Nuclear Localization Of NF-κB Research Articles

AKIP1 regulation of PKA and NF‐κB in the heart

Ischemic preconditioning, an endogenous protective mechanism to salvage ischemic myocardium, was described nearly 25 years ago; however, there has been little to no clinical translation. Further, there has been a critical lack of knowledge to understand the molecular mechanisms that bring about the protection. Nuclear factor‐kappa B (NF‐κB) and protein kinase A (PKA) are key regulators of gene transcription in the heart and have contradictory roles in cell death and survival. Activation of NF‐κB and PKA has been shown to be protective as well as promote cardiac cell death in the setting of ischemia or oxidant stress. The answer as to why these two important regulators of gene transcription and cardiac physiology produce such a dichotomous response upon stress induction may provide insights into how protective signaling in the heart is integrated. We have discovered a novel scaffolding protein known as A‐kinase interacting protein 1 (AKIP1) that is expressed at low levels in the heart and is induced by stress. Our data shows that AKIP1 binds to and regulates nuclear localization of PKA catalytic subunit (PKAc) and increases nuclear PKAc activity. Further, AKIP1 interacts with and enhances NF‐κB nuclear localization in a PKAc dependent manner where disruption of AKIP1 binding to PKAc enhances nuclear NF‐κB. In cardiomyocytes and in the heart, oxidant/ischemic stress caused an increase of AKIP1 levels. Further, our data suggests that overexpression of AKIP1 was protective to the heart. Therefore, we infer that AKIP1 may be a key molecular regulator/scaffold that assembles PKAc and NF‐κB signaling complexes to alter the physiologic response of the heart in the basal and stressed states. Understanding the dynamics and physiologic implications of the interaction of PKAc and NF‐κB with AKIP1 may provide a novel therapeutic control point for limiting cardiac injury associated with ischemic stress.

Saquinavir in steroid-dependent and -resistant nephrotic syndrome: a pilot study

Some difficult cases of idiopathic nephrotic syndrome (NS) have been treated with a HIV protease inhibitor provided with proteasome-inhibiting activity. The objective of this study was to limit nuclear factor κB (NF-κB) activation which is up-regulated in these patients, aiming at decreasing proteinuria and prednisone need. Ten cases with long-lasting (up to 15 years) history of NS with steroid dependence (six cases, of which three with secondary steroid resistance) or resistance to steroids (four cases) unsuccessfully treated with multiple immunosuppressive drugs, accepted a treatment with the protease inhibitor saquinavir. p50/p65 NF-κB nuclear localization and immunoproteasome/proteasome messenger RNA (mRNA) were monitored in peripheral blood mononuclear cells (PBMCs). The effects of saquinavir on NF-κB nuclear localization in cultured PBMCs and in immortalized human podocytes were assessed. After a median follow-up of 14.7 months (6-68.7), 1/4 primary steroid-resistant NS (SRNS) and 5/6 steroid-dependent NS or secondary SRNS became infrequent (5) or frequent (1) relapsers, with 63% prednisone reduction (from 25.3 to 8.4 mg/kg/month, P = 0.015). Saquinavir was effective in association with low doses of calcineurin inhibitors (cyclosporine 2 mg/kg/day or tacrolimus 0.01-0.06 mg/kg/day). No side effects were observed apart from transitory mild diarrhoea. In PBMCs, NF-κB was down-regulated, while MECL-1 immunoproteasome/beta2 proteasome mRNA ratio was reversed to normal values. In culture, saquinavir blunted NF-κB activation in human podocytes and in PBMCs. In this pilot study, a HIV antiprotease drug reduced proteinuria and had a steroid-sparing effect in some multidrug-resistant/-dependent NS. This observation warrants further investigation.

Butein impairs the protumorigenic activity of malignant pleural mesothelioma cells

Chronic inflammation appears to be a driving force behind cancer progression. NFκB and STAT3 activation plays a pertinent role in this process by mediating chemoresistance and the acquisition of mesenchymal features of protumorigenic cells. Epidemiological data and experimental observations suggest that Malignant Pleural Mesothelioma (MPM) is a prototype of chronic inflammation-driven cancer. Chemoresistance is a major feature of MPM. Thus, this paper explores the effect of butein (3,4,2',4'-tetrahydroxychalcone), a naturally occurring NFκB and STAT3 inhibitor, on the tumorigenic properties of MPM cells. MPM cells harbor high nuclear levels of NFκB and pSTAT3(Y705). Butein inhibits pSTAT3(Y705) phosphorylation, nuclear localization of NFκB and the physical interaction of NFκB and pSTAT3. This correlates with a downregulation of several genes involved in cancer progression (such as ICAM1, Vimentin, MMP9, Twist) of proangiogenic cytokines (VEGF) and of IL-6 and IL-8, key growth factors for MPM. Hence, butein inhibits the migration of MPM cells and strongly affects the clonogenicity of MPM cells in vitro. Finally, we show that the in vitro actions of butein translate into anticancer effects in vivo. In fact, butein treatment severely affects tumor engraftment and potentiates the anticancer effects of pemetrexed in mouse xenograft models. Butein does not significantly affect the viability of human, untransformed mesothelial cells in vitro, nor does it affect survival of tumor-free mice in vivo. The possibility of using butein as an additional treatment to current MPM therapies is discussed here.

Laminin-α1 LG4–5 domain binding to dystroglycan mediates muscle cell survival, growth, and the AP-1 and NF-κB transcription factors but also has adverse effects

In our previous studies, we showed laminin binds α-dystroglycan in the dystrophin glycoprotein complex and initiates cell signaling pathways. Here, differentiated C2C12 myocytes serve as a model of skeletal muscle. C2C12 cells have a biphasic response to the laminin-α(1) laminin globular (LG) 4-5 domains (1E3) dependent on the concentration used; at low concentrations of 1E3 (<1 μg/ml), myoblast proliferation is increased while higher concentrations (>1 μg/ml) cause apoptosis in myoblasts and differentiated myotubes. This alters the activation of the transcription factors activator protein-1 (AP-1) and NF-κB via laminin-dystrophin glycoprotein complex (DGC)-src-grb2-sos1-Rac1-Pak1-c-jun N-terminal kinase (JNK)p46 and laminin-DGC-Gβγ-phosphatidylinositol 3-kinase (PI3K)-Akt pathways, respectively. A specific antibody against Ser(63) phosphorylated c-jun completely blocks or supershifts the AP-1-DNA binding resulting from laminin binding but only partially blocks or supershifts the AP-1-DNA binding resulting from 1E3. This suggests that AP-1 contains phosphorylated c-jun in the presence of hololaminin but contains a different composition in the presence of 1E3. Nuclear NF-κB was only upregulated by a low concentration of 1E3 and is then diminished by a higher concentration; it also has a biphasic response. Nuclear localization of NF-κB is affected by PI3K/Akt signaling, and DGC associated PI3K activity also shows a biphasic response to 1E3. Furthermore, our data suggest that activation of c-jun N-terminal kinase participates in the cell survival pathway and suggest that NF-κB is involved in both survival and cell death. A model is presented which incorporates these observations.

Exendin-4 attenuates lipopolysaccharides induced inflammatory response but does not protects H9c2 cells from apoptosis

Background: Glucagon-like peptide-1 (GLP-1) and its analogues are reported to exert wide-ranging cardiovascular actions in preclinical and clinical studies. We thus investigated whether the GLP-1 receptor agonist, exendin-4, has inhibitory effects on LPS-stimulated inflammatory response in cardiomyoblasts.Methods: H9c2 cardiomyoblasts were exposed to LPS and treated with exendin-4. Expressions of proinflammatory mediators were assessed using quantitative real-time PCR. Nuclear localization of NF-κB was examined using immunoblotting. mRNA expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production were evaluated by q PCR and NO assay. Furthermore, anti-apoptotic effect of exendin-4 in LPS-stimulated H9c2 cells was determined using qPCR and immunoblot.Results: Exposure to LPS increased mRNA expressions of TNF-α, COX-2 and MMP-9 in H9c2 cells. It also caused increases in iNOS mRNA expression and NF-κB nuclear translocation. Exendin-4 dose-dependently downregulated mRNA levels of TNF-α, COX-2 and MMP-9 in LPS-stimulated H9c2 cells. It also reduced NF-κB nuclear translocation. Treatment with exendin-4 showed no effect on LPS-induced apoptosis in H9c2 cells.Conclusions: Exendin-4 exerts an effect on cardiomyoblast exposed to LPS by inhibiting mRNA expression of inflammatory mediators and suppressing NF-κB activation. These effects are consistent with some of the observed anti-inflammatory properties of exendin-4, as well as its beneficial actions on the cardiovascular system.

Target protein interactions of indole‐3‐carbinol and the highly potent derivative 1‐benzyl‐I3C with the C‐terminal domain of human elastase uncouples cell cycle arrest from apoptotic signaling

Elastase is the only currently identified target protein for indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin in cruciferous vegetables such as broccoli, cabbage, and Brussels sprouts that induces a cell cycle arrest and apoptosis of human breast cancer cells. In vitro elastase enzymatic assays demonstrated that I3C and at lower concentrations its more potent derivative 1-benzyl-indole-3-carbinol (1-benzyl-I3C) act as non-competitive allosteric inhibitors of elastase activity. Consistent with these results, in silico computational simulations have revealed the first predicted interactions of I3C and 1-benzyl-I3C with the crystal structure of human neutrophil elastase, and identified a potential binding cluster on an external surface of the protease outside of the catalytic site that implicates elastase as a target protein for both indolecarbinol compounds. The Δ205 carboxyterminal truncation of elastase, which disrupts the predicted indolecarbinol binding site, is enzymatically active and generates a novel I3C resistant enzyme. Expression of the wild type and Δ205 elastase in MDA-MB-231 human breast cancer cells demonstrated that the carboxyterminal domain of elastase is required for the I3C and 1-benzyl-I3C inhibition of enzymatic activity, accumulation of the unprocessed form of the CD40 elastase substrate (a tumor necrosis factor receptor family member), disruption of NFκB nuclear localization and transcriptional activity, and induction of a G1 cell cycle arrest. Surprisingly, expression of the Δ205 elastase molecule failed to reverse indolecarbinol stimulated apoptosis, establishing an elastase-dependent bifurcation point in anti-proliferative signaling that uncouples the cell cycle and apoptotic responses in human breast cancer cells.

Resveratrol ameliorates TNFα‐mediated suppression of erythropoiesis in human CD34+ cells via modulation of NF‐κB signalling

Overexpression of pro-inflammatory cytokines, including tumour necrosis factor alpha (TNFα), has been implicated in the pathogenesis of anaemia of inflammation. TNFα suppresses erythroid colony formation via both direct and indirect effects on haematopoietic progenitors, often involving activation of nuclear factor (NF)-κB signalling resulting in downregulation of transcription factors critical for erythropoiesis. There is a dearth of effective and safe therapies for many patients with inflammatory anaemia. Resveratrol is a flavanol found in red wine grapes that possesses potent anti-inflammatory properties, but studies of its impact on human erythropoiesis have proven contradictory. We investigated whether resveratrol ameliorates TNFα-mediated suppression of erythropoiesis in human CD34(+) haematopoietic progenitors. We found that resveratrol partially reverses the erythroid suppressive effects of TNFα, leading to significant recovery in burst forming unit-erythroid colony formation in human CD34(+) cells. CD34(+) cells pre-incubated with resveratrol for 72 h in the presence of TNFα inhibited NF-κB activation via decreased NF-κB nuclear localization without altering total NF-κB protein levels and independent of IκB degradation. Resveratrol also significantly restored the baseline expression of erythroid transcription factors NFE2 and the GATA1/GATA2 ratio in CD34(+) cells treated with TNFα. In conclusion, resveratrol may inhibit TNFα-mediated NF-κB activation and promote erythropoiesis in primary human CD34(+) cells.

CPEB control of NF-kappaB nuclear localization and interleukin-6 production mediates cellular senescence.

CPEB is a sequence-specific translational regulatory RNA binding protein that mediates cellular senescence in primary mouse and human cells. CPEB knockout mouse embryo fibroblasts (MEFs) bypass senescence and synthesize large amounts of interleukin-6 (IL-6) and many other cytokines, which is not the case with either wild-type MEFs immortalized by prolonged culture or p53-deficient MEFs. CPEB regulates the production of IL-6 at both the translational and transcriptional levels; in CPEB-depleted cells, aberrant IL-6 transcription is mediated by improper NF-κB p65 phosphorylation and nuclear localization. Although IL-6 strengthens the senescence of wild-type cells, it has no effect on CPEB-deficient cells, even though they produce prodigious amounts of the cytokine. IL-6-promoted entry into senescence requires p53; CPEB knockout MEFs, however, synthesize only ∼50% of the p53 of wild-type MEFs, which is insufficient to respond to IL-6. Thus, CPEB deficiency not only increases IL-6 production but also renders the cell incapable of a senescence-promoting response.

Tax-induced rapid senescence is mediated by both classical and alternative NF-κB pathways

HTLV-1 oncoprotein, Tax, is a potent activator of classical and alternative NF-κB signaling pathways and is thought to promote cell proliferation and transformation via NF-κB activation. We have shown recently that hyper-activation of NF-κB by Tax immediately triggers the cellular senescence checkpoint. Down-regulation of NF-κB activation, by contrast, rescues cells from Tax-induced rapid senescence (Tax-IRS). Here, we demonstrate that the IKKalpha and NEMO subunit of the IKK complex are absolutely essential to Tax-IRS. Repression of IKKalpha or NEMO expression by small hairpin RNAs abrogated Tax-mediated activation of both classical and alternative NF-κB pathways and rendered the knockdown cells resistant to Tax-IRS. IKKalpha deficiency moderately affected Tax-induced I-κB-alpha degradation and NF-κB nuclear localization, but drastically repressed NF-κB transcriptional activation. While IKKbeta and TAK1 knockdown significantly attenuated Tax-induced NF-κB transcriptional activation respectively, they only partially prevented Tax-IRS and did not display significant effect on the alternative pathway. Importantly, NIK knockdown yielded similar effects as IKKalpha and NEMO knockdown. These data suggest that Tax, through its interaction with NEMO, help recruit NIK and TAK1 for IKKalpha and IKKbeta activation respectively. Tax-mediated senescence as driven by the NEMO/NIK/IKKalpha complex involves both classical and alternative NF-κB pathways. Thus, the quality rather than the quantity of NF-κB activity is more important for promoting senescence.

Abstract 5396: LPS mimotopes: A novel class of TLR4 agonist

Abstract Adjuvant driven inflammation is crucial to mounting an antigen specific immune response and is initiated by the activation of toll like receptor (TLR) proteins. Currently, aluminum based adjuvants remain the only FDA approved adjuvants for vaccines. In an effort to develop a new class of adjuvants, TLR agonists, would be natural candidates. LPS, a major structural component of gram negative bacteria cell walls, has been well studied for its property to induce the systemic inflammation observed in septic shock. The objective of the present study was to identify peptide mimics of LPS that could activate TLR-4 and induce an inflammatory condition congenial to an adaptive immune response and in contrast to LPS not induce septic shock. To this end we used phage display peptide library to identify and isolate LPS peptide mimics. The activity of LPS-TLR-4 interaction was assessed by NF-κB translocation analyses in HEK-BLUE™-4 cells, a cell culture model that expresses only TLR-4. NF-κB nuclear translocation and inflammatory cytokine production was assayed by using the murine macrophage cell line RAW264.7. Increasing the NF-κB nuclear translocation is reflected by IκB-degradation within the cytoplasm. As expected, the concentration of cytoplasmic NF-κB was also decreased, which further attests to the shuttling of NF-κB within the nucleus. Similar results pertaining to increased nuclear localization of NF-κB with simultaneous decrease of cytoplasmic IκB-α and NF-κB were observed with HEK-BLUE™-4 cells. Thus, the results observed in this study suggest that the LPS peptide mimics identified act as adjuvants by virtue of their interaction with TLR-4 and thus serve as novel candidates to be considered as a new class of TLR-4 agonist adjuvants. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5396. doi:10.1158/1538-7445.AM2011-5396

Abstract 2109: Tumor progression locus 2 (Tpl2) knockout mice have a dysregulation in COX-2 signaling in two-stage skin carcinogenesis

Abstract Skin cancer is the most common form of cancer in the United States, with an estimated two million cases diagnosed annually. Inflammation is believed to be a critical component in skin cancer progression, and therefore understanding genes controlling inflammation is beneficial. MAP3K8 (Tpl2) is a protein kinase in the MAP Kinase signal transduction cascade. Previous research using a two-stage skin carcinogenesis model has revealed that Tpl2 -/- mice have significantly higher tumor incidence and inflammation than wild-type (WT) controls. Mechanistically, dysregulation in NF-κB signaling is a major contributor to the high degree of inflammation and tumorigenesis found in the Tpl2 -/- mice. Knockout animals have heightened NF-κB reporter activity, increased NF-κB dependent edema, and increased NF-κB nuclear localization. This study investigates how cyclooxygenase-2 (COX-2), an NF-κB regulated gene known to contribute to skin carcinogenesis, and COX-2 downstream factors might play a role in inflammation-mediated skin carcinogenesis in Tpl2 -/- mice. Keratinocytes from newborn WT or Tpl2 -/- mice were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for varying times over 24 hours. Western analysis revealed significant differences in COX-2 and COX-2 dependent prostanoids and prostanoid receptors. Additionally, in vivo experiments confirmed that COX-2 and COX-2 downstream factors were elevated in TPA-treated Tpl2 -/- skin, as well as in papillomas and squamous cell carcinomas from Tpl2-/- mice. These experiments illustrate that COX-2 induction in the absence of Tpl2 may be responsible for the increased tumorigenesis found in Tpl2-/- mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2109. doi:10.1158/1538-7445.AM2011-2109

Ibuprofen Inhibits Activation of Nuclear β-Catenin in Human Colon Adenomas and Induces the Phosphorylation of GSK-3β

Nonselective cyclooxygenase (COX) inhibitors target many of the same cancer-associated molecular pathways as COX-2-specific inhibitors. Although these nonsteroidal anti-inflammatory drugs (NSAIDs) are often associated with gastrointestinal toxicity, there is renewed interest in their use as colorectal cancer (CRC) chemopreventive agents due to the adverse side effects associated with long-term use of selective COX-2 inhibitors. In this study, we investigated the effects of long-term use (up to 25 years) of NSAIDs (ibuprofen or aspirin) on adenoma pathology and β-catenin-mediated signaling in sporadic human colon adenomas. Although NSAID use did not impact overall adenoma size or degree of dysplasia, it did cause a significant inhibition of nuclear β-catenin localization, which correlated with suppression of cyclin D1 expression. To further elucidate the effect of these agents in regulating β-catenin, we treated SW480 colon cancer cells with a panel of NSAIDs and determined their effects on β-catenin levels and cellular localization. In agreement with our in vivo results, both S-ibuprofen and aspirin were found to decrease total levels of β-catenin while increasing its phosphorylation. In addition, S-ibuprofen induced both degradation of IκBα and nuclear localization of NF-κB. Despite its nuclear localization, however, the activation of the NF-κB target genes, Bcl-2, survivin, and cyclin D1, was suppressed. This reduction in NF-κB transcriptional activity may be due to increased phosphorylation of GSK-3β following S-ibuprofen treatment. These data suggest that ibuprofen can effectively target both the Wnt/β-catenin and NF-κB pathways, and potentially uncovers a novel mechanism through which NSAIDS may exert their chemopreventive efficacy.

The Receptor Tyrosine Kinase FGFR4 Negatively Regulates NF-kappaB Signaling

BackgroundNFκB signaling is of paramount importance in the regulation of apoptosis, proliferation, and inflammatory responses during human development and homeostasis, as well as in many human cancers. Receptor Tyrosine Kinases (RTKs), including the Fibroblast Growth Factor Receptors (FGFRs) are also important in development and disease. However, a direct relationship between growth factor signaling pathways and NFκB activation has not been previously described, although FGFs have been known to antagonize TNFα-induced apoptosis.Methodology/Principal FindingsHere, we demonstrate an interaction between FGFR4 and IKKβ (Inhibitor of NFκB Kinase β subunit), an essential component in the NFκB pathway. This novel interaction was identified utilizing a yeast two-hybrid screen [1] and confirmed by coimmunoprecipitation and mass spectrometry analysis. We demonstrate tyrosine phosphorylation of IKKβ in the presence of activated FGFR4, but not kinase-dead FGFR4. Following stimulation by TNFα (Tumor Necrosis Factor α) to activate NFκB pathways, FGFR4 activation results in significant inhibition of NFκB signaling as measured by decreased nuclear NFκB localization, by reduced NFκB transcriptional activation in electophoretic mobility shift assays, and by inhibition of IKKβ kinase activity towards the substrate GST-IκBα in in vitro assays. FGF19 stimulation of endogenous FGFR4 in TNFα-treated DU145 prostate cancer cells also leads to a decrease in IKKβ activity, concomitant reduction in NFκB nuclear localization, and reduced apoptosis. Microarray analysis demonstrates that FGF19 + TNFα treatment of DU145 cells, in comparison with TNFα alone, favors proliferative genes while downregulating genes involved in apoptotic responses and NFκB signaling.Conclusions/SignificanceThese results identify a compelling link between FGFR4 signaling and the NFκB pathway, and reveal that FGFR4 activation leads to a negative effect on NFκB signaling including an inhibitory effect on proapoptotic signaling. We anticipate that this interaction between an RTK and a component of NFκB signaling will not be limited to FGFR4 alone.

Distinct Roles for the NF-κB Pathway In Myeloid and Lymphoid Transformation and Leukemogenesis by BCR-ABL.

AbstractAbstract 1225The BCR-ABL tyrosine kinase, product of the t(9;22) Ph chromosome, activates multiple signaling pathways in leukemic cells from patients with chronic myeloid leukemia (CML) and Ph+ B-cell acute lymphoblastic leukemia (B-ALL). Previous studies have shown that NF-κB is activated in BCR-ABL-expressing cell lines and contributes to transformation of primary B-lymphoid cells by BCR-ABL (Reuther et al., Genes Dev. 1998;12:968), but the mechanism of activation has not been defined (Kirchner et al., Exp. Hematol. 2003;31:504), and importance of NF-kB to myeloid and lymphoid leukemogenesis by BCR-ABL is unknown.To interrogate the role of NF-κB in BCR-ABL-mediated transformation, we utilized a super-repressor mutant form of IκBα (IκBαSR), which has been used to block NF-κB nuclear localization and transactivation by constitutively sequestering NF-κB in the cytoplasm. Using retrovirus co-expressing BCR-ABL and IκBαSR, we found that IκBαSR blocked nuclear p65/RelA expression and inhibited the IL-3 independent growth of Ba/F3 cells and primary B-lymphoid cells transformed by BCR-ABL. The effect of NF-κB inhibition was primarily on proliferation rather than on cell survival, as there was no increase in apoptosis in cells expressing IκBαSR. When primary bone marrow cells were transduced and transplanted under conditions favoring induction of B-ALL or CML-like myeloproliferative neoplasm in recipient mice, co-expression of IκBαSR significantly attenuated disease development and prolonged survival of diseased mice. Molecular analysis of these leukemias demonstrated that NF-κB inhibition decreased the frequency of leukemia-initiating (“stem”) cells in the CML model, but not in the B-ALL model, and was associated with decreased expression of c-Myc, an NF-κB target. To clarify the mechanism of activation of NF-κB in BCR-ABL-expressing cells, we targeted two upstream kinases that negatively regulate IκBα, IKKα/IKK1 or IKKβ/IKK2. To accomplish this, we engineered retroviruses co-expressing BCR-ABL and kinase-inactive, dominant-negative mutants of IKK1 (IKK1KM) or IKK2 (IKK2KM). Co-expression of either IKK mutant inhibited both B-lymphoid transformation and leukemogenesis by BCR-ABL, as well as induction of CML-like MPN, with IKK1 inhibition more effective than IKK2.Together, these results demonstrate that NF-κB is activated in part through the canonical IKK pathway in BCR-ABL-expressing leukemia cells, and that NF-κB signaling plays distinct roles in the pathogenesis of myeloid and lymphoid leukemias induced by BCR-ABL. In CML, NF-κB may play a role for in generation and/or maintenance of leukemic stem cells. These results validate IKKs as targets for therapy in Ph+ leukemias, and motivate the evaluation of small molecule IKK inhibitors in these diseases. Disclosures:No relevant conflicts of interest to declare.

7-Ketocholesteryl-9-carboxynonanoate induced nuclear factor-kappa B activation in J774A.1 macrophages

Aims 7-Ketocholesteryl-9-carboxynonanoate (oxLig-1), a lipid moiety of oxidized low-density lipoprotein (oxLDL), has been reported to be a crucial ligand of beta2-glycoprotein I (β 2-GPI), and plays a potential role in the development of atherosclerosis (AS), however, the role of the sole oxLig-1 in the development of AS remains unclear. Main methods Expression and phosphorylation levels of several proteins, such as nuclear factor-kappaB (NF-κB), protein kinase C (PKC), IκBα and inter-cellular adhesion molecule 1 (ICAM-1) were determined by Western blot; nuclear localization of NF-κB was studied by immunocytochemistry; NF-κB activation was assayed by electrophoretic mobility shift assay (EMSA); and expressions of genes associated with AS process were investigated by Mouse Atherosclerosis RT 2 Profiler PCR Array assay. Key findings The present work indicated that oxLig-1 induced IκBα phosphorylation and results in the nuclear translocation of NF-κB in J774A.1 macrophages. Moreover, oxLig-1-induced NF-κB DNA binding activity was detected by EMSA. Indeed, oxLig-1 led to the activation of PKC prior to activating NF-κB. The treatment of oxLig-1 in J774A.1 macrophages up-regulates the expression of NF-κB target genes including ICAM-1. Significance OxLig-1 on the oxLDL plays an important role in AS process, as evidenced by the NF-κB activation and up-regulation of genes involved in AS development in oxLig-1 challenged J774A.1 macrophages.

Homoisoflavanone inhibits UVB-induced skin inflammation through reduced cyclooxygenase-2 expression and NF-κB nuclear localization

Since the generation of reactive oxygen species (ROS) and release of inflammatory mediators play a major role in UVB-induced inflammation, vigorous attempts have been made for the pharmacological management of these molecules as well as for uncovering the molecular signaling pathways. Homoisoflavanone (5,7-dihydroxy-3-(3-hydroxy-4-methoxybenzyl)-chroman-4-one, HIF) extracted from Cremastra appendiculata has anti-angiogenic activities, but its effect on inflammation was unknown. To investigate the anti-inflammatory effects of HIF on the skin and the underlying molecular mechanisms. HaCaT cells were irradiated by UVB (10 mJ/cm(2)) with or without HIF. Prostaglandin E(2) (PGE(2)) level was measured by enzyme immunoassay. Activation of MAPK and production of cyclooxygenase-2 (COX-2) were determined by Western blot analysis. Localization of nuclear factor kappa B (NF-kappaB) was assessed by immunofluorescence microscopy. Hairless mice were stimulated with UVB or chemical stimulants to induce inflammatory responses in skin. Pretreatment with HIF inhibited the production of intracellular ROS induced by UVB irradiation in HaCaT cells. Further analysis revealed a decrease in the level of MAPK activation and down-regulation of COX-2 expression. In addition, HIF attenuated the nuclear localization of NF-kappaB, resulting in the suppression of inflammatory molecules such as IL-6, IL-8, and TNF-alpha. Finally, topical treatment with HIF inhibited ear edema induced by UVB, 12-O-tetradecanoylphorbol-13-acetate (TPA), arachidonic acid (AA), or croton oil. HIF has a strong protective effect against proinflammatory responses, implying the possibility of preventive application for inflammatory skin diseases.

Abstract 3652: HDAC inhibitors activate NF-κB in human leukemia cells through an ATM/NEMO-dependent DNA Damage-related pathway

Abstract The ability of HDAC inhibitors (HDACIs) to induce sustained activation of NF-κB (p65/RelA) has been attributed to reversible RelA acetylation (Chen et al., Science 293:1653-7, 2001), a phenomenon that diminishes HDACI antineoplastic activity (Dai et al., Mol Cell Biol. 25:5429-44, 2005). However, the process by which HDACIs initially activate NF-κB has yet to be elucidated. To address this question, mechanisms underlying HDAC inhibitor (HDACI)-mediated NF-κB activation were investigated in human myeloid leukemia cells. Exposure of U937 and other leukemia cells to the pan-HDACI LBH-589 induced reactive oxygen species (ROS) and p65/RelA activation, the NF-κB-dependent induction of Mn-SOD2 mRNA and protein accompanied by ROS elimination, and sequential induction of XRCC1 (single strand) followed by γ-H2A. X (double strand) DNA breaks. LBH-589 lethality was significantly attenuated by siRNA knockdown of the chromatin-linked DNA damage protein histone H1.2. U937 IκBα super-repressor cells lacking serine 32 and 36 IκBα phosphorylation sites displayed diminished HDACI-mediated NF-κB activation/Mn-SOD2 induction, accompanied by enhanced ROS accumulation, DNA damage, and apoptosis. In contrast, TRAF2 siRNA knockdown blocked TNFα- but not HDACI-mediated NF-κB activation and lethality. The Mn-SOD2 mimetic TBAP prevented HDACI-induced NF-κB activation and nuclear localization, while dramatically attenuating DNA damage and apoptosis. Notably, LBH-589 exposure activated ATM (on serine 1981) and increased its association with NEMO, events comprising a recently described “inside-out” nuclear NF-κB activation pathway (Wu et al., Science 311:1110-1, 2006). Notably, siRNA NEMO or ATM knockdown blocked HDACI-mediated NF-κB nuclear translocation/activation, diminished MnSOD2 induction, and potentiated oxidative injury, DNA damage and cell death. Similarly, SUMOylation-site mutant NEMO (K277A and/or K309A) cells exposed to LBH-589 displayed diminished association of ATM with NEMO, reduced NEMO and p65/RelA nuclear localization/activation, and MnSOD2 down-regulation. These events were accompanied by increased ROS production, γ-H2A. X formation, and enhanced lethality. Collectively, these findings indicate that in human leukemia cells, HDACIs activate the cytoprotective NF-κB pathway through an ATM/NEMO-dependent process involving the induction of ROS and DNA damage. They also raise the possibility that blocking NF-κB activation via the atypical DNA damage-related ATM/NEMO nuclear pathway has the potential to enhance HDACI antileukemic activity. 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 3652.

Lutein protects HT-29 cells against Deoxynivalenol-induced oxidative stress and apoptosis: Prevention of NF-κB nuclear localization and down regulation of NF-κB and Cyclo-Oxygenase – 2 expression

Increasing evidence suggests that oxidative stress is closely linked to toxic responses in cells. The tricothecene mycotoxin, Deoxynivalenol (DON), primarily affects cells of the immune system and the GI tract. DON′s cytotoxicity is closely linked to intracellular ROS, and it exerts its toxic effect by a mechanism known as ribotoxic stress response, which drives both cytokine expressions at low dosages and apoptosis at high dosages. Studies to alleviate DON′s toxicity are sparsely reported in literature. In the present study, the cytoprotective effect of lutein, was tested in HT-29 cells against DON-induced oxidative stress and cytotoxicity. MTT assay revealed IC 20 values of DON at 250 ng/ml. Pre-treatment of cells with 10 μM lutein resulted in 95% cell viability. Lutein combated DON-induced oxidative stress and downregulated expression of inflammatory genes, NF-κB and COX-2. Lutein also prevented DON-induced migration of NF-κB into the nucleus, as measured by immunofluorescence. Morphological studies by Electron microscopy and Cell cycle analysis by flow cytometry indicated that lutein prevented DON-induced apoptosis. The results of the present study demonstrate for the first time that lutein exerts a cytoprotective role in DON-induced toxicity.

The fixed structure of Licochalcone A by α, β-unsaturated ketone is necessary for anti-inflammatory activity through the inhibition of NF-κB activation

Glycyrrhiza inflata has been used as a traditional medicine with anti-inflammatory activity. Previously, we reported that a major component, Licochalcone A, potently inhibited TNFα-induced NF-κB activation by inhibiting IKKβ activation. In this study, we investigated whether the fixed structure of Licochalcone A by α, β-unsaturated ketone is required for its inhibitory effect of NF-κB activation. Interestingly, reduced Licochalcone A, which lacks a double bond, failed to inhibit TNFα-induced NF-κB activation. Whereas Licochalcone A potently inhibited TNFα-induced IKK activation, IκBα degradation, nuclear localization of NF-κB and its DNA binding activity, no inhibitory effect was observed by reduced Licochalcone A. In addition, TNFα-induced expression of inflammatory cytokines, CCL2/MCP-1 and CXCL1/KC, was clearly inhibited by Licochalcone A but not reduced Licochalcone A. As a result, culture media pretreated with Licochalcone A but not reduced Licochalcone A following TNFα stimulation significantly inhibited the chemotactic activity of neutrophils. Furthermore, acute carrageenan-induced paw edema in mice was markedly inhibited by administration of Licochalcone A but not reduced Licochalcone A. Taken together, it is suggested that Licochalcone A is a promising anti-inflammatory drug in vivo and its fixed structure is critical for anti-inflammatory activity.

Silencing of C5a receptor gene with siRNA for protection from Gram-negative bacterial lipopolysaccharide-induced vascular permeability

Endothelial barrier dysfunction leading to increased permeability and vascular leakage is an underlying cause of several pathological conditions. Whereas these changes have been shown to be associated with activation of the complement system, leading to the release of C5a and interaction of C5a–C5a receptor (C5aR), the role of C5aR in endothelial cells remain(s) ill-defined. Here, we report an essential role of C5aR in endothelial cell injury and vascular permeability through silencing of the C5aR gene using siRNA. In the cultured mouse dermal microvascular endothelial cells (MEMECs) monolayer transfected with C5aR-siRNA, endotoxin-induced cell injury by evaluated as transendothelial flux, cell detachment, and cytoskeletal disorganization was inhibited. Upregulation of vascular cell adhesion molecule-1 (VCAM-1) was also suppressed. Studies exploring the underlying mechanism of siRNA-mediated suppression in VCAM-1 expression were related to reduction of NF-κB activation and nuclear localization of both p50 and p65. The effect was associated with inhibition in activation of protein kinase Cδ(PKC-δ) and induction of PKC-mediated mitogen-activated protein kinase phosphatases-1 (MKP-1) leading to the increased activity of p42/p44 mitogen-activated protein (MAP) kinase cascade. In the model of mice administrated with C5aR-siRNA, endotoxin-induced plasma leakage was inhibited in local abdominal skin. Systemic administration of endotoxin to mice resulted in increased microvascular permeability in multiple organs was reduced. These studies demonstrate that the C5aR responsible for vascular endothelial cell injury and plasma permeability is an important factor, and that blockade of C5aR may be useful therapeutic targets for the prevention of vascular permeability in pathogenic condition.