Resorption of the extracellular matrix (ECM) is a major feature of physiological remodeling in development and growth and in pathological processes such as a rheumatoid arthritis. In the degradation of the ECM of articular and skeletal tissues, collagenase, a member of the matrix metalloproteinase (MMP) family, activated in a proteolytic cascade and modulated by natural inhibitors, plays a major role. The purposes of this proposal are to understand the nature of the proteolytic action of collagenase on native, helical collagen substrates and to define the roles of collagenase in embryonic development and growth and in models of pathological connective tissue resorption. The approach is to make mutations in the collagens substrates by site-directed mutagenesis of the type I collagen genes or, alternatively to make mutations in the collagenases genes, and express these mutations in cultured cells and later in transgenic mice to characterize remodeling in vivo.l The synovial pathology of human rheumatoid arthritis provides a paradigm for understanding how collagenases act to degrade the ECM in the joint that results in the major morbidity of this disease.
Specific Aims are: (1) To make mutations in the collagen substrates by site-directed mutagenesis of the collagen genes to define the structural requirements for proteolysis and elucidate the mechanisms whereby collagenase makes unique cleavages at a single locus across the three polypeptide chains of the collagen triple helices. Use will be made of murine genomic clones of the collagen alpha1(I) chain gene, Co11a1, to produce these mutations and express them in cell lines (Mov13) in which the endogenous gene is inactivated. Molecular modelling and methods that measure helical stability will be used to determine the effects of the amino acid substitutions. This information will be applied to the design of enzyme inhibitors. (2) To define the phenotype of transgenic mice that express these mutant collagens. One of the collagenase-resistant collagen mutations has been successfully expressed in transgenic mice and in vivo models for excessive degradation of the ECM will be constructed in order to determine the effects of these mutations. (3) To clone the murine collagenase gene in order to """"""""knock out"""""""" the gene in embryonic stem cells and express the mutations in transgenic mice. The murine collagenase gene differs structurally from the human gene. It must be shown whether the enzyme encoded by this gene also cleaves the collagen chains in the trimer at the single locus and how disrupting the gene alters remodeling of the ECM. (4) To determine whether mutations in the human collagen gene affect the function of collagenase and whether the presence of particular substitutions is correlated with the propensity to develop aggressive, destructive joint disease in patients with rheumatoid arthritis. The approach is to screen for mutations i the collagenase gene in samples of peripheral blood using the polymerase chain reaction coupled with sensitive analytical techniques to identify the mutations.
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