Aggrecanase versus matrix metalloproteinases in the catabolism of the interglobular domain of aggrecan in vitro

The importance of aggrecanase versus matrix metalloproteinase (MMP) enzymic activities in the degradation of aggrecan in normal and osteoarthritic (OA ) articular cartilage in vitro was studied in order to further our understa nding of the potential role of these two enzyme activities in aggrecan cata bolism during the pathogenesis of cartilage degeneration. Porcine and bovin e articular cartilage was maintained in explant culture for up to 20 days i n the presence or absence of the catabolic stimuli retinoic acid, interleuk in-l or tumour necrosis factor-a. Release of proteoglycan from cartilage wa s measured as glycosaminoglycan (GAG) release using a colorimetric assay. A nalysis of proteoglycan degradation products, both released into culture me dia and retained within the cartilage matrix, was performed by Western blot ting using antibodies specific for the N- and C-terminal neoepitopes genera ted by aggrecanase- and MMP-related catabolism of the interglobular domain of the aggrecan core protein (IGD), In addition, studies determining the mR NA expression for MMP-3 and MMP-13 in these same cultures were undertaken. These analyses indicated that all three catabolic agents stimulated the rel ease of > 80% of the GAG from the articular cartilage over 4 days. The degr ee of GAG release corresponded to an increase in aggrecanase-generated aggr ecan catabolites released into the media and retained within the cartilage. Importantly, there was no evidence for the release of MMP-generated aggrec an metabolites into the medium, nor the accumulation of MMP-generated catab olites within the tissue in these same cultures. Expression of the mRNAs fo r two MMPs known to be capable of degrading the aggrecan IGD, MMP-3 and MMP -13, was detected. However, increased expression of these MMPs was not corr elated with aggrecan degradation. Analyses using porcine cartilage, culture d with or without catabolic stimulation for 12 h to 20 days, indicated that primary cleavage of the IGD by aggrecanase was responsible for release of aggrecan metabolites at both the early and late time points of culture. Cul tures of late-stage OA human articular cartilage samples indicated that agg recanase activity was upregulated in the absence of catabolic Stimulation w hen compared with normal porcine or bovine cartilage. In addition, even in this late-stage degenerate cartilage, aggrecanase and not MMP activity was responsible for the release of the majority of aggrecan from the cartilage. This study demonstrates that the release of aggrecan from both normal and OA cartilage in, response to catabolic stimulation in vitro involves a prim ary cleavage by aggrecanase and not MMPs.