Thioredoxin reductase-1 knock down does not result in thioredoxin-1 oxidation

Abstract

The active site of thioredoxin-1 (Trx1) is oxidized in cells with increased reactive oxygen species (ROS) and is reduced by thioredoxin reductase-1 (TrxR1). The purpose of the present study was to determine the extent to which the redox state of Trx1 is sensitive to changes in these opposing reactions. Trx1 redox state and ROS generation were measured in cells exposed to the TrxR1 inhibitors aurothioglucose (ATG) and monomethylarsonous acid (MMA(III)) and in cells depleted of TrxR1 activity by siRNA knock down. The results showed that all 3 treatments inhibited TrxR1 activity to similar extents (90% inhibition), but that only MMA(III) exposure resulted in oxidation of Trx1. Similarly, ROS levels were elevated in response to MMA(III), but not in response to ATG or TrxR1 siRNA. Therefore, TrxR1 inhibition alone was not sufficient to oxidize Trx1, suggesting that Trx1-independent pathways should be considered when evaluating pharmacological and toxicological mechanisms involving TrxR1 inhibition.

Glutathione and Glutaredoxin Act as a Backup of Human Thioredoxin Reductase 1 to Reduce Thioredoxin 1 Preventing Cell Death by Aurothioglucose

“…It is likely that ATG will not reach the mitochondria. There was no increase in the production of ROS, as shown in this study and reported previously as well (23). Trx stayed reduced, and cell death did not occur.…”

“…As shown in Fig. 2B, in cells without treatment, the redox state of Trx1 was mostly fully reduced (lane 2), in line with previous reports (23,34,35). In contrast, as shown in lane 6, in cells treated with 2 M AF, 40% of the total Trx1 was in the fully reduced form, 36% of the total Trx1 migrated with the Ϫ4 mobility, representing a mixed disulfide intermediate with one active site cysteine as a free thiol and the other involved in a disulfide bond to a substrate.…”

“…These results suggested that the viability of HeLa cells was correlated to the redox shift of Trx1 and Trx2 but not TrxR activity. In addition, it seemed that there was no causative relation between loss of TrxR activity and oxidation of Trx, consistent with a previous study (23).…”

“…Cells treated with AF underwent apoptosis with inhibition of TrxR1 activity and increased oxidative stress, such as accumulation of high level of ROS (18). In contrast, when cells were treated with ATG, there was no change in the redox state of the cells (23,24). The mechanism by which the Trx1 redox state is maintained in TrxR1 activity-depleted cells is unclear so far.…”

Hepatocytes lacking thioredoxin reductase 1 have normal replicative potential during development and regeneration

“…However, naturally occurring mRNAs encoding these isoforms have been described, and overexpression of minigenes encoding these isoforms fused to GFP in cultured cells results in cytosolic accumulation of the fluorescent Txnrd2-GFP fusion proteins (Turanov et al, 2006). Moreover, in vitro, recombinant Txnrd2 can reduce Txn1 (Turanov et al, 2006) and diminution of Txnrd1 activity in cell cultures by RNAi or inhibitory drugs does not lead to Txn1 oxidation (Watson et al, 2008).…”