Abstract
The aim of this study is to investigate the molecular mechanisms underlying delayed progressive pulmonary fibrosis, a characteristic of subacute paraquat (PQ) poisoning. Epithelial-mesenchymal transition (EMT) has been proposed as a cause of organ fibrosis, and transforming growth factor-β (TGF-β) is suggested to be a powerful mediator of EMT. We thus examined the possibility that EMT is involved in pulmonary fibrosis during PQ poisoning using A549 human alveolar epithelial cells in vitro. The cells were treated with various concentrations of PQ (0–500 μM) for 2–12 days. Short-term (2 days) high-dose (>100 μM) treatments with PQ induced cell death accompanied by the activation of caspase9 as well as a decrease in E-cadherin (an epithelial cell marker), suggesting apoptotic cell death with the features of anoikis (cell death due to the loss of cell-cell adhesion). In contrast, long-term (6–12 days) low-dose (30 μM) treatments with PQ resulted in a transformation into spindle-shaped mesenchymal-like cells with a decrease of E-cadherin as well as an increase of α-smooth muscle actin (α-SMA). The mesenchymal-like cells also secreted the extracellular matrix (ECM) protein fibronectin into the culture medium. The administration of a TGF-β1 receptor antagonist, SB431542, almost completely attenuated the mesenchymal transformation as well as fibronectin secretion, suggesting a crucial role of TGF-β1 in EMT-like cellular response and subsequent fibrogenesis. It is noteworthy that despite the suppression of EMT-fibrogenesis, apoptotic death was observed in cells treated with PQ+SB431542. EMT-like cellular response and subsequent fibrogenesis were also observed in normal human bronchial epithelial (NHBE) cells exposed to PQ in a TGF-β1-dependent manner. Taken together, our experimental model reflects well the etiology of PQ poisoning in human and shows the involvement of EMT-like cellular response in both fibrogenesis and resistance to cell death during subacute PQ poisoning of pulmonary epithelial cells.
Highlights
Paraquat (PQ, 1,1'-dimethyl-4,4'-bipyridinium) is a highly toxic compound, and its use is rigorously restricted in many industrialized countries including the United States and the members of European Union, it is still widely used as a herbicide in many developing countries around the world
We hypothesized that Epithelial-mesenchymal transition (EMT) has important roles in both cell death and fibrogenesis in PQ-induced lung injury, and, we explored the relationships between PQ poisoning, EMT and cell death using A549 human alveolar epithelial carcinoma cells and NHBE normal human bronchial epithelial cells in vitro
Cytomorphology was observed under light microscopy: cells showing rounded morphology, aggregation, and flotation in the medium were observed after exposure to 300 or 500 μM PQ, suggesting the induction of cell death by high-dose and short-term exposure to PQ (Fig. 1A)
Summary
Paraquat (PQ, 1,1'-dimethyl-4,4'-bipyridinium) is a highly toxic compound, and its use is rigorously restricted in many industrialized countries including the United States and the members of European Union, it is still widely used as a herbicide in many developing countries around the world. PQ distributes in multiple tissues, it accumulates mainly in the lung and kidney. PQ accumulates at high levels in Clara cells, as well as in alveolar type I and II epithelial cells [1, 2]. The lung injury caused by PQ is characterized by epithelial cell destruction, followed by secondary alveolitis that is defined by pulmonary edema and inflammation. Delayed progressive pulmonary fibrosis is the most characteristic feature of subacute PQ poisoning, which occurs over a period from days to weeks after PQ ingestion [1]. PQ-induced pulmonary fibrosis is associated with high mortality, the molecular mechanism of its toxicity and effective antidotes are not established to date
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