The thioredoxin system consists of thioredoxin (Trx1) and thioredoxin reductase (TR1). The major function of Trx1 is to reduce oxidized protein substrates. TR1 regenerates the active site of Trx1 which becomes oxidized and temporarily inactive upon reduction of a substrate. Many of the substrates of the thioredoxin system are involved in antioxidant defense and in redox signaling, providing mechanistic links between the thioredoxin system and several diseases including cancer and diabetes.

The transcription factor NFκB controls inflammation, cell survival and proliferation, and is sensitive to compartment-specific redox regulation. Oxidizing conditions in the cytoplasm favor activation of the latent form of NFκB, while oxidizing conditions in the nucleus inhibit its activity. Accumulating evidence suggests that Trx1 increases the activity of nuclear NFκB by reducing an oxidized cysteine within the DNA binding domain. Because Trx1 is dependent on reducing equivalents from TR1, we hypothesized that inhibition of TR1 activity would inhibit NFκB activity through the accumulation of oxidized Trx1 resulting in an inability to reduce the DNA binding domain of NFκB.

In Chapter 2 of this dissertation we examined whether inhibition of TR1 leads to an accumulation of oxidized Trx1. Using siRNA against TR1 we demonstrated that Trx1 oxidation is not a direct or necessary result of loss of TR1 activity and that elevated levels of reactive oxygen species (ROS) are a better predictor of the redox status of Trx1 than TR1 activity. In Chapter 3 we probed the relationship between the thioredoxin system and NFκB activity using curcumin and 1,2-chlorodinitrobenzene (CDNB), two inhibitors of TR1. The data indicate that tumor necrosis factor-α (TNFα)-induced NFκB activity within the nucleus could be completely blocked by the two chemical TR1 inhibitors. Surprisingly, this NFκB inhibition was not mediated by oxidation of the DNA binding domain of NFκB, suggesting an alternative mechanism by which TR1 regulates NFκB. Building on the findings presented in Chapters 2 and 3, the relative contributions of TR1 and Trx1 toward NFκB regulation were elucidated in Chapter 4 using an siRNA-based approach. Knockdown of TR1 resulted in decreased TNFα-stimulated NFκB activity in the absence of either Trx1 oxidation or increased ROS generation, while knockdown of Trx1 had no effect on NFκB activity. Taken together, the studies reported here provide evidence in support of a novel Trx1-independent mechanism for the regulation of NFκB by TR1.