Fibronectin polymerization stimulates cell growth by RGD-dependent and -independent mechanisms

Many aspects of cell behavior are regulated by cell-extracellular matrix interactions, including cell migration and cell growth. We previously showed that the addition of soluble fibronectin to collagen-adherent fibronectin-null cells enhances cell growth. This growth-promoting effect of fibronectin depended upon the deposition of fibronectin into the extracellular matrix; occupancy and clustering of fibronectin-binding integrins was not sufficient to trigger enhanced cell growth. To determine whether the binding of integrins to fibronectin's RGD site is required for fibronectin-enhanced cell growth, the ability of fibronectin lacking the integrin-binding RGD site (FN(Delta)RGD) to promote cell growth was tested. FN(Delta)RGD promoted cell growth when used as an adhesive substrate or when added in solution to collagen-adherent fibronectin-null cells. Addition of FN(Delta)RGD to collagen-adherent fibronectin-null cells resulted in a 1.6-1.8x increase in cell growth in comparison with cells grown in the absence of fibronectin. The growth-promoting effects of FN(Delta)RGD and wild-type fibronectin were blocked by inhibitors of fibronectin polymerization, including the anti-fibronectin antibody, L8. In addition, FN(Delta)RGD-induced cell growth was completely inhibited by the addition of heparin, and was partially blocked by either heparitinase-treatment or by addition of recombinant fibronectin heparin-binding domain. Heparin and heparitinase-treatment also partially blocked the growth-promoting effects of wild-type fibronectin, as well as the deposition of wild-type fibronectin into the extracellular matrix. These data suggest that cell surface heparan-sulfate proteoglycans contribute to the growth-promoting effects of FN(Delta)RGD and wild-type fibronectin. Addition of heparin, treatment with heparitinase, or incubation with monoclonal antibody L8 all inhibited the formation of short linear FN(Delta)RGD fibrils on the cell surface. Inhibitory (beta)1 integrin antibodies had no effect on FN(Delta)RGD fibril formation, FN(Delta)RGD-induced cell growth, or cell adhesion on FN(Delta)RGD-coated substrates. These data suggest that fibronectin fibril formation can promote cell growth by a novel mechanism that is independent of RGD-integrin binding, and that involves cell surface proteoglycans.