Lysophospholipid Receptors (Signaling and Biochemistry) || Sphingosine 1-Phosphate (S1P) Receptors

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CHAPTER 2

Sphingosine 1-Phosphate (S1P) ReceptorsBONGNAM JUNG and TIMOTHY HLA

2.1. INTRODUCTION

Sphingolipids are one of the basic constituents of the biological membrane present in all eukaryotic cells. Sphingolipid metabolites including ceramide, sphingosine, ceramide-1-phosphate (C1P), and sphingosine 1-phosphate (S1P) have received considerable attention as regulators of cellular function and physiological processes (1). Especially, S1P has been demonstrated as a potent, bioactive lipid mediator that regulates cellular processes such as cell migration, proliferation, and survival as well as physiological events including angiogen-esis and immunity. Indeed, the identification, cloning, and analysis of genes encoding S1P receptors, metabolic enzymes, and transporters have contributed to this knowledge base (2). Furthermore, pharmacological tools that modu-late S1P-related proteins have allowed further understanding of physiological functions of S1P and its receptors in vivo, interconnecting the basis of in vitro findings (3). This chapter will focus on S1P-mediated biology, especially physi-ological actions of S1P receptors.

2.2. S1P METABOLISM/ENZYME, AND TRANSPORT

Sphingosine is a member of the sphingolipid family, comprised of an aliphatic chain with 18 carbon atoms, with hydroxyl groups on carbon atoms 1 and 3, and an amine moiety on carbon atom 2 (4). Sphingosine, derived from the N-deacetylation of ceramide by ceramidase, is phosphorylated by sphinosine kinases (SphKs) 1 and 2, generating S1P. S1P was first identified as a bioactive

Lysophospholipid Receptors: Signaling and Biochemistry, First Edition. Edited by Jerold Chun, Timothy Hla, Sara Spiegel, and Wouter Moolenaar.© 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

42 SPHINGOSINe 1-PHOSPHATe (S1P) RecePTORS

sphingolipid that induces intracellular calcium increase and cell proliferation (5). S1P level is regulated by a variety of enzymes such as sphingosine kinases, sphingosine phosphatases, and sphinogine lyase, which will be discussed in detail in the next sections.

2.2.1. S1P Metabolism and Enzymes

Cellular level of S1P is controlled by the concerted action of the enzymes responsible for its formation and degradation. De novo synthesis of S1P occurs at the cytoplasmic leaflet of the endoplasmic reticulum (ER) with condensa-tion of serine and palmitate by serine palmitoyltransferase that ultimately results in the formation of this lysophospholipid elsewhere in the cell. S1P can be also generated by degradation of plasma membrane sphingolipid such as sphingomyelin by sphingomyelinases, which yields the critical intermediate ceramide (6). Ceramide is further metabolized by ceramidase, resulting in the formation of sphingosine (7). Subsequently, biosynthesis of S1P is through the phosphorylation of sphingosine by SphKs.

2.2.2. Sphingosine Kinases

SphKs are evolutionarily well-conserved lipid kinases from unicellular to multicellular organisms. Mammalian SphKs have two isoforms, namely SphK1 and SphK2, with 80% sequence similarity (8). Although both SphKs are ubiquitously expressed in most tissues, Sphk1 expression was predominantly observed in lung and spleen, whereas Sphk2 in liver and heart (9). SphKs are distributed at various subcellular locations in the cell. For instance, SphK1 has its nuclear export activity and thus is present mostly in the cytoplasm. In con-trast, SphK2 can be found in the nucleus as well as the cytoplasm since it possesses both a nuclear import signal and nuclear export signals (10). SphKs do not have membrane-anchoring sequences and usually behave as soluble enzymes under normal conditions. Upon simulation, however, SphKs are known to undergo activation and translocation by mechanisms involving protein phosphorylation, protein–lipid binding, protein–protein interaction, and calcium/calmodulin (11).

The significant functions of SphKs have been revealed from in vivo studies using genetically ablated mouse model, which suggest that homeostatic S1P level is essential during vasculature development and neurogenesis. For instance, inducible deletion of both Sphk1 and Sphk2 gene in the adult resulted in undetectable level of circulating S1P. Moreover, double Sphk1/Sphk2 knockout (KO) mice displayed severe hemorrhages and edema due to incom-plete vascular smooth muscle cell (VSMC) coverage and impaired neural tube closure, which subsequently resulted in embryonic lethality (12). However, mice lacking either Sphk1 or Sphk2 gene were viable and fertile with no appar-ent phenotype (9), indicating that the two isoenzymes can compensate for each other.

S1P MeTABOLISM/eNzYMe, ANd TRANSPORT 43

Although roles of S1P in the central nervous system (CNS) have not been fully explored, accumulating evidence suggest that S1P signaling may be important for proper neuronal system development and axon guidance. Neurite extension and retraction are important processes in the neuronal network formation during development and are largely regulated by the orga-nization of the actin cytoskeleton. This process is controlled by the balance between the opposing actions of the small GTPases, Rho and Rac: Rac is essential for neurite outgrowth, whereas Rho induces collapse of growth cones and inhibition of neurite outgrowth (13). It turned out that glial cell line-derived neurotrophic factor/RET signaling transactivates SphK/S1P signaling and induces neurite extension through S1P1 (14). Additionally, double Sphk1/Sphk2 null embryos display exencephaly, a severe brain development defect (12). Furthermore, S1P was able to mediate growth cone collapse and repul-sive turning through S1P5 in Xenopus retinal neuron system (15).

Recent in vitro findings related to SphKs raise the possibility of S1P as a gene expression regulating factor. SphK1, which can bind to tumor necrosis factor (TNF)-associated factor 2 (TRAF2) (16), seems to participate in TNF-α-mediated nuclear factor-κB (NF-κB) activation by producing S1P which directly binds to the amino-terminal really interesting new gene (RING) domain of TRAF2. This process resulted in increase of its E3 ubiquitin ligase activity, thus polyubiquinated receptor interacting protein 1 (RIP1) that acti-vates IκB kinase and NF-κB activation (17). It was also reported that intracel-lular S1P could act on molecular targets to alter gene expression in the nucleus via shuttling SphK2 back and forth. Specifically, SphK2 and S1P form core-pressor complexes with histone deacetylases (HDAC), HDAC1 and HDAC2, which prevent deacetylation of lysine residues with the histone tail, thus block-ing its DNA binding and upregulation of p21 and c-fos (18). Further studies need to be performed to elucidate the precise mechanism of SphK2 in these processes such as translocation regulation to the nucleus, target genes con-trolled by S1P-containing HDAC complexes, occurrence using in vivo model, and relevance to human diseases. Such intracellular mechanisms of S1P signal-ing contrast sharply with its well-established extracellular mode of action via G protein-coupled receptors (GPCRs) (6) and warrant further investigation to assess physiological relevance.

2.2.3. S1P Phosphatases and S1P Lyase

Several enzymes bearing lipid phosphatase (LPP) activity can dephosphory-late S1P: sphingosine 1-phosphate phosphatases (SPP), namely SPP1 and SPP2, S1P lyase (SPL), or broad-spectrum LPPs). Its metabolic fate includes either the irreversible cleavage to trans-hexadecenal and ethanolamine phos-phate by a pyridoxal phosphate-dependent lyase or the hydrolytic removal of the phosphomonoester group by S1P phosphatase or lipid phosphate phosphatases (13). The SPPs seem to play an important role in the control of the sphingoid base flow into different metabolic pathways, and thus affect


transport of precursors of S1P including ceramide (19). In addition, it is likely that altered level of SPP1 and SPP2 has correlations with various diseases, indicating the role of SPPs regulating vascular tone and pathophysiologic conditions including inflammatory diseases and cancer (20).

As previously mentioned, SPL can also facilitate irreversible degradation of S1P that yields ethanolamine phosphate and hexadecenal, which are even-tually reused for phosphatidylethanolamine for lipid metabolism. SPL is widely expressed in intestine, thymus, and olfactory mucosa. Genetic deletion of SPL leads to postnatal death around weaning age, displaying anemia, myeloid cell hyperplasia, and multiple congenital anomalies (21, 22). These phenotypes might be partially due to accumulation of long chain bases and ceramide in neurons and vital organs, which could be cytotoxic (2). Further-more, Spl-null mice showed abnormal lipid metabolism (i.e., increased level of triglycerides in plasma) and lack of adipose tissues, suggesting the impor-tance of the sphingolipid regulation in control of global lipid homeostasis and diseases (2).

2.2.3.1. Sources and Transporter(s) of S1P S1P is present in relatively high concentration (up to micromolar) in plasma, whereas S1P levels are extremely low in most tissues, including lymphoid tissue (23, 24), creating a S1P gradient in vivo (25). It was believed that erythrocytes were the major source of S1P, although all cells are capable of generating S1P by sphingomy-elin metabolism (26). However, recent studies showed that hematopoietic stem cells (27), endothelial cells (28), and astrocytes (29) can also secrete S1P. At least the endothelial cells seem to be important for maintaining high S1P levels in plasma (28). Other cell types including platelets and mast cells are thought to produce S1P under pathological conditions (28, 30).

Once S1P is generated in the cell, delivery of S1P to the extracellular envi-ronment is considered to occur via specific transporters. Various adenosine triphosphate (ATP)-binding cassette (ABC)-type transporters such as ABCC1 (31), ABCA1 (32), and ABCG1 (33) have been proposed to export S1P. However, the precise mechanism by which S1P is transported by the ABC proteins remains to be confirmed in vivo. In addition, such proposals were made using nonspecific chemical inhibitors and therefore further studies are needed to demonstrate if ABC transporters are indeed relevant in S1P secre-tion. Interestingly, recent findings from zebrafish suggest that the spinster homologue, Spns2, might be the very specific transporter for S1P and phos-phate metabolite of fingolimod (34, 35). It is known that 98.5% of S1P is bound to lipoproteins such as high-density lipoprotein (HDL) and albumin (36). A recent study using transgenic animals showed that ApoM is indeed a specific carrier for S1P in vivo (37). Free S1P or S1P bound to serum albumin is more prone to be degraded than lipoprotein-bound S1P, suggesting that binding partners of S1P are a key determinant for the release of S1P as well as the uptake and/or intracellular degradation of S1P (38). Once a free form of S1P

S1P RecePTOR SUBTYPeS, ANd PHYSIOLOGIcAL FUNcTIONS 45

is available, it can transmit the signal through one of the S1P receptor family members that couples to G protein, which we will discuss more in detail.

2.3. S1P RECEPTOR SUBTYPES, AND PHYSIOLOGICAL FUNCTIONS

It has become apparent, through the work of Cyster and Schwab (39), that the S1P gradient between circulatory fluid and tissue, created by intricate mecha-nisms by activities of S1P metabolic enzymes, is essential for the maintenance of homeostasis and immunity. Physiological functions of S1P are mediated through the activation of its specific receptors. So far, five high-affinity S1P receptors have been identified: S1P1 (Edg-1), S1P2 (Edg-5), S1P3 (Edg-3), S1P4 (Edg-6), and S1P5 (Edg-8). S1P receptors consist of 7-transmembrane protein that couples with a variety of heterotrimeric G proteins. S1P receptors are differentially expressed in cells and tissues. Therefore, finely tuned, spatiotem-poral regulation of S1P receptor expression pattern would diversify the responsiveness toward S1P by differential activation of its downstream signal-ing pathways.

2.3.1. S1P1

S1P1 (Edg-1/LPB1) was first discovered by Hla et al. as an orphan GPCR termed endothelial differentiation gene-1 (Edg-1) from a differential screen for mRNAs induced during angiogenesis. Subsequently we found that S1P is the high affinity ligand for S1P (Kd ∼ 8 nM) (40, 41). S1pr1 transcripts were abundant in endothelial cells, but also detected in VSMCs, fibroblasts, mela-nocytes, and epithelial cells. S1P1 is also expressed by cells of the immune system including T and B cells, macrophages, dendritic cells, and NK cells (42). Ubiquitous expression of S1P1 is observed in a large number of tissues such as brain, heart, kidney, lung, intestine, ovary, testis, lymphoid tissue, and spleen. Upon S1P binding, S1P1 exclusively couples to pertussis toxin (PTX)-sensitive Gi protein and leads to extracellular signal-regulated kinase (ERK) activation, phospholipase C (PLC) activation, Ca2+ mobilization, and adenyl cyclase (AC) inhibition (1). Moreover, S1P1-mediated PI3K/Akt and Rac activation have been reported to be essential for cellular events such as cell proliferation, survival, migration, and cytoskeletal assembly (4, 7, 43).

Given the physiological importance of S1P on cell spreading and vascular barrier enhancement in vivo, it was clear that S1P regulates endothelial cell behavior through the activation of its receptor, and influences the strength of the vascular stability and integrity. Specifically, S1P-dependent vascular integrity is mediated via S1P1 and S1P3 that activate Gi/Rac/PAK signaling and influence vascular endothelial (VE)-cadherin-mediated adherens junction assembly and actin cytoskeleton rearrangement (44, 45), suggesting that S1P1-mediated signaling is protective against vascular permeability/leakage. In


contrast, activation of S1P2 in endothelial cells disrupts adherens junction and induces vascular paracelluar permeability (46). Additionally, S1P mediates endothelial cell barrier function by phosphorylation of cortical actin and by binding to myosin light-chain kinase (MLCK) (47), and redistribution of focal adhesion kinase and paxillin to the cell surface, which process facilitates cell–cell adhesion (48). Moreover, depletion of ZO-1 using siRNA abolished S1P-induced barrier function, indicating the important role of ZO-1 in S1P/S1P1-mediated vascular integrity in endothelial cells (49). Interestingly, Camerer et al. showed that systemic depletion of plasma S1P in mice resulted in significant increase of the basal and inflammation-mediated vascular leakage and poor survival of mice upon inflammatory challenge, and this phenotype was recovered by wild-type erythrocyte transplantation (50). These findings suggest that the interaction between plasma S1P and blood vascular endo-thelium via S1P1 is an essential mechanism for maintenance of vascular stability.

The significance of S1P1 in vasculature is denoted from in vivo animal model. Liu et al. demonstrated that global deletion of the S1pr1 gene in mouse leads to embryonic death at midgestation due to the vascular maturation defect, which results in massive intraembryonic hemorrhages and edema throughout the body and the limbs. Specifically, S1pr1-null embryos do not seem to have any apparent defects in vasculature formation. Although VSMCs were present at the ventral side of the aorta, VSMC failed to cover the devel-oping vessels (51). Endothelial-specific deletion of S1pr1 gene using Tie2-Cre mice displayed the same vascular defect, suggesting that S1P1 in endothelium is critical for directing vascular coverage by VSMC (52, 53). The mechanism behind this phenomenon involves endothelial cell-derived S1P1 that facilitates N-Cadherin-mediated junction assembly between endothelial cells and VSMCs which is required for the vessel integrity and stability (54).

In the cardiovascular system, S1P might be involved in heart rate control. For instance, agonism for S1P1 and S1P3 causes an acute reduction in heart rate prior to these receptors being internalized/desensitized (55). In humans, S1P1 is a major receptor expressed in ventricular, septal, and atrial cardiomyo-cytes and in endothelial cells of cardiac vessels, indicating that S1P1, rather than S1P3, takes a dominant part in control of arterial myocyte function and heart rate (56).

The brain contains the highest concentration of S1P among organs (57). S1P receptors are expressed in the CNS such as neurons, oligodendrocytes, astrocytes, and microglial cells (58). S1P through the activation of S1P recep-tors has been reported to regulate astrocyte motility, neurite extension, neu-ronal cell proliferation and survival, and oligodendrite extension (59). Although the exact mechanism of S1P functions in the CNS has not been well under-stood, it is likely that S1P/S1P1 axis might be crucial for physiologic and patho-logical functions as both S1pr1 KO and double Sphk1/Sphk2 KO mouse embryos display severe defects in neuronal system development (57, 60). Fur-thermore, S1P1 silencing using a short hairpin RNA interference technique


diminished neural stem cell migration toward the site of injury, where local concentrations of S1P was increased (61). Interestingly, expression level of SphK1 was proportional to poor survival rate of patients with glioblastoma multiforme (61). Taken together, these findings indicate that regulation of sphingosine metabolism and the signaling mechanism mediated by differential receptor expression in the brain are the key for its action in neuronal system, raising therapeutic potential for brain injuries.

Multiple sclerosis (MS) is a chronic autoimmune neurodegenerative disor-der and has been reported to affect approximately 2.5 million people in Europe, Canada, the United States, New Zealand, Australia, and northern Asia (3). MS is characterized by inflammation of the CNS, including astrogliosis, demyelination, and destruction of oligodendrocytes and neurons, resulting in severe neurological dysfunction (62). Modulation of S1P receptors by phar-macologic means using a compound called FTY720 (fingolimod) has shown efficacy in the treatment of MS (3). FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol) was first chemically synthesized from myriocin (ISP-1), a metabolite of the ascomycete Isaria sinclairii, which depletes lymphocytes from blood and lymph, preventing skin allograft rejection (27). FTY720 received Food and Drug Administration (FDA) approval as an oral therapy for relapsing drug of MS in September 2010 (3).

As mentioned earlier, S1P exists in higher concentrations in blood and lymph than in lymphoid organs, creating a gradient of S1P by differentially regulated activities of S1P metabolic enzymes including SphKs and SPL. FTY720, structurally similar to S1P, can be rapidly phosphorylated in vivo by SphK2 (27). FTY720-phosphate metabolite, FTY720-P, acts as a high-affinity agonist of four of five S1P receptors: S1P1, S1P3, S1P4, and S1P5 but not S1P2 (63). The mechanism of action is believed to be immunological as FTY720-P reduces cell surface expression of S1P1, which subsequently prevents lympho-cyte egress from lymphoid organs (64, 65). In addition, FTY720-P binding to S1P1 induces rapid internalization and degradation of the receptor in a ubiquitin-dependent process (66). Thus, FTY720-P serves as a functional S1P1 antagonist: treatment of FTY720 leads to S1P1 activation and in turn lympho-penia (63, 67). It should be noted that the discovery and usage of FTY720 have shed light on the physiological functions of S1P and S1P1 in lymphocyte traf-ficking and vascular biology (39).

“Immune surveillance” is the fundamental process for the maintenance of homeostasis and immunity: immune cells including lymphocytes continuously circulate sampling cognate antigens throughout the body. Therefore, regula-tion of egress of lymphocytes from primary and secondary lymphoid organs into circulatory fluids is critical. It is well established that the trafficking and positioning of lymphocytes are dependent on precisely regulated S1P level in circulation and in tissues (39). It is not surprising that immune cells utilize S1P receptor expression level on their cell surface for emigration from lymphoid organs into blood. In other words, S1P1 expression on the cell surface of lym-phocytes is responsible for their egress. Indeed, expression level of S1P1 is high


in mature T cells in the thymus, whereas activated T cells that are normally retained in peripheral lymphoid organs have low S1P1 level (64).

Recent in vitro studies from Oo et al. elucidated the mechanism of FTY720-P action as a functional S1P1 antagonist that promotes endogenous S1P1 degrada-tion. Specifically, FTY720-P was as potent as S1P inducing receptor internaliza-tion by phosphorylation of S1P1. Furthermore, FTY720-P was efficiently able to polyubiquitinylate S1P1 and facilitated proteosomal degradation of the receptor (66). FTY720-P-induced receptor internalization was abrogated when conserved, serine-rich motif at the C-terminal tail of the S1P1 was mutated to alanine (S5A), suggesting that this motif is critical for receptor desensitization and internalization (66). Given that, Thangada et al. generated internalization-deficient mice, called S1pr1

S5A/S5A, which are viable and fertile with no apparent vascular phenotype (68). Although T-cell trafficking was normal under homeo-static condition, S1pr1

S5A/S5A animals exhibited kinetic resistance to FTY720-induced lymphopenia (68).

Adoptive cell transfer experiments using hematopoietic cells lacking S1P1 provided evidence that cell surface expression of S1P1 in lymphocytes is critical for their egress from the lymphoid organs into blood (64). Interestingly, results using labeled S1pr1

S5A/S5A T-cells transplantation confirmed that intrinsic S1P1 expression in T cells, not vascular endothelium, is essential for T-cell egress kinetics (68). Along with this, a study using the gain of function of S1P1 mouse model would be useful to unravel cell type-specific, pathophysiological func-tions of S1P1 upon challenge: tumor angiogenesis, ischemic reperfusion, ath-erosclerosis, diabetes, and MS.

Although the FTY720 effect was due to immune modulatory events, addi-tional effects in the CNS may also explain its efficacy in autoimmune neuronal inflammatory events in an animal model. Choi et al. demonstrated that nonim-munological CNS mechanisms are required for FTY720 efficacy using geneti-cally modified mouse model, especially CNS cell type-specific S1P1 deletion followed by experimental autoimmune encephalomyelitis (EAE) challenge which recapitulates human MS. CNS-specific deletion of S1P1 mutant mice displayed normal lymphocyte trafficking and comparable response toward FTY720 treatment (69). However, astrocyte-specific S1pr1-null mice showed diminished level of EAE, suggesting that astrocytes bearing S1P1 are function-ally involved in FTY720 activity.

2.3.2. S1P2

S1P2 (Edg-5/AGR16/H218/LPb2) was first cloned as an orphan GPCR gene from rat cardiovascular and nervous system and later identified by many groups as a high-affinity S1P receptor (Kd = 16–27 nM) (41, 70). S1pr2 is widely expressed including brain, heart, lung, thymus, kidney, spleen, adipose tissues, and all other tissues tested in animal models (9). S1P2 couples with diverse heterotrimeric G-proteins such as Gi/o, Gq, and G12/13, and mediates S1P-induced cell proliferation, cell survival, cell rounding by serum response element (SRE)


activation, ERK, c-Jun N-terminal kinase (JNK), p38 activation, PLC activa-tion, and small GTPase Rho activation (71). Contrary to the action of S1P1, S1P2 activates PIP3 phosphatase (PTEN) as a downstream effector, inhibits S1P-induced Rac activity, and prevents cell migration (72). Additionally, over-expression of S1P2 by adenoviral system in endothelial cells disrupted adher-ens junction assembly and increased vascular permeability via Rho–Rho kinase (ROCK) and PTEN activation (46). JTE-013, a specific S1P2 antagonist, showed significantly enhanced barrier function in H2O2-induced rat lung edema model, indicating that S1P2 signaling is crucial in regulation of the vascular permeability (46).

Genetic deletion of S1P2 in mouse embryos does not exhibit any apparent vascular defect during development. However, a more severe phenotype was observed in S1pr1/S1pr2 double-KO embryos than S1pr1 single-null embryos, suggesting that S1P2 also takes part in embryonic vascular development (73). Interestingly, S1pr2-null animals showed deafness due to vascular abnormali-ties in the stria vasculris of the inner ear and degeneration of sensory hair cells in the organ of corti, implicating the involvement of S1P2 in proper functioning of the auditory and vestibular systems (74). Cardiac development defects (cardia bifida) was observed when the Mil gene in zebrafish (Danio rerio), the homologue of mammalian S1pr2, was mutated, showing that S1P2 is essential for proper heart organogenesis (75).

Although S1pr2-null mice are viable and grossly normal, S1P2 KO mice showed significantly decreased infiltration of the inflammatory cells and enhanced revascularization in retina under hypoxic condition (76). These results suggest that S1P2 plays a critical role activating inflammatory pathways that lead to vascular permeability and pathological angiogenesis. Recent find-ings from Michaud et al. provided evidence that S1P2 is involved in this process. Specifically, S1P inhibited macrophage recruitment through the action of S1P2 during inflammation, altering migratory speed but not direc-tionality toward chemoattractants (77). Furthermore, S1P2-null mice and S1P2 blockade by JTE-013 exhibited significantly reduced mast cell-mediated ana-phylactic responses and vascular leakage in lung, indicating a pivotal role of S1P2 in regulation of mast cell functions such as degranulation and cytokine release (78).

Dysregulation of the vascular endothelium can affect the balance between vasodilation and vasoconstriction, and develop risk factors for atherosclerosis, including hypertension and vascular remodeling (79). Atherosclerosis is a chronic inflammatory disease, influenced by a variety of components such as modified lipoproteins, monocyte-derived macrophages or foam cells, endothe-lial cells, and smooth muscle cells (SMCs). Accumulating data have elucidated the significant function of S1P in both the early and late phases of atheroscle-rosis, apart from its general progression. For instance, S1P and S1P-containing HDL augmented anti-inflammatory responses through S1P1, including inhibi-tion of leukocyte adhesion and proinflammatory cytokine production (80). Furthermore, FTY720, an agonist for four among 5 S1P receptors, dose


dependently inhibited atherosclerosis development in Ldlr-null mice (81). Recently, Skoura et al. demonstrated using S1pr2-null mice in Apoe−/− back-ground that S1P2 signaling promotes atherosclerosis by regulating macrophage retention and proinflammatory cytokine production: S1p2r−/−;Apoe−/− mice showed significantly decreased atherosclerotic plaque area with less number of foam cells and macrophages, and reduced serum level of proinflammatory cytokines including interleukin-1β (IL-1β) (82). These findings were consistent from Wang et al., confirming that S1P2 is indeed pro-atherogenic (83).

In the neuronal system, it is likely that S1P2-mediated signaling inhibits neurite extension and glioblastoma motility, contrary to S1P1. Interestingly, S1pr2-null mice exhibited progressive cochlear and vestibular defects with hair cell loss, which subsequently leads to deafness, suggesting that S1P2 is essential for functional maintenance as well as development of the auditory and ves-tibular systems (74, 84). Although the precise mechanism of S1P2 in neuronal excitability remains to be elucidated, genetic ablation of S1pr2 in mice displays spontaneous, sporadic lethal seizures with increases in excitatory postsynaptic currents, implicating the significant role of S1P2 in the CNS (13, 85).

2.3.3. S1P3

S1P3 (Edg-3/LPB3) was isolated as an orphan GPCR gene with a high-binding affinity to S1P (Kd = 23–26 nM) encoded on a single exon (86). Although S1P3 is more related to S1P1, the intracellular signaling mediated by S1P3 is similar to those by S1P2 except for its capability of Rac activation. S1P3 couples with Gi/o, Gq, G12/13; activates ERK, SRE, and Rho/Rac; and induces cell prolifera-tion, survival, migration, and cell rounding (86, 87).

S1P3 is widely present in brain, heart, lung, thymus, spleen, kidney, testis, and skeletal muscle. S1P3 is expressed on vascular endothelial cells, medial SMCs, and cardiomyocytes (88). Although genetic ablation of the S1pr3 in animals does not show any obvious abnormality, S1pr3 deletion could abolish a variety of S1P effects on cardiovascular system, including negative chrono-tropic and hypertensive effects, constriction of basilar artery, and endothelium-dependent vasodilation (89, 90).

S1P3 appears to be protective against vascular endothelial injury. Specifi-cally, S1P3-mediated intracellular Ca2+ increases and Akt activation were sig-nificantly reduced in cells from S1P3 KO mice and these effects were associated with nitric oxide (NO)-mediated vasodilation (91, 92). In addition, HDL-induced vasodilative effect was abrogated by S1P3 deficiency, suggesting the crucial role of S1P3 in vascular tone regulation (93). Recent studies using S1P3-specific antagonist from Murakami and colleagues, namely TY52156, confirms the important function of S1P3 in regulation of vasoconstriction (94). S1P-stimulated Rho activation and Ca2+ elevation through the action of S1P3 resulted in vascular contraction, which effect was attenuated by TY52156 in primary cells from canine cerebral arteries. Interestingly, pretreatment with S1P3 antagonist significantly reduced FTY720-induced bradycardia via S1P3 in


vivo. Furthermore, S1P-mediated reduction of coronary flow in rat heart was significantly recovered by TY52156, which is consistent with the previous finding that S1P-mediated coronary flow reduction is through the action of S1P3 (93). In pathological conditions such as myocardial ischemia/reperfusion, S1P and HDL showed protective effect through the activation of both S1P2 and S1P3, inhibiting neutrophil migration and cardiomyocyte apoptosis, and was NO dependent (95).

Role of S1P3 in immune system has been on vigorous investigation. Recent findings from Niessen et al. suggested that S1P3 acts as a downstream effector upon protease-activated receptor 1 (PAR1)-induced sepsis lethality that induces IL-1β production and tissue factor upon severe LPS challenge. Specifi-cally, dendritic PAR1-S1P3 signaling was critical for regulation of the dendritic cell accumulation into draining lymph nodes and propagation of inflammation, intravascular coagulation and lethality (96). Interestingly, S1P3 KO animals have revealed that S1P3 mediates marginal zone and follicular B cell chemo-taxis, although B-cell egress is not altered. FTY720 treatment, however, pre-vented B cells from crossing the endothelium by altering B-cell motility and the interaction between B cells and LYVE-1+cortical lymphatics, suggesting that cortical lymphatic sinusoids around the lymph node follicles serve as B-cell egress sites (97).

2.3.4. S1P4

S1P4 (Edg-6/LPC1) was identified from differentiated human and murine dendritic cells and exhibited a high binding affinity to S1P (Kd = 13–63 nM) (41, 86). S1P4 couples with Gi/o, G12/13, and possibly Gs, mediates S1P-triggered ERK activation, PLC activation, AC activation, Rho activation, the small Rho family GTPase Cdc42 activation, and influences stress fiber formation and cell migration (86, 87). Expression of S1pr4 is restricted to thymus, lymph node, spleen, and lung, indicating its involvement in regulation of the immune system.

Given that, Golfier et al. provided evidence that S1P4 regulates the blood cell lineage development using S1pr4-null animals (98). Mice lacking S1pr4 were viable and fertile. However, S1pr4-null mice displayed increased number of morphologically aberrant megakaryocytes, reduced proplatelet formation, and a defect in platelet repopulation after thrombocytopenia, suggesting that S1P4 plays a potent role in shaping the lateral phases of megakaryocyte dif-ferentiation and platelet production (98). S1P4 function in immune cells has been further characterized by the work of Allende and colleagues. Neutrophil trafficking as well as proinflammatory cytokine release were significantly impaired by genetic deletion of S1pr4, but not S1pr1, in Sgpl-null background. These results suggest that S1P4 signaling contributes to proinflammatory responses caused by SPL deficiency (22). Additionally, Rivera et al. showed that, in CD4+ T cells, S1P4 signaling can reduce both interferon (IFN)-γ and


IL-4 production, and increase IL-10 production, whereas S1P1 signaling either inhibits production of IFN-γ or induces IL-4 production (99).

Interesting questions of the fundamental role of S1P4, in terms of the precise mechanisms and signaling mediated by S1P4, need to be further addressed. For example, what is the precise mechanism of S1P4 signaling-mediated mega-karyocyte differentiation? What and how can S1P4 be regulated during this process? Can other S1P receptors contribute to megakaryocytopoiesis, as the expression pattern of S1P1 and S1P2 appears to be differentially regulated? What is the function of S1P4 in other cell types under normal and pathologic conditions (i.e., pathogen challenge, hypoxia) and cancer? What is the mecha-nism by which S1P4 expression level is regulated in neutrophils? How does S1P4 preferentially sense the increased level of S1P among five S1P receptors? Drug development for S1P4-specific agonist and/or antagonist would be needed to address these questions and also be beneficial for therapeutic purposes in hematopoietic and immune disorders.

2.3.5. S1P5

S1P5 (Edg-8/LPB4) was isolated from rat PC12 cells as a high affinity S1P receptor (Kd = 2–10 nM) (86, 100). S1pr5 is expressed in specific tissues such as brain, lung, spleen, and skin. Specifically, S1pr5 was highly expressed in white matter tracts throughout the brain and oligodendrocyte lineage cells in rat brain, raising a possibility that S1P5 may play a role in oligodendrocyte matura-tion and myelination (9, 101). S1P5, in response to S1P, was critical for preoli-godendrocyte retraction via Rho kinase, and mature oligodendrocyte survival by Akt activation (102). Interestingly, S1P through the action of S1P5 facili-tated rapid retraction of Xenopus retinal neuron, suggesting that S1P through S1P5 may provide an important signaling cue for navigating axon in the visual system (15). However, S1pr5-null mice do not exhibit any obvious phenotypic defects, including myelination abnormalities (102), possibly due to functional redundancies with other S1P receptors in S1P5 KO mice.

In vivo studies from S1P5 KO mice have revealed that S1P5 is important for immune cell trafficking and positioning. Specifically, S1P5 expression appears to be dominant in natural killer cells (NK cells). NK cells play a role in the adaptive immune response and are accumulated within draining lymph nodes after immunization and infection. Walzer et al. found that mouse and human NK cells highly express S1pr5 and tissue distribution of NK cell population was altered when S1pr5 gene was ablated, although homing capability of S1P5-null NK cells to inflamed organs was comparable (103). Furthermore, Jenne et al. demonstrated that T-bet, a T-box-containing transcription factor that is important for the final step of the NK cell maturation, can directly elevate S1P5 expression, and mediates efficient NK cell egress from lymph nodes and bone marrow (104). Although there are some discrepancies regarding S1P1 involvement in the process of the activated NK cell egress (103, 104), these

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findings suggest that S1P5 has an impact on the NK cell behavior and controls NK cell trafficking.

2.4. CONCLUDING REMARKS

Discovery of S1P receptors and subsequent realization that FTY720 works by modulating S1P signaling have brought enormous achievements in the field of immunology but also cardiovascular biology, emphasizing the significant roles of the lysophospholipids throughout species. Furthermore, animal models spe-cifically targeting cognate S1P metabolic enzymes and S1P receptors have empowered better understanding of the physiological and pathological func-tions of S1P and its receptors in vivo. However, mechanistic studies of how the diverse action of S1P and differential regulation of S1P receptors can be incorporated into immune and vascular systems have just begun to be uncov-ererd. For example, the effect of S1P levels on differentiation and maturation of immune cells as well as a shift of T-cell response in association with antigen presenting cells, and morphological regulation of vascular endothelium during postnatal and pathological angiogenesis, remain to be explored. Additionally, definitive in vivo evidence of the role of intracellular S1P as a transcriptional regulator and the involvement of nuclear S1P receptors has been still missing. Screening of binding partners/factors for nuclear S1P needs to be examined.

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Lysophospholipid Receptors (Signaling and Biochemistry) || Sphingosine 1-Phosphate (S1P) Receptors