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. 2000 Nov 20;192(10):1403-14.
doi: 10.1084/jem.192.10.1403.

Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation

V R Sutton  1 , J E Davis, M Cancilla, R W Johnstone, A A Ruefli, K Sedelies, K A Browne, J A Trapani
Affiliations

Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation

V R Sutton et al. J Exp Med. .

Abstract

The essential upstream steps in granzyme B-mediated apoptosis remain undefined. Herein, we show that granzyme B triggers the mitochondrial apoptotic pathway through direct cleavage of Bid; however, cleavage of procaspases was stalled when mitochondrial disruption was blocked by Bcl-2. The sensitivity of granzyme B-resistant Bcl-2-overexpressing FDC-P1 cells was restored by coexpression of wild-type Bid, or Bid with a mutation of its caspase-8 cleavage site, and both types of Bid were cleaved. However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis. Bid with a mutation preventing its interaction with Bcl-2 was cleaved but also failed to restore apoptosis. Rapid Bid cleavage by granzyme B (<2 min) was not delayed by Bcl-2 overexpression. These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2. In granzyme B-treated Jurkat cells, endogenous Bid cleavage and loss of mitochondrial membrane depolarization occurred despite caspase inactivation with z-Val-Ala-Asp-fluoromethylketone or Asp-Glu-Val-Asp-fluoromethylketone. Initial partial processing of procaspase-3 and -8 was observed irrespective of Bcl-2 overexpression; however, later processing was completely abolished by Bcl-2. Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B.

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Figures

Figure 1
Figure 1
The expression of Bcl-2 and Bid in FDC-P1 transfectant populations. Expression of human Bcl-2 (thick lines) and Bid (thin lines) after gene transfection. FDC-P1 cells expressing either Bcl-2 or Bid alone, or both molecules (middle) were permeabilized, stained, and analyzed by flow cytometry using mouse mAbs specific for human Bcl-2 or EE-tagged Bid. Fluorescence staining in each case is compared with that with the FITC-conjugated anti–mouse reagent alone (dotted lines).
Figure 2
Figure 2
Cooverexpression of Bid overcomes Bcl-2's block of granzyme B–mediated cell death. Cytofluorographic analysis of cells, showing DNA fragmentation (TUNEL assay) and changes in light scatter parameters (forward vs. side scatter) of cells treated with sublytic perforin (Pfp; top panels) or perforin in combination with granzyme B (GrB, 60 nM; bottom panels) for 90 min at 37°C. The cells were wild-type FDC-P1, or FDC-P1 cells stably expressing human Bcl-2, Bid, or both Bcl-2 and Bid (see Fig. 1). The numerals in each panel indicate the percentage of TUNEL-positive cells, to the nearest integer. The experiment shown is representative of four similar experiments.
Figure 3
Figure 3
Bid expression does not overcome Bcl-2's block of cell death due to growth factor withdrawal or staurosporine. (A) Wild-type FDC-P1 cells, or FDC-P1 cells stably expressing human Bcl-2, Bid, or both Bcl-2 and Bid (see Fig. 1) were washed extensively and plated in culture medium lacking IL-3. Cell viability was estimated at the times indicated by trypan blue exclusion. (B) The same cell populations were plated in medium containing staurosporine for 18 h, or in normal medium, and cell viability was estimated by trypan blue exclusion. The experiment shown is representative of four similar experiments.
Figure 6
Figure 6
Bid cleavage in Jurkat cells, in response to granzyme B (GrB) and anti-Fas (αFas) IgM. Western blot using anti-Bid antiserum of Jurkat cells either untreated (Cells), incubated with either sublytic Perforin (Pfp) or PLO (P) alone, Pfp/PLO in combination with granzyme B (60 nM) for 2 h, or exposed to anti-Fas IgM (250 ng/ml) for 4 h. Tubulin (T) staining has been used to ensure approximately equal protein loading in the samples.
Figure 4
Figure 4
Bid cleavage in Bcl-2–overexpressing FDC-P1 cells after exposure to granzyme B, but not after growth factor withdrawal or exposure to staurosporine (STS). (A) Western blot analysis of whole cell lysates of FDC-P1 cells stably expressing only Bid or both Bid and Bcl-2, after exposure to sublytic perforin alone (Pfp) or a combination of perforin and granzyme B (GrB) for 15–60 min. The signals for uncleaved and tBid are indicated with arrowheads. (B) Western blot of whole cell lysates of the same FDC-P1 cell transfectants as in A after withdrawal of growth factor or exposure to staurosporine for 18 h. Both blots were reprobed with an antiserum specific for tubulin (T; 50 kD) to assess protein loading. The migration of relevant molecular size markers is indicated in kilodaltons at left.
Figure 4
Figure 4
Bid cleavage in Bcl-2–overexpressing FDC-P1 cells after exposure to granzyme B, but not after growth factor withdrawal or exposure to staurosporine (STS). (A) Western blot analysis of whole cell lysates of FDC-P1 cells stably expressing only Bid or both Bid and Bcl-2, after exposure to sublytic perforin alone (Pfp) or a combination of perforin and granzyme B (GrB) for 15–60 min. The signals for uncleaved and tBid are indicated with arrowheads. (B) Western blot of whole cell lysates of the same FDC-P1 cell transfectants as in A after withdrawal of growth factor or exposure to staurosporine for 18 h. Both blots were reprobed with an antiserum specific for tubulin (T; 50 kD) to assess protein loading. The migration of relevant molecular size markers is indicated in kilodaltons at left.
Figure 7
Figure 7
Bid is cleaved early in Jurkat cells exposed to granzyme B (GrB), irrespective of Bcl-2 overexpression. Jurkat cells overexpressing Bcl-2 or transfected with vector backbone DNA only (Jurkat-pgk) were exposed for the times indicated to granzyme B (60 nM) and sublytic PLO. Control cells were exposed to no stimulus (C) or PLO alone (P). At each time point, the cells were flooded with ice-cold medium, pelleted in a microfuge, washed twice in ice-cold medium, then lysed for Western blot analysis with anti-Bid antiserum (see Materials and Methods). The migration of intact and tBid is indicated by the arrowheads.
Figure 5
Figure 5
The granzyme B cleavage site of Bid (D75) is essential to overcome Bcl-2's block of granzyme B–mediated cell death, but the caspase-8 cleavage site (D59) is not. (A) Cytofluorographic analysis, showing DNA fragmentation (TUNEL assay) and changes in light scatter parameters (forward vs. side scatter) of cells exposed to sublytic PLO with granzyme B (60 nM) for 60 min at 37°C. The cells were Bcl-2–overexpressing FDC-P1 cells also expressing wild-type Bid or Bid in which the P1 Asp at the granzyme B cleavage site was mutated to Glu (D75E), or in which the P1 Asp at the caspase-8 cleavage site was mutated to Glu (D59E). The numerals in each panel indicate the percentage of TUNEL-positive cells, to the nearest integer. The experiment shown is representative of three similar experiments performed with either PLO or perforin. No significant apoptosis was detected when the cells were exposed to either sublytic quantities of perforin/PLO or granzyme B alone (<2%; data not shown). (B) Western blot using anti-EE antiserum of D59E and D75E cells shown in A. The cells were incubated with sublytic PLO (P) with or without granzyme B (GrB; 30 nM) at 37°C for 15–60 min, as indicated. Tubulin (T) staining has been used to ensure approximately equal protein loading in the samples. (C) Western blot analysis of whole cell lysates of FDC-P1 cells stably expressing L90A-mutated Bid and Bcl-2, after exposure to sublytic PLO or a combination of PLO and granzyme B for 15–60 min. The signal for uncleaved Bid is indicated. Staining of the same samples with antitubulin (T) was used to normalize the amount of protein loaded in each lane.
Figure 5
Figure 5
The granzyme B cleavage site of Bid (D75) is essential to overcome Bcl-2's block of granzyme B–mediated cell death, but the caspase-8 cleavage site (D59) is not. (A) Cytofluorographic analysis, showing DNA fragmentation (TUNEL assay) and changes in light scatter parameters (forward vs. side scatter) of cells exposed to sublytic PLO with granzyme B (60 nM) for 60 min at 37°C. The cells were Bcl-2–overexpressing FDC-P1 cells also expressing wild-type Bid or Bid in which the P1 Asp at the granzyme B cleavage site was mutated to Glu (D75E), or in which the P1 Asp at the caspase-8 cleavage site was mutated to Glu (D59E). The numerals in each panel indicate the percentage of TUNEL-positive cells, to the nearest integer. The experiment shown is representative of three similar experiments performed with either PLO or perforin. No significant apoptosis was detected when the cells were exposed to either sublytic quantities of perforin/PLO or granzyme B alone (<2%; data not shown). (B) Western blot using anti-EE antiserum of D59E and D75E cells shown in A. The cells were incubated with sublytic PLO (P) with or without granzyme B (GrB; 30 nM) at 37°C for 15–60 min, as indicated. Tubulin (T) staining has been used to ensure approximately equal protein loading in the samples. (C) Western blot analysis of whole cell lysates of FDC-P1 cells stably expressing L90A-mutated Bid and Bcl-2, after exposure to sublytic PLO or a combination of PLO and granzyme B for 15–60 min. The signal for uncleaved Bid is indicated. Staining of the same samples with antitubulin (T) was used to normalize the amount of protein loaded in each lane.
Figure 5
Figure 5
The granzyme B cleavage site of Bid (D75) is essential to overcome Bcl-2's block of granzyme B–mediated cell death, but the caspase-8 cleavage site (D59) is not. (A) Cytofluorographic analysis, showing DNA fragmentation (TUNEL assay) and changes in light scatter parameters (forward vs. side scatter) of cells exposed to sublytic PLO with granzyme B (60 nM) for 60 min at 37°C. The cells were Bcl-2–overexpressing FDC-P1 cells also expressing wild-type Bid or Bid in which the P1 Asp at the granzyme B cleavage site was mutated to Glu (D75E), or in which the P1 Asp at the caspase-8 cleavage site was mutated to Glu (D59E). The numerals in each panel indicate the percentage of TUNEL-positive cells, to the nearest integer. The experiment shown is representative of three similar experiments performed with either PLO or perforin. No significant apoptosis was detected when the cells were exposed to either sublytic quantities of perforin/PLO or granzyme B alone (<2%; data not shown). (B) Western blot using anti-EE antiserum of D59E and D75E cells shown in A. The cells were incubated with sublytic PLO (P) with or without granzyme B (GrB; 30 nM) at 37°C for 15–60 min, as indicated. Tubulin (T) staining has been used to ensure approximately equal protein loading in the samples. (C) Western blot analysis of whole cell lysates of FDC-P1 cells stably expressing L90A-mutated Bid and Bcl-2, after exposure to sublytic PLO or a combination of PLO and granzyme B for 15–60 min. The signal for uncleaved Bid is indicated. Staining of the same samples with antitubulin (T) was used to normalize the amount of protein loaded in each lane.
Figure 8
Figure 8
Time course of Bid and procaspase cleavage in response to granzyme B (GrB) or Fas ligation. (A) Western blots of Jurkat cells incubated with anti-Fas IgM Ab (250 ng/ml) or with sublytic PLO (P) with or without granzyme B (60 nM) at 37°C for the times indicated. Cell lysates were fractionated on 15% SDS-PAGE, transferred to nylon, and probed with the indicated antisera (see Materials and Methods). (B) Western blot using anti–caspase-3 antiserum of Jurkat cells overexpressing Bcl-2 or transfected with vector backbone (Jurkat.pgk). The cells were untreated (C) or incubated with sublytic PLO (P) with or without granzyme B (60 nM) at 37°C for the times indicated. To facilitate comparison, signals for 32-kD procaspase-3 (Pro C3, top panels) are shown after a short exposure to x-ray film (15 s), and those for the cleavage products (bottom panels) are from longer exposure (2 min). (C) Western blot using anti–caspase-8 antiserum of Jurkat cells overexpressing Bcl-2 or transfected with vector backbone (Jurkat.pgk). The cells were untreated (C) or incubated with sublytic PLO (P) with or without granzyme B (60 nM) at 37°C for the times indicated. To facilitate comparison, signals for 55-kD procaspase-8 (Pro C8, top panels) are shown after a short exposure to x-ray film (25 s), and those for the cleavage products (bottom panels) are from longer exposure (3 min).
Figure 8
Figure 8
Time course of Bid and procaspase cleavage in response to granzyme B (GrB) or Fas ligation. (A) Western blots of Jurkat cells incubated with anti-Fas IgM Ab (250 ng/ml) or with sublytic PLO (P) with or without granzyme B (60 nM) at 37°C for the times indicated. Cell lysates were fractionated on 15% SDS-PAGE, transferred to nylon, and probed with the indicated antisera (see Materials and Methods). (B) Western blot using anti–caspase-3 antiserum of Jurkat cells overexpressing Bcl-2 or transfected with vector backbone (Jurkat.pgk). The cells were untreated (C) or incubated with sublytic PLO (P) with or without granzyme B (60 nM) at 37°C for the times indicated. To facilitate comparison, signals for 32-kD procaspase-3 (Pro C3, top panels) are shown after a short exposure to x-ray film (15 s), and those for the cleavage products (bottom panels) are from longer exposure (2 min). (C) Western blot using anti–caspase-8 antiserum of Jurkat cells overexpressing Bcl-2 or transfected with vector backbone (Jurkat.pgk). The cells were untreated (C) or incubated with sublytic PLO (P) with or without granzyme B (60 nM) at 37°C for the times indicated. To facilitate comparison, signals for 55-kD procaspase-8 (Pro C8, top panels) are shown after a short exposure to x-ray film (25 s), and those for the cleavage products (bottom panels) are from longer exposure (3 min).
Figure 8
Figure 8
Time course of Bid and procaspase cleavage in response to granzyme B (GrB) or Fas ligation. (A) Western blots of Jurkat cells incubated with anti-Fas IgM Ab (250 ng/ml) or with sublytic PLO (P) with or without granzyme B (60 nM) at 37°C for the times indicated. Cell lysates were fractionated on 15% SDS-PAGE, transferred to nylon, and probed with the indicated antisera (see Materials and Methods). (B) Western blot using anti–caspase-3 antiserum of Jurkat cells overexpressing Bcl-2 or transfected with vector backbone (Jurkat.pgk). The cells were untreated (C) or incubated with sublytic PLO (P) with or without granzyme B (60 nM) at 37°C for the times indicated. To facilitate comparison, signals for 32-kD procaspase-3 (Pro C3, top panels) are shown after a short exposure to x-ray film (15 s), and those for the cleavage products (bottom panels) are from longer exposure (2 min). (C) Western blot using anti–caspase-8 antiserum of Jurkat cells overexpressing Bcl-2 or transfected with vector backbone (Jurkat.pgk). The cells were untreated (C) or incubated with sublytic PLO (P) with or without granzyme B (60 nM) at 37°C for the times indicated. To facilitate comparison, signals for 55-kD procaspase-8 (Pro C8, top panels) are shown after a short exposure to x-ray film (25 s), and those for the cleavage products (bottom panels) are from longer exposure (3 min).
Figure 9
Figure 9
Cleavage of Bid in response to granzyme B (GrB) is independent of caspases, but is caspase dependent after Fas ligation. (A) Western blot of Jurkat cells which were untreated (C) or incubated with sublytic PLO (P) with or without granzyme B (60 nM) at 37°C for the times indicated. Cell lysates were fractionated on 15% SDS-PAGE, transferred to nylon, and probed with anti-Bid antiserum (see Materials and Methods). Before the experiment, the cells were preincubated with the caspase inhibitors z-DEVD-fmk or z-VAD-fmk, or with the control compound z-FA-fmk (20 μM) for 30 min at 37°C. (B) Western blot of Jurkat cells which were untreated (C) or incubated with sublytic PLO (P) with or without granzyme B (60 nM) for 2 h or anti-Fas (αFas) IgM (250 ng/ml) for 4 h at 37°C. Cell lysates were probed with anti-Bid antiserum as in A. Before the experiment, the cells were preincubated with z-VAD-fmk or z-FA-fmk (20 μM) for 30 min at 37°C.
Figure 9
Figure 9
Cleavage of Bid in response to granzyme B (GrB) is independent of caspases, but is caspase dependent after Fas ligation. (A) Western blot of Jurkat cells which were untreated (C) or incubated with sublytic PLO (P) with or without granzyme B (60 nM) at 37°C for the times indicated. Cell lysates were fractionated on 15% SDS-PAGE, transferred to nylon, and probed with anti-Bid antiserum (see Materials and Methods). Before the experiment, the cells were preincubated with the caspase inhibitors z-DEVD-fmk or z-VAD-fmk, or with the control compound z-FA-fmk (20 μM) for 30 min at 37°C. (B) Western blot of Jurkat cells which were untreated (C) or incubated with sublytic PLO (P) with or without granzyme B (60 nM) for 2 h or anti-Fas (αFas) IgM (250 ng/ml) for 4 h at 37°C. Cell lysates were probed with anti-Bid antiserum as in A. Before the experiment, the cells were preincubated with z-VAD-fmk or z-FA-fmk (20 μM) for 30 min at 37°C.
Figure 10
Figure 10
Loss of mitochondrial membrane depolarization in response to granzyme B is independent of caspases. Loss of rhodamine 123 (Rh123) accumulation in Jurkat cells after exposure to granzyme B (GrB, 60 nM) and sublytic PLO (P) for 2 h. The same cell samples were also analyzed for DNA fragmentation by TUNEL and for changes in forward and side scatter characteristics by flow cytometry at 2 h. Before commencement of the experiment, the cells were preincubated with the caspase inhibitors z-DEVD-fmk or z-VAD-fmk, or with the control compound z-FA-fmk (each 100 μM) for 30 min at 37°C. The numerals indicate the percentage of rhodamine-positive or -negative cells, or TUNEL-positive cells, to the nearest integer.
Figure 11
Figure 11
Release of cytochrome c from mitochondria in response to granzyme B is dependent on caspase activation. Jurkat cells were preincubated with 20 μM z-VAD-fmk or without inhibitor for 30 min at 37°C, then exposed to sublytic PLO (P) with or without granzyme B (GrB) for 2 h at 37°C. The cells were then washed, lysed, and cytosolic extracts devoid of mitochondria prepared by ultracentrifugation. The extracts were analyzed in Western blots using antisera for cytochrome c (cyt. c) or tubulin (T) (see Materials and Methods).
Figure 12
Figure 12
A unifying model of granzyme B–mediated cell death. Immediately upon accessing the cytosol, granzyme B principally cleaves Bid (thick arrow) and partially activates procaspase-3 (dotted arrows). However, caspase-3 is not fully processed unless tBid primarily disrupts the mitochondrion, a step that is regulated by Bcl-2 overexpression. Activation of other caspases is downstream of caspase-3 or related DEVD-fmk–inhibitable caspases. Mitochondrial perturbation by Bid results in cell death that is independent of caspases, and therefore is not blocked by p35. The mitochondrial factor responsible for full caspase activation is unknown (factor X), but is not cytochrome c, the release of which also requires further (secondary) mitochondrial damage resulting from full caspase activation (see text). We speculate, without any evidence, that this factor may be similar to DIABLO/Smac, a protein that, when released from mitochondria, displaces IAP inhibitors from some caspases to allow full activation.
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