NF-kappaB Site Research Articles

Interleukin (IL) 1β Induction of IL-6 Is Mediated by a Novel Phosphatidylinositol 3-Kinase-dependent AKT/IκB Kinase α Pathway Targeting Activator Protein-1

Here we describe a novel role for the phosphatidylinositol 3-kinase/AKT pathway in mediating induction of interleukin-6 (IL-6) in response to IL-1. Pharmacological inhibition of phosphatidylinositol 3-kinase (PI3K) inhibited IL-6 mRNA and protein production. Overexpression of either dominant-negative AKT or IkappaB kinase alpha mutant, IKKalphaT23A, containing a mutation in a functional AKT phosphorylation site, shown previously to be important for NFkappaB activation, completely abrogated IL-6 promoter activation in response to IL-1. However, mutation of the consensus NFkappaB site on the IL-6 promoter did not abrogate promoter activation by IL-1 in contrast to the AP-1 site mutation. IL-1 induces phosphorylation of IKKalpha on the NFkappaB inducing kinase (NIK) phosphorylation sites Ser(176)/Ser(180) and on the Thr(23) site, and although phosphorylation of IKKalphaT23 is inhibited both by LY294002 and wortmannin, phosphorylation of Ser(176)/Ser(180) is not. Neither inhibition of PI 3-kinase/AKT nor IKKalphaT23A overexpression affected IkappaBalpha degradation in response to IL-1. Only partial inhibition by dominant-negative AKT and no inhibitory effect of IKKalphaT23A was observed on an IL-6 promoter-specific NFkappaB site in contrast to significant inhibitory effects on the AP-1 site. Taken together, we have discovered a novel PI 3-kinase/AKT-dependent pathway in response to IL-1, encompassing PI 3-kinase/AKT/IKKalphaT23 upstream of AP-1. This novel pathway is a parallel pathway to the PI 3-kinase/AKT upstream of NFkappaB and both are involved in IL-6 gene transcription in response to IL-1.

Molecular characterization and expression patterns of AMP deaminase1 ( AMPD1) in porcine skeletal muscle

AMPD1 is the muscle-specific form of the AMPD multigene families in mammals and plays an important role in the purine nucleotide cycle and energy metabolism in skeletal muscle. In this study, we cloned and characterized AMPD1 from Sus scrofa muscle. The promoter of porcine AMPD1 contained several putative muscle-specific transcription factor binding sites (E box, myogenin, MEF2, Spl-CTF/NF-l), one RORα2 binding motif and NF-kappaB site. The deduced amino acid sequence of porcine AMPD1 contains an AMP deaminase signature sequence (SLSTDDP). RT-PCR analyses showed that AMPD1 was expressed specifically in skeletal muscle. Expression of AMPD1 was up-regulated during the muscle development and was higher in Yorkshire than in Meishan pigs. AMPD1 gene was expressed at higher levels in longissimus dorsi and bicepsfemoris muscles compared with soleus and masseter muscles in both Yorkshire and Meishan pigs. Moreover, we found that a single nucleotide polymorphism (SNP, T/C 426) in exon12 of the AMPD1 gene was significantly associated with loin muscle area trait ( p < 0.01), loin muscle height ( p < 0.01) and average backfat thickness ( p < 0.05). This result suggests that the AMPD1 gene might be a candidate gene of meat production trait and provides useful information for further studies on its roles in porcine skeletal muscle.

Requirements for Two Proximal NF-κB Binding Sites and IκB-ζ in IL-17A-induced Human β-Defensin 2 Expression by Conducting Airway Epithelium

Among a panel of 21 cytokines (IL-1α, -1β, -2–13, and -15–18; interferon-γ; granulocyte-macrophage colony-stimulating factor; and tumor necrosis factor α), we have recently observed that IL-17A is the most potent inducer for human β-defensin 2 (hBD-2) in conducting airway epithelial cells (Kao, C. Y., Chen, Y., Thai, P., Wachi, S., Huang, F., Kim, C., Harper, R. W., and Wu, R. (2004) J. Immunol. 173, 3482–3491). The molecular basis of this regulation is not known. In this study, we demonstrated a coordinated degradation of inhibitory κB(IκB)-α followed by a nuclear translocation of p50 and p65 NF-κB subunits and their binding to NF-κB sites of hBD-2 promoter region. With site-directed mutagenesis, we demonstrated the requirement of two proximal NF-κB binding sites (pκB1, -205 to -186; pκB2, -596 to -572) but not the distal site (dκB, -2193 to -2182) in supporting IL-17A-induced hBD-2 promoter activity. These results are consistent with the data of the chromatin immunoprecipitation assay, which showed enhanced p50 binding to these pκB sites but not the dκB site in cells after IL-17A treatment. We also found that the NF-κB binding cofactor, IκB-ζ, was up-regulated by IL-17A, and the knockdown of IκB-ζ significantly diminished the IL-17A-induced hBD-2 expression. This is the first demonstration of the involvement of two proximal NF-κB sites and IκB-ζ in the regulation of hBD-2 by IL-17A, two important genes responsible for host defense.

Molecular characterization of Helicobacter pylori VacA induction of IL-8 in U937 cells reveals a prominent role for p38MAPK in activating transcription factor-2, cAMP response element binding protein, and NF-kappaB activation.

Helicobacter pylori VacA induces multiple effects on susceptible cells, including vacuolation, mitochondrial damage, inhibition of cell growth, and enhanced cyclooxygenase-2 expression. To assess the ability of H. pylori to modulate the production of inflammatory mediators, we examined the mechanisms by which VacA enhanced IL-8 production by promonocytic U937 cells, which demonstrated the greatest VacA-induced IL-8 release of the cells tested. Inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059), IkappaBalpha ((E)-3-(4-methylphenylsulfonyl)-2-propenenitrile), Ca(2+) entry (SKF96365), and intracellular Ca(2+) channels (dantrolene) blocked VacA-induced IL-8 production. Furthermore, an intracellular Ca(2+) chelator (BAPTA-AM), which inhibited VacA-activated p38 MAPK, caused a dose-dependent reduction in VacA-induced IL-8 secretion by U937 cells, implying a role for intracellular Ca(2+) in mediating activation of MAPK and the canonical NF-kappaB pathway. VacA stimulated translocation of NF-kappaBp65 to the nucleus, consistent with enhancement of IL-8 expression by activation of the NF-kappaB pathway. In addition, small interfering RNA of activating transcription factor (ATF)-2 or CREB, which is a p38MAPK substrate and binds to the AP-1 site of the IL-8 promoter, inhibited VacA-induced IL-8 production. VacA activated an IL-8 promoter containing an NF-IL-6 site, but not a mutated AP-1 or NF-kappaB site, suggesting direct involvement of the ATF-2/CREB binding region or NF-kappaB-binding regions in VacA-induced IL-8 promoter activation. Thus, in U937 cells, VacA directly increases IL-8 production by activation of the p38 MAPK via intracellular Ca(2+) release, leading to activation of the transcription factors, ATF-2, CREB, and NF-kappaB.

Regulation of the cd38 promoter in human airway smooth muscle cells by TNF-α and dexamethasone

BackgroundCD38 is expressed in human airway smooth muscle (HASM) cells, regulates intracellular calcium, and its expression is augmented by tumor necrosis factor alpha (TNF-α). CD38 has a role in airway hyperresponsiveness, a hallmark of asthma, since deficient mice develop attenuated airway hyperresponsiveness compared to wild-type mice following intranasal challenges with cytokines such as IL-13 and TNF-α. Regulation of CD38 expression in HASM cells involves the transcription factor NF-κB, and glucocorticoids inhibit this expression through NF-κB-dependent and -independent mechanisms. In this study, we determined whether the transcriptional regulation of CD38 expression in HASM cells involves response elements within the promoter region of this gene.MethodsWe cloned a putative 3 kb promoter fragment of the human cd38 gene into pGL3 basic vector in front of a luciferase reporter gene. Sequence analysis of the putative cd38 promoter region revealed one NF-κB and several AP-1 and glucocorticoid response element (GRE) motifs. HASM cells were transfected with the 3 kb promoter, a 1.8 kb truncated promoter that lacks the NF-κB and some of the AP-1 sites, or the promoter with mutations of the NF-κB and/or AP-1 sites. Using the electrophoretic mobility shift assays, we determined the binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-κB, AP-1, and GRE sites, and the specificity of this binding was confirmed by gel supershift analysis with appropriate antibodies.ResultsTNF-α induced a two-fold activation of the 3 kb promoter following its transfection into HASM cells. In cells transfected with the 1.8 kb promoter or promoter constructs lacking NF-κB and/or AP-1 sites or in the presence of dexamethasone, there was no induction in the presence of TNF-α. The binding of nuclear proteins to oligonucleotides encoding the putative cd38 NF-κB site and some of the six AP-1 sites was increased by TNF-α, and to some of the putative cd38 GREs by dexamethasone.ConclusionThe EMSA results and the cd38 promoter-reporter assays confirm the functional role of NF-κB, AP-1 and GREs in the cd38 promoter in the transcriptional regulation of CD38.

A PPAR-independent pathway to PUFA-induced COX-2 expression

Polyunsaturated fatty acids (PUFAs) induce COX-2 in bovine endometrial stromal cells through activation of peroxisome-proliferator-activated receptor alpha (PPARalpha). We have investigated alternative (PPAR-independent) pathways to COX-2 induction using a reporter construct driven by a COX-2 gene promoter sequence lacking a PPAR response element. This construct was induced by PUFAs, but not by PPAR agonists. PPAR-independent reporter gene expression occurred 6h after PPAR-dependent induction of the endogenous COX-2 gene. In contrast to PPAR-dependent COX-2 induction, which is not affected by NF-kappaB inhibitors, the PPAR-independent pathway was blocked by the NF-kappaB inhibitor MG132 or following deletion of NF-kappaB sites in the COX-2 promoter. The PPAR-independent effect of PUFA was mimicked by the PKC activators 4beta-PMA and prostaglandin F(2alpha), but was not blocked by the PKC inhibitor RO318425. The results demonstrate a pathway to the induction of COX-2 by PUFAs requiring NF-kappaB but not PPAR or PKC.

B-cell Receptor Activation Induces BIC/miR-155 Expression through a Conserved AP-1 Element

microRNA-155 is an oncogenic microRNA that has been shown to be critical for B-cell maturation and immunoglobulin production in response to antigen. In line with its function in B-cell activation, miR-155, and its primary transcript, B-cell integration cluster (BIC), is induced by B-cell receptor (BCR) cross-linking. Using pharmacological inhibitors in the human B-cell line, Ramos, we show that activation of BIC and miR-155 expression by BCR signaling occurs through the extracellular signaling-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways but not the p38 pathway. BCR activation results in the induction of c-Fos, FosB, and JunB, and expression of these are suppressed by ERK and JNK inhibitors. Reporter analysis established a key role for a conserved AP-1 site approximately 40 bp upstream from the site of initiation but not an upstream NF-kappaB site or a putative c-Ets located at the site of initiation. Lastly, chromatin immunoprecipitation analysis demonstrated the recruitment of FosB and JunB to the miR-155 promoter following BCR activation. These results identify key determinants of BCR-mediated signaling that lead to the induction of BIC/miR-155.

Molecular characterization, chromosomal and expression patterns of three aquaglyceroporins (AQP3, 7, 9) from pig

As one subgroup of aquaporin, aquaglyceroporin including AQP3, 7, 9, 10 facilitates glycerol transport as well as water transport. In this study, we cloned the full length coding sequences of porcine (Sus scrofa) AQP3, 7 and 9 and the genomic sequence of AQP3 including 6 exons and 5 introns. Additionally, as a first step toward understanding the regulatory mechanisms of AQP9 in pig, we cloned and analyzed the upstream genomic sequence of the ATG translation initiation codon and found two negative insulin response elements (TGTTTTC and TATTTTG.), glucocorticoid-responsive elements, several CCAAT enhancer binding protein (C/EBP) sites, hepatocyte nuclear factor (HNF) sites, and NF-kappaB sites in this region. Subsequently, semi-quantitative analysis showed that AQP3 selectively expressed in spleen, stomach, kidney and lung. AQP7 and AQP9 were ubiquitously detected in all tissues examined and highly expressed in adipose tissue and liver, respectively. Finally, both AQP3 and AQP7 were assigned to chromosome 10q while AQP9 was mapped to chromosome 1q. This is the first report of molecular characterization of aquaglyceroporin in pig, which provides basic observations useful for future assessing and characterizing the role of aquaglyceroporin.

Genetic and Functional Characterization of the LTR of HIV-1 Subtypes A and C Circulating in India

Genetic analysis of HIV-1 sequences circulating in different parts of India have shown that the predominant proportion of HIV-1 subtypes circulating in India is type C and a small fraction are subtypes A, B, E, and CRFs. We sequenced the HIV-1 LTR promoter region of seven subtype C and five subtype A isolates obtained from two major cities in India. Sequence analysis of the complete promoter and TAR regions revealed conserved subtype-specific variability in several major binding sites. Three NF-kappaB sites were present in all subtype C isolates and two isolates contained an insertion in the MFNLP. The transcriptional activity of one of these isolates may have been hindered due to this insertion. Despite the apparent variability between the LTRs we did not observe any significant difference in the transcriptional activity between subtype C and subtype A. To our knowledge, this is the first study characterizing the genetic structure and functional attributes of subtype A LTRs from India.

Expression of a pathogen-response program in peripheral blood cells defines a subgroup of Rheumatoid Arthritis patients

Rheumatoid arthritis (RA) is a heterogeneous disease with unknown etiology. Here we aimed to distinguish RA subtypes based on peripheral blood (PB) gene expression profiles in comparison with a pathogen-response transcriptional program. PB was obtained from 35 RA patients and 15 healthy individuals. For expression profiling we used DNA microarrays. A combined cluster analysis of RA and control samples together with samples from a viral infection model revealed that the gene expression profile of a subgroup of RA patients (RA(A)) was reminiscent to that of poxvirus-infected macaques. Statistical analysis, followed by Gene Ontology analysis of the RA(A) patients confirmed that these patients form a distinct group, with activation of several host defense mechanisms that resemble a common host-pathogen response. Analysis of the promoter region of genes that were overexpressed in the RA(A) patients, revealed an enrichment of transcription factor binding sites for NF kappaB and interferon-activated transcription factors. Moreover, this subgroup of RA patients expressed significantly increased titers of anti-cyclic citrullinated peptide antibodies. We conclude that activation of a host-pathogen response defines a subgroup of RA patients characterized by increased autoreactivity against citrullinated proteins.

Varicella-zoster virus infection induces the secretion of interleukin-8

Interleukin-8 (IL-8) is an important mediator in neutrophil-mediated acute inflammation but has also a wide range of actions on various cells types. We demonstrated that infection of melanoma cells and fibroblasts with cell-associated varicella-zoster virus (VZV) and infection of a T cell line with cell-free VZV resulted in an induction of IL-8 secretion in vitro. The inhibition of the VZV replication with a drug interfering with its DNA replication had no effect on the IL-8 release. Since the IL-8 promoter contains binding sites for NF-kappaB and AP-1, melanoma cells and the T cell line were treated with inhibitors of NF-kappaB, JNK/SAPK or p38/MAPK prior to infection. In melanoma cells, the JNK/SAPK pathway was shown to be important for the IL-8 secretion during the VZV replication, whereas in the T cell line, not only the JNK/SAPK but also the p38/MAPK pathways were required for IL-8 secretion. The neutralisation of the IL-8 bioactivity had no significant consequence on the VZV replication, suggesting that IL-8 acts neither as a proviral nor as an antiviral cytokine during the VZV replication in vitro.

The Oct DNA motif participates in the alcohol inhibition of the inducible nitric oxide synthase gene promoter in rat C6 glioma cells

Induction of nitric oxide synthase-2 (iNOS) by cytokines and bacterial products is associated with protein binding at the proximal promoter and in an upstream enhancer region of the Nos2 gene. To clarify how ethanol suppresses rat iNOS activity, we constructed several deletion mutants of the Nos2 promoter fused to the luciferase gene and transfected the constructs into C6 glial cells. Acute ethanol exposure of stably transfected cells for 24 h inhibits induced activity of Nos2 promoter constructs containing deletions in the 5' flanking region, including a 94 bp promoter that lacks any known NF-kappaB site but which carries a C/EBPbeta and overlapping gamma-IRE, GAS and Oct motifs. Ethanol failed to inhibit the endogenous activity of a smaller, 78 bp promoter that lacks the C/EBPbeta and overlapping, gamma-IRE and GAS motifs and showed no inducible activity. As another approach, in vivo DNA footprinting was used and identified protein protections at five regions of the proximal Nos2 promoter in induced cells. Exposure to acute ethanol diminished protein occupation in the five promoter regions including the gamma-IRE/NF-kappaB and the overlapping gamma-IRE/GAS/Oct sites. Site-directed mutagenesis in the octamer domain of the gamma-IRE/GAS/Oct motifs was studied in a 1002 bp promoter to examine its role in ethanol inhibition of cytokine and lipopolysaccharide induced activity. The data indicate that ethanol failed to inhibit promoter activity when the Oct motif is missing. Electrophoretic mobility shift assays performed using a 22-mer probe containing the overlapping gamma-IRE/GAS/Oct sites showed three complexes with one of the complexes being competed by an octamer-1 antibody. These observations demonstrate the role of protein-DNA binding at the core promoter, and the likely involvement of the octamer motif, in ethanol modulation of cytokine and lipopolysaccharide induced iNOS expression.

TREM‐1 expression in macrophages is regulated at transcriptional level by NF‐κB and PU.1

Triggering receptor expressed on myeloid cells (TREM)-1 is a recently identified immunoglobulin receptor that is expressed on neutrophils and monocytes where it amplifies the acute inflammatory response to bacteria. We examined the transcriptional regulation of TREM-1 in macrophages. Treatment of RAW cells with Escherichia coli LPS or Pseudomonas aeruginosa led to the induction of TREM-1 within 1 h with an expression lasting up to at least 24 h in vitro as detected by RT-PCR. Since the promoter of TREM-1 has multiple binding sites for NF-kappaB and PU.1 (one of the members of the ets family of transcription factors), we investigated the role of these transcription factors in the induction of TREM-1. Treatment of cells with NF-kappaB inhibitors abolished the expression of message of TREM-1 induced by LPS and P. aeruginosa. In contrast, the expression of TREM-1 was increased after stimulation with LPS or P. aeruginosa in cells that had gene of PU.1 silenced. Additionally, over-expression of PU.1 led to inhibition of TREM-1 induction in response to LPS and P. aeruginosa. These data suggest that both these transcription factors are involved in the expression of TREM-1. NF-kappaB functions as a positive regulator whereas PU.1 is a negative regulator of the TREM-1 gene.

1,25-Dihydroxyvitamin D3 targeting of NF-κB suppresses high glucose-induced MCP-1 expression in mesangial cells

Macrophages accumulate in kidney glomeruli and interstitium of patients with diabetic nephropathy in response to monocyte chemoattractant protein-1 (MCP-1); a chemokine produced by both tubular epithelial and mesangial cells (MCs). Vitamin D and its analogs have been shown to have renoprotective effects; however, there are few studies involving diabetic nephropathy. We explored mechanisms by which 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) can be renoprotective by measuring MCP-1 expression in MCs. Using a luciferase reporter assay, we found that high glucose (HG)-induced MCP-1 transcription and that this induction is blocked by 1,25(OH)2D3. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that HG increased the p65/p50 binding to the two NF-kappaB sites within the promoter. This was suppressed by 1,25(OH)2D3, but this decrease was reversed by overexpression of p65. 1,25(OH)2D3 was found to stabilize IkappaBalpha leading to an inhibition of p65 translocation to the nucleus and subsequent reduction of NF-kappaB binding. In primary MCs prepared from vitamin D receptor knockout animals, basal MCP-1 levels were elevated but not affected by 1,25(OH)2D3. The analog paricalcitol inhibited the induction and activity of MCP-1 while ameliorating glomerulosclerosis in streptozotocin-diabetic mice. Our results suggest that 1,25(OH)2D3 might block hyperglycemia-induced renal injury by blunting NF-kappaB activation.

The Induction of Growth Arrest DNA Damage-Inducible Gene 45 β in Human Hepatoma Cell Lines by S-Adenosylmethionine

Down-regulation of GADD45beta, which is known to influence cell growth control, apoptosis, and cellular response to DNA damage, has been verified to be specific in hepatocellular carcinoma and consistent with the degree of malignancy. Here, we identified promoter elements for several transcriptional factors in the proximal promoter of GADD45beta using the luciferase assay. As a methyl donor for biological transmethylation reactions, S-adenosylmethionine (SAMe) could restore GADD45beta expression in HepG2 in Northern blot analyses and quantitative real-time polymerase chain reaction. Activity and binding capacity of nuclear factor (NF)-kappaB were confirmed to be specifically induced by SAMe, as evidenced by electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, and a decrease of IkappaBalpha in Western blot analyses. The most upstream NF-kappaB-binding site was crucial for transcriptional activation. In contrast to NF-kappaB, although there is an E2F-1-binding site adjacent to the NF-kappaB sites, treatment with SAMe could not induce E2F-1-binding activity. Despite showing a similar GADD45beta promoter regulatory pattern as HepG2 (p53 wild type), Hep3B (p53-null) did not exhibit GADD45beta induction by SAMe, and the induction could be partially recovered on reconstituting p53 in Hep3B. Thus, our results suggest that GADD45beta induction by SAMe via NF-kappaB may represent a novel mechanism of SAMe-mediated hepatoprotection, with p53 playing an important role.

Repression of Dmp1 and Arf transcription by anthracyclins: critical roles of the NF-κB subunit p65

Both genotoxic and oncogenic stress activates the nuclear factor-kappa B (NF-kappaB) and p53 proteins; however, the p53 activity is antagonized by NF-kappaB signaling. Dmp1 is a Myb-like transcription factor that activates the Arf-p53 pathway. The Dmp1 promoter was activated by a classical NF-kappaB activator tumor necrosis factor alpha, but repressed by treatment of cells with non-classical NF-kappaB activators, anthracyclins and UV-C. p65 and other subsets of NF-kappaB proteins were bound to the Dmp1 promoter following anthracyclin/UV-C treatment of rodent fibroblasts. This resulted in the downregulation of Dmp1 mRNA and protein. Repression of the Dmp1 transcription by anthracyclins depended on the unique NF-kappaB site on the promoter. Downregulation of p65 significantly attenuated the repression of the Dmp1 promoter by anthracyclins/UV-C. The amount of Dmp1 bound to the Arf promoter decreased significantly upon anthracyclin treatment; this, in turn, downregulated the Arf levels. Repression of the Arf promoter by p65 or anthracyclins depended on Dmp1, which was significantly attenuated in Dmp1(-/-) cells. Both Dmp1(-/-)and Arf(-/-)cells showed resistance to anthracyclin-induced cell death compared to wild-type cells; non-immortalized p65-knockdown cells were much more sensitive. Thus, the Dmp1-Arf pathway is repressed by p65 in response to genotoxic stress, which implicates a novel mechanism of p53 inactivation by NF-kappaB.

Geranylgeranylacetone Induces Cyclooxygenase-2 Expression in Cultured Rat Gastric Epithelial Cells Through NF-κB

Geranylgeranylacetone (GGA) effectively protects the gastric mucosa against noxious agents. The precise mechanisms underlying the gastroprotective actions of GGA are not known. To elucidate the precise mechanism of GGA, the effect of GGA treatment on COX-2 expression in rat gastric epithelial (RGM1) cells was investigated. We used a prostaglandin E2 (PGE2) enzyme-linked immunoassay kit and Western blot analysis to measure PGE2 production and COX-2 induction by GGA treatment in serum-starved RGM1 cells. Gel-shift assay, Western blot analysis, and a reporter assay were performed to determine which COX-2 promoter was involved in GGA-induced COX-2 expression. GGA treatment dose dependently increased COX-2 expression and PGE2 production. The nuclear factor (NF)-kappaB sites of the COX-2 gene promoter were critical for GGA-mediated COX-2 expression. GGA induces COX-2 expression and increases PGE2 production in serum-starved RGM1 cells via activation of the NF-kappaB sites of COX-2 gene promoters.

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.

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.