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Abstract
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.
Highlights
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 shown that the type II transmembrane serine proteinase (TTSP) matriptase is expressed in OA cartilage, and can induce and activate proMMP-1 and -35
We have previously reported roles for proteinase-activated receptor-2 (PAR2) in inflammatory joint disease[7] and OA8,9 and shown PAR2 to be important in the production of pro-inflammatory mediators known to drive ECM breakdown such as interleukin-(IL-)[1] and tumour necrosis factor α (TNFα)[10], two cytokines which upregulate PAR2 expression in cartilage[11]
Summary
To assess whether hepsin can activate proMMPs we used cytokine-stimulated bovine cartilage explant cultures In this ex vivo model of cartilage breakdown, addition of proMMP activators induces early collagen release[5,19]. The addition of hepsin led to a significant increase in collagenolysis which was observed at both day 7 and 10 of culture (Fig. 2A, left panel) These data correlated with the levels of active collagenase detected in the media of these cultures which were similar for both hepsin- and matriptase-treated cartilages (Fig. 2A, right panel). To determine whether hepsin cleaved within the pathologically-relevant aggrecan interglobular domain (IGD), incubation with recombinant G1-IGD-G2 (corresponding to amino acids Val20-Gly[675] of the aggrecan core protein) confirmed cleavage within this region (Fig. 3A, right panel) at sites distinct from known metalloproteinase cleavage sites (Fig. 3B, left panel) Matriptase processed this protein, albeit with markedly weaker efficiency. These structural data are consistent with the notion that hydrolysis of the PAR2 activation sequence (SKGR↓SLIG) by hepsin is considerably less favourable than that provided by matriptase
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