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. 2007 Jul;27(13):4844-62.
doi: 10.1128/MCB.02141-06. Epub 2007 Apr 30.

Sequential activation of poly(ADP-ribose) polymerase 1, calpains, and Bax is essential in apoptosis-inducing factor-mediated programmed necrosis

Rana S Moubarak  1 , Victor J Yuste, Cédric Artus, Aïda Bouharrour, Peter A Greer, Josiane Menissier-de Murcia, Santos A Susin
Affiliations

Sequential activation of poly(ADP-ribose) polymerase 1, calpains, and Bax is essential in apoptosis-inducing factor-mediated programmed necrosis

Rana S Moubarak et al. Mol Cell Biol. 2007 Jul.

Abstract

Alkylating DNA damage induces a necrotic type of programmed cell death through the poly(ADP-ribose) polymerases (PARP) and apoptosis-inducing factor (AIF). Following PARP activation, AIF is released from mitochondria and translocates to the nucleus, where it causes chromatin condensation and DNA fragmentation. By employing a large panel of gene knockout cells, we identified and describe here two essential molecular links between PARP and AIF: calpains and Bax. Alkylating DNA damage initiated a p53-independent form of death involving PARP-1 but not PARP-2. Once activated, PARP-1 mediated mitochondrial AIF release and necrosis through a mechanism requiring calpains but not cathepsins or caspases. Importantly, single ablation of the proapoptotic Bcl-2 family member Bax, but not Bak, prevented both AIF release and alkylating DNA damage-induced death. Thus, Bax is indispensable for this type of necrosis. Our data also revealed that Bcl-2 regulates N-methyl-N'-nitro-N'-nitrosoguanidine-induced necrosis. Finally, we established the molecular ordering of PARP-1, calpains, Bax, and AIF activation, and we showed that AIF downregulation confers resistance to alkylating DNA damage-induced necrosis. Our data shed new light on the mechanisms regulating AIF-dependent necrosis and support the notion that, like apoptosis, necrosis could be a highly regulated cell death program.

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Figures

FIG. 1.
FIG. 1.
MNNG-induced cell death is caspase-independent. (A) After the indicated time post-MNNG treatment, WT MEFs were stained with annexin V-FITC and PI, and the frequency of double-positive labeling was recorded and illustrated as a plot. The data presented in the bar chart are the means ± standard deviations (SDs) of the results of five independent experiments. In the cytometry panels, the percentages refer to the levels of double-positive staining. (B) At the indicated times, the level of extracellular LDH from MNNG-treated MEFs was assessed and expressed as a percentage of the total LDH released from Triton X-100-lysed cells. The data shown are the means ± SDs of the results (n = 4). (C) The cells were treated with MNNG for the indicated times and then labeled with TMRE and assessed for the loss of ΔΨm. The results shown are the means ± SDs of the results of three independent experiments. (D) Quantification of the intracellular ATP levels in WT MEFs treated with MNNG at the indicated times (left graph). Alternatively, the cells were cultured for 48 h in complete medium without glucose (−Glucose) or treated with oligomycin (+Oligom; 6 μM) for 30 min prior to MNNG treatment (1 h), and the ATP levels were determined (right graph). STS or H2O2 was used as an apoptotic or necrotic inducer, respectively. The basal ATP levels scored in control cells cultured in complete medium are referred to as 100%. The levels of cell death were measured by means of annexin V-FITC and PI staining in untreated cells (−) or cells treated with MNNG (+) for 9 h, as described in Materials and Methods. The data shown are the means ± SDs of the results (n = 4). (E) Assessment of oligonucleosomal DNA fragmentation in WT MEFs not treated (Co) or incubated with MNNG (9 and 12 h). STS (6 h) was used as a positive control. (F) Mitochondrial AIF (Mit) is cleaved into a lower-molecular-weight tAIF in the mitochondrial intermembrane space (IMS) upon atractyloside treatment. The cytosolic fractions recovered after MNNG treatment were blotted for AIF detection. As in isolated mitochondria, MNNG treatment induces both AIF cleavage into tAIF and time-dependent tAIF release to the cytosol. Actin was used as a loading control. (G) Cytosolic extracts from untreated (Co) or STS- or MNNG-treated (12 h) cells were tested for the release of cytochrome c, Omi/HtrA2, and Smac/DIABLO. The actin levels were analyzed as a loading control. (H) Total cell lysates from WT MEFs not treated (Co) or treated with STS or MNNG (12 h) were probed for the detection of caspase-9 or -3 or ICAD. Equal loading was confirmed by actin assessment. Molecular mass markers are on the right. (I) Fluorometric measurement of caspase-3 activities observed in cytosolic extracts obtained from WT MEFs not treated (Co) or treated with STS or MNNG (12 h) in the absence or presence of the pancaspase inhibitor QVD. The results shown are the means ± SDs of the results of three experiments. (J) WT, Casp-9−/−, and Casp-3−/− MEFs were not treated (Co) or treated with STS or MNNG (12 h), and the levels of cell death were measured by PI staining. The data shown are the means ± SDs of the results (n = 4). (K) The levels of tAIF release were detected in cytosolic extracts purified from WT, Casp-9−/−, and Casp-3−/− MEFs treated as described for panel J. Actin detection was used as a loading control. (L) Assessment of levels of cell death in WT MEFs preincubated with the pancaspase inhibitors QVD, z-VAD-fmk, or BAF or specific inhibitors of caspase-2 (z-VDVAD-fmk), -3 and -7 (z-DEVD-fmk), -6 (z-VEID-fmk), -8 and -10 (z-IETD-fmk), or -9 (z-LEHD-fmk) before the induction of cell death by STS or MNNG treatment (12 h). The results shown are the means ± SDs of the results of five independent experiments.
FIG. 2.
FIG. 2.
The MNNG-induced PCD results correlate with the AIF relocalization from mitochondria to the nucleus and the levels of 3′-OH DNA breaks. (A) WT MEFs were not treated (Control) or treated with MNNG (6 h), immunostained for AIF detection (green), and examined by confocal microscopy. Hoechst 33342 (blue) was used to visualize the nuclei. The graphs represent the green/blue fluorescence distribution detected in the indicated cell sections. This experiment was repeated four times, yielding similar results. Bar, 10 μm. (B) WT MEFs were treated with MNNG for the indicated times and stained with Hoechst 33342 to visualize the nuclei. The numbers of cells presenting stage I of chromatin condensation were quantified and plotted as percentages of total cells (left panel). The data shown are the means ± standard deviations (SDs) of the results (n = 5). Representative nuclei from untreated (Control) or MNNG-treated (9 h) cells are shown (right panel). Bar, 10 μm. (C) At the indicated times after MNNG treatment, WT MEFs were stained for the detection of 3′-OH DNA breaks and analyzed by flow cytometry. The data shown are the means ± SDs of the results of five independent experiments. (D) Purified MEF nuclei were incubated in the absence (Control) or presence of mouse recombinant tAIF (7 μg/ml) and the presence of 3′-OH DNA breaks was determined by cytofluorometry. (E) In an experiment similar to that described for panel D, nuclei were stained with Hoechst 33342 and the levels of blue and green (TUNEL-positive) fluorescence were visualized. Representative microphotographs from untreated (Control) or tAIF-treated nuclei are shown. Bar, 20 μm. (F) WT MEFs were transfected with an siRNA against lamin A (Control siRNA) or two siRNAs against mouse AIF (AIF siRNAs I and II). Seventy-two hours after the indicated transfections, the MEFs were treated with MNNG or not treated (Co), and then the levels of cell death were assessed by flow cytometry by means of PI staining (left panel). The data shown are the means ± SDs of the results (n = 5). Immunoblotting analysis demonstrated the efficacy of siRNA-mediated AIF knockdown (right panel). Equal loading was confirmed by actin detection.
FIG. 3.
FIG. 3.
Necrosis induced by DNA alkylation is PARP-1 dependent and PARP-2/p53 independent. (A) WT, PARP-1−/−, PARP-2−/−, and p53−/− MEFs were not treated (Co) or treated with STS or MNNG (12 h), and the levels of cell death were measured by analysis of PI staining. The results shown are the means ± standard deviations (SDs) of the results of three independent experiments. (B) WT, PARP-1−/−, PARP-2−/−, and p53−/− MEFs were not treated (Control) or treated with MNNG (5 min), immunostained for PAR detection (green), and visualized by confocal microscopy. Hoechst 33342 (blue) was used to visualize the nuclei. Representative micrographs of each cell type are shown. This experiment was repeated four times, yielding similar results. Bar, 10 μm. (C) WT, PARP-1−/−, PARP-2−/−, and p53−/− cells were treated with MNNG (15 min) and analyzed by measuring absorbance at 570 nm to assess the total NAD+ levels. The concentrations of NAD+ were normalized to those from untreated cells (Co). The results shown are the means ± SDs of the results of four experiments. (D) Quantification of the intracellular ATP levels in WT, PARP-1−/−, PARP-2−/−, and p53−/− MEFs treated with MNNG (15 min). STS or H2O2 was used as an apoptotic or necrotic inducer, respectively. The basal level of ATP scored in the control cells (Co) is referred to as 100%. The data shown are the means ± SDs of the results of five independent experiments. (E) WT and PARP-1−/− MEFs were not treated (Co) or treated with MNNG (12 h), stained for the detection of 3′-OH DNA breaks, and analyzed by flow cytometry. The data shown are the means ± SDs of the results (n = 6). (F) WT and PARP-1−/− MEFs were treated with MNNG for the indicated times and stained with Hoechst 33342 to visualize the nuclei. Representative nuclei of untreated cells (Control) or cells treated with MNNG (9 h) are shown (right panel). Bar, 10 μm. The numbers of cells presenting stage I of chromatin condensation were quantified and plotted as percentages of total cells (left panel). The data shown are the means ± SDs of the results (n = 5). (G) tAIF release was detected in cytosolic extracts purified from WT, PARP-1−/−, PARP-2−/−, and p53−/− MEFs treated as described for panel A. Actin detection was used as a loading control.
FIG. 4.
FIG. 4.
Calpains, but not cathepsins, regulate mitochondrial AIF release during MNNG-induced necrosis. (A) WT, Cath B−/−, and Cath L−/− MEFs were not treated (Co) or treated with MNNG (12 h), and the levels of cell death were measured by PI staining. The results shown are the means ± standard deviations (SDs) of the results of four independent experiments. (B) The levels of tAIF release were detected in cytosolic extracts purified from WT, Cath B−/−, and Cath L−/− MEFs treated as described for panel A. The actin levels were assessed as a loading control. (C) WT and Capn4−/− MEFs were not treated (Co) or treated with MNNG (12 h), and the levels of cell death were measured by PI staining. The data shown are the means ± SDs of the results (n = 6). (D) The levels of tAIF release were detected in cytosolic extracts purified from WT and Capn4−/− MEFs treated as described for panel C. Equal loading was verified by actin reblotting. (E) WT and Capn4−/− MEFs were treated as above, stained for the detection of 3′-OH DNA breaks, and analyzed by flow cytometry. The data shown are the means ± SDs of the results of three independent experiments. (F) Fluorescence assessment of calpain activity measured in WT and Capn4−/− MEFs not treated (Control) or treated with MNNG (1 h). Phase-contrast analysis was used to visualize cells. Representative micrographs of each treatment are shown. This experiment was repeated four times, yielding similar results. Bar, 10 μm. (G) Fluorometric analysis of calpain activity observed in cytosolic extracts obtained from WT and Capn4−/− MEFs treated with MNNG for the indicated times. The basal level of calpain activity observed in untreated WT cells is referred to as 1 unit. The data shown are the means ± SDs of the results (n = 4). (H) Immunoblotting assessment of levels of μ-calpain (μ-calp) in WT and Capn4−/− MEFs not treated or treated with MNNG (9 h). MNNG induced a significant decrease in the amount of the 80-kDa proform of μ-calpain in WT MEFs, but not in Capn4−/− MEFs.
FIG. 5.
FIG. 5.
PARP-1 is activated upstream of calpains during MNNG-induced cell death. (A) PAR detection by immunoblotting (115-kDa band) in total extracts from WT and Capn4−/− MEFs not treated (Co) or treated with MNNG (15 min). Molecular masses are shown on the right. The asterisk marks a nonspecific band. The membrane was stained with naphthol blue (NB) to assess protein loading. (B) WT and Capn4−/− MEFs were preincubated, or not, with the PARP inhibitor DHIQ and then treated with MNNG for the indicated times and analyzed by measuring absorbance at 570 nm to assess total NAD+ levels. The concentrations of NAD+ were normalized to those from untreated cells. The data shown are the means ± standard deviations of the results (n = 5) (C) Spectrin immunoblotting analysis was performed on total cell lysates from WT, PARP-1−/−, and Capn4−/− MEFs treated with MNNG or STS for the indicated times. Full-length spectrin (240 kDa) and both the calpain-dependent (150 and 147 kDa) and caspase-3-dependent (150 and 120 kDa) fragments are indicated. (D) Fluorometric analysis of the calpain activity observed in extracts obtained from WT and PARP-1−/− MEFs treated with MNNG for the indicated times. The basal level of calpain activity observed in untreated cells is referred to as 1 unit. The data shown are the means ± standard deviations of the results (n = 4).
FIG. 6.
FIG. 6.
Bax, but not Bak, is necessary for AIF release and MNNG-induced necrosis. (A) WT, Bax−/−, and Bak−/− cells were not treated (Control) or treated with MNNG (12 h), labeled with annexin V-FITC and PI, and analyzed by flow cytometry. Representative cytofluorometric plots are shown. The percentages refer to the frequencies of double-positive staining. (B) Kinetic analysis of PS exposure and loss of cell viability induced by MNNG in WT, Bax−/−, and Bak−/− MEFs (left panel). After the indicated times, the cells were stained as described for panel A, and the frequencies of annexin V and PI double-positive labeling were recorded and expressed as percentages. STS was used as an apoptosis inducer (right panel). The data shown are the means ± standard deviations (SDs) of the results of five independent experiments. (C) Cytosolic fractions from MNNG-treated WT, Bax−/−, and Bak−/− cells were blotted for tAIF detection. The actin levels were analyzed for a loading control. (D) WT, Bax−/−, and Bak−/− MEFs were not treated (Co) or treated with MNNG (12 h), stained for the detection of 3′-OH DNA breaks, and analyzed by flow cytometry. The data shown are the means ± SDs of four independent experiments. (E) WT, Bax−/−, and Bak−/− MEFs were treated with MNNG (9 h) and stained with Hoechst 33342 to visualize the nuclei. Representative nuclei from untreated (Control) or MNNG-treated cells are shown. Bar, 10 μm. (F) Bax−/− MEFs were transiently transfected with the indicated expression plasmids (pcDNA3 or pcDNA3-Bax) in the presence of QVD (10 μM) to prevent caspase-dependent PCD. Twelve hours after the indicated transfections, the MEFs were not treated (Control) or treated with MNNG (12 h). The levels of cell death were determined by PI staining. The data shown are the means ± SDs of the results (n = 4) (upper left panel). Representative microphotographs from each treatment are shown (upper right panel). Bar, 40 μm. The expression levels of Bax before and after the indicated transfections were assessed by immunoblotting (lower left panel). Cytosolic fractions from untreated or MNNG-treated WT, Bax−/−, Bax−/− transfected with pcDNA3, or pcDNA3-Bax were immunoblotted for tAIF detection (lower right panel). Equal loading was confirmed by actin assessment.
FIG. 7.
FIG. 7.
Calpains are activated downstream of PARP-1 but upstream of Bax during MNNG-induced necrosis. (A) Detection of PAR by immunoblotting (115-kDa band) in total extracts from WT and Bax−/− MEFs not treated (Co) or treated with MNNG (15 min) (upper panel). The asterisk marks a nonspecific band. The membrane was reblotted for actin to control for protein loading. Alternatively, WT and Bax−/− MEFs were preincubated or not with DHIQ, treated with MNNG for the indicated times, and analyzed by measuring absorbance at 570 nm to assess total NAD+ levels (lower panel). The concentrations of NAD+ were normalized to those of untreated cells. The results shown are the means ± standard deviations (SDs) of the results of four independent experiments. (B) A fluorometric analysis of the levels of calpain activities was performed for extracts obtained from WT and Bax−/− MEFs treated with MNNG for the indicated times. The basal level of calpain activity observed in untreated cells is referred to as 1 unit. The results shown are the means ± SDs of the results of three independent experiments. (C) WT and Capn4−/− MEFs were treated with MNNG for the indicated times, and the levels of Bax activation were measured by flow cytometry (right panel). The data shown are the means ± SDs of the results (n = 5). Representative cytofluorometric plots of the results for untreated (Control) and MNNG-treated (9 h) cells are shown (left panel). (D) WT and Capn4−/− MEFs were not treated (Co) or treated with MNNG (12 h), and then Bax was immunoprecipitated from the cell lysates by using an antibody specific for the active form of Bax. Samples were then analyzed by Bax immunoblotting. The unbound fraction for each condition was also blotted for Bax detection. The results illustrated are representative of three independent experiments. The molecular mass of Bax is shown on the left. (E) Immunofluorescence staining of activated Bax detected in WT and Capn4−/− MEFs not treated (Control) or treated with MNNG (9 h). A representative overlay of activated Bax and Hoechst 33342 nuclear staining is shown. Note that activated Bax was detected in MNNG-treated WT but not in Capn4−/−cells. This experiment was repeated five times, yielding similar results. Bar, 10 μm.
FIG. 8.
FIG. 8.
The Bcl-2 family of proteins regulates MNNG-mediated necrosis through opposing effects of Bax and Bcl-2 on mitochondria. (A) WT MEFs were left untreated (Control) or treated with MNNG for 6 h and stained for the detection of Bax and Cox IV (used as a mitochondrial marker) before being examined by fluorescence microscopy (left panel). These representative cells show that Bax has a cytosolic distribution in control cells, whereas it colocalizes with Cox IV in MNNG-treated cells. The experiment was repeated three times with low variability in the results (<5%). Bar, 10 μm. In contrast to WT cells, in PARP-1−/− and Capn4−/− MEFs, Bax remains cytosolic after MNNG treatment (right panel). Alternatively, the number of cells presenting low ΔΨm (measured by flow cytometry) or punctated Bax were quantified and plotted as percentages of total cells (graph). The data shown are the means ± SDs of the results of five independent experiments. Bar, 10 μm. (B) Mitochondrial and cytosolic extracts of cells not treated (Co) or treated with MNNG (6 h) were analyzed by Western blotting for the presence of Bax. Cox IV and actin were used as controls for fractionation quality and protein loading. This experiment was repeated four times, yielding comparable results. STS was used as a positive control. (C) Cells stably transfected with vector only (Neo) or the cDNA coding for Bcl-2 were left untreated or treated with MNNG at the indicated times and stained with annexin V-FITC and PI, and the frequencies of double-positive labeling were recorded and pictured as a plot. The data shown are the means ± SDs of the results of five independent experiments (left panel). In a similar experiment, WT or Bcl-2-overexpressing cells were treated with MNNG for the indicated times and then labeled with TMRE and assessed for the loss of ΔΨm. The results shown are the means ± SDs of the results of three independent experiments (middle panel). Bcl-2 overexpression was confirmed by immunoblotting (right panel). (D) tAIF detection in cytosolic extracts purified from WT and Bcl-2-overexpressing cells treated as described for panel C. Note that tAIF was detected in MNNG-treated WT cells but not in Bcl-2-overexpressing cells. Actin detection was used as a loading control. (E) WT and Bcl-2-overexpressing cells were not treated (Control) or treated with MNNG (15 min), immunostained for PAR detection (green) as described for Fig. 3, and examined by fluorescence microscopy (left panel). Representative micrographs of each cell type are shown. This experiment was repeated three times, yielding similar results. Bar, 10 μm. A fluorescence assessment of calpain activity measured in WT and Bcl-2-overexpressing cells not treated (Control) or treated with MNNG (1 h) as described for Fig. 4 was performed (right panel). Micrographs of representative cells after each treatment are shown. This experiment was repeated three times, yielding comparable results. Bar, 10 μm.
FIG. 9.
FIG. 9.
Schematic model for MNNG-induced cell death. MNNG-induced DNA damage leads through PARP-1 to calpain activation. Calpain in turn activates Bax, resulting in its translocation from the cytosol to mitochondria, where it facilitates the release of tAIF from the mitochondrial intermembrane space to the cytosol. The activated calpain also regulates AIF release by cleaving the membrane-anchored AIF to the soluble form tAIF. Once liberated in the cytosol, tAIF translocates to the nucleus, where it generates 3′-OH DNA breaks and stage I of chromatin condensation.
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