Classical NF-κB Signaling Research Articles

Abstract 5470: IKK-epsilon supports anchorage independent growth via alternative NF-kB signaling in triple-negative breast cancer

Abstract Metastatic breast cancer carries a poor prognosis despite the success of newly targeted therapies and treatment options remain especially limited for the subtype of triple negative breast cancer (TNBC). Several signaling pathways, including NF-κB, are altered in TNBC. Given that ΙΚΚε behaves as an oncogene in breast cancer we hypothesized that IKKε regulates classical or alternative NF-κB signaling to control diverse oncogenic functions in TNBC. Vector expression and RNA interference were used to investigate the functional role of IKKε in triple-negative breast cancer cells. Viability, protein expression, NF-κB binding activity, invasion, anoikis, and spheroid formation were examined in cells expressing high or low levels of IKKε in conjunction with alternative NF-κB p52 RNA interference or MEK inhibition. Our data show that p52 protein levels and binding activity are inversely proportional to ΙΚΚε. Moreover, knockdown of IKKε leads to increased expression of p52 and the p52-regulated gene CXCL1. This interaction was confirmed by CHiP-PCR experiments showing increased binding of p52 in the CXCL1 promoter of cells with IKKε knockdown relative to control. We further found that IKKε and MEK were required for growth in anchorage supportive conditions whereas p52 was required in anchorage resistant conditions. Western blots confirm diminished IKKε expression and enhanced p52 processing in anchorage resistant conditions relative to anchorage supportive conditions. In this model, IKKε and MEK cooperate to support growth of cancer cells in the attached, anchored environment, whereas p52 is required for growth in unattached 3-D environment. These studies reveal novel information about the role of IKKε in TNBC and highlight the adaptability of NF-κB signaling in maintaining cancer cell survival under different growth conditions. Current studies are underway to clarify the mechanism of IKKε regulation of p52. A better understanding of the diversity of NF-κB signaling may ultimately improve the development of novel therapeutic regimens for TNBC. Citation Format: Carrie D. House, Valentina Grajales, Michelle Ozaki, Elizabeth Jordan, Helmae Wubneh, Danielle Kimble, Marianne Kim, Christina M. Annunziata. IKK-epsilon supports anchorage independent growth via alternative NF-kB signaling in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5470.

Myeloid NF-κB signaling contributes to pathogenesis of immune-mediated nephritis

Abstract Immune-mediated glomerulonephritis is a serious end organ pathology that affects patients with systemic lupus erythematosus (SLE). A common murine model used to study lupus nephritis (LN) is nephrotoxic serum nephritis (NTN) in which mice are passively transferred nephrotoxic antibodies. We have previously shown that macrophages are important for the pathogenesis of LN. To further investigate the mechanism and determine if the contribution of macrophages is mediated via the NF-κB pathway we created B6 mice which had RelA knocked out in myeloid cells, thus inhibiting classical NF-κB signaling in this cell lineage. We induced NTN in this strain to assess the importance of macrophage derived NF-κB signaling in contributing to disease progression. Myeloid cell RelA knock out (KO) mice injected with nephrotoxic serum had significantly ameliorated proteinuria (p<0.01) and serum BUN levels (p<0.05) compared to control injected wildtype B6 mice (WT). Inhibiting myeloid NF-κB signaling also decreased inflammatory modulators within the kidneys. We measured significant decreases of IL-1a, IFNg, and IL-6 in kidneys from KO mice, but higher IL-10 expression. Flow cytometry revealed decreased numbers of kidney infiltrating classically activated macrophages in KO mice as well. Analysis of renal histopathology is pending. Taken together, our studies indicate that macrophage NF-κB signaling is instrumental in the contribution of this cell type to the pathogenesis of NTN. Our results suggest that while approaches which decrease macrophage numbers can be effective in immune mediated nephritis, more targeted treatments directed at modulating macrophage signaling and/or function could be beneficial, at least in the early stages of disease.

CD40L mediated alternative NF\u03baB-signaling induces resistance to BCR-inhibitors in patients with mantle cell lymphoma

Drug resistance is a significant obstacle in cancer treatment and therefore a frequent subject of research. Developed or primary resistance limits the treatment success of inhibitors of the B cell receptor (BCR) pathway in mantle cell lymphoma (MCL) patients. Recent research has highlighted the role of the nuclear factor-kappa B (NFκB) pathway in the context of resistance to BCR inhibitors in MCL. In this study, we analyzed the dependency of MCL cell lines on NFκB signaling and illustrated the ability of CD40L to activate the alternative NFκB pathway in MCL. This activation leads to independency of classical NFκB signaling and results in resistance to BCR inhibitors. Therefore, ligands (such as CD40L) and their activation of the alternative NFκB pathway have a major impact on the drug response in MCL. Furthermore, this study indicates a protective role for cells expressing specific ligands as microenvironmental niches for MCL cells and underlines the significance of therapeutically targeting alternative NFκB signaling in MCL.

Production and characterization of a novel site-specific-modifiable anti-OX40-receptor single-chain variable fragment for targeted drug delivery

OX40 receptor (tumor necrosis factor receptor superfamily, member 4; CD134) is a T-cell co-stimulatory molecule that plays an important role in T-cell activation and survival. OX40 receptor is activated by its ligand, OX40L; and modulation of the OX40–OX40L interaction is a promising target for the treatment of autoimmune diseases and cancers. Here, we generated a high-affinity anti-OX40 single-chain variable fragment carrying a C-terminal cysteine residue (scFvC). Physicochemical and functional analyses revealed that the scFvC bound to OX40-expressing cells and was internalized via OX40-mediated endocytosis without inducing phosphorylation of IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), an important complex in the classical NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling pathway. In addition, mutation of the 36th cysteine residue in variable region of light chain enabled site-specific chemical modification to carboxy terminal cysteine and improved the thermal stability of the scFvC. These results suggest that this novel high-affinity anti-OX40 scFvC may be useful as a transporter for targeted delivery of small compounds, proteins, peptides, liposomes, and nanoparticles, into OX40-expressing cells for the treatment of autoimmune diseases and cancers.

CP-25, a Novel Anti-inflammatory and Immunomodulatory Drug, Inhibits the Functions of Activated Human B Cells through Regulating BAFF and TNF-alpha Signaling and Comparative Efficacy with Biological Agents.

Paeoniflorin-6′-O-benzene sulfonate (code: CP-25) was the chemistry structural modifications of Paeoniflorin (Pae). CP-25 inhibited B cells proliferation stimulated by B cell activating factor belonging to the TNF family (BAFF) or Tumor necrosis factor alpha (TNF-alpha). CP-25, Rituximab and Etanercept reduced the percentage and numbers of CD19+ B cells, CD19+CD20+ B cells, CD19+CD27+ B cells and CD19+CD20+CD27+ B cells induced by BAFF or TNF-alpha. There was significant difference between CP-25 and Rituximab or CP-25 and Etanercept. CP-25 down-regulated the high expression of BAFFR, BCMA, and TACI stimulated by BAFF or TNF-alpha. The effects of Rituximab and Etanercept on BAFFR or BCMA were stronger than that of CP-25. CP-25, Rituximab and Etanercept down-regulated significantly the expression of TNFR1 and TNFR2 on B cell stimulated by BAFF or TNF-alpha. CP-25, Rituximab and Etanercept down-regulated the expression of MKK3, P-p38, P-p65, TRAF2, and p52 in B cells stimulated by BAFF and the expression of TRAF2 and P-p65 in B cells stimulated by TNF-alpha. These results suggest that CP-25 regulated moderately activated B cells function by regulating the classical and alternative NF-κB signaling pathway mediated by BAFF and TNF-alpha-TRAF2-NF-κB signaling pathway. This study suggests that CP-25 may be a promising anti-inflammatory immune and soft regulation drug.

NFκB Promotes Ovarian Tumorigenesis via Classical Pathways That Support Proliferative Cancer Cells and Alternative Pathways That Support ALDH+ Cancer Stem-like Cells.

Understanding the mechanisms supporting tumor-initiating cells (TIC) is vital to combat advanced-stage recurrent cancers. Here, we show that in advanced ovarian cancers NFκB signaling via the RelB transcription factor supports TIC populations by directly regulating the cancer stem-like associated enzyme aldehyde dehydrogenase (ALDH). Loss of RelB significantly inhibited spheroid formation, ALDH expression and activity, chemoresistance, and tumorigenesis in subcutaneous and intrabursal mouse xenograft models of human ovarian cancer. RelB also affected expression of the ALDH gene ALDH1A2 Interestingly, classical NFκB signaling through the RelA transcription factor was equally important for tumorigenesis in the intrabursal model, but had no effect on ALDH. In this case, classical signaling via RelA was essential for proliferating cells, whereas the alternative signaling pathway was not. Our results show how NFκB sustains diverse cancer phenotypes via distinct classical and alternative signaling pathways, with implications for improved understanding of disease recurrence and therapeutic response. Cancer Res; 77(24); 6927-40. ©2017 AACR.

Central immune tolerance depends on crosstalk between the classical and alternative NF-κB pathways in medullary thymic epithelial cells

Medullary thymic epithelial cells (mTECs) contribute to self-tolerance by expressing and presenting peripheral tissue antigens for negative selection of autoreactive T cells and differentiation of natural regulatory T cells. The molecular control of mTEC development remains incompletely understood. We here demonstrate by TEC-specific gene manipulation in mice that the NF-κB transcription factor subunit RelB, which is activated by the alternative NF-κB pathway, regulates development of mature mTECs in a dose-dependent manner. Mice with conditional deletion of Relb lacked mature mTECs and developed spontaneous autoimmunity. In addition, the NF-κB subunits RelA and c-Rel, which are both activated by classical NF-κB signaling, were jointly required for mTEC differentiation by directly regulating the transcription of Relb. Our data reveal a crosstalk mechanism between classical and alternative NF-κB pathways that tightly controls the development of mature mTECs to ensure self-tolerance.

Arsenic Trioxide Induces Apoptosis and Incapacitates Proliferation and Invasive Properties of U87MG Glioblastoma Cells through a Possible NF-κB-Mediated Mechanism.

Identification of novel therapeutics in glioblastoma remains crucial due to the devastating and infiltrative capacity of this malignancy. The current study was aimed to appraise effect of arsenic trioxide (ATO) in U87MG cells. The results demonstrated that ATO induced apoptosis and impeded proliferation of U87MG cells in a dosedependent manner and also inhibited classical NF-κB signaling pathway. ATO further upregulated expression of Bax as an important proapoptotic target of NF-κB and also inhibited mRNA expression of survivin, c-Myc and hTERT and suppressed telomerase activity. Moreover, ATO significantly increased adhesion of U87MG cells and also diminished transcription of NF-κB down-stream targets involved in cell migration and invasion, including cathepsin B, uPA, MMP-2, MMP-9 and MMP-14 and suppressed proteolytic activity of cathepsin B, MMP-2 and MMP-9, demonstrating a possible mechanism of ATO effect on a well-known signaling in glioblastoma dissemination. Taken together, here we suggest that ATO inhibits survival and invasion of U87MG cells possibly through NF-κB-mediated inhibition of survivin and telomerase activity and NF-κB-dependent suppression of cathepsin B, MMP-2 and MMP-9.

Heterozygous Mutation in IκBNS Leads to Reduced Levels of Natural IgM Antibodies and Impaired Responses to T-Independent Type 2 Antigens.

Mice deficient in central components of classical NF-κB signaling have low levels of circulating natural IgM antibodies and fail to respond to immunization with T-independent type 2 (TI-2) antigens. A plausible explanation for these defects is the severely reduced numbers of B-1 and marginal zone B (MZB) cells in such mice. By using an ethyl-N-nitrosourea mutagenesis screen, we identified a role for the atypical IκB protein IκBNS in humoral immunity. IκBNS-deficient mice lack B-1 cells and have severely reduced numbers of MZB cells, and thus resemble several other strains with defects in classical NF-κB signaling. We analyzed mice heterozygous for the identified IκBNS mutation and demonstrate that these mice have an intermediary phenotype in terms of levels of circulating IgM antibodies and responses to TI-2 antigens. However, in contrast to mice that are homozygous for the IκBNS mutation, the heterozygous mice had normal frequencies of B-1 and MZB cells. These results suggest that there is a requirement for IκBNS expression from two functional alleles for maintaining normal levels of circulating natural IgM antibodies and responses to TI-2 antigens.

MicroRNA-125b regulates microglia activation and motor neuron death in ALS.

Understanding the means by which microglia self-regulate the neuroinflammatory response helps modulating their reaction during neurodegeneration. In amyotrophic lateral sclerosis (ALS), classical NF-κB pathway is related to persistent microglia activation and motor neuron injury; however, mechanisms of negative control of NF-κB activity remain unexplored. One of the major players in the termination of classical NF-κB pathway is the ubiquitin-editing enzyme A20, which has recognized anti-inflammatory functions. Lately, microRNAs are emerging as potent fine-tuners of neuroinflammation and reported to be regulated in ALS, for instance, by purinergic P2X7 receptor activation. In this work, we uncover an interplay between miR-125b and A20 protein in the modulation of classical NF-κB signaling in microglia. In particular, we establish the existence of a pathological circuit in which termination of A20 function by miR-125b strengthens and prolongs the noxious P2X7 receptor-dependent activation of NF-κB in microglia, with deleterious consequences on motor neurons. We prove that, by restoring A20 levels, miR-125b inhibition then sustains motor neuron survival. These results introduce miR-125b as a key mediator of microglia dynamics in ALS.

Classical and alternative NF-κB signaling cooperate in regulating adipocyte differentiation and function.

Inflammation of adipose tissue (AT) is a central mediator of insulin resistance. However, the molecular mechanisms triggered by inflammatory cells are not fully understood. The aim of this study was to analyze the metabolic functions of lymphotoxin-β-receptor (LTβR)-mediated alternative NF-κB signaling in adipocytes and to reveal its effects on body weight and insulin sensitivity in vivo. RelB(FatKO) mice and littermate controls were treated with LTβR agonistic antibody (α-LTβR) or a LTβR antagonist (LTβR:Ig fusion protein) after feeding a high-fat diet or standard diet. Mice were analyzed by insulin tolerance and glucose tolerance tests prior to analysis by necropsy and qRT-PCR of abdominal white adipose tissue. 3T3-L1 preadipocytes and mouse embryonic fibroblasts were used for differentiation and expression analysis after treatment with α-LTβR and differentiation to adipocytes. The molecular mechanism was elucidated by chromatin immunoprecipitation and combinatorial treatment with α-LTβR and tumor necrosis factor (TNF). RelB(FatKO) mice showed improved insulin sensitivity despite increased adiposity and adipocyte hypertrophy. LTβR-induced activation of p52-RelB in 3T3-L1 cells attenuated adipogenesis and modulated adipocyte functions via transcriptional downregulation of peroxisome proliferator-activated receptor γ (PPARγ). This LTβR-mediated pathway was synergistically regulated via a TNF-induced increase in p100 and RelB expression and nuclear translocation. Our data describe an anti-adipogenic action of LTβR signaling and a novel synergism of alternative and classical NF-κB signaling in the regulation of adipocytes. In conclusion, this strong synergism between the two NF-κB pathways shows a method to inhibit adipocyte differentiation and to improve insulin sensitivity and can be a potential target to treat metabolic disorders more efficiently than with other known drugs.

606 NFKB1 Deficiency Alters Susceptibility to Helicobacter spp. Induced IL-1β Secretion in Bone Marrow Derived Dendritic Cells

Introduction Deletion of specific NF-κB subunits in mice alters the outcome of Helicobacter felis infection. Nfkb1 -/- mice developed more severe gastric atrophy than wild-type (WT) mice 6 weeks after H. felis infection, whereas Nfkb2 -/- mice were protected from this pathology. The mechanisms underlying these outcomes remain unclear, but gastric Il1b transcript abundance was increased in H. felis infected Nfkb1 -/- mice relative to WT, and polymorphisms at the IL1B locus have also been associated with gastric cancer in humans. Il1b transcription is regulated by classical pathway NF-κB signalling. IL-1β secretion also requires the formation of inflammasome complexes, which form following intracellular pathogen recognition, and lead to the autocatalysis of pro-caspase 1 and subsequent cleavage of IL-1β. We hypothesised that inflammasome signalling was altered in mice with abrogated NF-κB signalling and that this influenced H. felis induced pathology. We therefore investigated inflammasome mediated signalling in bone marrow derived dendritic cells (BMDCs) from mice lacking specific NF-κB subunits. Method Cells were harvested from C57BL/6, Nfkb1 -/- , Nfkb2 -/- and c-Rel -/- bone marrow. Dendritic cells were differentiation using 20 ng/ml GM-CSF for 7 days. BMDCs and WT derived gastric epithelial organoids were primed with 20 ng/ml LPS and exposed to 300 μg/ml silica, 5 mM ATP, H. pylori (ATCC 53726) or H. felis (ATCC 49179) (MOI 1:100) with or without a pan-caspase inhibitor (Z-VAD-fmk), an inhibitor of NADPH oxidase (APDC) or 50 mM KCl. Secreted IL-1β and TNF concentrations were measured by ELISA. Results LPS alone induced TNF, but not Il-1β, secretion in all genotypes of BMDCs. Both silica and ATP induced IL-1β secretion in WT BMDCs pre-stimulated with LPS (750 ± 65 and 530 ± 77 pg/ml). BMDCs derived from NF-κB deficient mice secreted similar amounts of Il-1β in response to these stimuli. Inflammasome inhibitors returned IL-1β secretion to unstimulated levels. Gastric epithelial organoid cultures treated with LPS and silica did not secrete either IL-1β or TNF. Following exposure to H. felis or H. pylori , Nfkb1 -/- BMDCs, but not other genotypes, exhibited a 2.6 fold increase in IL-1β secretion compared to untreated cells or cells stimulated with LPS. This was abrogated by Z-VAD-fmk. Conclusion These data identify potent inflammasome activation in NF-κB deficient BMDCs. H. pylori and H. felis also weakly stimulated inflammasome activation in NFkb1-/- BMDCs. This mechanism may contribute to the more severe gastric phenotype that is observed in NFkb1 -/- mice in vivo following H. felis infection. Further studies are required to identify how NF-κB1 deletion influences inflammasome formation, and whether altered IL-1β transcription is involved. Disclosure of interest None Declared.

NF-κB inhibitors impair lung epithelial tight junctions in the absence of inflammation

NF-κB (p50/p65) is the best characterized transcription factor known to regulate cell responses to inflammation. However, NF-κB is also constitutively expressed. We used inhibitors of the classical NF-κB signaling pathway to determine whether this transcription factor has a role in regulating alveolar epithelial tight junctions. Primary rat type II alveolar epithelial cells were isolated and cultured on Transwell permeable supports coated with collagen for 5 d to generate a model type I cell monolayer. Treatment of alveolar epithelial monolayers overnight with one of 2 different IκB kinase inhibitors (BAY 11–7082 or BMS-345541) resulted in a dose-dependent decrease in TER at concentrations that did not affect cell viability. In response to BMS-345541 treatment there was an increase in total claudin-4 and claudin-5 along with a decrease in claudin-18, as determined by immunoblot. However, there was little effect on the total amount of cell-associated claudin-7, occludin, junctional adhesion molecule A (JAM-A), zonula occludens (ZO)-1 or ZO-2. Moreover, treatment with BMS-345541 resulted in altered tight junction morphology as assessed by immunofluorescence microscopy. Cells treated with BMS-345541 had an increase in claudin-18 containing projections emanating from tight junctions (“spikes”) that were less prominent in control cells. There also were several areas of cell-cell contact which lacked ZO-1 and ZO-2 localization as well as rearrangements to the actin cytoskeleton in response to BMS-345541. Consistent with an anti-inflammatory effect, BMS-345541 antagonized the deleterious effects of lipopolysaccharide (LPS) on alveolar epithelial barrier function. However, BMS-345541 also inhibited the ability of GM-CSF to increase alveolar epithelial TER. These data suggest a dual role for NF-κB in regulating alveolar barrier function and that constitutive NF-κB function is required for the integrity of alveolar epithelial tight junctions.

Abstract 1673: Alternative NF-κB signaling promotes inflammatory cell recruitment and lung tumor formation

Abstract Nuclear factor-κB (NF-κB) is a transcription factor that can be activated through either the classical or alternative signaling pathways. In lung tumors, high levels of classical and alternative pathway components have been observed. While many studies have demonstrated that classical NF-κB signaling is a central pathway regulating inflammation and tumorigenesis, the role of alternative NF-κB signaling remains undefined. We hypothesize that alternative NF-κB signaling in the lung epithelium plays a key role in inflammation and lung tumor formation through paracrine signaling to the inflammatory microenvironment. To investigate alternative NF-κB signaling in the lung epithelium, we generated a transgenic mouse model with doxycycline (dox)-inducible expression of the alternative pathway component p52 in airway epithelial cells (designated ALTA for alternative pathway trans-activated). When placed on dox, nuclear p52 is increased in the lungs of ALTA mice with no effect on activation of other NF-κB components. Surprisingly, alternative pathway activation alone did not cause a significant increase in inflammatory cell recruitment. However, in conjunction with the inflammatory stimulus lipopolysaccharide (LPS), alternative pathway activation caused increased mortality, which was associated with a significant increase in inflammation and increased lung permeability. To determine the effect of alternative pathway activation on lung tumor formation, mice were injected with the lung carcinogen urethane. Six weeks after urethane administration, ALTA mice demonstrated an increased number of bronchoalveolar lavage neutrophils, and by 6 months post-urethane, ALTA mice had an increased number of tumors, increased tumor size, and more advanced lesions. Together, these data suggest that alternative NF-κB signaling plays an important role in stimulus-induced inflammatory cell recruitment and that alternative pathway activation can promote lung tumor formation and progression. By defining the role of alternative NF-κB signaling in tumor formation, we hope to gain insight into the role of inflammation in lung carcinogenesis and to identify novel pathways and signaling targets that could potentially serve as therapeutic targets. Citation Format: Jamie Alicyn Ausborn, Dong-Sheng Cheng, Vasiliy Polosukhin, Wei Han, Fiona Yull, Timothy Blackwell. Alternative NF-κB signaling promotes inflammatory cell recruitment and lung tumor formation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1673. doi:10.1158/1538-7445.AM2014-1673

Water extract of the fruits of Alpinia oxyphylla inhibits osteoclast differentiation and bone loss.

BackgroundExcessive bone resorption by osteoclasts causes pathological bone destruction, seen in various bone diseases. There is accumulating evidence that certain herbal extracts have beneficial effects on bone metabolism. The fruits of Alpinia oxyphylla has been traditionally used for the treatment of diarrhea and enuresis. In this study, we investigated the effects of water extract of the fruits of Alpinia oxyphylla (WEAO) on osteoclast differentiation and osteoclast-mediated bone destruction.MethodsFor osteoclast differentiation assay, mouse bone marrow-derived macrophages (BMMs) were cultured in the presence of RANKL and M-CSF. RANKL signaling pathways and gene expression of transcription factors regulating osteoclast differentiation were investigated by real-time PCR and Western blotting. A constitutively active form of NFATc1 was retrovirally transduced into BMMs. Bone resorbing activity of mature osteoclast was examined on a plate coated with an inorganic crystalline calcium phosphate. The in vivo effect against bone destruction was assessed in a murine model of RANKL-induced osteoporosis by micro-computed tomography and bone metabolism marker analyses.ResultsWEAO dose-dependently inhibited RANKL-induced osteoclast differentiation from BMMs by targeting the early stages of osteoclast differentiation. WEAO inhibited RANKL-induced expression of NFATc1, the master regulator of osteoclast differentiation. Overexpression of a constitutively active form of NFATc1 blunted the inhibitory effect of WEAO on osteoclast differentiation, suggesting that NFATc1 is a critical target of the inhibitory action of WEAO. WEAO inhibited RANKL-induced expression of c-Fos, an upstream activator of NFATc1, by suppressing the classical NF-κB signaling pathway. WEAO also inhibited RANKL-induced down-regulation of Id2 and MafB, negative regulators of NFATc1. WEAO does not directly affect bone resorbing activity of mature osteoclasts. In accordance with the in vitro results, WEAO attenuated RANKL-induced bone destruction in mice by inhibiting osteoclast differentiation.ConclusionsThis study demonstrates that WEAO exhibits a protective effect against bone loss by inhibiting RANKL-induced osteoclast differentiation. These findings suggest that WEAO might be useful for the prevention and treatment of bone diseases associated with excessive bone resorption.

The RelB alternative NF-kappaB subunit promotes autophagy in 22Rv1 prostate cancer cells in vitro and affects mouse xenograft tumor growth in vivo.

BackgroundThe involvement of NF-κB signaling in prostate cancer (PCa) has largely been established through the study of the classical p65 subunit. Nuclear localization of p65 in PCa patient tissues has been shown to correlate with biochemical recurrence, while in vitro studies have demonstrated that the classical NF-κB signaling pathway promotes PCa progression and metastatic potential. More recently, the nuclear location of RelB, a member of the alternative NF-κB signaling, has also been shown to correlate with the Gleason score. The current study aims to clarify the role of alternative NF-κB in PCa cells by exploring, in vitro and in vivo, the effects of RelB overexpression on PCa biology.MethodsUsing a lentivirus-expression system, we constitutively overexpressed RelB or control GFP into 22Rv1 cells and monitored alternative transcriptional NF-κB activity. In vivo, tumor growth was assessed after the injection of 22Rv1-derived cells into SCID mice. In vitro, the impact of RelB on 22Rv1 cell proliferation was evaluated in monolayer culture. The anchorage-independent cell growth of derived-22Rv1 cells was assessed by soft agar assay. Apoptosis and autophagy were evaluated by Western blot analysis in 22Rv1-derived cells cultured in suspension using poly-HEMA pre-coated dishes.ResultsThe overexpression of RelB in 22Rv1 cells induced the constitutive activation of the alternative NF-κB pathway. In vivo, RelB expression caused a lag in the initiation of 22Rv1-induced tumors in SCID mice. In vitro, RelB stimulated the proliferation of 22Rv1 cells and reduced their ability to grow in soft agar. These observations may be reconciled by our findings that, when cultured in suspension on poly-HEMA pre-coated dishes, 22Rv1 cells expressing RelB were more susceptible to cell death, and more specifically to autophagy controlled death.ConclusionsThis study highlights a role of the alternative NF-κB pathway in proliferation and the controlled autophagy. Thus, the interplay of these properties may contribute to tumor survival in stress conditions while promoting PCa cells growth contributing to the overall tumorigenicity of these cells.

Modulating the tumor necrosis factor-α induced inflammatory response in human colonic epithelial cells by inhibiting NF-κB signaling (CCR5P.258)

Abstract In colonic epithelia, tumor necrosis factor alpha (TNF-α) secreted by inflammatory cells is a key mediator of inflammatory bowel disease. TNF-α is a well-characterized activator of the NF-κB signaling pathway in intestinal epithelial cells and is known to up-regulate the expression of a subset of chemokines and cytokines. In this study, we used antibody arrays to profile the relative changes in chemokines secreted upon activation of the human colon carcinoma cell line HT-29. Elevated levels of CXCL1, CXCL7, CXCL8, CXCL10 and CCL20 were observed in conditioned media after TNF-α treatment of HT-29 cells. To better understand the contributions of the classical NF-κB signaling pathway to TNF-α activation of HT-29 cells, we used an inhibitor with high selectivity for IKK2 over IKK1, TPCA-1. In addition, both classical and the alternative NF-κB signaling pathways were inhibited at a level downstream of IKK1 and IKK2 by using the NEDD8 activating enzyme (NAE)-inhibitor MLN4924. TPCA-1 and MLN4924 both attenuated TNF-α activated secretion of these chemokines in a dose dependent manner in HT-29 cells. Confirmation of these array data by quantitative ELISAs supports the use of arrays for analysis of inhibitor biomarkers.

Noncanonical NF-κB Signaling Is Limited by Classical NF-κB Activity

Precise regulation of nuclear factor κB (NF-κB) signaling is crucial for normal immune responses, and defective NF-κB activity underlies a range of immunodeficiencies. NF-κB is activated through two signaling cascades: the classical and noncanonical pathways. The classical pathway requires inhibitor of κB kinase β (IKKβ) and NF-κB essential modulator (NEMO), and hypomorphic mutations in the gene encoding NEMO (ikbkg) lead to inherited immunodeficiencies, collectively termed NEMO-ID. Noncanonical NF-κB activation requires NF-κB-inducing kinase (NIK) and IKKα, but not NEMO. We found that noncanonical NF-κB was basally active in peripheral blood mononuclear cells from NEMO-ID patients and that noncanonical NF-κB signaling was similarly enhanced in cell lines lacking functional NEMO. NIK, which normally undergoes constitutive degradation, was aberrantly present in resting NEMO-deficient cells, and regulation of its abundance was rescued by reconstitution with full-length NEMO, but not a mutant NEMO protein unable to physically associate with IKKα or IKKβ. Binding of NEMO to IKKα was not required for ligand-dependent stabilization of NIK or noncanonical NF-κB signaling. Rather, an intact and functional IKK complex was essential to suppress basal NIK activity in unstimulated cells. Despite interacting with IKKα and IKKβ to form an IKK complex, NEMO mutants associated with immunodeficiency failed to rescue classical NF-κB signaling or reverse the accumulation of NIK. Together, these findings identify a crucial role for classical NF-κB activity in the suppression of basal noncanonical NF-κB signaling.

Classical NF-κB activation impairs skeletal muscle oxidative phenotype by reducing IKK-α expression

Background: Loss of quadriceps muscle oxidative phenotype (OXPHEN) is an evident and debilitating feature of chronic obstructive pulmonary disease (COPD). We recently demonstrated involvement of the inflammatory classical NF-κB pathway in inflammation-induced impairments in muscle OXPHEN. The exact underlying mechanisms however are unclear. Interestingly, IκB kinase α (IKK-α: a key kinase in the alternative NF-κB pathway) was recently identified as a novel positive regulator of skeletal muscle OXPHEN. We hypothesised that inflammation-induced classical NF-κB activation contributes to loss of muscle OXPHEN in COPD by reducing IKK-α expression.Methods: Classical NF-κB signalling was activated (molecularly or by tumour necrosis factor α: TNF-α) in cultured myotubes and the impact on muscle OXPHEN and IKK-α levels was investigated. Moreover, the alternative NF-κB pathway was modulated to investigate the impact on muscle OXPHEN in absence or presence of an inflammatory stimulus. As a proof of concept, quadriceps muscle biopsies of COPD patients and healthy controls were analysed for expression levels of IKK-α, OXPHEN markers and TNF-α.Results: IKK-α knock-down in cultured myotubes decreased expression of OXPHEN markers and key OXPHEN regulators. Moreover, classical NF-κB activation (both by TNF-α and IKK-β over-expression) reduced IKK-α levels and IKK-α over-expression prevented TNF-α-induced impairments in muscle OXPHEN. Importantly, muscle IKK-α protein abundance and OXPHEN was reduced in COPD patients compared to controls, which was more pronounced in patients with increased muscle TNF-α mRNA levels.Conclusion: Classical NF-κB activation impairs skeletal muscle OXPHEN by reducing IKK-α expression. TNF-α-induced reductions in muscle IKK-α may accelerate muscle OXPHEN deterioration in COPD.

TWEAK Inhibits TRAF2-Mediated CD40 Signaling by Destabilization of CD40 Signaling Complexes

We found recently that TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor-inducible-14 (Fn14) by virtue of their strong capability to reduce the freely available cytoplasmic pool of TNFR-associated factor (TRAF)2 and cellular inhibitors of apoptosis (cIAPs) antagonize the functions of these molecules in TNFR1 signaling, resulting in sensitization for apoptosis and inhibition of classical NF-κB signaling. In this study, we demonstrate that priming of cells with TWEAK also interferes with activation of the classical NF-κB pathway by CD40. Likewise, there was strong inhibition of CD40 ligand (CD40L)-induced activation of MAPKs in TWEAK-primed cells. FACS analysis and CD40L binding studies revealed unchanged CD40 expression and normal CD40L-CD40 interaction in TWEAK-primed cells. CD40L immunoprecipitates, however, showed severely reduced amounts of CD40 and CD40-associated proteins, indicating impaired formation or reduced stability of CD40L-CD40 signaling complexes. The previously described inhibitory effect of TWEAK on TNFR1 signaling has been traced back to reduced activity of the TNFR1-associated TRAF2-cIAP1/2 ubiquitinase complex and did not affect the stability of the immunoprecipitable TNFR1 receptor complex. Thus, the inhibitory effect of TWEAK on CD40 signaling must be based at least partly on other mechanisms. In line with this, signaling by the CD40-related TRAF2-interacting receptor TNFR2 was also attenuated but still immunoprecipitable in TWEAK-primed cells. Collectively, we show that Fn14 activation by soluble TWEAK impairs CD40L-CD40 signaling complex formation and inhibits CD40 signaling and thus identify the Fn14-TWEAK system as a potential novel regulator of CD40-related cellular functions.