The interplay between the Bcl-2 family and death receptor-mediated apoptosis

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1 Biochimica et Biophysica Acta 1644 (2004) Review The interplay between the Bcl-2 family and death receptor-mediated apoptosis Martin R. Sprick, Henning Walczak* Department of Apoptosis Regulation, DKFZ Heidelberg, D040, Tumour Immunology Program, Im Neuenheimer Feld 280, Heidelberg, Germany Received 7 July 2003; accepted 4 November 2003 Abstract Two principal pathways for apoptosis initiation exist. One pathway, which is also termed the extrinsic pathway, is mediated by death receptors, a subgroup of the TNF receptor superfamily. The second pathway, which is also referred to as the intrinsic pathway, is controlled by members of the Bcl-2 family. A long standing discussion revolves around the question of how these two pathways influence each other in regulating the decision about life or death of a cell. Here, we review our current knowledge about the interactions between these two pathways and discuss current models which could help to resolve previous apparently contradictory results. D 2003 Elsevier B.V. All rights reserved. Keywords: Apoptosis; Bcl-2 family; Death receptor Apoptosis is the physiological form of cell death which has evolved to allow for tissue remodeling and homeostasis and to remove superfluous and potentially dangerous cells from an organism. Processes involving apoptosis are, e.g. the shaping of the body, removal of activated immune cells after an infection and deletion of oncogenically or virally transformed cells [1 4]. Apoptosis is characterized by the ordered destruction of cellular proteins and DNA, rendering the cells ready to be taken up by phagocytosis [5]. Key to this ordered destruction is the activation of a class of proteases, known as caspases. 1. Two converging pathways of apoptosis initiation Two principal pathways for apoptosis initiation exist. One pathway which is also termed the extrinsic pathway is mediated by death receptors, a subgroup of the TNF receptor superfamily. The second pathway, which is also referred to as the intrinsic, or possibly more accurate, Bcl-2-controlled pathway is consequently controlled by members of the Bcl-2 family (for reviews see Refs. [6 8]). Despite their differing * Corresponding author. Tel.: ; fax: address: h.walczak@dkfz.de (H. Walczak). modes of initiation, both pathways have the same outcome: the activation of a cascade of proteolytic enzymes, members of the caspase family. All caspases are synthesized as zymogens (procaspases) sharing a common domain structure consisting of a large (p10) and a small (p20) catalytic subunit (for in-depth reviews of caspases, see Refs. [9 13]). The N-terminal prodomain, however, differs widely between the caspases. Two fundamentally different groups can be discerned according to the architecture of the prodomain [14,15]. The first group, also termed initiator caspases, is characterized by a long prodomain which provides a protein protein interaction platform. These prodomains allow for the recruitment of the procaspases into an activating protein complex. Long prodomain caspases are caspases-1, -2, -4, -5, -9, -11 and -12 with an N- terminal caspase-activating recruitment domain (CARD), and caspase-8 and -10 with an N-terminal death effector domain (DED). In contrast to the initiator caspases, the socalled executioner caspases-3, -6 and -7 lack the large N- terminal non-enzymatic domain. Their name stems from the observation that they are responsible for the majority of cellular destruction during apoptosis. The executioner caspases share their short prodomain with caspase-14, a caspase which is involved in keratinocyte maturation [16 18]. Caspases-1, -4, -5, -11 and -12 are mainly responsible for processes other than apoptosis and will not be discussed in detail /$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi: /j.bbamcr

2 126 M.R. Sprick, H. Walczak / Biochimica et Biophysica Acta 1644 (2004) Central to the apoptotic process is the activation of caspases in a proteolytic cascade. Long prodomain initiator caspases and the short prodomain executioner caspases differ in their modes of activation. Initiator caspases become activated in specialized protein complexes whose formation is triggered by distinct stimuli. The activation complex for caspase-9 is termed the apoptosome [19]. Caspase-8 and caspase-10 are recruited to and activated in a complex termed death-inducing signaling complex (DISC) (see below). A somewhat more enigmatic role is played by caspase-2 for which limited data is available. The existence of a caspase-2-specific activation complex has been proposed recently [20], and a role for caspase-2 in the apoptotic response after DNA damage has been described [21,22]. The executioner caspases in turn are activated by a twostep mechanism. First, an initial proteolytic cleavage is needed, which separates the large from the small subunit. This cleavage is usually carried out by an active initiator caspase, mainly DISC-activated caspase-8 and apoptosomeactivated caspase-9 [12]. However, the activity of the resulting enzyme is still inhibited by its own prodomain. Removal of the prodomain is a prerequisite for full enzymatic activity and occurs in an autocatalytic step [23]. Importantly, this autocatalytic maturation can be inhibited by cellular caspase-inhibitors, the IAPs [24]. IAPS were first characterized as viral proteins capable of inhibiting apoptosis. Several cellular homologs exist, with XIAP being the best characterized. XIAP can interact with caspase-3 and caspase-9 and thus inhibit their activation. XIAP inhibits caspase-3 activation by blocking the removal of the inhibitory prodomain. Thus, even if active initiator caspases are present in a cell to cleave the executioner caspases, the cell does not necessarily undergo apoptosis, as the second step necessary for maturation of executioner caspases maturation can still be inhibited by IAPs. 2. The death receptor pathway activation of caspases in the DISC In the extrinsic, receptor-mediated pathway, cell death is initiated by stimulation of certain members of the TNF receptor superfamily, the death receptors [25]. Stimulation of these receptors leads to the formation of the DISC [26]. Binding of the death receptors by their ligands leads to intracellular binding of the adaptor-protein FADD to the receptor. FADD in turn serves to recruit pro-caspases-8 and -10 into the complex. Close proximity of these proenzymes in the DISC leads to activation of their catalytic activity, presumably by an allosteric mechanism, involving dimerization between two caspase-8 or -10 molecules [27 30]. While proteolytic separation of the large and small subunits is not necessary for activation, it probably enhances the stability of the dimer once it is released from the DISC. Although both caspase-8 and caspase-10 are activated in the CD95 DISC as well as in the TRAIL DISC [31 33], they are not redundant in function [31]. Activation of caspases in the DISC can be inhibited by a cellular protein, FLICEinhibitory protein (cflip). Both known splicing variants of cflip, cflip L and cflip s can be recruited to the DISC and inhibit caspase-8 and -10 activation [34]. 3. The Bcl-2-controlled pathway The intrinsic, cell autonomous pathway is triggered by activation of caspases from inside the cell. Central to this intrinsic pathway is the formation of an intracellular caspase-9-activating complex, the apoptosome [19]. A key player in regulating apoptosome-formation is the mitochondrion. The apoptosome forms after cytochrome c is released from mitochondria. Cytochrome c, together with ATP or datp and Apaf-1 recruits caspase-9 to activate it, again by an allosteric mechanism triggered by dimerization or oligomerization of caspase-9 [27,35]. Activated caspase-9, like the DISC-activated caspases-8 and -10, can trigger the activation of the executioner caspases by executing the first catalytic cleavage imperative for their activation. Examples of events triggering apoptosome formation include genotoxic stress, cytokine withdrawal and detachment of adherent cells (anoikis) [6]. Common to all these events is that they lead to a breach in mitochondrial integrity, associated with an efflux of cytochrome c and other pro-apoptotic molecules [6]. The integrity of the mitochondrial membrane is regulated by proteins of the Bcl-2 family [7]. This protein family can be divided into three groups based on their domain architecture. It consists of anti-apoptotic members like Bcl-2, Bcl-X L, Bcl-w, Mcl-1, Bfl-1/A1, which are associated with the mitochondrial outer membrane, serving to maintain mitochondrial integrity. The so-called BH3-only proteins, a second group of Bcl-2 family members, act as sentinels over various cellular organelles and processes. Under normal conditions, the BH3-only proteins are inactive. Upon activation, they act as activators of the third group of Bcl-2 family members, the large pro-apoptotic members Bax, Bak, Bok (Mtd). These proteins associate with the outer mitochondrial membrane during apoptosis, breaching its integrity. Bax and Bak are essential for the BH3-only proteins to act on mitochondria, as cells deficient in these two proteins are resistant to apoptosis induced by overexpression of BH3-only proteins [36 38]. Different apoptotic stimuli lead to the activation of distinct BH3-only proteins. The mechanisms by which these proteins become activated are different for each family member. They include transcriptional upregulation for Noxa and PUMA (Bbc3), dephosphorylation of Bad after growth factor withdrawal or release from cytoskeletal elements induced by UV radiation or anoikis (Bmf) or cytokine withdrawal and Ca ++ -flux (Bim) (reviewed by Puthalakath and Strasser [39]).

3 M.R. Sprick, H. Walczak / Biochimica et Biophysica Acta 1644 (2004) The BH3-only protein Bid finally provides a link between the receptor-mediated pathway and the Bcl-2-controlled pathway [40,41]. The pro-apoptotic activity of Bid is activated by caspase-8-mediated cleavage which induces its translocation to the mitochondria. Mitochondrially translocated Bid in turn has a pro-apoptotic effect. Whether this is due to direct binding to and oligomerization of Bax/Bak [42,43] or possibly by binding to pro-survival Bcl-2 family members is still debated [6]. The concept that apoptosomedriven caspase-9 activation is an integral component of Bcl- 2 family-regulated apoptosis has recently been challenged. Marsden et al. [44] demonstrated that Bcl-2-controlled apoptosis still occurs in cells derived from mice deficient in either Apaf-1 or caspase-9. The definition of the identity of the proteins involved in this alternative pathway awaits further experiments. 4. How could a Bcl-2 controlled (mitochondrial) pathway influence death receptor signaling? A long-standing discussion revolves around the question whether a Bcl-2 family-controlled mitochondrial pathway is required during CD95L-induced apoptosis or not [45 48]. One school of thought classified cells into two types, type I and type II [45]. Type I cells are characterized by a strong CD95 DISC formation and subsequent caspase activation. In these cells, CD95L-mediated apoptosis cannot be inhibited by overexpression of Bcl-2 or Bcl-X L. In type II cells, a weak DISC formation and caspase activation is seen after stimulation of CD95. In these cells, CD95-mediated apoptosis can be inhibited by overexpression of Bcl-2 or Bcl-X L [45,46]. This classification was hotly debated, as some argued that the effects were an artifact caused by the agonistic CD95-reactive antibodies used for stimulation which do not reliably mimic the physiological ligand [47,48]. While the concept held true in experiments using an artificially stabilized trimeric form of the CD95 ligand [46], no effect was seen when using an antibody-crosslinked ligand or membrane-bound ligand expressed on transfected cells [49,50]. The initial explanation for two distinct cell types with regard to CD95L-induced apoptosis was based on the assumption that the apoptosome would form an amplification loop which would compensate for weak DISC formation. As described above, the maturation of executioner caspases can be inhibited by cellular IAPs, most notably XIAP. Thus, a scenario is possible, where DISC-activated initiator-caspases cleave caspase-3, yet apoptosis does not proceed because cellular XIAP blocks the second caspase-3 maturation step. Bid, which is also cleaved by the DISCactivated caspases, mediates cytochrome c release which leads to apoptosome formation and caspase-9 activation. Activated caspase-9, in turn, also cleaves caspase-3, raising the levels of primed caspase-3. The levels of primed caspase-3 finally out-titrate XIAP which leaves the additional primed caspase-3 uninhibited. The role of an apoptosome-mediated amplification loop, however, seemed important only in a few situations. In agreement with this concept, studies using Bid-deficient mice showed a resistance to CD95L-induced cell death, at least in some tissues as the liver [51]. However, arguing against an important role of the apoptosome in CD95L-induced apoptosis are the phenotypes of Apaf-1 / and caspase-9 / mice, as cells derived from these mice are not resistant to CD95L-induced apoptosis [52 55]. This different requirement for Bid in certain situations, while the apoptosome seemed dispensable, has long been a subject of discussion and remained an unresolved conundrum. Possible explanations emerged by the recent identification of new players in the apoptosis field (reviewed by Vaux and Silke [56]). It was the discovery of two XIAPantagonizing proteins, Smac/DIABLO [57,58] and HtrA2/ Omi [59 63] which can help to explain some of the conflicting results of the past. These proteins which, like cytochrome c, normally reside in the mitochondrial intermembrane space have a pro-apoptotic effect once released. The mechanism by which this is attained has been elucidated for Smac/DIABLO. This protein interacts with, and sequesters XIAP to remove its inhibition from caspase-9 and caspase-3. In the case of casape-3 this allows for the second proteolytic step which fully activates this enzyme. Less is known about the action of HtrA2/Omi, but it possibly acts by a similar mechanism as well by its inherent protease activity. What is the link between these proteins to death receptor-mediated caspase activation? Intriguingly, using hepatocytes from Bid / mice, Li et al. [64] have shown that the release of Smac/DIABLO depends on the action of Bid. Adrain et al. have shown that Smac/DIABLO release from mitochondria requires caspase activity and can be blocked by Bcl-2 [65]. In line with these results, Sun et al. [65] have shown that in type II cells, the release of mitochondrial Smac/DIABLO is the decisive cellular event regulated by Bcl-2/Bcl-X L. Integrating these findings in the overall scheme of death receptor- and Bcl-2 family-controlled apoptotic pathways, it becomes clear that the requirement for mitochondria in apoptosis is not only to provide apoptosome-activated caspase-9. Even more important might be the release of XIAP antagonists which remove the apoptosis-inhibiting block from caspase-3 (this concept has also recently been discussed by Bratton and Cohen [66]). One should be aware, however, that sensitivity to death receptor-induced apoptosis can be regulated by several factors, and it is the sum of these factors which decides between life and death of a cell. The expression levels of Bcl-2 family members and of IAPs is only one of them (Fig. 1). On the one end of the spectrum are cells which are very sensitive to death receptor-mediated apoptosis. In these cells, sensitivity may be due to high expression of death receptors, the intrinsic ability of these receptors to be efficiently stimulated and low or absent expression of

4 128 M.R. Sprick, H. Walczak / Biochimica et Biophysica Acta 1644 (2004) Fig. 1. Crosstalk between the Bcl-2 family controlled and death-receptor pathways to apoptosis. Death receptors activate the initiator caspase-8 (and 10) at the DISC, which in turn prime caspase-3 for its autocatalytic activation. Activation of caspases in the DISC can be inhibited by cflip which recruited to the DISC complex. Alternatively, low receptor levels, intrinsic properties of the receptors or the expression of decoy receptors may lead to sub-optimal cross-linking and, thus, DISC formation. At the mitochondrial level, apoptosis is initiated by caspase-9 activation in the apoptosome. Caspase-9 acts in a similar manner as the DISC activated caspases by priming caspase-3. Apoptosome formation is triggered by cytochrome c release from the mitochondrion. Mitochondrial integrity, in turn, is regulated by the balance of pro- and anti-apoptotic Bcl family members. The BH3-only protein Bid provides a crosstalk between death receptors and mitochondria, as it is cleaved and thereby activated by DISC-derived initiator caspases, Primed caspase-3 needs to be further processed in an autocatalytic manner for full enzymatic activity. This step can be inhibited by cellular inhibitors like XIAP which binds to executioner caspases. The XIAP antagonists Smac/DIABLO and HtrA2/Omi, when released from the mitochondria, relieve this inhibition and allow for apoptosis to proceed. inhibitory (decoy) receptors. Intracellular factors which contribute to reduced sensitivity, like FLIPs or IAPs are low or absent. For these cell types, weak stimuli are sufficient to commit these cells to death. A notable contribution of factors released from mitochondria is predicted to be absent in these cells and thus, no contribution of Bcl-2 family members can be experimentally noted. On the other end of the spectrum are cells which are hard to kill by death ligands. On the receptor level, this can be due to low expression of death receptors, their resistance to cross-linking and possibly expression of inhibitory decoy receptors. On the intracellular level, these cells are predicted to express high levels of anti-apoptotic proteins like FLIPs and IAPs. In these cells, stimulation of death receptors only results in the initial cleavage of caspase-3, with the autocatalytic cleavage step being inhibited. Under these circumstances mitochondrial release of pro-apoptotic factors is required for apoptosis to proceed. Thus, an influence of Bcl-2 family members can be noted experimentally. Overexpression of Bcl-2 or deletion of Bax would lead to relative resistance of these cells to a certain death stimulus. It will immediately become apparent that the experimental setting used to determine sensitivity versus resistance of a cell type is also important. Differences in apoptosis sensitivity are most faithfully determined if careful titrations of the death-inducing stimuli are performed. Otherwise, differences which might well be physiologically relevant could go unnoticed. Crosstalk does not only exist between the death receptor pathway and the Bcl-2-controlled pathway. It is important to note that elements which are upstream of the intrinsic pathway might also influence the death receptor pathways. Most prominent is the activation of the tumor-suppressor p53, which can lead to an upregulation of some death receptors and thus sensitization to apoptosis mediated via death receptors. Due to space-constraints, we refer the interested reader to some in-depth reviews of this issue [67,68].

5 M.R. Sprick, H. Walczak / Biochimica et Biophysica Acta 1644 (2004) How does this concept apply to TRAIL? TRAIL (Apo-2L) is a cytokine belonging to the TNFcytokine superfamily based on its homology to CD95L [69,70]. Four membrane-bound receptors belonging to the TNF receptor superfamily have been identified (reviewed by MacFarlane [71]). Two of these receptors, TRAIL-R1 and TRAIL-R2 can mediate an apoptotic signal by virtue of their intracellular death domain. Two other receptors, TRAIL-R3 and TRAIL-R4 have been proposed to act as decoy receptors. The initial events after triggering the two TRAIL-death receptors, TRAIL-R1 and TRAIL-2, are well characterized. With respect to the kinetics of assembly and the intracellular molecules involved, the TRAIL DISC is very similar to the CD95 DISC. The TRAIL DISC also utilizes the adaptor protein FADD/MORT1 and the initiator caspases-8 and -10 for apoptosis induction [31,33,72 74]. In addition, recruitment of cflip into the TRAIL-DISC has also been shown [75]. 6. Intracellular factors determining sensitivity to TRAIL Considering what is currently known about the signaling molecules used by TRAIL, it would not be surprising that the same concept that emerges for CD95 could also be applied to TRAIL. Several studies addressed the question of whether a Bcl-2 family-controlled pathway can also influence TRAIL-mediated apoptosis in a manner similar to CD95L-induced apoptosis. In a number of studies, ectopic overexpression of Bcl-2 or Bcl-X L has been found to inhibit TRAIL-induced apoptosis [76 82]. In certain situations overexpression of Bcl-2 or Bcl-X L was not able to prevent but rather delay TRAIL-induced apoptosis [83 85]. Further information was provided by studies utilizing tumor cells bearing a mutation in Bax. LeBlanc et al. [86] have shown that this mutation results in resistance to TRAIL and that treatment of xenografted Bax wt tumors with TRAIL selects for Bax mutations in vivo. These results are in line with the observations of Deng et al. [87] who also showed an absolute requirement for Bax in TRAIL-mediated apoptosis. In this study, it was also proposed that it is the mitochondrial release of Smac/DIABLO which is the important event mediated via Bax. Similarly, melanoma cells can obtain resistance to TRAIL by an XIAP-mediated block of caspase-3 activation [88]. Srinivasula et al. [89] have shown that in certain situations overexpression of anti-apoptotic Bcl-X L inhibits TRAIL-induced apoptosis, which in turn can be overcome by expression of cytosolic Smac/DIABLO. The ability of Smac/DIABLO to enhance TRAIL-induced apoptosis was confirmed by several other authors using ectopic overexpression of Smac/DIABLO [90 93]. Another approach demonstrated that cell-permeable SMAC-peptides could overcome TRAIL-resistance in malignant gliomas xenografted into mice [94]. These results suggest an important role of mitochondrially released Smac/DIABLO and possibly HtrA2/Omi in removing an XIAP-mediated block in caspase activation. Experiments using primary human keratinocytes in addition suggested a role for XIAP in regulating TRAIL resistance in non-transformed cells [95]. Together, these experiments hint at an important role for XIAP and its antagonists Smac/DIABLO, and possibly, Omi/HtrA2 in regulating TRAIL sensitivity. However, the generation of mice deficient for XIAP revealed no apparent sensitization to different pro-apoptotic stimuli in cells derived from these mice [96]. Several explanations, apart from the possibility that XIAP plays only a minor role in apoptosis regulation, could be provided. First, a marked upregulation of ciap-1 and ciap-2 which might compensate for the loss of XIAP was noted in the XIAP-deficient mice. Second, the only death receptor stimuli that were tested were TNF and CD95L. Additionally, no titrations of the ligands were performed and only a limited number of cell types were investigated. Thus, it could well be that potential differences in apoptosis sensitivity went unnoticed. Similarly, cells derived from mice deficient in Smac/ DIABLO did not show a marked resistance to death stimuli, although in an in vitro assay, caspase-3 activation was impaired [97]. However, for the analysis of TRAILinduced apoptosis, titrations were not performed. Whether the phenotype of these mice speak against an important role of XIAP or whether the loss of XIAP is compensated by related molecules awaits further experiments. Apart from the regulation at the Bcl-2-controlled level, other factors can also contribute to resistance versus sensitivity to TRAIL (and possibly other death ligands). It has been shown that high levels of cflip can contribute to resistance of primary B cell chronic lymphocytic leukaemia (B- CLL) to TRAIL by blocking caspase-8 activation at the DISC [71,98]. In contrast, in TRAIL-resistant melanoma cells the expression and localization of TRAIL death and decoy receptors seems to be determining resistance (reviewed by Hersey and Zhang [99]). 7. Final words The influence of Bcl-2 family members on death receptor-mediated apoptosis was first identified in the context of experiments which led to the formulation of the type I versus type II theory with regards to CD95- mediated apoptosis. It has now become clear that the concept is valid but that the molecular basis is possibly broader than initially thought (Fig. 1). In that respect, the very same cell that is type I for one cell death system can be of type II for another cell death system, as receptor levels or the ability of the receptors to be cross-linked may be different for, e.g. CD95 on the one hand, and TRAIL receptor system on the other hand. Such different receptor levels may translate in more or less initiator caspase

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