Impaired Contracture of 3D Collagen Constructs by Fibronectin-Deficient Murine Fibroblasts

Fibronectin (FN) is an extracellular matrix glycoprotein that is abundantly expressed by fibroblasts in contracting wounds, where it mediates cell adhesion, migration and proliferation. FN also efficiently binds to collagen. Therefore, we and others hypothesized that FN and its cellular receptor integrin α₅β₁ might be involved in collagen matrix contracture by acting as linkers. However, there are conflicting reports on this issue. Moreover, several publications suggest an important role of collagen-binding integrin receptors α₂β₁ and α₁₁β₁ in collagen matrix contracture. Therefore, the aim of the present study was to determine the contributions of FN-integrin α₅β₁ interactions relative to those of collagen receptors α₂β₁ and α₁₁β₁ in this process. To assess the role of cellular FN directly, we employed FN-deficient mouse fibroblasts, subjected them to a collagen gel contracture assay in vitro, and compared them to their wildtype counterparts. Exogenous FN was removed from serum-containing medium. For dissecting the role of major collagen receptors, we used two FN-deficient mouse fibroblast lines that both possess integrin α₅β₁ but differ in their collagen-binding integrins. Embryo-derived FN-null fibroblasts, which express α₁₁- but no α₂-integrin, barely spread and tended to cluster on collagen gels. Moreover, FN-null fibroblasts required exogenously added FN to assemble α₅β₁-integrin in fibrillar adhesion contacts, and to contract collagen matrices. In contrast, postnatal kidney fibroblasts were found to express α₂- but barely α₁₁-integrin. When FN expression was suppressed in these cells by shRNA transfection, they were able to spread on and partially contract collagen gels in the absence of exogenous FN. Also in this case, however, collagen contracture was stimulated by adding FN to the medium. Antibody to integrin α₅β₁ or RGD peptide completely abolished collagen contracture by FN-deficient fibroblasts stimulated by FN addition. We conclude that although collagen-binding integrins (especially α₂β₁) can mediate contracture of fibrillar collagen gels by murine fibroblasts to some extent, full activity is causally linked to the presence of pericellular FN and its receptor integrin α₅β₁.