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Department of Cellular Biology, Biology and Medicine Faculty, Lausanne University, Lausanne, Switzerland,1 Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin, Ireland,2 Maxim Pharmaceuticals, San Diego, California3
Received 3 April 2004/ Returned for modification 7 May 2004/ Accepted 3 September 2004
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ABSTRACT |
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INTRODUCTION |
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Until recently, it was assumed that activation of caspases would inexorably engage cells on the road leading to cell death (31, 40). While this is certainly true in most cases, recent evidence indicates that there are situations in which upstream or executioner caspases are activated to fulfill functions other than the induction of apoptosis (reviewed in references 2 and 30). For example, caspase 3 is required for skeletal-muscle differentiation (15) and caspase 8 is essential for lymphocyte activation and homeostasis (1, 25, 35). The physiological importance of these nonapoptotic functions of caspases is supported by the observation that some mutations in caspase 8 in humans are associated with defects in lymphocyte activation leading to immunodeficiency (8).
A critical question raised by the demonstration that upstream and executioner caspases participate in nonapoptotic cellular functions is how cells survive after activating their caspases. Several antiapoptotic factors have been described, including FLICE (caspase 8)-inhibitory proteins (FLIPs), Bcl2 family members, and inhibitor of apoptosis proteins (IAPs) (20, 24, 28, 32). It appears, however, that only the last can directly inhibit executioner caspases (28). Cells, therefore, may express basal levels of IAPs to confer constitutive resistance to low levels of caspase activation. On the other hand, expression of IAPs and other antiapoptotic factors may be induced following caspase activation to regulate cell death in a more dynamic manner. The observations that caspases are required for neuroprotection following preconditioning (29) and to counteract tumor necrosis factor toxicity (7, 44) support the latter hypothesis.
RasGAP, a regulator of Ras and Rho GTP-binding proteins, is a caspase substrate that bears two cleavage sites used sequentially as caspase activity increases in cells (47, 50, 53) (Fig. 1A). It has been shown that fragment N, one of the fragments generated by the partial cleavage of RasGAP, inhibits apoptosis when overexpressed in cells. This has led to the hypothesis that mild activation of caspases, rather than promoting apoptosis, might generate a protective response (53). Here, we demonstrate that low-stress conditions can activate executioner caspases without inducing cell death. Cell survival under these conditions cannot occur if the caspase 3-mediated cleavage of RasGAP into fragment N is prevented. Cells can therefore generate an antiapoptotic response to the moderate activation of executioner caspases.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Electroporation. The total quantity of DNA used in the electroporation procedure was kept to 30 µg (appropriate amounts of pcDNA3 were added when required). Plasmids were diluted in 60 µl of H2O, mixed with 300 µl of Dulbecco's modified Eagle's medium (DMEM) containing 10% newborn calf serum (NBCS) (2 x 106 cells) and electroporated at a voltage of 200 V and a capacitance of 1,050 µF. In the experiment (see Fig. 7H and I), 3 µg of pEGFP-C1, together with 13.5 µg of HA-Akt1(K179M), 13.5 µg of N-D157A.dn3, or a combination of both, was used. The electroporated cells were incubated in their culture medium for an additional 24-h period before being analyzed.
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Apoptosis assay. Apoptosis was determined by scoring cells displaying pycnotic nuclei (visualized with Hoechst 33342) (53).
Western blot analysis. Cells were lysed in monoQ-c buffer (53). Western blotting was performed as described previously (49) using a homemade enhanced chemiluminescence reagent (53). To improve the signal in some experiments, an enhanced chemiluminescence solution (Super Signal west Femto Maximum Sensitivity Substrate from Pierce [catalog no. 34095]) was mixed with the homemade reagent.
In vitro RasGAP cleavage assays. Preparation of cell extracts, depletion of caspases from these extracts, and activation of caspases in the extracts were performed as described previously (38).
Caspase 3 production. Active caspase 3 was produced in BL21(DE3)pLysS (Promega) containing the Casp3.rst plasmid encoding the six-His-tagged version of active hamster caspase 3. The bacteria were cultured overnight at 37°C in 500 ml of LB-amp (0.8% peptone, 0.8% yeast extract, 3.6 mM NaOH, 0.34 M NaCl, 100 µg of ampicillin/ml). They were then diluted in 1.5 liters of the same medium and induced with 2 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) for 2 h at 37°C. The bacteria were pelleted at 6,000 x g and lysed by sonication in 40 ml of buffer A (20 mM HEPES, 10% TX-100, 100 µM phenylmethylsulfonyl fluoride, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM EDTA, 0.5 mM EGTA, 0.5% ß-mercaptoethanol, and the protease inhibitor cocktail from Roche [catalog no. 1873580]) containing 2 mg of lysozyme/ml. The purification of active caspase 3 was performed by incubating the cell lysates for 2 h with 0.5 ml of a 1:1 slurry of Ni-nitrilotriacetic acid beads (QIAGEN). The beads were then harvested by centrifugation and transferred to Eppendorf tubes and washed three times with 1 ml of buffer A and two times with 1 ml of buffer A containing 10 mM imidazole. The beads were then incubated for 10 min with 600 µl of buffer A containing 50 mM imidazole, and the supernatant was harvested. This step was repeated once, and the two supernatants were pooled. The imidazole was eliminated by exclusion-diffusion chromatography using G25 Sephadex beads. The resulting purified caspase 3 was kept at –20°C in 50% glycerol, 50 mM HEPES, 2 mM EDTA, 0.1% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 5% sucrose, 0.2 mM dithiothreitol.
Measurement of caspase 3 activity. Caspase 3 activity was determined in 3 ml of caspase 3 buffer (100 mM HEPES, 1% sucrose, 0.1% CHAPS, 2 mM dithiothreitol) in the presence of 5 µM fluorogenic caspase 3 substrate AFC 138 (Enzyme System Product). After a 1-h incubation at 37°C, the extent of the cleavage of AFC 138 was measured using a Photon Technology International fluorimeter (excitation, 400 nm; emission, 505 nm).
Lentivirus.
Recombinant lentivirus was produced as described previously (12). Briefly, 293T cells were cotransfected using the calcium phosphate DNA precipitation method (23) with 10 µg of the lentiviral vector containing the cDNA of interest (e.g., N-D157A.lti), 2.5 µg of the envelope protein-coding plasmid (pMD.G), and 7.5 µg of the packaging construct (pCMV
R8.91). Two days after the transfection, the virus-containing medium was harvested. To determine how much of the virus preparation was needed to infect 100% of the MEFs, subconfluent wild-type MEFs seeded in six-well plates were cultured overnight with various volumes of fragment N- or Bcl2-encoding recombinant virus. After removal of the virus solution, the cells were maintained for two more days before fixation and immunocytochemical staining with antibodies directed at the protein expressed by the lentivirus. The lowest volumes of the lentiviral preparations required to infect 100% of the cells were chosen for further experiments.
Image acquisition of wounds in cell layers.
Cells were grown to confluency, wounds were generated, and the cells were fixed as described previously (26, 36). After fixation, the cells were mounted in Vectashield (catalog no. H-1000; Vector Laboratories, Burlingame, Calif.). Pictures of the wounds were taken at room temperature with a Zeiss Axioplan 2 imaging microscope equipped with a Plan-Neofluar 10x/0.30 
/– lens and a Zeiss AxioCam HRC camera using the Zeiss AxioVision acquisition software.
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RESULTS |
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RasGAP is a caspase 3 substrate cleaved in nonapoptotic cells. If RasGAP is cleaved by caspase 3 into fragment N to generate a survival pathway, it is anticipated that this cleavage should be observed in nondying cells and in the absence of significant cleavage of caspase substrates involved in the dismantling of the cells, such as ICAD (14, 34). We therefore analyzed the cleavage of RasGAP and ICAD in cells stimulated with the apoptotic inducer cisplatin. Figure 2A shows that the formation of fragment N could occur in situations (i.e., at cisplatin concentrations below 2.5 µM) that did not lead to detectable ICAD cleavage or induction of apoptosis (even on a long-term basis) (Fig. 3; also see Fig. 5). The exposure time allowing the detection of active caspase 3 in apoptotic cells by Western blot analysis was insufficient to reveal activation of caspase 3 in cells incubated with low cisplatin concentrations (Fig. 2A, top, compare the first three lanes with the last three lanes). Longer exposure, however, revealed that low-stress conditions (e.g., those induced by 0.5 µM cisplatin) induced very mild caspase 3 activity (see Fig. 7A, in which short and long exposures of the same Western blot are depicted).
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Long-term incubation of cells with low cisplatin concentrations also resulted in fragment N formation but not in further processing of fragment N into fragments N1 and N2 (Fig. 3A). Under these conditions, caspase 3 activity could be detected, but only after maximal exposure of the Western blots (Fig. 2 and 3; also see Fig. 7A). This weak caspase activation was nevertheless required for the production of fragment N, since RasGAP was not cleaved in cells incubated with low cisplatin concentrations in the presence of general caspase inhibitors, such as MX1013 (21, 56) (Fig. 3B) or z-VAD-fmk (53). Fragment N was further cleaved into fragments N2 and N1 (the latter is not recognized by the antibody used here) when cisplatin concentrations reached 
10 µM, conditions that also induced strong activation of caspase 3, the cleavage of ICAD, and apoptosis (Fig. 2A). The weak caspase 3 activity detected in extracts from cells stimulated with low cisplatin concentrations could result from a low level of caspase 3 activity in many surviving cells or a high level of caspase 3 activity in a small number of cells undergoing apoptosis. The observation that 0.5 µM cisplatin did not increase the basal apoptotic rate (Fig. 2A), however, supports the first possibility. Two further approaches were used to confirm that these low cisplatin concentrations do not induce cell death. First, we followed the fates of individual cells by video microscopy recording. Figure 3C shows that there were no more cells dying in response to 0.5 µM cisplatin than in the absence of the genotoxin. Second, we assessed the potential of untreated cells and cells incubated with 0.5 µM cisplatin to form clones. As 0.5 µM cisplatin did not decrease the ability of cells to generate clones (see Fig. 5D and E), it can be concluded that the viability of the cells is not affected by low cisplatin concentrations. Altogether, these data indicate that the first cleavage of RasGAP, although caspase dependent, occurs in nonapoptotic cells able to proliferate and generate clones and in the absence of the cleavage of caspase substrates that participate in the destructive phase of apoptosis.
RasGAP cleavage into fragment N is required for cell survival under adverse conditions. To investigate specifically the functional role of RasGAP cleavage by caspases, we generated MEFs derived from RasGAP knockout mice in which the wild-type RasGAP cDNA or a mutant that cannot be cleaved at position 455 (mutant D455A), was reexpressed at endogenous levels (Fig. 4A; compare the third lane with subsequent lanes). In response to increasing concentrations of cisplatin, cells expressing the wild-type RasGAP protein generated fragment N and then fragments N2 and N1 (N1 is not recognized by the antibody used here) (Fig. 4B). As expected, because the first cleavage of RasGAP at position 455 is required for the second cleavage to occur (53), cells expressing mutant D455A did not display any processing of RasGAP in response to cisplatin (Fig. 4B).
We next determined whether the mutation in the caspase cleavage site affected the function of the full-length RasGAP protein. RasGAP shortens the activation of Ras-dependent pathways by stimulating the intrinsic GTPase activity of Ras. Hence, MEFs lacking RasGAP generate stronger and more prolonged ERK MAPK activation in response to growth factors than in control cells (26, 43). RasGAP is also required for normal cell polarization and cell migration, and this involves the regulation of both Ras and Rho (26). Consequently, MEFs lacking RasGAP have a reduced capacity to fill and heal wounds (26). We therefore assessed the capacities of the clones expressing the wild-type and the cleavage-resistant forms of RasGAP to control ERK activation in response to serum and their abilities to migrate into wounds. The wild-type and the cleavage-resistant forms of RasGAP were similarly able to rescue the impaired control of ERK activity observed in RasGAP–/– cells and to restore the wound-healing capacities of these cells (Fig. 4C and D). Moreover, there was no difference among the expansion rates in the clones expressing the various forms of RasGAP (Fig. 4E). When these three criteria (regulation of ERK activation, wound healing, and clonal expansion) were considered, the RasGAP–/– clones, expressing the wild-type or the cleavage-resistant form of RasGAP, were in fact indistinguishable from RasGAP+/+ cells (Fig. 4C to E). The absence of the D455 caspase cleavage site, therefore, does not seem to affect the function of full-length RasGAP.
We then assessed cell survival in the presence of mild stresses when RasGAP cleavage was abolished. In contrast to the control RasGAP+/+ cells or the clones expressing the wild-type RasGAP, the clones expressing the RasGAP mutant that cannot be cleaved at position 455 underwent apoptosis in response to 0.5 µM cisplatin (Fig. 5A; also see Video S1 in the supplemental material). RasGAP–/– cells also underwent apoptosis under these conditions. The increased susceptibility to apoptosis under mild stress conditions correlated with the inability of the RasGAP D455A-expressing cells to expand (Fig. 5B) or to form colonies (Fig. 5D and E) in the presence of 0.5 µM cisplatin. Since the mutations removing the caspase cleavage sites in RasGAP did not alter the ability of the cells to expand under control conditions (Fig. 4E), these results demonstrate that generation of fragment N gives a selective survival advantage only in cells facing stressful conditions. Cells unable to generate fragment N also underwent apoptosis when subjected to low concentrations of FasL (Fig. 5C), indicating that cleavage of RasGAP into fragment N is a general means for cells to survive adverse conditions.
Fragment N is not a direct inhibitor of caspase 3. To assess whether fragment N could directly inhibit caspase 3, purified caspase 3 and a fluorescent caspase 3 substrate were incubated in vitro with increasing concentrations of recombinant fragment N. A caspase-resistant mutant of fragment N [fragment N(D157A)] was used to prevent its processing by caspase 3. In order to detect potential modulation of caspase 3 activity by fragment N, a nonsaturating dose of caspase 3 was used (Fig. 6A). As shown in Fig. 6B, recombinant fragment N was unable to modulate caspase 3 activity, indicating that its inhibition of caspase activity in cells is indirect.
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Ectopic expression of Fragment N in cells activates a survival pathway that is dependent on Ras activation (55). As Ras activation can lead to stimulation of the ERK MAPK pathway that can, under certain conditions, promote cell survival (22, 52), we assessed whether the ERKs could also play a role in protecting cells following RasGAP cleavage at position 455. Wild-type RasGAP-expressing cells, cells lacking RasGAP, or cells expressing the uncleavable mutant of RasGAP stimulated with low cisplatin concentrations for several days did not, however, display noticeable activation of the ERK MAPK pathway (Fig. 8A). In contrast, but only in wild-type RasGAP-expressing cells, Akt was strongly activated upon exposure to mild stress (Fig. 8A). The cell lines tested activated the ERKs and Akt in similar manners in response to serum stimulation (Fig. 8B). Their inability to activate the ERKs and, for those unable to cleave RasGAP, to stimulate Akt in response to a mild stress is therefore not a consequence of nonfunctional ERK and Akt pathways. These results suggest that ERK activation is not required to promote cell survival after the partial cleavage of RasGAP into fragment N.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells expressing a RasGAP mutant bearing a single point mutation abolishing its cleavage by caspases are killed by mild stresses that would otherwise not affect the viability of wild-type cells. This indicates that even if other antiapoptotic fragments are generated by caspases, they cannot compensate for the lack of RasGAP cleavage. Remarkably, therefore, the negative feedback control of caspases following their mild activation cannot operate if only one protein among the 280 described caspase substrates is not cleaved (16).
Fragment N is not a direct caspase inhibitor but rather protects cells by activating Akt, a well-known antiapoptotic kinase (55). An amino-terminal moiety of RasGAP, corresponding to fragment N plus the plekstrin homology domain of fragment C, can in fact interact directly with Akt (57). It is therefore likely that the binding of fragment N to Akt modulates the activity of the kinase. However, the exact mechanisms underlying the regulation of Akt by fragment N remain to be characterized.
Akt promotes cell survival by inactivating proapoptotic molecules, such as Bad, or by stimulating the mTOR or the NF-
B pathways (4, 48). However, fragment N, while protecting cells in an Akt-dependent manner, does not do so by stimulating NF-B activity (55). This could indicate that NF-B activation may not always participate in cell protection. Indeed, there are situations in which activation of NF-B is detrimental to the cell. For example, NF-B is required for p53-mediated apoptosis (33) and cytokine-induced apoptosis in ß cells of the pancreas (3, 17). Therefore, activating Akt in a way that precludes NF-B stimulation may be crucial for fragment N to be a general inhibitor of apoptosis.
It is unknown which effector proteins activated or induced by fragment N in an Akt-dependent manner mediate its protective function. Several inhibitors of caspases, such as cellular FLIPs and members of the IAP family, are potential candidates. FLIPs specifically prevent the activation of caspase 8 in response to Fas ligand or tumor necrosis factor but appear not to be involved in the regulation of other caspases (27). Consequently, it would not be anticipated that FLIPs protect cells once executioner caspases are activated. The IAP family of proteins can directly inhibit upstream and downstream caspases, including caspase 3 (11, 28). As these proteins seem to be the only endogenous inhibitors that can block apoptosis once caspases have been activated, they may be responsible for the protective effect induced by the formation of fragment N. However, the expression of many IAPs depends on NF-B activity (11, 46), and expression of at least one IAP family member, cIAP-2, does not rely on phosphatidylinositol 3-kinase activity (45). In contrast, fragment N-induced protection depends on phosphatidylinositol 3-kinase but not on NF-B (55), which raises the question of whether protection by fragment N may be mediated by proteins other than the IAPs.
RasGAP knockout mice die in utero as a result of abnormal development of the circulatory system (18). The RasGAP–/– embryos also display extensive neuronal death (18). Our data provide a plausible explanation for this phenotype: RasGAP–/– cells are more sensitive to apoptosis because they do not have the possibility of producing fragment N and consequently cannot activate the survival Akt pathway. RasGAP–/– cells are, however, not the ideal system to specifically assess the role of RasGAP cleavage because the full-length RasGAP may also regulate cell survival independently of its caspase cleavage fragments. In this context, it should be noted that RasGAP–/– cells are slightly less susceptible to mild stress-induced apoptosis than cells expressing an uncleavable form of RasGAP (Fig. 5). Because Ras activation may stimulate survival pathways (55), it is possible that the higher basal Ras activity in RasGAP–/– cells than in cells expressing full-length RasGAP (Fig. 4C) (43) gives them a slight survival advantage over cells expressing an uncleavable form of full-length RasGAP. However, the observation that overexpression of fragment N, but not full-length RasGAP, renders cells more resistant to apoptosis (54) further indicates that RasGAP induces a protective pathway only when it is cleaved at position 455.
Our results indicate that caspase 3 controls the level of its own activation by targeting RasGAP. In the absence of this safeguard mechanism, low caspase 3 activities are amplified inexorably until apoptosis is induced. There is therefore no constitutive cellular signal to protect cells such as MEFs from untimely caspase 3 activation. Rather, it is the caspases themselves that turn on a survival pathway. Presumably, this mechanism has arisen to protect cells against caspase activation occurring inopportunely or for other purposes than apoptosis.
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ACKNOWLEDGMENTS |
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This work is supported by the Swiss National Science Foundation (grant no. 3100-066797/1), the Oncosuisse Foundation (grant no. OCS 1110-2-2001), and the Botnar Foundation (Lausanne, Switzerland).
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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