title: The activation of M3 mAChR in airway epithelial cells promotes IL-8 and TGF-β1 secretion and airway smooth muscle cell migration.
abstract: BACKGROUND
  Muscarinic acetylcholine receptors (mAChRs) have been identified in airway epithelium, and epithelium-derived chemokines can initiate the migration of airway smooth muscle (ASM) cells. However, the mAChRs that are expressed in airway epithelium and the mechanism underlying the regulation of ASM cell migration are not clear. The aim of this study was to test whether the effects of the epithelium-derived chemokines on ASM cell migration could be modulated by mAChRs.
  
  
  METHODS
  Human epithelial cells (A549 cells) were stimulated with cigarette smoke extract (CSE) or the mAChRs agonist carbachol. IL-8 and TGF-β1 production were measured by ELISA, and human ASM cell migration was measured using the transwell migration assay and scratch assay. The mRNA levels of the mAChRs subtypes and the acetylcholine concentrations were measured using RT-PCR and LC-MS/MS, respectively.
  
  
  RESULTS
  ASM cell migration toward CSE-stimulated A549 cells was markedly reduced by Ac-RRWWCR-NH2 (IL-8 inhibitor) and SB431542 (TGF-β1 inhibitor). CSE-induced ASM cell migration was also suppressed by the mAChRs antagonist tiotropium. Interestingly, carbachol-stimulated A549 cells also induced ASM cell migration; this migration event was suppressed by tiotropium, Ac-RRWWCR-NH2 and SB431542. In addition, the effects of CSE on ASM cell migration were significantly and cooperatively enhanced by carbachol compared to CSE alone. Carbachol-induced ASM cell migration was reduced by selective inhibitors of PI3K/Akt (LY294002) and p38 (SB203580), suggesting that it occurred through p38 and Akt phosphorylation, which was inhibited by the M3 mAChR antagonist 4-DAMP.
  
  
  CONCLUSIONS
  These findings indicate that M3 mAChR may be important therapeutic target for obstructive airway diseases, as it regulates the effects of the epithelial-derived chemokines on ASM cell migration, which results in lung remodeling.
authors.affiliation: Department of Pharmacology, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China. lujuanjuan825@163.com.
authors.forename: Juan-Juan
authors.initials: JJ
authors.lastname: Lu
authors.affiliation: Department of Anatomy, Histology and Embryology, Shanghai Jiao Tong University School of Medicine, Shanghai, China. xuguangni1986@163.com.
authors.forename: Guang-Ni
authors.initials: GN
authors.lastname: Xu
authors.affiliation: Department of Pharmacology, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China. zjypwz@163.com.
authors.forename: Ping
authors.initials: P
authors.lastname: Yu
authors.affiliation: Department of Pharmacology, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China. songwanyi@126.com.
authors.forename: Yun
authors.initials: Y
authors.lastname: Song
authors.affiliation: Department of Pharmacology, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China. xlwang1979@aliyun.com.
authors.forename: Xiao-Lin
authors.initials: XL
authors.lastname: Wang
authors.affiliation: Department of Pharmacology, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China. zhuliang17@gemail.com.
authors.forename: Liang
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Department of Pharmacology, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China. hongzhuan_chen@hotmail.com.
authors.forename: Hong-Zhuan
authors.initials: HZ
authors.lastname: Chen
authors.affiliation: Department of Pharmacology, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai, 200025, China. yongyaocui@hotmail.com.
authors.forename: Yong-Yao
authors.initials: YY
authors.lastname: Cui
mesh_terms: D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000502:physiology*; D043587:Receptor, Muscarinic M3 / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D013638:Tars / Q000633:toxicity*; D053773:Transforming Growth Factor beta1 / Q000096:biosynthesis*
chemical_list: D016209:Interleukin-8; D043587:Receptor, Muscarinic M3; D013638:Tars; D053773:Transforming Growth Factor beta1; C024746:tobacco tar