Transglutaminase 2 cross-linking activity is linked to invadopodia formation and cartilage breakdown in arthritis

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.

Platelet-Derived Growth Factor Receptor Activation Promotes the Prodestructive Invadosome-Forming Phenotype of Synoviocytes from Patients with Rheumatoid Arthritis

“…Interestingly, TNF resistance has recently been associated with a distinct synovial fibroid phenotype enriched for gene sets associated with TGF-b/SMAD signaling, as well as cell projection processes (95). In this work, we show that blockade of the TbR1/PDGFR axis or downstream PI3K-AKT pathway impaired invadosome formation, a process previously linked to cartilage damage (5,6). Various small-molecule inhibitors of TGF-b are being evaluated in preclinical and clinical trials (96).…”

“…They were shown to contain actin components, signaling molecules, such as Src, and high levels of proteolytic enzymes known to be particularly efficient at inducing cartilage damage. Importantly, interference with the formation of invadosomes in arthritic FLS strongly inhibited matrix degradation in vitro and ex vivo as well as cartilage degradation in a rat model of arthritis (5,6). These observations suggested that invadosomes were directly involved in joint degradation, leading to the conclusion that an in-depth understanding of the mechanism of invadosome formation is of importance for development of joint protection strategies for the clinical management of RA.…”

“…This property correlates with elevated production of inflammatory cytokines and proteolytic enzymes that sustain inflammation and joint matrix degradation. We have reported that the ability of arthritic FLS to degrade the extracellular matrix depends on the formation of plasma membrane structures that resembled invadopodia in tumor cells (5,6). These structures were detected in fibroblast-like cells strategically located at the cartilage-synovial membrane interface.…”

“…We have recently reported that autocrine TGF-b production was part of the mechanism that explained the aggressive invadosomeforming phenotype of rat synoviocytes derived from collageninduced arthritis joints (6). TGF-b has also been shown to promote proliferation of smooth muscle cells and to induce epithelialmesenchymal transition in mammary epithelial cells through a PDGF-dependent mechanism (50, 51).…”

“…Invadosome formation and in vivo cartilage degradation capability of synovial cells of collageninduced arthritis rats were shown to depend on an autocrine activation loop that involved TGF-b (6). Analysis of the protein and mRNA in RA synovial tissues revealed that TGF-b was highly expressed in RA patients (7)(8)(9).…”