Thioredoxin as a modulator of transcription factor binding

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Abstract

Our laboratory is interested in the role of thioredoxin in regulathg cellular redox status in airway epithelium. Specifically, I have been examining how thioredoxin modulates binding of transcription factors. My previous data illustrated that thioredoxin enhances binding of NF-κB, and that this increased binding was un que to thioredoxin. Other thiols such as N-acetyl-cysteine and glutathione were unable to increase binding of NF-κB. I have extended this work for the transcription factor AP-1 and have obtained similar results. Using primary monkey tracheobronchial epithelial cells and an immortalized human tracheobronchial cell line, HBE1, we observed that TNF-α, PMA, and retinoic acid increase the binding activity of AP-1 as measured by gel mobility shift assays (GMSA). This increased binding was further enhanced by the addition of thioredoxin to nuclear extracts. Antibodies to the c-Jun component of AP-1 were used to confirm AP-1 specificity. Transactivation assays were performed in order to correlate the GMSA results with functional promoter activity. Two constructs were used utilizing a 161 base pair spr-1 promoter, which contains two AP-1 consensus sequences, attached to a chloramphenicol acetyltransferase reporter gene. The -162-spr1-CAT3 construct contained two funtional AP-1 sites, whereas the -162-spv1-double mutant-CAT3 construct had mutations at both AP-1 sites. Increased CAT activity, as measured by ELISA, was seen under stimulated conditions with the -162-spr1-CAT3 construct. This increased activity was abolished when the mutant construct was used. These results, together with our previous data, suggest that thioredoxin plays a unique role by increasing transcription factor binding in airway epithelial cells.

Original languageEnglish (US)
JournalJournal of Investigative Medicine
Volume47
Issue number2
StatePublished - Feb 1999

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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