Abstract
BackgroundThe redox-active pyocyanin (PCN) is a toxic, secondary metabolite secreted by the respiratory pathogen Pseudomonas aeruginosa (PA). Previously, we have shown that mouse lungs chronically exposed to PCN develop goblet cell hyperplasia and metaplasia (GCHM) and mucus hypersecretion, fibrosis and emphysema. These pathological features are commonly found in the airways of several chronic lung diseases, including cystic fibrosis (CF), as well as in mouse airways deficient in the forkhead box A2 (FOXA2), a transcriptional repressor of goblet GCHM and mucus biosynthesis. Furthermore, PCN inhibits FOXA2 by activating the pro-GCHM signaling pathways Stat6 and EGFR. However, it is not known whether PCN-generated reactive oxygen (ROS) and nitrogen (RNS) species posttranslationally modify and inactivate FOXA2.MethodsWe examined the posttranslational modifications of FOXA2 by PCN using specific antibodies against oxidation, nitrosylation, acetylation and ubiquitination. Electrophoretic mobility shift assay (EMSA) was used to examine the ability of modified FOXA2 to bind the promoter of MUC5B mucin gene. In addition, we used quantitative real time PCR, ELISA, immunofluorescence and mouse lung infection to assess whether the loss of FOXA2 function caused GCHM and mucin overexpression. Finally, we examined the restoration of FOXA2 function by the antioxidant glutathione (GSH).ResultsWe found that PCN-generated ROS/RNS caused nitrosylation, acetylation, ubiquitination and degradation of FOXA2. Modified FOXA2 had reduced ability to bind the promoter of the MUC5B gene. The antioxidant GSH alleviated the modification of FOXA2 by PCN, and inhibited the overexpression of MUC5AC and MUC5B mucins.ConclusionThese results suggest that PCN-mediated posttranslational modifications of FOXA2 are positively correlated with GCHM and overexpression of airway mucins. Furthermore, antioxidant treatment restores the function of FOXA2 to attenuate GCHM and mucus hypersecretion.
Highlights
The redox-active pyocyanin (PCN) is a toxic, secondary metabolite secreted by the respiratory pathogen Pseudomonas aeruginosa (PA)
Normal human bronchial epithelial (NHBE) cells at passages 2 to 4, and 16HBE cells were trypsinized and seeded onto the Costar Transwells® inserts with 0.4 μm pore size (Corning) (Tewksbury, MA, USA) at a density of 1.5 × 105 cells/cm2 in media comprised of 50% BEBM and 50% DMEM-F12 low glucose (Invitrogen, Grand Island, NY, USA) supplemented with the growth factors provided in the SingleQuot® kits and retinoic acid (50 nM)
Because H2O2 and O2 - are capable of interacting with nitric oxide (NO) within airways to produce the highly toxic peroxynitrite, we evaluated the production of total Reactive oxygen species (ROS)/reactive oxygen (ROS) and nitrogen (RNS) in NCIH292 cells following 24 hr exposure to different concentrations of PCN
Summary
The redox-active pyocyanin (PCN) is a toxic, secondary metabolite secreted by the respiratory pathogen Pseudomonas aeruginosa (PA). We have shown that mouse lungs chronically exposed to PCN develop goblet cell hyperplasia and metaplasia (GCHM) and mucus hypersecretion, fibrosis and emphysema. These pathological features are commonly found in the airways of several chronic lung diseases, including cystic fibrosis (CF), as well as in mouse airways deficient in the forkhead box A2 (FOXA2), a transcriptional repressor of goblet GCHM and mucus biosynthesis. PCN inhibits FOXA2 by activating the pro-GCHM signaling pathways Stat and EGFR. It is not known whether PCN-generated reactive oxygen (ROS) and nitrogen (RNS) species posttranslationally modify and inactivate FOXA2. Excessive ROS/RNS production and inhibition of antioxidative mechanisms by PCN overwhelm the antioxidant capacity of the tissue, leading to lung damage. Antioxidants detoxify PCN, suggesting that its virulence is redox dependent [22,26,28,29]
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