The present studies show that CS inhibits expression of Bik and thereby increases airway epithelial cell hyperplasia in patients with chronic bronchitis. Restoring Bik expression using adenoviral expression systems together with CS-activated ERK1/2 caused death of epithelial cells to reduce ECH.
Bik remained suppressed in the lungs of mice even 60 days after recovery, suggesting that the miRNA or the RNase that is activated by CS remains intact once induced even after cessation of CS exposure. However, Bik expression was suppressed not only in the biopsy and autopsy lung tissues of cigarette smokers compared with nonsmokers but also in lung tissues of former smokers with chronic bronchitis compared with former smokers without. These findings suggest that not only are Bik levels reduced by cigarette smoking, but the level of reduction is driven by factors that may be directly associated with susceptibility to developing chronic bronchitis. It appears that Bik is suppressed by CS more drastically in former smokers with chronic bronchitis, and this reduction allows increased number of mucous cells to be sustained compared with former smokers without chronic bronchitis. It is possible that airway cells in people who are genetically predisposed to chronic bronchitis either have reduced baseline Bik levels or, once exposed to CS, permanently and more efficiently degrade Bik mRNA compared with smokers without chronic bronchitis. Therefore, Bik may be a useful biomarker that predicts susceptibility to chronic bronchitis, and identification of the factors that differentially regulate Bik expression in subjects with chronic bronchitis may help elucidate the susceptibility factors that cause permanent changes in former smokers with chronic bronchitis.
Interestingly, suppression of Bik expression was associated with significant increases in MUC5AC mRNA levels both in human primary HAECs and in the lungs of mice exposed to CS. Although mucin gene expression and MCM can also occur without epithelial cell proliferation, as shown in cells overexpressing the SAM pointed domain-containing ETS transcription factor (SPDEF) (38
), the present studies demonstrate that in mice, MCM is also associated with an increased number of epithelial cells per millimeter of basal lamina compared with those exposed to filtered air as control. These findings suggest that the reduction of this proapoptotic protein allows sustained presence of hyperplastic epithelial cells that synthesize and secrete mucus. The fact that Bik knockout mice show normal epithelial cell numbers when not exposed to CS suggests that the regulation of epithelial cell numbers in normal development is regulated by other mechanisms. However, our findings suggest that once injury occurs, Bik is crucial for the resolution of hyperplastic epithelial cells. Furthermore, these findings are consistent with previous reports that smokers with chronic bronchitis and chronic airflow obstruction have increased levels of MUC5AC mRNA compared with nonsmoking control subjects (40
The fact that CS-induced MCM and ECH in differentiated primary HAEC cultures and in vivo
mice were reduced when Bik expression was restored suggests that Bik must be inducing death of mucin-producing cells. Our previous studies showed susceptibility of hyperplastic rather than resting nonproliferating cells to Bik-induced cell death (23
). Previous studies showed that proliferating basal cells give rise to a subpopulation of mucous cells that retain the ability to divide, whereas others lose this proliferative phenotype and become fully differentiated (41
). When proliferating cells were arrested in mitosis with colchicines after mechanical injury of the airway epithelium in hamsters, a larger proportion of secretory cells compared with basal cells were found to be in metaphase (42
). Therefore, susceptibility of only proliferating AECs to death signals may ensure that only hyperplastic AECs are removed during the resolution process without damaging the barrier function of the airway epithelium. Our previous studies also suggest that hyperplastic cells in airways appear to be primarily mucus-producing cells (8
). Hence, restoring Bik expression may reduce hyperplastic epithelial cells, thereby reducing MCM. This selective targeting of hyperplastic cells restores the normal proportions of cell types in airways without destroying the integral barrier function and innate protective mechanism of the airway epithelium. Extensive examination of lung tissues failed to identify epithelial cells with classic apoptotic morphology. Current studies are developing new biomarkers that may be useful to identify the cells undergoing cell death and to characterize the nature and morphology of cell death occurring in vivo
The observation that Bik-induced cell death was enhanced by CS- or IGF-1–induced ERK1/2 activation and that blocking ERK1/2 activation using U0126 alleviated Bik-induced cell death suggests that ERK1/2 activation may be an integral functional part of Bik-induced cell death. The fact that the BH3 domain of Bik was required to inhibit nuclear localization of phospho-ERK1/2 in airway epithelial cells demonstrates that Bik is essential for cytosolic retention of phospho-ERK1/2. Collectively, these findings show that sustained activation of ERK1/2 in the cytosol enhances cell death. ERK activation has generally been associated with cell survival and proliferation (43
); however, a number of studies show that depending on the stimuli and cell types involved, activation of ERK can mediate cell death (reviewed by Mebratu and Tesfaigzi [44
In conclusion, our results provide a new paradigm for epithelial remodeling that should be useful for developing a rational basis for therapies aimed at reducing hyperplastic AECs that are involved in mucous hypersecretion in chronic diseases. A strategy that uses an irreversible inhibitor of epidermal growth factor receptor tyrosine kinase activity that showed efficacy in preventing epithelial hyperplasia in a model of intestinal neoplasia (45
) is being developed. In that setting, the pharmacologic strategy was aimed at inhibiting epithelial proliferation, but our approach to restore the proapoptotic effect of Bik may prove to be more effective. These studies lay the foundation to investigate peptides derived from Bik that can be used to reduce the number of mucous cells, mucous hypersecretion, and phlegm production and ameliorate the symptoms of chronic bronchitis.