title: Activation of focal adhesion kinase via M1 muscarinic acetylcholine receptor is required in restitution of intestinal barrier function after epithelial injury.
abstract: Impairment of epithelial barrier is observed in various intestinal disorders including inflammatory bowel diseases (IBD). Numerous factors may cause temporary damage of the intestinal epithelium. A complex network of highly divergent factors regulates healing of the epithelium to prevent inflammatory response. However, the exact repair mechanisms involved in maintaining homeostatic intestinal barrier integrity remain to be clarified. In this study, we demonstrate that activation of M1 muscarinic acetylcholine receptor (mAChR) augments the restitution of epithelial barrier function in T84 cell monolayers after ethanol-induced epithelial injury, via ERK-dependent phosphorylation of focal adhesion kinase (FAK). We have shown that ethanol injury decreased the transepithelial electrical resistance (TER) along with the reduction of ERK and FAK phosphorylation. Carbachol (CCh) increased ERK and FAK phosphorylation with enhanced TER recovery, which was completely blocked by either MT-7 (M1 antagonist) or atropine. The CCh-induced enhancement of TER recovery was also blocked by either U0126 (ERK pathway inhibitor) or PF-228 (FAK inhibitor). Treatment of T84 cell monolayers with interferon-γ (IFN-γ) impaired the barrier function with the reduction of FAK phosphorylation. The CCh-induced ERK and FAK phosphorylation were also attenuated by the IFN-γ treatment. Immunological and binding experiments exhibited a significant reduction of M1 mAChR after IFN-γ treatment. The reduction of M1 mAChR in inflammatory area was also observed in surgical specimens from IBD patients, using immunohistochemical analysis. These findings provide important clues regarding mechanisms by which M1 mAChR participates in the maintenance of intestinal barrier function under not only physiological but also pathological conditions.
authors.affiliation: Division of Cellular Signal Transduction, Department of Biochemistry, Asahikawa Medical University, Asahikawa, Japan; Department of Pharmacy, University of Rajshahi, Rajshahi, Bangladesh.
authors.forename: Rafiqul Islam
authors.initials: RI
authors.lastname: Khan
authors.affiliation: Division of Cellular Signal Transduction, Department of Biochemistry, Asahikawa Medical University, Asahikawa, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Yazawa
authors.affiliation: Department of Pharmacy, University of Rajshahi, Rajshahi, Bangladesh.
authors.forename: Abu Syed Md
authors.initials: AS
authors.lastname: Anisuzzaman
authors.affiliation: Division of Cellular Signal Transduction, Department of Biochemistry, Asahikawa Medical University, Asahikawa, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Semba
authors.affiliation: Department of Medical Oncology, The First Hospital of China Medical University, Shenyang, China.
authors.forename: Yanju
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, University of Fukui, Fukui, Japan.
authors.forename: Junsuke
authors.initials: J
authors.lastname: Uwada
authors.affiliation: Laboratory of Physiology, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan.
authors.forename: Hisayoshi
authors.initials: H
authors.lastname: Hayashi
authors.affiliation: Laboratory of Physiology, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan; Division of Health and Nutrition, Sendai Shirayuri Women's College, Sendai, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Suzuki
authors.affiliation: Inflammatory Bowel Disease Center, Hyogo College of Medicine, Nishinomiya, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ikeuchi
authors.affiliation: Inflammatory Bowel Disease Center, Hyogo College of Medicine, Nishinomiya, Japan.
authors.forename: Motoi
authors.initials: M
authors.lastname: Uchino
authors.affiliation: Department of Gastrointestinal Immunology and Regenerative Medicine, Asahikawa Medical University, Asahikawa, Japan; Inflammatory Bowel Disease Center, Sapporo Higashi Tokushukai Hospital, Sapporo, Japan.
authors.forename: Atsuo
authors.initials: A
authors.lastname: Maemoto
authors.affiliation: Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, University of Fukui, Fukui, Japan; Organization for Life Science Advancement Programs, University of Fukui, Fukui, Japan; Research Center for Child Mental Development, University of Fukui, Fukui, Japan.
authors.forename: Ikunobu
authors.initials: I
authors.lastname: Muramatsu
authors.affiliation: Division of Cellular Signal Transduction, Department of Biochemistry, Asahikawa Medical University, Asahikawa, Japan. Electronic address: takotago@asahikawa-med.ac.jp.
authors.forename: Takanobu
authors.initials: T
authors.lastname: Taniguchi
mesh_terms: D045744:Cell Line, Tumor; D017097:Electric Impedance; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000032:analysis / Q000502:physiology; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000032:analysis / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D007371:Interferon-gamma / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000378:metabolism*; D010766:Phosphorylation; D043643:Receptor, Muscarinic M1 / Q000032:analysis / Q000502:physiology*
chemical_list: D043643:Receptor, Muscarinic M1; D007371:Interferon-gamma; D051416:Focal Adhesion Protein-Tyrosine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases
keywords: Epithelial barrier function; Focal adhesion kinase; IBD; mAChR