Cleavage of aggrecan between residues Glu(373)-Ala(374), which is believed to be a key event in aggrecan destruction in arthritic diseases, has been attributed to an enzymatic activity, aggrecanase. Two cartilage aggrecanases have been identified, aggrecanase-1 (ADAM-TS4) and aggrecanase-2 (ADAM-TS5) and both enzymes have been shown very efficiently to cleave soluble aggrecan at the Glu(373)-Ala(374) site.To determine whether ADAM-TS4 and/or ADAM-TS5 are the aggrecanases responsible for aggrecan catabolism following interleukin-1 (IL-1) and tumor necrosis factor (TNF) treatment of bovine articular cartilage.(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.