MAPK/FoxA2-mediated cigarette smoke-induced squamous metaplasia of bronchial epithelial cells

Abstract

Objective: To explore the effect of cigarette smoke (CS) on the development of squamous metaplasia in human airway epithelial cells and the role of MAPK- and FoxA2-signaling pathways in the process.

Materials and methods: In an in vitro study, we treated the bronchial epithelial cell line BEAS2B with CS extract, followed by treatment with the ERK inhibitor U0126, the JNK inhibitor SP600125, or the p38 inhibitor SB203580. In vivo, we used a CS-induced rat model. After treatment with CS with or without MAPK inhibitors for 90 days, lung tissues were harvested. p-ERK, p-p38 and p-JNK protein levels in cells and lung tissue were measured using enzyme-linked immunosorbent assays, mRNA- and protein-expression profiles of FoxA2, E-cadherin, CD44, and ZO1 were measured using quantitative real-time polymerase chain reaction and Western blotting, respectively, and morphological changes in bronchial epithelial cells were observed using lung-tissue staining.

Results: In both the in vitro and in vivo studies, phosphorylation of the ERK1/2, JNK, and p38 proteins was significantly increased (P<0.05) and mRNA and protein expression of E-cadherin and FoxA2 significantly decreased (P<0.05) compared with the control group. ERK, JNK, and p38 inhibitors reversed the CS-extract-induced changes in E-cadherin, CD44, and ZO1 mRNA and protein expression (P<0.05), decreased p-ERK, p-p38, and p-JNK protein levels in cells and lung tissue, suppressed bronchial epithelial hyperplasia and local squamous metaplasia, and decreased FoxA2 expression.

Conclusion: MAPK and FoxA2 mediate CS-induced squamous metaplasia. MAPK inhibitors upregulate FoxA2, resulting in a reduction in the degree of squamous metaplasia.

Keywords: FoxA2; MAPK; bronchial epithelial cell; cigarette smoke; squamous metaplasia.

Figure 1

Phosphorylated ERK1/2 (A), JNK (B), and p38 (C) in CSE-treated bronchial epithelial cells. Notes: BEAS2B cells were treated with 1% CSE for 1 week with or without ERK, JNK, or p38 inhibitors. Results presented as means ± SEM (n=40). *P<0.05 compared with control (C) group; ^#P<0.05 compared with CSE group. Abbreviations: CSE, cigarette-smoke extract; RFU, relative fluorescence unit; SEM, standard error of the mean.

Figure 2

mRNA expression of epithelial cell-differentiation markers E-cadherin (A), CD44 (B), and ZO1 (C) in CSE-treated bronchial epithelial cells. Notes: BEAS2B cells were treated with 1% CSE for 1 week in the presence or absence of ERK inhibitor, JNK inhibitor, or p38 inhibitor. Results presented as means ± SEM (n=4). *P<0.05 compared with control (C) group; ^#P<0.05 compared with CSE group. Abbreviations: CSE, cigarette-smoke extract; SEM, standard error of the mean.

Figure 3

Protein expression of epithelial cell-differentiation markers E-cadherin, CD44, and ZO1 in CSE-treated bronchial epithelial cells. Notes: BEAS2B cells were treated with 1% CSE for 1 week in the presence or absence of ERK inhibitor, JNK inhibitor, or p38 inhibitor. (A) Representative Western blot; (B) quantitative analysis of E-cadherin; (C) quantitative analysis of CD44; (D) quantitative analysis of ZO1. Results presented as means ± SEM (n=4). *P<0.05 compared with control (C) group; ^#P<0.05 compared with CSE group. Abbreviations: CSE, cigarette-smoke extract; SEM, standard error of the mean.

Figure 4

Representative micrography of lung tissue obtained from rats exposed to the CS-induced rat model. Notes: CS induced mild hyperplasia, increased the number of lymphoid follicles in the bronchial epithelial layer, and caused lymphocyte infiltration of the bronchial wall and local squamous metaplasia. Treatment with an ERK1/2 inhibitor, JNK inhibitor, or p38 inhibitor decreased airway inflammation and reversed squamous metaplasia. Abbreviation: CS, cigarette smoke.

Figure 5

Phosphorylation levels of ERK1/2 (A), JNK (B), and p38 (C) in lung tissue of rats exposed to CS. Notes: Results presented as means ± SEM (n=4). *P<0.05 compared with control (C) group. Abbreviations: CS, cigarette smoke; SEM, standard error of the mean; RFU, relative fluorescence unit.

Figure 6

E-cadherin expression at the protein (A) and mRNA (B) levels in lung tissue of rats exposed to the rat smoking model. Notes: Results presented as means ± SEM (n=4). *P<0.05 compared with control (C) group; ^#P<0.05 compared with CS group. Abbreviations: CS, cigarette smoke; SEM, standard error of the mean.

Figure 7

FoxA2 expression at the protein (A) and mRNA (B) levels in CS-treated bronchial epithelial cells. Notes: BEAS2B cells were treated with 1% CSE for 1 week in the presence or absence of ERK inhibitor, JNK inhibitor, or p38 inhibitor. Results presented as means ± SEM (n=4). *P<0.05 compared with control (C) group; ^#P<0.05 compared with CSE group. Abbreviations: CSE, cigarette-smoke extract; SEM, standard error of the mean.

Figure 8

FoxA2 expression at the protein (A) and mRNA (B) levels in the rat smoking model. Notes: Results presented as means ± SEM (n=4). *P<0.05 compared with control (C) group; ^#P<0.05 compared with CS group. Abbreviations: CS, cigarette smoke; SEM, standard error of the mean.

Figure 9

Effects of MAPK pathway in CS-induced epithelial metaplasia. Abbreviation: CS, cigarette smoke.