Background/Purpose:

Fibroblast like synoviocytes (FLS) in RA possesses unique transformed phenotype, such as cartilage invasion that is maintained independent of cytokines and other inflammatory cells. However, the molecular mechanisms that regulate FLS behavior in RA are poorly understood. The transcriptional coactivator Yes-associated protein (YAP) was recently shown that is dysregulated in RA compared with OA FLS. Increased YAP activity might contribute to persistent activation and pathogenic behavior of RA FLS. Therefore, we assessed role of YAP in synoviocyte invasion.

Methods:

We used siRNA knock down and chemical inhibition to assess effect of YAP iactivation on RA FLS invasion. We used Verteporfin to inhibit YAP transcriptional activity. For siRNA knockdown, YAP and control siRNA were transfected using AMAXA technology. FLS invasion was studied using Matrigel coated transwells, 3 days following transfection. YAP inhibitor or vehicle was added to the upper chamber and PDGF was used as chemoattractant in the lower chamber. The cytoskeleton and focal adhesions were visualized with confocal microscopy using Rhodamin phalloidin and anti-Vinculin staining. MMP expression was measured using qPCR. 

Results:

The YAP inhibitor Verteporfin significantly decreased number of invading cells in a concentration dependent manner, with 65+5% inhibition at 0.1 uM (p< 0.02, n=3). Similar results were obtained when YAP was knockdown by siRNA. Cells with reduced YAP activity displayed 52+6% (p< 0.04, n=3) decrease in invasion through Matrigel matrix compared with scramble control. To determine effect of YAP inactivation on cytoskeleton and focal adhesion formation, cells were stained with phalloidin and anti-Viculin. RA FLS treated with the YAP inhibitor Verteporfin displayed diminished stress fiber formation and decrease in focal adhesion formation by 56+5% (p<0.04, n=3) visualized with confocal microscopy. Similar findings were obtained when YAP was inactivated by siRNA knock down. To determine the effect on MMP production cells were stimulated with TNF and qPCR was performed for MMP1, 2, 3, 9 and MT1MMP. mRNA levels for MMP1 and MMP3 were significantly lower in YAP deficient cells compared with scramble control (P<0.03 and p<0.05, n=3), while MMP2,9 and MT1MMP mRNA were unchanged. Similar findings were obtained using chemical inhibition with Verteporfin.

Conclusion:

YAP is a major regulator of RA FLS invasion by modulating focal adhesion formation and MMP production. These observations, together with our previous findings that increased YAP activity in RA FLS compared with OA suggest that YAP activity might contribute to persistent activation and pathogenic behavior of RA FLS. Therefore, modulating YAP pathway might represent a novel therapeutic approach for RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-yap-pathway-regulates-fibroblast-like-synoviocyte-invasion/