The Thioredoxin System: Gene Structure and Expression Studies in Response to Oxidative Stress

Abstract

The thioredoxin system is comprised of thioredoxin and thioredoxin reductase and is widely distributed in prokaryotes and eukaryotes. The thioredoxin system plays an important role in maintaining the redox state of the cell and in protecting the cell against oxidative stress by scavenging reactive oxygen species through a variety of mechanisms. However, elevated levels of the thioredoxin system are also present in many forms of cancer and with persistent oxidative stress associated with many types of cancer, an understanding of how the expression of the thioredoxin system is controlled under basal conditions and in response to oxidative stress, may aid in elucidating the link between the thioredoxin system and cancer. Initial studies were focused on determining the structure of the thioredoxin reductase gene. A mouse genomic library was screened and a 15kb clone containing 11 exons was mapped and the important regions sequenced. The remaining exons were identified by data base analysis. The structure of the human thioredoxin reductase gene was mapped by data base searches and identified many 5' splice variants involved in an extremely complex regulation of gene expression. The human thioredoxin promoter region directs expression of the thioredoxin gene under both housekeeping conditions and during stimulation by oxidative stress reagents. Two potential transcription start regions had previously been described. In this project luciferase assays and Real-Time PCR analysis were used to characterise the core human thioredoxin promoter region containing these alternate transcription start sites and to analyse the action of other regulatory elements present within the human thioredoxin promoter region in basal expression and in response to oxidative stress. These studies revealed an overlapping promoter region that appears to utilise non-TATA and TATA box containing promoters in conjunction with Sp1 binding sites, oxidative and antioxidant response elements to elicit maximal gene induction to oxidative stress stimuli (tert-butylhydroquinone). Other studies involving human skin cancer cell lines enabled the levels of the thioredoxin system to be measured and correlated with cell pigmentation to ultimately investigate the effect of the thioredoxin system on melanogenesis, a cellular process influenced by levels of oxidative stress. These studies suggested that due to its reactive oxygen species scavenging activities, the thioredoxin system can affect melanogenesis by altering the production of pigment without affecting tyrosinase activity.