THU0039 B1-Integrin is a Critical Mediator of TLR2-Induced Migrational and Invasive Mechanisms in RA Synovial Fibroblast Cells
Authors: T. Mcgarry, U. Fearon, W. Gao, M. Jackson, J. McCormick, D. Veale, M. Connolly
Journal: Annals of the Rheumatic Diseases (2013)

<h3>Background</h3> Rheumatoid arthritis (RA) is a chronic, progressive autoimmune disease, characterized by synovial proliferation and destruction of cartilage and bone. Engagement of integrin receptors induces cell migration/invasion through attachment to the extracellular matrix and downstream activation of RhoGTPases [1]. TLR2 has been implicated in the pathogenesis of joint destruction in RA [2], however, the mechanisms involved have yet to be elucidated. <h3>Objectives</h3> This study investigates the role of β1-integrin in mediating TLR2-induced cytoskeletal rearrangement, cell migration and invasion processes <i>in vivo</i> and <i>in vitro</i>. <h3>Methods</h3> β1-integrin expression and cytoskeletal rearrangement were assessed by immunohistochemistry, F-actin immunoflourescent staining and RT-PCR. β1-integrin binding (β1-4, β6, avβ5, a5β1), cytoskeletal rearrangement and RhoGTPase Rac-1 activation in response to Pam3CSK4 (1μg/ml)(TLR2-ligand) in RASFC and HMVEC were assessed by a multiplex adhesion binding assay, F-actin immunofluorescent staining and Rac-1 pull down assays/Western blot. The effect of Pam3CSK4 on cell migration, invasion and Rac-1 activation in the presence of anti-β1-integrin or anti-IgG control was assessed by wound repair assays, <i>ex vivo</i> RA synovial explant matrigel outgrowths, transwell matrigel™ invasion chambers and Rac-1 pulldown/western blot. <h3>Results</h3> β1-integrin expression is significantly increased in RA synovial tissue lining layer, sub-lining layer and vasculature in comparison to OA and control tissue (p&lt;0.05). Pam3CSK4 specifically induced β1-integrin binding in RASFC (p&lt;0.05), with no effect observed for β2-4, β6, avβ5 or a5β1 binding. Pam3CSK4 significantly induced β1-integrin mRNA and protein expression in RASFC (p&lt;0.05), and promoted RASFC/HMVEC cytoskeletal disassembly, microspike formation and filopodia extensions. Downstream from β1-intgerin, Pam3CSK4 induced Rac-1 activation, critical for cytoskeletal dynamics and cell motility. Consistent with this Pam3CSK4 induced RASFC/HMVEC migration, invasion, RA synovial explant outgrowths and Rac-1 activation were inhibited in the presence of anti-β1-integrin (p&lt;0.05), with no effect observed for anti-IgG control. <h3>Conclusions</h3> TLR2 activation induces migrational and invasive mechanisms through β1-integrin-induced cytoskeletal pathways, processes which are critically involved in the pathogenesis of RA. <h3>References</h3> Brakebusch, C., Bouvard, D., Stanchi, F. et al (2002) <b>Integrins in Invasive Growth</b><i>J Clin Invest.</i> 109:999-1006 Seibl, R., Birchler, T., Loeliger, S. et al (2003) <b>Expression and Regulation of Toll-like Receptor 2 in rheumatoid arthritis synovium.</b><i>Am J Pathol</i> 162:1221-7 Brentano, F., Kyburz, D., Schorr, O. et al (2005) <b>The role of Toll-like receptor signalling in the pathogenesis of arthritis.</b><i>Cell Immunol</i> 233:90-6 <h3>Disclosure of Interest</h3> T. Mcgarry: None Declared, U. Fearon: None Declared, W. Gao: None Declared, M. Jackson: None Declared, J. McCormick: None Declared, D. Veale Grant/research support from: Abbott, Opsona, Pfizer, Roche, Consultant for: MSD, Pfizer, Roche, Speakers bureau: Janssen, MSD, Pfizer, UCB, M. Connolly: None Declared