Cigarette smoke exposure reveals a novel role for the MEK/ERK1/2 MAPK pathway in regulation of CFTR

Abstract

Background: Cystic fibrosis transmembrane conductance regulator plays a key role in maintenance of lung fluid homeostasis. Cigarette smoke decreases CFTR expression in the lung but neither the mechanisms leading to CFTR loss, nor potential ways to prevent its loss have been identified to date.

Methods: The molecular mechanisms leading to down-regulation of CFTR by cigarette smoke were determined using pharmacologic inhibitors and silencing ribonucleic acids (RNAs).

Results: Using human bronchial epithelial cells, here we show that cigarette smoke induces degradation of CFTR that is attenuated by lysosomal inhibitors, but not proteasome inhibitors. Cigarette smoke can activate multiple signaling pathways in airway epithelial cells, including the MEK/Erk1/2 MAPK (MEK: mitogen-activated protein kinase/ERK kinase Erk1/2: extracellular signal-regulated kinase 1/2 MAPK: Mitogen-activated protein kinase) pathway regulating cell survival. Interestingly, pharmacological inhibition of the MEK/Erk1/2 MAPK pathway prevented the loss of plasma membrane CFTR upon cigarette smoke exposure. Similarly, decreased expression of Erk1/2 using silencing RNAs prevented the suppression of CFTR protein by cigarette smoke. Conversely, specific inhibitors of the c-Jun N-terminal kinase (JNK) or p38 MAPK pathways had no effect on CFTR decrease after cigarette smoke exposure. In addition, inhibition of the MEK/Erk1/2 MAPK pathway prevented the reduction of the airway surface liquid observed upon cigarette smoke exposure of primary human airway epithelial cells. Finally, addition of the antioxidant N-acetylcysteine inhibited activation of Erk1/2 by cigarette smoke and precluded the cigarette smoke-induced decrease of CFTR.

Conclusions: These results show that the MEK/Erk1/2 MAPK pathway regulates plasma membrane CFTR in human airway cells.

General significance: The MEK/Erk1/2 MAPK pathway should be considered as a target for strategies to maintain/restore CFTR expression in the lung of smokers.

Keywords: Airway epithelial cell; CFTR; Cigarette smoke; MAPK pathway.

Figure 1. Effect of the lysosomal inhibitors leupeptin and chloroquine and the proteasomal inhibitor lactacystin on the expression of CFTR after exposure to cigarette smoke extract

16HBE14o- cells were treated with 10% cigarette smoke extract (CSE) with or without the lysosomal inhibitor leupeptin (LP, 50 µg/ml) or chloroquine (CQ, 10 µM), or the proteasome inhibitor lactacystin (LC, 5 µM) for 48 hrs. CFTR protein was detected by immunoblotting as described in Methods. CTRL, Control. N=4. *, p < 0.05; **, p < 0.001; NS, not significant.

Figure 2. Role of MAPK inhibitors on CFTR expression after cigarette smoke exposure

16HBE14o- cells were treated with 10% CSE with or without the MEK/Erk1/2 inhibitors UO126 (10 µM) or PD98059 (PD, 20 µM), the p38 inhibitor SB203580 (SB, 20 µM), the JNK inhibitor SP600125 (JNKi, 20 µM), or UO124 (10 µM) for 48 hrs. CFTR protein was detected by immunoblotting. CTRL, Control. N=4. *, p < 0.05; **, p < 0.001; NS, not significant.

Figure 3. Decreased expression of Erk1 and 2 prevents the CSE-induced suppression of CFTR

16HBE14o- cells were incubated with Erk1 and 2 siRNAs or control siRNA. Forty eight hours later 16BE14o- cells were incubated with 10% CSE for 24 hrs. CFTR and Erk 1 and 2 proteins were detected by immunoblotting. β-actin was detected to confirm equal loading between samples. CTRL, Control. N=4.*, p < 0.05; **, p < 0.001.

Figure 4. Inhibition of the MEK/Erk1/2 MAPK pathway prevents loss of plasma membrane CFTR after cigarette smoke exposure

(A) and (B) 16HBE14o- cells were treated with 10% Camel cigarette smoke extract (CSE) with or without UO126 (10 µM) for 48 hrs. CFTR expression (total (A) or plasma membrane (B)) was detected as described in Methods. CTRL, Control. N=4. (C) Primary human bronchial epithelial cells were pretreated with 10 µM UO126 and then exposed to air or cigarette smoke (CS) as described in Methods section. ASL was measured at the indicated time. N=6 from two normal donors. *, p < 0.05; **, p < 0.001

Figure 5. Effect of the E3 ligase c-Cbl on CFTR expression after exposure to CSE

16HBE14o- cells were transfected with c-Cbl or control siRNA for 48 hrs, followed by treatment with 10% Camel cigarette smoke extract (CSE) for 24 hrs. CFTR and c-Cbl were detected by immunoblotting. β-actin was detected to confirm equal loading between samples. CTRL, Control. N=4. *, p < 0.05; **, p < 0.001.

Figure 6. Effect of CSE on intracellular localization of CFTR

Representative confocal microscopic images of CFTR (Alexa488, green color) and LAMP-1 (Alexa594, red color)-stained cells. 16HBE14o- cells were treated with 10% CSE with or without the lysosomal inhibitor chloroquine (CQ, 20 µg/ml) and the MEK/Erk1/2 MAPK inhibitor UO126 (10 µM) for 24 hrs. Representative images showing the co-localization of CFTR and LAMP-1 are shown. CTRL: Control. Original magnification 630×.

Figure 7. The antioxidant N-Acetylcysteine (NAC) prevents suppression of CFTR upon cigarette smoke exposure

(A) 16HBE14o- cells were treated with 10% CSE with or without 0.5, 2, or 10 mM NAC. CFTR protein was detected by immunoblotting. N=4. *, p < 0.05; **, p < 0.001. (B) 16HBE14o- cells were treated with 10% CSE for the indicated times. PhosphoErk1/2 and total Erk1/2 were detected by immunoblotting. N=4. (C) 16HBE14o- cells were treated with 10% CSE with or without NAC (2 or 10 mM) for the indicated time. Phospho-Erk1/2 was detected by immunoblotting. Blots are representative of at least three experiments.