Roles And Mechanisms Of MTOR Signaling Pathway In Cigarette Smoke-induced Chronic Airway Inflammation And Emphysema

Chronic obstructive pulmonary disease (COPD) contributes significantly to the global burden of disease as the fourth leading cause of mortality worldwide, including in clinical phenotypes of emphysema and bronchitis. The pathogenesis of COPD remains incompletely unclear but may involve aberrant airway inflammation, oxidative stress, protease/antiprotease imbalances, microbial infection, autoimmunity and apoptosis in the lung in response to cigarette smoke (CS), the major hazardous and harmful factor for this disease. Clinical research indicates that reactive oxygen species(ROS) act on alveolar macrophages and epidermal cells, thus leading to the release of inflammatory factor and Epidermal Growth Factor(EGF).The mechanistic target of rapamycin (mTOR) is an atypical serine/threonine protein kinase that belongs to the phosphoinositide3-kinase (PI3K)-related kinase family and integrates a wide variety of environ mental cues to regulate organismal growth and homeostasis.The pathway also links with diverse major cellular processes and is involved in an growing number of pathological conditions, including tumor, endocrine system diseases, and neurodegeneration. Autophagy is a homeostatic process during which organelles or proteins are assimilated into two layer membrane autophagosomes and then transferred to the lysosomes for degradation. Our previous studies demonstrated the pivotal role of autophagy in the pathogenesis of COPD. Of interest, mTOR, a pivotal upstream signal of autophagy, is also well known to be universally expressed in the lungs. However, the role of mTOR and its pathway in cigarette smoke-induced airway inflammation and COPD pathogenesis remains unclear.In order to testify our hypothesis, we proposed in the current project to explore the role and mechanisms of mTOR in cigarette smoke-induced inflammation and emphysema. Human bronchial epithelial cells will be treated with cigarette smoking extract (CSE), and mice with mTOR specifically deleted in airway epithilium or aveolar type Ⅱ cells will be exposed to cigarette smoke. The expression of mTOR related molecules, autophagy and apoptosis, production of inflammatory cytokines, airway inflammation, mucus hypersecretion and mean linear intercepts (MLI) of mouse lungs will be measured. This studies will identify an important role of mTOR in cigarette smoke-induced airway inflammation and COPD development, which would shed new lights for prevention and treatment of chronic airway inflammation, and provide new therapeutic targets of durg development for COPD. Part Ⅰ The AMPK-TSCl/2-mTOR Signaling Pathway Regulates Cigarette Smoke-Induced Inflammation and Apoptosis in Airway Epithelial Cells Objective:To investigate the possible role of AMPK-TSC1/2-mTOR signaling pathway in CS-induced inflammation and apoptosis in airway epithelial cells. Methods:Human bronchial epithelial cells (HBECs) were silenced by small interfering (si) RNAs for TSC2, mTOR, Raptor and Rictor genes or administrated with autophagy inhibitors Bafilomycin (BA) and Chloroquine (CQ), and were then treated with CS extract (CSE). Expression of mTOR signaling related molecules (p-AMPK, TSC2, p-TSC2, mTOR, p-mTOR, p-S6) and autophagy were detected by RT-PCR, western blotting or immunocytochemistry. Inflammatory cytokines interleukin (IL)-6and IL-8were measured by RT-PCR and ELISA. Annexin V/PI staining and western blotting for cleaved caspase-9and-3were performed to evaluate apoptosis. Levels of p-S6expression in human lungs were analyzed by inmmunohistochemistry.Results:CSE induced notable activation of AMPKa and Tscl/2while markedly down-regulated mTOR In HBECs. Selective autophagy inhibitor (BA and CQ) or knockout of TSC2significantly protected against CSE-induced inflammation and apoptosis, whereas silencing mTOR expression with siRNA or starvation exerted converse effects. Moreover, mTOR activity, as revealed by levels of p-S6, was dramatically decreased in CS-exposed COPD lung tissues.Conclusion:mTOR signaling pathway plays an important role in CS-induced airway inflammation and apoptosis, eventually participating in chronic obstructive pulmonary disease. Our study provides further insights into the molecular pathogenesis of this disease and suggests new therapeutic targets for it.Part Ⅱ mTOR is an Essential Mediator of CigaretteSmoke-Induced Inflammation and Emphysema in vivoObjective:To establish mTOR specifically deleted in airway epithilium or aveolar type Ⅱ cells mice model by cigarette smoke exposure, and to investigate the possible role of mTOR signaling pathway in chronic airway inflammation and emphysema.Methods:C57BL/6mice were randomly divided into two groups, control group (CON) and cigarette smoke group (CS). The mice in each group were further divided into six subgroups according to three time points (0week,12weeks and24weeks). At each time point, some mice in each group were sacrificed to analyze mTOR related molecular changes in lung tissue by Western Blot. Next, airway epithilium or type II alveolar epithelial-specific mTOR-knockout mice were generated and exposed to cigarette smoke (CS) or room air (AR) to induce inflammation and emphysema. Expression of mTOR signaling related molecules (TSC2, p-mTOR, p-S6) and autophagy in mice lung tissue were detected by western blotting and immunocytochemistry. Inflammatory cytokines (i.e.KC and MIP-2) in BALF and lung homogenate were measured by ELISA. Western blotting for cleaved caspase-9and-3were performed to evaluate apoptosis. In addition, left lung was inflated by gravity with4%paraformaldehyde, held at a pressure of30cmH2O for15minutes and made pathological sections. Tissue samples were stained with haematoxylin and eosin (H&E) to examine peribronchial inflammation and emphysematous change.Results:We found significantly downregulated mTOR mRNA and protein expression(P<0.05) in lungs of mice exposed to CS for24week compared with normal lungs, however, TSC2protein were activated. Besides, mTOR-knockout mice have abnormal lung structure at10weeks of age when compared with wild-type littermates, on the basis of analyses of lung sections stained with H&E, elastin, type II cell prosurfactant protein C expression and terminal airway epithelial Clara cell antigen immunohistochemistry.Conclusion:Our results identify a novel role of mTOR in cigarette smoke-induced inflammation and emphysema as it promoted autophagy activation, alveolar inflammation, oxidative stress and apoptosis of alveolar septal cells, resulting in chronic obstructive pulmonary disease.