Dependent NF-κB Activation Research Articles

Z-100, extracted from Mycobacterium tuberculosis strain Aoyama B, promotes TNF-α production via nucleotide-binding oligomerization domain containing 2 (Nod2)-dependent NF-κB activation in RAW264.7 cells

Macrophages are a major component of the innate immune system, and the cytokines they secrete are involved in antitumor responses. Z-100 is obtained from hot-water extract of human-type Mycobacterium tuberculosis strain Aoyama B and activates the innate immune response. However, while Z-100 is known to modulate macrophage activity, the mechanism behind this modulation is not fully understood. We evaluated the effects of Z-100 on the murine macrophage cell line RAW264.7. Tumor necrosis factor-alpha (TNF-α) production from RAW264.7 cells was strongly induced by Z-100 and interferon-gamma (IFN-γ) stimulation but only weakly induced by Z-100 alone. Quantitative gene expression analysis showed that nucleotide-binding oligomerization domain containing 2 (Nod2) expression was up-regulated by IFN-γ treatment in RAW264.7 cells while Z-100-induced TNF-α production was attenuated by Nod2 gene silencing. Further, componential analysis demonstrated that muramic acid and amino acids distinctive of muramyl dipeptide (MDP) were contained within Z-100 and Z-100Fr I, the low-molecular-weight fraction containing components <3kDa in size. In addition, Z-100Fr I enhanced TNF-α production in RAW264.7 cells and promoted NOD2-dependent nuclear factor-kappa B (NF-κB) activation in murine NOD2-expressing SEAP reporter HEK293 (HEK-Blue-mNOD2) cells. Taken together, these results suggest that Z-100 contains MDP-like molecules and augments NF-κB signaling via the direct activation of Nod2 in macrophages, which might be one mechanism driving the innate immune responses induced by Z-100 in cancer immunotherapy.

Sqstm1 Is Required to Retain Hematopoietic Stem Cell/ Progenitors As a Negative Regulator of Macrophage-Dependent Inflammatory Signaling in the Bone Marrow Osteoblastic Niche

In the bone marrow (BM), hematopoietic stem cells and progenitors (HSC/P) reside in specific anatomical niches. Among these niches, a functional osteoblast (Ob)-macrophage (MΦ) niche has been described where Ob and MΦ (so called “osteomacs”) are in direct relationship. A connection between innate immunity surveillance and traffic of hematopoietic stem cells/progenitors (HSC/P) has been demonstrated but the regulatory signals that instruct immune regulation from MΦ and Ob on HSC/P circulation are unknown. The adaptor protein sequestosome 1 (Sqstm1), contains a Phox bemp1 (PB1) domain which regulates signal specificities through PB1-PB1 scaffolding and processes of autophagy. Using microenvironment and osteoblast-specific mice deficient in Sqstm1, we discovered that the deficiency of Sqstm1 results in macrophage contact-dependent activation of Ob IKK/NF-κB, in vitro and in vivo repression of Ccl4 (a CCR5 binding chemokine that has been shown to modulate microenvironment Cxcl12-mediated responses of HSC/P), HSC/P egress and deficient BM homing of wild-type HSC/P. Interestingly, while Ccl4 expression is practically undetectable in wild-type or Sqstm1-/- Ob, primary Ob co-cultured with wild-type BM-derived MΦ strongly upregulate Ccl4 expression, which returns to normal levels upon genetic deletion of Ob Sqstm1. We discovered that MΦ can activate an inflammatory pathway in wild-type Ob which include upregulation of activated focal adhesion kinase (p-FAK), IκB kinase (IKK), nuclear factor (NF)-κB and Ccl4 expression through direct cell-to-cell interaction. Sqstm1-/- Ob cocultured with MΦ strongly upregulated p-IKBα and NF-κB activity, downregulated Ccl4 expression and secretion and repressed osteogenesis. Forced expression of Sqstm1, but not of an oligomerization-deficient mutant, in Sqstm1-/- Ob restored normal levels of p-IKBα, NF-κB activity, Ccl4 expression and osteogenic differentiation, indicating that Sqstm1 dependent Ccl4 expression depends on localization to the autophagosome formation site. Finally, Ob Sqstm1 deficiency results in upregulation of Nbr1, a protein containing a PB1 interacting domain. Combined deficiency of Sqstm1 and Nbr1 rescues all in vivo and in vitro phenotypes of Sqstm1 deficiency related to osteogenesis and HSC/P egression in vivo. Together, this data indicated that Sqstm1 oligomerization and functional repression of its PB1 binding partner Nbr1 are required for Ob dependent Ccl4 production and HSC/P retention, resulting in a functional signaling network affecting at least three cell types. A functional 'MΦ-Ob niche’ is required for HSC/P retention where Ob Sqstm1 is a negative regulator of MΦ dependent Ob NF-κB activation, Ob differentiation and BM HSC/P traffic to circulation. DisclosuresStarczynowski:Celgene: Research Funding. Cancelas:Cerus Co: Research Funding; P2D Inc: Employment; Terumo BCT: Research Funding; Haemonetics Inc: Research Funding; MacoPharma LLC: Research Funding; Therapure Inc.: Consultancy, Research Funding; Biomedical Excellence for Safer Transfusion: Research Funding; New Health Sciences Inc: Consultancy.

Effect of exogenous MCP-1 on TLR-2 neutralized murine macrophages and possible mechanisms of CCR-2/TLR-2 and MCP-1 signalling during Staphylococcus aureus infection

It has been reported that Staphylococcus aureus survives within macrophages by hijacking host cell surface Toll-like receptor-2 (TLR-2). Moreover, S. aureus infection induced activation of TLR-2 has been reported to downregulate the expression of CC-chemokine receptor-2 (CCR-2), a receptor essential for binding of chemokines to propagate phagocytosis. Thus, we hypothesized that prior blocking of TLR-2 may help normal expression of CCR-2 on cell surface; thereby, administration of exogenous MCP-1 (a CCR-2 ligand) to bind to its free receptors might result in activation of downstream inflammatory signalling cascade. In order to address this, we compared the ability of S. aureus to modulate CCR-2 expression in TLR-2 free or neutralized macrophages in presence or absence of exogenous MCP-1 and associated downstream signalling. Exogenous MCP-1 by interacting CCR-2 leads to the release of nitric oxide and ROS that are important for bacterial clearance. In this experimental setup, the possible molecular pathway connecting an increase in proinflammatory cytokine levels with increased ROS/NO production, and therefore increased killing activity, possibly by involving either MyD88 dependent or RhoA GTPases dependent NF-κB activation or endogenous synthesis of MCP-1, independent of TLR-2-MyD88 pathway. Thus, induction of CCR-2/MCP-1 signalling by macrophages depending on the availability of MCP-1 during S. aureus infection may be important for regulation of septic shock by induction of reactive oxygen species and various cytokines.

Ficolin-3 activity towards the opportunistic pathogen, Hafnia alvei

Ficolin-3 (also called H-ficolin or Hakata antigen) is a complement-activating pattern recognition molecule, possessing a fibrinogen-like domain involved in carbohydrate binding. Amongst human ficolins, Ficolin-3 has the highest concentration in serum and is the most potent lectin pathway activator in vitro. Evidence for its physiological function is sparse, although its deficiency has been suggested to increase susceptibility to infections. The specificity of Ficolin-3 is poorly characterized and currently few ligands are known. Here we report agglutination of Hafnia alvei, a Gram-negative enteric commensal bacterium and opportunist pathogen, in the presence of recombinant Ficolin-3 and calcium. Ficolin-3 also augmented phagocytosis of H. alvei by macrophages and displayed bactericidal activity. Additionally, Ficolin-3 inhibited host cells’ response to TLR4/MD-2/CD14-LPS dependent NF-κB activation. This is the first demonstration of protective activity of Ficolin-3 against a human bacterial pathogen. Although human Ficolin-3 does not recognise and bind to common pathogenic bacteria, it could be an important component of innate immunity providing protection, for example, from commensal flora that can cause extraintestinal, opportunistic infections.

S100A1 is released from ischemic cardiomyocytes and signals myocardial damage via Toll-like receptor 4.

Members of the S100 protein family have been reported to function as endogenous danger signals (alarmins) playing an active role in tissue inflammation and repair when released from necrotic cells. Here, we investigated the role of S100A1, the S100 isoform with highest abundance in cardiomyocytes, when released from damaged cardiomyocytes during myocardial infarction (MI). Patients with acute MI showed significantly increased S100A1 serum levels. Experimental MI in mice induced comparable S100A1 release. S100A1 internalization was observed in cardiac fibroblasts (CFs) adjacent to damaged cardiomyocytes. In vitro analyses revealed exclusive S100A1 endocytosis by CFs, followed by Toll-like receptor 4 (TLR4)-dependent activation of MAP kinases and NF-κB. CFs exposed to S100A1 assumed an immunomodulatory and anti-fibrotic phenotype characterized i.e. by enhanced intercellular adhesion molecule-1 (ICAM1) and decreased collagen levels. In mice, intracardiac S100A1 injection recapitulated these transcriptional changes. Moreover, antibody-mediated neutralization of S100A1 enlarged infarct size and worsened left ventricular functional performance post-MI. Our study demonstrates alarmin properties for S100A1 from necrotic cardiomyocytes. However, the potentially beneficial role of extracellular S100A1 in MI-related inflammation and repair warrants further investigation.

Acute sleep deprivation enhances post-infection sleep and promotes survival during bacterial infection in Drosophila.

Sleep is known to increase as an acute response to infection. However, the function of this behavioral response in host defense is not well understood. To address this problem, we evaluated the effect of acute sleep deprivation on post-infection sleep and immune function in Drosophila. Laboratory. Drosophila melanogaster. Flies were subjected to sleep deprivation before (early DEP) or after (late DEP) bacterial infection. Relative to a non-deprived control, flies subjected to early DEP had enhanced sleep after infection as well as increased bacterial clearance and survival outcome. Flies subjected to late DEP experienced enhanced sleep following the deprivation period, and showed a modest improvement in survival outcome. Continuous DEP (early and late DEP) throughout infection also enhanced sleep later during infection and improved survival. However, improved survival in flies subjected to late or continuous DEP did not occur until after flies had experienced sleep. During infection, both early and late DEP enhanced NFκB transcriptional activity as measured by a luciferase reporter (κB-luc) in living flies. Early DEP also increased NFκB activity prior to infection. Flies that were deficient in expression of either the Relish or Dif NFκB transcription factors showed normal responses to early DEP. However, the effect of early DEP on post-infection sleep and survival was abolished in double mutants, which indicates that Relish and Dif have redundant roles in this process. Acute sleep deprivation elevated NFκB-dependent activity, increased post-infection sleep, and improved survival during bacterial infection.

RSF1 Is a Positive Regulator of NF-κB–Induced Gene Expression Required for Ovarian Cancer Chemoresistance

Overexpression or amplification of the RSF1 gene has been associated with poor prognosis in various human cancers, including ovarian cancer. In previous work, RSF1 was identified as an amplified gene that facilitated the development of paclitaxel-resistant ovarian cancer. In the present study, we further demonstrated that RSF1 expression inversely correlated with paclitaxel response in patients with ovarian cancer and the mouse xenograft model. In addition, RSF1-overexpressing paclitaxel-resistant ovarian cancer cell lines were found to express elevated levels of genes regulated by NF-κB, including some involved with the evasion of apoptosis (CFLAR, XIAP, BCL2, and BCL2L1) and inflammation (PTGS2). In addition, ectopic expression of RSF1 using Tet-off inducible SKOV3 cells significantly enhanced NF-κB-dependent gene expression and transcriptional activation of NF-κB. An RSF1 knockdown using short hairpin RNAs suppressed these same pathways. Moreover, pretreatment with NF-κB inhibitors or downregulation of NF-κB-regulated gene expression considerably enhanced paclitaxel sensitivity in RSF1-overexpressing OVCAR3 and/or RSF1-induced SKOV3 cells. A coimmunoprecipitation assay revealed that RSF1 interacts with NF-κB and CREB-binding protein, a ubiquitous coactivator for NF-κB. Recruitment of RSF1 to the NF-κB binding element in the PTGS2 and XIAP promoters was demonstrated by the chromatin immunoprecipitation assay. Furthermore, hSNF2H, a well-known binding partner of RSF1, was partially involved in the interaction between RSF1 and NF-κB. Taken together, these data suggest that RSF1 may function as a coactivator for NF-κB, consequently augmenting expression of genes necessary for the development of chemoresistance in ovarian cancer cells.

Characteristic and Functional Analysis of Toll-like Receptors (TLRs) in the lophotrocozoan, Crassostrea gigas, Reveals Ancient Origin of TLR-Mediated Innate Immunity

The evolution of TLR-mediated innate immunity is a fundamental question in immunology. Here, we report the characterization and functional analysis of four TLR members in the lophotrochozoans Crassostrea gigas (CgTLRs). All CgTLRs bear a conserved domain organization and have a close relationship with TLRs in ancient non-vertebrate chordates. In HEK293 cells, every CgTLR could constitutively activate NF-κB responsive reporter, but none of the PAMPs tested could stimulate CgTLR-activated NF-κB induction. Subcellular localization showed that CgTLR members have similar and dual distribution on late endosomes and plasma membranes. Moreover, CgTLRs and CgMyD88 mRNA show a consistent response to multiple PAMP challenges in oyster hemocytes. As CgTLR-mediated NF-κB activation is dependent on CgMyD88, we designed a blocking peptide for CgTLR signaling that would inhibit CgTLR-CgMyD88 dependent NF-κB activation. This was used to demonstrate that a Vibrio parahaemolyticus infection-induced enhancement of degranulation and increase of cytokines TNF mRNA in hemocytes, could be inhibited by blocking CgTLR signaling. In summary, our study characterized the primitive TLRs in the lophotrocozoan C . gigas and demonstrated a fundamental role of TLR signaling in infection-induced hemocyte activation. This provides further evidence for an ancient origin of TLR-mediated innate immunity.

MicroRNA miR-146a is a critical suppressor of arthritis in the mouse model of Lyme Disease (P1275)

Abstract Lyme Disease is caused by infection with the spirochete Borrelia burgdorferi, and is the most common vector-borne disease in the United States. Often, infection leads to acute arthritis in humans. Our lab and others have exploited the mouse model system to elucidate the mechanical mechanisms of Lyme Disease development. Because miRNAs have been shown to be important regulators of inflammatory and immune responses, we performed a microarray screen for expression of miRNAs in joint tissue from three infected mouse strains. This screen identified upregulation of miR-146a, a key negative regulator of NF-κB signaling. Others and we have previously demonstrated that Toll-like receptor 2/MyD88 dependent activation of NF-κB is a major regulator of immune response in mice and humans, but determining its role in arthritis development has remained elusive. We show that miR-146a plays a critical nonreduntant role in modulating Borrelia-induced NF- κB response to infection, and mice lacking this miRNA develop more severe arthritis without compromising immune response. Furthermore, miR-146a-/- mice have elevated transcript levels of NF-κB-regulated cytokines and chemokines in joint tissue, as well as increased myeloid cell infiltration, linking NF-κB dysregulation to arthritis development. Together, these data show that miR-146a is required for suppression of Lyme Arthritis, and provides a much-needed model for studying the role of NF-κB activation in arthritis development.

Advanced Oxidation Protein Products Induce Inflammatory Response in Fibroblast-Like Synoviocytes through NADPH Oxidase -Dependent Activation of NF-κB

Background: Advanced oxidation protein products (AOPPs), a marker of oxidative stress, are prevalent in many kinds of disorders. Rheumatoid arthritis (RA), mainly resulting from the dysfunction of fibroblast-like synoviocytes (FLSs), is related to oxidative stress. Although the increased levels of AOPPs in RA patients were reported, the effect of AOPPs on FLSs function still remains unclear. Therefore, our study aims to investigate whether AOPPs have an effect on the inflammatory response of FLSs in vitro. Methods: FLSs obtained from both knees of rats were treated with or without AOPPs-modified rat serum albumin (AOPPs-RSA) in vitro. The mRNA and protein expression of tumor necrosis factor (TNF)-a, interleukin(IL)-1ß, matrix metalloproteinases(MMP)-3, MMP-13 and vascular endothelial growth factor (VEGF) were measured by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. Reactive oxygen species (ROS) generation was detected by fluorescent microscope and fluorescence microplate reader. Immunoprecipitation, Co-Immunoprecipitation and western blot were performed to examine the activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nuclear factor kappa B (NF-κB). Results: Exposure of FLSs to AOPPs upregulated the mRNA and protein expression of TNF-a, IL-1ß, MMP-3, MMP-13 and VEGF in a concentration dependent manner. AOPPs treatment triggered ROS production in FLSs, which was significantly abolished by ROS scavenger N-acetyl-L-cysteine (NAC), superoxide dismutase (SOD), NADPH oxidase inhibitors diphenyleneiodonium (DPI) and apocynin. Challenged AOPPs induced phosphorylation of p47^phox, triggered an interaction between p47^phox, p22^phox and gp91^phox, and significantly upregulated expression of NADPH oxidase subunits p47^phox, p22^phox and gp91^phox. IκB degradation and nuclear translocation of NF-κB p65 induced by AOPPs were significantly blocked by SOD, NAC, DPI and apocynin. Conclusions: These data indicate that AOPPs induce inflammatory response in FLSs is medicated through NADPH oxidase-dependent activation of NF-κB.

Activation of the human immune system via toll‐like receptors by the oncolytic parvovirus H‐1

This study aimed to investigate the function of toll-like receptors (TLRs) during oncolytic parvovirus H-1 (H-1PV)-induced human immune responses. First, the role of TLRs in the activation of the NFκB transcription factor was characterized; second, the immunologic effects of H-1PV-induced tumor cell lysates (TCL) on human antitumor immune responses were evaluated. A human ex vivo model was used to study immune responses with dendritic cells (DCs). Human embryonic kidney cells (HEK293) transfected to stably express TLRs were used as potential human DC equivalents to further investigate the role of specific TLRs during immune activation. TLR3 and TLR9 were activated by H-1PV infection, which correlated with NFκB translocation to the nucleus and a reduced cytoplasmic IκB expression. Using a TLR-signaling reporter plasmid (pNiFty-Luc), NFκB activity was increased following H-1PV infection. In addition, human DCs coincubated with H-1PV-induced TCL demonstrated increased TLR3 and TLR9 expression. These data suggest that H-1PV-induced TCL stimulate human DCs at least in part through TLR-dependent signaling pathways. Thus, DC maturation occurred through exposure to H-1PV-induced TCL through TLR-signaling leading to NFκB-dependent activation of the adaptive immune system as indicated by the increased expression of CD86, TLR3 and TLR9. Furthermore, the transcription of various cytokines indicates the activation of immune response, therefore the production of the proinflammatory cytokine TNF-α was determined. Here, H-1PV-induced TCL significantly enhanced the TNF-α level by DCs after coculture. H-1PV oncolytic virotherapy enhances immune priming by different effects on DCs and generates antitumor immunity. These findings potentially offer a new approach to tumor therapy.

Lenalidomide Efficacy in Activated B-Cell-Like Subtype Diffuse Large B-Cell Lymphoma Is Dependent Upon IRF4 and Cereblon Expression

AbstractAbstract 3287 Background:Durable responses with lenalidomide monotherapy have been reported in patients with non-Hodgkin lymphoma. In relapsed/refractory diffuse large B-cell lymphoma (DLBCL), higher responses were observed in the activated B-cell-like (ABC) subtype than in the germinal centre B-cell (GCB)-like subtype (Czuczman, et al. British Journal of Haematology, 2011, 154, 477–481). Herein, the molecular mechanisms involved in the differential efficacy of lenalidomide in DLBCL subtypes were investigated. Methods:A panel of DLBCL cell lines, with 5 of ABC-subtype and 11 of non-ABC subtype, was collected and cell of origin subtype was confirmed based on literature, molecular and genetic analysis. The direct antiproliferative effect of lenalidomide on DLBCL cells was assessed using the 3H-thymidine incorporation assay and apoptosis analysis. The molecular mechanisms involved in the antiproliferative efficacy of lenalidomide in DLBCL subtypes were investigated by western blot, immunohistochemistry (IHC) and qRT-PCR analysis of key signaling events during B-cell receptor (BCR)-dependent NF-κB activation. The critical roles of interferon regulatory factor 4 (IRF4), and cereblon (CRBN) in lenalidomide efficacy were established by knock-in or knock-down of these proteins in sensitive ABC cells. Finally, a mouse xenograft model was used to confirm the antitumor effect of lenalidomide and the relevance of the molecular mechanism involved. Results:Using DLBCL cell lines, lenalidomide treatment was found to preferentially suppress proliferation of ABC-DLBCL cells in vitro at a concentration range of 0.01–100 μM (the median plasma concentration at Cmax for patients receiving 25 mg lenalidomide is 2.2 μM) and delay tumor growth in a human tumor xenograft model of OCI-Ly10 cells (lenalidomide 3–30 mg/kg, p.o. qdX28), with minimal effect on non-ABC-DLBCL cells.This tumoricidal effect of lenalidomide was associated with downregulation of IRF4, a survival factor in ABC-DLBCL cells. Treatment with lenalidomide for 1–3 days, similar to the inhibitors of PKCb and MALT1 (LY-333,531 and z-VRPR-fmk, respectively), was found to significantly (p<0.05) downregulate IRF4 protein levels in sensitive cell lines such as OCI-Ly10 and U2932. IRF4 inhibition by lenalidomide reduced CARD11-BCL-10-MALT1 complex activity of ABC-DLBCL cells (as measured by BCL-10 cleavage) and resulted in downregulation of B-cell receptor (BCR)-dependent NF-κB activity. An NF-κB-driven luciferase assay revealed that lenalidomide (1 μM) inhibited transcriptional activity of NF-κB up to 56% in the sensitive ABC-DLBCL cell lines OCI-Ly10 (p <0.05) and U2932 (p <0.01) after 2-day drug treatment. Lenalidomide also significantly (p <0.05) inhibited DNA binding by Rel A/p65, p50 and c-rel/p70 in 4 lines of ABC cells. While IRF4-specific siRNA mimicked the effects of lenalidomide reducing NF-κB activation, IRF4 overexpression conferred cell resistance to lenalidomide, indicating the crucial role of IRF4 inhibition in lenalidomide efficacy in ABC DLBCL.Furthermore, knockdown of CRBN in OCI-Ly10 (p <0.05) and U2932 (p <0.01) conferred resistance to lenalidomide as demonstrated by the abrogation of the inhibitory effects of lenalidomide on IRF4 expression, BCL-10 cleavage, NF-κB activity, and proliferation of these cells, whereas the activity of inhibitors to PKC β and IKKα/β (LY-333,531 and CC-415501, respectively) remained unaffected. These data indicate that antitumor effects of lenalidomide on ABC-DLBCL cells require the presence of cereblon. Conclusions:These data may provide a mechanism for the preferential efficacy of lenalidomide in ABC-DLBCL observed in clinical studies. These findings suggest that lenalidomide has direct antitumor activity against DLBCL cells, preferentially ABC-DLBCL cells, by blocking IRF4 expression and the BCR-NF-κB signaling pathway in a cereblon-dependent manner (also see Figure below). [Display omitted] Disclosures:Zhang:Celgene Corp: Employment, Equity Ownership. Kosek:Celgene Corp: Employment, Equity Ownership. Wang:Celgene Corporation: Employment, Equity Ownership. Heise:Celgene Corporation: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Chopra:Celgene Corporation: Employment, Equity Ownership.

The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-κB

Several members of the NLR family of sensors activate innate immunity. In contrast, we found here that NLRC3 inhibited Toll-like receptor (TLR)-dependent activation of the transcription factor NF-κB by interacting with the TLR signaling adaptor TRAF6 to attenuate Lys63 (K63)-linked ubiquitination of TRAF6 and activation of NF-κB. We used bioinformatics to predict interactions between NLR and TRAF proteins, including interactions of TRAF with NLRC3. In vivo, macrophage expression of Nlrc3 mRNA was diminished by the administration of lipopolysaccharide (LPS) but was restored when cellular activation subsided. To assess biologic relevance, we generated Nlrc3(-/-) mice. LPS-treated Nlrc3(-/-) macrophages had more K63-ubiquitinated TRAF6, nuclear NF-κB and proinflammatory cytokines. Finally, LPS-treated Nlrc3(-/-) mice had more signs of inflammation. Thus, signaling via NLRC3 and TLR constitutes a negative feedback loop. Furthermore, prevalent NLR-TRAF interactions suggest the formation of a 'TRAFasome' complex.

Another Selective Path to Destruction

Selective autophagy of Bcl10 inhibits T cell receptor–dependent activation of the transcription factor NF-κB.

Sharpin Contributes to TNFα Dependent NFκB Activation and Anti-Apoptotic Signalling in Hepatocytes

TNFα stimulates both pro- and anti-apoptotic signalling in hepatocytes. Anti-apoptotic signalling depends on a cascade of ubiquitylation steps leading to NFκB activation. Using Sharpin-deficient mice, we show that the ubiquitin binding protein Sharpin interacts with Hoip, an E3 ligase which generates linear ubiquitin chains. Sharpin-deficiency sensitized hepatocytes to induction of apoptosis by TNFα even in the absence of transcriptional inhibition. TNFα induced activation of NFκB was strongly reduced in hepatocytes from Sharpin-deficient mice, due to reduced and delayed phosphorylation and degradation of IκBα. Injection of TNFα-inducing lipopolysaccharides led to strongly exacerbated liver damage and premature death in Sharpin-deficient mice. Our findings point to an essential role of Sharpin in linear ubiquitin chain formation, NFκB activation, and protection of the liver against inflammatory damaging signals.

Essential role of Sharpin in TNFα dependent NFκB activation in primary hepatocytes

Backgrounds&aims: Tumour necrosis factor (TNF)-α triggers cell survival and apoptosis in hepatocytes. The cell survival pathway depends on post-translational modifications (PTM) leading to activation of NFκB. An essential PTM is ubiquitination that regulates NFκB activation at different steps. One important ubiquitination step depends on the linear ubiquitin chain assembly complex (LUBAC) that is responsible for ubiquitination of IKKγ (NEMO). As recently shown, LUBAC consists of HOIP, HOIL–1L and Sharpin. We analysed TNFα dependent NFκB activation in Sharpin-deficient primary hepatocytes (PH).

New tigliane diterpenes from Euphorbia grandicornis

Phorbol esters are specific acylated polyhydroxy Δ1,6-tigliane diterpenes, which occur in plants of Euphorbiaceae and Thymelaeaceae family. Phorbol derivatives are promising candidates of drug development for HIV therapy, because they reactivate HIV-1 latency by protein kinase C dependent NF-κB activation, and avoid the new infection of CD4+ cells by down-regulating the expression of the HIV 1 receptors [1,2]. Recently we reported the isolation and structure determination of three phorbol analogues from Euphorbia grandicornis Goebel, a succulent cactiform South African plant whose phytochemical investigation has not been reported previously [3]. In continuation of our investigations we report now on the isolation and structural characterization of further six diterpene polyesters. The chloroform-soluble phase of a methanol extract of the fresh aerial parts of E. grandicornis was fractionated by column chromatography on polyamide, then by vacuum liquid chromatography on silica gel. Selected fractions from these separations were further purified by preparative TLC and HPLC to yield six pure compounds, including three new natural products. The structure elucidation was performed by means of HRESIMS and advanced two-dimensional NMR methods, including 1H NMR, JMOD, 1H-1H COSY, NOESY, HSQC, and HMBC experiments. The three known compounds were identified as 12 deoxyphorbol mono- and diesters, acylated with acetic, isobutyric and 2-methylbutyric acids. All of the three new compounds are 12-deoxy-16-hydroxyphorbols; one of them was found to have an unusual parent alcohol with 5-ene-7-ol function.

Receptor-Mediated Endocytosis Is a Trojan Horse in Light-Chain Nephrotoxicity

Receptor-Mediated Endocytosis Is a Trojan Horse in Light-Chain Nephrotoxicity

Scaffolding Adaptor Protein Gab1 Is Required for TLR3/4- and RIG-I–Mediated Production of Proinflammatory Cytokines and Type I IFN in Macrophages

RIG-I-like helicases and TLRs are critical sensors in the induction of type I IFN and proinflammatory cytokines to initiate innate immunity against invading pathogens. However, the mechanisms for the full activation of TLR and RIG-I-triggered innate response remain to be fully investigated. Grb2-associated binder 1 (Gab1), a member of scaffolding/adaptor proteins, can mediate signal transduction from many receptors, however, whether and how Gab1 is required for TLR and RIG-I-triggered innate responses remain unknown. In this study, we demonstrated that Gab1 significantly enhances TLR4-, TLR3-, and RIG-I-triggered IL-6, IL-1beta, and IFN-alpha/beta production in macrophages. Gab1 knockdown in primary macrophages or Gab1 deficiency in mouse embryonic fibroblasts significantly suppresses TLR3/4- and RIG-I-triggered production of IL-6, IL-1beta, and IFN-alpha/beta. Consistently, Gab1 deficiency impairs vesicular stomatitis virus (VSV) infection-induced IFN-alpha/beta production. In addition to promoting both MyD88- and TLR/IL-1 receptor domain-containing adaptor protein inducing IFN-beta-dependent MAPKs and NF-kappaB activation, Gab1 enhances PI3K/Akt activation by directly binding p85 in TLR signaling and VSV infection. Accordingly, Gab1 inhibits VSV replication and VSV infection-induced cell damage by inducing type I IFNs and IFN-inducible gene expression via PI3K/Akt pathway. Therefore, Gab1 is needed for full activation of TLR3/4- and RIG-I-triggered innate responses by promoting activation of PI3K/Akt, MAPKs, and NF-kappaB pathways.

Abstract 1402: Molecular basis for soy isoflavone genistein in the prevention of posttreatment recurrence of neuroblastoma

Abstract Clinical and laboratory evidence suggests that neuroblastoma contain populations of rapidly proliferating clonogens that survive chemoradiation or conventional radiotherapy leading to radioresistance. To that note, radioresistance could be associated with survival advantage resulting in clonal selection and tumor re-growth. Recently, we demonstrated that IR activates NFκB that enhances cell survival by switching on genes that attenuate apoptosis. Coherently, studies have demonstrated Genistein (GEN) inhibits NFκB and radiosensitizes cancer cells. In this study, we investigated the functional mechanistic regulation of NFκB mediated survival advantage and, delineated the efficacy of GEN in this setting. Human SH-SY5Y, IMR-32 and SK-N-MC cells exposed to 2Gy (IR) with or without GEN (100μM) were examined after 15min through 72h. EMSA analysis showed a robust and persistent activation of NFκB at least up to 72h in IR exposed cells. Consistently, IR induced a sustained transactivation (QPCR) and intercellular (ELISA) TNFα in these cells. Forced inhibition of NFκB (SN50, RelA siRNA) or TNFα (TNFR1-Ab) completely and persistently inhibited IR-induced NFκB and/or TNFα demonstrating the IR-induced initiation of NFκB→TNFα→NFκB feed back mechanism (PFC). Consistently, IR simulation experiments with NFκB (SNP, p50/p65 transfection) or TNFα (rH-TNFα) over expression, persistently activated NFκB and/or TNFα exhibiting the occurrence of PFC. Furthermore, IR profoundly induced both mRNA (QPCR) and cellular localization (immunoblotting) of IAP1, IAP2 and Survivin. NFκB knock-out experiments portrait a NFκB dependent activation of IAP1, IAP2 and Survivin in these cells. Conversely, GEN treatment completely and persistently inhibited IR-induced NFκB and, both TNFα transactivation and intercellular levels. Moreover, NFκB and/or TNFα over expression studies with GEN demonstrate the disruption of PFC. In addition, GEN markedly, inhibited IR-induced NFκB dependent IAP1, IAP2 and Survivin and greatly conferred IR-induced inhibition of cell survival. Taken together these results suggests that the surviving neuroblastoma cells may result in the development of survival advantage mediated by persistent activation of NFκB through NFκB→TNFα→NFκB PFC. More importantly, disrupting IR-initiated PFC with GEN may significantly inhibit NFκB dependent survival advantage and, may serve as a potential deliverable in mitigating neuroblastoma relapse and progression after radiotherapy. Funding Support: ACS- IRG-05-066-01; Presbyterian Health Foundation Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1402.