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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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Increased S-glutathionylation of Fas, caspase-8 activity, and cell death in cells lacking Grx1. (A) Assessment of S-glutathionylation of Fas after knockdown of Grx1. C10 cells were transfected with Grx1 siRNA or control (Ctr) siRNA and treated with FasL + M2 for the indicated times. S-glutathionylated proteins were immunoprecipitated using antiglutathione antibody (IP: PSSG). Samples treated with 50 mM DTT to reduce S-glutathionylated proteins (+DTT) were used as reagent controls. The content of Fas, Grx1, and actin in whole cell lysates (WCL) used as the input for IP are shown in the bottom panels. (B) Assessment of S-glutathionylation of Fas after loading of cells with biotinylated glutathione. siRNA-transfected cells were labeled with 5 mM biotinylated glutathione ethyl ester for 1 h before treatment with FasL. After 2 h of FasL + M2 treatment, glutathionylated proteins in lysates were immunoprecipitated using antibiotin antibody followed by immunoblot detection of Fas. The bottom panel shows total Fas expression in whole cell lysates as a loading control. (C) Evaluation of caspase-8 and -3 enzymatic activities in cells after knockdown of Grx1. C10 cells were transfected with control or Grx1 siRNA before stimulation with FasL + M2 for 1 h, 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 respective control siRNA groups (ANOVA). (D) Impact of Grx1 knockdown on cell survival. Cells were transfected with control or Grx1 siRNA and exposed as described in A. Cell survival was assessed using the MTT assay. Results are expressed at a percent of survival compared with respective untreated control groups and are presented as mean values + SEM of triplicate values obtained from two independent experiments. Note that survival in the Grx1 siRNA–treated cells was 11% lower than the control siRNA–treated cells in the absence of stimulation with FasL. *, P < 0.05 compared with respective control siRNA groups (ANOVA). (E) Assessment of S-glutathionylation of Fas in lung fibroblasts lacking Glrx1. WT or Glrx1−/− cells were exposed to FasL + M2 for the indicated times, and S-glutathionylation of Fas was determined as described in A. All samples were run on the same gel, and the lanes were cut and reassembled for consistency. Note that S-glutathionylation of Fas in these primary cells is relatively protracted compared with results obtained in the C10 cell line. Black line indicates that intervening lanes have been spliced out. (F) Assessment of S-glutathionylation of Fas in CD4+ T lymphocytes lacking Glrx1. WT or Glrx1−/− cells were exposed to FasL + M2 for the indicated times, and S-glutathionylation of Fas was determined as in A. The bottom panels show content of Fas and Grx1 in whole cell lysates. (G) Caspase-8 and -3 activities in primary lung fibroblasts isolated from WT or Glrx1−/− mice in response to exposure to FasL + M2 for the indicated times. 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 respective WT groups (ANOVA). Note that activation of caspases in these primary cells is relatively protracted compared with results obtained in the C10 cell line. (H) Comparative assessment of FasL-induced cell death in WT or Glrx1−/− primary lung fibroblasts. Cells were exposed as indicated, and survival was assessed using the MTT assay. Results are expressed as a percent of survival compared with respective untreated control groups and are presented as mean values + SEM of triplicate values obtained from two independent experiments. Note that survival in the Glrx1−/− cells was 13% lower than their WT counterparts in the absence of stimulation with FasL. *, P < 0.05 compared with the WT FasL-treated group (ANOVA). Note that FasL-induced cell death in the primary cells is relatively protracted compared with results obtained in the C10 cell line.
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fig3: Increased S-glutathionylation of Fas, caspase-8 activity, and cell death in cells lacking Grx1. (A) Assessment of S-glutathionylation of Fas after knockdown of Grx1. C10 cells were transfected with Grx1 siRNA or control (Ctr) siRNA and treated with FasL + M2 for the indicated times. S-glutathionylated proteins were immunoprecipitated using antiglutathione antibody (IP: PSSG). Samples treated with 50 mM DTT to reduce S-glutathionylated proteins (+DTT) were used as reagent controls. The content of Fas, Grx1, and actin in whole cell lysates (WCL) used as the input for IP are shown in the bottom panels. (B) Assessment of S-glutathionylation of Fas after loading of cells with biotinylated glutathione. siRNA-transfected cells were labeled with 5 mM biotinylated glutathione ethyl ester for 1 h before treatment with FasL. After 2 h of FasL + M2 treatment, glutathionylated proteins in lysates were immunoprecipitated using antibiotin antibody followed by immunoblot detection of Fas. The bottom panel shows total Fas expression in whole cell lysates as a loading control. (C) Evaluation of caspase-8 and -3 enzymatic activities in cells after knockdown of Grx1. C10 cells were transfected with control or Grx1 siRNA before stimulation with FasL + M2 for 1 h, 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 respective control siRNA groups (ANOVA). (D) Impact of Grx1 knockdown on cell survival. Cells were transfected with control or Grx1 siRNA and exposed as described in A. Cell survival was assessed using the MTT assay. Results are expressed at a percent of survival compared with respective untreated control groups and are presented as mean values + SEM of triplicate values obtained from two independent experiments. Note that survival in the Grx1 siRNA–treated cells was 11% lower than the control siRNA–treated cells in the absence of stimulation with FasL. *, P < 0.05 compared with respective control siRNA groups (ANOVA). (E) Assessment of S-glutathionylation of Fas in lung fibroblasts lacking Glrx1. WT or Glrx1−/− cells were exposed to FasL + M2 for the indicated times, and S-glutathionylation of Fas was determined as described in A. All samples were run on the same gel, and the lanes were cut and reassembled for consistency. Note that S-glutathionylation of Fas in these primary cells is relatively protracted compared with results obtained in the C10 cell line. Black line indicates that intervening lanes have been spliced out. (F) Assessment of S-glutathionylation of Fas in CD4+ T lymphocytes lacking Glrx1. WT or Glrx1−/− cells were exposed to FasL + M2 for the indicated times, and S-glutathionylation of Fas was determined as in A. The bottom panels show content of Fas and Grx1 in whole cell lysates. (G) Caspase-8 and -3 activities in primary lung fibroblasts isolated from WT or Glrx1−/− mice in response to exposure to FasL + M2 for the indicated times. 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 respective WT groups (ANOVA). Note that activation of caspases in these primary cells is relatively protracted compared with results obtained in the C10 cell line. (H) Comparative assessment of FasL-induced cell death in WT or Glrx1−/− primary lung fibroblasts. Cells were exposed as indicated, and survival was assessed using the MTT assay. Results are expressed as a percent of survival compared with respective untreated control groups and are presented as mean values + SEM of triplicate values obtained from two independent experiments. Note that survival in the Glrx1−/− cells was 13% lower than their WT counterparts in the absence of stimulation with FasL. *, P < 0.05 compared with the WT FasL-treated group (ANOVA). Note that FasL-induced cell death in the primary cells is relatively protracted compared with results obtained in the C10 cell line.

Mentions: We next examined the impact of modulation of Grx1 on Fas-SSG and the subsequent sensitivity of cells to undergo apoptosis. Transfection of lung epithelial cells with a Grx1-specific siRNA caused a marked decrease in the cellular content of Grx1 (Fig. 3 A). Two independent approaches were used to assess Fas-SSG that encompassed IP with an antibody against glutathione (Fig. 3 A) or preloading of cells with a biotinylated version of glutathione followed by incubation with an antibiotin antibody to IP S-glutathionylated proteins (Fig. 3 B). Results demonstrate that FasL-induced Fas-SSG was enhanced in cells with a lowered expression of Grx1 as compared with controls (Fig. 3, A and B). We were also able to detect Fas-SSG under nonstimulated conditions and increases after knockdown of Grx1 only after labeling of cells with biotinylated glutathione (Fig. 3 B), presumably because of the enhanced sensitivity of detection of PSSG using the latter approach as compared with the antiglutathione antibody. In addition to increasing Fas-SSG, FasL-stimulated activation of caspase-8 and -3 was also enhanced in cells subjected to Grx1 siRNA knockdown compared with controls (Fig. 3 C). Cells with lowered Grx1 content were less viable compared with controls, and Grx1 siRNA–treated cells were more sensitive to FasL-induced death than controls (Fig. 3 D). Similarly, in comparison to C57BL6 (wild type [WT]) controls, primary cultures of lung fibroblasts (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200807019/DC1) or proliferating CD4+ T lymphocytes derived from Glrx1−/− mice (Ho et al., 2007) demonstrated a higher extent of Fas-SSG upon receptor ligation (Fig. 3, E and F). Although we were unable to detect S-glutathionylation of Fas in the absence of FasL in lung fibroblasts (Fig. 3 E), possibly as a result of the aforementioned detection limits of our assay, baseline Fas-SSG was detected in Glrx1−/− CD4+ T lymphocytes before stimulation with FasL (Fig. 3 F). Enhanced FasL-induced activation of caspase-8 and -3 was readily apparent in Glrx1−/− fibroblasts, which were also less viable before, or in response to Fas ligation compared with WT counterparts (Fig. 3, G and H).


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)

Increased S-glutathionylation of Fas, caspase-8 activity, and cell death in cells lacking Grx1. (A) Assessment of S-glutathionylation of Fas after knockdown of Grx1. C10 cells were transfected with Grx1 siRNA or control (Ctr) siRNA and treated with FasL + M2 for the indicated times. S-glutathionylated proteins were immunoprecipitated using antiglutathione antibody (IP: PSSG). Samples treated with 50 mM DTT to reduce S-glutathionylated proteins (+DTT) were used as reagent controls. The content of Fas, Grx1, and actin in whole cell lysates (WCL) used as the input for IP are shown in the bottom panels. (B) Assessment of S-glutathionylation of Fas after loading of cells with biotinylated glutathione. siRNA-transfected cells were labeled with 5 mM biotinylated glutathione ethyl ester for 1 h before treatment with FasL. After 2 h of FasL + M2 treatment, glutathionylated proteins in lysates were immunoprecipitated using antibiotin antibody followed by immunoblot detection of Fas. The bottom panel shows total Fas expression in whole cell lysates as a loading control. (C) Evaluation of caspase-8 and -3 enzymatic activities in cells after knockdown of Grx1. C10 cells were transfected with control or Grx1 siRNA before stimulation with FasL + M2 for 1 h, 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 respective control siRNA groups (ANOVA). (D) Impact of Grx1 knockdown on cell survival. Cells were transfected with control or Grx1 siRNA and exposed as described in A. Cell survival was assessed using the MTT assay. Results are expressed at a percent of survival compared with respective untreated control groups and are presented as mean values + SEM of triplicate values obtained from two independent experiments. Note that survival in the Grx1 siRNA–treated cells was 11% lower than the control siRNA–treated cells in the absence of stimulation with FasL. *, P < 0.05 compared with respective control siRNA groups (ANOVA). (E) Assessment of S-glutathionylation of Fas in lung fibroblasts lacking Glrx1. WT or Glrx1−/− cells were exposed to FasL + M2 for the indicated times, and S-glutathionylation of Fas was determined as described in A. All samples were run on the same gel, and the lanes were cut and reassembled for consistency. Note that S-glutathionylation of Fas in these primary cells is relatively protracted compared with results obtained in the C10 cell line. Black line indicates that intervening lanes have been spliced out. (F) Assessment of S-glutathionylation of Fas in CD4+ T lymphocytes lacking Glrx1. WT or Glrx1−/− cells were exposed to FasL + M2 for the indicated times, and S-glutathionylation of Fas was determined as in A. The bottom panels show content of Fas and Grx1 in whole cell lysates. (G) Caspase-8 and -3 activities in primary lung fibroblasts isolated from WT or Glrx1−/− mice in response to exposure to FasL + M2 for the indicated times. 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 respective WT groups (ANOVA). Note that activation of caspases in these primary cells is relatively protracted compared with results obtained in the C10 cell line. (H) Comparative assessment of FasL-induced cell death in WT or Glrx1−/− primary lung fibroblasts. Cells were exposed as indicated, and survival was assessed using the MTT assay. Results are expressed as a percent of survival compared with respective untreated control groups and are presented as mean values + SEM of triplicate values obtained from two independent experiments. Note that survival in the Glrx1−/− cells was 13% lower than their WT counterparts in the absence of stimulation with FasL. *, P < 0.05 compared with the WT FasL-treated group (ANOVA). Note that FasL-induced cell death in the primary cells is relatively protracted compared with results obtained in the C10 cell line.
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fig3: Increased S-glutathionylation of Fas, caspase-8 activity, and cell death in cells lacking Grx1. (A) Assessment of S-glutathionylation of Fas after knockdown of Grx1. C10 cells were transfected with Grx1 siRNA or control (Ctr) siRNA and treated with FasL + M2 for the indicated times. S-glutathionylated proteins were immunoprecipitated using antiglutathione antibody (IP: PSSG). Samples treated with 50 mM DTT to reduce S-glutathionylated proteins (+DTT) were used as reagent controls. The content of Fas, Grx1, and actin in whole cell lysates (WCL) used as the input for IP are shown in the bottom panels. (B) Assessment of S-glutathionylation of Fas after loading of cells with biotinylated glutathione. siRNA-transfected cells were labeled with 5 mM biotinylated glutathione ethyl ester for 1 h before treatment with FasL. After 2 h of FasL + M2 treatment, glutathionylated proteins in lysates were immunoprecipitated using antibiotin antibody followed by immunoblot detection of Fas. The bottom panel shows total Fas expression in whole cell lysates as a loading control. (C) Evaluation of caspase-8 and -3 enzymatic activities in cells after knockdown of Grx1. C10 cells were transfected with control or Grx1 siRNA before stimulation with FasL + M2 for 1 h, 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 respective control siRNA groups (ANOVA). (D) Impact of Grx1 knockdown on cell survival. Cells were transfected with control or Grx1 siRNA and exposed as described in A. Cell survival was assessed using the MTT assay. Results are expressed at a percent of survival compared with respective untreated control groups and are presented as mean values + SEM of triplicate values obtained from two independent experiments. Note that survival in the Grx1 siRNA–treated cells was 11% lower than the control siRNA–treated cells in the absence of stimulation with FasL. *, P < 0.05 compared with respective control siRNA groups (ANOVA). (E) Assessment of S-glutathionylation of Fas in lung fibroblasts lacking Glrx1. WT or Glrx1−/− cells were exposed to FasL + M2 for the indicated times, and S-glutathionylation of Fas was determined as described in A. All samples were run on the same gel, and the lanes were cut and reassembled for consistency. Note that S-glutathionylation of Fas in these primary cells is relatively protracted compared with results obtained in the C10 cell line. Black line indicates that intervening lanes have been spliced out. (F) Assessment of S-glutathionylation of Fas in CD4+ T lymphocytes lacking Glrx1. WT or Glrx1−/− cells were exposed to FasL + M2 for the indicated times, and S-glutathionylation of Fas was determined as in A. The bottom panels show content of Fas and Grx1 in whole cell lysates. (G) Caspase-8 and -3 activities in primary lung fibroblasts isolated from WT or Glrx1−/− mice in response to exposure to FasL + M2 for the indicated times. 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 respective WT groups (ANOVA). Note that activation of caspases in these primary cells is relatively protracted compared with results obtained in the C10 cell line. (H) Comparative assessment of FasL-induced cell death in WT or Glrx1−/− primary lung fibroblasts. Cells were exposed as indicated, and survival was assessed using the MTT assay. Results are expressed as a percent of survival compared with respective untreated control groups and are presented as mean values + SEM of triplicate values obtained from two independent experiments. Note that survival in the Glrx1−/− cells was 13% lower than their WT counterparts in the absence of stimulation with FasL. *, P < 0.05 compared with the WT FasL-treated group (ANOVA). Note that FasL-induced cell death in the primary cells is relatively protracted compared with results obtained in the C10 cell line.
Mentions: We next examined the impact of modulation of Grx1 on Fas-SSG and the subsequent sensitivity of cells to undergo apoptosis. Transfection of lung epithelial cells with a Grx1-specific siRNA caused a marked decrease in the cellular content of Grx1 (Fig. 3 A). Two independent approaches were used to assess Fas-SSG that encompassed IP with an antibody against glutathione (Fig. 3 A) or preloading of cells with a biotinylated version of glutathione followed by incubation with an antibiotin antibody to IP S-glutathionylated proteins (Fig. 3 B). Results demonstrate that FasL-induced Fas-SSG was enhanced in cells with a lowered expression of Grx1 as compared with controls (Fig. 3, A and B). We were also able to detect Fas-SSG under nonstimulated conditions and increases after knockdown of Grx1 only after labeling of cells with biotinylated glutathione (Fig. 3 B), presumably because of the enhanced sensitivity of detection of PSSG using the latter approach as compared with the antiglutathione antibody. In addition to increasing Fas-SSG, FasL-stimulated activation of caspase-8 and -3 was also enhanced in cells subjected to Grx1 siRNA knockdown compared with controls (Fig. 3 C). Cells with lowered Grx1 content were less viable compared with controls, and Grx1 siRNA–treated cells were more sensitive to FasL-induced death than controls (Fig. 3 D). Similarly, in comparison to C57BL6 (wild type [WT]) controls, primary cultures of lung fibroblasts (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200807019/DC1) or proliferating CD4+ T lymphocytes derived from Glrx1−/− mice (Ho et al., 2007) demonstrated a higher extent of Fas-SSG upon receptor ligation (Fig. 3, E and F). Although we were unable to detect S-glutathionylation of Fas in the absence of FasL in lung fibroblasts (Fig. 3 E), possibly as a result of the aforementioned detection limits of our assay, baseline Fas-SSG was detected in Glrx1−/− CD4+ T lymphocytes before stimulation with FasL (Fig. 3 F). Enhanced FasL-induced activation of caspase-8 and -3 was readily apparent in Glrx1−/− fibroblasts, which were also less viable before, or in response to Fas ligation compared with WT counterparts (Fig. 3, G and H).

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