A novel splice variant of interleukin-1 receptor (IL-1R)-associated kinase 1 plays a negative regulatory role in Toll/IL-1R-induced inflammatory signaling

The interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK1) is a member of the IRAK kinase family that plays a pivotal role in the Toll/IL-1 receptor (TIR) family signaling cascade. We have identified a novel splice variant, IRAK1c, which lacks a region encoded by exon 11 of the IRAK1 gene. IRAK1c expression was confirmed by both RNA and protein detection. Although both IRAK1 and IRAK1c are expressed in most tissues tested, IRAK1c is the predominant form of IRAK1 expressed in the brain. Unlike IRAK1, IRAK1c lacks kinase activity and cannot be phosphorylated by IRAK4. However, IRAK1c retains the ability to strongly interact with IRAK2, MyD88, Tollip, and TRAF6. Overexpression of IRAK1c suppressed NF-kappaB activation and blocked IL-1beta-induced IL-6 as well as lipopolysaccharide- and CpG-induced tumor necrosis factor alpha production in multiple cellular systems. Mechanistically, we provide evidence that IRAK1c functions as a dominant negative by failing to be phosphorylated by IRAK4, thus remaining associated with Tollip and blocking NF-kappaB activation. The presence of a regulated, alternative splice variant of IRAK1 that functions as a kinase-dead, dominant-negative protein adds further complexity to the variety of mechanisms that regulate TIR signaling and the subsequent inflammatory response.     

Summary and comparison of the signaling mechanisms of the Toll/interleukin-1 receptor family

The Toll/interleukin-1 (IL-1) receptor (TIR) family comprises two groups of transmembrane proteins, which share functional and structural properties. The members of the IL-1 receptor (IL-1R) subfamily are characterized by three extracellular immunoglobulin (Ig)-like domains. They form heterodimeric signaling receptor complexes consisting of receptor and accessory proteins. The members of the Toll-like receptor (TLR) subfamily recognize alarm signals that can be derived either from pathogens or the host itself. TLRs possess leucine-rich repeats in their extracellular part. TLRs can form dimeric receptor complexes consisting of two different TLRs or homodimers in the case of TLR4. The TLR4 receptor complex requires supportive molecules for optimal response to its ligand lipopolysaccharide (LPS). A hallmark of the TIR family is the cytoplasmic TIR domain that is indispensable for signal transduction. The TIR domain serves as a scaffold for a series of protein-protein interactions which result in the activation of a unique signaling module consisting of MyD88, interleukin-1 receptor associated kinase (IRAK) family members and Tollip, which is used exclusively by TIR family members. Subsequently, several central signaling pathways are activated in parallel, the activation of NFkappaB being the most prominent event of the inflammatory response. Recent developments indicate that in addition to the common signaling module MyD88/IRAK/Tollip, other molecules can modulate signaling by TLRs, especially of TLR4, resulting in differential biological answers to distinct pathogenic structures. Subtle differences in TLR signaling pathways are now becoming apparent, which reveal how the innate immune system decides at a very early stage the direction in which the adaptive immune response will develop. The creation of pathogen-specific mediator environments by dendritic cells defines whether a cellular or humoral response will be activated in response to the pathogen.     

TLR8-mediated NF-kappaB and JNK activation are TAK1-independent and MEKK3-dependent

TLR8-mediated NF-kappaB and IRF7 activation are abolished in human IRAK-deficient 293 cells and IRAK4-deficient fibroblast cells. Both wild-type and kinase-inactive mutants of IRAK and IRAK4, respectively, restored TLR8-mediated NF-kappaB and IRF7 activation in the IRAK- and IRAK4-deficient cells, indicating that the kinase activity of IRAK and IRAK4 is probably redundant for TLR8-mediated signaling. We recently found that TLR8 mediates a unique NF-kappaB activation pathway in human 293 cells and mouse embryonic fibroblasts, accompanied only by IkappaBalpha phosphorylation and not IkappaBalpha degradation, whereas interleukin (IL)-1 stimulation causes both IkappaBalpha phosphorylation and degradation. The intermediate signaling events mediated by IL-1 (including IRAK modifications and degradation and TAK1 activation) were not detected in cells stimulated by TLR8 ligands. TLR8 ligands trigger similar levels of IkappaBalpha phosphorylation and NF-kappaB and JNK activation in TAK1(-/-) mouse embryo fibroblasts (MEFs) as compared with wild-type MEFs, whereas lack of TAK1 results in reduced IL-1-mediated NF-kappaB activation and abolished IL-1-induced JNK activation. The above results indicate that although TLR8-mediated NF-kappaB and JNK activation are IRAK-dependent, they do not require IRAK modification and are TAK1-independent. On the other hand, TLR8-mediated IkappaBalpha phosphorylation, NF-kappaB, and JNK activation are completely abolished in MEKK3(-/-) MEFs, whereas IL-1-mediated signaling was only moderately reduced in these deficient MEFs as compared with wild-type cells. The differences between IL-1R- and TLR8-mediated NF-kappaB activation are also reflected at the level of IkappaB kinase (IKK) complex. TLR8 ligands induced IKKgamma phosphorylation, whereas IKKalpha/beta phosphorylation and IKKgamma ubiquitination that can be induced by IL-1 were not detected in cells treated with TLR8 ligands. We postulate that TLR8-mediated MEKK3-dependent IKKgamma phosphorylation might play an important role in the activation of IKK complex, leading to IkappaBalpha phosphorylation.     

Protective effects of the notoginsenoside R1 on acute lung injury by regulating the miR-128-2-5p/Tollip signaling pathway in rats with severe acute pancreatitis

Notoginsenoside R1 (NG-R1), the extract and the main ingredient of Panax notoginseng, has anti-inflammatory effects and can be used in treating acute lung injury (ALI). In this study, we explored the pulmonary protective effect and the underlying mechanism of the NG-R1 on rats with ALI induced by severe acute pancreatitis (SAP). MiR-128-2-5p, ERK1, Tollip, HMGB1, TLR4, IκB, and NF-κB mRNA expression levels were measured using real-time qPCR, and TLR4, Tollip, HMGB1, IRAK1, MyD88, ERK1, NF-κB65, and P-IκB-α protein expression levels using Western blot. The NF-κB and the TLR4 activities were determined using immunohistochemistry, and TNF-α, IL-6, IL-1β, and ICAM-1 levels in the bronchoalveolar lavage fluid (BALF) using ELISA. Lung histopathological changes were observed in each group. NG-R1 treatment reduced miR-128-2-5p expression in the lung tissue, increased Tollip expression, inhibited HMGB1, TLR4, TRAF6, IRAK1, MyD88, NF-κB65, and p-IκB-α expression levels, suppressed NF-κB65 and the TLR4 expression levels, reduced MPO activity, reduced TNF-α, IL-1β, IL-6, and ICAM-1 levels in BALF, and alleviated SAP-induced ALI. NG-R1 can attenuate SAP-induced ALI. The mechanism of action may be due to a decreased expression of miR-128-2-5p, increased activity of the Tollip signaling pathway, decreased activity of HMGB1/TLR4 and ERK1 signaling pathways, and decreased inflammatory response to SAP-induced ALI. Tollip was the regulatory target of miR-128-2-5p.     

Cooperation of Toll-like receptor 2 and 6 for cellular activation by soluble tuberculosis factor and Borrelia burgdorferi outer surface protein A lipoprotein: role of Toll-interacting protein and IL-1 receptor signaling molecules in Toll-like receptor 2 signaling.

Toll-like receptor 2 (TLR2) and TLR4 play important roles in innate immune responses to various microbial agents. We have previously shown that human dermal endothelial cells (HMEC) express TLR4, but very little TLR2, and respond to LPS, but not to Mycobacterium tuberculosis 19-kDa lipoprotein, unless transfected with TLR2. Here we report that HMEC are unresponsive to several additional biologically relevant TLR2 ligands, including, phenol-soluble modulin (PSM), a complex of three small secreted polypeptides from the skin commensal Staphylococcus epidermidis, soluble tuberculosis factor (STF), and Borrelia burgdorferi outer surface protein A lipoprotein (OspA-L). Expression of TLR2 renders HMEC responsive to all these ligands. We further characterized the signaling pathway in response to STF, OspA-L, and PSM in TLR2-transfected HMEC. The TLR2 signaling pathway for NF-kappaB trans-activation shares the IL-1R signaling molecules. Dominant negative constructs of TLR2 or TLR6 inhibit the responses of STF and OspA-L as well as PSM in TLR2-transfected HMEC, supporting the concept of functional cooperation between TLR2 and TLR6 for all these TLR2 ligands. Moreover, we show that Toll-interacting protein (Tollip) coimmunoprecipitates with TLR2 and TLR4 using HEK 293 cells, and overexpression of Tollip inhibits NF-kappaB activation in response to TLR2 and TLR4 signaling. Collectively, these findings suggest that there is functional interaction between TLR2 and TLR6 in the cellular response to STF and OspA-L in addition to S. epidermidis (PSM) Ags, and that engagement of TLR2 triggers a signaling cascade, which shares the IL-1R signaling molecules, similar to the TLR4-LPS signaling cascade. Our data also suggest that Tollip may be an important constituent of both the TLR2 and TLR4 signaling pathways.     

Immune complex-mediated cell activation from systemic lupus erythematosus and rheumatoid arthritis patients elaborate different requirements for IRAK1/4 kinase activity across human cell types

IL-1R-associated kinases (IRAKs) are important mediators of MyD88-dependent signaling by the TLR/IL-1R superfamily and facilitate inflammatory responses. IRAK4 and IRAK1 function as active kinases and as scaffolds for protein-protein interactions. We report that although IRAK1/4 kinase activity is essential for human plasmacytoid dendritic cell (pDC) activation, it is dispensable in B, T, dendritic, and monocytic cells, which is in contrast with an essential active kinase role in comparable mouse cell types. An IRAK1/4 kinase inhibitor abrogated TLR7/9-induced IFN-α responses in both mouse and human pDCs, but other human immune cell populations activated via TLR7/9 or IL-1R were refractory to IRAK4 kinase inhibition. Gene ablation experiments using small interfering RNA demonstrated an essential scaffolding role for IRAK1 and IRAK4 in MyD88-dependent signaling. Finally, we demonstrate that autoimmune patient (systemic lupus erythematosus and rheumatoid arthritis) serum activates both pDC and B cells, but IRAK1/4 kinase inhibition affects only the pDC response, underscoring the differential IRAK1/4 functional requirements in human immune cells. These data reveal important species differences and elaborate cell type requirements for IRAK1/4 kinase activity.     

Signaling events induced by lipopolysaccharide-activated toll-like receptor 2.

Human Toll-like receptor 2 (TLR2) is a signaling receptor that responds to LPS and activates NF-kappaB. Here, we investigate further the events triggered by TLR2 in response to LPS. We show that TLR2 associates with the high-affinity LPS binding protein membrane CD14 to serve as an LPS receptor complex, and that LPS treatment enhances the oligomerization of TLR2. Concomitant with receptor oligomerization, the IL-1R-associated kinase (IRAK) is recruited to the TLR2 complex. Intracellular deletion variants of TLR2 lacking C-terminal 13 or 141 aa fail to recruit IRAK, which is consistent with the inability of these mutants to transmit LPS cellular signaling. Moreover, both deletion mutants could still form complexes with wild-type TLR2 and act in a dominant-negative (DN) fashion to block TLR2-mediated signal transduction. DN constructs of myeloid differentiation protein, IRAK, TNF receptor-associated factor 6, and NF-kappaB-inducing kinase, when coexpressed with TLR2, abrogate TLR2-mediated NF-kappaB activation. These results reveal a conserved signaling pathway for TLR2 and IL-1Rs and suggest a molecular mechanism for the inhibition of TLR2 by DN variants.     

The critical role of IL-1 receptor-associated kinase 4-mediated NF-κB activation in modified low-density lipoprotein-induced inflammatory gene expression and atherosclerosis

Exciting discoveries related to IL-1R/TLR signaling in the development of atherosclerosis plaque have triggered intense interest in the molecular mechanisms by which innate immune signaling modulates the onset and development of atherosclerosis. Previous studies have clearly shown the definitive role of proinflammatory cytokine IL-1 in the development of atherosclerosis. Recent studies have provided direct evidence supporting a link between innate immunity and atherogenesis. Although it is still controversial about whether infectious pathogens contribute to cardiovascular diseases, direct genetic evidence indicates the importance of IL-1R/TLR signaling in atherogenesis. In this study, we examined the role of IL-1R-associated kinase 4 (IRAK4) kinase activity in modified low-density lipoprotein (LDL)-mediated signaling using bone marrow-derived macrophage as well as an in vivo model of atherosclerosis. First, we found that the IRAK4 kinase activity was required for modified LDL-induced NF-κB activation and expression of a subset of proinflammatory genes but not for the activation of MAPKs in bone marrow-derived macrophage. IRAK4 kinase-inactive knockin (IRAK4KI) mice were bred onto ApoE(-/-) mice to generate IRAK4KI/ApoE(-/-) mice. Importantly, the aortic sinus lesion formation was impaired in IRAK4KI/ApoE(-/-) mice compared with that in ApoE(-/-) mice. Furthermore, proinflammatory cytokine production was reduced in the aortic sinus region of IRAK4KI/ApoE(-/-) mice compared with that in ApoE(-/-) mice. Taken together, our results indicate that the IRAK4 kinase plays an important role in modified LDL-mediated signaling and the development of atherosclerosis, suggesting that pharmacological inhibition of IRAK4 kinase activity might be a feasible approach in the development of antiatherosclerosis drugs.     

Tollip regulates proinflammatory responses to interleukin-1 and lipopolysaccharide

Activation of interleukin-1 (IL-1) receptor (IL-1R), Toll-like receptor 2 (TLR2), and TLR4 triggers NF-kappaB and mitogen-activated protein kinase (MAPK)-dependent signaling, thereby initiating immune responses. Tollip has been implicated as a negative regulator of NF-kappaB signaling triggered by these receptors in in vitro studies. Here, deficient mice were used to determine the physiological contribution of Tollip to immunity. NF-kappaB, as well as MAPK, signaling appeared normal in Tollip-deficient cells stimulated with IL-1beta or the TLR4 ligand lipopolysaccharide (LPS). Similarly, IL-1beta- and TLR-driven activation of dendritic cells and lymphocytes was indistinguishable from wild-type cells. In contrast, the production of the proinflammatory cytokines, IL-6 and tumor necrosis factor alpha was significantly reduced after IL-1beta and LPS treatment at low doses but not at lethal doses of LPS. Tollip therefore controls the magnitude of inflammatory cytokine production in response to IL-1beta and LPS.     

Regulation of interleukin-1- and lipopolysaccharide-induced NF-kappaB activation by alternative splicing of MyD88

MyD88 is an adaptor protein that is involved in interleukin-1 receptor (IL-1R)- and Toll-like receptor (TLR)-induced activation of NF-kappaB. It is composed of a C-terminal Toll/IL-1R homology (TIR) domain and an N-terminal death domain (DD), which mediate the interaction of MyD88 with the IL-1R/TLR and the IL-1R-associated kinase (IRAK), respectively. The interaction of MyD88 with IRAK triggers IRAK phosphorylation, which is essential for its activation and downstream signaling ability. Both domains of MyD88 are separated by a small intermediate domain (ID) of unknown function. Here, we report the identification of a splice variant of MyD88, termed MyD88(S), which encodes for a protein lacking the ID. MyD88(S) is mainly expressed in the spleen and can be induced in monocytes upon LPS treatment. Although MyD88(S) still binds the IL-1R and IRAK, it is defective in its ability to induce IRAK phosphorylation and NF-kappaB activation. In contrast, MyD88(S) behaves as a dominant-negative inhibitor of IL-1- and LPS-, but not TNF-induced, NF-kappaB activation. These results implicate the ID of MyD88 in the phosphorylation of IRAK. Moreover, the regulated expression and antagonistic activity of MyD88(S) suggest an important role for alternative splicing of MyD88 in the regulation of the cellular response to IL-1 and LPS.     

Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes

Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-kappaB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-kappaB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-kappaB-luciferase activity. LPS-induced NF-kappaB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-kappaB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells.     

Poly(I-C)-induced Toll-like receptor 3 (TLR3)-mediated activation of NFkappa B and MAP kinase is through an interleukin-1 receptor-associated kinase (IRAK)-independent pathway employing the signaling components TLR3-TRAF6-TAK1-TAB2-PKR

Recent studies show that a member of the interleukin-1 (IL-1)/Toll receptor superfamily, Toll-like receptor 3 (TLR3), recognizes double-stranded RNA (dsRNA). Because of the similarity in their cytoplasmic domains, IL-1/Toll receptors share signaling components that associate with the IL-1 receptor, including IL-1 receptor-associated kinase (IRAK), MyD88, and TRAF6. However, we find that, in response to dsRNA, TLR3 can mediate the activation of both NFkappaB and mitogen-activated protein (MAP) kinases in IL-1-unresponsive mutant cell lines, including IRAK-deficient I1A and I3A cells, which are defective in a component that is downstream of IL-1R but upstream of IRAK. These results clearly indicate that TLR3 does not simply share the signaling components employed by the IL-1 receptor. Through biochemical analyses we have identified an IRAK-independent TLR3-mediated pathway. Upon binding of dsRNA to TLR3, TRAF6, TAK1, and TAB2 are recruited to the receptor to form a complex, which then translocates to the cytosol where TAK1 is phosphorylated and activated. The dsRNA-dependent protein kinase (PKR) is also detected in this signal-induced TAK1 complex. Kinase inactive mutants of TAK1 (TAK1DN) and PKR (PKRDN) inhibit poly(dI.dC)-induced TLR3-mediated NFkappaB activation, suggesting that both of these kinases play important roles in this pathway.     

IL-1 receptor-associated kinase 4 is essential for IL-18-mediated NK and Th1 cell responses

IL-18 is an important cytokine for both innate and adaptive immunity. NK T cells and Th1 cells depend on IL-18 for their divergent functions. The IL-18R, IL-1R, and mammalian Toll-like receptors (TLRs) share homologous intracellular domains known as the TLR/IL-1R/plant R domain. Previously, we reported that IL-1R-associated kinase (IRAK)-4 plays a critical role in IL-1R and TLR signaling cascades and is essential for the innate immune response. Because TLR/IL-1R/plant R-containing receptors mediate signal transduction in a similar fashion, we investigated the role of IRAK-4 in IL-18R signaling. In this study, we show that IL-18-induced responses such as NK cell activity, Th1 IFN-gamma production, and Th1 cell proliferation are severely impaired in IRAK-4-deficient mice. IRAK-4(-/-) Th1 cells also do not exhibit NF-kappaB activation or IkappaB degradation in response to IL-18. Moreover, AP-1 activation which is triggered by c-Jun N-terminal kinase activation is also completely inhibited in IRAK-4(-/-) Th1 cells. These results suggest that IRAK-4 is an essential component of the IL-18 signaling cascade.     

Two human MYD88 variants, S34Y and R98C, interfere with MyD88-IRAK4-myddosome assembly

Innate immune receptors detect microbial pathogens and subsequently activate adaptive immune responses to combat pathogen invasion. MyD88 is a key adaptor molecule in both Toll-like receptor (TLR) and IL-1 receptor superfamily signaling pathways. This is illustrated by the fact that human individuals carrying rare, naturally occurring MYD88 point mutations suffer from reoccurring life-threatening infections. Here we analyzed the functional properties of six reported non-synonymous single nucleotide polymorphisms of MYD88 in an in vitro cellular system. Two variants found in the MyD88 death domain, S34Y and R98C, showed severely reduced NF-κB activation due to reduced homo-oligomerization and IRAK4 interaction. Structural modeling highlights Ser-34 and Arg-98 as residues important for the assembly of the Myddosome, a death domain (DD) post-receptor complex involving the DD of MyD88, IRAK4, and IRAK2 or IRAK1. Using S34Y and R98C as functional probes, our data show that MyD88 homo-oligomerization and IRAK4 interaction is modulated by the MyD88 TIR and IRAK4 kinase domain, demonstrating the functional importance of non-DD regions not observed in a recent Myddosome crystal structure. The differential interference of S34Y and R98C with some (IL-1 receptor, TLR2, TLR4, TLR5, and TLR7) but not all (TLR9) MyD88-dependent signaling pathways also suggests that receptor specificities exist at the level of the Myddosome. Given their detrimental effect on signaling, it is not surprising that our epidemiological analysis in several case-control studies confirms that S34Y and R98C are rare variants that may drastically contribute to susceptibility to infection in only few individuals.     

MicroRNA-27a Negatively Modulates the Inflammatory Response in Lipopolysaccharide-Stimulated Microglia by Targeting TLR4 and IRAK4

microRNA, a family of small non-coding RNA, plays significant roles in regulating gene expression, mainly via binding to the 3′-untranslated region of target genes. Although the role of miRNA in regulating neuroinflammation via the innate immune pathway has been studied, its role in the production of inflammatory mediators during microglial activation is poorly understood. In this study, we investigated the effect of miR-27a on lipopolysaccharide (LPS)-induced microglial inflammation. miR-27a expression was found to be rapidly decreased in microglia by real-time polymerase chain reaction (real-time PCR) after LPS stimulation. Over-expression of miR-27a significantly decreased the production of inflammatory cytokines, such as interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and nitric oxide (NO), whereas knockdown of miR-27a increased the expression of these inflammatory factors. We also demonstrated by loss- and gain-of-function studies that miR-27a directly suppressed the expression of toll-like receptor 4 (TLR4) and interleukin-1 receptor-associated kinase 4 (IRAK4)—a pivotal adaptor kinase in the TLR4/MyD88 signaling pathway—by directly binding their 3′-UTRs: knocking down TLR4 or IRAK4 in microglia significantly decreased TLR4 or IRAK4 expression and inhibited the downstream production of inflammatory mediators. Moreover, the inflammatory cytokines IL-6 and IL-1β were regulated by IRAK4, whereas TNF-α and NO were more dependent on TLR4 activation. Thus, miR-27a might regulate the LPS-induced production of inflammatory cytokines in microglia independently of TLR4 and IRAK4. Taken together, our results suggest that miR-27a is associated with microglial activation and the inflammatory response.     

Syntenin negatively regulates TRAF6-mediated IL-1R/TLR4 signaling

Toll-like receptors are involved in host defense against invading pathogens. The two members of this superfamily, IL-1R and TLR4, activate overlapping NF-kappaB activate signaling pathway mediated by TRAF6. In this study, we identified syntenin as a negative regulator of IL-1R and TLR4 mediated NF-kappaB activation. Overexpressed syntenin inhibited IL-1- or LPS-, but not TNF- induced NF-kappaB activation and IL-8 mRNA expression in a dose dependent manner. Syntenin specifically interacted with TRAF6 in human 293 cells, and inhibited TRAF6 induced NF-kappaB and AP-1 activation. Syntenin also associated with TRAF6 under physiological condition, and dissociated from TRAF6 upon IL-1 stimulation. This might be due to a competition between syntenin and IRAK1, as overexpression of IRAK1 disrupted the interaction of syntenin with TRAF6, and rescued syntenin induced reduction of TRAF6 ubiquitination. Moreover, knockdown of syntenin potentiated IL-1- or LPS- triggered NF-kappaB activation and IL-8 mRNA expression. These findings suggest that syntenin is a physiological suppressor of TRAF6 and plays an inhibitory role in IL-1R- and TLR4- mediated NF-kappaB activation pathways.     

FADD negatively regulates lipopolysaccharide signaling by impairing interleukin-1 receptor-associated kinase 1-MyD88 interaction

Lipopolysaccharide (LPS) engages Toll-like receptor 4 (TLR4) on various cells to initiate inflammatory and angiogenic pathways. FADD is an adaptor protein involved in death receptor-mediated apoptosis. Here we report a role for FADD in regulation of TLR4 signals in endothelial cells. FADD specifically attenuates LPS-induced activation of c-Jun NH(2)-terminal kinase and phosphatidylinositol 3'-kinase in a death domain-dependent manner. In contrast, FADD-null cells show hyperactivation of these kinases. Examining physical associations of endogenous proteins, we show that FADD interacts with interleukin-1 receptor-associated kinase 1 (IRAK1) and MyD88. LPS stimulation increases IRAK1-FADD interaction and recruitment of the IRAK1-FADD complex to activated MyD88. IRAK1 is required for FADD-MyD88 interaction, as FADD does not associate with MyD88 in IRAK1-null cells. By shuttling FADD to MyD88, IRAK1 provides a mechanism for controlled and limited activation of the TLR4 signaling pathway. Functionally, enforced FADD expression inhibited LPS- but not vascular endothelial growth factor-induced endothelial cell sprouting, while FADD deficiency led to enhanced production of proinflammatory cytokines induced by stimulation of TLR4 and TLR2, but not TLR3. Reconstitution of FADD reversed the enhanced production of proinflammatory cytokines. Thus, FADD is a physiological negative regulator of IRAK1/MyD88-dependent responses in innate immune signaling.