Roles of tumor necrosis factor p55 and p75 receptors in TNF-alpha-induced vascular permeability

We have investigated the role of p55 andp75 tumor necrosis factor receptors 1 and 2 (TNFR1 and TNFR2,respectively) in TNF-induced alteration of endothelial permeability invitro and in vivo. Stimulation of TNFR1 with an agonist antibody or areceptor-selective TNF mutein increased the flux of¹²⁵I-albumin through endothelial cell monolayers. Anantagonist anti-TNFR1 antibody, but not antagonist anti-TNFR2antibodies, blocked the activity of TNF in vitro. Stimulation of TNFR1,but not TNFR2, induced cytoskeletal reorganization associated withincreased permeability. SB-203580, a p38 mitogen-activated proteinkinase inhibitor, blocked TNFR1-induced cytoskeletal reorganization and permeability. A selective mouse TNFR1 agonist and human TNF, which binds to murine TNFR1, increased the leakage of trypan blue-albumin from liver vessels in mice. These results indicate that stimulation ofTNFR1 is necessary and sufficient to increase endothelial permeability in vitro and in vivo. However, an antagonist anti-murine TNFR2 antibodypartially inhibited the effect of murine TNF on liver vessels,suggesting that TNFR2 also plays a role in the regulation ofTNF-induced vascular permeability in vivo.

     

Generation and Characterization of Small Single Domain Antibodies Inhibiting Human Tumor Necrosis Factor Receptor 1

The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named “TNF Receptor-One Silencer” (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC₅₀ values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients.     

Polyubiquitinated Tristetraprolin Protects from TNF-induced,Caspase-mediated Apoptosis

Binding of TNF to its receptor (TNFR1) elicits the spatiotemporal assembly of two signaling complexes that coordinate the balance between cell survival and cell death. We have shown previously that, following TNF treatment, the mRNA decay protein tristetraprolin (TTP) is Lys-63-polyubiquitinated by TNF receptor-associated factor 2 (TRAF2), suggesting a regulatory role in TNFR signaling. Here we demonstrate that TTP interacts with TNFR1 in a TRAF2-dependent manner, thereby initiating the MEKK1/MKK4-dependent activation of JNK activities. This regulatory function toward JNK activation but not NF-κB activation depends on lysine 105 of TTP, which we identified as the corresponding TRAF2 ubiquitination site. Disabling TTP polyubiquitination results in enhanced TNF-induced apoptosis in cervical cancer cells. Together, we uncover a novel aspect of TNFR1 signaling where TTP, in alliance with TRAF2, acts as a balancer of JNK-mediated cell survival versus death.     

Tumor necrosis factor inhibits ligand-stimulated EGF receptor activation through a TNF receptor 1-dependent mechanism

Tumor necrosis factor (TNF) and epidermal growth factor (EGF) are key regulators in the intricate balance maintaining intestinal homeostasis. Previous work from our laboratory shows that TNF attenuates ligand-driven EGF receptor (EGFR) phosphorylation in intestinal epithelial cells. To identify the mechanisms underlying this effect, we examined EGFR phosphorylation in cells lacking individual TNF receptors. TNF attenuated EGF-stimulated EGFR phosphorylation in wild-type and TNFR2(-/-), but not TNFR1(-/-), mouse colon epithelial (MCE) cells. Reexpression of wild-type TNFR1 in TNFR1(-/-) MCE cells rescued TNF-induced EGFR inhibition, but expression of TNFR1 deletion mutant constructs lacking the death domain (DD) of TNFR1 did not, implicating this domain in EGFR downregulation. Blockade of p38 MAPK, but not MEK, activation of ERK rescued EGF-stimulated phosphorylation in the presence of TNF, consistent with the ability of TNFR1 to stimulate p38 phosphorylation. TNF promoted p38-dependent EGFR internalization in MCE cells, suggesting that desensitization is achieved by reducing receptor accessible to ligand. Taken together, these data indicate that TNF activates TNFR1 by DD- and p38-dependent mechanisms to promote EGFR internalization, with potential impact on EGF-induced proliferation and migration key processes that promote healing in inflammatory intestinal diseases.     

Hepatitis C Virus Core Protein Binds to the Cytoplasmic Domain of Tumor Necrosis Factor (TNF) Receptor 1 and Enhances TNF-Induced Apoptosis

The hepatitis C virus (HCV) core protein is known to be a multifunctional protein, besides being a component of viral nucleocapsids. Previously, we have shown that the core protein binds to the cytoplasmic domain of lymphotoxin β receptor, which is a member of tumor necrosis factor receptor (TNFR) family. In this study, we demonstrated that the core protein also binds to the cytoplasmic domain of TNFR 1. The interaction was demonstrated both by glutathione S-transferase fusion protein pull-down assay in vitro and membrane flotation method in vivo. Both the in vivo and in vitro binding required amino acid residues 345 to 407 of TNFR 1, which corresponds to the “death domain” of this receptor. We have further shown that stable expression of the core protein in a mouse cell line (BC10ME) or human cell lines (HepG2 and HeLa cells) sensitized them to TNF-induced apoptosis, as determined by the TNF cytotoxicity or annexin V apoptosis assay. The presence of the core protein did not alter the level of TNFR 1 mRNA in the cells or expression of TNFR 1 on the cell surface, suggesting that the sensitization of cells to TNF by the viral core protein was not due to up-regulation of TNFR 1. Furthermore, we observed that the core protein blocked the TNF-induced activation of RelA/NF-κB in murine BC10ME cells, thus at least partially accounting for the increased sensitivity of BC10ME cells to TNF. However, NF-κB activation was not blocked in core protein-expressing HeLa or HepG2 cells, implying another mechanism of TNF sensitization by core protein. These results together suggest that the core protein can promote cell death during HCV infection via TNF signaling pathways possibly as a result of its interaction with the cytoplasmic tail of TNFR 1. Therefore, TNF may play a role in HCV pathogenesis.     

Azadirachtin Interacts with the Tumor Necrosis Factor (TNF) Binding Domain of Its Receptors and Inhibits TNF-induced Biological Responses

The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor κB (NF-κB) and also expression of NF-κB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-κB (IκBα) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IκBα kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-κB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.     

TNF mediates the sustained activation of Nrf2 in human monocytes

Modulation of monocyte function is a critical factor in the resolution of inflammatory responses. This role is mediated mainly by the production of TNF-α. Investigations of the actions of TNF have mostly focused on acute activation of other cell types such as fibroblasts and endothelial cells. Less is known about the effects of TNF on monocytes themselves, and little is known about the regulation of cell responses to TNF beyond the activation of NF-κB. In this study, we investigated the regulation of NF-E2-related factor 2 (Nrf2) cyctoprotective responses to TNF in human monocytes. We found that in monocytes TNF induces sustained Nrf2 activation and Nrf2 cytoprotective gene induction in a TNFR1-dependent manner. Under TNF activation, monocytes increased their expression of Nrf2-dependent genes, including NAD(P)H:quinone oxidoreductase 1 and glutamyl cysteine ligase modulatory, but not heme oxygenase-1. We also showed that autocrine TNF secretion was responsible for this sustained Nrf2 response and that Nrf2 activation by TNF was mediated by the generation of reactive oxygen species. Moreover, we showed that Nrf2-mediated gene induction can modulate TNF-induced NF-κB activation. These results show for the first time, to our knowledge, that TNF modulates prolonged Nrf2-induced gene expression, which in turn regulates TNF-induced inflammatory responses.     

TNFR2 increases the sensitivity of ligand-induced activation of the p38 MAPK and NF-κB pathways and signals TRAF2 protein degradation in macrophages

Tumour necrosis factor (p55 or p60) receptor (TNFR) 1 is the major receptor that activates pro-inflammatory signalling and induces gene expression in response to TNF. Consensus is lacking for the function of (p75 or p80) TNFR2 but experiments in mice have suggested neuro-, cardio- and osteo-protective and anti-inflammatory roles. It has been shown in various cell types to be specifically required for the induction of TNFR-associated factor-2 (TRAF2) degradation and activation of the alternative nuclear factor (NF)-kappaB pathway, and to contribute to the activation of mitogen-activated protein kinases (MAPK) and the classical NF-kappaB pathway. We have investigated the signalling functions of TNFR2 in primary human and murine macrophages. We find that in these cells TNF induces TRAF2 degradation, and this is blocked in TNFR2^−/− macrophages. TRAF2 has been previously reported to be required for TNF-induced activation of p38 MAPK. However, TRAF2 degradation does not inhibit TNF-induced tolerance of p38 MAPK activation. Neither TNF, nor lipopolysaccharide treatment, induced activation of the alternative NF-kappaB pathway in macrophages. Activation by TNF of the p38 MAPK and NF-kappaB pathways was blocked in TNFR1^−/− macrophages. In contrast, although TNFR2^−/− macrophages displayed robust p38 MAPK activation and IkappaBα degradation at high concentrations of TNF, at lower doses the concentration dependence of signalling was weakened by an order of magnitude. Our results suggest that, in addition to inducing TRAF2 protein degradation, TNFR2 also plays a crucial auxiliary role to TNFR1 in sensitising macrophages for the ligand-induced activation of the p38 MAPK and classical NF-kappaB pro-inflammatory signalling pathways.     

Etk/Bmx as a tumor necrosis factor receptor type 2-specific kinase: role in endothelial cell migration and angiogenesis

Tumor necrosis factor (TNF) is a cytokine that mediates many pathophysiologial processes, including angiogenesis. However, the molecular signaling involved in TNF-induced angiogenesis has not been determined. In this study, we examined the role of Etk/Bmx, an endothelial/epithelial tyrosine kinase involved in cell adhesion, migration, and survival in TNF-induced angiogenesis. We show that TNF activates Etk specifically through TNF receptor type 2 (TNFR2) as demonstrated by studies using a specific agonist to TNFR2 and TNFR2-deficient cells. Etk forms a preexisting complex with TNFR2 in a ligand-independent manner, and the association is through multiple domains (pleckstrin homology domain, TEC homology domain, and SH2 domain) of Etk and the C-terminal domain of TNFR2. The C-terminal 16-amino-acid residues of TNFR2 are critical for Etk association and activation, and this Etk-binding and activating motif in TNFR2 is not overlapped with the TNFR-associated factor type 2 (TRAF2)-binding sequence. Thus, TRAF2 is not involved in TNF-induced Etk activation, suggesting a novel mechanism for Etk activation by cytokine receptors. Moreover, a constitutively active form of Etk enhanced, whereas a dominant-negative Etk blocked, TNF-induced endothelial cell migration and tube formation. While most TNF actions have been attributed to TNFR1, our studies demonstrate that Etk is a TNFR2-specific kinase involved in TNF-induced angiogenic events.     

Role of SODD in regulation of tumor necrosis factor responses

Signaling from tumor necrosis factor receptor type 1 (TNFR1) can elicit potent inflammatory and cytotoxic responses that need to be properly regulated. It was suggested that the silencer of death domains (SODD) protein constitutively associates intracellularly with TNFR1 and inhibits the recruitment of cytoplasmic signaling proteins to TNFR1 to prevent spontaneous aggregation of the cytoplasmic death domains of TNFR1 molecules that are juxtaposed in the absence of ligand stimulation. In this study, we demonstrate that mice lacking SODD produce larger amounts of cytokines in response to in vivo TNF challenge. SODD-deficient macrophages and embryonic fibroblasts also show altered responses to TNF. TNF-induced activation of NF-kappaB is accelerated in SODD-deficient cells, but TNF-induced c-Jun N-terminal kinase activity is slightly repressed. Interestingly, the apoptotic arm of TNF signaling is not hyperresponsive in the SODD-deficient cells. Together, these results suggest that SODD is critical for the regulation of TNF signaling.     

Keratin-dependent, epithelial resistance to tumor necrosis factor-induced apoptosis

Tumor necrosis factor (TNF) is a cytokine produced by macrophages and T lymphocytes that acts through two distinct receptors, TNFR1 (60 kD, CD120a) and TNFR2 (80 kD, CD120b), to affect cellular proliferation, differentiation, survival, and cell death. In addition to its proinflammatory actions in mucosal tissue, TNF is important for liver regeneration. Keratin 8 (K8) and keratin 18 (K18) form intermediate filaments characteristic of liver and other single cell layered, internal epithelia and their derivative cancers. K8-deficient (K8(-)) mice, which escape embryonic lethality, develop inflammatory colorectal hyperplasia, mild liver abnormalities, and tolerate hepatectomy poorly. We show that normal and malignant epithelial cells deficient in K8 and K18 are approximately 100 times more sensitive to TNF-induced death. K8 and K18 both bind the cytoplasmic domain of TNFR2 and moderate TNF-induced, Jun NH(2)-terminal kinase (JNK) intracellular signaling and NFkappaB activation. Furthermore, K8(-) and K18(-) mice are much more sensitive to TNF dependent, apoptotic liver damage induced by the injection of concanavalin A. This moderation of the effects of TNF may be the fundamental function of K8 and K18 common to liver regeneration, inflammatory bowel disease, hepatotoxin sensitivity, and the diagnostic, persistent expression of these keratins in many carcinomas.     

TNF transactivation of EGFR stimulates cytoprotective COX-2 expression in gastrointestinal epithelial cells

TNF and epidermal growth factor (EGF) are well-known stimuli of cyclooxygenase (COX)-2 expression, and TNF stimulates transactivation of EGF receptor (EGFR) signaling to promote survival in colon epithelial cells. We hypothesized that COX-2 induction and cell survival signaling downstream of TNF are mediated by EGFR transactivation. TNF treatment was more cytotoxic to COX-2(-/-) mouse colon epithelial (MCE) cells than wild-type (WT) young adult mouse colon (YAMC) epithelial cells or COX-1(-/-) cells. TNF also induced COX-2 protein and mRNA expression in YAMC cells, but blockade of EGFR kinase activity or expression inhibited COX-2 upregulation. TNF-induced COX-2 expression was reduced and absent in EGFR(-/-) and TNF receptor-1 (TNFR1) knockout MCE cells, respectively, but was restored upon expression of the WT receptors. Inhibition of mediators of EGFR transactivation, Src family kinases and p38 MAPK, blocked TNF-induced COX-2 protein and mRNA expression. Finally, TNF injection increased COX-2 expression in colon epithelium of WT, but not kinase-defective EGFR(wa2) and EGFR(wa5), mice. These data indicate that TNFR1-dependent transactivation of EGFR through a p38- and/or an Src-dependent mechanism stimulates COX-2 expression to promote cell survival. This highlights an EGFR-dependent cell signaling pathway and response that may be significant in colitis-associated carcinoma.     

Treatment of TNF mediated diseases by selective inhibition of soluble TNF or TNFR1

The TNF signaling pathway is a valuable target in the therapy of autoimmune diseases, and anti-TNF drugs are successfully used to treat diseases such as rheumatoid arthritis, Crohn's disease and psoriasis. By their ability to interfere with inflammatory processes at multiple levels, these TNF blockers have become invaluable tools to inhibit the inflammation induced damage and allow recovery of the affected tissues. Unfortunately this therapy has some drawbacks, including increased risk of infection and malignancy, and remarkably, the onset of new auto-immune diseases. Some of these effects are caused by the unwanted abrogation of beneficial TNF signaling. More specific targeting of the pathological TNF-induced signaling might lead to broader applicability and improved safety. Specificity might be increased by inhibiting the soluble TNF/TNFR1 axis while leaving the often beneficial transmembrane TNF/TNFR2 signaling untouched. This approach looks promising because it inhibits the pathological effects of TNF and reduces the side effects, and it opens the way for the treatment of other diseases in which TNFR2 inhibition is detrimental. In this review we give an overview of in vivo mouse studies of TNF mediated pathologies demonstrating that the blockade or genetic deletion of sTNF or TNFR1 is preferable over total TNF blockade.     

Granulocyte macrophage-colony stimulating factor and interleukin-3 increase expression of type II tumour necrosis factor receptor, increasing susceptibility to tumour necrosis factor-induced apoptosis. Control of leukaemia cell life/death switching

Tumour necrosis factor (TNF) induces apoptosis in a range of cell types via its two receptors, TNFR1 and TNFR2. Here, we demonstrate that proliferation and TNFR2 expression was increased in human leukaemic TF-1 cells by granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), with TNFR1 expression unaffected. Consequently, they switch from a proliferative to a TNF-induced apoptotic phenotype. Raised TNFR2 expression and susceptibility to TNF-induced apoptosis was not a general effect of proliferation as IL-1beta and IFN-gamma both proliferated TF-1 cells with no effect on TNFR expression or apoptosis. Although raised TNFR2 expression correlated with the apoptotic phenotype, stimulation of apoptosis in GM-CSF-pretreated cells was mediated by TNFR1, with stimulation of TNFR2 alone insufficient to initiate cell death. However, TNFR2 did play a role in apoptotic and proliferative responses as they were blocked by the presence of an antagonistic TNFR2 antibody. Additionally, coincubation with cycloheximide blocked the mitotic effects of GM-CSF or IL-3, allowing only the apoptotic responses of TNF to persist. TNF life/death was also observed in K562, but not MOLT-4 and HL-60 human leukaemic cell types. These findings show a cooperative role of TNFR2 in the TNF life/death switching phenomenon.     

Targeting of Tumor Necrosis Factor Receptor 1 to Low Density Plasma Membrane Domains in Human Endothelial Cells

TNFR1 (tumor necrosis factor receptor 1) localizes to caveolae of human endothelial-derived EA.hy926 cells. Transduced TNFR1 molecules lacking amino acid residues 229–244 (spanning the transmembrane/intercellular boundary) are expressed on the cell surface equivalently to full-length TNFR1 molecules but incompletely localize to caveolae. A peptide containing this sequence pulls down CAV-1 (caveolin-1) and TNFR1 from cell lysates but fails to do so following disruption of caveolae with methyl-β-cyclodextrin. We previously reported that methyl-β-cyclodextrin eliminates caveolae and blocks tumor necrosis factor (TNF)-induced internalization of TNFR1 but not TNF-induced activation of NF-κB in EA.hy926 cells. Both CAV-1 and FLOT-2 (flotillin-2), organizing proteins of caveolae and lipid rafts, respectively, associate with caveolae in EA.hy926 cells. Small interfering RNA-mediated knockdown of CAV-1 but not FLOT-2 strikingly reduces caveolae number. Both knockdowns reduce total TNFR1 protein expression, but neither prevents TNFR1 localization to low density membrane domains, TNF-induced internalization of TNFR1, or NF-κB activation by TNF. Both CAV-1 and FLOT-2 knockdowns reduce TNF-mediated activation of stress-activated protein kinase (SAPK). However, both knockdowns reduce expression of TRAF2 (TNF receptor-associated factor-2) protein, and small interfering RNA targeting of TRAF2 also selectively inhibits SAPK activation. We conclude that TNFR1 contains a membrane-proximal sequence that targets the receptor to caveolae/lipid rafts. Neither TNFR1 targeting to nor internalization from these low density membrane domains depends upon CAV-1 or FLOT-2. Furthermore, both NF-κB and SAPK activation appear independent of both TNFR1 localization to low density membrane domains and to TNF-induced receptor internalization.     

Activation of TNFR2 sensitizes macrophages for TNFR1-mediated necroptosis

Macrophages express TNFR1 as well as TNFR2 and are also major producers of tumor necrosis factor (TNF), especially upon contact with pathogen-associated molecular patterns. Consequently, TNF not only acts as a macrophage-derived effector molecule but also regulates the activity and viability of macrophages. Here, we investigated the individual contribution of TNFR1 and TNFR2 to TNF-induced cell death in macrophages. Exclusive stimulation of TNFR1 showed no cytotoxic effect whereas selective stimulation of TNFR2 displayed mild cytotoxicity. Intriguingly, the latter was strongly enhanced by the caspase inhibitor zVAD-fmk. The strong cytotoxic activity of TNFR2 in the presence of zVAD-fmk was reversed by necrostatin-1, indicating necroptotic cell death. TNFR1- and TNF-deficient macrophages turned out to be resistant against TNFR2-induced cell death. In addition, the cIAP-depleting SMAC mimetic BV6 also enforced TNF/TNFR1-mediated necroptotic cell death in the presence of zVAD-fmk. In sum, our data suggest a model in which TNFR2 sensitizes macrophages for endogenous TNF-induced TNFR1-mediated necroptosis by the known ability of TNFR2 to interfere with the survival activity of TRAF2-cIAP1/2 complexes.Tumor necrosis factor (TNF) is a pleiotropic cytokine that occurs as a type II transmembrane protein but can be released from the plasma membrane by proteolytic processing.¹ Membrane-bound and soluble TNF both contain the characteristic carboxy-terminal TNF homology domain, which is responsible for self-assembly into trimeric molecules and receptor binding. Membrane-bound and soluble TNF strongly interact with two receptors, TNFR1 and TNFR2, but the two forms of TNF are differentially effective in receptor activation.¹ Whereas membrane-bound TNF activates TNFR1 and TNFR2 efficiently, soluble TNF is sufficient for TNFR1 activation but largely inactive upon binding to TNFR2. TNFR1 belongs to the death receptor subgroup of the TNF receptor family and can trigger apoptosis and necroptosis.^{2, 3, 4} However, cell death induction by TNFR1 is typically efficiently antagonized by concomitant activation of the cytoprotective classical NFκB pathway and/or ubiquitous expression of anti-apoptotic proteins.^{1, 2} The latter involve FLIP proteins which generally inhibit death receptor-induced caspase-8 activation but also complexes containing TRAF2, cIAP1 and cIAP2 which specifically interfere with caspase-8 activation in context of TNFR1 signaling.^{2, 3, 4} Worth mentioning, TRAF2-cIAP1/2 complexes also mediate K63-linked ubiquitination of RIP1 in the TNFR1 signaling complex, thereby facilitating TNFR1-mediated activation of the classical NFκB pathway. Indeed, TNFR1 signaling is predominantly pro-inflammatory as TNFR1-induced cell death is blocked as long as the aforementioned protective mechanisms are not impaired.In contrast to TNFR1, TNFR2 contains no cytoplasmic death domain. Upon ligand binding, TNFR2 recruits TRAF2 and various TRAF2-associated proteins, such as TRAF1, cIAP1 and cIAP2, but also interacts with other signaling proteins independently of TRAF2.^{1, 5} TNFR2 activation has been linked to a variety of immune regulatory functions, which, in contrast to the activities of TNFR1, often result in anti-inflammatory effects.⁶Murine models shed light on the complex interplay of the TNFR1–TNFR2 system in vivo, demonstrating additive, synergistic or even antagonistic effects. At the cellular level, several mechanisms for the cross-talk between TNFR1 and TNFR2 have been identified.¹ Besides the obvious competition for ligand binding, TNFR1 and TNFR2 can induce, for example, autocrine TNF production in a cell type-specific manner.¹ In context of TNFR1 activation by soluble TNF, subsequent induction of membrane-bound TNF results in costimulation of TNFR2, thereby converting the initially transient activation into sustained autocrine signaling. In addition, TNFR1 and TNFR2 compete for the cytoplasmic pool of TRAF2–cIAP1/2 complexes. By depletion and/or degradation of TRAF2, TNFR2 is capable to modulate TNFR1 signaling.¹ Moreover, TNFR2 but not TNFR1, stimulates the alternative NFκB pathway by triggering proteolytic processing of the inactive p100/RelB dimers into active p52/RelB NFκB complexes.⁷ Notably, TNFR2-induced alternative NFκB signaling can be enhanced by TNFR1-mediated induction of p100 and RelB expression via the classical NFκB pathway.⁷In macrophages, the complexity of the TNF-TNFR1/2 system is especially relevant. Macrophages on one hand co-express TNFR1 and TNFR2 and are on the other hand a pathophysiologically important source of TNF, for example, in response to a variety of pathogen-associated molecular patterns (PAMPs). TNF not only acts as a macrophage-derived effector molecule, but in an autocrine fashion also controls macrophage activation and survival, as seen for example during infection with mycobacteria.^{8, 9, 10, 11, 12, 13, 14, 15, 16, 17} However, the molecular mechanisms of TNF-induced cell death in macrophages are incompletely understood and were, therefore addressed in our study. Using macrophages genetically deficient for TNFR1, TNFR2 or TNF together with TNFR1- and TNFR2-specific TNF variants, we show that TNFR2 activation sensitizes macrophages for TNFR1-mediated necroptosis triggered by autocrine produced TNF and provide evidence that this is related to TNFR2-induced depletion/degradation of TRAF2-cIAP1/2 complexes.     

RIPK3 contributes to TNFR1-mediated RIPK1 kinase-dependent apoptosis in conditions of cIAP1/2 depletion or TAK1 kinase inhibition

Receptor-interacting protein kinase (RIPK) 1 and RIPK3 have emerged as essential kinases mediating a regulated form of necrosis, known as necroptosis, that can be induced by tumor necrosis factor (TNF) signaling. As a consequence, inhibiting RIPK1 kinase activity and repressing RIPK3 expression levels have become commonly used approaches to estimate the contribution of necroptosis to specific phenotypes. Here, we report that RIPK1 kinase activity and RIPK3 also contribute to TNF-induced apoptosis in conditions of cellular inhibitor of apoptosis 1 and 2 (cIAP1/2) depletion or TGF-β-activated kinase 1 (TAK1) kinase inhibition, implying that inhibition of RIPK1 kinase activity or depletion of RIPK3 under cell death conditions is not always a prerequisite to conclude on the involvement of necroptosis. Moreover, we found that, contrary to cIAP1/2 depletion, TAK1 kinase inhibition induces assembly of the cytosolic RIPK1/Fas-associated protein with death domain/caspase-8 apoptotic TNF receptor 1 (TNFR1) complex IIb without affecting the RIPK1 ubiquitylation status at the level of TNFR1 complex I. These results indicate that the recruitment of TAK1 to the ubiquitin (Ub) chains, and not the Ub chains per se, regulates the contribution of RIPK1 to the apoptotic death trigger. In line with this, we found that cylindromatosis repression only provided protection to TNF-mediated RIPK1-dependent apoptosis in condition of reduced RIPK1 ubiquitylation obtained by cIAP1/2 depletion but not upon TAK1 kinase inhibition, again arguing for a role of TAK1 in preventing RIPK1-dependent apoptosis downstream of RIPK1 ubiquitylation. Importantly, we found that this function of TAK1 was independent of its known role in canonical nuclear factor-κB (NF-κB) activation. Our study therefore reports a new function of TAK1 in regulating an early NF-κB-independent cell death checkpoint in the TNFR1 apoptotic pathway. In both TNF-induced RIPK1 kinase-dependent apoptotic models, we found that RIPK3 contributes to full caspase-8 activation independently of its kinase activity or intact RHIM domain. In contrast, RIPK3 participates in caspase-8 activation by acting downstream of the cytosolic death complex assembly, possibly via reactive oxygen species generation.     

Intestinal Epithelial-Derived TAK1 Signaling Is Essential for Cytoprotection against Chemical-Induced Colitis

Background

We have previously reported that intestinal epithelium-specific TAK1 deleted mice exhibit severe inflammation and mortality at postnatal day 1 due to TNF-induced epithelial cell death. Although deletion of TNF receptor 1 (TNFR1) can largely rescue those neonatal phenotypes, mice harboring double deletion of TNF receptor 1 (TNFR1) and intestinal epithelium-specific deletion of TAK1 (TNFR1KO/TAK1^IEKO) still occasionally show increased inflammation. This indicates that TAK1 is important for TNF-independent regulation of intestinal integrity.

Methodology/Principal Findings

In this study, we investigated the TNF-independent role of TAK1 in the intestinal epithelium. Because the inflammatory conditions were sporadically developed in the double mutant TNFR1KO/TAK1^IEKO mice, we hypothesize that epithelial TAK1 signaling is important for preventing stress-induced barrier dysfunction. To test this hypothesis, the TNFR1KO/TAK1^IEKO mice were subjected to acute colitis by administration of dextran sulfate sodium (DSS). We found that loss of TAK1 significantly augments DSS-induced experimental colitis. DSS induced weight loss, intestinal damages and inflammatory markers in TNFR1KO/TAK1^IEKO mice at higher levels compared to the TNFR1KO control mice. Apoptosis was strongly induced and epithelial cell proliferation was decreased in the TAK1-deficient intestinal epithelium upon DSS exposure. These suggest that epithelial-derived TAK1 signaling is important for cytoprotection and repair against injury. Finally, we showed that TAK1 is essential for interleukin 1- and bacterial components-induced expression of cytoprotective factors such as interleukin 6 and cycloxygenase 2.

Conclusions

Homeostatic cytokines and microbes-induced intestinal epithelial TAK1 signaling regulates cytoprotective factors and cell proliferation, which is pivotal for protecting the intestinal epithelium against injury.