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Investigating ADAMTS-mediated aggrecanolysis in mouse cartilage

Nature Protocols volume 6, pages 388–404 (2011)Cite this article

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Abstract

Proteolysis of the cartilage proteoglycan aggrecan is a feature of arthritis. We present a method for analyzing aggrecanolysis in in vitro cultures of 3-week-old mouse femoral head cartilage based on traditional methods developed for large animal species. Investigators can choose either a simple analysis that detects several aggrecan fragments released into culture medium only or a more comprehensive study that detects all fragments present in both the medium and the cartilage matrix. The protocol comprises (i) cartilage culture and optional cartilage extraction, (ii) a quick and simple colorimetric assay for quantitating aggrecan and (iii) neoepitope western blotting to identify specific aggrecan fragments partitioning to the medium or cartilage compartments. The crucial difference between the methods for mice and larger animals is that the proportion of aggrecan in a given sample is normalized to total aggrecan rather than to tissue wet weight. This necessary break from tradition arises because tiny volumes of liquid clinging to mouse cartilage can increase the apparent tissue wet weight, causing unacceptable errors. The protocol has broad application for the in vitro analysis of transgenic mice, particularly those with mutations that affect cartilage remodeling, arthritic disease and skeletal development. The protocol is robust, reliable and takes 7–11 d to complete.

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Figure 1: Aggrecanase cleavage sites and neoepitopes in the mouse aggrecan core protein.
Figure 2: Flow chart of the comprehensive and simple protocols.
Figure 3: Harvesting the mouse femoral head.
Figure 4: Analyzing aggrecan release by the dimethylmethylene blue assay.
Figure 5: Western blot analysis of aggrecanolysis by the comprehensive or simple protocol.
Figure 6: Aggrecan fragments identified in medium or cartilage extracts with neoepitope antibodies.

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Acknowledgements

We gratefully acknowledge the mouse husbandry support provided by L. Tutolo and the Disease Models Unit of the Murdoch Childrens Research Institute. We thank E. Arner and M. Tortorella (Pfizer) for the anti-AGEGP antibody. The work was funded by the National Health and Medical Research Council (Australia).

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  1. Christopher B Little

    Present address: Present address: University of Sydney Raymond Purves Bone and Joint Research Laboratories, St. Leonards, New South Wales, Australia.,

Authors and Affiliations

  1. University of Melbourne Department of Paediatrics, Parkville, Victoria, Australia

    Heather Stanton, Suzanne B Golub, Fraser M Rogerson, Karena Last, Christopher B Little & Amanda J Fosang

  2. Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville, Victoria, Australia

    Heather Stanton, Suzanne B Golub, Fraser M Rogerson & Karena Last

  3. Kolling Institute of Medical Research at the Royal North Shore Hospital, St. Leonards, New South Wales, Australia

    Christopher B Little

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Contributions

A.J.F. conceived the work and designed the experiments. H.S. and A.J.F. wrote the paper and the experiments were done by S.B.G. and K.L. C.B.L. established the original protocol for mouse cartilage culture from which the present protocols have evolved. F.M.R. further optimized the protocol for western blot analysis. All authors contributed to the editing of the final manuscript.

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Correspondence to Amanda J Fosang.

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Supplementary information

Supplementary Fig. 1: Full length view of the western blots shown in figure 5.

The high molecular weight bands visible in panel g are non-specific. The specific VTEGE fragment has Mr ∼65 kDa. The non-specific bands (>100 kDa) are unlikely to be keratin. On the occasions we have seen a non-specific keratin band it has been ∼50 kDa. (EPS 8788 kb)

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Stanton, H., Golub, S., Rogerson, F. et al. Investigating ADAMTS-mediated aggrecanolysis in mouse cartilage. Nat Protoc 6, 388–404 (2011). https://doi.org/10.1038/nprot.2010.179

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