NF-kappaB inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death

NF-kappaB downregulates tumor necrosis factor (TNF)-induced c-Jun N-terminal kinase (JNK) activation that promotes cell death, but the mechanism is not yet fully understood. By using murine embryonic fibroblasts (MEFs) that are deficient in TNF receptor-associated factor (TRAF) 2 and TRAF5 (DKO) or p65 NF-kappaB subunit (p65KO), we demonstrate here that TNF stimulation leads to accumulation of reactive oxygen species (ROS), which is essential for prolonged mitogen-activated protein kinase (MAPK) activation and cell death. Interestingly, dying cells show necrotic as well as apoptotic morphological changes as assessed by electron microscopy and flow cytometry, and necrotic, but not apoptotic, cell death is substantially inhibited by antioxidant. Importantly, TNF does not induce ROS accumulation or prolonged MAPK activation in wild-type MEFs, indicating that TRAF-mediated NF-kappaB activation normally suppresses the TNF-induced ROS accumulation that subsequently induces prolonged MAPK activation and necrotic cell death     

TRAF6 is a critical signal transducer in IL-33 signaling pathway

IL-33 has been shown to induce Th2 responses by signaling through the IL-1 receptor-related protein, ST2L. However, the signal transduction pathways activated by the ST2L have not been characterized. Here, we found that IL-33-induced monocyte chemoattractant protein (MCP)-1, MCP-3 and IL-6 expression was significantly inhibited in TNF receptor-associated Factor 6 (TRAF6)-deficient MEFs. IL-33 rapidly induced the formation of ST2L complex containing IL-1 receptor-associated kinase (IRAK), however, lack of TRAF6 abolished the recruitment of IRAK to ST2L. Consequently, p38, JNK and Nuclear factor-kappaB (NF-kappaB) activation induced by IL-33 was completely inhibited in TRAF6-deficient MEFs. On the other hand, IL-33-induced ERK activation was observed regardless of the presence of TRAF6. The introduction of TRAF6 restored the efficient activation of p38, JNK and NF-kappaB in TRAF6 deficient MEFs, resulting in the induction of MCP-1, MCP-3 and IL-6 expression. Moreover, IL-33 augmented autoubiquitination of TRAF6 and the reconstitution of TRAF6 mutant (C70A) that is defective in its ubiquitin ligase activity failed to restore IL-33-induced p38, JNK and NF-kappaB activation. Thus, these data demonstrate that TRAF6 plays a pivotal role in IL-33 signaling pathway through its ubiquitin ligase activity.     

The RING Domain of TRAF2 Plays an Essential Role in the Inhibition of TNFα-Induced Cell Death but Not in the Activation of NF-κB

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) and receptor-interacting protein 1 (RIP1) play critical roles in activating c-Jun N-terminal kinase (JNK) and inhibitor of κB kinase (IKK), as well as in inhibiting apoptosis induced by TNFα. The TRAF2 RING domain-mediated polyubiquitination of RIP1 is believed to be essential for TNFα-induced IKK activation, and the RING-domain-deleted TRAF2 (TRAF2-ΔR) has been widely used as a dominant negative in transient overexpression systems to block TNFα-induced JNK and IKK activation. Here, we report that stable expression of TRAF2-ΔR at a physiological level in TRAF2 and TRAF5 double knockout (TRAF2/5 DKO) cells almost completely restores normal TNFα-induced IKK activation, but not RIP1 polyubiquitination. In addition, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells efficiently inhibited the TNFα-induced later phase of prolonged JNK activation, yet failed to inhibit TNFα-induced cell death. Although the basal and inducible expression of anti-apoptotic proteins in TRAF2-ΔR-expressing TRAF2/5 DKO cells was normal, the cells remained sensitive to TNFα-induced cell death because anti-apoptotic proteins were not recruited to the TNFR1 complex efficiently. Moreover, stable expression of TRAF2-ΔR in TRAF2/5 DKO cells failed to suppress constitutive p100 processing in these cells. These data suggest that (i) the TRAF2 RING domain plays a critical role in inhibiting cell death induced by TNFα and is essential for suppressing the noncanonical nuclear factor κB pathway in unstimulated cells; (ii) RIP1 polyubiquitination is not essential for TNFα-induced IKK activation; and (iii) prolonged JNK activation has no obligate role in TNFα-induced cell death.     

TRAF2 Suppresses Basal IKK Activity in Resting Cells and TNFα Can Activate IKK in TRAF2 and TRAF5 Double Knockout Cells

Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) and TRAF5 are adapter proteins involved in TNFα-induced activation of the c-Jun N-terminal kinase and nuclear factor κB (NF-κB) pathways. Currently, TNFα-induced NF-κB activation is believed to be impaired in TRAF2 and TRAF5 double knockout (T2/5 DKO) cells. Here, we report instead that T2/5 DKO cells exhibit high basal IκB kinase (IKK) activity and elevated expression of NF-κB-dependent genes in unstimulated conditions. Although TNFα-induced receptor-interacting protein 1 ubiquitination is indeed impaired in T2/5 DKO cells, TNFα stimulation further increases IKK activity in these cells, resulting in significantly elevated expression of NF-κB target genes to a level higher than that in wild-type cells. Inhibition of NIK in T2/5 DKO cells attenuates basal IKK activity and restores robust TNFα-induced IKK activation to a level comparable with that seen in wild-type cells. This suggests that TNFα can activate IKK in the absence of TRAF2 and TRAF5 expression and receptor-interacting protein 1 ubiquitination. In addition, both the basal and TNFα-induced expression of anti-apoptotic proteins are normal in T2/5 DKO cells, yet these DKO cells remain sensitive to TNFα-induced cell death, due to the impaired recruitment of anti-apoptotic proteins to the TNFR1 complex in the absence of TRAF2. Thus, our data demonstrate that TRAF2 negatively regulates basal IKK activity in resting cells and inhibits TNFα-induced cell death by recruiting anti-apoptotic proteins to the TNFR1 complex rather than by activating the NF-κB pathway.     

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

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

Physical and functional interaction of filamin (actin-binding protein-280) and tumor necrosis factor receptor-associated factor 2

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is an intracellular protein involved in signal transduction from TNF receptor I and II and related receptors. TRAF2 is required for TNF-induced activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and TRAF2 can also mediate activation of NF-kappaB. Here we have identified the actin-binding protein Filamin (actin-binding protein-280) as a TRAF2-interacting protein. Filamin binds to the Ring zinc finger domain of TRAF2. Overexpressed Filamin inhibits TRAF2-induced activation of JNK/SAPK and of NF-kappaB. Furthermore, ectopically expressed Filamin inhibits NF-kappaB activation induced via TNF, interleukin-1, Toll receptors, and TRAF6 but not activation induced via overexpression of NIK, a downstream effector in these pathways. Importantly, TNF fails to activate SAPK or NF-kappaB in a human melanoma cell line deficient in Filamin. Reintroduction of Filamin into these cells restores the TNF response. The data imply a role for Filamin in inflammatory signal transduction pathways.     

AIP1/DAB2IP, a novel member of the Ras-GAP family, transduces TRAF2-induced ASK1-JNK activation

Previously we have shown that ASK-interacting protein 1 (AIP1, also known as DAB2IP), a novel member of the Ras-GAP protein family, mediates TNF-induced activation of ASK1-JNK signaling pathway. However, the mechanism by which TNF signaling is coupled to AIP1 is not known. Here we show that AIP1 is localized on the plasma membrane in resting endothelial cells (EC) in a complex with TNFR1. TNF binding induces release of AIP1 from TNFR1, resulting in cytoplasmic translocation and concomitant formation of an intracellular signaling complex comprised of TRADD, RIP1, TRAF2, and AIPl. A proline-rich region (amino acids 796-807) is critical for maintaining AIP1 in a closed form, which associates with a region of TNFR1 distinct from the death domain, the site of TNFR1 association with TRADD. An AIP1 mutant with deletion of this proline-rich region constitutively binds to TRAF2 and ASK1. A PERIOD-like domain (amino acids 591-719) of AIP1 binds to the intact RING finger of TRAF2, and specifically enhances TRAF2-induced ASK1 activation. At the same time, the binding of AIP1 to TRAF2 inhibits TNF-induced IKK-NF-kappaB signaling. Taken together, our data suggest that AIP1 is a novel transducer in TNF-induced TRAF2-dependent activation of ASK1 that mediates a balance between JNK versus NF-kappaB signaling.     

Activation of NF-kappaB by RANK requires tumor necrosis factor receptor-associated factor (TRAF) 6 and NF-kappaB-inducing kinase. Identification of a novel TRAF6 interaction motif

Various members of the tumor necrosis factor (TNF) receptor superfamily activate nuclear factor kappaB (NF-kappaB) and the c-Jun N-terminal kinase (JNK) pathways through their interaction with TNF receptor-associated factors (TRAFs) and NF-kappaB-inducing kinase (NIK). We have previously shown that the cytoplasmic domain of receptor activator of NF-kappaB (RANK) interacts with TRAF2, TRAF5, and TRAF6 and that its overexpression activates NF-kappaB and JNK pathways. Through a detailed mutational analysis of the cytoplasmic domain of RANK, we demonstrate that TRAF2 and TRAF5 bind to consensus TRAF binding motifs located in the C terminus at positions 565-568 and 606-611, respectively. In contrast, TRAF6 interacts with a novel motif located between residues 340 and 358 of RANK. Furthermore, transfection experiments with RANK and its deletion mutants in human embryonic 293 cells revealed that the TRAF6-binding region (340-358), but not the TRAF2 or TRAF5-binding region, is necessary and sufficient for RANK-induced NF-kappaB activation. Moreover, a kinase mutant of NIK (NIK-KM) inhibited RANK-induced NF-kappaB activation. However, RANK-mediated JNK activation required a distal portion (427-603) of RANK containing the TRAF2-binding domain. Thus, our results indicate that RANK interacts with various TRAFs through distinct motifs and activates NF-kappaB via a novel TRAF6 interaction motif, which then activates NIK, thus leading to NF-kappaB activation, whereas RANK most likely activates JNK through a TRAF2-interacting region in RANK.     

Tumor necrosis factor-induced nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation

The mechanism of tumor necrosis factor (TNF)-induced nonapoptotic cell death is largely unknown, although the mechanism of TNF-induced apoptosis has been studied extensively. In wild-type mouse embryonic fibroblast cells under a caspase-inhibited condition, TNF effectively induced cell death that morphologically resembled necrosis. In this study, we utilized gene knockout mouse embryonic fibroblasts cells and found that tumor necrosis factor receptor (TNFR) I mediates TNF-induced necrotic cell death, and that RIP, FADD, and TRAF2 are critical components of the signaling cascade of this TNF-induced necrotic cell death. Inhibitors of NF-kappaB facilitated TNF-induced necrotic cell death, suggesting that NF-kappaB suppresses the necrotic cell death pathway. JNK, p38, and ERK activation seem not to be required for this type of cell death because mitogen-activated protein kinase inhibitors did not significantly affect TNF-induced necrotic cell death. In agreement with the previous reports that the reactive oxygen species (ROS) may play an important role in this type of cell death, the ROS scavenger butylated hydroxyanisole efficiently blocked TNF-induced necrotic cell death. Interestingly, during TNF-induced necrotic cell death, the cellular ROS level was significantly elevated in wild type, but not in RIP(-/-), TRAF2(-/-), and FADD(-/-) cells. These results suggest that RIP, TRAF2, and FADD are crucial in mediating ROS accumulation in TNF-induced necrotic cell death.     

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.     

Interleukin-1-induced NF-kappaB activation is NEMO-dependent but does not require IKKbeta

Activation of NF-kappaB by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the IkappaB kinase (IKK) complex, which contains two kinases named IKKalpha and IKKbeta and a critical regulatory subunit named NEMO. Although we have previously demonstrated that NEMO associates with both IKKs, genetic studies reveal that only its interaction with IKKbeta is required for TNF-induced NF-kappaB activation. To determine whether NEMO and IKKalpha can form a functional IKK complex capable of activating the classical NF-kappaB pathway in the absence of IKKbeta, we utilized a panel of mouse embryonic fibroblasts (MEFs) lacking each of the IKK complex subunits. This confirmed that TNF-induced IkappaBalpha degradation absolutely requires NEMO and IKKbeta. In contrast, we consistently observed intact IkappaBalpha degradation and NF-kappaB activation in response to IL-1 in two separate cell lines lacking IKKbeta. Furthermore, exogenously expressed, catalytically inactive IKKbeta blocked TNF- but not IL-1-induced IkappaBalpha degradation in wild-type MEFs, and reconstitution of IKKalpha/beta double knockout cells with IKKalpha rescued IL-1- but not TNF-induced NF-kappaB activation. Finally, we have shown that incubation of IKKbeta-deficient MEFs with a cell-permeable peptide that blocks the interaction of NEMO with the IKKs inhibits IL-1-induced NF-kappaB activation. Our results therefore demonstrate that NEMO and IKKalpha can form a functional IKK complex that activates the classical NF-kappaB pathway in response to IL-1 but not TNF. These findings further suggest NEMO differentially regulates the fidelity of the IKK subunits activated by distinct upstream signaling pathways.     

TRAF6 is required for TRAF2-dependent CD40 signal transduction in nonhemopoietic cells

The emerging role of CD40, a tumor necrosis factor (TNF) receptor family member, in immune regulation, disease pathogenesis, and cancer therapy necessitates the analysis of CD40 signal transduction in a wide range of tissue types. In this study we present evidence that the CD40-interacting proteins TRAF2 and TRAF6 play an important physiological role in CD40 signaling in nonhemopoietic cells. Using mutational analysis of the CD40 cytoplasmic tail, we demonstrate that the specific binding of TRAF2 to CD40 is required for efficient signaling on the NF-kappaB, Jun N-terminal protein kinase (JNK), and p38 axis. In fibroblasts lacking TRAF2 or in carcinoma cells in which TRAF2 has been depleted by RNA interference, the CD40-mediated activation of NF-kappaB and JNK is significantly reduced, and the activation of p38 and Akt is severely impaired. Interestingly, whereas the TRAF6-interacting membrane-proximal domain of CD40 has a minor role in signal transduction, studies utilizing TRAF6 knockout fibroblasts and RNA interference in epithelial cells reveal that the CD40-induced activation of NF-kappaB, JNK, p38, and Akt requires the integrity of TRAF6. Furthermore, we provide evidence that TRAF6 regulates CD40 signal transduction not only through its direct binding to CD40 but also indirectly via its association with TRAF2. These observations provide novel insight into the mechanisms of CD40 signaling and the multiple roles played by TRAF6 in signal transduction.     

Targeted deletion of MKK4 gene potentiates TNF-induced apoptosis through the down-regulation of NF-kappa B activation and NF-kappa B-regulated antiapoptotic gene products

MAPK kinase 4 (MKK4) is a dual-specificity kinase that activates both JNK and p38 MAPK. However, the mechanism by which MKK4 regulates TNF-induced apoptosis is not fully understood. Therefore, we used fibroblasts derived from MKK4 gene-deleted (MKK4-KO) mice to determine the role of this kinase in TNF signaling. We found that when compared with the wild-type cells, deletion of MKK4 gene enhanced TNF-induced apoptosis, and this correlated with down-regulation of TNF-induced cell-proliferative (COX-2 and cyclin D1) and antiapoptotic (survivin, IAP1, XIAP, Bcl-2, Bcl-x(L), and cFLIP) gene products, all regulated by NF-kappaB. Indeed we found that TNF-induced NF-kappaB activation was abrogated in MKK4 gene-deleted cells, as determined by DNA binding. Further investigation revealed that TNF-induced I kappaB alpha kinase activation, I kappaB alpha phosphorylation, I kappaB alpha degradation, and p65 nuclear translocation were all suppressed in MKK4-KO cells. NF-kappaB reporter assay revealed that NF-kappaB activation induced by TNF, TNFR1, TRADD, TRAF2, NIK, and I kappaB alpha kinase was modulated in gene-deleted cells. Overall, our results indicate that MKK4 plays a central role in TNF-induced apoptosis through the regulation of NF-kappaB-regulated gene products.     

Protein kinase PKN1 associates with TRAF2 and is involved in TRAF2-NF-kappaB signaling pathway

PKN1 is a fatty acid and Rho-activated serine/threonine protein kinase whose catalytic domain is highly homologous to protein kinase C (PKC) family. In yeast two-hybrid screening for PKN1 binding proteins, we identified tumor necrosis factor alpha (TNFalpha) receptor-associated factor 2 (TRAF2). TRAF2 is one of the major mediators of TNF receptor superfamily transducing TNF signal to various functional targets, including activation of NF-kappaB, JNK, and apoptosis. FLAG-tagged PKN1 was co-immunoprecipitated with endogenous TRAF2 from HEK293 cell lysate, and in vitro binding assay using the deletion mutants of TRAF2 showed that PKN1 directly binds to the TRAF domain of TRAF2. PKN1 has the TRAF2-binding consensus sequences PXQX (S/T) at amino acid residues 580-584 (PIQES), and P580AQ582A mutant was not co-immunoprecipitated with TRAF2. Furthermore, the reduced expression of PKN1 by RNA interference (RNAi) down-regulated TRAF2-induced NF-kappaB activation in HEK293T cells. These results suggest that PKN1 is involved in TRAF2-NF-kappaB signaling pathway.