To Investigate the Mechanism of Ezh2-Mediated Fibrotic Protein Expression and Airway Fibrosis in Severe Asthma

There are about 10% of asthma patients are steroids-insensitive along with airway remodeling and airway obstruction, which is defined as severe asthma. Subepithelial fibrosis is the pathological character of airway remodeling in severe asthma. Loss of lung function is caused by deposition of extracellular matrix (ECM) in airway walls. The methyltransferase enhancer of zeste homolog 2 (EZH2) mediates transcription factor methylation, which promotes gene expression. Previous studies have highlighted the role of EZH2 in cancer progression. EZH2 promotes cell survival, proliferation, epithelial to mesenchymal, invasion, and drug resistance of cancer cells. In addition to cancer, EZH2 also participates in several fibrosis diseases. For example, EZH2 inhibition attenuates TGF-β dependent hepatic stellate cell activation and liver fibrosis. Besides, EZH2 was up-regulated in the lung fibroblasts of patients with idiopathic pulmonary fibrosis (IPF), which enhanced the differentiation of fibroblasts into myofibroblasts. Our preliminary data showed that transfection of EZH2 siRNA attenuated CTGF expression which is caused by TGF-β, ET-1, thrombin, or hypoxia stimulation in human lung fibroblasts. TGF-β-induced STAT3 methylation was also decreased by the transfection of EZH2 siRNA. TGF-β-induced EZH2 phosphorylation and STAT3 methylation were reduced by treatment of PI3K inhibitor LY294002. In addition, EZH2 protein level was significantly increased in the lung tissue from OVA-induced airway fibrosis model. These results suggest that EZH2-mediated STAT3 methylation may play an important role in the regulation of fibrotic protein expression in airway fibrosis. The Central Hypothesis is that EZH2 promotes the inflammatory response and fibrotic protein expression, which in turn causes the airway fibrosis in patients with severe asthma. The specific aims and hypotheses are described below:Specific Aim 1 (1st year): To investigate the underlying mechanism of EZH2-regulated fibrotic protein expression in human lung fibroblasts, and confirm the pathological role of EZH2 in the airway fibrosis of severe asthmaHypothesis 1: TGF-β induces the methylation of transcription factors, such as STAT3 and YY1, through PI3K/Akt-mediated EZH2 phosphorylation, which promote CTGF expression, myofibroblast differentiation, and ECM protein production in human lung fibroblasts. Specific Aim 2 (2nd~3rd years): Using EZH2 knockout mice to confirm the role of EZH2 in ovalbumin (OVA)-induced airway fibrosis Hypothesis 2: EZH2 mediates fibrotic protein expression and promotes OVA-induced airway fibrosisSpecific Aim 3 (3rd year): To investigate the role of EZH2-regulated inflammatory response in airway fibrosis of severe asthma Hypothesis 3: EZH2 regulates Th2- or Th17-related inflammatory cytokines release in airway epithelial cells from patients with severe asthma, which in turn caused airway fibrosis