title: Pyocyanin-induced mucin production is associated with redox modification of FOXA2.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pathobiology, University of Illinois at Urbana-Champaign 2001, Lincoln Avenue, Urbana, IL, 61802, United States of America. geelau@illinois.edu.
authors.forename: Yonghua
authors.initials: Y
authors.lastname: Hao
authors.forename: Zhizhou
authors.initials: Z
authors.lastname: Kuang
authors.forename: Ying
authors.initials: Y
authors.lastname: Xu
authors.forename: Brent E
authors.initials: BE
authors.lastname: Walling
authors.forename: Gee W
authors.initials: GW
authors.lastname: Lau
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001483:Base Sequence; D018277:Carcinoma, Mucoepidermoid / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005978:Glutathione / Q000494:pharmacology; D051542:Hepatocyte Nuclear Factor 3-beta / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D008168:Lung / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D023421:Models, Animal; D008969:Molecular Sequence Data; D055271:Mucin 5AC / Q000378:metabolism*; D055257:Mucin-5B / Q000378:metabolism*; D010084:Oxidation-Reduction; D011710:Pyocyanine / Q000494:pharmacology*; D026361:Reactive Nitrogen Species / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000975:Antioxidants; C496502:FOXA2 protein, human; C524678:MUC5AC protein, human; C524578:MUC5B protein, human; C524679:Muc5ac protein, mouse; C524579:Muc5b protein, mouse; D055271:Mucin 5AC; D055257:Mucin-5B; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D051542:Hepatocyte Nuclear Factor 3-beta; D011710:Pyocyanine; D005978:Glutathione
references.citation: Mucosal Immunol. 2011 Mar;4(2):158-71
references.pmid: 20962773
references.citation: Arch Physiol Biochem. 2007 Oct-Dec;113(4-5):234-58
references.pmid: 18158646
references.citation: Infect Immun. 1998 Dec;66(12):5777-84
references.pmid: 9826354
references.citation: BMC Microbiol. 2007 May 23;7:45
references.pmid: 17521417
references.citation: Appl Microbiol. 1969 Nov;18(5):936-7
references.pmid: 4984207
references.citation: Am J Physiol Lung Cell Mol Physiol. 2003 Nov;285(5):L1077-86
references.pmid: 12871859
references.citation: J Allergy Clin Immunol. 2000 Apr;105(4):739-45
references.pmid: 10756224
references.citation: J Infect Dis. 1983 Mar;147(3):494-503
references.pmid: 6220094
references.citation: Infect Immun. 1983 Oct;42(1):170-7
references.pmid: 6413410
references.citation: Infect Immun. 1994 Feb;62(2):596-605
references.pmid: 8300217
references.citation: Infect Immun. 1988 Sep;56(9):2515-7
references.pmid: 3137173
references.citation: J Bacteriol. 2005 Aug;187(15):5267-77
references.pmid: 16030221
references.citation: Infect Immun. 1992 Sep;60(9):3913-5
references.pmid: 1500199
references.citation: Ann N Y Acad Sci. 1994 Nov 15;744:320-2
references.pmid: 7825858
references.citation: J Appl Physiol (1985). 1989 Jul;67(1):316-23
references.pmid: 2759959
references.citation: J Immunol. 2010 Jun 1;184(11):6133-41
references.pmid: 20483781
references.citation: Pulm Pharmacol Ther. 2008 Aug;21(4):595-9
references.pmid: 18234534
references.citation: Am J Physiol Lung Cell Mol Physiol. 2004 May;286(5):L931-8
references.pmid: 14695120
references.citation: Infect Immun. 1992 Jun;60(6):2536-40
references.pmid: 1316878
references.citation: J Biol Chem. 2011 Sep 30;286(39):34199-214
references.pmid: 21832046
references.citation: Am J Pathol. 2009 Dec;175(6):2473-88
references.pmid: 19893030
references.citation: Development. 2004 Feb;131(4):953-64
references.pmid: 14757645
references.citation: Infect Immun. 1999 Mar;67(3):1207-12
references.pmid: 10024562
references.citation: Am J Physiol. 1998 Jun;274(6):L893-900
references.pmid: 9609727
references.citation: Clin Microbiol Rev. 2002 Apr;15(2):194-222
references.pmid: 11932230
references.citation: Respir Res. 2008 Mar 18;9:27
references.pmid: 18348727
references.citation: Am J Respir Crit Care Med. 1995 Dec;152(6 Pt 1):1910-6
references.pmid: 8520754
references.citation: Respirology. 2008 Mar;13(2):191-202
references.pmid: 18339016
references.citation: Physiol Rev. 2006 Jan;86(1):245-78
references.pmid: 16371599
references.citation: Radiology. 2009 Aug;252(2):534-43
references.pmid: 19703887
references.citation: Am J Respir Crit Care Med. 2004 Apr 1;169(7):822-8
references.pmid: 14726422
references.citation: J Cyst Fibros. 2011 Jul;10(4):265-71
references.pmid: 21463973
references.citation: Am J Respir Crit Care Med. 1994 Mar;149(3 Pt 1):687-93
references.pmid: 8118638
references.citation: Pulm Pharmacol Ther. 2007;20(1):9-22
references.pmid: 16458553
references.citation: Respir Res. 2012 Oct 31;13:98
references.pmid: 23113953
references.citation: Curr Opin Infect Dis. 2010 Apr;23(2):165-70
references.pmid: 19996746
references.citation: FEBS J. 2008 Feb;275(3):481-9
references.pmid: 18167142
references.citation: Am J Respir Cell Mol Biol. 2005 Dec;33(6):523-30
references.pmid: 16151054
references.citation: Infect Immun. 1995 Dec;63(12):4921-3
references.pmid: 7591157
references.citation: Trends Mol Med. 2004 Dec;10(12):599-606
references.pmid: 15567330
references.citation: Am J Physiol Lung Cell Mol Physiol. 2004 Jul;287(1):L94-103
references.pmid: 15020296
references.citation: J Immunol. 2005 Sep 15;175(6):4017-23
references.pmid: 16148150
references.citation: Pediatr Pulmonol. 2006 Jan;41(1):67-73
references.pmid: 16270327
references.citation: Am J Respir Crit Care Med. 2008 Nov 15;178(10):1033-9
references.pmid: 18776153
references.citation: Am J Respir Crit Care Med. 2008 Apr 15;177(8):853-60
references.pmid: 18202344
references.citation: Free Radic Biol Med. 1995 Apr;18(4):801-6
references.pmid: 7750803
references.citation: Thorax. 2007 Feb;62(2):153-61
references.pmid: 16928707
references.citation: Cell Microbiol. 2012 Mar;14(3):401-15
references.pmid: 22103442
references.citation: J Immunol. 2002 Feb 15;168(4):1861-8
references.pmid: 11823520
references.citation: Hybrid Hybridomics. 2004 Apr;23(2):93-9
references.pmid: 15165482
references.citation: Biochem Biophys Res Commun. 2008 Feb 8;366(2):513-9
references.pmid: 18073139
references.citation: J Immunol. 1987 May 15;138(10):3481-7
references.pmid: 2952718
references.citation: Trends Microbiol. 2005 Aug;13(8):389-97
references.pmid: 15951179
references.citation: Eur J Pharmacol. 2006 Mar 8;533(1-3):240-52
references.pmid: 16464450
references.citation: Eur J Pharmacol. 2012 Sep 5;690(1-3):207-13
references.pmid: 22766066
references.citation: Int J Rad Appl Instrum B. 1991;18(5):455-9
references.pmid: 1917514
references.citation: Am J Respir Crit Care Med. 2011 Jun 15;183(12):1674-9
references.pmid: 21297072
references.citation: Am J Respir Cell Mol Biol. 2007 Feb;36(2):244-53
references.pmid: 16980555
references.citation: Ann Surg Oncol. 2011 Jan;18(1):199-206
references.pmid: 20717734
references.citation: PLoS One. 2012;7(10):e47550
references.pmid: 23094061
references.citation: Biochim Biophys Acta. 1979 Jan 4;582(1):67-78
references.pmid: 760819
references.citation: J Immunol. 2005 Sep 15;175(6):4009-16
references.pmid: 16148149
references.citation: Eur J Pharmacol. 2001 Oct 19;429(1-3):251-62
references.pmid: 11698045
references.citation: Infect Immun. 2004 Jul;72(7):4275-8
references.pmid: 15213173