Sphingosine 1-Phosphate Receptor 1 Signaling in Mammalian Cells

The bioactive lipid, sphingosine 1-phosphate (S1P) binds to a family of G protein-coupled receptors, termed S1P1-S1P5. These receptors function in, for example, the cardiovascular system to regulate vascular barrier integrity and tone, the nervous system to regulate neuronal differentiation, myelination and oligodendrocyte/glial cell survival and the immune system to regulate T- and B-cell subsets and trafficking. S1P receptors also participate in the pathophysiology of autoimmunity, inflammatory disease, cancer, neurodegeneration and others. In this review, we describe how S1P1 can form a complex with G-protein and β-arrestin, which function together to regulate effector pathways. We also discuss the role of the S1P1-Platelet derived growth factor receptor β functional complex (which deploys G-protein/β-arrestin and receptor tyrosine kinase signaling) in regulating cell migration. Possible mechanisms by which different S1P-chaperones, such as Apolipoprotein M-High-Density Lipoprotein induce biological programmes in cells are also described. Finally, the role of S1P1 in health and disease and as a target for clinical intervention is appraised.


Introduction
The bioactive lipid, sphingosine 1-phosphate (S1P), is produced by phosphorylation of sphingosine, catalysed by two isoforms of sphingosine kinase (SK1 and SK2), whereas its degradation involves cleavage by S1P lyase to produce (E)-2-hexadecenal and phosphoethanolamine [1]. S1P is also reversibly dephosphorylated by S1P phosphatase to regenerate sphingosine, the levels of which are additionally controlled by flux through the de novo ceramide synthesis and sphingosine salvage pathways. S1P in the plasma is produced by erythrocytes, vascular endothelial cells and, to a lesser extent, platelets (which lack S1P lyase). Release of S1P from activated platelets requires calcium-dependent and ATP (adenosine triphosphate)-dependent transporters whereas S1P is constitutively released in an ATP-dependent manner from erythrocytes, likely involving an ATP-binding cassette (ABC) type transporter [2]. The Spinster homologue 2 (Spns2) transporter passively exports S1P from vascular endothelial cells [3] (reviewed in [4]) and Spns2 knockout mice are protected from airway inflammation, colitis, arthritis and experimental autoimmune encephalopathy (EAE) [5]. Thus, inhibitors of Spns2 may be usefully exploited to treat inflammatory diseases. Moreover, knockout of lymphatic endothelial Spns2 reduces pulmonary metastasis via a mechanism that involves induction of a lymphopenia and an increase in effector T cell and natural killer (NK) cell number to enhance tumour cell killing in the lung [6]. S1P released from cells functions to stimulate a family of G protein-coupled receptors (GPCR), the S1P receptors (S1P 1 -S1P 5 ) [7,8] on neighboring cells to induce biological responses such as growth, differentiation, cell migration and trafficking [1]. A major advance in understanding ligand binding endosomes. These workers also suggested that as class A receptors lack these Ser/Thr clusters in the C-terminal tail then weak binding of β-arrestin in the 'core' conformation (with limited C-terminal tail interaction) predominates and prevents G-protein interaction. In this regard, class A GPCRs can use G-protein or β-arrestin to separately regulate the ERK-1/2 pathway [23]. These receptors will therefore, exist either in a GPCR-G-protein or GPCR-β-arrestin conformation. Thus, β-arrestin-biased agonists stabilise the GPCR-β-arrestin conformation and have inverse agonist activity on the GPCR-G-protein conformation as these receptor conformations exist in equilibrium. For example, the β-arrestin-biased angiotensin 1 receptor (AT1R) agonist Sar 1 ,Ile 4 ,Ile 8 angiotensin II (SII) [24] stimulates ERK-1/2 activation and salt intake in the brain but blocks G protein-stimulated inositol phosphate formation and water intake [25].
It is possible that class B GPCRs might use both β-arrestin and G-protein βγ (Gβγ) sub-units together in an integrated manner to activate effector pathways. Indeed, β-arrestin 1 is positioned adjacent to the Gβγ subunits and several class averages indicate a direct interface between these proteins [22]. Moreover, biochemical analysis using the non-hydrolysable mimetic of guanosine triphosphate, guanosine diphosphate-tetrafluoroaluminate to dissociate Gα from Gβγ promotes interaction between β-arrestin 1 and Gβγ [22]. Therefore, it is possible that the association of Gβγ with β-arrestin might enable these proteins to function together in an integrated manner. Indeed, we demonstrated some years ago that S1P 1 uses Gβγ and β-arrestin as multipliers of signal output to regulate ERK-1/2 in HEK 293 cells and airway smooth muscle cells [26,27] and this likely requires both G-protein and β-arrestin to be accommodated on S1P 1 at the same time.
Clusters of Ser/Thr (e.g., SSS, SXSS, SSXS) in the C-terminal tail of GPCRs (which serve as phosphorylation acceptor sites) represent a barcode for formation of stable complexes between GPCR and β-arrestin [13]. In this regard, S1P 1 contains an SSS motif at the extreme C-terminus ( Figure 2). Furthermore, deletion of 12 amino-acids at the C-terminal tail reduces the S1P-stimulated phosphorylation of S1P 1 and abolishes endocytosis of the receptor [30]. These findings identify the last 12 amino acids in the C-terminal tail of S1P 1 as being critical for endocytosis and suggest that the SSS motif might represent a key β-arrestin interaction site. However, there is also evidence that stable GPCR-β-arrestin complexes can be formed without requirement for phosphorylation in the C-terminal tail. For instance, phosphorylation deficient receptor mutants for substance P [31], lutropin [32] and leukotriene B4 [33] still interact to form stable complexes with β-arrestin.
A key signal tranducer of S1P 1 is c-Src, which is recruited in response to S1P [26] and can receive stimulatory signals from Gβγ and β-arrestin, with the latter acting as an adapter for c-Src. S1P also stimulates a pertussis toxin-sensitive accumulation of dynamin-2 in lamellipodia in airway smooth muscle cells expressing S1P 1 [26] (Figure 1). In addition, β-arrestin is a clathrin adaptor [12], which functions to load endosomes with GPCR. This provides an alternative mechanism by which β-arrestin can function as a multiplier of signal output by virtue of its ability to regulate the formation of endosomes containing competent S1P 1 that can stimulate the ERK-1/2 pathway by a G-protein-dependent mechanism.

Figure 1.
Schematic demonstrating the role of inhibitory G-protein (Gi) and β-arrestin in regulating sphingosine 1-phosphate receptor 1 (S1P1) signaling in mammalian cells. β-Arrestin (Arr) associates with S1P1 and recruits c-Src to the receptor in response to S1P ligation of the receptor. G-protein βγ subunits are essential for subsequent activation of c-Src, Raf, MEK (mitogen activated protein kinase kinase) and ERK-1/2 (extracellular signal regulated kinase-1/2). S1P1 is internalised in endosomes via a β-arrestin-dependent mechanism and extracellular signal regulated kinase-1/2 (ERK-1/2) is recruited to the complex. . S1P1 C-terminal tail showing a Ser cluster at the extreme C-terminus possibly required for stable interaction with β-arrestin. The sequence underlined in the S1P1 C-terminal tail is essential for endocytosis of S1P1. Comparison is made with the class A β2 adrenergic receptor (β2AR) (that lacks Ser/Thr clusters) and the class B vasopressin receptor 2 (V2R) (which contains Ser/Thr clusters).
The role of β-arrestin/dynamin-2 in S1P1 signaling has recently been confirmed by others. Reeves et al. used spinning disc confocal fluorescence microscopy and flow cytometry to demonstrate that S1P1 function was inhibited in cells depleted of β-arrestin 1/β-arrestin 2 or clathrin or AP-2 or by treating cells with the dynamin-2 inhibitor, dynasore-OH [34]. In addition, dynamin-2 was shown to be essential for low concentrations of S1P to induce S1P1 internalisation and continuous signaling in Figure 1. Schematic demonstrating the role of inhibitory G-protein (G i ) and β-arrestin in regulating sphingosine 1-phosphate receptor 1 (S1P 1 ) signaling in mammalian cells. β-Arrestin (Arr) associates with S1P 1 and recruits c-Src to the receptor in response to S1P ligation of the receptor. G-protein βγ subunits are essential for subsequent activation of c-Src, Raf, MEK (mitogen activated protein kinase kinase) and ERK-1/2 (extracellular signal regulated kinase-1/2). S1P 1 is internalised in endosomes via a β-arrestin-dependent mechanism and extracellular signal regulated kinase-1/2 (ERK-1/2) is recruited to the complex. Schematic demonstrating the role of inhibitory G-protein (Gi) and β-arrestin in regulating sphingosine 1-phosphate receptor 1 (S1P1) signaling in mammalian cells. β-Arrestin (Arr) associates with S1P1 and recruits c-Src to the receptor in response to S1P ligation of the receptor. G-protein βγ subunits are essential for subsequent activation of c-Src, Raf, MEK (mitogen activated protein kinase kinase) and ERK-1/2 (extracellular signal regulated kinase-1/2). S1P1 is internalised in endosomes via a β-arrestin-dependent mechanism and extracellular signal regulated kinase-1/2 (ERK-1/2) is recruited to the complex. . S1P1 C-terminal tail showing a Ser cluster at the extreme C-terminus possibly required for stable interaction with β-arrestin. The sequence underlined in the S1P1 C-terminal tail is essential for endocytosis of S1P1. Comparison is made with the class A β2 adrenergic receptor (β2AR) (that lacks Ser/Thr clusters) and the class B vasopressin receptor 2 (V2R) (which contains Ser/Thr clusters).
The role of β-arrestin/dynamin-2 in S1P1 signaling has recently been confirmed by others. Reeves et al. used spinning disc confocal fluorescence microscopy and flow cytometry to demonstrate that S1P1 function was inhibited in cells depleted of β-arrestin 1/β-arrestin 2 or clathrin or AP-2 or by treating cells with the dynamin-2 inhibitor, dynasore-OH [34]. In addition, dynamin-2 was shown to be essential for low concentrations of S1P to induce S1P1 internalisation and continuous signaling in Figure 2. S1P 1 C-terminal tail showing a Ser cluster at the extreme C-terminus possibly required for stable interaction with β-arrestin. The sequence underlined in the S1P 1 C-terminal tail is essential for endocytosis of S1P 1 . Comparison is made with the class A β2 adrenergic receptor (β2AR) (that lacks Ser/Thr clusters) and the class B vasopressin receptor 2 (V2R) (which contains Ser/Thr clusters).
The role of β-arrestin/dynamin-2 in S1P 1 signaling has recently been confirmed by others. Reeves et al. used spinning disc confocal fluorescence microscopy and flow cytometry to demonstrate that S1P 1 function was inhibited in cells depleted of β-arrestin 1/β-arrestin 2 or clathrin or AP-2 or by treating cells with the dynamin-2 inhibitor, dynasore-OH [34]. In addition, dynamin-2 was shown to be essential for low concentrations of S1P to induce S1P 1 internalisation and continuous signaling in T cells, thereby regulating their egress from both thymus and lymph nodes [35]. In contrast, S1P was only capable of inducing a pulse of S1P 1 signaling in T cells deficient in dynamin-2 and this was insufficient to promote T cell egress [35]. Thus, activated S1P 1 uses a canonical route of clathrin-and dynamin-2-dependent endocytosis for persistent signaling.
We have proposed that the S1P 1 modulator SB649146 binds exclusively to and stabilises a low efficacy G i coupling conformation of S1P 1 and we demonstrated that SB649146 promotes endocytosis of S1P 1 and β-arrestin, typical of stable interaction [28,36]. The poor stimulatory effect of SB649146 on G i yields a low signal output resulting in a weak activation of ERK-1/2. It is possible that SB649146 induces the endocytosis of S1P 1 (to a very much lesser extent than induced by S1P, possibly as G i might also be involved in regulating loading of S1P 1 in endocytic vesicles) via a β-arrestin-dependent mechanism, but that G-protein activation is poor and therefore stimulation of ERK-1/2 is weak. One can consider SB649146 as inducing weak internalisation of compromised S1P 1 . We also proposed that S1P binds exclusively to a high efficacy G i and β-arrestin coupling conformation of S1P 1 , thus stabilising it and inducing a strong activation of ERK-1/2 [28,36]. Indeed, while we have demonstrated that SB649146 is a weak agonist for ERK-1/2 activation, it can competitively antagonise the S1P-stimulated activation of ERK-1/2 (by altering the equilibrium transition of each respective receptor conformation that can specifically bind S1P or SB649146 by mass action) [28,36]. Therefore, SB649146 might be a bona fide pharmacological modulator of stable GPCR-G-protein-β-arrestin complexes. Such pharmacological agents could function to 'dial up' or 'dial down' respective amounts of active G-protein and β-arrestin to determine signal output.
The S1P 1 modulator SB649146 might also reduce the availability of Gβγ subunits for use by the PDGFRβ (which is in a complex with the high efficacy G i /β-arrestin coupling conformation of S1P 1 ), thereby inhibiting PDGF-stimulated activation of ERK-1/2 and cell migration [28,36]. We have proposed that SB649146 binds to the low efficacy G i coupling conformation of S1P 1 , (which does not bind PDGFRβ) and reduces the concentration of the high efficacy G i /β-arrestin S1P 1 conformation associated with PDGFRβ by mass action [28,36]. We have demonstrated existence of the different conformational states of S1P 1 using immunofluorescent staining in airway smooth muscle cells. Treatment of cells with PDGF stimulates the endocytosis of the S1P 1 -PDGFRβ complex [26,27,36]. This pool of S1P 1 is likely to represent the high efficacy G i /β-arrestin coupling conformation. In contrast, SB649146 weakly stimulates the endocytosis of S1P 1 and this occurs in the absence of PDGFRβ [36]. This second pool of S1P 1 is likely to represent the low efficacy G i coupling conformation [28,36]. In addition, SB649146 inhibits the PDGF-stimulated endocytosis of the S1P 1 -PDGFRβ complex and also reduces the S1P-stimulated endocytosis of S1P 1 [36].
Interestingly, recent studies have shown that S1P 1 can be regulated by tyrosine phosphorylation, thereby providing additional evidence of a role for receptor tyrosine kinase (RTK) or c-Src in the regulation of S1P 1 function [41]. The Y143 site was shown to be required for S1P 1 internalization in response to S1P and this was associated with defective endothelial barrier enhancement induced by S1P. Overexpression of phosphorylation deficient (Y143F) or phosphorylation mimicking (Y143D) mutants failed to internalise or exhibited very high receptor internalisation respectively [41]. Therefore, Y143 regulates cell surface expression of S1P 1 and this is required for the endothelial barrier repair function of S1P.
There are other examples of S1P 1 forming complexes with other RTK. For example, in follicular thyroid carcinoma ML-1 cells, the vascular endothelial growth factor receptor-2 (VEGFR-2) forms a complex with S1P 1 . The S1P 1 -VEGFR-2 complex interacts with ERK-1/2 and protein kinase Cα [42]. In addition, S1P treatment of mouse embryonic stem (ES) cells promotes β-arrestin binding to S1P 1/3 and this leads to activation of c-Src [43]. This is associated with the stimulation of cell proliferation. S1P also increases the binding of S1P 1/3 with VEGFR-2 and promotes VEGFR-2 phosphorylation, which was blocked by β-arrestin siRNA, and the c-Src inhibitor, PP2 [43]. There are other examples of GPCR-RTK complexes. For instance, insulin-like growth factor 1 (IGF-1) is associated with the constitutively active G i coupled chemokine receptor type 4 (CXCR4). This complex promotes migration of MDA-MB-231 breast cancer cells [44]. Furthermore, constitutively active pituitary adenylate cyclase-activating peptide type 1 (PAC1) receptor associates with IGF-1R to regulate neuronal survival [45]. Interestingly, both CXCR4 and PAC1 are class B receptors based on the presence of serine clusters in the C-terminal tail required for stable β-arrestin interaction, and suggesting that along with S1P 1 , there might be specificity for class B receptors with RTKs.
Other S1P receptor sub-types, such as S1P 2 deploy endosomal signaling [43]. In this case, phosphorylation of ezrin (of the ezrin-radixin-moesin family of adapter molecules, required for cancer cell invasion) in response to EGF requires SK2 and intracellular S1P 2 and involves an intracrine action of intracellular S1P possibly made available by a close proximity localisation of Spns2 with S1P 2 in endosomes [46].

Sphingosine 1-Phosphate Receptor 1 and Regulator of G-Protein Signaling 12
We have previously shown that Regulator of G-protein Signaling 12 (RGS12) modulates PDGFRβ signaling [47]. Firstly, over-expression of RGS12, RGS12 (Post synaptic density protein (PDZ)/Phosphotyrosine binding domain (PTB) N-terminus or RGS12 PTB domain decreased ERK-1/2 activation in response to PDGF in airway smooth muscle cells. Secondly, the RGS12 PDZ/PTB domain N-terminus and RGS12 PDZ domain associate with the PDGFRβ [47]. In addition recombinant RGS12 and the isolated PDZ/PTB domain N-terminus co-localise with PDGFRβ in cytoplasmic vesicles [47]. Similarly, we show here that S1P 1 co-localises with recombinant RGS12 in these cytoplasmic vesicles and overexpression of RGS12 reduces S1P-stimulation of ERK-1/2 in airway smooth muscle cells ( Figure 3). . RGS-12 (Regulator of G-protein signaling-12) co-localises with the S1P1 in airway smooth muscle cells and reduces S1P-stimulation of the ERK-1/2 pathway. Airway smooth muscle cells were transfected with plasmid constructs encoding myc-tagged S1P1 and hemagglutinin (HA) tagged RGS12 and stimulated with S1P (1 μM, 5 min). The data shows that RGS12 co-localises with S1P1 in cytoplasmic vesicles. It remains to be determined whether S1P1 in these vesicles is competent to signal. However, RGS12 dampens the activation of ERK-1/2 by S1P in these cells. The results are representative of three independent experiments.

Sphingosine 1-Phosphate Receptor 1 and Sphingosine 1-Phosphate Carriers/Chaperones
Plasma S1P is associated with carrier/chaperone proteins, such as albumin and high density lipoprotein (HDL). Christofferson et al. [48] were the first to show that HDL-S1P was bound to apolipoprotein M (ApoM). S1P binds to an amphiphilic pocket in the lipocalin fold of ApoM. From a functional perspective, ApoM-HDL stimulated endocytosis of S1P1 and promoted activation of ERK-1/2, protein kinase B (PKB), endothelial cell migration and formation of adherent junctions. These studies revealed that S1P in ApoM-HDL was protective towards endothelial function. Recent studies have demonstrated that S1P1 signaling in endothelial cells is more sustained in response to HDLbound ApoM-S1P compared with albumin-bound S1P [49]. This might involve an HDL-bound S1P-S1P1 receptor/β-arrestin complex, resident at the plasma-membrane, which reduces tumour necrosis factor alpha (TNFα)-induced activation of nuclear factor kappa B (NF-κB) and intercellular adhesion molecule 1 (ICAM-1) expression. In contrast, albumin-bound S1P-S1P1 receptor is endocytosed and involves Gi-mediated signaling [50]. Since it is well established that S1P1 uses a β-arrestin-dependent mechanism to regulate endocytosis of S1P1, we suggest that HDL-bound S1P-S1P1 might be specifically trapped at the plasma membrane with β-arrestin. This could be achieved if HDL-bound S1P-S1P1 is associated with an accessory protein that prevents endocytosis of S1P1 and therefore initiates a plasma-membrane S1P1 receptor/β-arrestin signaling programme which is anti-inflammatory.
ApoM-S1P is involved in regulating specific cell biology. For instance, ApoM-S1P is dispensable for lymphocyte trafficking but limits lymphopoiesis via S1P1 expressed on bone marrow lymphocyte progenitors [51]. Proliferation of Lin(−) Sca-1(+) cKit(+) haematopoietic progenitor cells (LSKs) and common lymphoid progenitors (CLPs) in bone marrow is increased in mice that are deficient in ApoM. Moreover, overexpression of S1P1 suppresses proliferation of LSK and CLP cells in vivo and decreases lymphopoiesis in vitro. The failure to deliver S1P in Apom −/− mice to bone progenitors results in severe EAE. This is due to increased lymphocytes in the central nervous system (CNS) and breakdown of the blood-brain barrier [51]. In addition, activation of endothelial S1P1 by HDL-S1P Figure 3. RGS-12 (Regulator of G-protein signaling-12) co-localises with the S1P 1 in airway smooth muscle cells and reduces S1P-stimulation of the ERK-1/2 pathway. Airway smooth muscle cells were transfected with plasmid constructs encoding myc-tagged S1P 1 and hemagglutinin (HA) tagged RGS12 and stimulated with S1P (1 µM, 5 min). The data shows that RGS12 co-localises with S1P 1 in cytoplasmic vesicles. It remains to be determined whether S1P 1 in these vesicles is competent to signal. However, RGS12 dampens the activation of ERK-1/2 by S1P in these cells. The results are representative of three independent experiments.

Sphingosine 1-Phosphate Receptor 1 and Sphingosine 1-Phosphate Carriers/Chaperones
Plasma S1P is associated with carrier/chaperone proteins, such as albumin and high density lipoprotein (HDL). Christofferson et al. [48] were the first to show that HDL-S1P was bound to apolipoprotein M (ApoM). S1P binds to an amphiphilic pocket in the lipocalin fold of ApoM. From a functional perspective, ApoM-HDL stimulated endocytosis of S1P 1 and promoted activation of ERK-1/2, protein kinase B (PKB), endothelial cell migration and formation of adherent junctions. These studies revealed that S1P in ApoM-HDL was protective towards endothelial function. Recent studies have demonstrated that S1P 1 signaling in endothelial cells is more sustained in response to HDL-bound ApoM-S1P compared with albumin-bound S1P [49]. This might involve an HDL-bound S1P-S1P 1 receptor/β-arrestin complex, resident at the plasma-membrane, which reduces tumour necrosis factor alpha (TNFα)-induced activation of nuclear factor kappa B (NF-κB) and intercellular adhesion molecule 1 (ICAM-1) expression. In contrast, albumin-bound S1P-S1P 1 receptor is endocytosed and involves G i -mediated signaling [50]. Since it is well established that S1P 1 uses a β-arrestin-dependent mechanism to regulate endocytosis of S1P 1 , we suggest that HDL-bound S1P-S1P 1 might be specifically trapped at the plasma membrane with β-arrestin. This could be achieved if HDL-bound S1P-S1P 1 is associated with an accessory protein that prevents endocytosis of S1P 1 and therefore initiates a plasma-membrane S1P 1 receptor/β-arrestin signaling programme which is anti-inflammatory.
ApoM-S1P is involved in regulating specific cell biology. For instance, ApoM-S1P is dispensable for lymphocyte trafficking but limits lymphopoiesis via S1P 1 expressed on bone marrow lymphocyte progenitors [51]. Proliferation of Lin(−) Sca-1(+) cKit(+) haematopoietic progenitor cells (LSKs) and common lymphoid progenitors (CLPs) in bone marrow is increased in mice that are deficient in ApoM. Moreover, overexpression of S1P 1 suppresses proliferation of LSK and CLP cells in vivo and decreases lymphopoiesis in vitro. The failure to deliver S1P in Apom −/− mice to bone progenitors results in severe EAE. This is due to increased lymphocytes in the central nervous system (CNS) and breakdown of the blood-brain barrier [51]. In addition, activation of endothelial S1P 1 by HDL-S1P induces liver regeneration and suppresses fibrosis. Indeed, in mice deficient in HDL-S1P, liver regeneration after partial hepatectomy is reduced and associated with aberrant vascular remodelling, thrombosis and peri-sinusoidal fibrosis [52].
Studies of S1P 1 and its interaction with accessory proteins and/or co-receptors are being facilitated by in vivo reporters of S1P 1 signaling. For instance, a green fluorescent protein (GFP) expression reporter following activation of a S1P 1 /transcription factor fusion protein that is cleaved by a β-arrestin/protease fusion protein has been developed [53]. This mouse was used to demonstrate that lipopolysaccharide (LPS)-mediated systemic inflammation leads to the activation of S1P 1 in endothelial cells and hepatocytes in vivo [53]. Another model involves differential internalisation of a competent S1P 1 /GFP fusion protein compared with an S1P binding deficient S1P 1 :RFP fusion protein [54]. Therefore, these S1P 1 reporter mice will also allow the tissue-specific interrogation of S1P 1 activation in disease models.

Sphingosine 1-Phosphate Receptor 1 and Immune Function
The sphingosine-like molecule, FTY720 (or fingolimod, formulated as Gilenya TM ) is used as the first oral treatment for relapsing and remitting multiple sclerosis [55]. SK2 catalyses the phosphorylation of FTY720 and the FTY720-phosphate is released from cells to stimulate S1P receptors. Chronic exposure to FTY720 (FTY720-phosphate) induces a decrease in S1P 1 levels thereby reducing inflammatory T cell invasion of the CNS and reducing multiple sclerosis disease progression. FTY720 phosphate induces functional antagonism due to proteasomal degradation of S1P 1 and this prevents the egress of T-cells as these are S1P 1 null and are unable to respond to a critical S1P gradient between lymph and lymph nodes (Figure 4). Th17 cells found primarily within central memory T cells are reduced (including retinoid related orphan receptor γt (RORγt) and interleukin-17 (IL-17)-producing T cells) by >90% in response to FTY720 [56]. induces liver regeneration and suppresses fibrosis. Indeed, in mice deficient in HDL-S1P, liver regeneration after partial hepatectomy is reduced and associated with aberrant vascular remodelling, thrombosis and peri-sinusoidal fibrosis [52]. Studies of S1P1 and its interaction with accessory proteins and/or co-receptors are being facilitated by in vivo reporters of S1P1 signaling. For instance, a green fluorescent protein (GFP) expression reporter following activation of a S1P1/transcription factor fusion protein that is cleaved by a β-arrestin/protease fusion protein has been developed [53]. This mouse was used to demonstrate that lipopolysaccharide (LPS)-mediated systemic inflammation leads to the activation of S1P1 in endothelial cells and hepatocytes in vivo [53]. Another model involves differential internalisation of a competent S1P1/GFP fusion protein compared with an S1P binding deficient S1P1:RFP fusion protein [54]. Therefore, these S1P1 reporter mice will also allow the tissue-specific interrogation of S1P1 activation in disease models.

Sphingosine 1-Phosphate Receptor 1 and Immune Function
The sphingosine-like molecule, FTY720 (or fingolimod, formulated as Gilenya TM ) is used as the first oral treatment for relapsing and remitting multiple sclerosis [55]. SK2 catalyses the phosphorylation of FTY720 and the FTY720-phosphate is released from cells to stimulate S1P receptors. Chronic exposure to FTY720 (FTY720-phosphate) induces a decrease in S1P1 levels thereby reducing inflammatory T cell invasion of the CNS and reducing multiple sclerosis disease progression. FTY720 phosphate induces functional antagonism due to proteasomal degradation of S1P1 and this prevents the egress of T-cells as these are S1P1 null and are unable to respond to a critical S1P gradient between lymph and lymph nodes (Figure 4). Th17 cells found primarily within central memory T cells are reduced (including retinoid related orphan receptor γt (RORγt) and interleukin-17 (IL-17)-producing T cells) by >90% in response to FTY720 [56]  . Schematic demonstrating the role of S1P/S1P1 in T-cell trafficking in the immune system. Engagement with Dendritic Cells (DC) presenting antigen in a major histocompatibility class II (MHC class II) complex causes expansion of CD4 + (Cluster of Differentiation), which requires retention in lymph nodes and is achieved by the chemokine receptor 7 (CCR7) and CD69-mediated downregulation of S1P1. Newly formed effector T-cells then lose CCR7 and up-regulate S1P1 so that they can sense an S1P gradient between lymph nodes and lymph thereby allowing their egress into lymph.
Multiple sclerosis involves an unrestrained autoimmune Th17 response. Indeed, S1P enhances Th17 cell polarisation [57] found primarily within central memory T cells. The mechanism is promoted by an S1P/S1P1-dependent increase in signal transducer and activator of transcription 3 (STAT3) and IL-6 formation; this S1P1 dependent pro-inflammatory pathway was first, demonstrated . Schematic demonstrating the role of S1P/S1P 1 in T-cell trafficking in the immune system. Engagement with Dendritic Cells (DC) presenting antigen in a major histocompatibility class II (MHC class II) complex causes expansion of CD4 + (Cluster of Differentiation), which requires retention in lymph nodes and is achieved by the chemokine receptor 7 (CCR7) and CD69-mediated down-regulation of S1P 1 . Newly formed effector T-cells then lose CCR7 and up-regulate S1P 1 so that they can sense an S1P gradient between lymph nodes and lymph thereby allowing their egress into lymph.
The resistance of certain relapsing and remitting multiple sclerosis patients to FTY720 might be due to polymorphism in the S1P1 receptor gene. For instance, Ile45 to Thr and Gly305 to Cys mutations (found in patients) renders the S1P1 receptor resistant to FTY720-induced degradation [64]. In addition, phosphorylation on S351 in S1P1 has been identified as a critical regulator of receptor internalisation [65]. Indeed, mutant mice expressing phosphorylation-deficient receptors (S1P1(S5A)) develop severe EAE involving Th17 cells. S1P1 activates the Janus activated kinase (JAK)/STAT3 pathway via IL-6 thereby enhancing Th17 polarisation and worsening neuro-inflammation, which is likely to represent a key mechanism in multiple sclerosis [65]. These findings suggest that plasmamembrane S1P1 regulates Jak-STAT3/IL-6 as this is enhanced in cells where S1P1 is resistant to endocytosis. S1P1 is required for the exit of mature B cells from secondary lymphoid organs. In addition, S1P1 deficiency reduces the number of newly generated immature B cells in the blood [66]. This is due to enhanced apoptosis of immature B cells in contact with the vascular compartment. Forced expression of CD69, a negative regulator of S1P1 receptor expression also reduced the number of immature B cells in the blood. Chemokine receptor (CCR7 and CXCR4) recycling and S1P1 are also implicated in chronic lymphocytic leukemia pathogenesis and clinical outcome [67]. Figure 5. Schematic demonstrating the role of S1P/S1P1 in T-cell differentiation. S1P acting via S1P1 can promote an interleukin-6 (IL-6)-dependent polarisation of CD4 + cells to form T helper 17 (Th17) cells, which release IL-17. S1P binding to S1P1 also inhibits regulatory T cells (T(reg)) formation thereby exacerbating the polarisation of Th17 cells. Figure 5. Schematic demonstrating the role of S1P/S1P 1 in T-cell differentiation. S1P acting via S1P 1 can promote an interleukin-6 (IL-6)-dependent polarisation of CD4 + cells to form T helper 17 (Th17) cells, which release IL-17. S1P binding to S1P 1 also inhibits regulatory T cells (T(reg)) formation thereby exacerbating the polarisation of Th17 cells.
The resistance of certain relapsing and remitting multiple sclerosis patients to FTY720 might be due to polymorphism in the S1P 1 receptor gene. For instance, Ile45 to Thr and Gly305 to Cys mutations (found in patients) renders the S1P 1 receptor resistant to FTY720-induced degradation [64]. In addition, phosphorylation on S351 in S1P 1 has been identified as a critical regulator of receptor internalisation [65]. Indeed, mutant mice expressing phosphorylation-deficient receptors (S1P 1 (S5A)) develop severe EAE involving Th17 cells. S1P 1 activates the Janus activated kinase (JAK)/STAT3 pathway via IL-6 thereby enhancing Th17 polarisation and worsening neuro-inflammation, which is likely to represent a key mechanism in multiple sclerosis [65]. These findings suggest that plasma-membrane S1P 1 regulates Jak-STAT3/IL-6 as this is enhanced in cells where S1P 1 is resistant to endocytosis. S1P 1 is required for the exit of mature B cells from secondary lymphoid organs. In addition, S1P 1 deficiency reduces the number of newly generated immature B cells in the blood [66]. This is due to enhanced apoptosis of immature B cells in contact with the vascular compartment. Forced expression of CD69, a negative regulator of S1P 1 receptor expression also reduced the number of immature B cells in the blood. Chemokine receptor (CCR7 and CXCR4) recycling and S1P 1 are also implicated in chronic lymphocytic leukemia pathogenesis and clinical outcome [67].

Sphingosine 1-Phosphate Receptor 1 and the Nervous System
The S1P 1 modulator, FTY720 also reduces astrogliosis and supports nerve remyelination and recovery [68]. Moreover, the S1P 1 -specific agonist (AUY954) which reduces EAE in SJL/J autoimmune susceptible mice induces a decrease in lymphocyte numbers in the CNS without interfering with trafficking of plasmacytoid dendritic cells (pDCs) to the CNS [69]. pDCs are important in limiting the autoimmune responses during EAE. S1P 1 deficiency also delays differentiation of oligodendrocyte progenitors (OPCs) into oligodendroglial cells (OLGs); accompanied by decreased levels of myelin basic protein but not myelin-OLG glycoprotein [70]. S1P 1 -deficient OLGs exhibited slower process extension concomitant with reduced phosphorylated ERK-1/2 and p21-activated kinase (PAK) levels. Therefore, S1P 1 regulates OLG development, morphological maturation and early myelination. FTY720 phosphate binding to S1P 1 also reduces activated microglial production of pro-inflammatory mediators, TNFα, IL-1β and IL-6 and increases microglial production of brain-derived neurotrophic factor and glial cell-derived neurotrophic factor that are protective [71].

Sphingosine 1-Phosphate Receptor 1 and Neovascularisation
VEGF promotes sprouting of endothelial cells to produce capillary tubes that are then stabilised by S1P. In this regard, S1P binding to S1P 1 inhibits VEGFR2 signaling and angiogenesis in endothelial cells [72,73] by promoting stabilisation of VE-cadherin at endothelial junctions [72]. In other words, VEGF starts the process of blood vessel formation and S1P finishes it. In wet age-related macular degeneration, atrophy of the retinal pigment epithelium (which is a proposed source of S1P, and which can undergo Epithelial Mesenchymal Transition (EMT) to become contractile myofibroblasts [74]) and/or development of new unstable blood vessels results in death of photoreceptors and loss of central vision. VEGF promotes sprouting of endothelial cells in the choroidal region behind the retina, which leak/hemorrhage. This can be considered as pathological angiogenesis, where blood vessels have proceeded to some degree of maturity but have not fully matured. Removal of VEGF prevents sprouting and branching and induces regression of these unstable vessels. As VEGF is a survival signal, its removal can induce apoptosis of endothelial cells to regress blood vessels. Intervention with anti-VEGF therapeutics such as Avastin [75] (a humanised antibody that binds VEGF) halts vessel sprouting, regresses immature blood vessels and elicits a small improvement in visual acuity [75]. It follows that targeting S1P 1 could achieve the same therapeutic utility in terms of preventing full maturation and promoting vessel regression in response to anti-VEGF therapy. In support of this approach, anti-S1P monoclonal antibodies have been shown to markedly reduce choroidal neovascularisation lesion volume, sub-retinal fibrosis and pericyte recruitment in a murine model of laser-induced rupture of Bruch's membrane [76]. However, in a first clinical phase IIa study (Nexus trial), anti-S1P antibody treatment did not meet its primary or key secondary end points.

Sphingosine 1-Phosphate Receptor 1 and the Heart
S1P mediates multiple pathophysiological effects in the cardiovascular system. For instance, cardiac S1P levels are increased post-myocardial infarction (MI) and this is associated with increased SK1 and S1P 1 expression [77]. In addition, β1-adrenergic receptor stimulation of S1P/S1P 1 underlies the pro-inflammatory response in cardiomyocytes [77]. Administration of FTY720, to functionally antagonise S1P 1 , reduces chronic cardiac inflammation, and improves cardiac remodeling and dysfunction in vivo post-MI [77]. Moreover, S1P 1 is required for normal cardiac development. Thus, the conditional knockout of the S1P receptor 1 (S1pr1) results in ventricular non-compaction and ventricular septal defects leading to perinatal lethality [78].
The transition from beneficial hypertrophy (which enables the heart to tolerate high blood pressure) to heart failure is governed by the formation of new blood vessels that can reoxygenate the heart [79]. Angiogenesis can prevent the development of malfunctional hypertrophy and heart failure and this is regulated by the p53 gene [80]. Thus, hypoxia has been shown to increase p53 expression via a hypoxia-inducible factor 1 alpha (HIF1α)-dependent mechanism and p53 inhibits angiogenesis by reducing HIF1α in a negative feedback manner [80]. Therefore, high p53 expression is a causative factor in the development of heart failure, a result of apoptosis of cardiomyocytes. Therapeutic approaches that increase angiogenesis in the heart have been suggested as a means to prevent the transition from beneficial hypertrophy to heart failure. However, approaches using angiogenic factors have largely failed because this results in formation of immature vessels that cannot be sustained. In this case, stimulation of S1P 1 and recruitment of mural cells to enable full maturation of newly formed blood vessels is likely to normalise the heart vasculature and offer an improved efficacy with VEGF in preventing transition to heart failure. In addition, FTY720 has been employed to reduce cardiac remodelling post myocardial infarction in animal models [77]. Although this has been interpreted as being mediated through S1P 1 -dependent changes in inflammation [77], it is possible that the agonistic effect of FTY720 phosphate on S1P 1 might mature blood vessels to protect against deleterious cardiac remodelling.

Sphingosine 1-Phosphate Receptor 1 and Cancer
There is substantial evidence implicating S1P in cancer including SK1-induced transformation, EMT, invasiveness, regulation of cancer cell survival and replicative immortality, regulation of tumour neovascularisation and changes in metabolism [81]. In this regard, high expression of S1P 1 in tumours from estrogen receptor positive (ER + ) breast cancer patients is associated with shorter disease-specific survival [82]. S1P 1 has also been shown to co-localise with SK1 and filamin A in lamellipodia in filamin A-expressing A7 melanoma cells, and this is required for cell motility and is blocked by an S1P 1 antagonist [83]. S1P 1 is also implicated in the neovascularisation of tumours. For instance, FTY720 inhibited primary and metastatic tumour growth in a mouse model of melanoma growth [84]. This was associated with inhibited tumour-associated angiogenesis and decreased tumour cell proliferation. Recent studies have demonstrated that EMT of hepatocellular carcinoma cells involves S1P/S1P 1 -dependent phosphoinositol-3-kinase (PI3K)/PKB activation and increased expression of metalloproteinase 7 (MMP7) and shedding and loss of syndecan-1 [85]. This loss of syndecan-1 promotes a TGFβ (Transforming growth factor β)-dependent EMT that is implicated in hepatocellular carcinoma metastasis [86]. S1P/S1P 1 is also involved regulating the expression of hypoxia-inducible factor 2 alpha (HIF2α), which can drive aggressive cancer [87]. Thus, siRNA knockdown of S1P 1 and Spns2 blocks HIF2α accumulation, suggesting that S1P might exert so-called 'inside-out' signaling, where SK1 catalyses formation of S1P, which is released from cells to act on S1P 1 in an autocrine manner to regulate cancer cell growth. Similarly, estrogen (E2) activates SK1 and promotes internalisation of S1P 1 ; the latter is required for activation of PKB/endothelial nitric oxide synthase (eNOS) in endothelial cells and which regulates endothelial cell migration and tube formation [87]. Therefore endothelial cell S1P 1 might function as a nodal point in E2 signaling and play an important role in neovascularisation of estrogen-dependent tumours. SK1 expression is also increased in invasive cancer phenotypes compared with non-invasive cancer cells [88] and results in increased IL-6 levels. Moreover S1P 1 knockdown reduces IL-6/STAT3 signaling; this representing a pathway by which SK1/S1P regulates invasion [88]. HDL-S1P also promotes phosphorylation of STAT3 (at S727) and cell migration and this is reduced by S1P 1/3 and S1P 2 antagonists [89]. A link between S1P and STAT3 signaling is exemplified by studies showing that S1P enhances colitis associated cancer via a malicious amplification loop involving SK1, S1P 1 , NF-κB, STAT3 and IL-6 [90,91].

Conclusions
There is significant translational activity and potential in developing novel S1P 1 modulators. However, more subtle approaches appear available based on increased knowledge of S1P 1 signaling and its regulation. For instance, the existence of different pools of S1P 1 bound with accessory proteins or co-receptors raises the possibility of targeting these distinct S1P 1 receptor pools for therapeutics. Examples could include the use of modulators to manipulate S1P 1 -β-arrestin-G-protein complex, S1P 1 -Receptor Tyrosine Kinase complex and HDL-S1P signaling in pathogenic conditions such as auto-immune disease, cardiovascular disease and cancer. In this regard, the use of in vivo reporters of S1P 1 signaling seems poised to facilitate development of next generation small molecules and biologics that can be employed to therapeutically intervene in disease.