Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5

Fas (APO-1/CD95) and tumor necrosis factor receptor 1 (TNFR1) trigger apoptosis by recruiting the apoptosis initiator caspase-8 through the adaptor FADD. Fas binds FADD directly, whereas TNFR1 binds FADD indirectly, through TRADD. TRADD alternatively recruits the NF-kappaB-inducing adaptor RIP. The TNF homolog Apo2L/TRAIL triggers apoptosis through two distinct death receptors, DR4 and DR5; however, receptor over-expression studies have yielded conflicting results on the ligand's signaling mechanism. Apo2L/TRAIL induced homomeric and heteromeric complexes of DR4 and DR5 and stimulated recruitment of FADD and caspase-8 and caspase-8 activation in nontransfected cells. TRADD and RIP, which bound TNFR1, did not bind DR4 and DR5. Thus, Apo2L/TRAIL and FasL initiate apoptosis through similar mechanisms, and FADD may be a universal adaptor for death receptors.     

The A20 protein interacts with the Epstein-Barr virus latent membrane protein 1 (LMP1) and alters the LMP1/TRAF1/TRADD complex.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) interacts with the tumor necrosis factor receptor (TNFR)-associated factor (TRAF) molecules, which are important for LMP1-mediated signaling. Two domains of LMP1 can independently activate NF-kB, carboxyl-terminal activating region 1 (CTAR1) and CTAR2. The activation of NF-kB by CTAR1 occurs through direct interaction of LMP1 with the TRAF molecules, whereas CTAR2 interacts with the TNFR-associated death domain protein (TRADD) to activate NF-kB and the c-Jun N-terminal kinase (JNK). A20, which is induced by LMP1 through NF-kB, can block NF-kB activation from both domains of LMP1 and inhibit JNK activation from CTAR2. A20 also has been shown to associate with TRAF1 and TRAF2. In this study, an interaction between LMP1 and A20 was detected that was increased by TRAF2 overexpression. A20 did not affect the association of TRAF1 with TRAF2 but did displace TRAF1 from the LMP1 complex. The interaction of LMP1 and TRADD was decreased in the presence of A20, and the LMP1-A20 association was decreased by TRADD, suggesting that A20 and TRADD both interact with LMP1 and may compete for binding. These data indicate that A20 alters the interactions between LMP1 and the TRAF molecules and TRADD, affecting the activation of NF-kB, JNK, and perhaps other TRAF-mediated signaling events.     

Characterization of DISC formation and TNFR1 translocation to mitochondria in TNF-α-treated hepatocytes

Tumor necrosis factor receptor 1 (TNFR1) activation in hepatocytes can trigger apoptotic or inflammatory signaling. The factors that determine which signaling pathway dominates are not clear and are thought to relate to the efficiency of death-inducing signaling complex (DISC) formation. However, the steps involved in DISC formation in hepatocytes are poorly understood. In characterizing DISC formation within cultured hepatocytes, we demonstrated that TNF-α exposure leads to the rapid formation of a DISC involving TNF-α, the TNFR-associated death domain adaptor molecule (TRADD), the Fas-associated death domain adaptor molecule (FADD), caspase-8, TNFR-associated factor 2 (TRAF2), and receptor-interacting protein (RIP). The inclusion of the sensitizing agent actinomycin D both accelerated and amplified the appearance of the DISC. Notably, TNFR1 along with some DISC components also appeared within mitochondria within 30 minutes. Whereas TNFR1 consistently co-localized with the TRADD, FADD, the caspase-8, and TRAF2 in the cytosolic fraction, TNFR1 in the mitochondria was associated only with caspase-8 after TNF-α exposure. Similar observations were made in vivo using TNF-α with D-galactosamine. Actinomycin D alone also enhanced the appearance of DISC components in both cytosol and the mitochondria. Thus the DISC that includes TNFR1 forms in the cytosol of hepatocytes under both survival and pro-apoptotic conditions. The observations also suggest that TNF-α-mediated signaling includes the translocation of TNFR1 to mitochondria.     

Sensitivity of TLR4- and -7-induced NF kappa B1 p105-TPL2-ERK pathway to TNF-receptor-associated-factor-6 revealed by RNAi in mouse macrophages

Tumor necrosis factor (TNF)-receptor-associated-factor-6 (TRAF6) is an adaptor protein involved in Toll-like receptor (TLR) signaling. Recent studies using macrophages from TRAF6 knockout mice have revealed that TRAF6 is required for TLR7 signaling. However, an essential role of TRAF6 in TLR4 signaling and cytokine production is slightly controversial. Using an RNAi approach to reduce the cellular levels of TRAF6, we tested the role of this adaptor protein on the sensitivity of the various components of the ERK pathway mediated by TLR4 and -7 in Raw264.7, a mouse macrophage cell line. ERK activation in macrophages by TLR4 and -7 is mediated via a MAP3K, called TPL2/COT, which under unstimulated conditions is associated with NF kappa B1 p105, a member of the I kappa B family of proteins. Upon stimulation with TLR ligands, p105 is phosphorylated by I kappa B kinase (IKK) complex and partially degraded, which releases TPL2. The free TPL2 is active and stimulates the ERK pathway via MEK1/2. The free TPL2, however, is also unstable and is targeted for degradation. We demonstrate here that reduced level of TRAF6 ( approximately 80% decrease) in macrophages does not significantly affect any of the components of the TLR4-stimulated ERK pathway, including p105 phosphorylation, TPL2 degradation and ERK1/2 phosphorylation. Surprisingly, however, TLR4-induced JNK1/2 phosphorylation is significantly blocked by TRAF6 knockdown, suggesting that ERK and JNK pathways are differentially sensitive to TRAF6 levels. Furthermore, although TLR4-mediated IKK-induced p105 phosphorylation is not sensitive to TRAF6 knockdown, I kappa B alpha phosphorylation (also, IKK-induced) is significantly blocked, suggesting that TLR4 activation results in a TRAF6-sensitive and -insensitive IKK activation in macrophages. In contrast to TLR4 signaling, TLR7 activation of ERK, JNK pathways and phosphorylation of p105 and I kappa B alpha are completely inhibited in TRAF6 knockdown cells. Compared to the signaling data, while TLR4-induced TNFalpha mRNA expression is not significantly inhibited by TRAF6 knockdown, TLR7-induced TNFalpha mRNA is significantly blocked. In contrast, both TLR4- and TLR7-induced IL6 mRNA are significantly blocked by TRAF6 knockdown. These results suggest that while TRAF6 is absolutely essential for TLR7 activation of ERK, JNK and NF kappa B pathways, TLR4-induced ERK, JNK pathways and IKK-mediated phosphorylation of I kappa B family members as well as cytokine expression are differentially sensitive to the cellular levels of TRAF6. These results have important implications in terms of therapeutic targeting of TRAF6 complexes in diseases where TLR4 and -7 are involved.     

Cleavage of the death domain kinase RIP by Caspase-8 prompts TNF-induced apoptosis

Although the molecular mechanisms of TNF signaling have been largely elucidated, the principle that regulates the balance of life and death is still unknown. We report here that the death domain kinase RIP, a key component of the TNF signaling complex, was cleaved by Caspase-8 in TNF-induced apoptosis. The cleavage site was mapped to the aspartic acid at position 324 of RIP. We demonstrated that the cleavage of RIP resulted in the blockage of TNF-induced NF-kappaB activation. RIPc, one of the cleavage products, enhanced interaction between TRADD and FADD/MORT1 and increased cells' sensitivity to TNF. Most importantly, the Caspase-8 resistant RIP mutants protected cells against TNF-induced apopotosis. These results suggest that cleavage of RIP is an important process in TNF-induced apoptosis. Further more, RIP cleavage was also detected in other death receptor-mediated apoptosis. Therefore, our study provides a potential mechanism to convert cells from life to death in death receptor-mediated apoptosis.     

Decrease in Tumor Necrosis Factor-α Receptor-Associated Death Domain Results from Ubiquitin-Dependent Degradation in Obstructive Renal Injury in Rats

Increased expression levels of tumor necrosis factor-α (TNFα) is involved in tubulointerstitial cell proliferation and apoptosis in obstructive renal injury. Two TNFα receptors (TNFRs), TNFR1 and TNFR2, are known to exist. On TNFα binding, TNFR1 recruits TNFR-associated death domain (TRADD), an assembly platform to mediate TNFR1 signaling. We investigated postreceptor TRADD regulation in rat kidneys with unilateral ureteral obstruction (UUO). Whereas UUO was associated with increased expression levels of TNFα, TNFR1, TNFR2, and TRADD mRNAs, it resulted in the marked decrease of TRADD protein levels (which appeared at day 1 and persisted thereafter) and a slight decrease in TNFR1 protein levels at days 7 and 14. Both ubiquitination and degradation of TRADD were increased in UUO kidneys, degradation of TRADD was stimulated by TNFα in HK-2 cells, and TRADD degradation was suppressed by proteasome inhibitor. Inhibition of TNFα by soluble TNFR2, etanercept, reduced significantly, although transiently, tubular and interstitial cell proliferation, fibronectin expression, and apoptosis in UUO kidneys, and also suppressed TRADD degradation. These data suggest that the decrease in TRADD resulting from enhanced ubiquitin-dependent degradation is involved in obstructive renal injury. Since TRADD is not incorporated into TNFR2-mediated TNFα signaling, the persistent decrease in TRADD, associated with a mild decrease in TNFR1 levels, may function, at least in part, to divert TNFα signals toward a TNFR2-mediated pathway in UUO kidneys.Unilateral ureteral obstruction (UUO) is a well-established model of experimental renal injury characterized by significant renal tubular dilatation, proliferation, apoptotic cell death, and followed by tubulointerstitial fibrosis.^1,2 In the kidney, cell proliferation is believed to be a central response to injury and culminates in the development of fibrotic renal damage.³ An imbalance between cell proliferation and apoptosis leads to unchecked apoptosis, resulting in progressive cell loss, renal tubular atrophy, and interstitial fibrosis.⁴ Tumor necrosis factor-α (TNFα) is a highly pleiotropic cytokine that induces diverse cellular responses ranging from proliferation and differentiation to activation of apoptosis.⁵ Overexpression of TNFα is reported to be involved in proliferation and apoptosis of renal tubular and interstitial cells in obstructive renal injury.^6,7,8 However, little is known about the postreceptor regulation of TNFα signaling in renal lesions.TNFα binds to TNF receptors (TNFR) to elicit its biological functions. There are two different cell-surface TNFRs; TNFR1 and TNFR2, which originate from separate gene products.⁹ On binding of TNFα, TNFR1 recruits the adaptor protein, TNFR associated death domain (TRADD), directly to its cytoplasmic death domain. In turn, TRADD serves as an assembly platform to diverge TNFR1 signaling. Interaction of TRADD with receptor interacting protein and TNF receptor associated factor 2 (TRAF2) leads to the activation of nuclear factor κB (NFκB).¹⁰ Furthermore, TRADD is also involved in the recruitment of Fas-associated protein with death domain, resulting in the initiation of apoptosis through activation of the caspase-8/3 cascade.¹¹On the other hand, the precise mechanism of TNFR2-mediated signaling is not fully elucidated. One report demonstrated that the binding of TNFα to TNFR2 recruits TRAF2 and induces NFκB activation.¹² However, it was also shown that the binding of TNFα to TNFR2 causes ubiquitin-dependent degradation of TRAF2, resulting in the suppression of NFκB activation through the inhibition of TRADD, receptor interacting protein, and TRAF2 complex formation, and finally leading to TNFR1-mediated TNF-α signaling toward the pro-apoptotic direction.¹³At present, the differential contribution of TNFR1- and TNFR2-mediated TNFα signaling is not fully elucidated in renal lesions. Ramesh et al⁹ reported that renal injury induced by cisplatin was less severe in TNFR2-deficient mice than TNFR1-deficient mice. In contrast, Guo et al¹ reported that the renal lesions in UUO mice were less severe in TNFR1 knockout mice compared with TNFR2 knockout mice. There is no report on the involvement and regulation of TRADD, an assembly platform to diverge TNFR1 signaling, in the development of renal lesions. In the present study, we investigated the postreceptor regulation of TRADD in the UUO rat kidneys. The effect of TNFα inhibition by etanercept, a soluble TNFR2, was also studied in UUO rat kidneys.     

TRAF1 is a negative regulator of TNF signaling. enhanced TNF signaling in TRAF1-deficient mice.

TNF receptor-associated factor 1 (TRAF1) is a unique TRAF protein because it lacks a RING finger domain and is predominantly expressed in activated lymphocytes. To elucidate the function of TRAF1, we generated TRAF1-deficient mice. TRAF1(-/-) mice are viable and have normal lymphocyte development. TRAF1(-/-) T cells exhibit stronger than wild-type (WT) T cell proliferation to anti-CD3 mAb, which persisted in the presence of IL-2 or anti-CD28 antibodies. Activated TRAF1(-/-) T cells, but not TRAF1(+/+) T cells, responded to TNF by proliferation and activation of the NF-kappa B and AP-1 signaling pathways. This TNF effect was mediated by TNFR2 (p75) but not by TNFR1 (p55). Furthermore, skin from TRAF1(-/-) mice was hypersensitive to TNF-induced necrosis. These findings suggest that TRAF1 is a negative regulator of TNF signaling.     

JAB1 determines the response of rheumatoid arthritis synovial fibroblasts to tumor necrosis factor-alpha

Fibroblast-like synoviocytes (FLSs) of patients with rheumatoid arthritis (RA FLSs) exhibit prosurvival, rather than apoptotic, response to tumor necrosis factor (TNF)-alpha stimulation. Here, we show that JAB1 is a critical regulator of the TNF-alpha-mediated anti-apo-ptosis pathways in RA FLSs. We found that knockdown of JAB1 using small interfering (si)RNA led to restoration of the TNF-alpha-induced apoptosis response, reduction of nuclear factor-kappaB activity, delayed degradation of IkappaB-alpha, and inhibited phosphorylation of JNK. Analysis of the interactions of JAB1 by reciprocal co-immunoprecipitations and confocal microscopy revealed that JAB1 interacts with TNF receptor-associated-factor 2 (TRAF2). The generation of the anti-apoptotic signal on binding of TNF-alpha to the TNF receptor (TNFR)1 has been shown to be associated with the recruitment of TRAF2 to the TNFR1 in a process that requires ubiquitination of TRAF2 with lysine-63-linked polyubiquitin chains. We found that TNF-alpha stimulation of JAB1 siRNA-transfected RA FLSs failed to stimulate ubiquitination of TRAF2. Thus, we conclude that JAB1-regulated ubiquitination of TRAF2 is a novel mechanism whereby TNF-alpha can induce anti-apoptosis signaling and production of matrix metalloproteinases through activation of nuclear factor-kappaB and JNK in RA FLSs.     

Redox regulation of apoptosis by members of the TNF superfamily

Tumor necrosis factor (TNF), fibroblast-associated cell surface (Fas) ligand, and TNF-related apoptosisinducing ligand (TRAIL), all members of the TNF superfamily, are arguably the most potent inducers of cell death. These cytokines induce cell death through sequential recruitment by the death receptors TNFR1- associated death domain protein (TRADD), Fas-associated death domain protein (FADD), FADD-like interleukin-1beta-converting enzyme (FLICE), and downstream caspases. Increasing evidence indicates that mitochondria play a critical role in cytokine receptor-mediated apoptosis. There is also now ample evidence that apoptosis induced by TNF and its family members is mediated through the production of reactive oxygen intermediates (also known as reactive oxygen species). Here we review the evidence linking reactive oxygen intermediates to cytokine-induced cell death mediated by TNF-alpha/beta, Fas, TRAIL, TNF-like weak inducer of apoptosis (TWEAK), and vascular endothelial cell growth inhibitor (VEGI).     

Tumor necrosis factor receptor- associated factor 6 (TRAF6) regulation of development,function, and homeostasis of the immune system

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an adapter protein that mediates a wide array of protein–protein interactions via its TRAF domain and a RING finger domain that possesses non-conventional E3 ubiquitin ligase activity. First identified nearly two decades ago as a mediator of interleukin-1 receptor (IL-1R)-mediated activation of NFκB, TRAF6 has since been identified as an actor downstream of multiple receptor families with immunoregulatory functions, including members of the TNFR superfamily, the Toll-like receptor (TLR) family, tumor growth factor-β receptors (TGFβR), and T-cell receptor (TCR). In addition to NFκB, TRAF6 may also direct activation of mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and interferon regulatory factor pathways. In the context of the immune system, TRAF6-mediated signals have proven critical for the development, homeostasis, and/or activation of B cells, T cells, and myeloid cells, including macrophages, dendritic cells, and osteoclasts, as well as for organogenesis of thymic and secondary lymphoid tissues. In multiple cellular contexts, TRAF6 function is essential not only for proper activation of the immune system but also for maintaining immune tolerance, and more recent work has begun to identify mechanisms of contextual specificity for TRAF6, involving both regulatory protein interactions, and messenger RNA regulation by microRNAs.     

Compartmentalization of TNF receptor 1 signaling: internalized TNF receptosomes as death signaling vesicles

The molecular regulation of the recruitment of initial signaling complexes at the TNF-R1 is poorly defined. We demonstrate here that within minutes internalized TNF-R1 (TNF receptosomes) recruits TRADD, FADD, and caspase-8 to establish the "death-inducing signaling complex" (DISC). In addition, we identified the TNF-R1 internalization domain (TRID) required for receptor endocytosis and provide evidence that TNF-R1 internalization, DISC formation, and apoptosis are inseparable events. Analyzing cell lines expressing an internalization-deficient receptor (TNF-R1 DeltaTRID) revealed that recruitment of RIP-1 and TRAF-2 to TNF-R1 occurred at the level of the plasma membrane. In contrast, aggregation of TRADD, FADD, and caspase-8 to establish the TNF-R1-associated DISC is critically dependent on receptor endocytosis. Furthermore, fusion of TNF receptosomes with trans-Golgi vesicles results in activation of acid sphingomyelinase and cathepsin D. Thus, TNF receptosomes establish the different TNF signaling pathways by compartmentalization of plasma membrane-derived endocytic vesicles harboring the TNF-R1-associated DISC.     

Xaf1调节TNFR信号转导而诱导细胞凋亡的机制研究

目的： 利用基因开关调节的Xaf1-Saos诱导细胞株， 检测Xaf1对TNFR信号转导通路的影响，探索Xaf1与TNF-α协同诱导细胞凋亡的机制。方法： 以免疫印迹法和RT-PCR检测Xaf1对TNFR1 表达的影响，细胞周期 DNA 含量流式细胞术检测NF-κB对Xaf1诱导细胞凋亡的影响，gel mobility shift assay 检测NF-κB的DNA结合活性， luciferase 活性检测法及RT-PCR检测NF-κB的转录活性，激酶分析法检测SAPK/JNK 激酶的活性。结果： Xaf1不影响TNFR1蛋白及mRNA水平的表达，细胞内诱导活性的NF-κB可抑制Xaf1诱导的细胞凋亡， Xaf1的表达抑制TNF-α所介导的NF-κB的DNA结合活性和转录活性，也抑制了SAPK/JNK 激酶的活性。结论： Xaf1 对TNFR信号转导的抑制是Xaf1协同TNF-α诱导细胞凋亡的机制之一。     

Requirement for Casper (c-FLIP) in regulation of death receptor-induced apoptosis and embryonic development

Casper (c-FLIP) associates with FADD and caspase-8 in signaling complexes downstream of death receptors like Fas. We generated Casper-deficient mice and cells and noted a duality in the physiological functions of this molecule. casper-/- embryos do not survive past day 10.5 of embryogenesis and exhibit impaired heart development. This phenotype is reminiscent of that reported for FADD-/- and caspase-8-/- embryos. However, unlike FADD-/- and caspase-8-/- cells, casper-/- embryonic fibroblasts are highly sensitive to FasL- or TNF-induced apoptosis and show rapid induction of caspase activities. NF-kappaB and JNK/SAPK activation is intact in TNF-stimulated casper-/- cells. These results suggest that Casper has two distinct roles: to cooperate with FADD and caspase-8 during embryonic development and to mediate cytoprotection against death factor-induced apoptosis.