Identification and characterization of receptor-interacting protein 2 as a TNFR-associated factor 3 binding partner

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a highly versatile immune regulator that positively controls type I interferon production, but negatively regulates the activation of mitogen-activated protein kinase and alternative nuclear factor-κB signaling. The precise function of TRAF3 in different signaling pathways remains unclear. Thus, in a yeast two-hybrid assay, TRAF3 was used as the bait to screen a human spleen cDNA library for TRAF3 interactors that may potentially mediate TRAF3-regulated functions. Receptor-interacting protein 2 (RIP2) was identified as a TRAF3 binding partner. The interaction between TRAF3 and RIP2 was further confirmed by mammalian two-hybrid, co-immunoprecipitation and GST pull-down assays, and this interaction was also verified by immunoprecipitation of endogenous proteins in Ramos cells, a human B lymphoma cell line. RIP2 is an activator of NF-κB. We therefore examined the effect of TRAF3 in RIP2-induced NF-κB activation. The result showed that TRAF3 could inhibit RIP2-induced NF-κB activation. Given the high expression of RIP2 in the B lymphoma cell line and endogenous interaction between TRAF3 and RIP2 in Ramos cells, the role of RIP2 was further studied. The result demonstrated that RIP2 knockdown was capable of increasing the expression of TRAF3 and suppressing the activation of alternative NF-кB pathway in Ramos cells. These findings suggest that functional interactions between RIP2 and TRAF3 may provide some clues to the mechanisms of TRAF3-involvement in both positive and negative regulatory functions.     

Pellino proteins are more than scaffold proteins in TLR/IL-1R signalling: a role as novel RING E3-ubiquitin-ligases

Members of the Toll-like receptor (TLR) and IL-1 receptor (IL-1R) family initiate signalling pathways that shape innate immunity. Pellino proteins have recently been implicated as evolutionary conserved scaffold proteins in TLR/IL-1R signalling leading to nuclear factor-kappaB and mitogen activated protein kinase-dependent gene expression. We found that Pellino proteins contain a new RING-like motif. Because RING motifs are a feature of a subclass of E3-ubiquitin-ligases that target specific proteins for ubiquitination, we suggest that Pellino proteins are involved in TLR/IL-1R signalling not only as scaffold proteins but also as RING E3-ubiquitin-ligases. In support of this hypothesis we show that Pellino proteins induce IRAK-1 polyubiquitination in a RING-dependent manner. We further propose a model in which Pellino-mediated IRAK-1 polyubiquitination regulates TLR/IL-1R signalling.     

Discovering differential activation machinery of the Toll-like receptor 4 signaling pathways in MyD88 knockouts

To understand differential time activation of nuclear factor kappaB (NF-kappaB) and the temporal features of the downstream pro-inflammatory cytokines' [tumour-necrosis-factor-alpha (TNF-alpha) and IP-10] mRNA levels in myeloid differentiation primary-response protein 88 (MyD88) knockouts (KOs), I developed a computational model of the TLR4 pathway. The result suggests that the late phase expression of NF-kappaB activity observed in MyD88 KOs is possibly due to a number of novel intermediates acting along the MyD88-independent pathway. I also simulate that the TNF-alpha levels will increase at a longer time in MyD88 KOs, not previously mentioned.     

p38-Mediated phosphorylation of Eps15 endocytic adaptor protein

Epidermal growth factor receptor pathway substrate 15 (Eps15) has been suggested to be involved in the endocytosis of cell surface receptors, including epidermal growth factor receptor (EGFR). Eps15 is phosphorylated at Tyr-849 upon stimulation with EGF during endocytic processes. In the present study, we found that stimulation of HeLa cells with EGF or TNF-α induced transient phosphorylation of Eps15 at Ser-796. Inhibition of p38 completely blocked phosphorylation and recombinant p38α directly phosphorylated the residue. These results demonstrate a novel stress kinase-mediated signaling pathway to Eps15 endocytic adapter protein.     

Characterization of interleukin-1 receptor-associated kinase 1 binding protein 1 gene in small abalone Haliotis diversicolor

Interleukin receptor-associated kinase (IRAK)-1 binding protein 1 (IRAK1BP1) is a critical factor in preventing dangerous overproduction of proinflammatory cytokines by the innate immune system and in influencing the specificity of TLR responses. In this study, a first molluscan IRAK1BP1 gene, saIRAK1BP1, was cloned from the small abalone (Haliotis diversicolor). Its full-length cDNA sequence is 1047bp, with a 747bp open reading frame encoding a protein of 249 aa. The molecular mass of the deduced protein is approximately 28.1kDa with an estimated pI of 8.87, and shows highest identity (52%) to acorn worm Saccoglossus kowalevskii. Amino acid sequence analysis revealed that saIRAK1BP1 shares a conserved SIMPL domain. Quantitative real-time PCR was employed to investigate the tissue distribution of saIRAK1BP1 mRNA, and its expression in abalone under bacteria challenge and larvae at different developmental stages. The saIRAK1BP1 mRNA could be detected in all examined tissues, with the highest expression level in hemocytes, and was up-regulated in gills, kidneys and hemocytes after bacteria injection. Additionally, saIRAK1BP1 was constitutively expressed at all examined developmental stages. These results indicate that saIRAK1BP1 play an important role in the adult abalone immune system and might be essential in embryo and larval development in abalone.     

Aerobic glycolysis promotes tumor immune evasion by hexokinase2-mediated phosphorylation of IκBα

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1,25-dihydroxyvitamin D3 induces biphasic NF-kappaB responses during HL-60 leukemia cells differentiation through protein induction and PI3K/Akt-dependent phosphorylation/degradation of IkappaB

1,25-dihydroxyvitamin D(3) (VD(3)) induces differentiation in a number of leukemia cell lines and under various conditions is able to either stimulate or inhibit nuclear factor kappa B (NF-kappaB) activity. Here we report a time-dependent biphasic regulation of NF-kappaB in VD(3)-treated HL-60 leukemia cells. After VD(3) treatment there was an early approximately 4 h suppression and a late 8-72 h prolonged reactivation of NF-kappaB. The reactivation of NF-kappaB was concomitant with increased IKK activities, IKK-mediated IkappaBalpha phosphorylation, p65 phosphorylation at residues S276 and S536, p65 nuclear translocation and p65 recruitment to the NF-kappaB/vitamin D responsive element promoters. In parallel with NF-kappaB stimulation, there was an up-regulation of NF-kappaB controlled inflammatory and anti-apoptotic genes such as TNFalpha, IL-1beta and Bcl-xL. VD(3)-triggered reactivation of NF-kappaB was associated with PI3K/Akt phosphorylation. PI3K/Akt antagonists suppressed VD(3)-stimulated IkappaBalpha phosphorylation as well as NF-kappaB-controlled gene expression. The early approximately 4 h VD(3)-mediated NF-kappaB suppression coincided with a prolonged increase of IkappaBalpha protein which require de novo protein synthesis, lasted for as least 72 h and was insensitive to MAPK, IKK or PI3K/Akt inhibitors. Our data suggest a novel biphasic regulation of NF-kappaB in VD(3)-treated leukemia cells and our results may have provided the first molecular explanation for the contradictory observations reported on VD(3)-mediated immune-regulation.     

Endogenous macrophage migration inhibitory factor modulates glucocorticoid sensitivity in macrophages via effects on MAP kinase phosphatase-1 and p38 MAP kinase

The pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is induced by glucocorticoids (GCs), but it was not previously known if MIF regulates cellular sensitivity to GC. Here we show in GC and LPS-treated peritoneal macrophages derived from MIF-/- and wt mice that the absence of endogenous MIF is associated with increased sensitivity to GC of TNF release. This is associated with increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), concomitant decreased phosphorylation of p38 MAPK, but no effect of MIF on nuclear factor kappaB (NF-kappaB). These results demonstrate that MIF regulates GC sensitivity by phosphorylation of p38, and provides a cellular mechanism for this observation, indicating that MKP-1 is a central target of this regulation.     

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.     

Green tea polyphenol epigallocatechin-3-gallate inhibits TLR4 signaling through the 67-kDa laminin receptor on lipopolysaccharide-stimulated dendritic cells

Epigallocatechin-3-gallate (EGCG), a major active polyphenol of green tea, has been shown to down-regulate inflammatory responses in dendritic cells (DCs); however, the underlying mechanism has not been understood. Recently, we identified the 67-kDa laminin receptor (67LR) as a cell-surface EGCG receptor. In this study, we showed the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in DCs. The expressions of CD80, CD86, and MHC class I and II, which are molecules essential for antigen presentation by DCs, were inhibited by EGCG via 67LR. In addition, EGCG-treated DCs inhibited lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, and IL-6) and activation of mitogen-activated protein kinases (MAPKs), e.g., extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and nuclear factor κB (NF-κB) p65 translocation through 67LR. Interestingly, we also found that EGCG markedly elevated the expression of the Tollip protein, a negative regulator of TLR signaling, through 67LR. These novel findings provide new insight into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and consequent inflammatory responses that are implicated in the development and progression of many chronic diseases.     

The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice

TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF’s role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined the activation of NF-κB and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. NF-κB activity and phosphorylation of the inhibitor of kappa B (IκBα) increased in ischemic brains, but IRF3, inhibitor of κB kinase complex-ε (IKKε), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-κB activity or p-IκBα induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-κB signaling and brain injury after acute cerebral I/R.     

Naringenin suppresses macrophage infiltration into adipose tissue in an early phase of high-fat diet-induced obesity

Obese adipose tissue is characterized by increased macrophage infiltration, which results in chronic inflammation in adipose tissue and leads to obesity-related diseases such as type 2 diabetes mellitus and atherosclerosis. The regulation of macrophage infiltration into adipose tissue is an important strategy for preventing and treating obesity-related diseases. In this study, we report that naringenin, a citrus flavonoid, suppressed macrophage infiltration into adipose tissue induced by short-term (14 days) feeding of a high-fat diet in mice; although naringenin did not show any differences in high-fat diet-induced changes of serum biochemical parameters in this short administration period. Naringenin suppressed monocyte chemoattractant protein-1 (MCP-1) in adipose tissue, and this effect was mediated in part through inhibition of c-Jun NH₂-terminal kinase pathway. Naringenin also inhibited MCP-1 expression in adipocytes, macrophages, and a co-culture of adipocytes and macrophages. Our results suggest a mechanism by which daily consumption of naringenin may exhibit preventive effects on obesity-related diseases.     

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.     

1alpha,25-dihydroxyvitamin D3 stimulates phosphorylation of IkappaBalpha and synergizes with TPA to induce nuclear translocation of NFkappaB during monocytic differentiation of NB4 leukemia cells.

Treatment of NB4 acute promyelocytic leukemia cells with 1,25-dihydroxyvitamin D3 (1,25D3) or analogs 20-epi-22-oxa-24a,26a,27a-trihomo-1alpha,25-dihydroxyvitamin D3, 1,24-dihydroxy-22-ene-24-cyclopropylvitamin D3, 1alpha,25-dihydroxylumisterol3, or 1alpha,25(OH)2-d5-previtamin D3 in combination with TPA induces monocytic differentiation. The role of 1,25D3 in the induction of maturation has been shown to be a priming effect. Differentiation in response to these agents requires VDR-independent signaling of 1,25D3, PKC signaling, intracellular calcium, and calpain activity. In this study we identify the NFkappaB/IkappaB signaling pathway as a target of 1,25D3 and TPA action. One of the priming effects of 1,25D3 appears to be the rapid phosphorylation of serine residues on IkappaBalpha. On their own, 1,25D3, its analogs, and TPA do not alter IkappaBalpha expression; however, combinations of analogs with TPA result in a synergistic decrease in IkappaBalpha expression. Decreased expression of IkappaBalpha likely results from enhanced degradation, which allows the observed subsequent nuclear translocation of NFkappaB subunit p65. Since nuclear-localized NFkappaB was observed only in combination-treated cells, it is proposed that nuclear targets of NFkappaB are required for monocytic differentiation. Intracellular calcium and proteolytic activity are both necessary for the induction of IkappaB regulation and translocation of NFkappaB and are critical components of the nongenomic signaling cascades of the 1,25D3-induced differentiation pathway.     

A novel binding protein of single immunoglobulin IL-1 receptor-related molecule: Paralemmin-3

Previous studies have shown that single immunoglobulin IL-1 receptor-related molecule (SIGIRR) is a negative regulator of Toll-Interleukin-1 receptor signaling. Nevertheless, the molecular mechanism of the negatively regulatory effect of SIGIRR remains unknown. Using a yeast two-hybrid screen, we identified paralemmin-3 (PALM3) as a novel binding protein of SIGIRR. This interaction of SIGIRR with PALM3 was confirmed by coimmunoprecipitation in mammalian cells. In addition, the PALM3 mRNA expression was upregulated by lipopolysaccharide (LPS)-stimulation in a human alveolar epithelial cell line (A549 cells). Furthermore, silencing PALM3 by RNA interference inhibited the release of inflammatory cytokines in A549 cells after LPS-stimulation. These results suggest that PALM3 may function as an adaptor in the LPS- Toll-like receptor 4 signaling and the interaction of SIGIRR with PALM3 may partly account for the mechanism of the negatively regulatory effect of SIGIRR.     

Divergent effect of proteasome inhibition on interleukin-1beta and tumor necrosis factor alpha signaling in human astroglial cells

Impaired functioning of the proteasome pathway is one of the molecular mechanism underlying neurodegenerative changes in Alzheimer's disease. In this study, we report that dysfunction of the proteasome pathway in astroglial cells leads to decreased survival and dysregulation of chemokines by differential regulation of the nuclear factor kappa B and c-jun N-terminal kinase (JNK) pathways. We further demonstrated that proteasome inhibition augmented interleukin-1 beta- and tumor necrosis factor-alpha-induced activation of the IkappaBalpha kinase and MKK4/JNK/c-Jun pathway along with TAK1 activation. These results suggest that impaired function of the proteasome pathway may potentiate the immuno-pathologic role of secondarily activated astrocytes in the brain.