The activation of M3 mAChR in airway epithelial cells promotes IL-8 and TGF-β1 secretion and airway smooth muscle cell migration

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. Method: Human epithelial cells (A549 cells) were stimulated with cigarette smoke extract (CSE) or the mAChRs agonist carbachol. IL-8 and TGF-beta 1 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-beta 1 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.