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

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

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

Targeting the Metabolism and Receptors of

level of S1P synthesis.

synthesis, catabolism enzymes and S1P signalling in RA (Fig. 2).

**4.2 S1P expression and SphK activity in RA synovium and synovial fluids** 

Sphingosine-1-Phosphate for the Treatment of Rheumatoid Arthritis 199

however, that the activation of RA synoviocytes can be maintained in the absence of inflammatory cytokines (Muller-Ladner et al., 1996), indicating the involvement of biologically active mediators other than inflammatory cytokines in RA disease progression. Emerging evidence suggests a role for S1P metabolism and signalling in various aspects of the pathogenesis of RA. Recent *in vitro* and *in vivo* studies evaluated the potential role of S1P

Several studies reveal high levels of S1P in RA synovial tissues and fluids (Kitano et al., 2006; Lai et al., 2008). Kitano et al. (2006) reported elevated levels of S1P in synovial fluids of RA patients (1078 pM/ml) in comparison to those of osteoarthritis (OA) patients (765 pM/ml). The S1P content in RA synovial fluids was significantly higher than those in serum (400 pM/ml) or plasma (100 pM/ml) from normal people. Lai et al. (2008) also measured the amount of S1P by a competitive ELISA and detected up to 17 M S1P in RA synovial fluids, which was more than five fold higher than that found in OA synovial fluids (3 M). The increased level of S1P could be responsible for the recruitment and the infiltration of immune cells into the synovium (see section 4.2.2). Pi et al. (2006) also showed that peripheral blood B lymphoblastoid cell lines derived from patients with RA exhibit a high

SphK expression and activity were also demonstrated in RA. Increased SphK1 expression and activity were found in RA B lymphoblastoid cell lines, which were implicated as the underlying mechanism of impaired Fas-mediated death signalling in RA (Pi et al., 2006). More recently, SphK2 was shown to be expressed in rheumatoid synovial fibroblasts *in vivo* and *in vitro* and associated with the up-regulation of S1P production (Kamada et al., 2009). Surprisingly, FTY-720 (2-amino-2-[2-(4-octylphenyl) ethyl] propane-1, 3-diol hydrochloride), a sphingosine analog that is phosphorylated to the active metabolite FTY-720-phosphate (FTY-720-P) by SphK2 (Brinkmann et al., 2002; Mandala et al., 2002), induces apoptosis of synovial fibroblasts. This effect could be due, at least in part, to FTY-720-P-mediated degradation of specific S1P receptors (Matloubian et al., 2004). Corroborating this observation, we reported that S1P protects synovial fibroblasts from ongoing apoptosis (Zhao et al., 2008). Using the mouse model of collagen-induced arthritis (CIA), Lai et al. (2009) highlighted distinct roles for SphK1 and SphK2 in regulating cell growth and survival. The *in vivo* administration of SphK1 siRNA reduced inflammation whereas mice treated with SphK2 siRNA resulted in a more aggressive disease and greater secretion of proinflammatory cytokines (IL-6, TNF- and IFN-) by immune cells. How and why SphK1 and SphK2 exert distinct opposing roles in the regulation of inflammatory arthritis remains unclear and may be related to the location of S1P production (Maceyka et al., 2005). The role of SphK2 in chronic inflammatory diseases remains ambiguous since the selective inhibitor of SphK2 ABC249640 has been reported to reduce bone and cartilage degradation in the mouse models of adjuvant-induced arthritis (AIA) and of CIA (Fitzpatrick et al., 2011). The SphK1 signalling pathway is activated in RA (Limaye et al., 2009). In the mouse model of CIA, administration of a non-specific pharmacological inhibitor of SphKs, *N*,*N*dimethylsphingosine (DMS), and a siRNA approach to knockdown the SphK1 isoform, markedly suppressed joint pathologies such as adjacent cartilage and bone erosion, synovial hyperplasia, and infiltration of inflammatory cells into the joint compartment (Lai et al., 2008). Moreover, SphK1 deficiency in hTNF- transgenic mice that develop arthritis at an early age was related to less synovial inflammation and bone erosion (Baker et al., 2010). To

regulates stomatal aperture (Coursol et al., 2005) or stress responses and survival (Jenkins and Hannun, 2001), respectively. Similarly, in mammalian cells intracellular S1P regulates calcium release independently of inositol trisphosphate formation and of S1P receptor activation (Blom et al., 2005). S1P also signals within the nucleus by binding to and inhibiting histone deacetylases HDAC1 and HDAC2, leading to the epigenetic regulation of gene expression (Hait et al., 2009). Further studies are needed, however, to reveal the intracellular second messenger functions of S1P.
