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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 VP16-, paclitaxel- and staurosporine-induced, but not rtBid-induced, apoptosis by anti-VDAC antibodies. (A) Lack of inhibition of Bid- and Bik-induced cytochrome c release from isolated mitochondria 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, NRI, or 31HL) for 5 min, and then rBid (0.2 μg/μl) or rBik (0.2 μg/μl) was added. Cytochrome c release was measured at the indicated times (top two panels) or at 10 min (bottom panel) by Western blot analysis of supernatants obtained after centrifugation to remove the mitochondria. “Total” represents the total amount of cytochrome c in the same amount of mitochondria. Data are representative of two independent experiments. (B and C) Lack of effect of Ab#20 and Ab#25 on tBid-induced apoptosis. HeLa cells were microinjected with 12 μg/μl of Ab#25 (B; filled circles in C), Ab#20 (filled squares in C), or NRI (B; open circles in C). After 1 h, 2 μg/μl (C, left) or 0.5 μg/μl (B; C, right) of rtBid was microinjected into the same cells, and apoptosis was assessed from the cell morphology. More than 100 injected cells were analyzed. Data are representative of three independent experiments. Representative photographs taken at 12 h are shown in B. All cells shown in B were microinjected. (D–G) Inhibition of VP16-, paclitaxel-, and staurosporine-induced apoptosis by microinjection of Ab#25 and Ab#20. HeLa cells were microinjected with 12 μg/μl (D and E, left; F, and G) and 18 μg/μl (E, right) of antibodies together with 3 μg/μl of GFP. After 1 h, 200 μM of VP16 (D and E), 0.3 μM of paclitaxel (F), or 2 μM of staurosporine (G) was added. HeLa cells were also injected only with GFP as living cell control (no treatment in D). At 18 h, cells were stained with 1 μM Hoechst 33342 (Ho342), and photographed (D). In E–G, apoptosis was assessed from the cell morphology by fluorescence microscopy. More than 100 injected cells were analyzed. The open circles, filled circles, and filled squares correspond to NRI, Ab#25, and Ab#20, respectively. Data are representative of two or three independent experiments.
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Figure 6: Inhibition of VP16-, paclitaxel- and staurosporine-induced, but not rtBid-induced, apoptosis by anti-VDAC antibodies. (A) Lack of inhibition of Bid- and Bik-induced cytochrome c release from isolated mitochondria 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, NRI, or 31HL) for 5 min, and then rBid (0.2 μg/μl) or rBik (0.2 μg/μl) was added. Cytochrome c release was measured at the indicated times (top two panels) or at 10 min (bottom panel) by Western blot analysis of supernatants obtained after centrifugation to remove the mitochondria. “Total” represents the total amount of cytochrome c in the same amount of mitochondria. Data are representative of two independent experiments. (B and C) Lack of effect of Ab#20 and Ab#25 on tBid-induced apoptosis. HeLa cells were microinjected with 12 μg/μl of Ab#25 (B; filled circles in C), Ab#20 (filled squares in C), or NRI (B; open circles in C). After 1 h, 2 μg/μl (C, left) or 0.5 μg/μl (B; C, right) of rtBid was microinjected into the same cells, and apoptosis was assessed from the cell morphology. More than 100 injected cells were analyzed. Data are representative of three independent experiments. Representative photographs taken at 12 h are shown in B. All cells shown in B were microinjected. (D–G) Inhibition of VP16-, paclitaxel-, and staurosporine-induced apoptosis by microinjection of Ab#25 and Ab#20. HeLa cells were microinjected with 12 μg/μl (D and E, left; F, and G) and 18 μg/μl (E, right) of antibodies together with 3 μg/μl of GFP. After 1 h, 200 μM of VP16 (D and E), 0.3 μM of paclitaxel (F), or 2 μM of staurosporine (G) was added. HeLa cells were also injected only with GFP as living cell control (no treatment in D). At 18 h, cells were stained with 1 μM Hoechst 33342 (Ho342), and photographed (D). In E–G, apoptosis was assessed from the cell morphology by fluorescence microscopy. More than 100 injected cells were analyzed. The open circles, filled circles, and filled squares correspond to NRI, Ab#25, and Ab#20, respectively. Data are representative of two or three independent experiments.

Mentions: We next tested the effect of Ab#20 and Ab#25 on other forms of apoptosis. We have recently shown that BH3-only proteins, such as Bid (also tBid, an active form with an NH2-terminal truncation) and Bik, do not bind to the VDAC, suggesting that a non-VDAC mechanism is involved in Bid/Bik-induced cytochrome c release and cell death (Shimizu and Tsujimoto 2000). Consistently, addition of Ab#20 or Ab#25 did not inhibit rBid- and rBik-induced cytochrome c release from isolated mitochondria (Fig. 6 A, compare with Fig. 2 B). Similar results were obtained with rtBid (data not shown). Furthermore, rtBid-induced apoptosis was not inhibited by preinjection of Ab#20 or Ab#25 into HeLa cells, irrespective of the injected amount of tBid (Fig. 6B and Fig. C). The fact that the onset of apoptosis induced by 0.5 μg/μl of rtBid (Fig. 6 C) was slower than that induced by 2 μg/μl of rBax (Fig. 4 D) excluded the possibility that the amount of rtBid used supplied a death signal that overwhelmed the inhibitory capacity of Ab#25. These findings, taken together with our previous results (Shimizu and Tsujimoto 2000), indicated that the VDAC is not required for tBid-induced cytochrome c release and cell death. Failure of Ab#20 and Ab#25 to inhibit tBid-induced cytochrome c release and apoptosis also indicated that inhibition of Bax-induced apoptosis and cytochrome c release by the anti-VDAC antibodies was not due to a nonspecific effect.


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 VP16-, paclitaxel- and staurosporine-induced, but not rtBid-induced, apoptosis by anti-VDAC antibodies. (A) Lack of inhibition of Bid- and Bik-induced cytochrome c release from isolated mitochondria 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, NRI, or 31HL) for 5 min, and then rBid (0.2 μg/μl) or rBik (0.2 μg/μl) was added. Cytochrome c release was measured at the indicated times (top two panels) or at 10 min (bottom panel) by Western blot analysis of supernatants obtained after centrifugation to remove the mitochondria. “Total” represents the total amount of cytochrome c in the same amount of mitochondria. Data are representative of two independent experiments. (B and C) Lack of effect of Ab#20 and Ab#25 on tBid-induced apoptosis. HeLa cells were microinjected with 12 μg/μl of Ab#25 (B; filled circles in C), Ab#20 (filled squares in C), or NRI (B; open circles in C). After 1 h, 2 μg/μl (C, left) or 0.5 μg/μl (B; C, right) of rtBid was microinjected into the same cells, and apoptosis was assessed from the cell morphology. More than 100 injected cells were analyzed. Data are representative of three independent experiments. Representative photographs taken at 12 h are shown in B. All cells shown in B were microinjected. (D–G) Inhibition of VP16-, paclitaxel-, and staurosporine-induced apoptosis by microinjection of Ab#25 and Ab#20. HeLa cells were microinjected with 12 μg/μl (D and E, left; F, and G) and 18 μg/μl (E, right) of antibodies together with 3 μg/μl of GFP. After 1 h, 200 μM of VP16 (D and E), 0.3 μM of paclitaxel (F), or 2 μM of staurosporine (G) was added. HeLa cells were also injected only with GFP as living cell control (no treatment in D). At 18 h, cells were stained with 1 μM Hoechst 33342 (Ho342), and photographed (D). In E–G, apoptosis was assessed from the cell morphology by fluorescence microscopy. More than 100 injected cells were analyzed. The open circles, filled circles, and filled squares correspond to NRI, Ab#25, and Ab#20, respectively. Data are representative of two or three independent experiments.
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Figure 6: Inhibition of VP16-, paclitaxel- and staurosporine-induced, but not rtBid-induced, apoptosis by anti-VDAC antibodies. (A) Lack of inhibition of Bid- and Bik-induced cytochrome c release from isolated mitochondria 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, NRI, or 31HL) for 5 min, and then rBid (0.2 μg/μl) or rBik (0.2 μg/μl) was added. Cytochrome c release was measured at the indicated times (top two panels) or at 10 min (bottom panel) by Western blot analysis of supernatants obtained after centrifugation to remove the mitochondria. “Total” represents the total amount of cytochrome c in the same amount of mitochondria. Data are representative of two independent experiments. (B and C) Lack of effect of Ab#20 and Ab#25 on tBid-induced apoptosis. HeLa cells were microinjected with 12 μg/μl of Ab#25 (B; filled circles in C), Ab#20 (filled squares in C), or NRI (B; open circles in C). After 1 h, 2 μg/μl (C, left) or 0.5 μg/μl (B; C, right) of rtBid was microinjected into the same cells, and apoptosis was assessed from the cell morphology. More than 100 injected cells were analyzed. Data are representative of three independent experiments. Representative photographs taken at 12 h are shown in B. All cells shown in B were microinjected. (D–G) Inhibition of VP16-, paclitaxel-, and staurosporine-induced apoptosis by microinjection of Ab#25 and Ab#20. HeLa cells were microinjected with 12 μg/μl (D and E, left; F, and G) and 18 μg/μl (E, right) of antibodies together with 3 μg/μl of GFP. After 1 h, 200 μM of VP16 (D and E), 0.3 μM of paclitaxel (F), or 2 μM of staurosporine (G) was added. HeLa cells were also injected only with GFP as living cell control (no treatment in D). At 18 h, cells were stained with 1 μM Hoechst 33342 (Ho342), and photographed (D). In E–G, apoptosis was assessed from the cell morphology by fluorescence microscopy. More than 100 injected cells were analyzed. The open circles, filled circles, and filled squares correspond to NRI, Ab#25, and Ab#20, respectively. Data are representative of two or three independent experiments.
Mentions: We next tested the effect of Ab#20 and Ab#25 on other forms of apoptosis. We have recently shown that BH3-only proteins, such as Bid (also tBid, an active form with an NH2-terminal truncation) and Bik, do not bind to the VDAC, suggesting that a non-VDAC mechanism is involved in Bid/Bik-induced cytochrome c release and cell death (Shimizu and Tsujimoto 2000). Consistently, addition of Ab#20 or Ab#25 did not inhibit rBid- and rBik-induced cytochrome c release from isolated mitochondria (Fig. 6 A, compare with Fig. 2 B). Similar results were obtained with rtBid (data not shown). Furthermore, rtBid-induced apoptosis was not inhibited by preinjection of Ab#20 or Ab#25 into HeLa cells, irrespective of the injected amount of tBid (Fig. 6B and Fig. C). The fact that the onset of apoptosis induced by 0.5 μg/μl of rtBid (Fig. 6 C) was slower than that induced by 2 μg/μl of rBax (Fig. 4 D) excluded the possibility that the amount of rtBid used supplied a death signal that overwhelmed the inhibitory capacity of Ab#25. These findings, taken together with our previous results (Shimizu and Tsujimoto 2000), indicated that the VDAC is not required for tBid-induced cytochrome c release and cell death. Failure of Ab#20 and Ab#25 to inhibit tBid-induced cytochrome c release and apoptosis also indicated that inhibition of Bax-induced apoptosis and cytochrome c release by the anti-VDAC antibodies was not due to a nonspecific effect.

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