Low Molecular Weight Isoforms of the Aggrecanases Are Responsible for the Cytokine-Induced Proteolysis of Aggrecan in a Porcine Chondrocyte Culture System.

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 zy-mogens, of ADAMTS-4 and ADAMTS-5 were seques- 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.