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The release of aggrecan catabolites from cartilage is an early event in the pathogenesis of degenerative joint diseases. The enzymes involved in this process are unknown, controversial, and the subject of intense investigation. In this paper we have utilized a recombinant substrate containing the interglobular domain (IGD) of aggrecan to study specifically aggrecanase versus matrix metalloproteinase (MMP) catabolism in this domain of aggrecan. Our studies have shown that (i) there are species differences in the expression of latent versus active MMP activity on the aggrecan IGD; (ii) interleukin-1alpha exposure induces both aggrecanase and MMP activities, whereas retinoic acid induces only aggrecanase activity and inhibits the MMP activity on the aggrecan IGD; (iii) activators of latent MMP activity (p-aminophenylmercuric acetate and trypsin) significantly reduce aggrecanase activity; (iv) the time course of the appearance of aggrecanase versus the MMP catabolism of aggrecan IGD differs; (v) aggrecanase is a protease with metalloprotease characteristics; however (vi) the physiological (tissue) inhibitors of MMPs show weak inhibition (TIMP-1) or no inhibition (TIMP-2) of aggrecanase activity. Collectively, these studies show that aggrecanase and MMP catabolism of the aggrecan IGD are independent and uncoupled.

The recent detection of membrane type 1 matrix metalloproteinase (MT1-MMP) expression in human articular cartilage [Büttner, Chubinskaya, Margerie, Huch, Flechtenmacher, Cole, Kuettner, and Bartnik (1997) Arthritis Rheum. 40, 704-709] prompted our investigation of MT1-MMP's catabolic activity within the interglobular domain of aggrecan. For these studies we used rAgg1mut, a mutated form of the recombinant fusion protein (rAgg1) that has been used as a substrate to monitor 'aggrecanase' catabolism in vitro [Hughes, Büttner, Eidenmüller, Caterson and Bartnik (1997) J. Biol. Chem. 272, 20269-20274]. The rAgg1 was mutated (G332 to A) to avoid the generation of a splice variant seen with the original genetic construct, which gave rise to heterogeneous glycoprotein products. This mutation yielded a homogeneous recombinant product. Studies in vitro with retinoic acid-stimulated rat chondrosarcoma cells indicated that the rAgg1mut substrate was cleaved at the 'aggrecanase' site equivalent to Glu373-Ala374 (human aggrecan sequence enumeration) in its interglobular domain sequence segment. The differential catabolic activities of the recombinant catalytic domain (cd) of MT1-MMP and matrix metalloproteinases (MMPs) 3 and 8 were then compared by using this rAgg1mut as a substrate. Coomassie staining of rAgg1mut catabolites separated by SDS/PAGE showed similar patterns of degradation with all three recombinant enzymes. However, comparative immunodetection analysis, with neoepitope antibodies BC-3 (anti-ARGS...) and BC-14 (anti-FFGV...) to distinguish between 'aggrecanase' and MMP-generated catabolites, indicated that the catalytic domain of MT1-MMP exhibited strong 'aggrecanase' activity, cdMMP-8 weak activity and cdMMP-3 no activity. In contrast, cdMMP-3 and cdMMP-8 led to strongly BC-14-reactive catabolic fragments, whereas cdMT1-MMP resulted in weak BC-14 reactivity. N-terminal sequence analyses of the catabolites confirmed these results and also identified other potential minor cleavage sites within the interglobular domain of aggrecan. These results indicate that MT1-MMP is yet another candidate for 'aggrecanase' activity in vivo.

Objective: To detect the message for membrane type 1 (MT1) matrix metalloproteinase (MMP) in articular cartilage and chondrosarcoma cells, to study its expression in osteoarthritis (OA), and to determine whether interleukin-1beta (IL-1beta) influences its expression., Methods: Reverse transcription-polymerase chain reaction methods were used to detect message. Cloning and sequencing were applied to confirm the sequence. Northern blotting was used to quantify the message for MT1-MMP and to compare its expression in OA cartilage with or without IL-1beta treatment. In situ hybridization was utilized to show MT1-MMP transcripts in cartilage and to study the influence of IL-1beta., Results: The results show that MT1-MMP messenger RNA (mRNA) is expressed in chondrosarcoma cells and OA chondrocytes. Results of the in situ hybridization confirmed the expression in OA cartilage as well as in normal cartilage. The level of mRNA was not modulated following IL-1beta stimulation., Conclusion: This study shows that MT1-MMP is expressed by chondrocytes. The similarities in mRNA levels in OA and normal chondrocytes suggest that regulation of MT1-MMP mRNA may not be a primary factor in OA.