Abstract
Abstract: :
Purpose: We utilized human corneal epithelial cells (HCECs) functionally depleted of erbB2 to elucidate the role of erbB2 in epidermal growth factor receptor (EGFR)–dependent cell migration. Methods: Retrovirus pBabe–5R encoding an erbB2 single–chain antibody with an endoplasmic reticulum (ER)–targeting sequence and control pBabe–puro were used to infect HCECs (an SV40 immortalized cell line). Several cell lines expressing 5R were selected along with a pBabe–puro control line. The depletion of erbB2 was verified by cell surface biotinylation of proteins, followed by streptavidin precipitation and subsequent detection of erbB2 by immunoblotting. Activation of erbBs was analyzed by immunoprecipitation using phosphotyrosine antibody PY20, followed by Western blotting with erbB1 or erbB2 antibodies. Effects of erbB2 depletion on HB–EGF–induced cell migration were determined by Boyden chamber migration and scratch wound assay. Phosphorylation of extracellular signal–regulated kinase (ERK) and PI3 kinase was analyzed by Western blotting with antibodies specific to the phosphorylated proteins. Porcine corneal organ culture model was adopted to study the role of ERK and PI3 kinase during corneal epithelial wound closure. Results: Wounding induced erbB2 tyrosine phosphorylation. Expression of 5R in HCECs depleted cell surface expressing erbB2. Wounding resulted in a rapid increase in the phosphorylation of erbB1 in both 5R expressing cells and controls, while wound–induced erbB2 phosphorylation in 5R expressing cells was not detectable. Depletion of functional erbB2 attenuated chemotactic cell migration and healing of scratch wounds in the presence of HB–EGF. Expression of 5R affected both the intensity and duration of wound–induced, EGFR–elicited ERK and PI3 kinase activation. Inhibition of ERK or PI3 kinase pathways impaired corneal wound healing in porcine corneal organ culture. Conclusions: ErbB2 serves as a critical component that couples erbB receptor tyrosine kinases to the migratory machinery of HCECs.
Keywords: growth factors/growth factor receptors • signal transduction • cornea: epithelium