Inhibition Of NF-kappaB Nuclear Translocation Research Articles

Influence of silencing TRAF6 with shRNA on LPS/TLR4 signaling in vitro

This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide (LPS)/toll-like receptor (TLR)-4 signaling pathway in vitro. Four plasmids (pGCsi-TRAF6-shRNA1, 2, 3, 4) containing different shRNA sequences were designed and synthesized. The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay. Inflammatory cellular models were established by LPS stimulation. Levels of TNF-alpha, IL-1beta and TGF-beta1 in the supernatants, mRNA expressions of TRAF6, IL-6 and COX-2, protein expression of TRAF6 and translocation of NF-kappaB were assayed by ELISA, real-time quantitative PCR and Western blotting, respectively. The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h. The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1, 2 groups than in the TRAF6-shRNA3, 4 groups. Therefore, pGCsi-TRAF6-shRNA1, 2 were selected for the subsequent experiments. Our results still showed that pGCsi-TRAF6-shRNA1, 2 could significantly reduce the production of pro-inflammatory cytokines and mediators including TNF-alpha, IL-1beta, IL-6 and COX-2, and inhibit NF-kappaB nuclear translocation. Moreover, pGCsi-TRAF6-shRNA1, 2 could suppress the release of TGF-beta1 at the protein level. It was concluded that the recombinant plasmid pTRAF6-shRNA can, to some extent, inhibit inflammatory response stimulated by LPS at the initial phase. TRAF6 may become the potential therapeutic target of many inflammation-related diseases.

MiR-10a Contributes to Retinoid Acid-induced Smooth Muscle Cell Differentiation

MicroRNAs (miRs) have been reported to play a critical role in muscle differentiation and function. The purpose of this study is to determine the role of miRs during smooth muscle cell (SMC) differentiation from embryonic stem cells (ESCs). MicroRNA profiling showed that miR-10a expression is steadily increased during in vitro differentiation of mouse ESCs into SMCs. Loss-of-function approaches using miR-10a inhibitors uncovered that miR-10a is a critical mediator for SMC lineage determination in our retinoic acid-induced ESC/SMC differentiation system. In addition, we have documented for the first time that histone deacetylase 4 is a novel target of miR-10a and mediates miR-10a function during ESC/SMC differentiation. To determine the molecular mechanism through which retinoic acid induced miR-10a expression, a consensus NF-kappaB element was identified in the miR-10a gene promoter by bioinformatics analysis, and chromatin immunoprecipitation assay confirmed that NF-kappaB could bind to this element. Finally, inhibition of NF-kappaB nuclear translocation repressed miR-10a expression and decreased SMC differentiation from ESCs. Our data demonstrate for the first time that miR-10a is a novel regulator in SMC differentiation from ESCs. These studies suggest that miR-10a may play important roles in vascular biology and have implications for the diagnosis and treatment of vascular diseases.

The Negative Co-Signaling Molecule B7-H4 Is Expressed by Human Bone Marrow–Derived Mesenchymal Stem Cells and Mediates its T-Cell Modulatory Activity

Though experimental evidence shows that human bone marrow-derived mesenchymal stem cells (hBMSCs) are able to suppress T-cell activation and proliferation, the precise mechanisms are still not completely understood. Here, we investigated the role of the negative costimulatory molecule B7-H4 in the immunosuppressive effect of hBMSCs on T-cell activation. We showed that B7-H4 expresses abundantly on hBMSCs assessed by reverse transcription, immunofluorescence staining, and flow cytometric analysis. Further studies demonstrated that B7-H4 expressed on hBMSCs inhibits T-cell activation and proliferation via induction of cell cycle arrest and inhibition of NF-kappaB nuclear translocation. Blocking B7-H4 would decrease the secretion of transforming growth factor-beta1 (TGF-beta1) in the supernatant of activated T cells co-cultured with hBMSCs. Addition of neutralizing antibodies against B7-H4 significantly attenuated the inhibitory effects of hBMSCs on T-cells. Thus, our study established the novel role of B7-H4 molecule in the suppressive effect of hBMSCs on T-cell activation and proliferation. Taken together, these results highlight the complex role of hBMSCs in regulating the immune response, asserting the possibility of their therapeutic application in transplantation, the treatment of graft-versus-host disease (GVHD), and autoimmune diseases.

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.

Pranlukast Attenuates Ischemia-like Injury in Endothelial Cells Via Inhibiting Reactive Oxygen Species Production and Nuclear Factor-κB Activation

The anti-inflammatory effects of pranlukast, an antagonist of cysteinyl leukotriene receptor 1, may be rendered not only by antileukotriene activity but also by other pharmacological activities. Previous studies indicate that pranlukast reduces ischemic tissue injury partially through decreasing vascular permeability, but its effect on ischemic injury in endothelial cells is not known. Thus, in this study, we investigated the effect of pranlukast on ischemia-like injury induced by oxygen-glucose deprivation (OGD) in EA.hy926 cells, a human endothelial cell line, and the possible mechanisms. We found that cell viability was reduced, lactate dehydrogenase release was increased 4-8 hours after OGD, and necrosis was induced 8 hours after OGD. Production of reactive oxygen species (ROS) increased by 211%, 176%, and 128%, respectively, 0.5, 1, and 2 hours after OGD. Nuclear factor-kappaB (NF-kappaB) was translocated to the nuclei 4-8 hours after OGD. Pranlukast ameliorated the reduced viability, the increased lactate dehydrogenase release, and necrosis after OGD. It also reduced ROS production and inhibited NF-kappaB nuclear translocation after OGD. The ROS scavenger, edaravone, inhibited OGD-induced nuclear translocation of NF-kappaB as well. Edaravone and pyrrolidine dithiocarbamate (a specific NF-kappaB inhibitor) protected endothelial cells from the OGD-induced injury. However, zileuton, a 5-lipoxygenase inhibitor, did not affect the cell injury, ROS production, and NF-kappaB nuclear translocation after OGD. The exogenous leukotriene D4 did not induce cell injury, ROS production, and NF-kappaB translocation. Thus, we conclude that pranlukast protects endothelial cells from ischemia-like injury via decreasing ROS production and inhibiting NF-kappaB activation, which is leukotriene independent.

Small molecule inhibitors of Hsp90 potently affect inflammatory disease pathways and exhibit activity in models of rheumatoid arthritis

To evaluate the ability of SNX-7081, a novel small molecule inhibitor of Hsp90, to block components of inflammation, including cytokine production, protein kinase activity, and angiogenic signaling. A close analog was evaluated in preclinical in vivo models of rheumatoid arthritis (RA). SNX-7081 binding to Hsp90 was characterized in Jurkat cells and RA synovial fibroblasts (RASFs). Inhibition of NF-kappaB nuclear translocation was evaluated in cellular systems, using lipopolysaccharide (LPS), tumor necrosis factor alpha, or interleukin-1beta stimulation. Suppression of cytokine production in THP-1 cells, human umbilical vein endothelial cells, and RASFs was studied. Disruption of MAPK signaling cascades by SNX-7081 following growth factor stimulation was assessed. SNX-7081 was tested in 2 relevant angiogenesis assays: platelet-derived growth factor activation of fibroblasts and LPS-induced nitric oxide (NO) release in J774 macrophages. A close analog, SNX-4414, was evaluated in rat collagen-induced arthritis and adjuvant-induced arthritis, following oral treatment. SNX-7081 showed strong binding affinity to Hsp90 and expected induction of Hsp70. NF-kappaB nuclear translocation was blocked by SNX-7081 at nanomolar concentrations, and cytokine production was potently inhibited. Growth factor activation of ERK and JNK signaling was significantly reduced by SNX-7081. NO production was also sharply inhibited. In animal models, SNX-4414 fully inhibited paw swelling and improved body weight. Scores for inflammation, pannus formation, cartilage damage, and bone resorption returned to normal. The present results demonstrate that a small molecule Hsp90 inhibitor can impact inflammatory disease processes. The strong in vivo efficacy observed with SNX-4414 provides preclinical validation for consideration of Hsp90 inhibitors in the treatment of RA.

Therapeutic efficacy of Kangen-karyu against H<SUB>2</SUB>O<SUB>2</SUB>-induced premature senescence

The anti-aging potential of Kangen-karyu extract was investigated using the mechanisms of the cellular aging model of stress-induced premature senescence (SIPS) in TIG-1 human fibroblasts. SIPS was induced by a sublethal dose of H2O2 and chronic oxidative stress with repeat treatment of low-dose H2O2. Reactive oxygen species generation, lipid peroxidation, and senescence-associated beta-galactosidase activity were elevated in TIG-1 cells under SIPS induced by H2O2. However, Kangen-karyu extract led to significant declines in these parameters, suggesting its role in ameliorating oxidative stress-related aging. It was also observed that SIPS due to H2O2 treatment led to the loss of cell viability, whereas Kangen-karyu extract improved cell viability by attenuating H2O2-induced oxidative damage. TIG-1 cells under the condition of SIPS caused by sublethal and chronic low doses of H2O2 showed nuclear factor-kappaB (NF-kappaB) translocation to the nucleus from the cytosol, while Kangen-karyu extract inhibited NF-kappaB nuclear translocation, implying that Kangen-karyu extract could exert an anti-aging effect through NF-kappaB modulation. In addition, treatment with Kangen-karyu extract under H2O2-induced chronic oxidative stress normalized the cell cycle by reducing the number of cells in the G0/G1 phase and elevating the proportion of those in the S phase, indicating the role of Kangen-karyu extract in cell cycle regulation. On the other hand, Kangen-karyu extract did not exert such an effect on cell cycle regulation under acute oxidative stress induced by sublethal H2O2. Furthermore, treatment with Kangen-karyu extract prolonged the lifespan of TIG-1 cells under SIPS. The present study suggests that Kangen-karyu might play a therapeutic role against the aging process caused by oxidative stress.

Prednisolone inhibits PDGF-induced nuclear translocation of NF-kappaB in human pulmonary artery smooth muscle cells.

Pulmonary vascular remodeling, a major cause for the elevated pulmonary vascular resistance in patients with pulmonary arterial hypertension (PAH), is partially due to increased proliferation of pulmonary arterial smooth muscle cells (PASMC) in the media, resulting in vascular wall thickening. Platelet-derived growth factor (PDGF) is a potent mitogen that may be involved in the progression of PAH. Blockade of PDGF receptors has been demonstrated to have therapeutic potential for patients with severe pulmonary hypertension. Prednisolone is an immunosuppressant shown to have anti-inflammatory and antiproliferative effects on PASMC. This study was designed to investigate whether PDGF and prednisolone affect human PASMC proliferation by regulating the nuclear translocation of NF-kappaB (a transcription factor composed of 2 subunits, p50 and p65). Treatment of human PASMC with PDGF (10 ng/ml) significantly increased nuclear translocation of p50 and p65 subunits. Inhibition of NF-kappaB activation or nuclear translocation of p50/p65 significantly attenuated PDGF-induced PASMC proliferation (determined by [(3)H]thymidine incorporation). In the presence of prednisolone (200 microM), the PDGF-induced nuclear translocation of p50 and p65 subunits was markedly inhibited (P < 0.05 vs. the cells treated with PDGF alone). These results indicate that PDGF-induced nuclear translocation of NF-kappaB may play an important role in stimulating PASMC proliferation (and/or enhancing PASMC survival), whereas prednisolone may exert anti-inflammatory and antiproliferative effects on PASMC by inhibiting NF-kappaB nuclear translocation.

Anti-inflammatory activity of chondroitin sulfate

Osteoarthritis is primarily characterized by areas of destruction of articular cartilage and by synovitis. Articular damage and synovitis are secondary to local increase of pro-inflammatory cytokines (interleukin-1beta and tumor necrosis factor-alpha), enzymes with proteolytic activity (matrix metalloproteinases), and enzymes with pro-inflammatory activity (cyclooxygenase-2 and nitric oxide synthase-2). Enhanced expression of these proteins in chondrocytes and in synovial membrane appears associated to the activation and nuclear translocation of nuclear factor-kappaB (NF-kappaB). Chondroitin sulfate (CS) prevents joint space narrowing and reduces joint swelling and effusion. To produce these effects, CS elicits an anti-inflammatory effect at the chondral and synovial levels. CS and its disaccharides reduce NF-kappaB nuclear translocation, probably by diminishing extracellular signal-regulated kinase1/2, p38mitogen-activated protein kinase and c-Jun N-terminal kinase activation. This review discusses the evidence supporting that CS pleiotropic effects in chondrocytes and synoviocytes are primarily due to a common mechanism, e.g., the inhibition of NF-kappaB nuclear translocation.

ATL‐1, an analogue of aspirin‐triggered lipoxin A4, is a potent inhibitor of several steps in angiogenesis induced by vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is the most important proangiogenic protein. We have demonstrated that ATL-1, a synthetic analogue of aspirin-triggered lipoxin A(4), inhibits VEGF-induced endothelial cell (EC) migration. In the present study, we investigated the effects of ATL-1 in several other actions stimulated by VEGF. Human umbilical vein ECs were treated with ATL-1 for 30 min before stimulation with VEGF. Cell proliferation was measured by thymidine incorporation. Adherent cells were determined by fluorescence intensity using a Multilabel counter. Expression and activity of matrix metalloproteinases (MMP) were analysed by western blot and zymography. ATL-1 inhibited EC adhesion to fibronectin via interaction with its specific receptor. Furthermore, VEGF-induced MMP-9 activity and expression were reduced by pretreatment with ATL-1. Because the transcription factor NF-kappaB has been implicated in VEGF-mediated MMP expression and EC proliferation, we postulated that ATL-1 might modulate the NF-kappaB pathway and, indeed, ATL-1 inhibited NF-kappaB nuclear translocation. Pretreatment of EC with ATL-1 strongly decreased VEGF-dependent phosphorylation of phosphainositide 3-kinase (PI3-K) and extracellular signal-regulated kinase-2 (ERK-2), two signalling kinases involved in EC proliferation. Inhibition of VEGF-induced EC proliferation by ATL-1 was antagonized by sodium orthovanadate, suggesting that this inhibitory activity was mediated by a protein tyrosine phosphatase. This was confirmed by showing that ATL-1 inhibition of VEGF receptor-2 (VEGFR-2) phosphorylation correlates with SHP-1 association with VEGFR-2. The synthetic 15-epi-lipoxin analogue, ATL-1, is a highly potent molecule exerting its effects on multiple steps of the VEGF-induced angiogenesis.

Up-Regulation and Increased Activity of KV3.4 Channels and Their Accessory Subunit MinK-Related Peptide 2 Induced by Amyloid Peptide Are Involved in Apoptotic Neuronal Death

The aim of the present study was to investigate whether K(V)3.4 channel subunits are involved in neuronal death induced by neurotoxic beta-amyloid peptides (Abeta). In particular, to test this hypothesis, three main questions were addressed: 1) whether the Abeta peptide can up-regulate both the transcription/translation and activity of K(V)3.4 channel subunit and its accessory subunit, MinK-related peptide 2 (MIRP2); 2) whether the increase in K(V)3.4 expression and activity can be mediated by the nuclear factor-kappaB (NF-kappaB) family of transcriptional factors; and 3) whether the specific inhibition of K(V)3.4 channel subunit reverts the Abeta peptide-induced neurodegeneration in hippocampal neurons and nerve growth factor (NGF)-differentiated PC-12 cells. We found that Abeta(1-42) treatment induced an increase in K(V)3.4 and MIRP2 transcripts and proteins, detected by reverse transcription-polymerase chain reaction and Western blot analysis, respectively, in NGF-differentiated PC-12 cells and hippocampal neurons. Patch-clamp experiments performed in whole-cell configuration revealed that the Abeta peptide caused an increase in I(A) current amplitude carried by K(V)3.4 channel subunits, as revealed by their specific blockade with blood depressing substance-I (BDS-I) in both hippocampal neurons and NGF-differentiated PC-12 cells. The inhibition of NF-kappaB nuclear translocation with the cell membrane-permeable peptide SN-50 prevented the increase in K(V)3.4 protein and transcript expression. In addition, the SN-50 peptide was able to block Abeta(1-42)-induced increase in K(V)3.4 K(+) currents and to prevent cell death caused by Abeta(1-42) exposure. Finally, BDS-I produced a similar neuroprotective effect by inhibiting the increase in K(V)3.4 expression. As a whole, our data indicate that K(V)3.4 channels could be a novel target for Alzheimer's disease pharmacological therapy.

MAP kinase subtypes and Akt regulate diosgenin-induced apoptosis of rheumatoid synovial cells in association with COX-2 expression and prostanoid production

In the present study, we investigated the signalling pathways involved in diosgenin-induced apoptosis in human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) in vitro with particular interest on Akt and MAPKs activation in relation to arachidonic acid metabolism via COX-2 pathway. MAPK activation was measured by ELISA quantification in diosgenin-treated human RA FLS. Expression of Akt and phospho-Akt was analyzed by Western blot analysis. Nuclear factor-kappaB (NF-kappaB) translocation was evaluated by electromobility shift assay. The prostanoid production (COX-2 activity) was measured by quantitative ELISA. Diosgenin-induced apoptosis in the presence of MAPK or Akt inhibitors was detected by a quantitative determination of DNA fragmentation. Treatment of human RA FLS with 40 microM diosgenin caused an activation of p38 and JNK and an inhibition of ERK phosphorylation. Akt and NF-kappaB are potentially required for diosgenin-induced apoptosis in human RA FLS because 40 microM diosgenin abrogated Akt phosphorylation which correlated with an inhibition of NF-kappaB nuclear translocation. SB203580 and SP600125 (p38 and JNK inhibitors) reduced diosgenin-induced DNA fragmentation whereas U0126 and LY294002 (MEK and PI3 kinase/Akt inhibitors) caused an amplification of proapoptotic effect of diosgenin. Diosgenin increased COX-2 activity resulting in PGE2 and 6-keto-PGF1alpha overproduction in human RA FLS. All MAPK inhibitors markedly reduced diosgenin-induced PGE2 and 6-keto-PGF1alpha synthesis except for SP600125 on 6-keto-PGF1alpha production. These results provide, for the first time, strong evidence that a combined association implicating a MEK inhibitor (U0126) and diosgenin is the most effective in inducing very strong apoptosis with down-regulation of COX-2 expression and activity in human RA FLS.

RelA is required for IL-1β stimulation of Matrix Metalloproteinase-1 expression in chondrocytes

Interleukin-1beta (IL-1beta) stimulates collagenase-1 (Matrix Metalloproteinase-1 (MMP-1)) expression in articular chondrocytes, leading to cleavage of type II collagen and irreversible cartilage degradation. The nuclear factor-kappa B (NF-kappaB) pathway is potently activated in IL-1beta-stimulated cells and has been implicated as an intermediate in MMP-1 gene expression. However, the roles of individual NF-kappaB family members during IL-1beta-induced MMP-1 gene expression have not been defined. To address the relationship between the NF-kappaB pathway and MMP-1 gene activation in chondrocytes, primary cultured human articular chondrocyte cultures (HAC) and SW-1353 cells were stimulated with IL-1beta over a 24-h time course and MMP-1, NF-kappaB1, NF-kappaB2 and RelA gene expression was assayed. IL-1beta-induced MMP-1 expression was comparable in HAC and SW-1353 cells both temporally and quantitatively. MMP-1 gene expression was mirrored by increases in NF-kappaB gene expression, and inhibition of NF-kappaB nuclear translocation with dominant-negative IkappaBalpha reduced IL-1beta-dependent MMP-1 gene expression. IL-1beta activated the NF-kappaB pathway in chondrocytes, both through phosphorylation and transient degradation of IkappaBalpha, as well as through sustained phosphorylation of RelA. Small inhibitory RNAs (siRNA) specific for RelA resulted in significant reduction of MMP-1 mRNA, whereas siRNA for NF-kappaB1 and NF-kappaB2 augmented IL-1beta-induced MMP-1 expression. Our data demonstrate that IL-1beta activation of the NF-kappaB pathway is required for IL-1beta induction of MMP-1 in chondrocytes and that RelA can work independently of NF-kappaB1 or NF-kappaB2 to activate this gene expression program.

Aspirin induces apoptosis in oesophageal cancer cells by inhibiting the pathway of NF‐kappaB downstream regulation of cyclooxygenase‐2

Aspirin has potential in the prevention or treatment of oesophageal cancer, the seventh most common cancer in the world, but its mechanism of action is still not certain. The oesophageal squamous cell carcinoma cell line TE-13 was cultured with aspirin at different concentrations or for different times. Proliferation and apoptosis were measured by MTT reduction and flow cytometry. 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. Aspirin significantly inhibited cell proliferation and induced apoptosis at concentrations of 1, 4, 8 mmol/L. Aspirin dose-dependently decreased the levels of COX-2 mRNA, COX-2 protein and nuclear NF-kappaB protein and increased the cytoplasmic IkappaB protein. We conclude that aspirin 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. These results support the further investigation of the cyclooxygenase pathway in investigating the potential of aspirin and similar drugs in cancer prevention and therapy.

N-Substituted 4-Aminobenzamides (Procainamide Analogs): An Assessment of Multiple Cellular Effects Concerning Ion Trapping

Procainamide and related triethylamine-substituted 4-aminobenzamides, such as metoclopramide and declopramide, exert cellular effects potentially exploitable in oncology at millimolar concentrations (DNA demethylation, nuclear factor-kappaB inhibition, apoptosis) and display anti-inflammatory properties. However, these drugs induce massive cell vacuolization at similar concentrations, a response initiated by vacuolar (V-) ATPase-dependent ion trapping into and osmotic swelling of acidic organelles. We have examined whether this overlooked response might be related to the effects on cell proliferation and viability using cultured vascular smooth muscle cells and tumor-derived cell lines (Morris 7777 hepatoma, HT-1080 fibrosarcoma). Giant vacuole formation, of confirmed trans-Golgi origin (labeled with C5-ceramide, p230, golgin-97), is a cellular response to all tested amines in the series (> or = 2.5 mM), including triethylamine. These drugs and the V-ATPase inhibitor bafilomycin A1 inhibited smooth muscle cell proliferation, suggesting that acidification of a cellular compartment is essential to cell division. The cytotoxicity was maximal with metoclopramide, and this effect was minimally influenced by bafilomycin A1; furthermore, metoclopramide (2.5 mM) induced apoptosis in tumor cells as judged by poly(ADP-ribose)polymerase (PARP) cleavage. Triethylamine and procainamide exhibit a low level of cytotoxicity variably reduced by bafilomycin co-treatment. In Morris cells, the secretion of alpha-fetoprotein is inhibited by amines, consistent with the impairment of the secretory pathway. The most highly substituted 4-aminobenzamides are significant NF-kappaB inhibitors in smooth muscle cells. Although some effects of 4-aminobenzamides are independent of V-ATPase-driven ion trapping (inhibition of NF-kappaB nuclear translocation, agent-specific cytotoxicity, PARP cleavage), other effects are dependent on this phenomenon (vacuolization, a component of the cytotoxicity, inhibition of secretion).

Combination gene therapy for glioblastoma involving herpes simplex virus vector-mediated codelivery of mutant IκBα and HSV thymidine kinase

To improve the effectiveness of herpes simplex virus (HSV) thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy, the replication-defective HSV vector TOIkappaB expressing both HSV-TK and a mutant form of the NF-kappaB inhibitor IkappaBalpha (IkappaBalphaM) was developed. TOIkappaB was constructed by recombining the IkappaBalphaM gene into the U(L)41 locus of a replication-defective lacZ expression vector, TOZ.1. Expression of IkappaBalphaM was confirmed by Western blotting, and the ability of the mutant protein to inhibit NF-kappaB nuclear translocation was examined by electrophoretic mobility shift assay. In human glioblastoma U-87MG cells, the p50/p50 dimer of NF-kappaB was already translocated to the nucleus without receptor-dependent signaling by TNF-alpha. Following infection with TOIkappaB, nuclear translocation of NF-kappaB in U-87MG cells was significantly inhibited and caspase-3 activity increased compared with TOZ.1-infected cells. The cytotoxicity of TOIkappaB for U-87MG cells was investigated by colorimetric MTT assay. At an MOI of 3, TOIkappaB infection killed 85% of the cells compared to 20% killed by TOZ.1 infection. In the presence of GCV, these numbers increased to 95-100% for TOIkappaB and 80-85% for TOZ.1. TOIkappaB neurotoxicity measured on cultured murine neurons was relatively low and similar to that of TOZ.1. The survival of nude mice implanted into the brain with U-87MG tumor cells was markedly prolonged by intratumoral TOIkappaB injection and GCV administration. Survival of TOIkappaB+GCV group was significantly longer (P<.02, Wilcoxon test) than for the control groups (TOZ.1 or TOIkappaB only, PBS or PBS+GCV). These results suggest that IkappaBalphaM expression may be a safe enhancement of replication-defective HSV-based suicide gene therapy in vitro and in vivo.

Corticosterone modulates noradrenaline‐induced melatonin synthesis through inhibition of nuclear factor kappa B

In chronically inflamed animals, adrenal hormones exert a positive control on the secretion of melatonin by the pineal gland. In this paper, the mechanism of corticosterone as a modulator of melatonin and N-acetylserotonin (NAS) was determined. Rat pineal glands in culture, stimulated for 5 hr with noradrenaline (10 nm), were previously incubated with corticosterone (1.0 nm-1.0 microm) for 48 hr in the presence or absence of the glucocorticoid receptor (GR) antagonist, mifepristone (1.0 microm), the proteasome inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN, 12.5 microm) or the antagonist of the nuclear factor kappa B (NFkappaB), pyrrolidinedithiocarbamate (PDTC, 12.5 microm). Corticosterone potentiated noradrenaline-induced melatonin and NAS production in a bell-shaped manner. The increase in NAS (12.9 +/- 2.7, n=6 versus 34.3 +/- 8.3 ng per pineal) and melatonin (16.3 +/- 2.0, n=6 versus 44.3 +/- 12.9 ng per pineal) content induced by 1 microm corticosterone was blocked by mifepristone, and mimicked by ALLN and PDTC. The presence of GRs was shown by [3H]-dexamethasone binding (0.30 +/- 0.09 pmol/mg protein) and corticosterone inhibition of NFkappaB nuclear translocation was demonstrated by electromobility shift assay. Therefore, corticosterone potentiates noradrenaline-induced melatonin and NAS production through GR inhibition of NFkappaB nuclear translocation. To the best of our knowledge, this is the first time that this relevant pathway for passive and acquired immune response is shown to modulate melatonin production in pineal gland.

Fatty Acid-induced Insulin Resistance in L6 Myotubes Is Prevented by Inhibition of Activation and Nuclear Localization of Nuclear Factor κB

Recent studies have implicated inhibitor of kappaB kinase (IKK) in mediating fatty acid (FA)-induced insulin resistance. How IKK causes these effects is unknown. The present study addressed the role of nuclear factor kappaB (NFkappaB), the distal target of IKK activity, in FA-induced insulin resistance in L6 myotubes, an in vitro skeletal muscle model. A 6-h exposure of myotubes to the saturated FA palmitate reduced insulin-stimulated glucose uptake by approximately 30%, phosphatidylinositol-3 kinase and protein kinase B phosphorylation by approximately 40%, and stimulated inhibitor of kappaBalpha degradation and the nuclear translocation of NFkappaB. On the other hand, the Omega-3 polyunsaturated FA linolenate neither induced insulin resistance nor promoted nuclear localization of NFkappaB. Supporting the hypothesis that IKK acts through NFkappaB to cause insulin resistance, the IKK inhibitors acetylsalicylate and parthenolide prevented FA-induced reductions in insulin-stimulated glucose uptake and NFkappaB nuclear translocation. Most importantly, NFkappaB SN50, a cell-permeable peptide that inhibits NFkappaB nuclear translocation downstream of IKK, was sufficient to prevent palmitate-induced reductions in insulin-stimulated glucose uptake. Acetylsalicylate, but not NFkappaB SN50, prevented FA effects on phosphatidylinositol-3 kinase activity and protein kinase B phosphorylation. We conclude that FAs induce insulin resistance and activates NFkappaB in L6 cells. Furthermore, inhibition of NFkappaB activation, indirectly by preventing IKK activation or directly by inhibiting NFkappaB nuclear translocation, prevents the detrimental effects of palmitate on the metabolic actions of insulin in L6 myotubes.

NF-κB Site Interacts with Sp Factors and Up-regulates the NR1 Promoter during Neuronal Differentiation

The NR1 gene undergoes induction in neurogenesis mainly via promoter de-repression, and up-regulation during neuronal differentiation by undefined mechanism(s). Here, we show that in the distal region the NR1 promoter has an active NF-kappaB site sharing the consensus with the immunoglobulin (Ig)/human immunodeficiency virus NF-kappaB site. Mutation of this site significantly reduced NR1 promoter up-regulation during neuronal differentiation of P19 cells. Electrophoretic mobility shift assays revealed that P19 nuclei constitutively contained p50 and that neuronal differentiation not only increased nuclear p50 but also induced p65 nuclear translocation. Responding to this change was an up-regulation of NF-kappaB-dependent promoter activity. However, inhibition of NF-kappaB nuclear translocation by an IkappaBalpha super-repressor or decoy DNA only moderately inhibited NR1 promoter up-regulation. Interestingly, the NR1 NF-kappaB site strongly interacted with Sp3/Sp1, instead of NF-kappaB factors, in P19 nuclear extracts. This interaction was reduced for Sp3 following neuronal differentiation, accompanied by dynamic expression of Sp factors. Cotransfection of Sp factors (Sp1, 3, or 4) upregulated the NR1 NF-kappaB site dramatically in differentiated neurons, but only moderately in undifferentiated P19 cells. This up-regulation was strong for Sp1 in differentiated cells and for Sp3 in undifferentiated cells. Chromatin-immunoprecipitation assays further demonstrated that Sp1 and Sp3 interacted with the NR1 NF-kappaB site in situ, and Sp3 lost its interaction after neuronal differentiation. We conclude that the NF-kappaB site positively regulates the NR1 promoter during neuronal differentiation via interacting mainly with Sp factors and neuronal differentiation reduces the effect of Sp3 factor on this site.

Effects of chrisotherapeutic gold compounds on prostaglandin E2 production.

The mechanism of action of anti-rheumatic gold compounds on 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced prostaglandin E(2) (PGE(2)) production in rat peritoneal macrophages were examined. Auranofin (AF) at 3-10 muM inhibited TPA-induced PGE(2) production in a concentration-dependent manner. In the pharmacological experiments, prostaglandin G/H synthase (PGHS)-2-dependent PGE(2) production was inhibited by 10 muM of AF. The enzyme activities of both PGHS-1 and PGHS-2 were not affected by the 10 muM AF. Other gold compounds, aurothioglucose (ATG) and aurothiomalate (ATM) did not inhibit PGE2 production at 10 muM. AF decreased the PGHS-2 protein content, but had no effect on the PGHS-1 protein content. AF at 3-10 muM decreased the PGHS-2 messenger RNA (mRNA) level by RT-PCR determination. Then, the effect of AF on nuclear factor kappa B (NF-kappaB), one of the transcription factors known to regulate transcription of a group of proinflammatory proteins, was determined. AF at 1-10 muM inhibited nuclear translocation of NF-kappaB in a concentration-dependent manner. ATG and ATM at 10 muM did not inhibit NF-kappaB nuclear translocation, but with 20 h preincubation, ATG and ATM inhibited PGE(2) production and NF-kappaB nuclear translocation. AF, ATG, and ATM did not affect the binding of NF-kappaB to its specific DNA. These observations may suggest that the effects of gold compounds on the inhibition of NF-kappaB nuclear translocation plays one of the major role in its anti-inflammatory effects in rat peritoneal macrophages.