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, 9 (5), 550-5

Voltage-dependent Anion Channels Are Dispensable for Mitochondrial-Dependent Cell Death

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Voltage-dependent Anion Channels Are Dispensable for Mitochondrial-Dependent Cell Death

Christopher P Baines et al. Nat Cell Biol.

Abstract

Mitochondria are critically involved in necrotic cell death induced by Ca(2+) overload, hypoxia and oxidative damage. The mitochondrial permeability transition (MPT) pore - a protein complex that spans both the outer and inner mitochondrial membranes - is considered the mediator of this event and has been hypothesized to minimally consist of the voltage-dependent anion channel (Vdac) in the outer membrane, the adenine-nucleotide translocase (Ant) in the inner membrane and cyclophilin-D in the matrix. Here, we report the effects of deletion of the three mammalian Vdac genes on mitochondrial-dependent cell death. Mitochondria from Vdac1-, Vdac3-, and Vdac1-Vdac3-null mice exhibited a Ca(2+)- and oxidative stress-induced MPT that was indistinguishable from wild-type mitochondria. Similarly, Ca(2+)- and oxidative-stress-induced MPT and cell death was unaltered, or even exacerbated, in fibroblasts lacking Vdac1, Vdac2, Vdac3, Vdac1-Vdac3 and Vdac1-Vdac2-Vdac3. Wild-type and Vdac-deficient mitochondria and cells also exhibited equivalent cytochrome c release, caspase cleavage and cell death in response to the pro-death Bcl-2 family members Bax and Bid. These results indicate that Vdacs are dispensable for both MPT and Bcl-2 family member-driven cell death.

Figures

Figure 1
Figure 1
Mitochondrial permeability transition in VDAC1- and 3-deficient mitochondria. (a) Western blotting for VDAC1, VDAC2, VDAC3, ANT, and cyclophilin-D (CypD) in wildtype, Vdac1-, Vdac3-, and Vdac1/Vdac3-null mouse hearts. GAPDH was used to demonstrate equivalent loading. (b) Mitochondrial swelling assay measured by absolute absorbance at baseline and after Ca2+ (250 μM) addition (arrowhead) in isolated cardiac mitochondria from each of the indicated genotypes (V1/3 represents Vdac1/Vdac3 double null). (c) The data shown in panel b were also plotted as change in absorbance, which shows that loss of VDAC1 and/or VDAC3 protein does not compromise MPT. (d) Change in absorbance of mitochondria after Ca2+ stimulation in the presence of cyclosporine A (1 μM) for each of the indicated genotypes. (e) Change in absorbance of mitochondrial with a lower dosage of Ca2+ (100 μM). The results represent the average values of at least 4 independent experiments. Error bars indicate s.e.m.
Figure 2
Figure 2
Cytochrome c release induced by Ca2+, oxidative stress, Bax and tBid. (a-d) Isolated wildtype and Vdac1/Vdac3 null (V1/3−/−) liver mitochondria were incubated with 250 μM Ca2+ (a), 100 μM tert-butyl hydroperoxide (tBH; b), 1 μg GST-Bax (c) or 0.25 μg tBid (d) and incubated over time (in minutes). The control (Con) represents mitochondria treated with vehicle or GST over 180 min. The suspension was centrifuged and the resultant pellet (Mito) and supernatant (Sup) were subjected to Western blotting for cytochrome c (CytoC). COX-IV (cytochrome oxidase IV) was used as a control to show the integrity of the two protein fractions. The results shown are representative of 3 independent experiments.
Figure 3
Figure 3
Mitochondrial permeability transition and cell death in Vdac1- and Vdac3-null MEFs. (a) Western blotting for VDAC1, VDAC2, VDAC3, and GAPDH in wildtype, Vdac1-, Vdac3-, and Vdac1/Vdac3–null MEFs. (b) H2O2-induced MPT determined by calcein/CoCl2 fluorescence in Vdac1-, Vdac3-, Vdac1/Vdac3- and Ppif-null MEFs. Bar = 50 μm. (c, d) Propidium iodide (PI) staining in wildtype, Vdac1-, Vdac3-, and Vdac1/Vdac3-null MEFs treated with 10 μM ionomycin (c) for 18 h or 500 μM H2O2 (d) for 6 h. (e) PI staining in wildtype and Ppif-deficient MEFs treated with 10 μM ionomycin for 18 h or (f) 500 μM H2O2 for 6 h. Results are indicative of 3 independent experiments. Error bars are s.e.m., and the asterisk denotes P<0.05 versus wildtype.
Figure 4
Figure 4
Cell death in Vdac-deficient MEFs. (a) Propidium iodide (PI) staining in wildtype, Vdac1-, Vdac3-, and Vdac1/Vdac3-null MEFs infected with adenoviruses encoding inducible Bax (AdBax). (b) Western blotting for Bax, cleaved caspase-3 (Cl-Csp3), cleaved PARP (Cl-PARP), and actin was performed in parallel. (c) PI staining in wildtype, Vdac1-, Vdac3-, and Vdac1/Vdac3-null MEFs treated with 300 nM staurosporine (Stauro) for 18 h. (d) Western blotting for cleaved caspase-3, cleaved PARP, and actin was also analyzed in parallel. (e) PI staining in wildtype, Vdac1-, Vdac3-, and Vdac1/Vdac3-null MEFs treated with 3 ng/mL TNFα plus 0.1 μg/mL actinomycin-D for 18 h, (f) and corresponding western blotting for caspase-3 and PARP cleavage. (g) PI staining in wildtype and Bax/Bak double-null MEFs following 300 nM staurosporine (Stauro) treatment for 18 h. All graphs show the average of 4 independent experiments. Error bars are s.e.m., and the asterisk denotes P<0.05 versus wildtype.
Figure 5
Figure 5
MPT and cell death in Vdac2-null fibroblasts and in MEFs deficient in VDAC1/2/3 protein. (a, b) Calcein/CoCl2 fluorescence in wildtype and Vdac2-null MEFs treated with 100, 250, and 500 μM H2O2. Bar = 50 μm. Graph shows the average of 4 independent experiments. Error bars are s.e.m and asterisk denotes P<0.05 versus wildtype. (c) PI staining for cell death in wildtype and vdac2-null MEFs treated with 500 μM H2O2 for 6 h. (d) Western blotting for VDAC2 protein from Vdac1/Vdac3 double null MEFs treated with a control scrambled siRNA or an siRNA (#1) against VDAC2. COX4 blotting shows equal amounts of protein and mitochondria in each sample. (e) Calcein fluorescence-based quantitation of MPT in MEFs transfected with the indicated siRNAs and treated with the indicated dosages of H2O2 for 6 h. (f) PI staining for cell death in wildtype and Vdac1/Vdac3-null MEFs transfected with the indicated siRNAs and treated with the indicated dosages of H2O2 for 6 h, (g) ionomycin for 18 h, (h) staurosporine for 18 h, and (i) TNFα plus 0.1 μg/mL actinomycin-D for 18 h. Graphs show the average of 3 independent experiments. Error bars are s.e.m., and asterisks denote P<0.05 versus wildtype of the same treatment condition.

Comment in

  • Mitochondrial apoptosis without VDAC.
    Galluzzi L, Kroemer G. Galluzzi L, et al. Nat Cell Biol. 2007 May;9(5):487-9. doi: 10.1038/ncb0507-487. Nat Cell Biol. 2007. PMID: 17473857 No abstract available.

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