Tumor Necrosis Factor Receptor-associated Death Domain Protein Research Articles

FADD Is Upregulated in Relapsing Remitting Multiple Sclerosis

Objective: To elucidate the role of tumor necrosis factor (TNF) receptor signal transduction in multiple sclerosis (MS). Methods: We performed a cross-sectional analysis of the gene expression of TNF receptor-associated death domain protein (TRADD) and Fas-associated death domain protein (FADD) in peripheral blood leukocytes of 23 relapsing remitting (RR), 19 secondary progressive (SP) and 12 primary progressive (PP) MS patients, as well as of 29 healthy controls by quantitative RT-PCR. Additionally, we monitored a subgroup of 15 RR MS patients longitudinally every 3 months over the time period of 9 months. Results: FADD expression was significantly elevated in RR MS patients compared to the other disease courses (p < 0.048). The median of FADD expression was elevated in the RR MS patient groups compared to the healthy group, but this was not significant (p < 0.053). The median of TRADD expression was elevated in the patient groups compared to the healthy group, but this was not significant (p < 0.14). Neither variable changed significantly over the time course of 9 months. Conclusion: FADD elevation in leukocytes might be interpreted as the molecular equivalent of an elevated general inflammatory activity in RR MS patients compared to other disease courses. FADD elevation in RR MS reinforces the concept that different pathophysiological and immunological processes sustain RR MS and SP or PP MS.

UXT-V1 protects cells against TNF-induced apoptosis through modulating complex II formation

Proteins that directly regulate tumor necrosis factor (TNF) signaling have critical roles in determining cell death and survival. Previously we characterized ubiquitously expressed transcript (UXT)-V2 as a novel transcriptional cofactor to regulate nuclear factor-κB in the nucleus. Here we report that another splicing isoform of UXT, UXT-V1, localizes in cytoplasm and regulates TNF-induced apoptosis. UXT-V1 knockdown cells are hypersensitive to TNF-induced apoptosis. We demonstrated that UXT-V1 is a new component of TNF receptor signaling complex. We found that UXT-V1 binds to TNF receptor-associated factor 2 and prevents TNF receptor-associated death domain protein from recruiting Fas-associated protein with death domain. More importantly, UXT-V1 is a short-half-life protein, the degradation of which facilitates the formation of the apoptotic receptor complex II in response to TNF treatment. This study demonstrates that UXT-V1 is a novel regulator of TNF-induced apoptosis and sheds new light on the underlying molecular mechanism of this process.

Zfra is a small wizard in the mitochondrial apoptosis

Zfra (zinc finger-like protein that regulates apoptosis) is a naturally occurring short peptide consisting of 31 amino acids, which regulates tumor necrosis factor (TNF)-mediated cell death by interacting with receptor adaptor protein TRADD (TNF receptorassociated death domain protein) and downstream JNK (c-Jun N-terminal kinase), NF-κB (Nuclear factor kappa B) and WWOX/WOX1 (WW domain-containing oxidoreductase). Cytochrome c release is generally considered as a pivotal step in apoptosis. Remarkably, overexpressed Zfra induces apoptosis via the mitochondrial pathway, which involves suppression of Bcl-2 and Bcl-xL expression (without causing cytochrome c release), counteracting the apoptotic function of tumor suppressor p53 and WWOX, and dissipation of mitochondrial membrane potential for ultimately leading to cell death. Control of cellular aging and apoptosis by Zfra, p53 and WWOX is discussed.

Expression of tumor necrosis factor-α signaling adapter proteins in peripheral blood mononuclear cells from patients with systemic lupus erythematosus

To investigate the mRNA expression of the tumor necrosis factor (TNF)-adapter proteins, TNF receptor-associated death domain protein (TRADD), Fas-associated death domain protein (FADD), receptor-interacting protein 1 (RIP-1), and TNF receptor-associated factor-2 (TRAF-2) in peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE), and to explore the relationship between the expression of these adaptors and the SLE disease activity. PBMC were isolated from venous blood of 51 SLE patients and 17 healthy subjects. The mRNA expression of TRADD, FADD, RIP-1, TRAF-2, Caspase 3, and interleukin (IL)-1beta were analyzed by quantitative real time RT-PCR. Disease activity was measured using the SLE Disease Activity Index (SLEDAI). All healthy subjects showed mRNA expressions of TRADD, FADD, RIP-1, and TRAF-2. The mRNA expression levels of TRADD, FADD, RIP-1, and TRAF- in the PBMC from the patients were 0.38, 0.69, 0.59, and 0.55 tomes that from the control subjects (all P < 0.05). The expression levels of these 4 adapters of the SLE patients with the SLEDAI >/= 10 were significantly lower than those of the SLE patients with the SLEDAI < 10 (all P < 0.05). The Caspase3 mRNA expression of the SLE patients was significantly higher than that of the healthy controls (P < 0.01); however, the IL-1beta mRNA expression was not significantly different between the SLE and control subjects. The mRNA expression levels of TRADD, FADD, RIP-1 and TRAF-2 in the PBMC from the SLE patients were all negatively correlated with the SLE activity index with the coefficient correlation of -0.285, -0.280, -0.307, and -0.298 respectively (all P < 0.05). The mRNA expression levels of the TNF adapter molecules, such as TRADD, FADD, RIP-1, and TRAF-2, decrease significantly in the PBMC from the SLE patients, and are negatively correlated with the SLE activity index. These abnormalities may participate in the immunopathogenic injury mediated by the aberration TNFalpha signaling pathway in SLE.

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.

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.

JNK1 Physically Interacts with WW Domain-containing Oxidoreductase (WOX1) and Inhibits WOX1-mediated Apoptosis

Transient activation of c-Jun N-terminal kinase (JNK) promotes cell survival, whereas persistent JNK activation induces apoptosis. Bovine testicular hyaluronidase PH-20 activates JNK1 and protects L929 fibroblasts from staurosporine-mediated cell death. PH-20 also induces the expression of a p53-interacting WW domain-containing oxidoreductase (WOX1, also known as WWOX or FOR) in these cells. WOX1 enhances the cytotoxic function of tumor necrosis factor and mediates apoptosis synergistically with p53. Thus, the activated JNK1 is likely to counteract WOX1 in mediating apoptosis. Here it is demonstrated that ectopic JNK1 inhibited WOX1-mediated apoptosis of L929 fibroblasts, monocytic U937 cells, and other cell types. Also, JNK1 blocked WOX1 prevention of cell cycle progression. By stimulating cells with anisomycin or UV light, JNK1 became activated, and WOX1 was phosphorylated at Tyr(33). The activated JNK1 physically interacted with the phosphorylated WOX1, as determined by co-immunoprecipitation. Alteration of Tyr(33) to Arg(33) in WOX1 abrogated its binding interaction with JNK1 and its activity in mediating cell death, indicating that Tyr(33) phosphorylation is needed to activate WOX1. A dominant negative WOX1 was developed and shown to block p53-mediated apoptosis and anisomycin-mediated WOX1 phosphorylation but could not inhibit JNK1 activation. This mutant protein bound p53 but could not interact with JNK1, as determined in yeast two-hybrid analysis. Taken together, phosphorylation of JNK1 and WOX1 is necessary for their physical interaction and functional antagonism.

TRAF1 is a critical regulator of JNK signaling by the TRAF-binding domain of the Epstein-Barr virus-encoded latent infection membrane protein 1 but not CD40.

The oncogenic Epstein-Barr virus (EBV)-encoded latent infection membrane protein 1 (LMP1) mimics a constitutive active tumor necrosis factor (TNF) family receptor in its ability to recruit TNF receptor-associated factors (TRAFs) and TNF receptor-associated death domain protein (TRADD) in a ligand-independent manner. As a result, LMP1 constitutively engages signaling pathways, such as the JNK and p38 mitogen-activated protein kinases (MAPK), the transcription factor NF-kappaB, and the JAK/STAT cascade, and these activities may explain many of its pleiotropic effects on cell phenotype, growth, and transformation. In this study we demonstrate the ability of the TRAF-binding domain of LMP1 to signal on the JNK/AP-1 axis in a cell type- dependent manner that critically involves TRAF1 and TRAF2. Thus, expression of this LMP1 domain in TRAF1-positive lymphoma cells promotes significant JNK activation, which is blocked by dominant-negative TRAF2 but not TRAF5. However, TRAF1 is absent in many established epithelial cell lines and primary nasopharyngeal carcinoma (NPC) biopsy specimens. In these cells, JNK activation by the TRAF-binding domain of LMP1 depends on the reconstitution of TRAF1 expression. The critical role of TRAF1 in the regulation of TRAF2-dependent JNK signaling is particular to the TRAF-binding domain of LMP1, since a homologous region in the cytoplasmic tail of CD40 or the TRADD-interacting domain of LMP1 signal on the JNK axis independently of TRAF1 status. These data further dissect the signaling components used by LMP1 and identify a novel role for TRAF1 as a modulator of oncogenic signals.

Expression of TNF receptors and related signaling molecules in the bone marrow from patients with myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are characterized by peripheral blood cytopenias despite hypercellularity of the bone marrow regarded as the result of ineffective hematopoiesis mainly caused by apoptosis. In this study, we examined the role of tumor necrosis factor (TNF)-induced apoptosis in the bone marrow cells of MDS patients. The constitutive expression of mRNA for TNF receptors (TNFR), including TNFRI and TNFRII, and the adapter molecules, such as the TNF receptor-associated death domain protein (TRADD), Fas-associated death domain protein (FADD), receptor interacting protein (RIP) and TNF receptor-associated factor 2 (TRAF-2) were analyzed by reverse transcriptase (RT)-PCR in bone marrow samples from control, MDS and AML cases. In bone marrow cells from refractory anemia (RA) patients, there was a significant increase in TNFRI expression as compared with control subjects. The expression of TNFRII was also up-regulated in RA cases. In contrast, RA with excess of blasts (RAEB), RAEB in transformation (RAEB-T) and AML cases revealed increased expression of TNFRII, whereas the expression of TNFRI was comparable to control subjects. Immunohistochemical staining revealed that the TNFRI, as well as TNFRII of MDS bone marrow was expressed mainly in hematopoietic cells, but not in macrophage-lineage stromal cells at the protein level. An increased constitutive expression of mRNA for TRADD, FADD and RIP and decreased expression of mRNA for TRAF-2 were observed in bone marrow cells from MDS patients, especially from RA patients, as compared with controls, although the differences were not significant. In many of the AML bone marrow samples, strong expression of TRAF-2 mRNA was marked, while expression levels of other proteins were similar to those in control subjects. These data suggested enhanced signaling by the TNFRI–TRADD–FADD pathway and suppressed signaling by the TRAF-2 pathway in RA. Thus, TNF-α-induced apoptosis may play a role in ineffective hematopoiesis in “early stage MDS” bone marrow, although the regulatory mechanisms for TNF-α-induced signaling would be complicated and not be simply explained only by these pathways.

TRADD is Involved in Apoptosis Induction in Granulosa Cells during Atresia in Pig Ovaries.

Previously, we histochemically demonstrated the expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its receptors, death receptor-4 (DR4), death receptor-5 (DR5) and decoy receptor-1 (DcR1) in granulosa cells of porcine follicles. However, TRAIL can induce both cell death and cell proliferation. In the present study, reverse transcription polymerase chain reaction and in situ hybridization analyses revealed increased mRNA expression of TNF receptor-associated death domain protein (TRADD), which transmits the death signal from DR4 and/or DR5 to intracellular apoptosis-signal transduction components, in granulosa cells was demonstrated only in atretic follicles but not in healthy follicles. These findings indicate that TRADD is involved in induction of apoptosis in granulosa cells, and that the TRAIL-receptor system induces apoptosis in granulosa cells during atresia in porcine ovaries.

Lithium Sensitizes Tumor Cells in an NF-κB-independent Way to Caspase Activation and Apoptosis Induced by Tumor Necrosis Factor (TNF): EVIDENCE FOR A ROLE OF THE TNF RECEPTOR-ASSOCIATED DEATH DOMAIN PROTEIN

We have previously shown that lithium salts can considerably increase the direct cytotoxic effect of tumor necrosis factor (TNF) on various tumor cells in vitro and in vivo. However, the underlying mechanism has remained largely unknown. Here we show that the TNF-sensitizing effect of lithium chloride (LiCl) is independent of the type of cell death, either necrosis or apoptosis. In the case of apoptosis, TNF/lithium synergism is associated with an enhanced activation of caspases and mitochondrial cytochrome c release. Sensitization to apoptosis is specific for TNF-induced apoptosis, whereas Fas-mediated or etoposide-induced apoptosis remains unaffected. LiCl also potentiates cell death induced by artificial oligomerization of a fusion protein between FKBP and the TNF receptor-associated death domain protein. TNF-induced activation of NF-kappa B-dependent gene expression is not modulated by LiCl treatment. These results indicate that LiCl enhances TNF-induced cell death in an NF-kappa B-independent way, and suggest that the TNF receptor-associated death domain protein plays a crucial role in the TNF-sensitizing effect of LiCl.

N-Acetylcysteine suppresses TNF-induced NF-κB activation through inhibition of IκB kinases

Here, we used a reductant, N-acetyl-L-cysteine (NAC), to investigate the redox-sensitive step(s) in the signalling pathway from the tumor necrosis factor (TNF) receptor to nuclear factor κB (NF-κB). We found that NAC suppressed NF-κB activation triggered by TNF or by overexpression of either the TNF receptor-associated death domain protein, TNF receptor-associated factor 2, NF-κB-inducing kinase (NIK), or IκB kinases (IKKα and IKKβ). NAC also suppressed the TNF-induced activation of IKKα and IKKβ, phosphorylation and degradation of IκB, and nuclear translocation of NF-κB. Furthermore, NAC suppressed the activation of IKKα and IKKβ triggered by the overexpression of NIK. These results indicate that IKKα and IKKβ are subject to redox regulation in the cells, and that NAC inhibits NF-κB activation through the suppression of these kinases.

The zinc finger protein A20 inhibits TNF-induced NF-kappaB-dependent gene expression by interfering with an RIP- or TRAF2-mediated transactivation signal and directly binds to a novel NF-kappaB-inhibiting protein ABIN.

The zinc finger protein A20 is a tumor necrosis factor (TNF)- and interleukin 1 (IL-1)-inducible protein that negatively regulates nuclear factor-kappa B (NF-kappaB)-dependent gene expression. However, the molecular mechanism by which A20 exerts this effect is still unclear. We show that A20 does not inhibit TNF- induced nuclear translocation and DNA binding of NF-kappaB, although it completely prevents the TNF- induced activation of an NF-kappaB-dependent reporter gene, as well as TNF-induced IL-6 and granulocyte macrophage-colony stimulating factor gene expression. Moreover, NF-kappaB activation induced by overexpression of the TNF receptor-associated proteins TNF receptor-associated death domain protein (TRADD), receptor interacting protein (RIP), and TNF recep- tor-associated factor 2 (TRAF2) was also inhibited by expression of A20, whereas NF-kappaB activation induced by overexpression of NF-kappaB-inducing kinase (NIK) or the human T cell leukemia virus type 1 (HTLV-1) Tax was unaffected. These results demonstrate that A20 inhibits NF-kappaB-dependent gene expression by interfering with a novel TNF-induced and RIP- or TRAF2-mediated pathway that is different from the NIK-IkappaB kinase pathway and that is specifically involved in the transactivation of NF-kappaB. Via yeast two-hybrid screening, we found that A20 binds to a novel protein, ABIN, which mimics the NF-kappaB inhibiting effects of A20 upon overexpression, suggesting that the effect of A20 is mediated by its interaction with this NF-kappaB inhibiting protein, ABIN.