Human 55-kDa receptor for tumor necrosis factor coupled to signal transduction cascades.

The numerous biological activities of tumor necrosis factor (TNF) appear mediated by two types of receptors of 55 kDa (TR55) and 75 kDa (TR75) molecular mass. To test TR55 for its individual role in signaling across the membrane, a cDNA coding for the human TR55 was stably expressed in murine 70Z/3 pre-B cells, which lack binding sites for, and proved nonresponsive to human TNF. The transfected TR55 showed high affinity ligand binding and active internalization. It is demonstrated that the TNF signaling cascade, i.e. stimulation of protein kinase C, sphingomyelinase, and phospholipase A2, production of the second messengers diacylglycerol and ceramide, can occur completely through exclusive binding of TNF to TR55. The p55 TNF-binding site functions as an autonomous TNF receptor that mediates key signal transduction pathways, which may control the majority of TNF actions.     

The soluble glucocorticoid-induced tumor necrosis factor receptor causes cell cycle arrest and apoptosis in murine macrophages

In order to clarify the mechanism by which soluble GITR (sGITR) inhibits the survival of murine macrophages we examined its effect on the macrophage cell cycle. Soluble GITR induced G1 phase arrest followed by apoptosis. It also reduced the expression of cyclins D2 and A, and of cdk4, resulting in reduced cdk2 and cdk4 activities. These findings suggest that sGITR arrests division of the macrophages in G1 by lowering the activities of cdk2 and cdk4, and that this leads to apoptosis.     

Phosphorylation of the tumor necrosis factor receptor CD120a (p55) recruits Bcl-2 and protects against apoptosis

Ligation of the tumor necrosis factor alpha receptor CD120a initiates responses as diverse as apoptosis and the expression of NF-kappaB-dependent pro-survival genes. How these opposing responses are controlled remains poorly understood. Here we demonstrate that phosphorylation by p42(mapk/erk2) inhibits the apoptotic activity of CD120a while preserving its ability to activate NF-kappaB. Phosphorylated CD120a is re-localized from the Golgi complex to tubular structures of the endoplasmic reticulum wherein it recruits Bcl-2. Antisense-mediated down-regulation of Bcl-2 antagonized the localization of CD120a to tubular structures and reversed the protection from apoptosis conferred by receptor phosphorylation. We propose that phosphorylation of CD120a represents a novel, Bcl-2-dependent mechanism by which the apoptotic activity of the receptor may be regulated. Thus, oncogenic activation of p42(mapk/erk2) may serve to inhibit the apoptotic activity of this death receptor while preserving NF-kappaB-dependent responses and may thus indirectly contribute to a failure to eliminate cells bearing oncogenes of the Ras-Raf-MEK-p42(mapk/erk2) pathway.     

The CD95 receptor: apoptosis revisited

CD95 is the quintessential death receptor and, when it is bound by ligand, cells undergo apoptosis. Recent evidence suggests, however, that CD95 mediates not only apoptosis but also diverse nonapoptotic functions depending on the tissue and the conditions.     

CD95 death receptor and epidermal growth factor receptor (EGFR) in liver cell apoptosis and regeneration

Recent evidence suggests that signaling pathways towards cell proliferation and cell death are much more interconnected than previously thought. Whereas not only death receptors such as CD95 (Fas, APO-1) can couple to both, cell death and proliferation, also growth factor receptors such as the epidermal growth factor receptor (EGFR) are involved in these opposing kinds of cell fate. EGFR is briefly discussed as a growth factor receptor involved in liver cell proliferation during liver regeneration. Then the role of EGFR in activating CD95 death receptor in liver parenchymal cells (PC) and hepatic stellate cells (HSC), which represent a liver stem/progenitor cell compartment, is described summarizing different ways of CD95- and EGFR-dependent signaling in the liver. Here, depending on the hepatic cell type (PC vs. HSC) and the respective signaling context (sustained vs. transient JNK activation) CD95-/EGFR-mediated signaling ends up in either liver cell apoptosis or cell proliferation.     

Caspase inhibition activates HIV in latently infected cells. Role of tumor necrosis factor receptor 1 and CD95

Stimulation of tumor necrosis factor receptor 1 (TNF-R1) triggers both caspase-dependent and caspase-independent signaling activities. The caspase-dependent signaling pathway induces apoptotic cell death in susceptible cells, whereas the caspase-independent signaling cascade leads to activation of nuclear factor kappa B and induces antiapoptotic signaling activities. Stimulation of nuclear factor kappa B via TNF-R1 is known to activate human immunodeficiency virus (HIV) replication in infected cells. Here we show that the broad range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD) activates HIV replication in the chronically infected T-cell line ACH-2. Virus activation was caused by a sensitization of TNF-R1 toward endogenously produced tumor necrosis factor alpha (TNF-alpha). Neutralizing anti-TNF-alpha antibodies completely abolished the virus-inducing activity of ZVAD. Treatment of cells with TNF-alpha in the presence of ZVAD caused increased expression of TNF-alpha and induced enhanced virus replication. Activation of CD95, another member of the TNF receptor family, similarly triggered HIV replication, which was further enhanced in the presence of ZVAD. Our data show that caspase inhibitors sensitize both CD95 and TNF-R1 to mediate activation of HIV in latently infected cells. Activation of HIV replication in latent virus reservoirs is currently discussed as a therapeutic strategy to achieve eradication of HIV in patients treated with antiretroviral therapy. Our results point to a novel role for caspase inhibitors as activators of virus replication in vivo.     

Phosphorylation of tumor necrosis factor receptor 1 (p55) protects macrophages from silica-induced apoptosis

Macrophages play a fundamental role in silicosis in part by removing silica particles and producing inflammatory mediators in response to silica. Tumor necrosis factor alpha (TNFalpha) is a prominent mediator in silicosis. Silica induction of apoptosis in macrophages might be mediated by TNFalpha. However, TNFalpha also activates signal transduction pathways (NF-kappaB and AP-1) that rescue cells from apoptosis. Therefore, we studied the TNFalpha-mediated mechanisms that confer macrophage protection against the pro-apoptotic effects of silica. We will show that exposure to silica induced TNFalpha production by RAW 264.7 cells, but not by IC-21. Silica-induced activation of NF-kappaB and AP-1 was only observed in RAW 264.7 macrophages. ERK activation in response to silica exposure was only observed in RAW 264.7 macrophages, whereas activation of p38 phosphorylation was predominantly observed in IC-21 macrophages. No changes in JNK activity were observed in either cell line in response to silica exposure. Silica induced apoptosis in both macrophage cell lines, but the induction of apoptosis was significantly larger in IC-21 cells. Protection against apoptosis in RAW 264.7 cells in response to silica was mediated by enhanced NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNFalpha receptor. Inhibition of these two protective mechanisms by specific pharmacological inhibitors or transfection of dominant negative mutants that inhibit IkappaBalpha or ERK phosphorylation significantly increased silica-induced apoptosis in RAW 264.7 macrophages. These data suggest that NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNF receptor are important cell survival mechanisms in the macrophage response to silica exposure.     

Epidermal growth factor receptor transactivation mediates tumor necrosis factor-induced hepatocyte replication

Tumor necrosis factor (TNF) has multiple biological effects such as participating in inflammation, apoptosis, and cell proliferation, but the mechanisms of its effects on epithelial cell proliferation have not been examined in detail. At the early stages of liver regeneration, TNF functions as a priming agent for hepatocyte replication and increases the sensitivity of hepatocytes to growth factors such as transforming growth factor alpha (TGFalpha); however, the mechanisms by which TNF interacts with growth factors and enhances hepatocyte replication are not known. Using the AML-12 hepatocyte cell line, we show that TNF stimulates proliferation of these cells through transactivation of the epidermal growth factor receptor (EGFR). The transactivation mechanism involves the release of TGFalpha into the medium through activation of the metalloproteinase TNFalpha-converting enzyme (also known as ADAM 17). Binding of the ligand to EGFR initiates a mitogenic cascade through extracellular signal-regulated kinases 1 and 2 and the partial involvement of protein kinase B. TNF-induced release of TGFalpha and activation of EGFR signaling were inhibited by TNFalpha protease inhibitor-1, an agent that interferes with TNFalpha-converting enzyme activity. We suggest that TNF-induced transactivation of EGFR may provide an early signal for the entry of hepatocytes into the cell cycle and may integrate proliferative and survival pathways at the start of liver regeneration.     

Tumor necrosis factor receptor signaling. A dominant negative mutation suppresses the activation of the 55-kDa tumor necrosis factor receptor.

To investigate the signaling mechanism of the 55-kDa tumor necrosis factor (TNF) receptor a functional transfection based assay was developed. The human 55-kDa TNF receptor, stably expressed in mouse L929 cells, was demonstrated to be activated specifically by agonist antibodies and to initiate a signal for cellular cytotoxicity. A deletion mutant of the human TNF receptor lacking most of the cytoplasmic domain was found to be completely defective in generating the signal for cytotoxicity. Additionally, expression of the truncated receptor substantially suppressed signaling by endogenous mouse TNF receptors in response to TNF, but not in response to specific anti-murine TNF receptor antibodies. These results suggest that aggregation of 55-kDa TNF receptor intracellular domains, which are not associated in the absence of ligand, is an important component of the signal for cellular toxicity. This work also provides an example of a dominant negative mutation in a transmembrane receptor that lacks a tyrosine kinase domain, and suggests a more general utility of dominant negative mutations in the investigation of cytokine receptor function.     

Distinct intracellular signaling in tumor necrosis factor-related apoptosis-inducing ligand- and CD95 ligand-mediated apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in tumor cells but not in healthy cells. Similar to CD95 ligand (CD95L), TRAIL signaling requires ligand-receptor interaction; the downstream signaling molecules, such as Fas-associated death domain and caspase-8, also seem similar. Using cells stably expressing TRAIL and CD95L, we show that both TRAIL and CD95L induce apoptosis in the rat colon carcinoma cell line CC531. The mitochondrial damage (loss of mitochondrial membrane potential (MMP) and release of cytochrome c) observed after co-incubation with TRAIL-expressing cells occurs much earlier than that observed with CD95L-expressing cells. The decrease in MMP induced by both ligands was caspase-8-mediated; no difference in caspase-8 activation by TRAIL and CD95L was found. TRAIL, but not CD95L, induced activation of caspase-10. bcl-2 overexpression could not prevent TRAIL-induced mitochondrial dysfunction, whereas it completely prevented CD95L-mediated loss of MMP and cytochrome c release. The selective effect of TRAIL on tumor cells and the apparent inability of bcl-2 to block TRAIL-induced apoptosis suggest that TRAIL may offer a lead for cancer therapy in the future.     

Phosphorylation of 130- and 95-kDa substrates associated with tumor necrosis factor-alpha receptor CD120a (p55)

Cross-linking of CD120a (p55), a receptor for tumor necrosis factor alpha (TNFalpha), initiates downstream events, including the activation of protein Ser/Thr kinases. In this report, we have characterized two protein Ser/Thr kinase substrates that are intrinsically associated with CD120a (p55) in mouse macrophages, and we have investigated the mechanism involved in their phosphorylation. pp130 and pp95 were detected by co-immunoprecipitation with CD120a (p55) from lysates of mouse bone marrow-derived macrophages and were phosphorylated on Ser and Thr residues during in vitro kinase assays in the presence of [gamma-(32)P]ATP. The level of phosphorylation of pp130 and pp95 was rapidly and transiently increased in response to TNFalpha in [(32)P]orthophosphate-labeled macrophages, although the level of pp130 protein associated with CD120a (p55) remained unchanged as detected by [(35)S]methionine labeling. In contrast, pp130 and pp95 were efficiently phosphorylated in in vitro kinase assays of CD120a (p55) immunoprecipitates from unstimulated cells, and the level of phosphorylation was rapidly and transiently reduced in response to TNFalpha. Both pp130 and pp95 were sensitive to dephosphorylation with purified protein phosphatase 2A, and okadaic acid, a PP1/PP2A inhibitor, mimicked the ability of TNFalpha to stimulate the phosphorylation of pp130 and pp95 in intact (32)P-labeled macrophages. Collectively, these findings suggest that pp130 and pp95 are constitutively associated with CD120a (p55) and become inducibly phosphorylated in macrophages in response to TNFalpha. We propose that the underlying mechanism of their phosphorylation may involve the inactivation of a cytoplasmic pp130/pp95 Ser/Thr phosphatase.     

Role of p55 tumor necrosis factor receptor 1 in acetaminophen-induced antioxidant defense

Tumor necrosis factor (TNF)-alpha is a macrophage-derived proinflammatory cytokine implicated in hepatotoxicity. In the present studies, p55 TNF receptor 1 (TNFR1) -/- mice were used to assess the role of TNF-alpha in acetaminophen-induced antioxidant defense. Treatment of wild-type (WT) mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis and increased serum alanine transaminases. This was correlated with a rapid depletion of hepatic glutathione (GSH). Whereas in WT mice GSH levels returned to control after 6-12 h, in TNFR1-/- mice recovery was delayed for 48 h. Delayed induction of heme oxygenase-1 and reduced expression of CuZn superoxide dismutase were also observed in TNFR1-/- compared with WT mice. This was associated with exaggerated hepatotoxicity. In WT mice, acetaminophen caused a time-dependent increase in activator protein-1 nuclear binding activity and in c-Jun expression. This response was significantly attenuated in TNFR1-/- mice. Constitutive NF-kappaB binding activity was detectable in livers of both WT and TNFR1-/- mice. A transient decrease in this activity was observed 3 h after acetaminophen in WT mice, followed by an increase that was maximal after 6-12 h. In contrast, in TNFR1-/- mice, acetaminophen-induced decreases in NF-kappaB activity were prolonged and did not return to control levels for 24 h. These data indicate that TNF-alpha signaling through TNFR1 plays an important role in regulating the expression of antioxidants in this model. Reduced generation of antioxidants may contribute to the increased sensitivity of TNFR1-/- mice to acetaminophen.     

The p70 tumor necrosis factor receptor mediates cytotoxicity.

Tumor necrosis factor alpha (TNF) selectively kills tumor cells, but this specificity is not clearly understood. Two distinctly different cell surface receptors (TNFRs), proteins of 55 kd (p55) and 70-80 kd (p70), mediate TNF action. Mouse TA1 cells are not killed by human (h) TNF, but are killed by mouse (m) TNF alone. Since the mouse p70 TNFR is recognized only by mTNF, these results implicate p70 receptor action in TA1 cell killing. Human HeLa cells have mainly the p55 receptor and are not killed by hTNF alone. When transfected with the human p70 TNFR, HeLa p70 die within 24 hr. HeLa p70 cells also show reduced c-fos and manganous superoxide dismutase induction by TNF. NIH 3T3 mouse fibroblasts are sensitive to only mTNF, but overexpression of the human p70 receptor causes cell death by hTNF and increased sensitivity to mTNF. These results provide a direct function for the p70 TNFR in TNF-induced cytotoxicity.     

Death receptor activation-induced hepatocyte apoptosis and liver injury

The TNFalpha receptor super-family consists of several members sharing a sequence homology in a unique function domain, the death domain, which is located in the intracellular portion of the receptor. These so-called death receptors, including Fas, TNF-R1 and TRAIL-R1/TRAIL-R2, are expressed on hepatocytes. When stimulated by their ligands, FasL, TNFalpha or TRAIL, respectively, the death receptors can activate multiple death domain-initiated apoptosis programs, including both extrinsic and intrinsic pathways. A cascade of caspases is activated, which cleave proteins important for the cell structure and function. Activation of the intrinsic pathway also leads to mitochondrial release of several apoptotic proteins and mitochondrial dysfunction, which kill the cell through both caspase-dependent and caspase-independent mechanisms. Death receptor-induced hepatocyte apoptosis contributes to the development of a number of liver diseases, including viral hepatitis, inflammatory hepatitis, Wilson's disease, alcoholic liver disease, endotoxiemia-induced liver failure and ischemia/reperfusion-induced liver damage. This article comprehensively reviews the mechanisms of induction and regulation of death receptor-initiated apoptosis in hepatocytes, examines how these molecular events affect our understanding of the pathogenesis of these diseases and further discusses the potential therapeutic application of the knowledge. We hope we can provide a cohesive and integrated perspective on the many aspects of these complicated processes.     

Bid-independent mitochondrial activation in tumor necrosis factor alpha-induced apoptosis and liver injury

The death receptor apoptosis pathway is intimately connected with the mitochondrial apoptosis pathway. Bid is a BH3-only pro-death Bcl-2 family protein and is the major molecule linking the two pathways. Bid-mediated mitochondrial activation occurs early and is responsible for the prompt progress of tumor necrosis factor alpha (TNF-alpha)-induced apoptosis. However, in both cultured cells and animal models of TNF-alpha-induced injury, later-phase Bid-independent mitochondrial activation could be demonstrated. Consequently, bid-deficient mice are still susceptible to endotoxin-induced liver injury and mortality. Notably, embryonic hepatocyte apoptosis and lethality caused by TNF-alpha in the absence of p65relA cannot be rescued by the simultaneous deletion of bid. Further studies indicate that multiple mechanisms including reactive oxygen species, JNK, and permeability transition are critically involved in Bid-independent mitochondrial activation. Inhibition of these events suppresses TNF-alpha-induced mitochondrial activation and apoptosis in bid-deficient cells. These findings thus indicate that there are at least two sets of mechanisms of mitochondrial activation upon TNF-alpha stimulation. While the Bid-mediated mechanism is rapid and potent, the Bid-independent mechanism progresses gradually and involves multiple players. The critical involvement of Bid-independent mitochondrial activation in TNF-alpha-induced apoptosis demands the intervention of TNF-alpha-mediated tissue injury via multiple avenues.     

Species specificity of human and murine tumor necrosis factor. A comparative study of tumor necrosis factor receptors

Recombinant murine and human tumor necrosis factor (mTNF and hTNF, respectively) were radioiodinated to high specific activity using a solid-phase lactoperoxidase method. A single class of high affinity receptors for 125I-TNF was identified on TNF-sensitive murine L cells and human HeLa S2 cells. Competitive radioligand binding assays were used to study the species specificity of TNF preparations. Unlabeled hTNF competed 30-fold less effectively than mTNF for binding to L cell receptors, whereas mTNF competed to approximately the same extent as hTNF for binding to HeLa cell receptors. A similar species specificity was observed in cytotoxicity assays; hTNF was more cytotoxic for HeLa cells than mTNF. Conversely, mTNF was more growth inhibitory and cytotoxic for L cells than hTNF. mTNF. and hTNF.receptor complexes were compared by gel filtration chromatography and polyacrylamide gel electrophoresis before and after cross-linking with bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES). These complexes eluted in gel filtration at a position corresponding to a globular protein of 350,000 Mr. Gel autoradiographs of the fractions containing cross-linked complexes showed bands of 95,000 and 75,000 Mr as well as small amounts of higher Mr bands. mTNF and hTNF treated with BSOCOES formed cross-linked dimers and trimers. Therefore, we were unable to determine whether the 95,000 and 75,000 Mr bands represented two distinct subunits of receptors or one subunit to which either a dimer or a monomer of TNF was cross-linked. These results demonstrate species specificity in the TNF-receptor interaction. In addition, the affinity labeling studies in two species give an identical pattern for the TNF X receptor complexes, suggesting that the receptors have similar subunit composition.     

Identification of the binding site of 55kDa tumor necrosis factor receptor by synthetic peptides.

We have synthesized a series of peptides, which cover almost the whole range of the N-terminal extracellular domain of human 55kDa TNF receptor (55kDa TNF-R). The peptides were examined for the binding activity to TNF by solid phase binding assay and for the inhibition of TNF cytotoxicity to mouse L-M cells. The peptide 159-178 exhibited remarkably higher binding activity to TNF than other peptides did. The specificity of the TNF binding to the peptides was confirmed by their inability to bind other cytokines. The peptide 159-178 also inhibited TNF cytotoxicity. These results indicate that the specific binding site of 55kDa TNF-R to TNF might reside within the peptide segment of amino acid numbers 159 to 178 in the N-terminal extracellular domain.     

Epstein-Barr virus immediate-early protein BZLF1 inhibits tumor necrosis factor alpha-induced signaling and apoptosis by downregulating tumor necrosis factor receptor 1

Tumor necrosis factor alpha (TNF-alpha) is a key mediator of host immune and inflammatory responses and inhibits herpesvirus replication by cytolytic and noncytolytic mechanisms. TNF-alpha effects are primarily mediated through the major TNF-alpha receptor, TNF-R1, which is constitutively expressed in most cell types. Here we show that the Epstein-Barr virus (EBV) immediate-early protein BZLF1 prevents TNF-alpha activation of target genes and TNF-alpha-induced cell death. These effects are mediated by down-regulation of the promoter for TNF-R1. Additionally, we demonstrate that expression of TNF-R1 is downregulated during the EBV lytic replication cycle. Thus, EBV has developed a novel mechanism for evading TNF-alpha antiviral effects during lytic reactivation or primary infection.