Apoptosis mediated by the TNF-related cytokine and receptor families

T lymphocytes use several specialized mechanisms to induce apoptotic cell death. The tumor necrosis factor (TNF)-related family of membrane-anchored and secreted ligands represent a major mechanism regulating cell death and cell survival. These ligands also coordinate differentiation of tissue to defend against intracellular pathogens and regulate development of lymphoid tissue. Cellular responses are initiated by a corresponding family of specific receptors that includes two distinct TNFR (TNFR60 and TNFR80), Fas (CD95), CD40, p75NTF, and the recently identified lymphotoxin β-receptor (LTβR), among others. The MHC-encoded cytokines, TNF and LTα, form homomeric trimers, whereas LTβ assembles into heterotrimers with LTα, creating multimeric ligands with distinct receptor specificities. The signal transduction cascade is initiated by transmembrane aggregation (clustering) of receptor cytoplasmic domains induced by binding to their multivalent ligands. The TRAF family of Zn RING/finger proteins bind to TNFR80; CD40 and LTβR are involved in induction NFκB and cell survival. TNFR60 and Fas interact with several distinct cytosolic proteins sharing the “death domain” homology region. TNF binding to TNFR60 activates a serine protein kinase activity and phosphoproteins are recruited to the receptor forming a multicomponent signaling complex. Thus, TNFRs use diverse sets of signaling molecules to initiate and regulate cell death and survival pathways. © 1996 Wiley-Liss, Inc.     

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.     

Inhibition of receptor internalization by monodansylcadaverine selectively blocks p55 tumor necrosis factor receptor death domain signaling

The 55-kDa receptor for tumor necrosis factor (TR55) triggers multiple signaling cascades initiated by adapter proteins like TRADD and FAN. By use of the primary amine monodansylcadaverine (MDC), we addressed the functional role of tumor necrosis factor (TNF) receptor internalization for intracellular signal distribution. We show that MDC does not prevent the interaction of the p55 TNF receptor (TR55) with FAN and TRADD. Furthermore, the activation of plasmamembrane-associated neutral sphingomyelinase activation as well as the stimulation of proline-directed protein kinases were not affected in MDC-treated cells. In contrast, activation of signaling enzymes that are linked to the "death domain" of TR55, like acid sphingomyelinase and c-Jun-N-terminal protein kinase as well as TNF signaling of apoptosis in U937 and L929 cells, are blocked in the presence of MDC. The results of our study suggest a role of TR55 internalization for the activation of select TR55 death domain signaling pathways including those leading to apoptosis.     

Infection of epithelial cells with Chlamydia trachomatis inhibits TNF‐induced apoptosis at the level of receptor internalization while leaving non‐apoptotic TNF‐signalling intact

Chlamydia trachomatis is an obligate intracellular bacterial pathogen of medical importance. C. trachomatis develops inside a membranous vacuole in the cytosol of epithelial cells but manipulates the host cell in numerous ways. One prominent effect of chlamydial infection is the inhibition of apoptosis in the host cell, but molecular aspects of this inhibition are unclear. Tumour necrosis factor (TNF) is a cytokine with important roles in immunity, which is produced by immune cells in chlamydial infection and which can have pro‐apoptotic and non‐apoptotic signalling activity. We here analysed the signalling through TNF in cells infected with C. trachomatis. The pro‐apoptotic signal of TNF involves the activation of caspase‐8 and is controlled by inhibitor of apoptosis proteins. We found that in C. trachomatis‐infected cells, TNF‐induced apoptosis was blocked upstream of caspase‐8 activation even when inhibitor of apoptosis proteins were inhibited or the inhibitor of caspase‐8 activation, cFLIP, was targeted by RNAi. However, when caspase‐8 was directly activated by experimental over‐expression of its upstream adapter Fas‐associated protein with death domain, C. trachomatis was unable to inhibit apoptosis. Non‐apoptotic TNF‐signalling, particularly the activation of NF‐κB, initiates at the plasma membrane, while the activation of caspase‐8 and pro‐apoptotic signalling occur subsequently to internalization of TNF receptor and the formation of a cytosolic signalling complex. In C. trachomatis‐infected cells, NF‐κB activation through TNF was unaffected, while the internalization of the TNF–TNF‐receptor complex was blocked, explaining the lack of caspase‐8 activation. These results identify a dichotomy of TNF signalling in C. trachomatis‐infected cells: Apoptosis is blocked at the internalization of the TNF receptor, but non‐apoptotic signalling through this receptor remains intact, permitting a response to this cytokine at sites of infection.     

Interferon-gamma sensitizes the human salivary gland cell line, HSG, to tumor necrosis factor-alpha induced activation of dual apoptotic pathways

Activated immune cells secrete proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), interferon–gamma (IFN-gamma) and Fas ligand (FasL) and these cytokines have been reported to induce apoptosis in numerous cell types. Apoptotic cell death has been associated with the progression of numerous autoimmune diseases. Proinflammatory cytokines are reportedly involved in apoptosis in the salivary glands of patients with Sjögren’s syndrome (SS); an autoimmune disorder characterized by the destruction of salivary and lachrymal glands. In this study, we used the HSG cell line to determine if exposure to proinflammatory cytokines induces apoptosis in human salivary gland cells. In addition, we identified the mediators controlling the apoptotic process in response to TNF alpha and IFN gamma. TNF-alpha and IFN-gamma induced apoptosis in HSG cells and resulted in the activation of caspase 8 and the “death receptor” pathway. We further determined that caspase 9 and the “mitochondrial” pathway was also activated. Induction of the intrinsic and extrinsic pathways in HSG cells resulted in substrate cleavage by effector caspases, in particular the cleavage of alpha II spectrin, an autoantigen in Sjögren’s syndrome. Our results suggest that HSG cells provide a model system to study processes regulating proinflammatory cytokine-induced apoptotic cell death.     

Caspases are the main executioners of Fas-mediated apoptosis, irrespective of the ceramide signalling pathway

Tumor necrosis factor alpha (TNF) or cytotoxic anti-Fas antibodies lead to the activation of apoptotic proteases (caspases) and to sphingomyelinase-mediated ceramide generation. Caspases and ceramide are both known to induce apoptosis on its own, but their relative contribution to Fas- and TNF-induced cell death is not well established. We report here that rapid apoptosis induced by TNF in U937 cells or anti-Fas in Jurkat cells, in the presence of cycloheximide, induced only a very low increase (<20%) in the cell ceramide content. Neither treatment with inhibitors of sphingomyelinases nor incubation of cells with fumonisin B1, which inhibits de novo ceramide synthesis, prevented TNF and Fas-mediated apoptosis. Increasing or depleting the cell ceramide content by prolonged culture in the presence of monensin or fumonisin B1, respectively, did not prevent TNF and Fas-mediated apoptosis. Treatment of cells with sphingomyelinase inhibitors did not affect to the activation of CPP32 (caspase-3) induced by TNF or anti-Fas antibodies. Chromatin condensation and fragmentation in cells treated with anti-Fas or TNF was abrogated by peptide inhibitors of caspases, which also inhibited Fas-, but not TNF-induced cell death. These results indicate that while ceramide does not seem to act as a critical mediator of TNF and Fas-induced apoptosis, it is generated as a consequence of CPP32 activation and could contribute to the spread of the intracellular death signal.     

Tumor necrosis factor receptor-associated factor (TRAF) 2 and its role in TNF signaling

Tumor necrosis factor (TNF) is the prototypic member of the TNF ligand family and has a key role in the regulation of inflammatory processes. TNF exerts its functions by interaction with the death domain-containing TNF-receptor 1 (TNF-R1) and the non-death domain-containing TNF-receptor 2 (TNF-R2), both members of a receptor family complementary to the TNF ligand family. Due to the prototypic features of the TNF receptors and their importance for the regulation of inflammation, the signal transduction mechanisms utilized by these receptors have been extensively studied. Several proteins that interact directly or indirectly with the cytoplasmic domains of TNF-R1 and TNF-R2 have been identified in the recent years giving ideas how these receptors are connected to the apoptotic pathway and the signaling cascades leading to activation of NF-kappaB and JNK. Of special interest are TNF receptor-associated factor (TRAF) 1 and 2, which defines a novel group of adaptor proteins involved in signal transduction by most members of the TNF receptor family, of IL-1 receptor and IL-17 receptor as well as some members of the TOLL-like receptor family. TRAF 2 is currently the best-characterized TRAF family member, having a key role in mediating TNF-R1-induced activation of NF-kappaB and JNK. Moreover, recent studies suggest that TRAF 2 represents an integration point for pro- and antiapoptotic signals. This review focuses on the molecular mechanisms that underlay signal initiation by TNF-R1 and TNF-R2, with particular consideration of the role of TRAF 2, and highlights the importance of this molecule for the integration of such antagonizing pathways as death induction and NF-kappaB-mediated surviving signals.     

Apoptosis-inducing agents cause rapid shedding of tumor necrosis factor receptor 1 (TNFR1). A nonpharmacological explanation for inhibition of TNF-mediated activation

Several chemical compounds not known to interact with tumor necrosis factor (TNF) signal transducing proteins inhibit TNF-mediated activation of vascular endothelial cells (EC). Four structurally diverse agents, arachidonyl trifluoromethylketone, staurosporine, sodium salicylate, and C6-ceramide, were studied. All four agents caused EC apoptosis at concentrations that inhibited TNF-induced IkappaBalpha degradation. However, evidence of apoptosis was not evident until after several (e.g. 3-12) hours of treatment, whereas 2 h of treatment was sufficient to inhibit TNF responses. IL-1-induced IkappaBalpha degradation was unaffected by these treatments. Inhibition of TNF signaling could not be prevented with either of the broad spectrum caspase inhibitors zVADfmk or yVADcmk. The inhibition of p38 kinase with SB203580 prevented the inhibition of TNF signaling by all agents except arachidonyl trifluoromethylketone. No changes in the levels or molecular weights of the adaptor proteins TRADD (TNF receptor-associated death domain), RIP (receptor-interacting protein), or TRAF2 (TNF receptor-associated factor-2) were caused by apoptogenic drugs. However, TNF receptor 1 (TNFR1) surface expression was significantly reduced by all four agents. Furthermore, TNF-dependent recruitment of TRADD to surface TNFR1 was also inhibited. These data suggest that several putative inhibitors of TNF signaling work by triggering apoptosis and that an early event coincident with the initiation of apoptosis, preceding evidence of injury, is loss of TNFR1. Consistent with this hypothesis, cotreatment of EC with the metalloproteinase inhibitor Tapi (TNF-alpha proteinase inhibitor) blocked the reduction in surface TNFR1 by apoptogenic drugs and prevented inhibition of TNF-induced IkappaBalpha degradation without blocking apoptosis. TNFR1 loss could be a mechanism to limit inflammation in response to apoptotic cell death.     

Sam68 is required for both NF-κB activation and apoptosis signaling by the TNF receptor

The RNA-binding protein Sam68 is implicated in various cellular processes including RNA metabolism, apoptosis, and signal transduction. Here we identify a role of Sam68 in TNF-induced NF-κB activation and apoptosis. We found that Sam68 is recruited to the TNF receptor, and its deficiency dramatically reduces RIP recruitment and ubiquitylation. It also impairs cIAP1 recruitment and maintenance of recruited TRAF2 at the TNF receptor. In its absence, activation of the TAK1-IKK kinase complex is defective, greatly reducing signal transduction. Sam68 is also found as a part of the TNF-induced cytoplasmic caspase-8-FADD complex. RIP is not recruited to this complex in Sam68 knockout cells, and caspase activation is virtually absent. These findings delineate previously unknown functions for Sam68 in the TNF signaling pathway, where it acts as a signaling adaptor both in the membrane-associated complex I and in the cytoplasmic complex II, regulating both NF-κB activation and apoptosis.     

Fas activates NF-kappaB and induces apoptosis in T-cell lines by signaling pathways distinct from those induced by TNF-alpha

The p55 tumor necrosis factor (TNF) receptor and the Fas (CD95/APO-1) receptor share an intracellular domain necessary to induce apoptosis, suggesting they utilize common signaling pathways. To define pathways triggered by Fas and TNF-alpha we utilized human CEM-C7 T-cells. As expected, stimulation of either receptor induced apoptosis and TNF-alpha-induced signaling included the activation of NF-kappaB. Surprisingly, Fas-induced signaling also triggered the activation of NF-kappaB in T cells, yet the kinetics of NF-kappaB induction by Fas was markedly delayed. NF-kappaB activation by both pathways was persistent and due to the sequential degradation of IkappaB-alpha and IkappaB-beta. However, the kinetics of IkappaB degradation were different and there were differential effects of protease inhibitors and antioxidants on NF-kappaB activation. Signaling pathways leading to activation of apoptosis were similarly separable and were also independent of NF-kappaB activation. Thus, the Fas and TNF receptors utilize distinct signal transduction pathways in T-cells to induce NF-kappaB and apoptosis.     

Inhibition of ligand binding and antiproliferative effects of tumor necrosis factor and lymphotoxin by soluble forms of recombinant P60 and P80 receptors.

Tumor necrosis factor (TNF) is one of the mediators of inflammatory responses. Recently, the cDNA for two distinct receptors of TNF with predicted molecular masses of 60 kDa and 80 kDa, respectively, were isolated. In this report, we compare the inhibitory effects of these two forms of recombinant soluble TNF receptors (extracellular domains) on the ligand binding and on the antiproliferative effects of TNF and lymphotoxin (LT) in a human histiocytic lymphoma cell line (U-937). Our results show that the soluble form of the p60 receptor is approximately 100-fold more potent than the soluble form of the p80 receptor in inhibiting both the antiproliferative effects of TNF as well as in blocking TNF binding to U-937 cells. In contrast, the antiproliferative effects of LT and its binding to cells is inhibited equally by both the p60 and p80 forms of the soluble receptor. Thus, overall our results indicate that the two soluble receptors differ in their ability to inhibit TNF and LT. The impotance of these soluble receptors in blocking the harmful effects of TNF and LT is discussed.     

Homotypic FADD interactions through a conserved RXDLL motif are required for death receptor-induced apoptosis

Death receptors in the TNF receptor superfamily signal for apoptosis via the ordered recruitment of FADD and caspase-8 to a death-inducing signaling complex (DISC). However, the nature of the protein-protein interactions in the signaling complex is not well defined. Here we show that FADD self-associates through a conserved RXDLL motif in the death effector domain (DED). Despite exhibiting similar binding to both Fas and caspase-8 and preserved overall secondary structure, FADD RDXLL motif mutants cannot reconstitute FasL- or TRAIL-induced apoptosis and fail to recruit caspase-8 into the DISC of reconstituted FADD-deficient cells. Abolishing self-association can transform FADD into a dominant-negative mutant that interferes with Fas-induced apoptosis and formation of microscopically visible receptor oligomers. These findings suggest that lateral interactions among adapter molecules are required for death receptor apoptosis signaling and implicate self-association into oligomeric assemblies as a key function of death receptor adapter proteins in initiating apoptosis.     

TNFα and SOCS3 regulate IRS-1 to increase retinal endothelial cell apoptosis

Rates of diabetes are reaching epidemic levels. The key problem in both type 1 and type 2 diabetes is dysfunctional insulin signaling, either due to lack of production or due to impaired insulin sensitivity. A key feature of diabetic retinopathy in animal models is degenerate capillary formation. The goal of this present study was to investigate a potential mechanism for retinal endothelial cell apoptosis in response to hyperglycemia. The hypothesis was that hyperglycemia-induced TNFα leads to retinal endothelial cell apoptosis through inhibition of insulin signaling. To test the hypothesis, primary human retinal endothelial cells were grown in normal glucose (5 mM) or high glucose (25 mM) and treated with exogenous TNFα, TNFα siRNA or suppressor of cytokine signaling 3 (SOCS3) siRNA. Cell lysates were processed for Western blotting and ELISA analyses to verify TNFα and SOCS3 knockdown, as well as key pro- and anti-apoptotic factors, IRS-1, and Akt. Data indicate that high glucose culturing conditions significantly increase TNFα and SOCS3 protein levels. Knockdown of TNFα and SOCS3 significantly increases anti-apoptotic proteins, while decreasing pro-apoptotic proteins. Knockdown of TNFα leads to decreased phosphorylation of IRS-1(Ser307), which would promote normal insulin signaling. Knockdown of SOCS3 increased total IRS-1 levels, as well as decreased IR(Tyr960), both of which would inhibit retinal endothelial cell apoptosis through increased insulin signaling. Taken together, our findings suggest that increased TNFα inhibits insulin signaling in 2 ways: 1) increased phosphorylation of IRS-1(Ser307), 2) increased SOCS3 levels to decrease total IRS-1 and increase IR(Tyr960), both of which block normal insulin signal transduction. Resolution of the hyperglycemia-induced TNFα levels in retinal endothelial cells may prevent apoptosis through disinhibition of insulin receptor signaling.     

Necrosis-like death can engage multiple pro-apoptotic Bcl-2 protein family members

Necroptosis is a physiologically relevant mode of cell death with some well-described initiating events, but largely unknown executioners. Here we investigated necrostatin-1 (Nec-1) sensitive death elicited by different necroptosis stimuli in L929 mouse fibrosarcoma cells, mouse embryonic fibroblasts (MEF) and bone marrow-derived macrophages. We found that TNFα- or zVAD-induced necroptosis occurs independently of the recently implicated executioners Bmf or PARP-2, but can involve the Bcl-2 family proteins Bid and Bak. Furthermore, this type of necroptosis is associated with mitochondrial cytochrome c release and partly sensitive to cyclosporine A inhibition, suggesting a cross talk with the mitochondrial permeability transition pore. Necroptosis triggered by cadmium (Cd) exposure caused fully Nec-1-sensitive and caspase-independent death in L929 cells that was associated with autocrine TNFα-mediated feed-forward signalling. In MEF Cd-exposure elicited a mixed mode of cell death that was to some extent Nec-1-sensitive but also displayed features of apoptosis. It was partly dependent on Bmf and Bax/Bak, proteins typically considered to act pro-apoptotic, but ultimately insensitive to caspase inhibition. Overall, our study indicates that inducers of “extrinsic” and “intrinsic” necroptosis can both trigger TNF-receptor signalling. Further, necroptosis may depend on mitochondrial changes engaging proteins considered critical for MOMP during apoptosis that ultimately contribute to caspase-independent necrotic cell death.     

Structural basis for the regulation of phytohormone receptors

Phytohormones are central players in diverse plant physiological events, such as plant growth, development, and environmental stress and defense responses. The elucidation of their regulatory mechanisms through phytohormone receptors could facilitate the generation of transgenic crops with cultivation advantages and the rational design of growth control chemicals. During the last decade, accumulated structural data on phytohormone receptors have provided critical insights into the molecular mechanisms of phytohormone perception and signal transduction. Here, we review the structural bases of phytohormone recognition and receptor activation. As a common feature, phytohormones regulate the interaction between the receptors and their respective target proteins (also called co-receptors) by two types of regulatory mechanisms, acting as either “molecular glue” or an “allosteric regulator.” However, individual phytohormone receptors adopt specific structural features that are essential for activation. In addition, recent studies have focused on the molecular diversity of redundant phytohormone receptors.