Oxygen-evoked changes in transcriptional activity of the 5′-flanking region of the human amiloride-sensitive sodium channel (αENaC) gene: role of nuclear factor κB

Expression of the α-subunit of the amiloride-sensitive sodium channel (αENaC) is regulated by a number of factors in the lung, including oxygen partial pressure (Po2). As transcriptional activation is a mechanism for raising cellular mRNA levels, we investigated the effect of physiological changes in Po2 on the activity of the redox-sensitive transcription factor nuclear factor κB (NF-κB) and transcriptional activity of 5′-flanking regions of the human αENaC gene using luciferase reporter-gene vectors transiently transfected into human adult alveolar carcinoma A549 cells. By Western blotting we confirmed the presence of NF-κB p65 but not p50 in these cells. Transiently increasing Po2 from 23 to 42mmHg for 24h evoked a significant increase in NF-κB DNA-binding activity and transactivation of a NF-κB-driven luciferase construct (pGLNF-κBpro), which was blocked by the NF-κB activation inhibitor sulphasalazine (5mM). Transcriptional activity of αENaC-luciferase constructs containing 5′-flanking sequences (including the NF-κB consensus) were increased by raising Po2 from 23 to 142mm Hg if they contained transcriptional initiation sites (TIS) for exons 1A and 1B (pGL3E2.2) or the 3′ TIS of exon 1B alone (pGL3E0.8). Sulphasalazine had no significant effect on the activity of these constructs, suggesting that the Po2-evoked rise in activity was not a direct consequence of NF-κB activation. Conversely, the relative luciferase activity of a construct that lacked the 3′ TIS, a 3′ intron and splice site but still retained the 5′ TIS and NF-κB consensus sequence was suppressed significantly by raising Po2. This effect was reversed by sulphasalazine, suggesting that activation of NF-κB mediated Po2-evoked suppression of transcription from the exon 1A TIS of αENaC.

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Identification and Characterization of Two Nuclear Factor-κB Sites in the Regulatory Region of the Dopamine D2 Receptor

Endocrinology ◽

10.1210/en.2006-1618 ◽

2007 ◽

Vol 148 (5) ◽

pp. 2563-2570 ◽

Cited By ~ 35

Author(s):

Sandra Bontempi ◽

Chiara Fiorentini ◽

Chiara Busi ◽

Nicoletta Guerra ◽

PierFranco Spano ◽

...

Keyword(s):

Promoter Activity ◽

Transcriptional Activity ◽

Dopamine D2 Receptor ◽

Regulatory Region ◽

D2 Receptor ◽

Nuclear Factor Κb ◽

Site Directed Mutagenesis ◽

Luciferase Reporter ◽

Nuclear Factor

Regulation of D2 receptor (D2R) expression is crucial in the function of dopaminergic systems. Because alterations of D2R expression may contribute to the development of different disorders, it is important to elucidate the mechanisms regulating D2R gene transcription. We report the characterization of two putative nuclear factor-κB (NF-κB) motifs, referred to as D2-κB sites, in the human D2R promoter, and demonstrate that they bind NF-κB subunits and stimulate D2R promoter activity. D2-κB sites show different degrees of conservation and specificity, when compared with canonical kB sites. The D2-κB1 site (from −407 to −398) is highly conserved and binds p50/p65 and p50/c-Rel complexes, whereas D2-κB2 (from −513 to −504) is more degenerated and only binds p50/p65 heterodimers. Activation of D2-κB sites in COS-7 cells expressing a luciferase reporter vector containing the D2R promoter resulted in increased transcriptional activity. Site-directed mutagenesis of each D2-κB site differentially modified D2R promoter activity. In particular, mutation of the D2-κB1 motif did not affect D2R promoter response to p50/c-Rel complexes, whereas inactivation of the D2-κB2 site decreased it. Mutations of either D2-κB1 or D2-κB2 sites attenuated the D2R promoter transcriptional efficiency induced by p50/p65 complexes. Thus, D2R transcription mediated by p50/c-Rel is supported mainly by the D2-κB2 site, whereas both sites are necessary to support the full transcriptional activity mediated by p50/p65 complexes. A correlation was found between NF-κB activity and D2R expression in the pituitary and pituitary-derived cells but not in the striatum, suggesting that NF-κB regulation of D2R expression could be a pituitary-specific mechanism.

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Hepatocyte nuclear factor-1α regulates transcription of the guanylin gene

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1997.273.4.g833 ◽

1997 ◽

Vol 273 (4) ◽

pp. G833-G841 ◽

Cited By ~ 8

Author(s):

J. A. Hochman ◽

D. Sciaky ◽

T. L. Whitaker ◽

J. A. Hawkins ◽

M. B. Cohen

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Transcriptional Activation ◽

Consensus Sequence ◽

Luciferase Reporter ◽

Intestinal Cell ◽

Nuclear Factor ◽

Hepatocyte Nuclear Factor ◽

Luciferase Reporter Gene ◽

Intestinal Cell Lines

To study the molecular mechanisms controlling guanylin expression, we have cloned the mouse guanylin gene, including 2.7 kb of upstream sequence. We show that the first 133 base pairs (bp) of the upstream guanylin promoter are sufficient to drive near maximal (6-fold over basal) luciferase reporter gene expression in Caco-2 intestinal cells; at least 300 bp of upstream promoter are required for reporter gene expression in HT-29 intestinal cell lines. Using electromobility shift assays, we demonstrate that nuclear proteins bind to the hepatocyte nuclear factor-1 (HNF-1) consensus sequence in the guanylin promoter. The HNF-1 consensus sequence, located in the immediate 5′ flanking region, is required for transcriptional activation of the guanylin gene in both intestinal cell lines. Mutagenesis of the HNF-1 consensus sequence abolishes transcriptional activation of guanylin promoter-luciferase reporter gene constructs. Cotransfection of these constructs with HNF-1α augments transcriptional initiation of the reporter gene. In contrast, HNF-1β has no significant effect on transcription of the reporter gene. These experiments demonstrate that HNF-1α is an important regulatory element in the transcriptional activation of guanylin.

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Nuclear factor-κB regulates expression of platelet phospholipase C-β2 (PLCB2)

Thrombosis and Haemostasis ◽

10.1160/th15-09-0749 ◽

2016 ◽

Vol 116 (11) ◽

pp. 931-940 ◽

Cited By ~ 5

Author(s):

Guangfen Mao ◽

Jianguo Jin ◽

Satya P. Kunapuli ◽

A. Koneti Rao

Keyword(s):

Phospholipase C ◽

Healthy Subjects ◽

Consensus Sequence ◽

Nuclear Factor Κb ◽

Luciferase Reporter ◽

Upstream Region ◽

Nuclear Factor ◽

Nuclear Extracts ◽

Hel Cells ◽

Activation Mechanisms

SummaryPhospholipase C (PLC)-β2 (gene PLCB2) is a critical regulator of platelet responses upon activation. Mechanisms regulating of PLC-β2 expression in platelets/MKs are unknown. Our studies in a patient with platelet PLC-β2 deficiency revealed the PLCB2 coding sequence to be normal and decreased platelet PLC-β2 mRNA, suggesting a defect in transcriptional regulation. PLCB2 5’- upstream region of the patient revealed a heterozygous 13 bp deletion (-1645/-1633 bp) encompassing a consensus sequence for nuclear factor-κB (NF-κB). This was subsequently detected in three of 50 healthy subjects. To understand the mechanisms regulating PLC-β2, we studied the effect of this variation in the PLCB2. Gel-shift studies using nuclear extracts from human erythroleukaemia (HEL) cells or recombinant p65 showed NF-κB binding to oligonucleotide with NF-κB site; in luciferase reporter studies its deletion reduced PLCB2 promoter activity. PLCB2 expression was decreased by siRNA knockdown of NF-κB p65 subunit and increased by p65 overexpression. By immunoblotting platelet PLC-β2 in 17 healthy subjects correlated with p65 (r=0.76, p=0.0005). These studies provide the first evidence that NF-kB regulates MK/platelet PLC-β2 expression. This interaction is important because of the major role of PLC-β2 in platelet activation and of NF-κB in processes, including inflammation and atherosclerosis, where both are intimately involved.Supplementary Material to this article is available online at www.thrombosis-online.com.

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The nuclear factor κB inhibitor (E)-2-fluoro-4′-methoxystilbene inhibits firefly luciferase

Bioscience Reports ◽

10.1042/bsr20120043 ◽

2012 ◽

Vol 32 (6) ◽

pp. 531-537 ◽

Cited By ~ 16

Author(s):

Albert Braeuning ◽

Silvia Vetter

Keyword(s):

Photon Emission ◽

Gene Promoter ◽

Firefly Luciferase ◽

Nuclear Factor Κb ◽

Signalling Pathway ◽

Luciferase Reporter ◽

Nuclear Factor ◽

Reporter System ◽

Reporter Assays

Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4′-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4′-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4′-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4′-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.

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Co-activation of synovial fibroblasts by laminin-111 and transforming growth factor-β induces expression of matrix metalloproteinases 3 and 10 independently of nuclear factor-κB

Annals of the Rheumatic Diseases ◽

10.1136/ard.2007.073809 ◽

2007 ◽

Vol 67 (4) ◽

pp. 559-562 ◽

Cited By ~ 4

Author(s):

K Warstat ◽

T Pap ◽

G Klein ◽

S Gay ◽

W K Aicher

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Synovial Fibroblasts ◽

Transforming Growth Factor Β ◽

Nuclear Factor Κb ◽

Mrna Levels ◽

Nuclear Factor ◽

The Matrix ◽

Specific Tissue ◽

Significant Expression

We showed previously that the attachment of synovial fibroblasts to laminin (LM)-111 in the presence of transforming growth factor-β induces significant expression of the matrix metalloproteinase (MMP)-3. Here we go on to investigate the regulation of additional MMPs and their specific tissue inhibitors of matrix proteases (TIMPs). Changes in steady-state mRNA levels encoding TIMPs and MMPs were investigated by quantitative reverse transcription–polymerase chain reaction. Production of MMPs was monitored by a multiplexed immunoarray. Signal transduction pathways were studied by immunoblotting. Attachment of synovial fibroblasts to LM-111 in the presence of transforming growth factor-β induced significant increases in MMP-3 mRNA (12.35-fold, p<0.001) and protein (mean 62 ng/ml, sixfold, p<0.008) and in expression of MMP-10 mRNA (11.68-fold, p<0.05) and protein (54 ng/ml, 20-fold, p⩾0.02). All other TIMPs and MMPs investigated failed to show this LM-111-facilitated transforming growth factor-β response. No phosphorylation of nuclear factor-κB was observed. We conclude that co-stimulation of synovial fibroblasts by LM-111 together with transforming growth factor-β suffices to induce significant expression of MMP-3 and MMP-10 by synovial fibroblasts and that this induction is independent of nuclear factor-κB phosphorylation.

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Astragalus membranaceus modulates Th1/2 immune balance and activates PPARγ in a murine asthma model

Biochemistry and Cell Biology ◽

10.1139/bcb-2014-0008 ◽

2014 ◽

Vol 92 (5) ◽

pp. 397-405 ◽

Cited By ~ 21

Author(s):

Shih-Ming Chen ◽

Yau-Sheng Tsai ◽

Su-Wen Lee ◽

Ya-Hui Liu ◽

Shuen-Kuei Liao ◽

...

Keyword(s):

Airway Inflammation ◽

Chinese Herb ◽

A549 Cells ◽

Nuclear Hormone Receptor ◽

Astragalus Membranaceus ◽

Luciferase Reporter ◽

Mrna Levels ◽

Th2 Cytokines ◽

Asthma Model ◽

Murine Asthma Model

Astragalus membranaceus, a traditional Chinese herb, has been used to improve airway inflammation and asthma. The present study investigated whether A. membranaceus has immunotherapeutic effects on asthma, a chronic inflammatory mucosal disease that is associated with excess production of IgE, eosinophilia, T helper 2 (Th2) cytokines, and bronchial hyperresponsiveness. An ovalbumin (OVA)-induced, chronic inflammatory airway murine asthma model was used to examine the status of pulmonary inflammation after the administration of A. membranaceus. The IgE levels in serum and bronchoalveolar lavage fluid showed a tendency to decrease after the administration of A. membranaceus. The number of eosinophils decreased and infiltration of inflammatory cells and collagen deposition declined in lung sections after A. membranaceus administration. The RNA and protein levels of Th2 cytokines and the ratio of the GATA3/T-bet mRNA levels decreased after A. membranaceus treatment. Furthermore, the mRNA level of peroxisome proliferator-activated receptor γ (PPARγ), a nuclear hormone receptor, increased in the lung tissues of A. membranaceus–treated mice. Finally, an A. membranaceus water extract activated PPARγ activity in either human embryonic kidney 293 (HEK293) or A549 cells in a PPARγ-responsive element-containing luciferase reporter assay. These results indicate that A. membranaceus has an inhibitory effect on airway inflammation in a murine model of asthma through modulating the imbalanced relationship between Th1 and Th2 cytokines.

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Vasoactive intestinal peptide inhibits IL-8 production in human monocytes by downregulating nuclear factor κB-dependent transcriptional activity

Biochemical and Biophysical Research Communications ◽

10.1016/s0006-291x(03)00149-9 ◽

2003 ◽

Vol 302 (2) ◽

pp. 275-283 ◽

Cited By ~ 28

Author(s):

Mario Delgado ◽

Doina Ganea

Keyword(s):

Vasoactive Intestinal Peptide ◽

Transcriptional Activity ◽

Nuclear Factor Κb ◽

Nuclear Factor ◽

Human Monocytes

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UV stimulation induces nuclear factor κB (NF-κB) DNA-binding activity but not transcriptional activation

Biochemical Society Transactions ◽

10.1042/bst0290688 ◽

2001 ◽

Vol 29 (6) ◽

pp. 688-691 ◽

Cited By ~ 18

Author(s):

K. J. Campbell ◽

N. R. Chapman ◽

N. D. Perkins

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Cellular Response ◽

Uv Light ◽

Binding Protein ◽

Nuclear Factor Κb ◽

Camp Response Element Binding ◽

Binding Activity ◽

Nuclear Factor ◽

Dna Binding Activity

The cellular response to DNA-damaging agents is partly mediated by DNA-binding transcription factors such as p53 and nuclear factor κB (NF-κB). Typically NF-κB activation is associated with resistance to apoptosis. Following stimulation with UV light however, NF-κB activation has been shown to be required for programmed cell death. To study this effect further and to determine the relationship between NF-κB and p53 function, we have examined the effect of UV light on U2OS cells. UV stimulation resulted in the activation of NF-κB DNA-binding and the induction of p53. Surprisingly, and in contrast with tumour necrosis factor α stimulation, this UV-induced NF-κB was transcriptionally inert. These observations suggest a model in which the NF-κB switch from an anti-apoptotic to a pro-apoptotic role within the cell results from modulation of its ability to stimulate gene expression, possibly as a result of the ability of p53 to sequester transcriptional co-activator proteins such as p300/CREB (cAMP-response-element-binding protein)-binding protein.

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PPARγ inhibits airway epithelial cell inflammatory response through a MUC1-dependent mechanism

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00360.2011 ◽

2012 ◽

Vol 302 (7) ◽

pp. L679-L687 ◽

Cited By ~ 11

Author(s):

Yong Sung Park ◽

Erik P. Lillehoj ◽

Kosuke Kato ◽

Choon Sik Park ◽

Kwang Chul Kim

Keyword(s):

Epithelial Cells ◽

Culture Media ◽

A549 Cells ◽

Luciferase Reporter ◽

Muc1 Expression ◽

Mrna Levels ◽

Airway Epithelial ◽

Pparγ Agonist ◽

Tnf Α ◽

Primary Mouse

This study was conducted to examine the relationship between the peroxisome proliferator-associated receptor-γ (PPARγ) and MUC1 mucin, two anti-inflammatory molecules expressed in the airways. Treatment of A549 lung epithelial cells or primary mouse tracheal surface epithelial (MTSE) cells with phorbol 12-myristate 13-acetate (PMA) increased the levels of tumor necrosis factor (TNF)-α in cell culture media compared with cells treated with vehicle alone. Overexpression of MUC1 in A549 cells decreased PMA-stimulated TNF-α levels, whereas deficiency of Muc1 expression in MTSE cells from Muc1 null mice increased PMA-induced TNF-α levels. Treatment of A549 or MTSE cells with the PPARγ agonist troglitazone (TGN) blocked the ability of PMA to stimulate TNF-α levels. However, the effect of TGN required the presence of MUC1/Muc1, since no differences in TNF-α levels were seen between PMA and PMA plus TGN in MUC1/Muc1-deficient cells. Similarly, whereas TGN decreased interleukin-8 (IL-8) levels in culture media of MUC1-expressing A549 cells treated with Pseudomonas aeruginosa strain K (PAK), no differences in IL-8 levels were seen between PAK and PAK plus TGN in MUC1-nonexpressing cells. EMSA confirmed the presence of a PPARγ-binding element in the MUC1 gene promoter. Finally, TGN treatment of A549 cells increased MUC1 promoter activity measured using a MUC1-luciferase reporter gene, augmented MUC1 mRNA levels by quantitative RT-PCR, and enhanced MUC1 protein expression by Western blot analysis. These combined data are consistent with the hypothesis that PPARγ stimulates MUC1/Muc1 expression, thereby blocking PMA/PAK-induced TNF-α/IL-8 production by airway epithelial cells.

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