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

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.

Figure 1

Effect of tobacco smoke on MCC parameters in vitro. (a) Graph showing the experimental design. Abbreviations are: ASL (airway surface liquid), UC (Ussing chamber). (b) Upper left: representative traces of 10 µM CFTR_inh172-induced short circuit current (Isc) changes after 10 µM forskolin stimulation of HBECs exposed to 10 µM amiloride. Tobacco smoke exposure of fully differentiated HBECs via the VC-10 smoking robot using 24 puffs of a total of 4 cigarettes (Kentucky 3R4F) reduced CFTR conductance 1 h after cigarette smoke exposure. Middle and upper right: Representation of CFTR channel activities of cells from smokers and non-smokers exposed to the air (middle) and smoke (right). Lower left: time course of CFTR activity after smoke or air exposure. 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 smoke-exposed cells – control cells exposed to air. (c) Upper left: Representative traces of ATP-induced (10 µM) short circuit current (Isc) changes in basolaterally permeabilized HBECs cells exposed to a basolateral-to apical K⁺ gradient in the presence of 10 µM amiloride. Middle and upper right: Representation of BK channel activities of cells from smokers and non-smokers exposed to air (middle) and smoke (right). 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). Lower right: BK activity decrease is represented by the channel activity difference of smoke-exposed cells compared to air-exposed cells (ΔBK activity): ΔSmoke = BK activity of smoke-exposed cells – cells exposed to the air. (d) Smoke reduces ASL volume 2 h after exposure (meniscus scanning method) but (e) no change in transepithelial resistance (TER) is seen. All n ≥ 4 from ≥ 3 different lung donors. * indicates p < 0.05 compared to air exposure at the same time point.

Figure 2

Smoke induces Smad3 as well as p38 and HSP27 phosphorylation. (a) Western blot quantification of Smad3 phosphorylation showed an >2-fold increase 1 h after smoke exposure, an about 3-fold at 2 h and 4 h as well as 2-fold increase at 6 h. (n ≥ 20 from 12 lungs). Below each quantification, a representative western blot is shown and below the blot, single data points of cells from smokers and non-smokers. (b) p38 phosphorylation was increased 16.3-fold upon smoke exposure within 1 h, followed by a decrease to ~3.8 fold at 6 h (n ≥ 10 from 10 lungs). Below each quantification, a representative western blot is shown and below the blot, single data points of cells from smokers and non-smokers. (c) HSP27 phosphorylation (n = 4 from 4 lungs) was increased within 1 h to ~4.5 fold and to ~2.5 fold at 6 h after cigarette smoke exposure. Below each quantification, a representative western blot is shown and below the blot, single data points of cells from smokers and non-smokers. All: Ratios were quantified for the appropriate phosphorylated protein as a fraction of total protein, corrected for GAPDH and normalized to air control. *Indicates p < 0.05 compared to control (6 h air exposure).

Figure 3

Changes in phosphorylation patterns and channel functions after smoke exposure in the presence of Smad3 and p38 signalling inhibitors. (a) Schematic diagram of the TGF-β pathway activated by smoke exposure with subsequent phosphorylation of Smad3 and p38/HSP-27. Appropriate inhibitors of different pathways are shown. (b) Smad3 phosphorylation was inhibited by SIS3 (3 µM) 2 h after smoke exposure, while SIS3 had no effect on HSP-27 phosphorylation. SB203580 (10 µM) had not effect on Smad3 phosphorylation but reduced HSP-27 phosphorylation 1 and 2 h after cigarette smoke exposure. Below each quantification, a representative western blot is shown. Abbreviations are: S (Smoke) and SB (SB203580). (c) SIS3 ameliorated decreases in CFTR conductance and ASL volume at 2 h after smoke exposure but had no effect on BK activity at any time point. (d) SB203580 did not rescue CFTR function at any time point, but BK activity was less decreased from 1 to 6 h after smoke exposure. ASL volume also did not decrease at 4 and 6 h. Finally, the importance of BK channels for ASL rescue was shown in CF cells 4 h after smoke exposure (far right). 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 (air exposure). All n ≥ 4 from at least 3 lungs, except for the CF cells (duplicates measured in duplicates from one lung).

Figure 4

LY2157299 modulates TGF-β signalling after smoke exposure by decreasing Smad3 and p38 activation, thereby preserving CFTR and BK activities as well as ASL volume. (a) Schematic diagram showing LY2157299 (10 µM) effects on the TGF-β pathway (p38/HSP-27 and Smad3), activated by smoke exposure. (b) LY2157299 prevented smoke-induced Smad3 and HSP-27 phosphorylation at 2 h when compared to cells exposed to cigarette smoke alone. Below each quantification, a representative western blot is shown. Abbreviations are: S (Smoke) and LY (LY2157299). (c) LY2157299 improved CFTR and BK conductance at 2 h as well as ASL volume loss at 4 h after smoke exposure. ΔCFTR and ΔBK activity represent the difference of smoke exposed and air exposed cells, ΔSmoke + DMSO = Smoke + DMSO-exposed cells - average of control cells exposed to air + DMSO; ΔSmoke + LY = Smoke + LY2157299 - average of control cells exposed to air + LY. *Indicates p < 0.05 compared to control (6 h air exposure). All n ≥ 4 from at least 3 lungs.

Figure 5

Pirfenidone transiently reduces smoke-induced p38 signalling, thereby ameliorating BK dysfunction and ASL volume loss. (a) Pirfenidone (1 mg/mL) inhibits smoke-mediated activation of p38 signalling. (b) Protein analysis using western blots showed no reduction of smoke-activated Smad3 phosphorylation by pirfenidone but a reduction of HSP27 phosphorylation 1 h after smoke exposure. Below each quantification, a representative western  blot is shown. Abbreviations are: S (Smoke) and Pirf (Pirfenidone). (c) Ussing chamber data showed that smoke-induced reduction in CFTR activity was not rescued by pirfenidone, but BK activity was temporarily improved at 1 and 2 h after cigarette smoke exposure. Pirfenidone also ameliorated smoke-induced ASL volume loss at 2, 4 and 6 h after exposure. ΔCFTR and ΔBK activity represent the difference of smoke-exposed compared to air exposed cells; ΔSmoke = Smoke-exposed cells - average of control cells exposed to air; ΔSmoke + Pirf = Smoke + Pirf - average of control cells exposed to air + Pirf. *Indicates p < 0.05 compared to control (6 h air exposure). All n ≥ 4 from at least 3 lungs.

Figure 6

Schematic diagram of smoke effects on CFTR and BK activities as well as ASL volume in the absence or presence of different inhibitors. Smoke (black arrow), stimulates both Smad3 and p38 phosphorylation via TGF-β signalling, which in turn decreases CFTR and BK channel functions. Overall this causes ASL volume loss, resulting in mucociliary dysfunction. Inhibitors (dotted arrow) used for the presented experiments ameliorated CFTR and BK activities and thereby improved ASL volume loss.