DNA Binding Activity Of Nuclear Factor-kappa Research Articles

Regulatory Mechanism of Mast Cell Activation by Zinc Signaling.

Mast cells are hematopoietic-lineage cells that participate in immunoglobulin E (IgE)-associated immune responses, including allergic reactions and parasite resistance. Recent studies have shown that zinc (Zn) ion can behave as an intracellular signaling molecule and that Zn is involved in mast cell activation. We demonstrated that mast cells stimulated through the high-affinity IgE receptor (FcεRI) rapidly release intracellular Zn from the endoplasmic reticulum (ER), and we named this phenomenon the "Zn wave". Furthermore, we found that the L-type calcium channel (LTCC) is the gatekeeper for the Zn wave. LTCC antagonists inhibited the Zn wave, and an agonist was sufficient to induce it. Notably, LTCC was mainly localized to the ER rather than to the plasma membrane in mast cells, and the Zn wave was impaired by LTCC knockdown. We also found that the LTCC-mediated Zn wave positively controlled inflammatory cytokine gene induction by enhancing the DNA-binding activity of nuclear factor-kappa B (NF-κB). These findings indicated that the LTCC has a novel function as a gatekeeper for the Zn wave, which is involved in regulating NF-κB signaling. In this review, we describe our current understanding of Zn signaling, especially with regard to the Zn wave and the role of Zn signaling in mast cells.

Evodiamine Inhibits Zymosan-Induced Inflammation In Vitro and In Vivo: Inactivation of NF-κB by Inhibiting IκBα Phosphorylation.

Evodiamine (EVO), an important alkaloidal component extracted from the fruit of Evodiae fructus, has been known to possess anti-tumor, anti-inflammatory, anti-oxidative, and other therapeutic capabilities. In the present study, the effects of EVO on zymosan-induced inflammation and its underlying mechanism were investigated both in vitro and in vivo. Our results showed that EVO effectively suppressed both protein and mRNA expression of interleukin-1β, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in vitro. Zymosan-induced DNA-binding activity of nuclear factor-kappa B (NF-κB) was attenuated by EVO, which was achieved through inhibitory effects on the phosphorylation of inhibitory κB α and p65 nuclear translocation, but there was very little association with mitogen-activated protein kinase activation. In vivo, treatment with EVO markedly decreased TNF-α and IL-6 levels in plasma. EVO also repressed inflammatory cytokine expression and ameliorated the abnormal state in both lung and intestine tissues by inactivation of NF-κB. Furthermore, EVO significantly reduced the mortality caused by zymosan. In summary, these results suggested that EVO could effectively suppress inflammatory responses in vitro and in vivo, and may be a potential therapeutic agent against inflammatory disorders.

Fimasartan, Anti-hypertension Drug, Suppressed Inducible Nitric Oxide Synthase Expressions <i>via</i> Nuclear Factor-Kappa B and Activator Protein-1 Inactivation

Since inhibition of angiotensin II type 1 (AT1) receptor reduces chronic inflammation associated with hypertension, we evaluated the anti-inflammatory potential and the underlying mechanism of fimasartan, a Korean Food and Drug Administration approved anti-hypertension drug, in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Fimasartan suppressed the expressions of inducible nitric oxide synthase (iNOS) by down-regulating its transcription, and subsequently inhibited the productions of nitric oxide (NO). In addition, fimasartan attenuated LPS-induced transcriptional and DNA-binding activities of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1). These reductions were accompanied by parallel reductions in the nuclear translocation of NF-κB and AP-1. Taken together, our data suggest that fimasartan down-regulates the expression of the iNOS in macrophages via NF-κB and AP-1 inactivation.

Inhibition of TAK1 by kamebakaurin in dendritic cells

Kamebakaurin (KA) has anti-cancer and anti-inflammatory activities through direct inhibition of DNA-binding activity of nuclear factor-kappa B (NF-κB) p50. We suggest here another molecular target of KA by the use of lipopolysaccharide-treated dendritic cells. In cell- and enzyme-based assays, KA directly inhibited autophosphorylation and kinase activity of TAK1, followed by the inhibition of TAK1-downstream signaling cascades, such as IKK phosphorylation–IκBα degradation–nuclear translocation of NF-κB, phosphorylation of MEK3/6-p38 mitogen activated protein kinase (MAPK), and MKK4/7-c-Jun N-terminal kinase MAPK. These results demonstrated that TAK1 might be the direct molecular target of KA.

Intranasal brain-derived neurotrophic factor protects brain from ischemic insult via modulating local inflammation in rats

Inflammation plays a vital role in the pathogenesis of ischemic stroke. Brain-derived neurotrophic factor (BDNF) may protect brain tissues from ischemic injury. In this study, we investigated whether intranasal BDNF exerted neuroprotection against ischemic insult by modulating the local inflammation in rats with ischemic stroke. Rats were subjected to temporary occlusion of the right middle cerebral artery (120 min) and intranasal BDNF or vehicle was adminstrated 2 h after reperfusion. Infarct volume and neuron injury were measured using triphenyltetrazolium chloride, Nissl staining and TUNEL assay, respectively. Microglia were detected by immunohistofluorescence. Tumor necrosis factor-α, interleukin10 and mRNAs were evaluated by enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction. DNA-binding activity of nuclear factor-kappa B was measured by electrophoretic mobility shift assay. BDNF level in brain tissues was markedly raised following intranasal administration. There were more Nissl positive and less TUNEL positive neurons in BDNF group than in control group while intranasal BDNF did not reduce the infarct volume significantly (n=6, 0.27±0.04 vs. 0.24±0.05, P>0.05). BDNF increased the number of activated microglia (OX-42 positive) and phagocytotic microglia (ED1 positive). BDNF suppressed tumor necrosis factor-α and mRNA expression while increasing the interleukin10 and mRNA expression. BDNF also increased DNA-binding activity of nuclear factor-kappa B (n=6, 49.78±1.23 vs. 52.89±1.64, P<0.05). Our data suggest intranasal BDNF might protect the brain against ischemic insult by modulating local inflammation via regulation of the levels of cellular, cytokine and transcription factor in the experimental stroke.

Effects of Brain-Derived Neurotrophic Factor on Local Inflammation in Experimental Stroke of Rat

This study was aimed to investigate whether brain-derived neurotrophic factor (BDNF) can modulate local cerebral inflammation in ischemic stroke. Rats were subjected to ischemia by occluding the right middle cerebral artery (MCAO) for 2 hours. Rats were randomized as control, BDNF, and antibody groups. The local inflammation was evaluated on cellular, cytokine, and transcription factor levels with immunofluorescence, enzyme-linked immunosorbent assay, real-time qPCR, and electrophoretic mobility shift assay, respectively. Exogenous BDNF significantly improved motor-sensory, sensorimotor function, and vestibulomotor function, while BDNF did not decrease the infarct volume. Exogenous BDNF increased the number of both activated and phagocytotic microglia in brain. BDNF upregulated interleukin10 and its mRNA expression, while downregulated tumor necrosis factor α and its mRNA expression. BDNF also increased DNA-binding activity of nuclear factor-kappa B. BDNF antibody, which blocked the activity of endogenous BDNF, showed the opposite effect of exogenous BDNF. Our data indicated that BDNF may modulate local inflammation in ischemic brain tissues on the cellular, cytokine, and transcription factor levels.

The role of nuclear factor‐κB in rats of radiocontrast‐media‐induced nephropathy

This study was undertaken to examine the possible role of the DNA-binding activity of nuclear factor-kappa B (NF-kappaB) in rat of radiocontrast-media-induced nephropathy (RCIN) and to explore the characteristic of RCIN in rats and the role of NF-kappaB in its occurrence. Forty-eight adult male Sprague-Dawley (SD) rats were randomly divided into Groups A-D. Rats of Groups A and B were intravenously injected with NG-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg) and indomethacin (10 mg/kg), respectively. Rats of Groups C and D were intravenously injected with 1-M phosphate buffer (PH = 8.4 3 mL/kg) and normal saline (NS 2 mL/kg), respectively. After 30 min, Groups A and D were injected with NS (8 mL/kg) and Groups B and C were injected with diatrizoate (DTZ 8 mL/kg). After injected contrast media (CM) for 6 h, the serum creatinine and blood urea nitrogen of rat in Group B increased sharply as compared with Groups A, C, and D. After 48 h, the data recovered to 49.28 +/- 8.81 mumol/L and 6.72 +/- 2.75 mmol/L, respectively. Vacuolization of the tubule epithelial cells of the kidney was observed in Group A. Especially, these pathological changes were most obvious in outer medulla. Contrast to group A, the DNA-binding activity of NF-kappaB in rat kidney of Group B reached a peak at the 6th h and recovered to the normal level after the 48th h. CM mainly damages renal tubular-interstitial, which appears the earliest and most serious in the outer medulla. Activation of NF-kappaB of renal may be one of the mechanisms of RCIN occurrence.

Glycine attenuates endotoxin-induced liver injury by downregulating TLR4 signaling in Kupffer cells

Background Several experimental studies have observed better outcomes after glycine treatment in patients with endotoxin-induced liver injuries, but its molecular mechanism is not yet fully understood. The purpose of this study was to evaluate the hypothesis that glycine attenuates endotoxin-induced liver injury by affecting endotoxin signal transduction in liver macrophages. Methods An animal model of endotoxin-induced liver injury was established by intraperitoneally injecting mice with 10 mg/kg body weight endotoxin fed a pretreatment diet with or without 5% (w/w) glycine. Blood and liver samples were obtained for analysis of liver morphology and to determine concentrations of alanine aminotransferase, endotoxin receptor Toll-like receptor 4 (TLR4), tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-10 at various time points after injection. To investigate the effect of glycine on liver macrophages, Kupffer cells (KCs) were isolated and challenged by LPS (100 ng/mL), with or without glycine (4 mmol/l) pretreatment, and the expressions of TLR4, IL-10, and TNF-α were assayed at mRNA and protein levels. DNA-binding activity of nuclear factor-kappa B (NF-κB) was also analyzed using enzyme-linked immunosorbent assay. Results Dietary glycine significantly improved the survival rate of endotoxemic mice ( P < .05), whereas serum alanine aminotransferase and TNF-α levels were significantly decreased at different time points ( P < .05); IL-10 levels were increased ( P < .05). Concurrently, LPS-induced hepatic tissue injury was attenuated as indicated by morphologic analysis; secretion of IL-10 in liver tissue ( P < .05) was enhanced; and expression of TLR4 and TNF-α in liver tissue was downregulated ( P < .05). Consistent with these in vivo experiments, enhanced secretion of IL-10 and inhibited expression of TLR4 and TNF-α caused by glycine pretreatment were also observed in LPS-stimulated KCs. NF-κB DNA-binding activity was also significantly inhibited by glycine ( P < .05, respectively). Conclusions Dietary glycine improved survival rates and liver function in endotoxemic mice by regulating the production of proinflammatory or anti-inflammatory cytokines in liver. It attenuated liver injury by deactivating KCs through inhibiting TNF-α secretion and increasing IL-10 production. The downregulative effect of glycine on the endotoxin signaling pathway and TLR4/NF-κB/TNF-α may be a novel potential mechanism by which glycine inhibits KC activity.

Chemopreventive effect of plant originated glycoprotein on colitis-mediated colorectal cancer in A/J mice

This study was carried out to investigate the chemopreventive effects of glycoprotein (UDN glycoprotein, 116-kDa) isolated from Ulmus davidiana Nakai on colitis-mediated colorectal cancer (CRC) in A/J mice. UDN glycoprotein intake significantly reduced the incidence and the multiplicity of colorectal tumors, induced by combination treatment with 10 mg/kg 1,2-dimethylhydrazine (DMH) and 2% dextran sodium sulfate (DSS). We found that the abnormal levels of lactate dehydrogenase (LDH), thiobarbituric acid reactive substances (TBARS), and nitric oxide (NO) were significantly suppressed in proportion to the concentration of UDN glycoprotein (0.01% and 0.02%) in the mice serum. In addition, consumption of UDN glycoprotein attenuated the activities of proliferating cell nuclear antigen (PCNA), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), and inhibited the DNA-binding activities of nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1) in the mice colonic tissue. Interestingly, the results obtained from reverse transcription polymerase chain reaction (RT-PCR) assay showed that 0.02% UDN glycoprotein inhibited the expressions of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 mRNA in the mice. Collectively, these results suggest that UDN glycoprotein has chemopreventive activity via modulation of inflammation-related factors responsible for development of colitis-mediated CRC in A/J mice.

Tumor necrosis factor-α does not modulate ischemia/reperfusion injury in naïve myocardium but is essential for the development of late preconditioning

The role of tumor necrosis factor (TNF)-α in myocardial ischemia/reperfusion injury remains controversial. We used homozygous TNF-α null mice ( TNF-α –/– ) to determine whether TNF-α modulates myocardial ischemia/reperfusion injury. Mice were subjected to a 30-min coronary occlusion followed by 24 h of reperfusion. When wild-type mice were preconditioned with six cycles of 4-min coronary occlusion/4-min reperfusion 24 h before the 30-min occlusion, infarct size was reduced from 58.6 ± 1.9% of the risk region to 19.3 ± 3.6%, indicating a late preconditioning (PC) effect. In non-preconditioned TNF-α –/– mice, infarct size was similar to that observed in wild-type mice (55.5 ± 3.7%). However, in TNF-α –/– mice preconditioned with six occlusion/reperfusion cycles 24 h earlier, infarct size was not reduced (55.2 ± 5.7%), indicating that the late PC protection against infarction was completely abolished. While minimal TNF-α immunoreactivity was detected in sham-operated hearts, extensive TNF-α expression was noted in the cytoplasm of cardiomyocytes in the ischemic/reperfused region 30 min after the PC ischemia. At 30 min after PC, wild-type mice exhibited increased DNA-binding activity of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1) and nuclear translocation of p65, c-Jun and c-Fos; all of these changes were absent in TNF-α –/– mice. These data demonstrate that TNF-α does not modulate infarct size in the naïve (non-preconditioned) state but is essential for the development of the late phase of ischemic PC, possibly via the activation of NF-κB and AP-1 transcription factors.

Glycoprotein isolated from Solanum nigrum L. inhibits the DNA-binding activities of NF-κB and AP-1, and increases the production of nitric oxide in TPA-stimulated MCF-7 cells

Solanum nigrum L. (SNL) has been used in traditional folk medicine to treat numerous cancers. We isolated a glycoprotein (150 kDa) from SNL and tested its effect on the modulation of transcriptional factors (NF-kappa B and AP-1) and iNO production in TPA induced-MCF-7 cells, which are part of the human breast cancer cell line, without estrogen receptors. However, the mechanism of SNL glycoprotein in pharmacological and biochemical actions in cancer cells has not been studied. To test the effect of SNL glycoprotein on the DNA-binding activities of nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1), and nitric oxide (NO) production, these experiments were carried out using electrophoretic mobility shift assays (EMSA), western blot analysis, and the Griess method. Results in this experiment showed that SNL glycoprotein inhibits 12- O-Tetra decanoylphorbol-13-acetate (TPA; 100 nM)-induced DNA-binding activities of NF-κB and AP-1, and enhances NO production in MCF-7 cells. That is, our results indicated that SNL glycoprotein has the capacity to modulate the TPA-induced DNA-binding activities of transcription factors and NO production, which play a critical role with respect to cytotoxicity in MCF-7 cells. Therefore, SNL glycoprotein might be one of the agents that blocks TPA-mediated signal responses in tumor cells.

Cytotoxic effect of glycoprotein isolated from Solanum nigrum L. through the inhibition of hydroxyl radical-induced DNA-binding activities of NF-kappa B in HT-29 cells

Solanum nigrum L. (SNL) has been traditionally used as an herbal plant for a long time. In the present study, SNL glycoprotein showed a dose-dependent radical scavenging activity on radicals, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, hydroxyl radical ( OH), and superoxide anion (O 2 −). When the HT-29 cells were treated with 60 μg/ml SNL glycoprotein, the cytotoxic effect was induced in a time-dependent manner. More specifically, it was more than 60% ( P<0.01) after 4 h, compared to the control. On the other hand, the cells treated with 100 mU/ml glucose oxidase (GO) to generate the OH radical showed a cancer resistance up to 12 h. Furthermore, the addition of GO to the SNL glycoprotein caused a strong cytotoxic effect, rather than a radical scavenging effect. Interestingly, when the cells were exposed to 100 mU/ml GO for 4 h, the DNA-binding activity of nuclear factor-kappa B (NF-κB) was increased 4.15-fold ( P<0.01) compared to the control, whereas 40 μg/ml SNL glycoprotein completely blocked the DNA-binding activity of OH radical-induced NF-κB by electrophoretic mobility shift assays (EMSAs). Apoptosis, according to the apoptosis assay, increased as a result of treatment with 40 μg/ml SNL glycoprotein in a time-dependent manner, whereas they were weakly induced by GO in the cells. Consequently, the SNL glycoprotein may induce apoptosis through the inhibition of NF-κB activation, induced by oxidative stress in HT-29 cells.