- Email: [email protected]
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Orderingthe Multiple Pathways of Apoptosis David S. Park, Leonidas Stefanis, and Lloyd A. Greene
Apoptosis plays an important role in development, homeostasis, and disease. Cun-ent wo~k has suggested that apoptosis can be evoked by multiple stimuli that, in turn, initiate distinct death pathways. Recently, exciting advances have been made in the understanding of biochemical pathways that regulate apoptotic processes. These pathways contain both evolutionan”lyconserved elements and components that are dependent on the death stimulus and cell context. Accordingly, this review focuses on the compositions and relative ordering of the apoptotic pathways in four different death paradigms: activation of receptors of the Fas ligand, destruction by cytotoxic T lymphocytes, exposure to DNA damagingagents, and loss of support by neurotr-ophic factom. These examples illustrate the conservation and divergence in the ways that death pathways are composed and o~dered. (Trends Cardiovasc Ivled 1997;7:294–301). 01997, Elsevier Science Inc.
Apoptotic cell death plays major roles in a diversity of developmental processes as well as in numerous diseases. In general, two modes of cellular death have been described [see Wyllie et al. (1980) for review]. In contrast to necrotic cell death, which is usuallyaccompanied by swelling and disruption of cellularmembranes and
David S. Park and Lloyd A. Greene are at the Department of Pathology and Center for Neurobiology and Behavior,and Leonidas Stefanis is at the Departmentsof Pathology and Neurology and the Center for Neurobiology and Behavior, Columbia UniversityCollege of Physicians and Surgeons, New York, ~, 10032, USA.
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inflammation of adjacent tissue, apoptosis seems to be a regulated process of death typified by cell shrinkage and blebbing, clumping of chromatin, nuclear pyknosis and disruption, and formation of cytosol containing apoptotic bodies. In the cardiovascular system, it appears that apoptosis plays an important part in the development of the sinus and atrioventricular nodes and of the His bundle (James 1994). In addition, regulation of the number of cells in the intimal layer of the vascularwall occurs by an appropriate balance between cell growth and death, and decreased apoptosis may contribute to vascularlesion formation (Gibbons and
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Dzau 1996). Finally,apoptosis may play a critical role in ischemia and reperfusion injury of the heart (Thompson 1995). Initial descriptions of apoptotic cell death were defined by characteristic morphological changes [see Wyllie et al. (1980) for review]. Recently, however, a rapidly growing body of work has developed a more biochemical view of apoptosis, and exciting progress has been made in characterization of the molecules that regulate this process. Analysis of cell death in various systems has revealed several key points. First, there are certain conserved classes of molecules that regulate death in nearly all apoptotic paradigms. Second, apoptosis occurs by complex, multistep pathways of causally related events that culminate in cell destruction. Third, there are multiple means to trigger death, and these in turn initiate distinct apoptotic pathways. These pathways are formed by combination of what are seemingly conserved cell death components with diverse elements that depend on both cellular context and on the initiating cause of death. The purpose of this review is to describe what is currently known about the nature and relative order of the events that comprise the apoptotic pathways. First, the conserved elements of these pathways are briefly reviewed. Then, because there is no single route to death, the events that mediate apoptosis evoked by four distinct initiating causes are considered. This will highlight the degree to which there is both conservation and diversity in the composition and arrangement of the mechanistic pathways that mediate cell death.
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ConservedElements in Death and Survival
Much of our understanding of mammalian apoptosis began with genetic studies in Caenorhabditis elegans (Ellis et al. 1991). These revealed the death effecter genes ced-3 and ced-4, and a third gene, ced-9, which blocks death. Identification of ced-3 and ced-9 homologies in higher organisms has led to the belief that these gene families represent conserved and basal elements of the death pathway. The mammalian homologies of ced-3 constitute a family of aspartic acid– directed cysteine proteases now designated as caspases. The prototypic mam-
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malian caspase is the interleukin-con- capacity to form homodimers and hetverting enzyme (ICE) that was initially erodimers, and this has been suggested identified as the protease that converts to play a key role in their proapoptotic pro-ILl-@ into the active form of this and antiapoptotic activities (Gajewski cytokine (Yuan et al. 1993). Various clon- and Thompson 1996). Two major moding strategies have currently uncovered els of Bcl-2-family member function ex11 mammalian caspases, which can be ist. In the first, it is thought that the Bax divided into four classes by molecular homodimers are the trigger for death. homology (ICE-like, Ced-3/CPP32-like, Accordingly, the presence of the antiapoNedd2/Ich-l-like [Fraser and Evan 1996] ptotic members Bcl-2 and Bc1-xL would and those containing FADD-like death bind to Bax, preventing homodimerizadomains IBoldin et al. 1996, Muzio et al tion. In the second model, it is Bcl-2 and 1996, Fernandes-Alnemri et al. 19961), Bc1-xL homodimers that actively block which are discussed later here. Studies death [see Gajewski and Thompson by a variety of groups have established (1996) for review]. In support of the latthat expression of caspases in multiple ter model, some mutations in Bc1-xL cell types produces death and that their that affect binding to Bax do not abroinhibition blocks apoptosis induced by gate the antiapoptotic function of Bclvarious stimuli. The caspases are thus xL/Bcl-2 (Cheng et al. 1996). It is unclear considered to be the primary effecters of how Bcl-2 members interact with the apoptotic cell death (Fraser and Evan rest of the apoptotic pathway. Recently, 1996). some evidence suggests that Bcl-2 family Caspase family members are initially members interact with classical signal translated as inactive precursor polypeptransduction molecules such as Ras tides that are activated during the death (Fernandez-Sarabia and Bischoff 1993), process by cleavage of the prodomain Rafl (H.-G. Wang et al. 1996), and 14at specific aspartate residues to permit 3-3 (Zha et al. 1996). Both Bcl-2 and Bad assembly into active heterotetramers. are known to be phosphorylated under The primary mechanisms that regulate certain circumstances. In the case of caspase activation are currently unclear. Bc1-2,the function of phosphorylation is Multiple caspases maybe active within a controversial. Recent evidence suggests, single cell type. This has led to the sug- however, that Bad phosphorylation pregestion that a hierarchical cascade of vents interaction with Bc1-xL, presumcaspases (and perhaps other proteases) ably allowing for either homodimerizais involved in the apoptotic process tion or binding to Bax, both resulting in (Fraser and Evan 1996, Orth et al 1996). an antiapoptotic phenotype (Zha et al. An alternative view is that the caspases, 1996). In support of this, mutation of the which have differing substrate specificiBad serine phosphorylation sites enties, act in a parallel fashion to execute hanced its apoptotic characteristics. the apoptotic response collectively (La- Other have shown that Rafl is sequeszebnik et al. 1995). One major focus, tered to the mitochondria in a Bcl-2– then, of the cell death field has been to dependent fashion with a consequent identify the signals that are upstream of phosphorylation of Bad (H.-G. Wang et caspases in various apoptotic pathways al. 1996). Just as the interactions beand to determine how these promote tween family members and the postcaspase activation. translational modifications that control A growing family of mammalian genes them are beginning to be understood, has been identified that are homologous there is now emerging ideas of how to ced-9. The first discovered and protothese interactions directly or indirectly type is Bcl-2 (Hengartner and Horvitz lead to control of ICE family members. 1994), which is a membrane-bound pro- (See note added in proof.) Genetic studtein that protects a variety of cells from ies in the worm (Ellis et al. 1991), and apoptosis triggered by multiple initiat- recent biochemical studies in several difing causes. Additional members of the ferent contexts (Stefanis et al. 1996, Bcl-2 family, such as Bc1-xL and bfl-1, Chinnaiyan et al. 1996, Frisch et al. also have antiapoptotic activity, whereas 1996, Shimizu et al. 1996) have indiothers (Bax, Bad, Bak, Bclxs, Bik) are cated that Bcl-2 family members funcproapoptotic and generally promote tion upstream of caspase activation in death (D’Sa-Eipper et al. 1996). A salient various apoptotic pathways. In addition, feature of Bc1-2 family members is their recent seminal studies have shown that
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ced-4, a worm death gene unrelated in structure to ced-3 or ced-9, is able to bind simultaneously to Ced-9/Bcl-xL and Ced-3/ICE/FLICE (Chinnaiyan et al. 1997). This interaction could be blocked by overexpression of Bax. This suggests that ced-9/Bcl-2 members may function to promote survival by inhibiting the ability of ced-4 to activate certain caspases and that Bax-type proteins may promote apoptosis by competing with ced-9/Bcl-2 members for association with ced-4, thereby freeing the latter to promote caspase activation (Chinnaiyan et al. 1997). Recently, a mammalian ced-4 homologue has been identified (see note added in proof). In addition to the caspase and Bcl-2 family members that function as conserved elements in a variety of apoptotic pathways, a number of other additional molecules have been described that play roles in mediating cell death. The most important of these is considered here within the context of four diverse models of apoptotic death.
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Figure1. Conceptual scheme of death signaling pathways. Some of the known CaenoYhabdifis elegarzsand mammalian genes are listed. Apoptotic signaling can be viewed schematically as four distinct phases. In the first step, a diverse number of apoptotic stimuli initiate a complex series of events that amplify and propagate the apoptotic signal. Integration of these upstream input signals (step 2) determines whether the death effecter molecules, caspases, are activated (step 3). Once activated, caspases set in motion the processes required for death (step 4). These events may include cleavage of substrates directly by the caspases themselves and events which most likely occur further downstream, such as DNA fragmentation by endonucleases.
of a molecule termed FLICE/MACH/ Mch5 (Boldin et al. 1996, Muzio et al 1996, Fernandes-Alnemri et al. 1996). A very similar molecule, Mch4, has recently been identified (Fernandes-Alnemri et al. 1996). Like Fas, FADD, ● Pathwaysof Death FLICE, and Mch4 contain death domain Fas-A4ediated Death consensus sequences, and these appear Perhaps the best understood pathway of to mediate their mutual interactions. apoptosis is that mediated by a cell sur- FLICE and Mch4 also possess C-termiface receptor designated Fas. The Fas nal domains that share significant seligand (FasL), produced by the immune quence homology with the caspases. Insystem, binds to Fas and activates a deed, both possess caspaselike activity death program (Nagata and Golstein in vitro. FLICE has a CPP32-like activity and Mch4 cleaves and activates the pre1995). FasL binding participates in control of autoreactive T cells and T cell– form of CPP32 (Boldin et al. 1996, mediated killing of Fas-bearing target Muzio et al. 1996, Fernandes-Alnemri cells. Fas is a member of the tumor ne- 1996). Such findings suggest that FLICE crosis factor (TNF) receptor superfam- (and perhaps Mch4) may serve at the top ily, which includes the TNFR1 (Smith et of a caspase cascade; however, it real. 1994) and NGF p75 receptors (Casac- mains unknown how the recruitment of cia-Bonnefil et al. 1996) (which also in- FLICE to the Fas/FADDcomplex leads to duce death upon ligand binding under its activation. In consonance with the concept of certain conditions). All three receptors contain an 80 amino acid region called caspase cascades, following recruitment the death domain that is required for of FLICE to the Fas/FADD complex, activation of the apoptotic pathway and there is, in at least certain cell types, transient induction of ICE-like and that appears to participate in specific interactions with the death domains of CPP32-like caspase activities (Enari et al. 1996). The ICE-like activity appears other proteins (Smith et al. 1994). The Fas/FasL system is one of the to be upstream of the CPP32-likeactivity most direct apoptotic signaling path- in that a peptide inhibitor directed toways yet characterized. Upon binding of ward the former blocks the latter, but FasL, a molecule designated FADD/ not vice versa. The possibility that the MORT1 is recruited to Fas (Boldin et al. ICE-like activity is due to ICE itself is 1995, Chinnaiyan et al. 1995). The Fast supported by the observations that thyFADD complex in turn attracts binding mocytes from ICE-deficient mice are re-
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sistant to FasL-mediated apoptosis, and that an antisense construct directed against ICE inhibits FasL-promoted death (Kuida et al. 1995, Los et al. 1995). The CPP32-like activity may be comprised by several members of this caspase subfamily, including Mch2a, LAP3/Mch3, and CPP32 itself. Here, again, there is some evidence for a cascade in which Mch2 may act as a processor of the others, which may go on to act in a parallel or sequential fashion (Orth et al. 1996). Feedback loops that lead to amplification of the cascade may also be part of the picture. The short signaling pathway from ligand binding to caspase activation may explain why death induced by FasL is a rapid process requiring no protein synthesis (Nagata and Golstein 1995). There are, however, several additional molecules that may modulate or contribute to the apoptotic pathway in this paradigm. Bc1-xL and Bc1-2 (Jaattela et al. 1995, Itoh et al. 1993), in conjunction with its interacting protein Bag-1 (Takayama et al. 1995), have been reported to confer protection from FasL in certain systems and functions at some level prior to activation of CPP-32 (Armstrong et al. 1996). Further work has identified a protein phosphatase that interacts with the death domain of Fas and that in some cells confers protection from FasL by a mechanism that has yet to be defined (Sato et al. 1995). In addition, the death domain containing protein RIP binds to
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Figure 2. Schematic pathways of apoptosis in four different systems. (A) Apoptotic signaling pathways from Fas. Fas is normally active as a trimer, For convenience, however, the receptor is depicted in the figure as a monomer. (B) Pathways of granzyme B–mediated cytotoxicity. (C) Potential pathways controlling DNA damage mediated apoptosis. Asterisk indicates the two pathways leading to apoptosis, p53- and ceramide-mediated, are described. These pathways are depicted separately because the relationships between the two pathways are unknown; however, these two pathways most likely interact at some unknown level. (D) Model for the apoptotic signaling pathways regulating neuronal cell death due to trophic factor deprivation.
Fas and induces death when overexpressed (Stanger et al. 1995). The functional significance of RIP in the context of FLICE activation is at present unclear. Generation of ceramide from sphingomyelin has also been suggested as a component of the Fas-mediated apoptotic pathway [reviewed previously by Perry et al. 1996]. Experiments from several different laboratories have positioned ceramide both upstream (Smyth et al. 1996) and downstream of caspases (Gamen et al. 1996) and hence, if it does play a required role in Fas-evokecfdeath, it may act as a link between the initial and downstream elements of the caspase cascade. Finally, the JNK/p38 stress ki-
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nases have been reported to be activated upon FasL binding (Wilson et al 1996, Juo et al. 1997). Recent evidence, however, suggests that JNK is not an obligate component of the Fas death pathway (Lenczowski et al. 1997). Cytotoxic T Lymphocyte-Mediated
Death
Cytotoxic T lymphocytes (CTLS) induce apoptosis through the action of the transmembrane pore-forming molecule perforin and exocytotic release of the contents of cytoplasmic granules into target cells (Shiver et al. 1992). The serine protease granzyme B has been identified as a critical component for the toxicity of CTL cytoplasmic granules in
that CTLSfrom granzyme B–’– mice are defective in induction of apoptosis (Heusel et al. 1994). Granzyme B shares with caspases the ability to cleave substrates at aspartate residues and, at least in vitro, is able to process and directly activate a variety of caspases, including CPP32, LAP3/Mch3, Ich-3, LAP6/Mch6, FLICE and Mch4 (Darmon et al. 1995, Femandes-Alnemri et al. 1996, Wang et al. 1996, Duan et al. 1996, Muzio et al. 1996). Although granzyme B does not directly cleave ICE, certain cell types from ICE-’mice are resistant to granzyme B–induced apoptosis (Shi et al. 1996). This indicates both that CTLinduced death requires the action of ICE and that at least one other protease must lie between granzyme B and ICE. Both CPP32 and Ich-3 have been suggested as potential candidates, but their putative action on ICE should be indirect, given that neither of these two caspases is able to cleave ICE in vitro (S. Wang et al. 1996, Xue et al. 1996). The picture that thus emerges is one in which CTLS kill target cells rapidly by bypassing more complex upstream signaling events and directly inducing the effecter phase of apoptosis. It is interesting that in this paradigm, the relative ordering of ICE and CPP32 appears to be reversed compared with that of Fas-mediated cell death. In this paradigm, Bcl-2 has no protective effect (Vaux et al. 1992), which suggests that the antiapoptotic activities of this molecule in other death paradigms may not be due to direct caspase inhibition. It is also interesting to note that CTL-mediated apoptosis can be abrogated at a point downstream of caspases in the apoptotic pathway. It was recently reported (Shi et al. 1994 and 1996) that activation of the p34cdc2 kinase is required for CTL-evoked death and does not occur in ICE–’– B lymphoblasts. This thus places p34cdc2 activation at a point distal of at least ICE. DIVA Damage and p53 Response in Proliferating Cells Exposure to radiation as well as many chemotherapeutic agents induces apoptotic death by a mechanism that appears to be initiated by DNA damage (genotoxic stress). In such instances, the tumor suppressor p53 seems to play a pivotal role in the apoptotic pathway [see Levine (1997), for review]. p53 appears to serve as part of a “checkpoint” at
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which cells with damaged DNA are shunted toward apoptosis, thereby preventing the accumulation of potentially oncogenic or function-disabling mutations. One manner in which p53 may act
as a checkpoint switch is by serving as a transcriptional factor [for example, Sabbatini et al. (1995)], although the transactivation potential of p53 is not required in all cases of p53-mediated death [for example, Caelles et al. (1994)]. Because p53 acts as a “life or death” switch in the case of genotoxic stress, it can be placed upstream of conserved death effecters. The role of caspases in death due to DNA damage has been confirmed by a number of reports using various caspase inhibitors [for example, Nicholson et al. (1995)]. Very little is known, however, about the mechanisms by which DNA damage and p53 regulate caspase activation. Apoptotic death in response to genotoxic stress is alleviated by overexpression of Bcl-2 and BcIxL (Zhang et al. 1996, Dole et al. 1995). Although no direct evidence places Bc1-2 upstream of caspases, lessons from other systems suggest that this may be the case. In addition to p53 action and caspase activation,genotoxic damage is associated with provocation of a number of additional signalingresponses. This has raised some confusion in that not all such responses are necessarilyrelated to the apoptotic pathway. For instance, not all upstream signaling events may be targeted toward demise of the cell, but may rather function to initiate repair (perhaps as some form of a mammalian SOS response). Nevertheless,it can be expected that additional death signaling components will function both upstream and downstream of p53 and perhaps in parallel with it. Some of the candidate upstream regulators of p53 in genotoxic stress include (a) the ATM gene, which was discovered to be defective in patients with ataxia telangiectasia (Barlow et al. 1996); (b) poly-ADP-ribosyltransferease (PARP) (de Murcia et al. 1994); and (c) DNA-proteinkinase,which is activatedby DNA-strand breaks (Anderson 1993). Ml three proteins have been shown to stabilize or activate p53 [see Ashkenas and Werb (1996) for review].Anotherresponse to DNA damage that appears to be important for death is the generation of intracellularceramide via activationof sphingomyelinase.This in turn appears to activate
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the Jun kinase (JNK) cascade (Verheij et
al. 1996). Evidence has been presented that interferencewith eitherceramide formation or activation of JNK renders cells resistantto genotoxic stresses (Bose et al 1995,Santanaet al. 1996).At present, it is unknown whether or how the ceramideJNK pathway interacts with p53 or whether ceramide and p53 function in parallelpathways,each of which might be required, but not sufficient, for apoptosis. Neuronal Apoptosis Due to T~ophic Factor Deprivation Trophic factors regulate the survival and death of a variety of both proliferating and postmitotic cell types. The example of neurons is considered here, partly because it has been the subject of intensive studies and because these may serve as a model for other postmitotic cell types such as those in the myocardium. Neurons are often dependent on trophic factors [exemplified by nerve growth factor (NGF)] for their support, and withdrawal of such support during development or in the adult can lead to apoptotic death (Oppenheim 1991). As in other systems, caspases play a critical role in the apoptotic death pathway of trophic factor–deprived neurons (Gagliardini et al. 1994, Park et al. 1996a, Stefanis et al. 1996, Deshmukh et al. 1996). Overexpression of caspases induces neuronal death, and agents that specifically inhibit or suppress caspases block death of neurons evoked by withdrawal of trophic support. For instance, depletion of the caspase Nedd2 in sympathetic neurons by application of an appropriate antisense oligonucleotide inhibits apoptosis brought about by withdrawal of NGF (Troy et al. 1997). It has been suggested, but not yet established, that loss of trophic support activates a caspase cascade. The antiapoptotic Bc1-2and BcIxL proteins are also important modulators of neuronal death and their overexpression protects neuronalcells from trophic factor deprivation (Batistatou et al. 1993, Frankowski et al. 1995) At least in some neurons, expression of the proapoptotic protein Bax is required for death evoked by loss of trophic support, as indicated by their excess numbers in B=–’– mice and their capacity to survive in vitro in absence of trophic support (Deckworth et al. 1996).Bcl-2 appearsto function upstream of caspases, given that overexpression of
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Bc1-2 suppresses caspase activation after withdrawal of trophic support (Stefanis et
al. 1996). Studies of apoptosis induced by trophic factor deprivation in replicating cells have implicated, in addition to the basal cell death components, inappropriate activation of molecules involved in regulation of cell cycle (Meikrantz and Schlegel 1996). Cell cycle–related molecules also appear to play a role in the death of trophic factor–deprived neuronal cells. For example, pharmacological G1/S phase blockers (Farinelli and Greene 1996) or pharmacologic or biological inhibitors of cyclin dependent kinases (Park et al. 1996b, D.S. Park et al. 1997) suppress apoptosis of NGF-deprived neurons. Such agents also appear to block caspase activation (Stefanis et al. 1996). In contrast, inhibitors of caspases do not affect cell cycle (Stefanis et al. 1997). These observations place caspase activation downstream of cell cycle components. An additional point to note is that cell cycle control elements, which act in death induced by both trophic factor deprivation (Park et al. 1996a) and CTL (Shi et al. 1996), are positioned differently in the two apoptotic pathways. An additional response induced by NGF deprivation and one that seems to be required for death is activation of JNK (Xia et al. 1995). Interestingly, the transactivational activity of c-Jun, a known target of JNK, is also required for neuronal death induced by NGF deprivation (Ham et al. 1995, Estus et al 1994). It is tempting to speculate that activation of the JNK cascade signals to c-Jun, which then regulates genes required for neuronal death. Recent studies have begun to order other cell death components relative to JNK activation and c-Jun regulation. Bcl-2 blocks and therefore appears to be upstream of JNK activation (Park et al. 1996a). In contrast, inhibition of caspases does not affect either regulation of c-Jun or JNK activation (Park et al. 1996a, Deshmukh et al. 1996), indicating that c-Jun and JNK are either upstream of caspases or on a parallel apoptotic pathway.
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ConcludingRemarks
The purpose of this article has been to consider various regulators of cell death and to review the diverse means by which
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they may be ordered relative to one another to construct apoptotic pathways. It is clear from just the four specific paradigms reviewed here that despite the presence of conserved elements, the makeup and ordering of death pathways is strongly dependent on cell type and apoptotic stimulus. Thus, in mammalian cells, nature has both elaborated and rearranged the basic cell death pathway as it exists in C. elegans. It is also evident that aside from the continuing need to identify and order additional elements of apoptotic pathways, a vital objective of future work will be to understand the molecular mechanisms by which upstream and downstream components of apoptotic pathways are causally linked to one another. These will be especially important goals in the context of therapeutic applications in which it will be desirable to either promote or prevent apoptotic death in specific cell types without disrupting important homeostatic functions. The diverse nature of cell death mechanisms suggeststhat it should be possible to fulfill such aims by selectivelytargetingspecific components of individual apoptotic pathways.
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Note Added in Proof
Recent studies that appeared after completion of this brief review have offered new insights into the way bcl-2 family members may regulate caspase activation. In experiments performed both in vitro and in cells, bcl-2 blocks the mitochondrial release of cytochrome c that occurs following apoptotic stimuli. Cytochrome c in turn binds to Apaf-1, a molecule that shares sequence homology with ced-3 and ced-4, and thus is the
first mammalian homologue of ced-4 to be identified (Zou et al. 1997). Both Apaf-1 and cytochrome c are required for the in vitro activation of caspase-3. bcl-2 and its homologies are involved in pore formation in vitro, and thus a model is emerging in which bcl-2, through modulation of mitochondrial pores, retains cytochrome c in the mitochondria, thus preventing its interaction with Apaf- 1 and subsequent caspase activation and apoptosis. Conceivably, bcl-2 could also bind directly to Apaf-1, the way ced-9 binds to ced-4, and thus inhibit the caspase-Apaf-l interaction (reviewed by Reed, 1997 and Vaux, 1997). TCM
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Acknowledgment
This work was supported in part by grants from the National Institutes of Health (NS33689), March of Dimes, Blanchette Rockefeller Foundation, ALS Foundation, and the Aaron Diamond Foundation (L.A.G.). L.S. is supported by a grant from the Lucille P. Markey Trust. D.S.P. is an Aaron Diamond Foundation Fellow.
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NADH/NADPH Oxidase and Vascular Function Kathy K. Griendling and Masuko Ushio-Fukai
The vascular NADH/NADPHoxidase has been shown to be the major source of superoxide in the vessel wall. Recent work has provided insight into its structure and activity in vascular cells. This enzyme is involved in both vascular smooth muscle hypertrophy and in some forms of impaired endothelium-dependent relaxation. Because oxidative stress in general participates in the pathogenesis of hypertension and atherosclerosis, the enzymes that produce reactive oxygen species may be important determinants of the course of vascular disease. (Trends Cardiovasc Med 1997;7:301-307). 01997, Elsevier Science Inc.
Oxidative stress has been the subject of a vast amount of research over the years, initially because of its propensity to induce DNA damage. It has now become apparent, however, that reactive oxygen species may act as intercellular and intracellular second messengers in both normal and pathophysiologic responsiveness. In the vascular system, the sources of oxidative stress have not been clearly established. Recent work has shown that the traditional sources of oxidative molecules (xanthine oxidase, mitochondrial oxidases, arachidonic acid) play a relatively minor role in the production of reactive oxygen species in the vessel wall and that a nonmitochondrial, NADH/NADPH membrane-associated oxidase is the major source of superoxide (.02–) in vascular cells (Griendling et al. 1994, Mohazzab-H and Wolin 1994b, Rajagopalan et al. 1996a). .
The NADI-UNADPHOxidase as a Source of OxidantStress in VascularCells
Vascular cells are exposed to both paracrine and autocrine regulation by reac-
Kathy K. Griendling and Masuko Ushio-Fukai are at the Emory University School of Medicine, Division of Cardiology, Atlanta, GA 30322, USA.
tive oxygen species. Endothelial and smooth muscle cells produce .02– and H202 (Friedl et al. 1989, Pagano et al. 1993, Panus et al. 1993, Mohazzab-H and Wolin 1994b, Rajagopalan et al. 1996a), and are exposed to free radicals released by circulating blood cells and inflammatory cells. Cell associated-reactive oxygen species appear to derive mainly from an NADH/NADPH oxidase (Mohazzab-H et al. 1994, Rajagopalan et
al.
1996a).
Acetylcholine-insensitive
.02- generation from rabbit aorta was noted by Pagano et al. (1993). These investigators concluded that vascular .02– is derived mainly from a nonendothelial source and is modulated by endogenous superoxide dismutase. Subsequently, a .02--generating NADH oxidase was observed in pulmonary arteries by Mohazzab-H and Wolin (1994a). This microsomal NADH-dependent production of .02– is decreased by hypoxia and apparently utilizes a cytochrome b~~~electron transport system (see later here). Simultaneously, our laboratory demonstrated (Griendling et al. 1994) that angiotensin II increases NADH- and NADPH-dependent .02- production in cultured vascular smooth muscle cells (VSMC). This activity is membrane associated and is inhibited by diphenylene iodinium (DPI), an inhibitor of flavincontaining enzymes (Figure la). Pagano et al. (1995) reported an NADPH-depen-
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Orderingthe Multiple Pathways of Apoptosis David S. Park, Leonidas Stefanis, and Lloyd A. Greene
Apoptosis plays an important role in development, homeostasis, and disease. Cun-ent wo~k has suggested that apoptosis can be evoked by multiple stimuli that, in turn, initiate distinct death pathways. Recently, exciting advances have been made in the understanding of biochemical pathways that regulate apoptotic processes. These pathways contain both evolutionan”lyconserved elements and components that are dependent on the death stimulus and cell context. Accordingly, this review focuses on the compositions and relative ordering of the apoptotic pathways in four different death paradigms: activation of receptors of the Fas ligand, destruction by cytotoxic T lymphocytes, exposure to DNA damagingagents, and loss of support by neurotr-ophic factom. These examples illustrate the conservation and divergence in the ways that death pathways are composed and o~dered. (Trends Cardiovasc Ivled 1997;7:294–301). 01997, Elsevier Science Inc.
Apoptotic cell death plays major roles in a diversity of developmental processes as well as in numerous diseases. In general, two modes of cellular death have been described [see Wyllie et al. (1980) for review]. In contrast to necrotic cell death, which is usuallyaccompanied by swelling and disruption of cellularmembranes and
David S. Park and Lloyd A. Greene are at the Department of Pathology and Center for Neurobiology and Behavior,and Leonidas Stefanis is at the Departmentsof Pathology and Neurology and the Center for Neurobiology and Behavior, Columbia UniversityCollege of Physicians and Surgeons, New York, ~, 10032, USA.
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inflammation of adjacent tissue, apoptosis seems to be a regulated process of death typified by cell shrinkage and blebbing, clumping of chromatin, nuclear pyknosis and disruption, and formation of cytosol containing apoptotic bodies. In the cardiovascular system, it appears that apoptosis plays an important part in the development of the sinus and atrioventricular nodes and of the His bundle (James 1994). In addition, regulation of the number of cells in the intimal layer of the vascularwall occurs by an appropriate balance between cell growth and death, and decreased apoptosis may contribute to vascularlesion formation (Gibbons and
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Dzau 1996). Finally,apoptosis may play a critical role in ischemia and reperfusion injury of the heart (Thompson 1995). Initial descriptions of apoptotic cell death were defined by characteristic morphological changes [see Wyllie et al. (1980) for review]. Recently, however, a rapidly growing body of work has developed a more biochemical view of apoptosis, and exciting progress has been made in characterization of the molecules that regulate this process. Analysis of cell death in various systems has revealed several key points. First, there are certain conserved classes of molecules that regulate death in nearly all apoptotic paradigms. Second, apoptosis occurs by complex, multistep pathways of causally related events that culminate in cell destruction. Third, there are multiple means to trigger death, and these in turn initiate distinct apoptotic pathways. These pathways are formed by combination of what are seemingly conserved cell death components with diverse elements that depend on both cellular context and on the initiating cause of death. The purpose of this review is to describe what is currently known about the nature and relative order of the events that comprise the apoptotic pathways. First, the conserved elements of these pathways are briefly reviewed. Then, because there is no single route to death, the events that mediate apoptosis evoked by four distinct initiating causes are considered. This will highlight the degree to which there is both conservation and diversity in the composition and arrangement of the mechanistic pathways that mediate cell death.
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ConservedElements in Death and Survival
Much of our understanding of mammalian apoptosis began with genetic studies in Caenorhabditis elegans (Ellis et al. 1991). These revealed the death effecter genes ced-3 and ced-4, and a third gene, ced-9, which blocks death. Identification of ced-3 and ced-9 homologies in higher organisms has led to the belief that these gene families represent conserved and basal elements of the death pathway. The mammalian homologies of ced-3 constitute a family of aspartic acid– directed cysteine proteases now designated as caspases. The prototypic mam-
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malian caspase is the interleukin-con- capacity to form homodimers and hetverting enzyme (ICE) that was initially erodimers, and this has been suggested identified as the protease that converts to play a key role in their proapoptotic pro-ILl-@ into the active form of this and antiapoptotic activities (Gajewski cytokine (Yuan et al. 1993). Various clon- and Thompson 1996). Two major moding strategies have currently uncovered els of Bcl-2-family member function ex11 mammalian caspases, which can be ist. In the first, it is thought that the Bax divided into four classes by molecular homodimers are the trigger for death. homology (ICE-like, Ced-3/CPP32-like, Accordingly, the presence of the antiapoNedd2/Ich-l-like [Fraser and Evan 1996] ptotic members Bcl-2 and Bc1-xL would and those containing FADD-like death bind to Bax, preventing homodimerizadomains IBoldin et al. 1996, Muzio et al tion. In the second model, it is Bcl-2 and 1996, Fernandes-Alnemri et al. 19961), Bc1-xL homodimers that actively block which are discussed later here. Studies death [see Gajewski and Thompson by a variety of groups have established (1996) for review]. In support of the latthat expression of caspases in multiple ter model, some mutations in Bc1-xL cell types produces death and that their that affect binding to Bax do not abroinhibition blocks apoptosis induced by gate the antiapoptotic function of Bclvarious stimuli. The caspases are thus xL/Bcl-2 (Cheng et al. 1996). It is unclear considered to be the primary effecters of how Bcl-2 members interact with the apoptotic cell death (Fraser and Evan rest of the apoptotic pathway. Recently, 1996). some evidence suggests that Bcl-2 family Caspase family members are initially members interact with classical signal translated as inactive precursor polypeptransduction molecules such as Ras tides that are activated during the death (Fernandez-Sarabia and Bischoff 1993), process by cleavage of the prodomain Rafl (H.-G. Wang et al. 1996), and 14at specific aspartate residues to permit 3-3 (Zha et al. 1996). Both Bcl-2 and Bad assembly into active heterotetramers. are known to be phosphorylated under The primary mechanisms that regulate certain circumstances. In the case of caspase activation are currently unclear. Bc1-2,the function of phosphorylation is Multiple caspases maybe active within a controversial. Recent evidence suggests, single cell type. This has led to the sug- however, that Bad phosphorylation pregestion that a hierarchical cascade of vents interaction with Bc1-xL, presumcaspases (and perhaps other proteases) ably allowing for either homodimerizais involved in the apoptotic process tion or binding to Bax, both resulting in (Fraser and Evan 1996, Orth et al 1996). an antiapoptotic phenotype (Zha et al. An alternative view is that the caspases, 1996). In support of this, mutation of the which have differing substrate specificiBad serine phosphorylation sites enties, act in a parallel fashion to execute hanced its apoptotic characteristics. the apoptotic response collectively (La- Other have shown that Rafl is sequeszebnik et al. 1995). One major focus, tered to the mitochondria in a Bcl-2– then, of the cell death field has been to dependent fashion with a consequent identify the signals that are upstream of phosphorylation of Bad (H.-G. Wang et caspases in various apoptotic pathways al. 1996). Just as the interactions beand to determine how these promote tween family members and the postcaspase activation. translational modifications that control A growing family of mammalian genes them are beginning to be understood, has been identified that are homologous there is now emerging ideas of how to ced-9. The first discovered and protothese interactions directly or indirectly type is Bcl-2 (Hengartner and Horvitz lead to control of ICE family members. 1994), which is a membrane-bound pro- (See note added in proof.) Genetic studtein that protects a variety of cells from ies in the worm (Ellis et al. 1991), and apoptosis triggered by multiple initiat- recent biochemical studies in several difing causes. Additional members of the ferent contexts (Stefanis et al. 1996, Bcl-2 family, such as Bc1-xL and bfl-1, Chinnaiyan et al. 1996, Frisch et al. also have antiapoptotic activity, whereas 1996, Shimizu et al. 1996) have indiothers (Bax, Bad, Bak, Bclxs, Bik) are cated that Bcl-2 family members funcproapoptotic and generally promote tion upstream of caspase activation in death (D’Sa-Eipper et al. 1996). A salient various apoptotic pathways. In addition, feature of Bc1-2 family members is their recent seminal studies have shown that
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ced-4, a worm death gene unrelated in structure to ced-3 or ced-9, is able to bind simultaneously to Ced-9/Bcl-xL and Ced-3/ICE/FLICE (Chinnaiyan et al. 1997). This interaction could be blocked by overexpression of Bax. This suggests that ced-9/Bcl-2 members may function to promote survival by inhibiting the ability of ced-4 to activate certain caspases and that Bax-type proteins may promote apoptosis by competing with ced-9/Bcl-2 members for association with ced-4, thereby freeing the latter to promote caspase activation (Chinnaiyan et al. 1997). Recently, a mammalian ced-4 homologue has been identified (see note added in proof). In addition to the caspase and Bcl-2 family members that function as conserved elements in a variety of apoptotic pathways, a number of other additional molecules have been described that play roles in mediating cell death. The most important of these is considered here within the context of four diverse models of apoptotic death.
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Figure1. Conceptual scheme of death signaling pathways. Some of the known CaenoYhabdifis elegarzsand mammalian genes are listed. Apoptotic signaling can be viewed schematically as four distinct phases. In the first step, a diverse number of apoptotic stimuli initiate a complex series of events that amplify and propagate the apoptotic signal. Integration of these upstream input signals (step 2) determines whether the death effecter molecules, caspases, are activated (step 3). Once activated, caspases set in motion the processes required for death (step 4). These events may include cleavage of substrates directly by the caspases themselves and events which most likely occur further downstream, such as DNA fragmentation by endonucleases.
of a molecule termed FLICE/MACH/ Mch5 (Boldin et al. 1996, Muzio et al 1996, Fernandes-Alnemri et al. 1996). A very similar molecule, Mch4, has recently been identified (Fernandes-Alnemri et al. 1996). Like Fas, FADD, ● Pathwaysof Death FLICE, and Mch4 contain death domain Fas-A4ediated Death consensus sequences, and these appear Perhaps the best understood pathway of to mediate their mutual interactions. apoptosis is that mediated by a cell sur- FLICE and Mch4 also possess C-termiface receptor designated Fas. The Fas nal domains that share significant seligand (FasL), produced by the immune quence homology with the caspases. Insystem, binds to Fas and activates a deed, both possess caspaselike activity death program (Nagata and Golstein in vitro. FLICE has a CPP32-like activity and Mch4 cleaves and activates the pre1995). FasL binding participates in control of autoreactive T cells and T cell– form of CPP32 (Boldin et al. 1996, mediated killing of Fas-bearing target Muzio et al. 1996, Fernandes-Alnemri cells. Fas is a member of the tumor ne- 1996). Such findings suggest that FLICE crosis factor (TNF) receptor superfam- (and perhaps Mch4) may serve at the top ily, which includes the TNFR1 (Smith et of a caspase cascade; however, it real. 1994) and NGF p75 receptors (Casac- mains unknown how the recruitment of cia-Bonnefil et al. 1996) (which also in- FLICE to the Fas/FADDcomplex leads to duce death upon ligand binding under its activation. In consonance with the concept of certain conditions). All three receptors contain an 80 amino acid region called caspase cascades, following recruitment the death domain that is required for of FLICE to the Fas/FADD complex, activation of the apoptotic pathway and there is, in at least certain cell types, transient induction of ICE-like and that appears to participate in specific interactions with the death domains of CPP32-like caspase activities (Enari et al. 1996). The ICE-like activity appears other proteins (Smith et al. 1994). The Fas/FasL system is one of the to be upstream of the CPP32-likeactivity most direct apoptotic signaling path- in that a peptide inhibitor directed toways yet characterized. Upon binding of ward the former blocks the latter, but FasL, a molecule designated FADD/ not vice versa. The possibility that the MORT1 is recruited to Fas (Boldin et al. ICE-like activity is due to ICE itself is 1995, Chinnaiyan et al. 1995). The Fast supported by the observations that thyFADD complex in turn attracts binding mocytes from ICE-deficient mice are re-
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sistant to FasL-mediated apoptosis, and that an antisense construct directed against ICE inhibits FasL-promoted death (Kuida et al. 1995, Los et al. 1995). The CPP32-like activity may be comprised by several members of this caspase subfamily, including Mch2a, LAP3/Mch3, and CPP32 itself. Here, again, there is some evidence for a cascade in which Mch2 may act as a processor of the others, which may go on to act in a parallel or sequential fashion (Orth et al. 1996). Feedback loops that lead to amplification of the cascade may also be part of the picture. The short signaling pathway from ligand binding to caspase activation may explain why death induced by FasL is a rapid process requiring no protein synthesis (Nagata and Golstein 1995). There are, however, several additional molecules that may modulate or contribute to the apoptotic pathway in this paradigm. Bc1-xL and Bc1-2 (Jaattela et al. 1995, Itoh et al. 1993), in conjunction with its interacting protein Bag-1 (Takayama et al. 1995), have been reported to confer protection from FasL in certain systems and functions at some level prior to activation of CPP-32 (Armstrong et al. 1996). Further work has identified a protein phosphatase that interacts with the death domain of Fas and that in some cells confers protection from FasL by a mechanism that has yet to be defined (Sato et al. 1995). In addition, the death domain containing protein RIP binds to
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Figure 2. Schematic pathways of apoptosis in four different systems. (A) Apoptotic signaling pathways from Fas. Fas is normally active as a trimer, For convenience, however, the receptor is depicted in the figure as a monomer. (B) Pathways of granzyme B–mediated cytotoxicity. (C) Potential pathways controlling DNA damage mediated apoptosis. Asterisk indicates the two pathways leading to apoptosis, p53- and ceramide-mediated, are described. These pathways are depicted separately because the relationships between the two pathways are unknown; however, these two pathways most likely interact at some unknown level. (D) Model for the apoptotic signaling pathways regulating neuronal cell death due to trophic factor deprivation.
Fas and induces death when overexpressed (Stanger et al. 1995). The functional significance of RIP in the context of FLICE activation is at present unclear. Generation of ceramide from sphingomyelin has also been suggested as a component of the Fas-mediated apoptotic pathway [reviewed previously by Perry et al. 1996]. Experiments from several different laboratories have positioned ceramide both upstream (Smyth et al. 1996) and downstream of caspases (Gamen et al. 1996) and hence, if it does play a required role in Fas-evokecfdeath, it may act as a link between the initial and downstream elements of the caspase cascade. Finally, the JNK/p38 stress ki-
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nases have been reported to be activated upon FasL binding (Wilson et al 1996, Juo et al. 1997). Recent evidence, however, suggests that JNK is not an obligate component of the Fas death pathway (Lenczowski et al. 1997). Cytotoxic T Lymphocyte-Mediated
Death
Cytotoxic T lymphocytes (CTLS) induce apoptosis through the action of the transmembrane pore-forming molecule perforin and exocytotic release of the contents of cytoplasmic granules into target cells (Shiver et al. 1992). The serine protease granzyme B has been identified as a critical component for the toxicity of CTL cytoplasmic granules in
that CTLSfrom granzyme B–’– mice are defective in induction of apoptosis (Heusel et al. 1994). Granzyme B shares with caspases the ability to cleave substrates at aspartate residues and, at least in vitro, is able to process and directly activate a variety of caspases, including CPP32, LAP3/Mch3, Ich-3, LAP6/Mch6, FLICE and Mch4 (Darmon et al. 1995, Femandes-Alnemri et al. 1996, Wang et al. 1996, Duan et al. 1996, Muzio et al. 1996). Although granzyme B does not directly cleave ICE, certain cell types from ICE-’mice are resistant to granzyme B–induced apoptosis (Shi et al. 1996). This indicates both that CTLinduced death requires the action of ICE and that at least one other protease must lie between granzyme B and ICE. Both CPP32 and Ich-3 have been suggested as potential candidates, but their putative action on ICE should be indirect, given that neither of these two caspases is able to cleave ICE in vitro (S. Wang et al. 1996, Xue et al. 1996). The picture that thus emerges is one in which CTLS kill target cells rapidly by bypassing more complex upstream signaling events and directly inducing the effecter phase of apoptosis. It is interesting that in this paradigm, the relative ordering of ICE and CPP32 appears to be reversed compared with that of Fas-mediated cell death. In this paradigm, Bcl-2 has no protective effect (Vaux et al. 1992), which suggests that the antiapoptotic activities of this molecule in other death paradigms may not be due to direct caspase inhibition. It is also interesting to note that CTL-mediated apoptosis can be abrogated at a point downstream of caspases in the apoptotic pathway. It was recently reported (Shi et al. 1994 and 1996) that activation of the p34cdc2 kinase is required for CTL-evoked death and does not occur in ICE–’– B lymphoblasts. This thus places p34cdc2 activation at a point distal of at least ICE. DIVA Damage and p53 Response in Proliferating Cells Exposure to radiation as well as many chemotherapeutic agents induces apoptotic death by a mechanism that appears to be initiated by DNA damage (genotoxic stress). In such instances, the tumor suppressor p53 seems to play a pivotal role in the apoptotic pathway [see Levine (1997), for review]. p53 appears to serve as part of a “checkpoint” at
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which cells with damaged DNA are shunted toward apoptosis, thereby preventing the accumulation of potentially oncogenic or function-disabling mutations. One manner in which p53 may act
as a checkpoint switch is by serving as a transcriptional factor [for example, Sabbatini et al. (1995)], although the transactivation potential of p53 is not required in all cases of p53-mediated death [for example, Caelles et al. (1994)]. Because p53 acts as a “life or death” switch in the case of genotoxic stress, it can be placed upstream of conserved death effecters. The role of caspases in death due to DNA damage has been confirmed by a number of reports using various caspase inhibitors [for example, Nicholson et al. (1995)]. Very little is known, however, about the mechanisms by which DNA damage and p53 regulate caspase activation. Apoptotic death in response to genotoxic stress is alleviated by overexpression of Bcl-2 and BcIxL (Zhang et al. 1996, Dole et al. 1995). Although no direct evidence places Bc1-2 upstream of caspases, lessons from other systems suggest that this may be the case. In addition to p53 action and caspase activation,genotoxic damage is associated with provocation of a number of additional signalingresponses. This has raised some confusion in that not all such responses are necessarilyrelated to the apoptotic pathway. For instance, not all upstream signaling events may be targeted toward demise of the cell, but may rather function to initiate repair (perhaps as some form of a mammalian SOS response). Nevertheless,it can be expected that additional death signaling components will function both upstream and downstream of p53 and perhaps in parallel with it. Some of the candidate upstream regulators of p53 in genotoxic stress include (a) the ATM gene, which was discovered to be defective in patients with ataxia telangiectasia (Barlow et al. 1996); (b) poly-ADP-ribosyltransferease (PARP) (de Murcia et al. 1994); and (c) DNA-proteinkinase,which is activatedby DNA-strand breaks (Anderson 1993). Ml three proteins have been shown to stabilize or activate p53 [see Ashkenas and Werb (1996) for review].Anotherresponse to DNA damage that appears to be important for death is the generation of intracellularceramide via activationof sphingomyelinase.This in turn appears to activate
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the Jun kinase (JNK) cascade (Verheij et
al. 1996). Evidence has been presented that interferencewith eitherceramide formation or activation of JNK renders cells resistantto genotoxic stresses (Bose et al 1995,Santanaet al. 1996).At present, it is unknown whether or how the ceramideJNK pathway interacts with p53 or whether ceramide and p53 function in parallelpathways,each of which might be required, but not sufficient, for apoptosis. Neuronal Apoptosis Due to T~ophic Factor Deprivation Trophic factors regulate the survival and death of a variety of both proliferating and postmitotic cell types. The example of neurons is considered here, partly because it has been the subject of intensive studies and because these may serve as a model for other postmitotic cell types such as those in the myocardium. Neurons are often dependent on trophic factors [exemplified by nerve growth factor (NGF)] for their support, and withdrawal of such support during development or in the adult can lead to apoptotic death (Oppenheim 1991). As in other systems, caspases play a critical role in the apoptotic death pathway of trophic factor–deprived neurons (Gagliardini et al. 1994, Park et al. 1996a, Stefanis et al. 1996, Deshmukh et al. 1996). Overexpression of caspases induces neuronal death, and agents that specifically inhibit or suppress caspases block death of neurons evoked by withdrawal of trophic support. For instance, depletion of the caspase Nedd2 in sympathetic neurons by application of an appropriate antisense oligonucleotide inhibits apoptosis brought about by withdrawal of NGF (Troy et al. 1997). It has been suggested, but not yet established, that loss of trophic support activates a caspase cascade. The antiapoptotic Bc1-2and BcIxL proteins are also important modulators of neuronal death and their overexpression protects neuronalcells from trophic factor deprivation (Batistatou et al. 1993, Frankowski et al. 1995) At least in some neurons, expression of the proapoptotic protein Bax is required for death evoked by loss of trophic support, as indicated by their excess numbers in B=–’– mice and their capacity to survive in vitro in absence of trophic support (Deckworth et al. 1996).Bcl-2 appearsto function upstream of caspases, given that overexpression of
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Bc1-2 suppresses caspase activation after withdrawal of trophic support (Stefanis et
al. 1996). Studies of apoptosis induced by trophic factor deprivation in replicating cells have implicated, in addition to the basal cell death components, inappropriate activation of molecules involved in regulation of cell cycle (Meikrantz and Schlegel 1996). Cell cycle–related molecules also appear to play a role in the death of trophic factor–deprived neuronal cells. For example, pharmacological G1/S phase blockers (Farinelli and Greene 1996) or pharmacologic or biological inhibitors of cyclin dependent kinases (Park et al. 1996b, D.S. Park et al. 1997) suppress apoptosis of NGF-deprived neurons. Such agents also appear to block caspase activation (Stefanis et al. 1996). In contrast, inhibitors of caspases do not affect cell cycle (Stefanis et al. 1997). These observations place caspase activation downstream of cell cycle components. An additional point to note is that cell cycle control elements, which act in death induced by both trophic factor deprivation (Park et al. 1996a) and CTL (Shi et al. 1996), are positioned differently in the two apoptotic pathways. An additional response induced by NGF deprivation and one that seems to be required for death is activation of JNK (Xia et al. 1995). Interestingly, the transactivational activity of c-Jun, a known target of JNK, is also required for neuronal death induced by NGF deprivation (Ham et al. 1995, Estus et al 1994). It is tempting to speculate that activation of the JNK cascade signals to c-Jun, which then regulates genes required for neuronal death. Recent studies have begun to order other cell death components relative to JNK activation and c-Jun regulation. Bcl-2 blocks and therefore appears to be upstream of JNK activation (Park et al. 1996a). In contrast, inhibition of caspases does not affect either regulation of c-Jun or JNK activation (Park et al. 1996a, Deshmukh et al. 1996), indicating that c-Jun and JNK are either upstream of caspases or on a parallel apoptotic pathway.
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ConcludingRemarks
The purpose of this article has been to consider various regulators of cell death and to review the diverse means by which
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they may be ordered relative to one another to construct apoptotic pathways. It is clear from just the four specific paradigms reviewed here that despite the presence of conserved elements, the makeup and ordering of death pathways is strongly dependent on cell type and apoptotic stimulus. Thus, in mammalian cells, nature has both elaborated and rearranged the basic cell death pathway as it exists in C. elegans. It is also evident that aside from the continuing need to identify and order additional elements of apoptotic pathways, a vital objective of future work will be to understand the molecular mechanisms by which upstream and downstream components of apoptotic pathways are causally linked to one another. These will be especially important goals in the context of therapeutic applications in which it will be desirable to either promote or prevent apoptotic death in specific cell types without disrupting important homeostatic functions. The diverse nature of cell death mechanisms suggeststhat it should be possible to fulfill such aims by selectivelytargetingspecific components of individual apoptotic pathways.
●
Note Added in Proof
Recent studies that appeared after completion of this brief review have offered new insights into the way bcl-2 family members may regulate caspase activation. In experiments performed both in vitro and in cells, bcl-2 blocks the mitochondrial release of cytochrome c that occurs following apoptotic stimuli. Cytochrome c in turn binds to Apaf-1, a molecule that shares sequence homology with ced-3 and ced-4, and thus is the
first mammalian homologue of ced-4 to be identified (Zou et al. 1997). Both Apaf-1 and cytochrome c are required for the in vitro activation of caspase-3. bcl-2 and its homologies are involved in pore formation in vitro, and thus a model is emerging in which bcl-2, through modulation of mitochondrial pores, retains cytochrome c in the mitochondria, thus preventing its interaction with Apaf- 1 and subsequent caspase activation and apoptosis. Conceivably, bcl-2 could also bind directly to Apaf-1, the way ced-9 binds to ced-4, and thus inhibit the caspase-Apaf-l interaction (reviewed by Reed, 1997 and Vaux, 1997). TCM
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Acknowledgment
This work was supported in part by grants from the National Institutes of Health (NS33689), March of Dimes, Blanchette Rockefeller Foundation, ALS Foundation, and the Aaron Diamond Foundation (L.A.G.). L.S. is supported by a grant from the Lucille P. Markey Trust. D.S.P. is an Aaron Diamond Foundation Fellow.
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NADH/NADPH Oxidase and Vascular Function Kathy K. Griendling and Masuko Ushio-Fukai
The vascular NADH/NADPHoxidase has been shown to be the major source of superoxide in the vessel wall. Recent work has provided insight into its structure and activity in vascular cells. This enzyme is involved in both vascular smooth muscle hypertrophy and in some forms of impaired endothelium-dependent relaxation. Because oxidative stress in general participates in the pathogenesis of hypertension and atherosclerosis, the enzymes that produce reactive oxygen species may be important determinants of the course of vascular disease. (Trends Cardiovasc Med 1997;7:301-307). 01997, Elsevier Science Inc.
Oxidative stress has been the subject of a vast amount of research over the years, initially because of its propensity to induce DNA damage. It has now become apparent, however, that reactive oxygen species may act as intercellular and intracellular second messengers in both normal and pathophysiologic responsiveness. In the vascular system, the sources of oxidative stress have not been clearly established. Recent work has shown that the traditional sources of oxidative molecules (xanthine oxidase, mitochondrial oxidases, arachidonic acid) play a relatively minor role in the production of reactive oxygen species in the vessel wall and that a nonmitochondrial, NADH/NADPH membrane-associated oxidase is the major source of superoxide (.02–) in vascular cells (Griendling et al. 1994, Mohazzab-H and Wolin 1994b, Rajagopalan et al. 1996a). .
The NADI-UNADPHOxidase as a Source of OxidantStress in VascularCells
Vascular cells are exposed to both paracrine and autocrine regulation by reac-
Kathy K. Griendling and Masuko Ushio-Fukai are at the Emory University School of Medicine, Division of Cardiology, Atlanta, GA 30322, USA.
tive oxygen species. Endothelial and smooth muscle cells produce .02– and H202 (Friedl et al. 1989, Pagano et al. 1993, Panus et al. 1993, Mohazzab-H and Wolin 1994b, Rajagopalan et al. 1996a), and are exposed to free radicals released by circulating blood cells and inflammatory cells. Cell associated-reactive oxygen species appear to derive mainly from an NADH/NADPH oxidase (Mohazzab-H et al. 1994, Rajagopalan et
al.
1996a).
Acetylcholine-insensitive
.02- generation from rabbit aorta was noted by Pagano et al. (1993). These investigators concluded that vascular .02– is derived mainly from a nonendothelial source and is modulated by endogenous superoxide dismutase. Subsequently, a .02--generating NADH oxidase was observed in pulmonary arteries by Mohazzab-H and Wolin (1994a). This microsomal NADH-dependent production of .02– is decreased by hypoxia and apparently utilizes a cytochrome b~~~electron transport system (see later here). Simultaneously, our laboratory demonstrated (Griendling et al. 1994) that angiotensin II increases NADH- and NADPH-dependent .02- production in cultured vascular smooth muscle cells (VSMC). This activity is membrane associated and is inhibited by diphenylene iodinium (DPI), an inhibitor of flavincontaining enzymes (Figure la). Pagano et al. (1995) reported an NADPH-depen-
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