Abstract
IntroductionThe microenvironment surrounding inflamed synovium leads to the activation of fibroblast-like synoviocytes (FLSs), which are important contributors to cartilage destruction in rheumatoid arthritic (RA) joints. Transglutaminase 2 (TG2), an enzyme involved in extracellular matrix (ECM) cross-linking and remodeling, is activated by inflammatory signals. This study was undertaken to assess the potential contribution of TG2 to FLS-induced cartilage degradation.MethodsTransglutaminase (TGase) activity and collagen degradation were assessed with the immunohistochemistry of control, collagen-induced arthritic (CIA) or TG2 knockdown (shRNA)-treated joint tissues. TGase activity in control (C-FLS) and arthritic (A-FLS) rat FLSs was measured by in situ 5-(biotinamido)-pentylamine incorporation. Invadopodia formation and functions were measured in rat FLSs and cells from normal (control; C-FLS) and RA patients (RA-FLS) by in situ ECM degradation. Immunoblotting, enzyme-linked immunosorbent assay (ELISA), and p3TP-Lux reporter assays were used to assess transforming growth factor-β (TGF-β) production and activation.ResultsTG2 and TGase activity were associated with cartilage degradation in CIA joints. In contrast, TGase activity and cartilage degradation were reduced in joints by TG2 knockdown. A-FLSs displayed higher TGase activity and TG2 expression in ECM than did C-FLSs. TG2 knockdown or TGase inhibition resulted in reduced invadopodia formation in rat and human arthritic FLSs. In contrast, increased invadopodia formation was noted in response to TGase activity induced by TGF-β, dithiothreitol (DTT), or TG2 overexpression. TG2-induced increases in invadopodia formation were blocked by TGF-β neutralization or inhibition of TGF-βR1.ConclusionsTG2, through its TGase activity, is required for ECM degradation in arthritic FLS and CIA joints. Our findings provide a potential target to prevent cartilage degradation in RA.
Highlights
The microenvironment surrounding inflamed synovium leads to the activation of fibroblast-like synoviocytes (FLSs), which are important contributors to cartilage destruction in rheumatoid arthritic (RA) joints
We recently reported that the ability of arthritic FLS (A-FLS) to degrade extracellular matrix (ECM) is dependent on the formation of specialized structures resembling invadopodia in tumor cells, which are invasive structures involved in basement membrane degradation, or podosomes, the bone-resorbing structures found in osteoclasts [10,11]
Transglutaminase 2 (TG2) and TGase activity are associated with degradation of type II collagen in collagen-induced arthritis (CIA) joints Knee joints of control and CIA rats were prepared for immunohistochemistry (IHC) and labeled with an ε-(gglutamyl)-lysine bond-specific monoclonal antibody to obtain an estimation of the cross-linking activity of TGase or TG2-specific antibodies
Summary
The microenvironment surrounding inflamed synovium leads to the activation of fibroblast-like synoviocytes (FLSs), which are important contributors to cartilage destruction in rheumatoid arthritic (RA) joints. The selective pressure from the inflammatory environment generates intrinsic changes in RA-FLSs leading to an enhanced ability to attach to cartilage, invade through matrix, and synthesize degradative enzymes [9]. Characterization of the A-FLS structures indicated that they contained actin components, activated kinases (Scr), and the metalloproteinases MMP3 and MMP-13, which are known to be efficient at inducing cartilage degradation. They were found in cells at the cartilage/pannus junction, well positioned for cartilage degradation. Interference with the formation of invadopodia in A-FLSs by Src kinase inhibition impeded ECM degradation in vitro and cartilage degradation in a model of collagen-induced arthritis (CIA) [11], strongly suggesting that invadopodia are physiological structures involved in cartilage destruction
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