MTOR Signaling Pathway Contributes The Pathogenesis Of Pulmonary Fibrosis

Background:Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with poor prognosis and there is no definitive drug treatment. Pathogenesis of IPF is unclear yet. mTOR signaling pathway is a core cellular process which involves the cell growth, proliferation and survival. Relationships between mTOR pathway and fibrotic diseases have been elucidated in some kinds of organ fibrosis, like kidney, liver, intestine. However, the relationship between mTOR signaling pathway and idiopathic pulmonary fibrosis has not been well described.Objective:To investigate the underlying mechanisms of mTOR signaling pathway in pulmonary fibrosis.Methods:1) p-S6, a downstream effector of mTOR, was detected by immunohistochemistry staining in IPF lung tissue.2) p-S6was detected by immunohistochemistry staining in lungs of bleomycin-induced lung fibrosis mice.3) Primary lung fibroblasts (PLF) were isolated from normal human lung. After TGF-β1stimulation, p-S6was detected by western blot.4) In cell line MRC5, p-S6was detected by western blot after TGF-β1stimulation.5) To explore the role of mTOR in lung epithelial cell during pulmonary fibrosis process, we generated an inducible knockout mouse model by crossing alveolar epithelial cell-specific Cre transgenic mice SPC-rtTA/TetO-Cre with floxed-TSC1(TSC1f/f) mice to get the SPC-rtTA/TetO-Cre/TSClf/+CSTT) mice. In STT mice, conditional TSC1deletion in alveolar type2(AT2) epithelial cells occurred when the mice were treated with drink-water containing doxycycline, a tetracycline analogue, resulting in mTOR signaling pathway hyper-activation in AT2epithelial cells of lung. Then we used this TSC1gene deletion mouse in bleomycin-induced lung fibrosis model.6) Rapamycin, an mTOR specific inhibitor, was used as an intervention in bleomycin-induced lung fibrosis model in wildtype C57BL/6mice through two different ways:pre-treatment and late treatment.7) Chloroquine, an autophagy inhibitor, was used combining with rapamycin in bleomycin-induced lung fibrosis model in wildtype C57BL/6mice.Results:1) p-S6was strongly expressed in myofibroblasts of fibroblast foci of human lung tissue from IPF patient.2) p-S6was strongly expressed in lungs of bleomycin-induced lung fibrosis mice model.3) In both PLF and MRC5cell line, p-S6expressions were elevated in lung fibroblasts during fibroblast-myofibroblast transdifferentiation stimulated by TGF-β1.4) Conditional TSC1deletion (mTOR pathway hyper-activated) in lung alveolar epithelial cells worsened bleomycin-induced lung fibrosis with more severe lung histology injury and higher mortality.5) In wild-type C57mice, rapamycin attenuated bleomycin-induced mortality, lung histology injury when pre-treatment with rapamycin (5days before bleomycin injection) was administrated. However, late treatment with rapamycin (8days after bleomycin injection) could not achieve such benefits.6) Rapamycin induced autophagy in lungs of mice from bleomycin-induced mouse model. The benefit of rapamycin could be counterbalanced by chloroquine.Conclusion:In summary, mTOR signaling pathway contributes to pathogenesis of pulmonary fibrosis through multiple cells in lung, at least including cells like myofibroblasts, alveolar epithelial cells. Insufficient autophagy may also contribute to pathogenesis of pulmonary fibrosis. mTOR and autophagy signaling pathways may be new target treatment for IPF patients in the future.