RIPK3 contributes to TNFR1-mediated RIPK1 kinase-dependent apoptosis in conditions of cIAP1/2 depletion or TAK1 kinase inhibition

Receptor-interacting protein kinase (RIPK) 1 and RIPK3 have emerged as essential kinases mediating a regulated form of necrosis, known as necroptosis, that can be induced by tumor necrosis factor (TNF) signaling. As a consequence, inhibiting RIPK1 kinase activity and repressing RIPK3 expression levels have become commonly used approaches to estimate the contribution of necroptosis to specific phenotypes. Here, we report that RIPK1 kinase activity and RIPK3 also contribute to TNF-induced apoptosis in conditions of cellular inhibitor of apoptosis 1 and 2 (cIAP1/2) depletion or TGF-β-activated kinase 1 (TAK1) kinase inhibition, implying that inhibition of RIPK1 kinase activity or depletion of RIPK3 under cell death conditions is not always a prerequisite to conclude on the involvement of necroptosis. Moreover, we found that, contrary to cIAP1/2 depletion, TAK1 kinase inhibition induces assembly of the cytosolic RIPK1/Fas-associated protein with death domain/caspase-8 apoptotic TNF receptor 1 (TNFR1) complex IIb without affecting the RIPK1 ubiquitylation status at the level of TNFR1 complex I. These results indicate that the recruitment of TAK1 to the ubiquitin (Ub) chains, and not the Ub chains per se, regulates the contribution of RIPK1 to the apoptotic death trigger. In line with this, we found that cylindromatosis repression only provided protection to TNF-mediated RIPK1-dependent apoptosis in condition of reduced RIPK1 ubiquitylation obtained by cIAP1/2 depletion but not upon TAK1 kinase inhibition, again arguing for a role of TAK1 in preventing RIPK1-dependent apoptosis downstream of RIPK1 ubiquitylation. Importantly, we found that this function of TAK1 was independent of its known role in canonical nuclear factor-κB (NF-κB) activation. Our study therefore reports a new function of TAK1 in regulating an early NF-κB-independent cell death checkpoint in the TNFR1 apoptotic pathway. In both TNF-induced RIPK1 kinase-dependent apoptotic models, we found that RIPK3 contributes to full caspase-8 activation independently of its kinase activity or intact RHIM domain. In contrast, RIPK3 participates in caspase-8 activation by acting downstream of the cytosolic death complex assembly, possibly via reactive oxygen species generation.     

TWEAK induces apoptosis through a death-signaling complex comprising receptor-interacting protein 1 (RIP1), Fas-associated death domain (FADD), and caspase-8

The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TNFSF12, CD255) (TWEAK) can stimulate apoptosis in certain cancer cells. Previous studies suggest that TWEAK activates cell death indirectly, by inducing TNFα-mediated autocrine signals. However, the underlying death-signaling mechanism has not been directly defined. Consistent with earlier work, TWEAK assembled a proximal signaling complex containing its cognate receptor FN14, the adaptor TRAF2, and cellular inhibitor of apoptosis protein 1 (cIAP1). Neither the death domain adaptor Fas-associated death domain nor the apoptosis-initiating protease caspase-8 associated with this primary complex. Rather, TWEAK induced TNFα secretion and TNF receptor 1-dependent assembly of a death-signaling complex containing receptor-interacting protein 1 (RIP1), FADD, and caspase-8. Knockdown of RIP1 by siRNA prevented TWEAK-induced association of FADD with caspase-8 but not formation of the FN14-TRAF2-cIAP1 complex and inhibited apoptosis activation. Depletion of the RIP1 E3 ubiquitin ligase cIAP1 enhanced assembly of the RIP1-FADD-caspase-8 complex and augmented cell death. Conversely, knockdown of the RIP1 deubiquitinase CYLD inhibited these functions. Depletion of FADD, caspase-8, BID, or BAX and BAK but not RIP3 attenuated TWEAK-induced cell death. Pharmacologic inhibition of the NF-κB pathway or siRNA knockdown of RelA attenuated TWEAK induction of TNFα and association of RIP1 with FADD and caspase-8. These results suggest that TWEAK triggers apoptosis by promoting assembly of a RIP1-FADD-caspse-8 complex via autocrine TNFα-TNFR1 signaling. The proapoptotic activity of TWEAK is modulated by cIAP1 and CYLD and engages both the extrinsic and intrinsic signaling pathways.     

Caspase inhibition prolongs inflammation by promoting a signaling complex with activated RIPK1

Activation of inflammation by lipopolysaccharide (LPS) is an important innate immune response. Here we investigated the contribution of caspases to the LPS-mediated inflammatory response and discovered distinctive temporal roles of RIPK1 in mediating proinflammatory cytokine production when caspases are inhibited. We propose a biphasic model that differentiates the role of RIPK1 in early versus late phase. The early production of proinflammation cytokines stimulated by LPS with caspase inhibition is mediated by the NF-κB pathway that requires the scaffold function of RIPK1 but is kinase independent. Autocrine production of TNFα in the late phase promotes the formation of a novel TNFR1-associated complex with activated RIPK1, FADD, caspase-8, and key mediators of NF-κB signaling. The production of proinflammatory cytokines in the late phase can be blocked by RIPK1 kinase inhibitor Nec-1s. Our study demonstrates a mechanism by which the activation of RIPK1 promotes its own scaffold function to regulate the NF-κB–mediated proinflammatory cytokine production that is negatively regulated by caspases to restrain inflammatory signaling.     

Fas-associated death domain protein and caspase-8 are not recruited to the tumor necrosis factor receptor 1 signaling complex during tumor necrosis factor-induced apoptosis

Death receptors are a subfamily of the tumor necrosis factor (TNF) receptor subfamily. They are characterized by a death domain (DD) motif within their intracellular domain, which is required for the induction of apoptosis. Fas-associated death domain protein (FADD) is reported to be the universal adaptor used by death receptors to recruit and activate the initiator caspase-8. CD95, TNF-related apoptosis-inducing ligand (TRAIL-R1), and TRAIL-R2 bind FADD directly, whereas recruitment to TNF-R1 is indirect through another adaptor TNF receptor-associated death domain protein (TRADD). TRADD also binds two other adaptors receptor-interacting protein (RIP) and TNF-receptor-associated factor 2 (TRAF2), which are required for TNF-induced NF-kappaB and c-Jun N-terminal kinase activation, respectively. Analysis of the native TNF signaling complex revealed the recruitment of RIP, TRADD, and TRAF2 but not FADD or caspase-8. TNF failed to induce apoptosis in FADD- and caspase-8-deficient Jurkat cells, indicating that these apoptotic mediators were required for TNF-induced apoptosis. In an in vitro binding assay, the intracellular domain of TNF-R1 bound TRADD, RIP, and TRAF2 but did not bind FADD or caspase-8. Under the same conditions, the intracellular domain of both CD95 and TRAIL-R2 bound both FADD and caspase-8. Taken together these results suggest that apoptosis signaling by TNF is distinct from that induced by CD95 and TRAIL. Although caspase-8 and FADD are obligatory for TNF-mediated apoptosis, they are not recruited to a TNF-induced membrane-bound receptor signaling complex as occurs during CD95 or TRAIL signaling, but instead must be activated elsewhere within the cell.     

dsRNA induces apoptosis through an atypical death complex associating TLR3 to caspase-8

Toll-like receptor 3 (TLR3) is a pattern-recognition receptor known to initiate an innate immune response when stimulated by double-stranded RNA (dsRNA). Components of TLR3 signaling, including TIR domain-containing adapter inducing IFN-α (TRIF), have been demonstrated to contribute to dsRNA-induced cell death through caspase-8 and receptor interacting protein (RIP)1 in various human cancer cells. We provide here a detailed analysis of the caspase-8 activating machinery triggered in response to Poly(I:C) dsRNA. Engagement of TLR3 by dsRNA in both type I and type II lung cancer cells induces the formation of an atypical caspase-8-containing complex that is devoid of classical death receptors of the TNFR superfamily, but instead is physically associated to TLR3. The recruitment of caspase-8 to TLR3 requires RIP1, and is negatively modulated by cellular inhibitor of apoptosis protein (cIAP)2-TNF receptor-associated factor (TRAF)2-TNFR-associated death domain (TRADD) ubiquitin ligase complex, which regulates RIP1 ubiquitination. Intriguingly, unlike Fas- or TRAILR-dependent death signaling, caspase-8 recruitment and activation within the TLR3 death-signaling complex appears not to be stringently dependent on Fas-associated with death domain (FADD). Our findings uncover a novel aspect of the molecular mechanisms involved during apoptosis induced by the innate immune receptor TLR3 in cancer cells.     

Survival function of the FADD-CASPASE-8-cFLIP(L) complex

Caspase-8, the initiator caspase of the death receptor pathway of apoptosis, its adapter molecule, FADD, required for caspase-8 activation, and cFLIPL, a caspase-8-like protein that lacks a catalytic site and blocks caspase-8-mediated apoptosis, are each essential for embryonic development. Animals deficient in any of these genes present with E10.5 embryonic lethality. Recent studies have shown that development in caspase-8-deficient mice is rescued by ablation of RIPK3, a kinase that promotes a form of programmed, necrotic cell death. Here, we show that FADD, RIPK3 double-knockout mice develop normally but that the lethal effects of cFLIP deletion are not rescued by RIPK3 deficiency. Remarkably, in mice lacking FADD, cFLIP, and RIPK3, embryonic development is normal. This can be explained by the convergence of two cell processes: the enzymatic activity of the FADD-caspase-8-cFLIPL complex blocks RIPK3-dependent signaling (including necrosis), whereas cFLIPL blocks RIPK3-independent apoptosis promoted by the FADD-caspase-8 complex.     

Protein kinase C modulates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by targeting the apical events of death receptor signaling

We have further examined the mechanism by which phorbol ester-mediated protein kinase C (PKC) activation protects against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. We now report that activation of PKC targets death receptor signaling complex formation. Pre-treatment with 12-O-tetradecanoylphorbol-13-acetate (PMA) led to inhibition of TRAIL-induced apoptosis in HeLa cells, which was characterized by a reduction in phosphatidylserine (PS) externalization, decreased caspase-8 processing, and incomplete maturation and activation of caspase-3. These effects of PMA were completely abrogated by the PKC inhibitor, bisindolylmaleimide I (Bis I), clearly implicating PKC in the protective effect of PMA. TRAIL-induced mitochondrial release of the apoptosis mediators cytochrome c and Smac was blocked by PMA. This, together with the observed decrease in Bid cleavage, suggested that PKC activation modulates apical events in TRAIL signaling upstream of mitochondria. This was confirmed by analysis of TRAIL death-inducing signaling complex formation, which was disrupted in PMA-treated cells as evidenced by a marked reduction in Fas-associated death domain protein (FADD) recruitment, an effect that could not be explained by any change in FADD phosphorylation state. In an in vitro binding assay, the intracellular domains of both TRAIL-R1 and TRAIL-R2 bound FADD: activation of PKC significantly inhibited this interaction suggesting that PKC may be targeting key apical components of death receptor signaling. Significantly, this effect was not confined to TRAIL, because isolation of the native TNF receptor signaling complex revealed that PKC activation also inhibited TNF receptor-associated death domain protein recruitment to TNF-R1 and TNF-induced phosphorylation of IkappaB-alpha. Taken together, these results show that PKC activation specifically inhibits the recruitment of key obligatory death domain-containing adaptor proteins to their respective membrane-associated signaling complexes, thereby modulating TRAIL-induced apoptosis and TNF-induced NF-kappaB activation, respectively.     

Competitive control of independent programs of tumor necrosis factor receptor-induced cell death by TRADD and RIP1

Stimulation of tumor necrosis factor receptor 1 (TNFR1) can initiate several cellular responses, including apoptosis, which relies on caspases, necrotic cell death, which depends on receptor-interacting protein kinase 1 (RIP1), and NF-kappaB activation, which induces survival and inflammatory responses. The TNFR-associated death domain (TRADD) protein has been suggested to be a crucial signal adaptor that mediates all intracellular responses from TNFR1. However, cells with a genetic deficiency of TRADD are unavailable, precluding analysis with mature immune cell types. We circumvented this problem by silencing TRADD expression with small interfering RNA. We found that TRADD is required for TNFR1 to induce NF-kappaB activation and caspase-8-dependent apoptosis but is dispensable for TNFR1-initiated, RIP1-dependent necrosis. Our data also show that TRADD and RIP1 compete for recruitment to the TNFR1 signaling complex and the distinct programs of cell death. Thus, TNFR1-initiated intracellular signals diverge at a very proximal level by the independent association of two death domain-containing proteins, RIP1 and TRADD. These single transducers determine cell fate by triggering NF-kappaB activation, apoptosis, and nonapoptotic death signals through separate and competing signaling pathways.     

Impaired RIPK1 ubiquitination sensitizes mice to TNF toxicity and inflammatory cell death

Receptor-interacting protein 1 (RIP1; RIPK1) is a key regulator of multiple signaling pathways that mediate inflammatory responses and cell death. TNF-TNFR1 triggered signaling complex formation, subsequent NF-κB and MAPK activation and induction of cell death involve RIPK1 ubiquitination at several lysine residues including Lys376 and Lys115. Here we show that mutating the ubiquitination site K376 of RIPK1 (K376R) in mice activates cell death resulting in embryonic lethality. In contrast to Ripk1^K376R/K376R mice, Ripk1^K115R/K115R mice reached adulthood and showed slightly higher responsiveness to TNF-induced death. Cell death observed in Ripk1^K376R/K376R embryos relied on RIPK1 kinase activity as administration of RIPK1 inhibitor GNE684 to pregnant heterozygous mice effectively blocked cell death and prolonged survival. Embryonic lethality of Ripk1^K376R/K376R mice was prevented by the loss of TNFR1, or by simultaneous deletion of caspase-8 and RIPK3. Interestingly, elimination of the wild-type allele from adult Ripk1^K376R/cko mice was tolerated. However, adult Ripk1^K376R/cko mice were exquisitely sensitive to TNF-induced hypothermia and associated lethality. Absence of the K376 ubiquitination site diminished K11-linked, K63-linked, and linear ubiquitination of RIPK1, and promoted the assembly of death-inducing cellular complexes, suggesting that multiple ubiquitin linkages contribute to the stability of the RIPK1 signaling complex that stimulates NF-κB and MAPK activation. In contrast, mutating K115 did not affect RIPK1 ubiquitination or TNF stimulated NF-κB and MAPK signaling. Overall, our data indicate that selective impairment of RIPK1 ubiquitination can lower the threshold for RIPK1 activation by TNF resulting in cell death and embryonic lethality.Subject terms: Acute inflammation, Chronic inflammation     

TNF-alpha induces two distinct caspase-8 activation pathways

The inflammatory response of mammalian cells to TNF-alpha can be switched to apoptosis either by cotreatment with a protein synthesis inhibitor, cycloheximide, or Smac mimetic, a small molecule mimic of Smac/Diablo protein. Cycloheximide promotes caspase-8 activation by eliminating endogenous caspase-8 inhibitor, c-FLIP, while Smac mimetic does so by triggering autodegradation of cIAP1 and cIAP2 (cIAP1/2), leading to the release of receptor interacting protein kinase (RIPK1) from the activated TNF receptor complex to form a caspase-8-activating complex consisting of RIPK1, FADD, and caspase-8. This process also requires the action of CYLD, a RIPK1 K63 deubiquitinating enzyme. RIPK1 is critical for caspase-8 activation-induced by Smac mimetic but dispensable for that triggered by cycloheximide. Moreover, Smac mimetic-induced caspase-8 activation is not blocked by endogenous c-FLIP. These findings revealed that TNF-alpha is able to induce apoptosis via two distinct caspase-8 activation pathways that are differentially regulated by cIAP1/2 and c-FLIP.     

TNFR1-induced activation of the classical NF-κB pathway

The molecular mechanisms underlying activation of the IκB kinase (IKK) complex are presumably best understood in the context of tumor necrosis factor (TNF) receptor-1 (TNFR1) signaling. In fact, it seems that most, if not all, proteins relevant for this process have been identified and extensive biochemical and genetic data are available for the role of these factors in TNF-induced IKK activation. There is evidence that protein modification-independent assembly of a core TNFR1 signaling complex containing TNFR1-associated death domain, receptor interacting kinase?1, TNF receptor-associated factor?2 and cellular inhibitor of apoptosis protein?1 and 2 starts a chain of nondegrading ubiquitination events that culminate in the recruitment and activation of IKK complex-stimulating kinases and the IKK complex itself. Here, we sum up the known details of TNFR1-induced IKK activation, address arising contradictions and discuss possible explanations resolving the apparent discrepancies.     

RIPK1- and RIPK3-induced cell death mode is determined by target availability

Both receptor-interacting protein kinase 1 (RIPK1) and RIPK3 can signal cell death following death receptor ligation. To study the requirements for RIPK-triggered cell death in the absence of death receptor signaling, we engineered inducible versions of RIPK1 and RIPK3 that can be activated by dimerization with the antibiotic coumermycin. In the absence of TNF or other death ligands, expression and dimerization of RIPK1 was sufficient to cause cell death by caspase- or RIPK3-dependent mechanisms. Dimerized RIPK3 induced cell death by an MLKL-dependent mechanism but, surprisingly, also induced death mediated by FADD, caspase 8 and RIPK1. Catalytically active RIPK3 kinase domains were essential for MLKL-dependent but not for caspase 8-dependent death. When RIPK1 or RIPK3 proteins were dimerized, the mode of cell death was determined by the availability of downstream molecules such as FADD, caspase 8 and MLKL. These observations imply that rather than a ‘switch'' operating between the two modes of cell death, the final mechanism depends on levels of the respective signaling and effector proteins.Mammalian cells can use a number of mechanisms to kill themselves. The best characterized depends on the Bcl-2 family members Bax and Bak that work via mitochondria to activate caspases.¹ Some caspases, notably caspase 8, can be activated independently of Bcl-2 family members, for example, after stimulation of members of the TNF receptor superfamily.² Recently, it has become apparent that some of these receptors, including TNFR1, can activate a third suicide mechanism that does not require caspases, and in which the morphology of the dying cell differs from classical apoptosis. This form of cell death, termed ‘necroptosis'', can often be blocked by necrostatin-1 (nec-1), an inhibitor of the kinase activity of receptor-interacting protein kinase 1 (RIPK1).^{3, 4} Accordingly, observations from several groups have shown that in some cell types, expression of RIPK1 can signal cell death by caspase-independent necroptosis.⁵It has previously been revealed that RIPK1 could function downstream of death receptors, but in those cases, cell death was usually blocked by coexpression of the viral inhibitor of caspases 1 and 8, CrmA,⁶ and typically exhibited a classical ‘apoptotic'' morphology. It was revealed that RIPK1 engages FADD via homotypic binding of their death domains (DDs), and FADD in turn activates caspase 8.^{6, 7}RIPK3, like RIPK1, bears a kinase domain and RIP homology interaction motif (RHIM), but unlike RIPK1 does not have a DD.^{8, 9, 10, 11} RIPK3 is required for necroptosis.^{12, 13} Furthermore, RIPK1 appears to activate RIPK3 in this pathway, as cell death could be blocked by nec-1.¹⁴ RIPK3 activates, by phosphorylation, MLKL, a pseudokinase essential for this death pathway.^{15, 16, 17} Once activated, MLKL forms multimers that trigger breaches of the plasma membrane.^{18, 19, 20}Although RIPK3 is necessary for necroptosis, it is unclear whether activation of RIPK3 is sufficient for cell death, because TNF activates signaling by many pathways in addition to those controlled by RIPK1.²¹ It is also unclear whether RIPK3 can contribute to apoptosis. Despite some reports to this effect,^{8, 9, 22} RIPK3 has been described as the necroptotic ‘switch'', implying its activity precipitates necroptosis to the exclusion of apoptosis.^{23, 24, 25}Here, we have directly activated RIP kinases without the confounding effects of multiple signals emanating from the target cell''s cytokine receptors, allowing us to define more precisely the functions of RIPK1 and RIPK3. We activated RIP kinases by dimerization using inducible lentiviral vectors, each encoding a chimera of a RIP kinase with subunit B of E. coli DNA gyrase.²⁶ We infected mouse embryonic fibroblasts (MEFs) that lack genes for, or expression of, various cell death proteins, induced expression of the RIPK chimera, caused its dimerization by addition of the divalent antibiotic coumermycin (C) and quantitated the resulting cell death.Our results reveal that each of RIPK1 and RIPK3 can contribute to both apoptosis and necroptosis depending on the biochemical context. Furthermore, necroptosis can occur in the absence of caspase 8 and FADD, which shows that the ripoptosome, with core components caspase 8, FADD and RIPK1,^{27, 28} is not required for necroptosis. Instead, we propose that dimers of RIPK1 and/or RIPK3 are the pivotal complexes from which both forms of cell death can progress.     

The adaptor protein TRADD activates distinct mechanisms of apoptosis from the nucleus and the cytoplasm

TNFR1 associated death domain protein (TRADD) contains an N-terminal TRAF binding domain and a C-terminal death domain along with nuclear import and export sequences that cause shuttling between the cytoplasm and nucleus. The death domain of TRADD contains the nuclear import sequence and expression of the core death domain (nuclear TRADD) results in exclusive nuclear localization and activation of a distinct apoptotic pathway. Cytoplasmic TRADD activates apoptosis through Fas-associated death domain protein (FADD) and caspase-8 activation that was blocked by caspase inhibitors or dominant-negative FADD. These inhibitors did not inhibit death induced by nuclear TRADD, which could only be inhibited by combining caspase inhibitors and a serine protease inhibitor. The pathway activated by nuclear TRADD requires caspase-9 catalytic activity. However, apoptosis activating factor deficiency confers only partial protection from death. This pathway represents an alternate means by which TRADD can regulate cell death independently of FADD and caspase-8 that occurs from the nucleus rather than the cytoplasm.     

Fas- and tumor necrosis factor-mediated apoptosis uses the same binding surface of FADD to trigger signal transduction. A typical model for convergent signal transduction

FADD is known to function as a common signaling conduit in Fas- and tumor necrosis factor (TNF)-mediated apoptosis. The convergent death signals from the Fas receptor and TNF receptor 1 are transferred to FADD by death domain interactions triggering the same cellular event, caspase-8 activation. In this work, we investigated whether the same binding surface of FADD is used for both signaling pathways by using FADD death domain mutants. Mutations in helices alpha2 and alpha3 of the FADD death domain, the interacting surface with the Fas death domain, affected TNF-mediated apoptosis to various extents. This indicated that TNF-mediated apoptosis uses the same binding surface of the FADD death domain as Fas-mediated apoptosis. The binding specificity is not the same, however. Some mutations affected the binding affinity of the Fas death domain for the FADD death domain, but did not influence TNF-mediated apoptosis and vice versa. Interestingly, all mutants tested that affected TNF-mediated apoptosis have structural perturbations, implying that the structural integrity, involving helices alpha2 and alpha3 in particular, is critical in TNF-mediated apoptosis. Our results suggest that different signaling molecules use a similar structural interaction to trigger the same cellular event, such as caspase-8 recruitment, which could be typical in convergent signal transduction.     

Protein kinase C regulates FADD recruitment and death-inducing signaling complex formation in Fas/CD95-induced apoptosis

Activation of protein kinase C (PKC) triggers cellular signals that inhibit Fas/CD95-induced cell death in Jurkat T-cells by poorly defined mechanisms. Previously, we have shown that one effect of PKC on Fas/CD95-dependent cell death occurs through inhibition of cell shrinkage and K(+) efflux (Gómez-Angelats, M., Bortner, C. D., and Cidlowski, J. A. (2000) J. Biol. Chem. 275, 19609-19619). Here we report that PKC alters Fas/CD95 signaling from the plasma membrane to the activation of caspases by exerting a profound action on survival/cell death decisions. Specific activation of PKC with 12-O-tetradecanoylphorbol-13-acetate or bryostatin-1 induced translocation of PKC from the cytosol to the membrane and effectively inhibited cell shrinkage and cell death triggered by anti-Fas antibody in Jurkat cells. In contrast, inhibition of classical PKC isotypes with G?6976 exacerbated the effect of Fas activation on both apoptotic volume decrease and cell death. PKC activation/inhibition did not affect anti-Fas antibody binding to the cell surface, intracellular levels of FADD (Fas-associated protein with death domain), or c-FLIP (cellular FLICE-like inhibitory protein) expression. However, processing/activation of both caspase-8 and caspase-3 and BID cleavage were markedly blocked upon PKC activation and, conversely, were augmented during PKC inhibition, suggesting a role for PKC upstream of caspase-8 processing and activation. Analysis of death-inducing signaling complex (DISC) formation was carried out to examine the influence of PKC on recruitment of both FADD and procaspase-8 to the Fas receptor. PKC activation blocked FADD recruitment and caspase-8 activation and thus DISC formation in both type I and II cells. In contrast, inhibition of classical PKCs promoted the opposite effect on the Fas pathway by rapidly increasing FADD recruitment, caspase-8 activation, and DISC formation. Together, these data show that PKC finely modulates Fas/CD95 signaling by altering the efficiency of DISC formation.     

Antioxidant and food additive BHA prevents TNF cytotoxicity by acting as a direct RIPK1 inhibitor

Butylate hydroxyanisole (BHA) is a synthetic phenol that is widely utilized as a preservative by the food and cosmetic industries. The antioxidant properties of BHA are also frequently used by scientists to claim the implication of reactive oxygen species (ROS) in various cellular processes, including cell death. We report on the surprising finding that BHA functions as a direct inhibitor of RIPK1, a major signaling hub downstream of several immune receptors. Our in silico analysis predicts binding of 3-BHA, but not 2-BHA, to RIPK1 in an inactive DLG-out/Glu-out conformation, similar to the binding of the type III inhibitor Nec-1s to RIPK1. This predicted superior inhibitory capacity of 3-BHA over 2-BHA was confirmed in cells and using in vitro kinase assays. We demonstrate that the reported protective effect of BHA against tumor necrosis factor (TNF)-induced necroptotic death does not originate from ROS scavenging but instead from direct RIPK1 enzymatic inhibition, a finding that most probably extends to other reported effects of BHA. Accordingly, we show that BHA not only protects cells against RIPK1-mediated necroptosis but also against RIPK1 kinase-dependent apoptosis. We found that BHA treatment completely inhibits basal and induced RIPK1 enzymatic activity in cells, monitored at the level of TNFR1 complex I under apoptotic conditions or in the cytosol under necroptosis. Finally, we show that oral administration of BHA protects mice from RIPK1 kinase-dependent lethality caused by TNF injection, a model of systemic inflammatory response syndrome. In conclusion, our results demonstrate that BHA can no longer be used as a strict antioxidant and that new functions of RIPK1 may emerge from previously reported effects of BHA.Subject terms: Kinases, Apoptosis, Necroptosis, Target identification, Cell death and immune response     

Stat1 as a component of tumor necrosis factor alpha receptor 1-TRADD signaling complex to inhibit NF-kappaB activation

Activated tumor necrosis factor alpha (TNF-alpha) receptor 1 (TNFR1) recruits TNFR1-associated death domain protein (TRADD), which in turn triggers two opposite signaling pathways leading to caspase activation for apoptosis induction and NF-kappaB activation for antiapoptosis gene upregulation. Here we show that Stat1 is involved in the TNFR1-TRADD signaling complex, as determined by employing a novel antibody array screening method. In HeLa cells, Stat1 was associated with TNFR1 and this association was increased with TNF-alpha treatment. TNFR1 signaling factors TRADD and Fas-associated death domain protein (FADD) were also found to interact with Stat1 in a TNF-alpha-dependent process. Our in vitro recombinant protein-protein interaction studies demonstrated that Stat1 could directly interact with TNFR1 and TRADD but not with FADD. Interaction between Stat1 and receptor-interacting protein (RIP) or TNFR-associated factor 2 (TRAF2) was not detected. Examination of Stat1-deficient cells showed an apparent increase in TNF-alpha-induced TRADD-RIP and TRADD-TRAF2 complex formation, while interaction between TRADD and FADD was unaffected. As a consequence, TNF-alpha-mediated I-kappaB degradation and NF-kappaB activation were markedly enhanced in Stat1-deficient cells, whereas overexpression of Stat1 in 293T cells blocked NF-kappaB activation by TNF-alpha. Thus, Stat1 acts as a TNFR1-signaling molecule to suppress NF-kappaB activation.