RESEARCH PROJECTS

Cartilage-Restricted Fibronectin Isoform

Fibronectin is a large matrix glycoprotein present in most body tissues and fluids. Functionally, it is the classic example of an adhesive glycoprotein, binding and interconnecting extracellular matrix components with each other and to the surface of cells. Fibronectin has received less attention over the years than type II collagen, proteoglycans, and metalloproteinase enzymes from scientists interested in articular cartilage and osteoarthritis. However, in other areas of biology, fibronectin has generated great interest. The reason is that this protein is one of the most important molecules through which cells interact with their surrounding environment. The binding of fibronectin to cell surface integrin receptors plays a critical role in cell migration both developmentally and postnatally. Indeed, homozygous inactivation of the fibronectin gene by homologous recombination is a very early embryonic lethal resulting in defects of neurulation and mesodermally-derived tissues. In addition to cell movement, however, it is now very clear that most non-migratory cells also constitutively express integrin receptors that bind fibronectin. These interactions between cells and their pericellular environment are very important in the regulation of cellular differentiation and gene expression.

In 1995, my laboratory (in collaboration with Nancy Burton-Wurster and George Lust at Cornell University) identified and characterized a unique isoform of fibronectin restricted to the cartilaginous tissues of mammals. It is generated by an alternative RNA splicing pattern that internally deletes 771 nucleotides which would normally encode fibronectin protein segments V, III-15, and I-10 (Figures 1 and 2). Designated (V+C)- fibronectin, the cartilage-restricted expression pattern contrasts markedly to other fibronectin isoforms that occur in tissues and mesenchymal cell types throughout the body. This suggests that (V+C)- fibronectin has an important role in cartilage function and possibly in chondrocyte differentiation.

Figure 1. Schematic diagram of the primary structure of monomeric fibronectin. The cartilage-restricted (V+C)- fibronectin isoform has an internal deletion of the 771 nucleotides that would normally encode protein segments V, III-15, and I-10.

Figure 2. Expression of the (V+C)- fibronectin isoform in cartilage. A. Northern blot hybridization of equine articular cartilage RNA illustrating the major 7.3 kb band that encodes (V+C)- fibronectin. B. Silver stain protein gel (3%-8% gradient PAGE) illustrating 400 kDa dimers and 200 kDa monomers of (V+C)- fibronectin in equine articular cartilage.

Our current experiments are designed to test the hypothesis that the unique structure and restricted dimerization of (V+C)- fibronectin influences matrix organization and the cell/matrix interactions that regulate the differentiated phenotype of chondrocytes and the biomechanical properties of cartilage. We believe that (V+C)- fibronectin provides an important new experimental opportunity to define the specific functional role(s) of fibronectin in normal cartilage and determine how changes in fibronectin expression contribute to the molecular pathogenesis of osteoarthritis. In this regard, 10- to 20-fold elevations of fibronectin occur early in the development of osteoarthritic lesions. Now in addition to changes in total fibronectin expression, we can specifically analyze relative changes in isoform ratios between morphologically and functionally different types of cartilage, between healthy and diseased cartilage, and during chondrocytic differentiation. It is tempting to speculate that (V+C)- fibronectin may be necessary to support the normal differentiated phenotype of chondrocytes or for the structural integrity of cartilage matrix.