Degenerative joint disease, or osteoarthritis (OA) is one of the most common disabling diseases affecting middle-aged and older people. It is characterized by the breakdown of the unstable matrix network in cartilage. To better understand the pathogenic process of OA, it is necessary to examine how the matrix structure is stabilized in mature cartilage, and what causes the destabilization of such a structure. The long-term objective of this study is to analyze the molecular mechanisms of stabilizing matrix networks in mature cartilage. In particular, the role of cartilage matrix protein (CMP) in this process will be examined. This proposal is based on recent findings that CMP is synthesized exclusively by post-mitotic mature chondrocytes and distributed only in mature cartilage. There, CMP interacts with both type II collagen-containing fibrils, and non-collagenous matrix components, to form a filamentous network, and these interactions are further stabilized by covalent cross-linking. The hypothesis is, that CMP contributes to the stabilization of mature cartilage by a two-step process. First, it interacts with existing matrix components through specific adhesion sites located in specific domain(s) of CMP. Second, the interactions of CMP in the matrix are strengthened by covalent cross-links. Therefore, defects in either interaction, or cross-linking of CMP with matrix molecules may contribute to the destabilization of cartilage structure. This proposal contains three Specific Aims: 1) to characterize the interaction sites between CMP and matrix network. The location and properties of the adhesion sites will be determined by examining the interaction of a series of recombinant CMPs with matrix networks in a primary chondrocyte culture; 2) to analyze the nature of the cross-linking of CMP in cartilage matrix. A monoclonal antibody will be used to determine whether CMP is a substrate for tissue transglutaminase; 3) to determine whether the disruption of interaction and cross-linking of CMP will result in a mis-assembled or unstable matrix network that is subject to degeneration. The dominant negative CMP constructs will be expressed in cartilage by retroviral infection. The effects will be examined by immunohistochemical analysis. It is suggested by the applicant that these data will contribute not only to our basic understanding of cartilage matrix assembly, but also to the development of methods for prevention and treatment of cartilage degeneration.
