Cholesterol is an essential plasma membrane component, and altered cholesterol metabolism has been linked to cholesterol accumulation in the airways of COPD and cystic fibrosis patients. However, its role in airway epithelial differentiation is not well understood. Tandem mass spectrometry-based proteomic analysis of differentiating primary human bronchial epithelial cells (phBECs) revealed an overall inhibition of the cholesterol biosynthesis pathway. We hypothesized that excess cholesterol impairs the differentiation of phBECs into a fully functional bronchial epithelium. PhBECs were differentiated in the presence of 80 µM cholesterol for 21 days, the main airway cell type populations monitored using qRT-PCR and immunofluorescent stainings, and epithelial barrier integrity was analyzed via transepithelial electrical resistance measurements. Chronic cholesterol exposure led to a significant increase in CC10(+) secretory cells at the expense of ciliated cells. Pathway enrichment analysis suggested the tumor protein p53 as a master regulator of genes during normal differentiation of phBECs. Chronic cholesterol exposure drastically impaired the nuclear translocation of p53. Our findings suggest that this inhibition underlies the cholesterol-induced expansion of CC10(+) secretory cell populations at the expense of ciliated cells. In conclusion, we identify cholesterol as an important regulator of normal bronchial epithelial cell differentiation through inhibition of p53 nuclear translocation.
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