Studies on Induction and Functions of Thioredoxin Reductase in Stress Responses

硫氧还蛋白还原酶（TrxR）是一种具有还原硫氧还蛋白（Trx）能力的抗氧化酶。研究表明Trx在植物的应激反应中起重要作用，但TrxR在植物应激反应中的作用尚不清楚。迄今为止，已鉴定出三种TrxR同工酶，TrxR 1、TrxR 2和Trx 3。在这些同工酶中，只有TrxR 1受各种胁迫诱导。为了揭示TrxR在应激反应中的作用，本研究对TrxR 1的诱导机制和功能进行了如下研究：（1）应激诱导TrxR 1表达的调控--TNFα、PMA+A23187、Cd处理可上调牛动脉内皮细胞（BAEC）TrxR 1的表达，但TrxR 2和TrxR 3均未上调。首先，通过将荧光素酶报告载体瞬时转染到BAEC中来评估人TrxR 1基因的启动子区的作用。PMA+A23187和Cd均通过-646和46 bp之间的区域提高荧光素酶活性。另一方面，TNFα不影响荧光素酶活性，但使用放线菌素D的mRNA衰减实验表明，TNFα稳定TrxR 1 mRNA。这些结果表明，转录激活和转录后mRNA稳定化均可能参与了胁迫诱导TrxR 1的表达，其作用机制可能因胁迫种类而异。(2)TrxR 1在应激反应转录因子介导的基因表达中的作用-报告基因分析显示，TrxR 1的过表达增强了AP-1和NF-κ B依赖的基因表达。催化硒半胱氨酸残基的TrxR 1，这是必不可少的减少Trx，需要这种激活，和aurothiomalate，抑制剂的TrxR，抑制这种激活。这些结果表明，TrxR 1可能通过其减少Trx的能力作为应激反应转录因子AP-1和NF-κB的正调节因子。

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.