Sphingosine 1-phosphate receptors in health and disease: Mechanistic insights from gene deletion studies and reverse pharmacology

Abstract

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that is critically involved in the embryonic development of the cardiovascular and central nervous systems. In the adult, S1P can produce cytoskeletal re-arrangements in many cell types to regulate immune cell trafficking, vascular homeostasis and cell communication in the central nervous system. S1P is contained in body fluids and tissues at different concentrations, and excessive production of the pleiotropic mediator at inflammatory sites may participate in various pathological conditions. Gene deletion studies and reverse pharmacology (techniques aiming to identify both ligands and function of receptors) provided evidence that many effects of S1P are mediated via five G-protein-coupled S1P receptor subtypes, and novel therapeutic strategies based on interaction with these receptors are being initiated. The prototype S1P receptor modulator, FTY720 (fingolimod), targets four of the five S1P receptor subtypes and may act at several levels to modulate lymphocyte trafficking via lymphocytic and endothelial S1P1 and, perhaps, other inflammatory processes through additional S1P receptor subtypes. A recently completed Phase II clinical trial suggested that the drug may provide an effective treatment of relapsing–remitting multiple sclerosis. FTY720 is currently being evaluated in larger-scale, longer-term, Phase III studies. This review provides an overview on S1P activities and S1P receptor function in health and disease, and summarizes the clinical experience with FTY720 in transplantation and multiple sclerosis.

Introduction

Bioactive lysophospholipids and their receptors have been proposed as targets for rational drug design, thereby opening up the potential for an entire new class of lipidomic-based therapeutics (reviewed in Brinkmann and Lynch, 2002, Rosen and Goetzl, 2005, Chun and Rosen, 2006). The family of lysophospholipids comprises pleiotropic mediators including lysophosphatidic acid, lysophosphatidylcholine (lysolecithin), sphingosylphosphorylcholine, and sphingosine 1-phosphate (S1P) (Chun et al., 1999, Fukushima et al., 2001).

S1P is a bioactive sphingolipid that mediates diverse cellular responses such as proliferation, cytoskeletal organization and migration, adherence- and tight junction assembly, and morphogenesis (Hla, 2004, McVerry and Garcia, 2005, Lee et al., 2006). S1P is present at concentrations of 200–900 nanomolar (nM) in blood and, like most small lipids in extracellular spaces, is bound by albumin and other plasma proteins (Murata et al., 2000). This provides both a stable reservoir in extracellular fluids and efficient delivery to high-affinity cell-surface receptors. The S1P levels in different tissues are maintained by the coordinated activities of two biosynthetic sphingosine kinases (SphKs) and two biodegradative S1P phosphatases, one S1P lyase, and three lysophospholipid phosphatases (Le Stunff et al., 2002). S1P is produced by platelets during activation and thrombotic processes (English et al., 2000), mast cells during inflammatory reactions (Prieschl et al., 1999), and other non-haematopoietic cells such as endothelial cells (ECs) (Ancellin et al., 2002). It had been suggested that blood platelets store S1P abundantly, possibly due to the presence of highly active SphK and a lack of the S1P-degrading enzyme S1P lyase (Yatomi, 2006). However, a major contribution of platelets to plasma S1P levels has recently been challenged by the observation that transcription factor NF-E2-deficient mice have normal plasma S1P concentrations, despite having virtually no circulating platelets (Pappu et al., 2007). More recent data showed that erythrocytes possess weaker SphK activity compared to platelets, but lack both S1P-degrading enzymes (S1P lyase and S1P phosphohydrolase) (Ito et al., 2007), and therefore appear to be specialized cells for storing and supplying plasma S1P (Hanel et al., 2007). Studies with conditional SphK1/2-double deficient mice confirmed that plasma S1P is mainly erythrocytic in origin, but also showed that lymph S1P is from a distinct radiation-resistant source, presumably lymphatic endothelium (Pappu et al., 2007). SphK1 activity and S1P production are increased by many cytokines, including interleukin (IL)-1, tumour necrosis factor (TNF)α and vascular endothelial growth factor (VEGF), opening the possibility of a central role for sphingolipids in many inflammatory processes (Hla, 2004, Chalfant and Spiegel, 2005).

S1P binds with low nM affinity to the five related G-protein-coupled receptors (GPCRs), S1P1-5, formerly termed endothelial differentiation gene (EDG) receptor-1, -5, -3, -6, -8, respectively (Chun et al., 2002). The receptor subtypes S1P1, S1P2, and S1P3 are widely expressed and represent the dominant receptors in the cardiovascular system (Waeber et al., 2004). S1P1 is also a dominant receptor on lymphocytes and regulates their egress from secondary lymphatic organs (Matloubian et al., 2004). S1P4 receptors are expressed at low levels in the lymphoid system (Gräler et al., 1998, Gräler et al., 1999), and S1P5 is expressed in the white matter tracts of the central nervous system (CNS) (Im et al., 2000, Terai et al., 2003). The cell-type-specific expression of combinations of S1P receptors, together with a differential coupling to heterotrimeric G-proteins (guanine nucleotide-binding-proteins), and down-stream signaling pathways dramatically enhance the repertoire of any S1P stimulation. An additional layer of complexity has recently been added by the observation that cell membrane-associated vs. internalized/nuclearized S1P receptors may provide different regulatory signals to cells (Liao et al., 2007).

Despite the complex regulatory features of GPCRs, about 40% of pharmaceuticals in the market today target this receptor class, and it has been estimated that the human genome codes for more than 1000 GPCRs (> 2% of the genome) (Tyndall & Sandilya, 2005). Therapies include synthetic agonists and antagonists, and ‘agonist–antagonist’ drugs that target more than one GPCR of a single class and act as agonist at one receptor and as antagonist at another (i.e. opioid receptor ligands) (Hoskin and Hanks, 1991, Tyndall and Sandilya, 2005). GPCRs stimulated by agonistic ligands often display bell-shaped dose–response curves, switching from activation and signaling of the receptors to desensitization and internalization as a result of increased ligand concentration. The desensitization reduces the responsiveness to endogenous ligands, and this involves either phosphorylation of the GPCRs by kinases and an uncoupling from G-proteins realized by arrestins (Marie et al., 2006), an attenuation of signaling by regulator-of-G-protein signaling (RGS) proteins (Cho et al., 2003), or a receptor internalization and degradation (Marie et al., 2006). Emerging data suggest that a similar picture could arise for S1P receptors and synthetic ligands (Brinkmann et al., 2002, Brinkmann and Lynch, 2002), opening the possibility for a broad therapeutic application of this new receptor class. FTY720 (fingolimod; 2-amino-2[2-(4-octylphenyl)ethyl]-1,3-propanediol) represents the prototype of this new generation of S1P receptor modulators that could find use in autoimmune and demyelinating diseases (Brinkmann et al., 2004, Kappos et al., 2006). This review summarizes the biology of S1P and its GPCRs, focusing on S1P receptor gene deletion studies and in vivo reverse pharmacology techniques using synthetic S1P receptor agonists and antagonists.