title: SRC-family tyrosine kinases in wound- and ligand-induced epidermal growth factor receptor activation in human corneal epithelial cells.
abstract: OBJECTIVE
  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. In this study, the authors sought to identify the underlying mechanisms for EGFR transactivation in response to wounding in HCECs.
  
  
  METHODS
  SV40-immortalized HCEC (THCE) monolayer was wounded and allowed to heal in the presence or absence of a selective inhibitor of the Src family kinases PP2 and EGFR ligand heparin-binding EGF-like growth factor (HB-EGF). Wound closure was monitored by photographing of the injury immediately or 24 hours after wounding. Activation of EGFR in THCE cells and in primary HCECs was analyzed by immunoprecipitation of EGFR, followed by Western blotting with phosphotyrosine antibody. Phosphorylation of extracellular signal-regulated kinase (ERK), AKT (a major substrate of phosphatidylinositol 3'-kinase [PI3K]), Src at tyrosine Y416, and EGFR at Y845 was analyzed by Western blotting with antibodies specific to phosphorylated proteins. Effects of PP2 on THCE cell migration were determined by Boyden chamber migration assay.
  
  
  RESULTS
  Among several inhibitors tested, PP2 blocked wound-induced EGFR phosphorylation in THCE cells. PP2 at 12.5 microM effectively inhibited EGFR transactivation in response to wounding and to the phosphorylation of ERK and AKT in THCE cells and primary HCECs. Consistent with the inhibition of EGFR transactivation, PP2 also attenuated epithelial migration and wound closure with or without exogenously added HB-EGF. PP2 at a concentration as high as 50 microM exhibited no effects on HB-EGF induced ERK phosphorylation. On the other hand, AKT phosphorylation was much more sensitive to PP2 than ERK or EGFR phosphorylation because 3.13 microM PP2 effectively inhibited wound- or HB-EGF-induced AKT phosphorylation.
  
  
  CONCLUSIONS
  These results suggest that Src kinase mediates wound-induced EGFR transactivation and participates in a pathway to activate the PI3K-AKT pathway downstream of EGFR in HCECs.
authors.affiliation: Department of Ophthalmology, the Kresge Eye Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
authors.forename: Ke-Ping
authors.initials: KP
authors.lastname: Xu
authors.forename: Jia
authors.initials: J
authors.lastname: Yin
authors.forename: Fu-Shin X
authors.initials: FS
authors.lastname: Yu
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D019573:Epithelium, Corneal / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051058:Oncogene Protein v-akt / Q000378:metabolism; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D015533:Transcriptional Activation; D014945:Wound Healing / Q000502:physiology*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: C412373:AG 1879; D008024:Ligands; D011743:Pyrimidines; D066246:ErbB Receptors; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases; D051058:Oncogene Protein v-akt; D048049:Extracellular Signal-Regulated MAP Kinases