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 fr agments produced by cleavage at this site as compared to degradation o f proteoglycan as assessed by glycosaminoglycan (GAG) release from car tilage in response to cytokines and the ability of inhibitors to block this cleavage. Results: (1) There was a strong correlation between sp ecific cleavage at the Glu(373)-Ala(374) bond and the release of aggre can catabolites in response to interleukin-1 (IL-1) or tumour necrosis factor (TNF) stimulation. (2) This cleavage in the interglobular doma in of aggrecan was inhibited by the inclusion of cycloheximide, thus i ndicating 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 degrada tion as measured by GAG release from cartilage. (4) In contrast, compo unds which act through two different mechanisms to inhibit MMPs were e ffective in blocking both specific cleavage at the 'aggrecanase' site and proteoglycan degradation. Conclusions: Our data suggest that 'aggr ecanase' is primarily responsible for proteoglycan cleavage in these e xperimental systems and that tl-lis protease has properties in common with metalloproteases including members of the MMP and ADAM family. In hibition of 'aggrecanase' may have utility in preventing cartilage los s in arthritis.