Cartilage-Specific Fibronectin Isoform and Osteoarthritis

Fibronectin is an essential extracellular matrix glycoprotein and an important constituent of cartilage. We have discovered that 50-80% of the fibronectin in mammalian articular cartilage is encoded by a novel mRNA splice variant that is restricted to cartilage and rapidly lost when chondrocytes are removed from the matrix and placed under most culture conditions. This RNA splicing pattern creates an internal deletion of 771 nuc1eotides that would normally encode the variable (V) region and protein segments III-IS and 1-10. The resulting protein, designated (V+Cr fibronectin, is detected only in homodimeric and monomeric configurations, reflecting a very limited ability to heterodimerize with other fibronectin isoforms. This proposal is designed to test the hypothesis that the unique primary structure and restricted dimerization of cartilage-specific (V+Cr fibronectin influences matrix organization and the cell/cell and cell/matrix interactions that regulate the differentiated phenotype of chondrocytes and the biomechanica1 properties of cartilage. Three primary questions are addressed in the form of specific aims. First, why is the dimerization of (V+Cr fibronectin restricted to homodimer formation and does this restriction alter the structural features of an assembled fibronectin matrix? The role of protein segment 1-10 in dimerization will be assessed using constructs with targeted deletions and substitutions. Structural features and assembly kinetics of matrices generated from (V+Cr fibronectin will be compared to those of other isoforms. Second, how are structural features of (V+Cr fibronectin and resulting matrices related to the differentiated phenotype of chondrocytes? The relationship of cell proliferation, cell/cell contact, cell adhesion, and differential gene expression to (V+Cr fibronectin will be studied in different chondrocyte culture systems. In vitro relationships will be re-assessed in vivo by targeted mutagenesis of the fibronectin gene in mice to manipulate (V+Cr splicing. Third, does the unique structure and restricted dimerization profile of (V+Cr fibronectin alter the biomechanica1 properties of an extracellular matrix? Mechanical properties of tissue disks generated in vitro with different fibronectin isoform compositions will be compared. The 10ngterm objective of this project is to understand the function of fibronectin in normal cartilage and how the 10-20 fold increase in fibronectin observed in osteoarthritis contributes to the molecular pathogenesis of this disease.