GENE REGULATION AND INJURY IN AIRWAY BY THIOREDOXIN

Project: Research project

Project Details

Description

The conducting airway epithelial cells are the primary targets of various oxidant stress. Changes in gene expression are frequently observed in association with injury. However, the nature of oxidant/antioxidants that regulate airway epithelial cell injury and gene expression is not known. In this application, we focus on the role of thioredoxin (TRX) on the regulation of epithelial cell injury and gene expression by oxidant, such as ozone. Preliminary studies demonstrated that TRX can serve as an antioxidant to prevent injury from oxidative stress. In addition, TRX can also modulate the induced NF-kappaB activity by TNF-alpha in human cell lines. Based on these preliminary studies and other observations, such as enhanced resistance to H2O2 by an overexpression of TRX, we proposed to test the hypothesis that TRX plays an essential antioxidant role in protecting cells from injury, and it also acts as a regulator for change in gene expression due to oxidant injury. To test this hypothesis, we proposed to extend these initial findings to new experiments to precisely define the molecular nature of such an effect in vitro (Specific aim 1). Using the genomic footprinting technique, the DNA-protein interaction on the cis- elements of human IL-8 gene that is regulated by TNF-alpha and TRX will be elucidated. The TRX participation in the activity will be further confirmed by the transfection studies with dominant negative TRX mutant and wild type genes. This in vitro study will then be extended to in vivo in transgenic mouse model. Here, we will test whether or not that an overexpression of wild type TRX protein in airway epithelia of the transgenic mice can elevate the resistance to injury and altering the change of gene expression due to ozone exposure (Specific aim 2). This can be accomplished by the use of rat CC-10 promoter-based expression vector for targeting TRX gene expression on mouse airway epithelium. For change in gene expression, we will focus on mouse IL-8-like cytokine, MGSA/GROalpha, gene expression. Using the same approach, we will address whether or not an overexpression of the dominant negative TRX protein in transgenic mice airway epithelium can decrease the resistance to injury and also altering the change of gene expression by ozone (Specific aim 3). Finally, we will use the primary airway epithelial cell culture models derived from these transgenic mice to demonstrate that these changes in vivo are due to the difference in the TRX level (Specific aim 4). These studies should provide a fundamental basis of understanding of the function of an antioxidant, TRX, in the regulation of airway epithelial cell injury and gene expression.
StatusFinished
Effective start/end date5/15/994/30/05

Funding

  • National Institutes of Health: $244,101.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $238,163.00
  • National Institutes of Health: $210,598.00
  • National Institutes of Health: $216,563.00

ASJC

  • Environmental Science(all)
  • Medicine(all)

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