BID regulates AIF-mediated caspase-independent necroptosis by promoting BAX activation

Abstract

Alkylating DNA-damage agents such as N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG) trigger necroptosis, a newly defined form of programmed cell death (PCD) managed by receptor interacting protein kinases. This caspase-independent mode of cell death involves the sequential activation of poly(ADP-ribose) polymerase-1 (PARP-1), calpains, BAX and AIF, which redistributes from mitochondria to the nucleus to promote chromatinolysis. We have previously demonstrated that the BAX-mediated mitochondrial release of AIF is a critical step in MNNG-mediated necroptosis. However, the mechanism regulating BAX activation in this PCD is poorly understood. Employing mouse embryonic knockout cells, we reveal that BID controls BAX activation in AIF-mediated necroptosis. Indeed, BID is a link between calpains and BAX in this mode of cell death. Therefore, even if PARP-1 and calpains are activated after MNNG treatment, BID genetic ablation abolishes both BAX activation and necroptosis. These PCD defects are reversed by reintroducing the BID-wt cDNA into the BID(-/-) cells. We also demonstrate that, after MNNG treatment, BID is directly processed into tBID by calpains. In this way, calpain non-cleavable BID proteins (BID-G70A or BID-Δ68-71) are unable to promote BAX activation and necroptosis. Once processed, tBID localizes in the mitochondria of MNNG-treated cells, where it can facilitate BAX activation and PCD. Altogether, our data reveal that, as in caspase-dependent apoptosis, BH3-only proteins are key regulators of caspase-independent necroptosis.

Figure 1

MNNG-induced caspase-independent necroptosis. (a) After the indicated time post MNNG or STS treatment, WT MEFs were stained with Annexin V and PI, and the frequency of Annexin V and PI-positive labeling (% cell death) was recorded by flow cytometry and illustrated as a plot. Data are the means of six independent experiments±S.D. Representative cytofluorometric plots are shown. Percentages in MNNG-treated cells refer to double-positive staining, and in STS-treated cells to Annexin V and PI-positive labeling. When indicated, WT MEFs were pre-incubated (1 h) with the pan-caspase inhibitor QVD before induction of cell death. Note that QVD inhibits STS-induced caspase-dependent apoptosis but not MNNG-mediated caspase-independent necroptosis. (b) Fluorometric analysis of calpain activity observed in cytosolic extracts obtained from WT treated with MNNG for the indicated times. One unit refers to basal calpain activity observed in untreated WT cells. Data are the means of five independent experiments±S.E.M. (c) Immunofluorescent staining of activated BAX detected in WT MEFs untreated (control) or treated with MNNG (6 h). To visualize mitochondria, cells were incubated with Mitotracker Red before fixation. Hoechst 33 342 was used to stain DNA. A representative overlay of activated BAX, Mitotracker Red and Hoechst 33342 nuclear staining is shown. The number of cells presenting activated BAX were quantified and plotted as a percentage of total cells. Data are the means±S.D. (n=5). Bar: 10 μm. Alternatively, MEFs were treated with MNNG for the indicated times, and BAX activation was measured by flow cytometry and illustrated as a bar chart. Data are the means±S.D. (n=5). (d) Cells were treated with MNNG for the indicated time, then labeled with TMRE and assessed for ΔΨm by flow cytometry. Results are the means of six independent experiments±S.D. In cytometry panels, percentages refer to cells with ΔΨm loss. TMRE, tetramethylrhodamine ethyl ester. (e) Cytosolic fractions, recovered after MNNG treatment, were blotted for tAIF detection. MNNG treatment induces time-dependent tAIF release to cytosol. Actin (cytosolic marker) and Cox IV (mitochondrial marker) were used to control protein loading and fractionation quality. (f) At the indicated times after MNNG treatment, WT MEFs were stained for the detection of 3′-OH DNA breaks and analyzed by flow cytometry. Data are the means of five independent experiments±S.D. In representative cytofluorometric plots, percentages refer to TUNEL-positive cells

Figure 2

The activation of BAX is a key step in necroptosis. (a) WT and BAX^−/− MEFs were untreated (control) or treated with MNNG (9 h) or STS, labeled with Annexin V and PI, and analyzed by flow cytometry. Representative plots are shown. Percentages in MNNG-treated cells refer to double-positive staining, and in STS-treated cells to Annexin V and PI-positive labeling. The square highlights the absence of cell-viability loss recorded in BAX^−/− MEFs after MNNG treatment. (b) Cells were stained as in a, and the frequency of Annexin V and PI-positive labeling (% cell death) was recorded and expressed as a plot. Data are the means of five independent experiments±S.D. ^*P<0.05. (c) BAX immunoblotting detection in total extracts from WT MEFs untreated or treated with MNNG at different times. STS-treated cells were used as a positive control. The membrane was stained with naphtol blue (NB) to assess protein loading

Figure 3

BID but not BIM or BAD deficiency disabled BAX activation and MNNG-induced necroptosis. (a) WT, BIM^−/−, BAD^−/−, and BID^−/− MEFs were untreated (control) or treated with MNNG (9 h) or STS, labeled with Annexin V and PI, and analyzed by flow cytometry. Representative cytofluorometric plots are shown. The percentages refer to the frequencies of Annexin V and PI positive staining. (b) WT, BIM^−/−, BAD^−/−, and BID^−/− MEFs were untreated (control) or treated with MNNG (9 h), stained as in a, and the frequency of double-positive labeling was recorded and expressed as a percentage. Data are the mean±S.E.M. (n=5). ^*P<0.05. (c) WT, BIM^−/−, BAD^−/−, and BID^−/− MEFs were treated with MNNG, and BAX activation was recorded by flow cytometry with the help of an α-Bax antibody (clone 6A7) specifically designed against the active conformation of BAX. Data in bar chart are the means of six independent experiments±S.D. ^*P<0.05. Representative cytofluorometric plots of untreated (control) and MNNG-treated (9 h) cells are shown. Percentages correspond to cells with active BAX. Note that, in the absence of α-Bax antibody, the labeling with the secondary antibody (Neg. in cytofluorometric plots) yields negative results. The squares in a and c highlight the absence of cell-viability loss and BAX activation recorded in BID^−/− MEFs after MNNG treatment. Neg, negative. (d) Cytosolic fractions recovered from WT, BIM^−/−, BAD^−/−, and BID^−/− MEFs untreated (Co) or treated with MNNG (9 h) were probed for tAIF detection. Actin was used to control protein loading

Figure 4

Lentiviral transduction of BID^−/− MEFs with V5-tagged BID-wt cDNA restores BAX activation and MNNG-induced necroptosis. (a) WT, BID^−/−, and two selected clones of BID^−/− MEFs cells expressing BID-wt were untreated (control) or treated with MNNG (9 h), and labeled with Annexin V and PI. The frequency of double-positive labeling was recorded and expressed as a plot. Data are the mean±S.D. (n=4). ^*P<0.05. The expression level of BID in these cells was assessed by immunoblotting. Note the different apparent molecular mass of endogenous BID (∼22 kDa) and lentiviral-transduced BID-V5 (∼26 KDa). Actin was used to control protein loading. (b) MEFs treated as in a were labeled with TMRE, and assessed for ΔΨm by flow cytometry. The frequency of cells with ΔΨm loss was recorded and expressed as a plot. Data are the means of four independent experiments±S.D. ^*P<0.05. (c) The panel of MEFs used in a was untreated (control) or MNNG-treated (9 h), and BAX activation was measured by flow cytometry and illustrated as a bar chart. Data are the means of four independent experiments±S.D. In representative cytofluorometric plots, percentages correspond to cells with active BAX. TMRE, tetramethylrhodamine ethyl ester. (d) MEFs were untreated (control) or MNNG-treated (9 h), and the presence of 3′-OH DNA breaks was assessed by flow cytometry and illustrated as a plot. Data are the means of four independent experiments±S.D. In cytofluorometric plots, percentages correspond to TUNEL-positive cells

Figure 5

BID acts downstream of PARP-1 and calpains but upstream of BAX activation, mitochondrial damage, tAIF release, and DNA degradation in MNNG-induced PCD. (a) WT and BID^−/− MEFs were untreated (control) or treated with MNNG (15 min), immunostained for PAR detection (green), and visualized by fluorescent microscopy. Hoechst 33 342 (blue) was used to visualize the nuclei. Representative micrographs of each cell type are shown. Bar: 10 μm. After MNNG treatment, the entire WT and BID^−/− cell population (100% of cells) display PAR-positive labeling. This experiment was repeated six times, yielding similar results (the entire WT and BID^−/− cell population display PAR-positive labeling). (b) PAR immunoblotting detection in lysates from WT and BID^−/− MEFs untreated or treated with MNNG at different times. The membrane was stained with naphtol blue (NB) to assess protein loading. (c) WT and BID^−/− MEFs were treated with MNNG at the times indicated, and analyzed by measuring absorbance at 570 nm to assess total NAD⁺ levels. Concentrations of NAD⁺ were normalized to those from untreated cells. Results are the means of five independent experiments±S.E.M. A pharmacological PARP inhibitor, PJ34, helped to determine the specificity of the PARP-dependent NAD⁺ loss associated with MNNG treatment. (d) Quantification of the intracellular ATP levels in WT and BID^−/− MEFs treated with MNNG at different times. H₂O₂ was used as a positive control. A value of 100% refers to the basal level of ATP scored in untreated cells. Data are the means of five independent experiments±S.E.M. (e) Fluorescent assessment of calpain activity measured in WT and BID^−/− MEFs untreated (control) or treated with MNNG (1 h). Phase-contrast was used to visualize cells. Representative micrographs of each treatment are shown. After MNNG treatment both WT and BID^−/− MEFs (100%) display calpain-positive staining. This experiment was repeated four times, yielding similar results. Bar: 10 μm. (f) WT and BID^−/− MEFs were treated with MNNG for the indicated times, then labeled with TMRE and assessed for ΔΨm by flow cytometry. Results refer to cells with ΔΨm loss and are the means of six independent experiments±S.E.M. TMRE, tetramethylrhodamine ethyl ester. (g) Cytosolic fractions recovered from WT and BID^−/− MEFs after MNNG treatment at different times were probed for tAIF detection. Actin was used to assess protein loading. (h) WT and BID^−/− MEFs were untreated or treated with MNNG (9 h), stained for the detection of 3′-OH DNA breaks, and analyzed by flow cytometry. Percentages refer to TUNEL-positive cells. This experiment was repeated ten times, with low experimental variability

Figure 6

BID is cleaved into tBID via calpains and localizes in mitochondria during MNNG-induced necroptosis. (a) Lysates from MEFs treated by MNNG at the indicated times were prepared, and blotted for BID and tBID detection. STS-treated cells were used as a positive control. Alternatively, mitochondrial and cytosolic extracts of cells treated or not with MNNG at different times were analyzed by western blotting for the presence of tBID. STS-treated cells were used as a positive control. Cox IV (mitochondrial marker) and pan-ERK (cytosolic marker) were used to control fractionation quality and protein loading. This experiment was repeated three times, yielding comparable results. (b) BID and tBID immunoblotting detection in lysates from WT and CAPN4^−/− MEFs untreated (Co) or treated with MNNG (9 h) or STS. The membrane was reblotted for actin detection to control protein loading. Note the absence of tBID in MNNG-treated CAPN4^−/− MEFs. (c) WT and CAPN4^−/− MEFs were treated with MNNG (9 h) or STS, and BAX activation was measured by flow cytometry and illustrated as a bar chart. Data are the means of four independent experiments±S.D. ^*P<0.05. (d) WT and CAPN4^−/− MEFs were treated or not with MNNG (9 h) or STS, then labeled with TMRE and assessed for ΔΨm by flow cytometry. Results refer to cells with ΔΨm loss±S.D. (n=6). ^*P<0.05. TMRE, tetramethylrhodamine ethyl ester. (e) WT and CAPN4^−/− cells were untreated (control) or treated with MNNG (9 h) or STS, labeled with Annexin V and PI, and analyzed by flow cytometry. Representative cytofluorometric plots are shown. Percentages in MNNG-treated cells refer to double-positive staining, and in STS-treated cells to Annexin V and PI-positive labeling. The square highlights the absence of cell-viability loss recorded in CAPN4^−/− MEFs after MNNG treatment

Figure 7

Calpains cleave BID into tBID at Gly70 in MNNG-induced necroptosis. (a) Ribbon structure of BID and tBID. Caspases and calpains cleave BID at the D59 and G70 amino-acidic position, respectively, to generate the truncated form of BID (tBID). This processing, which provokes the removal of a part of the BID N terminus (red), discloses the BH3 α-helix (magenta) on tBID. Here, we represent the tBID generated by calpain processing at G70. (b) WT, BID^−/−, BID^−/− transduced with the pLVX-IRES-Zs-Green lentiviral empty vector (pLV), and two selected clones of BID^−/− MEFs cells expressing: (i) BID-wt, (ii) a caspase ncBID-D59A mutant, or (iii) two calpain ncBID mutants (BID-G70A and BID-Δ68-71) were untreated (control) or treated with MNNG (9 h) and labeled with Annexin V and PI. The frequency of double-positive labeling was recorded and expressed as a plot. Data are the mean±S.D. (n=4). The expression level of BID in these cells was assessed by immunoblotting with the help of a V5 monoclonal antibody. Actin was used to control protein loading. (c) MEFs were untreated (control) or MNNG-treated (9 h), and BAX activation was measured by flow cytometry and illustrated as a graph. Data are the means of four independent experiments±S.D. In representative cytofluorometric plots, percentages correspond to cells with active BAX. (d) The panel of MEFs used in b was untreated (control) or MNNG-treated (9 h), labeled with TMRE and assessed for ΔΨm by flow cytometry. The frequency of cells with ΔΨm loss was recorded and expressed as a bar chart. Data are the means of four independent experiments±S.D. TMRE, tetramethylrhodamine ethyl ester. (e) MEFs left untreated or treated with MNNG (9 h) were subjected to immunoblot detection of BID with the help of a V5 monoclonal antibody. BID-wt and the caspase ncBID-D59A were processed after MNNG treatment (∼80% of BID is cleaved after MNNG treatment, see Materials and Methods section for quantification details). In contrast, the calpain ncBID mutants BID-G70A and BID-D68-71 remained as precursor proteins. Similar results were observed in two independent clones. Actin was used to control protein loading

Figure 8

Representation of AIF-mediated necroptosis. MNNG-induced DNA damage leads through PARP-1 to NAD⁺, ATP depletion and calpain activation. Calpains cleave BID into tBID at Gly70. Once processed, tBID localizes in mitochondria, where it facilitates BAX activation. Furthermore, activated BAX provokes mitochondrial damage and favors the release of tAIF from mitochondria to the cytosol and nucleus. The anti-apoptotic protein BCL-2 can prevent this release. Upon transfer to the nucleus, tAIF associates with CypA and γH2AX to generate a DNA-degrading complex that promotes chromatinolysis and cell-viability loss