Pellino 1 Is Required for Interleukin-1 (IL-1)-mediated Signaling through Its Interaction with the IL-1 Receptor-associated Kinase 4 (IRAK4)-IRAK-Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Complex*

Zhengfan JiangDagger , H. Jan Johnson§, Huiqing NieDagger , Jinzhong QinDagger , Timothy A. Bird§, and Xiaoxia LiDagger

From the Dagger  Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 and § Amgen, Incorporated, Seattle, Washington 98101

Received for publication, November 27, 2002, and in revised form, December 19, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
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The signaling pathway downstream of the mammalian interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) is evolutionally conserved with that mediated by the Drosophila Toll protein. Toll initiates its signal through the adapter molecule Tube and the serine-threonine kinase Pelle. Pelle is highly homologous to members of the IL-1R-associated kinase (IRAK) family in mammals. Recently, a novel Pelle-interacting protein called Pellino was identified in Drosophila. We now report a mammalian counterpart of Pellino, termed Pellino 1, which is required for NFkappa B activation and IL-8 gene expression in response to IL-1, probably through its signal-dependent interaction with IRAK4, IRAK, and the tumor necrosis factor receptor-associated factor 6 (TRAF6). The Pellino 1-IRAK-IRAK4-TRAF6 signaling complex is likely to be intermediate, located between the IL-1 receptor complex and the TAK1 complex in the IL-1 pathway.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Interleukin-1 (IL-1),1 a major inflammatory cytokine, exerts its biological effects by activating the transcription of various responsive genes (1). The transcription factors activated by IL-1 include NFkappa B (Rel proteins), AP1, and ATF (2-4). The IL-1 receptor belongs to the IL-1R/TLR (Toll-like receptor) superfamily. Human TLRs have recently emerged as key components in the generation of immune and inflammatory responses because of their ability to recognize pathogen-associated molecules (5). The signaling pathway downstream of mammalian IL-1R/TLR is evolutionally conserved with that initiated by the Drosophila Toll protein (5, 6). Drosophila Toll controls dorsoventral axis formation in embryogenesis and has a role in the anti-fungal immune response in the adult fly (6).

Upon IL-1 stimulation, the adaptor molecules MyD88 and Tollip are recruited to the IL-1 receptor complex, which then recruits the serine-threonine kinases IRAK4 and IRAK. IRAK is hyperphosphorylated, mediating the recruitment of TRAF6 to the receptor complex (7-12). IRAK-TRAF6 then leaves the receptor complex to interact with pre-associated TAK1, TAB1, and TAB2 on the membrane. TAK1 and TAB2 are phosphorylated on the membrane, which facilitates the formation and translocation of the TRAF6-TAK1-TAB1-TAB2 complex from the membrane to the cytosol. TAK1 is subsequently activated in the cytosol, leading to the activation of Ikappa B kinase (IKK). Activated IKK causes the activation of NFkappa B through the phosphorylation and degradation of Ikappa Bs, which sequester NFkappa B prior to activation. Activation of TAK1 also results in the activation of mitogen-activated protein kinases and c-Jun NH2-terminal kinase.

Drosophila Toll is activated by the endogenous ligand Spaetzle, leading to the activation of Dif (Rel protein) through the degradation of Cactus, the Drosophila homologue of the mammalian Ikappa Bs, which sequester Dif prior to activation. Toll signals through the adapter molecule Tube and the serine-threonine kinase Pelle (13). Although no mammalian counterpart has been found for Tube, Pelle is highly homologous to IRAK4 and IRAK (7-10). Drosophila TRAF (dTRAF) has also been identified and shown to interact with Pelle (14). Therefore, the Toll-Pelle-dTRAF-Cactus-Dif cascade is quite analogous to the mammalian IL-1R/TLR-IRAK4/IRAK-TRAF6-Ikappa B-NFkappa B pathway (14). However, the downstream signaling events from Pelle-dTRAF to Cactus are not clear. When Pelle was used as bait in a yeast two-hybrid screening, a novel Pelle-interacting protein called Pellino was identified (15). Pellino was shown to only interact with the catalytically active Pelle but not with a kinase-dead Pelle mutant, suggesting that Pellino may play an important role in facilitating the function of Pelle. In this study, we report the identification of mammalian Pellino (termed Pellino 1) and its role in IL-1- mediated signaling.

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Biological Reagents and Cell Culture-- Recombinant human IL-1beta was provided by the NCI, National Institutes of Health. Anti-IRAK4 was a gift from Dr. Holger Wesche (Tularik, South San Francisco, CA). Anti-TRAF6, anti-IRAK, and anti-IL-1R were from Santa Cruz Biotechnology. Rabbit anti-TAK1, anti-TAB1, and anti-TAB2 polyclonal antibodies were kindly provided by Dr. Kunihiro Matsumoto (16, 17). Anti-FLAG (M2) was from Sigma. HEK293-TK/Zeo cells, I1A, and I3A (18) were maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum, penicillin G (100 µg/ml), and streptomycin (100 µg/ml).

Recombinant Plasmids and Stable Transfection-- pE-selectin-luc, an NFkappa B-dependent E-selectin-luciferase (E-selectin-luc) reporter plasmid, was described by Schindler and Baichwal (19). Dominant negative TAK1 (DNTAK1-K66W) was a kind gift from Dr. Kunihiro Matsumoto (Nagoya University, Nagoya, Japan). The vector pSUPER was obtained from Dr. Reuven Agami's group (Center for Biomedical Genetics, Leiden, The Netherlands). Pellino 1-pSUPER was constructed by cloning primers derived from Pellino 1 (47-66 bp) according to the methods described by Brummelkamp et al. (20). For stable transfections, 2 × 105 cells were seeded onto a 10-cm plate and cotransfected the following day by the calcium phosphate method with 10 µg of each expression vector and 1 µg of pBabePuro. After 48 h, the cells were selected with 1 µg/ml puromycin until clones appeared.

Co-immunoprecipitation and Immunoblotting-- 6 × 105 cells were seeded onto each 15-cm plate and transfected the following day by the calcium phosphate method with 15 µg of FLAG-tagged Pellino 1. After 48 h, cells untreated or treated with 100 units/ml of IL-1 were lysed in a Triton-containing lysis buffer (0.5% Triton X-100, 20 mM HEPES, pH 7.4, 150 mM NaCl, 12.5 mM beta -glycerophosphate, 1.5 mM MgCl2, 10 mM NaF, 2 mM dithiothreitol, 1 mM sodium orthovanadate, 2 mM EGTA, 20 µM aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Cell extracts were incubated with 1 µg of antibody or preimmune serum (negative control) for 2 h, followed by a 2-h incubation with 20 µl of protein A-Sepharose beads (pre-washed and resuspended in phosphate-buffered saline at a 1:1 ratio). After incubation, the beads were washed four times with lysis buffer, separated by SDS-PAGE, transferred to Immobilon-P membranes (Millipore), and analyzed by immunoblotting.

Reporter Assays-- 2 × 105 cells were transfected by the same procedure as described above with 1 µg of pE-selectin-luc, 1 µg of pSV2-beta -gal, and 100 ng of each expression construct. After 48 h, the cells were split onto two 35-mm plates and stimulated with IL-1 the next day for 4 h before harvest. Luciferase and beta -galactosidase activities were determined by using the luciferase assay system and chemiluminescent reagents from Promega (Madison, WI).

Gel Shift Assays-- An NFkappa B binding site (5'-GAGCAGAGGGAAATTCCGTAACTT-3') from the IP-10 gene was used as a probe (21). Complementary oligonucleotides, end-labeled with polynucleotide kinase (Roche Molecular Biochemicals) and gamma -32P-labeled ATP, were annealed by slow cooling. Approximately 20,000 cpm of probe were used per assay (22). Whole cell extracts were used for the assay. The binding reaction was carried out at 4 °C for 20 min in a total volume of 20 µl containing 20 mM Hepes buffer, pH 7.0, 10 mM KCl, 0.1% Nonidet P-40, 0.5 mM dithiothreitol, 0.25 mM phenylmethanesulfonyl fluoride, and 10% glycerol.

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IL-1 Induces the Interaction of Pellino 1 with IRAK4 and IRAK-- Human Pellino 1 cDNA, cloned from a human dermal fibroblast cDNA library, encodes an open reading frame of 418 amino acid residues (accession number NP_065702, Fig. 1a). To identify functionally important regions of Pellino 1, the mammalian Pellino 1 was aligned with those from other species (Fig. 1). There is a surprising degree of similarity between the human Pellino 1 sequence and the homologous protein from Caenorhabditis elegans, which share 44% amino acid identity and 53% similarity. The C-terminal portion of Pellino 1 contains two invariant Cys-Gly-His triplets, two invariant Cys-Pro-X-Cys motifs (where X is a hydrophobic residue), and a conserved Cys-Pro-Val motif (Fig 1a), reminiscent of the structure of the C3HC4 Ring finger subfamily of zinc finger domains (23), which mediate diverse protein-protein interactions of tumor suppressors, protooncogenes, and signaling molecules such as TRAFs. Pellino 1 is ubiquitously expressed; it is expressed highly in peripheral blood leukocytes, moderately in placenta, lung, liver, kidney, spleen, thymus, skeletal muscle, and brain, and at a low level in small intestine, colon and heart (Fig. 1b).


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Fig. 1.   Characterization of Pellino 1 cDNA and expression pattern. a, deduced amino acid sequences of Pellino 1 from human (Peli1.hu), mouse (Peli1.mu), C. elegans (Peli1.ce), and Drosophila melanogaster (Peli1.dm). The motifs that resemble the structure of the C3HC4 Ring finger are underlined. Identical residues are shaded gray. b, Northern analysis of the indicated mouse tissues using mouse Pellino 1 cDNA as a probe.

We examined whether mammalian Pellino 1 interacts with IRAK and IRAK4, which are the mammalian counterparts of Pelle (7, 9, 10, 24). Because IRAK and IRAK4 are essential signaling components in the IL-1 pathway, the interaction of Pellino 1 with IRAK and IRAK4 was studied upon IL-1 stimulation. Cell extracts prepared from HEK293 cells transfected with FLAG-tagged Pellino 1, either untreated or stimulated with IL-1, and immunoprecipitated with anti-FLAG M2 antibody followed by Western immunoblotting using antibodies against IRAK and IRAK4. Interestingly, Pellino 1 indeed forms an IL-1-dependent signaling complex with the endogenous IRAK and IRAK4, strongly suggesting that it plays a role in IL-1-mediated signaling (Fig. 2a).


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Fig. 2.   IL-1 signaling complexes. a and b, IL-1-induced interaction of Pellino 1 with IL-1 signaling components. Cell extracts from HEK293 cells (293; a) or HEK293 cells transfected with FLAG-Pellino 1 (293-Pellino 1; a and b) untreated (0) or stimulated with IL-1 for the indicated times were immunoprecipitated (IP) with M2 antibody (anti-FLAG; a and b), anti-TRAF6 (b) and a control antibody (anti-p27, 1h*; a) followed by Western analyses with antibodies against the FLAG tag on Pellino 1 (a and b), IRAK (a), IRAK4 (a), TRAF6 (a and b), IL-1R (b), TAK1 (b), and TAB2 (b). c, model for IL-1-mediated signaling pathway.

Pellino 1 Is Required for IL-1-induced NFkappa B Activation and IL-8 Gene Expression-- We used small interfering RNA (siRNA), a new gene knock-down technology, to investigate the functional role of Pellino 1 in IL-1-mediated signaling pathways. Recently, a mammalian expression vector was developed that directs the synthesis of siRNA-like transcripts (pSUPER, suppression of endogenous RNA) (20). Pellino 1-pSUPER has been generated and stably transfected into HEK293 cells. Northern analysis showed that 27% of the clones (clones 1, 3, 12, 16, and 18) transfected with Pellino 1-pSUPER had a 90% reduction of Pellino 1 mRNA (Fig. 3a), whereas the clones transfected with vector showed the same levels of Pellino 1 mRNA (data not shown). As shown in Fig. 3b, IL-1 induced much weaker NFkappa B activation in C-12 and C-16 (in which Pellino 1 expression is knocked down; Fig. 3a) than in C-9 (a clone from the same transfection in which Pellino 1 expression is not altered, Fig. 3a), strongly suggesting that Pellino 1 is required for IL-1-mediated NFkappa B activation. The same expression levels of NFkappa B were shown in these clones by immunoblotting with antibodies against p65 and p50 (Fig. 3b). On the other hand, the reduced expression of Pellino 1 had no effect on tumor necrosis factor-induced NFkappa B activation, indicating that Pellino 1 is specifically required for IL-1-dependent signaling. We also examined the effect of reduced Pellino 1 expression on IL-1-induced IL-8 gene expression, which was greatly reduced in C-12 and C-16 as compared with C-9 (Fig. 3c), confirming that Pellino 1 is indeed required for this IL-1-mediated signaling pathway.


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Fig. 3.   Reduced expression of Pellino 1 impairs IL-1 signaling. a, screening of clones with reduced expression of Pellino 1. Total RNA from HEK293 cells stably transfected with Pellino 1-pSUPER was analyzed by Northern procedure with a Pellino1 gene-specific probe. B, NFkappa B gel-shift assay. Cell extracts from clones C-9, C-12, and C16, untreated or stimulated with IL-1 (2, 4, and 8 h) and tumor necrosis factor (2 h), were analyzed by NFkappa B gel shift assay as described under "Experimental Procedures." The same extracts were analyzed by immunoblotting with anti-p65 and anti-p50. C, Northern analysis. RNA samples from C-9, C-12, and C16, untreated or stimulated with IL-1 (4, 8, 12, 24 h), were analyzed by using IL-8 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs as probes.

IL-1-induced Pellino 1-IRAK4-IRAK-TRAF6 Is an Intermediate Complex between IL-1 Receptor and TAK1 Complex-- Previous biochemical and genetic studies are consistent with a model for IL-1-mediated signaling (12) (Fig. 2c). To address the molecular mechanism by which Pellino 1 functions in IL-1 signaling, we investigated whether Pellino 1 interacts with IRAK and IRAK4 in the receptor complex (Complex I) or the TAK1-containing Complex II. Cell extracts prepared from HEK293 cells transfected with FLAG-tagged Pellino 1, either untreated or stimulated with IL-1, were immunoprecipitated with anti-FLAG M2 (Fig. 2, a and b), anti-TRAF6 (Fig. 2b), and anti-TAK1 (data not shown) and anti-TAB2 (data not shown) antibodies followed by immunoblotting with antibodies against IRAK, TRAF6, IL-1 receptor, TAK1, and TAB2. As shown previously (12), TRAF6 is present both in the receptor complex (interacting with IL-1R and IRAK, Fig. 2, b and c) and the TAK1 complex (interacting with TAK1 and TAB2, Fig. 2, b and c). However, whereas Pellino 1 formed a complex with IRAK (Fig. 2, a and b), IRAK4 (Fig. 2a), and TRAF6 (Fig. 2, a and b), it co-immunoprecipitated with neither the IL-1 receptor (Fig. 2b) nor the TAK1 complex (TAK1 and TAB2; Fig. 2b, and data not shown) upon IL-1 stimulation, suggesting that the IL-1-induced Pellino 1-IRAK4-IRAK-TRAF6 is likely to exist as an intermediate between the receptor complex (Complex I) and the TAK1-containing complex (Complex II) (Fig. 2, a-c).

Pellino 1 Functions Downstream of IRAK-- Because most of the signaling components in the IL-1-mediated pathway are able to constitutively activate NFkappa B upon overexpression, Pellino 1 was examined for this activity. Pellino 1 in a mammalian expression vector was co-transfected with NFkappa B-dependent E-selectin-luc into HEK293 cells followed by luciferase reporter assay. Pellino 1 can activate the E-selectin promoter activity in a dose-dependent manner (Fig. 4a). We have previously taken a genetic approach to studying IL-1-dependent signaling pathways through random mutagenesis, generating IL-1-unresponsive cell lines lacking specific components of the pathways. Mutant cell line I1A lacks both the IRAK protein and mRNA and is defective in IL-1-induced NFkappa B and c-Jun NH2-terminal kinase activation (18, 18, 25). As shown in Fig. 4a, whereas MyD88-mediated NFkappa B activation is completely abolished in IRAK deficient I1A cells, Pellino 1-mediated NFkappa B activation is intact in these cells, indicating that Pellino 1 functions downstream of IRAK. On the other hand, Pellino 1-induced NFkappa B activation was inhibited by a dominant negative, kinase-inactive TAK1 mutant (TAK1 DN), indicating that Pellino1 must function upstream of TAK1 (Fig. 4b). In support of this conclusion, TAK1 can still activate NFkappa B in C-12, where Pellino 1 expression is greatly knocked down by RNA interference (Fig. 4c). Taken together, the above results support the hypothesis that the Pellino 1-IRAK-IRAK4-TRAF6 signaling complex functions between the receptor complex (Complex I) and the TAK1 complex (Complex II).


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Fig. 4.   Pellino 1 functions upstream of TAK1 and downstream of IRAK. a, Pellino 1 leads to NFkappa B activation in the IRAK-deficient I1A cells. FLAG-MyD88 (100 ng) and FLAG-Pellino 1 (100 ng) expression constructs were co-transfected with E-selectin-luc (100 ng) into HEK293 (WT, wild type) and the IRAK-deficient I1A cells, followed by luciferase reporter assay. The fold induction is relative to that in cells transfected with vector DNA (100 ng) and reporter DNA (100 ng). Shown are the averages and standard deviation from three independent experiments. b, kinase-dead TAK1 mutant (TAK1DN) inhibits Pellino 1-induced NFkappa B activation. Increasing amounts of TAK1DN were co-transfected with FLAG-Pellino 1 (0.5 µg) and E-selectin-luc (0.5 µg) into HEK293 cells, followed by luciferase reporter assay. The fold induction is relative to that in cells transfected with only vector (3.5 µg) and reporter DNA (0.5 µg). Shown are the averages and standard deviation from three independent experiments. c, TAK1 leads to NFkappa B activation in clones transfected with Pellino 1-pSUPER. TAK1 (0.5, 1, and 3 µg of DNA) expression construct was co-transfected with E-selectin-luc (100 ng) into C9 (with normal Pellino 1 expression) and C12 (with reduced Pellino 1 expression), followed by luciferase reporter assay. The fold induction is relative to that in cells transfected with vector DNA (3 µg) and reporter DNA (100 ng). Shown are the averages and standard deviation from three independent experiments.


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One possible function for Pellino 1 is linking TRAF6 to IRAK in IL-1 signaling. However, several lines of evidence suggest that Pellino 1 is unlikely to be involved in the interaction between IRAK and TRAF6. First, several TRAF6 binding sites have been identified in both human and murine IRAK, and mutations in these sites abolish the ability of IRAK to activate NFkappa B, suggesting that TRAF6 is likely to interact with IRAK directly through these sites (26). Secondly, although our previous results have clearly shown that TRAF6 is recruited to the IL-1 receptor through its interaction with IRAK upon IL-1 stimulation (Fig. 2, b and c) (12), Pellino 1 was not detected in the IL-1-induced receptor complex (IL-1R-IRAK-TRAF6, Complex I; Fig. 2, b and c). Therefore, it is unlikely that Pellino 1 is responsible for the IL-1-induced interaction between IRAK and TRAF6.

Previous studies have shown that the release of phosphorylated IRAK from the receptor is essential for mediating downstream signaling events in response to IL-1 (27). However, it is unclear how the phosphorylated IRAK is released from the receptor. The fact that Pellino 1 forms a complex with the phosphorylated IRAK but not with the IL-1 receptor upon IL-1 stimulation (Fig. 2, a and b) suggests that Pellino 1 may play an important role in facilitating the release of phosphorylated IRAK from the receptor.

It is important to note that Pellino 1 does interact with TRAF6 upon IL-1 stimulation, whereas it does not interact with TAK1, suggesting that Pellino 1 probably functions downstream of TRAF6 and upstream of TAK1. In support of this, a dominant negative mutant of TAK1 inhibits Pellino 1-mediated NFkappa B activation (Fig. 4b). The fact that Pellino 1 can activate NFkappa B in IRAK-deficient cells (I1A) suggests that Pellino 1 may have a direct impact on the activation of TAK1. As a matter of fact, TAK1 is activated in I1A cells transfected with Pellino 1 (data not shown). It is quite possible that Pellino 1 has a transient interaction with the TAK1 complex, which could not be detected by the co-immunoprecipitation experiments (Fig. 2c).

We have previously proposed a model for the IL-1 pathway (12). Taking the above findings of Pellino 1 into consideration, we now propose a modified model (Fig. 2c). Upon IL-1 stimulation, adapter molecules MyD88 and Tollip are first recruited to the IL-1 receptor, which in turn recruits IRAK4, IRAK, and TRAF6, resulting in the formation of the receptor complex (Complex 1). During the formation of Complex I, IRAK4 is activated, leading to the hyperphosphorylation of IRAK, which creates an interface for the interaction of Pellino 1 with the IRAK4-IRAK-TRAF6 complex. The formation of Pellino 1-IRAK4-IRAK-TRAF6 causes conformational changes in the receptor complex (Complex I), which results in quick release of this intermediate complex (Pellino 1-IRAK4-IRAK-TRAF6) from the receptor. Through an unknown mechanism, the intermediate complex Pellino 1-IRAK4-IRAK-TRAF6 interacts with the membrane-bound pre-associated TAK1-TAB1-TAB2, resulting in the formation of Complex II (TAK1 complex, IRAK-TRAF6-TAK1-TAB1-TAB2). TRAF6-TAK1-TAB1-TAB2 (Complex III) is then translocated from the membrane to the cytosol where TAK1 is activated, eventually resulting in the activation of NFkappa B.

Although this study has shown the critical role of Pellino 1 in IL-1-mediated signaling, it was recently reported that a related protein, Pellino 2, modulates IL-1 and lipopolysaccharide (LPS) signaling (28). At present, the relationship between Pellino 1 and Pellino 2 and their specific functions in IL-1 signaling are still unclear. One possibility is that each Pellino has its specific interaction with IRAK and/or IRAK4. The elucidation of precise functions of Pellino proteins in IL-1/TLR-mediated signaling will be an exciting and important research area in the future.

    ACKNOWLEDGEMENTS

We thank Dr. Hoger Wesche for the IRAK4 antibodies and Drs. Jun Ninomiya-Tsuji and Kunihiro Matsumoto for dominant negative mutant TAK1 and antibodies against TAK1 and TAB2.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM 600020 (to X. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Immunology, Lerner Research Inst., Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-8706; Fax: 216-444-9329; E-mail: Lix@ccf.org.

Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M212112200

    ABBREVIATIONS

The abbreviations used are: IL-1, interleukin-1; IL-1R, IL-1 receptor; TLR, Toll-like receptor; NFkappa B, nuclear factor kappa B; IRAK, IL1R-associated kinase; TRAF, tumor necrosis factor receptor-associated factor; TAK, transforming growth factor beta -activated kinase; TAB, TAK-1-binding protein; Pellino 1, mammalian Pellino; HEK293, human embryonic kidney 293.

    REFERENCES
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INTRODUCTION
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REFERENCES

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