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

Endocrinology ◽  
2007 ◽  
Vol 148 (5) ◽  
pp. 2563-2570 ◽  
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.

scholarly journals 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

2002 ◽  
Vol 364 (2) ◽  
pp. 537-545 ◽  
Author(s):  
Deborah L. BAINES ◽  
Mandy JANES ◽  
David J. NEWMAN ◽  
Oliver G. BEST
Keyword(s):  
Sodium Channel ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Consensus Sequence ◽  
A549 Cells ◽  
Nuclear Factor Κb ◽  
Luciferase Reporter ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Α Subunit

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.

scholarly journals Nerve Growth Factor Regulates Dopamine D2 Receptor Expression in Prolactinoma Cell Lines via p75NGFR-Mediated Activation of Nuclear Factor-κB

2002 ◽  
Vol 16 (2) ◽  
pp. 353-366 ◽  
Author(s):  
Chiara Fiorentini ◽  
Nicoletta Guerra ◽  
Marco Facchetti ◽  
Alessandra Finardi ◽  
Laura Tiberio ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Cell Lines ◽  
Dopamine D2 Receptor ◽  
D2 Receptor ◽  
Nuclear Factor Κb ◽  
Receptor Expression ◽  
Nuclear Factor ◽  
Nerve Growth ◽  
Dopamine D2

scholarly journals Characterization of the human liver fructose-1,6-bisphosphatase gene promoter

2000 ◽  
Vol 351 (2) ◽  
pp. 385-392 ◽  
Author(s):  
Birger HERZOG ◽  
Mary WALTNER-LAW ◽  
Donald K. SCOTT ◽  
Klaus ESCHRICH ◽  
Daryl K. GRANNER
Keyword(s):  
Hormonal Regulation ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Nuclear Factor Κb ◽  
Site Directed Mutagenesis ◽  
Upstream Stimulatory Factor ◽  
Mobility Shift ◽  
Fructose 1,6 Bisphosphatase

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), an important gluconeogenic enzyme, catalyses the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and Pi. Enzyme activity is mainly regulated by the allosteric inhibitors fructose 2,6-bisphosphate and AMP. Although some observations about hormonal regulation of the enzyme have been published, the FBPase promoter has not been studied in detail. Here we report an in vitro characterization of the FBPase promoter with respect to the elements that are required for basal promoter activity. Transient transfection of H4IIE rat hepatoma cells, combined with site-directed mutagenesis, demonstrated that an enhancer box, three GC-boxes and a nuclear factor κB (NF-κB)-binding element are important for hepatic FBPase promoter activity. These elements are found in the region located between -405 to +25bp relative to the transcription start site. Electrophoretic-mobility-shift assays and supershift analysis confirmed that upstream stimulatory factor 1 (USF1)/USF2, specificity protein 1 (Sp1)/Sp3 and NF-κB respectively bind to these sites. The present study provides the basis for a more comprehensive screening for mutations in FBPase-deficient patients and for further studies of the transcriptional regulation of this gene.

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

2012 ◽  
Vol 32 (6) ◽  
pp. 531-537 ◽  
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.

scholarly journals Characterization of a Viral Phosphoprotein Binding Site on the Surface of the Respiratory Syncytial Nucleoprotein

2012 ◽  
Vol 86 (16) ◽  
pp. 8375-8387 ◽  
Author(s):  
Marie Galloux ◽  
Bogdan Tarus ◽  
Ilfad Blazevic ◽  
Jenna Fix ◽  
Stéphane Duquerroy ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Specific Interaction ◽  
Viral Rna ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Potential Candidate ◽  
Luciferase Reporter Gene ◽  
Interaction Domain ◽  
Rna Complex

The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (PCTD) and N. However, the P binding region on N remains to be identified. In this study, glutathioneS-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (NNTD) as a P binding domain. A biochemical characterization of the PCTDand molecular modeling of the NNTDallowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the PCTDinteraction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolishedin vitroandin vivoP-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.

scholarly journals Sequence of the 5′-flanking region and promoter activity of the human mucin gene MUC5B in different phenotypes of colon cancer cells

2000 ◽  
Vol 348 (3) ◽  
pp. 675-686 ◽  
Author(s):  
Isabelle VAN SEUNINGEN ◽  
Michaël PERRAIS ◽  
Pascal PIGNY ◽  
Nicole PORCHET ◽  
Jean-Pierre AUBERT
Keyword(s):  
Gene Expression ◽  
Transcription Start Site ◽  
Promoter Activity ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Start Site ◽  
Transcription Start ◽  
Mucin Gene ◽  
Intron 1 ◽  
Flanking Region

Control of gene expression in intestinal cells is poorly understood. Molecular mechanisms that regulate transcription of cellular genes are the foundation for understanding developmental and differentiation events. Mucin gene expression has been shown to be altered in many intestinal diseases and especially cancers of the gastrointestinal tract. Towards understanding the transcriptional regulation of a member of the 11p15.5 human mucin gene cluster, we have characterized 3.55 kb of the 5ʹ-flanking region of the human mucin gene MUC5B, including the promoter, the first two exons and the first intron. We report here the promoter activity of successively 5ʹ-truncated sections of 956 bases of this region by fusing it to the coding region of a luciferase reporter gene. The transcription start site was determined by primer-extension analysis. The region upstream of the transcription start site is characterized by the presence of a TATA box at bases -32/-26, DNA-binding elements for transcription factors c-Myc, N-Myc, Sp1 and nuclear factor ĸB as well as putative activator protein (AP)-1-, cAMP-response-element-binding protein (CREB)-, hepatocyte nuclear factor (HNF)-1-, HNF-3-, TGT3-, gut-enriched Krüppel factor (GKLF)-, thyroid transcription factor (TTF)-1- and glucocorticoid receptor element (GRE)-binding sites. Intron 1 of MUC5B was also characterized, it is 2511 nucleotides long and contains a DNA segment of 259 bp in which are clustered eight tandemly repeated GA boxes and a CACCC box that bind Sp1. AP-2α and GATA-1 nuclear factors were also shown to bind to their respective cognate elements in intron 1. In transfection studies the MUC5B promoter showed a cell-specific activity as it is very active in mucus-secreting LS174T cells, whereas it is inactive in Caco-2 enterocytes and HT-29 STD (standard) undifferentiated cells. Within the promoter, maximal transcription activity was found in a segment covering the first 223 bp upstream of the transcription start site. Finally, in co-transfection experiments a transactivating effect of Sp1 on to MUC5B promoter was seen in LS174T and Caco-2 cells.

Regulation of the transcriptional activity of the nuclear factor-κB p65 subunit

2002 ◽  
Vol 64 (5-6) ◽  
pp. 963-970 ◽  
Author(s):  
Linda Vermeulen ◽  
Gert De Wilde ◽  
Sofie Notebaert ◽  
Wim Vanden Berghe ◽  
Guy Haegeman
Keyword(s):  
Transcriptional Activity ◽  
Nuclear Factor Κb ◽  
Nuclear Factor

scholarly journals MicroRNA-218 Negatively Regulates Osteoclastogenic Differentiation by Repressing the Nuclear Factor-κB Signaling Pathway and Targeting Tumor Necrosis Factor Receptor 1

2018 ◽  
Vol 48 (1) ◽  
pp. 339-347 ◽  
Author(s):  
Weiwei Wang ◽  
Lei Yang ◽  
Dan Zhang ◽  
Chao Gao ◽  
Jie Wu ◽  
...  
Keyword(s):  
Bioinformatics Analysis ◽  
Tumor Necrosis Factor Receptor ◽  
Nuclear Factor Κb ◽  
Raw 264.7 Cells ◽  
Luciferase Reporter ◽  
Raw 264.7 ◽  
Nuclear Factor ◽  
Reporter Assay ◽  
Trap Staining ◽  
Necrosis Factor

Background/Aims: Postmenopausal osteoporosis is a common disease associated with estrogen deficiency leading to bone loss and bone tissue changes. The resultant bone fragility and increased risk of fracture has serious adverse effects on health and quality of life of the elderly, making it an important health issue. MicroRNA-218 (miR-218) is closely related to the development of osteoporosis. In this study, we investigated the regulatory mechanisms of miR-218 in osteoclastogenesis. Methods: We investigated miR-218 levels on differentiation of RAW 264.7 cells into osteoclasts compared with normal cells. Next, RAW 264.7 cells were transfected with miR-218 mimics or inhibitors to study the role of miR-218 in osteoclastogenic differentiation. Tartrate-resistant acid phosphatase (TRAP) staining was performed to determine osteoclastogenic differentiation. Bioinformatics analysis and luciferase reporter assay were used to identify and validate miR-218 target genes. Results: miR-218 was downregulated following RAW 264.7 cell differentiation into osteoclasts. miR-218 overexpression attenuated osteoclast differentiation, whereas low miR-218 expression promoted it as demonstrated by increased expression of osteoclast-specific genes and TRAP staining. Bioinformatics analysis and the luciferase reporter assay showed that tumor necrosis factor receptor 1 (TNFR1), a cell membrane receptor of TNF (TNF is an activator of nuclear factor-κB [NF-κB]), is a direct target of miR-218. Conclusions: Our findings indicate that miR-218 regulates osteoclastogenic differentiation negatively by repressing NF-κB signaling by targeting TNFR1, suggesting that targeting miR-218 may be a therapeutic approach in postmenopausal osteoporosis.

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