ATF3Regulates Cigarette Smoke-Induced Mucin Hypersecretion Via AP-1

Background:Mucus hypersecretion is an important pathological feature of chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) was importantly associated with the pathogenesis of COPD. Excess mucus production contributes to airway obstruction and results in a reduction in lung function. Activating transcription factor3(ATF3), an adaptive-response gene that participates in celluar processes, has been demonstrated to play dichotomous roles in the regulation of inflammation. Mucus hypersecretion is one of the most important characteristics of airway inflammation. However, little is known about whether ATF3is involved in the regulation of CS-induced MUC5AC (a major component of airway mucus) expression in airway epithelial cells.Objective:Our study investigated whether ATF3is involved in the regulation of MUC5AC expression in COPD and explore the molecular mechanisms. Methods:CS-induced ATF3and MUC5AC expression levels were examined in human bronchial epithelial (HBE) cells (normal bronchial epithelial cells), human NCI-H292cells (lung mucoepidermoid carcinoma cell line) and MTEC (primary mouse tracheal epithelial cells). ATF3small-interfering RNA(siRNA) and plasmid were used to investigate whether ATF3is involved in CS-induced MUC5AC expression. We used quantitative real time PCR, Western Blot and immunofluorescence to assess whether the loss or gain of ATF3function affected MUC5AC expression. Co-Immunoprecipitation (Co-IP) and chromatin immunoprecipitation (CHIP) were used to examine the ability of ATF3to bind the promoter of MUC5AC gene via activator protein-1(AP-1). In vivo, ATF3-/-mice were used to identify the role of ATF3in regulation the expression of MUC5AC.Results:1. Cigarette smoke promoted ATF3release and MUC5AC production in HBE cells, NCI-H292cells and mTECS. MUC5AC mRNA expression was significantly inhibited by specific knockdown using siRNA for ATF3. Conversely, the expression of iterleukin-6(IL-6) and interleukin-8(IL-8) induced by CS increased after knockdown of ATF3. Furthermore, Co-IP and ChIP assays confirmed that ATF3involved in the CS-induced MUC5AC overproduction through the AP-1signal pathways in HBE cells.2. In vivo, we have found the increased bronchial MUC5AC secretion in CS-treated wild type mice. However, there was no significant difference in CS-treated ATF3-/-mice compared with WT mice. Moreover, we observed a remarkably increased inflammatory cells, mainly neutrophils, accumulated around the airways and vessels in CS-treated ATF3-/-mice. Conclusion:Our study indicates that ATF3regulates CS-induced airway MUC5AC hypersecretion via the AP-1-dependent mechanisms in airway epithelial cells. Meanwhile, ATF3has also participated in the regulation of CS-induced IL-6and IL-8expression. It is possible that ATF3may be a potential target for pharmacotherapy of airway mucus overproduction in COPD.