Abstract Rationale Silicosis is an incurable and progressive fibrotic lung disease caused by inhalation of crystalline silica from materials like sand and rock, often related to occupational exposures. Although global incidence and mortality have declined, recent outbreaks have been reported in countries such as the United States. Inhaled silica microparticles can reach pulmonary alveoli, induce cytotoxicity and macrophage cell death leading to inflammatory responses. While alveolar macrophages are widely recognized as key players in silica-induced lung pathology, alveolar epithelial cell (AEC) injury and plasticity may also contribute to the pathogenesis of silicosis. In a previous study, we found that the interaction of endoplasmic reticulum (ER) stress with mammalian target of rapamycin (mTOR) signaling drives the aging phenotype and promotes the accumulation of transitional cells in stressed and injured alveolar epithelial type II (AT2) cells in fibrotic Grp78 KO mouse lungs and in human idiopathic pulmonary fibrosis (IPF) lungs. Since AT2 cells in silicosis lungs exhibit hypertrophy and epithelial-mesenchymal transition (EMT) features similar to those observed in IPF, we hypothesized that ER stress-mTOR signaling represents a common pathway promoting AEC plasticity in fibrotic diseases as in IPF. Methods We treated normal human lung slices or Grp78 KO precision cut lung slices (PCLS) (300 μm) ex vivo with two sizes of silicon dioxide (SD) that are reported to reach the alveoli, 5-20 nm and 0.5-10 μm (Sigma-Aldrich), for 3 days. We then performed Western blot analysis to examine the expression of markers for mesenchymal cells, transitional cells, basal cells, ER stress and mTOR signaling (Figure 1). Results In human normal lung slice ex vivo cultures (n = 2, Figure 1A), Western blot analysis revealed that treatment with both sizes of silica increased expression of mesenchymal markers, α-smooth muscle actin (α-SMA), vimentin and fibroblast specific protein 1 (FSP1)/S100A4, ER stress marker activated transcription factor 6 (Cl-ATF6) and mTOR effector eIF4E-BP1 phosphorylation as well as C/EBP homologous protein (CHOP), KRT5 and KRT8 compared to vehicle (V). Silica also increased expression of mesenchymal markers and activated ER stress and mTOR signaling compared to vehicle in Grp78 KO PCLS (n = 4-5), although changes in C/EBP homologous protein (CHOP) were equivocal (Figure 1B). Conclusions Our data suggest that silica activates ER stress-mTOR signaling, triggers the epithelial injury/repair response and induces fibrosis. This provides new insights that targeting ER stress-mTOR signaling could be a potential strategy for treating silica-induced fibrosis. This abstract is funded by: NHLBI, TRDRP and the USC Hastings Center for Pulmonary Research
Liebler et al. (Fri,) studied this question.