In this study, we have shown that cytochrome c can initiate a complex series of caspase activation events, ultimately resulting in apoptotic changes (nuclear condensation and fragmentation, degradation of several caspase substrates, DNA fragmentation) in cell extracts. Surprisingly, cytochrome c initiated activation of several of the so-called signaling (caspases-2, -8, -9, and -10) as well as effector (-3, -6, -7) caspases. In contrast, no detectable processing of the ICE subfamily caspases (-1, -4, -5) was observed in this context. All of these events were abrogated by removal of caspase-9 from the extracts, confirming that this protease is indispensable for cytochrome c–initiated triggering of the death program and occupies an apical point in the caspase cascade. In line with these observations, the CARD domain of Apaf-1 was shown to bind selectively to caspase-9. Inhibitory profiles generated with the caspase inhibitor DEVD-CHO suggested that caspases-3 and -7 were activated downstream of caspase-9 and that these caspases then went on to propagate the caspase cascade by activating caspases-2, -6, -8, and -10. This interpretation was confirmed and extended by immunodepleting caspases-3, -6, or -7 from the extracts and assessing the impact of their removal on the other caspase activation events. Interestingly, removal of caspase-3 revealed that this caspase was required for four other caspase activation events and also revealed a feedback loop in this pathway involving caspase-9.
In this study we have assessed caspase activation events in most cases (with the exception of caspases-1, -3, and -9) by adding 35S-labeled in vitro transcribed and translated caspases to the cell extracts. Clearly, this raises the issue of whether caspases endogenous to the extracts behave in the same way as their exogenously added counterparts. Where antibodies were available to us (caspases-2, -6, -7, and -8), we confirmed that the order and kinetics of endogenous caspase processing was essentially identical to that observed using exogenously added caspases (Slee, E.A., and S.J. Martin, data not shown). However, the use of radiolabeled caspases enabled us to track complete processing of each caspase whereas many of the available anticaspase antibodies recognized processed forms inefficiently or not at all.
Although the initial report that cytochrome c could trigger caspase-3 processing was surprising (Liu et al., 1996
), much evidence has accumulated to suggest that release of cytochrome c from mitochondria is an important control point in apoptosis (reviewed by Reed, 1997
). It is still unclear exactly how cytochrome c release is achieved, although recent reports suggest that death-promoting members of the Bcl-2 family such as Bax or Bid may play a role in this, possibly due to their ability to form ion or small protein channels (Jurgensmeier et al., 1998
; Li et al., 1998
; Luo et al., 1998
). Although opening of a permeability transition pore was proposed previously as one possible means of enabling cytochrome c escape to the cytosol (Kroemer et al., 1997
), cytochrome c release has been observed in situations where either no loss in mitochondrial transmembrane potential was observed or where changes in transmembrane potential occurred after cytochrome c efflux (Kluck et al., 1997a
; Yang et al., 1997
; Bossy-Wetzel et al., 1998
Irrespective of the exact mechanism of release, much evidence now exists to suggest that cytochrome c plays an important role as an initiator of the death machinery in cases where cellular damage is general (i.e., radiation, heat shock, cytotoxic drugs), or as an amplifier of death signals in cases where caspase activation is initiated by a membrane receptor such as Fas (Kuwana et al., 1998
; Li et al., 1998
; Luo et al., 1998
; Scaffidi et al., 1998
). Perhaps the most compelling argument for a central role for cytochrome c in apoptosis is the finding of Wang and colleagues that cytochrome c binds to and activates Apaf-1, the first human CED-4 homologue to be discovered (Zou et al., 1997
). Gene targeting experiments in mice have revealed that Apaf-1 plays a critical role in developmental-related cell death in the brain, as well as in cytotoxic drug-induced cell death in other cell lineages (Cecconi et al., 1998
; Yoshida et al., 1998
). At present, cytochrome c is the only known activator of Apaf-1, although it is possible that other pathways may harness the caspase-activating properties of this molecule.
Although numerous studies have appeared documenting the activation of individual caspases in the context of many different death-promoting stimuli, it is still unclear whether caspases are activated sequentially or in parallel in many of these contexts. Here, we provide evidence for a stepwise series of caspase activation events occurring in response to cytochrome c. Caspase-9 appears to be the first caspase to become activated in this context, almost certainly due to clustering of this protease via Apaf-1. Clustering of caspase-9 results in partial activation of this protease in an autocatalytic manner (Pan et al., 1998a
; Srinivasula et al., 1998
). Caspase-9 then initiates processing of caspase-3 as well as caspase-7. The activation of caspase-3 in this context appears to occur in a partly autocatalytic manner since the caspase-3 inhibitor, DEVD-CHO, arrested maturation of caspase-3 at an incompletely processed intermediate stage. The pattern of caspase-3 breakdown suggests that caspase-9 attacks this molecule between the large and small subunits and that caspase-3 subsequently removes its own prodomain by autocatalysis. Activated caspase-3 in turn activates caspases-2 and -6 and also appears to be capable of acting in a feedback loop on caspase-9 to ensure complete activation of the latter. Somewhat surprisingly, caspase-6 was found to be required for the activation of caspases-8 and -10 in this context (Fig. ).
Clearly, further work is necessary to determine whether the sequence of cytochrome c–inducible caspase activation events that take place in cell extracts also takes place in intact cells. However, recent gene targeting studies provide support for our model (Hakem et al., 1998
; Yoshida et al., 1998
null embryonic stem (ES) cells and embryonic fibroblasts were found to be resistant to multiple proapoptotic stimuli, but not to cytotoxic T lymphocyte or TNF-mediated killing, arguing that caspase-9 is required for forms of apoptosis that are thought to be routed along the mitochondrial pathway (Hakem et al., 1998
). As further evidence of this, CASP-9−/−
ES cells were found to be resistant to UV-induced death, although cytochrome c release still took place (Hakem et al., 1998
). Moreover, UV-induced caspase-3 and caspase-8 processing was impaired in CASP-9−/−
ES cells as was dexamethasone-induced processing of caspases-2, -7, and -8 in CASP-9−/−
thymocytes (Hakem et al., 1998
), supporting our observations that these caspases are activated downstream of caspase-9 in the cytochrome c pathway. Similar observations have also been made with respect to etoposide-induced caspase-2 and caspase-8 activation in thymocytes from APAF1−/−
mice (Yoshida et al., 1998
). Once again, these observations lend support to our observations that caspases-2 and -8 are activated downstream of cytochrome c/Apaf-1.
We have confirmed the observation of Wang and colleagues (Li et al., 1997
) that Apaf-1 directly binds to caspase-9 and have extended this observation to show that caspases-1, -2, -3, -6, -7, -8, and -10 do not bind to this molecule. These observations are at odds with recent findings that suggest that Apaf-1 can also form complexes with caspases-4 and -8 (Hu et al., 1998
). However, in the latter study no direct interaction between these caspases was demonstrated since coimmunoprecipitation from cell lysates was the criteria used to determine interaction. Thus, binding could have been mediated by an adaptor protein. Using a Gal4-based yeast two-hybrid system we have confirmed the interaction between the CED-3-homologous region of Apaf-1 and caspase-9 but again failed to detect direct binding of Apaf-1 to caspase-8 (Harte, M.T., C. Adrain, and S.J. Martin, unpublished data). In addition, the observation that removal of caspase-9 from cell extracts abolished all caspase activating activity of cytochrome c suggests that this caspase is indispensable for this pathway, irrespective of the ability of Apaf-1 to complex with other caspases.
Although it is generally believed that multiple caspases participate in the signaling and destruction phases of apoptosis, it is still unclear whether there is significant functional redundancy within this family of proteases. The observations that caspase-3, caspase-8, and caspase-9 knockout mice die in utero or soon after birth would argue against redundancy, at least in certain tissues (Kuida et al., 1996
; Hakem et al., 1998
; Varfolomeev et al., 1998
). In addition, in vitro studies that have used dominant negative forms of caspase-9, as well as data available from CASP-9
null mice, suggest that this caspase occupies a critical position in a major subset of cell death pathways since apoptosis was abrogated in the absence of this caspase in a number of contexts (Hakem et al., 1998
; Kuida et al., 1998
; Pan et al., 1998b
; Srinivasula et al., 1998
It is also commonly believed that caspases with long prodomains are upstream or signaling caspases whereas those with short prodomains are effector or executioner caspases. For example, studies on caspase-8 suggest that this protease is the most proximal caspase to become activated upon ligation of the CD95 (Fas/Apo-1) molecule since this caspase is directly recruited into the CD95 signaling complex upon receptor aggregation (Kischkel et al., 1995
; Boldin et al., 1996
; Fernandes-Alnemri et al., 1996
; Muzio et al., 1996
). However, recent studies have suggested that caspase-8 is not always activated early in the context of CD95 signaling (Scaffidi et al., 1998
). This has led to the suggestion that two distinct cellular types exist with respect to CD95 signaling: type I, cells that activate caspase-8 early (within seconds) of CD95 receptor aggregation and type II, cells that activate caspase-8 late and in a mitochondrial-dependent fashion (Scaffidi et al., 1998
In this study we also observed caspase-8 activation late in the cytochrome c–inducible caspase cascade. It is possible that caspase-8 activation is merely a bystander event in this model of apoptosis, since the caspase substrates examined (fodrin, PARP, U1snRNP) were almost completely cleaved within 60 min of addition of cytochrome c to the extracts (Fig. B) and nuclear destruction was largely complete by 90 min (Fig. A). In contrast, only a small portion of the [35
S]methionine-labeled caspase-8 that was added to the extracts had become processed by 60 min under similar conditions (Fig. ). However, as previously discussed, caspase-8 activation was also found to be impaired in cells from CASP-9−/−
as well as APAF-1−/−
mice in certain contexts, suggesting that this caspase may be activated downstream in some situations (Hakem et al., 1998
; Yoshida et al., 1998
In summary, our observations suggests that a branched cascade of caspase activation events, with at least one feedback loop, is initiated distal to entry of cytochrome c into the cytosol. Further studies are required to establish whether the sequence of caspase activation events we report is conserved between different cell types and in response to divergent death-promoting stimuli.