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Protease Analysis by Neoepitope Approach Reveals the Activation of MMP-9 Is Achieved Proteolytically in a Test Tissue Cartilage Model Involved in Bone Formation

Eunice R. Lee, Lisa Lamplugh, Beata Kluczyk, John S. Mort and Charles Philippe Leblond

Electron Microscopy Unit (ERL,LL,BK,CPL) and Joint Diseases Laboratory (JSM), Shriners Hospital for Children, Montreal, Canada, and Division of Surgical Research, Department of Surgery (ERL,JSM) and Department of Anatomy and Cell Biology (CPL), McGill University, Montreal, Canada

Correspondence to: Dr. Eunice R. Lee, Electron Microscopy Unit, Shriners Hospital for Children, 1529 Cedar Avenue, Montreal, Quebec, H3G 1A6, Canada. E-mail: elee{at}shriners.mcgill.ca

A principle of regulation of matrix metalloproteinase (MMP) activity has been introduced as the cysteine-switch mechanism of activation (Springman et al. 1990). According to this mechanism, a critical Cys residue found in the auto-inhibitory propeptide domain of latent proenzyme is important to determine whether or not activation is turned on or off. The mechanism further allows for multiple modes of activation. To determine whether or not activation is accomplished proteolytically within a rat test cartilage model, protease analysis by the neoepitope approach, which relies upon a set of antibodies, was applied. One is used to identify the MMP-9 proenzyme bearing the critical cysteine residue, the other to identify any enzyme present bearing a new NH₂-terminus ⁸⁹FQTFD. This is indicative of MMP-9 lacking the cysteine switch. The antibody set has been applied to frozen tissue sections and analyzed by light and electron microscopic methods. Results reveal that activation of the MMP-9 protease involves limited proteolysis resulting in propeptide domain release. Here we report the observed changes of protease form to indigenous cells and extracellular matrix, thereby making it possible to uncover the features of MMP-9 activation within a specified set of tissue circumstances where a cartilage model is transformed into definitive bone. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 54:965–980, 2006)

Key Words: bone growth and development • cartilage resorption • cartilage canals • matrix metalloproteinases • MMP activation • gelatinase B • MMP-9 • neoepitope antibodies • immunohistochemistry

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