Control of mitochondrial outer membrane permeabilization and Bcl-xL levels by thioredoxin 2 in DT40 cells
Control of mitochondrial outer membrane permeabilization and Bcl-xL levels by thioredoxin 2 in DT40 cells
复制标题
DOI：
10.1074/jbc.m509876200
复制
发表时间：
2006-03-17
影响因子：
4.8
通讯作者：
Yodoi, J
中科院分区：
文献类型：
--
作者：
Wang, DM;Masutani, H;Yodoi, J
线粒体在细胞凋亡的启动中起核心作用，细胞凋亡受多种因素如ATP合成、活性氧、氧化还原状态和外膜透化的调节。鸡硫氧还蛋白2（Trx 2），线粒体氧化还原调节蛋白的中断，导致细胞凋亡的DT 40。为了研究这种细胞凋亡的机制，我们在含有四环素抑制性Trx 2基因的条件Trx 2缺陷型DT 40细胞中制备了表达对照（DT 40-TRX 2-/-）、人硫氧还蛋白2（TRX 2）（DT 40-hTRX 2）或氧化还原失活的TRX 2（DT 40-hTRX 2CS）的转染子。Trx 2表达下调对ATP的产生无明显影响。在DT 40-TRX 2-/-中，通过下调Trx 2表达来增强活性氧的产生。出乎意料的是，这种变化在DT 40-hTRX 2和DT 40-hTRX 2CS细胞中都被阻断。Trx 2表达下调导致细胞色素c和凋亡诱导因子在第3天释放，并在第5天凋亡。这些变化在DT 40-hTRX 2和DT 40-hTRX 2CS细胞中也被抑制，表明TRX 2通过氧化还原活性位点半胱氨酸非依赖性机制调节线粒体外膜透化和凋亡。Trx 2表达下调导致Bcl-xL蛋白水平在第3天下降，而Bcl-2蛋白水平直到第4天才发生变化，而Bcl-xL mRNA水平不变。Bcl-xL的降低不被半胱天冬酶3抑制剂阻断，但在DT 40-hTRX 2和DT 40-hTRX 2CS中均被阻断。这些发现表明TRX 2的氧化还原活性位点半胱氨酸非依赖性作用与Bcl-xL在细胞凋亡调节中的水平之间存在联系。
Mitochondria play a central role in the initiation of apoptosis, which is regulated by various factors such as ATP synthesis, reactive oxygen species, redox status, and outer membrane permeabilization. Disruption of chicken thioredoxin 2 (Trx2), a mitochondrial redox-regulating protein, results in apoptosis in DT40 cells. To investigate the mechanism of this apoptosis, we prepared transfectants expressing control (DT40-TRX2-/-), human thioredoxin 2 (TRX2) (DT40-hTRX2), or redox-inactive TRX2 (DT40-hTRX2CS) in conditional Trx2-deficient DT40 cells containing a tetracycline-repressible Trx2 gene. Production of ATP was not significantly changed by down-regulation of Trx2 expression. The generation of reactive oxygen species was enhanced by the down-regulation of Trx2 expression in DT40-TRX2-/-. Unexpectedly, the change was blocked in both DT40-hTRX2 and DT40-hTRX2CS cells. The down-regulation of Trx2 expression caused the release of cytochrome c and apoptosis-inducing factor on day 3, and apoptosis on day 5. These changes were also suppressed in both DT40-hTRX2 and DT40-hTRX2CS cells, suggesting that TRX2 regulates mitochondrial outer membrane permeabilization and apoptosis by redox-active site cysteine-independent mechanisms. The downregulation of Trx2 expression caused a decrease in the protein level of Bcl-xL on day 3, whereas the protein level of Bcl-2 did not change until day 4, and the mRNA level of Bcl-xL was unchanged. The decrease in Bcl-xL was not blocked by a caspase 3 inhibitor but blocked in both DT40-hTRX2 and DT40-hTRX2CS. These findings indicate a link between the redox active site cysteine-independent action of TRX2 and the level of Bcl-xL in the regulation of apoptosis.