Abstract
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 down-regulation 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.
Highlights
Mitochondria play an essential role in the control of apoptotic death
Because Thioredoxin 2 (TRX2) is considered to play an important part in regulating the redox status of mitochondria, we analyzed the role of the redox reaction in the regulation of apoptosis by introducing human wild-type TRX2 or mutant TRX2 in which cysteines in the active site were replaced with serines (-WSGPS-), into conditional thioredoxin 2 (Trx2)-deficient chicken DT40 cells
Confocal microscopic images showed that both human wild-type TRX2 (hTRX2)-FLAG and hTRX2CS-FLAG proteins are expressed in mitochondria (Fig. 1D)
Summary
Mitochondria play an essential role in the control of apoptotic death. In the mitochondrial pathway of apoptosis, there is frequently a dissipation of mitochondrial outer membrane permeabilization (MOMP),3 accompanied by the release of apoptogenic proteins Disruption of chicken thioredoxin 2 (Trx2), a mitochondrial redox-regulating protein, results in apoptosis in DT40 cells. These changes were 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.
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