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TIMP-3 inhibits aggrecan breakdown in pig articular cartilage stimulated with IL-1

Degradation of articular cartilage seriously impairs the function of joints and it is a hallmark of various types of arthritides. The primary cause of this process is due to elevated proteolytic enzyme activities that degrade aggrecan proteoglycan and type II collagen fibrils, major components of the extracellular matrix in cartilage. While the network of collagen fibrils is degraded primarily by collagenases and possibly by other matrix metalloproteinases (MMPs), such as MMP-2 and MMP-14, aggrecan is degraded by MMPs and the more recently discovered 'aggrecanases' that belong to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family.

To investigate the relative contribution of MMPs and ADAMTSs in cartilage aggrecan degradation during the progression of joint destruction, we have used the pig articular cartilage explants treated with IL-1 as an in vitro cartilage degradation model and have tested the ability of tissue inhibitors of metalloproteinases (TIMPs), TIMP-1, TIMP-2 and TIMP-3, to block the release of aggrecan. MMPs are inhibited by all three TIMPs, but aggrecanases (ADAMTS-1, ADAMTS-4 and ADAMTS-5) are inhibited only by TIMP-3.

Treatment of pig articular cartilage in culture with IL-1 for 3 days resulted in approximately 70% of aggrecan degradation, and the fragments were released into the medium. The degradation of aggrecan was completely inhibited by the addition of recombinant N-terminal inhibitory domain of TIMP-3 (N-TIMP-3) at the concentration of 0.1 mM, but not by TIMP-1 or TIMP-2. This indicates that aggrecan degradation in this model is due to aggrecanases, but not due to MMPs. TIMP-3 is known to cause apoptosis of several cell types. N-TIMP-3 caused apoptosis of pig chondrocytes only at concentrations above 0.75 μM. Our mutagenesis studies of N-TIMP-3 also revealed that N-TIMP-3 contains the metalloproteinase reactive site common among TIMPs and the unique sites that interact with aggrecanases, suggesting that it may be possible to design TIMP variants that selectively inhibit aggrecanases.

Our studies suggest that TIMP-3 and the related molecules may be potential therapeutics to prevent articular cartilage from degradation during the progression of osteoarthritis and rheumatoid arthritis.


This work was supported by grants from the Wellcome Trust and the National Institutes of Health.

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Nagase, H., Gendron, C. & Kashiwagi, M. TIMP-3 inhibits aggrecan breakdown in pig articular cartilage stimulated with IL-1. Arthritis Res Ther 5 (Suppl 3), 48 (2003).

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