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Matrix metalloproteinase-13 (MMP-13) is a uniquely important collagenase that promotes the irreversible destruction of cartilage collagen in osteoarthritis (OA). Collagenase activation is a key control point for cartilage breakdown to occur, yet our understanding of the proteinases involved in this process is limited. Neutrophil elastase (NE) is a well-described proteoglycan-degrading enzyme which is historically associated with inflammatory arthritis, but more recent evidence suggests a potential role in OA. In this study, we investigated the effect of neutrophil elastase on OA cartilage collagen destruction and collagenase activation. Neutrophil elastase induced significant collagen destruction from human OA cartilage ex vivo, in an MMP-dependent manner. In vitro, neutrophil elastase directly and robustly activated pro-MMP-13, and N-terminal sequencing identified cleavage close to the cysteine switch at 72 MKKPR, ultimately resulting in the fully active form with the neo-N terminus of 85 YNVFP. Mole-per-mole, activation was more potent than by MMP-3, a classical collagenase activator. Elastase was detectable in human OA synovial fluid and OA synovia which displayed histologically graded evidence of synovitis. Bioinformatic analyses demonstrated that, compared with other tissues, control cartilage exhibited remarkably high transcript levels of the major elastase inhibitor, (AAT) alpha-1 antitrypsin (gene name SERPINA1), but these were reduced in OA. AAT was located predominantly in superficial cartilage zones, and staining enhanced in regions of cartilage damage. Finally, active MMP-13 specifically inactivated AAT by removal of the serine proteinase cleavage/inhibition site. Taken together, this study identifies elastase as a novel activator of pro-MMP-13 that has relevance for cartilage collagen destruction in OA patients with synovitis., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Serpins (serine proteinase inhibitors) are an ancient superfamily of structurally similar proteins, the majority of which use an elegant suicide inhibition mechanism to target serine proteinases. Despite likely evolving from a single common ancestor, the 36 human serpins have established roles regulating diverse biological processes, such as blood coagulation, embryonic development and extracellular matrix (ECM) turnover. Genetic mutations in serpin genes underpin a host of monogenic disorders - collectively termed the 'serpinopathies' - but serpin dysregulation has also been shown to drive pathological mechanisms in many common diseases. Osteoarthritis is a degenerative joint disorder, characterised by the progressive destruction of articular cartilage. This breakdown of the cartilage is driven by the metalloproteinases, and it has long been established that an imbalance of metalloproteinases to their inhibitors is of critical importance. More recently, a role for serine proteinases in cartilage destruction is emerging; including the activation of latent matrix metalloproteinases and cell-surface receptors, or direct proteolysis of the ECM. Serpins likely regulate these processes, as well as having roles beyond serine proteinase inhibition. Indeed, serpins are routinely observed to be highly modulated in osteoarthritic tissues and fluids by 'omic analysis, but despite this, they are largely ignored. Confusing nomenclature and an underappreciation for the role of serine proteinases in osteoarthritis (OA) being the likely causes. In this narrative review, serpin structure, biochemistry and nomenclature are introduced, and for the first time, their putative importance in maintaining joint tissues - as well as their dysregulation in OA - are explored., (© 2021 The Author(s).)

Objectives: 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., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Objective: To assess the role of glycogen synthase kinase 3 (GSK-3) as a regulator of cartilage destruction in human tissue and a murine model of osteoarthritis (OA)., Methods: Surgical destabilization of the medial meniscus (DMM) was performed to induce experimental murine OA, and joint damage was assessed histologically. Bovine nasal and human OA cartilage samples were incubated with interleukin-1 (IL-1) plus oncostatin M (OSM) and GSK-3 inhibitor. Collagen and proteoglycan release was assessed by hydroxyproline measurement and dye binding assay, collagenase activity was assessed by bioassay, and gene expression was analyzed by real-time polymerase chain reaction. Human articular chondrocytes were isolated by enzymatic digestion and cultured prior to gene silencing and immunoblotting of cell lysates and nuclear fractions., Results: Mice treated with GSK-3 inhibitor exhibited significantly greater cartilage damage compared with sham-operated control mice. GSK-3 inhibition in bovine cartilage dramatically accelerated IL-1 plus OSM-stimulated degradation, concomitant with a profound increase in collagenase activity. GSK-3 inhibitor induced collagen release from human OA cartilage in the presence of IL-1 plus OSM and increased proteoglycan loss. Gene expression profiling of resorbing OA cartilage revealed a marked procatabolic switch in gene expression upon GSK-3 inhibition. This was mirrored in human articular chondrocytes following GSK3 silencing, particularly with the GSK-3β isoform. GSK-3 inhibition or silencing led to enhanced IL-1 plus OSM-stimulated abundance and activity of Jun, and silencing of c-jun ameliorated GSK-3 inhibitor-mediated procatabolic gene expression., Conclusion: GSK-3 is an important regulator of matrix metalloproteinase (MMP)-mediated joint destruction, the inhibition of which by proinflammatory stimuli de-represses catabolic gene expression. Therapeutic strategies that maintain cartilage GSK-3 activity may therefore help curtail aberrant MMP activity during pathologic joint destruction., (Copyright © 2014 by the American College of Rheumatology.)

ObjectiveTo assess the ability of matriptase, a type II transmembrane serine proteinase, to promote aggrecan loss from the cartilage of patients with osteoarthritis (OA) and to determine whether its inhibition can prevent aggrecan loss and cartilage damage in experimental OA.MethodsAggrecan release from human OA cartilage explants and human stem cell-derived cartilage discs was evaluated, and cartilage-conditioned media were used for Western blotting. Gene expression was analyzed by real-time polymerase chain reaction. Murine OA was induced by surgical destabilization of the medial meniscus, and matriptase inhibitors were administered via osmotic minipump or intraarticular injection. Cartilage damage was scored histologically and aggrecan cleavage was visualized immunohistochemically using specific neoepitope antibodies.ResultsThe addition of soluble recombinant matriptase promoted a time-dependent release of aggrecan (and collagen) from OA cartilage, which was sensitive to metalloproteinase inhibition and protease-activated receptor 2 antagonism. Although engineered human (normal) cartilage discs failed to release aggrecan following matriptase addition, both matrix metalloproteinase- and aggrecanase-mediated cleavages of aggrecan were detected in human OA cartilage. Additionally, while matriptase did not directly degrade aggrecan, it promoted the accumulation of low-density lipoprotein receptor-related protein 1 (LRP-1) in conditioned media of the OA cartilage explants. Matriptase inhibition via neutralizing antibody or small molecule inhibitor significantly reduced cartilage damage scores in murine OA, which was associated with reduced generation of metalloproteinase-mediated aggrecan cleavage.ConclusionMatriptase potently induces the release of metalloproteinase-generated aggrecan fragments as well as soluble LRP-1 from OA cartilage. Therapeutic targeting of matriptase proteolytic activity reduces metalloproteinase activity, further suggesting that this serine proteinase may have potential as a disease-modifying therapy in OA.