Role of Rac1 in Fibronectin-Induced Adhesion and Motility of Human Corneal Epithelial Cells

Abstract

The fibronectin-integrin system plays an important role in adhesion and migration of corneal epithelial cells and thereby contributes to epithelial wound healing. The role of Rac1, a member of the Rho family of GTPases, in the intracellular signaling responsible for regulation of the adhesion and motility of corneal epithelial cells by fibronectin was examined. Simian virus 40-transformed human corneal epithelial (HCE) cells were plated on fibronectin or on bovine serum albumin as a control. Cell motility was monitored by time-lapse video microscopy. The actin cytoskeleton and focal adhesions were detected by staining of cells with rhodamine-phalloidin and antibodies to phosphotyrosine, respectively. The activation of Rac1 and phosphorylation of its effector PAK were evaluated with a pull-down assay and immunoblot analysis, respectively. The effects of mutant forms of Rac1 were determined by cell transfection. HCE cells plated on fibronectin manifested greater levels of cell adhesion and motility than did those plated on bovine serum albumin. Fibronectin also induced the accumulation of F-actin and the formation of focal adhesions at the cell periphery as well as the activation of Rac1 and the phosphorylation of PAK. Expression of the dominant negative mutant Asn17Rac1 inhibited the effects of fibronectin on cell adhesion and motility, the actin cytoskeleton, and focal adhesions. Expression of the constitutive active mutant Val12Rac1 mimicked the effects of fibronectin on F-actin and focal adhesions. Rac1 is necessary for the promotion of HCE cell adhesion and motility by fibronectin. It therefore probably plays an important role in corneal wound healing.