Increasing evidence implicates serine proteinases in the proteolytic cascades leading to the pathological destruction of extracellular matrices such as cartilage in osteoarthritis (OA). We have previously demonstrated that the type II transmembrane serine proteinase (TTSP) matriptase acts as a novel initiator of cartilage destruction via the induction and activation of matrix metalloproteinases (MMPs). Hepsin is another TTSP expressed in OA cartilage such that we hypothesized this proteinase may also contribute to matrix turnover. Herein, we demonstrate that addition of hepsin to OA cartilage in explant culture induced significant collagen and aggrecan release and activated proMMP-1 and proMMP-3. Furthermore, hepsin directly cleaved the aggrecan core protein at a novel cleavage site within the interglobular domain. Hepsin expression correlated with synovitis as well as tumour necrosis factor α expression, and was induced in cartilage by a pro-inflammatory stimulus. However, a major difference compared to matriptase was that hepsin demonstrated markedly reduced capacity to activate proteinase-activated receptor-2. Overall, our data suggest that hepsin, like matriptase, induces potent destruction of the extracellular matrix whilst displaying distinct efficiencies for the cleavage of specific substrates.

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.

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 72MKKPR, ultimately resulting in the fully active form with the neo‐N terminus of 85YNVFP. 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.

MicroRNAs are non-coding RNAs that act to downregulate the expression of target genes by translational repression and degradation of messenger RNA molecules. Individual microRNAs have the ability to specifically target a wide array of gene transcripts, therefore allowing each microRNA to play key roles in multiple biological pathways. miR-324 is a microRNA predicted to target thousands of RNA transcripts and is expressed far more highly in the brain than in any other tissue, suggesting that it may play a role in one or multiple neurological pathways. Here we present data from the first global miR-324-null mice, in which increased excitability and interictal discharges were identified in vitro in the hippocampus. RNA sequencing was used to identify differentially expressed genes in miR-324-null mice which may contribute to this increased hippocampal excitability, and 3′UTR luciferase assays and western blotting revealed that two of these, Suox and Cd300lf, are novel direct targets of miR-324. Characterisation of microRNAs that produce an effect on neurological activity, such as miR-324, and identification of the pathways they regulate will allow a better understanding of the processes involved in normal neurological function and in turn may present novel pharmaceutical targets in treating neurological disease.

miR-140 is selectively expressed in cartilage. Deletion of the entire Mir140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology; however, the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5 ′ ′ ′ ′ ′ and 3 ′ ′ ′ ′ ′ end, with >99% having one of two seed sequences (5 ′ ′ ′ ′ ′ bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes, respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3 ′ ′ ′ ′ ′ UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published data sets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140-3p.1 is more functional than original consensus miR-140-3p seed containing isomiR.