Nuclear factor-kappaB Binding Site Research Articles

580. Prevention of Airway Inflammation by Simultaneous Inhibition of NFkB and STST6 Using Chimeric Decoy Oligonucleotides in a Mouse Model of Asthma

Allergic asthma is a common inflammatory disease of the airways characterized by reversible airflow obstruction and airway hyperresponsiveness (AHR) to bronchoconstrictor stimuli. The pathophysiology in asthma mainly occurs as a consequence of overexpression of the TH2 cytokines. These inflammatory factors are tightly regulated by a transcriptional network system. Nuclear factor kappaB (NFkB) and STAT6 are well known to regulate a set of gene associated with inflammatory and immune responses, and is thought to play an important role in the induction of allergic asthma. Therefore, we focused on the simultaneous inhibition of these important transcription factors using a decoy strategy to develop a novel therapeutic approach for treating asthma. For increased efficacy of decoy oligodeoxynucleotides (ODN) in vivo, we employed chimeric decoy ODN containing consensus sequences of both NFkB and STAT6 binding sites. In addition, two strands of decoy ODN were combined by the chemical spacer to increase its resistance to endonuclease for intratracheal administration. The therapeutic effect of chimeric decoy ODN was investigated using OVA-induced experimental asthma in mice. Mice were sensitized by intraperitoneal injections of 20 ug of ovalbumin (OVA) and 2 mg aluminum hydroxide constituted in 0.1 ml of saline on days 0 and 14. On days 21, 22 and 23, mice were challenged with 1% OVA aerosol for 20 min. At 24 hours after the last challenge, AHR was measured by methacholine-induced airflow obstruction. Intratracheal administration of decoy ODN was performed in OVA-sensitized mice at 3 days before the first challenge with aerosolized OVA. FITC-labeled chimeric decoy ODN could be detected in macrophages and monocytes migrating into the lung and airway, and NFkB and STAT6 activity were simultaneously inhibited by chimeric decoy ODN. Twenty-four hours after the last OVA challenge, treatment with chimeric or single transfection of NFkB decoy ODN was protected from methacholine-induced AHR, while mice treated with scrambled decoy ODN or saline developed a significantly increase in airway reactivity to methacholine. Importantly, this inhibitory effect of chimeric decoy ODN on airway hyperresponsiveness was significantly greater than that of NFkB decoy ODN. Treatment with chimeric decoy ODN markedly suppressed airway inflammation after OVA sensitization and challenge as compared with control and scrambled decoy ODN treatment. Inflammatory infiltrate, such as macrophage, was significantly inhibited by chimeric decoy ODN through suppression of ICAM-1 and eotaxin expression. In addition, secretion of Th2 cytokines including IL-4, IL-5 and IL-13 in BALF, and histamine in the whole lung were reduced by chimeric decoy ODN. Furthermore, a significant reduction of mucin secretion was observed by chimeric decoy ODN treatment accompanied by a suppression of MUC5AC gene expression. However, intratracheal administration of chimeric decoy ODN did not affected IgE synthesis. The present study provides a novel strategy for treating bronchial asthma by the simultaneous inhibition of both NFkB and STAT6 using chimeric decoy ODN. Further modification of chimeric decoy ODN would be useful to treat asthma as a decoy-based therapy.

Molecular mechanisms of syndecan-4 upregulation by TNF- in the endothelium-like EAhy926 cells

Syndecan-4, a cell-surface heparan sulfate proteoglycan, can participate in inflammation and wound healing as a host defense molecule. Tumour necrosis factor (TNF)-α, one of the most potent proinflammatory cytokines, is known to upregulate syndecan-4 expression, but the precise mechanisms are unclear. To elucidate these mechanisms in detail, we examined syndecan-4 upregulation by TNF-α in the endothelium-like EAhy926 cell. Of the two putative nuclear factor kappa-B (NF-κB) binding sites in the syndecan-4 gene (SDC4) promoter, deletion or mutation of one or both sites significantly diminished the effects of TNF-α. Electrophoretic mobility shift assays showed that p65 and c-Rel, but not p50, bound to these NF-κB binding sites, whereas pull-down assays showed binding of all three NF-κB components. Chromatin immunoprecipitation assays clearly showed that p65 and phosphorylated p65, but not p50 or c-Rel, bound to the SDC4 promoter. An NF-κB inhibitor, p65 knockdown and a transcriptional elongation inhibitor completely blocked the effect of TNF-α on SDC4 promoter activity and significantly, but not completely, blocked that on SDC4 mRNA expression. These data suggest that NF-κB p65 could be a key mediator of syndecan-4 upregulation by TNF-α through two binding sites in the SDC4 promoter, but other NF-κB-p65 independent pathways might also be involved through transcriptional elongation.

Interleukin-1 alpha produced by human T-cell leukaemia virus type I-infected T cells induces intercellular adhesion molecule-1 expression on lung epithelial cells

The pathogenic mechanism of human T-cell leukaemia virus type I (HTLV-I)-related pulmonary disease, which involves overexpression of intercellular adhesion molecule-1 (ICAM-1) in lung epithelial cells, was investigated. The supernatant of HTLV-I-infected Tax(+) MT-2 and C5/MJ cells induced ICAM-1 expression on A549 cells, a human tumour cell line with the properties of alveolar epithelial cells. Neutralization of ICAM-1 partially inhibited HTLV-I-infected T-cell adhesion to A549 cells. Analysis of the ICAM-1 promoter showed that the nuclear factor-kappa B-binding site was important for supernatant-induced ICAM-1 expression. Induction of interleukin (IL)-1 alpha (IL-1α) expression in MT-2 and C5/MJ cells was observed compared with uninfected controls and HTLV-I-infected Tax-negative cell lines. The significance of IL-1α as a soluble messenger was supported by blocking the biological activities of MT-2 supernatant with an IL-1α-neutralizing mAb. Moreover, Tax and IL-1α expression was demonstrated in the bronchoalveolar lavage cells of patients with HTLV-I-related pulmonary disease. Immunohistochemistry confirmed ICAM-1 and IL-1α expression in lung epithelial cells and lymphocytes of patients with HTLV-I-related pulmonary diseases, and in a transgenic mouse model of Tax expression. These results suggest that IL-1α produced by HTLV-I-infected Tax(+) T cells is crucial for ICAM-1 expression in lung epithelial cells and subsequent adhesion of lymphocytes in HTLV-I-related pulmonary diseases.

Transforming growth factor‐β stimulates the expression of eotaxin/CC chemokine ligand 11 and its promoter activity through binding site for nuclear factor‐κB in airway smooth muscle cells

Chemokines ligands of CCR3 including eotaxin/CC chemokine ligand 11 (CCL11) may contribute to the pathogenesis of asthma. These chemokines and a growth factor (TGF-beta) may be involved in the process of airway remodelling. We analysed the effects of TGF-beta on the expression of CCR3 ligands in human airway smooth muscle (HASM) cells and investigated the mechanisms. HASM cells were cultured and treated with TGF-beta and Th2 cytokines IL-4 or IL-13. Expression of mRNA was analysed by real-time PCR. Secretion of CCL11 into the culture medium was analysed by ELISA. Transcriptional regulation of CCL11 was analysed by luciferase assay using CCL11 promoter-luciferase reporter plasmids. IL-4 or IL-13 significantly up-regulated the expression of mRNAs for CCL11 and CCL26. TGF-beta alone did not increase the expression of chemokine mRNAs, but enhanced the induction of only CCL11 by IL-4 or IL-13 among CCR3 ligands. Activity of the CCL11 promoter was stimulated by IL-4, and this activity was enhanced by TGF-beta. Activation by IL-4 or IL-4 plus TGF-beta was lost by mutation of the binding site for signal transducers and activators of transcription-6 (STAT6) in the promoter. Cooperative activation by IL-4 and TGF-beta was inhibited by mutation of the binding site for nuclear factor-kappaB (NF-kappaB) in the promoter. Pretreatment with an inhibitor of NF-kappaB and glucocorticoid fluticasone propionate significantly inhibited the expression of CCL11 mRNA induced by IL-4 plus TGF-beta, indicating the importance of NF-kappaB in the cooperative activation of CCL11 transcription by TGF-beta and IL-4. These results indicate that Th2 cytokines and TGF-beta may contribute to the pathogenesis of asthma by stimulating expression of CCL11. The transcription factors STAT6 and NF-kappaB may play pivotal roles in this process.

Helicobacter pylori CagL Activates ADAM17 to Induce Repression of the Gastric H, K-ATPase α Subunit

Infection with Helicobacter pylori represses expression of the gastric H, K-adenosine triphosphatase alpha-subunit (HKalpha), which could contribute to transient hypochlorhydria. CagL, a pilus protein component of the H pylori type IV secretion system, binds to the integrin alpha(5)beta1 to mediate translocation of virulence factors into the host cell and initiate signaling. alpha(5)beta1 binds a disintegrin and metalloprotease (ADAM) 17, a metalloenzyme that catalyzes ectodomain shedding of receptor tyrosine kinase ligands. We investigated whether H pylori-induced repression of HKalpha is mediated by CagL activation of ADAM17 and release of heparin-binding epidermal growth factor (HB-EGF).HKalpha promoter and ADAM17 activity were measured in AGS gastric epithelial cells transfected with HKalpha promoter-reporter constructs or ADAM17-specific small interfering RNAs and infected with H pylori. HB-EGF secretion was measured by enzyme-linked immunosorbent assay analysis, and ADAM17 interaction with integrins was investigated by coimmunoprecipitation analyses.Infection of AGS cells with wild-type H pylori or an H pylori cagL-deficient isogenic mutant that also contained a wild-type version of cagL (P12DeltacagL/cagL) repressed HKalpha promoter-Luc reporter activity and stimulated ADAM17 activity. Both responses were inhibited by point mutations in the nuclear factor-kappaB binding site of HKalpha or by infection with P12DeltacagL. Small interfering RNA-mediated silencing of ADAM17 in AGS cells inhibited the repression of wild-type HKalpha promoter and reduced ADAM17 activity and HB-EGF production, compared to controls. Coimmunoprecipitation studies of AGS lysates showed that wild-type H pylori disrupted ADAM17-alpha5beta1 complexes.During acute H pylori infection, CagL dissociates ADAM17 from the integrin alpha(5)beta1 and activates ADAM17-dependent, nuclear factor-kappaB-mediated repression of HKalpha. This might contribute to transient hypochlorhydria in patients with H pylori infection.

An IgE-associated polymorphism in STAT6 alters NF-κB binding, STAT6 promoter activity, and mRNA expression

The IL-4/IL-13 pathway is central for IgE regulation. Signal transducer and activator of transcription 6 (STAT6) is the major transcription factor within this pathway. STAT6 polymorphisms were recently associated with elevated total IgE levels in a genome-wide association study. This study aimed to assess biological mechanisms by which an IgE-associated genetic variation in STAT6 may potentially influence gene expression. STAT6 intron 2 carrying either the wild-type C or the polymorphic T allele of the putatively causal single nucleotide polymorphism rs324011 was cloned into STAT6 promoter vectors to investigate their influence on STAT6 promoter activity by in vitro luciferase assays. Transcription factor binding depending on rs324011 was examined by electrophoretic mobility shift assays in Jurkat T cells and primary CD4(+) T cells. Allele-specific STAT6 gene expression of 3 splice variants was studied ex vivo by real-time PCR in 239 individuals. STAT6 intron 2 acts as a silencer regulatory element. The polymorphic T allele at rs324011 (in linkage disequilibrium with the genome-wide association signal and consistently associated with elevated IgE levels in 3 previous studies) increases STAT6 promoter activity significantly in vitro (P < .00001) and gene expression of STAT6 splice variants ex vivo (P < .01) compared with the wild-type C allele. These effects correlate with the creation of a novel, T-allele-specific binding site for the transcription factor nuclear factor-kappaB in T cells. The consistently replicated effects of genetic variance in STAT6 on IgE regulation may be explained in part by allele-specific alterations in nuclear factor-kappaB binding at rs324011 and consecutive changes in STAT6 gene expression.

Homeobox Gene CDX2 Inhibits Human Pancreatic Cancer Cell Proliferation by Down-Regulating Cyclin D1 Transcriptional Activity

Homeobox gene caudal related homeobox gene 2 (CDX2) is an intestine-specific tumor suppressor gene. This study is intended to investigate the effect of CDX2 expression on cell proliferation and cyclin D1 expression in pancreatic cancer cells. Four pancreatic ductal adenocarcinoma cell lines (PancQGO-1, BxPC-3, MIAPaCa-2, CFPAC-1), 1 islet carcinoma cell line (QGP-1), and 1 adenosquamous carcinoma cell line (KP-3) were analyzed for CDX1 and CDX2 expression using real-time reverse transcription-polymerase chain reaction and Western blot analysis. Proliferation of pancreatic cancer cells was analyzed using WST-1 assay after CDX2 transfection. Luciferase assay was performed to examine the effects of CDX2 on cyclin D1 transcriptional activity. CDX2 was expressed at a significantly higher level in QGP-1 cells than in KP-3 cells. Moreover, CDX2 was expressed at a middle level in 4 pancreatic ductal adenocarcinoma cells. Cell proliferation and cyclin D1 mRNA level were inhibited significantly after CDX2 transfection in pancreatic cancer cells. Furthermore, CDX2 inhibited exogenous nuclear factor kappaB-p65-induced luciferase gene expression in a dose-dependent manner. In addition, CDX2 inhibited pGL2HIVD1kappaB2-luciferase activity. CDX2 might play a role in inhibiting cell proliferation and repressing cyclin D1 transcriptional activity through the proximal nuclear factor kappaB binding site in pancreatic cancer cells.

A Novel Mitogen-Activated Protein Kinase Phosphatase-1 and Glucocorticoid Receptor (GR) Interacting Protein-1-Dependent Combinatorial Mechanism of Gene Transrepression by GR

Glucocorticoids have major antiinflammatory effects. Because COX-2 is the rate-limiting enzyme for proinflammatory prostaglandins, this study investigated the combinatorial inhibitory role of glucocorticoid receptor (GR) in COX-2 gene induction in macrophages and sought to identify the molecular mechanisms for that inhibition. Glucocorticoid-activated GR repressed COX-2 gene induction by lipopolysaccharide (LPS). Activated GR inhibited LPS-induced activator protein 1 activity, which in turn decreased activating transcription factor 2/c-Jun phosphorylation. The inhibition of MAPK-dependent activating transcription factor 2/c-Jun phosphorylation by GR in COX-2 repression was a result of MAPK phosphatase-1 (MKP-1) induction. Although GR did not inhibit LPS-induced p65 phosphorylation or nuclear factor-kappaB DNA binding activity, deletion of the nuclear factor-kappaB binding site in the COX-2 gene suppressed the ability of glucocorticoid to attenuate COX-2 induction. Chromatin immunoprecipitation and transfection assays revealed that a p65 DNA complex involving GR-bound GR-interacting protein 1 (GRIP1) also contributed to COX-2 repression. Additional knockdown and transfection assays identified other inflammatory genes coordinately regulated by MKP-1 and GRIP1. In summary, activated GR was found to antagonize the LPS-dependent induction of the COX-2 gene via a novel combinatorial mechanism involving MKP-1-mediated activator protein 1 inhibition and GR/GRIP1 recruitment to the p65 DNA complex; moreover, this work facilitated the identification of other GR-responding MKP-1/GRIP1-regulated genes.

Involvement of nuclear factor‐kappa B in bcl‐xL‐induced interleukin 8 expression in glioblastoma

We recently reported that bcl-xL regulates interleukin 8 (CXCL8) protein expression and promoter activity in glioblastoma cells. In this paper we demonstrate that CXCL8 induction by bcl-xL is mediated through a nuclear factor-kappa B (NF-kB)-dependent mechanism. Mutational studies on the CXCL8 promoter showed that NF-kB binding site was required for bcl-xL-induced promoter activity and an enhanced nuclear expression of NF-kB subunits p65 and p50 was observed after bcl-xL over-expression. Electrophoretic mobility shift assay showed an increased DNA-binding activity of NF-kB in bcl-xL over-expressing cells and the use of specific antibodies confirmed the involvement of p65 and p50 in NF-kB activity on CXCL8 promoter sequence. NF-kB activity regulation by bcl-xL involved IkBalpha and IKK complex signaling pathway. In fact, bcl-xL over-expression induced a decrease of cytoplasmic expression of the IkBalpha protein, paralleled by an increase in the phosphorylation of the same IkBalpha and IKKalpha/beta. Moreover, the down-regulation of the ectopic or endogenous bcl-xL expression through RNA interference confirmed the ability of bcl-xL to modulate NF-kB pathway, and the transient expression of a degradation-resistant form of the cytoplasmic NF-kB inhibitor IkBalpha in bcl-xL transfectants confirmed the involvement of that inhibitor in bcl-xL-induced CXCL8 expression and promoter activity. In conclusion, our results demonstrate the role of NF-kB as the mediator of bcl-xL-induced CXCL8 up-regulation in glioblastoma cells.

Signaling pathway for TNF-α-induced MMP-9 expression: Mediation through p38 MAP kinase, and inhibition by anti-cancer molecule magnolol in human urinary bladder cancer 5637 cells

We investigated the molecular mechanisms involved in the expression of matrix metalloproteinase-9 (MMP-9) and the inhibition of MMP expression by magnolol in 5637 human urinary bladder cancer cells. Tumor necrosis factor-α (TNF-α) stimulated the secretion of MMP-9 in 5637 cells, as shown by zymography and promoter assay. The transcription factor nuclear factor kappaB (NF-κB) binding site was identified by gel-shift assay to be a cis-element for TNF-α activation of the MMP-9 promoter. Our results also demonstrated that TNF-α stimulates MMP-9 expression via the p38 MAP kinase signaling pathway in 5637 cells. Moreover, p38 MAP kinase-mediated MMP-9 gene regulation in response to TNF-α is involved in the NF-κB response element in 5637 cells. In addition, magnolol inhibited TNF-α-induced expression of the MMP-9, as determined by zymography and immunoblot, in 5637 cells. The TNF-α-induced invasion and migration of cells was inhibited by magnolol, as assessed by a modified boyden chamber and wound-healing assays, respectively. Finally, magnolol blocked MMP-9 expression, at least in part, by decreasing the binding of transcription factor NF-κB to DNA. In conclusion, TNF-α induced MMP-9 expression in 5637 cells by activating the transcription factor NF-κB, which is involved in the p38 MAP kinase-mediated control of MMP-9 regulation. Magnolol inhibited MMP-9 expression through the transcription factor NF-κB in TNF-α-induced 5637 cells.

Transcriptional Regulation of Deoxynivalenol-Induced IL-8 Expression in Human Monocytes

The trichothecene mycotoxin deoxynivalenol (DON), commonly present in contaminated grains worldwide, induces expression of the chemokine interleukin (IL)-8 in human monocytes. The purpose of this study was to test the hypothesis that DON modulates transcriptional and posttranscriptional regulation of IL-8 expression in the U937 human monocyte model. When U937 cells were transfected with a wild-type IL-8 promoter luciferase construct (-162/+44 IL-8 LUC) and incubated with DON (1 mug/ml) or the positive control, lipopolysaccharide (LPS) (1 mug/ml), there was a significant increase in luciferase expression. Mutation of the nuclear factor-kappaB (NF-kappaB) binding site significantly impaired both DON- and LPS-induced luciferase expression. In contrast, mutating the activator protein-1 binding site resulted in significantly increased DON- and LPS-induced luciferase expression. CCAAT/enhancer-binding protein beta, octamer-1, or NF-kappaB repressing factor binding site mutations did not affect DON-induced luciferase activity. Consistent with reporter studies, the NF-kappaB inhibitor caffeic acid phenethyl ester completely ablated both DON-induced IL-8 mRNA and protein expression. When NF-kappaB subunit binding to a specific IL-8 promoter probe was evaluated by enzyme-linked immunosorbent assay (ELISA), DON was observed to increase p65 binding by 21-fold, have no effect on p50 binding and decrease p52 binding. DON was not found to stabilize IL-8 mRNA in U937 cells. Taken together, these data suggest that DON-induced IL-8 expression is likely to be mediated at the transcriptional level by NF-kappaB, specifically p65, but does not appear to involve mRNA stabilization.

Transcriptional regulation of parathyroid hormone-related protein promoter P2 by NF-κB in adult T-cell leukemia/lymphoma

Parathyroid hormone-related protein (PTHrP) plays a primary role in the development of humoral hypercalcemia of malignancy (HHM) that occurs in the majority of patients with adult T-cell leukemia/lymphoma (ATLL) due to human T-cell lymphotropic virus type-1 (HTLV-1) infection. We previously showed that ATLL cells constitutively express high levels of PTHrP via activation of promoters P2 and P3, resulting in HHM. In this study, we characterized a nuclear factor-kappaB (NF-kappaB) binding site in the P2 promoter of human PTHrP. Using electrophoretic mobility shift assays, we detected a specific complex in Tax-expressing human T cells composed of p50/c-Rel, and two distinct complexes in ATLL cells consisting of p50/p50 homodimers and a second unidentified protein(s). Chromatin immunoprecipitation assays confirmed in vivo binding of p50 and c-Rel on the PTHrP P2 promoter. Using transient co-transfection with NF-kappaB expression plasmids and PTHrP P2 luciferase reporter-plasmid, we showed that NF-kappaB p50/p50 alone and p50/c-Rel or p50/Bcl-3 cooperatively upregulated the PTHrP P2 promoter. Furthermore, inhibition of NF-kappaB activity by Bay 11-7082 reduced PTHrP P2 promoter-initiated transcripts in HTLV-1-infected T cells. In summary, the data demonstrated that transcriptional regulation of PTHrP in ATLL cells can be controlled by NF-kappaB activation and also suggest a Tax-independent mechanism of activation of PTHrP in ATLL.

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.

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.

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-κB) binding site of pGL1.3-Luc and pNF-κB-Luc by lipofection method. Luciferase activity of the cells was measured in the presence or absence of sCD40L or sCD40L and an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC). Finally NF-κB 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-κB-Luc and pGL1.3-Luc was significantly elevated by stimulation with sCD40L and suppressed by PDTC. NF-κB 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-κB p50 in the SS SG cells.

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.

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.

Bangladeshi Medicinal Plant Extracts Inhibiting Molecular Interactions between Nuclear Factors and Target DNA Sequences Mimicking NF-kB Binding Sites

Several medicinal plants can be employed to produce extracts exhibiting biological effects. The aim of this work was to verify the ability of extracts derived from different medicinal plants of Bangladesh in interfering with specific DNA-protein interactions. The rationale for this study is based on the observation that alteration of gene transcription represents a very promising approach to control the expression of selected genes and could be obtained using different molecules acting on the interactions between DNA and transcription factors (TFs). We have analysed the antiproliferative activity of extracts from the medicinal plants Hemidesmus indicus, Polyalthia longifolia, Aphanamixis polystachya, Moringa oleifera, Lagerstroemia speciosa, Paederia foetida, Cassia sophera, Hygrophila auriculata and Ocimum sanctum. Antiproliferative activity was assayed on different human cell lines, including erythroleukemia K562, B-lymphoid Raji, T-lymphoid Jurkat and erythroleukemia HEL cell lines. We employed the electrophoretic mobility shift assay (EMSA) as a suitable technique for the identification of plant extracts altering the binding between transcription factors and the specific DNA elements. We found that low concentrations of Hemidesmus indicus, Polyalthia longifolia, Moringa oleifera and Lagerstroemia speciosa, and very low concentrations of Aphanamixis polystachya extracts inhibit the interactions between nuclear factors and target DNA elements mimicking sequences recognized by the nuclear factor kappaB (NF-kappaB). On the contrary, high amount of extracts from Paederia foetida, Cassia sophera, Hygrophila auriculata or Ocimum sanctum were unable to inhibit NF-kappaB/DNA interactions. Extracts inhibiting both NF-kappaB binding activity and tumor cell growth might be a source for anti-tumor compounds, while extracts inhibiting NF-kappaB/DNA interactions with lower effects on cell growth, could be of interest in the search of compounds active in inflammatory diseases, for which inhibition of NF-kappaB binding activity without toxic effects should be obtained.

Molecular cloning of TRAF2 binding protein gene and its promoter region from the grass carp Ctenopharyngodon idellus

A tumor necrosis factor receptor-associated factor 2 binding protein (T2BP) gene was isolated from the grass carp (Ctenopharyngodon idellus) by utilizing suppression subtractive hybridization (SSH) and rapid amplification of cDNA ends (RACE). The grass carp T2BP (GT2BP) gene contains an open reading frame of 579 nucleotide(s) (nt), encoding 193 amino acids, with 23 nt 5'-untranslated region and a long 3'-untranslated region of 434 nt including poly (A), 1 AUUUA motif and 4 AUUUUA motifs. No signal peptide has been detected in the predicted GT2BP, but a characteristic forkhead associated domain is present. The GT2BP mRNA shares 83% identity with the zebrafish DNA sequence, and they both have no introns in the genomic DNA. The putative transcription factor binding sites of GT2BP include two C/EBP alpha binding sites, and one c-Jun binding, one AP-1 binding, and one nuclear factor kappaB (NF kappaB) binding sites. Southern blot analysis revealed that the GT2BP was a single-copy gene. Individual difference was observed in GT2BP expression in examined organs of healthy grass carp. However, the expression of GT2BP in all examined organs in a fish with the highest copepod infection level and the significantly higher expression level in spleen and liver in infected fish may indicate its up-regulation with the parasite infection.