SRC-family tyrosine kinases in wound- and ligand-induced epidermal growth factor receptor activation in human corneal epithelial cells.

Corneal epithelial cells respond rapidly to injury, resulting in a healing process of cell migration as a sheet to cover the defect and to reestablish epithelial barrier function.1 Critical for wound healing are cell migration and proliferation, which are driven by growth factors and cytokines released coordinately into the injured bed. Prominent among these epithelium-derived factors are ligands for the epidermal growth factor receptor (EGFR), the EGF family.1 Numerous studies have shown that epithelial wound healing is, at least in part, mediated by EGFR-ligand (EGF) interactions in an autocrine fashion.1–3 We recently showed that wounding induces EGFR transactivation through ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF) and that this wound-induced activation of EGFR and its coreceptor, erbB2, is required for wound closure in cultured HCECs and in cultured porcine corneas.4,5 HB-EGF is synthesized as a type 1 transmembrane protein that can be shed enzymatically to release a soluble 14- to 20-kDa growth factor; the process has been termed ectodomain shedding.6–8 The transmembrane form of HB-EGF acts in a juxtacrine manner to signal neighboring cells.9 The soluble form of HB-EGF is a potent mitogen and chemoattractant for many types of cells, including keratinocytes and other epithelial cells.10,11 The released HB-EGF acts through the stimulation of specific cell-surface receptors.12,13 Four related receptor tyrosine kinases have been identified.14–16 These are EGFR/erbB1/HER1, erbB2/HER2/neu, erbB3/HER3, and erbB4/HER4.12 Shed EGFR ligands such as HB-EGF act as autocrine/paracrine EGFR ligands and stimulate EGFR activation. Phosphorylation of EGFR creates docking sites for adaptor proteins such as Grb2, Shc, and Gab1 and leads to activation (tyrosine phosphorylation) of effectors such as extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K). These two pathways have been shown to be involved in corneal epithelial wound healing.17–22 Ectodomain shedding is a highly regulated process.23 The mechanism by which intracellular signaling pathways lead to an increase in sheddase activity has not been fully established. PKC or intracellular Ca2+ pathways are shown to up-regulate the activity of sheddase.24–27 Src nonreceptor tyrosine kinases have also been suggested as upstream mediators of EGFR transactivation,28 particularly in response to the stimulation of G-protein–coupled receptor ligands.29,30 Recently, Src was found to be activated in cells along the wounding edge of cultured mouse corneal epithelial cells; blocking this activation with the Src kinase inhibitor, PP1, inhibited wound closure.31 c-Src is the prototype of a closely related family of nine genes encoding nonreceptor membrane–associated protein tyrosine kinases (PTKs). In a variety of cell types, Src family members participate in the regulation of diverse functions, including proliferation, cell cycle, migration, adhesion, and differentiation.32 Only three of the nine members of the Src family are found in epithelial cells, and these include Src, Fyn, and Yes, which are ubiquitously expressed.32 The members of the Src family have a similar structure and share common pathways of regulation and function.33,34 They are also integral components of the signal transduction apparatus used by growth factor receptor tyrosine kinases.35 To determine signaling pathways regulating wound-induced EGFR transactivation, we used several inhibitors and found that PP2, a selective inhibitor of the Src family kinases, attenuated wound-induced EGFR transactivation and wound closure in cultured human corneal epithelial cells with or without exogenously added EGFR ligands. We also investigated the effects of Src inhibition on EGFR downstream signaling pathways. The study suggests that Src mediates wound-induced EGFR transactivation and plays a role in growth factor–mediated corneal epithelial cell migration and wound closure.