TNFR-associated Death Domain Research Articles

Tumor necrosis factor α induces γ-glutamyltransferase expression via nuclear factor-κB in cooperation with Sp1

γ-Glutamyltransferase (GGT) cleaves the γ-glutamyl moiety of glutathione (GSH), an endogenous antioxidant, and is involved in mercapturic acid metabolism and in cancer drug resistance when overexpressed. Moreover, GGT converts leukotriene (LT) C4 into LTD4 implicated in various inflammatory pathologies. So far the effect of inflammatory stimuli on regulation of GGT expression and activity remained to be addressed. We found that the proinflammatory cytokine tumor necrosis factor alpha (TNFα) induced GGT promoter transactivation, mRNA and protein synthesis, as well as enzymatic activity. Remicade, a clinically used anti-TNFα antibody, small interfering RNA (siRNA) against p50 and p65 nuclear factor-kappaB (NF-κB) isoforms, curcumin, a well characterized natural NF-κB inhibitor, as well as a dominant negative inhibitor of kappaB alpha (IκBα), prevented GGT activation at various levels, illustrating the involvement of this signaling pathway in TNFα-induced stimulation. Over-expression of receptor of TNFα-1 (TNFR1), TNFR-associated factor-2 (TRAF2), TNFR-1 associated death domain (TRADD), dominant negative (DN) IκBα or NF-κB p65 further confirmed GGT promoter activation via NF-κB. Linker insertion mutagenesis of 536 bp of the proximal GGT promoter revealed NF-κB and Sp1 binding sites at −110 and −78 relative to the transcription start site, responsible for basal GGT transcription. Mutation of the NF-κB site located at −110 additionally inhibited TNFα-induced promoter induction. Chromatin immunoprecipitation (ChIP) assays confirmed mutagenesis results and further demonstrated that TNFα treatment induced in vivo binding of both NF-κB and Sp1, explaining increased GGT expression, and led to RNA polymerase II recruitment under inflammatory conditions.

Activation of Apoptosis by 1-Hydroxy-5,7-Dimethoxy-2-Naphthalene-Carboxaldehyde, a Novel Compound from Aegle marmelos

We have identified a natural compound that activates apoptosis of epithelial cancer cells through activation of tumor necrosis factor-alpha (TNF-alpha), TNF receptor (TNFR)-associated death domain (TRADD), and caspases. The molecule 1-hydroxy-5,7-dimethoxy-2-naphthalene-carboxaldehyde (HDNC, marmelin) was isolated and characterized from ethyl acetate fraction of extracts of Aegle marmelos. HDNC treatment inhibited the growth of HCT-116 colon cancer tumor xenografts in vivo. Immunostaining for CD31 showed that there was a significant reduction in microvessels in the HDNC-treated animals, coupled with decreased cyclooxygenase-2, interleukin-8, and vascular endothelial growth factor mRNA. Using hexoseaminidase assay, we determined that HDNC inhibits proliferation of HCT-116 colon and HEp-2 alveolar epithelial carcinoma cells. Furthermore, the cancer cells showed increased levels of activated caspase-3 and induced G(1) cell cycle arrest, which was suppressed by caspase-3 inhibitors. HDNC induced TNF-alpha, TNFR1, and TRADD mRNA and protein expression. Moreover, caspase-8 and Bid activation, and cytochrome c release, were observed, suggesting the existence of a cross-talk between death receptor and the mitochondrial pathways. HDNC inhibited AKT and extracellular signal-regulated kinase phosphorylation both in cells in culture and in tumor xenografts. In addition, electrophoretic mobility shift assay and luciferase reporter assays showed that HDNC significantly suppressed TNF-alpha-mediated activation and translocation of nuclear factor-kappaB (NF-kappaB). This was further confirmed by Western blot analysis of nuclear extracts wherein levels of RelA, the p65 component of NF-kappaB, were significantly less in cells treated with HDNC. Together, the data suggest that the novel compound HDNC (marmelin) is a potent anticancer agent that induces apoptosis during G(1) phase of the cell cycle and could be a potential chemotherapeutic candidate.

C-IAP1 and c-IAP2 Are Critical Mediators of Tumor Necrosis Factor α (TNFα)-induced NF-κB Activation

c-IAP1 and c-IAP2 Are Critical Mediators of Tumor Necrosis Factor α (TNFα)-induced NF-κB Activation

STAP-2 Negatively Regulates both Canonical and Noncanonical NF-κB Activation Induced by Epstein-Barr Virus-Derived Latent Membrane Protein 1

The signal-transducing adaptor protein 2 (STAP-2) is a recently identified adaptor protein that contains a pleckstrin homology (PH) and Src homology 2 (SH2)-like domains, as well as a proline-rich domain in its C-terminal region. In previous studies, we demonstrated that STAP-2 binds to MyD88 and IKK-alpha or IKK-beta and modulates NF-kappaB signaling in macrophages. In the present study, we found that ectopic expression of STAP-2 inhibited Epstein-Barr virus (EBV) LMP1-mediated NF-kappaB signaling and interleukin-6 expression. Indeed, STAP-2 associated with LMP1 through its PH and SH2-like domains, and these proteins interacted with each other in EBV-positive human B cells. We found, furthermore, that STAP-2 regulated LMP1-mediated NF-kappaB signaling through direct or indirect interactions with the tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) and TNFR-associated death domain (TRADD) proteins. STAP-2 mRNA was induced by the expression of LMP1 in human B cells. Furthermore, transient expression of STAP-2 in EBV-positive human B cells decreased cell growth. Finally, STAP-2 knockout mouse embryonic fibroblasts showed enhanced LMP1-induced cell growth. These results suggest that STAP-2 acts as an endogenous negative regulator of EBV LMP1-mediated signaling through TRAF3 and TRADD.

A New TRADDition in Intracellular Antiviral Signaling

To effectively defeat viral infection, a mammalian host must detect the presence of viruses and activate a robust antiviral response, which is mediated by cytokines known as type I interferons (IFNs). Much excitement has been generated by the discovery of the retinoic acid-induced gene 1-like helicase (RLH) pathway, which detects RNA-containing viruses in the cytoplasm and activates the type I IFN response. A study now describes a role for the tumor necrosis factor receptor (TNFR)-associated death domain (TRADD) in the antiviral response and sheds new light on how the RLH signaling complex is organized upon viral infection.

Function of TRADD in tumor necrosis factor receptor 1 signaling and in TRIF-dependent inflammatory responses

Tumor necrosis factor receptor 1 (TNFR1) and Toll-like receptors (TLRs) regulate immune and inflammatory responses. Here we show that the TNFR1-associated death domain protein (TRADD) is critical in TNFR1, TLR3 and TLR4 signaling. TRADD deficiency abrogated TNF-induced apoptosis, prevented recruitment of the ubiquitin ligase TRAF2 and ubiquitination of the adaptor RIP1 in the TNFR1 signaling complex, and considerably inhibited but did not completely abolish activation of the transcription factor NF-kappaB and mitogen-activated protein kinases 'downstream' of TNFR1. TRIF-dependent cytokine production induced by the synthetic double-stranded RNA poly(I:C) and lipopolysaccharide was lower in TRADD-deficient mice than in wild-type mice. Moreover, TRADD deficiency inhibited poly(I:C)-mediated RIP1 ubiquitination and activation of NF-kappaB and mitogen-activated protein kinase signaling in fibroblasts but not in bone marrow macrophages. Thus, TRADD is an essential component of TNFR1 signaling and has a critical but apparently cell type-specific function in TRIF-dependent TLR responses.

Pinitol targets nuclear factor-κB activation pathway leading to inhibition of gene products associated with proliferation, apoptosis, invasion, and angiogenesis

Pinitol (3-O-methyl-chiroinositol), a component of traditional Ayurvedic medicine (talisapatra), has been shown to exhibit anti-inflammatory and antidiabetic activities through undefined mechanisms. Because the transcription factor nuclear factor-kappaB (NF-kappaB) has been linked with inflammatory diseases, including insulin resistance, we hypothesized that pinitol must mediate its effects through modulation of NF-kappaB activation pathway. We found that pinitol suppressed NF-kappaB activation induced by inflammatory stimuli and carcinogens. This suppression was not specific to cell type. Besides inducible, pinitol also abrogated constitutive NF-kappaB activation noted in most tumor cells. The suppression of NF-kappaB activation by pinitol occurred through inhibition of the activation of IkappaBalpha kinase, leading to sequential suppression of IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and NF-kappaB-dependent reporter gene expression. Pinitol also suppressed the NF-kappaB reporter activity induced by tumor necrosis factor receptor (TNFR)-1, TNFR-associated death domain, TNFR-associated factor-2, transforming growth factor-beta-activated kinase-1 (TAK-1)/TAK1-binding protein-1, and IkappaBalpha kinase but not that induced by p65. The inhibition of NF-kappaB activation thereby led to down-regulation of gene products involved in inflammation (cyclooxygenase-2), proliferation (cyclin D1 and c-myc), invasion (matrix metalloproteinase-9), angiogenesis (vascular endothelial growth factor), and cell survival (cIAP1, cIAP2, X-linked inhibitor apoptosis protein, Bcl-2, and Bcl-xL). Suppression of these gene products by pinitol enhanced the apoptosis induced by TNF and chemotherapeutic agents and suppressed TNF-induced cellular invasion. Our results show that pinitol inhibits the NF-kappaB activation pathway, which may explain its ability to suppress inflammatory cellular responses.

Zfra is an inhibitor of Bcl-2 expression and cytochrome c release from the mitochondria

Zfra is a small size 31-amino-acid C2H2 zinc finger-like protein, which is known to interact with c-Jun N-terminal kinase 1 (JNK1), WW domain-containing oxidoreductase (WWOX, FOR or WOX1), TNF receptor-associated death domain protein (TRADD) and nuclear factor kappaB (NF-κB) during stress response. Here, we show that Zfra became phosphorylated at Ser8 (as determined by specific antibody) and translocated to the mitochondria in response to inducers of mitochondrial permeability transition (MPT) (e.g. staurosporine and betulinic acid). Overexpressed Zfra induced cell death. This event is associated, in part, with increased dissipation of mitochondrial membrane potential (MMP) and increased chromosomal DNA fragmentation. Intriguingly, Zfra significantly downregulated Bcl-2 and yet blocked cytochrome c release from the mitochondria. Overexpression of an S8G-Zfra mutant (Ser8 to Gly8 alteration) could not induce cell death, probably due to its failure of translocating to the mitochondria and causing MMP dissipation. Over-expressed proapoptotic WOX1 induced cytochrome c release from the mitochondria. Zfra bound and blocked the effect of WOX1. Taken together, Ser8 is essential for overexpressed Zfra to exert cell death via the mitochondrial pathway. Zfra downregulates Bcl-2 and induces MMP dissipation but causes no cytochrome c release, indicating a novel death pathway from the mitochondria.

Tumor necrosis factor receptor-associated death domain mediated neuronal death contributes to the glial activation and subsequent neuroinflammation in Japanese encephalitis

While a number of studies have documented the importance of microglia in central nervous system (CNS) response to injury, infection and in disease state, little is known regarding how the neuronal death initiates the cascades of secondary neuroinflammation. We have exploited an experimental model of Japanese encephalitis to better understand how neuronal death following viral infection initiates microglial activation following Japanese encephalitis virus infection. We have earlier shown that the altered expression of tumor necrosis factor receptor-1 (TNFR-1) and TNFR associated death domain (TRADD) following Japanese encephalitis virus infection regulates the downstream apoptotic cascades. Here we have reported that silencing TRADD expression with small-interfering RNA reduced neuronal apoptosis and subsequent microglial and astroglial activation and release of various pro-inflammatory mediators. Our findings suggest that the engagement of TNFR-1 and TRADD following Japanese encephalitis virus infection plays a crucial role in glial activation also and influences the outcome of viral pathogenesis.

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.

The NMR structure of the TRADD death domain, a key protein in the TNF signaling pathway

Tumor necrosis factor (TNF) is a member of a superfamily of cytokines that consists of more than 20 structurally related transmembrane and soluble proteins, which play a crucial role in various biological events by recruiting several intracellular adaptors, thus activating multiple signal transduction pathways (Wajant et al. 2003). TNF-a is a homotrimer produced by activated macrophages, and signals through two distinct cell surface receptor subgroups, TNFR1 and TNFR2. The cytoplasmic domain of these two receptors initiate intracellular events, with TNFR1 being the dominant receptor for TNF. The binding of TNF-a to TNFR1 triggers a series of intracellular events, involving many proteins which will recruit key enzymes to TNFR1 that are responsible for initiating signaling events, which leads to either apoptosis or activation of two transcription factors, NF-kB and AP-1. The cytoplasmic region of TNFR1 contains the death domain (DD) sequence, which forms a trimer upon formation of the TNF complex with TNFR1, recruiting other DD containing proteins such as TRADD (TNF receptor-associated DD), the adaptor protein RIP (receptor-interacting protein), and FADD (Fasassociated DD). These protein interactions occur mostly through their common domain, the DD, comprised of *90 amino acids, with low (*30%) sequence homology. The DD superfamily is comprised of several subfamilies: DD, caspase recruitment domain (CARD), death effector domain (DED) and pyrin domain (PYR). So far, about 86 proteins have been identified in humans (Park et al. 2007). The adaptor protein TRADD is a multifunctional 34 kDa protein that is recruited to TNFR1 upon ligand binding. It contains two separate domains, allowing it to bind to several protein partners and participate in different signaling pathways (Hsu et al. 1996). The C-terminal of TRADD contains a DD which is a central component in signaling for TNFR1 stimulation (Hsu et al. 1996). The DD of TRADD is recruited to TNFR1 through homotypic DD interactions, which then promotes subsequent binding to all other signal transducers. TRADD DD can associate to the death domains of TNFR1 and RIP (Hsu et al. 1996), while the N-terminal of TRADD interacts with TRAFs (TNF receptor associated factors), leading to NF-kB and MAP kinase activation. The signaling events generated from TNFR1 upon ligand binding ultimately result in the inflammatory and immunological responses. These responses contribute to both physiological homeostasis and can lead to inflammatory diseases when TNF-a is overproduced. While the role of TNF-a in inflammatory diseases such as rheumatoid arthritis is clinically validated by several anti-TNF therapies, the question of how it triggers these specific signaling responses from TNFR1 by the complex array of signaling proteins remains to be fully understood. Many investigations have focused on understanding the mechanism by which these protein–protein interactions occur, including structural studies of proteins in the TNFR1 pathway. The three dimensional structure of the FAS DD (Huang et al. 1996), FADD DD (Carrington et al. 2006), TNF DD (Sukits et al. D. H. H. Tsao (&) W.-T. Hum K. Malakian Structural Biology and Computational Chemistry, Chemical and Screening Sciences, Wyeth Research, 200 CambridgePark Drive, Cambridge, MA 02140, USA e-mail: dtsao@wyeth.com

SEPSIS-INDUCED MYOCARDIAL DYSFUNCTION

Despite the fact that septic patients exhibit altered cardiac function, it is not considered a major pathology during sepsis. Thus, the molecular mechanisms underlying sepsis-induced myocardial dysfunction have not been studied extensively. In a polymicrobial septic rat model, +dP/dt and -dP/dt on day 1 were not altered but found depressed later, i.e., at 3 and 7 days postsepsis. Diastolic dysfunction characterized by an elevation of the time constant of left ventricular relaxation, tau, was evident at 1, 3, and 7 days postsepsis. Recent data from our laboratory demonstrated that sepsis-induced cardiodynamic alterations correlated with upregulation of TNF receptor-associated death domain, Bax, Smac (both mitochondrial and cytosolic fractions), total nuclear factor kappaB expression, p38-mitogen-activated protein kinase and c-Jun N-terminal kinase phosphorylation, and cytochrome c levels in the rat heart at 3 and 7 days postsepsis. Data from various laboratories emphasized that molecular myocardial alteration, which occurs during early and late stages of sepsis, needs to be elucidated thoroughly. A poor understanding of myocardial signaling during early sepsis could be one of the main reasons for limited success of pharmacotherapeutic options for sepsis. We anticipate that an increased understanding of pathophysiological mechanisms leading to sepsis-induced myocardial dysfunction would generate new enthusiasm among various research groups in this area of research.

Optineurin Negatively Regulates TNFα- Induced NF-κB Activation by Competing with NEMO for Ubiquitinated RIP

NF-kappaB essential modulator (NEMO), the regulatory subunit of the IkappaB kinase (IKK) that activates NF-kappaB, is essential for NF-kappaB activation. NEMO was recently found to contain a region that preferentially binds Lys (K)63-linked but not K48-linked polyubiquitin (polyUb) chains, and the ability of NEMO to bind to K63-linked polyUb RIP (receptor-interacting protein) is necessary for efficient tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation. Optineurin is a homolog of NEMO, and mutations in the optineurin gene are found in a subset of patients with glaucoma, a neurodegenerative disease involving the loss of retinal ganglion cells. Although optineurin shares considerable homology with NEMO, in resting cells, it is not present in the high-molecular-weight complex containing IKKalpha and IKKbeta, and optineurin cannot substitute for NEMO in lipopolysaccharide (LPS)-induced NF-kappaB activation. On the other hand, the overexpression of optineurin blocks the protective effect of E3-14.7K on cell death caused by the overexpression of TNFalpha receptor 1 (TNFR1). Here we show that optineurin has a K63-linked polyUb-binding region similar to that of NEMO, and like NEMO, it bound K63- but not K48-linked polyUb. Optineurin competitively antagonized NEMO's binding to polyUb RIP, and its overexpression inhibited TNFalpha-induced NF-kappaB activation. This competition occurs at physiologic protein levels because microRNA silencing of optineurin resulted in markedly enhanced TNFalpha-induced NF-kappaB activity. These results reveal a physiologic role for optineurin in dampening TNFalpha signaling, and this role might provide an explanation for its association with glaucoma.

A Human scFv Antibody against TRAIL Receptor 2 Induces Autophagic Cell Death in Both TRAIL-Sensitive and TRAIL-Resistant Cancer Cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptotic cell death in a variety of tumor cells without significant cytotoxicity on normal cells. However, many cancer cells with apoptotic defects are resistant to treatment with TRAIL alone, limiting its potential as an anticancer therapeutic. Here, we report on the tumoricidal activity of a human single-chain fragment variable, HW1, which specifically binds to TRAIL receptor 2 (TR2) without competing with TRAIL for the binding. HW1 treatment as a single agent induces autophagic cell death in a variety of both TRAIL-sensitive and TRAIL-resistant cancer cells, but exhibits much less cytotoxicity on normal cells. The HW1-induced autophagic cell death was inhibited by an autophagy inhibitor, 3-methyladenine, or by RNA interference knockdown of Beclin-1 and Atg7. We also show that the HW1-mediated autophagic cell death occurs predominantly via the c-Jun NH(2)-terminal kinase pathway in a caspase-independent manner. Analysis of the death-inducing signaling complex induced by HW1 binding to TR2 exhibits the recruitment of TNF receptor-associated death domain and TNF receptor-associated factor 2, but not Fas-associated death domain, caspase-8, or receptor-interacting protein, which is distinct from that induced by TRAIL. Our results reveal a novel TR2-mediated signaling pathway triggering autophagic cell death and provides a new strategy for the elimination of cancer cells, including TRAIL-resistant tumors, through nonapoptotic cell death.

Evidence that genetic deletion of the TNF receptor p60 or p80 inhibits Fas mediated apoptosis in macrophages

Almost 19 members of the tumor necrosis factor (TNF) superfamily have been identified that interact with 29 different receptors. Whether these receptors communicate with each other is not understood. Recently, we have shown that receptor activator of NF-κB ligand signaling is modulated by genetic deletion of the TNF receptor. In the current report, we investigated the possibility of a cross-talk between Fas and TNF-α signaling pathway in macrophage cell lines derived from wild-type (WT) mice and from mice with genetic deletion of the type 1 TNF receptor (p60−/−), the type 2 TNF receptor (p80−/−), or both receptors (p60−/−p80−/−). We found that the macrophages expressing TNF receptors were highly sensitive to apoptosis induced by anti-Fas. The genetic deletion of TNF receptors, however, made the cells resistance to anti-Fas-induced apoptosis. Anti-Fas induced activation of caspase-3 and PARP cleavage in WT cells but not in TNF receptor-deleted cells. This difference was found to be independent of the expression of Fas, Fas-associated protein with death domain (FADD) or TNF receptor-associated death domain (TRADD). We found that anti-Fas induced recruitment of TNFR1 into Fas-complex. We also found that TRADD, which mediates TNF signaling, was constitutively bound to Fas receptor in TNF receptor-deleted cells but not in wild-type cells. Transient transfection of TNFR1 in TNFR1-deleted cells sensitized them to anti-Fas-induced apoptosis. Overall our results demonstrate that Fas signaling is modulated by the TNF receptors and thus provide the evidence of cross-talk between the receptors of two cytokines.

Tumor necrosis factor receptor‐1‐induced neuronal death by TRADD contributes to the pathogenesis of Japanese encephalitis

While a number of studies have documented the neurotropism of Japanese encephalitis virus (JEV), little is known regarding the molecular mechanism of neuronal death following viral infection. The tumor necrosis factor receptor (TNFR)-associated death domain (TRADD) has been suggested to be the crucial signal adaptor that mediates all intracellular responses from TNFR-1. Using mouse (Neuro2a) and human (SK-N-SH) neuroblastoma cell lines, we have shown that the altered expression of TNFR-1 and TRADD following JEV infection regulates the downstream apoptotic cascades. Activation of TRADD led to mitochondria-mediated neuronal apoptosis. As TRADD-knockout animals or deficient cell lines are unavailable, it has been difficult to definitively address the physiological role of TRADD in diseases pathology following JEV infection. We circumvented this problem by silencing TRADD expression with small-interfering RNA (siRNA) and have found that TRADD is required for TNFR-1-initiated neuronal apoptosis following in vitro infection with JEV. Interestingly, siRNA against TRADD also decreased the viral load in Neuro2a cells. Furthermore, siRNA against TRADD increased the survival of JEV-infected mice by altering the expression of pro apoptotic versus antiapoptotic molecules. These studies show that the engagement of TNFR-1 and TRADD following JEV infection plays a crucial role in neuronal apoptosis.

Intrinsic resistance to TNF-α-induced hepatocyte apoptosis in ICR mice correlates with expression of a short form of c-FLIP

The administration of tumor necrosis factor-α (TNF-α) or the anti-Fas antibody (Jo-2) to mice causes acute liver failure, which is lethal within hours as a result of the induction of apoptosis in hepatocytes. It was recently reported that nonobese diabetic (NOD) mice are less sensitive to TNF-α/D-galactosamine (GalN)-induced liver failure than C57BL/6J (B6) mice, whereas both NOD and B6 mice were sensitive to the lethal effect of Jo-2. In the present study, we investigated the differences between the apoptotic liver cell death induced by TNF-α/GalN and that induced by Jo-2. B6, NOD, and Jcl-Imperial Cancer Research (ICR) mice were injected intravenously with TNF-α/GalN or Jo-2. ICR mice were less sensitive to TNF-α/GalN-induced liver failure than NOD and B6 mice (P<0.0001). In contrast, ICR mice were more sensitive to Jo-2-induced liver failure than B6 mice (P=0.0003). The liver caspase-3, -8 activity, serum transaminase levels, and the number of apoptotic liver nuclei all decreased in ICR in comparison to B6 mice treated with TNF-α/GalN. The mRNA expression of TNFR-associated death domain, Fas associated protein with death domain, and Bcl family and nuclear factor-κB activation induced by TNF-α/GalN were similar in both mice. Interestingly, the short form of cellular FLICE/caspase-8-inhibitory protein (c-FLIPS) was constitutively upregulated in ICR mice. In conclusion, these results suggest that ICR mice have an intrinsic resistance to TNF-α-induced hepatocyte apoptosis, and that c-FLIPS may play a role in TNF-α/GalN-induced liver failure, but not in Fas-induced liver failure.

Epstein-Barr Virus (EBV) Latent Membrane Protein-1 Down-Regulates Tumor Necrosis Factor-α (TNF-α) Receptor-1 and Confers Resistance to TNF-α-Induced Apoptosis in T Cells: Implication for the Progression to T-Cell Lymphoma in EBV-Associated Hemophagocytic Syndrome

The infection of T cells by Epstein-Barr virus (EBV) may result in hemophagocytic syndrome (HPS) through enhanced cytokine secretion, particularly tumor necrosis factor-alpha (TNF-alpha), by EBV latent membrane protein-1 (LMP-1). One bewildering observation of HPS patients is relapsing disease or progression to T-cell lymphoma. This finding raises the question whether EBV LMP-1-expressing T cells may survive and proliferate in the cytokine milieu of HPS. To explore this possibility, we tested the sensitivity of LMP-1-expressing T cells to apoptosis in the presence of TNF-alpha. LMP-1 up-regulated TNF-alpha through TRAF2,5 and nuclear factor-kappaB pathway in T cells. The LMP-1-expressing T cells then became resistant to TNF-alpha-induced apoptosis. Interestingly, the expression of TNFR1 was remarkably down-regulated by LMP-1 in T cells. Furthermore, the TNF-alpha/TNFR1 downstream death signal TNFR1-associated death domain protein was constitutively recruited by LMP-1, and the activities of apoptotic caspases 3, 8, and 9 were suppressed. Reconstitution of TNFR1 successfully reversed the TNF-alpha-induced apoptotic cascades. Therefore, EBV LMP-1 not only activates T cells to proliferate but also confers resistance to TNF-alpha-mediated apoptosis via down-regulation of TNFR1 in the cytokine milieu of HPS. This finding provides a potential mechanism to explain the disease persistence or progression to T-cell lymphoma in HPS patients.

Morin (3,5,7,2′,4′-Pentahydroxyflavone) Abolishes Nuclear Factor-κB Activation Induced by Various Carcinogens and Inflammatory Stimuli, Leading to Suppression of Nuclear Factor-κB–Regulated Gene Expression and Up-regulation of Apoptosis

Morin is a flavone that exhibits antiproliferative, antitumor, and anti-inflammatory effects through a mechanism that is not well understood. Because of the role of transcription factor nuclear factor-kappaB (NF-kappaB) in the control of cell survival, proliferation, tumorigenesis, and inflammation, we postulated that morin mediates its effects by modulating NF-kappaB activation. We investigated the effect of morin on NF-kappaB pathway activated by inflammatory agents, carcinogens, and tumor promoters. The effect of this flavone on expression of NF-kappaB-regulated gene products involved in cell survival, proliferation, and invasion was also examined. We showed by DNA-binding assay that NF-kappaB activation induced by tumor necrosis factor (TNF), phorbol 12-myristate 13-acetate, lipopolysaccharide, ceramide, interleukin-1, and H(2)O(2) was suppressed by morin; the suppression was not cell type specific. The suppression of NF-kappaB by morin was mediated through inhibition of IkappaBalpha (inhibitory subunit of NF-kappaB) kinase, leading to suppression of phosphorylation and degradation of IkappaBalpha and consequent p65 nuclear translocation. Morin also inhibited the NF-kappaB-dependent reporter gene expression activated by TNF, TNF receptor (TNFR) 1, TNFR1-associated death domain, TNFR-associated factor 2, NF-kappaB-inducing kinase, IkappaB kinase, and the p65 subunit of NF-kappaB. NF-kappaB-regulated gene products involved in cell survival [inhibitor of apoptosis (IAP) 1, IAP2, X chromosome-linked IAP, Bcl-xL, and survivin], proliferation (cyclin D1 and cyclooxygenase-2), and invasion (matrix metalloproteinase-9) were down-regulated by morin. These effects correlated with enhancement of apoptosis induced by TNF and chemotherapeutic agents. Overall, our results indicate that morin suppresses the activation of NF-kappaB and NF-kappaB-regulated gene expression, leading to enhancement of apoptosis. This may provide the molecular basis for the ability of morin to act as an anticancer and anti-inflammatory agent.

Receptor-mediated Endocytosis Is Not Required for Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared with normal cells. Other members of the TNF family of death ligands (TNF, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signaling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but, rather, amplified the apoptotic signaling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization is required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin-dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors is required for transmission of its apoptotic signal, recruitment of FADD (Fas-associated death domain) and procaspase-8 to form the TRAIL-associated DISC occurred at 4 degrees C, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL receptor 1/2, unlike TNF-TNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8, or transmission of an apoptotic signal in BJAB type I cells.