Study of the common activating mechanism of apoptosis and epithelial-to-mesenchymal transition in alveolar type II epithelial cells
Introduction
Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) may result from infection and severe trauma including sepsis, pneumonia, acute pancreatitis, aspiration of gastric contents or injurious ventilation (Mokra and Kosutova, 2015; Ye et al., 2012; Zhang et al., 2019). Although knowledge about the mechanisms leading to ALI/ARDS has increased, no specific and successful treatment options exist to date; thus, the mortality rate remains high in patients with ALI/ARDS (Mart and Ware, 2020).
Apoptosis of alveolar epithelial cells is one of the main mechanisms of ALI/ARDS (Martin et al., 2003). Apoptosis of alveolar epithelial cells is detectable in mice with ALI/ARDS induced by lipopolysaccharide (Abadie et al., 2005; Fujita et al., 1998). Fas and FasL (an important death receptor-mediated extrinsic pathway of apoptosis) are increased in pulmonary odema fluid and in lung tissue of patients with ARDS (Albertine et al., 2002). Alveolar type II epithelial cells (AEC II) are an important component of lung epithelial cells; as a kind of stem cell, they can proliferate and differentiate into alveolar type I epithelial cells (AEC I), thus contributing to lung epithelial repair. In addition, they synthesize and secrete all components of surfactant, which regulates alveolar surface tension in the lungs. Moreover, AEC II play an active role in enhancing alveolar fluid clearance and reducing lung inflammation (Matthay et al., 2019). Therefore, apoptosis of AEC II plays an important role in the early stage of ALI/ARDS.
Moreover, there is a coordinated and concurrent initiation of pulmonary fibrosis, which is characterized by excessive deposition of the extracellular matrix (ECM) and impaired lung function (Lederer and Martinez, 2018). Fibrosis has been reported in 53% of lung biopsy specimens in mechanically ventilated ARDS patients (Papazian et al., 2007). Fifty-five percent of ALI/ARDS patients died of severe pulmonary fibrosis, and some survivors also develop severe pulmonary fibrosis, leading to a marked decline in quality of life (Bardales et al., 1996). Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells gradually acquire the phenotype and morphology of mesenchymal cells or fibroblasts. EMT has been well studied as an underlying cause of lung fibrosis (Salton et al., 2019; Loffredo et al., 2017; Yin et al., 2020). An increasing number of studies have found that AEC II can transdifferentiate into fibroblasts and myofibroblasts, forming fibrotic tissue (Kim et al., 2006), which may play an important role in pulmonary fibrosis.
Therefore, both EMT and apoptosis of AEC II have been considered essential components in the pathogenesis of ALI/ARDS. Our study found that pyocyanin (PCN) could induce both apoptosis and EMT in AEC II, which corresponded to the clinical phenomenon that severe Pseudomonas aeruginosa pneumonia can cause both ALI/ARDS and pulmonary fibrosis. We conjectured that there may be a common activating mechanism between EMT and apoptosis of AEC II.
PCN, a redox-active, blue-green phenazine pigment secreted by P. aeruginosa, is essential for its virulent toxic effects in a wide range of host cells (Hall et al., 2016; Li et al., 2019; Moayedi et al., 2018). Sputum from patients with cystic fibrosis colonized by P. aeruginosa has been shown to contain PCN at concentrations up to 27.3 μg/mL, and there was an inverse correlation between sputum PCN concentration and lung function (Carlsson et al., 2011). Increased reactive oxygen species (ROS) formation is a contributing factor to the cytotoxicity displayed by PCN (Carlsson et al., 2011). We speculated that ROS may be the common upstream stimulus between EMT and apoptosis of AEC II. Many factors cause lung injury through ROS production, including severe infection, severe insult hyperoxia, and chemotherapy drugs. It is of great value to explore effective clinical prevention and treatment for ALI/ARDS based on this mechanism.
The aim of this study was to elucidate whether ROS is a common upstream stimulating factor that induces apoptosis and EMT in alveolar epithelial cells and to further explore the underlying molecular mechanism. This study may provide future strategies for the treatment of lung injury and fibrosis.
Section snippets
Cell culture
The human alveolar type II epithelial cell line (A549) was obtained from the Chinese Academy of Sciences Cell Bank. A549 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 (Thermo Fisher, Milan, Italy) supplemented with 10 % foetal bovine serum (Thermo Fisher, USA) and 1% penicillin/streptomycin (Thermo Fisher, USA) in an atmosphere of 5% CO2 at 37 °C.
ROS detection
Intracellular ROS generation was assessed by the fluorescent probe 2′,7′-dichlordehydrofluorescein-diacetate (DCFH-DA) (Molecular
PCN induces ROS generation in A549 cells
To test whether PCN is causally linked to oxidative stress as previously suggested, we examined ROS levels in A549 cells using a DCFH-DA probe. As presented in Fig. 1A and B, PCN considerably (p < 0.05) elevated ROS in PCN‐treated cells in a dose-dependent manner. However, when A549 cells were pretreated with NAC (5 mM) for 1 h and then incubated with PCN (25 μg/mL) for 24 h, ROS was significantly reduced in A549 cells pretreated with the specific antioxidant of ROS, NAC (p < 0.05) (Fig. 1A PCN
Discussions
The overproduction of ROS by injured endothelial/epithelial cells plays an important role in ALI/ARDS progression. ROS can regulate a variety of cellular functions as intracellular signalling molecules and further induce multiple cell signalling pathways. Our study showed that apoptosis and EMT can occur in A549 cells upon PCN stimulation. We also found that PCN exposure can induce ROS generation in A549 cells. However, when an antioxidant was applied, PCN could not induce apoptosis of A549
Conclusions
In summary, our study confirms that PCN can induce ROS in alveolar epithelial cells, which in turn causes cell apoptosis and EMT. On the one hand, ROS induces apoptosis through the mitochondrial pathway or endoplasmic reticulum pathway; on the other hand, ROS induces EMT through activation of the TGF-β/Smad signalling pathway. Meanwhile, apoptosis and EMT are significantly positively correlated. Therefore, ROS production is the common activating mechanism of apoptosis and EMT in alveolar
Declaration of Competing Interest
There are no conflicts of interest.
Acknowledgements
This work was supported by the Scientific Research Common Program of Beijing Municipal Commission of Education (KM201710025017). Our study design, data analysis, and paper writing were independent of the funding.
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Jiali Wang and Tianjiao Xue contributed equally to this work.