Cytokine-induced cartilage proteoglycan degradation is mediated by aggrecanase

To evaluate the relationship between specific cleavage of aggrecan at the Glu(3730)-Ala(374) 'aggrecanase' site and degradation and release of proteoglycan catabolites from cartilage in explant cultures. Design: The monoclonal antibody, BC-3, which specifically recognizes the new N-terminus, ARGSVIL, generated by cleavage of aggrecan at the Glu(373)-Ala(374) 'aggrecanase' site, was used to follow the generation of fragments produced by cleavage at this site as compared to degradation of proteoglycan as assessed by glycosaminoglycan (GAG) release from cartilage in response to cytokines and the ability of inhibitors to block this cleavage. Results: (1) There was a strong correlation between specific cleavage at the Glu(373)-Ala(374) bond and the release of aggrecan catabolites in response to interleukin-1 (IL-1) or tumour necrosis factor (TNF) stimulation. (2) This cleavage in the interglobular domain of aggrecan was inhibited by the inclusion of cycloheximide, thus indicating a requirement for de novo protein synthesis in the induction of 'aggrecanase' activity. (3) The inhibitors. indomethacin, naproxen, tenidap, dexamethasone and doxycycline were ineffective in blocking either specific cleavage at the 'aggrecanase' site or aggrecan degradation as measured by GAG release from cartilage. (4) In contrast, compounds which act through two different mechanisms to inhibit MMPs were effective in blocking both specific cleavage at the 'aggrecanase' site and proteoglycan degradation. Conclusions: Our data suggest that 'aggrecanase' is primarily responsible for proteoglycan cleavage in these experimental systems and that tl-lis protease has properties in common with metalloproteases including members of the MMP and ADAM family. Inhibition of 'aggrecanase' may have utility in preventing cartilage loss in arthritis.