Control of Mitochondrial Outer Membrane Permeabilization and Bcl-xL Levels by Thioredoxin 2 in DT40 Cells

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 tetracyclinerepressible 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.

Prep1 Directly Regulates the Intrinsic Apoptotic Pathway by Controlling Bcl-XL Levels

“…The 60% decrease in Bcl-X L in Prep1 i/i MEFs is not marginal, since Bcl-x ϩ/Ϫ heterozygous male germ cells are apoptotic (21) and since cell lines in which the Bcl-X L level is downregulated undergo apoptosis because of an altered mitochondrial homeostasis (10,36).…”

Mitochondrial Thiols in the Regulation of Cell Death Pathways

“…Overexpression of Trx2 inhibits ASK1-induced apoptosis, while knockdown of Trx2 increases TNFa/ASK1-induced cytochrome c release (156,200). The mitochondrial permeability transition (MPT) plays significant roles in activation of apoptosis and necrosis (72,144,174,198): Trx2 protects isolated mitochondria from MPT induced by peroxide or Ca + + (61) and Trx2-deficient cells show lower mitochondrial membrane potential (182). Whether Trx2 regulates the MPT by either interacting with MPT pore or modulating the mitochondrial oxidant levels is still unclear (61).…”

Redox proteins thioredoxin 1 and thioredoxin 2 support retinal ganglion cell survival in experimental glaucoma

“…EGFP-tagged Trx1 and Trx2 expression constructs, pEGFP-C1-Trx1 51 and pCMV-hTrx2-EGFP, 47 were delivered to retinal cells by ELP 1 day before laser photocoagulation. Transcription of the Trx genes in both vectors is controlled by CMV (cytomegalovirus) promoter.…”

“…46 The antiapoptotic characteristics of Trx2 were associated with its roles in regulation of mitochondrial outer membrane permeability and the levels of proapoptotic Bcl-xL. 47 In summary, changes in Txnip and Trx1 levels after IOP elevation implicate these redox proteins in the RGC degenerative process in glaucomatous retinas. Txnip, Trx1 and Trx2 are shown to be abundantly expressed in RGC somas and nerve fiber layer.…”