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Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells.

Shimizu S, Matsuoka Y, Shinohara Y, Yoneda Y, Tsujimoto Y - J. Cell Biol. (2001)

Bottom Line: Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release.When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death.Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Osaka University Graduate School of Medicine, Biomedical Research Center, Department of Medical Genetics, Osaka 565-0871, Japan.

ABSTRACT
Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

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Inhibition of Bax-induced Δψ loss and cytochrome c release in isolated mitochondria by anti-VDAC antibodies. (A) Inhibition of Bax-induced Δψ loss by Ab#20 and Ab#25. Mitochondria (1 mg/ml) were preincubated with or without 0.6 μg/μl of the indicated antibodies (Ab#20, Ab#25, 31HL, or NRI) for 5 min, after which rBax (0.2 μg/μl) was added. Then Δψ was measured from the rhodamine 123 (Rh123) uptake over 25 min. When Δψ dropped, rhodamine 123 was released, resulting in an increase of rhodamine 123 intensity. Complete loss of Δψ was demonstrated by incubation of the mitochondria with 1 mM carbonylcyanide m-chlorophenylhydrazone (CCCP, protonophore). Data are representative of three independent experiments. (B) Inhibition of Bax-induced cytochrome c release by Ab#20 and Ab#25. Mitochondria (1 mg/ml) were preincubated with or without the indicated concentrations of antibodies (Ab#20, Ab#25-1, Ab#25-2, 31HL, or NRI) for 5 min, after which rBax (0.2 μg/μl) or Ca2+ (50 μM) was added (top 5 panels). Mitochondria preincubated with antibodies were also incubated with rBax in the presence of 0.2 mM EGTA (bottom panel). In the presence of EGTA, a higher concentration of rBax (1 μg/μl) was used to induce cytochrome c release comparable to that without EGTA. The extent of cytochrome c release was measured at 10 min (second, third, fifth, and bottom panels) or at the indicated times (top and fourth panels) by Western blot analysis of the supernatants. “Total” represents the total amount of cytochrome c in the same amount of mitochondria. Data are representative of two or three independent experiments. (C) Immunostaining of mitochondria with Ab#25. Mitochondria (1 μg/μl) were incubated with Ab#25 (open circles) or NRI (filled circle) at the indicated concentrations, and then stained with anti–rabbit IgG-Alexa488, after which the fluorescence was measured by flow cytometry as described in Materials and Methods. Data are shown as the mean ± SD for three independent experiments. (D) Lack of effect of Ab#20 and Ab#25 on mitochondrial respiration. Mitochondria (1 μg/μl) were incubated with 0.6 μg/μl of the indicated antibodies for 5 min, and then respiration was measured in the presence of 5 mM succinate (state IV; white bars) or succinate plus 0.3 mM ADP (state III; black bars). Data are representative of two independent experiments. (E) Lack of effect of Ab#20 and Ab#25 on mitochondrial association of Bax. Mitochondria were treated as described in A. At 10 min after addition of rBax, the mitochondria were spun, and the supernatants (sup) and pellets (pt) were subjected to Western blot analysis for Bax detection. (F) Lack of effect of Ab#25 on Bax–VDAC interaction. Mitochondria were treated as described in A. At 10 min after addition of rBax, mitochondria were lysed and immunoprecipitated with anti-Bax antibody (α-Bax) or NRI. Immune complexes were analyzed by Western blotting. “Total” represents 1/10 the amount of mitochondria used for the experiment.
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Figure 2: Inhibition of Bax-induced Δψ loss and cytochrome c release in isolated mitochondria by anti-VDAC antibodies. (A) Inhibition of Bax-induced Δψ loss by Ab#20 and Ab#25. Mitochondria (1 mg/ml) were preincubated with or without 0.6 μg/μl of the indicated antibodies (Ab#20, Ab#25, 31HL, or NRI) for 5 min, after which rBax (0.2 μg/μl) was added. Then Δψ was measured from the rhodamine 123 (Rh123) uptake over 25 min. When Δψ dropped, rhodamine 123 was released, resulting in an increase of rhodamine 123 intensity. Complete loss of Δψ was demonstrated by incubation of the mitochondria with 1 mM carbonylcyanide m-chlorophenylhydrazone (CCCP, protonophore). Data are representative of three independent experiments. (B) Inhibition of Bax-induced cytochrome c release by Ab#20 and Ab#25. Mitochondria (1 mg/ml) were preincubated with or without the indicated concentrations of antibodies (Ab#20, Ab#25-1, Ab#25-2, 31HL, or NRI) for 5 min, after which rBax (0.2 μg/μl) or Ca2+ (50 μM) was added (top 5 panels). Mitochondria preincubated with antibodies were also incubated with rBax in the presence of 0.2 mM EGTA (bottom panel). In the presence of EGTA, a higher concentration of rBax (1 μg/μl) was used to induce cytochrome c release comparable to that without EGTA. The extent of cytochrome c release was measured at 10 min (second, third, fifth, and bottom panels) or at the indicated times (top and fourth panels) by Western blot analysis of the supernatants. “Total” represents the total amount of cytochrome c in the same amount of mitochondria. Data are representative of two or three independent experiments. (C) Immunostaining of mitochondria with Ab#25. Mitochondria (1 μg/μl) were incubated with Ab#25 (open circles) or NRI (filled circle) at the indicated concentrations, and then stained with anti–rabbit IgG-Alexa488, after which the fluorescence was measured by flow cytometry as described in Materials and Methods. Data are shown as the mean ± SD for three independent experiments. (D) Lack of effect of Ab#20 and Ab#25 on mitochondrial respiration. Mitochondria (1 μg/μl) were incubated with 0.6 μg/μl of the indicated antibodies for 5 min, and then respiration was measured in the presence of 5 mM succinate (state IV; white bars) or succinate plus 0.3 mM ADP (state III; black bars). Data are representative of two independent experiments. (E) Lack of effect of Ab#20 and Ab#25 on mitochondrial association of Bax. Mitochondria were treated as described in A. At 10 min after addition of rBax, the mitochondria were spun, and the supernatants (sup) and pellets (pt) were subjected to Western blot analysis for Bax detection. (F) Lack of effect of Ab#25 on Bax–VDAC interaction. Mitochondria were treated as described in A. At 10 min after addition of rBax, mitochondria were lysed and immunoprecipitated with anti-Bax antibody (α-Bax) or NRI. Immune complexes were analyzed by Western blotting. “Total” represents 1/10 the amount of mitochondria used for the experiment.

Mentions: Using these neutralizing anti-VDAC antibodies, we first examined the role of the VDAC in Bax-induced apoptotic changes of isolated mitochondria. As shown in Fig. 2A and Fig. B (third panel from the top) NRI and a monoclonal anti-VDAC antibody (31HL: with an epitope corresponding to the NH2-terminal region of VDAC as shown in Fig. 1 A) (Babel et al. 1991) that had no influence on VDAC activity (Benz et al. 1992; data not shown) did not inhibit, but rather slightly enhanced both Bax-induced Δψ loss and cytochrome c release. In contrast, these mitochondrial changes were efficiently inhibited by Ab#20 and Ab#25 in a concentration-dependent manner (Fig. 2A and Fig. B, top three panels), indicating that the VDAC is required for Bax-induced cytochrome c release and Δψ loss in mammalian mitochondria under our experimental conditions, consistent with our previous findings in yeast mitochondria (Shimizu et al. 1999, Shimizu et al. 2000c). The amount of Ab#20 and Ab#25 used was near the saturation level for VDAC on the mitochondria, as judged from flow cytometric analysis of mitochondria stained with Ab#25 (Fig. 2 C) and also by comparing the amount of each antibody with the amount of VDAC in the mitochondria using PAGE (data not shown).


Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells.

Shimizu S, Matsuoka Y, Shinohara Y, Yoneda Y, Tsujimoto Y - J. Cell Biol. (2001)

Inhibition of Bax-induced Δψ loss and cytochrome c release in isolated mitochondria by anti-VDAC antibodies. (A) Inhibition of Bax-induced Δψ loss by Ab#20 and Ab#25. Mitochondria (1 mg/ml) were preincubated with or without 0.6 μg/μl of the indicated antibodies (Ab#20, Ab#25, 31HL, or NRI) for 5 min, after which rBax (0.2 μg/μl) was added. Then Δψ was measured from the rhodamine 123 (Rh123) uptake over 25 min. When Δψ dropped, rhodamine 123 was released, resulting in an increase of rhodamine 123 intensity. Complete loss of Δψ was demonstrated by incubation of the mitochondria with 1 mM carbonylcyanide m-chlorophenylhydrazone (CCCP, protonophore). Data are representative of three independent experiments. (B) Inhibition of Bax-induced cytochrome c release by Ab#20 and Ab#25. Mitochondria (1 mg/ml) were preincubated with or without the indicated concentrations of antibodies (Ab#20, Ab#25-1, Ab#25-2, 31HL, or NRI) for 5 min, after which rBax (0.2 μg/μl) or Ca2+ (50 μM) was added (top 5 panels). Mitochondria preincubated with antibodies were also incubated with rBax in the presence of 0.2 mM EGTA (bottom panel). In the presence of EGTA, a higher concentration of rBax (1 μg/μl) was used to induce cytochrome c release comparable to that without EGTA. The extent of cytochrome c release was measured at 10 min (second, third, fifth, and bottom panels) or at the indicated times (top and fourth panels) by Western blot analysis of the supernatants. “Total” represents the total amount of cytochrome c in the same amount of mitochondria. Data are representative of two or three independent experiments. (C) Immunostaining of mitochondria with Ab#25. Mitochondria (1 μg/μl) were incubated with Ab#25 (open circles) or NRI (filled circle) at the indicated concentrations, and then stained with anti–rabbit IgG-Alexa488, after which the fluorescence was measured by flow cytometry as described in Materials and Methods. Data are shown as the mean ± SD for three independent experiments. (D) Lack of effect of Ab#20 and Ab#25 on mitochondrial respiration. Mitochondria (1 μg/μl) were incubated with 0.6 μg/μl of the indicated antibodies for 5 min, and then respiration was measured in the presence of 5 mM succinate (state IV; white bars) or succinate plus 0.3 mM ADP (state III; black bars). Data are representative of two independent experiments. (E) Lack of effect of Ab#20 and Ab#25 on mitochondrial association of Bax. Mitochondria were treated as described in A. At 10 min after addition of rBax, the mitochondria were spun, and the supernatants (sup) and pellets (pt) were subjected to Western blot analysis for Bax detection. (F) Lack of effect of Ab#25 on Bax–VDAC interaction. Mitochondria were treated as described in A. At 10 min after addition of rBax, mitochondria were lysed and immunoprecipitated with anti-Bax antibody (α-Bax) or NRI. Immune complexes were analyzed by Western blotting. “Total” represents 1/10 the amount of mitochondria used for the experiment.
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Related In: Results  -  Collection

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Figure 2: Inhibition of Bax-induced Δψ loss and cytochrome c release in isolated mitochondria by anti-VDAC antibodies. (A) Inhibition of Bax-induced Δψ loss by Ab#20 and Ab#25. Mitochondria (1 mg/ml) were preincubated with or without 0.6 μg/μl of the indicated antibodies (Ab#20, Ab#25, 31HL, or NRI) for 5 min, after which rBax (0.2 μg/μl) was added. Then Δψ was measured from the rhodamine 123 (Rh123) uptake over 25 min. When Δψ dropped, rhodamine 123 was released, resulting in an increase of rhodamine 123 intensity. Complete loss of Δψ was demonstrated by incubation of the mitochondria with 1 mM carbonylcyanide m-chlorophenylhydrazone (CCCP, protonophore). Data are representative of three independent experiments. (B) Inhibition of Bax-induced cytochrome c release by Ab#20 and Ab#25. Mitochondria (1 mg/ml) were preincubated with or without the indicated concentrations of antibodies (Ab#20, Ab#25-1, Ab#25-2, 31HL, or NRI) for 5 min, after which rBax (0.2 μg/μl) or Ca2+ (50 μM) was added (top 5 panels). Mitochondria preincubated with antibodies were also incubated with rBax in the presence of 0.2 mM EGTA (bottom panel). In the presence of EGTA, a higher concentration of rBax (1 μg/μl) was used to induce cytochrome c release comparable to that without EGTA. The extent of cytochrome c release was measured at 10 min (second, third, fifth, and bottom panels) or at the indicated times (top and fourth panels) by Western blot analysis of the supernatants. “Total” represents the total amount of cytochrome c in the same amount of mitochondria. Data are representative of two or three independent experiments. (C) Immunostaining of mitochondria with Ab#25. Mitochondria (1 μg/μl) were incubated with Ab#25 (open circles) or NRI (filled circle) at the indicated concentrations, and then stained with anti–rabbit IgG-Alexa488, after which the fluorescence was measured by flow cytometry as described in Materials and Methods. Data are shown as the mean ± SD for three independent experiments. (D) Lack of effect of Ab#20 and Ab#25 on mitochondrial respiration. Mitochondria (1 μg/μl) were incubated with 0.6 μg/μl of the indicated antibodies for 5 min, and then respiration was measured in the presence of 5 mM succinate (state IV; white bars) or succinate plus 0.3 mM ADP (state III; black bars). Data are representative of two independent experiments. (E) Lack of effect of Ab#20 and Ab#25 on mitochondrial association of Bax. Mitochondria were treated as described in A. At 10 min after addition of rBax, the mitochondria were spun, and the supernatants (sup) and pellets (pt) were subjected to Western blot analysis for Bax detection. (F) Lack of effect of Ab#25 on Bax–VDAC interaction. Mitochondria were treated as described in A. At 10 min after addition of rBax, mitochondria were lysed and immunoprecipitated with anti-Bax antibody (α-Bax) or NRI. Immune complexes were analyzed by Western blotting. “Total” represents 1/10 the amount of mitochondria used for the experiment.
Mentions: Using these neutralizing anti-VDAC antibodies, we first examined the role of the VDAC in Bax-induced apoptotic changes of isolated mitochondria. As shown in Fig. 2A and Fig. B (third panel from the top) NRI and a monoclonal anti-VDAC antibody (31HL: with an epitope corresponding to the NH2-terminal region of VDAC as shown in Fig. 1 A) (Babel et al. 1991) that had no influence on VDAC activity (Benz et al. 1992; data not shown) did not inhibit, but rather slightly enhanced both Bax-induced Δψ loss and cytochrome c release. In contrast, these mitochondrial changes were efficiently inhibited by Ab#20 and Ab#25 in a concentration-dependent manner (Fig. 2A and Fig. B, top three panels), indicating that the VDAC is required for Bax-induced cytochrome c release and Δψ loss in mammalian mitochondria under our experimental conditions, consistent with our previous findings in yeast mitochondria (Shimizu et al. 1999, Shimizu et al. 2000c). The amount of Ab#20 and Ab#25 used was near the saturation level for VDAC on the mitochondria, as judged from flow cytometric analysis of mitochondria stained with Ab#25 (Fig. 2 C) and also by comparing the amount of each antibody with the amount of VDAC in the mitochondria using PAGE (data not shown).

Bottom Line: Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release.When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death.Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Osaka University Graduate School of Medicine, Biomedical Research Center, Department of Medical Genetics, Osaka 565-0871, Japan.

ABSTRACT
Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

Show MeSH
Related in: MedlinePlus