Background/Purpose: In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) undergo a stable transformation leading to an aggressive phenotype that mediates cartilage damage through increased levels of adhesion molecules. In this context, Lasp1 and the Arp2/3 complex are of interest as they modulate actin organization and turnover of focal adhesions. Therefore, we investigated the effects of Arp2/3 on cadherin-11 mediated cell-to-cell contact formation using the arthritic hTNFtg mouse model.

Methods: The expression levels of Lasp1 and Arp2/3 protein complex were examined in synovial tissue of wild type (wt) and hTNFtg hind paws by using Western blot analysis and their subcellular distribution was investigated by immunofluorescence stainings. In addition, lasp1^-/- mice were crossed with hTNFtg animals, and offspring were examined for disease progression and joint destruction. Primary FLS were isolated from wt, lasp1^-/-, hTNFtg and lasp1^-/-hTNFtg mice, and co-immunoprecipitation experiments with cell lysates using G-labelled Dynabeads were performed. Furthermore, the effects of the Arp2/3 inhibitor CK666 on the formation of cadherin-11 mediated cell-to-cell contacts in FLS from hTNFtg and lasp1^-/-hTNFtg mice were analyzed by immunofluorescence stainings. To determine the functional effects of CK666 on FLS migration, all genotypes were examined in a modified scratch assay, to also assess the effects on pathway activation, cells were stimulated with the growth factor PDGF.

Results: Elevated levels of Lasp1 were detected 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 wt and hTNFtg FLS, although Arp2/3 expression levels were not altered between genotypes. In vivo, assessment of lasp1^-/-hTNFtg mice revealed a milder arthritis score, less cartilage degradation and reduced FLS attachment to articular cartilage compared with hTNFtg mice.In vitro, β-catenin expression was found mainly at adhesion sites between adjacent cells, but the loss of Lasp1 resulted in an altered β-catenin pattern and marked changes in AJ arrangement. In hTNFtg FLS, these structures were characterized by a zipper-like pattern. In contrast, these structures were interrupted in lasp1^-/-hTNFtg FLS. Interestingly, CK666 induced zipper-like structures in hTNFtg FLS comparable to the pattern were found in lasp1^-/-hTNFtg cells. Quantification of scratch assay data revealed a significantly reduced migration rate of lasp1^-/-hTNFtg FLS compared to hTNFtg FLS (-75.2.%, p< 0.05) and even more prominently in the presence of CK666 the hTNFtg FLS showed a reduction in migration compared to controls (-42.7%, p< 0.05). Furthermore,lasp1^-/-hTNFtg FLS showed decreased Src phosphorylation following PDGF stimulation compared with hTNFtg FLS. Interestingly, the presence of CK666 also blocked the phosphorylation of Src in hTNFtg FLS.

Conclusion: Lasp1 represents an interesting target involved in RA-related joint destruction, as its loss leads to significantly reduced cartilage destruction and altered FLS contacts mediated by Arp2/3.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-focal-adhesion-protein-lasp1-links-the-arp2-3-complex-to-cell-to-cell-contact-formation-in-arthritic-fibroblast-like-synoviocytes/