Promotion by fibronectin of collagen gel contraction mediated by human corneal fibroblasts

Collagen contraction mediated by corneal fibroblasts (CFs) is implicated in the maintenance of corneal shape. Given that fibronectin is expressed at sites of corneal stromal wounding, we investigated the effect of fibronectin on CF-mediated collagen gel contraction. Human Us were cultured in a three-dimensional gel of type I collagen in the absence or presence of various extracellular matrix (ECM) components. The contraction of collagen gels mediated by CFs was evaluated by measurement of changes in gel diameter. The formation of stress fibers and focal adhesions in Us was examined by fluorescence microscopy. The abundance of paxillin, phosphorylated paxillin, integrins alpha 5, beta 1, and alpha 2, and alpha-smooth muscle actin in CFs was examined by immunoblot analysis. Fibronectin promoted CF-mediated collagen gel contraction in a concentration- and time-dependent manner. Other ECM proteins or glycosaminoglycans did not exhibit such an effect. Fibronectin also induced cell spreading, the formation of stress fibers, and the establishment of focal adhesions containing paxillin in CFs cultured in three-dimensional collagen gels. In addition, it increased the amounts of paxillin, phosphorylated paxillin, and integrins alpha 5 and beta 1 in these cells. The expression of integrin alpha 2 and alpha-smooth muscle actin was not affected by fibronectin, however. Furthermore, the peptide GRGDSP (an antagonist of fibronectin receptors) blocked the stimulatory effect of fibronectin on CF-mediated collagen gel contraction. These results suggest that fibronectin promoted CF-mediated collagen gel contraction in a manner dependent on the formation of stress fibers and focal adhesions, the activation of paxillin, and the up-regulation of integrin alpha 5 beta 1. Fibronectin may therefore contribute to the maintenance of corneal shape by Us during the healing of stromal wounds. (c) 2006 Elsevier Ltd. All rights reserved.