ABSTRACT
Silicosis, caused by the inhalation of crystalline silicon dioxide or silica, is one of the most severe occupational diseases. Persistent inflammation and progressive massive pulmonary fibrosis are the most common histological changes caused by silicosis. Association of epithelial-mesenchymal transition (EMT) of hyperplastic type II epithelial cells with the fibrotic events of pulmonary fibrosis has been suggested in in vitro silica-exposed cultured cell models, patients with idiopathic pulmonary fibrosis, and bleomycin-induced experimental models. Histological features of EMT, however, are not fully described in silicotic lungs in in vivo. The purpose of this study was to demonstrate EMT of hyperplastic type II epithelial cells in the developmental process of progressive massive pulmonary fibrosis in the lungs of rats exposed to silica. F344 female rats were intratracheally instilled with 20 mg of crystalline silica (Min-U-Sil-5), followed by sacrifice at 1, 3, 6, and 12 months after instillation. Fibrosis, characterized by the formation of silicotic nodules, progressive massive fibrosis, and diffuse interstitial fibrosis, was observed in the lungs of the treated rats; the effects of fibrosis intensified in a time-dependent manner. Hyperplasia of the type II epithelial cells, observed in the massive fibrotic lesions, dominated in the lungs of rats at 6 and 12 months after the treatment. Immunohistochemistry of the serial sections of the lung tissues demonstrated positive labeling for cytokeratin, vimentin, and α-smooth muscle actin in spindle cells close to the foci of hyperplasia of type II epithelial cells. Spindle cells, which exhibited features of both epithelial cells and fibroblasts, were also demonstrated with bundles of collagen fibers in the fibrotic lesions, using electron microscopy. Increased expression of TGF-ß was shown by Western blotting and immunohistochemistry in the lungs of the treated rats. These findings suggested that enhanced TGF-ß expression and EMT of hyperplastic type II epithelial cells are involved in the development process of progressive massive pulmonary fibrosis during silicosis.
ABSTRACT
In this study, we have characterized an essential gene ent1+ encoding fission yeast epsin, which is similar to mammalian and budding yeast endocytic protein epsins. The S. pombe Ent1p contains ENTH (epsin amino-terminal homology) domain at its amino terminus, two copies of a ubiquitin-interacting motif (UIM) immediately carboxyl-terminal to the ENTH domain, three NPF motifs in the carboxyl-terminal half, and the clathrin-binding motif at the carboxyl terminal. When repressed the expression of ent1+ gene, the conditional ent1 gene knockout cells showed a marked defect in internalization of fluorescent dyes, suggesting that Ent1p is essential for endocytosis. Changes in conserved amino acid residues within ENTH domain in ent1 mutant cells revealed temperature-sensitive defect in actin organization and cell morphology. The Ent1p bound PI(4,5)P2 and PI(3,5)P2 immobilized onto nitrocellulose in vitro and also weakly bound PI(3,4)P2, PI(3,4,5)P3, PI4P and PI5P. Surprisingly, the localization of Ent1p-GFP was not affected even in the its3-1 cells, in which the level of PI(4,5)P2 was severely reduced, suggesting that PI(4,5)P2 may not be essential for proper localization of Ent1p at endocytic sites. Our findings indicate that S. pombe Ent1p is an essential component in endocytic process, and involved in actin organization and cell morphogenesis.
