Objectives: To study the expression of cysteine proteinases, particularly cathepsin K, and their extracellular inhibitor cystatin C in articular cartilage of transgenic Del1 mice which harbour a short deletion mutation in a type II collagen transgene and are predisposed to early onset osteoarthritis.
Methods: Northern analysis was used to measure mRNA levels of cathepsins B, H, K, L, and S, and cystatin C in total RNA extracted from knee joints of Del1 mice, using their non-transgenic litter mates as controls. Immunohistochemistry and morphometry was used to study the distribution of cathepsin K and cystatin C in the knee joints.
Results: Up regulation of cathepsin K mRNA expression was seen in the knee joints of transgenic Del1 mice at the onset of cartilage degeneration. Cathepsin K was found near sites of matrix destruction in articular chondrocytes, particularly in clusters of proliferating cells, and in calcified cartilaginous matrix. In intact articular cartilage of control animals, cathepsin K was only seen in a small number of chondrocytes. Upon aging, control animals also developed osteoarthritis, which was accompanied by increased cathepsin K expression. Cystatin C was mostly localised in and around chondrocytes located in calcified cartilage, with no obvious association with the onset of cartilage degeneration.
Conclusion: The temporospatial distribution of cathepsin K in osteoarthritic cartilage suggests a role for this enzyme in the pathogenesis of osteoarthritis. Because cathepsin K can digest cartilage matrix components it may contribute to the development of osteoarthritic lesions. These data may provide new clues for the development of treatments aimed at preventing cartilage degeneration.
- cathepsin K
- cystatin C
- synovial tissue
- transgenic mice
- MMPs, matrix metalloproteinases
- PBS, phosphate buffered saline
- SDS, sodium dodecyl sulphate
- SSC, saline-sodium citrate
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- [View Figure] - Figure W1 Progression of articular cartilage degeneration in knee joints of transgenic Del1 mice and nontransgenic controls. Grading of osteoarthritic changes was performed as described earlier. 5 33 34 Closed symbols denote Del1 samples and open circles their control litter mates. (Mann-Whitney U test; n = 5�30; *** p <_0.001-- end="end" desc="desc" _636649figurew1.gif="_636649figurew1.gif" _--="_--">
- [View Figure] - Figure W2 Temporal expression of cysteine cathepsins and cystatin C mRNAs in developing, growing and aging mouse knee joints. Total RNAs were extracted from entire knee joints of Del1 and control mice at birth and at the age of 4 and 9 months (marked above lines; nb, newborn; mo, month; , control; +, Del1). The hybridisations were performed with cDNA probes detecting mRNAs for cathepsin B (Ctsb), cathepsin H (Ctsh), cathepsin K (Ctsk), cathepsin L (Ctsl) and cathepsin S (Ctss), as well as for cystatin C (Cst3) (marked on the left, respectively). The lowest panel 28S demonstrates the hybridisation of the filter with a probe for 28S rRNA. Hybridisation intensity was analysed using a phosphoimager. Transcript sizes are shown on the right.
- [View Figure] - Figure W3 Immunohistochemical detection of cathepsin K and cystatin C in the mouse synovial tissue at the age of 4 months. Tissue distribution is shown in sagittal sections of knee joints obtained from control (A, D, F) and Del1 mice (B, C, E). Immunostaining for cathepsin K (A, B) and cystatin C (D, E) is identified as a brown precipitate. There was no staining for cathepsin K (C) or cystatin C (F) when primary antibodies were omitted or replaced by normal mouse serum. The bar in panel A corresponds to 15 m for panels A-F. Paraffin sections; biotin-streptavidin complex with horseradish peroxidase label and diaminobenzidine detection, counterstaining with hematoxylin.
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