title: Tiotropium attenuates IL-13-induced goblet cell metaplasia of human airway epithelial cells.
abstract: BACKGROUND
  It has been shown that acetylcholine is both a neurotransmitter and acts as a local mediator, produced by airway cells including epithelial cells. In vivo studies have demonstrated an indirect role for acetylcholine in epithelial cell differentiation. Here, we aimed to investigate direct effects of endogenous non-neuronal acetylcholine on epithelial cell differentiation.
  
  
  METHODS
  Human airway epithelial cells from healthy donors were cultured at an air-liquid interface (ALI). Cells were exposed to the muscarinic antagonist tiotropium (10 nM), interleukin (IL)-13 (1, 2 and 5 ng/mL), or a combination of IL-13 and tiotropium, during or after differentiation at the ALI.
  
  
  RESULTS
  Human airway epithelial cells expressed all components of the non-neuronal cholinergic system, suggesting acetylcholine production. Tiotropium had no effects on epithelial cell differentiation after air exposure. Differentiation into goblet cells was barely induced after air exposure. Therefore, IL-13 (1 ng/mL) was used to induce goblet cell metaplasia. IL-13 induced MUC5AC-positive cells (5-fold) and goblet cells (14-fold), as assessed by histochemistry, and MUC5AC gene expression (105-fold). These effects were partly prevented by tiotropium (47-92%). Goblet cell metaplasia was induced by IL-13 in a dose-dependent manner, which was inhibited by tiotropium. In addition, tiotropium reversed goblet cell metaplasia induced by 2 weeks of IL-13 exposure. IL-13 decreased forkhead box protein A2 (FoxA2) expression (1.6-fold) and increased FoxA3 (3.6-fold) and SAM-pointed domain-containing ETS transcription factor (SPDEF) (5.2-fold) expression. Tiotropium prevented the effects on FoxA2 and FoxA3, but not on SPDEF.
  
  
  CONCLUSIONS
  We demonstrate that tiotropium has no effects on epithelial cell differentiation after air exposure, but inhibits and reverses IL-13-induced goblet cell metaplasia, possibly via FoxA2 and FoxA3. This indicates that non-neuronal acetylcholine contributes to goblet cell differentiation by a direct effect on epithelial cells.
authors.affiliation: Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands GRIAC Research Institute, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: Loes E M
authors.initials: LE
authors.lastname: Kistemaker
authors.affiliation: Department of Pulmonology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Pieter S
authors.initials: PS
authors.lastname: Hiemstra
authors.affiliation: Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands GRIAC Research Institute, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: I Sophie T
authors.initials: IS
authors.lastname: Bos
authors.affiliation: Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands GRIAC Research Institute, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: Susanne
authors.initials: S
authors.lastname: Bouwman
authors.affiliation: GRIAC Research Institute, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Department of Pulmonary Diseases, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Maarten
authors.initials: M
authors.lastname: van den Berge
authors.affiliation: GRIAC Research Institute, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Department of Pathology and Medical Biology, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Machteld N
authors.initials: MN
authors.lastname: Hylkema
authors.affiliation: Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands GRIAC Research Institute, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: Herman
authors.initials: H
authors.lastname: Meurs
authors.affiliation: GRIAC Research Institute, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands Department of Pulmonary Diseases, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Huib A M
authors.initials: HA
authors.lastname: Kerstjens
authors.affiliation: Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands GRIAC Research Institute, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: Reinoud
authors.initials: R
authors.lastname: Gosens
mesh_terms: D000109:Acetylcholine / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D018680:Cholinergic Antagonists / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000473:pathology; D005786:Gene Expression Regulation / Q000187:drug effects; D020397:Goblet Cells / Q000187:drug effects* / Q000473:pathology; D006801:Humans; D018793:Interleukin-13 / Q000008:administration & dosage / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D008679:Metaplasia / Q000139:chemically induced / Q000235:genetics / Q000473:pathology; D055271:Mucin 5AC / Q000096:biosynthesis / Q000235:genetics; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000473:pathology; D012602:Scopolamine Derivatives / Q000494:pharmacology*; D000069447:Tiotropium Bromide; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D018680:Cholinergic Antagonists; D018793:Interleukin-13; C524678:MUC5AC protein, human; D055271:Mucin 5AC; D012602:Scopolamine Derivatives; D014157:Transcription Factors; D000109:Acetylcholine; D000069447:Tiotropium Bromide
keywords: Airway Epithelium; Asthma Pharmacology