Evidence against a functional site for Bcl-2 downstream of caspase cascade in preventing apoptosis

Oncogene, Oct 1997

Apoptotic cell death is driven by ICE family proteases (caspases) and negatively regulated by Bcl-2 family proteins. Although it has been shown that Bcl-2 exerts anti-apoptotic activity by blocking a step(s) leading to the activation of caspases, a role for Bcl-2 and Bcl-xL downstream of the caspase cascade has remained unclear. Here, we show that purified active caspase-3 (CPP32/Yama/apopain) and caspase-1 (ICE) induces apoptosis when microinjected into the cytoplasm of cells, confirming our recent observations, and that the apoptosis is not at all prevented by Bcl-2 and Bcl-xL, which are overexpressed more than sufficiently to prevent Fas-mediated and overexpressed procaspase-1-mediated apoptosis. Thus, Bcl-2 and Bcl-xL do not act downstream of the caspase cascade.

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Evidence against a functional site for Bcl-2 downstream of caspase cascade in preventing apoptosis

Evidence against a functional site for Bcl-2 downstream of caspase cascade in preventing apoptosis Noriko Yasuhara 0 Setsuko Sahara 0 Shinji Kamada 0 Yutaka Eguchi 0 Yoshihide Tsujimoto 0 0 Department of Medical Genetics, Biomedical Research Center, Osaka University Medical School , 2-2 Yamadaoka, Suita, Osaka 565 Japan Apoptotic cell death is driven by ICE family proteases (caspases) and negatively regulated by Bcl-2 family proteins. Although it has been shown that Bcl-2 exerts anti-apoptotic activity by blocking a step(s) leading to the activation of caspases, a role for Bcl-2 and Bcl-xL downstream of the caspase cascade has remained unclear. Here, we show that puri?ed active caspase-3 (CPP32/Yama/apopain) and caspase-1 (ICE) induces apoptosis when microinjected into the cytoplasm of cells, con?rming our recent observations, and that the apoptosis is not at all prevented by Bcl-2 and Bcl-xL, which are overexpressed more than su?ciently to prevent Fas-mediated and overexpressed procaspase-1-mediated apoptosis. Thus, Bcl-2 and Bcl-xL do not act downstream of the caspase cascade. apoptosis; bcl-2; bcl-x; caspase; ICE; CPP32; Fas; signal transduction Introduction Apoptosis, a mechanism of cell death, plays important roles in a variety of biological events, including morphogenesis, maintenance of homeostasis of various tissues and removal of harmful cells (reviewed by Steller, 1995) . Apoptosis is characterized by chromatin condensation, nuclear fragmentation and formation of apoptotic bodies, which are phagocytosed by other cells (Wyllie et al., 1980) . Apoptosis can be induced by a variety of stimuli, including depletion of growth factors, hormones, heat shock, genotoxins and crosslinking of death factor receptors (reviewed by Thompson, 1995) . Apoptotic signal transduction pathways activated by various treatments converge into a common pathway, which is driven by ICE family proteases (reviewed by Martin and Green, 1995) , now called caspases (Alnemri et al., 1996) , and negatively regulated by anti-cell death proteins such as the Bcl-2 family (reviewed by Thompson, 1995) and the IAP family (Clem and Miller, 1994; Hay et al., 1995) . Caspase-1 (ICE)-like and caspase-3 (CPP32/Yama)-like proteases are implicated in apoptosis, because their tetrapeptide competitive inhibitors prevent apoptosis induced by a variety of stimuli (reviewed by Martin and Green, 1995) . Studies of ice-de?cient mice have shown that ICE (caspase-1) itself is directly involved in Fasmediated apoptosis of thymocytes (Kuida et al., 1995) , and the use of Ich-1s, an isoform of Ich-1, revealed the involvement of Ich-1L (caspase-2) in at least one form of apoptosis (Wang et al., 1994) . Accumulation of neuronal cells in CPP32-de?cient mice also indicates the involvement of CPP32/Yama (caspase-3) in apoptosis in nervous system (Kuida et al., 1996) . Caspases are translated in their precursor forms and activated by proteolytic cleavage. Some caspases including caspase-1 are likely activated autonomously when overexpressed (Miura et al., 1993; Wang et al., 1994; Kamada et al., 1997) , but others such as caspase-3 are not activated without apoptotic stimuli (Hasegawa et al., 1996) . Caspases have been suggested to constitute a protease cascade, based on observations that their active forms directly cleave the precursors of other family members in vitro to activate them (reviewed by Martin and Green, 1995) . We and others have recently reported that a protease cascade involving caspase-1-like and caspase3-like proteases operates to transduce apoptotic signals in vivo (Shimizu et al., 1996a; Enari et al., 1996) . Bcl-2 blocks the activation of caspases (Chinnaiyan et al., 1996; Shimizu et al., 1996a; Boulakia et al., 1996) , probably by preventing the cell death-induced loss of mitochondrial membrane potential (Zamzami et al., 1995; Shimizu et al., 1996b) . It has recently been described that Bcl-xL indirectly binds pro-FLICE (caspase-8) (Chinnaiyan et al., 1997) , raising the possibility that Bcl-xL prevents the activation of caspases by sequestering pro-caspases. Thus, Bcl-2 acts at least upstream of the activation of caspases. Bcl-2 localizes in multiple membrane compartments, including the nuclear envelope, endoplasmic reticulum and mitochondrial membranes (Hockenbery et al., 1990; Monaghan et al., 1992; Jacobson et al., 1993; Krajewski et al., 1993; Akao et al., 1994) , raising the possibility that Bcl-2 also acts downstream of caspases. Indeed an anti-cell death role for Bcl-2 exerted downstream of active ICE family proteases has been suggested by the observations that (1) using in vitro apoptosis system, apoptotic DNA ladder formation is prevented by a Bcl-2-containing cell lysate, even after an e?ector(s) downstream of caspases is activated, as judged by the failure of caspase inhibitors to prevent apoptotic changes in naked nuclei (Enari et al., 1995) and (2) apoptosis induced by transfection with caspase genes which encode precursor forms of proteases is prevented by overexpressed Bcl-2 (Miura et al., 1993; Wang et al., 1994) . Using microinjection procedures, we show here that Bcl-2 and Bcl-xL exert their anti-cell death activity only upstream and/or within but not downstream of the caspase cascade. 1922 Results Failure of overexpressed Bcl-2 and Bcl-xL to prevent apoptosis induced by active CPP32/Yama protease (caspase-3) In an e?ort to activate apoptotic process downstream of the caspase cascade, we microinjected puri?ed active a bcl-2 b bcl-xL RITC CPP32/Yama protease (caspase-3) into HeLa (D98AH2) cells in the presence or absence of overexpressed Bcl-2 or Bcl-xL. Overexpression of Bcl-2 and Bcl-xL was achieved by microinjection of bcl-2 and bclxL DNA into the nucleus. To identify microinjected cells, ?uorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) chemically conjugated with the synthetic nuclear localization signal (NLS) of SV40 Tantigen (see Materials and methods), was co-injected into cells together with bcl-2 or bcl-xL DNA. We ?rst examined the e?ects of Bcl-2 and Bcl-xL overexpressed through microinjection on Fas-mediated apoptosis, to verify their e?ective anti-apoptotic activity. Two hours after microinjection of the respective DNA into HeLa cell nucleus, when these proteins are expressed (Figure 1), the cells were treated with agonistic anti-Fas antibody for 4 h and were stained with Hoechst 33342 and visualized under a ?uorescence microscope to judge apoptosis by chromatin condensation, nuclear fragmentation, and cellular shrinkage and fragmentation. About 80% of vector DNA-injected cells underwent apoptosis, whereas overexpressed Bcl-2 and Bcl-xL completely inhibited Fas-mediated apoptosis (Figure 2 and Table 1). Even when one ?fth of DNA was microinjected, Fas-mediated apoptosis was completely prevented (data not shown), indicating that Bcl-2 and Bcl-xL overexpressed through microinjection are fully functional under the present conditions. We next examined the e?ectiveness of overexpressed Bcl-2 and Bcl-xL on apoptosis induced by active caspase-3. In this study, the puri?ed active caspase-3 at 232 units/ml was microinjected into the cytoplasm of the DNA-injected cells. The amount of microinjected a vector b bcl-2 c bcl-xL RITC Hoechst Number of apoptotic cells/total cell number and the extent (%) of apoptosis are given. The indicated DNAs were microinjected into D98AH2 cells as described in the legend for Figure 2. Two hours after microinjection, cells were treated with 1 mg/ml agonistic antiFas monoclonal antibody for 4 h, and apoptotic cells were identi?ed as described in Materials and methods. vec0.001 and vec0.1 indicate the microinjection of vector DNA at 0.001 mg/ml and 0.1 mg/ml, respectively caspase-3 was minimally su?cient to induce apoptosis. Given that at most 20 ? of the solution was microinjected into cells (see Materials and methods) and treatment of un-injected cells with anti-Fas antibody induced activation of caspase-3-like proteases up to 50 units/106 cells within 6 h (Figure 3), less than 4.6 units of caspase-3 were injected per 106 cells, which were one-order magnitude less than that of caspase-3-like proteases activated during Fas-mediated apoptosis. Rhodamine isothiocyanate (RITC)-labeled BSA was co-injected with active caspase-3, so that cells which were injected with both caspase-3 and DNA were identi?ed by RITC-?uorescence in cytoplasm and FITC-?uorescence in the nucleus. Two hours after microinjection of caspase-3, cells were analysed as described. Microinjection of DNAs and subsequent microinjection of bu?er did not enhance cell death, whereas microinjection of puri?ed active caspase-3 into vector DNA-injected cells induced apoptosis (Figure 4 and Table 2). Apoptosis was observed in 50 to 70% of cells microinjected with active caspase-3 in 2 h (Figure 4 and Table 2). Active caspase-3-induced cell death was inhibited in the presence of the tetrapeptide caspase-3 inhibitor Ac-DEVD-CHO at 100 mM in the medium (data not shown), indicating that the proteolytic activity of microinjected caspase-3 was responsible for the apoptosis. However, active caspase-3-induced apoptosis was not at all inhibited by overexpressed Bcl-2 and Bcl-xL (Figure 4 and Table 2), which is functional to prevent Fas-mediated apoptosis as described above. When caspase-3-injected cells were incubated for 1 h, observed apoptotic cells were about half of those after 2 h incubation regardless of the expression of Bcl-2 and Bcl-xL (Table 2, exp. 4), arguing against the possibility that Bcl-2 and Bcl-xL, signi?cantly delay the active caspase-3-induced apoptosis. When only half the amount of caspase-3 was microinjected, cells did not undergo apoptosis (data not shown), indicating that the microinjected amount of active caspase-3 was minimally su?cient to induce apoptosis and did not exceed the capacity of Bcl-2 and Bcl-xL. Furthermore, longer preincubation after microinjection of bcl-2 and bcl-xL DNA up to 6 h to allow possible further accumulation of Bcl-2 and Bcl-xL protein did not a?ect the active caspase-3-induced apoptosis (data not shown), and microinjection of tenfold more bcl-2 or bcl-xL DNA did not a?ect either (data not shown). Thus, active caspase-3-induced apoptosis is not prevented by overexpressed Bcl-2 and Bcl-xL, suggesting that no sites of Bcl-2 and Bcl-xL action exist downstream of active caspase-3. The same conclusion was reached using stably transfected cells overexpressing Bcl-2 (data not shown). Prevention of pro-caspase-1-mediated apoptosis by overexpressed Bcl-2 and Bcl-xL It was previously reported that apoptosis induced by introducing ice DNA encoding precursor form of caspase-1 (ICE) into cells by calcium-phosphate method is inhibited by overexpressed Bcl-2 (Miura et al., 1993) . To determine whether the inhibitory e?ects of overexpressed Bcl-2 and Bcl-xL against pro-caspase1-mediated apoptosis are also observed in microinjection system, ice DNA was injected into the nucleus of cells previously microinjected with bcl-2 or bcl-xL DNA to overexpress these proteins, and the extent of apoptosis was assessed. Overexpressed Bcl-2 and BclxL inhibited pro-caspase-1-mediated apoptosis by approximately 50% and nearly 100%, respectively (Figure 5 and Table 3), indicating that both proteins are functional in inhibiting pro-caspase-1-mediated apoptosis, consistent with previous observations (Miura et al., 1993) . None the less, apoptosis induced by minimally su?cient amounts of recombinant active caspase-1 was not at all prevented by Bcl-2 or Bcl-xL (Table 4), further supporting that no sites of Bcl-2 and Bcl-xL action exist downstream of active caspases, and that the inhibitory e?ects of overexpressed Bcl-2 and Bcl-xL against pro-caspase-1-mediated apoptosis are due to their action at steps leading to the activation of Number of apoptotic cells/total cell number and the extent (%) of apoptosis are given. The indicated reagents were microinjected into D98AH2 cells as described in the legend for Figure 4, except for exp. 4 in which caspase-3 injected cells were analysed after 1 h, and apoptotic cells were identi?ed as described in Materials and methods. vec0.001 and vec0.1 indicate the microinjection of vector DNA at 0.001 mg/ml and 0.1 mg/ml, respectively, and bcl-2 and bcl-xL DNAs were injected at 0.001 mg/ml and 0.1 mg/ml, respectively 1st injection Number of apoptotic cells/total cell number and the extent (%) of apoptosis are given. The indicated reagents were microinjected into D98AH2 cells as described in the legend for Figure 5, and apoptotic cells were identi?ed as described in Materials and methods. vec0.001 and vec0.1 indicate the microinjection of vector DNA at 0.001 mg/ml and 0.1 mg/ml, respectively, and bcl-2 and bcl-xL DNAs were injected at 0.001 mg/ml and 0.1 mg/ml, respectively Number of apoptotic cells/total cell number and the extent (%) of apoptosis are given. Vector, bcl-2 and bcl-xL DNA were microinjected into the nucleus of D98AH2 cells on glass coverslips together with FITC-conjugated BSA-NLS as in Figure 2. Two hours later, puri?ed active caspase-1 at indicated concentrations were microinjected into the cytoplasm of DNA-injected cells together with RITC-labeled BSA. After 2 h incubation, cells were ?xed and analysed as described in Materials and methods inactive pro-caspases but not due to the direct action downstream of the activated caspases. Discussion We and others have previously shown that Bcl-2 and Bcl-xL prevent a step(s) leading to the activation of ICE family proteases (caspases) (Shimizu et al., 1996a; Chinnaiyan et al., 1996; Boulakia 1996) . However, those data did not exclude the possibility that Bcl-2 and Bcl-xL also function downstream of the caspase cascade or a?ect some steps within the cascade. Here, we show that Bcl-2 and Bcl-xL, overexpressed more than su?cient to prevent Fas-mediated or pro-caspase1-mediated apoptosis, do not prevent apoptosis induced by microinjection of puri?ed active caspase-3 with one-order magnitude less amounts than that of caspase-3-like proteases activated during Fas-mediated apoptosis, as well as a minimal amount of puri?ed active caspase-1. Although we cannot exclude the possibility that the functional steps for Bcl-2/Bcl-xL could locate downstream of caspase family members other than caspase-3 and -1 themselves. However, since caspases possessing substrate speci?city similar to that of caspase-3 likely function most downstream in the caspase cascade, we conclude that there are no functional sites of Bcl-2 and Bcl-xL downstream of the caspase cascade. Although several groups have shown that Bcl-2 protein localizes in multiple membrane compartments (Hockenbery et al., 1990; Monaghan et al., 1992; Jacobson et al., 1993; Krajewski et al., 1993; Akao et al., 1994) , it has not been convincingly determined in which subcellular compartment Bcl-2 acts to prevent cell death. Since active caspase-3 induces apoptotic changes of nuclei in vivo when microinjected into the cytoplasm (Yasuhara et al., 1997, and this study) and also in vitro when incubated with naked nuclei and cell lysates (Enari et al., 1996), caspase-3-mediated death signals or the caspase itself must be transferred from the cytoplasm to the nucleus to induce apoptotic nuclear changes. Thus, the previously suggested possibility (Ryan et al., 1994; Martin et al., 1996) that Bcl-2 on the nuclear envelope protects the nucleus from apoptotic signals produced through the caspase cascade seems unlikely. Bcl-2 on the endoplasmic reticulum membrane has been suggested to regulate intracellular calcium levels (Ba?y et al., 1993; Lam et al., 1994; Marin et al., 1996) , but its importance in anti-apoptotic activity depends on the unsolved question of whether Bcl-2 regulates calcium levels upstream of the caspase cascade. We recently showed that Bcl-2 and Bcl-xL, but not inhibitors of caspases, prevent apoptotic and necrotic stimuli-induced loss of mitochondrial membrane potential, whereas both Bcl-2 family proteins and caspase inhibitors prevent cell death (Zamzami et al., 1995; Shimizu et al., 1996b) . The membrane potential loss leading to membrane permeability transition results in the release of an mitochondrial apoptogenic factor, AIF, which in turn, activates caspases as well as induces apoptotic change of the nucleus (Susin et al., 1996). It was also shown that Bcl-2 prevents the release of mitochondrial cytochrome c which along with another cytoplasmic factor activates caspase-3 (Yang et al., 1997; Kluck et al., 1997) . These previous suggestions that mitochondria are important targets of Bcl-2 function upstream of active caspases is consistent with the present data. Bcl-2 and Bcl-xL function only upstream of caspases and yet prevent apoptosis induced by overexpression of genes encoding the proform of caspases (Miura et al., 1993; Wang et al., 1994; this study) . Since proforms of caspases are zymogens and inactive, apoptotic signals from the upstream pathway, which are likely Bcl-2 inhibitable, must be required to activate the caspases. Such apoptotic signals might be generated by DNA transfection and microinjection procedures which stress cells and therefore likely activate apoptotic pathways to some extent. It is also conceivable that cells in culture su?er from various environmental stresses that generate chronic low-level apoptotic signals which are inhibited by Bcl-2. These signals might cooperate with other apoptotic stimuli to induce apoptosis. In either case, activated apoptotic pathways upstream of the caspase cascade might not be su?cient to induce apoptosis by themselves but might be ampli?ed by the overexpressed pro-caspases to induce cell death. Materials and methods DNA Human ice cDNA was isolated by screening a human B cell cDNA library with a probe prepared by PCR. Human bcl2 and chicken bcl-xL cDNAs have been described (Shimizu et al,. 1995). All cDNAs were subcloned in the expression vector pUC-CAGGS (Niwa et al., 1991) bearing a b-actin promoter and CMV enhancer. Preparation of active recombinant caspases Recombinant ICE (caspase-1) (Thornberry et al., 1992) was a generous gift from Dr NA Thornberry (Merck). Recombinant active CPP32b was used as a representative of active caspase-3, and was puri?ed from E. coli strain JM109 carrying an expression plasmid for the N-terminally (His)6-tagged CPP32b essentially as described (Lippke et al., 1996) . Puri?ed caspase-3 in transport bu?er (20 mM HEPES (pH 7.3), 110 mM potassium acetate, 2 mM magnesium acetate, 5 mM sodium acetate, 0.5 mM EGTA, 2 mM dithiothreitol) with a speci?c proteolytic activity of 232 units/ml for Ac-DEVD-MCA (acetyl-L-aspartyl-Lglutamyl-L-valyl-L-aspartyl - 4 - methyl - coumaryl - 7 -amide; Peptide Research Institute, Osaka, Japan) was used for microinjection. Activities of recombinant caspases-1 and -3 and cellular caspase-3-like proteases were measured as described (Shimizu et al., 1996a) . One unit was de?ned as the amount of enzyme required to release 1 pmole AMC (7-amino-4-methylcoumarin) per min at 378C. Microinjection and analysis of cell death Human carcinoma cell line HeLa (D98AH2) was maintained in RPMI-1640 medium containing 10% fetal bovine serum. Cells were seeded on glass coverslips in the medium. Overexpression of Bcl-2 and Bcl-xL was achieved by microinjecting bcl-2 and bcl-xL DNA into the nucleus with FITC-labeled BSA chemically conjugated with synthetic NLS (nuclear localization signal) of SV40 Tantigen, and incubating injected cells for 2 h. The concentration of microinjected bcl-2 and bcl-xL DNA was 0.001 mg/ml and 0.1 mg/ml, respectively, which exerts anti-cell death activity against Fas-mediated and procaspase-1-mediated apoptosis to the similar extent. As a control, vector DNA (0.1 mg/ml or 0.001 mg/ml) was microinjected. To induce apoptosis, active recombinant caspase-3 at 232 units/ml or caspase-1 at 0.23 units/ml was microinjected into the cytoplasm together with RITClabeled BSA, or ice DNA encoding pro-caspase-1 at 0.02 mg/ml was microinjected into the nucleus together with RITC-labeled BSA. The smallest e?ective amount of active caspases or ice DNA to induce apoptosis was used. In some experiments, cell death was induced by treatment with 1 mg/ml agonistic anti-Fas monoclonal antibody (CH-11: Medical and Biological Laboratories, Nagoya, Japan). Microinjection was performed using a Leica microinjection system. Where indicated, the tetrapeptide caspase-3 inhibitor Ac-DEVD-CHO (acetyl-L-aspartyl-Lglutamyl-L-valyl-L-aspartyl-aldehyde: Peptide Research Institute, Osaka, Japan) was included in the medium at 100 mM from 1 h prior to and throughout the experiments. After 4 h incubation for Fas-treatment, 1 h or 2 h incubation for caspase-3, or 3 h incubation for ice DNA, cells were ?xed with 4% formaldehyde solution for 20 min, stained with 10 mM Hoechst 33342 for 5 min and analysed under a ?uorescence microscope (BX-50, Olympus, Tokyo, Japan) with excitation at 480 nm for FITC, 530 nm for RITC and 360 nm for Hoechst 33342. Apoptosis was judged by nuclear fragmentation, chromatin condensation and cellular shrinkage and fragmentation. Immunostaining of microinjected cells HeLa (D98AH2) cells were seeded on glass coverslips in RPMI-1640 medium containing 10% FBS. Stable Bcl-2 and Bcl-xL transfectants of HeLa cell, which showed complete resistance against Fas-mediated apoptosis (data not shown), were used as positive controls. After microinjecting bcl-2 or bcl-xL DNA into the nucleus with FITC-labeled BSA-NLS, cells were incubated for 1, 2, 4 or 6 h, then ?xed with 4% formaldehyde, and immunostained with anti-human Bcl-2 monoclonal antibody (Pezzella et al., 1990) /RITC-labeled anti-mouse IgG antibody, or antichicken Bcl-xL polyclonal antibody (Shimizu et al., 1995) / RITC-labeled anti-rabbit IgG antibody, respectively. Cells were then examined under a ?uorescence microscope with excitation at 480 nm for FITC, and 530 nm for RITC. Estimation of volume of injected solution Injected volume was estimated essentially as described (Matsuoka et al., 1994) . Because a single molecule of diphtheria toxin fragment A (DTFA) introduced into a cell is su?cient to kill the cell (Yamaizumi et al., 1978) , serially diluted solutions of DTFA (a gift from Dr Mahito Nakanishi (Osaka University)) in PBS containing 1 mg/ ml BSA were microinjected into HeLa cell cytoplasm. 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Noriko Yasuhara, Setsuko Sahara, Shinji Kamada, Yutaka Eguchi, Yoshihide Tsujimoto. Evidence against a functional site for Bcl-2 downstream of caspase cascade in preventing apoptosis, Oncogene, 1997, 1921-1928, DOI: 10.1038/sj.onc.1201370