Low molecular weight isoforms of the aggrecanases are responsible for the cytokine‐induced proteolysis of aggrecan in a porcine chondrocyte culture system

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Abstract
Objective: The major proteases responsible for aggrecan turnover in articular cartilage are the aggrecanases (ADAMTS‐4 and ADAMTS‐5). Although several studies have demonstrated C‐terminal truncation of these aggrecanases, the mechanism and importance of this processing are poorly understood. The objective of this study was to further investigate ADAMTS‐4 and ADAMTS‐5 C‐terminal truncation in a porcine model in vitro culture system.Methods: Chondrocyte–agarose cultures with well‐established extracellular matrices were treated with or without interleukin‐1 (IL‐1), for a variety of different culture time periods. Cultures were analyzed for release of sulfated glycosaminoglycan, aggrecanase‐generated interglobular domain (IGD)–aggrecan cleavage, and the presence of ADAMTS‐4 and ADAMTS‐5 isoforms. Inhibition of aggrecanase activity with monoclonal antibodies, tissue inhibitor of metalloproteinases 3 (TIMP‐3), and cycloheximide pretreatment were used to identify ADAMTS isoforms involved in IGD–aggrecan catabolism.Results: Multiple isoforms, including possible zymogens, of ADAMTS‐4 and ADAMTS‐5 were sequestered within the extracellular matrix formed by 3‐week chondrocyte–agarose cultures. IL‐1 exposure induced production of a low molecular weight (37 kd) isoform of ADAMTS‐4. This isoform was capable of degrading exogenous aggrecan at the IGD–aggrecanase site, was inhibited by TIMP‐3, was blocked after preincubation with an antibody to a sequence in the catalytic domain of ADAMTS‐4, and required de novo synthesis in the presence of IL‐1 for its generation.Conclusion: In porcine chondrocyte–agarose cultures, a 37‐kd ADAMTS‐4 isoform appears to be the major matrix protease responsible for the IGD–aggrecanase activity detected in response to exposure to IL‐1. This conclusion contradicts that of recent studies of transgenic knockout mice and highlights the need to determine the roles of the different aggrecanase(s) in human disease.