Inhibition of furin‐like enzymes blocks interleukin‐1α/oncostatin M–stimulated cartilage degradation

Abstract

Objective. To investigate the role of furin-like enzymes in the proteolytic cascades leading to cartilage breakdown and to examine which collagenase(s) contribute to collagen degradation.Methods. Bovine nasal cartilage was stimulated to resorb with the addition of interleukin-1␣ (IL-1␣)/ oncostatin M (OSM) in the presence or absence of a furin inhibitor, Dec-RVKR-CH 2 Cl, or selective matrix metalloproteinase 1 (MMP-1) inhibitors. Collagen and proteoglycan levels were determined by assay of hydroxyproline and sulfated glycosaminoglycan, respectively. Collagenase and gelatinase activity were measured using 3 H-acetylated collagen and gelatin zymography, respectively.Results. The addition of Dec-RVKR-CH 2 Cl to stimulated cartilage reduced the release of collagen fragments and the levels of active collagenase and MMP-2, suggesting that furin-like enzymes are involved in the cascades leading to activation of procollagenases. At MMP inhibitor concentrations that selectively inhibit MMP-1, no inhibition of collagen release was observed, but increasing the concentration to the 50% inhibition concentration for MMP-13 resulted in a 50% blockage of collagen release. The addition of Dec-RVKR-CH 2 Cl to resorbing cartilage also partially blocked proteoglycan release, thus demonstrating a role for furin-activated enzymes in the pathways leading to proteoglycan degradation. Conclusion.Furin-like enzymes are involved in cascades leading to activation of procollagenases and degradation of collagen. MMP-13, which can be activated by furin-processed membrane-type 1 MMP-1, appears to be a major collagenase involved in collagen degradation induced by IL-1␣/OSM. Furin-like enzymes also appear to play a role in the pathways leading to proteoglycan degradation. These findings are of importance when considering proteinase inhibition as a target for therapeutic intervention in arthritic diseases.

Emerging roles of serine proteinases in tissue turnover in arthritis

“…In a model of cartilage resorption, we have shown that the addition of a chloromethylketone PC inhibitor reduces the levels of aggrecan and collagen breakdown, thus implicating a role for PCs in the cascades leading to cartilage resorption (148). In this assay, a reduction in the levels of active collagenase and MMP-2 was observed, suggesting that PCs are involved in proMMP activation (see below).…”

“…Although it is still too early to know whether distinct serine proteinase cascade(s) occur in RA and OA, it is evident that there is overlap in terms of collagenolysis (148,176,183). Further understanding of the complex roles of these enzymes, their substrate specificities, and identification of novel serine proteinases in the arthritic joint will provide new opportunities to identify tractable therapeutic targets that effectively prevent pathologic tissue turnover in RA and OA.…”

“…Activation of procollagenases in cartilage and other collagenous matrices is an important control point and a rate-limiting step in collagen resorption (176)(177)(178)(179)(180). We have shown that both furin-and trypsin-like serine proteinases are involved in the cascades that lead to such activation (148,176), although the precise proteinases involved remain unknown. It has long been established that plasmin can activate procollagenases (36), leading to the suggestion that the PA/plasmin cascade might function in various resorbing tissues to activate proMMPs, including procollagenases (37).…”

Proteinases involved in matrix turnover during cartilage and bone breakdown

“…2) have a furin recognition sequence between the propeptide and the catalytic domain and these enzymes are often activated within the Golgi. Recent data show that cartilage explant cultures, treated with cytokines and an inhibitor of furin, have reduced levels of active collagenases and low collagen release (Milner et al 2003). For those MMPs without a furin site, the proteolytic removal of the propeptide is likely to be achieved in a tightly controlled environment close to the cell surface.…”

Assay of collagenase activity for native triple-helical collagen using capillary gel electrophoresis with laser-induced fluorescence detection

“…The assay using the coumarin-labeled peptide substrate has been in popular use for HTS; however, the other estimation of inhibitors should be needed to confirm the inhibition of collagenase activity using the native substrate, because the labeled peptide substrate has different characteristics from the native one. At the second stage, the drug candidates that have passed through the first stage need to be checked for whether they can inhibit the cleavage of triple-helical collagen by collagenase, using sodium dodecylsulfate-polyacrylamide gel electrophoresis [6], zymography [7][8][9] and collagenolysis assay [10][11][12] with native, fluorescence-labeled or radioisotope-labeled collagen as a substrate. However, these assays have drawbacks such as troublesome procedure and poor quantification.…”