Differential regulation of epidermal growth factor receptor by hydrogen peroxide and flagellin in cultured lung alveolar epithelial cells

Eur J Pharmacol. 2015 Feb 5;748:133-42. doi: 10.1016/j.ejphar.2014.12.016. Epub 2014 Dec 24.


In previous studies, we found that stimulation of Toll-like receptor 5 (TLR5) by flagellin induced the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2) through activation of the p38 MAPK pathway in cultured alveolar epithelial A549 cells. Our studies strongly suggested that MAPKAPK-2 phosphorylated epidermal growth factor receptor (EGFR) at Ser1047. It has been reported that phosphorylation of Ser1047 after treatment with tumor necrosis factor α (TNFα) induced the internalization of EGFR. In the present study, we first found that treatment of A549 cells with hydrogen peroxide induced the activation of MAPKAPK-2 and phosphorylation of EGFR at Ser1047 within 30 min. This was different from flagellin treatment because hydrogen peroxide treatment induced the phosphorylation of EGFR at Tyr1173 as well as Ser1047, indicating the activation of EGFR. We also found that KN93, an inhibitor of CaM kinase II, inhibited the hydrogen peroxide-induced phosphorylation of EGFR at Ser1047 through inhibition of the activation of the p38 MAPK pathway. Furthermore, we examined the internalization of EGFR by three different methods. Flow cytometry with an antibody against the extracellular domain of EGFR and biotinylation of cell surface proteins revealed that flagellin, but not hydrogen peroxide, decreased the amount of cell-surface EGFR. In addition, activation of extracellular signal-regulated kinase by EGF treatment was reduced by flagellin pre-treatment. These results strongly suggested that hydrogen peroxide activated the p38 MAPK pathway via activation of CaM kinase II and that flagellin and hydrogen peroxide regulate the functions of EGFR by different mechanisms.

Keywords: A549 cells; CaM kinase II; Epidermal growth factor receptor; Flagellin; Hydrogen peroxide; TNFα.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcysteine / pharmacology
  • Cell Line
  • Cell Survival / drug effects
  • Enzyme Activation / drug effects
  • Epithelial Cells / cytology
  • Epithelial Cells / drug effects*
  • Epithelial Cells / metabolism*
  • ErbB Receptors / chemistry
  • ErbB Receptors / metabolism*
  • Flagellin / pharmacology*
  • HSP27 Heat-Shock Proteins / metabolism
  • Humans
  • Hydrogen Peroxide / pharmacology*
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Phosphorylation / drug effects
  • Protein Kinase Inhibitors / pharmacology
  • Protein Serine-Threonine Kinases / metabolism
  • Protein Transport / drug effects
  • Pulmonary Alveoli / cytology*
  • Serine / metabolism
  • Signal Transduction / drug effects
  • Tumor Necrosis Factor-alpha / pharmacology


  • HSP27 Heat-Shock Proteins
  • Intracellular Signaling Peptides and Proteins
  • Protein Kinase Inhibitors
  • Tumor Necrosis Factor-alpha
  • Flagellin
  • Serine
  • Hydrogen Peroxide
  • MAP-kinase-activated kinase 2
  • ErbB Receptors
  • Protein Serine-Threonine Kinases
  • Acetylcysteine