scholarly journals Endothelial nuclear lamina is not required for glucocorticoid receptor nuclear import but does affect receptor-mediated transcription activation

2013 ◽  
Vol 305 (3) ◽  
pp. C309-C322 ◽  
Author(s):  
Arman Nayebosadri ◽  
Julie Y. Ji
Keyword(s):  
Shear Stress ◽  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Protein Function ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Nuclear Lamina ◽  
Luciferase Reporter ◽  
Lamin A ◽  
Nuclear Mechanics

The lamina serves to maintain the nuclear structure and stiffness while acting as a scaffold for heterochromatin and many transcriptional proteins. Its role in endothelial mechanotransduction, specifically how nuclear mechanics impact gene regulation under shear stress, is not fully understood. In this study, we successfully silenced lamin A/C in bovine aortic endothelial cells to determine its role in both glucocorticoid receptor (GR) nuclear translocation and glucocorticoid response element (GRE) transcriptional activation in response to dexamethasone and shear stress. Nuclear translocation of GR, an anti-inflammatory nuclear receptor, in response to dexamethasone or shear stress (5, 10, and 25 dyn/cm2) was observed via time-lapse cell imaging and quantified using a Bayesian image analysis algorithm. Transcriptional activity of the GRE promoter was assessed using a dual-luciferase reporter plasmid. We found no dependence on nuclear lamina for GR translocation from the cytoplasm into the nucleus. However, the absence of lamin A/C led to significantly increased expression of luciferase under dexamethasone and shear stress induction as well as changes in histone protein function. PCR results for NF-κB inhibitor alpha (NF-κBIA) and dual specificity phosphatase 1 (DUSP1) genes further supported our luciferase data with increased expression in the absence of lamin. Our results suggest that absence of lamin A/C does not hinder passage of GR into the nucleus, but nuclear lamina is important to properly regulate GRE transcription. Nuclear lamina, rather than histone deacetylase (HDAC), is a more significant mediator of shear stress-induced transcriptional activity, while dexamethasone-initiated transcription is more HDAC dependent. Our findings provide more insights into the molecular pathways involved in nuclear mechanotransduction.

scholarly journals Xiao Yao San against Corticosterone-Induced Stress Injury via Upregulating Glucocorticoid Receptor Reaction Element Transcriptional Activity

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Guoping Cao ◽  
Shenglan Gong ◽  
Fengxue Zhang ◽  
Wenjun Fu
Keyword(s):  
Hippocampal Neurons ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Luciferase Reporter ◽  
Potential Candidate ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Stress Injury ◽  
Induced Stress

Previous studies have revealed that uncontrollable stress can impair the synaptic plasticity and firing property of hippocampal neurons, which influenced various hippocampal-dependent tasks including memory, cognition, behavior, and mood. In this work, we had investigated the effects and mechanisms of the Chinese herbal medicine Xiao Yao San (XYS) against corticosterone-induced stress injury in primary hippocampal neurons (PHN) cells. We found that XYS and RU38486 could increase cell viabilities and decrease cell apoptosis by MTT, immunofluorescence, and flow cytometry assays. In addition, we observed that XYS notably inhibited the nuclear translocation of GR and upregulated the mRNA and protein expressions levels of Caveolin-1, GR, BDNF, TrkB, and FKBP4. However, XYS downregulated the FKBP51 expressions. Furthermore, the results of the electrophoretic mobility shift assay (EMSA) and double luciferase reporter gene detection indicated that FKBP4 promotes the transcriptional activity of GR reaction element (GRE) by binding with GR, and FKBP51 processed the opposite action. Thein vivoexperiment also proved the functions of XYS. These results suggested that XYS showed an efficient neuroprotection against corticosterone-induced stress injury in PHN cells by upregulating GRE transcriptional activity, which should be developed as a potential candidate for treating stress injury in the future.

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 11βHSD2 Efficacy in Preventing Transcriptional Activation of the Mineralocorticoid Receptor by Corticosterone

2021 ◽  
Vol 5 (11) ◽  
Author(s):  
Yusuf Ali ◽  
Maniselvan Kuppusamy ◽  
Carolina Velarde-Miranda ◽  
Clara M Gomez-Sanchez ◽  
Maria Plonczynski ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Transcriptional Activation ◽  
Mineralocorticoid Receptor ◽  
Nuclear Translocation ◽  
Rat Kidney ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Dose Dependent Fashion ◽  
The Right ◽  
In Cells

Abstract Affinity of the mineralocorticoid receptor (MR) is similar for aldosterone and the glucocorticoids (GC) cortisol and corticosterone, which circulate at concentrations far exceeding those of aldosterone. 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) inactivation of GC within the immediate vicinity of the MR is credited with prereceptor specificity for aldosterone in cells coexpressing MR and 11βHSD2. 11βHSD2 efficacy is also critical to other recently described 11βHSD2 substrates. The aim of this work was to address doubts that low levels of expression of 11βHSD2 in aldosterone target tissues suffice to prevent the initiation of gene transcription by the MR activated by physiological concentrations of corticosterone. Cell models stably expressing an MR/Gaussia luciferase reporter and various levels of constitutive or induced 11βHSD2 at concentrations lower than those in rat kidney homogenates and microsomes were produced. Aldosterone and corticosterone were equipotent transactivators of the MR reporter gene in cells without 11βHSD2. Rate of conversion of tritiated corticosterone to 11-dehydrocorticosterone increased and corticosterone-induced nuclear translocation of MR decreased, as 11βHSD2 expression increased. The 50% maximal MR activation for the reporter gene stimulation by corticosterone rose with increasing 11βHSD2 expression, shifting the steroid dose-response curve for corticosterone-induced MR transactivation to the right. Several stable cell lines expressing an easily and reproducibly measured MR reporter system and consistent incremental amounts of 11βHSD2 protein were produced and used to document that 11βHSD2 within low physiological levels inactivates relevant concentrations of GC and decreases MR transactivation by GC in a dose-dependent fashion, laying to rest doubts of the efficacy of this enzyme.

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 A Novel Isoform of Human LZIP Negatively Regulates the Transactivation of the Glucocorticoid Receptor

2009 ◽  
Vol 23 (11) ◽  
pp. 1746-1757 ◽  
Author(s):  
Hyereen Kang ◽  
Yoon Suk Kim ◽  
Jesang Ko
Keyword(s):  
Amino Acids ◽  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Target Genes ◽  
Transmembrane Domain ◽  
Negative Regulator ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Basic Leucine Zipper

Abstract The human leucine zipper protein (LZIP) is a basic leucine zipper transcription factor that is involved in leukocyte migration, tumor suppression, and endoplasmic reticulum stress-associated protein degradation. Although evidence suggests a diversity of roles for LZIP, its function is not fully understood, and the subcellular localization of LZIP is still controversial. We identified a novel isoform of LZIP and characterized its function in ligand-induced transactivation of the glucocorticoid receptor (GR) in COS-7 and HeLa cells. A novel isoform of human LZIP designated as “sLZIP” contains a deleted putative transmembrane domain (amino acids 229–245) of LZIP and consists of 345 amino acids. LZIP and sLZIP were ubiquitously expressed in a variety of cell lines and tissues, with LZIP being much more common. sLZIP was mainly localized in the nucleus, whereas LZIP was located in the cytoplasm. Unlike LZIP, sLZIP was not involved in the chemokine-mediated signal pathway. sLZIP recruited histone deacetylases (HDACs) to the promoter region of the mouse mammary tumor virus luciferase reporter gene and enhanced the activities of HDACs, resulting in suppression of expression of the GR target genes. Our findings suggest that sLZIP functions as a negative regulator in glucocorticoid-induced transcriptional activation of GR by recruitment and activation of HDACs.

scholarly journals RelB/NF-κB2 Regulates Corticotropin-Releasing Hormone in the Human Placenta

2012 ◽  
Vol 26 (8) ◽  
pp. 1356-1369 ◽  
Author(s):  
Bingbing Wang ◽  
Nataliya Parobchak ◽  
Todd Rosen
Keyword(s):  
Human Placenta ◽  
Chromatin Immunoprecipitation ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Gene Promoter ◽  
Significant Inhibition ◽  
Luciferase Reporter ◽  
Nuclear Staining ◽  
Heterologous Promoter ◽  
P100 Processing

Placental CRH may be part of a clock that governs the length of human gestation. The mechanism underlying differential regulation of CRH in the human placenta is poorly understood. We report here that constitutively activated RelB/nuclear factor-κB2 (NF-κB)-2 (p100/p52) acts as an endogenous stimulatory signal to regulate CRH by binding to an NF-κB enhancer of CRH gene promoter in the human placenta. Nuclear staining of NF-κB2 and RelB in villous syncytiotrophoblasts and cytotrophoblasts was coupled with cytoplasmic CRH in syncytial knots of cytotrophoblasts. Chromatin immunoprecipitation identified that CRH gene associated with both RelB and NF-κB2 (p52). Dexamethasone increased synthesis and nuclear translocation of RelB and NF-κB2 (p52) and their association with the CRH gene. In contrast, progesterone, a down-regulator of placental CRH, repressed NF-κB2 (p100) processing, nuclear translocation of RelB and NF-κB2 (p52), and their association with the CRH gene. Luciferase reporter assay determined that the NF-κB enhancer of CRH was sufficient to regulate transcriptional activity of a heterologous promoter in primary cytotrophoblasts. RNA interference-mediated repression of RelB or NF-κB2 resulted in significant inhibition of CRH at both transcriptional and translational levels and prevented the dexamethasone-mediated up-regulation of CRH transcription and translation. These results suggest that the noncanonical NF-κB pathway regulates CRH production in the human placenta and is responsible for the positive regulation of CRH by glucocorticoids.

scholarly journals Compounds from Cynomorium songaricum with Estrogenic and Androgenic Activities Suppress the Oestrogen/Androgen-Induced BPH Process

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Xueni Wang ◽  
Rui Tao ◽  
Jing Yang ◽  
Lin Miao ◽  
Yu Wang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Immunofluorescence Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Lncap Cells ◽  
Mcf 7

Objective. To investigate the phytoestrogenic and phytoandrogenic activities of compounds isolated from CS and uncover the role of CS in prevention of oestrogen/androgen-induced BPH. Methods. Cells were treated with CS compounds, and immunofluorescence assay was performed to detect the nuclear translocation of ERα or AR in MCF-7 or LNCaP cells; luciferase reporter assay was performed to detect ERs or AR transcriptional activity in HeLa or AD293 cells; MTT assay was performed to detect the cell proliferation of MCF-7 or LNCaP cells. Oestrogen/androgen-induced BPH model was established in rat and the anti-BPH, anti-estrogenic, and anti-androgenic activities of CS in vivo were further investigated. Results. The nuclear translocation of ERα was stimulated by nine CS compounds, three of which also stimulated AR translocation. The transcriptional activities of ERα and ERβ were induced by five compounds, within which only ECG induced AR transcriptional activity as well. Besides, ECG stimulated the proliferation of both MCF-7 cells and LNCaP cells. CS extract suppressed oestrogen/androgen-induced BPH progress in vivo by downregulation of E2 and T level in serum and alteration of the expressions of ERα, ERβ, and AR in the prostate. Conclusion. Our data demonstrates that compounds from CS exhibit phytoestrogenic and phytoandrogenic activities, which may contribute to inhibiting the oestrogen/androgen-induced BPH development.

Mechanisms of Leukemogenesis by MLL-CALM: Characterization of a CALM-Derived Transcriptional Regulatory Domain.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3387-3387
Author(s):  
Mwe Mwe Chao ◽  
Emily J. Fox ◽  
Daniel S. Wechsler
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Nuclear Export ◽  
Transcriptional Activity ◽  
Hox Genes ◽  
Luciferase Activity ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Fusion Partner ◽  
Cytoplasmic Localization

Abstract Background: MLL translocations are common in infant leukemias, and >50 distinct translocation partners have been described. We recently identified the CALM gene as a novel MLL partner in an infant with aggressive AML. Interestingly, CALM was first discovered as a translocation partner for AF10, which had previously been identified as an MLL fusion partner in aggressive leukemias and lymphomas. The native CALM protein exhibits predominantly cytoplasmic localization, and participates in clathrin-dependent endocytosis and intracellular vesicle transport. We have previously shown that expression of MLL-CALM immortalizes murine hematopoietic progenitors, and that fusion of the carboxy terminus of CALM to MLL alters MLL transcriptional activity. We hypothesize that CALM possesses a specific transcriptional activation domain (TAD) which modulates MLL transcriptional activity of HOX genes, thereby contributing to leukemogenesis. Objectives: 1) To determine whether native CALM localizes to the nucleus, 2) To delineate specific CALM domains which constitute the CALM TAD, and 3) To determine whether MLL-CALM activates transcription through the murine HOXA7 promoter. Methods: Human fibroblast cells were treated with Leptomycin B (an antifungal antibiotic which specifically inhibits nuclear export) and stained with an anti-CALM antibody. We prepared a set of expression vectors in which various portions of CALM are fused to a GAL4 DNA-binding domain. These vectors were co-transfected with a GAL4-luciferase reporter plasmid into COS7 cells, and luciferase activity was measured 48 hours after transient transfection. Luciferase assays were also performed using MSCV-MLL-CALM or MSCV-CALM plasmids co-transfected with a HOXA7 promoter-luciferase reporter construct. Results: After inhibition of nuclear export, native CALM localized to both the nucleus and cytoplasm. Significant luciferase activity was only observed with constructs containing distal CALM carboxy amino acids (aa 436–660). Mutation of an NR (Nuclear Receptor) Box motif (aa 510–514) did not affect CALM-dependent transcription. We found that two endocytosis-related NPF domains play opposite roles: deletion of NPF#1 (aa 437–439) dramatically reduced, while mutation of NPF#2 (aa 639–641) increased transcriptional activity. Expression constructs lacking GAL4 DNA binding domains had no effect on transcription, and GAL4 binding sites were required for luciferase activity in this system. Finally, MLL-CALM activated transcription of the murine HOXA7 promoter in comparison with native CALM or empty vector. Conclusions: We have confirmed that native CALM is able to localize to the nucleus, and we have begun to identify specific critical residues in the CALM TAD. The presence of a CALM TAD in MLL-CALM suggests that altered transcriptional regulation of MLL-dependent HOX genes may play an important role in MLL-CALM dependent transformation. Our observations raise the possibility that other MLL partners with native cytoplasmic localization may possess unrecognized transcriptional activity, and provide new insight into both MLL-CALM and CALM-AF10 mediated leukemogenesis.

scholarly journals Tumor Suppressor Pten Inhibits Nuclear Accumulation of β-Catenin and T Cell/Lymphoid Enhancer Factor 1–Mediated Transcriptional Activation

2001 ◽  
Vol 153 (6) ◽  
pp. 1161-1174 ◽  
Author(s):  
Sujata Persad ◽  
Armelle A.Troussard ◽  
Timothy R. McPhee ◽  
David J. Mulholland ◽  
Shoukat Dedhar
Keyword(s):  
T Cell ◽  
Tumor Suppressor ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Glycogen Synthase ◽  
Regulation Of Gene Expression ◽  
Nuclear Accumulation ◽  
Luciferase Reporter ◽  
Independent Manner ◽  
Elevated Expression

β-Catenin is a protein that plays a role in intercellular adhesion as well as in the regulation of gene expression. The latter role of β-catenin is associated with its oncogenic properties due to the loss of expression or inactivation of the tumor suppressor adenomatous polyposis coli (APC) or mutations in β-catenin itself. We now demonstrate that another tumor suppressor, PTEN, is also involved in the regulation of nuclear β-catenin accumulation and T cell factor (TCF) transcriptional activation in an APC-independent manner. We show that nuclear β-catenin expression is constitutively elevated in PTEN null cells and this elevated expression is reduced upon reexpression of PTEN. TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity. Furthermore, the constitutively elevated expression of cyclin D1, a β-catenin/TCF–regulated gene, is also suppressed upon reexpression of PTEN. Mechanistically, PTEN increases the phosphorylation of β-catenin and enhances its rate of degradation. We define a pathway that involves mainly integrin-linked kinase and glycogen synthase kinase 3 in the PTEN-dependent regulation of β-catenin stability, nuclear β-catenin expression, and transcriptional activity. Our data indicate that β-catenin/TCF–mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression.

scholarly journals Androgen-stimulated DNA synthesis and cytoskeletal changes in fibroblasts by a nontranscriptional receptor action

2003 ◽  
Vol 161 (3) ◽  
pp. 547-556 ◽  
Author(s):  
Gabriella Castoria ◽  
Maria Lombardi ◽  
Maria Vittoria Barone ◽  
Antonio Bilancio ◽  
Marina Di Domenico ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Signaling Pathways ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Nuclear Translocation ◽  
Pi3 Kinase ◽  
Experimental Conditions ◽  
Nih3t3 Cells ◽  
Membrane Ruffling ◽  
Signaling Activation

In NIH3T3 cells, 0.001 nM of the synthetic androgen R1881 induces and stimulates association of androgen receptor (AR) with Src and phosphatidylinositol 3-kinase (Pl3-kinase), respectively, thereby triggering S-phase entry. 10 nM R1881 stimulates Rac activity and membrane ruffling in the absence of the receptor–Src–PI3-kinase complex assembly. The antiandrogen Casodex and specific inhibitors of Src and PI3-kinase prevent both hormonal effects, DNA synthesis and cytoskeletal changes. Neither low nor high R1881 concentration allows receptor nuclear translocation and receptor-dependent transcriptional activity in fibroblasts, although they harbor the classical murine AR. The very low amount of AR in NIH3T3 cells (7% of that present in LNCaP cells) activates the signaling pathways, but apparently is not sufficient to stimulate gene transcription. This view is supported by the appearance of receptor nuclear translocation as well as receptor-mediated transcriptional activity after overexpression of AR in fibroblasts. In addition, AR-negative Cos cells transiently transfected with a very low amount of hAR cDNA respond to low and high R1881 concentrations with signaling activation. Interestingly, they do not show significant transcriptional activation under the same experimental conditions. Fibroblasts are the first example of cells that respond to steroid hormones with activation of signaling pathways in the absence of endogenous receptor transcriptional activity. The data reported also show that hormone concentration can be crucial in determining the type of cell responsiveness.

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