Regulated cell death: signaling and mechanisms.

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Review in Advance first posted online on August 13, 2014. (Changes may still occur before final publication online and in print.)

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Regulated Cell Death: Signaling and Mechanisms Annu. Rev. Cell Dev. Biol. 2014.30. Downloaded from www.annualreviews.org by University of Sydney on 08/28/14. For personal use only.

Avi Ashkenazi1 and Guy Salvesen2 1

Genentech Inc., South San Francisco, California 94080; email: [email protected]

2

Sanford-Burnham Institute, La Jolla, California 92037; email: [email protected]

Annu. Rev. Cell Dev. Biol. 2014. 30:20.1–20.20

Keywords

The Annual Review of Cell and Developmental Biology is online at cellbio.annualreviews.org

apoptosis, necroptosis, death receptor, caspase, tumor necrosis factor, TNF, receptor interacting protein, RIP

This article’s doi: 10.1146/annurev-cellbio-100913-013226 c 2014 by Annual Reviews. Copyright All rights reserved

Abstract Cell turnover is a fundamental feature in metazoans. Cells can die passively, as a consequence of severe damage to their structural integrity, or actively, owing to a more confined biological disruption such as DNA damage. Passive cell death is uncontrolled and often harmful to the organism. In contrast, active cell death is tightly regulated and serves to support the organism’s life. Apoptosis—the primary form of regulated cell death—is relatively well defined. Necroptosis—an alternative, distinct kind of regulated cell death discovered more recently—is less well understood. Apoptosis and necroptosis can be triggered either from within the cell or by extracellular stimuli. Certain signaling components, including several death ligands and receptors, can regulate both processes. Whereas apoptosis is triggered and executed via intracellular proteases called caspases, necroptosis is suppressed by caspase activity. Here we highlight current understanding of the key signaling mechanisms that control regulated cell death.

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Contents

Annu. Rev. Cell Dev. Biol. 2014.30. Downloaded from www.annualreviews.org by University of Sydney on 08/28/14. For personal use only.

REGULATED CELL DEATH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.2 APOPTOSIS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.3 NECROPTOSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.5 DEATH RECEPTORS AS SIGNAL TRANSDUCERS OF REGULATED CELL DEATH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.6 THE DISC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.7 COMPLEX II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.8 THE NECROSOME . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.8 CONTROL OF CASPASE-8 ACTIVITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.9 Role of Dimerization in Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.9 Role of Proteolytic Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20.9 cFLIP: A Key Regulator of Caspase-8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20.10 ENHANCEMENT OF CASPASE-8 ACTIVITY BY STIMULATORY UBIQUITINATION AND INACTIVATION OF CASPASE-8 BY DEGRADATIVE UBIQUITINATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20.11 Nonproteolytic Roles of Caspase-8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20.12 Control of Regulated Death by Caspase-8: Knockout of DISC and Necrosome Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20.12 Lessons from Human Genetic Alterations of Caspase-8 or -10 . . . . . . . . . . . . . . . . . . . .20.13 CLOSING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20.13

REGULATED CELL DEATH Most metazoan cell types turn over at various rates throughout an organism’s lifespan. Cells may die either individually or in groups in an accidental or deliberate manner. If a cell suffers critical structural damage, e.g., owing to an extreme physicochemical insult, it passively disintegrates and dies. The most common form of accidental cell death is necrosis, after the Greek word nekros, meaning “dead body.” Necrosis is characterized by loss of plasma-membrane integrity, organelle and cell swelling, and ultimately, cell lysis (Vanlangenakker et al. 2012, Vercammen et al. 1998). The leakage of noxious intracellular components can damage neighboring cells and invariably triggers an inflammatory response. Alternatively, if a cell sustains damage that alters it in a nondetrimental manner, but poses an eventual threat to the whole organism, it may be eliminated deliberately through an active process of cell death. The most prevalent and extensively studied type of regulated cell death is apoptosis, named after the Greek word for “falling off,” e.g., of dead tree leaves (Danial & Korsmeyer 2004, Kerr et al. 1972). The process of apoptosis is characterized morphologically by cell shrinkage, plasma membrane blebbing, nuclear condensation, and internucleosomal DNA fragmentation. The dead cell is packaged into membrane-bound apoptotic bodies, which are engulfed and removed by neighboring cells or tissue phagocytes. Apoptosis is usually associated with minimal inflammation and serves mainly to facilitate cell elimination and cell-corpse removal. A second prominent mode of regulated cell death that has become a topic of great interest in the past decade is necroptosis. It is thus named because it shares some critical inductive-phase features with apoptosis but possesses many of the morphological characteristics of accidental necrosis. Like necrosis, necroptosis is characterized by swelling of the cell and its organelles, particularly the mitochondria, and by consequent cell implosion. However, unlike necrosis and more akin 20.2

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to apoptosis, the upstream molecular signaling pathways that trigger necroptosis are ordered and strictly controlled (Christofferson & Yuan 2010, Tait & Green 2008, Vanlangenakker et al. 2012). Accidental cell death in metazoans usually harms the organism’s health. In contrast, physiologically regulated cell death plays beneficial biological roles, both in embryonic development and in adult animal life. However, it also can become harmful and lead to pathological consequences if excessive or deficient (Thompson 1995). In the embryo, apoptosis contributes to the sculpting of various tissues and organs; in the adult animal, it supports the operation and maintenance of key physiological functions, including the immune, digestive, endocrine, and nervous systems. Genetic evidence suggests that necroptosis also may be operative during development, although it may have a more prominent postdevelopmental role in combating infection by intracellular pathogens. Regardless, both apoptosis and necroptosis must be kept tightly in check during all phases of metazoan life to prevent excessive cell loss. Indeed, unscheduled apoptosis of certain cell types contributes to diseases such as diabetes, immune deficiency, and neurodegeneration. Similarly, undue necroptosis may worsen tissue injury caused by ischemia reperfusion in the heart, brain, liver, or kidney and may exacerbate certain autoimmune inflammatory conditions, including Crohn’s disease (Vanlangenakker et al. 2012). Conversely, insufficient apoptosis can promote autoimmunity and cancer, whereas too little necroptosis may exacerbate infection by certain viruses (Kaiser et al. 2013).

APOPTOSIS Apoptosis is a process of programmed cell suicide, mediated by a group of specialized proteincleaving enzymes dubbed caspases (Thornberry & Lazebnik 1998). Kerr, Wyllie, and Currie first identified the unique morphology of this form of cell death in the 1970s (Kerr et al. 1972). A few decades later, studies by Horvitz and coworkers in Caenorhabditis elegans nematodes demonstrated that apoptosis is a gene-controlled cell death program (Ellis & Horvitz 1986, Hengartner & Horvitz 1994). The seminal discovery of specific cell death (CED) genes in nematodes enabled the identification of CED homologs in mammals, thus paving the way to biochemical investigation and mechanistic understanding of apoptotic cell death. One of these genes, CED-3, represents the caspase family of proteases, of which several members play crucial roles in apoptosis initiation or execution. Another gene, CED-9, represents the mammalian BCL-2 gene family, of which multiple members play critical roles in apoptotic control. Yet another gene, CED-4, is homologous to mammalian Apaf-1—a component of the apoptosome complex, which drives caspase activation in many contexts. Apoptosis is a latent built-in mechanism inherent to most nucleated metazoan cells. It is activated by default, when a cell is deprived of essential prosurvival factors. Alternatively, apoptosis may be elicited deliberately, either by certain types of severe cell distress or in response to extracellular signals carried through specific death-inducing ligands. The core apoptotic enzymatic machinery consists of caspases—a subset of the cysteinedependent aspartate-specific protease family (Thornberry & Lazebnik 1998). Apoptotic caspases fall into two major categories: initiator caspases (e.g., caspase-8 and -9) and executioner, or effector, caspases (e.g., caspase-3 and -7) (Salvesen & Ashkenazi 2011). Caspase proteins are constituent in the cytoplasm in zymogen form. Apoptotic stimuli induce activation of one or more of the initiator caspases through specific oligomerization platforms. The initiators then trigger a cascade-like proteolytic stimulation of effector caspase zymogens. In turn, the latter products drive the execution phase of the apoptotic death program by cleaving hundreds or even thousands of structurally and functionally critical proteins within the cell (Dix et al. 2008, Mahrus et al. 2008, Pham et al. 2012). Two major cellular signaling pathways in mammals control apoptotic caspase activation (Figure 1): (a) the intrinsic pathway, gated through proteins encoded by the BCL-2 gene family www.annualreviews.org • Regulated Cell Death


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FasL Apo2L/TRAIL

TNFα

Fas DR4, DR5

TNFR1

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Complex I TRADD

RIPK1

CUL3

Bid

IKK

CYLD

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FADD RIPK1


DISC Caspase-8

cFLIP

TRAF2 NEMO cIAP TAK1

FADD

Complex II

Caspase-8

TRADD

FADD cFLIP

Caspase-8

RIPK1

Bax/Bak

NEMO

NF-кB, JNK, ERK

Smac

RIPK1 RIPK3 Necrosome

Apoptosis

Damage sensors

TRAF2

Cytochrome C

Apaf1

Caspase-9

NF-кB, JNK, ERK

XIAP

Apoptosome

Caspase-3, -7 MLKL

Apoptosis Necroptosis

Figure 1 Death-receptor control of regulated cell death. Upon ligation, the death receptors Fas, DR4, or DR5 assemble a primary deathinducing signaling complex (DISC) at the plasma membrane, thereby activating caspase-8 and triggering apoptosis. This is called the cell-extrinsic pathway. In epithelial cells, K63-linked ubiquitination of caspase-8 mediated by CUL3 augments activation, whereas K48-linked ubiquitination mediated by TRAF2 promotes proteasomal inactivation of caspase-8. Caspase-8 directly stimulates the executioner proteases, caspase-3 and -7. It also engages the cell-intrinsic pathway by processing the BH3-only protein BID to amplify the activation of executioner caspases. BID activates BAX and BAK to induce release of cytochrome C and Smac/DIABLO from mitochondria. Cytochrome C associates with Apaf1 to form an apoptosome that drives activation of caspase-9, which then stimulates caspase-3 and -7. Smac further augments the apoptotic signal by preventing X-linked inhibitor of apoptosis protein (XIAP) from blocking caspase-3 and-7. The same receptors may also assemble a secondary cytoplasmic complex (Complex II), which mediates activation of prosurvival and proinflammatory pathways or other cell functions via NF-κB, JNK, and ERK. By contrast, upon ligation by TNFα, TNFR1 assembles a distinct primary complex (Complex I) at the plasma membrane. Complex I mediates NF-κB, JNK, and ERK signaling, supporting cell survival or other non-death functions. However, deubiquitination of RIPK1 by CYLD enables nucleation of a secondary cytoplasmic complex (Complex II), thereby activating caspase-8 homodimers and triggering apoptosis. Caspase-8, likely in its heterodimeric association with cFLIP, suppresses necroptosis by cleaving RIPK1, among other substrates; however, inhibition of caspase-8 permits RIPK1 to recruit RIPK3 and form a third complex called the necrosome. RIPK1 phosphorylates RIPK3, driving recruitment and phosphorylation of MLKL and thereby triggering necroptosis. Phosphorylation of MLKL by RIPK3 causes its oligomerization and translocation to the plasma membrane, where it perturbs membrane integrity, thereby promoting necroptotic cell death. Phosphorylation of RIPK1 is dispensable for Complex I activity but essential for necrosome activity; in some circumstances, e.g., in response to autocrine TNFα signaling driven by excessive DNA damage, RIPK1 phosphorylation is also important for assembly and proapoptotic activity of Complex II. The intrinsic pathway—controlled by the Bcl-2 protein family—is activated by various types of cell distress, including DNA or microtubule damage or metabolic stress. Damage sensors, such as the p53 protein or the lipid kinase AKT, activate specific BH3 proteins by inducing their mRNA transcription or post-translational modification. Activated BH3 proteins stimulate pro-apoptotic BAX and BAK, either directly or by counteracting their inhibition by anti-apoptotic Bcl-2 family members, such as Bcl-2 and Bcl-XL. BAX and BAK form pores in the mitochondrial outer membrane, facilitating release of cytochrome C and Smac/DIABLO to augment stimulation of caspase-3 and -7 and apoptotic cell demise.

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(Adams & Cory 1998), which control the release of specific caspase-activating factors from mitochondria, and (b) the extrinsic pathway, governed by specialized death receptors (Ashkenazi & Dixit 1998), which transmit signals from extracellular death ligands across the plasma membrane to engage the intracellular caspase machinery. These two pathways often act directly and/or indirectly to reinforce one another. The CED-9/BCL-2 gene family encodes two major subclasses of apoptosis-regulating factors, which contain different numbers of BCL-2 homology (BH) motifs: (a) single BH motif (BH3-only) proteins (e.g., BAD, BIK, BMF, BID, PUMA, and NOXA), which typically act as death agonists, and (b) multi-BH motif proteins, which possess three or four BH regions and act respectively as agonists (e.g., BAX, BAK, BOK) or antagonists (e.g., BCL-2, BCL-XL , BCL-w, A1, MCL-1) of apoptotic stimulation (Hardwick & Youle 2009). Interplay between members of these subgroups in each individual cell determines whether apoptosis is switched on or off. For example, DNA damage activates tumor suppressor p53 protein, which upregulates mRNA transcription of PUMA and NOXA. These BH3-only proteins then activate BAX and BAK, either through direct interaction or indirectly by binding to and sequestering BCL-2 or BCL-XL , tipping the balance toward apoptotic activation. BAX and BAK reside in the mitochondrial outer membrane; their homooligomerization upon activation creates membrane pores, releasing mitochondrial cytochrome C into the cytoplasm (Green & Reed 1998). Here, cytochrome C triggers a conformational change in Apaf-1, resulting in (d)ATP exchange and oligomerization to form the caspase-9 activating apoptosome (Martin & Green 1995). In turn, caspase-9 activates effector caspase-3 and -7 to execute the cell’s apoptotic demise. Caspase-3, -7, and -9 are kept in check by X-linked inhibitor of apoptosis protein (XIAP); however, BAX and BAK also release from mitochondria a protein called SMAC/DIABLO, which sequesters XIAP, thereby facilitating effector caspase activation (Deveraux & Reed 1999). Specific death-inducing proteins—often called death ligands—can stimulate the cell-extrinsic apoptosis pathway in susceptible cells through cognate death receptors (Nagata 1997, Peter & Krammer 2003, Wilson et al. 2009). Ligation of death receptors on susceptible target cells leads to activation of the apoptosis-initiating protease caspase-8. In a minority of cell types, caspase-8 can then directly activate caspase-3 and -7 to drive apoptosis execution. More often, however, caspase-8 drives apoptotic execution less directly, by cleaving and thereby stimulating the BH3only protein BID. Truncated BID then engages the cell-intrinsic pathway via BAX and BAK, activating effector caspases through the mitochondria. Caspase-10 is a close structural relative of caspase-8, present in zebrafish and primates but not in rodents (Eimon & Ashkenazi 2010). Yet another close relative of caspase-8 and -10 is cellular FLICE inhibitory protein (cFLIP) (Thome & Tschopp 2001). Present in all three aforementioned animal classes, cFLIP lacks protease function; it is an important regulator of apoptotic as well as nonapoptotic functions of caspase-8).

NECROPTOSIS As implied by its name, tumor necrosis factor α (TNFα) is capable of deliberately eliciting necrotic cell death (Vercammen et al. 1998). In 2000, the laboratories of Tschopp and Nagata described a similar, necrosis-like type of cell death, triggered in response to the death ligand FasL (Holler et al. 2000, Matsumura et al. 2000). Electron microscopic examination revealed a necrosis-like morphology of the dying cells. Surprisingly, this form of cell death was found to be independent of caspase activity. Moreover, it required receptor-interacting protein 1 (RIP1, also called RIPK1)—a serine/threonine kinase previously known to be involved in mediating nuclear factor-κB (NF-κB) activation by TNF receptor 1 (TNFR1). These findings therefore suggested the existence of a specific molecular mechanism for inducing regulated necrosis. Some years later, Degterev and www.annualreviews.org • Regulated Cell Death


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Yuan identified a small molecule compound, called Necrostatin-1 (Nec-1), that could inhibit this unusual type of cell death (Degterev et al. 2005); furthermore, they identified RIPK1 as the molecular target of Nec-1, thus reinforcing its importance in mediating necrotic death signaling (Degterev et al. 2008). The process of regulated necrosis is now referred to as necroptosis or programmed necrosis (Vandenabeele et al. 2010). Necroptosis occurs—and can be detected more readily—when caspase activity is suppressed. It is believed to provide an important innate mechanism of defense against infectious intracellular pathogens, including certain viruses that can block apoptosis activation (Kaiser et al. 2013). The signaling pathway that initiates necroptosis activation is relatively well defined, particularly in connection with TNFα and TNFR1. However, the biochemical events that mediate necroptotic death execution are less well understood. Nevertheless, evidence so far suggests that production of reactive oxygen species by mitochondria, as well as lysosomal leakage and lipid peroxidation, plays a role in necroptotic cell demise (Vanden Berghe et al. 2010). Several proapoptotic death ligands belonging to the TNF superfamily, including TNFα, FasL, and Apo2L/TRAIL, are capable of inducing necroptosis, particularly when caspase activation is prevented. In addition, damage-associated molecular patterns such as bacterial lipopolysaccharide or viral nucleic acid products and their cognate receptors, i.e., certain Toll-like receptors, NOD-like receptors, and viral DNA receptors, also are capable of inducing necroptotic signaling upon ligand engagement (Kaiser et al. 2013, Vanlangenakker et al. 2012), as are interferons (Dillon et al. 2014, Thapa et al. 2013). Several intracellular proteins involved in apoptotic signaling—especially components of the cell-extrinsic pathway—are also involved in controlling necroptotic stimulation, suggesting molecular coordination between the two types of regulated cell ablation.


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DEATH RECEPTORS AS SIGNAL TRANSDUCERS OF REGULATED CELL DEATH The TNFR superfamily consists of more than 20 proteins that typically possess a type-I transmembrane topology with two to four cysteine-rich extracellular domains. Death receptors form a subgroup within this superfamily, distinguished by the presence of an approximately 80-aminoacid-long death domain in the cytoplasmic portion (Ashkenazi & Dixit 1998, Nagata 1997). Six death receptors can regulate apoptosis either directly or indirectly: TNFR1 (TNFRSF1A; cognate ligands: TNFα/TNFSF2 and lymphotoxin α/TNFSF1), Fas (APO1/CD95/TNFRSF6; cognate ligand: FasL/CD95L/TNFSF6), DR3 (APO3/TNFRSF25; cognate ligand: TL1A/ TNFSF15), DR4 (TRAILR1/TNFRSF10A; cognate ligand: Apo2L/TRAIL/TNFSF10), DR5 (TRAILR2/TNFRSF10B; cognate ligand: Apo2L/TRAIL/TNFSF10), and DR6 (TNFRSF21) (Gonzalvez & Ashkenazi 2010, Wilson et al. 2009). The ectodysplasin A receptor EDAR (TNFRSF19) and the p75 neurotrophin receptor (an atypical TNFR superfamily member) also possess discernible death domain motifs. Ligand binding to some of the death receptors is counter-regulated by decoy receptors, e.g., DcR1 (TNFRSF10C) and DcR2 (TNFRSF10D), which bind to Apo2L/TRAIL, and DcR3 (TNFRSF6B), which binds to FasL (Ashkenazi 2002). Upon ligation, the death receptors Fas, DR4, and DR5 typically signal apoptosis in responsive cells. Alternatively, depending on the status of key intracellular signaling components, these receptors may induce necroptosis or even engage pathways that promote cell survival or other cellular functions. These latter pathways, including the IKK/NF-κB, JNK/c-Jun, and p38/MAPK/AP-1 cascades, are engaged more readily when apoptosis stimulation is intercepted (Imamura et al. 2004, Varfolomeev et al. 2005). On the other hand, TNFR1 mainly signals activation of the IKK/NF-κB, JNK/c-Jun, and p38/MAPK pathways. However, it can trigger apoptosis under specific alternative circumstances, namely (a) in the presence of transcriptional or translational 20.6

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inhibitors (Wallach et al. 1999), (b) when NF-κB activation is blocked, (c) upon depletion of cellular inhibitor of apoptosis proteins (cIAPs) (Vucic et al. 2011), or (d ) in the face of unmitigated DNA damage (Biton & Ashkenazi 2011). Furthermore, if caspase activation is blocked in conjunction with one of the above conditions, TNFR1 ligation by TNFα typically induces necroptotic cell death (Vanlangenakker et al. 2012). DR3 mainly regulates noncanonical NF-κB signaling but can activate apoptosis indirectly via TNFα (Ikner & Ashkenazi 2011, Schneider et al. 1999). Cells of the innate and adaptive immune systems are the main producers of death ligands such as FasL, Apo2L/TRAIL, and TNFα (Ehrlich et al. 2003, Halaas et al. 2000, Nagata 1997). These ligands are often expressed as type-II transmembrane proteins at the surface of activated immune cells; alternatively, they can be released as soluble ligands into the extracellular space through the action of specific cell-surface proteases. In turn, these ligands can deliberately kill target cells that express cognate death receptors and are primed for death. Target cells may be primed for apoptotic or necrotic stimulation as part of their normal differentiation program, or owing to damage, infection, or oncogenic transformation. Indeed, the most salient biological function of death receptors and their ligands is their involvement in regulation and activity of the innate and adaptive immune system (Wilson et al. 2009). Although death-receptor activation typically requires ligand binding, recent evidence uncovers ligand-independent intracellular activation of DR5 in response to unmitigated endoplasmic reticulum stress (Lu et al. 2014a).

THE DISC Fas, DR4, and DR5 directly trigger the apoptotic cascade by assembling a death-inducing signaling complex (DISC) (Figure 1), which minimally contains the adaptor protein Fas-associated death domain (FADD) and the apoptosis-initiating protease caspase-8 (Boldin et al. 1996; Kischkel et al. 1995, 2000; Sprick et al. 2000). The corresponding death ligands, FasL and Apo2L/TRAIL, are homotrimeric proteins; indeed, crystallographic studies suggest that the basic ligand-receptor complex consists of one trimeric ligand and three receptor molecules (Hymowitz et al. 1999, Scott et al. 2009). Upon ligation, death receptors multimerize in the cell membrane, leading to a conformational change in the receptors’ intracellular domain (Scott et al. 2009, Wang et al. 2010). This, in turn, enables recruitment of the adaptor protein FADD, which contains both a death domain that interacts with the receptor and a death effector domain (DED) that recruits caspase-8 or -10 and/or cFLIP. Importantly, caspase-8 dimers form the basic catalytic unit of the protease (see Figure 2 below). However, apoptosis commitment often requires further clustering of the initial ligand-receptor complex (Siegel et al. 2000, Wagner et al. 2007). Certain posttranslational modifications of death receptors can facilitate ligand-induced clustering and apoptosis activation. These include palmitoylation of Fas near its transmembrane domain (Feig et al. 2007) and O-linked glycosylation of DR4 and DR5 in their ectodomains (Wagner et al. 2007). Artificial cross-linking of the trimeric ligands, e.g., through an epitope tag (Lawrence et al. 2001, Wilson et al. 2012) or a leucine zipper (Ganten et al. 2006), augments DISC assembly, perhaps by mimicking the transmembrane presentation of the endogenous ligand to enhance receptor clustering. Clathrin-mediated internalization of DR5 upon ligation limits signaling and dampens caspase activation; however, caspase-8-mediated cleavage of the clathrin adaptor AP2α counteracts internalization, reinforcing apoptosis stimulation (Austin et al. 2006, Kohlhaas et al. 2007). In lymphoma cells, an excess of caspase-8 molecules over FADD is recruited to the DISC via sequential DED interactions of caspase-8 (Dickens et al. 2012, Schleich et al. 2012). In epithelial cells, the homotypic adhesion protein E-cadherin interacts with ligated DR4 and DR5; E-cadherin couples these death receptors to the actin cytoskeleton via its interacting www.annualreviews.org • Regulated Cell Death


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partner a-catenin, which augments DISC assembly (Lu et al. 2014b). When epithelial cancer cells undergo epithelial-to-mesenchymal transition, they down-regulate E-cadherin expression, which makes them less susceptible to apoptosis activation via DR4 and DR5. Furthermore, in epithelial cells an E3 ligase complex based on cullin 3 (CUL3) promotes caspase-8 activation at the DISC by mediating ubiquitination on the small catalytic domain of caspase-8 ( Jin et al. 2009). Indeed, CUL3 recruitment and caspase-8 ubiquitination are augmented by the interaction between E-cadherin and DR4/DR5 (Lu et al. 2014b).

COMPLEX II


Like Fas, DR4, and DR5, TNFR1 also relies on FADD and caspase-8 to trigger apoptosis (Figure 1). However, it engages these mediators indirectly, by inducing a secondary cytoplasmic complex (Complex II) downstream of the primary, membrane-proximal signaling assembly (Complex I) (Micheau & Tschopp 2003). Whereas Complex I transmits signals that drive potent NF-κB activation, Complex II can either mediate apoptosis activation via caspase-8 or give rise to a related but distinct complex (Complex IIb, also called necrosome) to elicit necroptosis (Vanlangenakker et al. 2012). Complex I recruits several adaptors and effectors, including RIPK1, TNFR-associated death domain, cIAP1 and -2, and TRAF2. K63-linked ubiquitination of RIPK1 by cIAP1/2 promotes recruitment of transforming growth factor β-activated kinase 1 (TAK1) and NEMO/IKKγ, promoting IKK activation via phosphorylation of TAK1 and ubiquitination of NEMO by linear ubiquitin chain assembly complex (Walczak 2011). After initial stimulation, several components of Complex I, most importantly RIPK1, move into the cytoplasm to nucleate Complex II, thereby recruiting FADD and caspase-8 (Biton & Ashkenazi 2011, Feoktistova et al. 2011, Tenev et al. 2011). This leads to caspase-8 activation, which triggers apoptosis under specific circumstances. Whereas RIPK1 undergoes K63-linked ubiquitination within Complex I, its deubiquitination—mediated by the deubiquitinase cylindromatosis (CYLD)—promotes Complex II assembly (Wang et al. 2008). Moreover, depletion of cIAPs, e.g., by smac mimetics, also augments Complex II formation (even in the absence of TNFα), most likely because it inhibits RIPK1 ubiquitination (Tenev et al. 2011). Furthermore, phosphorylation of RIPK1, which can be induced through feed-forward signaling by TNFα in response to unmitigated DNA damage, promotes Complex II formation and caspase-8 activation (Biton & Ashkenazi 2011). A critical negative regulator of Complex II is cFLIP, which inhibits caspase-8-dependent apoptosis initiation (Feoktistova et al. 2011, Micheau & Tschopp 2003), as well as RIPK1-mediated necroptosis stimulation (see section on The Necrosome, below). In certain contexts (e.g., high intracellular levels of cFLIP) stimulation by Apo2L/TRAIL induces the formation of a cytosolic signaling complex downstream of the DISC (Varfolomeev et al. 2005) (Figure 1). This complex contains RIPK1, TRAF2, and NEMO, among other components, and leads to activation of the NF-κB, JNK, and p38 MAPK cascades and to production of chemokines such as IL-8 and MCP-1. FasL induces a similar outcome, even within cells undergoing apoptosis, mediating an inflammatory response to facilitate clearance of the apoptotic corpses (Cullen et al. 2013).

THE NECROSOME Depending on the intracellular setting, Complex II can transmit a signal that drives necroptosis, rather than apoptosis, by giving rise to a distinct complex called the necrosome (Figure 1). Two key components of the necrosome are RIPK1 (Holler et al. 2000, Wang et al. 2008) and the related Ser/Thr kinase RIPK3, which binds to RIPK1 via a RIP homology interaction motif (He 20.8

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et al. 2009). RIPK1 autophosphorylation is required for recruitment and engagement of RIPK3 (Degterev et al. 2008, Holler et al. 2000, Wang et al. 2008). RIPK1 and RIPK3 form fibrillar structures akin to β-amyloids, which augment necrosome signaling and necroptosis activation (Li et al. 2012). RIPK1 cross-phosphorylates RIPK3, thereby promoting recruitment of yet another critical necroptosis-signaling protein: mixed lineage kinase domain-like (MLKL) (Sun et al. 2012). Recruitment of MLKL to the necrosome leads to its phosphorylation by RIPK3 (Chen et al. 2013). As a pseudokinase, MLKL does not phosphorylate any protein targets. However, phosphomimetic mutations in its pseudoactive site bypass the dependence on RIPK3 for necroptosis induction, whereas deletion of MLKL blocks necroptosis activation, thus establishing an essential role for MLKL in necroptosis signaling (Sun et al. 2012). The deacetylase SIRT2 was initially thought to inhibit necrosome formation (Narayan et al. 2012), but this was later refuted and retracted (Newton et al. 2014a) (Narayan et al. 2014). Caspase-8 acts as a homodimer (O’Donnell et al. 2011) or in heterodimers with cFLIP (Oberst et al. 2011) to suppress necroptosis by cleaving several critical signaling components, namely RIPK1, RIPK3, and CYLD. RIPK3 conversely augments caspase-8 activation in the context of cIAP depletion or TAK1 inhibition (Dondelinger et al. 2013). Consistent with this, TAK1 disruption favors apoptotic versus necroptotic signaling in response to TNFα (Morioka et al. 2014). Moreover, whereas kinase activity of RIPK3 was required for the pronecroptotic function of RIPK3 in mice, a kinase-dead D161N RIPK3 mutant promoted RIPK1- and caspase-8-dependent apoptosis, suggesting that RIPK3 acts as a kinase to drive necroptosis and perhaps as a scaffold to promote apoptosis (Newton et al. 2014b). As for the execution phase of necroptosis, there is evidence that the mitochondrial phosphatase PGAM5 (Wang et al. 2012) and acid sphingomyelinase (Roca & Ramakrishnan 2013) regulate this process downstream of the necrosome. Furthermore, phosphorylation of MLKL by RIPK3 drives its translocation to the plasma membrane and to intracellular membranes via binding to specific lipids; there, MLKL oligomerization leads to pore formation, which disrupts membrane integrity and mediates necrotic cell death (Dondelinger et al. 2014, Wang et al. 2014).

CONTROL OF CASPASE-8 ACTIVITY Role of Dimerization in Activation Caspases can be active only as dimers, because neither the active site dyad nor the substrate pocket is formed in the monomeric state (Pop & Salvesen 2009, Riedl & Shi 2004). Executioner procaspases, such as caspase-3 and -7, are constitutive dimers but undergo substantial rearrangement of crucial surface loops after the intersubunit linker has been cleaved, leading to formation of the active site (Figure 2). Initiator caspase-8, -9, and -10, however, are constituent monomers and must undergo dimerization and subsequent activation following recruitment to oligomeric multiprotein complexes, such as the DISC (Mace & Riedl 2010). From an enzyme-kinetic point of view, it is important to emphasize that the dimerization of caspase-8 or -10 within activation platforms such as the DISC is the key driver of catalytic activation (van Raam & Salvesen 2012). Nevertheless, higher-order oligomerization of the activating platform often augments apoptotic engagement, perhaps by affecting spatial rather than kinetic aspects of initiator caspase function, e.g., access to critical apoptotic substrates.

Role of Proteolytic Processing Although the caspase-8 dimer is active in the absence of proteolytic cleavage within the intersubunit linker, cleavage stabilizes the dimer and yields a caspase species with increased activity www.annualreviews.org • Regulated Cell Death


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Caspase-8 Prodomain linker

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Intersubunit linker

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cFLIPL

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DISC-tethered heterodimer: cleaved or uncleaved intersubunit linker

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d

DISC-tethered heterodimer: retained at DISC longer?

Figure 2 Activation modes of caspase-8. Caspase-8 and cFLIPL share the same molecular architecture, but FLIPL lacks catalytic residues. Both proteins possess caspase cleavage sites (Asp) between the large and small subunits of the catalytic domain, but FLIPL lacks the caspase cleavage site between the prodomain (DEDs) and the catalytic domain. Hetero- or homodimerization can lead to some of the species indicated above, but the relative abundance of these species remains unclear. Importantly, mutation of the caspase-8 interchain linker in mice abrogates apoptotic signaling, but survival signaling is retained, which suggests that cleavage at this site is necessary to promote apoptosis. Subsequent cleavage of the homodimer (c) and release separates the catalytic domains from the DISC, presumably allowing their decay by monomerization. The heterodimer would not be expected to dissociate from the DISC (d ) because the FLIPL unit lacks a caspase cleavage site in its prodomain linker.

(Boatright et al. 2003, Chang et al. 2003, Donepudi et al. 2003, Keller et al. 2009, Oberst et al. 2010, Pop et al. 2007). Importantly, caspase-8 also cleaves itself between the DEDs and the catalytic domain, resulting in release of a partly stabilized dimer from the DISC (Medema et al. 1997b) (Figure 2). Cleavage in the absence of dimerization does not lead to activation, but cleavage after dimerization increases both the activity and the temporary stability of the dimer (Pop et al. 2007). Caspase-8 may be activated by cleavage through another protease. The serine protease granzyme B, which is known to activate both caspase-3 and -7 during cytotoxic T cell–induced death, was shown to cleave caspase-8 as well (Martin et al. 1996, Medema et al. 1997a). Similarly, caspase-6 was shown to cleave caspase-8 between the large and small subunits (Cowling & Downward 2002). Finally, the lysosomal protease cathepsin D was reported to cleave and activate caspase-8 during neutrophil apoptosis (Conus et al. 2008). However, further studies on the role of caspase-8 cleavage by cathepsin D revealed the general principle that cleavage can enhance dimerization if a dimerization platform such as the DISC is present but cannot by itself activate the caspase-8 zymogen (Conus et al. 2012). In all of these cases, dimerization is most likely a prerequisite for caspase-8 activation, whereas cleavage stabilizes the dimers and increases their activity.

cFLIP: A Key Regulator of Caspase-8 Homologous to caspase-8 (also called FLICE), cellular FLICE-inhibitory protein (cFLIP) contains two DEDs and therefore can also be recruited to the DISC via FADD. However, cFLIP contains disruptive changes in key catalytic and substrate-binding amino acids and is therefore 20.10

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enzymatically dead. A short mRNA splicing variant of cFLIP (cFLIPS ) lacks the catalytic homology domain and therefore in principle can act as a competitive inhibitor of caspase-8 recruitment to the DISC. However, a long variant (cFLIPL ) contains the entire caspase-8 homology domain and confers polymorphic phenotypes upon cell transfection. Several groups independently identified cFLIPL ; however, their findings were highly divergent regarding whether its function was pro- or antiapoptotic. Some thought cFLIPL to be an inhibitor of DISC formation and caspase-8 activation, hence the name, and others thought it to be an independent caspase-8-like cell death inducer (Han et al. 1997, Hu et al. 1997, Irmler et al. 1997, Rasper et al. 1998, Shu et al. 1997). This controversy was settled when it was recognized that cFLIPL prevented cell death only upon overexpression, by occupying the majority of binding sites for caspase-8 at the DISC (Scaffidi et al. 1999). The field has now moved ahead, and it seems that cFLIP has an entirely different mechanism in securing cell survival. Heterodimerization with cFLIPL activates caspase-8 and -10 (Boatright et al. 2004, Dohrman et al. 2005, Micheau et al. 2002) because cFLIPL uses the same dimer interface between the catalytic domains (Yu et al. 2009). Thus, cFLIPL could be considered an activator of caspase-8 and -10. Perhaps more importantly, cFLIPL is actually a better activator of caspase-8 and -10 than are these caspases themselves, because the entropic barrier for heterodimerization is lower than for homodimerization (Boatright et al. 2004, Pop et al. 2011). Thus, one could imagine, although this is currently difficult to test, that in the presence of cFLIPL the first active caspase-8 enzyme at the DISC may actually be the heterodimer with cFLIPL . In purified systems, this heterodimer has a restricted substrate repertoire and reduced activity on the main proapoptotic targets downstream of caspase-8/10 activation, such as caspase-3 and BID, as demonstrated in two independent studies using in vitro–generated caspase-8/cFLIPL heterodimers (Hughes et al. 2009, Pop et al. 2011). Caspase-8 clearly has a wider array of functions than simply inducing apoptosis (see section on Nonproteolytic Roles of Caspase-8 below), and it appears to be the heterodimer, rather than the homodimer, that promotes the non-death functions of caspase-8 (Hughes et al. 2009, Oberst et al. 2011, Pop et al. 2011). Furthermore, the in utero lethal phenotype observed in caspase-8 knockout mice is shared with both the cFLIPL knockout and the FADD knockout (Yeh et al. 1998, 2000), and cFLIPL has been shown to be essential for cellular differentiation and activation in several studies (Dohrman et al. 2005, Huang et al. 2010, Lens et al. 2002, Oberst et al. 2011, Wu et al. 2004). This has led to the proposal that it is cFLIPL -activated caspase-8 that fulfills nonapoptotic functions, rather than the caspase-8 homodimer (Green et al. 2011, van Raam & Salvesen 2012). Importantly, the prosurvival role of caspase-8 requires proteolytic activity (Leverrier et al. 2011) but dispenses with the need for a cleavage between the large and small catalytic domains (Figure 2). Mutation of this cleavage site, rendering an uncleavable caspase-8, abrogates apoptosis but preserves the prosurvival role of caspase-8 in mice (Kang et al. 2008). In humans, mutation of this cleavage site suppresses caspase-8 homodimerization in favor of heterodimerization with FLIPL (Pop et al. 2011). Hence, it would seem that the heterodimer—in which caspase-8 remains uncleaved—is likely the survival-signaling enzyme, whereas the homodimer—in which caspase-8 is cleaved between its catalytic domains—is the apoptosis-signaling enzyme (Figure 2). However, this hypothesis has yet to be tested rigorously.

ENHANCEMENT OF CASPASE-8 ACTIVITY BY STIMULATORY UBIQUITINATION AND INACTIVATION OF CASPASE-8 BY DEGRADATIVE UBIQUITINATION C-terminal ubiquitination of caspase-8 is mediated in epithelial cells by the E3 ubiquitin ligase CUL3, which interacts with the DISC downstream of DR4 and DR5 ( Jin et al. 2009). www.annualreviews.org • Regulated Cell Death


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Ubiquitination of caspase-8 by K63-linked chains increased its proapoptotic potential, apparently by stabilizing the dimers. Moreover, ubiquitinated caspase-8 colocalized with the ubiquitinbinding protein p62 within intracellular foci, perhaps representing cytoplasmic centers of caspase-8 activity ( Jin et al. 2009). An important negative regulator of caspase-8 is TRAF2—an adaptor protein containing a RING E3 ligase domain that supports NF-κB signaling by several TNFR family members, including TNFR1 (Gonzalvez et al. 2012). TRAF2 associates with the DISC downstream to CUL3, where it mediates K48-linked ubiquitination of caspase-8 on the large catalytic subunit. Upon initial processing of caspase-8, the resulting ubiquitin-tagged products undergo rapid proteasomal degradation, thus curbing caspase-8 activation. Hepatic deletion of TRAF2 in wild-type and particularly BID knockout mice increases caspase-8 activation in response to Fas ligation, augmenting apoptosis-mediated lethality. Moreover, the TNFSF member TWEAK (TNFSF12) causes depletion of TRAF2, thereby augmenting Apo2L/TRAIL-induced caspase-8 activation and apoptosis. Hence, TRAF2 acts as an important checkpoint for cell-extrinsic apoptosis induction by setting a threshold for death receptor–mediated caspase-8 activation. Introduction of mutations in the cleavage sites between the DEDs and large catalytic domain of caspase-8 to prevent processing stabilized the protein and augmented apoptosis activation by diminishing the proteasomal degradation of K48-ubiquitinated caspase-8 (Gonzalvez et al. 2012).


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Nonproteolytic Roles of Caspase-8 This review focuses on the protease role of caspase-8, but it has become apparent that caspase-8 has roles independent of its well-known catalytic activity. Thus, DEDs accumulate during terminal differentiation and senescence of epithelial, endothelial, and myeloid cells (Mielgo et al. 2009). The pool of caspase-8 that participates in this function seems to decorate the cell periphery and interact with focal adhesions (Graf et al. 2014). For example, it seems that caspase-8 is necessary for the efficient activation of downstream events associated with epidermal growth factor signaling, perhaps explaining why it is rarely deleted in tumors that require this pathway for survival (Finlay et al. 2009). In some cases, this signaling seems to be dependent on tyrosine phosphorylation, with caspase-8 playing a potential scaffolding role rather than a catalytic role (Stupack 2010).

Control of Regulated Death by Caspase-8: Knockout of DISC and Necrosome Components Mouse embryos deficient in caspase-8, FADD, or cFLIP die in utero at approximately embryonic day 10.5 (Varfolomeev et al. 1998; Yeh et al. 1998, 2000; Zhang et al. 1998). These mice display remarkably similar defects in vascularization of the yolk sac (Sakamaki et al. 2002), implying a related role for the three DISC components in endothelial cell function or survival (Green et al. 2011). Much debate surrounded how absence of these proteins could result in lethality and why the phenotypes were so similar. A compelling answer came with the understanding that the necrosome and the DISC may be intimately connected (Figure 1). Thus, the lethal effect of not having caspase8 or FADD was substantially rescued by simultaneous deletion of RIPK3 (Kaiser et al. 2011, Lu et al. 2011, Oberst et al. 2011) or RIPK1 (Zhang et al. 2011)—both components of the necrosome. Although the developmental defect of caspase-8−/− mice was overcome, ablation of RIPK3 could not rescue the apoptotic defect, because caspase-8:RIPK3 doubly deficient mice developed a massive lymphoaccumulative phenotype reminiscent of mutations in Fas or FasL. Thus, it appears that one or more proteins in the necroptosis pathway are targeted by FADD-activated caspase-8 and must be cleaved to switch off the necrotic signal emanating from the necrosome. This topic requires 20.12

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far more discussion than can be accommodated here and is a rapidly developing field. Suffice it to say that caspase-8 has the ability, at least in mice, to drive death signals by mediating classic extrinsic apoptosis, as well as to drive survival signals by cleaving key mediator(s) in the necroptosis pathway. If the survival function of caspase-8 indeed operates through heterodimers with cFLIPL , then crossing cFLIP−/− mice with RIPK3−/− mice should not rescue embryonic lethality because these mice would lack the survival function of caspase-8/cFLIP heterodimers (van Raam & Salvesen 2012). Duly, such mice were generated and the hypothesis was supported (Dillon et al. 2012). Clear regions of apoptosis were observed in E9.5 cFLIP:RIPK3 double-knockout embryos but not in caspase-8−/− or FADD−/− embryos, indicating that the apoptotic function was engaged. Doubleknockout embryos died by apoptotic mechanisms, and as predicted, embryos lacking FADD, cFLIP, and RIPK3 developed normally. This study suggested that cross-regulation of RIPK3dependent signaling (including necroptosis) is controlled by the enzymatic activity of the FADDcaspase-8-cFLIPL complex and by cFLIP inhibition of RIPK3-independent apoptotic activity of the FADD-caspase-8 complex (Dillon et al. 2012). In other words, cFLIP can either activate caspase-8 or prevent its activation, although it remains unclear whether cFLIPL or cFLIPS serves the inhibitory function, or how regulatory balance is achieved. It is notable in this context that although RIPK1 plays a critical role in mediating apoptosis and necroptosis signaling downstream of TNFR1 and its associated embryonic death in mice, during early postnatal life deletion of RIPK1 actually augments lethality independent of specific gene disruption of NF-κB-inducing kinase, RIPK3, FADD or caspase-8; this suggests that during this period RIPK1 acts to suppress cell death signaling via the FADD-caspase-8 and RIPK3-MLKL pathways (Dillon et al. 2014).

Lessons from Human Genetic Alterations of Caspase-8 or -10 In humans, caspase-8 deficiency is compatible with development, as revealed by a family with a functionally detrimental mutation (R248W) in the coding region of the caspase-8 gene (Chun et al. 2002). In homozygous individuals, lymphocyte apoptosis and activation are impaired, but overall development is normal. A separate caspase-8 polymorphism, D302H, is associated with a lowered risk of ovarian and breast cancer (Engel et al. 2010), although the decreased breast cancer risk is seen primarily in BRCA1 but not BRCA2 mutation carriers. Additional caspase-8 mutations were detected in 1–2% of colorectal cancers (Lawrence et al. 2014). No mechanistic examination of these genetic alterations has been reported to date. Because humans also express the close caspase-8 paralog caspase-10, which has similar substrate specificity to caspase-8 (Wachmann et al. 2010), some of the survival roles of mouse caspase-8 may be subsumed by caspase-10 in humans. These caspases have been demonstrated to have some overlapping functions in vitro but cannot completely substitute for the loss of one or the other (Kischkel et al. 2001, Sprick et al. 2002). Mutations or deficiencies in human caspase-10 are associated with autoimmune lymphoproliferative syndrome (Grønbaek et al. 2000, Wang et al. 2001, Worth et al. 2006). Thus, whereas expression of either caspase-8 or caspase-10 is apparently sufficient for normal human development, both seem to be required to execute apoptotic functions, at least in lymphocytes. Of note, recent work implicates caspase-10 in nonapoptotic control of autophagy in multiple myeloma—a malignancy of B lymphocytes (Lamy et al. 2013).

CLOSING REMARKS In all multicellular organisms, accidental cell death occurs as a frequent consequence of exposure to external or internal physicochemical insults. However, metazoans have evolved a more specialized www.annualreviews.org • Regulated Cell Death


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ability to deliberately eliminate unwanted or damaged cells through two major forms of regulated death: apoptosis and necroptosis. The intrinsic and extrinsic apoptosis signaling pathways converge at the level of the executioner caspases. The intrinsic pathway, represented by several CED genes in C. elegans, is relatively ancient and plays a key role in directing cell elimination during development and in tissue homeostasis. In contrast, the extrinsic pathway, represented by a single TNF-like ligand and its cognate receptor in Drosophila, is a more recent evolutionary adaptation and is involved primarily in immune function. Less is known about the evolution of necroptosis. Nevertheless, the intimate connection of this cell death modality with signaling components of the extrinsic apoptosis pathway, as well as with other immune system–related genes, suggests that it may have evolved as a fail-safe mechanism to serve a similar function to that of extrinsic apoptosis. Although both of the apoptotic pathways clearly exist in species ranging from fruit flies to zebrafish and mammals, it remains to be established whether the necroptosis pathway as defined in murine and human cells also operates and is accessible to interrogation in other model organisms. Death ligands and their cognate receptors are categorized as such based on their capacity to induce regulated cell death under specific biological circumstances. Indeed, the recent advances in mechanistic understanding of necroptosis further reinforce this latter concept. However, given the intricate cross-regulation of intracellular signaling pathways, it is neither surprising nor counterintuitive that regulated death is not the only possible cellular outcome of death receptor signaling. Along similar lines, caspase-8 was first identified as the proteolytic conduit for death receptor– mediated apoptosis. However, it has since been found to be a more versatile molecule that can also impact cell survival and even other aspects of cellular activity, such as differentiation. During mouse embryonic development, a key role fulfilled by caspase-8—which it performs in conjunction with cFLIP—is to suppress necroptosis. Thus, further elucidation of the molecular parameters that determine cell fate in response to death receptor signaling and caspase-8 engagement is likely to be an important area for future scientific exploration. Regulated cell death might provide a fruitful target for therapeutic manipulation. On the one hand, its inhibition may help mitigate undue cell loss in disorders such as neurodegeneration, diabetes, stroke, or myocardial infarction. On the other hand, its activation may help eliminate unwanted cells in diseases such as cancer or chronic infection. TNFα is a major clinical target in conditions such as rheumatoid arthritis and inflammatory bowel disease, wherein efficacy is based primarily on amelioration of its proinflammatory pathological role. Other death ligands or receptors, as well as various components of the downstream signaling pathways, for example, caspase-8, RIPK1, and RIPK3, also may support valuable treatment approaches. However, better understanding of the underlying molecular mechanisms that toggle between apoptosis, necroptosis, and cell survival is needed in order to devise coordinated strategies that harness cell death regulation more effectively for therapeutic gain.


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DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

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Regulated cell death part A: apoptotic mechanisms. Preface.

Cell death in development: Signaling pathways and core mechanisms.

Regulated cell death in AKI.

Mechanisms of cell death.

Mechanisms in photodynamic therapy: part two-cellular signaling, cell metabolism and modes of cell death.

Redox Signaling and Myocardial Cell Death: Molecular Mechanisms and Drug Targets.

Regulated Forms of Cell Death in Fungi.

Mechanisms and functions of cell death.

Inhibition of regulated cell death by cell-penetrating peptides.

Cell death and cell death responses in liver disease: mechanisms and clinical relevance.

ERK Signaling Regulated Apoptosis.

The role of glycine in regulated cell death.

Developmental cell death: morphological diversity and multiple mechanisms.

Necroptotic cell death in failing heart: relevance and proposed mechanisms.

Ca(2+)-dependent mechanisms of cytotoxicity and programmed cell death.

Ras and rheb signaling in survival and cell death.

Identification of simvastatin-regulated targets associated with JNK activation in DU145 human prostate cancer cell death signaling.

Triggers and signaling cross-talk controlling cell death commitment.

Oxidative stress, redox signaling, and autophagy: cell death versus survival.

Signaling unmasked: Autophagy and catalase promote programmed cell death.

IAP family of cell death and signaling regulators.

Metabolism and iron signaling in ferroptotic cell death.

Cell death-associated molecular-pattern molecules: inflammatory signaling and control.

Molecular mechanisms of Ebola virus pathogenesis: focus on cell death.