Role of Smad3 and p38 Signalling in Cigarette Smoke-induced CFTR and BK dysfunction in Primary Human Bronchial Airway Epithelial Cells

Juliette Sailland,Andreas Schmid,John S Dennis,Astrid Grosche,Nathalie Baumlin,Matthias Salathe,Stefanie Krick

doi:10.1038/s41598-017-11038-x

Abstract

Mucociliary clearance (MCC) is a major airway host defence system that is impaired in patients with smoking-associated chronic bronchitis. This dysfunction is partially related to a decrease of airway surface liquid (ASL) volume that is in part regulated by apically expressed cystic fibrosis transmembrane conductance regulator (CFTR) and large-conductance, Ca2+-activated, and voltage dependent K+ (BK) channels. Here, data from human bronchial epithelial cells (HBEC) confirm that cigarette smoke not only downregulates CFTR activity but also inhibits BK channel function, thereby causing ASL depletion. Inhibition of signalling pathways involved in cigarette smoke-induced channel dysfunction reveals that CFTR activity is downregulated via Smad3 signalling whereas BK activity is decreased via the p38 cascade. In addition, pre-treatment with pirfenidone, a drug presently used to inhibit TGF-β signalling in idiopathic pulmonary fibrosis, ameliorated BK dysfunction and ASL volume loss. Taken together, our results highlight the importance of not only CFTR but also BK channel function in maintaining ASL homeostasis and emphasize the possibility that pirfenidone could be employed as a novel therapeutic regimen to help improve MCC in smoking-related chronic bronchitis.

Highlights

Lower right: Decreased CFTR activity is represented by the different channel activities of smoked cells and cells exposed to air (ΔCFTR activity): ΔSmoke = CFTR activity of smokeexposed cells – control cells exposed to air. (c) Upper left: Representative traces of ATP-induced (10 μM) short circuit current (Isc) changes in basolaterally permeabilized human bronchial epithelial cells (HBEC) cells exposed to a basolateral-to apical K+
ΔCFTR and ΔBK activity represent the difference of smoke exposed and air exposed cells; ΔSmoke = Smoke-exposed cells - average of control cells exposed to air; ΔSmoke + DMSO/SB/SIS3 = Smoke + DMSO/SB/SIS3 - average of control cells exposed to air + DMSO/SB/SIS3. *Indicates p < 0.05 compared to control
While we have shown that BK rescue in cystic fibrosis (CF) cells can ameliorate TGF-β1-induced airway surface liquid (ASL) volume loss, indicating the importance of BK channels, this is the first report to show that this is the case in HBECs and CF bronchial epithelial cells when exposed to cigarette smoke

Summary

Introduction

Lower left: Cigarette smoke exposure decreased BK activity within 1 h of exposure (ATP elicited Isc after basolateral permeabilization and a basolateral to apical K+ gradient). Exposure of a human airway epithelial cell line has been reported to regulate the loss of plasma membrane CFTR via MEK/ERK/MAPK but not via p3826. In cystic fibrosis (CF), TGF-β1 has been described to cause mucociliary dysfunction by reducing ASL volume via decreased apical BK channel activity[11] through a down-regulation of Leucine Rich Repeat Containing protein 26 (LRRC26), the γ subunit necessary for BK function in non-excitable tissues[30]. We tested the hypothesis that activation of canonical Smad[3] and p38 MAPK signalling by cigarette smoke differentially affects CFTR and BK channel functions and addressed whether either channel can uphold ASL volume homeostasis in HBECs

Methods

Results

Conclusion