Protection against murine osteoarthritis by inhibition of the 26S proteasome and lysine-48 linked ubiquitination.
OBJECTIVE
To determine whether the process of ubiquitination and/or activity of the 26S proteasome are involved in the induction of osteoarthritis (OA).


METHODS
Bovine cartilage resorption assays, chondrocyte cell-line SW1353 and primary human articular chondrocytes were used with the general proteasome inhibitor MG132 or vehicle to identify a role of the ubiquitin-proteasome system (UPS) in cartilage destruction and matrix metalloproteinase-13 (MMP13) expression. In vivo, MG132 or vehicle, were delivered subcutaneously to mice following destabilisation of the medial meniscus (DMM)-induced OA. Subsequently, DMM was induced in Lys-to-Arg (K48R and K63R) mutant ubiquitin (Ub) transgenic mice. Cytokine signalling in SW1353s was monitored by immunoblotting and novel ubiquitinated substrates identified using Tandem Ubiquitin Binding Entities purification followed by mass spectrometry. The ubiquitination of TRAFD1 was assessed via immunoprecipitation and immunoblotting and its role in cytokine signal-transduction determined using RNA interference and real-time RT-PCR for MMP13 and interleukin-6 (IL6).


RESULTS
Supplementation with the proteasome inhibitor MG132 protected cartilage from cytokine-mediated resorption and degradation in vivo in mice following DMM-induced OA. Using transgenic animals only K48R-mutated Ub partially protected against OA compared to wild-type or wild-type Ub transgenic mice, and this was only evident on the medial femoral condyle. After confirming ubiquitination was vital for NF-κB signalling and MMP13 expression, a screen for novel ubiquitinated substrates involved in cytokine-signalling identified TRAFD1; the depletion of which reduced inflammatory mediator-induced MMP13 and IL6 expression.


CONCLUSIONS
Our data for the first time identifies a role for ubiquitination and the proteasome in the induction of OA via regulation of inflammatory mediator-induced MMP13 expression. These data open avenues of research to determine whether the proteasome, or K48-linked ubiquitination, are potential therapeutic targets in OA.
Musculoskeletal Research Group, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle-upon-Tyne, UK Cardiff School of Biosciences, The Sir Martin Evans Building, Cardiff University, Cardiff, UK.
Marta
M
Radwan
Musculoskeletal Research Group, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle-upon-Tyne, UK.
David J
DJ
Wilkinson
Musculoskeletal Research Group, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle-upon-Tyne, UK.
Wang
W
Hui
Musculoskeletal Research Group, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle-upon-Tyne, UK.
Auriane P M
AP
Destrument
Musculoskeletal Research Group, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle-upon-Tyne, UK.
Sarah H
SH
Charlton
Musculoskeletal Research Group, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle-upon-Tyne, UK.
Matt J
MJ
Barter
Musculoskeletal Research Group, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle-upon-Tyne, UK.
Beth
B
Gibson
Centre for Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.
Josée
J
Coulombe
Musculoskeletal Research Group, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle-upon-Tyne, UK Centre for Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.
Douglas A
DA
Gray
Musculoskeletal Research Group, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle-upon-Tyne, UK.
Andrew D
AD
Rowan
Musculoskeletal Research Group, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle-upon-Tyne, UK.
David A
DA
Young
D000818:Animals; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004195:Disease Models, Animal; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D007976:Leupeptins / Q000493:pharmacokinetics*; D008239:Lysine / Q000378:metabolism; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010003:Osteoarthritis / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D054875:Ubiquitination / Q000502:physiology*; D016335:Zinc Fingers / Q000502:physiology
D015853:Cysteine Proteinase Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D007976:Leupeptins; D053509:Matrix Metalloproteinase 13; C507081:Mmp13 protein, mouse; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D008239:Lysine; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
Chondrocytes; Cytokines; Osteoarthritis