Fibronectin is an essential extracellular matrix glycoprotein and an important constituent of cartilage. The applicants have discovered that approximately 80% of fibronectin transcripts in adult articular cartilage are a new splice variant, designated (V+C)-, that deletes not only the variable (V) region, but also nucleotides that encode the 15th type III (III-15) and the 10th type I (I-10) homology repeats. Previously known splice variants were limited to three regions, ED-A, ED-B and V; consequently, this discovery may have important conceptual implications for the field of fibronectin biology. Tissue distribution studies provide strong evidence that this splice variant is associated specifically with the cartilage phenotype. Biochemical and immunological analyses confirm that the (V+C)- fibronectin isoform is translated and secreted by chondrocytes in articular cartilage. The hypothesis that guides this study states that expression of the new (V+C)- fibronectin isoform is tightly linked to chondrocyte differentiation and is required for the normal function and biomechanical properties of cartilage. It is proposed that the combined absence of the V, III-15, and I-10 segments is required for the appropriate interaction of fibronectin with other cartilage matrix constituents (e.g., type II collagen and aggrecan) and the regulation of chondrocyte gene expression and phenotype. The first Specific Aim is to determine how tightly linked expression of the (V+C)- fibronectin isoform is to the chondrocyte phenotype. Expression of the (V+C)- fibronectin isoform will be compared in different cartilaginous tissues, in chondrocytes cultured under conditions that modulate their phenotype, during chondrocytic differentiation, and as a function of both developmental and postnatal age. The second Specific Aim is to determine if the biochemical and functional properties of (V+C)- fibronectin differ from those of the V+,C+ and V-,C+ isoforms found in other tissues. Functional studies with (V+C)- fibronectin will include: interaction with type II collagen and proteoglycan; formation of a fibrillar fibronectin matrix; support of cell adhesion, spreading and migration; and regulation of chondrocyte metalloproteinase and matrix protein gene expression. The long-term objective of this study is to define the specific functional role(s) of fibronectin in normal cartilage. This knowledge will provide an essential basis for future research on how the 10- to 20-fold increase of fibronectin observed in osteoarthritic cartilage contributes to the molecular pathogenesis of this disease.
