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Published on: June 14, 2016
Pyocyanin-induced mucin production is associated with redox modification of FOXA2
Yonghua Hao1, Zhizhou Kuang, Ying Xu
1Department of Pathobiology, University of Illinois at Urbana-Champaign 2001, Lincoln Avenue, Urbana, IL, 61802, United States of America. geelau@illinois.edu.
Background:
The 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.
Methods:
We 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).
Results:
We 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.
Conclusion:
These 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.
Insights
Pyocyanin (PCN) from Pseudomonas aeruginosa causes lung damage by modifying the FOXA2 protein. Antioxidant treatment can restore FOXA2 function, reducing mucus overproduction and goblet cell hyperplasia.
Area of Science:
- Pulmonary Medicine
- Microbiology
- Biochemistry
Background:
- Pyocyanin (PCN), a toxin from Pseudomonas aeruginosa, induces lung pathology including goblet cell hyperplasia and mucus hypersecretion.
- These changes mimic features of chronic lung diseases like cystic fibrosis and are linked to reduced function of the FOXA2 protein.
- PCN inhibits FOXA2 via Stat6 and EGFR signaling, but its effect on FOXA2 posttranslational modification by reactive oxygen and nitrogen species was unknown.
Purpose of the Study:
- To investigate whether PCN-induced reactive oxygen and nitrogen species (ROS/RNS) posttranslationally modify and inactivate FOXA2.
- To determine the functional consequences of FOXA2 modification on MUC5B gene promoter binding and mucin expression.
- To assess the potential of antioxidant therapy to restore FOXA2 function and ameliorate PCN-induced lung pathology.
Main Methods:
- Examined FOXA2 posttranslational modifications (nitrosylation, acetylation, ubiquitination) using specific antibodies.
- Assessed FOXA2 binding to the MUC5B promoter using electrophoretic mobility shift assay (EMSA).
- Evaluated GCHM and mucin overexpression via qPCR, ELISA, immunofluorescence, and mouse lung infection models; tested antioxidant glutathione (GSH) effects.
Main Results:
- PCN-generated ROS/RNS induced nitrosylation, acetylation, ubiquitination, and degradation of FOXA2.
- Modified FOXA2 exhibited reduced binding to the MUC5B promoter.
- GSH treatment alleviated FOXA2 modification and inhibited MUC5AC/MUC5B mucin overexpression.
Conclusions:
- PCN-induced posttranslational modifications of FOXA2 correlate with goblet cell hyperplasia and metaplasia (GCHM) and airway mucin overexpression.
- Antioxidant intervention, specifically with GSH, restores FOXA2 function, thereby attenuating GCHM and mucus hypersecretion.
- These findings highlight a novel mechanism of PCN-induced lung injury and suggest a therapeutic strategy involving antioxidants.
