NF-kappaB Protein Research Articles

Redox Regulation of Nuclear Post-Translational Modifications During NF-κB Activation

The transcription factor NF-kappaB controls the expression of hundreds of genes involved in the regulation of the immune/inflammatory response, development, and apoptosis. In resting cells, NF-kappaB proteins are sequestered in the cytoplasm through their tight association with IkappaB proteins. NF-kappaB activation relies on the signal-induced IkappaB phosphorylation and degradation, thereby allowing the nuclear translocation of NF-kappaB proteins. In the nucleus, several post-translational modifications of NF-kappaB and chromatin remodeling of target genes are mandatory for NF-kappaB DNA binding and full transcription. Since 1991, reactive oxygen species (ROS) have been implicated in NF-kappaB activation. ROS enhance the cytoplasmic signaling pathways leading to NF-kappaB nuclear translocation, but reduction/oxidation (redox) also controls several key steps in the nuclear phase of the NF-kappaB program, including chromatin remodeling, recruitment of co-activators, and DNA binding. Here we describe the redox regulation of NF-kappaB activity in the nucleus.

The prolyl isomerase Pin1 regulates the NF-κB signaling pathway and interleukin-8 expression in glioblastoma

The brain tumor glioblastoma remains one of the most aggressive and devastating tumors despite decades of effort to find more effective treatments. A hallmark of glioblastoma is the constitutive activation of the NF-κB signaling pathway, which regulates cell proliferation, inflammation, migration, and apoptosis. The prolyl isomerase Pin1 has been found to bind directly to the NF-κB protein, p65, and cause increases in NF-κB promoter activity in a breast cancer model. We now present evidence that this interaction occurs in glioblastoma and that it has important consequences on NF-κB signaling. We demonstrate that Pin1 levels are enhanced in primary glioblastoma tissues compared to controls, and that this difference in Pin1 expression affects the migratory capacity of glioblastoma-derived cells. Pin1 knockdown decreases the amount of activated, phosphorylated p65 in the nucleus, resulting in inhibition of the transcriptional program of the IL-8 gene. Through the use of microarray, we also observed changes in the expression levels of other NF-κB regulated genes due to Pin1 knockdown. Taken together, these data suggest that Pin1 is an important regulator of NF-κB in glioblastoma, and support the notion of using Pin1 as a therapeutic target in the future.

NF-κB activation induced by Notch ligand stimulation in acute myeloid leukemia cells

There are conflicting reports regarding the effects of Notch activation on nuclear factor-kappaB (NF-kappaB) activity. The relationships are cell type-dependent and have not been fully elucidated. We examined the effects of Notch activation induced by a recombinant Notch ligand, Delta-like1 (Dll1), on the NF-kappaB activity in two acute myeloid leukemia (AML) cell lines. We found that Delta1-induced Notch activation activated the NF-kappaB pathway in THP-1 cells. Regarding the possible mechanisms, Dll1 stimulation increased the mRNA and protein expression levels of some components of the NF-kappaB pathway and induced phosphorylation of IKKalpha/beta, IkappaB and RelA proteins after 24 or 48 h of stimulation. Since the phosphorylation required a long time, it did not appear to be caused by physical interactions between Notch and NF-kappaB proteins, but rather by indirect effects. One possible mechanism for the indirect effects was the observed induction of IL-1beta expression by Dll1 stimulation. On the other hand, Notch activation did not affect NF-kappaB activity in TMD7 cells. RelA was phosphorylated without stimulation, indicating that NF-kappaB was constitutively activated in TMD7 cells. To the best of our knowledge, this is the first study to investigate AML cells and use a recombinant Notch ligand to activate Notch. The present findings lead to better understanding of Notch functions, which have not been fully elucidated.

Activation of nuclear factor kappa B and induction of migration inhibitory factor in tumors by surgical stress of laparotomy versus carbon dioxide pneumoperitoneum: an animal experiment

Surgical trauma may be associated with enhanced tumor growth and establishment. The authors studied the effect of carbon dioxide (CO(2)) pneumoperitoneum versus laparotomy on tumor necrosis factor-alpha (TNFalpha), migration inhibitory factor (MIF) expression, and nuclear factor kappa B (NFkappaB) activity in human gastric cancer. Nude mice were inoculated intraperitoneally with human gastric cancer cells (MKN45). Then laparotomy, CO(2) pneumoperitoneum, and anesthesia alone were performed randomly. Tumor growth and associated TNFalpha and MIF expression and NFkappaB activity were determined. Total tumor weight, especially at the anterior abdominal wall, was higher after laparotomy than after CO(2) pneumoperitoneum (p < 0.05). The mRNA expression of TNFalpha was higher 24 and 48 h after laparotomy than after CO(2) pneumoperitoneum (p < 0.05 and p < 0.01, respectively). At all the examined time points, MIF mRNA expression also was higher after laparotomy than after CO(2) pneumoperitoneum (p < 0.05 until 1 week or p < 0.01 at 2 weeks). The NFkappaB protein was more activated after laparotomy than after CO(2) pneumoperitoneum 6 h subsequent to surgical procedures. After CO(2) pneumoperitoneum, tumors have less TNFalpha and MIF expression and less NFkappaB activity than after laparotomy. This may be associated with less tumor growth, supporting minimal invasive techniques in gastrointestinal oncologic surgery.

Anti-inflammatory Activity of Flavokawain B from Alpinia pricei Hayata

Alpinia pricei (Zingiberaceae) is a spicy herb indigenous to Taiwan. A potent anti-inflammatory compound, flavokawain B (FKB), was obtained from A. pricei. FKB significantly inhibited production of NO and PGE(2) in LPS-induced RAW 264.7 cells. Moreover, it also notably decreased the secretion of TNF-alpha. Expression of iNOS and COX-2 proteins was also inhibited by FKB in a dose-dependent manner. FKB blocked the nuclear translocation of NF-kappaB induced by LPS, which was associated with prevention IkappaB degradation, and subsequently decreased NF-kappaB protein levels in the nucleus. Similar anti-inflammatory activities of FKB were observed in an animal assay. NO concentrations in mouse serum rose dramatically from 3.2 to 28.8 microM after mice were challenged with LPS; however, preadministration of 200 mg/kg FKB reduced the NO concentration to 3.8 microM after challenge with LPS. Moreover, FKB strongly suppressed LPS-induced iNOS, COX-2, and NF-kappaB proteins expression in mouse liver.

NF‐κB and estrogen receptor α interactions: Differential function in estrogen receptor‐negative and ‐positive hormone‐independent breast cancer cells

Estrogen receptor (ER)-positive breast cancer cells have low levels of constitutive NF-kappaB activity while ER negative (-) cells and hormone-independent cells have relatively high constitutive levels of NF-kappaB activity. In this study, we have examined the aspects of mutual repression between the ERalpha and NF-kappaB proteins in ER+ and ER- hormone-independent cells. Ectopic expression of the ERalpha reduced cell numbers in ER+ and ER- breast cancer cell lines while NF-kappaB-binding activity and the expression of several NF-kappaB-regulated proteins were reduced in ER- cells. ER overexpression in ER+/E2-independent LCC1 cells only weakly inhibited the predominant p50 NF-kappaB. GST-ERalpha fusion protein pull downs and in vivo co-immunoprecipitations of NF-kappaB:ERalpha complexes showed that the ERalpha interacts with p50 and p65 in vitro and in vivo. Inhibition of NF-kappaB increased the expression of diverse E2-regulated proteins. p50 differentially associated directly with the ER:ERE complex in LCC1 and MCF-7 cells by supershift analysis while p65 antibody reduced ERalpha:ERE complexes in the absence of a supershift. ChIP analysis demonstrated that NF-kappaB proteins are present on an endogenous ERE. Together these results demonstrate that the ER and NF-kappaB undergo mutual repression, which may explain, in part, why expression of the ERalpha in ER- cells does not confer growth signaling. Secondly, the acquisition of E2-independence in ER+ cells is associated with predominantly p50:p50 NF-kappaB, which may reflect alterations in the ER in these cells. Since the p50 homodimer is less sensitive to the presence of the ER, this may allow for the activation of both pathways in the same cell.

The IκBα gene is a peroxisome proliferator‐activated receptor cardiac target gene

The purpose of this study was to provide a better understanding of the regulatory role of the nuclear steroid receptor on the nuclear factor of kappa light polypeptide gene enhancer in B cells (NFkappaB) in mouse heart. NFkappaB regulates many nuclear genes and has been associated with many human cardiac diseases. NFkappaB's protein regulator gene, nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha gene (IkappaBalpha), was found in this study to be regulated by peroxisome proliferator-activated receptors (PPARs). PPARs, retinoid X receptors (RXRs) and thyroid hormone receptors (THRs) are members of the nuclear receptor superfamily, which consists of a large number of transcription factors whose activities are regulated by their cognate ligands. These steroid hormone receptors are important regulators of gene expression and differentiation in the heart. These receptors form homo-(RXR, THR) and hetero-(PPAR-RXR, RXR-THR) dimers that bind DNA at various response elements (PPAR, RXR and THR) in the promoter regions of target genes. The PPAR/RXR response elements in the promoter of IkappaBalpha are described in this article. A known PPAR activator (Wy14643) and dimethylsulfoxide (vehicle) were introduced into control (FVB) and delta337T thyroid hormone receptor (TRbeta) transgenic mice. The delta337T TRbeta transgenic mouse has a resistance to the thyroid hormone (RTH) phenotype. Affymetrix 430_2 chip gene expression was examined for four study groups (control, control with Wy14643, delta337T TRbeta and delta337T TRbeta with Wy14643), consisting of seven mice each. IkappaBalpha mRNA expression in the Wy14643 control and in transgenic mice was upregulated significantly in microarray (P < 0.05) and quantitative RT-PCR (P < 0.01) analyses. The increase in mRNA level was also accompanied by an increase in IkappaBalpha protein in cells, as measured by Western blot analysis. Duplex oligo-DNAs containing the putative PPAR/RXR motif (AGGTCA/TCCAGT) from the IkappaBalpha promoter were used in gel shift assays to verify the binding of PPAR and RXR to their response elements. pGL4.0 [Luc] constructs of the IkappaBalpha promoter, with and without the PPAR/RXR motifs, were co-transfected with mouse PPAR alpha, beta and gamma(1) into HepG2 cells and used in luciferase assays to verify gene activation. In conclusion, our study revealed that PPAR regulates the mouse cardiac IkappaBalpha gene in both control and transgenic mouse heart. The implications of this finding are discussed in relation to possible changes in cardiac function.

Effects of Salvia Miltiorrhizae on ICAM-1, TLR4, NF-κB and Bax Proteins Expression in Multiple Organs of Rats with Severe Acute Pancreatitis or Obstructive Jaundice

To observe the protective effects of Salvia miltiorrhizae injection on multiple organs of rats with SAP or OJ. Two hundred eighty-eight rats were used for SAP-associated experiments and OJ-associated experiments. The rats were randomly divided into sham-operated, model control and treated group. According to the different time points after operation, the SAP rats in each group were subdivided into 3, 6 and 12 h groups while the OJ rats were divided into 7, 14, 21 and 28 days groups. The mortality rates, pathological changes and ICAM-1 (only in lung), TLR4 (only in liver), Bax and NF-kappaB proteins expression in multiple organs (liver, kidney, lung, intestinal mucosa, spleen, thymus and lymph nodes) were observed, respectively. The mortality rates of treated groups decreased in both SAP and OJ experiments. Compared to model control group, the pathological changes can be seen in treated groups including; (1) the pathological changes of multiple organs in SAP and OJ experiments were improved, the pathological severity scores of kidney (at 6 h), intestinal mucosa (at 12 h), spleen (at 6 and 12 h), thymus (at 3 and 6 h) and lymph nodes (at 3 and 6 h) in SAP experiment and of liver (on 21 and 28 days), lung (21 days), intestinal mucosa (on 21 and 28 days) and spleen (on 21 and 28 days) in OJ experiment significantly declined (P < 0.05 or P < 0.01); (2) the positive rate of Bax protein in pancreas (at 3 h) and liver (on 6 and 12 h) in SAP experiment and in liver (on 28 days), kidney (on 21 days), and thymus (on 28 days) in OJ experiment were significantly lower (P < 0.05 or P < 0.01); (3) the positive rate of NF-kappaB in liver (at 12 h) and kidney (at 6 h) in SAP experiment and in liver (on 21 days), kidney (on 28 days), intestinal mucosa (on 28 days) and thymus (on 14 days) in OJ experiment significantly decreased (P < 0.05). (4) the positive rate of ICAM-1 in lungs (at 12 h) in SAP experiment was significantly less (P < 0.05). Salvia miltiorrhizae injection can protect multiple organs of SAP or OJ rats and inhibit the expression of ICAM-1, TLR4, NF-kappaB and regulate Bax proteins. Salvia miltiorrhizae may relieve the inflammation response and enhance the immunity function of SAP and OJ rats.

Inhibition of Vascular Smooth-Muscle Cell Proliferation and Arterial Restenosis by HO-3867, a Novel Synthetic Curcuminoid, through Up-Regulation of PTEN Expression

Phosphatase and tensin homolog (PTEN), a tumor suppressor gene, has been shown to play a vital role in vascular smooth muscle cell (SMC) proliferation and hence is a potential therapeutic target to inhibit vascular remodeling. The goal of this study was to evaluate the efficacy and mechanism of HO-3867 [((3E,5E)-3,5-bis[(4-fluorophenyl)methylidene]-1-[(1-hydroxy-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl]piperidin-4-one)], a new synthetic curcuminoid, in the inhibition of vascular SMC proliferation and restenosis. Experiments were performed using human aortic SMCs and a rat carotid artery balloon injury model. HO-3867 (10 microM) significantly inhibited the proliferation of serum-stimulated SMCs by inducing cell cycle arrest at the G(1) phase (72% at 24 h) and apoptosis (at 48 h). HO-3867 significantly increased the phosphorylated and total levels of PTEN in SMCs. Suppression of PTEN expression by PTEN-small interfering RNA transfection reduced p53 and p21 levels and increased extracellular signal-regulated kinase 1/2 phosphorylation, resulting in decreased apoptosis. Conversely, overexpression of PTEN by cDNA transfection activated caspase-3 and increased apoptosis. Furthermore, HO-3867 significantly down-regulated matrix metalloproteinase (MMP)-2, MMP-9, and nuclear factor (NF)-kappaB expressions in SMCs. Finally, HO-3867 inhibited arterial neointimal hyperplasia through overexpression of PTEN and down-regulation of MMPs and NF-kappaB proteins. HO-3867 is a potent drug, capable of overexpressing PTEN, which is a key target in the prevention of vascular remodeling, including restenosis.

Short-Term Starvation of Immune Deficient Drosophila Improves Survival to Gram-Negative Bacterial Infections

BackgroundPrimary immunodeficiencies are inborn errors of immunity that lead to life threatening conditions. These predispositions describe human immunity in natura and highlight the important function of components of the Toll-IL-1- receptor-nuclear factor kappa B (TIR-NF-κB) pathway. Since the TIR-NF-κB circuit is a conserved component of the host defence in higher animals, genetically tractable models may contribute ideas for clinical interventions.Methodology/Principal FindingsWe used immunodeficient fruit flies (Drosophila melanogaster) to address questions pertaining to survival following bacterial infection. We describe here that flies lacking the NF-κB protein Relish, indispensable for countering Gram-negative bacteria, had a greatly improved survival to such infections when subject to dietary short-term starvation (STS) prior to immune challenge. STS induced the release of Nitric Oxide (NO), a potent molecule against pathogens in flies, mice and humans. Administering the NO Synthase-inhibitory arginine analog N-Nitro-L-Arginine-Methyl-Ester (L-NAME) but not its inactive enantiomer D-NAME increased once again sensitivity to infection to levels expected for relish mutants. Surprisingly, NO signalling required the NF-κB protein Dif, usually needed for responses against Gram-positive bacteria.Conclusions/SignificanceOur results show that NO release through STS may reflect an evolutionary conserved process. Moreover, STS could be explored to address immune phenotypes related to infection and may offer ways to boost natural immunity.

Effects of Qingkailing effective components on nuclear factor-kappa B in an ischemia-reperfusion injury model of rat brain microvascular endothelial cells in vitro

To establish an ischemia-reperfusion injury model of rat cerebral microvascular endothelial cells (MVECs) in vitro, and to explore the relationship between nuclear factor-kappa B (NF-kappaB) and the protective effects of Qingkailing effective components (hyocholic acid, taurocholic acid, baicalin, jasminoidin, Pinctada martensii) on MVECs. Brain MVECs of male rats were digested with trypsin and subcultured, then the content of MVECs was adjusted to 1x10 (5)/mL and the MVECs were divided into normal control group, untreated group, hyocholic acid group, taurocholic acid group, baicalin group, jasminoidin group, Pinctada martensii group and nimodipine group, with six holes in each group. Except for the normal control group, the MVECs in the other groups were exposed in oxygen and glucose deprivation (OGD) circumstance in vitro to simulate ischemia-reperfusion injury. Immunocytochemical staining and image analysis system were used to observe the expression of NF-kappaB protein. Under a light microscope, the nuclei of MVECs in the normal control group were blank. Staining intensity of NF-kappaB protein in the nucleus in the untreated group was much deeper than that in the endochylema, with NF-kappaB shifted to nucleus after activation; a small quantity of NF-kappaB protein were expressed in the border of nucleus next to endochylema in groups of Qingkailing effective components, and the NF-kappaB protein expression was weaker than that in the untreated group. With the image analysis, we found that transmittance of nucleus and endochylema in the untreated group was significantly lower than that in the normal control group (P<0.01). Transmittance of nucleus and endochylema in the treated groups was higher than that in the untreated group (P<0.05, P<0.01). Qingkailing effective components have significant effect in inhibiting NF-kappaB protein transferring from endochylema to nucleus in vitro.

NS-398 Induces Apoptosis in Human Esophageal Cancer Cells Through Inhibition of NF-kappaB Downstream Regulation of Cyclooxygenase-2

Although non-steroidal anti-inflammatory drugs (NSAIDs) have been demonstrated to have cancer-preventive effects and induce apoptosis of cancer cells, the mechanism of their effects is not clearly known. We studied the mechanism in human esophageal cancer cell line TE13. The esophageal squamous cell carcinoma cell line TE-13 was cultured with NS-398 at different concentrations or for different times. Proliferation and apoptosis were measured by MTT reduction and flow cytometry. Prostaglandin F1α was determined with radioimmunoassay. Expression of COX-2 mRNA was measured by RT-PCR and COX-2 protein levels with Western blot analysis. Nuclear NF-kappaB and cytoplasmic IkappaB protein levels were determined by electrophoretic mobility shift assay and Western blot, respectively. NS-398 significantly inhibited cell proliferation and induced apoptosis at concentrations of 0.001, 0.01, 1, and 100 μ mol/L. NS-398 dose-dependently decreased the levels of COX-2 mRNA, COX-2 protein, nuclear NF-kappaB protein and production of PGF1α and increased the cytoplasmic IkappaB protein. In conclusion, NS-398 inhibits the proliferation of, and induced apoptosis in, the cultured TE-13 SCC cell line. These changes correlate with a reduction in COX-2 mRNA and protein expression, prostaglandin synthesis, an inhibition of NF-kappaB nuclear translocation, and an increase in cytoplasmic IkappaB.

Induction of apoptosis in human liver carcinoma HepG2 cell line by 5-allyl-7-gen-difluoromethylenechrysin

To investigate the effect of 5-allyl-7-gen-difluoromethylenechrysin (ADFMChR) on apoptosis of human liver carcinoma HepG2 cell line and the molecular mechanisms involved. HepG2 cells and L-02 cells were cultured in vitro and the inhibitory effect of ADFMChR on their proliferation was measured by MTT assay. The apoptosis of HepG2 cells was determined by flow cytometry (FCM) using propidium iodide (PI) fluorescence staining. DNA ladder bands were observed by DNA agarose gel electrophoresis. The influence of ADFMChR on the proxisome proliferator-activated receptor gamma (PPARgamma), NF-kappaB, Bcl-2 and Bax protein expression of HepG2 cells were analyzed by Western blotting. MTT assay showed that ADFMChR significantly inhibited proliferation of HepG2 cells in a dose-dependent manner, with little effect on growth of L-02 cells, and when IC(50) was measured as 8.45 micromol/L and 191.55 micromol/L respectively, the potency of ADFMChR to HepG2 cells, was found to be similar to 5-fluorouracil (5-FU, IC(50) was 9.27 micromol/L). The selective index of ADFMChR cytotoxicity to HepG2 cells was 22.67 (191.55/8.45), higher than 5-FU (SI was 7.05 (65.37/9.27). FCM with PI staining demonstrated that the apoptosis rates of HepG2 cells treated with 3.0, 10.0 and 30.0 micromol/L ADFMChR for 48 h were 5.79%, 9.29% and 37.8%, respectively, and were significantly higher when treated with 30.0 micromol/L ADFMChR than when treated with 30.0 micromol/L ChR (16.0%) (P < 0.05) and were similar to those obtained with 30.0 micromol/L 5-FU (41.0%). DNA agarose gel electrophoresis showed that treatment of HepG2 cells with 10.0 micromol/L ADFMChR for 48 h and 72 h resulted in typical DNA ladders which could be reversed by 10.00 micromol/L GW9662, a blocker of PPARgamma. Western blotting analysis revealed that after 24 h of treatment with 3.0, 10.0, 30.0 micromol/L ADFMChR, PPARgamma and Bax protein expression in HepG2 cells increased but Bcl-2 and NF-kappaB expression decreased; however, pre-incubation with 10.0 micromol/L GW9662 could efficiently antagonize and weaken the regulatory effect of 3.0, 30.0 micromol/L ADFMChR on PPARgamma and NF-kappaB protein expression in HepG2 cells. ADFMChR induces apoptosis of HepG2 cell lines by activating PPARgamma, inhibiting protein expression of Bcl-2 and NF-kappaB, and increasing Bax expression.

Influence of Baicalin and Octreotide on NF-κB and P-Selectin Expression in Liver and Kidney of Rats with Severe Acute Pancreatitis

To observe the influence of Baicalin and Octreotide on liver and kidney of rats with severe acute pancreatitis (SAP) and discuss the related mechanism. SAP rats were randomly divided into model control, Baicalin treated and Octreotide treated group (n = 45). The same number of normal rats were included in sham-operated group (n = 45). In all groups, the mortality rate, pathological changes as well as expression levels of NF-kappaB p65 and P-selectin protein in liver and kidney were observed at 3, 6 and 12 h after operation. The survival rate of treated group was 100% at 12 h significantly higher than that of model control group (P < 0.05). The pathological changes of liver and kidney in treated groups were alleviated to different degrees, the NF-kappaB protein expression levels and pathological severity scores in liver and kidney of treated groups were significantly lower than those of model control group (P < 0.05 or P < 0.001). The hepatic P-selectin protein expression level in Baicalin treated group was significantly lower than that of model control group at 3 h (P < 0.01), and renal P-selectin expression level in Baicalin treated group at 3 and 6 h were significantly lower than those of model control group and Octreotide treated group (P < 0.01). (1) Early treatment with Baicalin or Octreotide have obvious protecting effects on liver and kidney injuries in SAP with their mechanisms associated to inhibiting NF-kappaB and P-selectin expression of liver and kidney. (2) Comparing the pharmacologic effects of Octreotide and Baicalin, we believe Baicalin as a new drug with its protecting effects on liver and kidney of SAP rats similar to Octreotide is worth further studying. (3) The advantages of tissue microarrays in pathological examination include time and energy saving and highly efficient. But the restriction of small diameter weakens the representation of tissues to various extents, which may lead to the deviation of analysis.

Increased tissue transglutaminase expression in human atherosclerotic coronary arteries

Transglutaminase 2 (TGase 2) is a calcium-dependent cross-linking enzyme that catalyzes a covalent iso-peptide bond between two proteins. Interestingly, this catalysis can activate the nuclear factor-kappaB (NF-kappaB) through the polymerization of the inhibitory protein of NF-kappaB (I-kappaB). The objective of the present study was to investigate the expression of TGase 2 in the human atherosclerotic human coronary artery, and the possible roles of TGase 2 in NF-kappaB activation. We explored whether expressions of TGase 2 and NF-kappaB are associated in atherosclerosis. Using human samples, we found that TGase 2 was markedly higher than normal in the neointimal tissue of atherosclerotic coronary arteries with atherosclerosis progression. TGase 2 activity was also increased approximately two-fold in the atherosclerotic vascular wall. In immunofluorescence analysis, NF-kappaB, COX-2, and TNF-alpha were co-localized at TGase 2-positive neointimal smooth muscle cells. A promoter assay test showed that NF-kappaB activity increased in both the human monocyte and human breast carcinoma cell by TGase 2, and that TGase 2-mediated NF-kappaB activation was reversed by TGase 2 siRNA. According to these results, we suggest that TGase 2 may function as an activator in the NF-kappaB pathway; this effect may occur in the atherosclerotic vessel wall.

Glutamate-Induced NFκB Activation in the Retina

To determine the distribution and glutamate-mediated activation of nuclear factor (NF) kappaB members in the retina and pan-purified retinal ganglion cells (RGCs) and to characterize steps in the signal transduction events that lead to NFkappaB activation. Retinal expression patterns and RGCs were evaluated for five NFkappaB proteins with the aid of immunohistochemistry. Retinal explants or RGCs were treated with glutamate with or without the presence of the NDMA receptor antagonist memantine, the calcium chelator EGTA, or a specific inhibitor for calcium/calmodulin-dependent protein kinase-II (CaMKII). Characterizations of NFkappaB activation were performed with the aid of electrophoretic mobility shift assays and supershift assays. All five NFkappaB proteins were present in the retina and in the pan-purified RGCs. In response to a glutamate stimulus, all NFkappaB proteins except c-Rel were activated. P65 was unique in that it was not constitutively active but showed a glutamate-inducible activation in the retina and in the cultured RGCs. Memantine, EGTA, or autocamtide-2-related inhibitory peptide (AIP) inhibited NFkappaB activation in the retina. Furthermore, AIP significantly reduced the level of glutamate-induced degradation of IkappaBs. These data indicate that glutamate activates distinct NFkappaB proteins in the retina. P65 activation may be especially important with regard to RGC responses to glutamate given that its activity is induced by conditions known to lead to the death of these cells. The NMDA receptor-Ca(2+)-CaMKII signaling pathway is involved in glutamate-induced NFkappaB activation. Because AIP blocks the degradation of IkappaB, its regulation is clearly downstream of CaMKII.

Homocysteine-induced enhanced expression of tissue factor in human vascular smooth muscle cells

The homocysteine (Hcy)-induced tissue factor (TF) expression in human vascular smooth muscle cells (VSMCs) and the effect of Hcy on the activity of nuclear factor-kappaB (NF-kappaB) and the expression of inducible nitric oxide synthase (iNOS) were investigated. Human umbilical artery VSMCs were cultured by tissue explanting method, identified by alpha-actin immunohistochemistry, and incubated with different concentrations of Hcy/PTDC (NF-kappaB inhibitor). Semi-quantitative RT-PCR was performed to detect the expression of TF mRNA in VSMCs. Flow cytometry was used to assay the expression of TF protein on the surface of VSMCs and the expression of iNOS in VSMCs. Western blot was carried out to detect the expression of NF-kappaB protein in nuclei. The results showed that Hcy could induce VSMCs expressing TF mRNA significantly after the VSMCs were incubated with Hcy at concentrations of 10, 100, 500 micromol/L respectively. There was low expression level of TF protein on the surface of the resting VSMCs and Hcy could also induce VSMCs expressing TF protein on the cell surface in different concentrations. Additionally, Hcy could rapidly induce the activation of NF-kappaB and this effect could be significantly inhibited by PDTC. Hcy alone could not induce the expression of iNOS in VSMCs. It was concluded that Hcy could significantly induce the expression of TF in VSMCs and enhance the activation of NF-kappaB, subsequently mediate TF gene expression and protein synthesis. NF-kappaB-mediated expression of TF in VSMCs might be the important mechanism of atherosclerosis and thrombosis induced by Hcy.

Study of the Protective Effects of Dexamethasone on Ileum Mucosa Injury in Rats With Severe Acute Pancreatitis

To study the protecting effects of dexamethasone on ileum mucosa injury of rats with severe acute pancreatitis (SAP). The SAP rats were prepared by improved Aho's methods. The plasma endotoxin and inflammatory mediators in serum were determined. The rat mortality, pathological changes of terminal ileum, nuclear factor kappa B (NF-kappaB), apoptotic indexes, and apoptotic related protein expression were observed. The plasma endotoxin, inflammatory mediators, and NF-kappaB protein expression as well as pathological scores of the treatment group of ileum mucosa were lower than those of the model group at different time points. P selectin in model group significantly exceeded the dexamethasone treatment group at 3 and 6 hours (P < 0.01, P < 0.05). Caspase-3 protein expression in dexamethasone treatment group significantly exceeded the model group at 3 and 6 hours (P < 0.05), and apoptotic indexes were higher than those of the model group at 6 hours (P < 0.05), but Bax protein has shown no marked difference among groups. Dexamethasone can reduce the endotoxin level and inflammatory mediators and down-regulate NF-kappaB protein expression of ileum mucosa, and ileum mucosa epithelial cell apoptosis induction was involved as well. The tissue microarrays technique is of advantage in SAP study.

Effects of tetrandrine on nuclear factor-kappaB expression in leukemia multidrug-resistant cell line K562/A02

To investigate the effects of tetrandrine (Tet) on nuclear factor kappaB (NF-kappaB) expression in leukemia cell line K562 and multidrug-resistant K562/A02 cell line. The activations of NF-kappaB in K562 and K562/A02 cells and the effects of 1 micromol/L Tet on NF-kappaB expression were determined by immunocytochemistry and Western blot assay. Tet had no effect on NF-kappaB expression in K562 cells after 6- and 12-hour treatment (P>0.05), and K562/A02 cells displayed higher level of NF-kappaB protein expression than their parental K562 cells (P<0.01). Tet could significantly down-regulate NF-kappaB protein expression and nuclear translocation in K562/A02 cells shown by immunocytochemistry and Western blot, and this decrease became more significant after 12-hour treatment than after at 6-hour treatment (P<0.05). Activation of NF-kappaB may be related to multidrug resistance of K562/A02 cell line. And the inhibition of NF-kappaB activation by Tet leads to multidrug resistance reversal in K562/A02 cell line.

Blockage of interleukin-6 signaling with 6-amino-4-quinazoline synergistically induces the inhibitory effect of bortezomib in human U266 cells

The transcription factor nuclear factor-kappa B (NF-kappaB) regulates the transcription of a number of genes involved in a variety of cellular responses, including cell survival, inflammation, and differentiation. NF-kappaB is activated by a variety of stimuli, proinflammatory cytokines, mitogens, growth factors, and stress-inducing agents. Aberrant NF-kappaB expression is considered to be one of the oncogenic factors of cancer and the constitutive activation of NF-kappaB is observed in several hematologic disorders [classic Hodgkin's lymphoma, diffuse large B cell lymphoma, and multiple myeloma (MM)], and the modulation of NF-kappaB activation is emerging as a promising novel anticancer therapeutic strategy.Therefore, we focused on the regulation of NF-kappaB activation in MM. When U266 cells were treated with 6-amino-4-quinazoline, an NF-kappaB activation inhibitor, we determined that it most effectively blocked the interleukin (IL)-6-induced activation of MAPK and JAK/STAT pathways among different signaling inhibitors. The results of the luciferase assay indicated that 6-amino-4-quinazoline inhibited NF-kappaB activation with diminished NF-kappaB protein bound to NF-kappaB DNA binding sites. In addition, 6-amino-4-quinazoline suppressed the production of IL-6, which affected MM cell proliferation. Furthermore, combined treatment with bortezomib and 6-amino-4-quinazoline effectively inhibited the IL-6 and soluble IL-6R-induced activation of STAT3 and extracellular signal-regulated kinase phosphorylation. Our data showed that the inhibition of NF-kappaB activation abrogated MM cell proliferation induced by the IL-6 pathway, and might represent a promising therapeutic strategy for the treatment of MM.