with PTGS2 at three additional sites at the maternal:fetal interface throughout pregnancy. Treatment of cultured predecidualized stromal cells with S1P resulted in upregulation of Ptgs2 mRNA and PTGS2 protein, but not the downstream enzyme prostacyclin synthase. These combined results suggest the existence of a link between the sphingolipid and prostanoid signaling pathways in uterine physiology, and that, based on their expression pattern, S1P receptors function to coordinate uterine mesometrial angiogenesis during the implantation phase of early gestation.     

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.     

Synthesis and biological properties of novel sphingosine derivatives

Sphingosine-1-phosphate (S-1P) derivatives such as threo-(2S,3S)-analogues, which are C-3 stereoisomers of natural erythro-(2S,3R)-S-1P, have been synthesized starting from l-serine or (1S,2S)-2-amino-1-aryl-1,3-propanediols (6). threo-(1S,2R)-2-Amino-1-aryl-3-bromopropanols (HBr salt) have also been prepared from 6. The threo-S-1Ps and the threo-amino-bromide derivatives have shown potent inhibitory activity against Ca(2+) ion mobilization in HL60 cells induced by erythro-S-1P, suggesting that these compounds would compete with cell surface EDG/S1P receptors.     

Sphingosine 1-phosphate signalling and termination at lipid phosphate receptors

Sphingosine 1-phosphate (S1P) is a polar lysophospholipid metabolite that is stored in platelets and released upon their activation. However, diverse stimuli such as growth factors, cytokines, G-protein coupled receptor (GPCR) agonists and antigens have been shown to increase sphingosine kinase activity and S1P formation in other cell types, such as smooth muscle. Indeed, S1P has been implicated in the regulation of several important cellular processes, such as proliferation, differentiation, apoptosis and migration in these cells. Over the past few years, there has been a major advance in our understanding of how S1P can act as an intercellular mediator by binding to a new class of G-protein coupled receptors to regulate cell function. This review focuses on the enzymatic regulation of S1P formation and degradation and its interaction with a novel tethered receptor complex containing the S1P receptor (S1P(1)) and the platelet-derived growth factor (PDGF) beta receptor. This tethered receptor complex enables coincident integrative signalling to p42/p44 MAPK. This is compared with a sequential model in which PDGF promotes S1P release, which in turn acts on S1P(1) to promote Rac signalling.     

Targeting sphingosine-1-phosphate signaling for cancer therapy

Sphingosine-1-phosphate(S1P) is a potent pleotropic bioactive lipid mediator involved in immune cell trafficking, cell survival,cell proliferation, cell migration, angiogenesis and many other cellular processes. S1 P either activates S1 P receptors(S1PR1-5) through "inside-out signaling" or acts directly on intracellular targets to regulate various cellular processes. In the past two decades, much progress has been made in exploring S1 P signaling and its pathogenic roles in diseases as well as in developing modulators of S1 P signaling, including S1 P agonists, S1 P antagonists and sphingosine kinase(SphK) inhibitors.Ceramide and S1 P have been defined as reciprocal regulators of cell fate, and S1 P signaling has been shown to be crucial for the pathogenesis of various diseases, including autoimmune diseases, inflammation and cancer; therefore, targeting S1 P signaling may curtail the process of pathogenesis and serve as a potential therapeutic target for the treatment of these diseases. In this review, we describe recent advances in our understanding of S1 P signaling in cancer development(particularly in inflammationassociated cancer) as well as in innate and adaptive immunity, and we also discuss modulators of S1 P signaling in cancer treatment.     

Sphingosine 1-phosphate signalling in cancer

There is an increasing body of evidence demonstrating a critical role for the bioactive lipid S1P (sphingosine 1-phosphate) in cancer. S1P is synthesized and metabolized by a number of enzymes, including sphingosine kinase, S1P lyase and S1P phosphatases. S1P binds to cell-surface G-protein-coupled receptors (S1P1-S1P5) to elicit cell responses and can also regulate, by direct binding, a number of intracellular targets such as HDAC (histone deacetylase) 1/2 to induce epigenetic regulation. S1P is involved in cancer progression including cell transformation/oncogenesis, cell survival/apoptosis, cell migration/metastasis and tumour microenvironment neovascularization. In the present paper, we describe our research findings regarding the correlation of sphingosine kinase 1 and S1P receptor expression in tumours with clinical outcome and we define some of the molecular mechanisms underlying the involvement of sphingosine kinase 1 and S1P receptors in the formation of a cancer cell migratory phenotype. The role of sphingosine kinase 1 in the acquisition of chemotherapeutic resistance and the interaction of S1P receptors with oncogenes such as HER2 is also reviewed. We also discuss novel aspects of the use of small-molecule inhibitors of sphingosine kinase 1 in terms of allosterism, ubiquitin-proteasomal degradation of sphingosine kinase 1 and anticancer activity. Finally, we describe how S1P receptor-modulating agents abrogate S1P receptor-receptor tyrosine kinase interactions, with potential to inhibit growth-factor-dependent cancer progression.