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Apopain
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
221, 340–345 (1996)
0597
Bcl-2 Overexpression Blocks Activation of the Death Protease CPP32/Yama/Apopain Laurent Monney,* Isabelle Otter,* Reynald Olivier,* Ulla Ravn,* Hengameh Mirzasaleh,* Isabelle Fellay,* Guy G. Poirier,† and Christoph Borner*,1 *Institute of Biochemistry, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland; and †Poly (ADP) Ribose Metabolism Group, Laboratory of Molecular Endocrinology, Centre Hospitalier de l’Université Laval Research Center and Laval University, Sainte-Foy, Quebec G1V 4G2, Canada Received February 27, 1996 The C. elegans gene product ced-9 inhibits programmed cell death by negatively regulating the deathmediating protease ced-3. The mammalian hormolog of ced-9 is the oncoprotein Bcl-2. Overexpression of Bcl-2 spares mammalian and nematodal cells from dying and prevents ectopic cell death in ced-9 loss-of-function mutants. Although Bcl-2 has been shown to act as an antioxidant under certain conditions, additional functions have emerged from studies under low oxygen pressure. Here we show that Bcl-2 overexpression impairs activation of the interleukin-1b converting enzyme-related death protease CPP32/Yama/apopain, the mammalian homolog of ced-3. When U937 monocytes undergo programmed cell death in response to tumor necrosis factor a, the inactive CPP32 precursor is cleaved into its active forms. As a consequence poly(ADP ribose) polymerase, a major substrate of CPP32, is faithfully cleaved into a 85 kD fragment. Bcl-2 overexpressing cells are protected from tumor necrosis factor a-induced death and display neither CPP32 maturation nor PARP cleavage. The inhibitory effect of Bcl-2 on CPP32 activation is indirect since no physical interaction between the two proteins could be detected. These results indicate that Bcl-2 neutralizes an unknown cellular activator of CPP32 to save cells from programmed cell death. © 1996 Academic Press, Inc.
Programmed cell death (PCD) is an essential mechanism to maintain homeostasis in multicellular organisms (1). In contrast to accidental cell lysis due to plasma membrane damage (necrosis), PCD is characterized by a programmed, orchestrated fragmentation of intracellular constituents (protein, DNA, organelles) (1,2). The degraded products are packed into membrane-enclosed cellular bodies which are subsequently engulfed by neighboring cells without provoking inflammatory reactions (1). Studies on the development of the nematode Caenorhabditis elegans have given insights into the genetic programme which drives PCD. Two genes products, called ced-4 and ced-3 are necessary for PCD (3). The ced-3 protein encodes a cysteine protease which cleaves peptide substrates after aspartic acid residues (Aspase) (4). A series of mammalian ced-3 homologs have been identified (2,5–14) and shown to belong to a family of proteins whose prototype is the IL-1b converting enzyme (ICE) (5,7). Most of the ICE-like enzymes induce PCD when overexpressed (6–8,11,13). This is probably due to the proteolysis of cellular proteins which thereafter either lose cell survival activity or gain death effector functions (2). Although none of the identified cellular substrates of ICE-like proteases have so far been shown to be involved in PCD, some such as the poly(ADPribose) polymerase (PARP) serve as marker to assay the endogenous activation of ICE-like proteases during the death process (9–14). PARP is a specific substrate of a subgroup of ICE-like proteases which include CPP32/Yama/apopain (9–12), ICE-LAP3 (13) and Mch-3 (14). These enzymes appear to be the key ICE-like proteases involved in the induction of PCD because a specific tetrapeptide inhibitor, called Ac-DEVD-CHO, effectively blocked PCD in vivo (11,12) and 1
To whom correspondence should be sent. Fax: ++41-37-29 97 35. E-mail: [email protected]. Abbreviations: TNFa, tumor necrosis factor a; PCD, programmed cell death; Act, actinomycin D; ICE, IL-1b converting enzyme; PARP, poly(ADP ribose) polymerase; ROS, reactive oxygen species. 340 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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in vitro (12,15). ICE-like enzymes, including CPP32/Yama/apopain exist in an inactive pro-form in unstressed cells (6,10,11). Upon the induction of PCD, the immature 32 kD CPP32 protease (pro-CPP32) is cleaved into 12 and 17 kD proteins which together form the active protease (6,10,11). Although ICE-like proteases have the capacity for autoprocessing (16), it is believed that the initial step of maturation/activation of a given ICE protease is performed by a heterologous, upstream-acting protease (15,17). However other activators of the processing of ICE-like proteases have been suggested, notably a putative mammalian homolog of ced-4 which in C. elegans acts upstream of ced-3 (3). PCD in C. elegans is negatively regulated by ced-9 (18). Genetic analysis of ced mutants has allowed the identification of an ordering of the cell death pathway (18). In this scenario, ced-9 acts upstream of ced-4 and ced-3 to inhibit cell death. It is conceivable to propose a similar ordering of the cell death pathway in mammalian cells. However formal proof for this has so far been missing. The mammalian homolog of ced-9 is the proto-oncogene product Bcl-2 (19). The two proteins are functionally interchangeable such that the Bcl-2 suppresses a ced-9 loss-of-function phenotype in C. elegans (19). Numerous experiments have implicated Bcl-2 in the protection of various types of mammalian cells from PCD induced by different stimuli (20). How Bcl-2 works is still unknown (21). It has been proposed that Bcl-2 has either an antioxidant activity (22) or the capacity to interfere with intracellular calcium signalling (23). Alternatively, analogous to ced-9, Bcl-2 may impair the proper functioning of ICE/ced-3 proteases. Here we show for the first time that in U937 monocytes treated with tumor necrosis factor a (TNFa) cell death protection by Bcl-2 correlates with a blockage of the maturation/activation of the ICE-like protease CPP32/Yama/apopain. MATERIALS AND METHODS Reagents. TNFa and hygromycin B were purchased from Juro Supply/Calbiochem. Actinomycin D and protein ASepharose were from Sigma, DNase-free RNase from Boehringer Mannheim and Enhance from DuPont-Nemours. Mouse monoclonal anti-human CPP32 was bought from Transduction Laboratories and peroxidase-labeled sheep anti-mouse antibodies from Jackson Immuno Research Laboratories, Inc. [35S]methionine/cysteine (TRAN-label) was purchased from ICN. Cells. Vector control U937pMEP and Bcl-2 overexpressing U937Bcl-2 cells were generated as previously described (24). Both were mixed cell populations expressing either the episomal pMEPhygro vector (Invitrogen, Corp.) or murine Bcl-2 (Bcl-2). They were cultured in RPMI 1640 containing 10% fetal calf serum and 50 mg/ml hygromycin (to maintain Bcl-2 expression) at 5%CO2/37°C. DNA fragmentation. 2 × 106 cell were harvested by centrifugation, washed three times in phosphate buffered saline (PBS) and then immediately lysed in Sevag (phenol:chloroform:isoamylalcohol 4 25:24:1). The aqueous phase containing nucleic acids was treated with 0.1 mg/ml RNase at 37°C for 30 min and then loaded onto a 2% agarose gel containing ethidium bromide to visualize DNA fragmentation. Cell viability. Cells were seeded at 2 × 106 in triplicates into 35 mm wells. The next day, the cells were either treated with the solvent DMSO (0.1%) or 20 ng/ml TNFa plus 50 ng/ml actinomycin D (time point zero). At the times indicated, viable cells were counted by a trypan blue exclusion assay. Protein extraction/immunoblots. Total cellular extracts were prepared as follows: Cells were harvested by centrifugation, washed three times in PBS and then lysed in H8 (20 mM Tris-HCl 7.5, 2 mM EDTA, 2 mM EGTA, 6mM b-mercaptoethanol, 10mg/ml aprotinin, 2 mg/ml leupeptin) containing 1% SDS followed by sonication. Protein was determined by Lowry as previously described (25). Equal amounts of protein were loaded onto 12% polyacrylamide gels, subjected to SDS-PAGE and transferred to PVDF as described (25). Human pro-CPP32 was immunodetected by a mouse monoclonal anti-human CPP32 antibody (Transduction Laboratories) at a titer of 1:1000, PARP by a mouse monoclonal anti-human PARP antibody (C-2-10) at 1:109000 (26). Secondary antibodies were peroxidase-coupled goat anti-mouse antibodies (Jackson Laboratories). The detection system was enhanced chemiluminescence (ECL) (Amersham). Immunoprecipitations. 3 × 106 U937pMEP or U937Bcl-2 cells were labeled with 150 mCi/ml [35S]methionine/cysteine in methionine/cysteine-free RPMI medium overnight. 6, 12 or 24 hr before extraction, cells were either treated with 0.1% solvent (DMSO) or 20 ng/ml TNFa plus 50 ng/ml actinomycin D. Cells were harvested by centrifugation, washed once in PBS and once in buffer A (10 mM Hepes, pH 7.2, 143 mM KCl, 5 mM MgCl2, 1 mM EGTA, 100 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mg/ml pepstatin, 200 mM di-isopropyl fluorophosphate) and lysed in buffer A plus 0.2% Nonidet P-40. After leaving on ice for 30 min, the cell lysate was cleared by centrifugation, supplemented with 2.5 mg/ml ovalbumine and subjected to immunoprecipitation using an polyclonal anti-mouse Bcl-2 antibody (25). Following antibody incubation for 2 hrs at 4°C, 50 ml of a 50% protein A-Sepharose suspension was added and immuncomplexes were captured on a 341
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end-over-end wheel at 4°C for 60 min. Immuncomplexes were pelleted by centrifugation, washed three times in buffer A and then boiled in SDS-sample buffer for SDS-PAGE analysis. The gels were fixed in 40% methanol/10% acetic acid, treated with Enhance (DuPont-Nemours), washed in H2O, dried and subjected to fluorography.
RESULTS Exposure of vector control U937pMEP monocytes to 20 ng/ml TNFa plus 50 ng/ml actinomycin D (Act) led to a rapid decrease in cell viability (Fig. 1A). This was due to PCD since genomic DNA was cleaved into nucleosome-sized fragments (Fig. 1B). U937 cells transfected with a murine Bcl-2 cDNA (U937Bcl-2) expressed high levels of the Bcl-2 protein (Fig. 3). These cells were less susceptible to TNFa/Act-induced DNA fragmentation (Fig. 1B) and cell death (Fig. 1A). It has been reported that TNFa-induced PCD is accompanied by the activation of ICE-like proteases (10,13). We therefore tested whether the 32 kD pro-form of CPP32 is cleaved into its mature/active forms during a cellular treatment with TNFa/Act. CPP32 activation was assayed by the proteolytic cleavage of its substrate PARP using a specific anti-PARP antibody. Anti-CPP32 and anti-PARP immunoblots of total cellular extracts revealed that both U937pMEP and U937Bcl-2 cells expressed high levels of pro-CPP32 (32 kD) (Fig. 2A) and PARP (116 kD) (Fig. 2B). Following a cellular treatment with TNFa/Act pro-CPP32 gradually disappeared from U937pMEP (Fig. 2A). However, in U937Bcl-2 cells it remained at high levels throughout the stress period (Fig. 2A). Since the anti-CPP32 antibody used only recognized pro-CPP32, and no other antibody was available, we could not detect the actual generation of the mature 17 and 12 kD CPP32 forms in TNFa/Act-treated U937pMEP cells. Anti-PARP immunoblots however revealed that CPP32 must have been activated under these conditions because its major substrate PARP (116 kD) was typically cleaved into a 85 kD species (9–14). In contrast, TNFa/Act-treated U937Bcl-2 cells did not reveal any PARP cleavage (Fig. 2B). These results indicate that Bcl-2 interferes with the maturation/activation of CPP32 and the cleavage of its substrate PARP. We propose that the inhibitory effect of Bcl-2 on CPP32 activation is responsible for the delay in the onset of DNA fragmentation and subsequent PCD in TNFa/Act-treated U937Bcl-2 cells. A similar interference of Bcl-2 with CPP32 and PARP was found in cells which survive an apoptotic stress induced by staurosporine (data not shown).
FIG. 1. Cell survival and DNA fragmentation in response to TNFa/Act. 2 × 106 vector control U937pMEP (vector) or Bcl-2 overexpressing U937Bcl-2 (Bcl-2) mixed cell populations were treated with 20 ng/ml TNFa plus 50 ng/ml actinomycin D. After the times indicated cells were counted and assayed for viability by trypan blue exclusion (A) or for the appearance of DNA fragmentation by 2% agarose gel electrophoresis (B) as described in Materials and Methods. Data represent the means of three independent experiments ± SD. 342
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FIG. 2. Anti-CPP32 and anti-PARP immunoblots of extracts from untreated and TNFa/Act-treated cells. 2 × 106 U937pMEP (vector) or U937Bcl-2 (Bcl-2) cells were either untreated or treated with 20 ng/ml TNFa plus 50 ng/ml actinomycin D for 6 and 12 hr. Following total protein extraction, identical amounts of protein were subjected to 12% SDS-PAGE, blotted to PVDF and probed with either anti-CPP32 (A) or anti-PARP (B) antibodies. Immunodetection was by ECL.
A plausible explanation for the inhibitory action of Bcl-2 on CPP32 maturation/activation is that Bcl-2 directly binds the 32 kD proform of CPP32 to prevent its autoprocessing or processing by another protease. If this is the case, pro-CPP32 and Bcl-2 should associate in cellular extracts prepared from TNFa/Act-treated U937Bcl-2 cells. To test this notion, we subjected [35S]methionine/cysteine-labeled extracts from cells treated with TNFa/Act for 6-24 hr to anti-Bcl-2 immunoprecipitations. As shown in Fig. 3, no protein corresponding to the molecular mass of pro-CPP32 (32 kD) co-immunoprecipitated with Bcl-2 from extracts of TNFa/Act-treated U937Bcl-2 cells. Shorter TNFa/Act exposures did not change the protein pattern of the immunoprecipitates either. An anti-CPP32 Western blot of anti-Bcl-2 immunoprecipitates confirmed that Bcl-2 does not
FIG. 3. Anti-Bcl-2 immunoprecipitation of extracts from untreated and TNFa/Act-treated cells. 3 × 106 U937pMEP (vector) or U937Bcl-2 cells were radiolabeled and treated with 20 ng/ml TNFa plus 50 ng/ml actinomycin D for 6, 12 and 24 hr. Protein extracts were prepared and subjected to anti-Bcl-2 immunoprecipitation. Immunoprecipitates were analyzed by 15% SDS-PAGE and fluorographed. The 21 kD band represents Bax as tested by an anti-Bax immunoblot of the anti-Bcl-2 immunoprecipitates (data not shown). 343
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directly associate with the CPP32 precursor (data not shown). Other Bcl-2-associating proteins which could encode proteases were also not detected in anti-Bcl-2 immunoprecipitates. However, Bax was consistently co-immunoprecipitated indicating that the immunoprecipitation protocol worked (Fig. 3). This finding indicates that Bcl-2 does not directly block CPP32 maturation but acts on a enigmatic activator of CPP32 processing. DISCUSSION We show here the evolutionary conservation of the ordering of the death pathway between C. elegans and mammals. As ced-9 blocks the action of the ICE-like, ced-3 protease to mediate developmentally regulated PCD, Bcl-2 interferes with the activation of the ced-3-related, ICE-like protease CPP32/Yama/apopain to mediate TNFa/Act-induced PCD. Since we have also found blockage of CPP32 maturation/activation by Bcl-2 in response to other apoptotic agents (i.e. staurosporine), we propose that the negative impact of Bcl-2 on ICE-like proteases is a general function of Bcl-2. Similar results have been obtained by Chinnaiyan et. al. (27). From our immunoprecipitation studies we invoke that Bcl-2 acts rather on an activator of CPP32 than on CPP32 itself. Such an activator remains to be discovered. Since in the molecular ordering the ced-4 protein functions as a cell death inducer between ced-9 and ced-3 (3) it is conceivable to propose that Bcl-2 impinges on a mammalian ced-4 homolog to held CPP32 in check. However we have not found any Bcl-2 interacting protein under TNFa/Act-induced stress in anti-Bcl-2 immunoprecipitates with the notable exception of Bax. In addition, neither the biochemical function nor any mammalian homolog of ced-4 have so far been uncovered. Alternatively, Bcl-2 may impinge on a non-protein activator of CPP32. Possible substances could be calcium, ceramide or reactive oxygen species (ROS). They have all been implicated in various forms of apoptosis (28-30). Increased intracellular calcium may activate calpain-like proteases to convert CPP32 into mature forms (31). How ceramide or ROS act on the maturation/activation of CPP32 is less clear. If these substances played a crucial role in the activation of CPP32, it would corroborate the functions so far proposed for Bcl-2: antioxidant (22), inhibitor of intracellular calcium signalling (23) and blocker of ceramideinduced PCD (32, 33). Further studies are in progress to determine whether these are the true molecular functions of Bcl-2. ACKNOWLEDGMENTS This work was supported by the Swiss National Science Foundation (31-34600.92 and 31-36152.92) and the Swiss Cancer League (421).
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Duvall, E., and Wyllie, A. H. (1986) Immunology Today 7, 115–119. Martin, S. J., and Green, D. R. (1995) Cell 82, 349–352. Ellis, H. M., and Horvitz, H. R. (1986) Cell 44, 817–829. Hugunin, M., Quintal, L. J., Mankovich, J. A., and Ghayur, T. (1996) J. Biol. Chem. 271, 3517–3522. Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M., and Horvitz, H. R. (1993) Cell 75, 641–652. Fernandes-Alnemri, T., Litwack, G., and Alnemri, E. S. (1994) J. Biol. Chem. 269, 30761–30764. Wang, L., Miura, M., Bergeron, L., Zhu, H., and Yuan, J. (1994) Cell 78, 739–750. Miura, M., Zhu, H., Rotello, R., Hartwieg, E. A., and Yuan, J. (1993) Cell 75, 653–660. Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W. C. (1994) Nature 371, 346–347. Tewari, M., Quan, L. T., O’Rourke, K., Desnoyers, S., Zeng, Z., Beidler, D. R., Poirier, G. G., Salvesen, G. S., and Dixit, V. M. (1995) Cell 81, 801–809. 11. Nicholson, D. W., Ali, A., Thornberry, N. A., Vaillancourt, J. P., Ding, C. K., Gallant, M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A., Munday, N. A., Raju, S. M., Smulson, M. E., Yamin, T.-T., Yu, V. L., and Miller, D. K. (1995) Nature 376, 37–43. 12. Schlegel, J., Peters, I., Orrenius, S., Miller, D. K., Thornberry, N. A., Yamin, T. T., and Nicholson, D. W. (1996) J. Biol. Chem. 271, 1841–1844. 13. Duan, H., Chinnaiyan, A. M., Hudson, P. L., Wing, J. P., He, W.-W., and Dixit, V. M. (1996) J. Biol. Chem. 271, 1621–1625. 344
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14. Fernandes-Alnemri, T., Takahashi, A., Armstrong, R., Krebs, J., Fritz, L., Tomaselli, K. J., Wang, L., Yu, Z., Croce, C. M., Salvesen, G., Earnshaw, W. C., Litwack, G., and Alnemri, E. S. (1995) Cancer Res. 55, 6045–6052. 15. Darmon, A. J., Nicholson, D. W., and Bleackley, R. C. (1995) Nature 377, 446–448. 16. Ramage, P., Cheneval, D., Chvei, M., Graff, P., Hemmig, R., Heng, R., Kocher, H. P., Mackenzie, A., Memmert, K., Revesz, L., and Wishart, W. (1995) J. Biol. Chem. 270, 9378–9383. 17. Kumar, S. (1995) Trends Biol. Sci. 20, 198–202. 18. Hengartner, M. O., and Horvitz, H. R. (1994) Phil. Trans. R. Soc. Lond. B 345, 243–246. 19. Hengartner, M. O., and Horvitz, H. R. (1994) Cell 76, 665–676. 20. Reed, J. C. (1994) J. Cell Biol. 124, 1–6. 21. Haecker, G., and Vaux, D. L. (1995) Current Biology 5, 622–624. 22. Hockenbery, D. M., Oltvai, Z. N., Yin, X.-M., Milliman, C. L., and Korsmeyer, S. J. (1993) Cell 75, 241–251. 23. Lam, M., Dubyak, G., Chen, L., Nunez, G., Miesfeld, R. L., and Distelhorst, C. W. (1994) Proc. Natl. Acad. Sci. USA 91, 6569–6573. 24. Borner, C. (1996) J. Biol. Chem., in press. 25. Borner, C., Martinou, I., Mattmann, C., Irmler, M., Schaerer, E., Martinou, J.-C., and Tschopp, J. (1994) J. Cell. Biol. 126, 1059–1068. 26. Lamarre, D., Talbot, B., de Murcia, G., Laplante, C., Leduc, Y., Mazen, A., and Poirier, G. G. (1988) Biochem. Biophys. Acta 950, 147–160. 27. Chinnaiyan, A. M., Orth, K., O’Rourke, K., Duan, H., Poirier, G. G., and Dixit, V. M. (1996) J. Biol. Chem., in press. 28. Hannun, Y. A., and Obeid, L. M. (1995) Trends Biol. Sci. 20, 73–77. 29. Buttke, T. M., and Sandstrom, P. A. (1994) Immunology Today 15, 7–10. 30. McConkey, D. J., Hartzell, P., Nicotera, P., and Orrenius, S. (1989) FASEB J. 3, 1843–1849. 31. Squier, M. K. T., Miller, A. C. L., Malkinson, A. M., and Cohen, J. J. (1994) J. Cell Physiol. 159, 229–237. 32. Martin, S. J., Takayama, S., McGahon, A. J., Miyashita, T., Corbeil, J., Kolesnick, R. N., Reed, J. C., and Green, D. R. (1995) Cell Death and Differentiation 2, 253–257. 33. Zhang, J., Alter, N., Reed, J. C., Borner, C., Obeid, L. M., and Hannun, Y. A. (1996) Proc. Natl. Acad. Sci. USA in press.
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Bcl-2 Overexpression Blocks Activation of the Death Protease CPP32/Yama/Apopain Laurent Monney,* Isabelle Otter,* Reynald Olivier,* Ulla Ravn,* Hengameh Mirzasaleh,* Isabelle Fellay,* Guy G. Poirier,† and Christoph Borner*,1 *Institute of Biochemistry, University of Fribourg, Rue du Musée 5, CH-1700 Fribourg, Switzerland; and †Poly (ADP) Ribose Metabolism Group, Laboratory of Molecular Endocrinology, Centre Hospitalier de l’Université Laval Research Center and Laval University, Sainte-Foy, Quebec G1V 4G2, Canada Received February 27, 1996 The C. elegans gene product ced-9 inhibits programmed cell death by negatively regulating the deathmediating protease ced-3. The mammalian hormolog of ced-9 is the oncoprotein Bcl-2. Overexpression of Bcl-2 spares mammalian and nematodal cells from dying and prevents ectopic cell death in ced-9 loss-of-function mutants. Although Bcl-2 has been shown to act as an antioxidant under certain conditions, additional functions have emerged from studies under low oxygen pressure. Here we show that Bcl-2 overexpression impairs activation of the interleukin-1b converting enzyme-related death protease CPP32/Yama/apopain, the mammalian homolog of ced-3. When U937 monocytes undergo programmed cell death in response to tumor necrosis factor a, the inactive CPP32 precursor is cleaved into its active forms. As a consequence poly(ADP ribose) polymerase, a major substrate of CPP32, is faithfully cleaved into a 85 kD fragment. Bcl-2 overexpressing cells are protected from tumor necrosis factor a-induced death and display neither CPP32 maturation nor PARP cleavage. The inhibitory effect of Bcl-2 on CPP32 activation is indirect since no physical interaction between the two proteins could be detected. These results indicate that Bcl-2 neutralizes an unknown cellular activator of CPP32 to save cells from programmed cell death. © 1996 Academic Press, Inc.
Programmed cell death (PCD) is an essential mechanism to maintain homeostasis in multicellular organisms (1). In contrast to accidental cell lysis due to plasma membrane damage (necrosis), PCD is characterized by a programmed, orchestrated fragmentation of intracellular constituents (protein, DNA, organelles) (1,2). The degraded products are packed into membrane-enclosed cellular bodies which are subsequently engulfed by neighboring cells without provoking inflammatory reactions (1). Studies on the development of the nematode Caenorhabditis elegans have given insights into the genetic programme which drives PCD. Two genes products, called ced-4 and ced-3 are necessary for PCD (3). The ced-3 protein encodes a cysteine protease which cleaves peptide substrates after aspartic acid residues (Aspase) (4). A series of mammalian ced-3 homologs have been identified (2,5–14) and shown to belong to a family of proteins whose prototype is the IL-1b converting enzyme (ICE) (5,7). Most of the ICE-like enzymes induce PCD when overexpressed (6–8,11,13). This is probably due to the proteolysis of cellular proteins which thereafter either lose cell survival activity or gain death effector functions (2). Although none of the identified cellular substrates of ICE-like proteases have so far been shown to be involved in PCD, some such as the poly(ADPribose) polymerase (PARP) serve as marker to assay the endogenous activation of ICE-like proteases during the death process (9–14). PARP is a specific substrate of a subgroup of ICE-like proteases which include CPP32/Yama/apopain (9–12), ICE-LAP3 (13) and Mch-3 (14). These enzymes appear to be the key ICE-like proteases involved in the induction of PCD because a specific tetrapeptide inhibitor, called Ac-DEVD-CHO, effectively blocked PCD in vivo (11,12) and 1
To whom correspondence should be sent. Fax: ++41-37-29 97 35. E-mail: [email protected]. Abbreviations: TNFa, tumor necrosis factor a; PCD, programmed cell death; Act, actinomycin D; ICE, IL-1b converting enzyme; PARP, poly(ADP ribose) polymerase; ROS, reactive oxygen species. 340 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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in vitro (12,15). ICE-like enzymes, including CPP32/Yama/apopain exist in an inactive pro-form in unstressed cells (6,10,11). Upon the induction of PCD, the immature 32 kD CPP32 protease (pro-CPP32) is cleaved into 12 and 17 kD proteins which together form the active protease (6,10,11). Although ICE-like proteases have the capacity for autoprocessing (16), it is believed that the initial step of maturation/activation of a given ICE protease is performed by a heterologous, upstream-acting protease (15,17). However other activators of the processing of ICE-like proteases have been suggested, notably a putative mammalian homolog of ced-4 which in C. elegans acts upstream of ced-3 (3). PCD in C. elegans is negatively regulated by ced-9 (18). Genetic analysis of ced mutants has allowed the identification of an ordering of the cell death pathway (18). In this scenario, ced-9 acts upstream of ced-4 and ced-3 to inhibit cell death. It is conceivable to propose a similar ordering of the cell death pathway in mammalian cells. However formal proof for this has so far been missing. The mammalian homolog of ced-9 is the proto-oncogene product Bcl-2 (19). The two proteins are functionally interchangeable such that the Bcl-2 suppresses a ced-9 loss-of-function phenotype in C. elegans (19). Numerous experiments have implicated Bcl-2 in the protection of various types of mammalian cells from PCD induced by different stimuli (20). How Bcl-2 works is still unknown (21). It has been proposed that Bcl-2 has either an antioxidant activity (22) or the capacity to interfere with intracellular calcium signalling (23). Alternatively, analogous to ced-9, Bcl-2 may impair the proper functioning of ICE/ced-3 proteases. Here we show for the first time that in U937 monocytes treated with tumor necrosis factor a (TNFa) cell death protection by Bcl-2 correlates with a blockage of the maturation/activation of the ICE-like protease CPP32/Yama/apopain. MATERIALS AND METHODS Reagents. TNFa and hygromycin B were purchased from Juro Supply/Calbiochem. Actinomycin D and protein ASepharose were from Sigma, DNase-free RNase from Boehringer Mannheim and Enhance from DuPont-Nemours. Mouse monoclonal anti-human CPP32 was bought from Transduction Laboratories and peroxidase-labeled sheep anti-mouse antibodies from Jackson Immuno Research Laboratories, Inc. [35S]methionine/cysteine (TRAN-label) was purchased from ICN. Cells. Vector control U937pMEP and Bcl-2 overexpressing U937Bcl-2 cells were generated as previously described (24). Both were mixed cell populations expressing either the episomal pMEPhygro vector (Invitrogen, Corp.) or murine Bcl-2 (Bcl-2). They were cultured in RPMI 1640 containing 10% fetal calf serum and 50 mg/ml hygromycin (to maintain Bcl-2 expression) at 5%CO2/37°C. DNA fragmentation. 2 × 106 cell were harvested by centrifugation, washed three times in phosphate buffered saline (PBS) and then immediately lysed in Sevag (phenol:chloroform:isoamylalcohol 4 25:24:1). The aqueous phase containing nucleic acids was treated with 0.1 mg/ml RNase at 37°C for 30 min and then loaded onto a 2% agarose gel containing ethidium bromide to visualize DNA fragmentation. Cell viability. Cells were seeded at 2 × 106 in triplicates into 35 mm wells. The next day, the cells were either treated with the solvent DMSO (0.1%) or 20 ng/ml TNFa plus 50 ng/ml actinomycin D (time point zero). At the times indicated, viable cells were counted by a trypan blue exclusion assay. Protein extraction/immunoblots. Total cellular extracts were prepared as follows: Cells were harvested by centrifugation, washed three times in PBS and then lysed in H8 (20 mM Tris-HCl 7.5, 2 mM EDTA, 2 mM EGTA, 6mM b-mercaptoethanol, 10mg/ml aprotinin, 2 mg/ml leupeptin) containing 1% SDS followed by sonication. Protein was determined by Lowry as previously described (25). Equal amounts of protein were loaded onto 12% polyacrylamide gels, subjected to SDS-PAGE and transferred to PVDF as described (25). Human pro-CPP32 was immunodetected by a mouse monoclonal anti-human CPP32 antibody (Transduction Laboratories) at a titer of 1:1000, PARP by a mouse monoclonal anti-human PARP antibody (C-2-10) at 1:109000 (26). Secondary antibodies were peroxidase-coupled goat anti-mouse antibodies (Jackson Laboratories). The detection system was enhanced chemiluminescence (ECL) (Amersham). Immunoprecipitations. 3 × 106 U937pMEP or U937Bcl-2 cells were labeled with 150 mCi/ml [35S]methionine/cysteine in methionine/cysteine-free RPMI medium overnight. 6, 12 or 24 hr before extraction, cells were either treated with 0.1% solvent (DMSO) or 20 ng/ml TNFa plus 50 ng/ml actinomycin D. Cells were harvested by centrifugation, washed once in PBS and once in buffer A (10 mM Hepes, pH 7.2, 143 mM KCl, 5 mM MgCl2, 1 mM EGTA, 100 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mg/ml pepstatin, 200 mM di-isopropyl fluorophosphate) and lysed in buffer A plus 0.2% Nonidet P-40. After leaving on ice for 30 min, the cell lysate was cleared by centrifugation, supplemented with 2.5 mg/ml ovalbumine and subjected to immunoprecipitation using an polyclonal anti-mouse Bcl-2 antibody (25). Following antibody incubation for 2 hrs at 4°C, 50 ml of a 50% protein A-Sepharose suspension was added and immuncomplexes were captured on a 341
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end-over-end wheel at 4°C for 60 min. Immuncomplexes were pelleted by centrifugation, washed three times in buffer A and then boiled in SDS-sample buffer for SDS-PAGE analysis. The gels were fixed in 40% methanol/10% acetic acid, treated with Enhance (DuPont-Nemours), washed in H2O, dried and subjected to fluorography.
RESULTS Exposure of vector control U937pMEP monocytes to 20 ng/ml TNFa plus 50 ng/ml actinomycin D (Act) led to a rapid decrease in cell viability (Fig. 1A). This was due to PCD since genomic DNA was cleaved into nucleosome-sized fragments (Fig. 1B). U937 cells transfected with a murine Bcl-2 cDNA (U937Bcl-2) expressed high levels of the Bcl-2 protein (Fig. 3). These cells were less susceptible to TNFa/Act-induced DNA fragmentation (Fig. 1B) and cell death (Fig. 1A). It has been reported that TNFa-induced PCD is accompanied by the activation of ICE-like proteases (10,13). We therefore tested whether the 32 kD pro-form of CPP32 is cleaved into its mature/active forms during a cellular treatment with TNFa/Act. CPP32 activation was assayed by the proteolytic cleavage of its substrate PARP using a specific anti-PARP antibody. Anti-CPP32 and anti-PARP immunoblots of total cellular extracts revealed that both U937pMEP and U937Bcl-2 cells expressed high levels of pro-CPP32 (32 kD) (Fig. 2A) and PARP (116 kD) (Fig. 2B). Following a cellular treatment with TNFa/Act pro-CPP32 gradually disappeared from U937pMEP (Fig. 2A). However, in U937Bcl-2 cells it remained at high levels throughout the stress period (Fig. 2A). Since the anti-CPP32 antibody used only recognized pro-CPP32, and no other antibody was available, we could not detect the actual generation of the mature 17 and 12 kD CPP32 forms in TNFa/Act-treated U937pMEP cells. Anti-PARP immunoblots however revealed that CPP32 must have been activated under these conditions because its major substrate PARP (116 kD) was typically cleaved into a 85 kD species (9–14). In contrast, TNFa/Act-treated U937Bcl-2 cells did not reveal any PARP cleavage (Fig. 2B). These results indicate that Bcl-2 interferes with the maturation/activation of CPP32 and the cleavage of its substrate PARP. We propose that the inhibitory effect of Bcl-2 on CPP32 activation is responsible for the delay in the onset of DNA fragmentation and subsequent PCD in TNFa/Act-treated U937Bcl-2 cells. A similar interference of Bcl-2 with CPP32 and PARP was found in cells which survive an apoptotic stress induced by staurosporine (data not shown).
FIG. 1. Cell survival and DNA fragmentation in response to TNFa/Act. 2 × 106 vector control U937pMEP (vector) or Bcl-2 overexpressing U937Bcl-2 (Bcl-2) mixed cell populations were treated with 20 ng/ml TNFa plus 50 ng/ml actinomycin D. After the times indicated cells were counted and assayed for viability by trypan blue exclusion (A) or for the appearance of DNA fragmentation by 2% agarose gel electrophoresis (B) as described in Materials and Methods. Data represent the means of three independent experiments ± SD. 342
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FIG. 2. Anti-CPP32 and anti-PARP immunoblots of extracts from untreated and TNFa/Act-treated cells. 2 × 106 U937pMEP (vector) or U937Bcl-2 (Bcl-2) cells were either untreated or treated with 20 ng/ml TNFa plus 50 ng/ml actinomycin D for 6 and 12 hr. Following total protein extraction, identical amounts of protein were subjected to 12% SDS-PAGE, blotted to PVDF and probed with either anti-CPP32 (A) or anti-PARP (B) antibodies. Immunodetection was by ECL.
A plausible explanation for the inhibitory action of Bcl-2 on CPP32 maturation/activation is that Bcl-2 directly binds the 32 kD proform of CPP32 to prevent its autoprocessing or processing by another protease. If this is the case, pro-CPP32 and Bcl-2 should associate in cellular extracts prepared from TNFa/Act-treated U937Bcl-2 cells. To test this notion, we subjected [35S]methionine/cysteine-labeled extracts from cells treated with TNFa/Act for 6-24 hr to anti-Bcl-2 immunoprecipitations. As shown in Fig. 3, no protein corresponding to the molecular mass of pro-CPP32 (32 kD) co-immunoprecipitated with Bcl-2 from extracts of TNFa/Act-treated U937Bcl-2 cells. Shorter TNFa/Act exposures did not change the protein pattern of the immunoprecipitates either. An anti-CPP32 Western blot of anti-Bcl-2 immunoprecipitates confirmed that Bcl-2 does not
FIG. 3. Anti-Bcl-2 immunoprecipitation of extracts from untreated and TNFa/Act-treated cells. 3 × 106 U937pMEP (vector) or U937Bcl-2 cells were radiolabeled and treated with 20 ng/ml TNFa plus 50 ng/ml actinomycin D for 6, 12 and 24 hr. Protein extracts were prepared and subjected to anti-Bcl-2 immunoprecipitation. Immunoprecipitates were analyzed by 15% SDS-PAGE and fluorographed. The 21 kD band represents Bax as tested by an anti-Bax immunoblot of the anti-Bcl-2 immunoprecipitates (data not shown). 343
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directly associate with the CPP32 precursor (data not shown). Other Bcl-2-associating proteins which could encode proteases were also not detected in anti-Bcl-2 immunoprecipitates. However, Bax was consistently co-immunoprecipitated indicating that the immunoprecipitation protocol worked (Fig. 3). This finding indicates that Bcl-2 does not directly block CPP32 maturation but acts on a enigmatic activator of CPP32 processing. DISCUSSION We show here the evolutionary conservation of the ordering of the death pathway between C. elegans and mammals. As ced-9 blocks the action of the ICE-like, ced-3 protease to mediate developmentally regulated PCD, Bcl-2 interferes with the activation of the ced-3-related, ICE-like protease CPP32/Yama/apopain to mediate TNFa/Act-induced PCD. Since we have also found blockage of CPP32 maturation/activation by Bcl-2 in response to other apoptotic agents (i.e. staurosporine), we propose that the negative impact of Bcl-2 on ICE-like proteases is a general function of Bcl-2. Similar results have been obtained by Chinnaiyan et. al. (27). From our immunoprecipitation studies we invoke that Bcl-2 acts rather on an activator of CPP32 than on CPP32 itself. Such an activator remains to be discovered. Since in the molecular ordering the ced-4 protein functions as a cell death inducer between ced-9 and ced-3 (3) it is conceivable to propose that Bcl-2 impinges on a mammalian ced-4 homolog to held CPP32 in check. However we have not found any Bcl-2 interacting protein under TNFa/Act-induced stress in anti-Bcl-2 immunoprecipitates with the notable exception of Bax. In addition, neither the biochemical function nor any mammalian homolog of ced-4 have so far been uncovered. Alternatively, Bcl-2 may impinge on a non-protein activator of CPP32. Possible substances could be calcium, ceramide or reactive oxygen species (ROS). They have all been implicated in various forms of apoptosis (28-30). Increased intracellular calcium may activate calpain-like proteases to convert CPP32 into mature forms (31). How ceramide or ROS act on the maturation/activation of CPP32 is less clear. If these substances played a crucial role in the activation of CPP32, it would corroborate the functions so far proposed for Bcl-2: antioxidant (22), inhibitor of intracellular calcium signalling (23) and blocker of ceramideinduced PCD (32, 33). Further studies are in progress to determine whether these are the true molecular functions of Bcl-2. ACKNOWLEDGMENTS This work was supported by the Swiss National Science Foundation (31-34600.92 and 31-36152.92) and the Swiss Cancer League (421).
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Duvall, E., and Wyllie, A. H. (1986) Immunology Today 7, 115–119. Martin, S. J., and Green, D. R. (1995) Cell 82, 349–352. Ellis, H. M., and Horvitz, H. R. (1986) Cell 44, 817–829. Hugunin, M., Quintal, L. J., Mankovich, J. A., and Ghayur, T. (1996) J. Biol. Chem. 271, 3517–3522. Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M., and Horvitz, H. R. (1993) Cell 75, 641–652. Fernandes-Alnemri, T., Litwack, G., and Alnemri, E. S. (1994) J. Biol. Chem. 269, 30761–30764. Wang, L., Miura, M., Bergeron, L., Zhu, H., and Yuan, J. (1994) Cell 78, 739–750. Miura, M., Zhu, H., Rotello, R., Hartwieg, E. A., and Yuan, J. (1993) Cell 75, 653–660. Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W. C. (1994) Nature 371, 346–347. Tewari, M., Quan, L. T., O’Rourke, K., Desnoyers, S., Zeng, Z., Beidler, D. R., Poirier, G. G., Salvesen, G. S., and Dixit, V. M. (1995) Cell 81, 801–809. 11. Nicholson, D. W., Ali, A., Thornberry, N. A., Vaillancourt, J. P., Ding, C. K., Gallant, M., Gareau, Y., Griffin, P. R., Labelle, M., Lazebnik, Y. A., Munday, N. A., Raju, S. M., Smulson, M. E., Yamin, T.-T., Yu, V. L., and Miller, D. K. (1995) Nature 376, 37–43. 12. Schlegel, J., Peters, I., Orrenius, S., Miller, D. K., Thornberry, N. A., Yamin, T. T., and Nicholson, D. W. (1996) J. Biol. Chem. 271, 1841–1844. 13. Duan, H., Chinnaiyan, A. M., Hudson, P. L., Wing, J. P., He, W.-W., and Dixit, V. M. (1996) J. Biol. Chem. 271, 1621–1625. 344
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14. Fernandes-Alnemri, T., Takahashi, A., Armstrong, R., Krebs, J., Fritz, L., Tomaselli, K. J., Wang, L., Yu, Z., Croce, C. M., Salvesen, G., Earnshaw, W. C., Litwack, G., and Alnemri, E. S. (1995) Cancer Res. 55, 6045–6052. 15. Darmon, A. J., Nicholson, D. W., and Bleackley, R. C. (1995) Nature 377, 446–448. 16. Ramage, P., Cheneval, D., Chvei, M., Graff, P., Hemmig, R., Heng, R., Kocher, H. P., Mackenzie, A., Memmert, K., Revesz, L., and Wishart, W. (1995) J. Biol. Chem. 270, 9378–9383. 17. Kumar, S. (1995) Trends Biol. Sci. 20, 198–202. 18. Hengartner, M. O., and Horvitz, H. R. (1994) Phil. Trans. R. Soc. Lond. B 345, 243–246. 19. Hengartner, M. O., and Horvitz, H. R. (1994) Cell 76, 665–676. 20. Reed, J. C. (1994) J. Cell Biol. 124, 1–6. 21. Haecker, G., and Vaux, D. L. (1995) Current Biology 5, 622–624. 22. Hockenbery, D. M., Oltvai, Z. N., Yin, X.-M., Milliman, C. L., and Korsmeyer, S. J. (1993) Cell 75, 241–251. 23. Lam, M., Dubyak, G., Chen, L., Nunez, G., Miesfeld, R. L., and Distelhorst, C. W. (1994) Proc. Natl. Acad. Sci. USA 91, 6569–6573. 24. Borner, C. (1996) J. Biol. Chem., in press. 25. Borner, C., Martinou, I., Mattmann, C., Irmler, M., Schaerer, E., Martinou, J.-C., and Tschopp, J. (1994) J. Cell. Biol. 126, 1059–1068. 26. Lamarre, D., Talbot, B., de Murcia, G., Laplante, C., Leduc, Y., Mazen, A., and Poirier, G. G. (1988) Biochem. Biophys. Acta 950, 147–160. 27. Chinnaiyan, A. M., Orth, K., O’Rourke, K., Duan, H., Poirier, G. G., and Dixit, V. M. (1996) J. Biol. Chem., in press. 28. Hannun, Y. A., and Obeid, L. M. (1995) Trends Biol. Sci. 20, 73–77. 29. Buttke, T. M., and Sandstrom, P. A. (1994) Immunology Today 15, 7–10. 30. McConkey, D. J., Hartzell, P., Nicotera, P., and Orrenius, S. (1989) FASEB J. 3, 1843–1849. 31. Squier, M. K. T., Miller, A. C. L., Malkinson, A. M., and Cohen, J. J. (1994) J. Cell Physiol. 159, 229–237. 32. Martin, S. J., Takayama, S., McGahon, A. J., Miyashita, T., Corbeil, J., Kolesnick, R. N., Reed, J. C., and Green, D. R. (1995) Cell Death and Differentiation 2, 253–257. 33. Zhang, J., Alter, N., Reed, J. C., Borner, C., Obeid, L. M., and Hannun, Y. A. (1996) Proc. Natl. Acad. Sci. USA in press.
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