Abstract 366: Increased Nuclear Localization of Thioredoxin by Physiological Concentrations of Reactive Oxygen Species Induces Glutathione S Transferase P1 Expression and Blocks Apoptosis
The redox regulator thioredoxin-1 (TRX) is a potent anti-oxidative enzyme and exerts important functions in cell growth and apoptosis. Physiological concentrations of reactive oxygen species (ROS) act as second messenger to activate signalling pathways. Low doses of ROS (10 μM H2O2) reduced apoptosis under basal and serum-depleted conditions in endothelial cells. Since the anti-apoptotic activity of 10 μM H2O2 was dependent on the expression of TRX, we further elucidated the underlying mechanisms. First, we investigated the localization of thioredoxin after 10 μM H2O2. ROS induced a nuclear import of TRX. We identified karyopherin α and karyopherin β as the receptor for nuclear import of TRX. Analysis of the TRX amino acid sequence revealed possible karyopherin α recognition sites at positions: lysines 81, 82, 85 and lysines 94 and 96. We mutated the basic lysines (K) to the acidic glutamate (E) and investigated the ability of nuclear import. TRX(K94/96E) and TRX(K85E) were imported into the nucleus, whereas TRX(K81/82E) showed a reduction in nuclear import and TRX(K81/82/85E) was entirely retained in the cytosol and induced apoptosis under basal conditions. Overexpression of TRX(K81/82E) abolished the anti-apoptotic capacity of ROS. To determine, which transcription factors are regulated by nuclear localized TRX, immunoprecipitates from nuclear extracts of TRXwt and TRX(K81/82E) were used in a DNA-protein binding array. Among more than 400 transcription factors, the ARE element showed significantly increased activity only in TRXwt extracts (TRXwt: 5.3 +/− 1.2 fold activity; TRX(K81/82E): 0.1 +/- 0.1 fold activity). In - silico analysis to identify proteins containing ARE elements in their promoter revealed the glutathione S-transferase P1 (GSTP1) as a potential candidate. Indeed, physiological concentrations of ROS increased protein expression of GSTP1 (3.2 +/− 0.5 fold). Strikingly, genetic ablation by siRNA against GSTP1 abolished the anti-apoptotic effect of ROS without inhibiting the nuclear import of TRX. These data demonstrate that the nuclear import of TRX and increased GSTP1 expression are required for the anti-apoptotic function of physiological concentrations of ROS.