The authors have previously demonstrated that wounding of human corneal epithelial cells (HCECs) transactivates epidermal growth factor (EGF) receptor (EGFR) and its downstream signaling pathways and that this EGFR signaling is required for epithelial wound healing. 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 (Glp1)-Apelin-13 Src family have a similar structure and share common pathways of regulation and function.33 34 They are also integral components of the transmission 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. Materials and Methods Materials Keratinocyte basic medium (KBM) and keratinocyte growth medium (KGM; KBM supplemented with bovine pituitary extract epinephrine hydrocortisone transferrin insulin and EGF) were from BioWhittaker (Walkersville MD). Human recombinant HB-EGF was obtained from R&D Systems (Minneapolis MN). Antibodies against human EGFR ERK2 (Glp1)-Apelin-13 (p42 MAPK) phospho-ERK1/2 (p42/p44) c-Src (Src 2) pY20 and pY99 were from Santa Cruz Biotechnology (Santa Cruz CA). Antibodies against a major substrate of PI3K-AKT phospho-AKT and phospho-Src (Y416)-were obtained from Cell Signaling (Beverly MA). Rabbit anti-EGFR (Y845) was from Biosource (Camarillo CA). The EGFR inhibitor tyrphostin AG (Glp1)-Apelin-13 1478 (Glp1)-Apelin-13 was from Sigma-Aldrich (St. Louis MO). The Src inhibitor PP2 (4-amino-5-(4-chloro-phenyl)-7-(t-butyl) pyrazolo [3 4 pyrimidine) the phospholipase C (PLC) inhibitor “type”:”entrez-nucleotide” attrs :”text”:”U73122″ term_id :”4098075″ term_text :”U73122″U73122 and the PI3K inhibitor LY294002 were from Calbiochem (La Jolla CA). Boyden chamber and polycarbonate membranes (14-and known to be catalyzed by ADAM (a disintegrin and metalloprotease) proteins.42-45 Pro-HB-EGF has been shown to be a common EGFR ligand subjected to ectodomain shedding leading to the transactivation of EGFR in a variety of cells.37 46 47 Using the matrix metalloproteinase inhibitor GM6001 and the HB-EGF inhibitor CRM 197 we previously showed that wound-induced Rabbit Polyclonal to TAS2R10. EGFR activation occurred through shedding of pro-HB-EGF by a metalloprotease-sensitive process in corneal epithelial cells.5 Because PP2 does not inhibit EGFR activation induced by exogenously added HB-EGF we suggest that Src is an upstream signaling molecule for HB-EGF ectodomain shedding in wounded HCECs. Consistent with the essential role of EGFR transactivation in mediating corneal epithelial wound healing we observed that PP2 (Glp1)-Apelin-13 blocked scrape wound closure in cultured HCECs. A previous study31 revealed that Src was activated in corneal epithelial cells along the wound edge and that blocking this activation with PP1 inhibited wound closure. Furthermore it created a complex with Cdk5 a member of the cyclin-dependent kinase family to regulate epithelial..