The serine proteinase hepsin is an activator of pro-matrix metalloproteinases: molecular mechanisms and implications for extracellular matrix turnover

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.

Serine proteinases in the turnover of the cartilage extracellular matrix in the joint: implications for therapeutics

“…A related TTSP, hepsin, can activate proMMP1 and proMMP3 and also induce cartilage destruction when added to human OA cartilage cultures. However, the lower levels of collagen release compared to matriptase are likely attributable to the markedly reduced capacity of hepsin for PAR2 activation (Wilkinson et al ., ), further highlighting how even related proteinases can have overlapping yet distinct substrate repertoires (Qiu et al ., ).…”

Collagenolytic matrix metalloproteinases antagonize proteinase-activated receptor-2 activation, providing insights into extracellular matrix turnover

“…The kinetic profiles of PAR2 cleavage by the collagenases were explored and compared with matriptase, a potent activator of PAR2 (12). Unsurprisingly, all three collagenases were less efficient at cleaving the substrate compared to matriptase (which exhibited similar kinetics to previously published findings (32,33)), primarily as a result of lower k cat values, with broadly similar K M values.…”

“…Non-linear regression analysis was performed to generate kinetic constants K M and V max (GraphPad Prism Software), and k cat calculated. Conditioned medium from an IL-1+OSMstimulated bovine nasal cartilage explant cultures was generated as previously (12).…”

“…SDS-PAGE and Immunoblotting -Whole cell lysates were generated using RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, 10 mM sodium fluoride and 1 mM sodium orthovanadate, supplemented with a cOmplete Mini Protease Inhibitor Cocktail tablet). Samples were prepared and SDS-PAGE performed as previously (12). Proteins were transferred to PVDF membrane using a Trans-Blot SD Semi-Dry Transfer Cell (BioRad, Watford, UK) according to the manufacturer's instructions, and incubated with primary antibody overnight at 4°C.…”

“…The importance of PAR2 in the pathogenesis of both inflammatory arthritis and OA has recently emerged, and PAR2 deficiency provides protection in experimental arthritis models (4)(5)(6)(7)(8)(9). We have demonstrated that an activator of PAR2, matriptase, induces cartilage destruction in a PAR2-and metalloproteinasedependent manner (10,11) and that a related serine proteinase, hepsin, can also activate PAR2 albeit in a markedly less efficient manner (12). Indeed, in complex disease environments (such as arthritis) multiple proteinases could be present to act on PAR2 making our understanding of how PAR2 is activated and regulated important (reviewed in (13)).…”