Background/Purpose:

Modulating molecular pathways that regulate pathogenic behavior fibroblast-like synoviocytes (FLS) could lead to new therapies for rheumatoid arthritis (RA). Targeting transcription factors are especially interesting, because they regulate the expression of multiple genes and therefore regulate diverse pathways that contribute to disease. The transcriptional coactivator YAP is emerging as a major regulator of proliferation, organ size and oncogenesis. Several downstream targets of YAP such as surviving and CTGF are implicated in different aspect of RA; however the YAP pathway itself has not been characterized yet in inflammatory synovitis. Therefore, we explored expression and function of YAP in RA synoviocytes.

Methods: For confocal microscopy cells were starved overnight, fixed and stained for YAP, DAPI, and with phalloidin for F-actin. For quantification, YAP signal was scored as nuclear/nuclear+cytoplasmic versus cytoplasmic only by analyzing 150-200 cells for each cell line. For qPCR analysis the relative abundance of mRNA was calculated by normalizing to GAPDH. For siRNA knockdown, YAP and control siRNA weretransfected using AMAXA technology. To quantify cell proliferation, 5 days following transfection cell were plated in 1% FBS containing media in the presence of absence of TNF (10 ng/ml) and cell number was assayed with MTT on day 0, 2, 4 and 6.

Results:

Using Western blot analysis, YAP protein was abundant and levels were similar in RA and OA FLS (n=4). Next we assayed the subcellular localization of YAP in RA and OA FLS using confocal microscopy. 80% of the RA FLS had nuclear YAP compared with only 40% of OA cells (n=4, p<0.035). These observations suggested that YAP is constitutively active in RA FLS and is increased compared with OA. To confirm YAP transcriptional activity, qPCR was performed for YAP target genes survivin, CTGF and CCN1. mRNA levels for all three were significantly greater in RA compared with OA (n=3, P<0.04 for each). When RA FLS were stimulated with TNF, YAP was dephosphorylated (S473) and underwent nuclear translocation. This was followed by a 2.5 fold (P<0.04) increase in survivin and 1.5 fold (P<0.03) in CTGF mRNA levels (n=3). To determine critical role of YAP in TNF signaling, YAP was knocked down by siRNA and cells were stimulated with TNF. Proliferation increased 1.8 fold in control siRNA treated cells but not in YAP deficient cells (n=3, P<0.03).

Conclusion: This is the first study to demonstrate that YAP is expressed in FLS and that YAP activity is dysregulated in RA compared with OA FLS. Increased YAP activity might contribute to persistent activation and pathogenic behavior of RA FLS, especially TNF-regulated functions. Therefore, reprogramming RA FLS by modulating the YAP pathway represents a novel therapeutic approach for RA.

 

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/expression-and-function-of-a-yap-a-novel-pathway-in-fibroblast-like-synoviocyte-in-rheumatoid-arthritis/