Protein Tyrosine Phosphatase Shp2Regulates Cigarette Smoke-induced Pulmonary Inflammation

Background and objective:Cigarette smoke (CS), the major cause of chronic obstructive pulmonary disease (COPD), contains a variety of oxidative components that were implicated in the regulation of Shp2activity. However, the contribution of Shp2enzyme to COPD pathogenesis remains unclear. We investigated the role of Shp2enzyme in blockading the CS-induced pulmonary inflammation.Methods:Shp2levels were assessed in smoker and nonsmoker individuals and lung tissues of mice exposed to CS or air. Mice were exposed to cigarette smoke extract (CSE) to induce epithelial injury and inflammation. PHPS1, a specific inhibitor of Shp2enzyme, was used to treat pulmonary inflammation in CSE-exposed mice. Endogenous inactivation of Shp2was achieved using the genetic strategy that the Shp2gene was selectively abolished in pulmonary epithelia. Pulmonary epithelia treated with PHPS1inhibitor or siRNA-based knockdown were used to regulate CSE-induced IL-8release and inflammation through a potential Shp2mechanism.Results:(1) Cigarette smoke-induced lung inflammation:to examine the effects of CSE on cytokine production in lung epithelia, pulmonary epithelial cells were treated with CSE followed by analysis of a panel of chemokines and cytokines. We found a remarkable elevation of IL-8in lung epithelia cells in vitro. Further studies suggest that IL-8release is induced by CSE in concentration-and time-dependent manner. (2) Cigarettet smoke-mediated increased levels of Shp2expression in the lung:the increased Shp2level was triggered by CSE in pulmonary epithelial cells in vitro. CSE concentration-dependently triggered Shp2production in early stage of CSE-exposure. In addition, we measured the activity of Shp2enzyme in lung epithelial cells in response to CSE stimulation in vitro. We noticed enhanced levels of the phosphor site (Y-542) of Shp2in a concentration-dependent manner. Similarly, we found that cigarette smoking elevated Shp2expression in vivo. Lungs of smokers showed increased levels of Shp2, as compared with nonsmokers.(3) PHPS1attenmuates cigarette smoki-incuded lung inflammation:to assess the potential role of Shp2in regulating inflammation, we examined the effects of CSE on IL-8gene expression and production in pulmonary epithelial cells with the administration of PHPS1, a Shp2inhibitor. Pharmacological inhibition of Shp2results in marked reduction of IL-8in both mRNA and protein levels. To further probe the effect of Shp2in CS exposure in vivo, C57B1/6mice were exposed to cigarette smoke. Our findings suggested that the IL-8release was significantly reduced in PHPS1-pretreated mice. Counting the total inflammatory cells in BALF collected from mice suggested a remarkable reduction of macrophages, and neutrophils in the treatment with PHPS1compared with controls. H&E staining revealed that PHPS1administration alleviated the influx of inflammatory cells compared to CS exposure, characterized as a significant infiltration of macrophages and neutrophils into alveolar spaces. These observations reveal a potential role for Shp2in IL-8-modulated recruitment of inflammatory cells in acute mouse models of cigarette smoke.(4) Endogenous Inactivation of Shp2Exhibits Decreased Inflammation in Cigarette Smoke-induced Lung Injuries:using siRNA-mediated knockdown of Shp2in pulmonary epithelial cells, we found inactivation of Shp2decreased CSE-induced IL-8release. We next generated lung-specific Shp2knockout mice (Shp2KO). Mice exposed to CS for4consecutive days showed a remarkable elevation of IL-8in controls but not in Shp2(KO) mice. Additionally, the inflammatory cells, especially macrophages, neutrophils and lymphocytes, were decreased in Shp2(KO) mice. Pathological analysis of the lungs using H&E staining exhibited a remarkable decrease in infiltration of macrophages and neutrophils into alveolar spaces compared with controls.(5) Shp2Regulates CSE-induced EGFR/Gabs/MAPK Axis, Contributing to IL-8Release:in NCI-H292cells we found that, upon CSE exposure, Shp2was recruited to an EGFR-dependent complex that involves Gabl docking proteins. Our data suggest that the direct interaction between cytoplasmic Shp2-docking proteins Gabl may increase activation of EGFR initiated by CSE. This notion has been further supported by increased levels of active ERK following CSE treatment. Pharmacological inhibition of Shp2and ERK both decreased the CSE-mediated IL-8production; meanwhile, wild type mice pretreated with PHPS1and Shp2(KO) mice revealed decreased activation of Erk1/2induced by CS. Taken together, our results identify the importance of Shp2in regulating IL-8release in lung epithelia in response to cigarette smoke exposure via EGFR/Gabs/MAPK, which potentially contributes to the development of a novel therapeutic target for lung inflammatory diseases.Conclusions:Our data suggest an important role of Shp2in the pathological alteration associated with CS-mediated inflammation. Shp2may be a potential target for therapeutic intervention of inflammation in COPD.