NF-κB Signal Transduction Research Articles

Mechanism and In Vitro Pharmacology of TAK1 Inhibition by (5Z)-7-Oxozeaenol

Transforming growth factor-β activated kinase-1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that regulates several signaling pathways including NF-κB signal transduction and p38 activation. TAK1 deregulation has been implicated in human diseases including cancer and inflammation. Here, we show that, in addition to its kinase activity, TAK1 has intrinsic ATPase activity, that (5Z)-7-Oxozeaenol irreversibly inhibits TAK1, and that sensitivity to (5Z)-7-Oxozeaenol inhibition in hematological cancer cell lines is NRAS mutation status and TAK1 pathway dependent. X-ray crystallographic and mass spectrometric studies showed that (5Z)-7-Oxozeaenol forms a covalent complex with TAK1. Detailed biochemical characterization revealed that (5Z)-7-Oxozeaenol inhibited both the kinase and the ATPase activity of TAK1 following a bi-phase kinetics, consistent with the irreversible inhibition mechanism. In DoHH2 cells, (5Z)-7-Oxozeaenol potently inhibited the p38 phosphorylation driven by TAK1, and the inhibition lasted over 6 h after withdrawal of (5Z)-7-Oxozeaenol. Profiling (5Z)-7-Oxozeaenol in a panel of hematological cancer cells showed that sensitive cell lines tended to carry NRAS mutations and that genes in TAK1 regulated pathways were enriched in sensitive cell lines. Taken together, we have elucidated the molecular mechanism of a TAK1 irreversible inhibitor and laid the foundation for designing next generation TAK1 irreversible inhibitors. The NRAS-TAK1-Wnt signaling network discerned in our study may prove to be useful in patient selection for TAK1 targeted agents in hematological cancers.

Cytotoxic and Inflammatory Effects of Contact Lens Multipurpose Solutions on Human Corneal Epithelial Cells

Multipurpose solutions (MPSs) are the leading method for cleaning and disinfecting soft contact lenses (CLs). During recent years, numerous clinical studies have evaluated the MPS damage to the ocular surface. This study examined the cytotoxic and the inflammatory effects of MPSs and hydrogen peroxide disinfection system (H202) compared to appropriate controls on human corneal epithelial (HCE) cells. Primary cultured HCE cells were exposed to eight different commercially available MPS products (MPS A, ReNu MultiPlus®; MPS B, Opti Free® EverMoist; MPS C, Solo-care Aqua®; MPS-D, Complete®; MPS-E, Unica Sensitive®; MPS-F, Options Multi®; MPS-G, Biotrue®; MPS-H, COMPLETE® RevitaLens). Morphological changes and cytotoxic effects were examined with FITC-Annexin V/PI and MTT assays. The protein contents of the inflammatory cytokines interleukin (IL)-1β, TNF-α, IL-6 and IL-8 were examined by multiplex fluorescent bead immunoassay (FBI), and the mRNA expression was examined by real time PCR. Lipopolysaccharide (LPS) with 500 ng/ml CD14 and 500 ng/ml LBP (LPS complex), polyinosinic: polycytidylic acid (Poly I:C) and un-neutralized H202 served as positive controls, respectively. Phosphate-buffered saline (PBS) was added as a negative control. The study demonstrated that most of the MPSs induced varying degrees of cytotoxicity to HCE cells, and increased production of pro-inflammatory cytokines compared to the negative control. In addition, several MPS increased the mRNA level of inhibitory factor-κBα (1-κBα). Among the various MPSs, MPS-H induced the highest protein contents of the pro-inflammatory cytokines (14.37±2.2-fold for TNF-α, 41.39±2.5-fold for IL-1β and 5.24±0.6-fold for IL-6) compared to the negative control (p<0.05). In contrast, no significant differences were noted between the neutralized H202 and the negative control. We conclude that most of the currently used MPSs induce significant damage and inflammatory response in corneal epithelial cells. MPS-induced inflammation was mediated through NF-κB signal transduction. This study demonstrates for the first time inflammatory responses at the molecular level in primary HCE cells following exposure to a large series of commercially available and commonly used MPSs. These findings strongly suggest that certain MPSs may be partially involved in the pathogenesis of contact lens intolerance. Therefore, we recommend that practitioners advise patients as to the preferable disinfecting contact lens solutions, and to consider using the hydrogen peroxide disinfection systems instead.

Gold nanoparticles induce transcriptional activity of NF-κB in a B-lymphocyte cell line.

Gold nanoparticles (Au-NPs) have been designated as superior tools for biological applications owing to their characteristic surface plasmon absorption/scattering and amperometric (electron transfer) properties, in conjunction with low or no immediate toxicity towards biological systems. Many studies have shown the ease of designing application-based tools using Au-NPs but the interaction of this nanosized material with biomolecules in a physiological environment is an area requiring deeper investigation. Immune cells such as lymphocytes circulate through the blood and lymph and therefore are likely cellular components to come in contact with Au-NPs. The main aim of this study was to mechanistically determine the functional impact of Au-NPs on B-lymphocytes. Using a murine B-lymphocyte cell line (CH12.LX), treatment with citrate-stabilized 10 nm Au-NPs induced activation of an NF-κB-regulated luciferase reporter, which correlated with altered B lymphocyte function (i.e. increased antibody expression). TEM imaging demonstrated that Au-NPs can pass through the cellular membrane and therefore could interact with intracellular components of the NF-κB signaling pathway. Based on the inherent property of Au-NPs to bind to -thiol groups and the presence of cysteine residues on the NF-κB signal transduction proteins IκB kinases (IKK), proteins specifically bound to Au-NPs were extracted from CH12.LX cellular lysate exposed to 10 nm Au-NPs. Electrophoresis identified several bands, of which IKKα and IKKβ were immunoreactive. Further evaluation revealed activation of the canonical NF-κB signaling pathway as evidenced by IκBα phosphorylation at serine residues 32 and 36 followed by IκBα degradation and increased nuclear RelA. Additionally, expression of an IκBα super-repressor (resistant to proteasomal degradation) reversed Au-NP-induced NF-κB activation. Altered NF-κB signaling and cellular function in B-lymphocytes suggests a potential for off-target effects with in vivo applications of gold nanomaterials and underscores the need for more studies evaluating the interactions of nanomaterials with biomolecules and cellular components.

IL4-induced IgE class switching is regulated by NFkB alternative pathway

Event Abstract Back to Event IL4-induced IgE class switching is regulated by NFkB alternative pathway Kano Tanabe1, Hitomi Sakamoto1 and Seiji Inui1* 1 Graduate School of Health Sciences, Kumamoto University, Department of Immunology and Hematology, Japan Alpha4 was identified as a signal transduction molecule which associated with Ig-alpha, a component of B cell antigen receptor (BCR) complex. Alpha4 was ubiquitously expressed and gene targeting experiment revealed that alpha4 was essential for cell survival. B cell specific gene targeting confirmed that alpha4 was not only essential for BCR signal transduction but also for CD40, IL4 and LPS signals. At the same time, we found that alpha4 associated with a protein phosphatase PP2A catalytic subunit and regulated its enzymatic activity. Neuron specific gene targeting of alpha4 revealed that alpha4/PP2Ac associated with CaMKII to regulate learning and memory. In this study, we show that IL4 receptor signaling activates alternative as well as canonical NFkB signal transduction pathway. IL4 induced the serine and threonine phosphorylation of alpha4. Both alternative NFkB pathway and threonine phosphorylation of alpha4 were inhibited by a potent CaMKII inhibitor KN93. These results suggest that alpha4/CaMKII molecules are involved in IL4 receptor-induced NFkB alternative pathway. These molecules also regulate IgE class switching induced by IL4 and CD40. Establishment of M12 B cell transfectant which inducibly express TRAF3 will be also discussed. Keywords: class switching, TNF Receptor-Associated Factor 3, NF-kappa B, alpha4, IgE Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune receptors and signaling Citation: Tanabe K, Sakamoto H and Inui S (2013). IL4-induced IgE class switching is regulated by NFkB alternative pathway. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00611 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Seiji Inui, Graduate School of Health Sciences, Kumamoto University, Department of Immunology and Hematology, Kumamoto, Kumamoto, 862-0976, Japan, inui@kumamoto-u.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Kano Tanabe Hitomi Sakamoto Seiji Inui Google Kano Tanabe Hitomi Sakamoto Seiji Inui Google Scholar Kano Tanabe Hitomi Sakamoto Seiji Inui PubMed Kano Tanabe Hitomi Sakamoto Seiji Inui Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Murine tumor necrosis factor α–induced adipose‐related protein (tumor necrosis factor α–induced protein 9) deficiency leads to arthritis via interleukin‐6 overproduction with enhanced NF‐κB, STAT‐3 signaling, and dysregulated apoptosis of macrophages

To elucidate the role of tumor necrosis factor α-induced adipose-related protein (TIARP; or tumor necrosis factor α-induced protein 9 [TNFAIP-9]) in the development and pathogenesis of arthritis. We generated TIARP-deficient (TIARP(-/-) ) mice and investigated several organs in aged mice. Peritoneal macrophages were collected and cultured with lipopolysaccharide (LPS) and TNFα, and then the production of cytokines and subsequent NF-κB signal transduction were analyzed. We also examined the susceptibility of young TIARP(-/-) mice to collagen-induced arthritis (CIA). Draining lymph nodes and splenocytes were isolated and cultured, and serum levels of anti-type II collagen (anti-CII) antibodies, interleukin-6 (IL-6), and TNFα on day 60 were measured. We further investigated the effects of anti-IL-6 receptor monoclonal antibody (mAb) on the development of arthritis in TIARP(-/-) mice. IL-6/STAT-3 signaling was also analyzed using TIARP(-/-) macrophages. TIARP(-/-) mice developed spontaneous enthesitis and synovitis, had high serum levels of IL-6, had increased CD11b+ cell counts in the spleen, and showed enhanced LPS- and TNFα-induced IL-6 expression in macrophages. Sustained degradation of IκBα with dysregulated apoptosis was also noted in TIARP(-/-) macrophages. CIA was clearly exacerbated in TIARP(-/-) mice, accompanied by marked neutrophil and macrophage infiltration in joints. The levels of anti-CII antibodies in serum were unchanged, whereas autoreactive Th1 cell and Th17 cell responses were higher in TIARP(-/-) mice. Treatment with anti-IL-6 receptor mAb prevented the development of CIA in TIARP(-/-) mice, and TIARP(-/-) macrophages showed increased IL-6-induced STAT-3 phosphorylation. These findings suggest that TIARP acts as a negative regulator of arthritis by suppressing IL-6 production, its signaling and TNFα-induced NF-κB signaling, resulting in enhanced apoptosis in macrophages.

NF-κB pathway mediates vascular smooth muscle response to nicotine

Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) play important roles in nicotine-induced cardiovascular disease. To elucidate the mechanism underlying the abnormal SMC behavioral response to nicotine, we investigated the activation of the NF-κB signal transduction pathway and cell adhesion molecular (CAM) expression on SMCs. Also we used different cell culture manner of SMC sole and EC–SMC co-culture with a 0.4 or a 3μm membrane pore, to observe whether there is a crosstalk between EC/SMC involved in the process of NF-κB pathway activation. Nicotine-induced effects were observed in SMCs by both monoculture and co-culture with the 3μm-pore size, including the phosphorylation of IKK and IκB, the shift of transcription factor NF-κB, and the enhancement of SMC cytoskeleton protein expression and migration ability, but none were observed by co-culture with the 0.4μm-pore size. All of the actions could be distinctly blocked by α-bungarotoxin (α7 nicotinic receptor inhibitor) or PDTC (NF-κB suppressor). Flow cytometry analysis showed that the adhesion molecules ICAM-1 and LFA-1 and VCAM-1 and VLA-4 were better expressed similarly on the surface of SMCs in the monoculture and 3μm-pore size co-culture system vs. the 0.4μm co-culture way. The results imply that nicotine induces SMC cytoskeleton protein up-expression and migration via the NF-κB signaling pathway and that EC–SMC crosstalk via CAM facilitates its response to nicotine.

Induction of the NFκ-B signal transduction pathway in response to Corynebacterium diphtheriae infection

Corynebacterium diphtheriae, the causative agent of diphtheria, has been thoroughly studied with respect to toxin production and pili formation, while knowledge on host responses to C. diphtheriae infection is limited. In this study, we studied adhesion to and invasion of epithelial cells by different C. diphtheriae isolates. When NFκ-B reporter cell lines were used to monitor the effect of C. diphtheriae infection on human cells, strain-specific differences were observed. While adhesion to host cells had no effect, a correlation of invasion rate with NFκ-B induction was found, which indicates that internalization of bacteria is crucial for NFκ-B induction. Immunofluorescence microscopy experiments used to support the reporter assays showed that translocation of p65, as a hallmark of NFκ-B induction, was only observed in association with cell invasion by C. diphtheriae. Our data indicate that the response of epithelial cells to C. diphtheriae infection is determined by internalization of bacteria and that invasion of these cells is an active process; tetracycline-treated C. diphtheriae was still able to attach to host cells, but lost its ability to invade the cytoplasm. Recognition of pathogen-associated molecular patterns such as pili subunits by membrane-bound receptors facing the outside of the cell is not sufficient for NFκ-B induction.

Altered IkBα expression promotes NF-kB activation in monocytes from primary Sjögren’s syndrome patients

To study the importance of IκBα in NF-κB signal transduction, we analysed the IκBα expression in monocytes from Sjögren's syndrome (SS) patients versus healthy controls. Monocytes were obtained from the peripheral blood of 30 SS patients and 23 healthy subjects. IκBα expression was studied by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), real-time PCR, immunoblotting, flow cytometry and enzyme linked immunosorbent assay (ELISA). Analysis of the gene and protein expression profiles of SS monocytes revealed a down-regulation of IκBα, and in all the Sjögren's syndrome cases examined, serum IκBα levels were significantly decreased in comparison with controls. Our findings clearly demonstrate changes in the levels of IκBα in SS monocytes, suggesting that the attenuated expression of IκBα could contribute to the deregulation of NF-κB pathways in the SS pathogenesis. Decreased expression of IκBα may specifically amplify cytokines production and inflammatory response linked to Sjögren's syndrome.

Ciliary neurotrophic factor activates NF-κB to enhance mitochondrial bioenergetics and prevent neuropathy in sensory neurons of streptozotocin-induced diabetic rodents

Diabetes causes mitochondrial dysfunction in sensory neurons that may contribute to peripheral neuropathy. Ciliary neurotrophic factor (CNTF) promotes sensory neuron survival and axon regeneration and prevents axonal dwindling, nerve conduction deficits and thermal hypoalgesia in diabetic rats. In this study, we tested the hypothesis that CNTF protects sensory neuron function during diabetes through normalization of impaired mitochondrial bioenergetics. In addition, we investigated whether the NF-κB signal transduction pathway was mobilized by CNTF. Neurite outgrowth of sensory neurons derived from streptozotocin (STZ)-induced diabetic rats was reduced compared to neurons from control rats and exposure to CNTF for 24 h enhanced neurite outgrowth. CNTF also activated NF-κB, as assessed by Western blotting for the NF-κB p50 subunit and reporter assays for NF-κB promoter activity. Conversely, blockade of NF-κB signaling using SN50 peptide inhibited CNTF-mediated neurite outgrowth. Studies in mice with STZ-induced diabetes demonstrated that systemic therapy with CNTF prevented functional indices of peripheral neuropathy along with deficiencies in dorsal root ganglion (DRG) NF-κB p50 expression and DNA binding activity. DRG neurons derived from STZ-diabetic mice also exhibited deficiencies in maximal oxygen consumption rate and associated spare respiratory capacity that were corrected by exposure to CNTF for 24 h in an NF-κB-dependent manner. We propose that the ability of CNTF to enhance axon regeneration and protect peripheral nerve from structural and functional indices of diabetic peripheral neuropathy is associated with targeting of mitochondrial function, in part via NF-κB activation, and improvement of cellular bioenergetics.

Effect of IL-8 on integrin αvβ3 expression and cell motility via PI3K, Akt, and NF-κB-dependent pathway in human breast cells.

158 Background: αvβ3 integrin-overexpression in tumor associated vasculature is a marker of poor prognosis in breast cancer. A positive correlation between αvβ3 integrin and overexpression of Interleukin-8 (IL-8), a multifunctional cytokine associated with breast cancer aggressiveness was recently demonstrated. Here, we addressed the effects of IL-8 in migration and integrin expression in breast cancer cells. Methods: The IL-8 overexpressing breast cancer cells MDA-MB-231 and HS 578t were treated with IL-8 siRNA and evaluated for cells motility and alterations in the expression of αvβ3. Western Blotting to identify the status of PI3K, Akt and NF-κB. Results: In this study, we found that αvβ3 expression was decreased by IL-8 siRNA, especially the expression of β3. Treatment with αvβ3-blocking antibody LM609 and β3 siRNA significantly reduced invasion in the IL-8 overexpressing cells MDA-MB-231 and HS 578t. The phosphorylation of PI3K, Akt and NF-κB was abrogated after IL-8 siRNA treatment. The IL-8 siRNA can also decrease the binding of β3 promoter and the NF-κB-P65. Conclusions: These results indicate that IL-8 enhances the motility of highly invasive breast cancer cells by regulating αvβ3 expression partially through PI3K/Akt/ NF-κB signal transduction pathway.

Interleukin‐11 Increases Cell Motility and Up‐Regulates Intercellular Adhesion Molecule‐1 Expression in Human Chondrosarcoma Cells

Interleukin-11 (IL-11) was originally identified as the cytokine that could induce the proliferation of human cells. Recent studies have shown that IL-11 plays a critical role in tumor growth, angiogenesis, and metastasis. Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effects of IL-11 on human chondrosarcoma cells are largely unknown. Here, we found that IL-11 increased the migration and expression of intercellular adhesion molecule-1 (ICAM)-1 in human chondrosarcoma cells. We also found that human chondrosarcoma tissues had significant expression of the IL-11 which was higher than that in primary chondrocytes. The phosphatidylinositol 3-kinase (PI3K), Akt, and NF-κB pathways were activated by IL-11 treatment, and the IL-11-induced expression of ICAM-1 and migration activity were inhibited by the specific inhibitors and mutant forms of PI3K, Akt, and NF-κB cascades. Taken together, our results indicate that IL-11 enhanced the migration of the chondrosarcoma cells by increasing ICAM-1 expression through the IL-11Rα receptor, PI3K, Akt, and NF-κB signal transduction pathway.

The molecular mechanisms of the effect of Dexamethasone and Cyclosporin A on TLR4 /NF-κB signaling pathway activation in oral lichen planus

Toll-like receptors (TLRs) and the nuclear factor-kappa B (NF-κB) signaling transduction pathway play important roles in the pathogenesis of several chronic inflammatory diseases, but its function in oral lichen planus (OLP) remains unclear. In this study, we examined the expression of TLR4 and NF-κB-p65 and inflammatory cytokines TNF-α and IL-1β by immunohistochemistry in OLP tissues, and found that TLR4 and NF-κB-p65 were significantly upregulated in OLP compared to normal oral mucosa (P<0.05). We used keratinocytes HaCaT stimulated with lipopolysaccharide (LPS) to simulate the local OLP immune environment to some extent. RT-PCR and immunoblotting analyses showed significant activation of TLR4 and NF-κB-p65 in the circumstance of LPS-induced inflammatory response. The high expression of TLR4 and NF-κB-p65 are correlated with expression of cytokines TNF-α and IL-1β (P<0.05). We further showed that NF-κB could act as an anti-apoptotic molecule in OLP. We conclude that TLR4 and the NF-κB signaling pathway may interact with the perpetuation of OLP. Steroids and cyclosporine are effective in the treatment of symptomatic OLP. However, there was some weak evidence for the mechanism over Dexamethasone (DeX) and Cyclosporine A (CsA) for the palliation of symptomatic OLP. In the present study, we found that Dexamethasone and Cyclosporine A negatively regulated NF-κB signaling pathway under LPS simulation in HaCaT cells by inhibiting TLR4 expression, on the other hand, Cyclosporine A could inhibit HaCaT cell proliferation by the induction of the apoptosis of HaCaT cells to protect OLP from the destruction of epidermal cells effectively.

Dietary supplementation with geranylgeraniol suppresses lipopolysaccharide-induced inflammation via inhibition of nuclear factor-κB activation in rats

The isoprenoid geranylgeraniol (GGOH) inhibits nuclear factor-kappa B (NF-κB) activation in the liver, yet the mechanism remains unclear. We investigated the modulation and inhibition of lipopolysaccharide (LPS)-induced NF-κB signaling in the liver of rats fed a GGOH-supplemented diet. Rats were fed a diet supplemented with or without GGOH for 10 days. Rats were then intraperitoneally injected with 0.5 mg/kg LPS or vehicle (sterilized saline) and fasted for 18 h. Plasma levels of the inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, and the liver damage indicators alanine and aspartate aminotransferases (ALT and AST) were assessed. Liver mRNA and proteins were assayed for changes in NF-κB target genes and signal transduction genes. Rats fed a high-dose, GGOH-supplemented diet showed significantly lower levels of plasma inflammatory cytokines and ALT and AST activities. In the liver, GGOH significantly suppressed NF-κB activation and mRNA expression of its pro-inflammatory target genes. Furthermore, GGOH supplementation substantially suppressed mRNA expression of signal transducer genes upstream of the IκB kinase complex. Western blotting of liver extracts further demonstrated the substantial decrease in total IL-1 receptor-associated kinase 1 (IRAK1) and TNF receptor-associated factor 6 (TRAF6), leading to lower signal transduction and inhibition of NF-κB after LPS. A 10-day, high-dose, GGOH-supplemented diet was sufficient to inhibit LPS-induced inflammation and activation of NF-κB in rat livers. GGOH significantly modulated NF-κB signaling molecules, inhibiting its signal transduction and activation in the liver, thus protecting against liver damage.

Blockade of JNK and NFAT pathways attenuates orthopedic particle‐stimulated osteoclastogenesis of human osteoclast precursors and murine calvarial osteolysis

Particles released from orthopedic implants attract immune host defense cells to the bone-implant interface and contribute to development of inflammation. The inflammatory microenvironment supports recruitment and differentiation of osteoclasts, the primary culprit of osteolysis. Therefore, understanding the complex signals that contribute to osteoclastogenesis and osteolysis is a sensible approach to design strategies to inhibit bone loss. The signaling cascades that coordinate osteoclastogenesis have been widely investigated. These include MAP kinases, Akt/PI3K pathway, NF-κB signal transduction pathway, and NFAT pathway. We have recently reported that polymethylmethacrylate (PMMA) particles activate the NFAT pathway in murine osteoclast precursors and that NFAT inhibitors dose-dependently block PMMA-induced osteoclastogenesis. In the current study, we examined the role of JNK and NFATc1 in mice in response to PMMA particles using murine calvaria model. We show that locally administered MAPK/JNK inhibitor SP600125 and calcineurin/NFAT inhibitor cyclosporine-A effectively blocked PMMA-induced osteolysis in murine calvaria. To buttress the clinical relevance of JNK/NFATc1-based regulation of PMMA-induced osteoclastogenesis, we evaluated the effect of PMMA using human macrophages. We demonstrate that SP600125 and cyclosporine-A abolished particle-induced osteoclastogenesis in human osteoclast progenitors retrieved from patients undergoing total hip replacement. Thus JNK and NFATc1 appear to act as significant mediators of orthopedic particle-induced osteolysis in humans.

Serine/threonine acetylation of TGFβ-activated kinase (TAK1) by Yersinia pestis YopJ inhibits innate immune signaling

The Gram-negative bacteria Yersinia pestis, causative agent of plague, is extremely virulent. One mechanism contributing to Y. pestis virulence is the presence of a type-three secretion system, which injects effector proteins, Yops, directly into immune cells of the infected host. One of these Yop proteins, YopJ, is proapoptotic and inhibits mammalian NF-κB and MAP-kinase signal transduction pathways. Although the molecular mechanism remained elusive for some time, recent work has shown that YopJ acts as a serine/threonine acetyl-transferase targeting MAP2 kinases. Using Drosophila as a model system, we find that YopJ inhibits one innate immune NF-κB signaling pathway (IMD) but not the other (Toll). In fact, we show YopJ mediated serine/threonine acetylation and inhibition of dTAK1, the critical MAP3 kinase in the IMD pathway. Acetylation of critical serine/threonine residues in the activation loop of Drosophila TAK1 blocks phosphorylation of the protein and subsequent kinase activation. In addition, studies in mammalian cells show similar modification and inhibition of hTAK1. These data present evidence that TAK1 is a target for YopJ-mediated inhibition.

Role of β2-adrenoceptor-β-arrestin2-nuclear factor-κB signal transduction pathway and intervention effects of oxymatrine in ulcerative colitis

To investigate the β2-adrenoceptor (β2AR)-β-arrestin2-nuclear factor-κB (NF-κB) signal transduction pathway and the intervention effects of oxymatrine in a rat model of ulcerative colitis. Forty SD rats were randomly divided into four groups, which included the normal control group, the model group, the mesalazine group and the oxymatrine treatment group, with 10 rats per group. Experimental colitis induced with trinitrobenzene sulfonic acid (TNBS) was established in each group except the normal control group. The rats in the oxymatrine treatment group were treated with intramuscular injection of oxymatrine 63 mg/(kg·d) for 15 days and the rats in the mesalazine group were treated with mesalazine solution 0.5 g/(kg·d) by gastric lavage for 15 days. The rats in the normal control group and model group were treated with 3 mL water by gastric lavage for 15 days. Diarrhea and bloody stool were carefully observed. Histological changes in colonic tissue were examined on day 7 in 2 rats per group that were randomly selected. The expression of β2AR, β-arrestin2 and NF-κB p65 in colon tissue and spleen lymphocytes were detected with immunohistochemistry and Western immunoblotting techniques on day 16 after fasting for 24 h. Six rats died of lavage with 2 each in the normal control, the model group and the mesalazine group; and were not included in the analysis. The rats in the model group suffered from looser stool and bloody purulent stool after modeling. But in the oxymatrine and mesalazine groups, looser stool and bloody purulent stool reduced after treatment. And the colonic wall in the model group was thickened and the colon length shortened. The colon mucosa was congested in multiple areas with edema, erosion, superficial or linear ulcer and scar formation, while the intestinal mucosa injury reduced in the mesalazine and oxymatrine groups (P<0.01). In colonic mucosa and in spleen lymphocytes, compared with the normal control group, the expression of NF-κBp65 were significantly increased (P<0.01) in the model group while the expressions of β 2AR and β-arrestin2 were significantly decreased (P<0.01). Compared with the model group, the expression of NF-κ Bp65 was significantly decreased in the mesalazine group (P<0.01) and oxymatrine treatment group (P<0.01) while the expressions of β2AR and β-arrestin2 were significantly increased (P<0.01). There were no statistically significant differences in the expression of β2AR, β-arrestin2 and NF-κBp65 between the mesalazine group and oxymatrine group (P>0.05). The β2AR-β-arrestin2-NF-κB signal transduction pathway participated in the pathologic course of ulcerative colitis. Oxymatrine attenuated ulcerative colitis through regulating the β2AR-β-arrestin2-NF-κB signal transduction pathway.

The bone anabolic carotenoid p-hydroxycinnamic acid promotes osteoblast mineralization and suppresses osteoclast differentiation by antagonizing NF-κB activation

Numerous plant derived nutritional factors including p-hydroxycinnamic acid (HCA), a member of the carotenoid family, have long been held to possess bone protective properties. Studies in animals have provided a mechanistic basis for these observations by demonstrating the capacity of HCA to promote bone formation and suppress bone resorption in vivo. However, the molecular mechanism by which HCA achieves these effects remains unclear. We have demonstrated that a centralized mechanism by which several other nutritional factors achieve similar effects is through modulation of the nuclear factor-κB (NF-κB) signal transduction pathway. NF-κB activation is essential for osteoclast formation and resorption but potently antagonizes osteoblast differentiation and mineralization. In this study we demonstrate that HCA does indeed antagonize the activation of NF-κB by the key osteoclastogenic cytokine receptor activator of NF-κB (RANKL) in RAW264.7 osteoclast precursors, suppressing their differentiation into osteoclasts. Furthermore, HCA augmented the in vitro differentiation of MC3T3 preosteoblastic cells into mineralizing osteoblasts and relieved the inhibitory action of tumor necrosis factor-α (TNF-α)-induced NF-κB signaling on transforming growth factor-β (TGF-β)- or bone morphogenetic protein-2 (BMP-2)-induced Smad activation, an important pathway in osteoblast commitment and differentiation. Our data provide a mechanism to explain the dual pro-anabolic and anti-catabolic activities of HCA.

The interaction between hepatitis B virus X protein and AIB1 oncogene is required for the activation of NFκB signal transduction

We identified the interaction between HBV X (HBx) protein and the oncogene AIB1 (amplified in breast cancer 1). A serine/proline motif (SSPSPS) in HBx was found to be required for the interaction. Two LXD motifs [LLXX(X)L, X means any amino acids], LLRNSL and LLDQLHTLL in AIB1 were also found to be involved in the HBx–AIB1 interaction. The HBx–AIB1 interaction was important for the activation of NFκB signal transduction, the HBx mutant that did not interact with AIB1showed dramatically lower NFκB activation activity than the WT HBx. These findings contribute to the new understanding on signal transduction activation mechanisms of HBx.

Inhibition of Type I Interferon-Mediated Antiviral Action in Human Glioma Cells by the IKK Inhibitors BMS-345541 and TPCA-1

The nuclear factor-kappa B (NFκB) signal transduction pathway plays an important role in immunity, inflammation, cell growth, and survival. Since dysregulation of this pathway results in high, constitutive NFκB activation in various cancers and immune disorders, the development of specific drugs to target this pathway has become a focus for treating these diseases. NFκB regulates various aspects of the cellular response to interferon (IFN). However, the role of the upstream regulator of the NFκB signaling pathway, the inhibitor of κB kinase (IKK) complex, on IFN function has not been examined. In the present study, we examined the effects of 2 IKK inhibitors, N-(1,8-Dimethylimidazo[1,2-a]quinoxalin-4-yl)-1,2-ethanediamine hydrochloride (BMS-345541) and 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1), on IFN action in several human glioma cell lines. IKK inhibitors inhibit glioma cell proliferation, as well as TNF-induced RelA (p65) nuclear translocation and NFκB-dependent IL8 gene expression. Importantly, BMS-345541 and TPCA-1 differentially inhibit IFN-induced gene expression, completely suppressing MX1 and GBP1 gene expression, while having only a minor effect on ISG15 expression. Furthermore, these IKK inhibitors displayed marked differences in blocking IFN-induced antiviral action against cytopathic effects and replication of vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV). Our results show that the IKK complex plays an important function in IFN-induced gene expression and antiviral activity. Since VSV and EMCV are oncolytic viruses used in cancer therapy, our results indicate the potential synergy in combining IKK inhibitors with oncolytic viruses.

Abstract 4737: Resveratrol targeting of carcinogen-induced brain endothelial cell inflammation biomarkers MMP-9 and COX-2 is Sirt1-independent

Abstract Background: The occurrence of a functional relationship between the release of metalloproteinases (MMPs) and the expression of cyclooxygenase (COX)-2, two inducible pro-inflammatory biomarkers with important pro-angiogenic effects, has recently been inferred. While brain endothelial cells play an essential role as structural and functional components of the blood-brain barrier (BBB), increased BBB breakdown is thought to be linked to neuroinflammation. Chemopreventive mechanisms targeting both MMPs and COX-2 however remain poorly investigated. In this study, we evaluated the pharmacological targeting of Sirt1 by the diet-derived and antiinflammatory polyphenol resveratrol. Methods: Total RNA, cell lysates, and conditioned culture media from human brain microvascular endothelial cells (HBMEC) were analyzed using qRT-PCR, immunoblotting, and zymography respectively. Results: Tissue scan microarray analysis of grade I-IV brain tumours cDNA revealed increased gene expression of Sirt-1 from grade I-III but surprisingly not in grade IV brain tumours. HBMEC were treated with a combination of resveratrol and phorbol 12-myristate 13-acetate (PMA), a carcinogen known to increase MMP-9 and COX-2 through NF-κB. We found that resveratrol efficiently reversed the PMA-induced MMP-9 secretion and COX-2 expression. Gene silencing of Sirt1, a critical modulator of angiogenesis and putative target of resveratrol, did not lead to significant reversal of MMP-9 and COX-2 inhibition. Decreased resveratrol inhibitory potential of carcinogen-induced IαB phosphorylation in siSirt1-transfected HBMEC was however observed. Conclusion: Our results suggest that resveratrol may prevent BBB disruption during neuroinflammation by inhibiting MMP-9 and COX-2 and act as a pharmacological NF-κB signal transduction inhibitor independent of Sirt1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4737. doi:1538-7445.AM2012-4737