Studies on the Role of ORMDL3 in Thrombin-Induced IL-8/CXCL8 Expression and Airway Inflammation

Asthma affects 7% of the population, and about 250,000 people die due to asthma attach each year. Asthma is characterized by airway inflammation and airway modeling. Thrombin is a well-known coagulation factor generated during vascular injury and plays an important role in airway inflammation. Previous studies demonstrated that the levels of thrombin and IL-8/CXCL8 are increased in bronchoalveolar lavage (BAL) fluid from asthma patients, which are related to the severe degree in asthma. Orosomucoid 1-like 3 (ORMDL3) was strongly linked with asthma and its expression could be induced by allergen in lung epithelial cells. In mice, ovalbumin induces airway inflammation via ORMDL3-dependent ATF6 pathway in mouse model of allergen-induced asthma. Previous study showed that AP-1 plays a circuital role in the regulation of IL-8/CXCL8 expression. However, it remains unclear whether ORMDL3-dependent ATF6 signaling pathway plays a role in thrombin-induced AP-1 activation and IL-8/CXCL8 expression in lung epithelial cells. Several studies indicated that ORMDL3 expression is mediated by CREB and STAT6 pathways. However, the role of PDK1/RSK1-dependent C/EBPactivation in thrombin-induced ORMDL3 expression is still unknown. In our preliminary data show that thrombin-induced IL-8/CXCL8-luciferase activity or IL-8/CXCL8 release were inhibited by cells transfeted with AP-1 mutation of IL-8 construct, ORMDL3 siRNA, ATF6 siRNA, curcumin (AP-1 inhibitor), and c-Jun siRNA in lung epithelial cells. Thrombin-induced c-Jun phosphorylation and c-Jun expression were inhibited by ATF6 siRNA. Thrombin induced c-Jun and ATF6 complex formation. Thrombin-induced ORMDL3 protein expression was inhibited by siRNAs of PDK1, RSK1, and C/EBP. Therefore, the Central Hypothesis of this project is that ORMDL3 mediates thrombin-induced IL-8/CXCL8 expression and airway inflammation. Specific Aim 1 (1st year): To establish the role of ORMDL3 in thrombin-induced AP-1 activation and IL-8/CXCL8 expression in lung epithelial cells Hypothesis 1: ORMDL3-mediated ATF6 activation is involved in thrombin-induced AP-1 activation and IL-8/CXCL8 expression in lung epithelial cells 1.1. To examine the ORMDL3, ATF6, and c-Jun expression upon thrombin stimulation 1.2. Using inhibitors, dominant-negative mutant constructs, and siRNA to examine the role of ORMDL3, ATF6, and AP-1 in thrombin-induced IL-8/CXCL8 expression 1.3. To examine the thrombin-induced AP-1 activation occurs through ORMLD3/ATF6 or JNK Specific Aim 2 (2nd year): To investigate the role of PDK1/RSK1-dependnet C/EBPactivation is involved in thrombin-induced ORMDL3 expression in lung epithelial cells Hypothesis 2: The PDK1/RSK1-dependent C/EBPactivation mediates thrombin-induced ORMDL3 expression in lung epithelial cells 2.1.To study effect of siRNAs of PDK1, RSK1, and C/EBP in thrombin-induced ORMDL3 expression 2.2.Using deletion and point mutant analysis to identify one putative C/EBPbinding site on ORMDL3 promoter (-586/+1) is required for thrombin-induced ORMDL3 expression 2.3.To asses the activation of PDK1, RSK1, and C/EBPupon thrombin stimulation Specific Aim 3 (2nd～3rd years): To confirm the role of ORMDL3 in thrombin-induced airway inflammation in mouse model of allergic asthma Hypothesis 3: The ORMDL3 mediates thrombin-induced airway inflammation in asthmatic mice 3.1.To compare the levels of ORMDL3 in lung in thrombin-induced airway inflammation in wild type and C/EBPknockout mice 3.2.To validate the levels of KC and MIP-2 (functional of homogenous of human IL-8/CXCL8) in BAL fluid in thrombin-induced airway inflammation using ORMDL3 nkRNA by MicroSprayerTM aeroliser in mouse model of allergic asthma 3.3.To analyze the expressions of ORMDL3 and c-Jun, and contents of number of lymphocytes, eosinophils, and goblet cells in lung in thrombin-induced airway inflammation using naked ORMDL3 nkRNA by MicroSprayerTM aeroliser in mouse model of allergic asthma