Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization

Abstract

Mitochondrial outer-membrane permeabilization by pro-apoptotic Bcl-2 family members plays a crucial role in apoptosis induction. However, whether this directly causes the release of the different mitochondrial apoptogenic factors simultaneously is currently unknown. Here we report that in cells or with isolated mitochondria, pro-apoptotic Bcl-2 proteins cause the release of cytochrome c, Smac/Diablo and HtrA2/Omi but not endonuclease G (EndoG) and apoptosis-inducing factor (AIF). In cells treated with Bax/Bak-dependent pro-apoptotic drugs, neither the caspase inhibitor zVAD-fmk nor loss of Apaf-1 affected the efflux of cytochrome c, Smac/Diablo and HtrA2/Omi, but both prevented the release of EndoG and AIF. Our findings identify the mitochondrial response to pro-apoptotic stimuli as a selective process leading to a hierarchical ordering of the effectors involved in cell death induction. Moreover, as in Caenorhabditis elegans, EndoG and AIF act downstream of caspase activation. Thus EndoG and AIF seem to define a 'caspase-dependent' mitochondria-initiated apoptotic DNA degradation pathway that is conserved between mammals and nematodes.

Fig. 1. Recombinant pro-apoptotic Bcl-2 members induce the release of cytochrome c, Smac/Diablo and HtrA2/Omi but not that of EndoG and AIF from isolated mitochondria. (A) Mitochondria isolated from HeLa cells were incubated for 30 min at 30°C with different concentrations (nM) of recombinant oligomeric Bax. Mitochondrial pellets and supernatant fractions were separated by SDS–PAGE (Tricine Gel), and their respective cytochrome c, Smac/Diablo, HtrA2/Omi, EndoG and AIF contents were analyzed by western blotting. (B) Mitochondria isolated from HeLa cells were incubated with 200 nM oligomeric recombinant Bax or with control buffer (none) at 30°C and the mitochondrial pellet and supernatant were analyzed at different time points (min), as in (A). (C) Mitochondria isolated from HeLa cells were incubated for 15 min with different concentrations (nM) of recombinant tBid and the mitochondrial pellet and supernatant were analyzed as in (A). In (A), (B) and (C), equal loading of the mitochondrial pellet was controlled using VDAC. The asterisks in (A), (B) and (C) indicate an additional band due to the previous VDAC detection.

Fig. 1. Recombinant pro-apoptotic Bcl-2 members induce the release of cytochrome c, Smac/Diablo and HtrA2/Omi but not that of EndoG and AIF from isolated mitochondria. (A) Mitochondria isolated from HeLa cells were incubated for 30 min at 30°C with different concentrations (nM) of recombinant oligomeric Bax. Mitochondrial pellets and supernatant fractions were separated by SDS–PAGE (Tricine Gel), and their respective cytochrome c, Smac/Diablo, HtrA2/Omi, EndoG and AIF contents were analyzed by western blotting. (B) Mitochondria isolated from HeLa cells were incubated with 200 nM oligomeric recombinant Bax or with control buffer (none) at 30°C and the mitochondrial pellet and supernatant were analyzed at different time points (min), as in (A). (C) Mitochondria isolated from HeLa cells were incubated for 15 min with different concentrations (nM) of recombinant tBid and the mitochondrial pellet and supernatant were analyzed as in (A). In (A), (B) and (C), equal loading of the mitochondrial pellet was controlled using VDAC. The asterisks in (A), (B) and (C) indicate an additional band due to the previous VDAC detection.

Fig. 1. Recombinant pro-apoptotic Bcl-2 members induce the release of cytochrome c, Smac/Diablo and HtrA2/Omi but not that of EndoG and AIF from isolated mitochondria. (A) Mitochondria isolated from HeLa cells were incubated for 30 min at 30°C with different concentrations (nM) of recombinant oligomeric Bax. Mitochondrial pellets and supernatant fractions were separated by SDS–PAGE (Tricine Gel), and their respective cytochrome c, Smac/Diablo, HtrA2/Omi, EndoG and AIF contents were analyzed by western blotting. (B) Mitochondria isolated from HeLa cells were incubated with 200 nM oligomeric recombinant Bax or with control buffer (none) at 30°C and the mitochondrial pellet and supernatant were analyzed at different time points (min), as in (A). (C) Mitochondria isolated from HeLa cells were incubated for 15 min with different concentrations (nM) of recombinant tBid and the mitochondrial pellet and supernatant were analyzed as in (A). In (A), (B) and (C), equal loading of the mitochondrial pellet was controlled using VDAC. The asterisks in (A), (B) and (C) indicate an additional band due to the previous VDAC detection.

Fig. 2. ΔΨm analysis in isolated mitochondria incubated with recombinant pro-apoptotic Bcl-2 members. Mitochondria isolated from HeLa cells were incubated with different concentrations (nM) of (A) recombinant oligomeric Bax or (B) recombinant tBid as in Figure 1. Then, ΔΨm was measured by flow cytometry using Rh123 (50 nM) as a probe. Incubation of isolated mitochondria with CCCP (10 µM) was used as a control. As in Figure 1, a fraction of mitochondrial pellets and supernatant fractions was also analyzed by western blotting. Blue arrows indicate a significant release of cytochrome c, Smac/Diablo and HtrA2/Omi (>50% release) and red arrows indicate complete or nearly complete release (>90% release).

Fig. 2. ΔΨm analysis in isolated mitochondria incubated with recombinant pro-apoptotic Bcl-2 members. Mitochondria isolated from HeLa cells were incubated with different concentrations (nM) of (A) recombinant oligomeric Bax or (B) recombinant tBid as in Figure 1. Then, ΔΨm was measured by flow cytometry using Rh123 (50 nM) as a probe. Incubation of isolated mitochondria with CCCP (10 µM) was used as a control. As in Figure 1, a fraction of mitochondrial pellets and supernatant fractions was also analyzed by western blotting. Blue arrows indicate a significant release of cytochrome c, Smac/Diablo and HtrA2/Omi (>50% release) and red arrows indicate complete or nearly complete release (>90% release).

Fig. 3. Caspase inhibition by zVAD-fmk prevents the mitochondrial release of EndoG and AIF but not that of cytochrome c and Smac/Diablo during Bax overexpression. (A) GFP-Bax expression and immunostaining of cytochrome c, Smac/Diablo, EndoG and AIF together with nuclear Hoechst staining in HeLa cells 18 h after transient transfection with a vector encoding GFP-Bax in the absence or in the presence of the caspase inhibitor z-VAD-fmk (100 µM). (B) Quantitative analysis of the numbers of GFP-Bax transfected cells with intracytosolic release of cytochrome c, Smac/Diablo, EndoG or AIF in the absence or presence of z-VAD-fmk. Each histogram indicates mean ± SD of three fields of at least 100 cells within a representative experiment.

Fig. 3. Caspase inhibition by zVAD-fmk prevents the mitochondrial release of EndoG and AIF but not that of cytochrome c and Smac/Diablo during Bax overexpression. (A) GFP-Bax expression and immunostaining of cytochrome c, Smac/Diablo, EndoG and AIF together with nuclear Hoechst staining in HeLa cells 18 h after transient transfection with a vector encoding GFP-Bax in the absence or in the presence of the caspase inhibitor z-VAD-fmk (100 µM). (B) Quantitative analysis of the numbers of GFP-Bax transfected cells with intracytosolic release of cytochrome c, Smac/Diablo, EndoG or AIF in the absence or presence of z-VAD-fmk. Each histogram indicates mean ± SD of three fields of at least 100 cells within a representative experiment.

Fig. 3. Caspase inhibition by zVAD-fmk prevents the mitochondrial release of EndoG and AIF but not that of cytochrome c and Smac/Diablo during Bax overexpression. (A) GFP-Bax expression and immunostaining of cytochrome c, Smac/Diablo, EndoG and AIF together with nuclear Hoechst staining in HeLa cells 18 h after transient transfection with a vector encoding GFP-Bax in the absence or in the presence of the caspase inhibitor z-VAD-fmk (100 µM). (B) Quantitative analysis of the numbers of GFP-Bax transfected cells with intracytosolic release of cytochrome c, Smac/Diablo, EndoG or AIF in the absence or presence of z-VAD-fmk. Each histogram indicates mean ± SD of three fields of at least 100 cells within a representative experiment.

Fig. 4. Cell-stress-associated EndoG and AIF release is prevented by zVAD-fmk, unlike cytochrome c, Smac/Diablo and HtrA2/Omi. (A) Percentages of cells with apoptotic nuclei (percentage of apoptotic nuclei) in HeLa cells treated for 7 and 9 h with staurosporine (STS, 2 µM) or actinomycin D (ActD, 20 µM) in the absence or presence of zVAD-fmk (100 µM). (B) Cells were treated as in (A). Total cell extracts were analyzed by western blotting for caspase-9 and caspase-3 processing and PARP cleavage. (C) HeLa cells were treated as in (A). Cytosolic fraction and heavy membrane fraction were analyzed by western blotting for the presence of cytochrome c, Smac/Diablo, HtrA2/Omi, EndoG and AIF. As control for loading, actin was used in the cytosolic fraction and Cox IV in the heavy membrane fraction. (D) HeLa cells were treated as in (A) and then ΔΨm was assessed by flow cytometry using DiOC₆ (50 nM). Top: % indicates percentage of ΔΨm loss. Bottom: histogram showing the percentage of ΔΨm loss in three independent experiments. The asterisks indicate additional non-specific bands.

Fig. 4. Cell-stress-associated EndoG and AIF release is prevented by zVAD-fmk, unlike cytochrome c, Smac/Diablo and HtrA2/Omi. (A) Percentages of cells with apoptotic nuclei (percentage of apoptotic nuclei) in HeLa cells treated for 7 and 9 h with staurosporine (STS, 2 µM) or actinomycin D (ActD, 20 µM) in the absence or presence of zVAD-fmk (100 µM). (B) Cells were treated as in (A). Total cell extracts were analyzed by western blotting for caspase-9 and caspase-3 processing and PARP cleavage. (C) HeLa cells were treated as in (A). Cytosolic fraction and heavy membrane fraction were analyzed by western blotting for the presence of cytochrome c, Smac/Diablo, HtrA2/Omi, EndoG and AIF. As control for loading, actin was used in the cytosolic fraction and Cox IV in the heavy membrane fraction. (D) HeLa cells were treated as in (A) and then ΔΨm was assessed by flow cytometry using DiOC₆ (50 nM). Top: % indicates percentage of ΔΨm loss. Bottom: histogram showing the percentage of ΔΨm loss in three independent experiments. The asterisks indicate additional non-specific bands.

Fig. 4. Cell-stress-associated EndoG and AIF release is prevented by zVAD-fmk, unlike cytochrome c, Smac/Diablo and HtrA2/Omi. (A) Percentages of cells with apoptotic nuclei (percentage of apoptotic nuclei) in HeLa cells treated for 7 and 9 h with staurosporine (STS, 2 µM) or actinomycin D (ActD, 20 µM) in the absence or presence of zVAD-fmk (100 µM). (B) Cells were treated as in (A). Total cell extracts were analyzed by western blotting for caspase-9 and caspase-3 processing and PARP cleavage. (C) HeLa cells were treated as in (A). Cytosolic fraction and heavy membrane fraction were analyzed by western blotting for the presence of cytochrome c, Smac/Diablo, HtrA2/Omi, EndoG and AIF. As control for loading, actin was used in the cytosolic fraction and Cox IV in the heavy membrane fraction. (D) HeLa cells were treated as in (A) and then ΔΨm was assessed by flow cytometry using DiOC₆ (50 nM). Top: % indicates percentage of ΔΨm loss. Bottom: histogram showing the percentage of ΔΨm loss in three independent experiments. The asterisks indicate additional non-specific bands.

Fig. 5. Cell-stress-associated cytochrome c and Smac/Diablo release is insensitive to zVAD-fmk, unlike EndoG and AIF. HeLa cells were treated for 8 h with actinomycin D (20 µM) or staurosporine (2 µM) in the presence of zVAD-fmk (100 µM). The cells were immunostained with (A) anti-cytochrome c and anti-EndoG antibodies or (B) anti-cytochrome c and anti-AIF or (C) anti-cytochrome c and anti-Smac/Diablo together with Hoechst nuclear staining. Then a quantitative analysis of the numbers of actinomycin D- or staurosporine-treated cells in the presence of z-VAD-fmk (100 µM) with intracytosolic release of (A) cytochrome c and/or EndoG or (B) AIF or (C) Smac/Diablo was performed. Each histogram indicates mean ± SD of three fields of at least 100 cells within a representative experiment.

Fig. 5. Cell-stress-associated cytochrome c and Smac/Diablo release is insensitive to zVAD-fmk, unlike EndoG and AIF. HeLa cells were treated for 8 h with actinomycin D (20 µM) or staurosporine (2 µM) in the presence of zVAD-fmk (100 µM). The cells were immunostained with (A) anti-cytochrome c and anti-EndoG antibodies or (B) anti-cytochrome c and anti-AIF or (C) anti-cytochrome c and anti-Smac/Diablo together with Hoechst nuclear staining. Then a quantitative analysis of the numbers of actinomycin D- or staurosporine-treated cells in the presence of z-VAD-fmk (100 µM) with intracytosolic release of (A) cytochrome c and/or EndoG or (B) AIF or (C) Smac/Diablo was performed. Each histogram indicates mean ± SD of three fields of at least 100 cells within a representative experiment.

Fig. 5. Cell-stress-associated cytochrome c and Smac/Diablo release is insensitive to zVAD-fmk, unlike EndoG and AIF. HeLa cells were treated for 8 h with actinomycin D (20 µM) or staurosporine (2 µM) in the presence of zVAD-fmk (100 µM). The cells were immunostained with (A) anti-cytochrome c and anti-EndoG antibodies or (B) anti-cytochrome c and anti-AIF or (C) anti-cytochrome c and anti-Smac/Diablo together with Hoechst nuclear staining. Then a quantitative analysis of the numbers of actinomycin D- or staurosporine-treated cells in the presence of z-VAD-fmk (100 µM) with intracytosolic release of (A) cytochrome c and/or EndoG or (B) AIF or (C) Smac/Diablo was performed. Each histogram indicates mean ± SD of three fields of at least 100 cells within a representative experiment.

Fig. 6. Bax/Bak-mediated mitochondrial permeabilization does not induce the release of nuclear effectors of apoptosis. (A) HeLa cells were either left untreated (Control) or treated for 8 h with staurosporine (2 µM) or actinomycin D (20 µM) in the presence of zVAD-fmk (100 µM). Then cells were immunostained with a sheep anti-cytochrome c, a mouse anti-AIF and a rabbit anti-EndoG together with Hoechst nuclear staining. (B) Flow cytometry analysis of DNA degradation in isolated nuclei from CEM cells after incubation for 6 h with supernatants from isolated HeLa mitochondria that had been incubated for 30 min with either 200 nM oligomeric recombinant Bax (Mitochondria + Bax) or control buffer (Control). As a positive control for mitochondrial effectors of DNA degradation, whole mitochondria extracts (Mitochondrial Rupture) were used (% indicates percentage of DNA degradation). (C) EndoG and AIF are not soluble in the mitochondrial inner-membrane space. HeLa mitochondria, mitoplasts and mitoplasts treated with sodium carbonate (Na₂CO₃) were resolved by SDS–PAGE, and their respective contents in cytochrome c, EndoG, AIF and Cox IV were analyzed by western blotting. To show that EndoG is not simply precipitated by carbonate, mitoplasts were also treated with sodium carbonate and proteinase K.

Fig. 6. Bax/Bak-mediated mitochondrial permeabilization does not induce the release of nuclear effectors of apoptosis. (A) HeLa cells were either left untreated (Control) or treated for 8 h with staurosporine (2 µM) or actinomycin D (20 µM) in the presence of zVAD-fmk (100 µM). Then cells were immunostained with a sheep anti-cytochrome c, a mouse anti-AIF and a rabbit anti-EndoG together with Hoechst nuclear staining. (B) Flow cytometry analysis of DNA degradation in isolated nuclei from CEM cells after incubation for 6 h with supernatants from isolated HeLa mitochondria that had been incubated for 30 min with either 200 nM oligomeric recombinant Bax (Mitochondria + Bax) or control buffer (Control). As a positive control for mitochondrial effectors of DNA degradation, whole mitochondria extracts (Mitochondrial Rupture) were used (% indicates percentage of DNA degradation). (C) EndoG and AIF are not soluble in the mitochondrial inner-membrane space. HeLa mitochondria, mitoplasts and mitoplasts treated with sodium carbonate (Na₂CO₃) were resolved by SDS–PAGE, and their respective contents in cytochrome c, EndoG, AIF and Cox IV were analyzed by western blotting. To show that EndoG is not simply precipitated by carbonate, mitoplasts were also treated with sodium carbonate and proteinase K.

Fig. 6. Bax/Bak-mediated mitochondrial permeabilization does not induce the release of nuclear effectors of apoptosis. (A) HeLa cells were either left untreated (Control) or treated for 8 h with staurosporine (2 µM) or actinomycin D (20 µM) in the presence of zVAD-fmk (100 µM). Then cells were immunostained with a sheep anti-cytochrome c, a mouse anti-AIF and a rabbit anti-EndoG together with Hoechst nuclear staining. (B) Flow cytometry analysis of DNA degradation in isolated nuclei from CEM cells after incubation for 6 h with supernatants from isolated HeLa mitochondria that had been incubated for 30 min with either 200 nM oligomeric recombinant Bax (Mitochondria + Bax) or control buffer (Control). As a positive control for mitochondrial effectors of DNA degradation, whole mitochondria extracts (Mitochondrial Rupture) were used (% indicates percentage of DNA degradation). (C) EndoG and AIF are not soluble in the mitochondrial inner-membrane space. HeLa mitochondria, mitoplasts and mitoplasts treated with sodium carbonate (Na₂CO₃) were resolved by SDS–PAGE, and their respective contents in cytochrome c, EndoG, AIF and Cox IV were analyzed by western blotting. To show that EndoG is not simply precipitated by carbonate, mitoplasts were also treated with sodium carbonate and proteinase K.

Fig. 7. EndoG and AIF release requires caspase activation downstream of cytochrome c release. Apaf +/– or –/– MEFs were treated for 24 h with staurosporine (STS, 10 µM) or actinomycin D (ActD, 40 µM). Next, the cells were immunostained with either (A) anti-cytochrome c and anti-EndoG antibodies or (B) anti-cytochrome c and anti-AIF together with Hoechst nuclear staining. Then a quantitative analysis of the numbers of actinomycin D or staurosporine-treated MEFs with intracytosolic release of cytochrome c and/or (A) EndoG or (B) AIF was performed. Each histogram indicates mean ± SD of three fields of at least 100 cells within a representative experiment.

Fig. 7. EndoG and AIF release requires caspase activation downstream of cytochrome c release. Apaf +/– or –/– MEFs were treated for 24 h with staurosporine (STS, 10 µM) or actinomycin D (ActD, 40 µM). Next, the cells were immunostained with either (A) anti-cytochrome c and anti-EndoG antibodies or (B) anti-cytochrome c and anti-AIF together with Hoechst nuclear staining. Then a quantitative analysis of the numbers of actinomycin D or staurosporine-treated MEFs with intracytosolic release of cytochrome c and/or (A) EndoG or (B) AIF was performed. Each histogram indicates mean ± SD of three fields of at least 100 cells within a representative experiment.

Fig. 7. EndoG and AIF release requires caspase activation downstream of cytochrome c release. Apaf +/– or –/– MEFs were treated for 24 h with staurosporine (STS, 10 µM) or actinomycin D (ActD, 40 µM). Next, the cells were immunostained with either (A) anti-cytochrome c and anti-EndoG antibodies or (B) anti-cytochrome c and anti-AIF together with Hoechst nuclear staining. Then a quantitative analysis of the numbers of actinomycin D or staurosporine-treated MEFs with intracytosolic release of cytochrome c and/or (A) EndoG or (B) AIF was performed. Each histogram indicates mean ± SD of three fields of at least 100 cells within a representative experiment.

Fig. 7. EndoG and AIF release requires caspase activation downstream of cytochrome c release. Apaf +/– or –/– MEFs were treated for 24 h with staurosporine (STS, 10 µM) or actinomycin D (ActD, 40 µM). Next, the cells were immunostained with either (A) anti-cytochrome c and anti-EndoG antibodies or (B) anti-cytochrome c and anti-AIF together with Hoechst nuclear staining. Then a quantitative analysis of the numbers of actinomycin D or staurosporine-treated MEFs with intracytosolic release of cytochrome c and/or (A) EndoG or (B) AIF was performed. Each histogram indicates mean ± SD of three fields of at least 100 cells within a representative experiment.

Fig. 8. EndoG and AIF define a caspase-dependent mitochondria-initiated apoptotic DNA degradation pathway that is conserved between mammals and C.elegans. The genetic control of apoptosis leading to caspase activation is conserved between C.elegans and mammals (Horvitz, 1999; Hengartner, 2000). In both models, active caspases trigger the mitochondrial release of EndoG and AIF (Cps-6 and Wah-1 respectively in C.elegans) that are involved in DNA degradation. In mammals, CAD and Acinus, two other factors involved in nuclear apoptosis are activated by caspases (Sahara et al., 1999; Nagata, 2000). The dashed line indicates that the inhibition of Apaf-1 by Bcl-2 or Bcl-XL is not direct.