Glutaredoxin-1 regulates TRAF6 activation and the IL-1 receptor/TLR4 signalling

Glutaredoxin-1 (GRX-1) is a cytoplasmic enzyme that highly contributes to the antioxidant defense system. It catalyzes the reversible reduction of glutathione-protein mixed disulfides, a process called deglutathionylation. Here, we investigated the role of GRX-1 in the pathway triggered by interleukin-1/Toll-like receptor 4 (IL-1R/TLR4) by using RNA interference (RNAi) in HEK293 and HeLa cells. TNF receptor-associated factor 6 (TRAF6) is an intermediate signalling molecule involved in the signal transduction by members of the interleukin-1/Toll-like receptor (IL-1R/TLR) family. TRAF6 has an E3 ubiquitin ligase activity which depends on the integrity of an amino-terminal really interesting new gene (RING) finger motif. Upon receptor activation, TRAF6 undergoes K63-linked auto-polyubiquitination which mediates protein-protein interactions and signal propagation. Our data showed that IL-1R and TLR4-mediated NF-κB induction was severely reduced in GRX-1 knockdown cells. We found that the RING-finger motif of TRAF6 is S-glutathionylated under normal conditions. Moreover, upon IL-1 stimulation TRAF6 undergoes deglutathionylation catalyzed by GRX-1. The deglutathionylation of TRAF6 is essential for its auto-polyubiquitination and subsequent activation. Taken together, our findings reveal another signalling molecule affected by S-glutathionylation and uncover a crucial role for GRX-1 in the TRAF6-dependent activation of NF-κB by IL-1R/TLRs.     

PECAM-1 ligation negatively regulates TLR4 signaling in macrophages

Uncontrolled TLR4 signaling may induce excessive production of proinflammatory cytokines and lead to harmful inflammation; therefore, negative regulation of TLR4 signaling attracts much attention now. PECAM-1, a member of Ig-ITIM family, can mediate inhibitory signals in T cells and B cells. However, the role and the mechanisms of PECAM-1 in the regulation of TLR4-mediated LPS response in macrophages remain unclear. In this study, we demonstrate that PECAM-1 ligation with CD38-Fc fusion protein negatively regulates LPS-induced proinflammatory cytokine TNF-alpha, IL-6, and IFN-beta production by inhibiting JNK, NF-kappaB, and IFN regulatory factor 3 activation in macrophages. In addition, PECAM-1 ligation-recruited Src homology region 2 domain-containing phosphatase 1 (SHP-1) and Src homology region 2 domain-containing phosphatase 2 (SHP-2) may be involved in the inhibitory effect of PECAM-1 on TLR4 signaling. Consistently, silencing of PECAM-1 enhances the macrophage response to LPS stimulation. Taken together with the data that PECAM-1 is constitutively expressed in macrophages and its expression is up-regulated by LPS stimulation, PECAM-1 might function as a feedback negative regulator of LPS inflammatory response in macrophages. This study may provide a potential target for intervention of inflammatory diseases.     

Procyanidin dimer B2-mediated IRAK-M induction negatively regulates TLR4 signaling in macrophages

Polyphenolic compounds have been found to possess a wide range of physiological activities that may contribute to their beneficial effects against inflammation-related diseases; however, the molecular mechanisms underlying this anti-inflammatory activity are not completely characterized, and many features remain to be elucidated. In this study, we investigated the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by procyanidin dimer B2 (Pro B2) in macrophages. Pro B2 markedly elevated the expression of the interleukin (IL)-1 receptor-associated kinase (IRAK)-M protein, a negative regulator of TLR signaling. Lipopolysaccharide (LPS)-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II) and production of pro-inflammatory cytokines (tumor necrosis factor-α, IL-1β, IL-6, and IL-12p70) were inhibited by Pro B2, and this action was prevented by IRAK-M silencing. In addition, Pro B2-treated macrophages inhibited LPS-induced activation of mitogen-activated protein kinases such as extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase and the translocation of nuclear factor κB and p65 through IRAK-M. We also found that Pro B2-treated macrophages inactivated naïve T cells by inhibiting LPS-induced interferon-γ and IL-2 secretion through IRAK-M. These novel findings provide new insights into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and the immune-pharmacological role of Pro B2 in the immune response against the development and progression of many chronic diseases.     

Protein phosphatase 4 negatively regulates LPS cascade by inhibiting ubiquitination of TRAF6

TRAF6 is an E3 ubiquitin ligase that transduces signals from members of the TLR/IL-1R family. Multiple molecules have been found to associate with TRAF6 and exert their functions in this pathway. Herein, by yeast two-hybrid screen using TRAF6 as bait, we identified PP4 as a potential TRAF6-interacting protein. PP4 physically interacted with TRAF6 and was recruited to TLR4 complex upon LPS stimulation. PP4 negatively regulated LPS-induced and TRAF6-mediated NF-kappaB activation by inhibiting the ubiquitination of TRAF6. LPS stimulation also induced the expression of PP4. Taken together, our findings suggest that PP4 is a negative feedback regulator of LPS/TLR4 pathway.     

USP15 negatively regulates lung cancer progression through the TRAF6-BECN1 signaling axis for autophagy induction

TNF receptor-associated factor 6 (TRAF6)-BECN1 signaling axis plays a pivotal role in autophagy induction through ubiquitination of BECN1, thereby inducing lung cancer migration and invasion in response to toll-like receptor 4 (TLR4) stimulation. Herein, we provide novel molecular and cellular mechanisms involved in the negative effect of ubiquitin-specific peptidase 15 (USP15) on lung cancer progression. Clinical data of the TCGA and primary non-small cell lung cancer (NSCLC) patients (n = 41) revealed that the expression of USP15 was significantly downregulated in lung cancer patients. Importantly, USP15-knockout (USP15KO) A549 and USP15KO H1299 lung cancer cells generated with CRISPR-Cas9 gene-editing technology showed increases in cancer migration and invasion with enhanced autophagy induction in response to TLR4 stimulation. In addition, biochemical studies revealed that USP15 interacted with BECN1, but not with TRAF6, and induced deubiquitination of BECN1, thereby attenuating autophagy induction. Notably, in primary NSCLC patients (n = 4) with low expression of USP15, 10 genes (CCNE1, MMP9, SFN, UBE2C, CCR2, FAM83A, ETV4, MYO7A, MMP11, and GSDMB) known to promote lung cancer progression were significantly upregulated, whereas 10 tumor suppressor genes (FMO2, ZBTB16, FCN3, TCF21, SFTPA1B, HPGD, SOSTDC1, TMEM100, GDF10, and WIF1) were downregulated, providing clinical relevance of the functional role of USP15 in lung cancer progression. Taken together, our data demonstrate that USP15 can negatively regulate the TRAF6-BECN1 signaling axis for autophagy induction. Thus, USP15 is implicated in lung cancer progression.Subject terms: Non-small-cell lung cancer, Cell invasion     

CKIP-1 regulates macrophage proliferation by inhibiting TRAF6-mediated Akt activation

Macrophages play pivotal roles in development, homeostasis, tissue repair and immunity. Macrophage proliferation is promoted by macrophage colony-stimulating factor (M-CSF)-induced Akt signaling; yet, how this process is terminated remains unclear. Here, we identify casein kinase 2-interacting protein-1 (CKIP-1) as a novel inhibitor of macrophage proliferation. In resting macrophages, CKIP-1 was phosphorylated at Serine 342 by constitutively active GSK3β, the downstream target of Akt. This phosphorylation triggers the polyubiquitination and proteasomal degradation of CKIP-1. Upon M-CSF stimulation, Akt is activated by CSF-1R-PI3K and then inactivates GSK3β, leading to the stabilization of CKIP-1 and β-catenin proteins. β-catenin promotes the expression of proliferation genes including cyclin D and c-Myc. CKIP-1 interacts with TRAF6, a ubiquitin ligase required for K63-linked ubiquitination and plasma membrane recruitment of Akt, and terminates TRAF6-mediated Akt activation. By this means, CKIP-1 inhibits macrophage proliferation specifically at the late stage after M-CSF stimulation. Furthermore, CKIP-1 deficiency results in increased proliferation and decreased apoptosis of macrophages in vitro and CKIP-1^−/− mice spontaneously develop a macrophage-dominated splenomegaly and myeloproliferation. Together, these data demonstrate that CKIP-1 plays a critical role in the regulation of macrophage homeostasis by inhibiting TRAF6-mediated Akt activation.     

Cigarette smoke-induced pulmonary inflammation is TLR4/MyD88 and IL-1R1/MyD88 signaling dependent

Acute cigarette smoke exposure of the airways (two cigarettes twice daily for three days) induces acute inflammation in mice. In this study, we show that airway inflammation is dependent on Toll-like receptor 4 and IL-1R1 signaling. Cigarette smoke induced a significant recruitment of neutrophils in the bronchoalveolar space and pulmonary parenchyma, which was reduced in TLR4-, MyD88-, and IL-1R1-deficient mice. Diminished neutrophil influx was associated with reduced IL-1, IL-6, and keratinocyte-derived chemokine levels and matrix metalloproteinase-9 activity in the bronchoalveolar space. Further, cigarette smoke condensate (CSC) induced a macrophage proinflammatory response in vitro, which was dependent on MyD88, IL-1R1, and TLR4 signaling, but not attributable to LPS. Heat shock protein 70, a known TLR4 agonist, was induced in the airways upon smoke exposure, which probably activates the innate immune system via TLR4/MyD88, resulting in airway inflammation. CSC-activated macrophages released mature IL-1beta only in presence of ATP, whereas CSC alone promoted the TLR4/MyD88 signaling dependent production of IL-1alpha and pro-IL-1beta implicating cooperation between TLRs and the inflammasome. In conclusion, acute cigarette exposure results in LPS-independent TLR4 activation, leading to IL-1 production and IL-1R1 signaling, which is crucial for cigarette smoke induced inflammation leading to chronic obstructive pulmonary disease with emphysema.     

Targeting TLR4 signaling by TLR4 Toll/IL-1 receptor domain-derived decoy peptides: identification of the TLR4 Toll/IL-1 receptor domain dimerization interface

Agonist-induced dimerization of TLR4 Toll/IL-1R (TIR) domains initiates intracellular signaling. Therefore, identification of the TLR4-TIR dimerization interface is one key to the rational design of therapeutics that block TLR4 signaling. A library of cell-permeating decoy peptides, each of which represents a nonfragmented patch of the TLR4 TIR surface, was designed such that the peptides entirely encompass the TLR4 TIR surface. Each peptide was synthesized in tandem with a cell-permeating Antennapedia homeodomain sequence and tested for the ability to inhibit early cytokine mRNA expression and MAPK activation in LPS-stimulated primary murine macrophages. Five peptides--4R1, 4R3, 4BB, 4R9, and 4αE--potently inhibited all manifestations of TLR4, but not TLR2 signaling. When tested for their ability to bind directly to TLR4 TIR by F?rster resonance energy transfer using time-resolved fluorescence spectroscopy, Bodipy-TMR-X-labeled 4R1, 4BB, and 4αE quenched fluorescence of TLR4-Cerulean expressed in HeLa or HEK293T cells, whereas 4R3 was partially active, and 4R9 was least active. These findings suggest that the area between the BB loop of TLR4 and its fifth helical region mediates TLR4 TIR dimerization. Moreover, our data provide direct evidence for the utility of the decoy peptide approach, in which peptides representing various surface-exposed segments of a protein are initially probed for the ability to inhibit protein function, and then their specific targets are identified by F?rster resonance energy transfer to define recognition sites in signaling proteins that may be targeted therapeutically to disrupt functional transient protein interactions.     

UNC93B1 physically associates with human TLR8 and regulates TLR8-mediated signaling

Toll-like receptors (TLRs) 3, 7, 8, and 9 are localized to intracellular compartments where they encounter foreign or self nucleic acids and activate innate and adaptive immune responses. The endoplasmic reticulum (ER)-resident membrane protein, UNC93B1, is essential for intracellular trafficking and endolysosomal targeting of TLR7 and TLR9. TLR8 is phylogenetically and structurally related to TLR7 and TLR9, but little is known about its localization or function. In this study, we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants. UNC93B1 physically associated with human TLR8, similar to TLRs 3, 7, and 9, and played a critical role in TLR8-mediated signaling. Localization analyses of TLR8 tail-truncated mutants revealed that the transmembrane domain and the Toll/interleukin-1 receptor domain were required for proper targeting of TLR8 to the early endosome. Hence, although UNC93B1 participates in intracellular trafficking and signaling for all nucleotide-sensing TLRs, the mode of regulation of TLR localization differs for each TLR.     

TRAF6 and Src kinase activity regulates Cot activation by IL-1

Cot is one of the MAP kinase kinase kinases that regulates the ERK1/ERK2 pathway under physiological conditions. Cot is activated by LPS, by inducing its dissociation from the inactive p105 NFkappaB-Cot complex in macrophages. Here, we show that IL-1 promotes a 10-fold increase in endogenous Cot activity and that Cot is the only MAP kinase kinase kinase that activates ERK1/ERK2 in response to this cytokine. Moreover, in cells where the expression of Cot is blocked, IL-1 fails to induce an increase in IL-8 and MIP-1betamRNA levels. The activation of Cot-MKK1-ERK1/ERK2 signalling pathway by IL-1 is dependent on the activity of the transducer protein TRAF6. Most important, IL-1-induced ERK1/ERK2 activation is inhibited by PP1, a known inhibitor of Src tyrosine kinases, but this tyrosine kinase activity is not required for IL-1 to activate other MAP kinases such as p38 and JNK. This Src kinases inhibitor does not block the dissociation and subsequently degradation of Cot in response to IL-1, indicating that other events besides Cot dissociation are required to activate Cot. All these data highlight the specific requirements for activation of the Cot-MKK1-ERK1/ERK2 pathway and provide evidence that Cot controls the functions of IL-1 that are mediated by ERK1/ERK2.     

TRAF6 promotes ubiquitination and regulated intramembrane proteolysis of IL-1R1

It has recently been shown that Interleukin-1 receptor, type 1, an essential regulator of inflammation and inate immunity, undergoes regulated intramembrane proteolysis (RIP). Although IL-1R1-mediated intracellular signalling has been well studied, very little is known about how RIP of IL-1R1 is modulated. In this study, by using wild-type TRAF6 and TRAF6 mutants that are defective in its ubiquitin ligase activity, we show for the first time that TRAF6 induces ubiquitination of IL-1R1. We further demonstrate that of all TRAF family members examined, TRAF6 preferentially ubiquitinates IL-1R1. Moreover, we show that TRAF6 ubiquitin ligase activity and ubiquitination of IL-1R1 are positively correlated with IL-1R1 ectodomain shedding and subsequent gamma-secretase cleavage. Our results indicate that TRAF6-mediated ubiquitination of IL-1R1 has a decisive role in IL-1R1 signalling and propose a molecular mechanism whereby TRAF6 promotes ubiquitination and RIP of IL-1R1 through its ubiquitin ligase activity.     

Intracellular heat shock protein-70 negatively regulates TLR4 signaling in the newborn intestinal epithelium

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal-related mortality in premature infants, and it develops under conditions of exaggerated TLR4 signaling in the newborn intestinal epithelium. Because NEC does not develop spontaneously, despite the presence of seemingly tonic stimulation of intestinal TLR4, we hypothesized that mechanisms must exist to constrain TLR4 signaling that become diminished during NEC pathogenesis and focused on the intracellular stress response protein and chaperone heat shock protein-70 (Hsp70). We demonstrate that the induction of intracellular Hsp70 in enterocytes dramatically reduced TLR4 signaling, as assessed by LPS-induced NF-κB translocation, cytokine expression, and apoptosis. These findings were confirmed in vivo, using mice that either globally lacked Hsp70 or overexpressed Hsp70 within the intestinal epithelium. TLR4 activation itself significantly increased Hsp70 expression in enterocytes, which provided a mechanism of autoinhibition of TLR4 signaling in enterocytes. In seeking to define the mechanisms involved, intracellular Hsp70-mediated inhibition of TLR4 signaling required both its substrate-binding EEVD domain and association with the cochaperone CHIP, resulting in ubiquitination and proteasomal degradation of TLR4. The expression of Hsp70 in the intestinal epithelium was significantly decreased in murine and human NEC compared with healthy controls, suggesting that loss of Hsp70 protection from TLR4 could lead to NEC. In support of this, intestinal Hsp70 overexpression in mice and pharmacologic upregulation of Hsp70 reversed TLR4-induced cytokines and enterocyte apoptosis, as well as prevented and treated experimental NEC. Thus, a novel TLR4 regulatory pathway exists within the newborn gut involving Hsp70 that may be pharmacologically activated to limit NEC severity.     

TLR5-mediated phosphoinositide 3-kinase activation negatively regulates flagellin-induced proinflammatory gene expression

Epithelial cells detect motile pathogens via TLR5 ligation of flagellin, resulting in rapid induction of antibacterial/proinflammatory gene expression. Although such flagellin-induced gene expression is quite transient, likely to avoid the negative consequences of inflammation, little is known regarding the molecular mechanisms that mediate its shutdown. We hypothesized that, analogous to the case for TLR4, phosphoinositide 3-kinase (PI3K) might negatively regulate TLR5 signaling. However, because PI3K is an essential positive mediator of some pathways of TLR-mediated gene expression, the opposite hypothesis was also considered. Herein, we observed that flagellin stimulation of epithelial cells indeed induced rapid (<30 min) PI3K activation, as evidenced by Akt phosphorylation, via a TLR5-mediated mechanism. Blockade of PI3K with wortmannin resulted in marked enhancement of flagellin-induced gene expression as assessed by measuring levels of inducible NO synthase, IL-6, and IL-8. Such enhancement of gene expression by PI3K inhibition correlated with prolonged activation of MAPK (p38 and ERK1/2) and was ablated under MAPK inhibition. Such effect of inhibiting PI3K with wortmannin was mimicked by the PI3K inhibitor LY294002, and, conversely, a constitutively active PI3K prevented p38 activation in response to flagellin. Last, to test the significance of these results in vivo, we measured flagellin-induced gene expression in PI3K knockout mice. PI3K-null mice displayed increased levels of flagellin-induced serum IL-6, KC (IL-8 homolog), and nitrite as compared with heterozygous littermates. Thus, TLR5's rapid activation of PI3K serves to limit MAPK signaling, thus limiting proinflammatory gene expression and reducing the potential negative consequences of proinflammatory gene expression.     

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

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 NF kappa 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.     

Caspase-cleaved TRAF1 negatively regulates the antiapoptotic signals of TRAF2 during TNF-induced cell death

Tumor necrosis factor (TNF) signaling leads to pleiotropic responses in a wide range of cell types, in part by activating antiapoptotic and proapoptotic pathways. Previous studies have suggested that TNF receptor-associated factor (TRAF) 2 can mediate crucial antiapoptotic signals during TNF stimulation. However, it is unclear how the antiapoptotic signals via TRAF2 in TNF-R1 signaling is regulated. Here we show that TRAF1 is cleaved by caspase-8 into two fragments during apoptosis induced by TNF. Overexpression of the C-terminal cleavage product, TRAF1-c, increased TNF-induced cell death of hybridoma T cells. Importantly, we demonstrate that the cleavage product of TRAF1 coimmunoprecipitates with TRAF2 that is released from the TNF-R1 complex in response to prolonged TNF treatment. These results indicate that caspase-dependent cleavage of TRAF1 generates TRAF1-c fragments that are able to bind TRAF2, and then sequester TRAF2 from the TNF-R1 complex, rendering cells, at least in part, sensitive to TNF.