Jin Gohda

Cutting Edge: TNFR-Associated Factor (TRAF) 6 Is Essential for MyD88-Dependent Pathway but Not Toll/IL-1 Receptor Domain-Containing Adaptor-Inducing IFN-β (TRIF)-Dependent Pathway in TLR Signaling

Signaling pathways from TLRs are mediated by the Toll/IL-1R (TIR) domain-containing adaptor molecules. TNF receptor-associated factor (TRAF) 6 is thought to activate NF-κB and MAPKs downstream of these TIR domain-containing proteins to induce production of inflammatory cytokines. However, the precise role of TRAF6 in signaling from individual TLRs has not been appropriately addressed. We analyzed macrophages from TRAF6-deficient mice and made the following observations. In the absence of TRAF6, 1) ligands for TLR2, TLR5, TLR7, and TLR9 failed to induce activation of NF-κB and MAPKs or production of inflammatory cytokines; 2) TLR4 ligand-induced cytokine production was remarkably reduced and activation of NF-κB and MAPKs was observed, albeit with delayed kinetics; and 3) in contrast with previously reported findings, TLR3 signaling was not affected. These results indicate that TRAF6 is essential for MyD88-dependent signaling but is not required for TIR domain-containing adaptor-inducing IFN-β (TRIF)-dependent signaling.

NF-κB activation in development and progression of cancer

Nuclear factor‐κΒ (NF‐κB) binds specifically to NF‐κB‐binding sites (κB sites, 5′‐GGGRNNYYCC‐3′; R, purine; Y, pyrimidine; N, any nucleotide) present in enhancer regions of various genes. Binding of various cytokines, growth factors and pathogen‐associated molecular patterns to specific receptors activates NF‐κB and expression of genes that play critical roles in inflammation, innate and acquired immunity, bone remodeling and generation of skin appendices. Activation of NF‐κB is also involved in cancer development and progression. NF‐κB is activated in cells that become malignant tumors and in cells that are recruited to and constitute the tumor microenvironment. In the latter scenario, the TLR‐TRAF6‐NF‐kB pathways seem to play major roles, and NF‐κB activation results in production of cytokines, which in turn induce NF‐κB activation in premalignant cells, leading to expression of genes involved abnormal growth and malignancy. Furthermore, NF‐κB activation is involved in bone metastasis. Osteoclasts, whose generation requires the RANK‐TRAF6‐NF‐κB pathways, release various growth factors stored in bone, which results in creation of microenvironment suitable for proliferation and colonization of cancer cells. Therefore, NF‐κB and molecules involved its activation, such as TRAF6, are attractive targets for therapeutic strategies against cancer. (Cancer Sci 2007; 98: 268–274)

Recruitment of Tumor Necrosis Factor Receptor-associated Factor Family Proteins to Apoptosis Signal-regulating Kinase 1 Signalosome Is Essential for Oxidative Stress-induced Cell Death

Apoptosis signal-regulating kinase 1 (ASK1) plays a pivotal role in oxidative stress-induced cell death. Reactive oxygen species disrupt the interaction of ASK1 with its cellular inhibitor thioredoxin and thereby activates ASK1. However, the precise mechanism by which ASK1 freed from thioredoxin undergoes oligomerization-dependent activation has not been fully elucidated. Here we show that endogenous ASK1 constitutively forms a high molecular mass complex including Trx (∼1,500-2,000 kDa), which we designate ASK1 signalosome. Upon H2O2 treatment, the ASK1 signalosome forms a higher molecular mass complex at least in part because of the recruitment of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. Consistent with our previous findings that TRAF2 and TRAF6 activate ASK1, H2O2-induced ASK1 activation and cell death were strongly reduced in the cells derived from Traf2-/- and Traf6-/- mice. A novel signaling complex including TRAF2, TRAF6, and ASK1 may thus be the key component in oxidative stress-induced cell death.

RANK-mediated amplification of TRAF6 signaling leads to NFATc1 induction during osteoclastogenesis

RANK and CD40 activate NF‐κB and MAPKs to similar levels via TRAF6. Even though overexpression of TRAF6 results in osteoclast formation, RANK but not CD40 promotes osteoclastogenesis. To understand the molecular basis for RANK‐specific activity in osteoclastogenesis, we created an osteoclast formation system driven by anti‐human CD40 antibody‐mediated stimulation of a chimeric receptor, h40/mRK, which consists of the extracellular domain of human CD40 and the transmembrane and cytoplasmic domains of mouse RANK. By introducing mutations into three TRAF6‐binding sites of RANK, we found that h40/mRK with a single TRAF6‐binding site efficiently induced Ca2+ oscillation and expression of NFATc1, a master switch in osteoclastogenesis, whereas CD40 carrying a single TRAF6‐binding site did not. However, expression of CD40 that was approximately 100 times greater than that of h40/mRK resulted in osteoclast formation, indicating that the RANK–TRAF6 signal is more potent than the CD40–TRAF6 signal in terms of NFATc1 activation and osteoclastogenesis. These results suggest that RANK may harbor a specific domain that amplifies TRAF6 signaling.

The Shigella flexneri effector OspI deamidates UBC13 to dampen the inflammatory response

Many bacterial pathogens can enter various host cells and then survive intracellularly, transiently evade humoral immunity, and further disseminate to other cells and tissues. When bacteria enter host cells and replicate intracellularly, the host cells sense the invading bacteria as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) by way of various pattern recognition receptors. As a result, the host cells induce alarm signals that activate the innate immune system. Therefore, bacteria must modulate host inflammatory signalling and dampen these alarm signals. How pathogens do this after invading epithelial cells remains unclear, however. Here we show that OspI, a Shigella flexneri effector encoded by ORF169b on the large plasmid and delivered by the type ΙΙΙ secretion system, dampens acute inflammatory responses during bacterial invasion by suppressing the tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6)-mediated signalling pathway. OspI is a glutamine deamidase that selectively deamidates the glutamine residue at position 100 in UBC13 to a glutamic acid residue. Consequently, the E2 ubiquitin-conjugating activity required for TRAF6 activation is inhibited, allowing S. flexneri OspI to modulate the diacylglycerol–CBM (CARD–BCL10–MALT1) complex–TRAF6–nuclear-factor-κB signalling pathway. We determined the 2.0 Å crystal structure of OspI, which contains a putative cysteine–histidine–aspartic acid catalytic triad. A mutational analysis showed this catalytic triad to be essential for the deamidation of UBC13. Our results suggest that S. flexneri inhibits acute inflammatory responses in the initial stage of infection by targeting the UBC13–TRAF6 complex.

Two Mechanistically and Temporally Distinct NF-κB Activation Pathways in IL-1 Signaling

The cooperative activation of NF-κB by two distinct pathways that diverge at TRAF6 critically contributes to the IL-1–dependent inflammatory response. A One-Two Punch Members of the interleukin-1 (IL-1) family of cytokines stimulate proinflammatory responses through their activation of the transcription factors nuclear factor κB (NF-κB) and activating protein 1 (AP-1). The binding of IL-1 to its receptor complex triggers the activation of the E3 ubiquitin ligase and scaffold protein TRAF6 and its association with the mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) TAK1, which leads to the activation of NF-κB. Another MAP3K, MEKK3, is also involved in IL-1–mediated activation of NF-κB, but how TAK1 and MEKK3 might physically or functionally interact has been unclear. Yamazaki et al. showed that early IL-1 signaling induced the formation of a transient complex of TRAF6, TAK1, and MEKK3. Formation of this complex, which was dependent on TRAF6-mediated ubiquitination of TAK1, led to NF-κB activation. In a later phase of IL-1 signaling, TRAF6 activated NF-κB in a MEKK3-dependent, TAK1-independent manner. Together, these two pathways resulted in the prolonged activation of NF-κB required to trigger an effective proinflammatory response. The cytokine interleukin-1 (IL-1) mediates immune and inflammatory responses by activating the transcription factor nuclear factor κB (NF-κB). Although transforming growth factor–β–activated kinase 1 (TAK1) and mitogen-activated protein kinase (MAPK) kinase kinase 3 (MEKK3) are both crucial for IL-1–dependent activation of NF-κB, their potential functional and physical interactions remain unclear. Here, we showed that TAK1-mediated activation of NF-κB required the transient formation of a signaling complex that included tumor necrosis factor receptor–associated factor 6 (TRAF6), MEKK3, and TAK1. Site-specific, lysine 63–linked polyubiquitination of TAK1 at lysine 209, likely catalyzed by TRAF6 and Ubc13, was required for the formation of this complex. After TAK1-mediated activation of NF-κB, TRAF6 subsequently activated NF-κB through MEKK3 independently of TAK1, thereby establishing continuous activation of NF-κB, which was required for the production of sufficient cytokines. Therefore, we propose that the cooperative activation of NF-κB by two mechanistically and temporally distinct MEKK3-dependent pathways that diverge at TRAF6 critically contributes to immune and inflammatory systems.

Identification of TIFA as an Adapter Protein That Links Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) to Interleukin-1 (IL-1) Receptor-associated Kinase-1 (IRAK-1) in IL-1 Receptor Signaling

Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals from members of the Toll/interleukin-1 (IL-1) receptor family by interacting with IL-1 receptor-associated kinase-1 (IRAK-1) after IRAK-1 is released from the receptor-MyD88 complex upon IL-1 stimulation. However, the molecular mechanisms underlying regulation of the IRAK-1/TRAF6 interaction are largely unknown. We have identified TIFA, a TRAF-interacting protein with a forkhead-associated (FHA) domain. The FHA domain is a motif known to bind directly to phosphothreonine and phosphoserine. In transient transfection assays, TIFA activates NFκΒ and c-Jun amino-terminal kinase. However, TIFA carrying a mutation that abolishes TRAF6 binding or mutations in the FHA domain that are known to abolish FHA domain binding to phosphopeptide fails to activate NFκΒ and c-Jun amino-terminal kinase. TIFA, when overexpressed, binds both TRAF6 and IRAK-1 and significantly enhances the IRAK-1/TRAF6 interaction. Furthermore, analysis of endogenous proteins indicates that TIFA associates with TRAF6 constitutively, whereas it associates with IRAK-1 in an IL-1 stimulation-dependent manner in vivo. Thus, TIFA is likely to mediate IRAK-1/TRAF6 interaction upon IL-1 stimulation.

Characteristics and Biological Functions of TRAF6

TRAF6 is divergent from other members of the TRAF family. Therefore, TRAF6 was expected to play physiological roles distinct from those of other TRAFs. In this chapter, we focused on the physiological functions specific to TRAF6 but not other TRAFs in immune system, formation of skin appendices, and nervous system development by describing abnormal phenotypes observed in TRAF6-deficient mice. The role of TRAF6 in osteoclastogenesis and the molecular mechanisms ofTRAF6-mediated signal transduction are described in other chapters.

TRAF6 Establishes Innate Immune Responses by Activating NF-κB and IRF7 upon Sensing Cytosolic Viral RNA and DNA

Background In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor κB (NF-κB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-κB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed. Principal Findings Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-κB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFβ-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-κB activation, were not essential for RLH-mediated NF-κB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-κB and IRF7. Conclusions/Significance Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection.

Human lactoferrin activates NF‐κB through the Toll‐like receptor 4 pathway while it interferes with the lipopolysaccharide‐stimulated TLR4 signaling

Lactoferrin (LF) has been implicated in innate immunity. Here we reveal the signal transduction pathway responsible for human LF (hLF)‐triggered nuclear factor‐κB (NF‐κB) activation. Endotoxin‐depleted hLF induces NF‐κB activation at physiologically relevant concentrations in the human monocytic leukemia cell line, THP‐1, and in mouse embryonic fibroblasts (MEFs). In MEFs, in which both tumor necrosis factor receptor‐associated factor 2 (TRAF2) and TRAF5 are deficient, hLF causes NF‐κB activation at a level comparable to that seen in wild‐type MEFs, whereas TRAF6‐deficient MEFs show significantly impaired NF‐κB activation in response to hLF. TRAF6 is known to be indispensable in leading to NF‐κB activation in myeloid differentiating factor 88 (MyD88)‐dependent signaling pathways, while the role of TRAF6 in the MyD88‐independent signaling pathway has not been clarified extensively. When we examined the hLF‐dependent NF‐κB activation in MyD88‐deficient MEFs, delayed, but remarkable, NF‐κB activation occurred as a result of the treatment of cells with hLF, indicating that both MyD88‐dependent and MyD88‐independent pathways are involved. Indeed, hLF fails to activate NF‐κB in MEFs lacking Toll‐like receptor 4 (TLR4), a unique TLR group member that triggers both MyD88‐depependent and MyD88‐independent signalings. Importantly, the carbohydrate chains from hLF are shown to be responsible for TLR4 activation. Furthermore, we show that lipopolysaccharide‐induced cytokine and chemokine production is attenuated by intact hLF but not by the carbohydrate chains from hLF. Thus, we present a novel model concerning the biological function of hLF: hLF induces moderate activation of TLR4‐mediated innate immunity through its carbohydrate chains; however, hLF suppresses endotoxemia by interfering with lipopolysaccharide‐dependent TLR4 activation, probably through its polypeptide moiety.