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.2009 Aug;16(8):1135-45.
doi: 10.1038/cdd.2009.28. Epub 2009 Mar 20.

PUMA- and Bax-induced autophagy contributes to apoptosis

Affiliations

PUMA- and Bax-induced autophagy contributes to apoptosis

K S Yee et al. Cell Death Differ.2009 Aug.

Abstract

The p53-inducible BH3-only protein PUMA is a key mediator of p53-dependent apoptosis, and PUMA has been shown to function by activating Bax and mitochondrial outer membrane permeabilization. In this study, we describe an ability of PUMA to induce autophagy that leads to the selective removal of mitochondria. This function of PUMA depends on Bax/Bak and can be reproduced by overexpression of Bax. The induction of autophagy coincides with cytochrome c release, and taken together the results suggest that PUMA functions through Bax to induce mitochondrial autophagy in response to mitochondrial perturbations. Surprisingly, inhibition of PUMA or Bax-induced autophagy dampens the apoptotic response, suggesting that under some circumstances the selective targeting of mitochondria for autophagy can enhance apoptosis.

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Figures

Figure 1
Figure 1
PUMA induces perinuclear accumulation and loss of mitochondria in different cell types. A) U2OS cells were transfected with GFP-tagged PUMA expression plasmid and maintained in the presence of z-VAD-fmk to inhibit apoptosis. 24 hours later cells were fixed and subject to immunofluorescence analysis. Mitochondria were stained using anti-Tom20 antibody. The top panels show normal mitochondrial morphology in control transfected U2OS cells. B) Saos-2 TetOn PUMA cells were treated with doxycycline to induce PUMA. 24 hours later PUMA was detected using an anti-Flag antibody while mitochondria were stained as above. C) U2OS or MEF cells were co-transfected with a combination of pEGFP plasmid and PUMA expression plasmid and maintained in z-VAD-fmk. 24 hours later, cells were fixed and immunostained with anti-Tom20 antibody. GFP positivity indicated cells that co-expressed PUMA and showed loss of mitochondria (detected with anti-Tom20). D) U2OS cells were transfected with GFP-PUMA and maintained without z-VAD-fmk. After 12 hours, mitochondrial clustering was evident in GFP-PUMA positive cells, with some cells (an example is indicated by *) showing loss of the mitochondrial marker.
Figure 2
Figure 2
PUMA induces accumulation of GFP-LC3 puncta, which preferentially colocalize with mitochondria. A) Saos-2 TetOn PUMA cells were infected with GFP-LC3 expressing Adenovirus, left for 16 hours and then treated with doxycycline. Cells were maintained in the presence of z-VAD-fmk throughout the experiment. 24 hours later, cells were fixed and immunostained with anti-Tom20 antibody to visualize the mitochondria. B) U2OS cells were infected with GFP-LC3 expressing Adenovirus, left for 16 hours and then transfected with PUMA expression plasmid. Cells were maintained in the presence of z-VAD-fmk throughout the experiment. 24 hours later, cells were fixed and immunostained with anti-Tom20 (upper panel) or anti-GM130 (lower panel). Cells exhibiting GFP-LC3 puncta were then observed under a fluorescence microscope to compare the co-localization of GFP-LC3 puncta with mitchondria (Tom20) or Golgi (GM130). C) U2OS cells were transfected with vector control or PUMA expression plasmid and maintained in the presence of z-VAD-fmk throughout the experiment. Cells were harvested 24 or 48 hours after transfection and cell lysates were immunoblotted with the indicated antibodies to detect levels of mitochondria, Golgi and ER proteins.
Figure 3
Figure 3
The ability of PUMA to affect mitochondria and LC3-punctae depends on Bax/Bak A) U2OS cells were transfected with PUMAΔLRR expression plasmid and subjected to immunofluorescence analysis 24 hours later. PUMAΔLRR was detected using anti-FLAG antibody whilst mitochondria were stained with anti-Tom20 antibody. B) Quantitation of GFP-LC3 puncta formation. U2OS cells were infected with GFP-LC3 expressing Adenovirus and then transfected with expression plasmids for the indicated proteins. Cells were maintained in the presence of z-VAD-fmk throughout the experiment. 24 hours later, cells were fixed and observed under a fluorescence microscope. Results are presented as mean percentage of GFP-LC3 positive cells displaying puncta. Data are mean and SD of three independent experiments. (**P<0.001, ***P<0.005 compared to control transfected cells). C) Bax/Bak DKO MEFs were transfected with plasmids expressing the indicated proteins and subjected to immunofluorescence analysis after 24 hours. In the upper panel, PUMA was detected using an anti-Flag antibody whilst mitochondria were stained using anti-Tom20 antibody. In the lower panel, GFP positivity indicated cells that co-expressed PUMA and showed maintenance of mitochondria (detected with anti-Tom20).
Figure 4
Figure 4
Bax functions like PUMA to induce mitochondrial loss and accumulation of LC3 punctae. A) Bax induces mitochondrial perinuclear accumulation and loss. U2OS and WT MEFs were transfected with Bax expression plasmid and subjected to immunofluorescence analysis 24 hours later. Cells were maintained in the presence of z-VAD-fmk throughout the experiment. Bax was detected using anti-Bax antibody whilst mitochondria were stained using anti-Tom20 antibody. B) Saos-2 TetOn Bax cells were treated with doxycycline to induce Bax expression and maintained in the presence of z-VAD-fmk throughout the experiment. Cell were harvested 24 or 48 hours after doxycycline treatment and cell lysates were immunoblotted with the indicated antibodies to detect levels of mitochondria, Golgi and ER proteins. C) U2OS cells were infected with GFP-LC3 expressing Adenovirus and then transfected with Bax expression plasmid. Cells were maintained in the presence of z-VAD-fmk throughout the experiment. 24 hours later, cells were fixed, stained for Bax and observed under a fluorescence microscope. D) Quantitation of GFP-LC3 puncta. Saos-2 TetOn Bax cells were infected with GFP-LC3 expressing Adenovirus and then treated with doxycycline for 24 hours. Cells were maintained in the presence of z-VAD-fmk throughout the experiment. Cells were fixed and analysed using a fluorescence microscope. Results are presented as mean percentage of GFP-LC3 positive cells displaying puncta. Data are mean and SD of three independent experiments (**P<0.001 compared to control cells).
Figure 5
Figure 5
PUMA promotes autophagosome formation and loss of mitochondrial markers, but not general degradation of long-lived proteins A) HeLa cells stably expressing GFP-LC3 were transfected with plasmids expressing the indicated proteins, maintained in the presence of z-VAD-fmk and harvested after 24 hours. Cell lysates were then subjected to Western blotting using anti-GFP antibodies to detect GFP-LC3-I and the faster migrating GFP-LC3-II. Calnexin was used as a loading control. B) Inhibition of autophagosome degradation enhances levels of PUMA-induced GFP-LC3-II. Saos2 TetOn PUMA cells were infected with GFP-LC3 expressing Adenovirus and then treated with doxycycline for 24 hours. 30μM chloroquine was then added and cells were harvested after 9 hour. Cells were maintained in the presence of z-VAD-fmk throughout the experiment. Cell lysates were subjected to Western blotting using anti-GFP antibodies to detect GFP-LC3-I and the faster migrating GFP-LC3-II. Calnexin was used as a loading control. C) Protein stability was assessed in Saos2 TetOn PUMA cells following doxycycline treatment to induce PUMA or starvation (# No significant difference; **P-value<0.001 compared to untreated cells). Western blot for COXIV protein levels in these samples is shown below. Actin was used as a loading control
Figure 6
Figure 6
Inhibition of autophagy partially rescues the mitochondria loss and apoptosis induced by Bax and PUMA. A) Saos-2 TetOn Bax cells were transfected with non-targeting siRNA or siRNA against Atg5. 72 hours later, doxycycline was added to induce Bax expression in the presence of z-VAD-fmk. 24 hours after induction, cells were harvested for Western blot analysis. Cell lysates were immunoblotted and probed with antibodies against the indicated proteins. Anti-Atg12 was used to detect Atg5-12 complex to confirm the decrease in Atg5 expression. Anti-COXIV antibody was used to detect mitochondria protein levels. B) Saos-2 TetOn Bax cells were transfected with non-targeting siRNA as well as siRNA against Atg5, Atg7 and Atg10. 72 hours later, doxycycline was added to induce Bax expression. After 24 hours, cells were harvested and apoptosis was evaluated by flow cytometry. Results are the mean and standard deviation for at least three independent experiments. (*P-value<0.05, **P-value<0.001 compared with non-targeted siRNA transfected cells) C) Saos-2 TetOn PUMA cells were transfected with non-targeted siRNA or siRNA against Atg5, Atg7 and Atg 10. 72 hours later, doxycycline was added to induce PUMA expression. After 24 hours, cells were harvested and apoptosis was evaluated by flow cytometry. Results are the mean and standard deviation for at least three independent experiments (*P-value<0.05, **P-value<0.001 compared with non-targeted siRNA transfected cells)
Figure 7
Figure 7
Inhibition of autophagy inhibits cytochrome c release and decrease of mitochondrial membrane potential induced by Bax. Saos-2 TetOn Bax cells were transfected with non-targeting siRNA or siRNA against Atg5. 72 hours later, doxycycline was added to induce Bax expression in the presence of z-VAD-fmk. A) 24 hours after Bax induction, cells were harvested and subjected to subcellular fractionation. Equivalent amounts of each fraction were subjected to Western blot analysis to detect cytochrome c levels and marker proteins; COXIV for membrane fraction and Hsp70 for cytosolic fraction. B) 24 hours after Bax induction, cells were stained with TMRE to determine alterations in mitochondrial membrane potential (Δψm). A decrease in Δψm is demonstrated by the appearance of a peak with lower fluorescence intensity. The experiment was carried out at least three times and gave similar results.
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