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Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas.

Anathy V, Aesif SW, Guala AS, Havermans M, Reynaert NL, Ho YS, Budd RC, Janssen-Heininger YM - J. Cell Biol. (2009)

Bottom Line: In this study, we demonstrate that stimulation with Fas ligand (FasL) induces S-glutathionylation of Fas at cysteine 294 independently of nicotinamide adenine dinucleotide phosphate reduced oxidase-induced ROS.As a result, death-inducing signaling complex formation is also increased, and subsequent activation of caspase-8 and -3 is augmented.These results define a novel redox-based mechanism to propagate Fas-dependent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Vermont, Burlington, VT 05405, USA.

ABSTRACT
Reactive oxygen species (ROS) increase ligation of Fas (CD95), a receptor important for regulation of programmed cell death. Glutathionylation of reactive cysteines represents an oxidative modification that can be reversed by glutaredoxins (Grxs). The goal of this study was to determine whether Fas is redox regulated under physiological conditions. In this study, we demonstrate that stimulation with Fas ligand (FasL) induces S-glutathionylation of Fas at cysteine 294 independently of nicotinamide adenine dinucleotide phosphate reduced oxidase-induced ROS. Instead, Fas is S-glutathionylated after caspase-dependent degradation of Grx1, increasing subsequent caspase activation and apoptosis. Conversely, overexpression of Grx1 attenuates S-glutathionylation of Fas and partially protects against FasL-induced apoptosis. Redox-mediated Fas modification promotes its aggregation and recruitment into lipid rafts and enhances binding of FasL. As a result, death-inducing signaling complex formation is also increased, and subsequent activation of caspase-8 and -3 is augmented. These results define a novel redox-based mechanism to propagate Fas-dependent apoptosis.

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Overexpression of Grx1 decreases S-glutathionylation of Fas, caspase-8 and -3 activities, and cell death. (A) Assessment of Grx1 expression. C10 cells were transfected with pcDNA3 or Flag-Grx1 plasmids (1 µg/4 × 105 cells). Cells were stimulated with FasL + M2 for 2 h, and lysates were analyzed by immunoblotting for Grx1 expression. The bottom panel shows β-actin as a loading control. (B) Assessment of S-glutathionylation of Fas after overexpression of Grx1. pcDNA3 or Flag-Grx1–transfected cells were stimulated with FasL + M2, and after 2 h, S-glutathionylated proteins were immunoprecipitated using an antibody directed against glutathione (IP: PSSG). +DTT samples were used as reagent controls. The bottom panel represents Fas content in whole cell lysates (WCL). (C) Assessment of caspase-8 and -3 activities in cells after overexpression of Grx1. C10 cells were transfected with pcDNA3 or Flag-Grx1 plasmids and stimulated with FasL + M2. At the indicated times, cells were harvested, and lysates were prepared for evaluation of caspase activity. Results are expressed as mean ± SEM relative luminescence units (RLU)/20,000 cells. The graph represents triplicate values obtained from two independent experiments. *, P < 0.05 compared with FasL-treated pcDNA3-treated control groups (ANOVA). (D) Assessment of FasL-induced death in cells overexpressing Grx1. Cells were transfected with the indicated amounts of pcDNA3 or Flag-Grx1 plasmid (total DNA content 1.25 µg/4 × 105 cells) and stimulated with FasL + M2. After 2 h, cell survival was assessed using the MTT assay. Results represent triplicate values (mean ± SEM) from two independent experiments. *, P < 0.05 compared with the pcDNA3-transfected group (Student's t test).
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fig4: Overexpression of Grx1 decreases S-glutathionylation of Fas, caspase-8 and -3 activities, and cell death. (A) Assessment of Grx1 expression. C10 cells were transfected with pcDNA3 or Flag-Grx1 plasmids (1 µg/4 × 105 cells). Cells were stimulated with FasL + M2 for 2 h, and lysates were analyzed by immunoblotting for Grx1 expression. The bottom panel shows β-actin as a loading control. (B) Assessment of S-glutathionylation of Fas after overexpression of Grx1. pcDNA3 or Flag-Grx1–transfected cells were stimulated with FasL + M2, and after 2 h, S-glutathionylated proteins were immunoprecipitated using an antibody directed against glutathione (IP: PSSG). +DTT samples were used as reagent controls. The bottom panel represents Fas content in whole cell lysates (WCL). (C) Assessment of caspase-8 and -3 activities in cells after overexpression of Grx1. C10 cells were transfected with pcDNA3 or Flag-Grx1 plasmids and stimulated with FasL + M2. At the indicated times, cells were harvested, and lysates were prepared for evaluation of caspase activity. Results are expressed as mean ± SEM relative luminescence units (RLU)/20,000 cells. The graph represents triplicate values obtained from two independent experiments. *, P < 0.05 compared with FasL-treated pcDNA3-treated control groups (ANOVA). (D) Assessment of FasL-induced death in cells overexpressing Grx1. Cells were transfected with the indicated amounts of pcDNA3 or Flag-Grx1 plasmid (total DNA content 1.25 µg/4 × 105 cells) and stimulated with FasL + M2. After 2 h, cell survival was assessed using the MTT assay. Results represent triplicate values (mean ± SEM) from two independent experiments. *, P < 0.05 compared with the pcDNA3-transfected group (Student's t test).

Mentions: Because Grx1 deficiency caused an increase in FasL-induced caspase activation, Fas-SSG, and enhanced cell death, we speculated that overexpression of Grx1 would reduce levels of Fas-SSG and afford protection from FasL-induced apoptosis. Cells transfected with Flag-Grx1 were stimulated with FasL and examined for the S-glutathionylation status of Fas (Fas-SSG) by IP with antiglutathione antibody. Compared with pcDNA3 vector–transfected cells, overexpression of Grx1 (Fig. 4 A) markedly reduced Fas-SSG (Fig. 4 B) and markedly lowered the activities of caspase-8 and -3 (Fig. 4 C) induced in response to FasL. Overexpression of Grx1 also markedly enhanced survival (Fig. 4 D) in response to ligation of Fas as compared with controls.


Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas.

Anathy V, Aesif SW, Guala AS, Havermans M, Reynaert NL, Ho YS, Budd RC, Janssen-Heininger YM - J. Cell Biol. (2009)

Overexpression of Grx1 decreases S-glutathionylation of Fas, caspase-8 and -3 activities, and cell death. (A) Assessment of Grx1 expression. C10 cells were transfected with pcDNA3 or Flag-Grx1 plasmids (1 µg/4 × 105 cells). Cells were stimulated with FasL + M2 for 2 h, and lysates were analyzed by immunoblotting for Grx1 expression. The bottom panel shows β-actin as a loading control. (B) Assessment of S-glutathionylation of Fas after overexpression of Grx1. pcDNA3 or Flag-Grx1–transfected cells were stimulated with FasL + M2, and after 2 h, S-glutathionylated proteins were immunoprecipitated using an antibody directed against glutathione (IP: PSSG). +DTT samples were used as reagent controls. The bottom panel represents Fas content in whole cell lysates (WCL). (C) Assessment of caspase-8 and -3 activities in cells after overexpression of Grx1. C10 cells were transfected with pcDNA3 or Flag-Grx1 plasmids and stimulated with FasL + M2. At the indicated times, cells were harvested, and lysates were prepared for evaluation of caspase activity. Results are expressed as mean ± SEM relative luminescence units (RLU)/20,000 cells. The graph represents triplicate values obtained from two independent experiments. *, P < 0.05 compared with FasL-treated pcDNA3-treated control groups (ANOVA). (D) Assessment of FasL-induced death in cells overexpressing Grx1. Cells were transfected with the indicated amounts of pcDNA3 or Flag-Grx1 plasmid (total DNA content 1.25 µg/4 × 105 cells) and stimulated with FasL + M2. After 2 h, cell survival was assessed using the MTT assay. Results represent triplicate values (mean ± SEM) from two independent experiments. *, P < 0.05 compared with the pcDNA3-transfected group (Student's t test).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig4: Overexpression of Grx1 decreases S-glutathionylation of Fas, caspase-8 and -3 activities, and cell death. (A) Assessment of Grx1 expression. C10 cells were transfected with pcDNA3 or Flag-Grx1 plasmids (1 µg/4 × 105 cells). Cells were stimulated with FasL + M2 for 2 h, and lysates were analyzed by immunoblotting for Grx1 expression. The bottom panel shows β-actin as a loading control. (B) Assessment of S-glutathionylation of Fas after overexpression of Grx1. pcDNA3 or Flag-Grx1–transfected cells were stimulated with FasL + M2, and after 2 h, S-glutathionylated proteins were immunoprecipitated using an antibody directed against glutathione (IP: PSSG). +DTT samples were used as reagent controls. The bottom panel represents Fas content in whole cell lysates (WCL). (C) Assessment of caspase-8 and -3 activities in cells after overexpression of Grx1. C10 cells were transfected with pcDNA3 or Flag-Grx1 plasmids and stimulated with FasL + M2. At the indicated times, cells were harvested, and lysates were prepared for evaluation of caspase activity. Results are expressed as mean ± SEM relative luminescence units (RLU)/20,000 cells. The graph represents triplicate values obtained from two independent experiments. *, P < 0.05 compared with FasL-treated pcDNA3-treated control groups (ANOVA). (D) Assessment of FasL-induced death in cells overexpressing Grx1. Cells were transfected with the indicated amounts of pcDNA3 or Flag-Grx1 plasmid (total DNA content 1.25 µg/4 × 105 cells) and stimulated with FasL + M2. After 2 h, cell survival was assessed using the MTT assay. Results represent triplicate values (mean ± SEM) from two independent experiments. *, P < 0.05 compared with the pcDNA3-transfected group (Student's t test).
Mentions: Because Grx1 deficiency caused an increase in FasL-induced caspase activation, Fas-SSG, and enhanced cell death, we speculated that overexpression of Grx1 would reduce levels of Fas-SSG and afford protection from FasL-induced apoptosis. Cells transfected with Flag-Grx1 were stimulated with FasL and examined for the S-glutathionylation status of Fas (Fas-SSG) by IP with antiglutathione antibody. Compared with pcDNA3 vector–transfected cells, overexpression of Grx1 (Fig. 4 A) markedly reduced Fas-SSG (Fig. 4 B) and markedly lowered the activities of caspase-8 and -3 (Fig. 4 C) induced in response to FasL. Overexpression of Grx1 also markedly enhanced survival (Fig. 4 D) in response to ligation of Fas as compared with controls.

Bottom Line: In this study, we demonstrate that stimulation with Fas ligand (FasL) induces S-glutathionylation of Fas at cysteine 294 independently of nicotinamide adenine dinucleotide phosphate reduced oxidase-induced ROS.As a result, death-inducing signaling complex formation is also increased, and subsequent activation of caspase-8 and -3 is augmented.These results define a novel redox-based mechanism to propagate Fas-dependent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Vermont, Burlington, VT 05405, USA.

ABSTRACT
Reactive oxygen species (ROS) increase ligation of Fas (CD95), a receptor important for regulation of programmed cell death. Glutathionylation of reactive cysteines represents an oxidative modification that can be reversed by glutaredoxins (Grxs). The goal of this study was to determine whether Fas is redox regulated under physiological conditions. In this study, we demonstrate that stimulation with Fas ligand (FasL) induces S-glutathionylation of Fas at cysteine 294 independently of nicotinamide adenine dinucleotide phosphate reduced oxidase-induced ROS. Instead, Fas is S-glutathionylated after caspase-dependent degradation of Grx1, increasing subsequent caspase activation and apoptosis. Conversely, overexpression of Grx1 attenuates S-glutathionylation of Fas and partially protects against FasL-induced apoptosis. Redox-mediated Fas modification promotes its aggregation and recruitment into lipid rafts and enhances binding of FasL. As a result, death-inducing signaling complex formation is also increased, and subsequent activation of caspase-8 and -3 is augmented. These results define a novel redox-based mechanism to propagate Fas-dependent apoptosis.

Show MeSH
Related in: MedlinePlus