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 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.


CONCLUSIONS
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.
Powell
Alison J
AJ
Cardiff University, Cardiff, UK.
Little
Christopher B
CB
Hughes
Clare E
CE
D051722:ADAM Proteins / Q000032:analysis / Q000502:physiology*; D000071121:ADAMTS4 Protein; D000071116:ADAMTS5 Protein; D053674:Aggrecans / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D019902:Chondrocytes / Q000201:enzymology*; D007527:Isoenzymes / Q000502:physiology; D011348:Procollagen N-Endopeptidase / Q000032:analysis / Q000502:physiology*; D013552:Swine
D053674:Aggrecans; D007527:Isoenzymes; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human; C000603919:Adamts4 protein, mouse