Rapid Activation of Transforming Growth Factor β–Activated Kinase 1 in Chondrocytes by Phosphorylation and K⁶³‐Linked Polyubiquitination Upon Injury to Animal Articular Cartilage

Abstract

ObjectiveMechanical injury to cartilage predisposes to osteoarthritis (OA). Wounding of the articular cartilage surface causes rapid activation of MAP kinases and NF‐κB, mimicking the response to inflammatory cytokines. This study was undertaken to identify the upstream signaling mechanisms involved.MethodsCartilage was injured by dissecting it from the articular surface of porcine metacarpophalangeal (MCP) joints or by avulsing murine proximal femoral epiphyses. Protein phosphorylation was assayed by Western blotting of cartilage lysates. Immunolocalization of phosphorylated activating transcription factor 2 (ATF‐2) and NF‐κB/p65 was detected by confocal microscopy. Messenger RNA (mRNA) was measured by quantitative reverse transcriptase–polymerase chain reaction (qRT‐PCR). Receptor associated protein 80 (RAP‐80) ubiquitin interacting motif agarose was used in a pull‐down assay to obtain K63‐polyubiquitinated proteins. Ubiquitin linkages on immunoprecipitated transforming growth factor β–activated kinase 1 (TAK‐1) were analyzed with deubiquitinases.ResultsSharp injury to porcine cartilage caused rapid activation of JNK and NF‐κB pathways and the upstream kinases MKK‐4, IKK, and TAK‐1. Pharmacologic inhibition of TAK‐1 in porcine cartilage abolished JNK and NF‐κB activation and reduced the injury‐dependent inflammatory gene response. High molecular weight species of phosphorylated TAK‐1 were induced by injury, indicating its ubiquitination. An overall increase in K63‐linked polyubiquitination was detected upon injury, and TAK‐1 was specifically linked to K63‐ but not K48‐polyubiquitin chains. In mice, avulsion of wild‐type femoral epiphyses caused similar intracellular signaling that was reduced in cartilage‐specific TAK‐1–null mice. Epiphyseal cartilage of MyD88‐null and TRAF‐6–null mice responded to injury, suggesting the involvement of a ubiquitin E3 ligase other than TRAF‐6.ConclusionActivation of TAK‐1 by phosphorylation and K63‐linked polyubiquitination by injury indicates its role in driving cell activation. Further studies are needed to identify the upstream ubiquitination mechanisms, including the E3 ligase involved.

Reciprocal inhibition of YAP/TAZ and NF-κB regulates osteoarthritic cartilage degradation

“…However, both YAP and TAK1 have been implicated in response to mechanical stimuli. TAK1 is activated upon cartilage injury 48 and YAP activity is inhibited upon mechanical stress 33 . It seems that the trigger on TAK1/YAP action is multifactorial in OA development, in which both mechanical stress and inflammatory stimuli will result in similar actions on TAK/YAP.…”

Mechanoadaptation: articular cartilage through thick and thin

“…; Ismail et al . ). The receptor proximal mechanism of activation for most of these remains unknown.…”

“…The upstream activator of TAK1 remains elusive and does not appear to be mediated by a soluble factor (Ismail et al . ). Exceeding the injury threshold could occur either when the mechanical load is increased, or when the load is moderate but the joint has lost its mechanoprotective mechanisms.…”

Of mice and men: converging on a common molecular understanding of osteoarthritis

“…Osteoarthritis is a mechanically driven disease. This notion is compellingly described in the epidemiological literature 6 and confirmed in basic science studies, which have shown the highly mechanosensitive nature of joint tissues, 7 , 8 , 9 , 10 , 11 , 12 the activation of inflammatory signalling by mechanical injury, 12 , 13 the dependence on mechanics in preclinical osteoarthritis, 14 , 15 and the involvement of mechanosensing mechanisms in in-vivo pathogenesis. 16 , 17 Several other important causal factors—such as obesity, age, and genetics—affect the ability of joint tissues to withstand mechanical stress over a lifetime and affect the ability to repair damaged tissues.…”