**4.4 S1P signalling in RA pathophysiology**

#### **4.4.1 S1P signalling in migration of synovial fibroblasts and osteoclasts**

As constituents of the synovial pannus in RA, synovial fibroblasts have long been considered as key players in the aggressive destruction of cartilage and bone (Shiozawa et al., 1983). Using an *in vitro* wound-closing assay, we previously reported that S1P could induce synoviocyte migration (Zhao et al., 2008). S1P-induced cell migration is mediated through the activation of extracellular signal-regulated kinase-1 and -2 (ERK1/2), as well as p38 mitogen-activated protein kinase (MAPK) and of Rho kinase. S1P-driven motility of synoviocytes was mainly via S1P1 and S1P3 receptor activation since the effect of S1P was mimicked by the S1P1 receptor agonist (SEW2871) but abolished by S1P1/3 (VPC23019) and S1P3 (CAY10444) receptor antagonists.

S1P has also been shown to induce chemotaxis and regulate migration of osteoclasts and their precursors *in vitro* and *in vivo* (Ishii et al., 2009). Cells bearing the properties of osteoclast precursors express S1P1 receptors and migrate along an S1P gradient *in vitro*. The S1P1 agonist, SEW2871, stimulated motility of osteoclast precursors-containing monocytoid populations *in vivo*. In addition, osteoclast/monocyte lineage-specific conditional S1P1 knockout mice show osteoporotic changes due to increased osteoclast attachment to the bone surface, suggesting a crucial role of the S1P-S1P1 receptor axis in regulating the migratory behaviour of osteoclast precursors and bone mineral homeostasis.

#### **4.4.2 S1P signalling in the secretion of cytokines/chemokines and lipid mediators**

In the inflamed RA synovium, large amounts of pro-inflammatory cytokines and chemokines may contribute directly to cartilage and bone erosion by promoting matrix metalloproteinase (MMP) production and suppressing chondrocyte/osteoclast functions (Feldmann et al., 1996b). Previous studies from our group and others demonstrated that exogenously applied S1P could stimulate synovial fibroblasts to release various

our knowledge the effect of specific inhibitors of SphK1, such as BML-258 (Paugh et al.,

At present, evidence for roles of SPPs and SPL in RA is limited. However, up-regulation of SPP2 was detected in samples of skin lesions from patients with psoriasis, a chronic inflammatory skin disease (Mechtcheriakova et al., 2007). Interestingly, elevated mRNA expression of SPP1 and SPL was observed in RA synoviocytes compared to non-arthritic

In RA, the abnormal growth and erosive activity of synovial fibroblasts suggest that these cells are important contributors to chronic inflammation. During RA disease progression, synovial fibroblasts become hyperplasic and closely interact with infiltrated immune cells to form the aggressive pannus tissue that eventually promotes joint destruction (reviewed in (Feldmann et al., 1996a)). Synovial fibroblasts have been reported to express three of five known S1P receptors, S1P1-3 (Kitano et al., 2006; Nochi et al., 2008; Zhao et al., 2008). Expression of S1P1 in RA synovial tissues was significantly higher compared to that of OA synovial tissues (Kitano et al., 2006). Moreover, pretreatment of synovial fibroblasts with TNF-, the well-recognized critical cytokine in RA, resulted in up-regulation of S1P3 receptor expression in synovial fibroblasts, which likely contributes to the synergistic production of inflammatory

As constituents of the synovial pannus in RA, synovial fibroblasts have long been considered as key players in the aggressive destruction of cartilage and bone (Shiozawa et al., 1983). Using an *in vitro* wound-closing assay, we previously reported that S1P could induce synoviocyte migration (Zhao et al., 2008). S1P-induced cell migration is mediated through the activation of extracellular signal-regulated kinase-1 and -2 (ERK1/2), as well as p38 mitogen-activated protein kinase (MAPK) and of Rho kinase. S1P-driven motility of synoviocytes was mainly via S1P1 and S1P3 receptor activation since the effect of S1P was mimicked by the S1P1 receptor agonist (SEW2871) but abolished by S1P1/3 (VPC23019) and

S1P has also been shown to induce chemotaxis and regulate migration of osteoclasts and their precursors *in vitro* and *in vivo* (Ishii et al., 2009). Cells bearing the properties of osteoclast precursors express S1P1 receptors and migrate along an S1P gradient *in vitro*. The S1P1 agonist, SEW2871, stimulated motility of osteoclast precursors-containing monocytoid populations *in vivo*. In addition, osteoclast/monocyte lineage-specific conditional S1P1 knockout mice show osteoporotic changes due to increased osteoclast attachment to the bone surface, suggesting a crucial role of the S1P-S1P1 receptor axis in regulating the

2008), has not been tested in animal models of arthritis.

**4.3 S1P receptor expression in RA synovial fibroblasts** 

cytokines/chemokines upon subsequent exposure to S1P (Zhao et al., 2008).

**4.4.1 S1P signalling in migration of synovial fibroblasts and osteoclasts** 

migratory behaviour of osteoclast precursors and bone mineral homeostasis.

**4.4.2 S1P signalling in the secretion of cytokines/chemokines and lipid mediators**  In the inflamed RA synovium, large amounts of pro-inflammatory cytokines and chemokines may contribute directly to cartilage and bone erosion by promoting matrix metalloproteinase (MMP) production and suppressing chondrocyte/osteoclast functions (Feldmann et al., 1996b). Previous studies from our group and others demonstrated that exogenously applied S1P could stimulate synovial fibroblasts to release various

synoviocytes (Zhao et al., unpublished data).

**4.4 S1P signalling in RA pathophysiology** 

S1P3 (CAY10444) receptor antagonists.

inflammatory mediators, including cytokines, chemokines, and prostaglandin E2 (PGE2) (Kitano et al., 2006; Zhao et al., 2008). S1P administration strongly stimulated the secretion of IL-8 (interleukin-8), IL-6, MCP-1 (monocyte chemotactic protein-1), and RANTES (regulated on activation normal T cells expressed and secreted) via S1P2 and S1P3 receptors. The signalling pathways involved in S1P-mediated cytokine/chemokine secretion were dependent on ERK1/2, p38 MAPK, and Rho kinase activation. These cytokines/chemokines may subsequently increase the recruitment of inflammatory cells such as neutrophils into the synovium, as chemokines such as IL-8 exhibit selective chemotactic activity for neutrophils, whereas MCP-1, MIP-1 (macrophage inflammatory protein-1), MIP-1 (macrophage inflammatory protein-1), and RANTES primarily attract monocytes (Koch, 2005). S1P may therefore contribute to the regulation and maintenance of the inflammatory response in RA, in part through stimulation of multiple cytokine/chemokine secretion by synovial fibroblasts. SphK/S1P signalling was also implicated in cell-contact-mediated proinflammatory cytokine/chemokine secretion in RA synovium without additional exogenous stimulation. Lai et al. (2008) used peripheral blood mononuclear cells from RA patients in cell-cell contact experiments. They discovered that activated peripheral T lymphocytes from RA patients induced substantial production of TNF-, IL-1, IL-6, MCP-1 and MMP-9 by autologous peripheral monocytes. Importantly, treatment with a potent SphK inhibitor, DMS, significantly suppressed production of these cytokines. The results suggest the importance of SphK/S1P signalling in cell-contact-mediated inflammatory mediators, which is relevant to RA pathology.

Regarding PGE2, S1P was reported to stimulate COX-2 expression and super-production of PGE2 by RA synovial fibroblasts (Kitano et al., 2006; Nochi et al., 2008). PGE2 is an autocrine lipid mediator derived from arachidonic acid metabolism by cyclooxygenases (COX-1 or COX-2) (Ghosh et al., 2004). High levels of PGE2 were detected in synovial fluids of RA patients (Hidaka et al., 2001; Lettesjo et al., 1998). PGE2 has pro-inflammatory properties and contributes to the pathogenesis of arthritis, since PGE2 stimulates angiogenesis in the rheumatoid synovium (Ben-Av et al., 1995) and triggers bone resorption by osteoclasts (Mino et al., 1998). Thus, S1P may aggravate synovial hyperplasia, inflammation and angiogenesis through the induction of COX-2 and PGE2 in RA synovial tissues. Indeed, the COX-2-PGE2 axis has been suggested to play an important role in arthritic diseases by stimulating inflammation, angiogenesis, and osteoclastic bone destruction (Martel-Pelletier et al., 2003).

#### **4.4.3 S1P signalling in the proliferation and survival of RA synovial fibroblasts, B lymphoblastoid cells and chondrocytes**

Abnormal proliferation and resistance to apoptosis of synovial fibroblasts have been suggested to contribute directly to the hyperplasia of the rheumatoid synovium (Ospelt et al., 2004). Indeed, S1P appears capable of increasing cell survival and inhibiting apoptosis of various cell types, including B lymphoblastoid cells derived from RA patients (Pi et al., 2006). We previously demonstrated that S1P, through S1P1, protected synovial fibroblasts from apoptosis (Zhao et al., 2008). In our study we were not able to demonstrate a significant effect of S1P on RA synoviocyte proliferation, although Kitano et al. (2006) reported a small stimulatory effect of S1P on cell proliferation. Nonetheless these studies suggest that S1P can induce the proliferation of synovial fibroblasts, which may contribute to synovial hyperplasia (Knedla et al., 2007). S1P was also reported to aggravate arthritis by inducing chondrocyte proliferation through the stimulation of COX-2 and PGE2 production and via the activation of ERK (Kim et al., 2006; Masuko et al., 2007).

Targeting the Metabolism and Receptors of

siSphK1 Reduces S1P

SPL inhibitor Downregulates

**Mechanism of** 

Reduces S1P formation

Reduces S1P formation

formation

S1PR activity

extracellular S1P

Down-regulates S1PRs and renders cells unresponsive to

Table 1. Targeting the SphK/S1P/S1PR signalling pathway in RA.

Depletes

S1P

patients (Bagdanoff et al., 2010).

Block S1P binding to S1PR

**action** 

**Approach and reagent** 

dimethylsphingo sine (DMS, SphK

*Inhibition of SPL* 

*Sequestration of*

*Blocking S1PR*  FTY-720 (sphingosine analogue)

S1P specific antibody

VPC23019 (S1P1/3 antagonist), CAY10444 (S1P3 antagonist) and JTE-013 (S1P2 antagonist)

*Inhibition of SphK activity* 

inhibitor)

*activity* 

*S1P* 

ABC249640 (SphK2 inhibitor)

*N*,*N*-

Sphingosine-1-Phosphate for the Treatment of Rheumatoid Arthritis 203

SphK1 and SphK2

S1P1, S1P3, S1P4 and S1P5

and induces premature internalization of the exit-signal-sensing S1P1 receptor on lymphocytes (Schwab et al., 2005). S1P1 receptor internalization renders lymphocytes unresponsive to S1P, preventing their egress from the thymus and lymph nodes (Lo et al., 2005). One physiological outcome of this systemic redistribution of lymphocytes is potent immunosuppression, which offers new opportunities for developing immunoregulatory agents to treat autoimmune and inflammatory diseases (Gardell et al., 2006; Huwiler and Pfeilschifter, 2008; Mandala et al., 2002; Rosen et al., 2003; Zhang and Schluesener, 2007). Indeed, SPL-deficient mice showed resistance to various inflammatory and autoimmune challenges (Bagdanoff et al., 2009; Bandhuvula et al., 2007; Vogel et al., 2009). The evaluation of a synthetic SPL inhibitor, LX2931, is currently underway in phase II clinical trials in RA

**Target Experimental** 

**system** 

SphK2 *In vivo* Fitzpatrick et al.,

SphK1 *In vivo* Lai et al., 2008

SPL Clinical trial Bagdanoff et al.,

S1P *In vivo* Visentin et al.,

S1P1-3 *In vitro* Kitano et al.,

**Reference** 

*In vivo* Lai et al., 2008

2011

2010

2006

2007

2008

2000; Tsunemi et al.; Wang et al.,

2006; Zhao et al.,

*In vivo* Matsuura et al.,
