A Small Molecule BH3-mimetic Suppresses Cigarette Smoke-Induced Mucous Expression in Airway Epithelial Cells

Abstract

Cigarette smoke (CS) exposure is one of the primary risk factors associated with the chronic mucous hypersecretion (CMH). The antiapoptotic protein, Bcl-2 sustains hyperplastic mucous cells, and the airway epithelium of ex-smokers with CMH as well as mice exposed to chronic CS showed increased Bcl-2 expression. Therefore, we investigated whether Bcl-2 plays a role in CS-induced mucous expression. Primary airway epithelial cells (AECs) of murine and human origin were treated with CS extract (CSE), and there was a concentration- and time-dependent increase in secretory mucin (MUC5AC), mucous regulator (SPDEF) and Bcl-2 expression. Using differentiated human AECs cultured on air-liquid interface, EGFR and ERK1/2 pathways were interrogated. Bcl-2 activity was blocked using a small molecule BH3 mimetic ABT-263 that disrupts the Bcl-2 interaction with pro-apoptotic proteins. The ABT-263 treatment resulted in the downregulation of CSE-induced mucus expression and disrupted the EGFR-signaling while inducing the apoptosis and the pro-apoptotic protein, Bik expression. This strategy significantly suppressed the mainstream CS-induced mucous phenotype in a 3-D human airway epithelium model. Therefore, the present study suggests that CS induces Bcl-2 expression to help promote mucous cell survival; and small molecule BH3 mimetics targeting Bcl-2 could be useful in suppressing the CS-induced mucous response.

Figure 1

CS exposure induces mucous phenotype and Bcl-2 levels in murine airway epithelial cells (AECs). Primary murine AECs were treated with cigarette smoke extract (CSE) at 0, 1, 10, and 100 µg/ml for 24 h and the mRNA levels of Muc5ac (A) Spdef (B) and Bcl-2 (C) were analyzed by qRT-PCR. Murine AECs treated with 10 µg/ml CSE and cells were harvested at 0, 3, 24, 48 and 72 h and mRNA levels of Muc5ac (D) Spdef (E) and Bcl-2 (F) were quantified. (G) Representative micrographs showing Muc5ac (red) and Spdef (green) expression in murine AECs following CSE treatment in comparison with non-treated (NT) cells. Murine AECs were treated with CSE (10 µg/ml) for 48 h and immunostained for Muc5ac and Spdef, and the nuclei (blue) were stained with DAPI (Scale – 5 µ). (H) Representative micrographs showing Muc5ac (red) and Bcl-2 (green) expression in CSE-treated and treated (NT) murine AECs, nuclei were stained with DAPI (Scale – 5 µ). (I) Quantification of Muc5ac, Spdef and Bcl-2 immunopositive cells following CSE exposure. Approximately 300 cells from each treatment were analyzed to calculate the percentage of Muc5ac-positive (Muc5ac⁺), Spdef-positive (Spdef⁺) and Bcl-2-positive (Bcl-2⁺) cells. Data shown as mean ± SEM (n ≥ 3); *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

CS exposure induces MUC5AC, SPDEF, and Bcl-2 levels in human AEC monolayers. Primary human AECs grown in submerged cultured conditions were treated with 0, 1, 10, and 100 µg/ml of CSE for 24 h and the mRNA levels of MUC5AC (A) SPDEF (B) and Bcl-2 (C) were analyzed by qRT-PCR. (D) Immunoblot analyses of Bcl-2 expression following CSE exposure. Differentiated human AECs were treated with 10 µg/ml CSE and cell lysates were analyzed at 0, 24 and 48 h post CSE exposure by western blot analysis for Bcl-2 protein levels with β-actin levels detected as the loading controls. (E) Relative quantities of Bcl-2 protein as determined by densitometric analysis and normalized to β-actin levels. (F) Representative micrographs showing MUC5AC (green) and Bcl-2 (red) immunopositivity in HAECs treated with CSE or left non-treated (NT). Human AECs were treated with CSE (10 µg/ml) for 48 h and immunostained for Bcl-2 and MUC5AC, and nuclei were stained with DAPI (Scale – 5 µ). (G) Quantification of human AECs immunopositive for MUC5AC and Bcl-2 following CSE exposure. The percentage of human AECs immunopositive for Bcl-2 (Bcl-2⁺) and MUC5AC (MUC5AC⁺) were calculated. Data shown as mean ± SEM (n ≥ 3); *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

CSE engages EGFR and ERK1/2 pathways to induce mucous phenotype and Bcl-2 levels in differentiated human AECs. Primary human AECs differentiated for 3 weeks on air-liquid interface were treated with 10 µg/ml of CSE for 48 h and the mRNA levels of MUC5AC (A), SPDEF (B) and Bcl-2 (C) were analyzed by qRT-PCR. (D) A 3-D representation of a micrograph showing cilia (green) and MUC5AC (red) immunopositivity in differentiated human AECs treated with CSE. Differentiated human AECs were treated with 10 µg/ml CSE for 48 h and were immunostained for acetylated-tubulin (ACT, in green) for cilia and MUC5AC (red), and nuclei were stained with DAPI (Scale – 5 µ). (E) Quantification of MUC5AC immunopositive cells following CSE exposure. Approximately 300 cells from each treatment were analyzed to calculate the percentage of MUC5AC-positive (MUC5AC⁺) cells. (F) Immunoblot analyses of EGFR and ERK1/2 signaling pathway following CSE exposure. Differentiated human AECs were treated with 10 µg/ml CSE and cell lysates were analyzed at 0, 24 and 48 h post CSE exposure by western blot analysis for phosphorylated ERK1/2, total ERK1/2, phosphorylated EGFR and total EGFR with β-actin levels as the loading controls. (G) Relative quantities of pERK1/2, ERK1/2, pEGFR and EGFR as determined by densitometric analysis where protein quantities were normalized to β-actin levels. (H) Fold-change in EGFR mRNA levels in human AECs treated with 10 µg/ml of CSE for 48 h. Data shown as mean ± SEM (n = 3); *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

Blocking Bcl-2 with ABT-263 suppresses the CSE-induced mucous phenotype by suppressing EGFR signaling and inducing apoptosis and Bik expression. ABT-263 treatment suppresses CSE-induced MUC5AC (A), SPDEF (B) and EGFR (C) mRNA levels. Differentiated human AECs were treated with 1 µM ABT-263 for 2 h before treating with 10 µg/ml CSE and cells were harvested at 48 h post treatment. (D) Immunoblot analyses of pERK1/2, ERK1/2, pEGFR and EGFR following CSE exposure and ABT-263 treatment of differentiated human AECs. ALI-differentiated human AECs were treated with ABT-263 (1 µM) for 2 h before treating with 10 µg/ml CSE and cells were harvested at 48 h post treatment. (E) Relative quantities of pERK1/2, ERK1/2, pEGFR and EGFR as determined by densitometric analysis with protein quantities normalized to β-actin levels. (F) ABT-263 treatment augments apoptosis in CSE-exposed human AECs. Differentiated human AECs treated with ABT-263, CSE and CSE + ABT were harvested at 48 h post treatment, and analyzed for Annexin V-FITC and propidium iodide staining by Flow cytometry. (G) ABT-263 treatment induces the proapoptotic Bik mRNA levels that are suppressed by CSE exposure. (H) ABT-263 treatment of differentiated murine AECs suppresses the CSE-induced mucous secretory phenotype by modulating cell survival/death pathways. Primary murine AECs differentiated for 3 weeks on ALI were treated with ABT-263 (1 µM) and/or CSE (10 µg/ml) and the mRNA levels of Muc5ac, FoxA3, Egfr and Bik were analyzed by qRT-PCR. Data shown as mean ± SEM (n ≥ 3); *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

BH3 mimetic ABT-263 blocks the CS-induced mucous phenotype in 3-D human airway tissue model via inducing apoptosis and Bik expression. 3-D tissue cultures of human airways were exposed to CS using a SCIREQ smoke machine (Montreal, QC, Canada) for 3 consecutive days and one group was treated with 1 µM ABT-263 2 h before exposures. ABT-263 treatment inhibited the CS-induced MUC5AC (A) and SPDEF (B) mRNA expression. (C) Representative micrographs showing cilia (green) and MUC5AC (red) immunopositivity in 3-D airway tissue culture treated with CS and/or ABT-263 compared to non-treated (NT) ones. 3-D airway tissues were immunostained for acetylated-tubulin (ACT, in green) for cilia and MUC5AC (red), and nuclei were stained with DAPI (Scale – 5 µ). Quantification of MUC5AC + mucous cells (D) and ACT + ciliated cells (E) where more than 300 cells from each treatment were analyzed. (F) ABT-263 treatment results in increased TUNEL-positivity in CSE-exposed human AECs. Human AECs treated with ABT-263 (1 µM) and/or CSE (10 µg/ml) were stained for TUNEL (green) and MUC5AC (red). (G) Quantification of human AECs immunopositive for MUC5AC (MUC5AC⁺) or TUNEL (TUNEL⁺) or both (MUC5AC⁺/TUNEL⁺) following ABT-263 and CSE treatment. (H) ABT-263 increases Bik expression and caspase 3 activation in CSE-exposed AECs. Human AECs were immunostained for Bik (red) and cleaved caspase 3 (CC3, shown in green). (I) Quantification of human AECs immunopositive for Bik (Bik⁺) or CC3 (CC3⁺) or both (Bik⁺/CC3⁺) following ABT-263 and CSE treatment. Data shown as mean ± SEM (n ≥ 3); *p < 0.05; **p < 0.01; ***p < 0.001.