Insights into the pharmacological relevance of lysophospholipid receptors

Introduction

Lysophosphospholipids are cell membrane lipid derivatives that also act as extracellular signals, joining other lipid mediators that include prostaglandins, leukotrienes, platelet-activating factors, and endocannabinoids. Lysophospholipids (LP) are small (around 400 Da), 3-carbon backbone phospholipids derived from glycerophospholipids or sphingolipids, containing a single carbon chain of varied length and saturation. Examples of LPs include LPA (lysophosphatidic acid), S1P (sphingosine 1-phosphate), LPC (lysophosphatidylcholine), SPC (sphingosylphosphorylcholine), LPS (lysophosphatidylserine) and LPE (lysophosphatidylethanolamine) (Figure 1). This review will focus on LPA and S1P, both of which have been analysed extensively with respect to both ligand and receptor. However, it is notable that other LPs likely have distinct physiologies that remain to be elucidated, such as LPC, that is present at high concentration in blood (several hundred µM) (Croset et al., 2000), and has been reported to modulate cardiac potassium channels through several G proteins (Ding et al., 2011), suggesting involvement of one or more unidentified receptors. LPA and S1P have well documented in vivo functions mediated by cognate G protein-coupled receptors (GPCRs), despite their comparatively low concentrations (low µM range) (Okajima, 2002; Aoki, 2004). LPs are usually bound to lipoproteins in vivo. Serum LPA is bound by albumin, gelsolin and other proteins (Moolenaar et al., 2004). S1P is bound by HDL and albumin (Okajima, 2002; Levkau et al., 2004; Nofer et al., 2004; Theilmeier et al., 2006; Sato et al., 2007). These lipoproteins stabilize LPs in the hydrophilic environment and possibly protect them from overly rapid degradation. LPs in isolation are unstable and are rapidly degraded within a few hours or less in vivo (Pages et al., 2001). Dephosphorylation by lipid phosphate phosphatases (LPPs) inactivates LPA and S1P by conversion to monoacylglycerol (MAG) and sphingosine respectively (Morris et al., 2009). S1P can be permanently degraded to hexadecanal and phosphoethanolamine by S1P lyase (Brindley and Pilquil, 2009). LPA can also be converted to phosphatidic acid by an LPA acyltransferase, and the long chain fatty acyl group can be hydrolysed by lysophospholipases (Lin et al., 2010). Ligand availability, concentration and half-life may influence cellular responses mediated by LP receptors. The stabilization effect of lipoproteins is currently being studied (Moumtzi et al., 2007).

Figure 1

The molecular structure of major lysophospholipids. Space-filling molecular models and structural formulas of LPA, S1P, LPC, SPC, LPS and LPE and some major analogues are shown. High-affinity LP receptors are indicated in parentheses under the name of each ligand.

Initially, LPs were shown to be precursors and metabolites in the de novo biosynthesis of phospholipids. It was previously thought that the effect of LPs and their mechanisms of action were largely non-specific. However, other bioactive properties were subsequently discovered. For instance, LPA was shown to function as an anti-hypertensive agent (Sen et al., 1968; Tokumura et al., 1978). LPA also acts as a cell growth and motility factor present in serum, and the signalling cascades mediated by LPA were shown to involve G proteins (van Corven et al., 1989), suggesting that LPA's effects were mediated through GPCRs, although other GPCR-independent mechanisms were also possible considering the high concentrations often employed combined with a lack of identified receptors.

The first LP receptor was cloned from mouse brain cDNA by degenerate PCR with primers designed against GPCRs (Hecht et al., 1996). This receptor, originally designated VZG-1, and now called LPA₁, was the first LP receptor discovered. Within several years of this initial report, several members of an orphan GPCR receptor family, called ‘endothelial differentiation genes (Edg)’, as well as other names were identified as GPCRs for both LPA and S1P (An et al., 1997; Lee et al., 1998; Im et al., 2000a; Van Brocklyn et al., 2000; Ishii et al., 2004). For the LPA receptors, another group of less similar GPCR genes have also been identified (Noguchi et al., 2003; Kotarsky et al., 2006; Lee et al., 2006). This latter group is more closely related to the family of P2Y purinergic receptor genes, indicating that LPA receptors have evolved via two distinct lineages in the rhodopsin GPCR family. To date, 11 bona fide LP receptors have been reported, 6 for LPA (LPA_1–6) and 5 for S1P (S1P_1–5), with additional putative LP G protein-coupled receptors (GPCRs) existing in the literature (Anliker and Chun, 2004b; Ishii et al., 2004; Lee et al., 2006; Rivera and Chun, 2008). All of these LP receptors are GPCRs capable of interacting with a number of heterotrimeric G proteins. The current nomenclature reflects the receptor's cognate ligand and chronological order of the relevant receptor's identification (Chun et al., 2010) (Table 1). Protein receptor names are LPA_1–6 and S1P_1–5. Gene names for humans are LPAR1-6 and S1PR1-5, or, for mice, Lpar1-6 and S1pr1-5 (Chun et al., 2010). This nomenclature will be used throughout this review.

The synthesis of lipid mediators and subsequent receptor activation is tightly regulated under normal physiological conditions, and enzyme and/or receptor dysfunction can lead to a variety of disease conditions. LP signalling itself can also influence other signalling pathways, such as signalling crosstalk with S1P, and other pro-angiogenic growth factors, such as VEGF, EGF, IGF, PDGF, bFGF and IL-8 have been reported (Schwartz et al., 2001; Spiegel and Milstien, 2003; Pyne et al., 2007). Thus, the manipulation of lipid mediator signalling through enzyme inhibitors or receptor antagonists and agonists has great potential as a therapeutic approach to disease. This review summarizes our current knowledge of LP lipid mediators and the function of their cognate receptors. It also discusses the effects of genetic ablation or pharmacological inhibition of enzyme or receptor function on various pathophysiological processes. We will also discuss the normal physiological functions of LPA and S1P mediated by their cognate receptors and discuss diseases associated with these bioactive LP molecules and their pharmaceutical application or potential.

LP Receptors and GPCRs

LP receptor genes are distributed throughout the genome. The coding regions for the LPA receptors in the genomes of humans and mice are found in multiple exons (LPA_1–3,6) or single exons (LPA₄ and LPA₅), while the coding region of each of the 5 S1P receptors is contained within a single exon, with only non-coding exon(s) upstream (Contos and Chun, 2001; Contos et al., 2000b; 2002). Several structural characteristics are shared between LPA and S1P receptors, including an extracellular N-terminus, seven a-helical transmembrane helices, and an intracellular C-terminus (Pierce et al., 2002). No formal structural data have been reported for this family of GPCRs, although non-G protein binding, fusion-protein structures should be available for some receptors in the near future.

LP receptors each have a heterogeneous spatiotemporal gene expression pattern and the same cell may express multiple receptors. These data were derived by examining mRNA for the presence of specific receptor transcripts and combined with functional assays. Notably, no antibodies or antisera have been clearly proven for use in immunohistochemical studies of native proteins, although many can identify overexpressed proteins in cell lines. Some reports have suggested a nuclear localizing protein fraction of LPA₁, but more physiological studies are still needed to elucidate the biological relevance of this fraction (Gobeil et al., 2006).

LPA and S1P receptors couple to heterotrimeric G proteins, which consist of a Gα and associated Gβγ subunits. The heterotrimeric G proteins are thought to bind to the inner surface of the cell membrane. One receptor may couple to several different types of Gα protein subunits to form a complex signalling network (Figure 2). LPA_1,2,4,5,6 and S1P_2–5 all signal through Gα_12/13 to activate RhoA, a member of the family of Rho GTPases. LPA_1–5 and S1P_2,3 couple to Gα_q/11 to activate phospholipase C (PLC). LPA_1–4,6 and S1P_1–5 also couple with Gα_i to activate PLC, Ras, phosphoinositide-3 Kinase (PI3K), and to inhibit adenylyl cyclase (AC), but LPA₄ can also couple to Gαs to activate AC (Etienne-Manneville and Hall, 2002; Neves et al., 2002; Chun et al., 2010). When a ligand binds to a receptor, it exchanges GDP for GTP on the Gα subunit, and then Gα-GTP and Gβγ can activate the effector molecule complex for each signalling cascade. The desensitization of receptors is probably mediated by known mechanisms in other systems including phosphorylation of GPCRs by kinases and/or an uncoupling from G proteins by arrestins, followed by receptor internalization, recycling and/or degradation (Lefkowitz and Shenoy, 2005).

Figure 2

The network of LPA and S1P signalling through G protein-coupled receptors. Each LPA and S1P receptor couples to specific classes of G proteins. Ligand-binding activates or inhibits downstream second messenger molecules, and the most prominent cellular effects are illustrated. Rock, Rho-associated kinase; SRF, serum response factor; IP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C; DAG, diacylglycerol; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; PI3K, phosphoinositol 3-kinase; DAG, diacylglycerol.

Endogenous regulation of LPA and S1P in vivo

It was previously thought that the main source of lysophospholipids was from blood. Reported concentrations of LPA and S1P vary in the literature, however most publications report around 1000 nM (200–5000 nM) in blood, and 0.2–100 nmol·g ^{in tissues under basal conditions (}Das and Hajra, 1989; Eichholtz et al., 1993; Olivera et al., 1994; Yatomi et al., 1997; Murata et al., 2000; Min et al., 2002; Okajima, 2002; Aoki, 2004; Berdyshev et al., 2005; Bielawski et al., 2006). Platelets contain large amounts of LPA and S1P, both of which can be released following platelet activation (Benton et al., 1982; Yatomi et al., 1997). For this reason, it was believed that platelets were the major source of S1P. However, it has been shown that erythrocytes can synthesize S1P by enzymatic pathways (Stoffel et al., 1970), and it is now clear that sphingosine kinases (SPHKs) present in erythrocytes are responsible for S1P in blood (Pappu et al., 2007). In addition, it has been shown that erythrocytes are able to import and store S1P, which can then be actively released upon stimulation (Hanel et al., 2007). It was also believed that LPA in blood is mainly derived from activated platelets; however, it was reported that an LPA producing enzyme, lysophospholipase D (lysoPLD) in plasma, may also contribute to the total amount of LPA found in blood (Aoki, 2004). The plasma lysoPLD activity was directly measured, and half of this activity is attributed to autotaxin (ATX), one of the major lysoPLDs (Tanaka et al., 2006). In addition to the aforementioned pathway, there might be local LP synthesis in specific tissues. For example, the highest expression of ATX is found in the floor plate of the developing embryo and in the choroid plexus and osteoblasts throughout development (Bachner et al., 1999). Neurons are also a potential source of LPA in the developing brain, as nanomolar concentrations of LPA are found in conditioned medium from embryonic brain primary cultures as estimated in a biological activity assay (Fukushima et al., 2000).

As noted above, there is a wide concentration range of LPA and S1P reported in both blood and tissues. Possible reasons for the differences in concentration may include differences in platelet activation during blood collection, the presence or absence of catabolic enzymes in different tissues and the potential conversion of LPC to LPA by ATX. The inherently sticky nature of LPA also makes it particularly difficult to quantify, especially when levels are already low. LPA is also difficult to extract and differences in the pH of the extraction buffer can produce dramatic differences in sample recovery. It is also possible that LPA and S1P may vary with age, gender or strains of mice used in experiments.

As signalling mediators in vivo, the production and degradation of lysophospholipids is tightly controlled. LPs are produced by multi-step enzymatic pathways that are initiated by the de-esterification of membrane phospholipids. The metabolism of LPA has been partially characterized and involves a number of convergent biosynthetic pathways and enzymes of varied specificity (Meyer zu Heringdorf and Jakobs, 2007). To date, lysoPLD, ATX, phospholipase A1 (PLA1), phospholipase A2 (PLA2) and acylglycerol kinase (AGK) are enzymes reported to be involved in LP synthesis (Chun and Rosen, 2006). These enzymes, through multiple pathways, are responsible for LPA production (Meyer zu Heringdorf and Jakobs, 2007).

S1P metabolism involves a number of specific and highly conserved enzymes (Saba and Hla, 2004). Two SPHK isoforms, SPHK1 and SPHK 2, produce S1P from sphingosine (Kohama et al., 1998; Liu et al., 2000). Recently, it was shown by specific genetic removal of SPHK1 and 2 in erythrocytes that these cells are the major source of S1P in blood (Pappu et al., 2007). SPHK activity is not only present in blood, but also in most mouse tissues (with high activity in thymus and lung) (Fukuda et al., 2003).

The duration and strength of LP signalling likely depends, at least locally, on the activity of synthetic and degradative enzymes and their localization relative to the LP receptors. For example, it has been reported that S1P lyase (SPL) has an important role in maintaining a steep gradient of S1P between blood and tissues, which in part controls lymphocyte localization within lymphoid organs, particularly within lymph nodes where a low-to-high gradient exists in moving from the parenchyma to the efferent lymphatics (Schwab et al., 2005; Schwab and Cyster, 2007). The fine distribution and potential LP gradients in other tissues remain to be elucidated. Pharmacological and molecular manipulation of LP metabolic enzyme activity is also an intriguing approach for cancer therapy or other clinical treatments.

LP receptors and clinical relevance

LPA₁

In adult tissues, LPA₁ shows broad gene expression, with mRNA present in brain, lung, heart, uterus, and other organs of both human and mouse (Choi et al., 2010). Expression of the mouse homologue, Lpar1, is more spatially restricted during embryonic development, and is enriched in the brain (Table 2). Lpar1 was initially called ‘ventricular zone gene-1 (Vzg-1)’ because of its enrichment in the neural progenitor zone of the mouse embryonic cerebral cortex, the ‘ventricular zone’ (Hecht et al., 1996). The effects of Lpar1 gene deletion are seen in studies of the CNS (Contos et al., 2000a,b; Kingsbury et al., 2003; Estivill-Torrus et al., 2008; Matas-Rico et al., 2008; Santin et al., 2009; Castilla-Ortega et al., 2010; Dubin et al., 2010). Furthermore, the deletion of Lpar1 in mice causes a reduction in litter size with 50% perinatal death (Contos et al., 2000a,b; 2002). Intrathecal injection of LPA, but not S1P, initiates behavioural, morphological and biochemical symptoms of neuropathic pain via an LPA₁-mediated Rho/Rho-kinase (Inoue et al., 2004; 2006; 2008). LPC conversion to LPA by ATX is necessary for these processes in vivo (Nagai et al., 2010). LPA signalling modulation may be relevant to some forms of neuropathic pain, an area of significant, unmet medical need (Dworkin et al., 2007). LPA₁ has also been linked to multiple disease processes, including cancer (Hama et al., 2004), fibrosis in kidney (Pradere et al., 2007) and lung (Tager et al., 2008), and male infertility (Ye et al., 2008), in conjunction with loss of other LPA receptors. Recent data has identified LPA₁ signalling as a mediator of hypoxic damage in the fetal brain (Herr et al., 2011) and in the initiation of fetal hydrocephalus (Yung et al., 2011).

LPA₂

LPA₂ has relatively high gene expression in human testis and leukocytes and moderate expression in prostate, spleen, thymus, and pancreas. Compared to the expression pattern of LPA₁, LPA₂ has a more restricted spatiotemporal expression pattern (An et al., 1998; Contos et al., 2000b; Choi et al., 2010). In mice, Lpar2 is highly expressed in kidney, uterus and testis, and moderately expressed in lung while lower expression levels are found in stomach, spleen, thymus, brain and heart (Contos and Chun, 2000; Anliker and Chun, 2004a). Expression is also detected in embryonic brain but decreases within a week after birth (Contos et al., 2000b). In several cancer cell types, aberrant expression of LPA₂ has been reported, suggesting a possible tumour-promoting role for LPA₂ (Goetzl et al., 1999a; Contos et al., 2000b; Schulte et al., 2001; Kitayama et al., 2004; Shida et al., 2004; Yamashita et al., 2006; Hope et al., 2009). Constitutive loss of Lpar2 in mice does not cause a grossly abnormal phenotype. However, in combination with deletion of other LPA receptors, reproductive defects resulting from a decrease in germ cell survival are apparent in male mice (Ye et al., 2008). In ex vivo experiments, this receptor is also partially responsible for neural progenitor cell intracellular calcium responses, LPA-induced cortical folding (Kingsbury et al., 2003; Dubin et al., 2010), and contributes to synaptic functions in slice cultures derived from the adult CNS (Trimbuch et al., 2009). A reduced incidence of tumour formation in LPA₂ deficient mice in a mouse model of colitis-associated cancer demonstrates a link to this form of cancer (Lin et al., 2009). In addition, reduced eosinophil infiltration and prostaglandin E2 levels in the lung fluid of Lpar2 heterozygous antigen-challenged mice compared with that of control mice suggests that Lpar2 plays a role in proinflammatory responses (Zhao et al., 2009).

LPA₃

LPA₃ gene expression is broad in human tissues with transcripts present in heart, testis, prostate, pancreas, lung and ovary (Bandoh et al., 1999; Im et al., 2000b). In mice, Lpar3 is most abundant in uterus, testis, kidney, lung, stomach, spleen, brain and thymus (Contos et al., 2000b).

Interestingly, in the murine uterus, Lpar3 mRNA is exclusively expressed in the luminal endometrial epithelium at a crucial time in the embryo implantation window (Ye et al., 2005; Hama et al., 2007; Ye, 2008; Ye and Chun, 2010). Lpar3 expression appears to be positively and negatively regulated by progesterone and oestrogen and plays a critical role in blastocyst implantation and proper embryo spacing through COX-2, which generates prostaglandins (PGs) E₂ and I₂ (Shah and Catt, 2005; Ye et al., 2005; Hama et al., 2006; Hama et al., 2007). It was recently reported that the expression of several matrix metalloproteases are reduced in the Lpar₃ deficient uterus, which may lead to abnormal collagen subtype turnover and improper extracellular matrix remodelling which may also contribute to the observed implantation defect (Diao et al., 2010).

LPA₄

LPA₄ mRNA is more ubiquitously expressed than other LPA receptors; however, it is highly abundant in both human and mouse ovary (Noguchi et al., 2003). In mice, Lpar4 is also expressed in heart, skin, thymus, developing brain and embryonic fibroblasts (Lee et al., 2007a; 2008). Whole mount in situ hybridization confirmed expression in the developing brain, as well as in limb buds, somites and facial processes (Ohuchi et al., 2008). Gene deletion studies in mice show that Lpar4 can affect embryonic viability; however, this effect appears to be dependent on genetic background (Lee et al., 2008; Sumida et al., 2010). The identified partially penetrant effect on embryonic lethality is due to a failure of cellular recruitment to the developing blood and lymphatic vessels (Sumida et al., 2010).

Mouse embryo fibroblast (MEF) cells isolated from Lpar4 deficient mice are hypersensitive to LPA-induced cell motility (Lee et al., 2008). This result indicates that Lpar4 plays an inhibitory role in LPA-dependent migratory responses. This inhibitory effect was reported to inhibit LPA-induced migratory effects mediated through Lpar1. Interestingly, a human cancer cell line ectopically expressing Lpar4 was prevented from invading a Matrigel Basement Membrane Matrix (BD Biosciences, San Jose, CA, USA) (Lee et al., 2008).

LPA₅

In humans, gene expression for LPA₅ is observed in spleen, heart, small intestine, placenta, liver and colon. Lpar5 is relatively broadly expressed in murine tissues, including embryonic brain, small intestine, skin, spleen, stomach, thymus, lung, heart, liver and embryonic stem cells (Kotarsky et al., 2006; Lee et al., 2006). In addition to LPA, farnesyl pyrophosphate (FPP) and N-arachidonylglycine (NAG) have been reported as LPA₅ ligands (Oh et al., 2008). However, independent studies gave support to LPA₅ as being a legitimate LPA receptor (Williams et al., 2009; Yin et al., 2009). To date, constitutive Lpar5 gene knockout mice have not been reported. However, expression in immune tissues and dorsal root ganglion suggests a role for Lpar5 in these tissues (Kotarsky et al., 2006; Lee et al., 2006; Oh et al., 2008).

LPA₆

The orphan receptor P2Y5 was recently supported as a legitimate sixth LPA receptor and designated as LPA₆ by the International Union of Basic and Clinical Pharmacology (IUPHAR) committee on the basis of the emerging literature (Lee et al., 2007b; Pasternack et al., 2008; Yanagida et al., 2009; Chun et al., 2010). LPA₆ is more closely related to LPA₄ and LPA₅, which are members of the purinoreceptor (P2Y) family of receptors. Its gene structure is that of a nested gene located within intron 17 of the retinoblastoma gene, and it was reported that 2-acyl-LPA rather than 1-acyl-LPA is the preferred ligand of LPA₆ (Yanagida et al., 2009). It is noteworthy that homozygous inactivation of LPA₆ has been implicated in bladder cancer development through unclear mechanisms that may reduce the activity of the tumour suppressor gene, RB1 (Lee et al., 2007b). Human genetic studies indicate that LPA₆ influences forms of human hair growth (Pasternack et al., 2008; Shinkuma et al., 2010). Gene targeting of Lpar6 in mice has not been reported, but loss of the receptor may reflect the human hair-growth phenotype.

S1P₁

S1P₁ gene expression is observed in many tissues, with abundance in spleen, brain, heart, lung, adipose tissues, liver, thymus, kidney and skeletal muscle (Zhang et al., 1999; Ishii et al., 2001). Mice engineered to express the beta-galactosidase gene knocked into the S1pr1 locus show that S1pr1 protein exists in brain, lung, spleen, heart, vasculature and kidney in adult mice (Chae et al., 2004). In humans, lymphocytes/leukocytes also strongly express S1P₁ (Balthasar et al., 2006).

Constitutive S1P₁-null mice have severe defects in vascular maturation, and die between E12.5 to E14.5 in utero because of haemorrhaging (Liu et al., 2000). Endothelial cell-specific deletion of S1P₁ showed that this abnormality was due to a maturation defect in vascular endothelial cells (Allende et al., 2003). S1P₁ expression is localized to myocytes and perivascular SMCs, and bradycardia and hypertension in primates, including humans, is driven by S1P₁ activation (Brinkmann et al., 2010; Chun et al., 2010).

T cell-specific deletion showed that S1P₁ is crucial for egress of mature T cells from the thymus to the periphery (Allende et al., 2004a). S1P₁ expression on lymphocytes also plays a profound role in the control of lymphocyte recirculation. Elegant studies with S1P₁ conditional or SPHK knockout mice showed that S1P₁ and S1P are necessary for the egress of thymocytes from the thymus and for egress of T and B cells from lymphoid tissues (Brinkmann et al., 2004; Matloubian et al., 2004; Schwab and Cyster, 2007).

The proper S1P gradient between efferent lymphatics and lymph node, together with lymphocyte receptor expression, has been proposed to be important for lymphocyte migration (Schwab et al., 2005). It has been demonstrated that S1P stimulates migration of naïve T-cells via S1P₁. Following activation, T-cells temporarily suppress receptor expression which results in the loss of the S1P mediated migration response and retention of T cells in lymphoid organs. Terminally differentiated effector T-cells then up-regulate S1P₁ to egress from lymph nodes (Graeler and Goetzl, 2002; Matloubian et al., 2004; Schwab and Cyster, 2007).

FTY720, a precursor compound that upon in vivo phosphorylation acts acutely as an S1P receptor agonist, but chronically as a functional antagonist, has been shown to produce selective retention of T cells (and B-cells) in peripheral lymphoid organs. Several reports have shown that FTY720 is phosphorylated in vivo by SPHK2 but not SPHK1 to produce FTY720-P (Allende et al., 2004b; Kharel et al., 2005), and this phosphorylated form inhibits T and B-cell egress from lymph nodes by modulating S1P signalling (Zemann et al., 2006; Schwab and Cyster, 2007). In a recent study involving patients with relapsing MS, FTY720 was found to prevent the egress of CCR7-positive naive T cells and central memory T cells (TCM) from the lymph nodes, but not CCR7-negative effector memory T cells (TEMs) (Mehling et al., 2008; Brinkmann et al., 2010). Because FTY720 does not generally impair lymphocyte proliferation and/or functions, it is distinct from classical immunosuppressive therapies that are accompanied by opportunistic infections and cancers (Gardell et al., 2006; Rivera and Chun, 2007). Interestingly, in experimental autoimmune encephalomyelitis (EAE), a mouse MS model, nervous system specific-deletion of S1P₁– particularly from astrocytes – attenuates FTY720 efficacy suggesting the possibility of a non-immunological target for S1P-related MS therapies (Choi et al., 2011).

S1P₂

S1pr2 is abundantly expressed in the lung and heart, but less so in the brain of adult mice and rats (Okazaki et al., 1993; Zhang et al., 1999). However, during embryonic development, S1pr2 is prominently expressed in the brain (MacLennan et al., 1994).

S1P₂-null mice show a progressive loss of hearing and balance due to degeneration of vestibular and cochlear hair cells (MacLennan et al., 2006; Herr et al., 2007; Kono et al., 2007). This suggests that it may be possible to prevent the degeneration of hair cells with a selective S1P signalling modulator. These studies are ongoing, and may offer novel treatment modalities for the prevention of age-related and ototoxic hearing loss.

S1P₂-null mutants in the C57Bl/6J background have also been reported to show electrophysiological defects and develop seizures (MacLennan et al., 2001). These defects may be due to impairment of calcium-regulated signal transduction, but the S1P₂ pathways involved in neuronal excitability have not yet been determined (An et al., 1999).

In zebrafish, a single-point mutation in the S1P₂-related mil gene leads to abnormal heart development (Kupperman et al., 2000), however, this defect is not recapitulated in S1P₂ knockout mice (Ishii et al., 2002).

S1P levels are up-regulated in the airways of asthmatic patients following allergen exposure. Cross-linking of IgE receptors on mast cells activates SPHK1 and increases S1P levels. Activation of S1P₂, and to a lesser extent S1P₁, promotes degranulation and chemotaxis of mast cells (Jolly et al., 2002; 2004). Also, airway SMC express S1P_1–4, and they could modulate the SMC contraction and proliferation through the G_12/13 and G_i/o pathways (Jolly et al., 2002).

S1P₃

In mice, S1pr3 is highly expressed in heart, lung, spleen, kidney, intestine, diaphragm and certain cartilagnous regions (Ishii et al., 2001). LPA and S1P have vaso-regulatory functions, such as regulation of heart rate, blood pressure, platelet aggregation and smooth muscle contraction (Siess et al., 2000; Karliner, 2004). HDL, which includes S1P as a component, induces vasodilation, in part, by activation of Akt through S1P₃ (Levkau et al., 2004; Nofer et al., 2004). Importantly, in an in vivo ischemia/reperfusion (I/R) mouse model of cardiac injury, HDL, and to a greater extent S1P, protected the heart from injury. HDL and S1P reduced infarction size by inhibiting inflammation caused by polymorphonuclear leukocyte recruitment and cardiomyocyte apoptosis in a NO-dependent manner (Theilmeier et al., 2006). In another I/R mouse study, it was shown that infarct size is significantly increased in mice lacking both S1pr2 and S1pr3, but not in mice deficient in only one of these receptors. Furthermore, S1P-induced cardiomyocyte Akt activation was only ablated when both receptors were deleted, suggesting a redundant protective role for S1pr2 and S1pr3 (Means et al., 2007). These studies demonstrated that vasorelaxation, myocardial cell survival, and protection from ischemic damage are mediated by the S1P₃-Akt-eNOS/NO pathway (Levkau et al., 2004; Nofer et al., 2004; Theilmeier et al., 2006; Means et al., 2007). S1P₃ influences on cardiac fibrosis (Takuwa et al., 2009) and myofibroblasts have also been reported (Keller et al., 2007; Niessen et al., 2008).

S1P₄

S1P₄ gene expression is enriched in lymphoid tissues, including immune system cells (Graler et al., 1998; Graeler and Goetzl, 2002). It is also present in SMCs of the human lung (Jolly et al., 2002). Despite wide expression of S1pr4 throughout the cells of the immune system, no obvious immune cell deficiencies have been reported in S1pr4 null mutant mice, and the only apparent immune cell abnormality is a perturbation in megarkaryocyte cell morphology. However, in vitro megakaryocytes from S1pr4-deficient mice have reduced pro-platelet formation, and platelet recovery is delayed in an experimental thrombocytopenia model (Golfier et al., 2010). These data suggest that in vivo S1pr4 plays a role in platelet formation; however, functional redundancies with other S1P receptors likely exist. Crosses of S1p4 mice with other S1P receptor knockout mice may reveal other roles for S1pr4 in the immune system.

S1P₅

S1P₅ gene expression is enriched in the spleen and white matter tracts of the CNS, primarily on oligodendrocytes (Im et al., 2000a; Malek et al., 2001; Jaillard et al., 2005). In a recent study using S1pr5-constitutive null-mutant mice, it was found that this receptor influences natural killer (NK) cell egress through a T-bet/Tbx21 transcription factor mechanism involving various immunological compartments (Jenne et al., 2009). The functions of S1pr5 in oligodendrocytes under normal and pathological conditions remain to be determined.

Possible relevance of LP signalling to human diseases

Both LPA and S1P have been shown to act as immunomodulators in the regulation of T cells, B cells and macrophages. Overlapping expression patterns suggest that immune cells are likely regulated by combinations of LP receptors. For example, LPA and S1P acting through LPA_1,2 and S1P_2,3, respectively, may serve as survival factors for T-cells by suppressing Bax (Goetzl et al., 1999b). LPA induces migration and suppression of IL-2 production in unstimulated T cells via LPA₂, however, following T cell stimulation, LPA inhibits cell migration but activates IL-2 production via LPA₁ (Zheng et al., 2000; 2001). LP receptor expression can also dynamically vary with changes in cell activation (Zheng et al., 2000; Graler and Goetzl, 2002; Rosen et al., 2003).

As mentioned previously, LP signalling has relevance to cancer. One of the better-characterized possible cancer links is to ovarian cancer. LPA elevation in the ascites of patients was reported to elicit growth factor-like activity (Mills et al., 1988), although there is controversy over the reproducibility of this initial report. It has also been shown that LP receptors and the enzymes involved in LPA and S1P metabolism are highly expressed in multiple cancer types (e.g. ovarian cancer and glioblastoma cells have high levels LPA₂ and LPA₃) (Murph et al., 2006). S1P has both positive and negative effects on cancer cell growth (Hong et al., 1999). FTY720 has anti-tumour effects in vitro and in vivo, and this may be due to a direct effect on tumour cells themselves, and also to indirect effects, like the inhibition of angiogenesis (Azuma et al., 2002; Ho et al., 2005; LaMontagne et al., 2006).

S1P can act to prevent intrinsic-, chemical- and irradiation-induced oocyte apoptosis. S1P injection into the ovarian bursa, ahead of ionizing irradiation, improves the rate of successful pregnancy in irradiated mice (Morita et al., 2000; Tilly, 2001). These data complement effects of LPA signalling on implantation and spermatogenesis (Ye et al., 2005; 2008) and support possible roles in the treatment of human infertility.

LPA and S1P in blood may enter the brain during CNS injury and cause pathological effects. Experimentally induced brain haemorrhage can provide an influx of 1–10 µM of LPA in the CSF (Tigyi et al., 1995). In cerebral infarction, platelet aggregation can release micromolar concentrations of LPA and can also lead to increased LPA levels in CSF (Eichholtz et al., 1993). Indeed, intracranial injection of LPA or S1P causes astrogliosis in vivo (Sorensen et al., 2003). Reactive astrogliosis is a prominent component of CNS injury, including diseases like multiple sclerosis, and recent data support S1P signalling as a therapeutically tractable mechanism in reducing astrogliosis (Choi et al., 2011). Ongoing studies of LP signalling should reveal novel aspects of this therapeutic approach.

LPA pharmacological tools

Over 1% of genes in the human genome are estimated to encode GPCRs (over 1000). Their therapeutic value is proven, but not yet optimally realized because drugs in the clinic target only around 30 of the approximately 400 orphan GPCRs in the genome (Klabunde and Hessler, 2002). Even so, GPCRs are the target of half of all the drugs currently on the market, and remain the focus of major pharmaceutical companies (Klabunde and Hessler, 2002; Gloriam et al., 2009). Chemical tools have been developed towards providing experimental uses and proof-of-concept data. Several LPA receptor agonists or antagonists have been reported, although most show modest selectivity and a relative lack of in vivo validation, which must be considered in any experimental usage and particularly for in vivo studies.

One LPA receptor agonist is N-acyl ethanolamide phosphate (NAEPA), an LPA analogue that has an ethanol amine backbone (Lynch et al., 1997). To identify receptor selective compounds, a screen with 2-oleoyl LPA, a derivative that had a pyran ring to stabilize the head group, was performed, and one LPA₁-selective agonist, two LPA₃-selective LPA agonists and one LPA₃-selective antagonist were identified (Tamaruya et al., 2004).

Other LPA receptor antagonists have also been reported. For example, antagonists to both LPA₁ and LPA₃ include VPC-12449, a compound that can protect against LPA₃-mediated renal ischemia-reperfusion injury in a mouse model (Okusa et al., 2003), a natural lipid metabolite, diacylglycerol pyrophosphate (DGPP) (Fischer et al., 2001), and Ki16425, an antagonist with little resemblance to LPA (Ohta et al., 2003) that can inhibit breast cancer cell proliferation and metastasis in mice (Boucharaba et al., 2006). A unique LPA analogue, diastereoisomaeric α-bromophosphonates (BrP-LPA) showed antagonistic activity to LPA_1–4, and notably inhibited lysophospholipase D activity of autotaxin at a nanomolar level. The anti-isomer of BrP-LPA showed superior efficacy to paclitaxel in reducing the size and blood vessel density of tumours in a mouse orthotopic breast cancer xenograft model (Zhang et al., 2009). Recently, two oral LPA₁ selective antagonists, with chemical structures having little resemblance to LPA, were reportedly used for treatment of lung fibrosis in a rodent model. One compound, AM095, showed anti-fibrotic activity without affecting wound-healing processes (Swaney et al., 2010, 2011).

S1P pharmacological tools

FTY720 (fingolimod) is the best-characterized S1P receptor tool and deserves special consideration (Brinkmann, 2009; Brinkmann et al., 2010; Chun and Hartung, 2010). The US Food and Drug Administration recently approved FTY720 (fingolimod) as an orally active immunomodulatory drug for the treatment of relapsing forms of multiple sclerosis (Novartis). It binds 4 of the 5 identified S1P receptors (S1P₁ and S1P_3–5) with high affinity (0.3–3.1 nmol·L^{). A unique feature of FTY720 is that, following its} in vivo phosphorylation, it first binds S1P receptors and transiently activates downstream pathways; however with continued exposure, it down-regulates the S1P receptor by irreversible internalization and proteasomal degradation (Graler and Goetzl, 2004; Oo et al., 2007). Therefore, FTY720 produces S1P receptor functional antagonism. FTY720 was initially reported to originate from the fungal species known as Cordyceps, part of the larger division Ascomycota (Ascomycetes) in 1995 (Adachi et al., 1995; Suzuki et al., 1996).

Another S1P₁ agonist, AAL-(R), is a non-selective S1P receptor agonist with structural and functional similarities to FTY720 (Brinkmann et al., 2002; Rosen et al., 2003). An S1P₁ partially selective agonist, KRP-203, sequesters circulating lymphocytes into peripheral lymphoid organs (Shimizu et al., 2005), and prevents allograft rejection, but does not affect S1P₃ signalling (Fujishiro et al., 2006). KRP203 (S1P₁ > S1P₃) is currently in Phase II clinical trials for the treatment of subacute cutaneous lupus erythematosus (Novartis). An S1P₁-specific antagonist called W146 can induce loss of capillary integrity (Sanna et al., 2006). SEW2871 and AUY954 are S1P₁ specific agonists and have been shown to functionally prevent lymphocyte egress and inhibit allograft rejection, respectively (Sanna et al., 2004; Pan et al., 2006). AUY954 inhibits receptor recycling following internalization, similar to phosphorylated FTY720, thus acting as a functional antagonist for S1P₁ (Choi et al., 2011). These data suggest that at least some FTY720-related agonists can also remove receptors from further agonism, thus producing functional antagonism, as described above.

Aside from mild bradycardia, FTY720 produces few undesirable side effects in humans. In mice, bradycardia is mediated through S1P₃; however, several unpublished reports in primates, including humans, using S1P₁^{selective antagonists show that it is mediated through S1P}₁ (Chun and Hartung, 2010). The mechanism through which FTY720 operates in multiple sclerosis remains unclear, but likely involves both immunological as well as CNS effects (Chun, 2007; Choi et al., 2011), and this remains an active research area. FTY720 represents the first generation of LP receptor modulators that may have therapeutic value. Other data concerning the efficacy of FTY720 administration for the treatment of type I diabetes, uveoretinitis, thyroiditis, myocarditis, systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis in animal models have been reported (Suzuki et al., 1998; Hozumi et al., 1999; Kurose et al., 2000; Matsuura et al., 2000; Okazaki et al., 2002; Fujino et al., 2003; Webb et al., 2004).

For the other S1P receptors, JTE-013 is an S1P₂ specific antagonist (Yokoo et al., 2004). Recently, it was reported that JTE-013 ameliorates pancreatic Beta cell failure in a mouse diabetes model (Imasawa et al., 2010). In addition, there are a number of agonists/antagonists that have been described with varying affinities for the different receptor subtypes (Im et al., 2001; Clemens et al., 2003; 2004; Davis et al., 2005).

Several oral-S1P lyase inhibitors have been developed and clinical trials for the treatment of rheumatoid arthritis have begun. These compounds can increase tissue S1P levels predominantly in lymphoid tissue without other overt physiological effects in rodents. Human clinical trials for the S1P lyase inhibitor LX2931 are being pursued (Bagdanoff et al., 2010).

Another way to inhibit S1P signalling may be through the use of S1P specific monoclonal antibodies. Anti-S1P mAbs have been reported to arrest tumour-associated angiogenesis in a mouse xenograft model (Visentin et al., 2006). Validation of the mechanism and efficacy profile await further study.

Current and future efforts to modulate LP signalling should provide attractive and tractable tools and potentially drugs for the treatment of medically important diseases (Chun and Rosen, 2006; Delgado et al., 2007; Herr and Chun, 2007).