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.     

Focal adhesion kinase determines the fate of death or survival of cells in response to TNFα in the presence of actinomycin D

We speculated that focal adhesion kinase (FAK) might play a critical role in the TNFα-induced cell death. In this study, we found that FAK−/− cells are more sensitive to TNFα-induced apoptosis in the presence of actinomycin D (Act D) compared to FAK+/− cells. Prosurvival pathways are activated by the rapid recruitment of complex I, comprising TNFR1, TRADD, RIP and TRAF2, which leads to the activation of the NF-κB pathway. On the other hand, proapoptotic pathways are activated by complex II, the death-inducing signaling complex (DISC), which contains TNFR1, TRADD, RIP, and FADD, and procaspase-8 proteins. As TNFR1, TRADD, and RIP are included in both Complex I and DISC, we speculated that RIP might be a key protein. Coimmunoprecipitation assays revealed that RIP is included in complex I in FAK+/− cells, and FAK was associated with RIP. On the other hand, RIP is included in DISC in FAK−/− cells. FAK might be a key protein in the formation of complex I and the activation of NF-κB. Furthermore, Akt was activated in FAK+/− cells, but not FAK−/− cells. In conclusion, we first demonstrated that FAK determines the pathway leading to death or survival in TNFα/ActD-stimulated fibroblasts.     

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.     

Tumor necrosis factor-induced nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation

The mechanism of tumor necrosis factor (TNF)-induced nonapoptotic cell death is largely unknown, although the mechanism of TNF-induced apoptosis has been studied extensively. In wild-type mouse embryonic fibroblast cells under a caspase-inhibited condition, TNF effectively induced cell death that morphologically resembled necrosis. In this study, we utilized gene knockout mouse embryonic fibroblasts cells and found that tumor necrosis factor receptor (TNFR) I mediates TNF-induced necrotic cell death, and that RIP, FADD, and TRAF2 are critical components of the signaling cascade of this TNF-induced necrotic cell death. Inhibitors of NF-kappaB facilitated TNF-induced necrotic cell death, suggesting that NF-kappaB suppresses the necrotic cell death pathway. JNK, p38, and ERK activation seem not to be required for this type of cell death because mitogen-activated protein kinase inhibitors did not significantly affect TNF-induced necrotic cell death. In agreement with the previous reports that the reactive oxygen species (ROS) may play an important role in this type of cell death, the ROS scavenger butylated hydroxyanisole efficiently blocked TNF-induced necrotic cell death. Interestingly, during TNF-induced necrotic cell death, the cellular ROS level was significantly elevated in wild type, but not in RIP(-/-), TRAF2(-/-), and FADD(-/-) cells. These results suggest that RIP, TRAF2, and FADD are crucial in mediating ROS accumulation in TNF-induced necrotic cell death.     

Apoptosis-inducing agents cause rapid shedding of tumor necrosis factor receptor 1 (TNFR1). A nonpharmacological explanation for inhibition of TNF-mediated activation

Several chemical compounds not known to interact with tumor necrosis factor (TNF) signal transducing proteins inhibit TNF-mediated activation of vascular endothelial cells (EC). Four structurally diverse agents, arachidonyl trifluoromethylketone, staurosporine, sodium salicylate, and C6-ceramide, were studied. All four agents caused EC apoptosis at concentrations that inhibited TNF-induced IkappaBalpha degradation. However, evidence of apoptosis was not evident until after several (e.g. 3-12) hours of treatment, whereas 2 h of treatment was sufficient to inhibit TNF responses. IL-1-induced IkappaBalpha degradation was unaffected by these treatments. Inhibition of TNF signaling could not be prevented with either of the broad spectrum caspase inhibitors zVADfmk or yVADcmk. The inhibition of p38 kinase with SB203580 prevented the inhibition of TNF signaling by all agents except arachidonyl trifluoromethylketone. No changes in the levels or molecular weights of the adaptor proteins TRADD (TNF receptor-associated death domain), RIP (receptor-interacting protein), or TRAF2 (TNF receptor-associated factor-2) were caused by apoptogenic drugs. However, TNF receptor 1 (TNFR1) surface expression was significantly reduced by all four agents. Furthermore, TNF-dependent recruitment of TRADD to surface TNFR1 was also inhibited. These data suggest that several putative inhibitors of TNF signaling work by triggering apoptosis and that an early event coincident with the initiation of apoptosis, preceding evidence of injury, is loss of TNFR1. Consistent with this hypothesis, cotreatment of EC with the metalloproteinase inhibitor Tapi (TNF-alpha proteinase inhibitor) blocked the reduction in surface TNFR1 by apoptogenic drugs and prevented inhibition of TNF-induced IkappaBalpha degradation without blocking apoptosis. TNFR1 loss could be a mechanism to limit inflammation in response to apoptotic cell death.     

Focal adhesion kinase determines the fate of death or survival of cells in response to TNFalpha in the presence of actinomycin D

We speculated that focal adhesion kinase (FAK) might play a critical role in the TNFalpha-induced cell death. In this study, we found that FAK-/- cells are more sensitive to TNFalpha-induced apoptosis in the presence of actinomycin D (Act D) compared to FAK+/- cells. Prosurvival pathways are activated by the rapid recruitment of complex I, comprising TNFR1, TRADD, RIP and TRAF2, which leads to the activation of the NF-kappaB pathway. On the other hand, proapoptotic pathways are activated by complex II, the death-inducing signaling complex (DISC), which contains TNFR1, TRADD, RIP, and FADD, and procaspase-8 proteins. As TNFR1, TRADD, and RIP are included in both Complex I and DISC, we speculated that RIP might be a key protein. Coimmunoprecipitation assays revealed that RIP is included in complex I in FAK+/- cells, and FAK was associated with RIP. On the other hand, RIP is included in DISC in FAK-/- cells. FAK might be a key protein in the formation of complex I and the activation of NF-kappaB. Furthermore, Akt was activated in FAK+/- cells, but not FAK-/- cells. In conclusion, we first demonstrated that FAK determines the pathway leading to death or survival in TNFalpha/ActD-stimulated fibroblasts.     

Solution structure of N-TRADD and characterization of the interaction of N-TRADD and C-TRAF2, a key step in the TNFR1 signaling pathway

TRADD is a multifunctional signaling adaptor protein that is recruited to TNFR1 upon ligand binding. The C-terminal of TRADD comprises the "death domain" that is responsible for association of TNFR1 and other death domain-containing proteins such as FADD and RIP. The N-terminal domain (N-TRADD) promotes the recruitment of TRAF2 to TNFR1 by binding to the C-terminal of TRAF2, leading to the activation of JNK/AP1 and NF-kappa B. The solution structure of N-TRADD was determined, revealing a novel protein fold. A combination of NMR, BIAcore, and mutagenesis experiments was used to help identify the site of interaction of N-TRADD with C-TRAF2, providing a framework for future attempts to selectively inhibit the TNF signaling pathways.     

A role for tumor necrosis factor receptor-2 and receptor-interacting protein in programmed necrosis and antiviral responses

Members of the tumor necrosis factor (TNF) receptor (TNFR) superfamily are potent regulators of apoptosis, a process that is important for the maintenance of immune homeostasis. Recent evidence suggests that TNFR-1 and Fas and TRAIL receptors can also trigger an alternative form of cell death that is morphologically distinct from apoptosis. Because distinct molecular components including the serine/threonine protein kinase receptor-interacting protein (RIP) are required, we have referred to this alternative form of cell death as "programmed necrosis." We show that TNFR-2 signaling can potentiate programmed necrosis via TNFR-1. When cells were pre-stimulated through TNFR-2 prior to subsequent activation of TNFR-1, enhanced cell death and recruitment of RIP to the TNFR-1 complex were observed. However, TNF-induced programmed necrosis was normally inhibited by caspase-8 cleavage of RIP. To ascertain the physiological significance of RIP and programmed necrosis, we infected Jurkat cells with vaccinia virus (VV) and found that VV-infected cells underwent programmed necrosis in response to TNF, but deficiency of RIP rescued the infected cells from TNF-induced cytotoxicity. Moreover, TNFR-2-/- mice exhibited reduced inflammation in the liver and defective viral clearance during VV infection. Interestingly, death effector domain-containing proteins such as MC159, E8, K13, and cellular FLIP, but not the apoptosis inhibitors Bcl-xL, p35, and XIAP, potently suppressed programmed necrosis. Thus, TNF-induced programmed necrosis is facilitated by TNFR-2 signaling and caspase inhibition and may play a role in controlling viral infection.     

Sam68 is required for both NF-κB activation and apoptosis signaling by the TNF receptor

The RNA-binding protein Sam68 is implicated in various cellular processes including RNA metabolism, apoptosis, and signal transduction. Here we identify a role of Sam68 in TNF-induced NF-κB activation and apoptosis. We found that Sam68 is recruited to the TNF receptor, and its deficiency dramatically reduces RIP recruitment and ubiquitylation. It also impairs cIAP1 recruitment and maintenance of recruited TRAF2 at the TNF receptor. In its absence, activation of the TAK1-IKK kinase complex is defective, greatly reducing signal transduction. Sam68 is also found as a part of the TNF-induced cytoplasmic caspase-8-FADD complex. RIP is not recruited to this complex in Sam68 knockout cells, and caspase activation is virtually absent. These findings delineate previously unknown functions for Sam68 in the TNF signaling pathway, where it acts as a signaling adaptor both in the membrane-associated complex I and in the cytoplasmic complex II, regulating both NF-κB activation and apoptosis.     

AIP1/DAB2IP, a novel member of the Ras-GAP family, transduces TRAF2-induced ASK1-JNK activation

Previously we have shown that ASK-interacting protein 1 (AIP1, also known as DAB2IP), a novel member of the Ras-GAP protein family, mediates TNF-induced activation of ASK1-JNK signaling pathway. However, the mechanism by which TNF signaling is coupled to AIP1 is not known. Here we show that AIP1 is localized on the plasma membrane in resting endothelial cells (EC) in a complex with TNFR1. TNF binding induces release of AIP1 from TNFR1, resulting in cytoplasmic translocation and concomitant formation of an intracellular signaling complex comprised of TRADD, RIP1, TRAF2, and AIPl. A proline-rich region (amino acids 796-807) is critical for maintaining AIP1 in a closed form, which associates with a region of TNFR1 distinct from the death domain, the site of TNFR1 association with TRADD. An AIP1 mutant with deletion of this proline-rich region constitutively binds to TRAF2 and ASK1. A PERIOD-like domain (amino acids 591-719) of AIP1 binds to the intact RING finger of TRAF2, and specifically enhances TRAF2-induced ASK1 activation. At the same time, the binding of AIP1 to TRAF2 inhibits TNF-induced IKK-NF-kappaB signaling. Taken together, our data suggest that AIP1 is a novel transducer in TNF-induced TRAF2-dependent activation of ASK1 that mediates a balance between JNK versus NF-kappaB signaling.     

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.     

TNF受体家族介导的细胞凋亡信号转导

肿瘤坏死因子（TNF）家族是一类多功能的细胞因子,具有诱导细胞凋亡、抗病毒、免疫调节等多种生物学活性.其中一些成员可以通过和细胞膜上相应受体（即TNF受体家族成员）结合,启动细胞内的凋亡机制,而诱导细胞凋亡.一些蛋白质（如TRADD、FADD、RIP、RAIDD等）参与这些信号传递过程.越来越多的TNF家族成员、TNF受体以及与细胞凋亡相关的Caspase蛋白酶家族成员被人们发现.     

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.     

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.     

Regulatory roles and molecular signaling of TNF family members in osteoclasts

The tumor necrosis factor (TNF) family has been one of the most intensively studied families of proteins in the past two decades. The TNF family constitutes 19 members that mediate diverse biological functions in a variety of cellular systems. The TNF family members regulate cellular functions through binding to membrane-bound receptors belonging to the TNF receptor (TNFR) family. Members of the TNFR family lack intrinsic kinase activity and thus they initiate signaling by interacting intracellular signaling molecules such as TNFR associated factor (TRAF), TNFR associated death domain (TRADD) and Fas-associated death domain (FADD). In bone metabolism, it has been shown that numerous TNF family members including receptor activator of nuclear factor kappaB ligand (RANKL), TNF-alpha, Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) play pivotal roles in the differentiation, function, survival and/or apoptosis of osteoclasts, the principal bone-resorbing cells. These TNF family members not only regulate physiological bone remodeling but they are also implicated in the pathogenesis of various bone diseases such as osteoporosis and bone loss in inflammatory conditions. This review will focus on our current understanding of the regulatory roles and molecular signaling of these TNF family members in osteoclasts.     

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.