Molecular Cell

Review The Two Faces of Receptor Interacting Protein Kinase-1 Ricardo Weinlich1 and Douglas R. Green1,* 1Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2014.11.001

Receptor Interacting Protein Kinase-1 (RIPK1), a key player in inflammation and cell death, assumes opposite functions depending on the cellular context and its posttranslational modifications. Genetic evidence supported by biochemical and cellular biology approaches sheds light on the circumstances in which RIPK1 promotes or inhibits these processes. ‘‘If he be Mr. Hyde,’’ he had thought, ‘‘I shall be Mr. Seek.’’ –R.L. Stevenson RIPK1 as a Signaling Node in Cell Death and Inflammation ‘‘You start a question, and it’s like starting a stone. You sit quietly on the top of a hill; and away the stone goes, starting others.’’ Among the 28,000 or so proteins that comprise a mammalian body, there are only some that are so crucial to the organismal machinery that their loss results in irrevocable death during development. In many cases, such proteins are integral to fundamental processes such as cell division and metabolism or are central to the development of essential tissues. But in other cases, the obligate role for the protein relates to its placement as a signaling ‘‘node’’ within or between cells. And in rare instances in mammals, the early lethality of a null or mutant allele can be suppressed by mutation of one or more other genes in the signaling network. The analysis of these effects, especially when combined with biochemical and cell biological approaches, highlights its power to define the fundamental structures and functions of such networks. Receptor interacting protein kinase-1 (RIPK1) represents an essential signaling node. Mice with homozygous deletion of RIPK1 die shortly after birth, with widespread inflammation and cell death in several tissues (Kelliher et al., 1998). In contrast, mice with homozygous mutations that destroy the kinase activity of RIPK1 are developmentally normal (Berger et al., 2014; Newton et al., 2014; Polykratis et al., 2014). RIPK1 functions in response to a variety of input signals, including those generated in response to the TNF family cytokines, ligands for some Tolllike receptors (TLRs), sensors of viral infection, and interferons. The output of the network controlled by RIPK1 includes the activation of MAP kinases and NF-kB and both apoptotic and necrotic cell death. How these functions of RIPK1 prevent the early postnatal lethality caused by the homozygous null allele is far from simple, and new properties of RIPK1 signaling are emerging as the genetics of these animals are analyzed. Recent studies, based on the RIPK1 signaling pathways, have revealed a striking rescue of the postnatal lethality of RIPK1-null

mice. These are summarized in Figure 1. Ablation of both caspase-8 and RIPK3, a kinase that is closely related to RIPK1, permits normal development and maturation of RIPK1-null animals (Dillon et al., 2014; Kaiser et al., 2014; Rickard et al., 2014). Similarly, animals lacking RIPK1, RIPK3, and FADD (an adaptor protein required for the activation of caspase-8) also develop and mature normally (Dillon et al., 2014). No such rescue is seen in animals lacking RIPK1 and any one of these players (FADD, caspase-8, or RIPK3) (Dillon et al., 2014; Kaiser et al., 2014; Rickard et al., 2014). Formally, then, we can state that RIPK1 functions to suppress early postnatal lethality caused by two concurrent pathways, one mediated by FADD and caspase-8 and the other by RIPK3 (Figure 1). In this review, we will parse these findings in the context of the signaling network in which RIPK1 serves as a node. If we consider only these results, we can envision RIPK1 as the good Dr. Jekyll, in R.L. Stephenson’s ‘‘The Strange Case of Dr. Jekyll and Mr. Hyde,’’ serving to block lethal pathways engaged by FADD-caspase-8 and RIPK3. FADD-caspase-8 interactions are known to be essential for apoptosis triggered by the death receptors (a subset of the TNF-receptor [TNFR] superfamily, which includes TNFR1, CD95, and the TRAIL-receptors) (Boldin et al., 1996; Muzio et al., 1996). RIPK3 functions in another cell death pathway, that of necroptosis, a form of regulated necrosis (Weinlich et al., 2011). It would be relatively simple to assert that RIPK1 blocks both and thereby preserves cellular survival, and in doing so preserves the health of the animal. But Dr. Jekyll is also the evil Mr. Hyde, something we can similarly observe using genetic approaches. Ablation of caspase-8 or FADD causes an early embryonic lethality (at E10.5), in which one feature is a failure of the yolk sac to vascularize (Sakamaki et al., 2002; Varfolomeev et al., 1998; Yeh et al., 1998). This early lethality is fully rescued by ablation of RIPK3, such that the double-deficient animals develop normally and mature (Dillon et al., 2012; Kaiser et al., 2011; Oberst et al., 2011). Remarkably, ablation of RIPK1 also allows the normal embryonic development of caspase-8- or FADD-deficient animals, although these mice succumb to early postnatal lethality (as do RIPK1-null mice) (Dillon et al., 2014; Kaiser et al., 2014; Zhang et al., 2011). Therefore, based only on these results, we can assert that the RIPK1 promotes the lethality of the FADD- or caspase-8 null alleles (Figure 1). Indeed, as we will see, experiments in cells support Molecular Cell 56, November 20, 2014 ª2014 Elsevier Inc. 469

Molecular Cell

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Figure 1. Dual Function of ripk1 Genetics of ripk1 highlight the dual function of this molecule: by promoting organismal lethality, ripk1 is the evil Mr. Hyde; by averting it, ripk1 is the good Dr. Jekyll. Lifespan of mice with different combinations of gene deletions is shown.

the idea that catalytically active RIPK1 can trigger both FADDcaspase-8-mediated apoptosis and RIPK3-mediated necroptosis. In addition, RIPK1 has important roles in promoting inflammatory effects that can be independent of cell death. Therefore, if we are to solve the strange case of RIPK1 and its Jekyll/Hyde duality, we will have to understand how these and other genetic results (discussed below) can be resolved in the context of RIPK1 signaling. RIPK1 in FADD-Caspase-8-Dependent Apoptosis ‘‘I saw that, of the two natures...I was radically both.’’ Ligation of TNFR1 induces the recruitment of the adaptor molecule TRADD, which in turn binds another adaptor, FADD. FADD then binds to caspase-8, and the dimerization of the latter triggers its proteolytic activity, promoting apoptosis (Hsu et al., 1995; Juo et al., 1998; Yeh et al., 1998). RIPK1, independently of its kinase activity, has a scaffolding function that contributes to the activation of NF-kB and MAP kinases following ligation of TNFR1 (Ea et al., 2006). While some studies suggest that RIPK1 is required for NF-kB activation via TNFR1, other studies suggest that the effect is subtler, contributing to an amplification of NF-kB activation (Ea et al., 2006; Lee et al., 2004; Li et al., 2006; Wong et al., 2010). These differences might be explained by differences in the cell type used as well as the type and 470 Molecular Cell 56, November 20, 2014 ª2014 Elsevier Inc.

strength of the activating signal. Nevertheless, RIPK1-mediated NF-kB activity may be important for the expression of c-Flar/cFLIPL/FLIP, a caspase-8-like protein (without catalytic activity) that forms a complex with FADD-caspase-8 and inhibits proapoptotic activity (Dillon et al., 2014; Irmler et al., 1997; Kreuz et al., 2001; Micheau et al., 2001) (Figure 2A). Indeed, mouse embryonic fibroblasts (MEFs) from RIPK1-deficient animals display an increased sensitivity to TNF-induced, caspase-8dependent apoptosis, while MEFs bearing a kinase-inactive RIPK1 (RIPK1D138N) do not (Lee et al., 2004). This ‘‘Dr. Jekyll’’ role for RIPK1 in blocking TNFR1- and caspase-8-dependent apoptosis is highlighted in observations involving conditional ablation of RIPK1 in intestinal epithelium (Table 1). Mice without intestinal RIPK1 display postnatal lethality that is prevented by conditional deletion of FADD or caspase-8 in the same cells (Dannappel et al., 2014; Takahashi et al., 2014). The lethality is accompanied by extensive apoptosis in the intestine, with ensuing inflammation, and this is largely resolved in animals lacking intestinal FADD or caspase-8. This protective effect of RIPK1 in intestinal epithelial cells (IECs) does not appear to be through optimal NF-kB activation, which is actually unaltered or even elevated in RIPK1-deficient IECs (unlike MEFs), but is instead via stabilization of several RIPK1-associated proteins, including FLIP, TRAF2, and cIAP1 (Dannappel et al., 2014; Takahashi et al., 2014). One of the functions of both TRAF2 and cIAP1 is to inhibit NIK stabilization and the subsequent activation of the noncanonical NF-kB pathway and the production of TNF (Gentle et al., 2011; Vallabhapurapu et al., 2008; Varfolomeev et al., 2007; Vince et al., 2007; Zarnegar et al., 2008). Thus, upon loss of RIPK1, IECs produce abundant TNF, which in turn triggers FADD-caspase-8-dependent apoptosis, which is not antagonized by the reduced levels of FLIP. In contrast, mice with a homozygous, kinase-inactive RIPK1 show no such apoptosis, suggesting that this inhibitory function of RIPK1 is independent of its kinase activity and instead relies on its scaffolding function. Further, mice lacking TNFR1 are partially protected from the effects of IEC-specific ablation of RIPK1 (Dannappel et al., 2014) (Table 1). Therefore, RIPK1 promotes resistance to TNFR1-, FADD-caspase-8 mediated apoptosis. This is consistent with the ‘‘FADDcaspase-8’’ arm of the rescue of lethality in RIPK1-deficient mice (Figure 1): the requirement for FADD or caspase-8 ablation can largely be replaced by ablation of TNFR1, such that tnfr1 / , ripk1 / , and ripk3 / mice wean at Mendelian frequencies (Dillon et al., 2014). However, RIPK1 can also promote the activation of caspase-8 in a FADD-dependent manner. RIPK1 binds to FADD via a death domain (DD) homotypic interaction, whereupon FADD activates caspase-8. This so-called ‘‘Complex II’’ or ‘‘Ripoptosome’’ can be triggered by ligation of death receptors, genotoxic stress, depletion of IAPs, and the ligation of some TLRs, particularly TLR3 and TLR4 (Feoktistova et al., 2011; Geserick et al., 2009; Tenev et al., 2011). The latter do so via the TLR adaptor molecule TRIF. TRIF has a RHIM protein interaction motif that may directly bind to the RHIM in RIPK1 (Cusson-Hermance et al., 2005; Kaiser and Offermann, 2005; Meylan et al., 2004). This proapoptotic, ‘‘Mr. Hyde’’ role for RIPK1 is highlighted in studies of animals with a kinase-inactive mutation in RIPK3

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in MLKL-deficient MEFs (see below), the enforced dimerization of RIPK3 induced RIPK1-, caspase-8-dependent apoptosis (Cook et al., 2014). Deletion of TNFR1 or TRIF did not affect the E10.5 lethality in ripk3D161N mice, however (Newton et al., 2014); therefore, this inducing effect of RIPK3D161N may be constitutive. Indeed, cells expressing this mutant only survive in the presence of a pan-caspase inhibitor (Mandal et al., 2014). However, mice bearing the kinase-inactive RIPK3K51A are viable and fertile, raising the possibility that the charge change in the RIPK3D161N mutant results in a gain-of-function that induces RHIM oligomerization. Indeed, a neutral substitution of the same residue (RIPK3D161G) did not result in a spontaneous apoptosis-inducing molecule (Mandal et al., 2014). Therefore, while currently available pharmacologic inhibitors of RIPK3 promote apoptosis, (Mandal et al., 2014), it may well be possible to block the kinase activity of RIPK3 in order to suppress necroptosis (see below) without inducing apoptosis. Nonetheless, it is unclear why the effects of the RIPK3D161N mutant manifest only at E10.5, as all of these proteins are expressed well before this developmental stage. It remains possible that there is an unknown RIPK3 activator that only surfaces at this developmental stage. RIPK1 in RIPK3-MLKL Mediated Necroptosis ‘‘Hitherto it was his ignorance.that had swelled his indignation; now, by a sudden turn, it was his knowledge.’’

Figure 2. Antiapoptotic and Proapoptotic Roles of RIPK1 (A) Upon TNFR-1 ligation, RIPK1 stabilizes TNF-R1-induced complex I preventing transition to the proapoptotic complex II. Also, RIPK1 facilitates the activation of the canonical NF-kB pathway, promoting the expression of antiapoptotic molecules, such as FLIP. FLIP inhibits FADD-induced caspase-8 homodimerization in complex II, blocking apoptosis. RIPK1, TAK1, and TAB1 promote, but are not always required for, NF-kB activation in response to TNFR ligation. (B) In the absence of IAPs, the expression of a kinase-inactive RIPK3 mutant (RIPK3D161N), the addition of a RIPK3 kinase inhibitor, or after enforced RIPK3 dimerization (dimRIPK3) in the absence of MLKL, RIPK1 can promote apoptosis via the recruitment of FADD and caspase-8.

(Newton et al., 2014). Mice with homozygous ripk3D161N die at E10.5 but are completely protected by germline ablation of caspase-8. In this case, ablation of RIPK1 results in ripk3D161N mice that survive to birth but then succumb to perinatal lethality (Newton et al., 2014). RIPK3 and RIPK1 both contain RHIM domains that can bind as homo- or hetero-oligomers in b-amyloid structures (Li et al., 2012; Wu et al., 2014). The kinase-inactive RIPK3 (achieved by mutations or use of RIPK3 kinase inhibitors) binds to RIPK1 via RHIM-RHIM interactions, and this, in turn, recruits FADD-caspase-8-cFLIPL to cause apoptosis (Figure 2B) (Mandal et al., 2014). This recruitment seems to be independent of RIPK1 kinase activity, as the kinase-inactive RIPK1D138N did not prevent RIPK3D161N lethality (Newton et al., 2014). Likewise,

Necroptosis is a form of regulated necrosis that depends on the activation of RIPK3 and its phosphorylation of a pseudokinase, Mixed lineage kinase-like (MLKL) (Kaiser et al., 2013; Murphy et al., 2013; Sun et al., 2012; Wu et al., 2013; Zhao et al., 2012). MLKL contains a kinase-like domain that is phosphorylated by RIPK3 on what would be the activation loop of a bona fide kinase, and this appears to derepress the N-terminal region that contains a coiled-coil bundle that oligomerizes and interacts with the plasma membrane through binding to phosphoinositides (Cai et al., 2014; Chen et al., 2014; Dondelinger et al., 2014; Wang et al., 2014a). The N-terminal region of MLKL is capable of disrupting lipid vesicles and therefore may directly permeabilize the plasma membrane (Dondelinger et al., 2014; Wang et al., 2014a) or may engage ion channels to effect necrosis (Cai et al., 2014; Chen et al., 2014). RIPK1 plays a ‘‘Mr. Hyde’’ role in promoting necroptosis in response to ligation of death receptors (e.g., TNFR1, CD95, and TRAIL-receptors), provided caspase-8 activation is blocked or disrupted (Ch’en et al., 2011; Holler et al., 2000; Vercammen et al., 1998; Zhang et al., 1998) (Figure 3A). Kinase-active RIPK1 binds to RIPK3 via RHIM-RHIM interactions, and this promotes subsequent RIPK3 activation and necroptosis. In the absence of RIPK1 or its kinase activity, no necroptosis is observed in response to death receptor ligation (Berger et al., 2014; Dillon et al., 2014; Polykratis et al., 2014). Necrostatins inhibit RIPK1 kinase activity and effectively block death-receptor-induced necroptosis (Degterev et al., 2008; Takahashi et al., 2012). It remains unclear, however, how RIPK1 kinase activity controls necroptosis. In vitro experiments have shown that RIPK1 can phosphorylate itself but not RIPK3 (Cho et al., Molecular Cell 56, November 20, 2014 ª2014 Elsevier Inc. 471

Molecular Cell

Review Table 1. Consequences of RIPK1 Ablation in the Gut and the Skin Apoptosis

Necrosis

Inflammation

Wasting

Lethality

+

+

+

+

+

+

+

+

RIPK1 Ablation in the Gut ripk1IEC-KOF ripk1

IEC-KOF

+ + ripk3

/

+

ripk1IEC-KOF + casp8IEC-KOF

localized

ripk1IEC-KOF + germ-free

+

ripk1IEC-KOF + faddIEC-KOF ripk1IEC-KOF + faddIEC-KOF + ripk3 ripk1IEC-KOF + tnfr1 fadd

localized localized

localized

n.d.U

less

localized

localized

localized

localized

+

+ +

/

/

IEC-KO

faddIEC-KOF + ripk1D138NJ

less

laterp

+

+

+

+

+

+

+

less

later

later

later

earlyq

earlyq

earlyq

earlyq

+

+

+

+

ripk1D138NJ RIPK1 Ablation in the Skin ripk1E-KOs

+

ripk1E-KOs + ripk3

/

+

ripk1E-KOs + faddE-KOs ripk1E-KOs + faddE-KOs + ripk3 ripk1E-KOs + tnfr1 fadd

E-KOs

fadd

E-KOs

+ ripk1

/

/

D138NJ

ripk1D138NJ F

IEC-KO: conditional deletion of the respective gene in the IECs. n.d.: not determined. p later: lethality delayed in average from the third week to the third month. J ripk1D138N: kinase-inactive ripk1 mutant. s E-KO: conditional deletion of the respective gene in skin epithelial cells. q early: symptoms appear perinatally. U

2009); this raises the possibility that RIPK1 autophosphorylation is important to attain its open configuration in order to recruit RIPK3 via RHIM-RHIM interactions, although no strong evidence for this yet exists. Alternatively, there may be other substrates that are important for control of necroptosis, but these have not been identified. Studies employing enforced heterodimerization and homodimerization of RIPK1 and RIPK3 help to elucidate the interactions leading to MLKL-dependent necroptosis (Figure 3B). Homodimerization of the N termini of RIPK1 but not RHIM-deficient RIPK1 (RIPK1DRHIM) promotes RIPK3dependent cell death, as does heterodimerization of RIPK1DRHIM and RIPK3, while heterodimerization of RIPK1DRHIM and RIPK3DRHIM does not (Orozco et al., 2014). Homo-oligomerization, but not homo-dimerization, of the C termini of RIPK3DRHIM similarly promotes necroptosis (Orozco et al., 2014). Curiously, however, homodimerization of the N termini of RIPK3DRHIM is sufficient to induce necroptosis (Wu et al., 2014). As the kinase domain of RIPK3 is situated near the N terminus of the protein, these data suggest that it is the proximity of RIPK3 kinase domains that autoactivates RIPK3 to promote necroptosis, and RIPK1 can trigger RIPK3-RIPK3 interactions to induce this effect. This requisite role for RIPK1 in promoting death-receptorinduced necroptosis helps to explain how ablation of RIPK1 rescues the early embryonic lethality (E10.5) of FADD- or caspase8-deficient mice (Dillon et al., 2014; Kaiser et al., 2014; Zhang 472 Molecular Cell 56, November 20, 2014 ª2014 Elsevier Inc.

et al., 2011). Animals lacking TNFR1 and FADD similarly survive this developmental stage (Dillon et al., 2014) (Figure 1). Some stimuli for necroptosis do not depend on RIPK1 to engage RIPK3 and MLKL (collectively referred to as ‘‘signal 2’’) (Dillon et al., 2014). TRIF, activated in response to ligation of TLR3 or TLR4 (Dillon et al., 2014; Kaiser et al., 2013), and DNA-dependent activator of IFN-regulatory factors (DAI) (Upton et al., 2010; 2012) both directly bind to RIPK3 and induce necroptosis in the absence of RIPK1 (Figure 3C). Similarly, type I and II interferons, perhaps via PKR, engage RIPK3- and MLKLdependent necroptosis in the absence of RIPK1 (Dillon et al., 2014). In the presence of RIPK1, however, the induction of necroptosis by TLR ligation or interferons requires inhibition or disruption of FADD-caspase-8-FLIP activity (Dillon et al., 2014; Thapa et al., 2013). Therefore, RIPK1 also acts as ‘‘Dr. Jekyll’’ to inhibit RIPK3, MLKL-dependent necroptosis, and it does so in two different ways. The first is via recruitment of FADD-caspase-8-FLIP by the binding of the DD of both RIPK1 and FADD (see above). The caspase-8-FLIP heterodimer is catalytically active (Pop et al., 2011), and while it does not promote apoptosis (Irmler et al., 1997; Salvesen and Walsh, 2014), it can cleave both RIPK1 and RIPK3 (Feng et al., 2007; Lin et al., 1999; Rajput et al., 2011) and other components of the pathway (O’Donnell et al., 2011), disrupting the MLKL-activating complex (O’Donnell et al., 2011; Oberst et al., 2011).This effect is

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Figure 3. Roles of RIPK1 in Necroptosis (A) Ligation of death receptors or loss of IAPs induce necroptosis by the formation of a complex containing RIPK1, RIPK3, and MLKL under conditions of disruption of the inhibitory complex formed by FADD, Caspase-8, and FLIP. (B) Enforced dimerization of RIPK1 or its dimerization with RIPK3 can induce necroptosis only when it can seed the formation of RIPK3 oligomers. DRHIM indicates that the molecule has a defective RHIM and therefore cannot interact via this motif. dim, dimerization domain. (C) Different ‘‘signal 2s’’ (e.g., TLR3/4, DAI, and interferons) can induce necroptosis via recruitment of RIPK3 in the absence of RIPK1. RIPK1 blocks necroptosis either through recruitment of the inhibitory complex formed by FADD, caspase8, and FLIP or when it assumes a kinase-inactive conformation (by the mutation D138N or by Nec-1 inhibition).

C

readily observed upon enforced dimerization of RIPK3; in the presence of RIPK1, necroptosis is enhanced by inhibition or silencing of caspase-8 (Orozco et al., 2014). In the absence of RIPK1, no enhancement of RIPK3 dimerization-induced necroptosis is observed upon blockade or silencing of caspase activity. But the ‘‘Dr. Jekyll’’ effect of RIPK1 to block necroptosis induced by signal 2 goes beyond recruitment of FADD-caspase-8-FLIP to disrupt RIPK3-MLKL interactions. In the presence but not in the absence of RIPK1, necrostatins inhibit necroptosis induced by caspase inhibition and TLR ligation (Dillon et al., 2014; He et al., 2011; Kaiser et al., 2013; Kearney et al., 2014) or treatment with interferons (Dillon et al., 2014; Kaiser et al., 2014). Importantly, necrostatin-inhibited RIPK1 coprecipitates with RIPK3 but not MLKL upon treatment with a TLR3 agonist and caspase inhibitor (Dillon et al., 2014). Similarly, necroptosis induced by TLR3 ligation plus caspase inhibition is not observed in cells expressing kinase-inactive RIPK1 (Polykratis et al., 2014). It appears, therefore, that kinase-inactive RIPK1 blocks the activation of RIPK3-MLKL, even when RIPK3 is directly engaged by TRIF or interferons. The inhibitory effects of RIPK1 on necroptosis are highlighted by studies in mice in which RIPK1 is conditionally deleted in the skin. Such animals display a profound skin inflammation and pathology that is dramatically reduced by germline ablation of RIPK3 or MLKL (Dannappel et al., 2014). Animals with kinaseinactive RIPK1 do not display this pathology (Berger et al., 2014; Dannappel et al., 2014; Polykratis et al., 2014; Takahashi

et al., 2014). Therefore, it appears that a ‘‘signal 2’’ in the skin promotes RIPK3and MLKL-dependent pathology that is blocked by RIPK1, independent of its kinase activity. Moreover, it suggests that high levels of apoptosis induced by loss of RIPK1 in the skin (unaltered by ablation of RIPK3 or MLKL) are not sufficient to drive the disease. However, animals in which FLIP is conditionally deleted in skin display loss of tissue homeostasis and severe inflammation (Panayotova-Dimitrova et al., 2013; Weinlich et al., 2013), which is not rescued by deletion of RIPK3 (Weinlich et al., 2013), suggesting that in the skin, the mode of cell death—necroptosis or apoptosis—is not relevant for disease onset. Differences in the extent to which FLIP expression is lost may account for the different outcomes of ablation of FLIP or RIPK1 in skin. This idea, that kinase-inactive RIPK1 blocks necroptosis by interfering with RIPK3-MLKL interactions, is challenged by an observation in mice with a skin-specific deletion of FADD. Such animals display fulminant skin damage that is prevented by coablation of RIPK3 (Dannappel et al., 2014). However, homozygous kinase-inactive RIPK1D138N did not effectively prevent cell death and inflammation in these mice (Dannappel et al., 2014). One potential explanation for this is that in the skin both inhibitory functions of RIPK1 are necessary; without FADD, caspase-8-FLIP cannot be recruited via RIPK1 to RIPK3 to suppress the necroptotic complex. Alternatively, higher levels of RIPK1 may be required to interfere with RIPK3-MLKL interactions (when RIPK1 is kinase inactive or inhibited by necrostatins) than for RIPK1-mediated induction of NF-kB, MAP kinases, or necroptosis. If so, we may find differential effects of RIPK1 inhibition on cell death in different tissues, based on its availability (either expression or regulation; see below). We can now begin to make some sense of the genetics of RIPK1 as both Dr. Jekyll and Mr. Hyde (Figure 1). Upon loss of RIPK1, FADD-caspase-8-dependent apoptosis promotes disruption of tissues, including the intestinal epithelium. Molecular Cell 56, November 20, 2014 ª2014 Elsevier Inc. 473

Molecular Cell

Review Meanwhile, loss of RIPK1 also promotes RIPK3-MLKL-dependent necroptosis to disrupt other tissues, including the skin. Other affected tissues include the hematopoietic lineages and the endothelium (Rickard et al., 2014), and perhaps others, although the relative contributions of either or both pathways of cell death to their disruption are currently unresolved. If RIPK1 only functions to control cell death, then these effects can explain the lethality of RIPK1 ablation and the abilities of additional mutations to rescue this lethality. However, RIPK1 does not only function in the regulation of cell death. RIPK1 in Inflammation ‘‘They have only differed on some point of science,’’ he thought.‘‘It is nothing worse than that!’’ As briefly mentioned above, RIPK1 functions in a kinase-independent manner in the activation of NF-kB in response to TNFR1 ligation through its recruitment of NEMO/IKKg (Ea et al., 2006). Similarly, RIPK1 promotes TRIF-mediated activation of NF-kB upon TLR3 or TLR4 ligation (Cusson-Hermance et al., 2005; Kaiser and Offermann, 2005; Meylan et al., 2004). RIPK1 also promotes the activation of IRF-3 and NF-kB response to intracellular sensing of dsRNA, Sendai virus, or influenza virus by RIG-I and MDA-5 (Balachandran et al., 2004; Kawai et al., 2005; Rajput et al., 2011), as well as to immunostimulatory DNA or MCMV virus by DAI (Kaiser et al., 2008; Rebsamen et al., 2009). Thus, RIPK1 acts as ‘‘Mr. Hyde’’ to promote inflammation in response to infection. RIPK1-mediated, RIG-I-induced IRF3 activation appears to be controlled by recruitment of caspase-8 and FLIP to the RIG-I-MAVS complex, as cells lacking caspase-8 (Rajput et al., 2011) or FLIP (Handa et al., 2011) displayed dramatically elevated IRF3 activation in response to Sendai virus or transfected dsRNA. A mutant of caspase-8 that does not bind FADD did not inhibit such IRF3 activation (Rajput et al., 2011). In contrast, however, cells lacking or bearing mutated FADD (Balachandran et al., 2004; Kawai et al., 2005) or TRADD (Michallet et al., 2008) reportedly produced less IRF3 than wild-type cells upon Sendai virus infection or transfected Poly (I:C). It is possible that RIG-I recruits caspase-8 and FLIP directly, as in-vitro-transcribed and -translated caspase-8 and RIG-I were observed to associate (Rajput et al., 2011), although this remains to be tested in cells. Alternatively, TRADD and FADD may be required for recruitment of RIPK1 to RIG-I-MAVS, as neither of the latter proteins bears domains that could bind directly to RIPK1. RIPK3 was found to have opposing effects on the RIPK1 proinflammatory function; while its overexpression can inhibit RIPK1-mediated, TRIF-induced NF-kB activation by competitive binding to TRIF (Meylan et al., 2004), silencing of RIPK3 decreases DAI-induced NF-kB activation (Rebsamen et al., 2009). In the latter case, RIPK3 kinase activity seems to be required, as overexpression of the wild-type, but not the kinase-inactive, RIPK3 synergized with RIPK1 to promote NF-kB (Kaiser et al., 2008; Rebsamen et al., 2009). Unfortunately, however, roles for cell death were not thoroughly interrogated in these studies, and therefore, it is difficult to fully assess the conclusions. Another striking example of this ‘‘Mr. Hyde’’ role for RIPK1 in inflammation comes from studies of the LUBAC, a complex 474 Molecular Cell 56, November 20, 2014 ª2014 Elsevier Inc.

that contains the proteins HOIL-1, HOIP, and SHARPIN, which targets RIPK1 and functions as a Met-1 linear ubiquitinating complex (Gerlach et al., 2011; Ikeda et al., 2011; Tokunaga et al., 2011). Humans who carry loss-of-function HOIL-1 alleles suffer from a fatal autoinflammatory disease (Boisson et al., 2012). Likewise, mice lacking Sharpin present with severe inflammatory skin disease (Seymour et al., 2007), which is absent when these mice are also homozygous for kinase-inactive RIPK1K45A (Berger et al., 2014), demonstrating that the kinase activity of RIPK1 is necessary for the inflammatory effects of Sharpin deficiency. It is likely that RIPK1-dependent necroptosis, and its proinflammatory consequences, is responsible for the skin lesions associated with Sharpin deficiency, as proposed (Berger et al., 2014). On the other hand, ptpn6spin mice, which bear a loss-of-function mutation in the phosphatase SHP1, spontaneously develop a severe inflammatory skin disease that is driven by NF-kB activation and dysregulated IL-1a-mediated events (Lukens et al., 2013). Pharmacological inhibition of RIPK1 or its genetic ablation in hematopoietic cells greatly ameliorated the disease while RIPK3 deficiency did not, suggesting that in this model the pronecroptotic function of RIPK1 is dispensable for its proinflammatory role. Intriguingly, RIPK1, together with RIPK3, also appears to promote the activation of the NLRP3 inflammasome in some cases in a manner that depends on MLKL and is suppressed by caspase-8 (Kang et al., 2013). In this study, evidence that this was not an effect of necroptosis was provided, and no release of NLRP3-activating molecules was detected in supernatants. Interestingly, one study has suggested that MLKL-mediated necroptosis may involve the opening of transient receptor potential melastatin (TRPM) ion channels (Cai et al., 2014), and TRPM ion channels can enhance NLRP3 inflammasome activity (Zhong et al., 2013). Alternatively, reactive oxygen species generated in response to RIPK3 and MLKL activation appear to be important for NLRP3-dependent and -independent inflammasome activation (Vince et al., 2012), although no elevated ROS levels were detected during NLRP3 assembly in caspase-8-deficient dendritic cells (Kang et al., 2013). Activation of NLRP3 via RNA viruses, however, is independent of MLKL and instead appears to rely on mitochondrial damage caused by the activation of DRP1 through a RIPK1-RIPK3 complex (Wang et al., 2014b).Thus, RIPK1 (again, as ‘‘Mr. Hyde’’) may facilitate ripoptosome assembly to promote inflammation via NLRP3 and other signaling complexes. Despite these ‘‘Mr. Hyde’’ roles for RIPK1 in promoting inflammation, RIPK1 clearly acts as ‘‘Dr. Jekyll’’ to prevent inflammation, as seen in animals with germ-line (Dillon et al., 2014; Kaiser et al., 2014; Kelliher et al., 1998; Rickard et al., 2014), conditional (Dannappel et al., 2014; Takahashi et al., 2014), or acute (Takahashi et al., 2014) deletion of RIPK1. In each case, the observed inflammation was associated with cell death and was prevented by combined ablation of RIPK3 (or MLKL) and either caspase-8 or FADD. While necrotic cell death is proinflammatory, apoptotic cell death generally is not (Green, 2011), although a loss of barrier function (in the intestine or skin) can promote subsequent inflammation due to microbial exposure. However, the inflammation associated with IEC-specific deletion of RIPK1 was not ameliorated under germ-free

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Review A

B

C

Figure 4. RIPK1 Function Is Regulated by Ubiquitination (A) TRAF2, TRAF6, and the E3 ligases cIAP1 and cIAP2 act in concert to add K63-linked ubiquitin chains (K63-Ub) to RIPK1, as does Pel-1. The LUBAC complex, formed by HOIL-1, HOIP, and Sharpin, ubiquitinates RIPK1 with Met-1-linked ubiquitin chains (Met1-Ub). Both modifications stabilize RIPK1 in complex I, activating the NF-kB pathway and inducing the expression of antiapoptotic proteins, such as c-FLIP. (B) CYLD and OTULIN are deubiquinating enzymes that remove K63-Ub and linear Met1-Ub linkages from RIPK1, respectively, promoting either apoptosis or necroptosis. (C) RIPK1 modified with K48-linked ubiquitin chains (K48-Ub) is targeted for proteasomal degradation, which sensitizes cells to TNF-induced apoptosis (and possibly signal 2-mediated necroptosis, which is untested). This process is mediated by A20, a ubiquitin-editing enzyme that removes K63-Ub from RIPK1 while adding K48-linked ubiquitin chains, and TRIAD3, a K48-Ub E3 ligase.

conditions (Dannappel et al., 2014), suggesting that the inflammation may be a consequence of the response to cell death. If so, we are left with a puzzle: the lethal inflammation seen in IEC-specific deletion of RIPK1 was not observed in animals lacking RIPK1 and FADD (Dannappel et al., 2014) or caspase-8 (Takahashi et al., 2014), suggesting that rather than RIPK3dependent necrosis, it is caspase-8-dependent apoptosis that mediates these effects, independently of microbial infiltration. The alternative interpretation is that in the absence of RIPK1, a proinflammatory effect of FADD-caspase-8 mediates the subsequent damage. Intriguingly, several reports have suggested that FADD-caspase-8 can promote IL-1b processing and secretion in a manner that can be independent of the caspase-1 inflammasome (Bossaller et al., 2012; Gringhuis et al., 2012; Gurung et al., 2014; Maelfait et al., 2008; Vince et al., 2012). It is possible (although untested) that RIPK1 somehow blocks this process. Alternatively, it is conceivable that activation of NF-kB, which in some settings can negatively regulate pro-IL1b processing (Greten et al., 2007), may play a role in this effect. The Regulation of RIPK1 and Its Effects ‘‘It was the curse of mankind that these.polar twins should be continuously struggling.’’ Clearly, RIPK1 has two faces in the regulation of cell death and inflammation. What then is the ‘‘elixir’’ that changes Dr. Jekyll into Mr. Hyde and back? A clue to the answer came from a single amino acid change in the position 377 of RIPK1 (lysine to arginine: RIPK1K377R), a major acceptor site for K63-linked ubiquitin chains. Cells bearing RIPK1K377R are defective in activating NFkB and MAPK pathways and are more susceptible to apoptosis and necroptosis when stimulated by TNF, TLR3, or TLR4 (Deg-

terev et al., 2008; Ea et al., 2006; Feoktistova et al., 2011; Li et al., 2006; O’Donnell et al., 2007; Tenev et al., 2011). Ligation of TNFR1 induces the recruitment of TRADD and RIPK1 and subsequently TRAF2 (Hsu et al., 1995, 1996a, 1996b). Both TRAF2 and RIPK1 (the latter via its kinase-independent scaffold function) recruit the E3 ligases cIAP1 and cIAP2, which in turn add K63-linked ubiquitin (K63-Ub) chains to TRAF2 and RIPK1 (Bertrand et al., 2008; Lee et al., 2004; Varfolomeev et al., 2008; Vince et al., 2009). Likewise, stimulation of TLR3 and TLR4 recruits TRAF6, RIPK1, and IAPs to a complex nucleated by TRIF, in which RIPK1 is also K63 ubiquitinated (Cusson-Hermance et al., 2005). Polyubiquitinated RIPK1 in the K377 position and TRAF2 are the major docking sites for NEMO, TAK1, and TAB2, which activate the NF-kB and MAP kinase signaling pathways, inducing the expression of antiapoptotic molecules, such as FLIP and IAPs (Ea et al., 2006; Li et al., 2006; Micheau et al., 2001; O’Donnell et al., 2007) (Figure 4A). Deubiquitination or destabilization of this complex (called Complex I) favors the formation of a different complex (called Complex II) containing RIPK1, FADD, and caspase-8 or RIPK1, RIPK3, and MLKL (when caspase-8 activity is absent), which induce apoptosis and necroptosis, respectively (Hsu et al., 1996b; Micheau and Tschopp, 2003; Moquin et al., 2013; Moulin et al., 2012; Zhang et al., 2009) (Figure 4). Loss of IAPs, either by genetic ablation, genotoxic stress, or the use of Smac mimetics, compounds that target the IAPs for autodegradation, induces cell death by favoring the formation of complex II (Belz et al., 2014; Feoktistova et al., 2011; Moulin et al., 2012; Tenev et al., 2011). In some contexts in which there is loss of IAPs, the formation of complex II is independent of TNFR1 ligation, and in these cases, the RIPK1-containing complex is termed the ‘‘ripoptosome’’ (Belz et al., 2014; Feoktistova Molecular Cell 56, November 20, 2014 ª2014 Elsevier Inc. 475

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Review et al., 2011; Tenev et al., 2011). Ablation of c-IAP1 plus either c-IAP2 or XIAP promotes E10.5 lethality that is partially rescued (to later embryonically lethal checkpoints) by ablation of RIPK1, RIPK3, or TNFR1 (Moulin et al., 2012). Thus, the IAPs are important for restricting lethality induced by TNFR1-mediated cell death at E10.5. While IAP-mediated ubiquitination of RIPK1 and consequent NF-kB activation can clearly contribute to inflammation, loss of these E3 ligases can also be inflammatory. Both c-IAP1 and c-IAP2, recruited via TRAF2, induce the degradation of NIK, such that when these E3 ligases are inhibited or lost, NIK activates noncanonical NF-kB to trigger production of TNF and other inflammatory cytokines (Gentle et al., 2011; Vallabhapurapu et al., 2008; Varfolomeev et al., 2007; Vince et al., 2007; Zarnegar et al., 2008). Genetic ablation of TRAF2 induces lethality due to overactivation of noncanonical NF-kB, which can be rescued by NIK deficiency (Vallabhapurapu et al., 2008; Zarnegar et al., 2008). RIPK1 deficiency increases NIK activity, most likely through the downregulation of TRAF2 and IAP expression; however, NIK deficiency does not rescue from the postbirth lethality of RIPK1-deficient animals (Dillon et al., 2014), which is in accordance with the fundamental role of RIPK1 in inhibiting both FADD-caspase-8- and RIPK3-MLKLmediated cell death. Ubiquitination of RIPK1 may not be restricted to IAPs activity. Pellino-1 is an E3 ubiquitin ligase that physically binds and adds K63-Ub chains to RIPK1 upon TLR3 or TLR4 ligation (Chang et al., 2009) (Figure 4A). Pellino-1-null mice are resistant to LPS- and Poly (I:C)-induced shock due to lower NF-kB activation and production of proinflammatory cytokines (Chang et al., 2009). The role of Pellino-1 in RIPK1 ubiquitination downstream of TNFR1 is unknown. Another E3 ligase, TRIAD3A, directly interacts with and ubiquitinates RIPK1, most likely with K48-Ub linkage, targeting it for proteasomal degradation (Fearns et al., 2006). Overexpression of TRIAD3A reduces RIPK1-mediated NF-kB activation while increasing the levels of apoptosis in response to TNF (Chen et al., 2002). TRIAD3 also reduces the expression levels of TRIF and TLR4, and therefore the response to LPS, presumably by targeting these proteins for degradation (Chuang and Ulevitch, 2004; Fearns et al., 2006). How TRIAD3A impacts necroptosis has not been assessed. In addition to the K48- and K63-linked ubiquitin chains, RIPK1 (and other components of complex I) can also be modified with Met1-linear ubiquitin chains by LUBAC (Gerlach et al., 2011; Ikeda et al., 2011; Tokunaga et al., 2011) (Figure 4A). Linear ubiquitination further stabilizes the complex, facilitating the recruitment of NEMO and the activation of NF-kB and reducing apoptosis levels (Gerlach et al., 2011; Ikeda et al., 2011; Tokunaga et al., 2011). Sharpin-deficient mice, as noted above, develop a severe inflammatory skin phenotype associated with cell death via necroptosis, which is prevented by homozygous kinase-inactive RIPK1 (Berger et al., 2014). Another component of the ‘‘elixir,’’ which regulates RIPK1 functions, is provided by a set of enzymes that modifies the ubiquitination status of RIPK1, including Cylindromatosis (CYLD), A20/TNFAIP3, and OTULIN/FAM105B (Figures 4B and 4C). CYLD removes K63-Ub chains linked to lysine 377 of RIPK1, 476 Molecular Cell 56, November 20, 2014 ª2014 Elsevier Inc.

facilitating its transition from complex I to complex II (Hitomi et al., 2008; Moquin et al., 2013; O’Donnell et al., 2011). CLIPR-59, an adaptor protein, facilitates the interaction between CYLD and RIPK1 (Fujikura et al., 2012). CYLD-deficient cells are protected from RIPK1-mediated necroptosis in response to TNF plus caspase inhibition with or without Smac mimetic (Hitomi et al., 2008; Moquin et al., 2013; O’Donnell et al., 2011). Moreover, animals expressing a catalytically inactive CYLD are partially protected from the severe skin damage and inflammation produced by conditional deletion of FADD in the skin (Bonnet et al., 2011). CYLD is a substrate for caspase-8, and mutation of the caspase cleavage site of CYLD sensitizes cells for necroptosis induced by TNF even without caspase inhibition (O’Donnell et al., 2011). Interestingly, CYLD is a negative regulator of the RIG-I/MAVS pathway, as silencing of CYLD enhances the IRF3 response to sendai virus (Friedman et al., 2008). Although RIPK1 is involved in RIG-I-induced IRF3 activation, it seems that deubiquitination of RIG-I and TBK-1 accounts for most of the CYLD effect on this pathway (Friedman et al., 2008; Zhang et al., 2008). A20 reduces TNFR1- and TLR-induced RIPK1-mediated NF-kB activation and cell death by converting RIPK1 K63-Ub chains to K48-Ub chains, thus targeting RIPK1 and its associated proteins for proteasomal degradation (He and Ting, 2002; Lin et al., 2008; Wertz et al., 2004). Moreover, A20 can interfere in the E3 ligase activities of cIAP1 by disrupting their interaction with the E2 ubiquitin-conjugating enzymes Ubc13 and UbcH5c, thus decreasing the levels of K63-linked ubiquitination (Shembade et al., 2010). Ablation of A20 results in a late postnatal lethality with extensive inflammation and hypersensitivity to TNF and LPS (Lee et al., 2000); however, the specific role of RIPK1 and its interactions in this effect is yet to be determined. OTULIN, in turn, is a deubiquinating enzyme that specifically hydrolyzes Met1-linked linear ubiquitination added by the LUBAC complex (Fiil et al., 2013; Keusekotten et al., 2013). Ectopic expression of OTULIN decreases NF-kB activation and cytokine production after TNF, LPS, and Poly (I:C) ligation; decreases RIPK1 interaction with NEMO; and sensitizes cells to TNF-induced cell death (Keusekotten et al., 2013). RIPK1 is also modified by other posttranslational events, including phosphorylation and SUMOylation; however their impact on RIPK1 function remains elusive. RIPK1 can be SUMOylated by the ATM kinase and the inhibitor of activated STATy (PIASy) upon etoposide treatment, and this modification seems to be required for optimal NF-kB activation after DNA damage (Yang et al., 2011). RIPK1 SUMOylation and its effects in response to death receptors or TLR ligation are unknown. Phosphorylation of RIPK1 after TNF treatment is a well-documented event. It was proposed that upon phosphorylation, RIPK1 transitions from a closed, inactive conformation to an open, kinase-active conformation and that Necrostatin-1 blocks RIPK1 activity by allosterically blocking its phosphorylation. Indeed, the mutation of the serine 161 to glutamic acid (S161E), which should create an ‘‘open conformation,’’ renders RIPK1 insensitive to necrostatin-1 (Degterev et al., 2008). However, the mutation of this residue to an alanine (S161A) even in combination with a second mutation (S166A), which should

Molecular Cell

Review keep RIPK1 locked in the ‘‘closed conformation,’’ only slightly decreased RIPK1 kinase activity (Degterev et al., 2008; McQuade et al., 2013). Additional mutations of serine residues to alanine were performed, but none were found to block RIPK1 kinase activity (McQuade et al., 2013). The specific roles for RIPK1 phosphorylation (or autophosphorylation) therefore remain an open question. Concluding Remarks Genetic evidence demonstrating that RIPK1 exerts a fundamental role in vivo to suppress cell death and inflammation while also functioning to promote the same processes supports RIPK1 as a ‘‘node’’ in a cell death and inflammation signaling network. And, as with all other such ‘‘nodes,’’ the regulation of RIPK1 expression and function is complex. It involves several layers of modifying enzymes; it is defined by the availability of the other players of this network, and it also depends on the cellular context. We are left, however, with the major question of why this signaling network has been selected to function in this way. Clearly, under normal conditions, the system is balanced such that the catastrophic events seen when RIPK1 is absent are efficiently regulated to allow homeostasis. Two clues to the answer may be the upstream ligands and receptors that trigger the effects and the tissues that are affected. Sensors of infection (e.g., TRIF-associated TLRs, nucleotide sensors) and receptors for inflammatory cytokines (e.g., TNFR, interferon receptors) trigger the effects controlled by RIPK1. Tissues are largely limited to hematopoietic, endothelial, and epithelial cells, all of which represent barriers to infection. It seems likely then that RIPK1, acting as either Dr. Jekyll or Mr. Hyde (depending on its modifications), is a fundamental component in the response to infections. The effects can range from inflammatory responses to cell death (an ultimate control of obligate intracellular infections), depending on the conditions and the extent to which the balance sustained by RIPK1 and its interactions is perturbed. This is an idea that awaits further testing. ‘‘Someday.we may perhaps come to learn the right and wrong of this.’’

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ACKNOWLEDGMENTS

Cook, W.D., Moujalled, D.M., Ralph, T.J., Lock, P., Young, S.N., Murphy, J.M., and Vaux, D.L. (2014). RIPK1- and RIPK3-induced cell death mode is determined by target availability. Cell Death Differ. 21, 1600–1612.

All quotations are from R.L. Stevenson’s ‘‘The Strange Case of Dr. Jekyll and Mr. Hyde.’’ D.R.G. is supported by grants from the U.S. National Institutes of Health and by the American Lebanese Syrian Associated Charities.

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