Aggrecan Protects Cartilage Collagen from Proteolytic Cleavage

Abstract

The matrix components responsible for cartilage mechanical properties, type II collagen and aggrecan, are degraded in osteoarthritis through proteolytic cleavage by matrix metalloproteinases (MMPs) and aggrecanases, respectively. We now show that aggrecan may serve to protect cartilage collagen from degradation. Although collagen in freeze-thawed cartilage depleted of aggrecan was completely degraded following incubation with MMP-1, collagen in cartilage with intact aggrecan was not. Using interleukin-1-stimulated bovine nasal cartilage explants where aggrecan depletion occurs during the first week of culture, followed by collagen loss during the second week, we evaluated the effect of selective MMP and aggrecanase inhibitors on degradation. A selective MMP inhibitor did not block aggrecan degradation but caused complete inhibition of collagen breakdown. Similar inhibition was seen with inhibitor addition following aggrecan depletion on day 6 -8, suggesting that MMPs are not causing significant collagen degradation prior to the second week of culture. Inclusion of a selective aggrecanase inhibitor blocked aggrecan degradation, and, in addition, inhibited collagen degradation. When the inhibitor was introduced following aggrecan depletion, it had no effect on collagen breakdown, ruling out a direct effect through inhibition of collagenase. These data suggest that aggrecan plays a protective role in preventing degradation of collagen fibrils, and that an aggrecanase inhibitor may impart overall cartilage protection.Cartilage plays a critical role in joint function, where it acts as a shock absorber during joint loading and motion. The extracellular matrix molecules, type II collagen and aggrecan, provide the properties that enable the cartilage to serve this shock-absorbing function. Type II collagen is present as triple helical fibrils that provide cartilage with its structure and tensile strength (3). Aggrecan monomers consist of a core protein with three globular domains. The amino-terminal region contains two of these globular domains, G1 and G2, separated by an interglobular domain. The G2 is followed by a long region along which are attached the sulfated glycosaminoglycan side chains, keratan sulfate and chondroitin sulfate and by the C-terminal globular domain, G3 (4). Multiple aggrecan monomers bind to hyaluronan through their G1 domains to form huge aggregates that are trapped within the collagen network. Because of their high negative charge and water-binding capacity, the aggrecan molecules provide the mechanical properties of compressibility and elasticity to the cartilage. The mechanical properties of the matrix as a whole depend on both major components and their stabilization by intermolecular interactions. The ability of the cartilage to withstand mechanical stress, therefore, is dependent upon its structural integrity and upon the various interactions between the matrix components.Cartilage degradation leading to a loss of joint function in osteoarthritis involves proteolytic cleavage of both aggr...

Selective and non-selective metalloproteinase inhibitors reduce IL-1-induced cartilage degradation and loss of mechanical properties

“…Interestingly, the aggrecanase-selective inhibitor delayed and reduced hydroxyproline release to the media and loss from the explant by 50%. These results are consistent with a previously proposed model in which aggrecan protects the collagen network from proteolytic attack in bovine nasal cartilage (Pratta, et al, 2003b).…”

“…Interestingly, the aggrecanase-selective inhibitor also conferred some protection of the collagen network. Using a different small-molecule aggrecanase inhibitor, Pratta et al observed a similar result and hypothesized that aggrecan molecules can prevent MMPs from reaching their substrates on collagen fibers, perhaps by steric exclusion (Pratta, et al, 2003b). Treatment of IL-1-stimulated cartilage with the aggrecanase-selective inhibitor reduced cumulative collagen release by 50% through day 24 of the experiment, and delayed but did not prevent aggrecan release over the same period.…”

“…Many cartilage explant studies have shown that aggrecanolysis preceeds collagenolysis (Billinghurst, et al, 1997;Kuroki, et al, 2005;Pratta, et al, 2003b;Zwerina, et al, 2004), and it has been suggested (Pratta, et al, 2003b) that aggrecan protects the collagen network from proteolytic attack.…”

“…Inhibitors of glycophosphatidyl inositol-anchor formation, including mannosamine and glucosamine, interfere with MMP-17-mediated activation of ADAMTS-4 and reduce IL-1-induced sGAG release and loss of material properties in cartilage explants (Patwari, et al, 2000). Synthetic aggrecanase inhibitors can delay sGAG and collagen release from IL-1-stimulated nasal cartilage, and preservation of aggrecan using an aggrecanase inhibitor protected the collagen network from proteolytic attack (Pratta, et al, 2003b). Collectively, these studies suggest that inhibitors of aggrecanases specifically or metalloproteinases generally (ADAMTSs and MMPs) can attenuate cell-mediated aggrecan catabolism and loss of tissue function associated with arthritic disease.…”

The role of proteases in pathologies of the synovial joint

“…The reversible nature of aggrecan loss from the tissue suggests that the key pathological activity is the degradation of type II collagen. However, recent studies have suggested that aggrecan protects the collagen network from proteolysis and have demonstrated that prevention of aggrecan cleavage diminishes both aggrecan loss and cartilage erosion in arthritic model systems (Little et al, 2007;Pratta et al, 2003). Aggrecan proteolysis may therefore be a critical initiating step in these pathologies.…”