TRAF6 distinctively mediates MyD88- and IRAK-1-induced activation of NF-kappaB

MyD88 and IL-1R-associated kinase 1 (IRAK-1) play crucial roles as adaptor molecules in signal transduction of the TLR/IL-1R superfamily, and it is known that expression of these proteins leads to the activation of NF-kappaB in a TNFR-associated factor 6 (TRAF6)-dependent manner. We found in this study, however, that a dominant-negative mutant of TRAF6, lacking the N-terminal RING and zinc-finger domain, did not inhibit IRAK-1-induced activation of NF-kappaB in human embryonic kidney 293 cells, although the TRAF6 mutant strongly suppressed the MyD88-induced activation. The dominant-negative mutant of TRAF6 did not affect the IRAK-1-induced activation, regardless of the expression level of IRAK-1. In contrast, small interfering RNA silencing of TRAF6 expression inhibited MyD88-induced and IRAK-1-induced activation, and supplementation with the TRAF6 dominant-negative mutant did not restore the IRAK-1-induced activation. Expression of IRAK-1, but not MyD88, induced the oligomerization of TRAF6, and IRAK-1 and the TRAF6 dominant-negative mutant were associated with TRAF6. These results indicate that TRAF6 is involved but with different mechanisms in MyD88-induced and IRAK-induced activation of NF-kappaB and suggest that TRAF6 uses a distinctive mechanism to activate NF-kappaB depending on signals.     

Litopenaeus vannamei tumor necrosis factor receptor-associated factor 6 (TRAF6) responds to Vibrio alginolyticus and white spot syndrome virus (WSSV) infection and activates antimicrobial peptide genes

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a key signaling adaptor protein not only for the TNFR superfamily but also for the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) superfamily. To investigate TRAF6 function in invertebrate innate immune responses, Litopenaeus vannamei TRAF6 (LvTRAF6) was identified and characterized. The full-length cDNA of LvTRAF6 is 2823 bp long, with an open reading frame (ORF) encoding a putative protein of 594 amino acids, including a RING-type Zinc finger, two TRAF-type Zinc fingers, a coiled-coil region, and a meprin and TRAF homology (MATH) domain. The overall amino acid sequence identity between LvTRAF6 and other known TRAF6s is 22.2-33.3%. Dual luciferase reporter assays in Drosophila S2 cells revealed that LvTRAF6 could activate the promoters of antimicrobial peptide genes (AMPs), including Drosophila Attacin A and Drosomycin, and shrimp Penaeidins. Real-time quantitative PCR (qPCR) indicated that LvTRAF6 was constitutively expressed in various tissues of L. vannamei. After Vibrio alginolyticus and white spot syndrome virus (WSSV) challenge, LvTRAF6 was down-regulated, though with different expression patterns in the intestine compared to other tissues. After WSSV challenge, LvTRAF6 was up-regulated 2.7- and 2.3-fold over the control at 3 h in gills and hepatopancreas, respectively. These results indicated that LvTRAF6 may play a crucial role in antibacterial and antiviral responses via regulation of AMP gene expression.     

The TRAF6, but not the TRAF2/3, binding domain of CD40 is required for cytokine production in human lung fibroblasts

Fibroblasts are key effector cells in inciting inflammation, wound healing, and scarring. CD40, a member of the TNF receptor superfamily, mediates intercellular communication between fibroblasts and cells that express CD154 (CD40L), including T lymphocytes and platelets. To better understand the mechanisms by which CD40 regulates fibroblast function in inflammation and scarring, we examined the ability of CD40 cytoplasmic tail regions (CD40ct) containing the TRAF6 or the TRAF2/3 binding domains to regulate cytokine and chemokine expression by primary human lung fibroblasts. The full-length human CD40ct, the first 35 amino acids of the CD40ct encompassing the TRAF6 binding site (1-35), and amino acids 35-53 containing the TRAF2/TRAF3 binding domain were expressed in human lung fibroblasts as fusion proteins with the extracellular domain of human CD8alpha by retroviral transduction. The TRAF6, but not the TRAF2/3, binding domain was found to regulate IL-8 and IL-6 production, and induce activation of NF-kappaB and Jun kinase in lung fibroblasts, demonstrating for the first time that CD40ct domains can function independently to regulate pro-inflammatory responses of primary human fibroblasts. Thus, targeting TRAF6 function through pharmacological intervention may represent a viable strategy for modulating localized inflammation.     

Sensitivity of TLR4- and -7-induced NF kappa B1 p105-TPL2-ERK pathway to TNF-receptor-associated-factor-6 revealed by RNAi in mouse macrophages

Tumor necrosis factor (TNF)-receptor-associated-factor-6 (TRAF6) is an adaptor protein involved in Toll-like receptor (TLR) signaling. Recent studies using macrophages from TRAF6 knockout mice have revealed that TRAF6 is required for TLR7 signaling. However, an essential role of TRAF6 in TLR4 signaling and cytokine production is slightly controversial. Using an RNAi approach to reduce the cellular levels of TRAF6, we tested the role of this adaptor protein on the sensitivity of the various components of the ERK pathway mediated by TLR4 and -7 in Raw264.7, a mouse macrophage cell line. ERK activation in macrophages by TLR4 and -7 is mediated via a MAP3K, called TPL2/COT, which under unstimulated conditions is associated with NF kappa B1 p105, a member of the I kappa B family of proteins. Upon stimulation with TLR ligands, p105 is phosphorylated by I kappa B kinase (IKK) complex and partially degraded, which releases TPL2. The free TPL2 is active and stimulates the ERK pathway via MEK1/2. The free TPL2, however, is also unstable and is targeted for degradation. We demonstrate here that reduced level of TRAF6 ( approximately 80% decrease) in macrophages does not significantly affect any of the components of the TLR4-stimulated ERK pathway, including p105 phosphorylation, TPL2 degradation and ERK1/2 phosphorylation. Surprisingly, however, TLR4-induced JNK1/2 phosphorylation is significantly blocked by TRAF6 knockdown, suggesting that ERK and JNK pathways are differentially sensitive to TRAF6 levels. Furthermore, although TLR4-mediated IKK-induced p105 phosphorylation is not sensitive to TRAF6 knockdown, I kappa B alpha phosphorylation (also, IKK-induced) is significantly blocked, suggesting that TLR4 activation results in a TRAF6-sensitive and -insensitive IKK activation in macrophages. In contrast to TLR4 signaling, TLR7 activation of ERK, JNK pathways and phosphorylation of p105 and I kappa B alpha are completely inhibited in TRAF6 knockdown cells. Compared to the signaling data, while TLR4-induced TNFalpha mRNA expression is not significantly inhibited by TRAF6 knockdown, TLR7-induced TNFalpha mRNA is significantly blocked. In contrast, both TLR4- and TLR7-induced IL6 mRNA are significantly blocked by TRAF6 knockdown. These results suggest that while TRAF6 is absolutely essential for TLR7 activation of ERK, JNK and NF kappa B pathways, TLR4-induced ERK, JNK pathways and IKK-mediated phosphorylation of I kappa B family members as well as cytokine expression are differentially sensitive to the cellular levels of TRAF6. These results have important implications in terms of therapeutic targeting of TRAF6 complexes in diseases where TLR4 and -7 are involved.     

Threonine 66 in the death domain of IRAK-1 is critical for interaction with signaling molecules but is not a target site for autophosphorylation

Ligand binding in the TLR/IL-1R family results in the transient formation of an intracellular signaling complex, which contains, amongst others, the serine/threonine-specific kinase IL-1R-associated kinase 1 (IRAK-1). Concomitantly, the kinase function of IRAK-1 becomes activated, resulting in massive autophosphorylation and finally in the dissociation of the initially constituted signaling complex. The death domain (DD) of IRAK-1 mediates the interaction with other molecules of the signaling complex, e.g., the adaptor MyD88, the silencer Tollip, and the activator kinase IRAK-4. The conserved threonine at position 66 (T66), located within the DD, is a putative autophosphorylation target site. Here, we provide evidence that T66 critically impacts the secondary structure of the IRAK-1 DD. Thereby, it ensures the transient manner of interactions between IRAK-1 and the other signaling molecules. This essential role, however, is not regulated by phosphorylation of T66 itself.     

Toll-Like Receptors’ Pathway Disturbances are Associated with Increased Susceptibility to Infections in Humans

Toll-like receptors (TLRs) sense microbial products and play an important role in innate immunity. Currently, 11 members of TLRs have been identified in humans, with important function in host defense in early steps of the inflammatory response. TLRs are present in the plasma membrane (TLR1, TLR2, TLR4, TLR5, TLR6) and endosome (TLR3, TLR7, TLR8, TLR9) of leukocytes. TLRs and IL-1R are a family of receptors related to the innate immune response that contain an intracellular domain known as the Toll-IL-1R (TIR) domain that recruits the TIR-containing cytosolic adapters MyD88, TRIF, TIRAP and TRAM. The classical pathway results in the activation of both nuclear factor κB and MAPKs via the IRAK complex, with two active kinases (IRAK-1 and IRAK-4) and two non-catalytic subunits (IRAK-2 and IRAK-3/M). The classical pro-inflammatory TLR signaling pathway leads to the synthesis of inflammatory cytokines and chemokines, such as IL-1β, IL-6, IL-8, IL-12 and TNF-α. In humans, genetic defects have been identified that impair signaling of the TLR pathway and this may result in recurrent pyogenic infections, as well as virus and fungi infections. In this review, we discuss the main mechanisms of microbial recognition and the defects involving TLRs.     

IRAK-4 as the central TIR signaling mediator in innate immunity

Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns and members of the proinflammatory interleukin-1 receptor (IL-1R) family, share homologies in their cytoplasmic domains. Engagement of members of both of these families initiates a common intracellular signaling cascade, in which MyD88 and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) are key adaptor proteins. Signaling between MyD88 and TRAF6 is mediated by members of the IL-1R-associated kinase (IRAK) family; however, the exact function of each IRAK protein remains controversial. IRAK-1 is required for the optimal transduction of IL-1R- and TLR-mediated signals, but IRAK-1 can be replaced by other IRAKs. Surprisingly, gene targeting studies show that the newest IRAK protein, IRAK-4, has an essential role in mediating signals initiated by IL-1R and TLR engagement. The kinase activity of IRAK-4 might be necessary to functionally modify IRAK-1 and perhaps other signal transducing substrates. Understanding the role of IRAK-4 in innate immunity will enable us to design novel strategies for therapeutic intervention in human infectious disease.     

Hepatitis C virus core protein enhances FADD-mediated apoptosis and suppresses TRADD signaling of tumor necrosis factor receptor

Hepatitis C virus (HCV) core protein has been shown to interact with the death domain (DD) of tumor necrosis factor receptor-1 (TNFR1). In this study, we further examined the interaction of the core protein with the signaling molecules of TNFR1, including FADD, TRADD, and TRAF2, in a human embryonic kidney cell line, HEK-293, that overexpresses the HCV core protein. This core protein-expressing cell line exhibited enhanced sensitivity to TNF-induced apoptosis. By in vitro binding and in vivo coimmunoprecipitation assays, we showed that the HCV core protein interacted with the DD of FADD and enhanced apoptosis induced by FADD overexpression. This enhancement could be blocked by a dominant-negative mutant of FADD. In contrast, the core protein did not directly interact with the DD of TRADD, but could disrupt the binding of TRADD to TNFR1. TRAF2 recruitment to the TNFR1 signaling complex was also disrupted by the core protein. Correspondingly, TRAF2-dependent activation of the protein kinase JNK was suppressed in the core protein-expressing cells. However, NF kappa B activation by TNF was not significantly altered by the HCV core protein, suggesting the existence of TRAF2-independent pathways for NF kappa B activation. These results combined indicate that the HCV core protein sensitizes cells to TNF-induced apoptosis primarily by facilitating FADD recruitment to TNFR1. The inhibition of JNK activation by the HCV core protein may also contribute to the increased propensity of cells for apoptosis. These results, in comparison with other published studies, suggest that the effects of the HCV core protein and their underlying mechanisms vary significantly among cells of different origins.     

NOD2 downregulates colonic inflammation by IRF4-mediated inhibition of K63-linked polyubiquitination of RICK and TRAF6

It is well established that polymorphisms of the caspase activation and recruitment domain 15 (CARD15) gene, a major risk factor in Crohn's disease (CD), lead to loss of nucleotide-binding oligomerization domain 2 (NOD2) function. However, a molecular explanation of how such loss of function leads to increased susceptibility to CD has remained unclear. In a previous study exploring this question, we reported that activation of NOD2 in human dendritic cells by its ligand, muramyl dipeptide (MDP), negatively regulates Toll-like receptor (TLR)-mediated inflammatory responses. Here we show that NOD2 activation results in increased interferon regulatory factor 4 (IRF4) expression and binding to tumor necrosis factor receptor associated factor 6 (TRAF6) and RICK (receptor interacting serine–threonine kinase). We then show that such binding leads to IRF4-mediated inhibition of Lys63-linked polyubiquitination of TRAF6 and RICK and thus to downregulation of nuclear factor (NF)-κB activation. Finally, we demonstrate that protection of mice from the development of experimental colitis by MDP or IRF4 administration is accompanied by similar IRF4-mediated effects on polyubiquitination of TRAF6 and RICK in colonic lamina propria mononuclear cells. These findings thus define a mechanism of NOD2-mediated regulation of innate immune responses to intestinal microflora that could explain the relation of CARD15 polymorphisms and resultant NOD2 dysfunction to CD.     

Tumor necrosis factor receptor- associated factor 6 (TRAF6) regulation of development,function, and homeostasis of the immune system

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an adapter protein that mediates a wide array of protein–protein interactions via its TRAF domain and a RING finger domain that possesses non-conventional E3 ubiquitin ligase activity. First identified nearly two decades ago as a mediator of interleukin-1 receptor (IL-1R)-mediated activation of NFκB, TRAF6 has since been identified as an actor downstream of multiple receptor families with immunoregulatory functions, including members of the TNFR superfamily, the Toll-like receptor (TLR) family, tumor growth factor-β receptors (TGFβR), and T-cell receptor (TCR). In addition to NFκB, TRAF6 may also direct activation of mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and interferon regulatory factor pathways. In the context of the immune system, TRAF6-mediated signals have proven critical for the development, homeostasis, and/or activation of B cells, T cells, and myeloid cells, including macrophages, dendritic cells, and osteoclasts, as well as for organogenesis of thymic and secondary lymphoid tissues. In multiple cellular contexts, TRAF6 function is essential not only for proper activation of the immune system but also for maintaining immune tolerance, and more recent work has begun to identify mechanisms of contextual specificity for TRAF6, involving both regulatory protein interactions, and messenger RNA regulation by microRNAs.     

A novel domain in the CD30 cytoplasmic tail mediates NFkappaB activation

About 100 amino acid residues in the C-terminal region are conserved among human, rat and murine CD30, a member of the tumor necrosis factor receptor (TNFR) superfamily, and can be separated into three subdomains with relatively higher conservation (D1, D2 and D3). Activation of NFkappaB by CD30 was shown to be mediated through interaction of TNFR- associated factor (TRAF) 1, 2 and 5 with the D2 and D3 subdomains. However, the function of the other conserved subdomain, D1, remained to be determined. Deletion of the D2 and D3 subdomains abolished interactions with TRAF2 and 5 but it did not affect NFkappaB activation. Reporter gene assays using deletion and mutant constructs of CD30 revealed that the D1 subdomain is sufficient for NFkappaB activation, without interaction with TRAF2 or 5, and that each subdomain alone can activate NFkappaB. Electrophoretic mobility shift assays revealed constitutive and CD30-induced NFkappaB activation in stable transformants of 293 cells expressing CD30 or a deletion mutant lacking D2 and D3 subdomains. Deletion of C-terminal 19 amino acid residues of the D1 subdomain abolished activation of NFkappaB. Substitution of alanine for one of the two threonine residues (amino acid position 524 and 529), one of which is a potential phosphorylation site in the D1 subdomain, also abolished the NFkappaB activation. Overexpression of the TRAF domain of TRAF2 or 5 had a dominant negative effect on the NFkappaB activation mediated by the D1 subdomain, thereby suggesting involvement of TRAF proteins in the signaling. Thus, the C- terminal 100 amino acid region of CD30 is composed of three independent functional subdomains, two of which contain binding sites for TRAF proteins. A novel domain in the cytoplasmic tail mediates NFkappaB activation, without direct interaction of TRAF2 or 5. Our observations suggest involvement of an unknown TRAF protein(s) in the signal transduction pathway of CD30.      

IRAK-4 kinase activity is required for IRAK-4-dependent innate and adaptive immune responses

Interleukin-1 receptor-associated kinase (IRAK)-4 is a serine-threonine kinase that plays an important role in innate and adaptive immune responses. While the requirement of IRAK-4 kinase activity has been studied in the context of IL-1R signaling, it is not clear whether IRAK-4 requires its kinase function for all of its roles in the immune system. IRAK-4 kinase-dead knock-in (IRAK-4KD/KD) mice were generated to further elucidate whether IRAK-4 kinase activity is required for IRAK-4 to induce cytokine production. IRAK-4KD/KD mice were impaired in their ability to produce cytokines in response to in vivo challenge with lipopolysaccharide (LPS), a potent TLR4 ligand. Cytokine production was also reduced in macrophages and dendritic cells from IRAK-4KD/KD mice in response to LPS and other TLR ligands. In addition, adaptive immune responses were impaired in IRAK-4KD/KD mice. Although in vitro T cell proliferation in response to TCR activation was unaffected in IRAK-4-deficient mice, in vivo T cell responses to lymphocytic choriomeningitits virus infection were significantly impaired in IRAK-4-knockout mice or mice expressing the kinase-dead mutant of IRAK-4. Collectively, these results indicate that IRAK-4 kinase activity is required for IRAK-4-dependent signaling in innate and adaptive immunity.     

Prolonged Toll-like receptor stimulation leads to down-regulation of IRAK-4 protein

Interleukin-1 receptor-associated kinase (IRAK)-4 is a key mediator in the Toll-like receptor (TLR) signaling. We found that stimulation of TLR2, TLR4, or TLR9, but not TLR3, caused a decrease in IRAK-4 protein without affecting its mRNA level in a mouse macrophage cell line, RAW 264. The decrease in IRAK-4 was accompanied by the appearance of a smaller molecular weight protein (32 kD), which was recognized by an anti-IRAK-4 antibody raised against the C-terminal region. The decrease in IRAK-4 and the appearance of the 32-kD protein occurred with slower kinetics than the activation of IRAK-1 and were suppressed by inhibitors of the proteasome, inducible inhibitor of kappaBalpha phosphorylation or protein synthesis, but not by caspase inhibitors. These results indicate that prolonged stimulation of TLR2, TLR4, or TLR9 causes a down-regulation of IRAK-4 protein, which may be mediated through cleavage of IRAK-4 by a protease induced by the activation of nuclear factor-kappaB.