title: Combination of glycopyrronium and indacaterol inhibits carbachol-induced ERK5 signal in fibrotic processes.
abstract: Airway fibrosis is one of the pathological features of chronic obstructive pulmonary disease (COPD), and recent studies revealed that acetylcholine plays an important role in the development of airway remodeling by stimulating proliferation and collagen synthesis of lung fibroblasts. This study was designed to examine the effects of a long-acting muscarinic receptor antagonist (LAMA) glycopyrronium and a long-acting β2 adrenergic receptor agonist (LABA) indacaterol on acetylcholine-mediated fibrotic responses in lung fibroblasts.
  
  
  
  After carbachol (CCh) or transforming growth factor-β1 (TGF-β1) exposure, the response to glycopyrronium and indacaterol was determined in vitro in fibroblasts isolated from mild-to-moderate COPD lung tissue. The ability of fibroblasts to mediate the contraction of collagen gels was assessed. The expression of α-smooth muscle actin (α-SMA) and the phosphorylation of extracellular-signal-regulated kinase 5 (ERK5) were determined by immunoblot. TGF-β1 was quantified by ELISA and acetylcholine was quantified by liquid chromatography tandem-mass spectrometry.
  
  
  
  CCh stimulated fibroblast-mediated collagen gel contraction and α-SMA expression and TGF-β1 release by fibroblasts. Blockade of autocrine TGF-β1 attenuated CCh-mediated fibrotic responses, while TGF-β1 did not stimulate acetylcholine release. Glycopyrronium plus indacaterol significantly attenuated CCh- and TGF-β1-mediated fibrotic responses through inhibition of ERK5 phosphorylation. Notably, the magnitudes of CCh- and TGF-β1-stimulated gel contraction, CCh-induced TGF-β1 release, and ERK5 phosphorylation were greater in fibroblasts isolated from COPD subjects than in those from non-smokers.
  
  
  
  CCh induced TGF-β1 self-sustaining signaling loops by potentiating ERK5 signaling and promoted myofibroblast activity. This autocrine signaling mechanism may be an attractive therapeutic target to block the fibrotic response, which was modulated by the combination of glycopyrronium and indacaterol.
authors.affiliation: Division of Respiratory Medicine, Juntendo University Faculty of Medicine & Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.|Research Institute for Diseases of Old Ages, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Namba
authors.affiliation: Division of Respiratory Medicine, Juntendo University Faculty of Medicine & Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan. shinsaku@juntendo.ac.jp.|Research Institute for Diseases of Old Ages, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan. shinsaku@juntendo.ac.jp.
authors.forename: Shinsaku
authors.identifier: 0000-0002-6291-9034
authors.initials: S
authors.lastname: Togo
authors.affiliation: Division of Respiratory Medicine, Juntendo University Faculty of Medicine & Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.|Research Institute for Diseases of Old Ages, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.
authors.forename: Miniwan
authors.initials: M
authors.lastname: Tulafu
authors.affiliation: Division of Respiratory Medicine, Juntendo University Faculty of Medicine & Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.|Research Institute for Diseases of Old Ages, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.
authors.forename: Kotaro
authors.initials: K
authors.lastname: Kadoya
authors.affiliation: Division of Respiratory Medicine, Juntendo University Faculty of Medicine & Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.|Research Institute for Diseases of Old Ages, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.
authors.forename: Kumi Yoneda
authors.initials: KY
authors.lastname: Nagahama
authors.affiliation: Laboratory of Proteomics and Biomolecular Science, Research Support Center, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.
authors.forename: Hikari
authors.initials: H
authors.lastname: Taka
authors.affiliation: Laboratory of Proteomics and Biomolecular Science, Research Support Center, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Kaga
authors.affiliation: Departments of Pathology and Oncology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Orimo
authors.affiliation: Pulmonary Critical Care and Sleep Medicine, University of Nebraska Medical Center, Omaha, NE, USA.
authors.forename: Xiangde
authors.initials: X
authors.lastname: Liu
authors.affiliation: Division of Respiratory Medicine, Juntendo University Faculty of Medicine & Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.|Research Institute for Diseases of Old Ages, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Takahashi
mesh_terms: D058672:Adrenergic beta-2 Receptor Antagonists / Q000008:administration & dosage; D000368:Aged; D002217:Carbachol; D004305:Dose-Response Relationship, Drug; D004359:Drug Therapy, Combination / Q000379:methods; D005260:Female; D006024:Glycopyrrolate / Q000008:administration & dosage*; D006801:Humans; D007189:Indans / Q000008:administration & dosage*; D008297:Male; D008875:Middle Aged; D048054:Mitogen-Activated Protein Kinase 7 / Q000378:metabolism*; D018727:Muscarinic Antagonists / Q000008:administration & dosage; D029424:Pulmonary Disease, Chronic Obstructive / Q000139:chemically induced / Q000378:metabolism* / Q000517:prevention & control*; D011658:Pulmonary Fibrosis / Q000139:chemically induced / Q000378:metabolism* / Q000517:prevention & control*; D015363:Quinolones / Q000008:administration & dosage*; D015398:Signal Transduction / Q000187:drug effects; D016896:Treatment Outcome
chemical_list: D058672:Adrenergic beta-2 Receptor Antagonists; D007189:Indans; D018727:Muscarinic Antagonists; D015363:Quinolones; C510790:indacaterol; D002217:Carbachol; D048054:Mitogen-Activated Protein Kinase 7; D006024:Glycopyrrolate
keywords: Acetylcholine; Extracellular-signal-regulated kinase 5 (ERK5); Long-acting muscarinic receptor antagonist; Long-acting β2-adrenergic receptor agonist; Transforming growth factor-β1