scholarly journals Estrogen Up-Regulates Hepatic Expression of Suppressors of Cytokine Signaling-2 and -3 in Vivo and in Vitro

Endocrinology ◽  
2004 ◽  
Vol 145 (12) ◽  
pp. 5525-5531 ◽  
Author(s):  
Gary M. Leong ◽  
Sofia Moverare ◽  
Jesena Brce ◽  
Nathan Doyle ◽  
Klara Sjögren ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Hepatoma Cells ◽  
Cytokine Signaling ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Wild Type ◽  
Hepatic Expression ◽  
Suppressors Of Cytokine Signaling

Abstract Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine action. We recently reported that estrogen stimulates SOCS-2 expression and inhibits GH signaling in kidney cells. The effects of estrogen on SOCS expression in other tissues are unclear. The aim of this study was to investigate in vivo and in vitro whether estrogen affected SOCS expression in the liver, a major target organ of GH. The in vivo hepatic effects of estrogen on ovariectomized mice lacking estrogen receptor (ER)-α, ERβ, or both and their wild-type littermates were examined by DNA microarray analysis. In vitro, the effects of estrogen on SOCS expression in human hepatoma cells were examined by reverse transcription quantitative PCR. Long-term (3 wk) estrogen treatment induced a 2- to 3-fold increase in hepatic expression of SOCS-2 and -3 in wild-type and ERβ knockout mice but not in those lacking ERα or both ER subtypes. Short-term treatment (at 24 h) increased the mRNA level of SOCS-3 but not SOCS-2. In cultured hepatoma cells, estrogen increased SOCS-2 and -3 mRNA levels by 2-fold in a time- and dose-dependent manner (P < 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P < 0.05) in constructs containing a region between nucleotides −1862 and −855. Moreover, estrogen and GH had additive effects on the SOCS-3 promoter activity. In summary, estrogen, via ERα, up-regulated hepatic expression of SOCS-2 and -3, probably through transcriptional activation. This indicates a novel mechanism of estrogen regulation of cytokine action.

scholarly journals Tumor-Stroma Crosstalk Enhances REG3A Expressions that Drive the Progression of Hepatocellular Carcinoma

2020 ◽  
Vol 21 (2) ◽  
pp. 472 ◽  
Author(s):  
Yuri Cho ◽  
Min Ji Park ◽  
Koeun Kim ◽  
Jae-Young Park ◽  
Jihye Kim ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Microarray Analysis ◽  
Cdna Microarray ◽  
Tumor Stroma ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Cdna Microarray Analysis ◽  
Hcc Cells

Abstract: Background: Crosstalk between tumors and their microenvironment plays a crucial role in the progression of hepatocellular carcinoma (HCC). However, there is little existing information about the key signaling molecule that modulates tumor-stroma crosstalk. Methods: Complementary DNA (cDNA) microarray analysis was performed to identify the key molecule in tumor-stroma crosstalk. Subcutaneous xenograft in vivo murine model, immunoblotting, immunofluorescence, and real-time polymerase chain reaction using HCC cells and tissues were performed. Results: The key molecule, regenerating gene protein-3A (REG3A), was most significantly enhanced when coculturing HCC cells and activated human hepatic stellate cells (HSCs) (+8.2 log) compared with monoculturing HCC cells using cDNA microarray analysis. Downregulation of REG3A using small interfering RNA significantly decreased the proliferation of HSC-cocultured HCC cells in vitro and in vivo, and enhanced deoxycholic acid-induced HCC cell apoptosis. Crosstalk-induced REG3A upregulation was modulated by platelet-derived growth factor ββ (PDGF-ββ) in p42/44-dependent manner. REG3A mRNA levels in human HCC tissues were upregulated 1.8-fold compared with non-tumor tissues and positively correlated with PDGF-ββ levels. Conclusions: REG3A/p42/44 pathway/PDGF-ββ signaling plays a significant role in hepatocarcinogenesis via tumor-stroma crosstalk. Targeting REG3A is a potential novel therapeutic target for the management of HCCs by inhibiting crosstalk between HCC cells and HSCs.

scholarly journals Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

2021 ◽  
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Wen-Hsin Lee ◽  
Sandhya Bangaru ◽  
Andrew B Ward ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Mouse Model ◽  
Neutralizing Antibodies ◽  
Dependent Manner ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

Abstract 5566: Recovery of Erk Signaling Restores Defective Angiogenesis and Arteriogenesis in Synectin-Deficient Animals

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Bin Ren ◽  
Arpita Mukhopadhyay* ◽  
Anthony A Lanahan ◽  
Zhen W Zhuang ◽  
Karen L Moodie ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Branching Morphogenesis ◽  
Erk Signaling ◽  
Dependent Manner ◽  
Wild Type ◽  
Erk Activation ◽  
Arterial Development ◽  
Constitutively Active

Background : Arterial morphogenesis is an important and poorly understood process. We have previously demonstrated that disruption of synectin gene expression in mice and zebrafish results in impaired arterial development and branching morphogenesis. Synectin null endothelial cells demonstrate reduced VEGF responsiveness in terms of migration, proliferation and differentiation and ERK-1/2 activation (Chittenden et al, Dev Cell 2006). Since ERK has been established as major participants in the regulation of cell growth and differentiation and Erk activation has been previously linked to arterial morphogenesis, we evaluated whether activation of Erk signaling in synectin disrupted mice and zebrafish as well as synectin KO arterial endothelial cells (ECs) would restore defective migration, arterial differentiation, angiogenesis and arteriogenesis. To stimulate ERK signaling we used partial inhibition of PI3-K activity to reduce Akt-dependent suppression of Raf1 activation or introduction of constitutively active ERK construct. Methods : In vitro studies were conducted with primary arterial ECs isolated from synectin wild type (WT) and knock out (KO) mice. In vivo studies were carried out in WT and synectin deficient mice and synectin knockdown zebrafish embryos. Results: Exposure of synectin −/− arterial EC to two selective PI3K inhibitors GS4898 or LY294002 in vitro restored ERK activation in a dose-dependent manner and returned cell migration and in vitro branching morphogenesis to wild type levels. Transduction of a constitutively active ERK construct in vitro or in a Matrigel model in vivo had similar effect. Systemic treatment of synectin −/− mice with GS4898 fully restored impaired angiogenesis and arterial morphogenesis in adult animals in the setting of hindlimb ischemia. Similar treatment nearly completely restored arterial development defects in zebrafish treated with a synectin morpholino. Conclusions: ERK activation plays a key role in arteriogenesis both in adult tissues and during embryonic development. Activation of compromised ERK-1/2 signaling may be a novel therapeutic intervention to stimulate arteriogenesis.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

scholarly journals Differential transcriptional regulation of the human thrombin receptor gene by the Sp family of transcription factors in human endothelial cells

1998 ◽  
Vol 330 (3) ◽  
pp. 1469-1474 ◽  
Author(s):  
Yaxu WU ◽  
Johannes RUEF ◽  
N. Gadiparthi RAO ◽  
Cam PATTERSON ◽  
S. Marschall RUNGE
Keyword(s):  
Endothelial Cells ◽  
Promoter Activity ◽  
Receptor Gene ◽  
Thrombin Receptor ◽  
Wild Type ◽  
Mobility Shift ◽  
Human Thrombin ◽  
Gc Box

The mitogenic effects of thrombin are mediated by a G-protein-coupled receptor. Because the effects of thrombin are strongly influenced by the expression of its receptor, an understanding of its regulatory mechanisms is essential. To identify mechanisms of human thrombin receptor (HTR) gene regulation, a series of HTR-promoter-luciferase constructs were made and transfected into human microvascular endothelial cells for analysis. Deletion from bp -303 to -164 abolished reporter gene expression. Dimethyl sulphate treatment in vivo and DNase I footprinting in vitro demonstrated that a cluster of three GC box consensus sites was occupied, and electrophoretic mobility-shift assays established that Sp1 and Sp3 both bind to this 3ʹ GC box cluster. We mutated each of the three GC boxes individually and all three collectively within this 3ʹ cluster. Basal promoter activity was decreased to 46%, 78% and 29% of control for each of the GC boxes mutated individually, and to 6% when the three were mutated collectively. To test the individual abilities of Sp1 and Sp3 to activate or repress HTR transcription, we conducted co-transfection experiments with wild-type or mutated HTR-promoter-luciferase constructs. Co-transfection with Sp1 significantly augmented wild-type HTR promoter activity. Sp3 alone did not affect activity, and inhibited Sp1-mediated activation. Competition for shared binding sites by Sp1 and Sp3 might differentially regulate HTR expression in vascular endothelial cells.

scholarly journals Inhibition of Cellular Proteasome Activities Enhances Hepadnavirus Replication in an HBX-Dependent Manner

2004 ◽  
Vol 78 (9) ◽  
pp. 4566-4572 ◽  
Author(s):  
Zhensheng Zhang ◽  
Ulrike Protzer ◽  
Zongyi Hu ◽  
James Jacob ◽  
T. Jake Liang
Keyword(s):  
Recombinant Adenovirus ◽  
Hepatitis Virus ◽  
Proteasome Inhibitors ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Dependent Manner ◽  
Type Virus ◽  
Wild Type

ABSTRACT The X protein (HBX) of the hepatitis B virus (HBV) is not essential for the HBV life cycle in vitro but is important for productive infection in vivo. Our previous study suggests that interaction of HBX with the proteasome complex may underlie the pleiotropic functions of HBX. With the woodchuck model, we demonstrated that the X-deficient mutants of woodchuck hepatitis virus (WHV) are not completely replication defective, possibly behaving like attenuated viruses. In the present study, we analyzed the effects of the proteasome inhibitors on the replication of wild-type and X-negative HBV and WHV. Recombinant adenoviruses or baculoviruses expressing replicating HBV or WHV genomes have been developed as a robust and convenient system to study viral replication in tissue culture. In cells infected with either the recombinant adenovirus-HBV or baculovirus-WHV, the replication level of the X-negative construct was about 10% of that of the wild-type virus. In the presence of proteasome inhibitors, the replication of the wild-type virus was not affected, while the replication of the X-negative virus of either HBV or WHV was enhanced and restored to the wild-type level. Our data suggest that HBX affects hepadnavirus replication through a proteasome-dependent pathway.

scholarly journals Ets-2 Repressor Factor Silences Extrasynaptic Utrophin by N-Box–mediated Repression in Skeletal Muscle

2007 ◽  
Vol 18 (8) ◽  
pp. 2864-2872 ◽  
Author(s):  
Kelly J. Perkins ◽  
Utpal Basu ◽  
Murat T. Budak ◽  
Caroline Ketterer ◽  
Santhosh M. Baby ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Neuromuscular Junctions ◽  
Protein Product ◽  
Mrna Levels ◽  
Laser Capture Microscopy ◽  
Erk Phosphorylation ◽  
Duchenne's Muscular Dystrophy

Utrophin is the autosomal homologue of dystrophin, the protein product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin expression is temporally and spatially regulated being developmentally down-regulated perinatally and enriched at neuromuscular junctions (NMJs) in adult muscle. Synaptic localization of utrophin occurs in part by heregulin-mediated extracellular signal-regulated kinase (ERK)-phosphorylation, leading to binding of GABPα/β to the N-box/EBS and activation of the major utrophin promoter-A expressed in myofibers. However, molecular mechanisms contributing to concurrent extrasynaptic silencing that must occur to achieve NMJ localization are unknown. We demonstrate that the Ets-2 repressor factor (ERF) represses extrasynaptic utrophin-A in muscle. Gel shift and chromatin immunoprecipitation studies demonstrated physical association of ERF with the utrophin-A promoter N-box/EBS site. ERF overexpression repressed utrophin-A promoter activity; conversely, small interfering RNA-mediated ERF knockdown enhanced promoter activity as well as endogenous utrophin mRNA levels in cultured muscle cells in vitro. Laser-capture microscopy of tibialis anterior NMJ and extrasynaptic transcriptomes and gene transfer studies provide spatial and direct evidence, respectively, for ERF-mediated utrophin repression in vivo. Together, these studies suggest “repressing repressors” as a potential strategy for achieving utrophin up-regulation in DMD, and they provide a model for utrophin-A regulation in muscle.

Comparison of the Effects on Reversal of Multi-Drug Resistance of 5-Bromotetrandrine and Tetrandrine in K562/A02 Cell Line in Vivo and in Vitro

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5059-5059
Author(s):  
Bao-An Chen ◽  
Jue-qiong Wang ◽  
Jian Cheng ◽  
Feng Gao ◽  
Wen-lin Xu ◽  
...  
Keyword(s):  
Cell Line ◽  
Nude Mice ◽  
Leukemia Cell Line ◽  
Multi Drug Resistance ◽  
Human Leukemia ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Intracellular Accumulation

Abstract Objective This study was to compare the reversal effect of 5-bromotetrandrine (BrTet) with Tetrandrine (Tet) when combined with ADM on multidrug resistance cell line K562/A02 and to investigate the reversal mechanism of this new derivative. Methods The protein levels of P-glycoprotein (P-gp) were detected by fluorospectrophotometry and Western blot. The mRNA levels of P-gp were determined by RT-PCR. The in vivo effect of Tet was investigated using nude mice grafted with sensitive human leukemia cell line K562 and MDR cell line K562/A02. Results Flow cytometry assay showed that 1.0 μMol/L BrTet significantly increased the apoptosis percentage. BrTet also enhanced the intracellular accumulation of ADM in K562/A02 cells and its potency was greater than that of Tet at the same concentrations. BrTet inhibited the overexpression of P-gp and down regulated MDR1 mRNA expression in K562/A02 cells in a dose-dependent manner. In nude mice bearing K562 xenografts on the left flank and K562/A02 xenografts on the right flank, i.p. injection of 10 mg/kg BrTet significantly enhanced the antitumor activity of ADM against K562/A02 xenografts with inhibitory rates of 26.1%, while ADM alone inhibited the growth of KBv200 xenografts by only 5.8%. Conclusion BrTet showed significant MDR reversal activity in vitro and in vivo. Its activity may be related to the inhibition of P-gp overexpression and the increase in intracellular accumulation of anticancer drugs, which lead to more K562/A02 cells apoptosis.

scholarly journals Mutations in RNA Polymerase II and Elongation Factor SII Severely Reduce mRNA Levels in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (10) ◽  
pp. 5771-5779 ◽  
Author(s):  
J. Cale Lennon ◽  
Megan Wind ◽  
Laura Saunders ◽  
M. Benjamin Hock ◽  
Daniel Reines
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genetic Interaction ◽  
Elongation Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Mutant Cells ◽  
Rna Levels

ABSTRACT Elongation factor SII interacts with RNA polymerase II and enables it to transcribe through arrest sites in vitro. The set of genes dependent upon SII function in vivo and the effects on RNA levels of mutations in different components of the elongation machinery are poorly understood. Using yeast lacking SII and bearing a conditional allele of RPB2, the gene encoding the second largest subunit of RNA polymerase II, we describe a genetic interaction between SII and RPB2. An SII gene disruption or therpb2-10 mutation, which yields an arrest-prone enzyme in vitro, confers sensitivity to 6-azauracil (6AU), a drug that depresses cellular nucleoside triphosphates. Cells with both mutations had reduced levels of total poly(A)+ RNA and specific mRNAs and displayed a synergistic level of drug hypersensitivity. In cells in which the SII gene was inactivated, rpb2-10 became dominant, as if template-associated mutant RNA polymerase II hindered the ability of wild-type polymerase to transcribe. Interestingly, while 6AU depressed RNA levels in both wild-type and mutant cells, wild-type cells reestablished normal RNA levels, whereas double-mutant cells could not. This work shows the importance of an optimally functioning elongation machinery for in vivo RNA synthesis and identifies an initial set of candidate genes with which SII-dependent transcription can be studied.

scholarly journals Identification of a Bidirectional Splicing Enhancer: Differential Involvement of SR Proteins in 5′ or 3′ Splice Site Activation

1999 ◽  
Vol 19 (11) ◽  
pp. 7347-7356 ◽  
Author(s):  
Cyril F. Bourgeois ◽  
Michel Popielarz ◽  
Georges Hildwein ◽  
James Stevenin
Keyword(s):  
Splice Site ◽  
Sr Proteins ◽  
Dependent Manner ◽  
Wild Type ◽  
Adenovirus E1a ◽  
Differential Involvement ◽  
Splicing Enhancer

ABSTRACT The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5′ splice sites and of one major or one minor 3′ splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5′ splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5′ splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3′ splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5′ splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity.

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