The RIPK1 death domain restrains ZBP1- and TRIF-mediated cell death and inflammation.

RIPK1 is a multi-functional kinase that regulates cell death and inflammation and has been implicated in the pathogenesis of inflammatory diseases. RIPK1 acts in a kinase-dependent and kinase-independent manner to promote or suppress apoptosis and necroptosis, but the underlying mechanisms remain poorly understood. Here, we show that a mutation (R588E) disrupting the RIPK1 death domain (DD) caused perinatal lethality induced by ZBP1-mediated necroptosis. Additionally, these mice developed postnatal inflammatory pathology, which was mediated by necroptosis-independent TNFR1, TRADD, and TRIF signaling, partially requiring RIPK3. Our biochemical mechanistic studies revealed that ZBP1- and TRIF-mediated activation of RIPK3 required RIPK1 kinase activity in wild-type cells but not in Ripk1R588E/R588E cells, suggesting that DD-dependent oligomerization of RIPK1 and its interaction with FADD determine the mechanisms of RIPK3 activation by ZBP1 and TRIF. Collectively, these findings revealed a critical physiological role of DD-dependent RIPK1 signaling that is important for the regulation of tissue homeostasis and inflammation.

tRNA-derived fragment 3'tRF-AlaAGC modulates cell chemoresistance and M2 macrophage polarization via binding to TRADD in breast cancer.

BACKGROUND: Drug resistance, including Adriamycin-based therapeutic resistance, remains a challenge in breast cancer (BC) treatment. Studies have revealed that macrophages could play a pivotal role in mediating the chemoresistance of cancer cells. Accumulating evidence suggests that tRNA-Derived small RNAs (tDRs) are associated the physiological and pathological processes in multiple cancers. However, the underlying mechanisms of tDRs on chemoresistance of BC in tumor-associated macrophages remain largely unknown. METHODS: The high-throughput sequencing technique was used to screen tDRs expression profile in BC cells. Gain- and loss-of-function experiments and xenograft models were performed to verify the biological function of 3'tRF-Ala-AGC in BC cells. The CIBERSORT algorithm was used to investigate immune cell infiltration in BC tissues. To explore the role of 3'tRF-Ala-AGC in macrophages, M2 macrophages transfected with 3'tRF-Ala-AGC mimic or inhibitor were co-cultured with BC cells. Effects on Nuclear factor-κb (NF-κb) pathway were investigated by NF-κb nuclear translocation assay and western blot analysis. RNA pull-down assay was performed to identify 3'tRF-Ala-AGC interacting proteins. RESULTS: A 3'tRF fragment of 3'tRF-AlaAGC was screened, which is significantly overexpressed in BC specimens and Adriamycin-resistant cells. 3'tRF-AlaAGC could promote cell malignant activity and facilitate M2 polarization of macrophages in vitro and in vivo. Higher expression of M2 macrophages were more likely to have lymph node metastasis and deeper invasion in BC patients. Mechanistically, 3'tRF-AlaAGC binds Type 1-associated death domain protein (TRADD) in BC cells, and suppression of TRADD partially abolished the enhanced effect of 3'tRF-AlaAGC mimic on phenotype of M2. The NF-κb signaling pathway was activated in BC cells co-cultured with M2 macrophages transfected with 3'tRF-AlaAGC mimic. CONCLUSIONS: 3'tRF-AlaAGC might modulate macrophage polarization via binding to TRADD and increase the effect of M2 on promoting the chemoresistance in BC cells through NF-κb signaling pathway.

Molecular signatures in prion disease: altered death receptor pathways in a mouse model.

BACKGROUND: Prion diseases are transmissible and fatal neurodegenerative diseases characterized by accumulation of misfolded prion protein isoform (PrPSc), astrocytosis, microgliosis, spongiosis, and neurodegeneration. Elevated levels of cell membrane associated PrPSc protein and inflammatory cytokines hint towards the activation of death receptor (DR) pathway/s in prion diseases. Activation of DRs regulate, either cell survival or apoptosis, autophagy and necroptosis based on the adaptors they interact. Very little is known about the DR pathways activation in prion disease. DR3 and DR5 that are expressed in normal mouse brain were never studied in prion disease, so also their ligands and any DR adaptors. This research gap is notable and investigated in the present study. METHODS: C57BL/6J mice were infected with Rocky Mountain Laboratory scrapie mouse prion strain. The progression of prion disease was examined by observing morphological and behavioural abnormalities. The levels of PrP isoforms and GFAP were measured as the marker of PrPSc accumulation and astrocytosis respectively using antibody-based techniques that detect proteins on blot and brain section. The levels of DRs, their glycosylation and ectodomain shedding, and associated factors warrant their examination at protein level, hence western blot analysis was employed in this study. RESULTS: Prion-infected mice developed motor deficits and neuropathology like PrPSc accumulation and astrocytosis similar to other prion diseases. Results from this research show higher expression of all DR ligands, TNFR1, Fas and p75NTR but decreased levels DR3 and DR5. The levels of DR adaptor proteins like TRADD and TRAF2 (primarily regulate pro-survival pathways) are reduced. FADD, which primarily regulate cell death, its level remains unchanged. RIPK1, which regulate pro-survival, apoptosis and necroptosis, its expression and proteolysis (inhibits necroptosis but activates apoptosis) are increased. CONCLUSIONS: The findings from the present study provide evidence towards the involvement of DR3, DR5, DR6, TL1A, TRAIL, TRADD, TRAF2, FADD and RIPK1 for the first time in prion diseases. The knowledge obtained from this research discuss the possible impacts of these 16 differentially expressed DR factors on our understanding towards the multifaceted neuropathology of prion diseases and towards future explorations into potential targeted therapeutic interventions for prion disease specific neuropathology.

Structural basis of NF-κB signaling by the p75 neurotrophin receptor interaction with adaptor protein TRADD through their respective death domains.

The p75 neurotrophin receptor (p75NTR) is a critical mediator of neuronal death and tissue remodeling and has been implicated in various neurodegenerative diseases and cancers. The death domain (DD) of p75NTR is an intracellular signaling hub and has been shown to interact with diverse adaptor proteins. In breast cancer cells, binding of the adaptor protein TRADD to p75NTR depends on nerve growth factor and promotes cell survival. However, the structural mechanism and functional significance of TRADD recruitment in neuronal p75NTR signaling remain poorly understood. Here we report an NMR structure of the p75NTR-DD and TRADD-DD complex and reveal the mechanism of specific recognition of the TRADD-DD by the p75NTR-DD mainly through electrostatic interactions. Furthermore, we identified spatiotemporal overlap of p75NTR and TRADD expression in developing cerebellar granule neurons (CGNs) at early postnatal stages and discover the physiological relevance of the interaction between TRADD and p75NTR in the regulation of canonical NF-κB signaling and cell survival in CGNs. Our results provide a new structural framework for understanding how the recruitment of TRADD to p75NTR through DD interactions creates a membrane-proximal platform, which can be efficiently regulated by various neurotrophic factors through extracellular domains of p75NTR, to propagate downstream signaling in developing neurons.

Knockdown of circROBO2 attenuates acute myocardial infarction through regulating the miR-1184/TRADD axis.

BACKGROUND: Studies have found that circular RNAs (circRNAs) play key roles in cardiovascular diseases. However, the function of circROBO2 in acute myocardial infarction (AMI) is unclear. This study aimed to investigate the pathogenesis of circROBO2 in AMI. METHODS: qRT-PCR and Western blot were used to determine the expression levels of circROBO2, miR-1184, and TRADD in AMI and sham-operated mouse models at mRNA and protein level, respectively. The relationship among miR-1184, circROBO2 and TRADD was evaluated by RNA immunoprecipitation (RIP) analysis and luciferase reporter gene analysis. The roles of circROBO2, miR-1184, and TRADD in myocardial cell apoptosis were evaluated using flow cytometry. Ultrasound echocardiography, serum creatine kinase MB (CK-MB) and lactate dehydrogenase (LDH), myocardial infarction area, and myocardial cell apoptosis were measured to examine the effects of circROBO2 on myocardial injury. RESULTS: The expression levels of miR-1184 were significantly reduced, and the expression levels of circROBO2 and TRADD were significantly increased in MI group. CircROBO2 acted as a sponge for miR-1184 by upregulating the expression of TRADD. In addition, overexpression of miR-1184 enhanced the protective effect of knockdown of circROBO2 by partially inhibiting the expression of TRADD in vivo and in vitro. CONCLUSION: Knockdown of circROBO2 reduced the apoptosis of cardiomyocytes by increasing the expression levels of miR-1184, which in turn decreased the expression levels of TRADD in the myocardium post-MI.

Black carp TRADD suppresses MAVS/IFN signaling during the innate immune activation.

Tumor necrosis factor receptor 1 (TNFR1) associated death domain protein (TRADD) is a pivotal adaptor in TNF signaling pathway and up-regulates MAVS/IFN signaling pathway in human and mammal. However, the role of TRADD in teleost fish remains obscure. To reveal the function of teleost TRADD in the innate immune response, the TRADD homologue (bcTRADD) of black carp (Mylopharyngodon piceus) has been cloned and the function of bcTRADD is investigated in this study, which shares similar functional domain to its mammalian counterpart. bcTRADD mRNA expression level increased in response to different stimuli, including LPS, poly (I:C) and virus infection in host cells. bcTRADD activated the transcriptional activity of NF-κB promoter in the reporter assay; however, showed hardly any effect on the transcriptional activity of IFN promoter. It was interesting that black carp mitochondria antiviral signaling protein (bcMAVS)-activated IFN promoter transcription were dramatically depressed by bcTRADD and the C-terminal death domain of bcTRADD was indispensable for its regulation of bcMAVS. Accordingly, the plaque assay result showed that EPC cells co-expressing bcMAVS and bcTRADD presented much attenuated antiviral activity than EPC cells expressing bcMAVS alone. Knockdown of bcTRADD slightly promoted the antiviral ability of the host cells against SVCV. The current data support the conclusion that bcTRADD suppresses MAVS-mediated antiviral signaling, which is different to its mammalian counterpart.

Protein Kinase-Mediated Decision Between the Life and Death.

Protein kinases are intracellular signaling enzymes that catalyze the phosphorylation of specific residues in their target substrate proteins. They play important role for regulation of life and death decisions. The complexity of the relationship between death receptors and protein kinases' cell death decision-making mechanisms create many difficulties in the treatment of various diseases. The most of fifteen different cell death pathways, which are reported by Nomenclature Committee on Cell Death (NCCD) are protein kinase signal transduction-mediated negative or positive selections. Tumor necrosis factor (TNF) as a main player of death pathways is a dual-functioning molecule in that it can promote both cell survival or cell death. All apoptotic and necrotic signal transductions are conveyed through death domain-containing death receptors, which are expressed on the surface of nearly all human cells. In humans, eight members of the death receptor family have been identified. While the interaction of TNF with TNF Receptor 1 (TNFR1) activates various signal transduction pathways, different death receptors activate three main signal transduction pathways: nuclear factor kappa B (NF-ĸB)-mediated differentiation or pro-inflammatory cytokine synthesis, mitogen-activated protein kinase (MAPK)-mediated stress response and caspase-mediated apoptosis. The link between the NF-ĸB and the c-Jun NH2-terminal kinase (JNK) pathways comprise another check-point to regulate cell death. TNF-α also promotes the "receptor-interacting serine/threonine protein kinase 1" (RIPK1)/RIPK3/ mixed lineage kinase domain-like pseudokinase (MLKL)-dependent necrosis. Thus, necrosome is mainly comprised of MLKL, RIPK3 and, in some cases, RIPK1. In fact, RIPK1 is at the crossroad between life and death, downstream of various receptors as a regulator of endoplasmic reticulum stress-induced death. TNFR1 signaling complex (TNF-RSC), which contains multiple kinase activities, promotes phosphorylation of transforming growth factor ß-activated kinase 1 (TAK1), inhibitor of nuclear transcription factor κB (IκB) kinase (IKK) α/IKKß, IκBα, and NF-κB. IKKs affect cell-survival pathways in NF-κB-independent manner. Toll-like receptor (TLR) stimulation triggers various signaling pathways dependent on myeloid differentiation factor-88 (MyD88), Interleukin-1 receptor (IL-1R)-associated kinase (IRAK1), IRAK2 and IRAK4, lead to post-translational activation of nucleotide and oligomerization domain (NLRP3). Thereby, cell fate decisions following TLR signaling is parallel with death receptor signaling. Inhibition of IKKα/IKKß or its upstream activators sensitize cells to death by inducing RIPK1-dependent apoptosis or necroptosis. During apoptosis, several kinases of the NF-κB pathway, including IKK1 and NF-κB essential modulator (NEMO), are cleaved by cellular caspases. This event can terminate the NF-κB-derived survival signals. In both canonical and non-canonical pathways, IKK is key to NF-κB activation. Whereas, the activation process of IKK, the functions of NEMO ubiquitination, IKK-related non-canonical pathway and the nuclear transportation of NEMO and functions of IKKα are still debated in cell death. In addition, cluster of differentiation 95 (CD95)-mediated non-apoptotic signaling and CD95- death-inducing signaling complex (DISC) interactions are waiting for clarification.

Molecular Evidence of the Inhibitory Potential of Melatonin against NaAsO2-Induced Aging in Male Rats.

Arsenic (As) poisoning is widespread due to exposure to pollution. The toxic level of (As) causes oxidative stress-induced aging and tissue damage. Since melatonin (MLT) has anti-oxidant and anti-aging properties, we aimed to evaluate the protective effect of MLT against the toxicity of sodium arsenite (NaAsO2). Healthy male NMRI mice were divided into eight different groups. The control group received a standard regular diet. Other groups were treated with varying diets, including MLT alone, NaAsO2, and NaAsO2 plus MLT. After one month of treatment, biochemical and pathological tests were performed on blood, heart, and lung tissue samples. NaAsO2 increased the levels of TNF-α, 8-hydroxy-2-deoxy guanosine (8OHdG), malondialdehyde (MDA), reactive oxygen species (ROS), and high mobility group box 1 (HMGB1), increased the expression of TNF receptor type 1-associated death domain (TRADD) mRNA and telomerase reverse transcriptase, and decreased the expression of Klotho (KL) mRNA in both plasma and tissues. In contrast, MLT reduced MDA, ROS, HMGB1, lactate, and TNF-α enhanced the mRNA expression of KL, and suppressed the mRNA expression of the TERT and TRADD genes. Thus, MLT confers potent protection against NaAsO2- induced tissue injury and oxidative stress.

Comparison of Target Recognition by TRAF1 and TRAF2.

Although TRAF1 and TRAF2 share common receptors and have extremely conserved amino acid residues, recent studies have shown that key differences in receptor binding preferences with different affinities exist, which might be important for their different functions in TRAF-mediated signal transduction. To better understand TRAF1 and TRAF2 signaling, we analyzed and compared their receptor binding-affinities. Our study revealed that TRADD, TANK, and caspase-2 bind to both TRAF1 and TRAF2 with different affinities in vitro. Sequence and structural analyses revealed that S454 on TRAF2 (corresponding to A369 of TRAF1) is critical for the binding of TRADD, and F347 on TRAF1 (corresponding to L432 of TRAF2) is a critical determinant for high affinity binding of TANK and caspase-2.

MutY DNA Glycosylase Protects Cells From Tumor Necrosis Factor Alpha-Induced Necroptosis.

Numerous studies have implied that mutY DNA glycosylase (MYH) is involved in the repair of post-replicative mispairs and plays a critical role in the base excision repair pathway. Recent in vitro studies have shown that MYH interacts with tumor necrosis factor receptor type 1-associated death domain (TRADD), a key effector protein of tumor necrosis factor receptor-1 (TNFR1) signaling. The association between MYH and TRADD is reversed during tumor necrosis factor alpha (TNF-α)- and camptothecin (CPT)-induced apoptosis, and enhanced during TNF-α-induced survival. After investigating the role of MYH interacts with various proteins following TNF-α stimulation, here, we focus on MYH and TRADD interaction functions in necroptosis and its effects to related proteins. We report that the level of the MYH and TRADD complex was also reduced during necroptosis induced by TNF-α and zVAD-fmk. In particular, we also found that MYH is a biologically important necrosis suppressor. Under combined TNF-α and zVAD-fmk treatment, MYH-deficient cells were induced to enter the necroptosis pathway but primary mouse embryonic fibroblasts (MEFs) were not. Necroptosis in the absence of MYH proceeds via the inactivation of caspase-8, followed by an increase in the formation of the kinase receptor- interacting protein 1 (RIP1)-RIP3 complex. Our results suggested that MYH, which interacts with TRADD, inhibits TNF-α necroptotic signaling. Therefore, MYH inactivation is essential for necroptosis via the downregulation of caspase-8. J. Cell. Biochem. 118: 1827-1838, 2017. © 2017 Wiley Periodicals, Inc.

Loss of TRADD attenuates pressure overload-induced cardiac hypertrophy through regulating TAK1/P38 MAPK signalling in mice.

We investigated the role of tumour necrosis factor receptor (TNFR)-associated death domain (TRADD) on pressure overload-induced cardiac hypertrophy and the underlying molecular mechanisms by using a TRADD deficiency mice model. 6-8 weeks wild-type and TRADD knockout mice were performed to transverse aorta constriction (TAC) or sham operation (6-8 mice for each group). 14 days after TAC, cardiac function was measured by echocardiography, as well as by pathological and molecular analyses of heart samples. The expressions of cardiac hypertrophic and fibrotic markers were detected by qPCR. Phosphorylated and total TAK1, Akt, and p38 MAPK levels were examined by Western blotting. The ratios of lung or heart/body weight, wall thickness/chamber diameter of left ventricular and cross area of cardiomyocyte were significantly reduced in TRADD knockout (KO) mice than those of wild-type mice after TAC. Moreover, cardiac hypertrophic and fibrotic markers were downregulated in TRADD knockout mice than those of wild-type mice following TAC. Protein expression analysis showed phosphorylated TAK1, p38 MAPK and AKT were upregulated after TAC in both wild-type and TRADD KO mice, phosphorylation of TAK1 and p38 MAPK was reduced more remarkably after TRADD deficiency, while phosphorylated AKT expression was similar between TRADD KO and wild-type mice following TAC. Our data suggest that TRADD KO blunts pressure overload-induced cardiac hypertrophy through mediating TAK1/p38 MAPK but not AKT phosphorylation in mice.

Identification of TRADD as a potential biomarker in human uterine leiomyoma through iTRAQ based proteomic profiling.

Recurrent and refractory leiomyoma of uterus is one of the most common diseases in women of reproductive age. Despite its benign nature, uterine leiomyoma has presented an extremely deleterious impact on public health. The etiology of uterine leiomyoma remains unclear and clinical management remains suboptimal, leaving radical hysterectomy the only effective approach. Delineating the molecular mechanism underlying the leiomyoma initiation and progression remains an unmet clinical need. To screen proteins that were differentially expressed in uterine leiomyoma versus normal myometrium, we examined proteomic profile by isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with liquid chromatography - tandem mass spectrometry (LC-MS/MS). 72 proteins have been identified as differentially expressed in uterine leiomyoma, including the downregulation of TRADD (tumor necrosis factor receptor type 1-associated DEATH domain protein), which dominates the dysfunctional extrinsic apoptosis pathway and deregulated inflammatory responses. The reduction of TRADD was further validated by Western blot and immunohistochemistry in independent sample cohorts. Our data thus suggest potential biological significance of TRADD mediated inflammatory response in the development of uterine leiomyoma.

Synthesis of a GlcNAcylated arginine building block for the solid phase synthesis of death domain glycopeptide fragments.

Herein we describe the synthesis of glycopeptide fragments from the death domains of TRADD and FADD bearing the recently discovered Nω-GlcNAc-ß-arginine post-translational modification. TRADD and FADD glycopeptides were accessed through the use of a suitably protected synthetic glycosylamino acid 'cassette' that could be directly incorporated into conventional solid phase peptide synthesis (SPPS) protocols.

Hydrogen sulfide protects against TNF-α induced neuronal cell apoptosis through miR-485-5p/TRADD signaling.

BACKGROUND AND OBJECTIVE: Increasing studies suggest that miRNAs are served as responders and regulators for pathological change in human. miR-485-5p is such a miRNA that has been proved to be affected by spinal cord I/R injury. This study was to investigate the functional involvement and mechanism of miR-485-5p in sulfuretted hydrogen (H2S) protecting neural cell from injury. METHODS: In this study, serum tumor necrosis factor (TNF-α) and miR-485-5p were detected in 20 patients with spinal cord ischemia/reperfusion (I/R) injury and in 20 healthy control. H2S was administered by GYY4137 treatment. Two TNF-α-stimulated neural human cell lines, AGE1.HN and SY-SH-5Y, were used for in vitro I/R experiments. Quantitative RT-PCR was performed to determine miR-485-5p expression. QRT-PCR and western blot were respectively performed to evaluate expression of tumor necrosis factor receptor type 1-associated DEATH domain protein (TRADD). RESULTS: The result showed that serum TNF-α was significantly reduced in patients compared with healthy control. In vitro TNF-α treatment dose dependently caused GE1.HN and SY-SH-5Y apoptosis, whereas this promotion action was reversed by CYY4137. Moreover, we found that H2S protected neuronal cell against apoptosis via TRADD dependent. By luciferase reporting gene assay, western blot and qRT-PCR, we confirmed that TRADD expression was regulated by miR-485-5p. Such miR-485-5p/TRADD axis was proved to be involved in GE1.HN and SY-SH-5Y neural cell-protective process of H2S. CONCLUSION: In summary, our data for the first time identifies miR-485-5p/TRADD axis in hydrogen sulfide protecting against TNF-α-induced neuronal cell apoptosis.

Verproside inhibits TNF-α-induced MUC5AC expression through suppression of the TNF-α/NF-κB pathway in human airway epithelial cells.

Airway mucus secretion is an essential innate immune response for host protection. However, overproduction and hypersecretion of mucus, mainly composed of MUC5AC, are significant risk factors in asthma and chronic obstructive pulmonary disease (COPD) patients. Previously, we reported that verproside, a catalpol derivative iridoid glycoside isolated from Pseudolysimachion rotundum var. subintegrum, is a potent anti-asthmatic candidate drug in vivo. However, the molecular mechanisms underlying the pharmacological actions of verproside remain unknown. Here, we found that verproside significantly reduces the expression levels of tumor necrosis factor alpha (TNF-α)-induced MUC5AC mRNA and protein by inhibiting both nuclear factor kappa B (NF-κB) transcriptional activity and the phosphorylation of its upstream effectors such as IκB kinase (IKK)ß, IκBα, and TGF-ß-activated kinase 1 (TAK1) in NCI-H292 cells. Moreover, verproside attenuated TNF-α-induced MUC5AC transcription more effectively when combined with an IKK (BAY11-7082) or a TAK1 (5z-7-oxozeaenol) inhibitor than when administered alone. Importantly, we demonstrated that verproside negatively modulates the formation of the TNF-α-receptor (TNFR) 1 signaling complex [TNF-RSC; TNFR1-recruited TNFR1-associated death domain protein (TRADD), TNFR-associated factor 2 (TRAF2), receptor-interacting protein kinase 1 (RIP1), and TAK1], the most upstream signaling factor of NF-κB signaling. In silico molecular docking studies show that verproside binds between TRADD and TRAF2 subunits. Altogether, these results suggest that verproside could be a good therapeutic candidate for treatment of inflammatory airway diseases such as asthma and COPD by blocking the TNF-α/NF-κB signaling pathway.

TNF-α is involved in apoptosis triggered by grass carp reovirus infection in vitro.

Grass carp reovirus (GCRV) infection causes apoptosis in Ctenopharyngodon idella kidney cells (CIK). However, the cause of GCRV-induced apoptosis and its signaling pathways remain unknown. This study investigated the role of TNF-α-induced capase-8 pathways in mediating GCRV-induced apoptosis in the grass carp (Ctenopharyngodon idella). Recombinant TNF-α was expressed and purified from Escherichia. coli. The western blot assay indicated that TNF-α expression level in kidney and spleen was higher than that in liver. In apoptosis assay, recombinant TNF-α triggered significant apoptosis in CIK cells, which was characterized by increased mRNA levels of TNF-α, TRADD or caspase-8, and enhanced caspase-8 activity in CIK cells. To confirm the biological activity of TNF-α during GCRV infection, significant apoptosis in CIK cells was induced by GCRV correlating with enhanced caspase-8 activity, increased mRNA level of TNF-α, TRADD or caspase-8, increased protein level of TNF-α in CIK cells and cell supernatant, suggesting that TNF-α-induced capase-8 pathways might be involved in GCRV-triggered apoptosis. Furthermore, treatment with an anti-TNF-α polyclonal antibody significantly decreased the degree of apoptosis in infected CIK cells compared with cells treated with a control antibody, which confirmed that TNF-α was a key mediator involved in GCRV-induced apoptosis. Taken together, these results indicated that GCRV might trigger apoptosis via TNF-α induced capase-8 pathways in CIK cells.

TNFα triggers release of extracellular vesicles containing TNFR1 and TRADD, which can modulate TNFα responses of the parental cells.

Tumor necrosis factor-α (TNFα)-induced reactions are effective to maintain homeostasis; however, excessive responses play progressive roles in the pathogenesis of various chronic inflammatory diseases. We demonstrate that TNFα triggered the release of its receptor TNFR1 as a content of extracellular vesicles (EVs) from the human bronchial epithelial cell, BEAS-2b. The TNFR1 cytoplasmic domain binding partner, TNFR-associated death domain (TRADD), was released by TNFα treatment along with TNFR1. TNFα-triggered release of EVs was decreased in the presence of amitriptyline, an inhibitor of acid sphingomyelinase (A-SMase), or of GW4869, an inhibitor of neutral sphingomyelinase (N-SMase), indicating that EVs containing TNFR1 and TRADD are released through A-SMase and N-SMase dependent manners. From sucrose density gradient analysis, each sphingomyelinase is involved in the generation of distinct populations of EVs. Inhibition of A-SMase or N-SMase resulted in significantly increased responses to TNFα in parental cells. Given that TRADD serves as a platform for the assembly of subsequent signaling molecules, the TNFα triggered release of TNFR1 and TRADD might be an effective strategy for down regulation of the TNFα responses of parental cells.

A novel TLR2-triggered signalling crosstalk synergistically intensifies TNF-mediated IL-6 induction.

Toll-like receptors (TLR) recognize pathogens and trigger the production of vigorous pro-inflammatory cytokines [such as tumour necrosis factor (TNF)] that induce systemic damages associated with sepsis and chronic inflammation. Cooperation between signals of TLR and TNF receptor has been demonstrated through the participation of TNF receptor 1 (TNFR) adaptors in endotoxin tolerance. Here, we identify a TLR2-mediated synergy, through a MyD88-independent crosstalk, which enhances subsequent TNF-mediated nuclear factor-kappa B activation and interleukin-6 induction. Membrane-associated adaptor MAL conduces the link between TNF receptor-associated factor 6 (TRAF6) and TNFR-associated death domain, leading to a distinctive K63-ubiquitinylated TRAF6 recruitment into TNFR complex. In summary, our results reveal a novel route of TLR signal that synergistically amplifies TNF-mediated responses, indicating an innovative target for inflammation manipulation.

Crosstalk between apoptosis, necrosis and autophagy.

Apoptosis and necrosis are the two major modes of cell death, the molecular mechanisms of which have been extensively studied. Although initially thought to constitute mutually exclusive cellular states, recent findings reveal cellular contexts that require a balanced interplay between these two modes of cellular demise. Several death initiator and effector molecules, signaling pathways and subcellular sites have been identified as key mediators in both processes, either by constituting common modules or alternatively by functioning as a switch allowing cells to decide which route to take, depending on the specific situation. Importantly, autophagy, which is a predominantly cytoprotective process, has been linked to both types of cell death, serving either a pro-survival or pro-death function. Here we review the recent literature that highlights the intricate interplay between apoptosis, necrosis and autophagy, focusing on the relevance and impact of this crosstalk in normal development and in pathology. This article is part of a Special Section entitled: Cell Death Pathways.

Death domain complex of the TNFR-1, TRADD, and RIP1 proteins for death-inducing signaling.

Apoptosis can be induced by an extrinsic pathway involving the ligand-mediated activation of death receptors such as tumor necrosis factor receptor-1 (TNFR-1). TNFR-1-associated death domain (TRADD) protein is an adapter molecule that bridges the interaction between TNFR-1 and receptor-interacting serine/threonine-protein kinase 1 (RIP1). However, the molecular mechanism of the complex formation of these proteins has not yet been identified. Here, the binding among TNFR-1, TRADD, and RIP1 was identified using a GST pull-down assay and Biacore biosensor experiment. This study showed that structural characterization and formation of the death-signaling complex could be predicted using TNFR-1, TRADD, and RIP1. In addition, we found that the structure-based mutations of TNFR-1 (P367A and P368A), TRADD (F266A), and RIP1 (M637A and R638A) disrupted formation of the death domain (DD) complex and prevented stable interactions among those DDs.