Role of β-Pix in Corneal Epithelial Cell Migration on Fibronectin

Abstract

Corneal epithelial migration during wound healing is important for maintenance of corneal transparency, and fibronectin plays a key role in regulation of the adhesion and migration of corneal epithelial cells. The role of β-Pix in intracellular signaling that underlies the stimulatory effects of fibronectin on the adhesion and migration of corneal epithelial cells was examined. Simian virus 40-transformed human corneal epithelial (HCE) cells were cultured on fibronectin or on bovine serum albumin as a control. The localization and tyrosine phosphorylation of β-Pix were examined by immunofluorescence and immunoprecipitation analyses, respectively. The actin cytoskeleton and focal adhesions were detected by staining of cells with rhodamine-phalloidin and antibodies to phosphotyrosine, respectively. The effects of depletion of β-Pix on HCE cell adhesion and migration on fibronectin were investigated by cell transfection with a small interfering RNA specific for β-Pix mRNA. Fibronectin induced the tyrosine phosphorylation of β-Pix as well as its apparent accumulation at focal adhesions in HCE cells. Depletion of β-Pix inhibited the effects of fibronectin on remodeling of the actin cytoskeleton and the formation of focal adhesions. It also inhibited the migration of HCE cells on fibronectin in an in vitro model of wound healing, but it did not affect cell adhesion to fibronectin. β-Pix contributes to the regulation of the formation of focal adhesions as well as that of cell migration by fibronectin in HCE cells. This protein therefore likely plays an important role in signal transduction underlying corneal epithelial wound healing.