Studies on Induction and Functions of Thioredoxin Reductase in Stress Responses

硫氧还蛋白还原酶(TrxR)是一种抗氧化酶，具有还原硫氧还蛋白(Trx)的能力。研究表明，Trx在应激反应中起重要作用，但TrxR在应激反应中的作用尚不清楚。到目前为止，已鉴定出三种TrxR同工酶：TrxR1、TrxR2和Trx3。在这些同工酶中，只有TrxR1被各种胁迫诱导。在本研究中，为了揭示TrxR在应激反应中的作用，本研究对TrxR1的诱导机制和功能进行了如下研究：(1)应激诱导牛动脉内皮细胞TrxR1表达的调节--用肿瘤坏死因子α、PMA+A23187或CD刺激牛动脉内皮细胞，上调TrxR1的表达，但不上调TrxR2和TrxR3的表达。首先，通过瞬时将荧光素酶报告载体导入BAEC，研究了人TrxR1基因启动子区域的作用。PMA+A23187和Cd均通过-646~46bp区域提高荧光素酶活性。另一方面，肿瘤坏死因子α对荧光素酶活性没有影响，但用放线菌素D进行的信使核糖核酸衰变实验表明，肿瘤坏死因子α稳定TrxR1mRNA。这些结果表明，TrxR1的转录激活和转录后稳定都可能参与了应激诱导的TrxR1mRNA的表达，其作用机制可能因应激类型的不同而不同。(2)TrxR1在应激反应转录因子介导的基因表达中的作用-报告基因分析表明，TrxR1mRNA的过表达促进了AP-1和NF-κB依赖的基因表达。TrxR1的催化硒半胱氨酸残基是还原Trx所必需的，这是这种激活所必需的，而TrxR的抑制剂金硫马酸抑制了这种激活。这些结果表明，TrxR1可能通过降低Trx的能力，作为应激反应转录因子AP-1和NF-κB的正向调节因子。

Thioredoxin reductase (TrxR) is an antioxidant enzyme that has an ability to reduce thioredoxin (Trx). It is indicated that Trx plays an important role in stress responses, but the role of TrxR in the stress responses remains unclear. To date, three TrxR isozymes, TrxR1, TrxR2 and Trx3, have been identified. Among these isozymes, only TrxR1 is induced by various kinds of stresses. In this study, in order to reveal the role of TrxR in stress responses, the mechanism of induction and the functions of TrxR1 were studied as follows:(1) Regulation of stress-induced TrxR1 expression - Exposure of bovine arterial endothelial cells (BAEC) to TNFα, PMA+A23187, or Cd upregulated the expression of TrxR1 but neither TrxR2 nor TrxR3. First, role of the promoter region in the huma TrxR1 gene was evaluated by the transient transfection of luciferase reporter vectors into BAEC. Both PMA+A23187 and Cd elevated luciferase activity via the region between-646 and 46 bp. On the other hand, TNFα did not affect the luciferase activity, but the mRNA decay experiments using actinomycin D showed that TNFα stabilized TrxR1 mRNA. These results indicate that both transcriptional activation and posttranscriptional mRNA stabilization may contribute to the stress-induced expression of TrxR1, and that the contributive mechanisms may vary with kinds of stresses.(2) Role of TrxR1 in stress-responsive transcription factor-mediated gene expression - Reporter gene analysis revealed that the overexpression of TrxR1 enhanced AP-1- and NF-κB-dependent gene expression. The catalytic selenocysteine residue of TrxR1, which is essential for reducing Trx, was required for this activation, and aurothiomalate, an inhibitor of TrxR, suppressed this activation. These results suggest that TrxR1 may act as a positive regulator of stress-responsive transcription factors, AP-1 and NF-κB via its ability to reduce Trx.