NF-kappaB Binding Sites Research Articles

Differential regulation of BACE1 promoter activity by nuclear factor‐κB in neurons and glia upon exposure to β‐amyloid peptides

The brains of Alzheimer's disease (AD) patients display cerebrovascular and parenchymal deposits of beta-amyloid (A beta) peptides, which are derived by proteolytic processing by the beta-site APP-cleaving enzyme 1 (BACE1) of the amyloid precursor protein (APP). The rat BACE1 promoter has a nuclear factor-kappaB (NF-kappaB) binding site. Deletion studies with a BACE1 promoter/luciferase reporter suggest that the NF-kappaB binding DNA consensus sequence plays a suppressor role, when occupied by NF-kappaB, in the regulation of neuronal brain BACE1 expression. Here we characterize a signal transduction pathway that may be responsible for the increases in A beta associated with AD. We propose that the transcription factor NF-kappaB acts as a repressor in neurons but as an activator of BACE1 transcription in activated astrocytes present in the CNS under chronic stress, a feature present in the AD brain. The activated astrocytic stimulation of BACE1 may in part account for increased BACE1 transcription and subsequent processing of Ab eta in a cell-specific manner in the aged and AD brain. As measured by reporter gene promoter constructs and endogenous BACE1 protein expression, a functional NF-kappaB site was stimulatory in activated astrocytes and A beta-exposed neuronal cells and repressive in neuronal and nonactivated astrocytic cells. Given the evidence for increased levels of activated astrocytes in the aged brain, the age- and AD-associated increases in NF-kappaB in brain may be significant contributors to increases in A beta, acting as a positive feedback loop of chronic inflammation, astrocyte activation, increased p65/p50 activation of BACE1 transcription, and further inflammation.

Inhibition of mouse macrophages interleukin-12 production : Suppression of nuclear factor-κB binding activity by a specific factor isolated fromscapharca broughtonii

Pharmacological inhibition of interleukin-12 (IL-12) production may allow a therapeutic strategy for preventing development and progression of disease in experimental models of autoimmunity. In this study we investigated the effects of an ethanol fraction of the Scapharca broughtonii, on the production of IL-12 by mouse macrophages stimulated with lipopolysaccharides (LPS). The ethanol fraction (S3) prepared from Scapharca broughtonii potently inhibited LPS-induced IL-12 production in the RAW264.7 monocyte cell-line in a dose-dependent manner. The activation effect of the ethanol fraction (S3) on the IL-12 gene promoter was analyzed by transfecting RAW264.7 cells with IL-12 gene promoter/luciferase constructs. The repressive effect mapped to a region in the IL-12 gene promoter that contained a binding site for NF-kappaB. Furthermore, activation of macrophages by LPS resulted in markedly enhanced binding activity to the NF-kappaB site, which significantly decreased upon addition of the ethanol fraction, indicating that the ethanol fraction of the blood shell inhibited IL-12 production in LPS-activated macrophages via inhibition of NF-kappaB binding activity.

CD40-CD40 ligand signal induces the intercellular adhesion molecule-1 expression through nuclear factor-kappa B p50 in cultured salivary gland epithelial cells from patients with Sjögren's syndrome

The purpose of this study is to clarify signal transduction of expression of the intercellular adhesion molecule-1 (ICAM-1) via CD40-CD40 ligand in salivary glands of patients with Sjögren's syndrome (SS). We used cultured salivary gland epithelial cells (SG cells) from 15 SS patients and 8 controls obtained by labial minor salivary gland biopsy. First, ICAM-1 expression was determined with reverse transcriptase-polymerase chain reaction and flow cytometry in the presence or absence of soluble CD40L (sCD40L). Next, SG cells were transfected with plasmids of pGL1.3-Luc inserted with promoter region of ICAM-1, pGL1.3kB(-)-Luc mutated in nuclear factor kappa-B (NF-kappaB) binding site of pGL1.3-Luc and pNF-kappaB-Luc by lipofection method. Luciferase activity of the cells was measured in the presence or absence of sCD40L or sCD40L and an NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC). Finally NF-kappaB family proteins of cell nuclear extracts were determined. ICAM-1 expression was significantly enhanced with sCD40L at the mRNA and protein level. Activity of pNF-kappaB-Luc and pGL1.3-Luc was significantly elevated by stimulation with sCD40L and suppressed by PDTC. NF-kappaB p50 protein level was elevated by stimulation with sCD40L and suppressed by PDTC. Our results suggest that sCD40L enhances the ICAM-1 expression by activation of NF-kappaB p50 in the SS SG cells.

Recognition of interferon-inducible sites, promoters, and enhancers

BackgroundComputational analysis of gene regulatory regions is important for prediction of functions of many uncharacterized genes. With this in mind, search of the target genes for interferon (IFN) induction appears of interest. IFNs are multi-functional cytokines. Their effects are immunomodulatory, antiviral, antibacterial, and antitumor. The interaction of the IFNs with their cell surface receptors produces an activation of several transcription factors. Four regulatory factors, ISGF3, STAT1, IRF1, and NF-κB, are essential for the function of the IFN system. The aim of this work is the development of computational approaches for the recognition of DNA binding sites for these factors and computer programs for the prediction of the IFN-inducible regions.ResultsWe developed computational approaches to the recognition of the binding sites for ISGF3, STAT1, IRF1, and NF-κB. Analysis of the distribution of these binding sites demonstrated that the regions -500 upstream of the transcription start site in IFN-inducible genes are enriched in putative binding sites for these transcription factors. Based on selected combinations of the sites whose frequencies were significantly higher than in the other functional gene groups, we developed methods for the prediction of the IFN-inducible promoters and enhancers. We analyzed 1004 sequences of the IFN-inducible genes compiled using microarray data analyses and also about 10,000 human gene sequences from the EPD and RefSeq databases; 74 of 1,664 human genes annotated in EPD were significantly IFN-inducible.ConclusionAnalyses of several control datasets demonstrated that the developed methods have a high accuracy of prediction of the IFN-inducible genes. Application of these methods to several datasets suggested that the number of the IFN-inducible genes is approximately 1500–2000 in the human genome.

Up‐regulation of TFF1 (pS2) expression by TNF‐α in gastric epithelial cells

TFF1 (pS2) is expressed at a high level in gastric epithelial cells and plays an important role in protecting the gastric mucosa. However, the regulatory mechanisms of TFF1 expression are not fully understood. The aim of this study was to investigate the effect of TNF-alpha, a representative proinflammatory cytokine, on TFF1 expression. MKN45 and AGS cells, derived from human gastric carcinoma, were used. Endogenous TFF1 mRNA expression was analyzed by real-time quantitative RT-PCR. The sequences of the human TFF1 promoter were cloned into the pGL3-basic vector and reporter gene assays were performed. Nuclear factor (NF)-kappaB activity was monitored using a reporter vector that contained multiple copies of NF-kappaB responsive element upstream of the luciferase gene. Interaction between NF-kappaB and TFF1 cis-element was examined by electophoretic mobility shift assay (EMSA). TNF-alpha activated NF-kappaB and up-regulated endogenous TFF1 mRNA expression as well as the transcription of the TFF1 reporter genes in a dose-dependent manner. IL-1beta, another proinflammatory cytokine, also up-regulated TFF1 expression. TNF-alpha responsive element was mapped between -342 and -147 of the human TFF1 promoter and a putative NF-kappaB binding site was identified at -231. When this element was deleted, the reporter genes became almost insensitive to TNF-alpha treatment. EMSA showed binding of NF-kappaB to this element. Inflammatory stimuli that activate NF-kappaB appear to up-regulate TFF1 expression in gastric epithelial cells. This mechanism may aid in the protection of the gastric mucosa under inflammatory conditions.

NF-κB-dependent Regulation of Matrix Metalloproteinase-9 Gene Expression by Lipopolysaccharide in a Macrophage Cell Line RAW 264.7

Matrix metalloproteinase-9 (MMP-9) plays a pivotal role in the turnover of extracellular matrix (ECM) and in the migration of normal and tumor cells in response to normal physiologic and numerous pathologic conditions. Here, we show that the transcription of the MMP-9 gene is induced by lipopolysaccharide (LPS) stimulation in cells of a macrophage lineage (RAW 264.7 cells). We provide evidence that the NF-kappaB binding site of the MMP-9 gene contributes to its expression in the LPS-signaling pathway, since mutation of NF-kappaB binding site of MMP-9 promoter leads to a dramatic reduction in MMP-9 promoter activation. In addition, the degradation of IkappaBalpha, and the presences of myeloid differentiation protein (MyD88) and tumor necrosis factor receptor-associated kinase 6 (TRAF6) were found to be required for LPS-activated MMP-9 expression. Chromatin immunoprecipitation (ChIP) assays showed that functional interaction between NF-kappaB and the MMP-9 promoter element is necessary for LPS-activated MMP-9 induction in RAW 264.7 cells. In conclusion, our observations demonstrate that NF-kappaB contributes to LPS-induced MMP-9 gene expression in a mouse macrophage cell line.

MicroRNA gene expression during retinoic acid-induced differentiation of human acute promyelocytic leukemia

MicroRNAs (miRNAs) are small non-coding RNAs of 19-25 nucleotides that are involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation. However, little is known about the role of miRNAs in granulopoiesis. Here, we report the expression of miRNAs in acute promyelocytic leukemia patients and cell lines during all-trans-retinoic acid (ATRA) treatment by using a miRNA microarrays platform and quantitative real time-polymerase chain reaction (qRT-PCR). We found upregulation of miR-15a, miR-15b, miR-16-1, let-7a-3, let-7c, let-7d, miR-223, miR-342 and miR-107, whereas miR-181b was downregulated. Among the upregulated miRNAs, miR-107 is predicted to target NFI-A, a gene that has been involved in a regulatory loop involving miR-223 and C/EBPa during granulocytic differentiation. Indeed, we have confirmed that miR-107 targets NF1-A. To get insights about ATRA regulation of miRNAs, we searched for ATRA-modulated transcription factors binding sites in the upstream genomic region of the let-7a-3/let-7b cluster and identified several putative nuclear factor-kappa B (NF-kappaB) consensus elements. The use of reporter gene assays, chromatin immunoprecipitation and site-directed mutagenesis revealed that one proximal NF-kappaB binding site is essential for the transactivation of the let-7a-3/let-7b cluster. Finally, we show that ATRA downregulation of RAS and Bcl2 correlate with the activation of known miRNA regulators of those proteins, let-7a and miR-15a/miR-16-1, respectively.

Differential regulation of RANTES and IL-8 expression in lung adenocarcinoma cells

In lung adenocarcinoma, expression of Regulated upon Activation, Normal T cell Expressed and presumably Secreted (RANTES) is a predictor of survival while that of interleukin (IL)-8 is associated with a poor prognosis. In several models, tumorigenesis is abolished by RANTES, while it is facilitated by IL-8. We studied the regulation of RANTES and IL-8 expression in A549 lung adenocarcinoma cells. The effects of tumor necrosis factor (TNF)-alpha and regulators of protein kinases C (PKC)alpha/beta were tested because these have been shown to modulate cancer development and progression. TNF-alpha stimulated expression of both chemokines, while the PKCalpha/beta activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced only expression of IL-8 and inhibited TNF-alpha-induced RANTES expression. The PKCalpha/beta inhibitor Gö 6976 increased TNF-alpha-induced RANTES production and prevented its down-regulation by TPA. In contrast, it decreased TNF-alpha or TPA-induced IL-8 release. The differential regulation of RANTES and IL-8 expression was further analyzed. Site-directed mutagenesis indicated that regulation of RANTES promoter activity required two nuclear factor (NF)-kappaB response elements but not its activator protein (AP)-1 binding sites. An AP-1 and a NF-kappaB recognition sites were necessary for full induction of IL-8 promoter activity by TNF-alpha and TPA. Moreover, electrophoretic mobility shift assays demonstrated that NF-kappaB response elements from the RANTES promoter were of lower affinity than that from the IL-8 promoter. Immunoblotting experiments showed that TPA was more potent than TNF-alpha to induce in a PKCalpha/beta dependent manner the p44/p42 mitogen-activated protein kinases (MAPK) signaling cascade which controls AP-1 activity. Conversely, TPA inhibited TNF-alpha-induced NF-kappaB signaling and was a weak activator of this pathway. Thus, TPA did not sufficiently activate NF-kappaB to increase transcription through the low affinity NF-kappaB binding sites on RANTES promoter and its inhibitory effect on TNF-alpha-induced NF-kappaB signaling resulted in a reduced transcription rate. On IL-8 promoter, increased transcription through the high affinity NF-kappaB binding site occurred even with poorly activated NF-kappaB and the functional AP-1 response element compensated any loss of transcription rate. These data provide a mechanistic insight into the differential regulation of IL-8 and RANTES expression by PKCalpha/beta in lung adenocarcinoma cells.

Pneumococcal Peptidoglycan‐Polysaccharides Induce the Expression of Interleukin‐8 in Airway Epithelial Cells by way of Nuclear Factor‐κB, Nuclear Factor Interleukin‐6, or Activation Protein‐1 Dependent Mechanisms

Cell envelope compounds of bacteria trigger immune and inflammatory reactions by way of chemokines/cytokines. In this study, we demonstrated that pneumococcal peptidoglycan-polysaccharides (PGPS) induced the production of interleukin (IL)-8 by way of nuclear factor (NF)-kappaB, nuclear factor interleukin (NF-IL)6, and activation protein (AP)-1 dependent mechanisms in the human bronchial epithelial cells (NL-20) in a dose- and time-dependent manner in vitro, and the mutation of either the NF-kappaB, NF-IL6, or AP-1 binding sites in the promoter of IL-8 abrogated the IL-8 transcriptional activity. In a similar way, lipopolysaccharides induced the promoter activation of IL-8 in NL-20. However, the PGPS-induced IL-8 promoter activation in rodent middle ear epithelial cells required NF-kappaB and NF-IL6 but not AP-1.

Genome-wide identification of novel expression signatures reveal distinct patterns and prevalence of binding motifs for p53, nuclear factor-κB and other signal transcription factors in head and neck squamous cell carcinoma

Microarray profiling of ten head and neck cancer lines revealed novel p53 and NF-κB transcriptional gene expression signatures which distinguished tumor cell subsets in association with their p53 status.

Id1 induces the proliferation of cochlear sensory epithelial cells via the nuclear factor‐κB/cyclin D1 pathway in vitro

Inhibitors of differentiation (Id) play an essential role in the neurogenesis of the central nervous system. However, the expression and function of Id in the development of cochlear sensory epithelial cells have yet to be elucidated. In this study, we demonstrate the Id1 gene was expressed in the rapidly growing otocyst on embryonic day 12 (E12) and in the organ of Corti, spiral ganglions, and stria vascularis on postnatal day 1 (P1) by cellular and molecular biologic techniques. Knockdown of the Id1 gene with short interfering RNA (siRNA) in a cochlear sensory epithelial cell line (OC1) significantly reduced its proliferation, whereas overexpression of Id1 in OC1 significantly increased the proliferation of OC1, suggesting a role of Id1 in the development of cochlear sensory epithelial cells. The proliferative action of Id1 on OC1 was mediated by nuclear factor-kappaB (NF-kappaB) and cyclin D1 (a downstream molecule of NF-kappaB). Blockage of the NF-kappaB activity with pyrrolidine dithiocarbamate (PDTC) or enhancement of the NF-kappaB activity with p65 (a subunit of NF-kappaB) in OC1 significantly inhibited or increased, respectively, the cell proliferation and transcription of cyclin D1 induced by Id1. Truncation of the NF-kappaB binding site in the cyclin D1 promoter fully abrogated the transcription of cyclin D1, suggesting that the cyclin D1 transcription is dependent on NF-kappaB. We concluded from this study that Id1 induces the proliferation of OC1 via the NF-kappaB/cyclin D1 pathway.

Aberrant intestinal expression and allelic variants of mucin genes associated with inflammatory bowel disease

Loss of intestinal mucosa integrity is an important factor in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to characterize expression changes and allelic variants of genes related to intestinal epithelial barrier function in this disease. Therefore, ileal and colonic mucosal biopsies from nonaffected regions of patients with ulcerative colitis (UC) and Crohn's disease (CD), as well as non-IBD probands, were subjected to Affymetrix DNA-microarray analysis. Real-time reverse transcription polymerase chain reaction was used for verification in larger IBD sample numbers. Disturbed mRNA expression was identified for several mucin genes in both disease groups and tissues. A significant downregulation in the colon was obtained for MUC2 in CD and MUC12 in CD and UC. Expression analysis of all dysregulated mucins in a broad human tissue panel revealed dominant epithelial tissue-specific transcription. In silico analysis of the regulatory regions of these mucins indicated nuclear factor kappaB (NFkappaB) binding sites in each promoter. Furthermore, NFkappaB was overrepresented in mucin promoters and a component of a specific combination of transcription factors (composite module). In vivo stimulation experiments in the adenocarcinoma cell line LS174T showed inducible mucin expression by the cytokines tumor necrosis factor-alpha and transforming growth factor-beta, which could be blocked by NFkappaB signaling inhibitors. Allelic discrimination screening obtained statistically significant associations for the MUC2-V116M (P = 0.003) polymorphism with CD and for MUC4-A585S (P = 0.025), as well as MUC13-R502S (P = 0.0003) with UC. These data suggest that the disturbed expression of mucin genes and the connection to the NFkappaB pathway may influence the integrity of the intestine and therefore contribute to the pathophysiology of IBD.

Interleukin‐10 Plasmid Construction and Delivery for the Prevention of Type 1 Diabetes

Studies of animals with spontaneous autoimmune diabetes have revealed that autoreactive T cells that mediate islet beta cell destruction can be manipulated by the administration of Th(2) cytokines. In this article, the effect of interleukin-10 (IL-10) gene delivery was evaluated in vitro and in vivo with a novel IL-10 plasmid, pSI-IL-10-NFkappaB. In pSI-IL-10-NFkappaB, the expression of the IL-10 gene was driven by the SV40 promotor/enhancer. The nuclear factor kappaB (NFkappaB) binding sites were also introduced to facilitate nuclear transport of the plasmid in the cell. In vitro transfection assay with pSI-IL-10-NFkappaB showed a similar expression level of IL-10 to the plasmid without NFkappaB binding sites (pSI-IL-10). pSI-IL-10-NFkappaB and pSI-IL-10 were intravenously injected into 5-week-old nonobese diabetic (NOD) mice using polyethylenimine (PEI) as a gene carrier. Both groups had persistent gene expression, longer than 5 weeks, and secreted the similarly high IL-10 serum levels. Interestingly, the degree of insulitis in the pSI-IL-10-NFkappaB group was improved over the pSI-IL-10 group, PEI-only group, and noninjected controls. The serum glucose levels showed that single injection of pSI-IL-10-NFkappaB prevented the development of diabetes in 100% of the pSI-IL-10-NFkappaB-injected animals (5/5), while that of pSI-IL-10 prevented diabetes in 40% of the treated animals (2/5). These results suggest that pSI-IL-10-NFkappaB with PEI can effectively reduce the incidence of insulitis and type 1 diabetes in NOD mice.

Aldo-keto reductase 1 family B7 is the gene induced in response to oxidative stress in the livers of Long-Evans Cinnamon rats

The Long-Evans Cinnamon (LEC) rat strain (Atp7b m/m), which accumulates copper in the liver due to mutations in the Atp7b gene, is a useful model for investigating the relationship between oxidative stress and hepatocarcinogenesis. To determine the effect of this mutation on oxidative stress marker genes, we performed oligonucleotide array analysis (Affymetrix), and compared the results in Atp7b m/m rats with those of a sibling line with the Atp7b w/w genotype. We focused our studies on the expression of the aldo-keto reductase 1 family B7 (AKR1B7)-like protein gene, since this gene codes for reductase enzymes involved in the detoxification of oxidizing compounds (e.g., aldehydes) and was differentially expressed in Atp7b m/m and Atp7b w/w rat liver. Akr1B7 mRNA expression was significantly increased in comparison with the expression of 4 other known oxidative stress responsive genes, haem-oxygenase-1 (HO-1), thioredoxin (Trx), aldehyde reductase (AKR1A1), and glucose-6-phosphate dehydrogenase (G6PDH). By searching binding motifs, five nuclear factor kappa B (NF-kappaB) binding sites were located in the 5'-upstream region of the akr1b7 gene. Transient co-transfection with both I-kappaBalpha and the Akr1b7 6 kb promoter (p6.0-AKR-Luc) inhibited luciferase activity of p6.0-AKR-Luc in HepG2 cells. Cuprous ion however did not affect the transcription activity induced by p6.0-AKR-luc. Gel-shift assay showed that the DNA binding activity of NF-kappaB increased in the livers of LEC rats, suggesting that the oxidative stress is mediated through NF-kappaB. The results indicate conclusively that in LEC rat liver, akr1b7 might be up-regulated by oxidative stress mediated through NF-kappaB, but not that mediated directly by copper.

Molecularly cloned SHIV-1157ipd3N4: a highly replication- competent, mucosally transmissible R5 simian-human immunodeficiency virus encoding HIV clade C Env.

Human immunodeficiency virus type 1 (HIV-1) clade C causes >50% of all HIV infections worldwide, and an estimated 90% of all transmissions occur mucosally with R5 strains. A pathogenic R5 simian-human immunodeficiency virus (SHIV) encoding HIV clade C env is highly desirable to evaluate candidate AIDS vaccines in nonhuman primates. To this end, we generated SHIV-1157i, a molecular clone from a Zambian infant isolate that carries HIV clade C env. SHIV-1157i was adapted by serial passage in five monkeys, three of which developed peripheral CD4(+) T-cell depletion. After the first inoculated monkey developed AIDS at week 137 postinoculation, transfer of its infected blood to a naïve animal induced memory T-cell depletion and thrombocytopenia within 3 months in the recipient. In parallel, genomic DNA from the blood donor was amplified to generate the late proviral clone SHIV-1157ipd3. To increase the replicative capacity of SHIV-1157ipd3, an extra NF-kappaB binding site was engineered into its 3' long terminal repeat, giving rise to SHIV-1157ipd3N4. This virus was exclusively R5 tropic and replicated more potently in rhesus peripheral blood mononuclear cells than SHIV-1157ipd3 in the presence of tumor necrosis factor alpha. Rhesus macaques of Indian and Chinese origin were next inoculated intrarectally with SHIV-1157ipd3N4; this virus replicated vigorously in both sets of monkeys. We conclude that SHIV-1157ipd3N4 is a highly replication-competent, mucosally transmissible R5 SHIV that represents a valuable tool to test candidate AIDS vaccines targeting HIV-1 clade C Env.

Analysis of interleukin‐1β‐modulated mRNA gene transcription in human gingival keratinocytes by epithelia‐specific cDNA microarrays

Proinflammatory cytokines such as interleukin-1beta are known to be synthesized in oral gingivitis and periodontitis and lead to the activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Although numerous effects of interleukin-1beta on mesenchymal cells are known, e.g. up-regulation of intercellular adhesion molecule-1 in endothelial cells, little is known of the effects of interleukin-1beta on oral keratinocytes. The purpose of the present study was to seek interleukin-1beta-mediated alterations in mRNA gene transcription and a putative activation of NF-kappaB in oral gingival keratinocytes. As an in vitro model for gingivitis and periodontitis, immortalized human gingival keratinocytes (IHGK) were stimulated with the proinflammatory cytokine interleukin-1beta. An epithelia-specific cDNA microarray was used to analyze mRNA expression profiles from IHGK cells treated with 200 units interleukin-1beta/ml for 3, 6, 9, 12, and 24 h. Indirect immunofluorescence was carried out to detect NF-kappaB in IHGK following interleukin-1beta treatment. Detailed analysis revealed distinct patterns of time-dependent changes, including genes induced or repressed early (3-6 h) or late (12-24 h) after interleukin-1beta treatment. Differentially expressed genes were involved in (i) cell stress, (ii) DNA repair, (iii) cell cycle and proliferation, (iv) anti-pathogen response, (v) extracellular matrix turnover, and (vi) angiogenesis. A large number of genes were responsive to NF-kappaB and induction was concomitant with nuclear translocation of the p65 RelA subunit of NF-kappaB. Interestingly, many of these genes contain multiple NF-kappaB binding sites in their promoters. Analysis of altered gene expression allows identification of gene networks associated with inflammatory responses. In addition to a number of well-known genes involved in gingivitis and periodontitis, we identified novel candidates that might be associated with the onset and maintenance of an inflammatory disease.

Ca2+ Influx Induced by Protease-activated Receptor-1 Activates a Feed-forward Mechanism of TRPC1 Expression via Nuclear Factor-κB Activation in Endothelial Cells

Thrombin activation of protease-activated receptor-1 induces Ca(2+) influx through store-operated cation channel TRPC1 in endothelial cells. We examined the role of Ca(2+) influx induced by the depletion of Ca(2+) stores in signaling TRPC1 expression in endothelial cells. Both thrombin and a protease-activated receptor-1-specific agonist peptide induced TRPC1 expression in human umbilical vein endothelial cells, which was coupled to an augmented store-operated Ca(2+) influx and increase in endothelial permeability. To delineate the mechanisms of thrombin-induced TRPC1 expression, we transfected in endothelial cells TRPC1-promoter-luciferase (TRPC1-Pro-Luc) construct containing multiple nuclear factor-kappaB (NF-kappaB) binding sites. Co-expression of dominant negative IkappaBalpha mutant prevented the thrombin-induced increase in TRPC1 expression, indicating the key role of NF-kappaB activation in mediating the response. Using TRPC1 promoter-deletion mutant constructs, we showed that NF-kappaB binding sites located between -1623 and -871 in the TRPC1 5'-regulatory region were required for thrombin-induced TRPC1 expression. Electrophoretic mobility shift assay utilizing TRPC1 promoter-specific oligonucleotides identified that the DNA binding activities of NF-kappaB to NF-kappaB consensus sites were located in this domain. Supershift assays using NF-kappaB protein-specific antibodies demonstrated the binding of p65 homodimer to the TRPC1 promoter. Inhibition of store Ca(2+) depletion, buffering of intracellular Ca(2+), or down-regulation of protein kinase Calpha downstream of Ca(2+) influx all blocked thrombin-induced NF-kappaB activation and the resultant TRPC1 expression in endothelial cells. Thus, Ca(2+) influx via TRPC1 is a critical feed-forward pathway responsible for TRPC1 expression. The NF-kappaB-regulated TRPC1 expression may be an essential mechanism of vascular inflammation and, hence, a novel therapeutic target.

Involvement of Nuclear Factor-κB in the Inhibition of Pro-Inflammatory Mediators by Pinosylvin

Nuclear factor-kappaB (NF-kappaB) is a critical transcription factor for maximal expression of many of the cytokines that are involved in the pathogenesis of inflammatory diseases. In this study, we found that pinosylvin, a natural stilbenoid that is a component of the pine leaf (Pinus densiflora), significantly inhibited LPS-induced NF-kappaB activation in a concentration-dependent manner. Additionally, pinosylvin was found to inhibit the LPS-induced phosphorylation and degradation of IkappaB alpha in THP-1 cells. Therefore, we have attempted to determine whether pinosylvin can inhibit the expression of cytokines possessing NF-kappaB binding sites in their promoter regions. In a consistent result, pinosylvin inhibited LPS-induced production of TNF alpha and interleukin-8 (IL-8). Taken together, these results show that pinosylvin suppresses the production of pro-inflammatory mediators through the inhibition of the NF-kappaB signaling pathway.

Competition between glucocorticoid receptor and NFκB for control of the human FasL promoter

Glucocorticoids mediate a variety of biological effects via binding their intracellular receptor. Ligand-bound glucocorticoid receptor (GR) translocates to the nucleus and regulates gene transcription in a DNA binding-dependent or independent manner. The predominant biological effect of glucocorticoids on peripheral T cells is immunosupression via transcriptional repression of genes induced during T cell activation. Glucocorticoids have been implicated in the inhibition of activation-induced T cell apoptosis by virtue of their down-regulation of Fas ligand (fasL) expression. It is believed that FasL, similar to other cytokines, is repressed by glucocorticoids via GR interaction with other transcription factors, interfering with their transactivation ability. Here, we show that human fasL is directly regulated by GR in a DNA binding-dependent manner. A negative GR element found at position -990 in the fasL promoter binds GR in vitro as well as in the chromatin context. This negative glucocorticoid response element overlaps with a known NFkappaB binding site. GR down-regulates fasL promoter by competing with NFkappaB for binding to the common response element. Thus, fasL is the first gene described whose repression by GR is mediated by sterical occlusion of NFkappaB DNA binding. This type of repression represents an additional mechanism for the GR-NFkappaB mutual antagonism.

Nuclear Translocation and DNA-Binding Activity of NFKB (NF-κB) after Exposure of Human Monocytes to Pulsed Ultra-wideband Electromagnetic Fields (1 kV/cm) Fails to Transactivate κB-Dependent Gene Expression

The objective of this study was to investigate whether exposure of human monocytes to a pulsed ultra-wideband electromagnetic field (EMF) of 1 kV/cm average peak power triggers a signaling pathway responsible for the transcriptional regulation of NFKB (NF-kappaB)-dependent gene expression. Human Mono Mac 6 (MM6) cells were exposed intermittently to EMF pulses for a total of 90 min. The pulse width was 0.79+/-0.01 ns and the pulse repetition rate was 250 pps. The temperature of the medium was maintained at 37 degrees C in both sham- and EMF-exposed flasks. Total NFKB DNA-binding activity was measured in the nuclear extracts by the electrophoretic mobility shift assay. Cells exposed to the EMFs and incubated for 24 h postexposure showed a 3.5+/-0.2-fold increase in the NFKB DNA-binding activity. Since activation of NFKB was observed, the possibility of kappaB-dependent gene expression in response to exposure to the EMFs was investigated using NFKB signal-specific gene arrays. The results revealed no difference in the NFKB-dependent gene expression profiles at 8 or 24 h postexposure, indicating that activated NFKB does not lead to the differential expression of kappaB-dependent target genes. To determine whether the absence of the kappaB-dependent gene expression was due to compromised transcriptional regulation of NFKB, the functional activity of NFKB was examined in cells transiently transfected with Mercury Pathway constructs containing 4x NFKB binding sites associated either with the luciferase reporter system or a control vector. Pulsed EMF exposure did not induce NFKB-driven luciferase activity in these cells, indicating that the activation of NFKB at 24 h after the 1 kV/cm EMF exposure is functionally inactive. From these results, it is clear that the EMF-induced NFKB activation is only a transient response, with minimal or no downstream effect.