Abstract
Excessive secretion of airway mucus and fluid accumulation are the common features of many respiratory diseases, which, in turn, induce cell hypoxia in the airway epithelium, resulting in epithelial–mesenchymal transition (EMT) and ultimately fibrosis. However, the mechanisms of EMT induced by hypoxia in the airway are currently unclear. To mimic the status of edematous fluid retention in the airway, we cultured primary mouse tracheal epithelial cells (MTECs) in a liquid–liquid interface (LLI) mode after full differentiation in a classic air–liquid interface (ALI) culture system. The cell hypoxia was verified by the physical characteristics and lactate production in cultured medium as well as HIF expression in MTECs cultured by LLI mode. EMT was evidenced and mainly mediated by basal cells, supported by flow cytometry and immunofluorescence assay. The differently expressed genes of basal and other airway epithelial cells were found to be enriched in the ribosome by our analysis of an MTEC single-cell RNA sequencing data set and Myc, the global regulator of ribosome biogenesis was identified to be highly expressed in basal cells. We next separated basal cells from bulk MTECs by flow cytometry, and the real-time PCR results showed that ribosome biogenesis was significantly upregulated in basal cells, whereas the inhibition of ribosome biogenesis alleviated the phosphorylation of the mammalian target of rapamycin/AKT and abrogated hypoxia-induced EMT in MTECs. Collectively, these observations strongly suggest that basal cells in the airway epithelium may mediate the process of hypoxia-induced EMT, partly through enhancing ribosome biogenesis.
Highlights
Oxygen is essential for human survival, deficiency in the supplement of which may lead to the injury of cell, tissue, and organ, even organism death
We explore the mechanism of the hypoxia-induced epithelial–mesenchymal transition (EMT) process by analyzing the single-cell sequencing data set of mouse tracheal epithelial cells (MTECs) and culturing them in a liquid–liquid interface (LLI) mode to mimic the pathological status of respiratory diseases
To protect the airway from pathogens, the thin surface liquid layer lining on the mammalian epithelium of the airway is necessary for cilium beating and contaminant clearance, which exists in MTECs
Summary
Oxygen is essential for human survival, deficiency in the supplement of which may lead to the injury of cell, tissue, and organ, even organism death. Lowering the oxygen level in a CO2 incubator or chamber is the optimal method to induce hypoxia, which has been widely used in in vitro and in vivo experiments as an important physical method for the decrease of oxygen concentration (Polosukhin et al, 2011; Yee et al, 2016; Chen et al, 2020). As a replacement method for the abovementioned hypoxia incubator in our experiment, excessive culture medium can mimic the retention of fluid in the airway under pathological conditions, which generates a hypoxic condition for cells by increasing the thickness (Δx) of the medium and decreasing the diffusion velocity (F) of oxygen (Gerovac et al, 2014)
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