| Mucin overproduction is a hallmark of chronic inflammatory airway diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, while currently there is no effective treatment for mucin overproduction. Elucidating the mechanisms underlying the regulation of airway mucin gene expression could have important significance for finding potential therapy targets and developing new drugs against airway mucin overproduction.MUC5AC mucin is a major component of airway mucus, so it is very important to investigate the mechanisms underlying the regulation of MUC5AC. Many kinds of bacterias can induce upregulation of MUC5AC mucin, including Pseudomonas aeruginosa(PA) and its lipopolysaccharide(LPS) which is a main cause of respiratory infection. However, the molecular mechanisms underlying PA-LPS-induced MUC5AC overproduction still remain largely unknown. Previous studies have demonstrated that Reactive oxygen species (ROS) can induce TACE activation, and cigarette smoke and neutrophil elastase(NE) upregulate airway MUC5AC expression via signaling pathway ROS→TACE→TGF-a→EGF. Our study found that PA-LPS induces production of ROS through protein kinase C (PKC)-NADPH oxidase signaling pathway in human epithelial cells. Subsequently, ROS generation by PA-LPS releases transforming growth factor-a (TGF-a), which in turn, leads to up-regulate MUC5AC expression.In all,they have confirmed that oxidant stress played an important role in the airway MUC5AC overexpression by PA-LPS.Nuclear factor-erythroid-2-related factor 2 (Nrf2) plays a critical role in the regulation of the cellular redox status. Under normal homeostatic conditions, Nrf2 transcription is repressed by its negative regulator Kelch-like ECH-associated protein 1 (Keap1). Nrf2 is sequestered in the cytoplasm as an inactive complex with Keapl,and maintains low basal levels by constantly ubiquitin-mediated protein degradation. However, upon exposure to oxidative stress, Nrf2 dissociates from cytosolic Keapl and translocates to the nucleus, where it forms a heterodimer with other transcription factors,such as small Maf, which in turn binds to the 5'-upstream cis-acting regulatory sequence,termed antioxidant response elements (ARE), located in the promoter region of genes encoding various antioxidant and phase 2 detoxifying enzymes.Thereby,Nrf2 plays a crucial role in the coordinated induction of those genes encoding many stress-responsive and cytoptotective enzymes and related proteins. These include NAD(P)H:-quinone oxidoreductase-1, glutamate cysteine ligase, glutathione S-transferase, heme oxygenase-1. Nrf2-induced antioxidant protection involves in cancer, aging, athma,acute lung injury, lung fibrosis, atherosclerosis, neurodegeneration, chronic inflammation,autoimmune disease and so on. One study showed synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) in mice models of CF have significantly reduced airway inflammatory responses to LPS. These include the ability of CDDO to reduce NF-κB activation while increasing Nrf2 activity.Disruption of Nrf2 in experimental sepsis dramatically increased the mortality of mice in response to LPS induced septic shock. Temporal analysis of pulmonary global gene expression after LPS challenge revealed augmented expression of large numbers of proinflammatory genes associated with the innate immune response at as early as 30 minutes in lungs of Nrf2-deficient mice.Though,they all showed Keapl/Nrf2 signaling pathway can effectively reduce LPS-induced lung inflammation,there is no report on the role it plays in the regulation of aiway MUC5AC mucin expression by Pseudomonas aeruginosa lipopolysaccharide.In conclusion, we investigated the role of Keapl/Nrf2 signaling pathway in PA-LPS-induced MUC5AC mucin expression in human airway epithelial cells. Part 1 PA-LPS induces MUC5AC mucin expression and Nrf2 downstream antioxidant gene expression in airway epithelial cellsObjective:To investigate the inductive role of PA-LPS in MUC5AC mucin expression and Nrf2 downstream antioxidant gene expression in human airway epithelial cells.Methods:A549 cells (a human pulmonary mucoepidermoid carcinoma cell line) were stimulated with different concentration (5ug/mL,10 ug/mL and 15 ug/mL) of PA-LPS for different times(oh,8h,12h and 24h) to measure MUC5AC mRNA expression by real time PCR(Q-PCR),in order to certain the inductive effect of PA-LPS and find appropriate concentration and time.Subsequently,A549 cells were stimulated with PA-LPS for 24h to measure MUC5AC mRNA and protein expression,and Nrf2 downstream antioxidant gene mRNA and protein expression.Results:After stimulated with PA-LPS for 24h, the MUC5AC mRNA and protein expression in A549 cells were all significantly upregulated(p<0.01), as well as Keapl/Nrf2 signaling pathway downstream antioxidant gene,suah as NQO1,GCLC and GST-pi,especially NQO1(p<0.05).However, the mRNA and protein expression of HO-1 was down-regulated(p<0.05).Conclusion:①PA-LPS can induce MUC5AC mucin expression in human airway epithelial cells.②PA-LPS regulates Keapl/Nrf2 signaling pathway and downstream antioxidant gene expression.③This study suggests that Keap1/Nrf2 signaling pathway may play an important role in PA-LPS-induced airway mucin MUC5AC overproduction.Part 2 The role of Keapl/Nrf2 signaling pathway in PA-LPS-induced MUC5AC mucin expression in airway epithelial cellsObjective:To investigate the role of Keapl/Nrf2 signaling pathway in PA-LPS-induecd MUC5AC mucin expression in human airway epithelial cells.Methods:A549 cells were respectively transfected with different doses of Nrf2 siRNA or pCDNA3-Myc3-Nrf2 or negative control for 24h and 48h,then harvested for Nrf2 mRNA assay to estimate the knockdown or overexpression effects. At 24h after transfection, the A549. cells were serum-starved overnight to maintain low basal levels of MUC5AC expression,and then they were stimulated with PA-LPS(10ug/mL) for various time to measure Nrf2 and MUC5AC mRNA expression by Q-PCR.Once the appropriate transfected dose and stimulated time were determined,the cells were harvested for MUC5AC,Keapl/Nrf2 and downstream antioxidant gene mRNA and protein expression assay.Results:After transfection with Nrf2 siRNA, the Nrf2 mRNA expression in A549 cells were down-regulated about 82%, and the MUC5AC mRNA and protein expression in A549 cells in response to PA-LPS were all significantly augmented (p<0.01).On the another hand, after transfection with pCDNA3-Myc3-Nrf2, the Nrf2 mRNA expression in A549 cells were up-regulated over 200 times, and the MUC5AC mRNA and protein expression in A549 cells in response to PA-LPS were all significantly blocked (p<0.01).Conclusion:This study suggests that Keapl/Nrf2 signaling pathway is involved in the regulation of PA-LPS-induced MUC5AC expression in human airway epithelial cells.
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