**1. Introduction**

194 Rheumatoid Arthritis – Treatment

Yudoh K, Matsuno H & Kimura T. (1999) Plasma adrenomedullin in rheumatoid arthritis compared with other rheumatic diseases. *Arthritis Rheum*, Vol. 42, pp. 1297-8. Zagariya A, Bhat R, Navale S, Chari G & Vidyasagar D. (2006) Inhibition of meconium-

*Pediatrics.* Vol. 117, pp. 1722–1727

induced cytokine expression and cell apoptosis by pretreatment with captopril.

Sphingolipids belong to a major class of lipids that are ubiquitously synthesized by eukaryotic cells and are characterized by their sphingoid backbone and primary structural roles in membrane formation. Over the past two decades, sphingolipids have emerged as a source of important signalling molecules in addition to their structural functions (Merrill et al., 1997). In particular, sphingosine-1-phosphate (S1P), is a unique signalling molecule that has the ability to act as an intracellular second messenger, as well as an extracellular stimulus through specific G protein-coupled receptor (GPCRs) (Pyne and Pyne, 2000; Spiegel and Milstien, 2003; Van Brocklyn et al., 1998). S1P has been shown to mediate a variety of fundamental biological processes including cell proliferation, migration, invasion, angiogenesis, vascular maturation and lymphocyte trafficking. The intracellular level of S1P is tightly regulated by the equilibrium between its synthesis by sphingosine kinases (SphKs) and its degradation by S1P phosphatases (SPPs) and S1P lyase (SPL) (Saba and Hla, 2004). Activation of SphKs by a variety of agonists, including growth factors, cytokines and hormones, increases intracellular S1P. Once generated, S1P can either act intracellularly as a second messenger or be secreted out of the cell and act extracellularly by binding to and signalling through S1P receptors expressed on the surface of the same cell or nearby cells in autocrine and/or paracrine manners (Alvarez et al., 2007). Many of the biological effects of S1P are mediated by its binding to and activation of its specific receptors S1P1–S1P5 (Ishii et al., 2004). Alterations in S1P signalling, as well as in the enzymes involved in its synthesis and metabolism, have been observed in many types of pathological situations such as angiogenesis, metastasis, and autoimmunity.

The role played by S1P in the autoimmune disease rheumatoid arthritis (RA) has recently emerged. Activated SphK and elevated levels of S1P have been detected in the synovium and synovial fluids of patients with RA, respectively (Kamada et al., 2009; Kitano et al., 2006; Lai et al., 2008). In addition, exogenously applied S1P in cultured synovial fibroblasts from RA patients causes cell migration, production of cytokines/chemokines, expression of cyclooxygenase-2 and prostaglandins, as well as cell proliferation and survival (Kitano et al., 2006; Zhao et al., 2008). These results suggest a biological function for the SphK/S1P/S1P receptor (S1PR) axes in RA disease progression. In this review we summarize the current understanding of how S1P metabolism and signalling impact the biology of inflammation in

Targeting the Metabolism and Receptors of

important for export of S1P out of cells.

**3. S1P receptors and downstream signalling pathways** 

discussed in previous reviews (Choi et al., 2008; Rosen et al., 2009).

Sphingosine-1-Phosphate for the Treatment of Rheumatoid Arthritis 197

phosphorylation and translocation to the plasma membrane, 2) interaction with acidic phospholipids such as phosphatidic acid produced by phospholipase D (Delon et al., 2004), and 3) possible association with other proteins. All or parts of these mechanisms may be required for full activation of SphK1 (Takabe et al., 2008). Phosphorylation of SphK1 leads to its translocation to the plasma membrane where its substrate sphingosine is located, resulting in the production of S1P (Johnson et al., 2002; Pitson et al., 2005). S1P, in turn, activates specific S1P receptors present on the surface of the same cell or on nearby cells in autocrine and/or paracrine manners (Alvarez et al., 2007). The "inside-out" export of

The mechanism by which S1P is exported from the inside to the outside of cells after synthesis is not fully understood. Several lines of evidence suggest the involvement of the ATP-binding cassette (ABC) family of transporters in S1P secretion (Kobayashi et al., 2006; Mitra et al., 2006; Sato et al., 2007). Mitra et al. (2006) revealed that the release of S1P from mast cells is regulated by ABCC1. Similarly, the ABCA1 transporter is critical for the release of S1P from astrocytes (Sato et al., 2007). In breast cancer the export of S1P mediated by ABCC1 and ABCG2 transporters is stimulated by estrogen receptor- (Takabe et al., 2010). Altogether, these studies suggest that members of the family of ABC transporters may be

S1P evokes diverse biological functions by binding to its ubiquitously-expressed and specific cell surface receptors. So far, five S1P receptors, designated as S1P1/Edg1, S1P2/Edg5, S1P3/Edg3, S1P4/Edg6, and S1P5/Edg8, have been identified (An et al., 1997; Im et al., 2000; Lee et al., 1998; Van Brocklyn et al., 2000; Yamazaki et al., 2000). These receptors were initially named ''Edg'' receptors as they belong to the so-called endothelial differentiation gene (Edg) family; however ''S1P receptors'' is now preferred (Chun et al., 2002). S1P receptors are GPCRs that share high similarity with each other and with LPA receptors. S1P receptors exhibit variable tissue distribution, for example, S1P1, S1P2, and S1P3 are widely expressed in various tissues, whereas the expression of S1P4 and S1P5 is more restricted to cells of the immune system and nervous system, respectively (reviewed in (Sanchez and Hla, 2004)). Upon binding to S1P receptors, S1P activates downstream signalling pathways, leading to a variety of cellular responses such as proliferation, cell migration, actin cytoskeletal rearrangement, and adherens junction assembly (Kluk and Hla, 2002; Taha et al., 2004). Each S1P receptor is coupled to a specific heterotrimeric G protein (Gi/o, Gq and G12/13), which, when activated, dissociates into its and subunits and transduces signals toward the downstream pathways. Particularly, S1P1 is coupled predominantly to Gi/o, through which it activates: 1) Rho kinase and tyrosine kinases, leading to cytoskeletal rearrangement (Garcia et al., 2001); 2) MAPK, leading to angiogenesis (Lee et al., 1999); and 3) Akt, leading to cell chemotaxis (Lee et al., 2001). On the other hand, S1P2 and S1P3 are linked predominantly to Gq and G12/13, through which they activate phospholipase C leading to Ca2+ mobilization (Ancellin and Hla, 1999) and positive or negative modulation of Rho and thus of cell migration (Sugimoto et al., 2003). More detailed information on the various signalling pathways turned on by S1P receptor activation is

It is known that S1P can also act intracellularly to enhance cell proliferation and suppress apoptosis independently of S1P receptors (Rosenfeldt et al., 2001; Van Brocklyn et al., 1998). In plants and yeast, which do not express S1P receptors, for instance, intracellular S1P

intracellularly generated S1P is crucial for many S1P functions (Takabe et al., 2008).

RA. We also discuss the potential therapeutic benefit of modulators of S1P metabolism and S1P receptors, including SphK/SPL inhibitors, FTY-720, S1P receptor antagonists, and anti-S1P monoclonal antibodies, in the treatment of RA.
