If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
To whom correspondence should be addressed: Div. of Cellular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121. Tel.: 619-558-3515; Fax: 619-558-3525;
* This is manuscript 261 from La Jolla Institute for Allergy and Immunology. This research was supported by National Institutes of Health Grants AI40646 and CA69381.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Supported in part by The Children Leukemia Research Project fellowship. ¶ Supported by the Swiss National Science Foundation (Fellowship 823A-046638).
A growing body of evidence supports a role for mitochondria and mitochondria-derived factors in the cell death process. In particular, much attention has focused on cytochromec, a key component of the electron transport chain, that has been reported to translocate from the mitochondria to the cytosol in cells undergoing apoptosis. The mechanism for this release is, as yet, unknown. Here we report that ectopic expression of Bax induces apoptosis with an early release of cytochrome c preceding many apoptosis-associated morphological alterations as well as caspase activation and subsequent substrate proteolysis. A loss of mitochondrial transmembrane potential was detected in vivo, although no mitochondrial swelling or loss of transmembrane potential was observed in isolated mitochondria treated with Bax in vitro. Caspase inhibitors, such as endogenous XIAP and synthetic peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk), although capable of altering the kinetics and perhaps mode of cell death, had no influence on this release, suggesting that if cytochromec plays a role in caspase activation it must precede this step in the apoptotic process. Mitochondrial permeability transition was also shown to be significantly prevented by caspase inhibition, indicating that the translocation of cytochrome c from mitochondria to cytosol is not a consequence of events requiring mitochondrial membrane depolarization. In contrast, Bcl-xL was capable of preventing cytochrome c release while also significantly inhibiting cell death. It would therefore appear that the mitochondrial release of factors such as cytochrome c represents a critical step in committing a cell to death, and this release is independent of permeability transition and caspase activation but is inhibited by Bcl-xL.
), all features that promote the physiologically silent removal of the cell by its phagocytic neighbors. A large body of evidence supports the idea that these events are mediated by the activation of several cytosolic proteases, the caspases, which then orchestrate apoptosis via the cleavage of key substrates (reviewed in Refs.
). For example, specific cleavage of two such substrates, PAK2 and DNA fragmentation factor, activate these proteins, mediating membrane blebbing and DNA fragmentation, respectively, without further requirements for the proteases (for these events) (
But how are the caspases activated during apoptosis? Recent studies have delineated one key mechanism responsible for initiating the executioner phase of apoptosis. Early in the process, mitochondria release cytochrome c (
). In the presence of dATP or ATP, this complex processes and activates the caspase, which in turn can now trigger a cascade by processing and activating other caspases (in particular, caspases-3, -6, and -7) (
). This release was reportedly dependent upon induction of the mitochondrial permeability transition, an event that is associated with disruption of the mitochondrial inner transmembrane potential (ΔΨm) (
). Furthermore, in both cell-free systems and in cells undergoing apoptosis, the release of cytochromec can occur independently of changes in ΔΨm. We therefore examined the ability of Bax to induce the release of cytochrome c and apoptosis and evaluated the relationships between caspase activation, ΔΨm, and the effects of anti-apoptotic Bcl-2-family proteins.
Bax promotes apoptosis induced by removal of growth factors and other stimuli (
). We monitored caspase activation in individual cells using a cell-permeable, fluorescent substrate (as described under “Materials and Methods”). As shown in Fig. 1A, Bax-transfected cells exhibited both morphological and biochemical characteristics of apoptosis. Cells induced to die upon Bax expression appeared rounded and blebbed while displaying condensed chromatin, fragmented nuclei, and active DEVD-cleaving caspases. We then examined whether expression of Bax could induce the release of mitochondrial cytochromec. Fig. 1B shows that as the levels of Bax protein increased, cytochrome c could be detected in the cytosol. In other experiments including earlier time points before 9 h (not shown), negligible Bax expression was observed with neither cytochrome c nor caspase activation detectable.
We previously observed that the release of cytochrome cinduced by staurosporine or UVB irradiation occurs before and independently of caspase activation and subsequent apoptosis (
), zVAD-fmk significantly inhibited apoptosis during the period studied (24 h) (Fig. 2A). As shown in Fig.2B, this was not because of any effect on Bax expression levels. Bax-induced apoptosis corresponded to the activation of caspases and the subsequent cleavage of fodrin and poly(ADP-ribose) polymerase, two caspase substrates previously shown to be cleaved during apoptosis (
), whereas the caspase inhibitor zVAD-fmk efficiently blocked this. Nevertheless, Bax-induced cytochromec release proceeded with the same kinetics with or without caspase inhibition (Fig. 2B) as measured by cell fractionation and immunoblot analysis. This was confirmed by densitometric analysis of the cytochrome c immunoblots. At all time points, the cytosolic cytochrome c in the presence of Bax plus zVAD-fmk was ≥ that of Bax alone (data not shown).
As another approach to inhibiting caspase activation, we co-transfected Bax and a construct for expression of XIAP. Recent studies have shown that this molecule is a potent inhibitor of caspase function, including caspase activation by cytochrome c (
). As shown in Fig. 2, XIAP coexpression completely blocked apoptotic cell death (Fig.2C) and caspase substrate cleavage (Fig. 2D) triggered by Bax. Nevertheless, XIAP expression had no effect on Bax-induced cytochrome c release (Fig. 2D). Once again zVAD-fmk was demonstrated to be capable of blocking caspase activation but had no effect on cytochrome c release.
Cell-free systems have been extremely valuable for the analysis of apoptosis mechanisms, including cytochrome c release from mitochondria (
). We therefore asked whether recombinant Bax protein can induce cytochrome c releasein vitro and whether this is a direct or indirect effect of the protein. As shown in Fig. 3, the addition of Bax to cytosolic extracts containing mitochondria induced a rapid release of cytochrome c. Similarly, the addition of Bax to isolated mitochondria rapidly induced cytochrome crelease, which was even more pronounced than that seen in the presence of cytosol. It appears, therefore, that this release is a direct effect of Bax on the mitochondria, as observed by others (
). Interestingly, the presence of cytosol appeared to delay the Bax-induced release of cytochrome c from mitochondriain vitro. It is possible that this can be simply explained by levels of inhibitors in the cytosol, such as Bcl-2 or other Bax-binding proteins, which may sequester Bax and thereby interfere with its activity.
In many systems, apoptosis is associated with a loss of mitochondrial inner membrane potential (ΔΨm), which may correspond to the opening of an outer membrane pore (permeability transition pore). It has been suggested that this event is responsible for cytochrome crelease (
) have shown that such release can occur in the absence of a decrease in ΔΨm. Furthermore, we have shown that inhibition of caspase activation by zVAD-fmk can block early changes in ΔΨm without affecting cytochromec release. In contrast, Xiang et al. (
) observed that the decrease in ΔΨm induced by Bax is unaffected by caspase inhibition with zVAD-fmk.
Therefore, we examined ΔΨm in our system using two fluorochromes, DiOC6(3) and MitoTracker Orange. As shown in Fig.4A, Bax expression induced a loss of ΔΨm, regardless of the fluorochrome employed. The addition of zVAD-fmk substantially decreased the number of cells displaying this loss, although some cells continued to show a loss of ΔΨm in the presence of zVAD-fmk, consistent with the observations of Xianget al. (
). Similar results were obtained when caspases were inhibited by co-transfection with XIAP (Fig. 4C). Despite the persistence of this phenotype in some cells, these data suggest that the loss of ΔΨm is not required for Bax-induced cytochrome c release, because no change in this release was observed upon caspase inhibition (Fig. 2). The mitochondrial uncoupler, mCICCP, was used as a positive control for ΔΨm disruption (Fig. 4,B and D). To confirm this observation, we examined changes in ΔΨm using Rh123 and cytochrome crelease by immunoblot over time in isolated mitochondria in the presence or absence of recombinant Bax. As seen in Fig.5A, Bax alone cannot induce changes in ΔΨm. Despite this, a rapid release of cytochromec could be detected in the presence of Bax (Fig.5B). The uncoupler FCCP, although inducing a dramatic change in ΔΨm, had no effect on this cytochrome c release. Similar results were seen when ΔΨm was monitored using DiOC6(3) (data not shown).
) provided evidence that during apoptosis there is a disruption of the mitochondrial outer membrane, which may be responsible for the release of cytochromec. One way this could happen would be through induction of swelling of the outer membrane until it breaks the outer membrane, as was suggested (
). Although both CaCl2 and atractyloside induced rapid swelling, we were unable to detect Bax-induced swelling of mitochondria in vitro (Fig. 5C), suggesting either that other mechanisms are involved or that any swelling is too transient to be detected by this technique.
Altogether this suggests that Bax can induce alterations in ΔΨmin vivo, but it does so indirectly and requires cytosolic participation. Caspases are proposed as one likely candidate for this phenomenon. One of the best indicators of the mitochondrial permeability transition is swelling of the organelle, which can be readily observed in isolated mitochondria. Our results (discussed above), which failed to show such swelling, provide further evidence against an irreversible permeability transition induced by Bax in vitro.
Recently two groups reported that Bcl-2 proteins can block apoptosis by acting downstream of cytochrome c (
). In light of this we therefore examined whether Bcl-xL, a member of the Bcl-2 family, could inhibit Bax-induced death and cytochrome c release. Bcl-xL inhibited cell death (Fig.6A) when co-transfected with Bax, and this was accompanied by a prevention or delay in cytochromec release (Fig. 6B). Caspase activation as determined by poly(ADP-ribose) polymerase cleavage was also inhibited by Bcl-xL.
The ability of Bcl-2-family members to interfere with Bax-induced apoptosis was also observed in vitro. Mitochondria were prepared from CEM cells or CEM with ectopic expression of Bcl-2. Although Bax readily induced cytochrome c release from mitochondria isolated from the parental line, the mitochondria isolated from the Bcl-2-expressing cells were relatively resistant to this effect of Bax (Fig. 6C).
Therefore, the induction of cytochrome c release from mitochondria by Bax is inhibited by Bcl-2 and Bcl-xL.
In this paper we have shown that ectopic expression of Bax induces mitochondria to release cytochrome c, caspase activation, and apoptosis. Although apoptosis depends upon caspase function, cytochrome c release does not, suggesting that if cytochromec plays a role in caspase activation, it must precede this step in the apoptotic process. Several studies have shown that cytochrome c can trigger caspase activation in cell-free extracts (
In some systems, signals leading to apoptosis result in the transcription of key genes, which in turn lead to the death of the cell. Bax can function as one such gene. For example, DNA damage in some cells induces p53, which can activate the Bax promoter such that DNA damage results in elevation of Bax levels (
). Thus, upon growth factor withdrawal, Bcl-xL becomes sequestered, and Bax may then be free to induce cytochrome c release and apoptosis. Not surprisingly, then, elevated levels of Bax exacerbate the effects of growth factor deprivation in cells (
) upon apoptosis induction. In cells overexpressing Bax, we similarly observed that this molecule remained mostly cytoplasmic for several h and then localized to the mitochondria around the time of cytochrome c release (approximately 12 h, data not shown). The signals and mechanisms responsible for this change in Bax distribution are not known, although our results would suggest that they can be caspase-independent, because caspase inhibitors did not block Bax-induced cytochrome crelease (Fig. 2). Because Bax can clearly promote apoptosis, the nature of the translocation signal leading to induction of mitochondrial release of cytochrome c potentially takes on significance as a major apoptotic signaling pathway.
Once localized to the mitochondria, how does Bax induce the release of cytochrome c? Our studies support the idea that this can occur independently of a decrease in ΔΨm. Although Bax induced a dramatic decrease in mitochondrial transmembrane potential, inhibition of caspases by XIAP or zVAD-fmk significantly reduced the number of cells displaying such a loss while not affecting the extent of cytochrome c release. It is possible, however, that loss of ΔΨm occurs in two stages, a minor caspase-independent loss followed by a more dramatic caspase-dependent loss. Although this remains a possibility, it is noteworthy that we failed to detect any decrease in mitochondrial transmembrane potential in isolated mitochondria treated with Bax, despite the release of cytochromec (Fig. 5).
) have suggested that during apoptosis, a hyperpolarization of the mitochondrial inner membrane causes a swelling that might act to puncture the outer membrane without necessarily disrupting ΔΨm in the short term. In this model, Bcl-xL acts as an ion channel (
), and thus, our results are consistent with the possibility that Bax promotes inner membrane swelling and outer membrane puncture. However, we were unable to detect the expected mitochondrial swelling (Fig. 4B). Either this is a transient effect that we were simply unable to capture or else Bax promotes a loss of mitochondrial outer membrane integrity via a different mechanism. A similar failure to detect Bax-induced mitochondrial swelling in vitro was recently described by Jürgensmeier et al. (
) have shown that inducible Bax expression triggers a rapid caspase-dependent apoptosis, but if caspase activity is inhibited, a slower nonapoptotic death proceeds that has been associated with generation of reactive oxygen species (whether or not these are responsible for the subsequent death still remains unknown). Bax-induced cytochrome c release helps to explain these observations. First, cytochrome c can trigger caspase activation and apoptosis (
). In addition, however, and independently of caspase activation, the release of cytochrome c might be expected to result in disruption of electron transport, as has been observed in Fas-induced apoptosis. The resulting loss of ATP and generation of reactive oxygen species may ultimately cause cell death even in the absence of caspase function. This, of course, does not exclude the possibility that Bax has additional death-promoting activities. Nevertheless, the ability of Bax to induce the disruption of the mitochondrial outer membrane and the release of cytochrome c represents an important step in Bax-induced cell death.
Controversy over the mechanism of action of Bcl-2 and its homologue Bcl-xL has arisen recently with reports that these two anti-apoptotic oncogenes can block apoptosis by acting downstream of cytochromec in the cell death pathway (
). Bcl-2 has long been known to block cell death. Despite the vast literature dealing with this family of oncogenes, little is known about how the mechanisms used by these molecules prevent apoptosis. Some clues were provided by the observations that the structure of Bcl-xL resembles diphtheria toxin, which is able to form channels in cellular membranes (
). Previously it had been demonstrated that high levels of Bcl-2 can prevent the release of cytochrome c and, thus, caspase activation in response to a number of apoptosis-inducing stimuli, such as UVB, staurosporine, and etoposide (
) showed that redistribution of cytochromec is an early event in apoptosis that is inhibitable by Bcl-xL, but microinjection of cytochrome c overcomes this apoptotic inhibition. Together these studies proposed that a possible anti-apoptotic mechanism of Bcl-2 and its anti-apoptotic members was to inhibit cytochrome c translocation from mitochondria to the cytosol, thereby preventing caspase activation and subsequent apoptosis.
However Bcl-2 is not restricted exclusively to the mitochondrial membrane (
), and therefore, the possibility that this protein may have multiple anti-apoptotic mechanisms must be considered. To complicate matters, the Bcl-2 family members have been shown to bind to several proteins (
) may possibly explain how apoptosis induced by cytochromec microinjection could be inhibited in cells overexpressing Bcl-2. It remains possible that cells with high levels of Bcl-2 may have a large cytoplasmic fraction that is available to bind and thereby sequester exogenously added cytochrome c and, in doing so, quench its pro-apoptotic activity.
In contrast, we observed that Bcl-xL can significantly delay the release of cytochrome c from mitochondria (and subsequent apoptosis) in response to ectopic Bax. These results parallel similar observations reported in yeast studies (
), which would interfere with the availability and translocation of the Bax protein from the cytoplasm to the mitochondria. Alternatively, counteracting ion channels at the level of the mitochondria may help to explain the antagonistic nature of these proteins (
In conclusion, we have shown that Bax induces the release of cytochromec in conjunction with apoptosis and that caspase inhibition, although altering the kinetics and perhaps mode of cell death, has no effect on this release. In contrast, members of the anti-apoptotic oncogene family, Bcl-2 and Bcl-xL, are capable of inhibiting or delaying this release while significantly preventing cell death. This suggests that Bcl-2 family members may play a modulating role in blocking the mammalian cell death machinery by acting upstream of caspase function and upstream or at the mitochondrial level.
We thank Drs. A. Gross and S. Korsmeyer for their pGEX-KG-BaxΔC19 construct and advice in the isolating Bax protein. We also thank Dr. Ruth Kluck for helpful discussions.