Inhibition of ADAMTS-7 and ADAMTS-12 degradation of cartilage oligomeric matrix protein by alpha-2-macroglobulin

Summary Objective As we previously reported, ADAMTS-7 and ADAMTS-12, two members of ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family, degrade cartilage oligomeric matrix protein (COMP) in vitro and are significantly induced in the cartilage and synovium of arthritic patients [Liu CJ, Kong W, Ilalov K, Yu S, Xu K, Prazak L, et al. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. FASEB J 2006;20(7):988–90; Liu CJ, Kong W, Xu K, Luan Y, Ilalov K, Sehgal B, et al. ADAMTS-12 associates with and degrades cartilage oligomeric matrix protein. J Biol Chem 2006;281(23):15800–8]. The purpose of this study was to determine (1) whether cleavage activity of ADAMTS-7 and ADAMTS-12 of COMP are associated with COMP degradation in osteoarthritis (OA); (2) whether alpha-2-macroglobulin (a2 M) is a novel substrate for ADAMTS-7 and ADAMTS-12; and (3) whether a2 M inhibits ADAMTS-7 or ADAMTS-12 cleavage of COMP. Methods An in vitro digestion assay was used to examine the degradation of COMP by ADAMTS-7 and ADAMTS-12 in the cartilage of OA patients; in cartilage explants incubated with tumor necrosis factor-alpha (TNF-α) or interleukin-1-beta (IL-1β) with or without blocking antibodies; and in human chondrocytes treated with specific small interfering RNA (siRNA) to knockdown ADAMTS-7 or/and ADAMTS-12. Digestion of a2 M by ADAMTS-7 and ADAMTS-12 in vitro and the inhibition of ADAMTS-7 or ADAMTS-12-mediated digestion of COMP by a2 M were also analyzed. Results The molecular mass of the COMP fragments produced by either ADAMTS-7 or ADAMTS-12 were similar to those observed in OA patients. Specific blocking antibodies against ADAMTS-7 and ADAMTS-12 dramatically inhibited TNF-α- or IL-1β-induced COMP degradation in the cultured cartilage explants. The suppression of ADAMTS-7 or ADAMTS-12 expression by siRNA silencing in the human chondrocytes also prevented TNF-α- or IL-1β-induced COMP degradation. Both ADAMTS-7 and ADAMTS-12 were able to cleave a2 M, giving rise to 180- and 105-kDa cleavage products, respectively. Furthermore, a2 M inhibited both ADAMTS-7- and ADAMTS-12-mediated COMP degradation in a concentration (or dose)-dependent manner. Conclusion Our observations demonstrate the importance of COMP degradation by ADAMTS-7 and ADAMTS-12 in vivo . Furthermore, a2 M is a novel substrate for ADAMTS-7 and ADAMTS-12. More significantly, a2 M represents the first endogenous inhibitor of ADAMTS-7 and ADAM