Am. J. Respir. Cell Mol. Biol., Volume 25, Number 2, August, 2001 178-185

Activation of Nuclear Factor-B Transcriptional Activity in Airway Epithelial Cells by Thioredoxin but Not by N-Acetyl-Cysteine and Glutathione

Richart Harper, Kai Wu, Mary M. J. Chang, Ken Yoneda, Raiquin Pan, Sekhar P.-M Reddy,^* and Reen Wu

Center for Comparative Respiratory Biology and Medicine; Division of Pulmonary and Critical Care Medicine, School of Medicine; and Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California at Davis, Davis, California

Increasing evidence indicates that intracellular redox status modulates the activity of various transcriptional factors, including nuclear factor (NF)-B and activator protein-1. Our laboratory has been interested in characterizing the role thioredoxin (TRX) plays in regulating cellular redox status in airway epithelium. TRX is a small, ubiquitous protein with two redox-active half-cysteine residues, -Cys-Gly-Pro-Cys, in its active center. Using primary passage-1 human tracheobronchial epithelial cell cultures and an immortalized human bronchial epithelial cell line, HBE1, we observed that tumor necrosis factor (TNF)- enhanced NF-B transcriptional activity. This observation was based on gel mobility shift assays and interleukin (IL)-8 promoter-reporter gene transfection studies. TNF- activation coincided with translocation of NF-B p65 from the cytoplasm to the nucleus. Pretreatment with N-acetyl-cysteine (NAC) (1 to 10 mM) or glutathione (1 to 10 mM) inhibited TNF--induced activation of NF-B transcriptional activity and IL-8 promoter-mediated reporter gene expression. In contrast, elevated TRX protein levels in cells enhanced TNF--dependent NF-B transcriptional activity and IL-8 promoter activity. This observation was independent of the manner in which TRX was elevated in cells (e.g., by cotransfection with a FLAG-TRX expression clone, or by direct exposure to commercially available human TRX protein). Localization of TRX protein by anti-TRX antibody indicated an accumulation of TRX protein in the nucleus after TNF- treatment. The nuclear localization phenomenon was different from the major cytosolic accumulation of glutathione and NAC. This is the first known report demonstrating movement of TRX into the nucleus of airway epithelial cells after an inflammatory stress. These results suggest a compartment effect of thiol chemicals in the regulation of redox-dependent transcriptional activity.

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