DEGRADATION OF TYPE-II COLLAGEN, BUT NOT PROTEOGLYCAN, CORRELATES WITH MATRIX METALLOPROTEINASE ACTIVITY IN CARTILAGE EXPLANT CULTURES

Objective. To determine the contribution of certain matrix metalloprot einases (MMPs) to the degradation of proteoglycan and type II collagen in cartilage. Methods. Bovine nasal and articular cartilage explants were cultured with recombinant human interleukin-1 alpha (IL-1 alpha) for up to 4 weeks. Release of proteoglycan and type II collagen into t he medium was determined by colorimetric assay and immunoassay, respec tively. The activity of MMPs in the medium was assayed using a quenche d fluorescent substrate, as well as with a collagen fibril assay, by z ymography, and in Western immunoblots. In some experiments, the effect s of specific MMP inhibitors on type II collagen degradation were stud ied. Results. In cultures of nasal cartilage with IL-1 alpha, almost a ll the proteoglycan was released within the first week, whereas there was no detectable release of type II collagen for the first 2 weeks of culture. A rapid period of almost complete dissolution of the collage n occurred in the third or fourth week, MMP activity measured using a quenched fluorescent substrate was negligible during the first 2 weeks of culture but was substantially increased in the third week of cultu re, at the time of collagen degradation. Similarly, there was a large increase in collagenolytic activity (by collagen fibril assay) and gel atinolytic activity, (by zymography) during the third week of culture. Articular cartilage cultured with IL-1 alpha lost proteoglycan progre ssively during the 4-week period; however, there was no loss of type I I collagen from the matrix in that time and no significant increase in MMP activity. The loss of type II collagen from nasal cartilage stimu lated with IL-1 alpha was inhibited by BB87, an inhibitor of both coll agenases and gelatinases, and by BB3003, a selective inhibitor of gela tinase A. In Western immunoblots, procollagenase and active interstiti al collagenase could be readily detected in nasal cartilage cultures. Some procollagenase 3 and active collagenase 3 was also shown to be pr esent. Conclusion. MMP activity correlates with degradation of type II collagen, but not proteoglycan, in cartilage cultures. Interstitial c ollagenase, collagenase 3, and gelatinases are all likely to contribut e to cleavage and removal of collagen from the cartilage matrix. The p roteinase(s) responsible for aggrecan breakdown remains unclear.