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Objectives Mechanical injury to cartilage predisposes to osteoarthritis (OA). Wounding the articular cartilage surface causes rapid activation of MAP kinases and NFκB mimicking the response to inflammatory cytokines. Here, we identify the upstream signalling mechanisms involved. Methods Cartilage was injured by dissecting it from the articular surface of porcine metacarpophalangeal joints (MCP) or by avulsing murine proximal femoral epiphyses. Protein phosphorylation was assayed by Western blotting of cartilage lysates. Immunolocalisation of ph-ATF2 and NFkB-p65 was detected by confocal microscopy. mRNA was measured by qRT-PCR. K63-polyubiquitinated proteins were pulled down with Rap80-UIM agarose. Ubiquitin linkages on immunoprecipitated TAK1 were analysed with deubiquitinases. Results Sharp injury to porcine cartilage caused rapid activation of JNK and NFκB pathways and the upstream kinases MKK4, IKK, and TAK1. Pharmacological inhibition of TAK1 in porcine cartilage abolished JNK and NFkB activation and reduced the injury-dependent inflammatory gene response. High molecular weight species of phosphorylated TAK1 were induced by injury indicating its ubiquitination. An overall increase in K63-linked polyubiquitination was detected upon injury and TAK1 was specifically linked to K63- but not K48-polyubiquitin chains. In mice, avulsion of wildtype femoral epiphyses caused similar intracellular signalling that was reduced in cartilage-specific TAK1-null mice. Epiphyseal cartilage of MyD88-null and TRAF6-null mice responded to injury suggesting the involvement of an ubiquitin E3 ligase other than TRAF6. Conclusions Activation of TAK1 by phosphorylation and K63-linked polyubiquitination by injury indicates its role in driving the cell activation. Further investigation is required to identify the upstream ubiquitination machinery including the E3 ligase involved. This article is protected by copyright. All rights reserved.

Original publication

DOI

10.1002/art.39965

Type

Journal article

Journal

Arthritis Rheumatol

Publication Date

21/10/2016