COLLAGEN IN OSTEOARTHRITIC CARTILAGE

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It has been shown that the phenotypic expression of rabbit articular chondrocytes may be modulated either by continued subculture or by treatment of primary chondrocytes with retinoic acid, as monitored by changes in the collagen phenotype. Although the two modulated phenotypes differ, both are reversed by treatment with the microfilament modifying drug dihydrocytochalasin B, suggesting a common mechanism for the reexpression of the chondrocyte phenotype, we will investigate the possibility that modifications in microfilament structure are physically transmit:ed into the nucleus, by way of cytoskeletal-nuclear matrix interactions, and result in changes in gene expression, and the possibility that modification may generate chemical signals mediated by altered membrane association and phosphorylation by protein kinase C or tyrosine kinases. Multiple processes and molecular associations will be tested for involvement in the microfilament mechanism. Alterations in the compartmentalization of cellular proteins will be investigated using high resolution 2-D protein mapping techniques. Monoclonal antibodies to cytoskeletal proteins will be used in their identification and quantitation. The cellular distribution and architectural patterns of cytoskeletal/nuclear-matrix component will also be investigated using indirect immunofluorescence and transmission electron microscopy. Crosslinking analysis will be used to identify both actin- and DNA-associated proteins, both to establish the presence or absence of actin within the nucleus and to study changes in proteins that are likely to be involved in the mechanism of reexpression. Additional studies will test the role of poly-ADP ribosylation, DNA supercoiling, and the requirement for a serum-derived factor in the reexpression mechanism. The proposed studies are part of a long term strategy to understand and manipulate the pathogenesis of osteoarthritis. Developing an understanding of the mechanisms of phenotypic change, using this unique model system, will greatly enhance the probability of future clinical intervention at natural control points.