Abstract
IntroductionEngagement of the homotypic cell-to-cell adhesion molecule cadherin-11 on rheumatoid arthritis (RA) synovial fibroblasts with a chimeric molecule containing the cadherin-11 extracellular binding domain stimulated cytokine, chemokine, and matrix metalloproteinases (MMP) release, implicating cadherin-11 signaling in RA pathogenesis. The objective of this study was to determine if cadherin-11 extracellular domain fragments are found inside the joint and if a physiologic synovial fibroblast cleavage pathway releases those fragments.MethodsCadherin-11 cleavage fragments were detected by western blot in cell media or lysates. Cleavage was interrupted using chemical inhibitors or short-interfering RNA (siRNA) gene silencing. The amount of cadherin-11 fragments in synovial fluid was measured by western blot and ELISA.ResultsSoluble cadherin-11 extracellular fragments were detected in human synovial fluid at significantly higher levels in RA samples compared to osteoarthritis (OA) samples. A cadherin-11 N-terminal extracellular binding domain fragment was shed from synovial fibroblasts after ionomycin stimulation, followed by presenilin 1 (PSN1)-dependent regulated intramembrane proteolysis of the retained membrane-bound C-terminal fragments. In addition to ionomycin-induced calcium flux, tumor necrosis factor (TNF)-α also stimulated cleavage in both two- and three-dimensional fibroblast cultures. Although cadherin-11 extracellular domains were shed by a disintegrin and metalloproteinase (ADAM) 10 in several cell types, a novel ADAM- and metalloproteinase-independent activity mediated shedding in primary human fibroblasts.ConclusionsCadherin-11 undergoes ectodomain shedding followed by regulated intramembrane proteolysis in synovial fibroblasts, triggered by a novel sheddase that generates extracelluar cadherin-11 fragments. Cadherin-11 fragments were enriched in RA synovial fluid, suggesting they may be a marker of synovial burden and may function to modify cadherin-11 interactions between synovial fibroblasts.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0647-9) contains supplementary material, which is available to authorized users.
Highlights
Engagement of the homotypic cell-to-cell adhesion molecule cadherin-11 on rheumatoid arthritis (RA) synovial fibroblasts with a chimeric molecule containing the cadherin-11 extracellular binding domain stimulated cytokine, chemokine, and matrix metalloproteinases (MMP) release, implicating cadherin-11 signaling in RA pathogenesis
We previously observed that a recombinant cadherin-11 extracellular domain Fc protein activated multiple signaling pathways in synovial fibroblasts, inducing expression of IL6 and other mediators important in RA pathogenesis
We determined if intact, soluble cadherin-11 extracellular domains could be isolated from patient synovial fluid specimens
Summary
Engagement of the homotypic cell-to-cell adhesion molecule cadherin-11 on rheumatoid arthritis (RA) synovial fibroblasts with a chimeric molecule containing the cadherin-11 extracellular binding domain stimulated cytokine, chemokine, and matrix metalloproteinases (MMP) release, implicating cadherin-11 signaling in RA pathogenesis. Synovial fibroblasts are joint stromal cells with important roles in the normal and inflammatory synovium [1, 2]. Inhibitor-treated cells showed a dosedependent increase in CTF1 after ionomycin stimulation (Fig. 4a-b), suggesting that CTF1 is further processed by the γ-secretase complex, as predicted in our cleavage model (Fig. 2a). Lactacystin-treated cells showed a dose-dependent accumulation of a low molecular fragment with a size consistent for CTF2 (Fig. 4c-d). In this experiment, CTF1 and CTF2 were detected in both unstimulated and ionomycin-stimulated fibroblasts. As shown earlier (Fig. 2f), long exposure times detect a level of constitutive cleavage in unstimulated cells
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