Gefitinib, an EGFR Tyrosine Kinase inhibitor, Prevents Smoke-Mediated Ciliated Airway Epithelial Cell Loss and Promotes Their Recovery

Abstract

Cigarette smoke exposure is a major health hazard. Ciliated cells in the epithelium of the airway play a critical role in protection against the noxious effects of inhaled cigarette smoke. Ciliated cell numbers are reduced in smokers which weakens host defense and leads to disease. The mechanisms for the loss of ciliated cells are not well understood. The effects of whole cigarette smoke exposure on human airway ciliated ciliated cells were examined using in vitro cultures of normal human bronchial epithelial cells and a Vitrocell® VC 10® Smoking Robot. These experiments showed that whole cigarette smoke causes the loss of differentiated ciliated cells and inhibits differentiation of ciliated cells from undifferentiated basal cells. Furthermore, treatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, Gefitinib, during smoke exposure prevents ciliated cell loss and promotes ciliated cell differentiation from basal cells. Finally, restoration of ciliated cells was inhibited after smoke exposure was ceased but was enhanced by Gefitinib treatment. These data suggest that inhibition of EGFR activity may provide therapeutic benefit for treating smoke related diseases.

Fig 1. Whole cigarette smoke exposure causes an acute reduction of FoxJ1 positive cells.

(A-D) Representative confocal micrographs of differentiated human airway epithelial cells treated with Air (A, C) or Smoke (B, D) stained for FoxJ1 (green), acetylated-tubulin (white) or nuclei (blue) (A, B). C and D represent the same microscopic fields as A and B respectively showing only FoxJ1 and nuclear staining. Scale bar = 36 μm (E) Quantification of FoxJ1 positive (i.e. ciliated cells) in Air and Smoke treated samples showing the mean and SEM from 3 independent lung donors. * P<0.05, two-tailed Student’s t test. (F, G) Duplex qRT-PCR MCIDAS mRNA relative to B2M mRNA (F) and FoxJ1 mRNA relative to GAPDH mRNA (G) in cells treated with Air (white bars) and Smoke (grey bars). The bars show the mean and SEM of RNA from 3 independent lung donors. P values calculated using two-tailed Student’s t test are shown above the columns.

Fig 2. Inhibition of EGFR signaling prevents WCS induced loss of ciliated cells.

(A–F), Representative confocal micrographs of differentiated human airway epithelial cells treated with Air (A, D), Smoke (B, E) or Smoke + 500 nM Gefitinib (C, F) stained for FoxJ1 (green), acetylated-tubulin (white) or nuclei (blue) (A, B, C). D, E and F represent the same microscopic fields as A, B and C, respectively showing only the FoxJ1 and nuclear staining. Scale bar = 25 μm G, Quantification of the percent FoxJ1 positive cells relative to the Air treated samples. N = 7 air and smoke, N = 5 smoke + Gefitinib; * P<0.05, one way ANOVA.

Fig 3. Whole cigarette smoke exposure induces phosphorylation of both EGFR and Erk1/2 that is inhibited by Gefitinib.

Differentiated NHBE cells were treated with air or WCS from 2 cigarettes with and without Gefitinib. After 24 h total cell protein lysates were prepared and equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes and probed with anti-phospho-EGFR (Tyr1068) (A) or anti-Phospho-Erk1/2 (Thr202/Tyr204) (C) antibodies and anti-β actin antibody as a loading control (A, C). Graphs of the quantification of the signals for phospho-EGFR (B) from 3 different lungs and for phospho-Erk1/2 from 5 different lungs (D) are shown. * P<0.05, one-way ANOVA.

Fig 4. Whole cigarette smoke exposure inhibits ciliated cell differentiation.

(A–F) Representative confocal micrographs of human airway epithelial cells treated with Air (A, B, C) or Smoke (D, E, F) for 14 d (A, D), 21 d (B, E) or 27 d (C, F) during differentiation using ALI conditions and stained for FoxJ1 (green), acetylated-tubulin (white) or nuclei (blue). Scale bar = 30 μm. (G) Quantification of FoxJ1 positive cells treated with Air (white bars) or Smoke (grey bars) for 27 d during differentiation in ALI conditions. N = 3 different lung donors, * P = 0.06, two-tailed Student’s t test. (H) Quantitation of the average number of nuclei / microscopic field in cells treated with Air (white bar) or WCS (grey bar) for 27 days during differentiation. N = 3 different lung donors, N.S., not significant, two-tailed Student’s t-test. (I) Duplex qRT-PCR of FoxJ1 mRNA relative to GAPDH mRNA in cells treated with Air or Smoke for 27 d during differentiation using ALI conditions. N = 8 different lung donors, * P<0.05, two-tailed Student’s t-test. (J) Duplex qRT-PCR of MCIDAS mRNA relative to B2M in human airway epithelial cells treated with Air (white bars) or Smoke (grey bars) for 27 d of differentiation in ALI conditions. N = 8 different lung donors, N.S., not significant, two-tailed Student’s t-test.

Fig 5. Inhibition of EGFR signaling restores ciliated cell differentiation during whole cigarette smoke exposure.

(A–F) Representative confocal micrographs of human airway epithelial cells after treatment with Air (A, D), Smoke (B, E) or Smoke + Geftinib (C, F) during 21 d of differentiation using ALI conditions, stained for FoxJ1 (green), acetylated-tubulin (white) or nuclei (blue) (A, B, C). Panels D, E and F represent the same microscopic fields as A, B and C, respectively showing only the FoxJ1 and nuclear staining. Scale bar = 30 μm (G) Quantification of the percent FoxJ1 positive cells after 21 d of differentiation using ALI conditions treated with Air, Smoke or Smoke + Gefitinib. N = 7 different lung donors, * P<0.05, one-way ANOVA.

Fig 6. Inhibition of EGFR signaling promotes ciliated cell restoration after smoke exposure.

(A–D) Representative confocal micrographs of WCS treated differentiated human airway epithelial cells treated without (A, B) or with (C, D) Gefitinib for 7 d (A, C), or 14 d (B, D) after WCS treatment was stopped and stained for FoxJ1 (green), acetylated-tubulin (white) or nuclei (blue). Scale bar = 30 μm. (E) Quantification of the percent FoxJ1 positive cells cultured 7 and 14 d without (white bars) or with Gefitinib (grey bars) after WCS treatment was stopped. N = 2 for 7 d, N = 4 for 14 d. * P<0.05, Two tailed Student’s t test.