Mycobacterial infection in MyD88-deficient mice

MyD88 is an adaptor protein that plays a major role in TLR/IL-1 receptor family signaling. To understand the role of MyD88 in the development of murine tuberculosis in vivo, MyD88 knockout (KO) mice aerially were infected with Mycobacterium tuberculosis. Infected MyD88 mice were not highly susceptible to M. tuberculosis infection, but they developed granulomatous pulmonary lesions with neutrophil infiltration which were larger than those in wild-type (WT) mice (P < 0.01). The pulmonary tissue levels of mRNA for iNOS and IL-18 were slightly lower, but levels of mRNA for IL-1 beta, IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TGF-beta were higher in MyD88 KO mice. IFN-gamma, TNF-alpha, IL-1 beta, and IL-12 also were high in the sera of MyD88 KO mice. There were no statistically significant differences in the expression of TNF-alpha, IL-12, and ICAM-1 mRNA between MyD88 KO and WT mice. Thus, MyD88 deficiency did not influence the development of murine tuberculosis. NF-kappa B activity was similar in the alveolar macrophages from the lung tissues of MyD88 KO and WT mice. Also, there may be a TLR2-specific, MyD88-independent IL-1 receptor/TLR-mediated pathway to activate NF-kappa B in the host defense against mycobacterial infection.     

Flavopiridol inhibits lipopolysaccharide-induced TNF-α production through inactivation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway

Flavopiridol is a cyclin-dependent kinase inhibitor and inhibits the growth of various cancer cells. The effect of flavopiridol on lipopolysaccharide (LPS)-induced proinflammatory mediator production was examined in RAW 264.7 macrophage-like cells. Flavopiridol significantly reduced the production of tumor necrosis factor-α and, to a lesser extent, nitric oxide in LPS-stimulated cells. Flavopiridol inhibited the activation of nuclear factor-κB and IκB kinase in response to LPS. Flavopiridol also inhibited the activation of a series of mitogen-activated protein kinases, such as p38, stress-activated protein kinase/c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in response to LPS. However, flavopiridol did not alter the expression of tumor necrosis factor receptor-associated factor 6, myeloid differentiation factor 88 (MyD88) or CD14/toll-like receptor (TLR) 4. Flavopiridol inhibited nitric oxide production induced by a MyD88-dependent TLR2 ligand, but not a MyD88-independent TLR3 ligand. Further, flavopiridol did not alter the phosphorylation of interferon regulatory factor 3 in the MyD88-independent pathway. Therefore, it was suggested that flavopiridol exclusively inhibited the activation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway. Flavopiridol might be useful for the prevention of LPS-induced inflammatory response.     

TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway

Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.     

TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation caused by proteinuria

Proteinuria is an important risk factor for chronic kidney diseases (CKD). Several studies have suggested that proteinuria initiates tubulointerstitial inflammation, while the mechanisms have not been fully understood. In this study, we hypothesized whether the activation of the TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation induced by proteinuria. We observed expression of TLR2, MyD88, NF-κB, as well as TNF-α and IL-6 detected by immunohistostaining, Western blotting and real-time PCR in albumin-overloaded (AO) nephropathy rats. In vitro, we observed these markers in HK-2 cells stimulated by albumin. We used TLR2 siRNA or the NF-κB inhibitor BAY 11-7082 to observe the influence of TNF-α and IL-6 expression caused by albumin overload. Finally, we studied these markers in non-IgA mesangioproliferative glomerulonephritis (MsPGN) patients with different levels of proteinuria. It was demonstrated that expression of TLR2, MyD88 and NF-κB were significantly increased in AO rats and in non-IgA MsPGN patients with high levels of proteinuria, and TNF-α and IL-6 expressions were increased after NF-κB activation. Furthermore, TNF-α and IL-6 expression was positively correlated with the level of proteinuria. Albumin-overload induced TNF-α and IL-6 secretions by the TLR2–MyD88–NF-κB pathway activation, which could be attenuated by the TLR2 siRNA or BAY 11-7082 in HK-2 cells. In summary, we demonstrated that proteinuria may exhibit an endogenous danger-associated molecular pattern (DAMP) that induces tubulointerstitial inflammation via the TLR2–MyD88–NF-κB pathway activation.     

Stearoyl-CoA Desaturase 1 (SCD1) is a key factor mediating diabetes in MyD88-deficient mice

Saturated fatty acids, acting as ligands for toll-like receptor 4 (TLR4), induce inflammation and mediate the development of insulin resistance. Myeloid differentiation factor 88 (MyD88) is an adaptor protein for TLR4. Previously, we found MyD88-deficient mice fed a high-fat diet (HFD) exhibited a severe diabetic phenotype. Stearoyl-CoA Desaturase 1 (SCD1) is the rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids and known as a risk factor of diabetes. In the present study, we found SCD1 was dramatically increased in HFD-fed MyD88-deficient mice liver. This finding showed the novel linkage between MyD88 and SCD1 in the development of diabetes mellitus.     

NF-kappaB activation but not PI3K/Akt is required for dexamethasone dependent protection against TNF-alpha cytotoxicity in L929 cells

Tumor necrosis factor alpha (TNF-alpha) is one of the best-described cell death promoters. In murine L929 fibroblasts, dexamethasone inhibits TNF-alpha-induced cytotoxicity. Since phosphatidyl inositol 3 kinase (PI3K) and nuclear factor kappa B (NF-kappaB) proteins regulate several survival pathways, we evaluated their participation in dexamethasone protection against TNF-alpha cell death. We interfered with these pathways by overexpressing a negative dominant mutant of PI3K or a non-degradable mutant of inhibitor of NF-kappaB alpha (IkappaBalpha) (the cytoplasmic inhibitor of NF-kappaB) in L929 cells. The mutant IkappaB, but not the mutant PI3K, abrogated dexamethasone-mediated protection. The loss of dexamethasone protection was associated with a diminished accumulation in XIAP and c-IAP proteins.     

Identification of TNF-alpha-responsive NF-kappaB p65-binding element in the distal promoter of the mouse serine protease inhibitor SerpinE2

Serine protease inhibitor SerpinE2 is known as a cytokine-inducible gene. Here, we investigated whether tumor necrosis factor alpha-(TNF-alpha)-induced expression of SerpinE2 is mediated by the nuclear factor-kappaB (NF-kappaB) p65 subunit. Both steady state and TNF-alpha-induced expression of SerpinE2 mRNA were abrogated in p65-/- murine embryonic fibroblasts (MEFs). Reconstitution with wild-type p65 rescued SerpinE2 mRNA expression in an IkappaB kinase beta-dependent manner. Electrophoresis mobility shift assay and ChIP assay demonstrated that p65 bound to the kappaB-like DNA sequence located at approximately -9 kbp in the SerpinE2 promoter. In addition, TNF-alpha stimulated luciferase gene expression driven by the kappaB-like element in the reconstituted MEFs, but not in p65-/- MEFs. These results indicated that activation of NF-kappaB p65 plays an important role in TNF-alpha-induced expression of SerpinE2.     

The influence of the signal dynamics of activated form of IKK on NF-kappaB and anti-apoptotic gene expressions: a systems biology approach

NF-kappaB activation plays a crucial role in anti-apoptotic responses in response to the apoptotic signaling during tumor necrosis factor (TNF)-alpha stimulation. TNF-alpha induces apoptosis sensitive to the hepatitis B virus (HBV) infected cells, despite sustained NF-kappaB activation. Our results indicate that the HBV infection induces sustained NF-kappaB activation, in a manner similar to the TNF-alpha stimulation. However, these effects are not merely combined. Computational simulations show that the level of form of the IKK complex activated by phosphorylation (IKK-p) affects the dynamic pattern of NF-kappaB activation during TNF-alpha stimulation in the following ways: (i) the initial level of IKK-p determines the incremental change in IKK-p at the same level of TNF-alpha stimulation, (ii) the incremental change in IKK-p determines the amplitudes of active NF-kappaB oscillation, and (iii) the steady state level of IKK-p after the incremental change determines the period of active NF-kappaB oscillation. Based on experiments, we observed that the initial level of IKK-p was upregulated and the active NF-kappaB oscillation showed smaller amplitudes for a shorter period in HepG2.2.15 cells (HBV-producing cells) during TNF-alpha stimulation, as was indicated by the computational simulations. Furthermore, we found that during TNF-alpha stimulation, NF-kappaB-regulated anti-apoptotic genes were upregulated in HepG2 cells but were downregulated in HepG2.2.15 cells. Based on the previously mentioned results, we can conclude that the IKK-p-level changes induced by HBV infection modulate the dynamic pattern of active NF-kappaB and thereby could affect NF-kappaB-regulated anti-apoptotic gene expressions. Finally, we postulate that the sensitive apoptotic response of HBV-infected cells to TNF-alpha stimulation is governed by the dynamic patterns of active NF-kappaB based on IKK-p level changes.     

TAK1 lysine 158 is required for TGF-β-induced TRAF6-mediated Smad-independent IKK/NF-κB and JNK/AP-1 activation

Lys63-linked TAK1 polyubiquitination plays an essential role in the regulation of TAK1 activation. TRAF6-mediated Lys63-linked polyubiquitylation of TAK1 has been shown to be required for TGF-β-induced TAK1 activation. However, it remains unclear which lysine residue on TAK1 is TRAF6-mediated TAK1 polyubiquitination acceptor site in TGF-β signaling pathway. Here we report that lysine 158 on TAK1 is required for TGF-β-induced TRAF6-mediated TAK1 polyubiquitination and TAK1-mediated IKK, JNK and p38 activation. Notably, in contrast to TAK1 wild-type and K34R mutant, TAK1 K158R mutant co-overexpression with TAB1 failed to induce Lys63-linked TAK1 polyubiquitination. TRAF6-induced K63-linked TAK1 polyubiquitination was blocked by TAK1 K158R mutation, but not by K34R mutation. Furthermore, TGF-β-induced TAK1 polyubiquitination was inhibited by TAK1 K158R mutation, but not by K34R mutation in HeLa cells. Reconstitution of TAK1-deficient mouse embryo fibroblast cells with TAK1 wild-type, K158R mutant, or K34R mutant reveals that TAK1 lysine 158 residue is required for TGF-β-induced IKK, p38 and JNK activation.     

MAPPIT analysis of TLR adaptor complexes

Toll-like receptors (TLRs) are crucial components of the innate immune system, coupling pathogen recognition to a cellular response. We used the MAPPIT mammalian two-hybrid technique to investigate protein-protein interactions in the early steps in TLR signalling. A partial TLR-adaptor interaction map was constructed confirming several known but also documenting novel interactions. We show that the TLR adaptor Mal is critical for linking Myeloid Differentiation primary response protein 88 (MyD88) to TLR2 and TLR4. Analysis of the contributions of the different sub-domains of MyD88-adaptor-like protein (Mal) and MyD88 in adaptor homo- and hetero-dimerisation provides an initial mechanistic insight in this bridging function of Mal.     

Expression of APOBEC2 is transcriptionally regulated by NF-kappaB in human hepatocytes

Apolipoprotein B mRNA-editing enzyme catalytic subunit 2 (APOBEC2) is a member of the nucleic-acid-editing enzymes. However, the physiological function of APOBEC2 remains unclear. We demonstrate that APOBEC2 expression is strongly enhanced in response to both tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta. Inhibition of NF-kappaB activation invariably blocks TNF-alpha-induced APOBEC2 expression. The promoter region of APOBEC2 contains functional NF-kappaB response elements in the 5' untranslated region of the gene at -625/-616. These results show that APOBEC2 expression is regulated by pro-inflammatory cytokines via NF-kappaB activation and suggest a possible role of APOBEC2 in the pathophysiology of hepatic inflammation.     

TIRAP mediates endotoxin-induced NF-kappaB activation and apoptosis in endothelial cells

Bacterial lipopolysaccharide (LPS) initiates multiple signaling events in vascular endothelial cells that can result in activation and/or cell death. LPS-induced activation of endothelial cells elicits a wide array of vascular endothelial responses, many of which are dependent on NF-kappaB activation. Several of the signaling molecules that mediate LPS-induced NF-kappaB activation, including Tlr-4, MyD88, and IRAK-1, have been similarly reported to mediate LPS pro-apoptotic signaling. Recently, a new signaling molecule, TIRAP, has been identified that mediates LPS-induced NF-kappaB signaling in monocytes and macrophages. Using a TIRAP dominant negative construct, we have identified a role for TIRAP in mediating LPS-induced NF-kappaB activation and apoptosis in human endothelial cells. These data identify TIRAP as a dual functioning signaling molecule and suggest the presence of a MyD88-independent LPS signaling pathway in human endothelial cells.     

Hsp70 inhibits lipopolysaccharide-induced NF-kappaB activation by interacting with TRAF6 and inhibiting its ubiquitination

Inducible heat shock protein 70 (Hsp70) is one of the most important HSPs for maintenance of cell integrity during normal cellular growth as well as pathophysiological conditions. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial signaling transducer that regulates a diverse array of physiological and pathological processes and is essential for activating NF-kappaB signaling pathway in response to bacterial lipopolysaccharide (LPS). Here we report a novel mechanism of Hsp70 for preventing LPS-induced NF-kappaB activation in RAW264.7 macrophage-like cells. Our results show that Hsp70 can associate with TRAF6 physically in the TRAF-C domain and prevent TRAF6 ubiquitination. The stimulation of LPS dissociates the binding of Hsp70 and TRAF6 in a time-dependent manner. Hsp70 inhibits LPS-induced NF-kappaB signaling cascade activation in heat-shock treated as well as Hsp70 stable transfected RAW264.7 cells and subsequently decreases iNOS and COX-2 expression. Two Hsp70 mutants, Hsp70DeltaC(1-428aa) with N-terminal ATPase domain and Hsp70C(428-642aa) with C-terminal domain, lack the ability to influence TRAF6 ubiquitination and TRAF6-triggered NF-kappaB activation. Taken together, these findings indicate that Hsp70 inhibits LPS-induced NF-kappaB activation by binding TRAF6 and preventing its ubiquitination, and results in inhibition of inflammatory mediator production, which provides a new insight for analyzing the effects of Hsp70 on LPS-triggered inflammatory signal transduction pathways.     

Disruption of membrane cholesterol stimulates MyD88-dependent NF-kappaB activation in immature B cells

Agents that extract or sequester membrane cholesterol stimulate IkappaB degradation and lead to NF-kappaB activation in a subset of B cells. Although the extraction of cholesterol by methyl-beta-cyclodextrin is the most potent stimulus of NF-kappaB, other agents that sequester cholesterol have similar effects. B cells and B cell lines with an immature phenotype are significantly more sensitive to the effects of cholesterol perturbation than their mature B cell counterparts. NF-kappaB activation does not involve signaling from the B cell receptor complex. Instead, the disruption of membrane cholesterol activates NF-kappaB through a MyD88-dependent pathway involving the pattern recognition receptor, Toll-like receptor 4. We suggest that lipid raft microdomains may serve not only to orchestrate receptor signaling, but to sequester signaling components one from one another, which serves to prevent receptor-mediated signaling from occurring. A role for this process during B cell development is suggested.     

NF-kappaB-dependent regulation of tumor necrosis factor-alpha gene expression by CpG-oligodeoxynucleotides

Immunostimulatory activities of synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have gained attention as potentially useful immunotherapeutics. However, CpG-ODNs induce harmful and lethal shock effects because they greatly enhance the sequence-dependent induction of tumor necrosis factor-alpha (TNF-alpha). We have shown that phosphorothioate-modified oligodeoxynucleotides (PS-ODNs) of the CpG-ODN 1826 stimulate TNF-alpha gene expression, TNF-alpha promoter activity, IkappaB degradation, and NF-kappaB activation at higher levels compared with its phosphodiester ODN (PO-ODN). In contrast to the effects of CpG-ODN 1826, PS-ODN of the CpG-ODN 2006 showed lower stimulatory activities than its PO-ODN. Using transient transfection, it was found that myeloid differentiation protein (MyD88) and tumor necrosis factor receptor-associated factor 6 are commonly required for activation of the TNF-alpha promoter by various CpG-ODNs with different potencies. These results strongly suggest a possibility to optimally activate the innate immune responses by modulating the potency of CpG-ODNs via sequence rearrangement and phosphorothioate backbone modification.     

Expression differences of miRNAs and genes on NF-κB pathway between the healthy and the mastitis Chinese Holstein cows

In order to discover the variation of microRNAs and genes associated with NF-κB signaling pathway between the healthy and the mastitis Chinese Holstein cows, Illumina Deep Sequencing and qRT-PCR are applied to detect 25 kinds of miRNAs (miR-16, miR-125b, miR-15, miR-29a, miR-23b, miR-146, miR-301a, miR-181b, let-7, miR-30b, miR-21, miR-223, miR-27b, miR-10a, miR-143, etc.) expression levels in blood samples and 14 genes (RelA, RelB, Rel, p105, p100, IκBα, IκBβ, IκBδ, IκBε, IκBζ, Bcl-3, IKKα, IKKβ, IKKγ/NEMO) relative expression levels in nine tissues. The total number of miRNAs is declining, and RelA, Rel, p105, p100, IκBα, IκBβ, IκBδ, IκBζ, Bcl-3, and IKKα expressions are rising in mastitis individuals. So, we suppose that NF-κB pathway is active in mastitis individuals as a result of the decrease inhibition of miRNAs. While in healthy ones, the NF-κB pathway is inactive, because of the miRNAs enhanced inhibition action. However, the specific regulatory mechanism of miRNAs on NF-κB pathway in mastitis Holstein cows needs further investigation. Moreover, due to obvious expression differences, some miRNAs, especially miR-16 and miR-223, may be used as new markers for the dairy mastitis prognosing.     

Enhanced myeloid differentiation factor 88 promotes tumor metastasis via induction of epithelial–mesenchymal transition in human hepatocellular carcinoma

Metastasis is the leading cause of death in patients with hepatocellular carcinoma (HCC) after curative resection. Therefore, it is critical to understand the mechanisms underlying tumor metastasis in HCC. We have previously shown that elevated expression of myeloid differentiation factor 88 (MyD88) may promote tumor growth and metastasis in HCC. In this study, we reported that enhanced expression of MyD88 promoted epithelial–mesenchymal transition (EMT) properties and tumor-initiating capabilities in HCC cells. MyD88 was found to be able to interact with p85, a regulatory subunit of phosphoinositide 3-kinase (PI3-K), independent of TLR/IL-1R-mediated response and caused PI3-K/v-akt murine thymoma viral oncogene homolog (Akt) activation, which resulted in subsequent phosphorylation of glycogen synthase kinase-3β and stabilization of Snail, a critical EMT mediator. Consistently, we observed a significant correlation between MyD88 expression and p-Akt levels in a cohort of HCC patients, and found that the combination of these two parameters have better prognostic value for HCC patients. Taken together, these results suggest that elevated MyD88 may facilitate HCC metastasis by promoting EMT properties and tumor-initiating capabilities via PI3–K/Akt pathway.     

Nucleoredoxin Negatively Regulates Toll-like Receptor 4 Signaling via Recruitment of Flightless-I to Myeloid Differentiation Primary Response Gene (88)

We previously characterized nucleoredoxin (NRX) as a negative regulator of the Wnt signaling pathway through Dishevelled (Dvl). We perform a comprehensive search for other NRX-interacting proteins and identify Flightless-I (Fli-I) as a novel NRX-binding partner. Fli-I binds to NRX and other related proteins, such as Rod-derived cone viability factor (RdCVF), whereas Dvl binds only to NRX. Endogenous NRX and Fli-I in vivo interactions are confirmed. Both NRX and RdCVF link Fli-I with myeloid differentiation primary response gene (88) (MyD88), an important adaptor protein for innate immune response. NRX and RdCVF also potentiate the negative effect of Fli-I upon lipopolysaccharide-induced activation of NF-κB through the Toll-like receptor 4/MyD88 pathway. Embryonic fibroblasts derived from NRX gene-targeted mice show aberrant NF-κB activation upon lipopolysaccharide stimulation. These results suggest that the NRX subfamily of proteins forms a link between MyD88 and Fli-I to mediate negative regulation of the Toll-like receptor 4/MyD88 pathway.