Objective. Recent published studies have shown that cartilage from ADAMTS-5-knockout mice, but not ADAMTS-4-or ADAMTS-1-knockout mice, is significantly protected from degradation. The present study was undertaken to evaluate the respective roles of these enzymes in human cartilage breakdown, using a small interfering RNA (siRNA) approach to assess the effects of inhibition of each enzyme in normal and osteoarthritic (OA) explants.Methods. The activities of siRNA specifically targeting ADAMTS-1, -4, and -5 were assessed by transfection into primary human chondrocytes and cultured human cartilage explants. At 24 hours, a cytokine stimulus was applied to normal, but not OA, samples to initiate a catabolic response. At designated times, total RNA was isolated and gene expression was measured by quantitative real-time reverse transcription-polymerase chain reaction. Aggrecan release and aggrecanasegenerated neoepitope formation were determined by dye binding analysis and Western blotting, respectively.Results. Human chondrocytes and explants were efficiently transfected with siRNA that specifically decreased the expression of each targeted gene. Suppression of ADAMTS-4 and ADAMTS-5, individually or in combination, attenuated the degradation of aggrecan in cytokine-stimulated normal cartilage. A reduction in aggrecan degradation was also observed following siRNA-mediated knockdown of either gene in unstimulated OA cartilage. In contrast, knockdown of ADAMTS-1 failed to inhibit aggrecan loss.Conclusion. Despite the apparent dominant role of ADAMTS-5 in genetically modified mice, our data suggest that both ADAMTS-4 and ADAMTS-5 contribute to the structural damage that characterizes human OA.
(1) IL-1- and TNF-stimulated release of aggrecan was associated with cleavage of aggrecan within the C-terminus at the ADAM-TS4 and ADAM-TS5-sensitive sites, Glu(1480)-Gly(1481), Glu(1667)-Gly(1668), and Glu(1871)-Leu(1872). (2) The order of cleavage following IL-1 stimulation of cartilage explants was the same as when soluble aggrecan is digested with recombinant human ADAM-TS4 and ADAM-TS5. (3) Both constitutive and stimulated cleavage of aggrecan at the ADAM-TS4 and ADAM-TS5-sensitive sites in cartilage was blocked by a general metalloproteinase inhibitor but not by a MMP-specific inhibitor, and this inhibition correlated with inhibition of aggrecan release from cartilage. (4) PCR and Western blot analysis indicated that both ADAM-TS proteases are expressed in cartilage explants; ADAM-TS5 is constitutively expressed whereas ADAM-TS4 is induced following IL-1 and TNF treatment. (5) Immunodepletion of both ADAM-TS4 and ADAM-TS5 from bovine articular cartilage cultures following IL-1 stimulation resulted in a 90% reduction of aggrecanase activity in the culture medium.
Osteoarthritis is a degenerative joint disorder characterized by breakdown of articular cartilage. Degradation of aggrecan, which together with type II collagen provides cartilage with its unique characteristics of compressibility and elasticity, is an early and sustained feature of osteoarthritis. The present work was set up to identify the enzyme(s) responsible for aggrecan breakdown in osteoarthritis. We found that the two cartilage aggrecanases, ADAM-TS4 and ADAM-TS5, are present in osteoarthritic cartilage and that they are responsible for aggrecan degradation without the participation of matrix metalloproteinases. This is based on 1) neoepitopes found on aggrecan fragments in osteoarthritis (OA) cartilage explants in vitro, 2) aggrecan fragments detected in synovial fluid of OA patients, 3) the observation that an aggrecanase inhibitor, BB-16, blocked aggrecan degradation in OA cartilage in vitro, whereas the matrix metalloproteinase inhibitor XS309 did not, and 4) the presence of mRNA and protein for ADAM-TS4 and ADAM-TS5 in OA cartilage. These results suggest that ADAM-TS4 and ADAM-TS5 represent a potential target for the treatment of osteoarthritis.
Osteoarthritis is a chronic and painful disease of synovial joints. Chondrocytes, synovial cells and other cells in the joint can express and respond to cytokines and chemokines, and all of these molecules can also be detected in synovial fluid of patients with osteoarthritis. The presence of inflammatory cytokines in the osteoarthritic joint raises the question whether they may directly participate in pain generation by acting on innervating joint nociceptors. Here, we first provide a systematic discussion of the known proalgesic effects of cytokines and chemokines that have been detected in osteoarthritic joints, including TNF-α, IL-1, IL-6, IL-15, IL-10, and the chemokines, MCP-1 and fractalkine. Subsequently, we discuss what is known about their contribution to joint pain based on studies in animal models. Finally, we briefly discuss limited data available from clinical studies in human osteoarthritis.
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