Hongli Wu

Glutaredoxin 2 knockout increases sensitivity to oxidative stress in mouse lens epithelial cells

Glutaredoxin belongs to the oxidoreductase family, with cytosolic glutaredoxin 1 (Grx1) and mitochondrial glutaredoxin 2 (Grx2) isoforms. Of the two isozymes, the function of Grx2 is not well understood. This paper describes the effects of Grx2 deletion on cellular function using primary lens epithelial cell cultures isolated from Grx2 gene knockout (KO) and wild-type (WT) mice. We found that both cell types showed similar growth patterns and morphology and comparable mitochondrial glutathione pool and complex I activity. Cells with deleted Grx2 did not show affected Grx1 or thioredoxin expression but exhibited high sensitivity to oxidative stress. Under treatment with H(2)O(2), the KO cells showed less viability, higher membrane leakage, enhanced ATP loss and complex I inactivation, and weakened ability to detoxify H(2)O(2) in comparison with the WT cells. The KO cells had higher glutathionylation in the mitochondrial proteins, particularly the 75-kDa subunit of complex I. Recombinant Grx2 deglutathionylated complex I and restored most of its activity. We conclude that Grx2 has a function that protects cells against H(2)O(2)-induced injury via its peroxidase and dethiolase activities; particularly, Grx2 prevents complex I inactivation and preserves mitochondrial function.

Glutaredoxin 2 prevents H2O2-induced cell apoptosis by protecting complex I activity in the mitochondria☆

Glutaredoxin 2 (Grx2) belongs to the oxidoreductase family and is an isozyme of glutaredoxin 1 (Grx1) present in the mitochondria, however its function is not well understood. The purpose of this study is to evaluate the potential anti-apoptotic function of Grx2 by examining its ability to protect complex I in the mitochondrial electron transport system using human lens epithelial cells as a model. We found that cells treated with 200muM hydrogen peroxide (H(2)O(2)) for 24h exhibited decreased viability and became apoptotic with corresponding Bax up-regulation, Bcl-2 down-regulation, caspase 3 activation and mitochondrial cytochrome c leakage. Grx2 over-expression (OE) could protect cells against H(2)O(2)-induced damage while Grx2 knockdown (KD) showed the opposite effect. Under the same conditions, H(2)O(2) treatment caused 50% inactivation of complex I activity in control cells (vector only), 75% in Grx2 KD cells but only 20% in Grx2 OE cells. Furthermore, the inactivated complex I in the H(2)O(2)-treated cells could be protected mostly by importing the purified nascent Grx2 protein, but not the Grx2 protein mutated at the active site with C70S, or C73S, or with C70S plus C73S. Immunoprecipitation study also revealed that Grx2 co-precipitated with complex I, but not complex II, in the mitochondrial lysate. Thus, the mechanism of Grx2 protection against H(2)O(2)-induced apoptosis is likely associated with its ability to preserve complex I.

Overexpression of thioredoxin-binding protein 2 increases oxidation sensitivity and apoptosis in human lens epithelial cells.

Thioredoxin (Trx) is an important redox regulator with cytosolic Trx1 and mitochondrial Trx2 isozymes. Trx has multiple physiological functions in cells and its bioavailability is negatively controlled through active-site binding to a specific thioredoxin-binding protein (TBP-2). This paper describes the delicate balance between TBP-2 and Trx and the effect of overexpression of TBP-2 in human lens epithelial cells. Cells overexpressing TBP-2 (TBP-2 OE) showed a sevenfold increase in TBP-2 and a nearly 40% suppression of Trx activity but no change in Trx expression. The TBP-2 OE cells grew slower and their population decreased to 30% by day 7. Cell cycle analysis showed that TBP-2 OE cells arrested at the G2/M stage and that they displayed low expression of the cell cycle elements P-cdc2(Y15), cdc2, cdc25A, and cdc25C. Furthermore, TBP-2 OE cells were more sensitive to oxidation. Under H2O2 (200μM, 24h) treatment, these cells lost 80% viability and became highly apoptotic. Brief oxidative stress (200μM, 30min) to TBP-2 OE cells disrupted the Trx antiapoptotic function by dissociating the cytosolic and mitochondrial Trx-ASK binding complexes. The same H2O2-treated cells also showed activated ASK (P-ASK), increased Bax, lowered Bcl-2, cytochrome c release, and elevated caspase 3/7 activity. We conclude from these studies that high cellular levels of TBP-2 can potentially suppress Trx bioavailability and increase oxidation sensitivity. Overexpression of TBP-2 also causes slow growth by mitotic arrest and apoptosis by activating the ASK death pathway.