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

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.

Figure 1

Inhibition of VDAC activity by anti-VDAC antibodies. (A) Putative model of human VDAC1 topology. The epitopes of the three anti-VDAC antibodies (Ab#20, Ab#25, and 31HL) are shown by boxes. (B) Specificity of Ab#20 and Ab#25. Rat liver mitochondria (Mt) lysate (15 μg) and HeLa cell lysate (10 μg) were subjected to Western blotting using Ab#20 and Ab#25. (C) Inhibition of both VDAC activity and Bax-induced enhancement of VDAC activity by Ab#20 and Ab#25. 20 μl of plain liposomes or VDAC liposomes was incubated with 0.2 μg/μl of the indicated antibodies for 3 min, and then were incubated with 5 μl of [¹⁴C]sucrose (97%; 200 μCi/ml) in the presence (black bar) or absence (white bar) of rBax (0.2 μg/μl) at 25°C for the 6 min. The [¹⁴C]sucrose incorporated into the liposomes was measured as described in Materials and Methods. Data are shown as the mean ± SD for three independent experiments. (D) Lack of influence of Ab#25 on Bax channel activity. Irrelevant control protein liposomes and Bax liposomes were incubated with 0.2 μg/μl of Ab#25 or NRI for 5 min, and then incubated with 5 μl of [³H]glucose (97%; 20 Ci/mmol) at 25°C for 5 min. The [³H]glucose incorporated into the liposomes was measured as described in Materials and Methods. Data are shown as the mean ± SD for three independent experiments.

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.

Figure 3

Effect of the microinjection of anti-VDAC antibodies. (A) Localization of injected Ab#25. HeLa cells were microinjected with 3 μg/μl of Ab#25. After fixing, the cells were incubated with anti-cytochrome c antibody for 12 h at 4°C, and then with anti–rabbit IgG-Alexa488 (which reacted with Ab#25) and anti–mouse IgG-Alexa568 (which reacted with anti-cytochrome c antibody), followed by observation under a fluorescence microscope. (B) Lack of inhibition of mitochondrial respiration by Ab#25. HeLa cells were microinjected with 12 μg/μl of Ab#25. Then noninjected and injected cells were incubated in glucose-containing (+ glc) or glucose-free (− glc) medium in the presence or absence of 10 μM oligomycin (oligo). After 24 h, cells were stained with JC-1 dye and observed under a fluorescence microscope. The dye gave an orange color to cells with a high Δψ and a green color to cells with a low Δψ.

Figure 4

Inhibition of rBax-induced apoptosis by injection of anti-VDAC antibodies. (A) Induction of apoptosis by microinjection of rBax. rBax (1 μg/μl) or the equivalent amount of irrelevant control protein was microinjected into the cytoplasm of HeLa cells with GFP (3 μg/μl). After 8 h, cell morphology was assessed by transmission microscopy (TM) and fluorescence microscopy (GFP). Cells were also stained with annexin V and Hoechst 33342, and observed under a fluorescence microscope. The color photographs were taken from the same field. The arrowhead indicates an example of cells at the terminal stage of apoptosis, showing weak annexin V staining with no Hoechst 33342 staining. (B–E) Inhibition of rBax-induced apoptosis by microinjection of Ab#20 and Ab#25. (B) HeLa cells were microinjected with 12 μg/μl of the indicated antibodies. After 1 h, 2 μg/μl of rBax was microinjected into the same cells, and after 15 h, cells were examined under a transmission microscope. All of the cells shown were microinjected. Data are representative of seven independent experiments. (C) The same procedure shown in B was followed, except that 3 μg/μl of GFP was coinjected with rBax. Cell morphology was assessed at the indicated times under a fluorescence microscope. Data are representative of three independent experiments. (D) HeLa cells were microinjected with Ab#20 (filled squares), Ab#25 (filled circles), or NRI (open circles) at the indicated concentrations. After 1 h, rBax at the indicated concentrations was microinjected into the same cells, and apoptosis was investigated under a transmission microscope. More than 100 injected cells were analyzed. Data are representative of two or seven independent experiments. (E) HeLa cells were microinjected with 12 μg/μl of the indicated antibodies. After 1 h, the cells were injected with 1 μg/μl of rBax, and apoptosis was assessed at 12 h under a transmission microscope. NMI indicates normal mouse IgG used as a control for 31HL. Data are representative of two independent experiments.

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.

Figure 5

Inhibition of rBax-induced cytochrome c release by anti-VDAC antibodies. HeLa cells were microinjected with 12 μg/μl of NRI or Ab#25 plus 0.4 μg/μl of Cy5-mouse IgG (to identify the injected cells), and then 1 μg/μl of rBax was injected. After 12 h, the intracellular distribution of cytochrome c and that of F₁ ATPase (as a mitochondrial marker) was assessed by immunostaining. Fluorescence images of cytochrome c and F₁ ATPase are merged in the right panels (overlay).

Figure 8

Functional interaction of Bcl-2/Bcl-x_L and Bax/Bak, but not Bid/Bik, with the VDAC. Bax/Bak directly opens the VDAC to induce cytochrome c release, while Bcl-2/Bcl-x_L closes this channel. Bid/Bik does not interact with the VDAC, but probably has the ability to open an unidentified channel(s) that is involved in cytochrome c release. The VDAC is a component of the PT pore, which contains ANT and cyclophilin D (CyD) and would explain the concomitant induction of Δψ loss by Bax/Bak and inhibition of Bax/Bak-induced cytochrome c and Δψ loss by PT inhibitors. Bcl-2/Bcl-x_L inhibits Bid/Bik-induced cytochrome c release, probably through heterodimerization with Bid/Bik or by closing an unidentified channel(s).

Figure 7

Inhibition of rBax-induced release of hemoglobin from RBCs by addition of anti-VDAC antibodies. (A–D) Detection of VDAC on the RBC plasma membrane. (A) RBCs, at 0.25% (vol/vol), were incubated with 0.4 μg/μl of Ab#25 or NRI for 30 min. After washing twice, anti–rabbit IgG-Alexa488 was added to the cells for 30 min and then the RBCs were analyzed using a flow cytometer. (B) Whole lysates of RBCs (40 μg) and HeLa cells (3 μg) were subjected to Western blot analysis using anti-VDAC antibody (31HL and Ab#25) and anticytochrome c antibody. (C and D) RBCs (40 μl), at 15% (vol/vol), were incubated with (+) or without (−) 1% trypsin for 1 h at 25°C, followed by incubation with 3% trypsin inhibitor for 30 min (C). RBCs were also incubated with an equivalent volume of distilled water (D.W.+) to produce ghost RBCs or 0.9% NaCl (D.W.−) for 30 min (D). After brief centrifugation, half of trypsin-treated RBCs and ghost RBC lysates were subjected to Western blot analysis using anti-VDAC antibody (31HL) and anti-GPDH antibody. (E) Interaction of rBax with VDAC on RBCs. RBCs (100 μl), at 15% (vol/vol), were incubated with 50 μg of rBax for 15 min at 25°C. Then, RBCs were lysed and immunoprecipitated with anti-Bax antibody (α-Bax), anti-VDAC antibody (31HL) (α-VDAC), NRI, or normal mouse IgG. Immune complexes were analyzed by Western blotting. “Total” represents 1/10 the amount of lysates used for the experiment. (F–H) Inhibition of Bax- and Bak-induced release of hemoglobin from RBCs by Ab#20 and Ab#25. All data are indicated as mean ± SD for three independent experiments. (F) RBCs, at 2.5% (vol/vol), were preincubated for 5 min with Ab#25 (filled circles), or NRI (open circles) at 0.8 μg/μl, or were preincubated without antibodies (open triangles), and then were incubated with rBax (1 μg/μl). RBCs were also incubated with an equivalent amount of irrelevant protein (open squares) for the indicated times. Then, the RBCs were spun, and free hemoglobin was detected at OD₅₄₃ using a spectrophotometer. The total hemoglobin content was estimated after hypotonic lysis of RBCs (filled triangle). (G) A similar procedure as described in F was performed with the indicated concentrations of Ab#20 (filled squares), Ab#25 (filled circles), or NRI (open circles) for 5 min, followed by incubation with rBax (1 μg/μl) for 1 h. The total hemoglobin content was estimated after hypotonic lysis of RBCs (filled triangle). (H) A similar procedure as described in F was performed with Ab#25 (black bars), or NRI (white bars) for 5 min at 0.8 μg/μl, followed by incubation with rBak (1 μg/μl) or an equivalent amount of irrelevant protein for 1 h. Free hemoglobin was assessed. (I) Flow cytometric analysis of RBCs stained with Ab#25. RBCs, at 2.5% (vol/vol), were incubated with Ab#25 (open circles) or NRI (filled circle) at the indicated concentrations, and then stained with anti–rabbit IgG-Alexa488. Fluorescence was measured by flow cytometry as described in Materials and Methods. Data are indicated as mean ± SD for three independent experiments. (J) Inhibition of Bak-induced release of hemoglobin from RBCs by Bcl-x_L BH4 peptide. A similar procedure as described in H was performed with the BH4 peptide (open circles) or BH4 mutant ΔFL peptide (filled circles) for 5 min at the indicated concentrations, followed by incubation with rBak (1 μg/μl) for 1 h. Data are indicated as mean ± SD for three independent experiments. (K) Lack of effect of Ab#20 and Ab#25 on hemolysin-induced release of hemoglobin from RBCs. RBCs, at 2.5% (vol/vol), were preincubated with Ab#20, Ab#25, or NRI at 0.8 μg/μl for 5 min, and then were incubated with Kanagawa (K) hemolysin at the indicated concentrations for 1 h. Free hemoglobin was detected at OD₅₄₃ using a spectrophotometer. The total hemoglobin content was estimated after hypotonic lysis (filled triangle). Data are indicated as mean ± SD for three independent experiments.