The extra domain-A (EDA), present in fibronectin (FN) molecules arising from alternatively spliced transcripts, appears only during specific biological and pathogenic processes. However, its function is poorly understood. To define the physiologic role of this domain in joint connective tissue, the biological effects on rabbit cartilage explants, chondrocytes, and synovial cells were studied. A recombinant EDA protein (rEDA) increased proteoglycan release (3.6-fold) in cartilage explant cultures and markedly induced production of matrix metalloproteinase (MMP)-1 in chondrocytes. In addition, rEDA induced MMP-1, MMP-3, and MMP-9 in synovial cells. These effects were elicited only by rEDA, while its neighboring type III repeats, III 11 or III 12 , scarcely had any such effects. Interestingly, reorganization of F-actin stress fibers accompanied MMP-1 expression in synovial cells treated with rEDA, suggesting alteration of cellular phenotype. Subsequent Northern blotting revealed expression of pro-inflammatory cytokines, including interleukin (IL)-1␣ and IL-1, was induced by rEDA prior to MMP-1 expression. Delayed MMP-1 expression suggests that rEDA-induced IL-1s promote MMP-1 expression in an autocrine manner. This hypothesis is supported by the reduction of EDA-induced MMP-1 production by IL-1 receptor antagonist. The effect of EDA on MMP-1 production was reduced by connection with an adjacent type III repeat on either the NH 2 or COOH side of EDA and was abolished by connection on both sides of EDA, suggesting that exposure of either the NH 2 or COOH terminus of EDA domain by proteolytic cleavage releases the inducing activity. In agreement with these results, full-length cellular FN did not induce MMP-1 production. Furthermore, a 160-kDa EDA-positive FN fragment, which was purified from human placental tissue and corresponds to the region from NH 2 terminus through the EDA, induced MMP-1 production. Taken together, these results suggest that the EDA in FN fragments triggers alterations of cell physiology and plays a role in matrix degradation in joint connective tissue.
Fibronectin (FN)1 is a multifunctional glycoprotein abundant in plasma and widely distributed in the extracellular matrix (1). It is a dimer of subunits cross-linked by disulfide bonds. Each FN monomer is comprised of three types of repeating units designated type I, II, and III (2). Some of these repeats bind to cell surface and extracellular matrix components such as integrins, collagens, heparin, and fibrin. Several of these binding activities have been assigned to the motif sequences in FN, including the Arg-Gly-Asp (RGD) motif in the type III 10 domain (3), the Pro-His-Ser-Arg-Asn (PHSRN) motif in the type III 9 domain (4), and the CS-1 sequence in the III-CS region (5). Consequently, this multifunctional glycoprotein mediates a variety of cellular functions including cell adhesion, cell migration, and cell differentiation. FN molecules have multiple isoforms generated from a single gene by alternative splicing of combinations of 3 exons: e...