FRI0004 14-3-3ETA as a novel invadosome regulatory molecule in rheumatoid arthritis

Background Progressive cartilage destruction, mostly mediated by invasive fibroblast-like synoviocytes (FLS), is a central feature in the pathogenesis of rheumatoid arthritis (RA). We have reported that the ability of arthritic FLS to degrade the extracellular matrix depends on the formation of actin-rich plasma membrane invadosomal structures detected in cells strategically located at the cartilage-synovial membrane interface. Interference with the formation of invadosomes in RA FLS impeded matrix degradation in vitro and cartilage degradation in a model of collagen-induced arthritis, suggesting that invadosomes are important physiological structures involved in cartilage destruction. The chaperonin molecule 14–3-3η has been detected in the joints of patients with early and established RA and that its concentration in both serum and synovial fluid correlated with elevated expression of extracellular matrix (ECM) degrading enzymes and erosive damage. Extracellular 14–3-3η has therefore been proposed to be a novel biomarker for joint damage and a potential drug target for the personalized treatment of connective tissue-associated diseases but the direct relationship between 14–3-3η and joint damage remains a key area of research. Objectives To evaluate the role of 14–3-3η in the ability of synoviocytes to degrade the ECM. Methods mRNA from primary synoviocytes of healthy individuals (N=3) and RA patients (N=5) was extracted and the relative level of 14–3-3 isoforms and MMP gene expression was determined by qPCR. The ability of the synovial cell lines to degrade ECM was assessed by in situ invadosome assays using fluorescent cross-linked gelatine. Confocal microscopy was used to determine the cellular localization of 14–3-3η. Results We found a significant increase in 14–3-3η, MMP1 and MMP3 mRNA levels in synoviocytes from rheumatoid arthritis patients compared to cells from non-arthritis individuals. A strong correlation between 14–3-3η expression levels and the ability of synoviocyte cell lines to form invadosomes was observed (r2=0.8299). Knockdown of 14–3-3η decreased the ability of arthritic synoviocytes to form invadosomes indicating a role of 14–3-3η in extracellular matrix degrading ability. Confocal microscopy revealed that 14–3-3η staining was mostly found as small punctated structures in the cytoplasm and at the cell periphery of arthritic synoviocytes where they colocalized with leading edge F-actin and discrete patches of the exocyst component, Exo70. Conclusions The finding of the role of 14–3-3η in invadosome formation points to a previously unappreciated facet of how 14–3-3η influences joint ECM remodelling and reinforces its role as a marker of RA progression and joint damage. How 14–3-3η is involved in the regulation of MMP production/secretion and the possible role it plays in remodelling of actin-rich subcellular structures is the subject of ongoing studies. Disclosure of Interest C. Lalanne: None declared, R. Lavoie: None declared, M. Charbonneau: None declared, J. Savill Employee of: Augurex Life Sciences Corp., A. Marotta Employee of: Augurex Life Sciences Corp., C. Dubois: None declared