NF-kappaB DNA Binding Activity Research Articles

Regulation of German cockroach extract‐induced IL‐8 expression in human airway epithelial cells

Cockroaches have been known as a cause of respiratory allergies such as asthma. IL-8 plays an integral role in the coordination and persistence of the inflammatory process in the chronic inflammation of the airways in asthma. We investigated the mechanism by which German cockroach extract (GCE) triggers IL-8 release from human airway epithelial cells. Chemical inhibitors were pretreated before addition of GCE for promoter activity and protein synthesis of IL-8. The Transcriptional activity of IL-8 promoter was analysed by mutational, deletional anaylsis and electrophoretic mobility shift assay (EMSA). Stimulation of H292 cells with GCE resulted in a time- and concentration-dependent induction of IL-8 transcription and protein synthesis. IL-8 promoter deletion analysis indicated that position -132 to +41 was essential for GCE-induced IL-8 transcription, and mutants with substitutions in activator protein (AP)-1, nuclear factor (NF)-IL6 and NF-kappaB-binding sites revealed a requirement for NF-kappaB and NF-IL6, but not AP-1, in GCE-induced activation of the IL-8 promoter. The DNA-binding activities of NF-kappaB and NF-IL6 were induced by GCE, as determined by EMSA. The chemical inhibition of extracellular signal-regulated kinase (ERK) attenuated GCE-induced transcriptional activity and protein synthesis. In addition, through aprotinin treatment and PAR2 small interfering RNA transfection, it was proven that protease of GCE is consistent with the regulation of GCE-induced IL-8. We conclude that GCE with protease activity-induced IL-8 expression is regulated by transcriptional activation of NF-kappaB and NF-IL6 coordinating with the ERK pathway in human airway epithelial cells.

Chronic treatment of rats with sodium valproate downregulates frontal cortex NF‐κB DNA binding activity and COX‐2 mRNA1

Valproic acid (VPA) is used to treat bipolar disorder, but its mechanism of action is not clear. VPA shares many cellular and molecular targets with lithium, including reducing arachidonic acid turnover in rat brain phospholipids and cyclooxygenase-2 (COX-2) protein level and activity in rat brain. We examined the effect of chronic VPA administration (200 mg/kg body weight for 30 days) to produce therapeutically relevant plasma concentrations, on transcription factors (NF-kappaB, AP-1, AP-2, C/EBP, CREB, and ETS) that are known to regulate the COX-2 gene. Chronic VPA significantly increased AP-1 DNA binding activity and decreased NF-kappaB DNA binding activity, p50 subunit protein and mRNA expression of COX-2 in frontal cortex compared with untreated control rats. It did not alter AP-2, C/EBP, ETS or CREB DNA binding activity. VPA downregulates NF-kappaB DNA binding activity, likely by decreasing the p50 protein levels. This effect may explain its downregulation of COX-2 mRNA. The decrease in NF-kappaB activity by chronic VPA may affect other NF-kappaB-regulated genes and may be related to VPA's action in bipolar disorder. Chronic VPA may decrease the reported increased brain NF-kappaB components in bipolar patients.

Regulation of TNF‐α‐activated PKC‐ζ signaling by the human biliverdin reductase: identification of activating and inhibitory domains of the reductase

Human biliverdin reductase (hBVR) is a dual function enzyme: a catalyst for bilirubin formation and a S/T/Y kinase that shares activators with protein kinase C (PKC) -zeta, including cytokines, insulin, and reactive oxygen species (ROS). Presently, we show that hBVR increases PKC-zeta autophosphorylation, stimulation by TNF-alpha, as well as cytokine stimulation of NF-kappaB DNA binding and promoter activity. S149 in hBVR S/T kinase domain and S230 in YLS230F in hBVR's docking site for the SH2 domain of signaling proteins are phosphorylation targets of PKC-zeta. Two hBVR-based peptides, KRNRYLS230F (#1) and KKRILHC281 (#2), but not their S-->A or C-->A derivatives, respectively, blocked PKC-zeta stimulation by TNF-alpha and its membrane translocation. The C-terminal-based peptide KYCCSRK296 (#3), enhanced PKC-zeta stimulation by TNF-alpha; for this, Lys296 was essential. In metabolically 32P-labeled HEK293 cells transfected with hBVR or PKC-zeta, TNF-alpha increased hBVR phosphorylation. TNF-alpha did not stimulate PKC-zeta in cells infected with small interfering RNA for hBVR or transfected with hBVR with a point mutation in the nucleotide-binding loop (G17), S149, or S230; this was similar to the response of "kinase-dead" PKC-zeta(K281R). We suggest peptide #1 blocks PKC-zeta-docking site interaction, peptide #2 disrupts function of the PKC-zeta C1 domain, and peptide #3 alters ATP presentation to the kinase. The findings are of potential significance for development of modulators of PKC-zeta activity and cellular response to cytokines.

Hyperlipidemia induced by a cholesterol-rich diet aggravates necrotizing pancreatitis in rats

The aim of the present study was to investigate whether hyperlipidemia can cause acute pancreatitis or alter its severity. Male Wistar rats were fed a 3% cholesterol-enriched diet or a normal diet for 16 weeks. Edematous and necrotizing pancreatitis was induced with 3x75 mug/kg body weight of cholecystokinin s.c. and 2x2 g/kg body weight of L-arginine i.p., respectively, in separate groups of normal and hyperlipidemic rats. The severity of the pancreatitis was assessed. We studied the influence of hyperlipidemia on the formation of oxygen-derived free radicals, endogenous scavengers, nitric oxide synthases (NOS), peroxynitrite (ONOO(-)), heat shock protein 72 (HSP72) and nuclear factor-kappa B (NF-kappaB) activation in the pancreas during acute edematous and necrotizing pancreatitis. Hyperlipidemia did not worsen edematous, but aggravated necrotizing pancreatitis. The cholesterol-enriched diet significantly reduced the catalase and Mn-superoxide dismutase (SOD) and constitutive NOS (cNOS) activities and increased the inducible NOS (iNOS) in the pancreas relative to those in the rats on the normal diet. The pancreatic nitrotyrosine level, as a marker of ONOO(-), and the NF-kappaB DNA-binding activity in the pancreas, were significantly elevated in the cholesterol-fed rats. The pancreatic HSP72 expression during necrotizing pancreatitis was not influenced by the hyperlipidemia. The pancreatic Mn-SOD, Cu, Zn-SOD, glutathione peroxidase, total glutathione and cNOS activities were significantly reduced, while the catalase, iNOS and NF-kappaB DNA-binding activities were significantly increased in the animals with necrotizing pancreatitis on the cholesterol diet as compared with those with pancreatitis and receiving the normal diet. Hyperlipidemia induced with this cholesterol-enriched diet leads to decreases in endogenous scavenger and cNOS activities, results in iNOS and NF-kappaB activation and stimulates ONOO(-) generation in the pancreas, which may be responsible for the aggravation of acute necrotizing pancreatitis.

Transcriptional up-regulation of nitric oxide synthase II by nuclear factor-kappaB at rostral ventrolateral medulla in a rat mevinphos intoxication model of brain stem death.

As the origin of a 'life-and-death' signal that reflects central cardiovascular regulatory failure during brain stem death, the rostral ventrolateral medulla (RVLM) is a suitable neural substrate for mechanistic delineation of this vital phenomenon. Using a clinically relevant animal model that employed the organophosphate pesticide mevinphos (Mev) as the experimental insult, we evaluated the hypothesis that transcriptional up-regulation of nitric oxide synthase I or II (NOS I or II) gene expression by nuclear factor-kappaB (NF-kappaB) on activation of muscarinic receptors in the RVLM underlies brain stem death. In Sprague-Dawley rats maintained under propofol anaesthesia, co-microinjection of muscarinic M2R (methoctramine) or M4R (tropicamide), but not M1R (pirenzepine) or M3R (4-diphenylacetoxy-N-dimethylpiperidinium) antagonist significantly reduced the enhanced NOS I-protein kinase G signalling ('pro-life' phase) or augmented NOS II-peroxynitrite cascade ('pro-death' phase) in ventrolateral medulla, blunted the biphasic increase and decrease in baroreceptor reflex-mediated sympathetic vasomotor tone that reflect the transition from life to death, and diminished the elevated DNA binding activity or nucleus-bound translocation of NF-kappaB in RVLM neurons induced by microinjection of Mev into the bilateral RVLM. However, NF-kappaB inhibitors (diethyldithiocarbamate or pyrrolidine dithiocarbamate) or double-stranded kappaB decoy DNA preferentially antagonized the augmented NOS II-peroxynitrite cascade and the associated cardiovascular depression exhibited during the 'pro-death' phase. We conclude that transcriptional up-regulation of NOS II gene expression by activation of NF-kappaB on selective stimulation of muscarinic M2 or M4 subtype receptors in the RVLM underlies the elicited cardiovascular depression during the 'pro-death' phase in our Mev intoxication model of brain stem death.

Human chorionic gonadotrophin attenuates NF-κB activation and cytokine expression of endometriotic stromal cells

Recently, a clinical study provided evidence that treatment of endometriotic women with human chorionic gonadotrophin (hCG) alleviates disease-related pain and sleeplessness suggesting therapeutic effects of the hormone. Since endometriosis is associated with aberrant concentrations of inflammatory mediators in the peritoneal fluid, we investigated whether hCG may affect cytokine-dependent activation of the key-regulatory transcription factor NF-kappaB and expression of two nuclear factor kappa B (NF-kappaB)-inducible genes, tumour necrosing factor (TNF-alpha) and interleukin (IL)-1beta, in stromal cells isolated from ectopic endometriotic tissues. Electrophoretic mobility shift assay revealed that treatment of these cultures with the urinary preparation hCG-A suppressed TNF-alpha- or IL-1beta-induced NF-kappaB DNA-binding activity, whereas another urinary hCG preparation (hCG-B) was less effective. Recombinant alphahCG or epidermal growth factor (EGF), a contaminant of some urinary hCG preparations, did not alter cytokine-dependent NF-kappaB activation. Immunofluorescene of its p65 subunit revealed that pre-incubation with hCG-A strongly decreased TNF-alpha-dependent nuclear expression of NF-kappaB. Accordingly, hCG-A diminished IL-1beta-induced TNF-alpha transcript levels and protein release measured by quantitative real-time PCR and enzyme-linked immunosorbent assay. The hormone also attenuated TNF-alpha-dependent mRNA expression of IL-1beta. Western blot analyses revealed that hCG-A impaired TNF-alpha-mediated phosphorylation and degradation of the inhibitor IkappaBalpha suggesting that the hormone may reduce nuclear import of NF-kappaB by stabilizing its inhibitor. The data suggest that hCG attenuates inflammation-dependent NF-kappaB activation and cytokine expression that could provide one explanation for the beneficial role of the hormone in endometriotic patients.

Nuclear factor-kappa B is constitutively activated in peritoneal endometriosis

Red (active), black and white endometriotic lesions are characteristic of peritoneal endometriosis. The transcription factor nuclear factor-kappa B (NF-kappaB) activates proinflammatory, proliferative and antiapoptotic genes in many cell types. To determine whether NF-kappaB is activated in peritoneal endometriosis in women, and further ascertain the differential inflammatory status of endometriotic implants, NF-kappaB activation and intercellular adhesion molecule (ICAM)-1 expression were investigated in peritoneal endometriotic lesions according to their type. Furthermore, p65 and p50 subunits of active NF-kappaB dimers were evaluated in endometriotic lesions to gain some insight into NF-kappaB-implicated pathways. Thirty-six biopsies of peritoneal endometriotic lesions were analyzed. Constitutive NF-kappaB activation, involving p65- and p50-containing dimers, was demonstrated in peritoneal endometriotic lesions by electrophoretic mobility shift assays and supershift analyses, as well as NF-kappaB (p65) DNA-binding activity immunodetection assays. NF-kappaB activation and ICAM-1 expression (evaluated by immunoblotting) were significantly higher in red lesions than black lesions, whereas IkappaBalpha (NF-kappaB inhibitory protein) expression was constant, as shown by western blot analysis. This is the first study to demonstrate constitutive NF-kappaB activation in peritoneal endometriosis in women. NF-kappaB activation and ICAM-1 expression in red lesions confirm the more extensive inflammatory pattern of these lesions compared with black lesions. The involvement of p50/p65 dimers in NF-kappaB activation suggests implication of the classic NF-kappaB activation pathway, making it an attractive therapeutic target in endometriosis.

Platelet-Activating Factor Overturns the Transcriptional Repressor Disposition of Sp1 in the Expression ofMMP-9in Human Corneal Epithelial Cells

Matrix metalloproteinase (MMP)-9 is induced in corneal epithelial cells stimulated with platelet-activating factor (PAF), and interferes with the normal reepithelialization of wounded cornea. Here the transcriptional regulation of MMP-9 gene expression by PAF was investigated in human corneal epithelial cells (HCECs). DNA-binding activity of NFkappaB, Sp1, and AP-1 was determined in quiescent and PAF-stimulated HCECs by electrophoretic mobility shift assay (EMSA). A series of 5' deleted human MMP-9 promoter-luciferase reporter constructs was transiently transfected into HCECs, and luciferase activity was examined after stimulation with PAF. Mutagenesis and specific deletions of some elements in the MMP-9 promoter were also introduced and analyzed. Phosphorylation of Sp1 and MEK/ERK pathway proteins was examined by Western blot analysis. Activation of Sp1 and MMP-9 was also determined by ELISA and zymography, respectively, in the absence or presence of the MEK inhibitor PD98059. DNA-binding activity of NFkappaB, Sp1, and AP-1 was upregulated by PAF with a peak at 1 hour after stimulation. A region spanning -670 to -460 relative to the transcription start point was required for the induction of the MMP-9 promoter by PAF. Mutation of the -79AP-1 or -600NFkappaB motif reduced the activity of MMP-9 promoter and the induction of gene expression by PAF. In untreated HCECs, mutation of the -558Sp1 motif upregulated gene expression, but it caused a significant decrease in the promoter activity induced by PAF. Inhibition of MEK activity eliminated the PAF-induced phosphorylation and activation of Sp1 and abolished the upregulation of MMP-9 expression and activity. These findings demonstrate that collaboration between several regulatory elements is required for the induction of MMP-9 promoter activity by PAF and that PAF overturns the repressor effect of Sp1 through activation of the MEK/ERK signaling cascade.

Knocking Out Peroxisome Proliferator-Activated Receptor (PPAR) α Inhibits Radiation-Induced Apoptosis in the Mouse Kidney through Activation of NF-κB and Increased Expression of IAPs

Peroxisome proliferator-activated receptor (PPAR) alpha, a member of the ligand-activated nuclear receptor superfamily, plays an important role in lipid metabolism and glucose homeostasis and is highly expressed in the kidney. The present studies were aimed at testing the hypothesis that PPARalpha knockout mice would exhibit decreased radiation-induced apoptosis due to exacerbated activation of NF-kappaB (NFKB) and expression of pro-survival factors. Thirty wild-type mice (29S1/SvImJ) and 30 PPARalpha knockout mice were irradiated with a single total-body dose 10 Gy of (137)Cs gamma rays; controls were sham-irradiated. Tissue samples were collected at 3, 6, 12, 24 and 48 h postirradiation. Apoptosis was quantified using immunohistochemical staining for apoptotic bodies and cleaved caspase 3. Radiation-induced apoptosis was observed in both mouse strains in a time-dependent manner. However, the level of apoptosis was significantly suppressed in PPARalpha knockout mice compared with wild-type mice at 6 h postirradiation (P < 0.05). This inhibition of radiation-induced apoptosis was associated with time-dependent increases in NF-kappaB DNA-binding activity, IkappaBalpha phosphorylation, and expression of other antiapoptosis factors in the PPARalpha knockout mouse kidneys but not in wild-type animals. These data support the hypothesis that the loss of PPARalpha expression leads to the suppression of radiation-induced apoptosis in the mouse kidney, mediated through activation of NF-kappaB and up-regulation of anti-apoptosis factors.

A New Proteasome Inhibitor, TP-110, Induces Apoptosis in Human Prostate Cancer PC-3 Cells

Proteasome inhibitors are useful in the treatment of cancer. Recently, we found a new proteasome inhibitor, TP-110, derived from tyropeptin A produced by Kitasatospora sp. Here we report that TP-110 induces apoptosis in human prostate cancer PC-3 cells. TP-110 showed strong cytotoxicity to PC-3 cells (IC(50)=0.05 muM). It increased the number of cells in the G(2)-M phase and increased the accumulated amounts of the p21 and p27 proteins, which are negative regulators of cell cycle progression. Furthermore, it induced apoptosis along with chromatin condensation and DNA fragmentation in PC-3 cells, and TP-110-induced apoptosis appeared to be associated with caspase activation. Additionally, TP-110 inhibited not only the degradation of IkappaB and the nuclear translocation of nuclear factor-kappaB (NF-kappaB), but also the DNA binding activity of NF-kappaB. These results indicate that TP-110 shows a strong growth inhibition and apoptosis in PC-3 cells.

Disruption of ZAS3 in Mice Alters NF-κB and AP-1 DNA Binding and T-Cell Development

The large zinc finger proteins, ZAS, regulate the transcription of a variety of genes involved in cell growth, development, and metastasis. They also function in the signal transduction of the TGF-beta and TNF-alpha pathways. However, the endogenous protein of a representative member, ZAS3, is rapidly degraded in primary lymphocytes, which limits the determination of its physiological function in vitro. Therefore, we have generated mice with targeted disruption of ZAS3. Oligonucleotide-based microarray analyses revealed subtle but consistent differences in the expression of genes, many of which are associated with receptor or signal transduction activities between ZAS3+/+ and ZAS3-/- thymi. Gel mobility shift assays showed altered DNA binding activities of NF-kappaB and AP-1 proteins in ZAS3-deficient tissues, including the thymus. Lymphocyte analysis suggested a subtle but broad function of ZAS3 in regulating T-cell development and activation. In CD3+ ZAS3-/- thymocytes, the CD4/ CD8 ratio was decreased and CD69 expression was decreased. In peripheral CD4+ ZAS3-/- lymphocytes we observed an increased number of memory T cells.

Flutamide Attenuates Pro-inflammatory Cytokine Production and Hepatic Injury Following Trauma-Hemorrhage via Estrogen Receptor-related Pathway

To determine the mechanism by which flutamide administration following trauma-hemorrhage (T-H) decreases cytokine production and hepatic injury under those conditions. Although studies have demonstrated that flutamide administration following T-H improves hepatic and immune functions, the mechanism by which flutamide produces the salutary effects remains unknown. Male Sprague-Dawley rats underwent a 5-cm laparotomy and hemorrhagic shock (40 mm Hg for approximately 90 minutes), followed by resuscitation with 4 times the shed blood volume in the form of Ringer's lactate. Flutamide (25 mg/kg body weight, sc) was administered at the middle of resuscitation and animals were killed 2 hours thereafter. To block estrogen receptor (ER), ER antagonist ICI 182,780 was administrated with flutamide. Hepatic injury, myeloperoxidase activity, nuclear factor-kappaB (NF-kappaB) DNA binding activity and protein expression of intercellular adhesion molecule-1, and cytokine-induced neutrophil chemoattractant (CINC-1 and CINC-3) markedly increased following T-H. Hepatic mRNA and plasma IL-6 levels were also elevated following T-H. The alterations in these parameters induced by T-H were significantly attenuated by flutamide administration. The decreased plasma estradiol levels following T-H were restored to sham levels in the flutamide-treated T-H animals. Coadministration of ICI 182,780 prevented those salutary effects of flutamide administration on pro-inflammatory responses and hepatic injury following T-H. These findings suggest that the reduction in the production of pro-inflammatory mediators and hepatic injury produced by flutamide administration following T-H is likely due to the down-regulation in hepatic NF-kappaB DNA binding activity. Moreover, the salutary effects of flutamide administration appear to be mediated at least in part via ER-related pathway.

A neutrophil elastase inhibitor, sivelestat, ameliorates lung injury after hemorrhagic shock in rats

Hemorrhagic shock followed by resuscitation (HSR) causes neutrophil sequestration in the lung which leads to acute lung injury (ALI). Neutrophil elastase (NE) is thought to play a pivotal role in the pathogenesis of ALI. This study investigated whether sivelestat, a specific NE inhibitor, can attenuate ALI induced by HSR in rats. Male Sprague-Dawley rats were subjected to hemorrhagic shock by withdrawing blood so as to maintain a mean arterial blood pressure of 30+/-5 mm Hg for 60 min followed by resuscitation with the shed blood. HSR-treated animals received a bolus injection of sivelestat (10 mg/kg) intravenously at the start of resuscitation followed by continuous infusion for 60 min (10 mg/kg/h) during the resuscitation phase, or the vehicle. Lung injury was assessed by pulmonary histology, lung wet-weight to dry-weight (W/D) ratio, myeloperoxidase (MPO) activity, gene expression of tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS), DNA binding activity of nuclear factor (NF)-kappaB, and immunohistochemical analysis of intercellular adhesion molecule (ICAM)-1. HSR treatment induced lung injury, as demonstrated by pulmonary edema with infiltration of neutrophils, the increase in lung W/D ratio, MPO activity, gene expression of TNF-alpha and iNOS, and DNA-binding activity of NF-kappaB, and enhanced expression of ICAM-1. In contrast, sivelestat treatment significantly ameliorated the HSR-induced lung injury, as judged by the marked improvement in all these indices. These results indicate that sivelestat attenuated HSR-induced lung injury at least in part through an inhibition of the inflammatory signaling pathway, in addition to the direct inhibitory effect on NE.

Suberosin inhibits proliferation of human peripheral blood mononuclear cells through the modulation of the transcription factors NF-AT and NF-kappaB.

Extracts of Plumbago zeylanica containing suberosin exhibit anti-inflammatory activity. We purified suberosin from such extracts and studied its effects on a set of key regulatory events in the proliferation of human peripheral blood mononuclear cells (PBMC) stimulated by phytohemagglutinin (PHA). Proliferation of PBMC in culture was measured by uptake of 3H-thymidine; production of cytokines and cyclins by Western blotting and RT-PCR. Transcription factors NF-AT and NF-kappaB were assayed by immunocytochemistry and EMSA. Suberosin suppressed PHA-induced PBMC proliferation and arrested cell cycle progression from the G1 transition to the S phase. Suberosin suppressed, in activated PBMC, transcripts of interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and cyclins D3, E, A, and B. DNA binding activity and nuclear translocation of NF-AT and NF-kappaB induced by PHA were blocked by suberosin. Suberosin decreased the rise in intracellular Ca2+ concentration ([Ca2+]i) in PBMC stimulated with PHA. Suberosin did not affect phosphorylation of p38 and JNK but did reduce activation of ERK in PHA-treated PBMC. Pharmacological inhibitors of NF-kappaB, NF-AT, and ERK decreased expression of mRNA for the cyclins, IL-2, and IFN-gamma and cell proliferation in PBMC activated by PHA. The inhibitory effects of suberosin on PHA-induced PBMC proliferation, were mediated, at least in part, through reduction of [Ca2+]i, ERK, NF-AT, and NF-kappaB activation, and early gene expression in PBMC including cyclins and cytokines, and arrest of cell cycle progression in the cells. Our observations provide an explanation for the anti-inflammatory activity of P. zeylanica.

UVB activation of NF-κB in normal human keratinocytes occurs via a unique mechanism

The transcription factor nuclear factor-kappaB (NF-kappaB) is comprised of a family of proteins that are implicated in a wide variety of cellular functions, including the control of cell proliferation, cell survival, and cellular differentiation. Although NF-kappaB is activated in response to inflammatory signals or cellular stress, in the skin NF-kappaB is also implicated to play a role in normal epidermal homeostasis. Often the cellular consequences of NF-kappaB activation are dependent on the specific triggering stimuli. Thus, we have compared the activation mechanism and the function of NF-kappaB following two common stimuli of normal human keratinocytes, inflammatory mediators (tumor necrosis factor alpha (TNFalpha)), and cellular stress (ultraviolet light B (UVB) irradiation). These experiments indicate that although both TNFalpha and UVB stimulate NF-kappaB DNA-binding activity in normal human keratinocytes, the mechanisms of NF-kappaB activation by each stimulus is different. In contrast to the NF-kappaB response following TNFalpha, activation of NF-kappaB by UVB is independent of Ikappa Balpha degradation. Analyses of NF-kappaB-dependent gene expression following TNFalpha or UVB treatment demonstrate that each of these stimulatory signals results in a specific subset of genes that are activated or repressed. These studies provide further evidence of the stimuli and cell-type specific nature of NF-kappaB function.

Arginase Modulates NF-κB Activity via a Nitric Oxide–Dependent Mechanism

NF-kappaB is a versatile transcription factor that regulates a wide array of processes, including inflammation and survival, and plays a critical role in the etiology of inflammatory lung diseases. Nitric oxide (NO) has been suggested to play an antiinflammatory role through S-nitrosation of components of NF-kappaB pathway. NO production can be modulated by changing the availability of its substrate, L-arginine. Arginases compete with NO synthases (NOSs) for their common substrate, L-arginine, and thereby have the potential to alter the signaling function of NO. The goal of the present study was to determine the impact of arginase manipulation on NO, and subsequent effects on NF-kappaB activation, in lung epithelial cells. Our results demonstrate that reduction of arginase activity enhanced cellular content of NO and S-nitrosated proteins, and resulted in decreases in TNF-alpha- or LPS-stimulated NF-kappaB DNA binding and transcriptional activity, in association with enhanced S-nitrosation of p50. The effects of arginase inhibition on NF-kappaB were reversed by the generic NOS inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME), suggesting a causal role for NO in the attenuation of NF-kappaB induced by arginase suppression. Conversely, overexpression of arginase I decreased cellular S-nitrosothiol content and enhanced IkappaB kinase activity and NF-kappaB DNA binding, and decreased S-nitrosation of p50. Collectively, our data point to a regulatory mechanism wherein NF-kappaB is controlled through arginase-dependent regulation of NO levels, which may impact on chronic inflammatory diseases that are accompanied by NF-kappaB activation and upregulation of arginases.

Ergosterol peroxide from an edible mushroom suppresses inflammatory responses in RAW264.7 macrophages and growth of HT29 colon adenocarcinoma cells

5alpha,8alpha-Epidioxy-22E-ergosta-6, 22-dien-3beta-ol (ergosterol peroxide) is a major antitumour sterol produced by edible or medicinal mushrooms. However, its molecular mechanism of action has yet to be determined. Here, we examine the anticancer and anti-inflammatory effects of ergosterol peroxide. After treating RAW264.7 macrophages with LPS and purified ergosterol peroxide or ergosterol, we determined LPS-induced inflammatory cytokines, nuclear DNA binding activity of transcription factors and phosphorylation of MAP kinases (MAPKs). HT29 colorectal adenocarcinoma cells were treated with ergosterol peroxide for 5 days. To investigate the antitumour properties of ergosterol peroxide, we performed DNA microarray and RT-PCR analyses and determined the reactive oxygen species (ROS) in HT29 cells. Ergosterol peroxide suppressed LPS-induced TNF-alpha secretion and IL-1alpha/beta expression in RAW264.7 cells. Ergosterol peroxide and ergosterol suppressed LPS-induced DNA binding activity of NF-kappaB and C/EBPbeta, and inhibited the phosphorylation of p38, JNK and ERK MAPKs. Ergosterol peroxide down-regulated the expression of low-density lipoprotein receptor (LDLR) regulated by C/EBP, and HMG-CoA reductase (HMGCR) in RAW264.7 cells. In addition, ergosterol peroxide showed cytostatic effects on HT29 cells and increased intracellular ROS. Furthermore, ergosterol peroxide induced the expression of oxidative stress-inducible genes, and the cyclin-dependent kinase inhibitor CDKN1A, and suppressed STAT1 and interferon-inducible genes. Our results suggest that ergosterol peroxide and ergosterol suppress LPS-induced inflammatory responses through inhibition of NF-kappaB and C/EBPbeta transcriptional activity, and phosphorylation of MAPKs. Moreover, ergosterol peroxide appears to suppress cell growth and STAT1 mediated inflammatory responses by altering the redox state in HT29 cells.

Anti-Inflammatory Effect of Dohongsamultang through Inhibition of Nuclear Factor-κB Activation in Peripheral Blood Mononuclear Cells of Patients with Cerebral Infarction

The Korean indigenous medicine "Dohongsamultang (DHSMT)" has long been used for various cerebrovascular diseases. However, the exact mechanism for the anti-inflammatory effect of DHSMT is not completely understood. The aim of the present study is to elucidate how DHSMT modulates the inflammatory reaction in lipopolysaccaride (LPS)-stimulated peripheral mononuclear cells from cerebral infarction (CI) patients. Production and expression of cytokine was measured via the ELISA and RT-PCR methods. The level of nuclear factor-kappa B (NF-kappaB)/Rel A protein and NF-kappaB DNA binding activity were determined via the Western blot analysis and transcription factor enzyme-linked immunoassay. It showed that DHSMT inhibited the production of TNF-alpha, IL-1beta, and IL-6 induced by LPS in a dose-dependent manner (p < 0.05). The maximal inhibition rates for TNF-alpha, IL-1beta, and IL-6 production by DHSMT were about 50.18%, 32.13%, and 38.03%, respectively. DHSMT inhibited the TNF-alpha mRNA expression in a dose-dependent manner. We also showed that the inhibitory effect of DHSMT is through the suppression of the NF-kappaB pathway. The study suggests an important molecular mechanism by GMGHT to reduce inflammation, which might explain its beneficial effect in the regulation of inflammatory reactions.

Persistent activation of NF-kappaB related to IkappaB's degradation profiles during early chemical hepatocarcinogenesis

BackgroundTo define the NF-kappaB activation in early stages of hepatocarcinogenesis and its IkappaB's degradation profiles in comparison to sole liver regeneration.MethodsWestern-blot and EMSA analyses were performed for the NF-kappaB activation. The transcriptional activity of NF-kappaB was determined by RT-PCR of the IkappaB-α mRNA. The IkappaB's degradation proteins were determined by Western-blot assay.ResultsWe demonstrated the persistent activation of NF-kappaB during early stages of hepatocarcinogenesis, which reached maximal level 30 min after partial hepatectomy. The DNA binding and transcriptional activity of NF-kappaB, were sustained during early steps of hepatocarcinogenesis in comparison to only partial hepatectomy, which displayed a transitory NF-kappaB activation. In early stages of hepatocarconogenesis, the IkappaB-α degradation turned out to be acute and transitory, but the low levels of IkappaB-β persisted even 15 days after partial hepatectomy. Interestingly, IkappaB-β degradation is not induced after sole partial hepatectomy.ConclusionWe propose that during liver regeneration, the transitory stimulation of the transcription factor response, assures blockade of NF-kappaB until recovery of the total mass of the liver and the persistent NF-kappaB activation in early hepatocarcinogenesis may be due to IkappaB-β and IkappaB-α degradation, mainly IkappaB-β degradation, which contributes to gene transcription related to proliferation required for neoplasic progression.

Role of human LZIP in differential activation of the NF‐κB pathway that is induced by CCR1‐dependent chemokines

Human leucine zipper protein (LZIP) associates with CC chemokine receptor 1 (CCR1) and this protein-protein interaction should play an important role in leukocyte cell mobility. LZIP is known to regulate leukotactin-1 (Lkn-1)-dependent cell migration without affecting the chemotactic activities of other CC chemokines that bind to CCR1. Since Lkn-1 is engaged in the transcriptional activation of nuclear factor kappaB (NF-kappaB) and subsequent activation of the chemoattractant ability of leukocytes, we investigated the regulatory role of LZIP in the NF-kappaB pathway that is induced by CCR1-dependent chemokines. LZIP increased NF-kappaB-dependent luciferase activity in response to Lkn-1 in HOS/CCR1 cells and THP-1 cells. However, the NF-kappaB-dependent luciferase activities induced by other CCR1-dependent chemokines were not affected by LZIP overexpression. LZIP also increased Lkn-1-induced chemotactic activity through activation of the NF-kappaB pathway, whereas LZIP did not affect either the transactivation of NF-kappaB or the chemotactic activities induced by other CCR1-dependent chemokines. Western blot analysis showed that LZIP increased the degradation of IkappaBalpha induced by Lkn-1 but not by other CCR1-dependent chemokines. Results from electrophoretic mobility shift assay (EMSA) showed that LZIP enhanced the Lkn-1-induced DNA-binding activity of NF-kappaB. These data indicate that LZIP functions as a positive regulator in the NF-kappaB activation pathway that is triggered by Lkn-1 without affecting the transcriptional activation of NF-kappaB induced by other CCR1-dependent chemokines.