Objective We aimed to understand the role of the tyrosine phosphatase PTPN14—which in cancer cells modulates the Hippo pathway by retaining YAP in the cytosol—in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).
Methods Gene/protein expression levels were measured by quantitative PCR and/or Western blotting. Gene knockdown in RA FLS was achieved using antisense oligonucleotides. The interaction between PTPN14 and YAP was assessed by immunoprecipitation. The cellular localisation of YAP and SMAD3 was examined via immunofluorescence. SMAD reporter studies were carried out in HEK293T cells. The RA FLS/cartilage coimplantation and passive K/BxN models were used to examine the role of YAP in arthritis.
Results RA FLS displayed overexpression of PTPN14 when compared with FLS from patients with osteoarthritis (OA). PTPN14 knockdown in RA FLS impaired TGFβ-dependent expression of MMP13 and potentiation of TNF signalling. In RA FLS, PTPN14 formed a complex with YAP. Expression of PTPN14 or nuclear YAP—but not of a non-YAP-interacting PTPN14 mutant—enhanced SMAD reporter activity. YAP promoted TGFβ-dependent SMAD3 nuclear localisation in RA FLS. Differences in epigenetic marks within Hippo pathway genes, including YAP, were found between RA FLS and OA FLS. Inhibition of YAP reduced RA FLS pathogenic behaviour and ameliorated arthritis severity.
Conclusion In RA FLS, PTPN14 and YAP promote nuclear localisation of SMAD3. YAP enhances a range of RA FLS pathogenic behaviours which, together with epigenetic evidence, points to the Hippo pathway as an important regulator of RA FLS behaviour.
- rheumatoid arthritis
- fibroblast-like synoviocytes
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AB and DJW contributed equally.
Handling editor Josef S Smolen
Contributors AB, DJW, CP and NB conceived, designed and supervised the research, analysed data and wrote the paper. AB, DJW, RA, MLR, BB, MB, KMD, VZ, CS, MZ, AL, XL, LL, DLB, DH, T-CM, MC, SMS, ML, WW, GF, YK-G contributed to acquisition and/or analysis of data. All authors approved the final version of the paper.
Funding This study was funded in part by the National Institutes of Health (NIH) Grants R01 AR066053 and AI070544 and Department of Defense (DOD) grant # W81XWH-16-1-0751 (to NB). AB and DJW were supported by NIH Training Grant T32 AR064194.
Competing interests None declared.
Patient consent for publication Not required.
Ethics approval All animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of the La Jolla Institute (#AP140-NB4) and UC SAN DIEGO (#S16098). The generation and banking of FLS lines from arthroplasties was approved by the UC San Diego IRB (#140175). Ethical approval for the PEAC cohort was granted by the King’s College Hospital Research Ethics Committee (REC 05/Q0703/198).
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement There are no additional unpublished data.
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