Extracellular matrices (ECMs) function in the structure and support of most body tissues. In many cases, connective tissue diseases result from defective ECM formation. The proposed studies are designed to elucidate the biochemical and morphological properties of synthesis, secretion, and interactions of chondroitin sulfate proteoglycan (CSPG) and other molecules in the formation of the ECM. The research is based on the hypothesis that normal ECM formation is regulated through synthetic and processing events which occur in distinct subcellular compartments prior to the secretion of ECM constituents. Experiments will be directed toward characterizing the intracellular compartments of chondrocytes involved in the synthesis and processing of CSPG and other matrix molecules. Chordrocytes in culture will provide a well-controlled and readily manipulated living model system for the study of biosynthesis and interactions among ECM constituents. Cytoplasmic vesicles involved in matrix biosynthesis will be isolated by subcellular fractionation and characterized biochemically and morphologically. Properties of the co- and post-translational processing of CSPG monomer, type II collagen, and link protein will be determined. Information about processing and biosynthetic intermediates will be obtained from the kinetic, biochemical and morphological analyses of chondrocytes pulse-labeled and chased in culture. Results will be correlated with cell-free translation studies. The simultaneous immunolocalization of two products will be analyzed using light and electron microscopy to visualize coordinate biosynthesis and ECM deposition for specific gene products. In order to provide further insight into the biosynthesis of matrix molecules and their interrelationships, chondrocytes will be grown in culture in the presence of agents which 1) block expression of the cartilage phenotype, and 2) alter the synthesis or processing of matrix constituents. Nanomelic chondrocytes will be used as a model for abnormal CSPG production. The combination of immunohistochemical, biochemical and molecular approaches will be used to generate a broad view of ECM biosynthesis and interactions. These studies will provide fundamental information about the formation and regulation of cartilage ECM and a firm basis for the examination of proteoglycans and their interactions with ECM components in non-cartilage connective tissues.
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