**3.1 Anti-inflammatory signaling**

One of the first evidences of anti-inflammatory signalling in chondrocytes was the elucidation of the α5β1 integrin/IL-4 pathway. In this pathway, mechanical stimulation of normal chondrocytes acts through the α5β1 integrin to release IL-4, which acts in an autocrine/paracrine manner. Following IL-4 release, there is a decrease in MMP-3 and an increase in aggrecan mRNA, resulting in a net increase in cartilage extracellular matrix production (Millward-Sadler and Salter 2004). The transcription factor Signal Transducer and Activator of Transcription 6 (STAT6) plays a principal role in IL-4 signaling as demonstrated in mice lacking STAT6 that show a similar phenotype as mice lacking the IL-4 receptor alpha (IL-4Rα) (Takeda et al. 1996). IL-4 stimulates intracellular signaling pathways including the recruitment of STAT6 to the IL-4Rα. STAT6 binds to specic phosphotyrosine residues within the IL-4Rα (Ryan et al. 1998). In this complex, STAT6 is quickly phosphorylated by a JAK-dependent mechanism. After phosphorylation, STAT6 leaves the receptor, dimerizes, and migrates to the nucleus where it binds to specic DNA sequences in the promoter of genes (Darnell 1997). It is believed that STAT6 is tightly regulated, because in the absence of IL-4 stimulation, STAT6 is quickly deactivated (Andrews et al. 2002). Methylation of STAT6 is a regulator of STAT6 activity, necessary for optimal STAT6 phosphorylation, nuclear translocation, and DNA-binding activity (Chen et al. 2004). Accumulating evidence suggests that IL-4 STAT6 is a central anti-rheumatoid signaling pathway because it upregulates three factors known to antagonize the actions of specific pro-inflammatory agents implicated in RA: (1) soluble IL-1 receptor antagonist (sIL-1ra); (2) tristetraprolin (TTP), which antagonizes TNF-α; and (3) the β3 integrin, which was shown to antagonize the angiogenic actions of vascular endothelial growth factor (VEGF) (Vannier et al. 1992; McHugh et al. 2001; Suzuki et al. 2003).

While the mechanisms underlying the anti-inflammatory effects of IL-10 are largely unknown in chondrocytes, studies which overexpress IL-10 provide insight on the downstream targets of IL-10. IL-10 treatment in an antigen-induced arthritis animal model resulted in a marked reduction of TNF-α levels (Lubberts et al. 1998). In human chondrocytes treated with TNF-α, IL-10 overexpression ssuppressed MMP-13 levels and antagonized the TNF-α-mediated suppression of aggrecan (Muller et al. 2008). It has been hypothesized that IL-10 may exert its effects by stimulating the production of endogenous TNF-α inhibitors such as soluble TNF-α receptors (Fernandes et al. 2002).

#### **3.2 Anti-catabolic signaling**

*In vivo*, motion-based therapies have been demonstrated to mitigate joint inflammation in animal models of antigen-induced arthritis. Mechanical signals generated from these passive joint motion therapies were reported to be potent inhibitors of pro-inflammatory gene induction and inhibit expression of catabolic mediators, e.g., IL-1β, COX-2, and MMP-1 (Ferretti et al. 2005; Ferretti et al. 2006). At low magnitudes *in vitro*, biomechanical signals inhibit IL-1β- or TNF-α-induced transcriptional activation of COX-2, MMPs, IL-1β, and

exercise are commonly characterized as anti-inflammatory and anti-catabolic. Each of these components is mediated by distinct signalling pathways and evidence indicates crosstalk

One of the first evidences of anti-inflammatory signalling in chondrocytes was the elucidation of the α5β1 integrin/IL-4 pathway. In this pathway, mechanical stimulation of normal chondrocytes acts through the α5β1 integrin to release IL-4, which acts in an autocrine/paracrine manner. Following IL-4 release, there is a decrease in MMP-3 and an increase in aggrecan mRNA, resulting in a net increase in cartilage extracellular matrix production (Millward-Sadler and Salter 2004). The transcription factor Signal Transducer and Activator of Transcription 6 (STAT6) plays a principal role in IL-4 signaling as demonstrated in mice lacking STAT6 that show a similar phenotype as mice lacking the IL-4 receptor alpha (IL-4Rα) (Takeda et al. 1996). IL-4 stimulates intracellular signaling pathways including the recruitment of STAT6 to the IL-4Rα. STAT6 binds to specic phosphotyrosine residues within the IL-4Rα (Ryan et al. 1998). In this complex, STAT6 is quickly phosphorylated by a JAK-dependent mechanism. After phosphorylation, STAT6 leaves the receptor, dimerizes, and migrates to the nucleus where it binds to specic DNA sequences in the promoter of genes (Darnell 1997). It is believed that STAT6 is tightly regulated, because in the absence of IL-4 stimulation, STAT6 is quickly deactivated (Andrews et al. 2002). Methylation of STAT6 is a regulator of STAT6 activity, necessary for optimal STAT6 phosphorylation, nuclear translocation, and DNA-binding activity (Chen et al. 2004). Accumulating evidence suggests that IL-4 STAT6 is a central anti-rheumatoid signaling pathway because it upregulates three factors known to antagonize the actions of specific pro-inflammatory agents implicated in RA: (1) soluble IL-1 receptor antagonist (sIL-1ra); (2) tristetraprolin (TTP), which antagonizes TNF-α; and (3) the β3 integrin, which was shown to antagonize the angiogenic actions of vascular endothelial growth factor (VEGF) (Vannier et al. 1992; McHugh et al. 2001;

While the mechanisms underlying the anti-inflammatory effects of IL-10 are largely unknown in chondrocytes, studies which overexpress IL-10 provide insight on the downstream targets of IL-10. IL-10 treatment in an antigen-induced arthritis animal model resulted in a marked reduction of TNF-α levels (Lubberts et al. 1998). In human chondrocytes treated with TNF-α, IL-10 overexpression ssuppressed MMP-13 levels and antagonized the TNF-α-mediated suppression of aggrecan (Muller et al. 2008). It has been hypothesized that IL-10 may exert its effects by stimulating the production of endogenous

*In vivo*, motion-based therapies have been demonstrated to mitigate joint inflammation in animal models of antigen-induced arthritis. Mechanical signals generated from these passive joint motion therapies were reported to be potent inhibitors of pro-inflammatory gene induction and inhibit expression of catabolic mediators, e.g., IL-1β, COX-2, and MMP-1 (Ferretti et al. 2005; Ferretti et al. 2006). At low magnitudes *in vitro*, biomechanical signals inhibit IL-1β- or TNF-α-induced transcriptional activation of COX-2, MMPs, IL-1β, and

TNF-α inhibitors such as soluble TNF-α receptors (Fernandes et al. 2002).

between these pathways (see Figure 1 for hypothesized pathways/mechanisms).

**3.1 Anti-inflammatory signaling** 

Suzuki et al. 2003).

**3.2 Anti-catabolic signaling** 

other pro-inflammatory molecules (Chowdhury et al. 2003; Agarwal et al. 2004; Ferretti et al. 2005; Chowdhury et al. 2006; Deschner et al. 2006).

Exposure of meniscal or articular chondrocytes to proinflammatory cytokines (e.g. IL-1β and TNF-α) is reported to result in the expression of cyclo-oxygenase 2, inducible nitric oxide synthase, and genes involved in cartilage catabolism, such as matrix metalloproteinases 9 and 13 (Gassner et al. 1999). By contrast, when cells are subjected to mechanical stimuli in the form of cyclic tensile strain, they display a blunted response to cytokine exposure, thereby antagonizing the proinflammatory and catabolic effects of these cytokines (Ferretti et al. 2006; Madhavan et al. 2006). Interestingly, this anti-catabolic response seems to be mediated by inhibition of nuclear translocation of Nuclear factor-kappa B (NF-κB) and modulation of upstream signaling events associated with NF-κB, suggesting that mechanical activity can act at multiple points within the proinflammatory signaling network to counteract cytokine-induced proinflammatory gene expression (Dossumbekova et al. 2007). NF-*κ*B transcription factors regulate a wide range of pro-inflammatory and anti-apoptotic genes, and are involved in both acute and chronic inflammatory responses. NF-*κ*B is a rapid response, multiple-stimuli inducible transcription factor that is controlled by sequential signal activation cascades (Seguin and Bernier 2003). In the classical NF-*κ*B signaling pathway, binding of pro-inflammatory mediators, such as IL-1β, TNF-*α*, and/or LPS to their cognate receptors leads to activation of a series of receptor-associated signaling molecules leading to activated NF-*κ*B, which translocates to the nucleus, where it binds to the consensus sequences of several genes including pro-inflammatory cytokines and mediators (Ghosh and Karin 2002; Hoffmann et al. 2002; Liacini et al. 2003). Mechanical signals of low/physiological magnitudes block the IL-1β-induced transcriptional activity of NF-κB by intercepting multiple steps in the NF-κB signaling cascade. In chondrocytes, cyclic tensile strain of low magnitudes does not appear to inhibit IL-1β, TNF-α, or LPS receptor-mediated pro-inflammatory gene induction (Agarwal et al. 2004; Dossumbekova et al. 2007; Madhavan et al. 2007). These findings suggest that mechanical signals use specific target sites to trigger NF-κB signaling.

Another transcriptional regulator which plays a critical role in cartilage homeostasis is CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2). CITED2 expression is increased by moderate flow shear (5 dyn/cm2), intermittent hydrostatic pressure (1-5 MPa), and joint motion (Yokota et al. 2003; Leong et al. 2011). The induction of CITED2 *in vivo* by joint motion loading was correlated with the downregulation of MMP-1 and the maintenance of cartilage matrix integrity (Leong et al. 2011), suggesting it plays a key role in mediating the anti-catabolic effects of moderate loading. The induction of CITED2 by physiologic loading was mediated by mitogen-activated protein kinase (MAPK) p38δ, and CITED2 regulated the transcription of MMPs (ie. MMP-1) by competing with MMP transactivator ETS-1 for binding to limiting amounts of co-activator p300 (Leong et al. 2011).

#### **3.3 Crosstalk between anti-inflammatory and anti-catabolic responses**

There is also evidence of crosstalk between the anti-inflammatory and anti-catabolic pathways. CITED2, induced by p38δ, has also been demonstrated to be upregulated in response to IL-4 (Sun et al. 1998), raising the possibility these two pathways could work in synergy. Furthermore, treatment strategies involving gene transfer of IL-4 or IL-10

Molecular Effects of Exercise in Rheumatoid Arthritis 321

Agarwal, S., J. Deschner, P. Long, A. Verma, C. Hofman, C. H. Evans, & N. Piesco (2004).

Alamanos, Y., & A. A. Drosos (2005). Epidemiology of adult rheumatoid arthritis.

Andrews, R. P., M. B. Ericksen, C. M. Cunningham, M. O. Daines, & G. K. Hershey (2002).

Baillet, A., E. Payraud, V. A. Niderprim, M. J. Nissen, B. Allenet, P. Francois, L. Grange, P.

Baillet, A., N. Zeboulon, L. Gossec, C. Combescure, L. A. Bodin, R. Juvin, M. Dougados, & P.

Bijlsma, J. W. (2010). Optimal treatment of rheumatoid arthritis: EULAR recommendations

Bilberg, A., M. Ahlmen, & K. Mannerkorpi (2005). Moderately intensive exercise in a

Brentano, M. A., & L. F. Martins Kruel (2011). A review on strength exercise-induced muscle

Brorsson, S., M. Hilliges, C. Sollerman, & A. Nilsdotter (2009). A six-week hand exercise

Brukner, P. D., & W. J. Brown (2005). 3. Is exercise good for you? *Med J Aust* Vol.183, No.10, (Nov 2005), pp. (538-41), ISSN 0025-729X (Print), 0025-729X (Linking) Cairns, A. P., & J. G. McVeigh (2009). A systematic review of the effects of dynamic exercise

Chen, W., M. O. Daines, & G. K. Hershey (2004). Methylation of STAT6 modulates STAT6

Vol.62, No.7, (Jul 2010), pp. (984-92), ISSN 2151-4658 (Electronic)

ISSN 1897-9483 (Electronic), 0032-3772 (Linking)

8), ISSN 0004-3591 (Print), 0004-3591 (Linking)

Role of NF-kappaB transcription factors in antiinflammatory and proinflammatory actions of mechanical signals. *Arthritis Rheum* Vol.50, No.11, (Nov 2004), pp. (3541-

*Autoimmun Rev* Vol.4, No.3, (Mar 2005), pp. (130-6), ISSN 1568-9972 (Print), 1568-

Analysis of the life cycle of stat6. Continuous cycling of STAT6 is required for IL-4 signaling. *J Biol Chem* Vol.277, No.39, (Sep 2002), pp. (36563-9), ISSN 0021-9258

Casez, R. Juvin, & P. Gaudin (2009). A dynamic exercise programme to improve patients' disability in rheumatoid arthritis: a prospective randomized controlled trial. *Rheumatology (Oxford)* Vol.48, No.4, (Apr 2009), pp. (410-5), ISSN 1462-0332

Gaudin (2010). Efficacy of cardiorespiratory aerobic exercise in rheumatoid arthritis: meta-analysis of randomized controlled trials. *Arthritis Care Res (Hoboken)*

for clinical practice. *Pol Arch Med Wewn* Vol.120, No.9, (Sep 2010), pp. (347-53),

temperate pool for patients with rheumatoid arthritis: a randomized controlled study. *Rheumatology (Oxford)* Vol.44, No.4, (Apr 2005), pp. (502-8), ISSN 1462-0324

damage: applications, adaptation mechanisms and limitations. *J Sports Med Phys Fitness* Vol.51, No.1, (Mar 2011), pp. (1-10), ISSN 0022-4707 (Print), 0022-4707

programme improves strength and hand function in patients with rheumatoid arthritis. *J Rehabil Med* Vol.41, No.5, (Apr 2009), pp. (338-42), ISSN 1651-2081

in rheumatoid arthritis. *Rheumatol Int* Vol.30, No.2, (Dec 2009), pp. (147-58), ISSN

phosphorylation, nuclear translocation, and DNA-binding activity. *J Immunol*

**6. References** 

9972 (Linking)

(Print), 0021-9258 (Linking)

(Electronic), 1462-0324 (Linking)

(Print), 1462-0324 (Linking)

(Electronic), 1650-1977 (Linking)

1437-160X (Electronic), 0172-8172 (Linking)

(Linking)

combined with mechanical stimulation may augment the chondroprotective effects of exercise. However, these hypotheses still require further investigation.

Fig. 1. Hypothesized anti-inflammatory and anti-catabolic mechanisms underlying the effects of exercise in suppressing cartilage destruction in arthritis.
