Background In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) undergo a stable transformation resulting in an aggressive phenotype mediating cartilage damage by increased levels of adhesion molecules. In this context, Lasp1 and the Arp2/3 complex are of interest because they modulate actin organization and focal adhesion turnover.
Objectives In this study, the effects of Arp2/3 on cadherin-11 mediated cell-to-cell contact formation have been investigated using the arthritic hTNFtg mouse model.
Methods Expression levels of Lasp1 and Arp2/3 protein complex were investigated in synovial tissue of wild type (wt) and hTNFtg hind paws by immunohistochemistry. Primary FLS were analysed, respectively and co-immunoprecipitation experiments were performed. In addition, lasp1-/- mice were interbred with hTNFtg animals and offspring were evaluated for disease progression and joint destruction. To further study the role of Arp2/3 in the function of the cadherin-11 adhesion complex, the effects of an Arp2/3 inhibitor (CK666) on cell-to-cell contact formation in FLS derived from hTNFtg and lasp1-/-hTNFtg mice were investigated by stainings. To assess signaling pathway activation, cells were stimulated with the growth factor PDGF.
Results Upregulated Lasp1 levels were found in synovial tissue and FLS of hTNFtg compared to wt mice. Assays showed that Arp2/3 is part of the adherens junction (AJ) machinery in FLS although Arp2/3 expression levels were not changed between the genotypes. In vivo evaluation of lasp1-/-hTNFtg mice revealed a milder arthritis score, less cartilage degradation and reduced FLS attachment to articular cartilage compared to hTNFtg mice. In vitro, the loss of Lasp1 led to clear alterations in AJ arrangement indicated by altered β-catenin pattern. As expected, β-catenin expression was mainly located at adhesion sites between adjacent cells. In hTNFtg FLS, these structures were characterized by a zipper-like pattern. In contrast, these structures were disrupted in lasp1-/-hTNFtg FLS. Interestingly, CK666 induced zipper-like structures in hTNFtg FLS comparable to the pattern found in lasp1-/-hTNFtg cells. Furthermore, lasp1-/-hTNFtg FLS showed decreased Src phosphorylation following PDGF stimulation in comparison to hTNFtg FLS.
Conclusion Lasp1 represents an interesting target involved in RA-caused joint destruction, because its loss results in significantly reduced cartilage destruction and altered FLS contacts mediated by Arp2/3.
Disclosure of Interests None declared.
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