Takahito Kondo

Glutaredoxin (GRX) is a small dithiol protein involved in various cellular functions, including the redox regulation of certain enzyme activities. GRX functions via a disulfide exchange reaction by utilizing the active site Cys-Pro-Tyr-Cys. Here we demonstrated that overexpression of GRX protected cells from hydrogen peroxide (H2O2)-induced apoptosis by regulating the redox state of Akt. Akt was transiently phosphorylated, dephosphorylated, and then degraded in cardiac H9c2 cells undergoing H2O2-induced apoptosis. Under stress, Akt underwent disulfide bond formation between Cys-297 and Cys-311 and dephosphorylation in accordance with an increased association with protein phosphatase 2A. Overexpression of GRX protected Akt from H2O2-induced oxidation and suppressed recruitment of protein phosphatase 2A to Akt, resulting in a sustained phosphorylation of Akt and inhibition of apoptosis. This effect was reversed by cadmium, an inhibitor of GRX. Furthermore an in vitro that GRX Akt in with and GRX an in cells from apoptosis by regulating the redox state of Akt. Glutaredoxin (GRX) is a small dithiol protein involved in various cellular functions, including the redox regulation of certain enzyme activities. GRX functions via a disulfide exchange reaction by utilizing the active site Cys-Pro-Tyr-Cys. Here we demonstrated that overexpression of GRX protected cells from hydrogen peroxide (H2O2)-induced apoptosis by regulating the redox state of Akt. Akt was transiently phosphorylated, dephosphorylated, and then degraded in cardiac H9c2 cells undergoing H2O2-induced apoptosis. Under stress, Akt underwent disulfide bond formation between Cys-297 and Cys-311 and dephosphorylation in accordance with an increased association with protein phosphatase 2A. Overexpression of GRX protected Akt from H2O2-induced oxidation and suppressed recruitment of protein phosphatase 2A to Akt, resulting in a sustained phosphorylation of Akt and inhibition of apoptosis. This effect was reversed by cadmium, an inhibitor of GRX. Furthermore an in vitro that GRX Akt in with and GRX an in cells from apoptosis by regulating the redox state of Akt. redox of is to cellular functions the and of and regulation of the and of and the cellular redox cells the phosphatase protein protein and protein phosphatase protein protein and protein and the (GRX) was a hydrogen in GRX functions via a disulfide exchange reaction by utilizing the active site and the of disulfide GRX is to the by with the formation of disulfide and of by with and the of GRX with from disulfide and is by and of between the GRX a redox with GRX to an in and apoptosis the of the effect of GRX Akt is a of an that and apoptosis of Akt is and phosphorylation and of Akt via dephosphorylation of the phosphorylation by protein phosphatase 2A Akt to the of hydrogen peroxide cells the of Akt dephosphorylation and the of H2O2-induced dephosphorylation of Akt is the of an a disulfide bond in that Akt is a we a the effect of GRX via regulation of the redox state of Akt disulfide bond between Cys-297 and Cys-311 of Akt in cardiac H9c2 cells with Overexpression of GRX oxidation of Akt and protected cells from GRX was from the of a with a of GRX was from and from was from and Akt protein and from from and from was from was from and from a from from H9c2 cells and cells in with in a of and GRX was GRX was by the to the of GRX and a and the to the of the GRX and an was with and and then the GRX the with the was with and and then and was with and and then and by with an was to of the various and to in to and and was the a and to the by with an and of was to the was to the GRX. 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GRX is to the by with the formation of disulfide and of by with and the of GRX with from disulfide and is by and of between the GRX a redox with GRX to an in and apoptosis the of the effect of GRX Akt is a of an that and apoptosis of Akt is and phosphorylation and of Akt via dephosphorylation of the phosphorylation by protein phosphatase 2A Akt to the of hydrogen peroxide cells the of Akt dephosphorylation and the of H2O2-induced dephosphorylation of Akt is the of an a disulfide bond in that Akt is a Here we a the effect of GRX via regulation of the redox state of Akt disulfide bond between Cys-297 and Cys-311 of Akt in cardiac H9c2 cells with Overexpression of GRX oxidation of Akt and protected cells from apoptosis. 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Oxidative stress is believed to be a cause of aging and cardiovascular disorders. In response to inflam-mation or endothelial cell injury, production of reactive oxygen species (ROS) is enhanced in vascular cells. These changes contribute to the initiation of atherosclerosis. Vascular cells possess anti-oxidant systems to protect against oxidative stress, in addition to the redox system. The redox status of pro-tein thiols is important for cellular functions. The Akt signaling pathway exerts effects on survival and apoptosis, and is regulated by the glutathione (GSH)/glutaredoxin (GRX)-dependent redox sys-tem. Sex hormones such as estrogens protect against oxidative stress by protecting the Akt signaling pathway but the physiological role of the extracellular GSH/GRX system has not been clarified, although found an increase in the levels of S-glutathionylated serum proteins in patients with athero-sclerosis obliterans. The results suggested that impaired serum redox potential is a marker of the development vascular dysfunction and estrogen has a possible role in the prevention of atherosclerosis.

To elucidate the pathological metabolism of glutathione synthesis in diabetic endothelial cells, we studied the expression of γ-glutamylcysteine synthetase (γ-GCS) using a mouse vascular endothelial cell line.Exposing normoglycemic endothelial cells to tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) increased the activity and the mRNA expression of γ-GCS. The addition of inhibitors for nuclear factor κB (NF-κB) to the cells caused a loss of the γ-GCS mRNA expression in response to TNF-α.A shift of the concentration of glucose in the medium from 5.5 to 28 mM glucose and a following incubation for 7 days decreased the expression of γ-GCS mRNA. These cells showed no apparent responses of γ-GCS mRNA or the activity of NF-κB to TNF-α or IL-β. Increase in the GSH concentration of the cells treated with 28 mM glucose restored the expression of γ-GCS mRNA and its response to TNF-α or IL-β, suggesting that redox regulation is involved in the expression of γ-GCS.In summary, the expression of γ-GCS is regulated by TNF-α or IL-1β in endothelial cells mediated by NF-κB stimulation, and impairment of the regulation of γ-GCS in hyperglycemic cells may be a cause of medical complications that develop in diabetes mellitus. To elucidate the pathological metabolism of glutathione synthesis in diabetic endothelial cells, we studied the expression of γ-glutamylcysteine synthetase (γ-GCS) using a mouse vascular endothelial cell line. Exposing normoglycemic endothelial cells to tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) increased the activity and the mRNA expression of γ-GCS. The addition of inhibitors for nuclear factor κB (NF-κB) to the cells caused a loss of the γ-GCS mRNA expression in response to TNF-α. A shift of the concentration of glucose in the medium from 5.5 to 28 mM glucose and a following incubation for 7 days decreased the expression of γ-GCS mRNA. These cells showed no apparent responses of γ-GCS mRNA or the activity of NF-κB to TNF-α or IL-β. Increase in the GSH concentration of the cells treated with 28 mM glucose restored the expression of γ-GCS mRNA and its response to TNF-α or IL-β, suggesting that redox regulation is involved in the expression of γ-GCS. In summary, the expression of γ-GCS is regulated by TNF-α or IL-1β in endothelial cells mediated by NF-κB stimulation, and impairment of the regulation of γ-GCS in hyperglycemic cells may be a cause of medical complications that develop in diabetes mellitus.