Induced NF-κB DNA Binding Activity Research Articles

Glutathione peroxidase-1 overexpressing transgenic mice are protected from neurotoxicity induced by microcystin-leucine-arginine.

Although it has been well-recognized that microcystin-leucine-arginine (MCLR), the most common form of microcystins, induces neurotoxicity, little is currently known about the underlying mechanism for this neurotoxicity. Here, we found that MCLR (10 ng/μL/mouse, i.c.v.) induces significant neuronal loss in the hippocampus of mice. MCLR-induced neurotoxicity was accompanied by oxidative stress, as shown by a significant increase in the level of 4-hydroxynonenal, protein carbonyl, and reactive oxygen species (ROS). Superoxide dismutase-1 (SOD-1) activity was significantly increased, but glutathione peroxidase (GPx) level was significantly decreased following MCLR insult. In addition, MCLR significantly inhibited GSH/GSSG ratio, and significantly induced NFκB DNA binding activity. Because reduced activity of GPx appeared to be critical for the imbalance between activities of SODs and GPx, we utilized GPx-1 overexpressing transgenic mice to ascertain the role of GPx-1 in this neurotoxicity. Genetic overexpression of GPx-1 or NFκB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly attenuated MCLR-induced hippocampal neuronal loss in mice. However, PDTC did not exert any additive effect on neuroprotection mediated by GPx-1 overexpression, indicating that NFκB is a neurotoxic target of MCLR. Combined, these results suggest that MCLR-induced neurotoxicity requires oxidative stress associated with failure in compensatory induction of GPx, possibly through activation of the transcription factor NFκB.

Dead bacteria reverse antibiotic-induced host defense impairment in burns.

Burn patients can incur high rates of hospital-acquired infections. The mechanism of antibiotic exposure on inducing infection vulnerability has not been determined. This study aimed to examine the effects of antibiotic treatment on host defense mechanisms. First we treated C57/BL6 mice with combined antibiotic treatment after 30% to 35% total body surface area burn. Animals were sacrificed at 48 hours after sham or thermal injury treatment. Bacterial counts in intestinal lumen and mucosa were measured. Next, we treated animals with or without oral dead Escherichia coli or Staphylococcus aureus supplementation to stimulate Toll-like receptor in the intestinal mucosa. Toll-like receptor 4, antibacterial protein expression, nuclear factor (NF)-κB DNA-binding activity, and bacteria-killing activity in the intestinal mucosa; intestinal permeability; bacterial translocation to mesenteric lymph nodes; Klebsiella pneumoniae translocation; interleukin-6 in the blood; and phagocytic activity of alveolar macrophages, were assessed. Thermal injury increased microflora and NF-κB DNA-binding activity of the intestine. Systemic antibiotic treatment decreased gut microflora and increased bacterial translocation to mesenteric lymph nodes, intestinal permeability, and interleukin-6 levels in the blood. Antibiotic treatment also decreased bacteria-killing activity in intestinal mucosa and phagocytic activity of alveolar macrophages. Oral dead E coli and S aureus supplementation induced NF-κB DNA-binding activity, Toll-like receptor 4, and antibacterial protein expression of the intestinal mucosa. Taken together with the fact that dead bacteria reversed antibiotic-induced K pneumoniae translocation and intestinal and pulmonary defense impairment, we conclude that combined antibiotic treatment results in systemic host defense impairment in burns through the decrease in intestinal flora. We suggest that dead bacteria supplementation could induce nondefensin protein expression and reverse antibiotic-induced gut and lung defense impairment in burn patients.

Crebanine, an aporphine alkaloid, sensitizes TNF-α-induced apoptosis and suppressed invasion of human lung adenocarcinoma cells A549 by blocking NF-κB-regulated gene products.

Crebanine is an alkaloid known to exhibit anticancer, but its mechanism is not well understood. Besides, the nuclear factor-kappa B (NF-κB) transcription factor has been correlated with inflammation, carcinogenesis, tumor cell survival, invasion, and angiogenesis. In this study, we investigated the effects of crebanine on tumor necrosis factor alpha (TNF-α)-induced NF-κB activation and the expression of NF-κB-regulated gene products. We found that crebanine reduced the cell proliferation of lung, ovarian, and breast cancer cells. Crebanine also potentiated TNF-α-induced apoptosis which correlated with the suppression of the gene products linked to cell survival, B cell lymphoma-extra large, and proliferation, cyclin D1. In addition, crebanine affected TNF-α-induced activation of caspase-8, caspase-3, and poly(ADP-ribose) polymerase cleavage, indicating that the apoptotic effects of TNF-α were enhanced by crebanine. Moreover, crebanine reduced TNF-α-induced A549 cell invasion and migration. Furthermore, crebanine suppressed the TNF-α-mediated expression of proteins that involved cancer cell invasion (matrix metalloproteinase 9 urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor and intercellular adhesion molecule 1) and angiogenesis (COX-2 and VEGF), all of which are known to be regulated by NF-κB. We also demonstrated that TNF-α induced NF-κB DNA-binding activity, which was inhibited by crebanine. Moreover, crebanine suppressed the TNF-α-induced degradation of inhibitor of NF-κB alpha (IκBa), which led to reduced NF-κB translocation to the nucleus. Taken together, our results demonstrated that crebanine reduced TNF-α-induced cancer cell proliferation, invasion, and survival by suppressing NF-κB activity and expression profile of its downstream genes.

Toll-Like Receptor Stimulation Induces Nondefensin Protein Expression and Reverses Antibiotic-Induced Gut Defense Impairment

Prior antibiotic exposure is associated with increased mortality in Gram-negative bacteria-induced sepsis. However, how antibiotic-mediated changes of commensal bacteria promote the spread of enteric pathogenic bacteria in patients remains unclear. In this study, the effects of systemic antibiotic treatment with or without Toll-like receptor (TLR) stimulation on bacterium-killing activity, antibacterial protein expression in the intestinal mucosa, and bacterial translocation were examined in mice receiving antibiotics with or without oral supplementation of dead Escherichia coli or Staphylococcus aureus. We developed a systemic ampicillin, vancomycin, and metronidazole treatment protocol to simulate the clinical use of antibiotics. Antibiotic treatment decreased the total number of bacteria, including aerobic bacteria belonging to the family Enterobacteriaceae and the genus Enterococcus as well as organisms of the anaerobic genera Lactococcus and Bifidobacterium in the intestinal mucosa and lumen. Antibiotic treatment significantly decreased the bacterium-killing activity of the intestinal mucosa and the expression of non-defensin-family proteins, such as RegIIIβ, RegIIIγ, C-reactive protein-ductin, and RELMβ, but not the defensin-family proteins, and increased Klebsiella pneumoniae translocation. TLR stimulation after antibiotic treatment increased NF-κB DNA binding activity, nondefensin protein expression, and bacterium-killing activity in the intestinal mucosa and decreased K. pneumoniae translocation. Moreover, germfree mice showed a significant decrease in nondefensin proteins as well as intestinal defense against pathogen translocation. Since TLR stimulation induced NF-κB DNA binding activity, TLR4 expression, and mucosal bacterium-killing activity in germfree mice, we conclude that the commensal microflora is critical in maintaining intestinal nondefensin protein expression and the intestinal barrier. In turn, we suggest that TLR stimulation induces nondefensin protein expression and reverses antibiotic-induced gut defense impairment.

Abscopal effect of low-LET γ-radiation mediated through Rel protein signal transduction in a mouse model of nontargeted radiation response

Ascertaining the ionizing radiation (IR)-induced bystander response and its preceding molecular regulation would increase our understanding of the mechanism of acute and delayed radiobiological effects. Recent evidence clearly prompted that radiation-induced nuclear factor kappa B (NF-κB) would play a key role in bystander responses in nontargeted cells. Accordingly, we investigated the orchestration of NF-κB signaling after IR in a nontargeted distant organ. Heart tissues from C57/BL6 mice either mock irradiated or exposed (limited to lower abdomen 1 cm diameter) to single-dose IR (SDR: 2 or 10 Gy) or fractionated IR (FIR, 2 Gy per day for 5 days) were examined for onset of abscopal NF-κB signal transduction, translated activity, downstream functional signaling and associated DNA damage. Radiation significantly induced NF-κB DNA binding activity in nontargeted heart. Transcriptional profiling showed that 51, 46 and 26 of 88 genes were significantly upregulated after 2 Gy, 10 Gy and FIR. Of these genes, 22 showed dose- and fractionation-independent upregulation. Immunohistochemistry revealed a robust increase in p65 and cMyc expression in distant heart after SDR and FIR. Immunoblotting revealed increased phosphorylation of p38 after 2 Gy and extracellular signal-regulated kinases 1/2 after 10 Gy in nontargeted heart. In addition, IR exposure significantly enhanced DNA fragmentation in nontargeted heart. Together, these data clearly indicated an induced abscopal response in distant organ after clinically relevant IR doses. More importantly, the results imply that orchestration of NF-κB signal transduction in nontargeted tissues may serve as an effector and could play a key role in induced abscopal responses.

Blocking TNF-α enhances Pseudomonas aeruginosa-induced mortality in burn mice through induction of IL-1β

PurposeTumor necrosis factor (TNFα) is a proinflammatory cytokine and has been a target for intervention in human sepsis. However, inhibition of TNF-α with a high dose of a TNF-receptor fusion protein in patients with septic shock worsened patient survival. This study was designed to investigate whether blocking TNF-α enhances mortality in infected burn mice through the induction of IL-1β. MethodsWT or Tnfrsf1a−/− mice received Pseudomonas aeruginosa injection in the back at 8h after burn injury. The animals were sacrificed at 24h after burn and lung tissues were harvested and examined for determining myeloperoxidase (MPO) activity, pulmonary microvascular dysfunction, NF-κB DNA binding activity, and IL-1β expression. Also, the lung and blood were harvested for bacterial count assay. ResultThermal injury alone induced NF-κB DNA binding activity and neutrophil infiltration in the lung in WT but not in Tnfrsf1a−/− mice. A 50% total body surface area (TBSA) burn induced a significant increase of mortality in WT compared with Tnfrsf1a−/− mice. In contrast, P. aeruginosa injection with a 30% TBSA burn pretreatment enhanced IL-1β expression, bacterial counts in lung and blood, pulmonary microvascular dysfunction, and mortality in Tnfrsf1a−/− mice compared with WT mice. Injection of the IL-1 receptor antagonist, Anakinra, reduced P. aeruginosa infection with burn pretreatment-induced blood bacterial counts, IL-1β levels as well as permeability of lung, and mortality in Tnfrsf1a−/− mice. ConclusionsOur findings suggest that thermal injury induces lung NF-κB activation and neutrophil sequestration through TNFα signaling. However, blocking TNF-α enhances P. aeruginosa infection-induced lung damage in burn mice via induction of IL-1β. Using an IL-1 receptor antagonist combined with the neutralization of TNF-α could be a useful strategy for decreasing P. aeruginosa infection-induced mortality in burn patients.

The effects of bifidobacterial secretory adhesin on the stress response of intestinal epithelial cells

Objective To observe the effects of bifidobacterial sectory adhesin on NF-κB DNA binding activity,cytoplasm IκB,nuclei NF-κBp65,TNF-α,IL-1β,and IL-8 expression in intestinal epithelial cell post stress response.Methods The cultured intestinal epithelial cell line Lovo cells were divided into 5 groups,which included directly stimulated by lypopolysaccharide (LPS) (100 ng/ml) or H2O2 (200 μmol/L) for 3 hours groups,bifidobacterial sectory adhesin (30μg/ml) pre-incubation for 30 minutes and then treated by LPS (100 ng/ml) or H2O2 (200 μmol/L) for 3 hours groups and control group with no treatment.NF-κB DNA binding activity was evaluated by electrophoretic mobility shift assay (EMSA).The cytoplasm IκB and nuclei NF-κBp65 expression were determined by Western blot.The expressions of TNF-α,IL-1β and IL-8 at mRNA level were detected by semi-quantatitive assay of RT-PCR.Results The expressions of NF-κB DNA binding activity [(6.20±0.35) times and (4.16 ± 0.52) times of that of non treated control group,respectively] and nuclei NF-κBp65 [ (0.64±0.05) and (0.67±0.06)] were higher in cells after LPS or H2()2 stimulated,however the cytoplasm IκB expression [ (0.28 ± 0.10) and (0.39 ± 0.12) respectively] was weaker.After bifidobacterial sectory adhesin pre-incubation,the expressions of NF-κB DNA binding activity and nuclei NF-κBp65decreased obviously,but cytoplasm IκB expression increased.After treated with LPS or H2O2,the mRNA expressions of TNF-α,IL-1β and IL-8 were significantly increased [LPS treated group2 (0.92±0.10)、(0.38±0.03)、(1.44±0.25),H2O2 treated group:(0.89±0.13)、(0.36±0.06)、(1.42±0.18)].After bifidobacterial sectory adhesin pre-incubation,their expressions decreased.The mRNA expressions of TNF-α,IL-1β and IL-8 have significantly positive correlation with NF-κB DNA binding activity.Conclusions There is a significant inhibition role of bifidobacterial sectory adhesin in LPS and H2O2 induced NF-κB DNA binding activity in intestinal epithelia cells.The activation of NFκB may associate with the regulation of TNF-α,IL-1β and IL-8 proinflammatory cytokines expression. Key words: Bifidobacterium; Cell adhesion; Tumor cells, cultured; Intestines; Lipopolysaccharides; Hydrogen peroxide; Nuclear factor-κB; Cytokines

Radiation-triggered Tumor Necrosis Factor (TNF) α-NFκB Cross-signaling Favors Survival Advantage in Human Neuroblastoma Cells

Induced radioresistance in the surviving cancer cells after radiotherapy could be associated with clonal selection leading to tumor regrowth at the treatment site. Previously we reported that post-translational modification of IκBα activates NFκB in response to ionizing radiation (IR) and plays a key role in regulating apoptotic signaling. Herein, we investigated the orchestration of NFκB after IR in human neuroblastoma. Both in vitro (SH-SY5Y, SK-N-MC, and IMR-32) and in vivo (xenograft) studies showed that IR persistently induced NFκB DNA binding activity and NFκB-dependent TNFα transactivation and secretion. Approaches including silencing NFκB transcription, blocking post-translational NFκB nuclear import, muting TNF receptor, overexpression, and physiological induction of either NFκB or TNFα precisely demonstrated the initiation and occurrence of NFκB → TNFα → NFκB positive feedback cycle after IR that leads to and sustains NFκB activation. Selective TNF-dependent NFκB regulation was confirmed with futile inhibition of AP-1 and SP-1 in TNF receptor muted cells. Moreover, IR increased both transactivation and translation of Birc1, Birc2, and Birc5 and induced metabolic activity and clonal expansion. This pathway was further defined to show that IR-induced functional p65 transcription (not NFκB1, NFκB2, or c-Rel) is necessary for activation of these survival molecules and associated survival advantage. Together, these results demonstrate for the first time the functional orchestration of NFκB in response to IR and further imply that p65-dependent survival advantage and initiation of clonal expansion may correlate with an unfavorable prognosis of human neuroblastoma.

Abstract 574: Mechanism of radiation-induced bystander effect: Role of NFκß pathway

Abstract Evidence from our lab and others clearly delineated that the transcriptional regulator NFκB plays a key role in induced responses in non-targeted bystander cells. We further reported curcumin impedes radiation (IR)-induced NFκB. However, full potential of curcumin is yet to be realized because of its poor bioavailability. This prompted us to investigate the efficacy of a more potent synthetic analogue of curcumin, EF24 in this setting. In this study, we investigated the orchestration of NFκB signaling after IR in an organ (lung) distant from the exposure field and elucidated the effect of EF24 in reverting the induced response. C57/BL6 mice either mock irradiated, exposed to single dose IR (SDR: 2 or 10Gy) or fractionated IR (FIR, 2 Gy/day for 5d) with or without EF24 (i.p. 200μg/Kg, 3h prior to IR) treatment were sacrificed after 24h. All IR exposures were limited to the lower abdomen area (1cm diameter), while the rest of the animal was protected by a specially designed cerrobend shield. EMSA analysis was used to examine the changes in NFκB DNA binding activity in lungs. Transcriptional activation of 88 NFκB pathway molecules was assessed using QPCR profiling. Phosphorylation of ERK1/2 and p38 that play a key role in NFκB signal transduction were examined using immunoblotting. DNA fragmentation was measured using TdT nick end labeling. A known NFκB dependent oxidative stress response SOD2 localization was examined using immunohistochemistry. IR significantly induced NFκB DNA binding activity in lungs. On the other hand, EF24 markedly suppressed IR-bystander effect (BSE)-induced NFκB DNA binding activity. QPCR profiling showed a significant and differential modulation in NFκB pathway transcriptome. To that end, of the 88 genes analyzed, 86, 86 and 70 genes were upregulated after 2Gy, 10Gy and FIR, respectively. Of these, 85, 80 and 63 genes were significantly (≫ 2 fold) upregulated. Conversely, EF24 completely suppressed 51, 52 and 43 SDR (2Gy, 10Gy) and FIR upregulated genes respectively in the non-targeted lungs. While IR profoundly phosphorylated ERK1/2 and p38 in distant lungs, EF24 treatment completely inhibited the phosphorylation of these kinases. Furthermore, IR-induced SOD2 levels in distant lung after all three IR-dose regimens were notably conferred by EF24 treatment. In addition, IR-enhanced DNA fragmentations in the lung tissue were significantly reduced with EF24. Taken together, these data clearly indicated that (i) an induced non-targeted effect occurs in mice lungs after clinically relevant doses of IR exposure; (ii) the orchestration of NFκB signal transduction may play a key role in induced abscopal responses; and (iii) EF24 treatment may attenuate NFκB and thereby alleviate radiation-induced abscopal effect at the distant organ. 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 574. doi:10.1158/1538-7445.AM2011-574

Abstract 486: Abscopal effect of low-LET ionizing radiation on Rel protein signal transduction

Abstract Ascertaining the ionizing radiation (IR)-induced bystander response and identifying its mechanistic regulation would raise a paradigm shift in our understanding of the radiobiological effects. Recent evidence from our lab and others clearly prompted that the transcriptional regulator NFκB would play a key role in induced responses in non-targeted bystander cells. In this study, for the first time, we investigated the orchestration of NFκB signaling after IR in an organ (heart) distant from the exposure field. The C57/BL6 mice either mock irradiated, exposed to single dose IR (SDR: 2 or 10Gy) or fractionated IR (FIR, 2 Gy/day for 5 days) were sacrificed 24h post-irradiation. All IR exposures were limited to the lower abdomen area (1cm diameter), while the rest of the animal was protected by a specially designed cerrobend shield. EMSA analysis was used to examine the changes in NFκB DNA binding activity in the heart. Transcriptional activation of 88 mediators associated with NFκB signaling pathway was assessed using QPCR profiling. Phosphorylation of ERK1/2 and p38 that play a key role in NFκB signal transduction were examined using immunoblotting. Induced DNA fragmentation was measured using TdT nick end labeling. IR significantly induced NFκB DNA binding activity in the heart. Further more, real-time QPCR profiling showed a significant and differential modulation in the transcriptional responses of NFκB signaling pathway molecules. To that end, of the 88 genes analyzed, 28, 51 and 41 genes were upregulated after 2Gy, 10Gy and FIR, respectively. More importantly, at these exposures13 out of 28, 27 out of 51 and 25 out of 41 induced mediators of NFkB pathway were significantly upregulated. Conversely, lower abdominal IR exposure markedly suppressed 44, 20 and 30 genes after 2 Gy, 10Gy and FIR, respectively in the heart of the exposed animals. Compared to 2Gy, FIR significantly induced 26 genes, while 10Gy upregulated 27 genes. In addition, 6 and 15 genes were significantly upregulated after acute dose of 2 and 10Gy as opposed to fractionated dose. Consistently, immunoblotting analysis revealed a profound phosphorylation of both ERK1/2 and p38 in the heart. Interestingly, distant IR exposure significantly enhanced DNA fragmentation in the heart tissue. Taken together, these data clearly indicated an induced abscopal response in mice heart after clinically relevant doses of IR exposure. More importantly, these data implies that orchestration of NFκB signal transduction may play a key role in induced abscopal responses. Funding Support: Presbyterian Health Foundation, OKC. 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 486.

NFκB activity and transcriptional responses in human breast adenocarcinoma cells after single and fractionated irradiation

Radiotherapy is considered mandatory for breast cancer patients undergoing conservative surgery and for women at high risk of recurrence. However, relapse due to radio-resistance affects the success of radiotherapy. Ascertaining the fractionated radiation (FIR) modulated molecular targets is important to make tumors more susceptible to molecular targeted therapy. Accordingly, we investigated the (i) expression of 84 genes representing six functional pathways; (ii) NFκB DNA binding activity and; (iii) expression of radio-responsive molecules after single dose (10Gy) radiation (SDR) and FIR (2Gyx5). MCF-7 cells exposed to SDR or FIR were analyzed for alterations in gene expression using QPCR-profiling. NFκB DNA binding activity was analyzed using EMSA and pIκB using immunoblotting. Expression of TNFα, IL-1α, pAKT, IAP1, IAP2, XIAP, survivin, MnSOD, BID and Bak were determined using QPCR and/or immunoblotting. Compared to SDR, FIR significantly induced 60 genes and completely suppressed 14 genes. Furthermore, FIR induced NFκB-DNA binding activity and IκBα phosphorylation. Like-wise, FIR induced the expression of IAP1, IAP2, XIAP Survivin, MnSOD, TNFα, pAKT and IL-1α. The results of the study clearly show distinct differences in the molecular response of cells between SDR and FIR exposures. We identified several potential targets that may affect radio-resistance following FIR.

The Role of TRAF2 Binding to the Type I Interferon Receptor in Alternative NFκB Activation and Antiviral Response

Type I interferons (IFNs) play critical roles in the host defense by modulating gene expression through the IFN-dependent activation of STAT and NFkappaB transcription factors. Previous studies established that IFN activates NFkappaB through a classical NFkappaB pathway that results in IkappaBalpha degradation and formation of p50-containing NFkappaB complexes, as well as an alternative pathway that involves NFkappaB-inducing kinase and TRAF2, which results in the formation of p52-containing NFkappaB complexes. In this study, we examined the interaction of TRAF proteins with the type I IFN receptor. We found that TRAF2 was directly coupled to the signal-transducing IFNAR1 subunit of the IFN receptor. By immunoprecipitation, overexpression of epitope-tagged IFNAR1 constructs, and glutathione S-transferase pulldown experiments, we demonstrate that TRAF2 rapidly binds to the IFNAR1 subunit of the IFN receptor upon IFN binding. The membrane proximal half of the IFNAR1 subunit was found to directly bind TRAF2. Moreover, analysis of mouse embryo fibroblasts derived from TRAF2 knock-out mice demonstrated that TRAF2 plays a critical role in the activation of the alternative NFkappaB pathway by IFN, but not the classical NFkappaB pathway, as well as in the antiviral action of IFN. Our results place TRAF2 directly in the signaling pathway transduced through the IFNAR1 subunit of the IFN receptor. These findings provide an important insight into the molecular mechanisms by which IFN generates signals to induce its biological effects.

Curcumin protects against acute liver damage in the rat by inhibiting NF-κB, proinflammatory cytokines production and oxidative stress

Curcumin, an anti-inflammatory and antioxidant compound, was evaluated for its ability to suppress acute carbon tetrachloride-induced liver damage. Acute hepatotoxicity was induced by oral administration of CCl 4 (4 g/kg, p.o.). Curcumin treatment (200 mg/kg, p.o.) was given before and 2 h after CCl 4 administration. Indicators of necrosis (alanine aminotransferase) and cholestasis (γ-glutamyl transpeptidase and bilirubins) resulted in significant increases after CCl 4 intoxication, but these effects were prevented by curcumin treatment. As an indicator of oxidative stress, GSH was oxidized and the GSH/GSSG ratio decreased significantly by CCl 4, but was preserved within normal values by curcumin. In addition to its antioxidants properties, curcumin is capable of preventing NF-κB activation and therefore to prevent the secretion of proinflammatory cytokines. Therefore, in this study we determined the concentrations of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) mRNA, and NF-κB activation. CCl 4-administered rats depicted significant increases in TNF-α, IL-1β, and IL-6 production, while curcumin remarkably suppressed these mediators of inflammation in liver damage. These results were confirmed by measuring TNF-α, and IL-1β protein production using Western Blot analysis. Accordingly, these proteins were increased by CCl 4 and this effect was abolished by curcumin. Administration of CCl 4 induced the translocation of NF-κB to the nucleus; CCl 4 induced NF-κB DNA binding activity was blocked by curcumin treatment. These findings suggest that curcumin prevents acute liver damage by at least two mechanisms: acting as an antioxidant and by inhibiting NF-κB activation and thus production of proinflammatory cytokines.

High glucose induced NF-κB DNA-binding activity in HAEC is maintained under low shear stress but inhibited under high shear stress: role of nitric oxide

In the present study, we investigated whether low shear (LS, 2 dyn/cm 2) favors high glucose (HG, 30 mM) induced nuclear factor kappa B (NF-κB) activity by regulating NO release in human aortic endothelial cells (HAEC). The results show that (i) under LS, the NF-κB activity of HAEC exposed to HG was significantly higher than HAEC in normal glucose (NG, 5.5 mM) ( P<0.05). In contrast, under HS, the activation of NF-κB in HAEC exposed to HG showed no significant difference compared to that of NG. (ii) The NF-κB activity induced by HG is suppressed by high shear (HS) in the absence of a NO synthase inhibitor, Nω-nitro- l-arginine methyl ester ( l-NAME) but restored in its presence, while LS+HG induced NF-κB activity remains the same in the presence or absence of l-NAME. (iii) Endothelial nitric oxide synthase (eNOS) protein expression and quantitative detection of NO indicated that high shear stress significantly induced higher eNOS expression and NO production compared to low shear stress condition. Collectively, these data suggest that HS exerts a protective effect on HG induced NF-κB activity through NO mediated signaling. LS, on the other hand, may down-regulate eNOS expression resulting in reduced NO release, and thereby maintain high glucose induced NF-κB DNA-binding activity. These observations explain, in part, the mechanism by means of which hyperglycemia accelerates the focal development of atherosclerotic lesions in low shear (lesion prone) areas of the arterial tree.

Constitutive nuclear factor-kappa B mRNA, protein overexpression and enhanced DNA-binding activity in thymidylate synthase inhibitor-resistant tumour cells.

In this study, the gene copy number, mRNA and protein expression levels and nuclear DNA-binding activity of nuclear factor kappa B (NF-κB) were compared in a panel of five pairs of thymidylate synthase (TS) inhibitor-resistant and wild-type parent cancer cell lines. High constitutive NF-κB DNA-binding activity was detected in all chemoresistant cell lines. The upregulated NF-κB activity was composed of NF-κB subunits p50 and p65. Four out of five resistant cell lines constitutively overexpressed NF-κB p50 and p65 mRNA and protein. One resistant cell line with the highest NF-κB DNA-binding activity showed normal p50 and p65 protein expression. No NF-κB gene amplification was detected in resistant cell lines. Transient exposure of wild-type RKOWT and H630WT cells to 5-FU induced NF-κB DNA-binding activity but had no effect on NF-κB protein expression in these cells. Our results indicate that high constitutive NF-κB activity caused by its gene overexpression is an intrinsic character of TS inhibitor-resistant cells. NF-κB can antagonise anticancer drug-induced apoptosis. High NF-κB expression and nuclear activity in TS inhibitor-resistant cancer cells may play an important role in the chemoresistance.

High glucose induced nuclear factor kappa B mediated inhibition of endothelial cell migration

Delayed wound healing and accelerated atherosclerosis are common vascular complications of diabetes mellitus. Although elevated blood glucose level is the major contributing factor, mechanisms that mediate these complications are not clearly understood. In the present study, we have demonstrated that elevated glucose inhibits endothelial cell migration, thereby delaying wound healing. Our results clearly indicated that high glucose (10 or 30 mM) induced activation of nuclear factor kappa B (NF-κB) inhibited endothelial cell migration ( P<0.05). High glucose induced NF-κB DNA binding activity may mediate this inhibition of migration by regulating intracellular nitric oxide. In vitro wound healing model in human aortic endothelial cells (HAEC) were used to evaluate cell migration under the influence of high glucose. The migration inhibited by high glucose was restored by NF-κB inhibitors (including E3-4-methylphenyl sulfonyl-2-propenenitrile, N-tosyl-Lys-chloromethylketone (TLCK), or over-expression of inhibitor subunit of κB) and endothelial nitric oxide synthase inhibitors ( N-methyl- l-arginine ( l-NMMA); and Nω-nitro- l-arginine methyl ester ( l-NAME)). Furthermore, NF-κB inhibitors attenuated high glucose induced eNOS expression and intracellular nitric oxide (NO) production. Cytoskeletal immunofluorescence staining confirmed differences in actin distribution in HAEC incubated in high glucose in the presence or absence of NF-κB and NO inhibitors, explaining the differences observed in migration. In summary, our results for the first time suggest therapeutic strategies involving inhibition of NF-κB activation induced by high glucose, which may improve wound healing and help avoid some of the vascular complications of diabetes.

P38 Mitogen-Activated Protein Kinase Mediates Tumor Necrosis Factor- -Induced Apoptosis in Rat Fetal Brown Adipocytes

Tumor necrosis factor-α (TNFα) induces apoptosis and cell growth inhibition in primary rat fetal brown adipocytes. Here, we examine the role played by some members of the mitogen-activated protein kinase (MAPK) superfamily. TNFα activates extracellular regulated kinase-1/2 (ERK1/2) and p38MAPK. Inhibition of p38MAPK by either SB203580 or SB202190 highly reduces apoptosis induced by TNFα, whereas ERK inhibition potentiates it. Moreover, cotransfection of an active MKK3 mutant and p38MAPK induces apoptosis. p38MAPK inhibition also prevents TNFα-induced cell cycle arrest, whereas MEK1 inhibition enhances this effect, which correlates with changes in proliferating cell nuclear antigen expression, but not in cyclin D1. c-Jun and activating transcription factor-1 are potential downstream effectors of p38MAPK and ERKs upon TNFα treatment. Thus, TNFα-induced c-Jun messenger RNA expression requires ERKs activation, whereas p38MAPK inhibition enhances its expression. In addition, TNFα-induced activating transcription factor-1 phosphorylation is extensively decreased by SB203580. However, TNFα- induced NF-κB DNA-binding activity is independent of p38MAPK and ERK activation. On the other hand, C/EBP homology protein does not appear to mediate the actions of TNFα, because its expression is almost undetectable and even reduced by TNFα. Finally, although TNFα induces c-Jun N-terminal kinase (JNK) activation, transfection of a dominant negative of either JNK1 or JNK2 had no effect on TNFα-induced apoptosis. These results suggest that p38MAPK mediates TNFα-induced apoptosis and cell cycle arrest, whereas ERKs do the opposite, and JNKs play no role in this process of apoptosis.

P38 Mitogen-Activated Protein Kinase Mediates Tumor Necrosis Factor-α-Induced Apoptosis in Rat Fetal Brown Adipocytes*

Tumor necrosis factor-alpha (TNFalpha) induces apoptosis and cell growth inhibition in primary rat fetal brown adipocytes. Here, we examine the role played by some members of the mitogen-activated protein kinase (MAPK) superfamily. TNFalpha activates extracellular regulated kinase-1/2 (ERK1/2) and p38MAPK. Inhibition of p38MAPK by either SB203580 or SB202190 highly reduces apoptosis induced by TNFalpha, whereas ERK inhibition potentiates it. Moreover, cotransfection of an active MKK3 mutant and p38MAPK induces apoptosis. p38MAPK inhibition also prevents TNFalpha-induced cell cycle arrest, whereas MEK1 inhibition enhances this effect, which correlates with changes in proliferating cell nuclear antigen expression, but not in cyclin D1. c-Jun and activating transcription factor-1 are potential downstream effectors of p38MAPK and ERKs upon TNFalpha treatment. Thus, TNFalpha-induced c-Jun messenger RNA expression requires ERKs activation, whereas p38MAPK inhibition enhances its expression. In addition, TNFalpha-induced activating transcription factor-1 phosphorylation is extensively decreased by SB203580. However, TNFalpha-induced NF-kappaB DNA-binding activity is independent of p38MAPK and ERK activation. On the other hand, C/EBP homology protein does not appear to mediate the actions of TNFalpha, because its expression is almost undetectable and even reduced by TNFalpha. Finally, although TNFalpha induces c-Jun N-terminal kinase (JNK) activation, transfection of a dominant negative of either JNK1 or JNK2 had no effect on TNFalpha-induced apoptosis. These results suggest that p38MAPK mediates TNFalpha-induced apoptosis and cell cycle arrest, whereas ERKs do the opposite, and JNKs play no role in this process of apoptosis.

Invasive Shigella flexneri activates NF-kappa B through a lipopolysaccharide-dependent innate intracellular response and leads to IL-8 expression in epithelial cells.

The pathogenesis of Shigella flexneri infection centers on the ability of this organism to invade epithelial cells and initiate an intense inflammatory reaction. Because NF-kappa B is an important transcriptional regulator of genes involved in inflammation, we investigated the role of this transcription factor during S. flexneri infection of epithelial cells. Infection of HeLa cells with invasive S. flexneri induced NF-kappa B DNA-binding activity; noninvasive S. flexneri strains did not lead to this activation. The pathway leading to NF-kappa B activation by invasive S. flexneri involved the kinases, NF-kappa B-inducing kinase, I kappa B kinase-1, and I kappa B kinase-2. NF-kappa B activation was linked to inflammation, because invasive S. flexneri activated an IL-8 promoter-driven reporter gene, and the kappa B site within this promoter was indispensable for its induction. Microinjection of bacterial culture supernatants into HeLa cells suggested that LPS is responsible for NF-kappa B activation by S. flexneri infection. In conclusion, the eukaryotic transcription factor NF-kappa B was activated during S. flexneri infection of epithelial cells, which suggests a role for this transcriptional regulator in modulating the immune response during infection in vivo.

Insulin-like Growth Factor-II, Phosphatidylinositol 3-Kinase, Nuclear Factor-κB and Inducible Nitric-oxide Synthase Define a Common Myogenic Signaling Pathway

Insulin-like growth factors (IGFs) are potent inducers of skeletal muscle differentiation and phosphatidylinositol (PI) 3-kinase activity is essential for this process. Here we show that IGF-II induces nuclear factor-kappaB (NF-kappaB) and nitric-oxide synthase (NOS) activities downstream from PI 3-kinase and that these events are critical for myogenesis. Differentiation of rat L6E9 myoblasts with IGF-II transiently induced NF-kappaB DNA binding activity, inducible nitric-oxide synthase (iNOS) expression, and nitric oxide (NO) production. IGF-II-induced iNOS expression and NO production were blocked by NF-kappaB inhibition. Both NF-kappaB and NOS activities were essential for IGF-II-induced terminal differentiation (myotube formation and expression of skeletal muscle proteins: myosin heavy chain, GLUT 4, and caveolin 3), which was totally blocked by NF-kappaB or NOS inhibitors in rat and human myoblasts. Moreover, the NOS substrate L-Arg induced myogenesis in the absence of IGFs in both rat and human myoblasts, and this effect was blocked by NOS inhibition. Regarding the mechanisms involved in IGF-II activation of NF-kappaB, PI 3-kinase inhibition prevented NF-kappaB activation, iNOS expression, and NO production. Moreover, IGF-II induced, through a PI 3-kinase-dependent pathway, a decrease in IkappaB-alpha protein content that correlated with a decrease in the amount of IkappaB-alpha associated with p65 NF-kappaB.