Evidence of a novel aggrecan-degrading activity in cartilage: Studies of mice deficient in both ADAMTS-4 and ADAMTS-5

Authors

  • Fraser M. Rogerson,

    Corresponding author
    1. University of Melbourne Department of Paediatrics, and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia
    • University of Melbourne Department of Paediatrics and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria 3052, Australia
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  • Heather Stanton,

    1. University of Melbourne Department of Paediatrics, and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia
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  • Charlotte J. East,

    1. University of Melbourne Department of Paediatrics, and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia
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  • Suzanne B. Golub,

    1. University of Melbourne Department of Paediatrics, and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia
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  • Leonie Tutolo,

    1. University of Melbourne Department of Paediatrics, and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia
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  • Pamela J. Farmer,

    1. Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia
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  • Amanda J. Fosang

    1. University of Melbourne Department of Paediatrics, and Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia
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    • Assoc. Prof. Fosang holds a patent for a monoclonal antibody (aggrecan neoepitope); royalties for the sales of this monoclonal antibody are assigned to the University of Melbourne.


Abstract

Objective

To characterize aggrecan catabolism and the overall phenotype in mice deficient in both ADAMTS-4 and ADAMTS-5 (TS-4/TS-5 Δ-cat) activity.

Methods

Femoral head cartilage from the joints of TS-4/TS-5 Δ-cat mice and wild-type mice were cultured in vitro, and aggrecan catabolism was stimulated with either interleukin-1α (IL-1α) or retinoic acid. Total aggrecan release was measured, and aggrecanase activity was examined by Western blotting using neoepitope antibodies for detecting cleavage at EGE373–374ALG, SELE1279–1280GRG, FREEE1467–1468GLG, and AQE1572–1573AGEG. Aggrecan catabolism in vivo was examined by Western blotting of cartilage that had been extracted immediately ex vivo.

Results

TS-4/TS-5 Δ-cat mice were viable, fertile, and phenotypically normal. TS-4/TS-5 Δ-cat cartilage explants did not release aggrecan in response to IL-1α, and there was no detectable increase in aggrecanase neoepitopes. TS-4/TS-5 Δ-cat cartilage explants released aggrecan in response to retinoic acid. There was no retinoic acid–stimulated cleavage at either EGE373–374ALG or AQE1572–1573AGEG. There was a low level of cleavage at SELE1279–1280GRG and major cleavage at FREEE1467–1468GLG. Ex vivo, cleavage at FREEE1467–1468GLG was substantially reduced, but still present, in TS-4/TS-5 Δ-cat mouse cartilage compared with wild-type mouse cartilage.

Conclusion

An aggrecanase other than ADAMTS-4 and ADAMTS-5 is expressed in mouse cartilage and is up-regulated by retinoic acid but not IL-1α. The novel aggrecanase appears to have different substrate specificity from either ADAMTS-4 or ADAMTS-5, cleaving E–G bonds but not E–A bonds. Neither ADAMTS-4 nor ADAMTS-5 is required for normal skeletal development or aggrecan turnover in cartilage.

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