Nuclear Localization Of NF-κB Research Articles

The role of TLR4-mediated PTEN/PI3K/AKT/NF-κB signaling pathway in neuroinflammation in hippocampal neurons

This study investigated a possible role for a toll-like receptor 4 (TLR4)-mediated PTEN/PI3K/AKT/NF-κB signaling pathway in neuroinflammation in rat hippocampal neurons. Cultured neurons were treated with lipopolysaccharide (LPS), a TLR4 ligand, or pre-treated with TLR4 antibodies to block TLR4 signaling. Neurons were also treated with dipotassium bisperoxo (pyridine-2-carboxyl) oxovanadate [bpV(pic)] and pyrrolidine dithiocarbamate (PDTC), selective inhibitors of PTEN and NF-κB, respectively, in the presence of LPS. The levels of PTEN, AKT, and their phosphorylated forms were evaluated, along with the nuclear localization of NF-κB, to assess pathway activation. The induction of a neuroinflammatory response was determined by measuring TNF-α and IL-1β mRNA and protein levels. The results indicated that while LPS stimulation had no effect on PTEN and AKT expression, the levels of phosphorylated PTEN (pPTEN) decreased, while phosphorylated AKT (pAKT) levels increased. Moreover, LPS treatment induced the nuclear translocation of NF-κB, and increased the expression and secretion of TNF-α and IL-1β. Blocking TLR4 increased the levels of pPTEN and decreased the levels of pAKT, while pre-treatment with bpV(pic) led to a reduction in levels of pPTEN and pAKT. Furthermore, treatment with TLR4 antibody, bpV(pic), and PDTC decreased LPS-induced nuclear translocation of NF-κB, and resulted in a downregulation of TNF-α and IL-1β expression. Taken together, these results provide evidence for a TLR4-mediated PTEN/PI3K/AKT/NF-κB signaling pathway in rat hippocampal neurons, which is associated with the activation of a neuroinflammatory response.

Fish Oil Suppresses Cell Growth and Metastatic Potential by Regulating PTEN and NF-κB Signaling in Colorectal Cancer

Homeostasis in eukaryotic tissues is tightly regulated by an intricate balance of the prosurvival and antisurvival signals. The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10), a dual-specificity phosphatase, plays a functional role in cell cycle arrest and apoptosis. NF-κB and its downstream regulators (such as VEGF) play a central role in prevention of apoptosis, promotion of inflammation and tumor growth. Therefore, we thought to estimate the expression of PTEN, Poly-ADP-ribose polymerase (PARP), NF-κBp50, NF-κBp65 and VEGF to evaluate the effect of supplementation of fish oil on apoptotic and inflammatory signaling in colon carcinoma. Male wistar rats in Group I received purified diet while Group II and III received modified diet supplemented with FO∶CO(1∶1)&FO∶CO(2.5∶1) respectively. These were further subdivided into controls receiving ethylenediamine-tetra acetic-acid and treated groups received dimethylhydrazine-dihydrochloride (DMH)/week for 4 weeks. Animals sacrificed 48 hours after last injection constituted initiation phase and that sacrificed after 16 weeks constituted post-initiation phase. We have analysed expression of PTEN, NF-κBp50, NF-κBp65 by flowcytometer and nuclear localization of NF-κB by immunofluorescence. PARP and VEGF were assessed by immunohistochemistry. In the initiation phase, animals receiving DMH have shown increased % of apoptotic cells, PTEN, PARP, NF-κBp50, NF-κBp65 and VEGF however in post-initiation phase no significant alteration in apoptosis with decreased PTEN and increased PARP, NF-κBp50, NF-κBp65 and VEGF were observed as compared to control animals. On treatment with both ratios of fish oil in both the phases, augmentation in % of apoptotic cells, decreased PTEN, PARP, NF-κBp50, NF-κBp65 and VEGF were documented with respect to DMH treated animals with effect being more exerted with higher ration in post-initiation phase. Hence, fish oil activates apoptosis, diminishes DNA damage and inhibits inflammatory signalling in a dose and time dependent manner so as to inhibit progression of colon cancer.

Tyrosine Phosphorylation Allows Integration of Multiple Signaling Inputs by IKKβ

Signaling regulated by NFκB and related transcription factors is centrally important to many inflammatory and autoimmune diseases, cancer, and stress responses. The kinase that directly regulates the canonical NFκB transcriptional pathway, Inhibitor of κB kinase β (IKKβ), undergoes activation by Ser phosphorylation mediated by NIK or TAK1 in response to inflammatory signals. Using titanium dioxide-based phosphopeptide enrichment (TiO2)-liquid chromatography (LC)-high mass accuracy tandem mass spectrometry (MS/MS), we analyzed IKKβ phosphorylation in human HEK293 cells expressing IKKβ and FGFR2, a Receptor tyrosine kinase (RTK) essential for embryonic differentiation and dysregulated in several cancers. We attained unusually high coverage of IKKβ, identifying an abundant site of Tyr phosphorylation at Tyr169 within the Activation Loop. The phosphomimic at this site confers a level of kinase activation and NFκB nuclear localization exceeding the iconic mutant S177E/S181E, demonstrating that RTK-mediated Tyr phosphorylation of IKKβ has the potential to directly regulate NFκB transcriptional activation.

Experimental Diabetes Induces Structural, Inflammatory and Vascular Changes of Achilles Tendons

This study aims to demonstrate how the state of chronic hyperglycemia from experimental Diabetes Mellitus can influence the homeostatic imbalance of tendons and, consequently, lead to the characteristics of tendinopathy. Twenty animals were randomly divided into two experimental groups: control group, consisting of healthy rats and diabetic group constituted by rats induced to Diabetes Mellitus I. After twenty-four days of the induction of Diabetes type I, the Achilles tendon were removed for morphological evaluation, cellularity, number and cross-sectional area of blood vessel, immunohistochemistry for Collagen type I, VEGF and NF-κB nuclear localization sequence (NLS) and nitrate and nitrite level. The Achilles tendon thickness (µm/100g) of diabetic animals was significantly increased and, similarly, an increase was observed in the density of fibrocytes and mast cells in the tendons of the diabetic group. The average number of blood vessels per field, in peritendinous tissue, was statistically higher in the diabetic group 3.39 (2.98) vessels/field when compared to the control group 0.89 (1.68) vessels/field p = 0.001 and in the intratendinous region, it was observed that blood vessels were extremely rare in the control group 0.035 (0.18) vessels/field and were often present in the tendons of the diabetic group 0.89 (0.99) vessels/field. The immunohistochemistry analysis identified higher density of type 1 collagen and increased expression of VEGF as well as increased immunostaining for NFκB p50 NLS in the nucleus in Achilles tendon of the diabetic group when compared to the control group. Higher levels of nitrite/nitrate were observed in the experimental group induced to diabetes. We conclude that experimental DM induces notable structural, inflammatory and vascular changes in the Achilles tendon which are compatible with the process of chronic tendinopathy.

Calpastatin Prevents NF-κB–Mediated Hyperactivation of Macrophages and Attenuates Colitis

Calpain enzymes proteolytically modulate cellular function and have been implicated in inflammatory diseases. In this study, we found that calpain levels did not differ between intestinal tissues from inflammatory bowel disease (IBD) patients and healthy controls, but IBD tissues showed increased levels of the endogenous calpain inhibitor, calpastatin (CAST). To investigate the role of CAST in the immune system during IBD, mice were x-ray irradiated, reconstituted with either CAST-knockout (KO) or wild-type (WT) bone marrow, and subjected to dextran sulfate sodium-induced colitis. CAST-KO recipients with induced colitis exhibited more severe weight loss, bloody diarrhea, and anemia compared with WT controls. Histological evaluation of colons from KO recipients with colitis revealed increased inflammatory pathology. Macrophages purified from the colons of KO recipients had higher IL-6, TNF-α, and IFN-γ mRNA levels compared with WT controls. Mechanistic investigations using small interfering RNA and KO bone marrow to generate CAST-deficient macrophages showed that CAST deficiency during activation with bacterial pathogen associated molecular patterns, including heat-killed Enterococcus faecalis or CpG DNA, led to increased IκB cleavage, NF-κB nuclear localization, and IL-6 and TNF-α secretion. Thus, CAST plays a central role in regulating macrophage activation and limiting pathology during inflammatory disorders like IBD.

Calpastatin prevents NF-κB mediated hyperactivation of macrophages and attenuates colitis (P3151)

Abstract Calpain enzymes proteolytically modulate cellular function and have been implicated in inflammatory diseases. In this study, we found that calpain levels did not differ between intestinal tissues from inflammatory bowel disease (IBD) patients and healthy controls, but IBD tissues showed increased levels of the endogenous calpain inhibitor, calpastatin (CAST). To investigate the role of CAST in the immune system during IBD, mice were x-ray irradiated and reconstituted with either CAST knockout (KO) or wild-type (WT) bone marrow and subjected to dextran sulfate sodium (DSS)-induced colitis. CAST KO recipients with induced colitis exhibited more severe weight loss, bloody diarrhea, and anemia compared to WT controls. Histological evaluation of colons from KO recipients with colitis revealed increased inflammatory pathology. Macrophages purified from the colons of KO recipients had higher IL-6, TNFα, and IFNγ mRNA levels compared to WT controls. Mechanistic investigations using siRNA and KO bone marrow to generate CAST deficient macrophages showed that CAST deficiency during activation with bacterial PAMPs including heat-killed E. faecalis or CpG DNA led to increased IκB cleavage, NF-κB nuclear localization, and IL-6 and TNFα secretion. Thus, CAST plays a central role in regulating macrophage activation and limiting pathology during inflammatory disorders like IBD.

Peptide Inhibitor of NF-κB Translocation Ameliorates Experimental Atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the arterial wall. NF-κB is a major regulator of inflammation that controls the expression of many genes involved in atherogenesis. Activated NF-κB was detected in human atherosclerotic plaques, and modulation of NF-κB inflammatory activity limits disease progression in mice. Herein, we investigate the anti-inflammatory and atheroprotective effects of a cell-permeable peptide containing the NF-κB nuclear localization sequence (NLS). In vascular smooth muscle cells and macrophages, NLS peptide specifically blocked the importin α-mediated nuclear import of NF-κB and prevented lipopolysaccharide-induced pro-inflammatory gene expression, cell migration, and oxidative stress. In experimental atherosclerosis (apolipoprotein E-knockout mice fed a high-fat diet), i.p., 0.13 μmol/day NLS peptide administration for 5 weeks attenuated NF-κB activation in atherosclerotic plaques. NLS peptide significantly inhibited lesion development at both early (age 10 weeks) and advanced (age 28 weeks) stages of atherosclerosis in mice, without affecting serum lipid levels. Plaques from NLS-treated mice contained fewer macrophages of pro-inflammatory M1 subtype than those from respective untreated controls. By contrast, the relative smooth muscle cell and collagen content was increased, indicating a more stable plaque phenotype. NLS peptide also attenuated pro-inflammatory gene expression and oxidative stress in aortic lesions. Our study demonstrates that targeting NF-κB nuclear translocation hampers inflammation and atherosclerosis development and identifies cell-permeable NLS peptide as a potential anti-atherosclerotic agent.

Abstract 2320: Oncogenic K-ras and loss of Smad4 mediate invasion by activating a NF-kappaB/EGFR axis that induces expression of MMP9/uPA in human pancreas progenitor cells.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an extremely fatal and highly metastatic in nature. Several pathways and mutations contribute to the development and pathogenesis of PDAC. Activating K-ras mutations and inactivating mutations of Smad4 are two common genetic alterations that occur in the development and progression of PDAC. To further study the role of these two mutations in the pathogenesis of PDAC, immortalized human pancreas nestin postive cells (HPNE) were genetically modified by expressing oncogenic K-ras (GD12) and /or by shRNA knock down of Smad4. Present studies show that these two mutations caused increase in expression of EGFR and erbB2 growth factor receptors and increased expression and nuclear translocation of NF-κB. The increase in EGFR expression and nuclear translocation of NF-κB was associated with development of an invasive phenotype as measured by matrigel assays. Our data also demonstrated that MMP9 and uPA were substantially up regulated in HPNE cells that express K-ras or along with loss of Smad4 activities. MMP-9 and uPA are two extracellular matrix (ECM)-degrading proteinases known to play important roles in driving invasion and metastasis. Zymography studies confirm the expression and enzymatic activities of uPA and MMP-9 in the progenitor cells. The mechanism underlying this uPA/MMP9 induction is linked with NF-κB activation. Immuno-fluorescence, Western blot and chromatin immuno-precipitation (ChIP) analysis confirm the nuclear translocation of NF-κB RelA (p65) sub-unit, by induction of K-ras. Following signaling cascade, EGFR activated by the K-ras mediated NF-κB nuclear localization and further activation induced by loss of Smad4. Our results also suggest that the EGFR pathway as a critical mediator connecting Ras to NF-κB in proteolytic enzyme secretion as well as cancer invasion. In conclusion, the present study reveals a new insight in pancreatic progenitor cell invasion which includes signal transduction of K-ras activity and Smad4 inactivation in regulation of NF-κB/EGFR axis to induce expression of uPA and MMP9. Therefore, induction of K-ras and loss of Smad4 could play important roles in PDAC invasion through NF-κB/EGFR activation and subsequent induced expression of MMP9 and uPA, and might be suitable molecular candidates for the development of new therapeutic strategies for pancreatic cancer. Citation Format: Alakesh Bera, Shujie Zhao, Lin Cao, James W. Freeman. Oncogenic K-ras and loss of Smad4 mediate invasion by activating a NF-kappaB/EGFR axis that induces expression of MMP9/uPA in human pancreas progenitor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2320. doi:10.1158/1538-7445.AM2013-2320

Angiotensin II activates NF-κB through AT1A receptor recruitment of β-arrestin in cultured rat vascular smooth muscle cells

Activation of the angiotensin type 1A receptor (AT1AR) in rat aorta vascular smooth muscle cells (RASMC) results in increased synthesis of the proinflammatory enzyme cyclooxygenase-2 (COX-2). We previously showed that nuclear localization of internalized AT1AR results in activation of transcription of the gene for COX-2, i.e., prostaglandin-endoperoxide synthase-2. Others have suggested that ANG II stimulation of COX-2 protein synthesis is mediated by NF-κB. The purpose of the present study was to examine the interrelationship between AT1AR activation, β-arrestin recruitment, and NF-κB activation in the ability of ANG II to increase COX-2 protein synthesis in RASMC. In the present study we utilized RASMC, inhibitors of the NF-κB pathway, β-arrestin knockdown, radioligand binding, immunoblotting, and immunofluorescence to characterize the roles of AT1AR internalization, NF-κB activation, and β-arrestin in ANG II-induced COX-2 synthesis. Ro-106-9920 or parthenolide, agents that inhibit the initial steps of NF-κB activation, blocked ANG II-induced p65 NF-κB nuclear localization, COX-2 protein expression, β-arrestin recruitment, and AT1AR internalization without inhibiting ANG II-induced p42/44 ERK activation. Curcumin, an inhibitor of NF-κB-induced transcription, blocked ANG II-induced COX-2 protein expression without altering AT1AR internalization, ANG II-induced p65 NF-κB nuclear localization, or p42/44 ERK activation. Small interfering RNA-induced knockdown of β-arrestin-1 and -2 inhibited ANG II-induced p65 NF-κB nuclear localization. In vascular smooth muscle cells, internalization of the activated AT1AR mediated by β-arrestins activates the NF-κB pathway, producing nuclear localization of the transcription factor and initiation of COX-2 protein synthesis, thereby linking internalization of the receptor with the NF-κB pathway.

Propolis changes the anticancer activity of temozolomide in U87MG human glioblastoma cell line

BackgroundPropolis is a honey bee product which contains many active compounds, such as CAPE or chrysin, and has many beneficial activities. Recently, its anti-tumor properties have been discussed. We have tested whether the ethanolic extract of propolis (EEP) interferes with temozolomide (TMZ) to inhibit U87MG cell line growth.MethodsThe U87MG glioblastoma cell line was exposed to TMZ (10-100 μM), EEP (10-100 μg/ml) or a mixture of TMZ and EEP during 24, 48 or 72 hours. The cell division was examined by the H3-thymidine incorporation, while the western blot method was used for detection of p65 subunit of NF-κB and ELISA test to measure the concentration of its p50 subunit in the nucleus.ResultsWe have found that both, TMZ and EEP administrated alone, had a dose- and time-dependent inhibitory effect on the U87MG cell line growth, which was manifested by gradual reduction of cell viability and alterations in proliferation rate. The anti-tumor effect of TMZ (20 μM) was enhanced by EEP, which was especially well observed after a short time of exposition, where simultaneous usage of TMZ and EEP resulted in a higher degree of growth inhibition than each biological factor used separately. In addition, cells treated with TMZ presented no changes in NF-κB activity in prolonged time of treatment and EEP only slightly reduced the nuclear translocation of this transcription factor. In turn, the combined incubation with TMZ and EEP led to an approximately double reduction of NF-κB nuclear localization.ConclusionsWe conclude that EEP presents cytotoxic properties and may cooperate with TMZ synergistically enhancing its growth inhibiting activity against glioblastoma U87MG cell line. This phenomenon may be at least partially mediated by a reduced activity of NF-κB.

Catalpol suppresses advanced glycation end-products-induced inflammatory responses through inhibition of reactive oxygen species in human monocytic THP-1 cells

Advanced glycation end-products (AGEs) play a pivotal role in the development of diabetic complications by inducing inflammation. We previously reported that the fresh roots of Rehmannia glutinosa Libosch., which have been used for the treatment of diabetes in traditional Korean medicine, also have the potential to suppress AGE-mediated inflammatory response in THP-1 cells. In the present study, we isolated catalpol from R. glutinosa, and examined whether it has anti-inflammatory effects on AGE-stimulated THP-1 cells. Catalpol reduced the expression of pro-inflammatory mediates, such as monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), inducible NO synthase (iNOS), and receptor for AGE (RAGE). Promoter and electromobility shift assays showed that transcriptional activation of NF-κB was significantly reduced by catalpol treatment, while AP-1 was not. Catalpol also suppressed AGE-induced phosphorylation of mitogen activated protein (MAP) kinases, degradation of IκBα and the nuclear localization of NF-κB. Moreover, the production of intracellular reactive oxygen species (ROS) elicited by AGE was also suppressed by catalpol treatment, through dual action of reducing ROS itself and inhibiting NADPH oxidase activity. Our findings indicate that catalpol suppresses AGE-mediated inflammation by inhibiting ROS production and NF-κB activity. We suggest that catalpol, a major constituent of the fresh roots of R. glutinosa, contributes to the prevention of AGE-mediated diabetic complications.

The anti-inflamm-aging and hepatoprotective effects of huperzine A in d-galactose-treated rats

Oxidative stress contributes to a chronic inflammatory process referred to as “inflamm-aging”. Acetylcholinesterase inhibitors (AChEI) can enhance cholinergic transmission and act as anti-inflammatory agents via immunocompetent cells expressing α-7 acetylcholine receptors (AChR). The present study explores the possible role of huperzine A, a reversible and selective AChEI, against d-gal-induced oxidative damage, cell toxicity and inflamm-aging in rat livers. In two-month-old rats with normal liver function, an 8-week administration of d-gal (300mg/kg subcutaneously (s.c.) injected), significantly increased hepatic impairment, ROS generation and oxidative damage, hepatic senescence, nuclear factor-kappa B (NF-κB) activation and inflammatory responses. An 8-week co-administration of both d-gal (300mg/kg s.c.) and huperzine A (0.1mg/kg s.c.) not only significantly decreased hepatic function impairment, ROS generation, oxidative damage, but also suppressed inflamm-aging by inhibiting hepatic replicative senescence, AChE activity, IκBα degradation, NF-κB p65 nuclear translocation and inflammatory responses. The expression levels of pro-inflammatory cytokine mRNA and proteins, such as TNFα, IL-1β and IL-6 decrease significantly, and the protein levels of the anti-inflammatory cytokine IL-10 display an obvious increase. These findings indicated that d-gal-induced hepatic injury and inflamm-aging in the rat liver was associated with the development of a pro-inflammatory phenotype in this organ. d-gal induced damage-associated molecular patterns (DAMPs) because oxidative damages might play an important role in d-gal-induced hepatic sterile inflammation. Huperzine A exhibited protective effects against d-gal-induced hepatotoxicity and inflamm-aging by inhibiting AChE activity and via the activation of the cholinergic anti-inflammatory pathway. The huperzine A mechanism might be involved in the inhibition of DAMPs-mediated NF-κB nuclear localization and activation.

Pathways Involved in the Synergistic Activation of Macrophages by Lipoteichoic Acid and Hemoglobin

Lipoteichoic acid (LTA) is a Gram-positive cell surface molecule that is found in both a cell-bound form and cell-free form in the host during an infection. Hemoglobin (Hb) can synergize with LTA, a TLR2 ligand, to potently activate macrophage innate immune responses in a TLR2- and TLR4-dependent way. At low levels of LTA, the presence of Hb can result in a 200-fold increase in the secretion of IL-6 following macrophage activation. Six hours after activation, the macrophage genes that are most highly up-regulated by LTA plus Hb activation compared to LTA alone are cytokines, chemokines, receptors and interferon-regulated genes. Several of these genes exhibit a unique TLR4-dependent increase in mRNA levels that continued to rise more than eight hours after stimulation. This prolonged increase in mRNA levels could be the result of an extended period of NF-κB nuclear localization and the concurrent absence of the NF-κB inhibitor, IκBα, after stimulation with LTA plus Hb. Dynasore inhibition experiments indicate that an endocytosis-dependent pathway is required for the TLR4-dependent up-regulation of IL-6 secretion following activation with LTA plus Hb. In addition, interferon-β mRNA is present after activation with LTA plus Hb, suggesting that the TRIF/TRAM-dependent pathway may be involved. Hb alone can elicit the TLR4-dependent secretion of TNF-α from macrophages, so it may be the TLR4 ligand. Hb also led to secretion of high mobility group box 1 protein (HMGB1), which synergized with LTA to increase secretion of IL-6. The activation of both the TLR2 and TLR4 pathways by LTA plus Hb leads to an enhanced innate immune response.

Phyllostachys edulis Compounds Inhibit Palmitic Acid-Induced Monocyte Chemoattractant Protein 1 (MCP-1) Production

Background Phyllostachys edulis Carriere (Poaceae) is a bamboo species that is part of the traditional Chinese medicine pharmacopoeia. Compounds and extracts from this species have shown potential applications towards several diseases. One of many complications found in obesity and diabetes is the link between elevated circulatory free fatty acids (FFAs) and chronic inflammation. This study aims to present a possible application of P. edulis extract in relieving inflammation caused by FFAs. Monocyte chemoattractant protein 1 (MCP-1/CCL2) is a pro-inflammatory cytokine implicated in chronic inflammation. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein 1 (AP-1) are transcription factors activated in response to inflammatory stimuli, and upregulate pro-inflammatory cytokines such as MCP-1. This study examines the effect of P. edulis extract on cellular production of MCP-1 and on the NF-κB and AP-1 pathways in response to treatment with palmitic acid (PA), a FFA.Methodology/Principal FindingsMCP-1 protein was measured by cytometric bead assay. NF-κB and AP-1 nuclear localization was detected by colorimetric DNA-binding ELISA. Relative MCP-1 mRNA was measured by real-time quantitative PCR. Murine cells were treated with PA to induce inflammation. PA increased expression of MCP-1 mRNA and protein, and increased nuclear localization of NF-κB and AP-1. Adding bamboo extract (BEX) inhibited the effects of PA, reduced MCP-1 production, and inhibited nuclear translocation of NF-κB and AP-1 subunits. Compounds isolated from BEX inhibited MCP-1 secretion with different potencies.Conclusions/SignificancePA induced MCP-1 production in murine adipose, muscle, and liver cells. BEX ameliorated PA-induced production of MCP-1 by inhibiting nuclear translocation of NF-κB and AP-1. Two O-methylated flavones were isolated from BEX with functional effects on MCP-1 production. These results may represent a possible therapeutic application of BEX and its compounds toward alleviating chronic inflammation caused by elevated circulatory FFAs.

HTLV-1 Tax-Induced Rapid Senescence Is Driven by the Transcriptional Activity of NF-κB and Depends on Chronically Activated IKKα and p65/RelA

The HTLV-1 oncoprotein Tax is a potent activator of classical and alternative NF-κB pathways and is thought to promote cell proliferation and transformation via NF-κB activation. We showed recently that hyperactivation of NF-κB by Tax triggers a cellular senescence response (H. Zhi et al., PLoS Pathog. 7:e1002025, 2011). Inhibition of NF-κB activation by expression of I-κBα superrepressor or by small hairpin RNA (shRNA)-mediated knockdown of p65/RelA rescues cells from Tax-induced rapid senescence (Tax-IRS). Here we demonstrate that Tax-IRS is driven by the transcriptional activity of NF-κB. Knockdown of IKKγ, the primary Tax target, by shRNAs abrogated Tax-mediated activation of both classical and alternative NF-κB pathways and rendered knockdown cells resistant to Tax-IRS. Consistent with a critical role of IKKα in the transcriptional activity of NF-κB, IKKα deficiency drastically decreased NF-κB trans-activation by Tax, although it only modestly reduced Tax-mediated I-κBα degradation and NF-κB nuclear localization. In contrast, although IKKβ knockdown attenuated Tax-induced NF-κB transcriptional activation, the residual NF-κB activation in IKKβ-deficient cells was sufficient to trigger Tax-IRS. Importantly, the phenotypes of NIK and TAK1 knockdown were similar to those of IKKα and IKKβ knockdown, respectively. Finally, double knockdown of RelB and p100 had a minor effect on senescence induction by Tax. These data suggest that Tax, through its interaction with IKKγ, helps recruit NIK and TAK1 for IKKα and IKKβ activation, respectively. In the presence of Tax, the delineation between the classical and alternative NF-κB pathways becomes obscured. The senescence checkpoint triggered by Tax is driven by the transcriptional activity of NF-κB, which depends on activated IKKα and p65/RelA.

Magnesium Decreases Inflammatory Cytokine Production: A Novel Innate Immunomodulatory Mechanism

MgSO(4) exposure before preterm birth is neuroprotective, reducing the risk of cerebral palsy and major motor dysfunction. Neonatal inflammatory cytokine levels correlate with neurologic outcome, leading us to assess the effect of MgSO(4) on cytokine production in humans. We found reduced maternal TNF-α and IL-6 production following in vivo MgSO(4) treatment. Short-term exposure to a clinically effective MgSO(4) concentration in vitro substantially reduced the frequency of neonatal monocytes producing TNF-α and IL-6 under constitutive and TLR-stimulated conditions, decreasing cytokine gene and protein expression, without influencing cell viability or phagocytic function. In summary, MgSO(4) reduced cytokine production in intrapartum women, term and preterm neonates, demonstrating effectiveness in those at risk for inflammation-associated adverse perinatal outcomes. By probing the mechanism of decreased cytokine production, we found that the immunomodulatory effect was mediated by magnesium and not the sulfate moiety, and it was reversible. Cellular magnesium content increased rapidly upon MgSO(4) exposure, and reduced cytokine production occurred following stimulation with different TLR ligands as well as when magnesium was added after TLR stimulation, strongly suggesting that magnesium acts intracellularly. Magnesium increased basal IĸBα levels, and upon TLR stimulation was associated with reduced NF-κB activation and nuclear localization. These findings establish a new paradigm for innate immunoregulation, whereby magnesium plays a critical regulatory role in NF-κB activation, cytokine production, and disease pathogenesis.

Assessment of network perturbation amplitudes by applying high-throughput data to causal biological networks

BackgroundHigh-throughput measurement technologies produce data sets that have the potential to elucidate the biological impact of disease, drug treatment, and environmental agents on humans. The scientific community faces an ongoing challenge in the analysis of these rich data sources to more accurately characterize biological processes that have been perturbed at the mechanistic level. Here, a new approach is built on previous methodologies in which high-throughput data was interpreted using prior biological knowledge of cause and effect relationships. These relationships are structured into network models that describe specific biological processes, such as inflammatory signaling or cell cycle progression. This enables quantitative assessment of network perturbation in response to a given stimulus.ResultsFour complementary methods were devised to quantify treatment-induced activity changes in processes described by network models. In addition, companion statistics were developed to qualify significance and specificity of the results. This approach is called Network Perturbation Amplitude (NPA) scoring because the amplitudes of treatment-induced perturbations are computed for biological network models. The NPA methods were tested on two transcriptomic data sets: normal human bronchial epithelial (NHBE) cells treated with the pro-inflammatory signaling mediator TNFα, and HCT116 colon cancer cells treated with the CDK cell cycle inhibitor R547. Each data set was scored against network models representing different aspects of inflammatory signaling and cell cycle progression, and these scores were compared with independent measures of pathway activity in NHBE cells to verify the approach. The NPA scoring method successfully quantified the amplitude of TNFα-induced perturbation for each network model when compared against NF-κB nuclear localization and cell number. In addition, the degree and specificity to which CDK-inhibition affected cell cycle and inflammatory signaling were meaningfully determined.ConclusionsThe NPA scoring method leverages high-throughput measurements and a priori literature-derived knowledge in the form of network models to characterize the activity change for a broad collection of biological processes at high-resolution. Applications of this framework include comparative assessment of the biological impact caused by environmental factors, toxic substances, or drug treatments.

The Role of Nuclear Factor- B in Oxidative Stress Associated with Rabies Virus Infection (P03.263)

Objective: To investigate the role of NF-κB in rabies virus-induced oxidative stress in neurons. Background Recent studies in an experimental mouse model of rabies have shown that there are major structural changes in the brain involving neuronal processes that are associated with severe clinical disease. Cultured adult dorsal root ganglion (DRG) neurons are a good in vitro model for studying mechanisms involved in rabies virus – induced degeneration of neurites, because, unlike other neuronal cell types, these neurons are permissive to rabies virus infection. DRG neurons infected with the CVS strain of rabies virus show axonal swellings and immunostaining for 4-hydroxy-2-nonenal (4-HNE), indicating evidence of lipid peroxidation associated with oxidative stress, and also reduced axonal growth vs. mock-infected DRG neurons. Design/Methods: The expression and localization of NF-κB were evaluated by Western immunoblotting and immunofluorescence, respectively, in mock- versus CVS-infected rat DRG neurons. The effects of ciliary neurotrophic factor (CNTF), a potent activator of NF-κB, and SN50, a peptide inhibitor of NF-κB, were also evaluated. Results: CNTF had no effect on mock-infected rat DRG neurons, and was neuroprotective for CVS-neurons in reducing the number of 4-HNE-labeled puncta. SN50 and CVS infection had an additive effect in producing axonal swellings, suggesting that NF-κB is neuroprotective. CVS infection induced the expression of protein for the p50 subunit of NF-κB. The fluorescent signal for p50 was quantitatively evaluated in the nucleus and cytoplasm of mock- and CVS-infected rat DRG neurons. At 24 hrs p.i. there was a significant increase in the nucleus:cytoplasm ratio, indicating increased transcriptional activity of NF-kB, perhaps as a response to stress, whereas at both 48 and 72 hrs p.i. there was significantly reduced nuclear localization of NF-kB. Conclusions: CVS infection may induce oxidative stress by preventing nuclear activation of NF-κB. A rabies virus protein may directly inhibit NF-kB activity. Supported by: Canadian Institutes of Health Research. Disclosure: Dr. Jackson has nothing to disclose. Dr. Kammouni has nothing to disclose. Dr. Hasan has nothing to disclose. Dr. Fernyhough has nothing to disclose.

Abstract 310: Oncogene-specific regulation of immune responses during tumorigenesis

Abstract Oncogenes can induce inflammatory signaling, leading to the recruitment of immune cells to tumor site. Immune cells can further influence tumor development by either restraining or facilitating tumorigenesis. Here, we investigated how distinct oncogenes shape the immune responses during tumorigenesis in vivo. We utilized several established mouse models of liver cancer in which tumorigenesis is driven by the overexpression of MYC, MET, or AKT+RAS oncogenes. We evaluated the immune cells at early vs. late stages of tumor development by immunohistochemical staining of liver tissues using lineage-specific immune cell markers. In addition, we assessed activation of the pro-inflammatory NFκB pathway by immunoblot detection of IkBα and phospho-Ser(536)-NFκB levels in liver lysates, as well as by immunohistochemical analysis of NFκB (p65) nuclear localization. Histological analysis revealed leukocytic clustering around hepatocytes in pre-neoplastic livers in MYC and AKT+RAS, but not in the MET liver tumor model. Moreover, the leukocyte profiles identified in pre-neoplastic livers and liver tumors were different in MYC vs. AKT+RAS model. Finally, we found that the NFκB pathway was inactive in MYC and AKT+RAS tumors, but hyperactivated in MET tumors. In summary, our results suggest that the type of tumor-initiating oncogenic lesion profoundly influences the identity of recruited immune cells and activation of NFκB pathway in the course of tumorigenesis. We are currently in the process of elucidating the mechanisms behind oncogene-mediated regulation of immune responses, which may reveal novel targets for anti-cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 310. doi:1538-7445.AM2012-310

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.