title: Differential regulation of epidermal growth factor receptor by hydrogen peroxide and flagellin in cultured lung alveolar epithelial cells.
abstract: 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.
authors.affiliation: Department of Biochemistry, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan; Department of Anesthesiology, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nishi
authors.affiliation: Department of Biochemistry, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Maeda
authors.affiliation: Department of Biochemistry, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan; Department of Anesthesiology, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Izumi
authors.affiliation: Department of Biochemistry, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.
authors.forename: Sayomi
authors.initials: S
authors.lastname: Higa-Nakamine
authors.affiliation: Department of Biochemistry, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.
authors.forename: Seikichi
authors.initials: S
authors.lastname: Toku
authors.affiliation: Department of Anesthesiology, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Kakinohana
authors.affiliation: Department of Anesthesiology, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Sugahara
authors.affiliation: Department of Biochemistry, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan. Electronic address: hideyuki@med.u-ryukyu.ac.jp.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Yamamoto
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism*; D005408:Flagellin / Q000494:pharmacology*; D055551:HSP27 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011650:Pulmonary Alveoli / Q000166:cytology*; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D000099190:MAP-Kinase-Activated Kinase 2
chemical_list: D000111:Acetylcysteine; D066246:ErbB Receptors; D005408:Flagellin; D055551:HSP27 Heat-Shock Proteins; D006861:Hydrogen Peroxide; D047908:Intracellular Signaling Peptides and Proteins; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D012694:Serine; D014409:Tumor Necrosis Factor-alpha; D000099190:MAP-Kinase-Activated Kinase 2
keywords: A549 cells; CaM kinase II; Epidermal growth factor receptor; Flagellin; Hydrogen peroxide; TNFα