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mAChRs activation induces epithelial-mesenchymal transition on lung epithelial cells.

Yang K, Song Y, Tang YB, Xu ZP, Zhou W, Hou LN, Zhu L, Yu ZH, Chen HZ, Cui YY - BMC Pulm Med (2014)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Shanghai JiaoTong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China. hongzhuan_chen@hotmail.com.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

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Carbachol induces EMT-related changes in A549 cells. Cells were stimulated with TGF-β1 (5 ng/mL) or carbachol (0.1–10 μM) for 72 h (A), carbachol (1 μM) for the indicated times (B), or with the addition of pirenzepine (10 μM), methoctramine (10 μM), or 4-DAMP (1 μM) administered 1 h before carbachol (1 μM) for 72 h (C). Cell lysates were assayed for EMT markers and GAPDH. Representative western blots of E-cadherin, vimentin, α-SMA and GAPDH are shown. (D) Cells were treated with 4-DAMP (1 μM) 1 h before stimulation with carbachol (1 μM) for 72 h. Cells were fixed and stained with E-cadherin, vimentin and α-SMA antibodies, and then incubated with Alexa Fluor-conjugated secondary antibodies and DAPI for nuclei labeling. E-cadherin (green), vimentin (purple), α-SMA (red) and nuclei (blue) were visualized using confocal fluorescence microscopy. Data are expressed as mean ± SEM of 3–5 independent experiments after densitometric analysis. #p < 0.05, ##p < 0.01 vs. control; *p < 0.05, **p < 0.01 vs. carbachol.
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Figure 3: Carbachol induces EMT-related changes in A549 cells. Cells were stimulated with TGF-β1 (5 ng/mL) or carbachol (0.1–10 μM) for 72 h (A), carbachol (1 μM) for the indicated times (B), or with the addition of pirenzepine (10 μM), methoctramine (10 μM), or 4-DAMP (1 μM) administered 1 h before carbachol (1 μM) for 72 h (C). Cell lysates were assayed for EMT markers and GAPDH. Representative western blots of E-cadherin, vimentin, α-SMA and GAPDH are shown. (D) Cells were treated with 4-DAMP (1 μM) 1 h before stimulation with carbachol (1 μM) for 72 h. Cells were fixed and stained with E-cadherin, vimentin and α-SMA antibodies, and then incubated with Alexa Fluor-conjugated secondary antibodies and DAPI for nuclei labeling. E-cadherin (green), vimentin (purple), α-SMA (red) and nuclei (blue) were visualized using confocal fluorescence microscopy. Data are expressed as mean ± SEM of 3–5 independent experiments after densitometric analysis. #p < 0.05, ##p < 0.01 vs. control; *p < 0.05, **p < 0.01 vs. carbachol.

Mentions: If endogenous ACh is involved in TGF-β1-induced EMT, the application of an exogenous mAChR agonist should have the same effect as endogenous ACh. As shown in Figure 3A, B, C, carbachol dramatically decreased E-cadherin expression, and increased expression of vimentin and α-SMA in A549 cells in a concentration-dependent manner (Figure 3A). The expression levels of E-cadherin, vimentin and α-SMA significantly changed at 48 h and peaked at 72 h (Figure 3B). It is interesting to note that carbachol at concentrations as low as 0.1 μM was sufficient to induce EMT phenotypic markers with a maximal response at 10 μM. Furthermore, carbachol-induced EMT can be abrogated by pirenzepine (M1 mAChR antagonist, 10 μM) and diphenyl-acetoxy-4-methylpiperidine methiodide (4-DAMP, an M3 mAChR antagonist, 1 μM), but not methoctramine (an M2 mAChR antagonist, 10 μM) (Figure 3C).


mAChRs activation induces epithelial-mesenchymal transition on lung epithelial cells.

Yang K, Song Y, Tang YB, Xu ZP, Zhou W, Hou LN, Zhu L, Yu ZH, Chen HZ, Cui YY - BMC Pulm Med (2014)

Carbachol induces EMT-related changes in A549 cells. Cells were stimulated with TGF-β1 (5 ng/mL) or carbachol (0.1–10 μM) for 72 h (A), carbachol (1 μM) for the indicated times (B), or with the addition of pirenzepine (10 μM), methoctramine (10 μM), or 4-DAMP (1 μM) administered 1 h before carbachol (1 μM) for 72 h (C). Cell lysates were assayed for EMT markers and GAPDH. Representative western blots of E-cadherin, vimentin, α-SMA and GAPDH are shown. (D) Cells were treated with 4-DAMP (1 μM) 1 h before stimulation with carbachol (1 μM) for 72 h. Cells were fixed and stained with E-cadherin, vimentin and α-SMA antibodies, and then incubated with Alexa Fluor-conjugated secondary antibodies and DAPI for nuclei labeling. E-cadherin (green), vimentin (purple), α-SMA (red) and nuclei (blue) were visualized using confocal fluorescence microscopy. Data are expressed as mean ± SEM of 3–5 independent experiments after densitometric analysis. #p < 0.05, ##p < 0.01 vs. control; *p < 0.05, **p < 0.01 vs. carbachol.
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Figure 3: Carbachol induces EMT-related changes in A549 cells. Cells were stimulated with TGF-β1 (5 ng/mL) or carbachol (0.1–10 μM) for 72 h (A), carbachol (1 μM) for the indicated times (B), or with the addition of pirenzepine (10 μM), methoctramine (10 μM), or 4-DAMP (1 μM) administered 1 h before carbachol (1 μM) for 72 h (C). Cell lysates were assayed for EMT markers and GAPDH. Representative western blots of E-cadherin, vimentin, α-SMA and GAPDH are shown. (D) Cells were treated with 4-DAMP (1 μM) 1 h before stimulation with carbachol (1 μM) for 72 h. Cells were fixed and stained with E-cadherin, vimentin and α-SMA antibodies, and then incubated with Alexa Fluor-conjugated secondary antibodies and DAPI for nuclei labeling. E-cadherin (green), vimentin (purple), α-SMA (red) and nuclei (blue) were visualized using confocal fluorescence microscopy. Data are expressed as mean ± SEM of 3–5 independent experiments after densitometric analysis. #p < 0.05, ##p < 0.01 vs. control; *p < 0.05, **p < 0.01 vs. carbachol.
Mentions: If endogenous ACh is involved in TGF-β1-induced EMT, the application of an exogenous mAChR agonist should have the same effect as endogenous ACh. As shown in Figure 3A, B, C, carbachol dramatically decreased E-cadherin expression, and increased expression of vimentin and α-SMA in A549 cells in a concentration-dependent manner (Figure 3A). The expression levels of E-cadherin, vimentin and α-SMA significantly changed at 48 h and peaked at 72 h (Figure 3B). It is interesting to note that carbachol at concentrations as low as 0.1 μM was sufficient to induce EMT phenotypic markers with a maximal response at 10 μM. Furthermore, carbachol-induced EMT can be abrogated by pirenzepine (M1 mAChR antagonist, 10 μM) and diphenyl-acetoxy-4-methylpiperidine methiodide (4-DAMP, an M3 mAChR antagonist, 1 μM), but not methoctramine (an M2 mAChR antagonist, 10 μM) (Figure 3C).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Shanghai JiaoTong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China. hongzhuan_chen@hotmail.com.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus