Department of Pharmacology, Shanghai JiaoTong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China.
BMC Pulm Med. 2014 Mar 31;14:53. doi: 10.1186/1471-2466-14-53.
Epithelial-mesenchymal transition (EMT) has been proposed as a mechanism in the progression of airway diseases and cancer. Here, we explored the role of acetylcholine (ACh) and the pathway involved in the process of EMT, as well as the effects of mAChRs antagonist.
Human lung epithelial cells were stimulated with carbachol, an analogue of ACh, and epithelial and mesenchymal marker proteins were evaluated using western blot and immunofluorescence analyses.
Decreased E-cadherin expression and increased vimentin and α-SMA expression induced by TGF-β1 in alveolar epithelial cell (A549) were significantly abrogated by the non-selective mAChR antagonist atropine and enhanced by the acetylcholinesterase inhibitor physostigmine. An EMT event also occurred in response to physostigmine alone. Furthermore, ChAT express and ACh release by A549 cells were enhanced by TGF-β1. Interestingly, ACh analogue carbachol also induced EMT in A549 cells as well as in bronchial epithelial cells (16HBE) in a time- and concentration-dependent manner, the induction of carbachol was abrogated by selective antagonist of M1 (pirenzepine) and M3 (4-DAMP) mAChRs, but not by M2 (methoctramine) antagonist. Moreover, carbachol induced TGF-β1 production from A549 cells concomitantly with the EMT process. Carbachol-induced EMT occurred through phosphorylation of Smad2/3 and ERK, which was inhibited by pirenzepine and 4-DAMP.
Our findings for the first time indicated that mAChR activation, perhaps via M1 and M3 mAChR, induced lung epithelial cells to undergo EMT and provided insights into novel therapeutic strategies for airway diseases in which lung remodeling occurs.
上皮-间充质转化（EMT）被提出作为气道疾病和癌症进展的一种机制。在这里，我们探讨了乙酰胆碱（ACh）的作用及其在 EMT 过程中涉及的途径，以及 mAChR 拮抗剂的影响。
用卡巴胆碱（ACh 的类似物）刺激人肺上皮细胞，使用 Western blot 和免疫荧光分析评估上皮和间充质标记蛋白。
TGF-β1 诱导的肺泡上皮细胞（A549）中 E-钙黏蛋白表达减少，波形蛋白和α-SMA 表达增加，这一现象被非选择性 mAChR 拮抗剂阿托品显著阻断，并被乙酰胆碱酯酶抑制剂毒扁豆碱增强。单独使用毒扁豆碱也会引起 EMT 事件。此外，TGF-β1 增强了 A549 细胞的 ChAT 表达和 ACh 释放。有趣的是，ACh 类似物卡巴胆碱也以时间和浓度依赖的方式诱导 A549 细胞和支气管上皮细胞（16HBE）发生 EMT，M1（哌仑西平）和 M3（4-DAMP）mAChR 的选择性拮抗剂可阻断卡巴胆碱诱导的 EMT，但 M2（甲磺酸美托咪定）拮抗剂则不能。此外，卡巴胆碱诱导 A549 细胞产生 TGF-β1 的同时伴随着 EMT 过程。卡巴胆碱诱导的 EMT 发生是通过 Smad2/3 和 ERK 的磷酸化，这一过程被哌仑西平和 4-DAMP 抑制。
我们的研究结果首次表明，mAChR 激活，可能通过 M1 和 M3 mAChR，诱导肺上皮细胞发生 EMT，并为发生肺重塑的气道疾病提供了新的治疗策略。
