Aggrecan Protects Cartilage Collagen from Proteolytic Cleavage

Michael A Pratta,Wenqing Yao,Carl Decicco,Micky D Tortorella,Riu-Qin Liu,Robert A Copeland,Ronald Magolda,Robert C Newton,James M Trzaskos,Elizabeth C Arner

doi:10.1074/jbc.m303737200

Michael A Pratta, Wenqing Yao + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m303737200

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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.

Highlights

Cartilage plays a critical role in joint function, where it acts as a shock absorber during joint loading and motion
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 matrix metalloproteinases (MMPs) and aggrecanase inhibitors on degradation
Collagenase Digestion of Cartilage—To determine whether the presence of aggrecan matrix affects the ability of collagenase to degrade cartilage collagen, freeze-thawed bovine nasal cartilage explants were treated with trypsin (5 ␮g/ml) for 16 h to deplete the aggrecan content of the tissue

Summary

EXPERIMENTAL PROCEDURES

Materials—Dulbecco’s modified Eagle’s medium, fetal calf serum, trypsin, and penicillin/streptomycin were purchased from Invitrogen. Cartilage pieces were incubated 16 h with trypsin (5 ␮g/ml) in phosphate-buffered saline to deplete the matrix aggrecan or with buffer alone as a control (data not shown). At the end of the incubation 5% fetal calf serum was added for 60 min to inactivate the trypsin, and the disks were washed 4ϫ with phosphate-buffered saline. Each slice was weighed and placed into a well of a 96-well plate in a total volume of 180 ␮l of Dulbecco’s modified Eagle’s medium containing 5% fetal calf serum and 1ϫ antibiotic/antimycotic (Invitrogen) and equilibrated for at least 3 days prior to treatment. Forty microliters of chloramine T reagent (0.282 g of chloramine T, 1 ml of water, 8 ml of pH 6 buffer, 1 ml of 1-propanol) was added, and the samples were allowed to shake for 15 min at room temperature. Values shown are mean Ϯ S.E. for glycosaminoglycan release (n ϭ 8); mean Ϯ S.D. for hydroxyproline levels (n ϭ 3)

RESULTS

10 Ͼ5000 Ͼ5000

DISCUSSION