scholarly journals Physical and functional interaction between the ID1 and p65 for activation of NF-κB

2012 ◽  
Vol 303 (3) ◽  
pp. C267-C277 ◽  
Author(s):  
Xiao Peng ◽  
Yuna Wang ◽  
Swapna Kolli ◽  
Junpeng Deng ◽  
Li Li ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Transcriptional Activation ◽  
Nuclear Translocation ◽  
Luciferase Assay ◽  
Physical Interaction ◽  
Site Mutation ◽  
Transcriptional Activation Domain ◽  
Apoptosis Protein

Inhibitor of differentiation or DNA binding-1 (ID1) is an important helix-loop-helix (HLH) transcription factor involved in diverse biological functions including cell differentiation, proliferation, apoptosis, and senescence. Recently, it was reported that ID1 can activate the NF-κB signaling pathway in a variety of cancer cells and a T cell line, but the mechanisms involved in ID1-mediated transactivation of NF-κB are not clear. In this study, we demonstrate by both in vitro pull-down assays and a cell-based in vivo two-hybrid system that ID1-mediated NF-κB activation is due to its physical interaction with p65. We have identified that the transcriptional activation domain (TAD) in p65 and the HLH domain in ID1 are vital for their interaction. Interestingly, a single site mutation (Leu76) in the HLH domain of ID1 protein drastically decreased its ability to bind with p65. Using a dual-luciferase assay, we demonstrated that the interaction between ID1 and p65 modulates activation of the NF-κB signaling pathway in vivo. In addition, we demonstrated that, by affecting the nuclear translocation of p65, ID1 is essential in regulating TNF-α-induced p65 recruitment to its downstream target, the cellular inhibitor of apoptosis protein 2 (cIAP2) promoter.

Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain

1994 ◽  
Vol 14 (9) ◽  
pp. 6056-6067
Author(s):  
M Tanaka ◽  
W Herr
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Transcriptional Activation Domain ◽  
Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

scholarly journals HPV-18 E6 Oncoprotein and Its Spliced Isoform E6*I Regulate the Wnt/β-Catenin Cell Signaling Pathway through the TCF-4 Transcriptional Factor

2018 ◽  
Vol 19 (10) ◽  
pp. 3153 ◽  
Author(s):  
J. Muñoz-Bello ◽  
Leslie Olmedo-Nieva ◽  
Leonardo Castro-Muñoz ◽  
Joaquín Manzo-Merino ◽  
Adriana Contreras-Paredes ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Cell Proliferation ◽  
Signaling Pathway ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Promote Cell Proliferation ◽  
E6 And E7 ◽  
Hpv 18

The Wnt/β-catenin signaling pathway regulates cell proliferation and differentiation and its aberrant activation in cervical cancer has been described. Persistent infection with high risk human papillomavirus (HR-HPV) is the most important factor for the development of this neoplasia, since E6 and E7 viral oncoproteins alter cellular processes, promoting cervical cancer development. A role of HPV-16 E6 in Wnt/β-catenin signaling has been proposed, although the participation of HPV-18 E6 has not been previously studied. The aim of this work was to investigate the participation of HPV-18 E6 and E6*I, in the regulation of the Wnt/β-catenin signaling pathway. Here, we show that E6 proteins up-regulate TCF-4 transcriptional activity and promote overexpression of Wnt target genes. In addition, it was demonstrated that E6 and E6*I bind to the TCF-4 (T cell factor 4) and β-catenin, impacting TCF-4 stabilization. We found that both E6 and E6*I proteins interact with the promoter of Sp5, in vitro and in vivo. Moreover, although differences in TCF-4 transcriptional activation were found among E6 intratype variants, no changes were observed in the levels of regulated genes. Furthermore, our data support that E6 proteins cooperate with β-catenin to promote cell proliferation.

scholarly journals Meloxicam, a Selective COX-2 Inhibitor, Mediates Hypoxia-Inducible Factor- (HIF-) 1α Signaling in Hepatocellular Carcinoma

2020 ◽  
Vol 2020 ◽  
pp. 1-15
Author(s):  
Yinghong Zhou ◽  
Xiaofeng Dong ◽  
Peng Xiu ◽  
Xin Wang ◽  
Jianrong Yang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Transcriptional Activation ◽  
Mitochondrial Membrane ◽  
Hypoxia Inducible Factor ◽  
Luciferase Assay ◽  
Cox 2

Hepatocellular carcinoma (HCC) is regarded as a leading cause of cancer-related deaths, and its progression is associated with hypoxia and the induction of hypoxia-inducible factor (HIF). Meloxicam, a selective cyclooxygenase-2 (COX-2) inhibitor, induces cell death in various malignancies. However, the underlying mechanism remains to be elucidated in HCC, especially under hypoxic conditions. The alteration of COX-2 and HIF-1α oncogenicity was evaluated in HCC specimens by tissue microarray. Cell viability, angiogenesis assays, and xenografted nude mice were used to evaluate the effects of meloxicam, along with flow cytometry to detect the cell cycle, apoptosis, and mitochondrial membrane potential (ΔΨm) of HCC. qRT-PCR, Western blotting, immunofluorescence, immunohistochemistry, luciferase assay, and RNAi were carried out to determine the HIF-1α signaling affected by meloxicam. In this study, we showed that meloxicam exerts antiproliferative and antiangiogenesis efficacy in vitro and in vivo and causes disruption of mitochondrial membrane potential (ΔΨm), thus leading to caspase-dependent apoptosis under hypoxic environments. Exposure to meloxicam significantly reduced HIF-1α transcriptional activation and expression through sequestering it in the cytoplasm and accelerating degradation via increasing the von Hippel-Lindau tumor suppressor protein (pVHL) in HCC. These data demonstrated that inhibition of HIF-1α by meloxicam could suppress angiogenesis and enhance apoptosis of HCC cells. This discovery highlights that COX-2 specific inhibitors may be a promising therapy in the treatment of HCC.

scholarly journals MO069HIF-1Α C-TERMINAL TRANSCRIPTIONAL ACTIVATION DOMAIN MEDIATED KLF5 SIGNALING DRIVES AKI TO CKD PROGRESSION

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Zuolin Li ◽  
Jia-ling Ji ◽  
Linli Lv ◽  
Yan Yang ◽  
Tao-tao Tang ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Ischemic Injury ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Mice Model ◽  
Ckd Progression ◽  
Transcriptional Activation Domain ◽  
Tubular Cells

Abstract Background and Aims Acute kidney injury (AKI) is increasingly recognized as a major risk factor for progression to CKD. However, the mechanisms governing AKI to CKD progression are poorly understood. Hypoxia is a key player in the pathophysiology of the AKI to CKD transition. Thus, we aimed to investigate the exact mechanisms of AKI to CKD progression mediated by hypoxia. Method Mild ischemic injury and severe ischemic injury (AKI-to-CKD transition) were established by clamping renal pedicle for 30 and 40 minutes, respectively. Meanwhile, the mice model of AKI-to-CKD transition was treated with HIF-1α inhibitor, PX-478. In vitro, PHD inhibition and combined PHD with FIH inhibition mimic the HIF-1α activation caused by mild or severe hypoxia, respectively. Besides the human proximal tubular epithelial cell line HK-2, tubular cells were isolated from mice for primary culture. KLF5 knockdown, FIH and HIF-1α C-terminal transcriptional activation domain (C-TAD) overexpression in tubular cells were achieved by Lentiviral transfection. Immunocoprecipitation was used to explore the relationship between the HIF-1α and FIH-1. Luciferase reporter assay was used to investigate whether KLF5 was regulated transcriptionally by HIF-1α C-TAD. To explore the roles of FIH-1 and HIF-1α C-TAD in vivo, FIH-1 and HIF-1α C-TAD overexpression (Lentivirus-mediated) was given after severe ischemic injury or mild ischemic injury via tail vein injection, respectively. Results AKI to CKD progression was highly associated with the time-course expression of tubular HIF-1α in severe ischemia/reperfusion injury. Interestingly, ameliorated AKI-to-CKD transition was observed by treating PX-478, which destabilized HIF-1α. In vitro, fibrogenesis could be induced by combined PHD with FIH inhibitor treatment in TEC. More interestingly, alleviated fibrogenesis could be achieved by knockdown of KLF5 and overexpression of FIH, respectively, while HIF-1α C-TAD overexpression promoted fibrogenesis in tubular cells. Immunocoprecipitation results indicated that HIF-1α and FIH-1 are interactive. Furthermore, we demonstrated that KLF5 could be regulated transcriptionally by HIF-1α C-TAD by luciferase reporter assay. In vivo, AKI to CKD progression was ameliorated significantly when mice model of AKI-to-CKD transition intervened with FIH-1 overexpression (Lentivirus-mediated). However, treatment of HIF-1α C-TAD (Lentivirus-mediated) in mild ischemic injury model could promote progression of CKD significantly. Conclusion FIH-1 mediated HIF-1α C-TAD activation was the key mechanism of AKI to CKD transition by transcriptionally regulating the KLF5 pathway in tubules. Blockade of FIH-1 mediated HIF-1α C-TAD in tubules may serve as a novel therapeutic approach to ameliorate AKI to CKD progression.

scholarly journals Synergistic transcriptional activation by CTF/NF-I and the estrogen receptor involves stabilized interactions with a limiting target factor.

1991 ◽  
Vol 11 (6) ◽  
pp. 2937-2945 ◽  
Author(s):  
E Martinez ◽  
Y Dusserre ◽  
W Wahli ◽  
N Mermod
Keyword(s):  
Estrogen Receptor ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Limiting Factor ◽  
Gene Promoters ◽  
Protein Coding ◽  
Transcriptional Activation Domain

Transcription initiation at eukaryotic protein-coding gene promoters is regulated by a complex interplay of site-specific DNA-binding proteins acting synergistically or antagonistically. Here, we have analyzed the mechanisms of synergistic transcriptional activation between members of the CCAAT-binding transcription factor/nuclear factor I (CTF/NF-I) family and the estrogen receptor. By using cotransfection experiments with HeLa cells, we show that the proline-rich transcriptional activation domain of CTF-1, when fused to the GAL4 DNA-binding domain, synergizes with each of the two estrogen receptor-activating regions. Cooperative DNA binding between the GAL4-CTF-1 fusion and the estrogen receptor does not occur in vitro, and in vivo competition experiments demonstrate that both activators can be specifically inhibited by the overexpression of a proline-rich competitor, indicating that a common limiting factor is mediating their transcriptional activation functions. Furthermore, the two activators functioning synergistically are much more resistant to competition than either factor alone, suggesting that synergism between CTF-1 and the estrogen receptor is the result of a stronger tethering of the limiting target factor(s) to the two promoter-bound activators.

scholarly journals Glaucocalyxin A Attenuates Allergic Responses by Inhibiting Mast Cell Degranulation through p38MAPK/NrF2/HO-1 and HMGB1/TLR4/NF-κB Signaling Pathways

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Yihua Piao ◽  
Jingzhi Jiang ◽  
Zhiguang Wang ◽  
Chongyang Wang ◽  
Shan Jin ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Signaling Pathway ◽  
Nuclear Translocation ◽  
Calcium Influx ◽  
Mast Cell Degranulation ◽  
Therapeutic Drug ◽  
High Mobility Group Protein

Glaucocalyxin A (GLA) has various pharmacological effects like antioxidation, immune regulation, and antiatherosclerosis. Here, in this study, the effect and mechanism of GLA on mast cell degranulation were studied. The results of the anti-DNP IgE-mediated passive cutaneous anaphylaxis (PCA) showed that GLA dramatically inhibited PCA in vivo, as evidenced by reduced Evans blue extravasation and decreased ear thickness. In addition, GLA significantly reduced the release of histamine and β-hexosaminidase, calcium influx, cytokine (IL-4, TNF-α, IL-1β, IL-13, and IL-8) production in the RBL-2H3 (rat basophilic leukemia cells), and RPMCs (peritoneal mast cells) in vitro. Moreover, we further investigated the regulatory mechanism of GLA on antigen-induced mast cells by Western blot, which showed that GLA inhibited FcεRI-mediated signal transduction and invalidated the phosphorylation of Syk, Fyn, Lyn, Gab2, and PLC-γ1. In addition, GLA inhibited the recombinant mouse high mobility group protein B1- (HMGB1-) induced mast cell degranulation through limiting nuclear translocation of NF-κBp65. Treatment of mast cells with siRNA-HMGB1 significantly inhibited HMGB1 levels, as well as MyD88 and TLR4, decreased intracellular calcium levels, and suppressed the release of β-hexosaminidase. Meanwhile, GLA increased NrF2 and HO-1 levels by activating p38MAPK phosphorylation. Consequently, these data suggest that GLA regulates the NrF2/HO-1 signaling pathway through p38MAPK phosphorylation and inhibits HMGB1/TLR4/NF-κB signaling pathway to reduce mast cell degranulation and allergic inflammation. Our findings could be used as a promising therapeutic drug against allergic inflammatory disease.

scholarly journals Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Adrian L Sanborn ◽  
Benjamin T Yeh ◽  
Jordan T Feigerle ◽  
Cynthia V Hao ◽  
Raphael J L Townshend ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Structural Constraints ◽  
Sequence Motifs ◽  
Activation Domains ◽  
Activator Proteins ◽  
Transcriptional Activation Domain ◽  
Shape Complementarity ◽  
And Function

Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity ('fuzzy' binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

Regulation of the chondrocyte phenotype by β-catenin

Development ◽  
2002 ◽  
Vol 129 (23) ◽  
pp. 5541-5550
Author(s):  
Je-Hwang Ryu ◽  
Song-Ja Kim ◽  
Seon-Hee Kim ◽  
Chun-Do Oh ◽  
Sang-Gu Hwang ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Monolayer Culture ◽  
Nuclear Translocation ◽  
Articular Chondrocytes ◽  
Glycogen Synthase ◽  
Ectopic Expression ◽  
Activator Protein 1 ◽  
Chondrocyte Phenotype

β-Catenin regulates important biological processes, including embryonic development and tumorigenesis. We have investigated the role ofβ-catenin in the regulation of the chondrocyte phenotype. Expression ofβ-catenin was high in prechondrogenic mesenchymal cells, but significantly decreased in differentiated chondrocytes both in vivo and in vitro. Accumulation of β-catenin by the inhibition of glycogen synthase kinase-3β with LiCl inhibited chondrogenesis by stabilizing cell-cell adhesion. Conversely, the low level of β-catenin in differentiated articular chondrocytes was increased by post-translational stabilization during phenotypic loss caused by a serial monolayer culture or exposure to retinoic acid or interleukin-1β. Ectopic expression of β-catenin or inhibition of β-catenin degradation with LiCl or proteasome inhibitor caused de-differentiation of chondrocytes. Transcriptional activation ofβ-catenin by its nuclear translocation was sufficient to cause phenotypic loss of differentiated chondrocytes. Expression pattern of Jun, a known target gene of β-catenin, is essentially the same as that of β-catenin both in vivo and in vitro suggesting that Jun and possibly activator protein 1 is involved in the β-catenin regulation of the chondrocyte phenotype.

scholarly journals The actin-binding protein EPS8 binds VE-cadherin and modulates YAP localization and signaling

2015 ◽  
Vol 211 (6) ◽  
pp. 1177-1192 ◽  
Author(s):  
Costanza Giampietro ◽  
Andrea Disanza ◽  
Luca Bravi ◽  
Miriam Barrios-Rodiles ◽  
Monica Corada ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Adherens Junction ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Intracellular Signals ◽  
Transcriptional Cofactors

Vascular endothelial (VE)–cadherin transfers intracellular signals contributing to vascular hemostasis. Signaling through VE-cadherin requires association and activity of different intracellular partners. Yes-associated protein (YAP)/TAZ transcriptional cofactors are important regulators of cell growth and organ size. We show that EPS8, a signaling adapter regulating actin dynamics, is a novel partner of VE-cadherin and is able to modulate YAP activity. By biochemical and imaging approaches, we demonstrate that EPS8 associates with the VE-cadherin complex of remodeling junctions promoting YAP translocation to the nucleus and transcriptional activation. Conversely, in stabilized junctions, 14–3-3–YAP associates with the VE–cadherin complex, whereas Eps8 is excluded. Junctional association of YAP inhibits nuclear translocation and inactivates its transcriptional activity both in vitro and in vivo in Eps8-null mice. The absence of Eps8 also increases vascular permeability in vivo, but did not induce other major vascular defects. Collectively, we identified novel components of the adherens junction complex, and we introduce a novel molecular mechanism through which the VE-cadherin complex controls YAP transcriptional activity.

scholarly journals The Novel Thienopyrimidine Derivative, RP-010, Produces Its Efficacy in Prostate Cancer Cells by Inducing the Fragmentation of β-catenin

2019 ◽  
Author(s):  
Haneen Amawi ◽  
Noor Hussein ◽  
Sai HS Boddu ◽  
Chandrabose Karthikeyan ◽  
Frederick E. Williams ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Cells ◽  
Signaling Pathway ◽  
Nuclear Translocation ◽  
Biologically Active ◽  
Beta Catenin ◽  
Lead Compound ◽  
Prostate Cancer Cells

Thienopyrimidines are a versatile group of compounds that contain a biologically active pharmacophore and reported to have anticancer efficacy in vitro. Here, we report for the first time, that thieno[3,2-d]pyrimidine - based compounds, designated the RP series, have efficacy in prostate cancer cells. The lead compound, RP-010, was efficacious in PC3 and DU-145 prostate cancer (PC) cells (IC50< 1µM). The cytotoxicity of RP-010 was significantly lower in normal cells. RP-010 (0.5, 1, 2, and 4 µM) arrested prostate cancer cells in the G2 phase of the cell cycle, induced mitotic catastrophe and apoptotic signaling in both PC cell lines. Mechanistic studies suggested that RP-010 (1 and 2 µM) inhibits the wingless-type MMTV (Wnt)/β-catenin signaling pathway, mainly by inducing β-catenin fragmentation, while down regulating important proteins in the pathway, i.e. LRP-6, DVL3, and c-Myc. Interestingly, RP-010 (1 and 2 µM) induced the nuclear translocation of the negative feedback proteins, Naked 1 and Naked 2, in the signaling pathway. In addition, RP-010 (0.5, 1, 2, and 4 µM) significantly decreased the migration and invasiveness of PC cells in vitro. Finally, RP-010 did not produce significant toxic effects in zebrafish at concentrations up to 6 µM. In conclusion, RP-10 is a promising anticancer compound in metastatic prostate cancer and did not produce overt toxicity in an in vivo zebrafish model. Future mechanistic and efficacy studies are needed in-vivo to optimize the lead compound RP-010 for clinical use.

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