<b>BACKGROUNDb>Nuclear factor kappaB (NF-kappaB) overactivation, requiring phosphorylation and degradation of its inhibitor IkappaBalpha, is the basis for chronicity of airway inflammation in asthma. Based on our previous plasmid pShuttle-IkappaBalpha, carrying an IkappaBalpha gene from human placenta, we optimized a novel IkappaBalpha mutant (IkappaBalphaM) gene, constructed and characterized its replication-deficient recombinant adenovirus (AdIkappaBalphaM), and tested whether AdIkappaBalphaM-mediated overexpression of IkappaBalphaM could inhibit the NF-kappaB activation in endothelial cells.

<b>METHODSb>IkappaBalphaM gene (203 - 1003 bp) encoding 267 amino acids, acquired by site-directed deleting N-terminal phosphorylation sites of serine 32/36, was subcloned into the pShuttle and pGEM-T vectors for further polymerase chain reaction (PCR), restriction digestion, deoxyribonucleic acid (DNA) sequencing and homology analyses. Subsequent to inserting the expression unit of pShuttle-IkappaBalphaM, containing cytomegalovirus (CMV) promoter, IkappaBalphaM complementary DNA (cDNA) and polyadenylic acid (PolyA) signals, into the type 5 adenovirus (Ad5) vector, the resultant AdIkappaBalphaM was packaged in human embryonic kidney (HEK) 293 cells by cotransfection with lipofectamine. Western blot analysis and electrophoretic mobility shift assay were utilized to detect the AdIkappaBalphaM-mediated overexpression of IkappaBalphaM in HEK293 cells and its suppressive effect on phorbol 12-myristate 13-acetate (PMA)-induced NF-kappaB activation in human umbilical vein endothelial (ECV304) cells, respectively.

<b>RESULTSb>The relevant nucleotides and deduced amino acids of 801 bp IkappaBalphaM gene were consistent with those of IkappaBalpha gene (GenBank accession number: M69043). The titer of the prepared AdIkappaBalphaM was 4.0 x 10 (12) plaque-forming units (pfu)/L. Moreover, the IkappaBalphaM gene was overexpressed in HEK293 cells, and potently inhibited the PMA-induced NF-kappaB activation in ECV304 cells dose-dependently.

<b>CONCLUSIONSb>AdIkappaBalphaM is a novel vector for both efficient transfer and specific overexpression of IkappaBalphaM gene, as well as potent inhibition of NF-kappaB activity, providing a promising strategy for gene therapy of asthma.

Adenoviridae ; genetics ; Cell Line ; Endothelial Cells ; metabolism ; Genetic Therapy ; Humans ; I-kappa B Proteins ; genetics ; Mutation ; NF-KappaB Inhibitor alpha ; NF-kappa B ; antagonists & inhibitors ; Tetradecanoylphorbol Acetate ; pharmacology

Berberine (BBR) is an isoquinoline alkaloid extracted from Rhizoma coptidis and has been used for treating type 2 diabetes mellitus (T2DM) in China. The development of T2DM is often associated with insulin resistance and impaired glucose uptake in peripheral tissues. In this study, we examined whether BBR attenuated glucose uptake dysfunction through the cholinergic anti-inflammatory pathway in HepG2 cells. Cellular glucose uptake, quantified by the 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-2-deoxy-D-glucose (2-NBDG), was inhibited by 21% after HepG2 cells were incubated with insulin (10(-6) mol/L) for 36 h. Meanwhile, the expression of alpha7 nicotinic acetylcholine receptor (α7nAChR) protein was reduced without the change of acetylcholinesterase (AChE) activity. The level of interleukin-6 (IL-6) in the culture supernatant, the ratio of phosphorylated I-kappa-B kinase-β (IK&kappa;β) Ser181/IKKβ and the expression of nuclear factor-kappa B (NF-&kappa;B) p65 protein were also increased. However, the treatment with BBR enhanced the glucose uptake, increased the expression of α7nAChR protein and inhibited AChE activity. These changes were also accompanied with the decrease of the ratio of pIKKβ Ser181/IKKβ, NF-&kappa;B p65 expression and IL-6 level. Taken together, these results suggest that BBR could enhance glucose uptake, and relieve insulin resistance and inflammation in HepG2 cells. The mechanism may be related to the cholinergic anti-inflammatory pathway and the inhibition of AChE activity.
Berberine ; pharmacology ; Glucose ; metabolism ; Hep G2 Cells ; Humans ; Hypoglycemic Agents ; pharmacology ; I-kappa B Kinase ; metabolism ; I-kappa B Proteins ; metabolism ; Insulin ; metabolism ; Insulin Resistance ; Interleukin-6 ; metabolism ; Transcription Factor RelA ; metabolism ; alpha7 Nicotinic Acetylcholine Receptor ; genetics ; metabolism

<b>OBJECTIVEb>To investigate the role of NF-kappaB/IkappaB signal pathway in mediating the expression of monocyte chemoattractant protein-1 (MCP-1) in experimental rat glomerulonephritis.

<b>METHODSb>Nephrotoxic serum nephritis (NTN) was induced by injection of anti-GBM antibody into the tail veins of rats. Electrophoretic mobility shift assay (EMSA) and Western Blot were used to detect the activation of NF-kappaB, nuclear translocation of p65 subunit and degradation of IkappaBalpha and IkappaBbeta in rat renal tissue. MCP-1 expression in glomeruli and renal tubules was also assessed by immunohistochemistry and ribonuclease protection assay. This was further correlated with the activation of NF-kappaB.

<b>RESULTSb>There was an obvious expression of MCP-1 in glomeruli and renal tubules. Significant up-regulation of NF-kappaB activation, nuclear translocation of p65 subunit, and degradation of IkappaBalpha and IkappaBbeta were also observed in NTN rat renal tissue, as compared to the control group. A positive correlation was noted between NF-kappaB activation and MCP-1 expression.

<b>CONCLUSIONSb>NF-kappaB/IkappaB signal pathway may play an important pathogenetic role in glomerulonephritis, with mediating the expression of MCP-1.

Animals ; Blotting, Western ; Chemokine CCL2 ; genetics ; metabolism ; Glomerulonephritis ; chemically induced ; genetics ; metabolism ; I-kappa B Proteins ; metabolism ; Kidney Glomerulus ; metabolism ; pathology ; Kidney Tubules ; metabolism ; pathology ; Male ; NF-kappa B ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

<b>OBJECTIVEb>To observe the histopathological features, nuclear factor-&kappa;B (NF&kappa;B) and IKB expressions as well as calcium deposition of atherosclerosis plaques (AS) in apolipoprotein E (ApoE) and low density lipoprotein receptor (LDLR) knockout mice (ApoE(-/-), LDLR(-/-)fed high-fat diet.

<b>METHODSb>Eight C57BL/6J mice fed with normal diet were used as control, 32 ApoE(-/-) mice and LDLR(-/-) mice were divided into normal diet and high-fat diet groups (n = 8 each). After 4 months, aorta was collected for morphologic (HE, Oil Red O, Von Kossa) and immunohistochemistry (nuclear factor-&kappa;B, IKB, macrophage surface molecule-3, α-smooth action protein) analysis.

<b>RESULTSb>Degree of AS in ApoE(-/-) and LDLR(-/-) mice fed with high-fat diet were significantly severer than those fed with normal diet and AS was more significant in ApoE(-/-) mice than in LDLR(-/-) mice. NF&kappa;B and IKB expressions in high-fat diet group were significantly higher than the normal diet group (P < 0.05). Double-labeling of NF&kappa;B revealed dominant expression in smooth muscle cells. Calcium deposition was significantly more in ApoE(-/-) mice fed with high-fat diet than mice fed with normal diet (P < 0.05) and was similar in LDLR(-/-) mice fed with high and normal diet (P > 0.05).

<b>CONCLUSIONb>High-fat diet contributes to the formation of AS plagues in ApoE(-/-) and LDLR(-/-) mice joined by upregulated NF&kappa;B and IKB expressions and calcium deposition.

Animals ; Apolipoproteins E ; genetics ; metabolism ; Calcium ; metabolism ; Female ; I-kappa B Proteins ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; NF-kappa B ; metabolism ; Plaque, Atherosclerotic ; metabolism ; pathology ; Receptors, LDL ; genetics ; metabolism

The inhibitive effects of all-trans retinoic acid (ARTA) on airway inflammation in asthmatic rats and its mechanism on the basis of the regulation of nuclear factor kappaB (NF-kappaB) were explored. Thirty-two SD rats were randomly divided into 4 groups: control group, asthma group, dexamethasone treatment group and retinotic acid treatment group. The total and differential cell counts in the collected bronchoalveolar lavage fluid (BALF) were measured. The pathological changes in lung tissues were estimated by scoring. The expression of NF-kappaB inhibitor (IkappaBa), NF-kappaB, intercellular adhering molecule-1 (ICAM-1) in lung tissue was detected by immunohistochemical method. The results showed that in the two treatment groups, the total cell counts and proportion of inflammatory cells in BALF were significantly reduced, but there was no significant difference in differential cell counts in BALF between them. The pathological changes in lung tissues in the treatment groups were significantly attenuated as compared with asthma group. Except the epithelial injury in retinotic acid treatment group was milder than in dexamethasone treatment group, the remaining lesions showed no significant difference between them. In the two treatment groups, the expression of IkappaBa was increased, while the expression of NF-kappaB and ICAM-1 decreased with the difference between the two groups being not significant. It was concluded that the similar anti-inflammatory effects and mechanism of ATRA on airway in asthmatic rats to those of dexamethasone were contributed to the increase of cytoplasmic IkappaBa content and suppression of NF-kappaB activation and expression.
Animals ; Asthma ; metabolism ; pathology ; Bronchoalveolar Lavage Fluid ; cytology ; Dexamethasone ; pharmacology ; I-kappa B Proteins ; biosynthesis ; genetics ; Inflammation ; Intercellular Adhesion Molecule-1 ; biosynthesis ; genetics ; Male ; NF-kappa B ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Respiratory System ; Tretinoin ; pharmacology

<b>OBJECTIVEb>To investigate the inhibition consequence of NF-kappaB activity and cell viability by transfecting mutated inhibitor kappa B alpha (mI(kappa)B(alpha)) into liver cancer cell line of SMMC-7721 cells.

<b>METHODSb>The nucleic proteins of SMMC-7721 cells transfected with mI(kappa)B(alpha) plasmid and cells with empty pcDNA3 vector were used to determine not only the binding of the 32P-labelled kappaB probes by EMSA, but also the expression of NF-kappaB by western blot. Cell viability was also analyzed.

<b>RESULTSb>NF-kappaB nuclear translocation was inhibited remarkably in SMMC-7721 cells transfected with mI(kappa)B(alpha) at 0, 24, 48 and 96 hours. Furthermore, NF-kappaB was not detected in the nucleic protein of mI(kappa)B(alpha) -transfected cells at the same intended time by western blot. Compared with that of control cells, the growth of SMMC-7721 cells transfected with mI(kappa)B(alpha) was suppressed evidently, especially on the second day, the cpm values of mI(kappa)B(alpha) -transfected cells, pcDNA3-transfected cells, and control cells were 5,092.63+/-541.41, 7,851.87+/-72.76, and 8,240.8+/-603.26 respectively (t = 14.29, P<0.01; t = 10.99, P<0.01).

<b>CONCLUSIONb>Stable expression of mI(kappa)B(alpha) in SMMC-7721 cells transfected with mI(kappa)B(alpha) plasmid inhibits NF-kappaB nuclear translocation, then suppresses the cell growth.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Division ; Cell Line, Tumor ; Humans ; I-kappa B Proteins ; biosynthesis ; genetics ; physiology ; Liver Neoplasms ; metabolism ; pathology ; Mutation ; NF-KappaB Inhibitor alpha ; NF-kappa B ; antagonists & inhibitors ; physiology ; Transfection ; Translocation, Genetic

Human immunodeficiency virus type 1 (HIV-1) transcription is a crucial step in the viral replication cycle, which is considered to be a potential target for inhibition of HIV-1 replication. Among the factors involved in this step, the cellular protein nuclear factor NF-kappaB is the most powerful inducer of HIV-1 transcription. HIV-1 transcription is initiated by the binding of NF-kappaB to the enhancer region in the long terminal repeat (LTR) of HIV-1. Several compounds suppress HIV-1 transcription through the inhibition of NF-kappaB activation. The mechanisms of NF-kappaB in the transcription of HIV-1 and progress of the current inhibitors of NF-kappaB are reviewed.
Anti-HIV Agents ; pharmacology ; HIV Long Terminal Repeat ; HIV-1 ; genetics ; Humans ; I-kappa B Kinase ; metabolism ; I-kappa B Proteins ; metabolism ; NF-KappaB Inhibitor alpha ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Nicotinic Acids ; pharmacology ; Nitriles ; pharmacology ; Transcription, Genetic ; drug effects ; Virus Replication

<b>OBJECTIVEb>To investigate the role of TNF receptor-associated factor 2 (TRAF-2) and TRAF6 in CD40-induced nuclear factor-kappaB (NF-kappaB) signaling pathway and whether CD40 signaling requires TRAF2.

<b>METHODSb>Human B cell lines were transfected with plasmids expressing wild type TRAF2 or dominant negative TRAF2, TRAF2-shRNA, or TRAF6-shRNA. The activation of NF-kappaB was detected by Western blot, kinase assay, transfactor enzyme-linked immunosorbent assay (ELISA), and fluorescence resonance energy transfer (FRET). Analysis of the role of TRAF-2 and TRAF-6 in CD40-mediated NF-kappaB activity was examined following stimulation with recombinant CD154.

<b>RESULTSb>TRAF2 induced activity of IkappaB-kinases (IKKalpha, IKKi/epsilon), phosphorylation of IkappaBalpha, as well as nuclear translocation and phosphorylation of p65/RelA. In contrast, TRAF6 strongly induced NF-kappaB activation and nuclear translocation of p65 as well as p50 and c-Rel. Engagement of CD154-induced nuclear translocation of p65 was inhibited by a TRAF6-shRNA, but conversely was enhanced by a TRAF2-shRNA. Examination of direct interactions between CD40 and TRAFs by FRET documented that both TRAF2 and TRAF6 directly interacted with CD40. However, the two TRAFs competed for CD40 binding.

<b>CONCLUSIONSb>These results indicate that TRAF2 can signal in human B cells, but it is not essential for CD40-mediated NF-kappaB activation. Moreover, TRAF2 can compete with TRAF6 for CD40 binding, and thereby limit the capacity of CD40 engagement to induce NF-kappaB activation.

Animals ; B-Lymphocytes ; cytology ; physiology ; CD40 Antigens ; metabolism ; Cell Line ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Fluorescence Resonance Energy Transfer ; Humans ; I-kappa B Kinase ; metabolism ; NF-kappa B ; genetics ; metabolism ; Proto-Oncogene Proteins c-fos ; metabolism ; Signal Transduction ; physiology ; TNF Receptor-Associated Factor 2 ; genetics ; metabolism ; TNF Receptor-Associated Factor 6 ; genetics ; metabolism ; Transcription Factor RelA ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-kappaB (p65) in Caco-2 cells. However, IkappaB, an inhibitor of NF-kappaB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-kappaB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-kappaB and STATs in colonic epithelial cells, which ultimately accelerates cell death.
Caco-2 Cells ; Calcium-Binding Proteins ; Calpain/genetics/metabolism ; Caspase 3/genetics/metabolism ; Caspases ; *Cell Death ; Colon/cytology ; Entamoeba histolytica/*physiology ; Epithelial Cells/cytology/parasitology ; Humans ; I-kappa B Proteins/metabolism ; Intestinal Mucosa/cytology ; NF-kappa B/genetics/*metabolism ; RNA Interference ; RNA, Small Interfering ; STAT3 Transcription Factor/genetics/*metabolism ; STAT5 Transcription Factor/genetics/*metabolism ; Signal Transduction

<b>OBJECTIVEb>To investigate the efficacy of transfection with a mutant IkappaBalpha gene (mIkappaBalpha) in enhancing the radiosensitivity of hepatocellular carcinoma cells.

<b>METHODSb>The hepatocellular carcinoma Hep G2 cells were divided into 3 group and transfected with the adenovirus containing mIkappaBalpha vector (Ad-mIkappaBalpha group), Ad vector (Adv group), or without treatment (parental control cells). Before and after irradiation of the cells with 6 Gy high-energy X ray, Western blotting was performed to measure the expression level of IkappaBalpha in the cytoplasm, and electrophoresis mobility shift assay (EMSA) carried out to evaluate the nuclear factor-kappaB (NF- kappa;B) activity in the cell nuclei, with the cell apoptosis detected using TUNEL assay. The radiosensitivity of the HepG2 cells were determined by comparison between the 3 groups in term of the surviving cell fractions (SF2) after 2-Gy X-ray exposure, and of the Do and Dq values obtained using the single-hit multi target model.

<b>RESULTSb>Before the X-ray exposure, the Hep G2 cells in Adv group and the control group showed low levels of IkappaBalpha absorbance in the cytoplasm, which were further decreased after the exposure. The NF-kappaB activity in the nuclei of the cells in the two groups was positive (+) before irradiation, and substantially enhanced (++) after the exposure and maintained the stably activated state. The apoptotic index of the cells in the two groups was 1.4 and 1.6 before irradiation, and increased to 8.9 and 11.7 after the irradiation, respectively. The cells in the Ad-mIkappaBalpha group, however, exhibited high levels of IkappaBalpha absorbance either before or after the irradiation, which were approximately 3 times that of the Adv group, but the NF-kappaB activity remained negative irrespective of the irradiation. The apoptotic index of the cells in the Ad-mIkappaBalpha group was 18.2 before irradiation, was increased to 88.3 after the irradiation. Among the 3 groups, Ad-mIkappaBalpha group had the smallest SF(2) value of 0.301 but the highest sensitivity enhancement ratio (SER) of2.99, with the lowest Do and Dq values (1.468 and 0.709, respectively).

<b>CONCLUSIONb>mIkappaBalpha gene transfection into HepG2 cells inhibits the anti-apoptotic activity of NF-kappaB to enhance the radiosensitivity of the cells.

Adenoviridae ; genetics ; Apoptosis ; genetics ; physiology ; radiation effects ; Blotting, Western ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Genetic Vectors ; Humans ; I-kappa B Proteins ; genetics ; metabolism ; physiology ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Mutation ; NF-KappaB Inhibitor alpha ; Transfection