P65 Subunit Of NF-kappaB Research Articles

The novel triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces apoptosis of human diffuse large B-cell lymphoma cells through a peroxisome proliferator-activated receptor γ-independent pathway

Ligands for the transcription factor peroxisome proliferator-activated receptor gamma (PPAR gamma) are emerging as a new class of antitumor agents. Herein, we investigated the triterpenoid CDDO, a PPAR gamma ligand, for its potential as an anticancer agent on human diffuse large B-cell lymphoma (DLBCL) cells. The ability of CDDO to induce apoptosis in human DLBCL cells of both the germinal center and activated B-cell subtypes was determined by MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) assay, (3)H-thymidine incorporation, and Annexin-V/propidium iodide staining. Small molecule antagonists of PPAR gamma, transfection assays, DNA binding assays, immunofluorescence, Western blotting, and NF-kappaB inhibitors were utilized to determine the contribution of PPAR gamma and NF-kappaB to the cytotoxic effects of CDDO. Human DLBCL cells express PPAR gamma and PPAR gamma is activated by CDDO. In both subtypes of DLBCL cells CDDO inhibited proliferation, was cytotoxic, and induced apoptosis. The ability of CDDO to kill DLBCL cells was found to be independent of PPAR gamma activation. Interestingly, CDDO exposure resulted in activation of the p50 and p65 subunits of NF-kappaB. Moreover, the combination of CDDO with NF-kappaB inhibitors resulted in enhanced DLBCL cell death, indicating that NF-kappaB activation was a prosurvival signal. These findings support the potential of CDDO, alone or in combination with NF-kappaB inhibitors, as a novel therapy for patients with DLBCL.

Anti-apoptotic signalling by the Dot/Icm secretion system ofL. pneumophila

The Dot/Icm type IV secretion system of Legionella pneumophila triggers robust activation of caspase-3 during early and exponential stages of proliferation within human macrophages, but apoptosis is delayed till late stages of infection, which is novel. As caspase-3 is the executioner of the cell, we tested the hypothesis that L. pneumophila triggers anti-apoptotic signalling within the infected human macrophages to halt caspase-3 from dismantling the cells. Here we show that during early and exponential replication, L. pneumophila-infected human monocyte-derived macrophages (hMDMs) exhibit a remarkable resistance to induction of apoptosis, in a Dot/Icm-dependent manner. Microarray analyses and real-time PCR reveal that during exponential intracellular replication, L. pneumophila triggers upregulation of 12 anti-apoptotic genes that are linked to activation of the nuclear transcription factor kappa-B (NF-kappaB). Our data show that L. pneumophila induces a Dot/Icm-dependent sustained nuclear translocation of the p50 and p65 subunits of NF-kappaB during exponential intracellular replication. Bacterial entry is essential both for the anti-apoptotic phenotype of infected hMDMs and for nuclear translocation of the p65. Using p65-/- and IKKalpha-/- beta-/- double knockout mouse embryonic fibroblast cell lines, we show that nuclear translocation of NF-kappaB is required for the resistance of L. pneumophila-infected cells to apoptosis-inducing agents. In addition, the L. pneumophila-induced nuclear translocation of NF-kappaB requires the activity of IKKalpha and/or IKKbeta. We conclude that although the Dot/Icm secretion system of L. pneumophila elicits an early robust activation of caspase-3 in human macrophages, it triggers a strong anti-apoptotic signalling cascade mediated, at least in part by NF-kappaB, which renders the cells refractory to external potent apoptotic stimuli.

HEPATIC ISCHEMIA/REPERFUSION UPREGULATES THE SUSCEPTIBILITY OF HEPATOCYTES TO CONFER THE INDUCTION OF INDUCIBLE NITRIC OXIDE SYNTHASE GENE EXPRESSION

During hepatic ischemia/reperfusion (I/R), proinflammatory cytokines such as tumor necrosis factor alpha and interleukin (IL) 1beta stimulate the induction of inducible nitric oxide synthase (iNOS) in hepatocytes, followed by massive production of nitric oxide. We hypothesized that I/R upregulated the susceptibility of hepatocytes to confer the induction of iNOS gene expression. This study was designed to investigate whether cell susceptibility occurs in response to I/R and to delineate the mechanisms underlying the susceptibility. Hepatocytes were isolated from rats with hepatic I/R or sham, cultured, and treated with IL-1beta. The iNOS induction and its signal including inhibitor kappaB (IkappaB) kinase/nuclear factor kappaB (NF-kappaB) and Akt/type 1 interleukin 1 receptor (IL-1R1) were analyzed. Hepatocytes isolated from rats with I/R markedly increased the production of nitric oxide when stimulated by IL-1beta as compared with sham control. Ischemia/R also increased the levels of iNOS protein and its messenger RNA. Furthermore, I/R enhanced the activation of transcription factor NF-kappaB and the transactivation of iNOS promoter. However, I/R had no effects on the degradation of IkappaB and the nuclear translocation of p65 subunit of NF-kappaB. In contrast, I/R increased the phosphorylation of Akt and the upregulation of IL-1R1 induction, which is essential signal for the transcriptional activation of iNOS in addition to IkappaB kinase/NF-kappaB. These results demonstrate that I/R may augment hepatocyte susceptibility for the induction of iNOS gene expression through the enhancement of IL-1R1.

NF-κB represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2

The transcription factor nuclear factor kappa B (NF-kappaB) is constitutively active in both cancer cells and stromal cells of breast cancer; however, the precise role of activated NF-kappaB in cancer progression is not known. Using parental MCF10A cells and a variant that expresses the myoepithelial marker p63 stably overexpressing the constitutively active p65 subunit of NF-kappaB (MCF10A/p65), we show that NF-kappaB suppresses the expression of epithelial specific genes E-cadherin and desmoplakin and induces the expression of the mesenchymal specific gene vimentin. P65 also suppressed the expression of p63 and the putative breast epithelial progenitor marker cytokeratin 5/6. MCF10A/p65 cells were phenotypically similar to cells undergoing epithelial to mesenchymal transition (EMT). MCF10A/p65 cells failed to form characteristic acini in three-dimensional Matrigel. Analysis of parental and MCF10A/p65 cells for genes previously shown to be involved in EMT revealed elevated expression of ZEB-1 and ZEB-2 in MCF10A/p65 cells compared to parental cells. In transient transfection assays, p65 increased ZEB-1 promoter activity. Furthermore, MCF10A cells overexpressing ZEB-1 showed reduced E-cadherin and p63 expression and displayed an EMT phenotype. The siRNA against ZEB-1 or ZEB-2 reduced the number of viable MCF10A/p65 but not parental cells, suggesting the dependence of MCF10A/p65 cells to ZEB-1 and ZEB-2 for cell cycle progression or survival. MCF10A cells chronically exposed to tumor necrosis factor alpha (TNFalpha), a potent NF-kappaB inducer, also exhibited the EMT-like phenotype and ZEB-1/ZEB-2 induction, both of which were reversed following TNFalpha withdrawal.

Absence of NF‐κB subunit p50 improves heart failure after myocardial infarction

NF kappa B (NF-kappaB) is a ubiquitous transcription factor activated by various stimuli implicated in heart failure progression including reactive oxygen species (ROS), hypoxia, and inflammatory cytokines. Although NF-kappaB is involved in ischemic preconditioning, unstable angina pectoris, and atherogenesis, its role in heart failure has not been determined. Therefore, we investigated left ventricular remodeling in mice with a targeted deletion of the NF-kappaB subunit p50/NF-kappaB1 after myocardial infarction. p50 knockout (KO) and wild-type (WT) animals underwent coronary artery ligation. Transthoracic echocardiography was performed at days 0, 21, and 56 at midpapillary levels. Early mortality was significantly lower in KO than in WT animals. Moreover, p50 KOs exhibited significantly reduced ventricular dilatation over 8 wk compared to WT controls (end-systolic diameters by transthoracic echocardiography, WT vs. KO, 0.55+/-0.04 vs. 0.34+/-0.03 cm) and preserved left ventricular contractility. Collagen content and matrixmetalloproteinase (MMP) -9 expression were significantly lower in KO mice after myocardial infarction and may account for improved left ventricular remodeling. Absence of the NF-kappaB subunit p50 improves early survival and reduces left ventricular dilatation after myocardial infarction. NF-kappaB might therefore be an attractive target to treat heart failure.

Artemisolide fromArtemisia asiatica: Nuclear Factor-κB (NF-κB) inhibitor suppressing prostaglandin E2 and nitric oxide production in macrophages

Aerial parts of Artemisia asiatica (Compositae) have been traditionally used as an oriental medicine for the treatment of inflammatory and ulcerogenic diseases. In the present study, artemisolide was isolated as a nuclear factor (NF)-kappaB inhibitor from A. asiatica by activity-guided fractionation. Artemisolide inhibited NF-kappaB transcriptional activity in lipopolysaccharide (LPS)-stimulated macrophages RAW 264.7 with an IC50 value of 5.8 microM. The compound was also effective in blocking NF-kappaB transcriptional activities elicited by the expression vector encoding the NF-kappaB p65 or p50 subunits bypassing the inhibitory kB degradation signaling NF-kappaB activation. The macrophages markedly increased their PGE2 and NO production upon exposure to LPS alone. Artemisolide inhibited LPS-induced PGE2 and NO production with IC50 values of 8.7 microM and 6.4 microM, respectively, but also suppressed LPS-induced synthesis of cyclooxygenase (COX)-2 or inducible NO synthase (iNOS). Taken together, artemisolide is a NF-kappaB inhibitor that attenuates LPS-induced production of PGE2 or NO via down-regulation of COX-2 or iNOS expression in macrophages RAW 264.7. Therefore, artemisolide could represent and provide the anti-inflammatory principle associated with the traditional medicine, A. asiatica.

Human Gene Profiling in Response to the Active Protein Kinase, Interferon-induced Serine/threonine Protein Kinase (PKR), in Infected Cells: INVOLVEMENT OF THE TRANSCRIPTION FACTOR ATF-3 IN PKR-INDUCED APOPTOSIS

The interferon-induced serine/threonine protein kinase (PKR) has an essential role in cell survival and cell death after viral infection and under stress conditions, but the host genes involved in these processes are not well defined. We used human cDNA microarrays to identify, in infected cells, genes differentially expressed after PKR expression and analyzed the requirement of catalytic activity of the enzyme. To express PKR, we used vaccinia virus (VV) recombinants producing wild type PKR (VV-PKR) and the catalytically inactive mutant K296R (VV-PKR-K296R). Most regulated genes were classified according to biological function, including apoptosis, stress, defense, and immune response. Transcriptional changes detected by microarray analysis were confirmed for selected genes by quantitative real time reverse transcription PCR. A total of 111 genes were regulated specifically by PKR catalytic activity. Of these, 97 were up-regulated, and 14 were down-regulated. The ATF-3 transcription factor, involved in stress-induced beta-cell apoptosis, was up-regulated. Activation of endogenous PKR with a VV mutant lacking the viral protein E3L (VVDeltaE3L), a PKR inhibitor, triggered an increase in ATF-3 expression that was not observed in PKR(-/-) cells. Using null cells for ATF-3 and for the p65 subunit of NF-kappaB, we showed that induction of apoptosis by PKR at late times of infection was dependent on ATF-3 expression and regulated by NF-kappaB activation. Here, we identified human genes selectively induced by expression of active PKR in infected cells and linked ATF-3 to a novel mechanism used by PKR to induce apoptosis.

Midazolam Inhibits Proinflammatory Mediators in the Lipopolysaccharide-activated Macrophage

Midazolam, a benzodiazepine, has a hypnotic effect and is widely used as a sedative. The role of midazolam in activation of macrophages during sepsis is not known. The aim of this study was to evaluate the antiinflammatory actions of midazolam in cultured macrophages. Using a macrophage cell line, RAW264.7 cells, the effect of midazolam on proinflammatory mediators and activation of mitogen-activated protein kinase was measured by Western blot. Nuclear factor-kappaB (NF-kappaB) activation and translocation of p65 subunit of NF-kappaB was measured using luciferase assay and immunocytochemistry. Superoxide production was measured by lucigenin chemiluminescence. Midazolam significantly inhibited lipopolysaccharide-induced up-regulation of both cyclooxygenase 2 and inducible nitric oxide synthase in a dose-dependent manner (approximately 3-30 microm). IkappaB-alpha degradation and NF-kappaB transcriptional activity induced by lipopolysaccharide were also suppressed by the midazolam. Nuclear translocation of the p65 subunit of NF-kappaB was inhibited by midazolam. Furthermore, midazolam suppressed phosphorylation of p38 mitogen-activated protein kinase and also inhibited lipopolysaccharide-induced superoxide production in macrophages. These results suggest that midazolam has an antiinflammatory action by inhibiting inducible nitric oxide synthase and cyclooxygenase-2 expression, possibly through suppression of NF-kappaB and p38 mitogen-activated protein kinase activation.

15-Deoxy-Δ12,14-prostaglandin J2 induces renal epithelial cell death through NF-κB-dependent and MAPK-independent mechanism

The peroxisome proliferator-activated receptor-gamma (PPARgamma) ligand 15d-PGJ2 induces cell death in renal proximal tubular cells. However, the underlying molecular mechanism(s) remains unidentified. The present study was undertaken to examine the roles of reactive oxygen species (ROS), mitogen-activated protein kinase, and NF-kappaB in opossum kidney (OK) cell death induced by 15d-PGJ2. Treatment of OK cells with 15d-PGJ2 resulted in a concentration- and time-dependent cell death, which was largely attributed to apoptosis. 15d-PGJ2 increased ROS production and the effect was inhibited by catalase and N-acetylcysteine. The 15d-PGJ2-induced cell death was also prevented by these antioxidants, suggesting that the cell death was associated with ROS generation. The PPARgamma antagonist GW9662 did not prevent the 15d-PGJ2-induced cell death. 15d-PGJ2 caused a transient activation of extracellular signal-regulated kinase (ERK). However, inhibitors (PD98059 and U0126) of MEK, an ERK upstream kinase, did not alter the 15d-PGJ2-induced cell death. Transfection with constitutively active MEK and dominant-negative MEK had no effect on the cell death. 15d-PGJ2 inhibited the NF-kappaB transcriptional activity, which was accompanied by an inhibition of nuclear translocation of the NF-kappaB subunit p65 and impairment in DNA binding. Inhibition of NF-kappaB with a NF-kappaB specific inhibitor pyrrolidinecarbodithioate and transfection with IkappaBalpha (S32A/36A) caused cell death. These results suggest that the 5d-PGJ2-induced OK cell death was associated with ROS production and NF-kappaB inhibition, but not with MAPK activation.

Cyclooxygenase‐1 and ‐2 enzymes differentially regulate the brain upstream NF‐κB pathway and downstream enzymes involved in prostaglandin biosynthesis

We have recently reported that cyclooxygenase (COX)-2-deficiency affects brain upstream and downstream enzymes in the arachidonic acid (AA) metabolic pathway to prostaglandin E2 (PGE2), as well as enzyme activity, protein and mRNA levels of the reciprocal isozyme, COX-1. To gain a better insight into the specific roles of COX isoforms and characterize the interactions between upstream and downstream enzymes in brain AA cascade, we examined the expression and activity of COX-2 and phospholipase A2 enzymes (cPLA2 and sPLA2), as well as the expression of terminal prostaglandin E synthases (cPGES, mPGES-1, and - 2) in wild type and COX-1(-/-) mice. We found that brain PGE2 concentration was significantly increased, whereas thromboxane B2 (TXB2) concentration was decreased in COX-1(-/-) mice. There was a compensatory up-regulation of COX-2, accompanied by the activation of the NF-kappaB pathway, and also an increase in the upstream cPLA2 and sPLA2 enzymes. The mechanism of NF-kappaB activation in the COX-1(-/-) mice involved the up-regulation of protein expression of the p50 and p65 subunits of NF-kappaB, as well as the increased protein levels of phosphorylated IkappaBalpha and of phosphorylated IKKalpha/beta. Overall, our data suggest that COX-1 and COX-2 play a distinct role in brain PG biosynthesis, with basal PGE2 production being metabolically coupled with COX-2 and TXB2 production being preferentially linked to COX-1. Additionally, COX-1 deficiency can affect the expression of reciprocal and coupled enzymes, COX-2, Ca2+ -dependent PLA2, and terminal mPGES-2, to overcome defects in brain AA cascade.

Biological Evaluation, Chelation, and Molecular Modeling Studies of Novel Metal-Chelating Inhibitors of NF-κB-DNA Binding: Structure Activity Relationships

Previously, we have reported that aurintricarboxylic acid (ATA) is one of the most potent inhibitors of the DNA binding of transcription factor NF-kappaB. We now report the NF-kappaB-DNA binding inhibitory activity of ATA analogues. An electrophoretic mobility shift assay has shown that bromopyrogallol red (BPR) is the most effective inhibitor of NF-kappaB-DNA binding among the studied analogues. The molecular modeling studies showed that BPR makes a strong network of hydrogen bonds with the DNA-binding region of the p50 subunit of NF-kappaB and has electronegative potential on its peripheral surface. Because zinc has been reported to influence the DNA binding of NF-kappaB, the interaction of these analogues with zinc was studied. Chemical speciation and formation-constant studies showed that BPR forms the most stable 1:1 complex with zinc. BPR has also been found to be the most potent antioxidant among the studied analogues.

Anti‐inflammatory effect of buddlejasaponin IV through the inhibition of iNOS and COX‐2 expression in RAW 264.7 macrophages via the NF‐κB inactivation

Buddlejasaponin IV isolated from Pleurospermum kamtschatidum is an anti-inflammatory compound that inhibits NO, PGE(2) and TNF-alpha production. Here, we studied the mode of action of this compound. Buddlejasaponin IV (2.5-10 microM) reduced lipopolysaccaride (LPS (1 microg ml(-1)))-induced levels of iNOS and COX-2 at the protein levels, and iNOS, COX-2, TNF-alpha, interleukin (IL)-1beta and IL-6 mRNA expression in RAW 264.7 macrophages in a concentration-dependent manner, as determined by Western blotting and RT-PCR, respectively. Buddlejasaponin IV inhibited the LPS-induced activation of nuclear factor-kappaB (NF-kappaB), a transcription factor necessary for proinflammatory mediators, iNOS, COX-2, TNF-alpha, IL-1beta and IL-6 expression. This effect was accompanied by a parallel reduction in IkappaB-alpha degradation and phosphorylation, and by the nuclear translocation of the NF-kappaB p65 subunit. The effects of buddlejasaponin IV on acute phase inflammation were studied on serotonin- and carrageenan-induced paw edema. The antiedematous effect of buddlejasaponin IV was compared with 10 mg kg(-1) of indomethacin p.o. Maximum inhibitions of 26 and 41% were noted at a dose of 20 mg kg(-1) for serotonin- and carrageenan-induced paw edema, respectively. The analgesic effect of buddlejasaponin IV was evaluated using acetic acid-induced writhing and hot-plate tests. Buddlejasaponin IV (10 and 20 mg kg(-1), p.o.) was found to have a marked analgesic effect in both models. These results suggest that the inhibitions of the expressions of iNOS, COX-2, TNF-alpha, IL-1beta and IL-6 by blocking NF-kappaB activation, are responsible for the anti-inflammatory effects of buddlejasaponin IV isolated from P. kamtschatidum.

Bile Acids Induce Ectopic Expression of Intestinal Guanylyl Cyclase C Through Nuclear Factor-κB and Cdx2 in Human Esophageal Cells

Although progression to adenocarcinoma at the gastroesophageal junction reflects exposure to acid and bile acids associated with reflux, mechanisms mediating this transformation remain undefined. Guanylyl cyclase C (GC-C), an intestine-specific tumor suppressor, may represent a mechanism-based marker and target of transformation at the gastroesophageal junction. The present studies examine the expression of GC-C in normal tissues and tumors from esophagus and stomach and mechanisms regulating its expression by acid and bile acids. Gene expression was examined by reverse-transcription polymerase chain reaction, promoter analysis, immunohistochemistry, immunoblotting, and functional analysis. Promoter transactivation was quantified by using luciferase constructs and mutational analysis. DNA binding of transcription factors was examined by electromobility shift analysis. GC-C mRNA and protein were ectopically expressed in approximately 80% of adenocarcinomas arising in, but not in normal, esophagus and stomach. Similarly, in OE19 human esophageal cancer cells, deoxycholate and acid induced expression of GC-C. This was associated with the induction of expression of Cdx2, a transcription factor required for GC-C expression. In turn, induction of Cdx2 expression by deoxycholate was mediated by binding sites in the proximal promoter for nuclear factor kappaB (NF-kappaB). Furthermore, deoxycholate increased NF-kappaB activity, associated with nuclear translocation and Cdx2 promoter binding of the NF-kappaB subunit p50. Moreover, a dominant negative construct for NF-kappaB prevented deoxycholate-induced p50 nuclear translocation and activation of the Cdx2 promoter. Transformation associated with reflux at the gastroesophageal junction reflects activation by bile acid and acid of a transcriptional program involving NF-kappaB and Cdx2, which mediate intestinal metaplasia and ectopic expression of GC-C.

Nitric oxide transcriptionally down‐regulates specific isoforms of divalent metal transporter (DMT1) via NF‐κB

Studies were performed to examine the affect of nitric oxide (NO) on expression of the divalent metal transporter (DMT1) in undifferentiated P19 embryonic carcinoma cells. DMT1 has four known isoforms which differ in both the N- and C-terminals. Results demonstrate that exposure of P19 cells to the NO precursor, sodium nitro-prusside (SNP), resulted in a decrease in expression of both positive (+) and negative (-) IRE isoforms of DMT1 with no change in the 1A species. Regulation was not as a result of decreased stability of message but was caused by reduction in transcription of the DMT1 1B isoforms. Similar results were observed in other cell lines, including PC12 and SH-SY5Y cells and rat primary sympathetic neurons. Nuclear NF-kappaB was decreased after SNP treatment, suggesting that NF-kappaB may mediate this response. Luciferase reporter assays with normal and NF-kappaB mutated constructs of the 1B promoter confirm that the NF-kappaB site between -23 to -19 upstream from the transcription start site was responsible for regulating expression. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays further demonstrate that the p65 subunit of NF-kappaB and not p50 binding is specifically decreased by NO treatment. Results of these studies provide a general mechanism responsible for regulating DMT1 expression induced by stress-related signaling processes in vivo.

Reduction of experimental colitis in the rat by inhibitors of glycogen synthase kinase‐3β

The effects of the inhibitors of glycogen synthase kinase-3beta (GSK-3beta), TDZD-8 and SB 415286, which can substantially reduce the systemic inflammation associated with endotoxic shock in vivo, have now been investigated on the acute colitis provoked by trinitrobenzene sulphonic acid (TNBS) in the rat. Administration of the GSK-3beta inhibitor TDZD-8 (0.1, 0.33 or 1.0 mg kg-1, s.c., b.i.d., for 3 days) caused a dose-dependent reduction in the colonic inflammation induced by intracolonic TNBS assessed after 3 days, both as the area of macroscopic involvement and as a score using 0-10 scale. Likewise, following administration of the GSK-3beta inhibitor SB 415286 (0.1, 0.33 or 1.0 mg kg-1, s.c., b.i.d., for 3 days), the extent and degree of the TNBS-provoked colonic inflammation was reduced. Administration of either TDZD-8 or SB 415286 reduced the fall in body weight following challenge with TNBS at each dose level studied. The increase in myeloperoxidase activity, an index of neutrophil infiltration into the TNBS-induced inflamed colon, was significantly inhibited by both TDZD-8 and SB 415286 at each dose level. The increase in the levels of the proinflammatory cytokine, TNF-alpha, in the inflamed colon was also significantly inhibited by either compound at the highest doses evaluated. The elevated levels of the transcription factor NF-kappaB subunit p65, as determined by Western blot in the nuclear extracts from the TNBS-provoked inflamed colonic tissue, were dose-dependently reduced by TDZD-8 or SB 415286 treatment. These findings demonstrate that two chemically distinct selective inhibitors of the activity of GSK-3beta reduce the inflammation and tissue injury in a rat model of acute colitis. The mechanisms underlying this anti-inflammatory action may be related to downregulation of NF-kappaB activity, involved in the generation of proinflammatory mediators.

Cadmium-Induced Apoptosis in Rat Kidney Epithelial Cells Involves Decrease in Nuclear Factor-Kappa B Activity

Renal epithelial cells undergo apoptosis upon exposure to cadmium (Cd). Transcription factors, such as nuclear factor-kappa B (NF-kappaB), mediate the expression of a number of genes involved in apoptosis. The present study was designed to examine the involvement of this transcription factor in Cd-induced apoptosis. Rat kidney proximal tubular epithelial cells, NRK-52E, were incubated with up to 20 microM CdCl2 in serum-free medium for 5 h followed by incubation in serum-containing medium (without Cd) for an additional 12 h. The cells accumulated 582 +/- 19 ng Cd/mg protein after 5-h exposure to 20 microM Cd. As a result of Cd exposure, the DNA-binding activity of the p65 subunit of NF-kappaB was decreased in a concentration- and time-dependent manner. The activity of tumor necrosis factor-alpha-induced inhibitor of kappa B (IkappaB) kinase alpha was also inhibited by Cd. In addition, the phosphorylation of IkappaB-alpha and NF-kappaB p65, as well as the levels of NF-kappaB target gene products, cIAP-1 and cIAP-2, were reduced. Pretreatment of the cells with the antioxidant U83836E or butylated hydroxytoluene preserved the DNA-binding activity and blocked the Cd-induced decease in IkappaB-alpha phosphorylation. Cd exposure caused the activation of caspase-3, -7, and -9 and DNA fragmentation. By flow cytometry, 14.6 and 30.5% apoptosis was detected at 6 and 12 h after stopping the Cd exposure. Overexpression of NF-kappaB p65 by transient transfection protected the cells from the Cd-induced apoptosis. Conversely, attenuation of NF-kappaB activity by pretreatment with SN50, an NF-kappaB nuclear translocation inhibitor, potentiated apoptosis. These results suggest that Cd-induced apoptosis involves suppression of NF-kappaB activity which may be mediated by oxidative stress.

Kruppel-like factor 5 is an important mediator for lipopolysaccharide-induced proinflammatory response in intestinal epithelial cells

Lipopolysaccharide (LPS) is a bacterially-derived endotoxin that elicits a strong proinflammatory response in intestinal epithelial cells. It is well established that LPS activates this response through NF-κB. In addition, LPS signals through the mitogen-activated protein kinase (MAPK) pathway. We previously demonstrated that the Krüppel-like factor 5 [KLF5; also known as intestine-enriched Krüppel-like factor (IKLF)] is activated by the MAPK. In the current study, we examined whether KLF5 mediates the signaling cascade elicited by LPS. Treatment of the intestinal epithelial cell line, IEC6, with LPS resulted in a dose- and time-dependent increase in KLF5 messenger RNA (mRNA) and protein levels. Concurrently, mRNA levels of the p50 and p65 subunits of NF-κB were increased by LPS treatment. Pretreatment with the MAPK inhibitor, U0126, or the LPS antagonist, polymyxin B, resulted in an attenuation of KLF5, p50 and p65 NF-κB subunit mRNA levels from LPS treatment. Importantly, suppression of KLF5 by small interfering RNA (siRNA) resulted in a reduction in p50 and p65 subunit mRNA levels and NF-κB DNA binding activity in response to LPS. LPS treatment also led to an increase in secretion of TNF-α and IL-6 from IEC6, both of which were reduced by siRNA inhibition of KLF5. In addition, intercellular adhesion molecule-1 (ICAM-1) levels were increased in LPS-treated IEC6 cells and this increase was associated with increased adhesion of Jurkat lymphocytes to IEC6. The induction of ICAM-1 expression and T cell adhesion to IEC6 by LPS were both abrogated by siRNA inhibition of KLF5. These results indicate that KLF5 is an important mediator for the proinflammatory response elicited by LPS in intestinal epithelial cells.

Inhibition of inducible nitric oxide synthesis by azathioprine in a macrophage cell line

Azathioprine is used as an anti-inflammatory agent. Although there are numerous data demonstrating cytotoxic and immunosuppressive properties of azathioprine and its metabolite 6-mercaptopurine, the mechanism of the anti-inflammatory action of azathioprine has not yet been fully clarified. During our study, we investigated the effects of azathioprine on the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated murine macrophages (RAW 264.7) by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), and NO production (nitrite levels). Azathioprine (0–210 μM) induces a concentration dependent inhibition of inducible nitric oxide synthesis (IC50: 33.5 μM). iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of azathioprine. Azathioprine decreases iNOS mRNA levels as shown by semiquantitative competitive RT-PCR. In contrast, 6-mercaptopurine showed no inhibition of inducible nitric oxide synthesis. Azathioprine did not reduce iNOS mRNA stability after the addition of actinomycin D. Enzymatic activity assays with increasing concentrations of azathioprine (0–210 μM) showed no statistically significant inhibition of iNOS enzyme activity compared to cell lysates without azathioprine. Nuclear translocation of NF-kappaB p65 subunit and binding of NF-kappaB p50 subunit from nuclear extracts to a biotinylated-consensus sequence was unaffected by azathioprine treatment. iNOS inhibition by azathioprine was associated with a decreased expression of IRF-1 (interferon regulatory factor 1) and IFN-beta (beta-interferon) mRNA. Azathioprine induced iNOS inhibition seems to be associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic anti-inflammatory agents by replacing the 6-mercaptopurine component of azathioprine with other substituents. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of azathioprine.

Induction of cell death in adult T-cell leukemia cells by a novel IκB kinase inhibitor

NF-kappaB is constitutively activated in adult T-cell leukemia (ATL) and is considered responsible for cell growth and prevention of cell death. In this study, we demonstrate that NF-kappaB is constitutively activated in various HTLV-1-infected T-cell lines and ATL-derived cell lines irrespectively of Tax expression as evidenced by the phosphorylation of IkappaBalpha and p65 subunit of NF-kappaB, activation of NF-kappaB DNA binding, and upregulation of various target genes including bcl-xL, bcl-2, XIAP, c-IAP1, survivin, cyclinD1, ICAM-1 and VCAM-1. The effects of a novel IkappaB kinase (IKK) inhibitor, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile (ACHP), were examined on cell growth of these cell lines and fresh ATL leukemic cells. We found that ACHP could inhibit the phosphorylation of IkappaBalpha and p65, as well as NF-kappaB DNA-binding, associated with downregulation of the NF-kappaB target genes and induce cell growth arrest and apoptosis in these cells. When Tax-active and Tax-inactive cell lines were compared, ACHP could preferentially inhibit cell growth of Tax-active cells. Moreover, ACHP exhibited strong apoptosis-inducing activity in fresh ATL cells. These findings indicate that ACHP and its derivatives are effective in inducing ATL cell death and thus feasible candidates for the treatment of ATL.

Identification of Novel Pharmacological Activities of an Antifungal Agent, Nystatin, to Promote Dendritic Cell Maturation

As an unbiased functional screen to identify agents activating dendritic cells (DCs), we recently developed a DC-based biosensor system, in which a stable murine DC line XS106 was engineered to express the yellow fluorescent protein (YFP) gene under the control of the IL-1beta promoter. Here we report that nystatin (NYT), an antifungal drug of the family of polyene macrolide antibiotics, elevated YFP expression by the resulting XS106-pIL1-YFP DC biosensor clone in a dose-dependent fashion. With respect to the underlying mechanisms, NYT activated the NFkappaB p65 and c-Rel subunits in the parental XS106 DC line. Moreover, NYT dose-dependently increased the surface expression of major histocompatibility complex (MHC) class II (MHC II), CD40, CD54, CD80, and CD86 by murine bone marrow-derived DCs and triggered their robust production of IL-1beta, IL-6, IL-12, tumor necrosis factor alpha, and macrophage inflammatory protein-1alpha. Our results document previously unrecognized pharmacological activities of the most commonly used antifungal drug to promote DC maturation.