A requirement for thioredoxin in redox-sensitive modulation of T-cadherin expression in endothelial cells

2008 ◽  
Vol 416 (2) ◽  
pp. 271-280 ◽  
Author(s):  
Manjunath B. Joshi ◽  
Danila Ivanov ◽  
Maria Philippova ◽  
Emmanouil Kyriakakis ◽  
Paul Erne ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Nuclear Extract ◽  
Mobility Shift ◽  
Protein Levels ◽  
Nuclear Extracts ◽  
Nucleoprotein Complex ◽  
Thioredoxin 1 ◽  
Mobility Shift Assay ◽  
Reporter Gene Analysis

T-cad (T-cadherin), a glycosylphosphatidylinositol-anchored cadherin superfamily member, is expressed widely in the brain and cardiovascular system, and absent, decreased, or even increased, in cancers. Mechanisms controlling T-cad expression are poorly understood. The present study investigated transcriptional regulation of T-cad in ECs (endothelial cells). Conditions of oxidative stress (serum-deprivation or presence of H2O2) elevate T-cad mRNA and protein levels in ECs. Reporter gene analysis, using serially deleted T-cad promoter stretches ranging from −99 to −2304 bp, located the minimal promoter region of T-cad within −285 bp from the translation start site. Reporter activity in ECs transfected with the −285 bp construct increased under conditions of oxidative stress, and this was normalized by antioxidant N-acetylcysteine. An electrophoretic-mobility-shift assay revealed a specific nucleoprotein complex unique to −156 to −203 bp, which increased when nuclear extracts from oxidatively stressed ECs were used, suggesting the presence of redox-sensitive binding element(s). MS analysis of the nucleoprotein complex unique to −156 to −203 bp after streptavidin–agarose pull-down detected the presence of the redox-active protein thioredoxin. The presence of thioredoxin-1 in a nuclear extract from oxidatively stressed ECs was demonstrated after immunoprecipitation and immunoblotting. Transfection of ECs with thioredoxin-1 small interfering RNA abrogated oxidative-stress-induced up-regulation of T-cad transcripts and protein. We conclude that thioredoxin-1 is an important determinant of redox-sensitive transcriptional up-regulation of T-cad in ECs.

scholarly journals Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver

2005 ◽  
Vol 70 (5) ◽  
pp. 705-712 ◽  
Author(s):  
Miroslava Vujcic ◽  
Natasa Terzic ◽  
Aleksandra Ristic-Fira ◽  
Dusan Kanazir ◽  
Sabera Ruzdijic
Keyword(s):  
Dna Interaction ◽  
Binding Activity ◽  
Regulatory Proteins ◽  
Nuclear Proteins ◽  
Middle Aged ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Supershift Assay ◽  
Mobility Shift Assay ◽  
Oligonucleotide Analogues

Abstract: In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p < 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing. .

DNA-binding factors of B lymphoid cells are susceptible to limited proteolytic cleavage during nuclear extract preparation

1988 ◽  
Vol 8 (4) ◽  
pp. 1812-1815
Author(s):  
E L Mather
Keyword(s):  
Dna Binding ◽  
Nuclear Extract ◽  
Lymphoid Cells ◽  
Chain Gene ◽  
Immunoglobulin Heavy Chain Gene ◽  
Mobility Shift ◽  
Lymphoid Lines ◽  
Dna Binding Factors ◽  
Mobility Shift Assay ◽  
B Lymphoid

DNA-binding proteins that interact with the 3' end of the mouse mu immunoglobulin heavy chain gene were identified by the electrophoretic mobility shift assay. Complexes of distinctly different mobilities were formed by extracts prepared from B lymphoid lines representing different stages of maturation. The apparent stage-specific differences are shown to be due to proteolytic events that occurred during extract preparation.

Abstract 447: Ac-SDKP Suppresses TNFα-induced ICAM-1 Expression In Endothelial Cells Via Inhibition Of IκB Kinase And NF-κB Activation

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Liping Zhu ◽  
Xiao-Ping Yang ◽  
Pablo Nakagawa ◽  
Nour-Eddine Rhaleb ◽  
Pamela Harding ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Types ◽  
Intercellular Adhesion Molecule ◽  
Intercellular Adhesion Molecule 1 ◽  
Human Coronary Artery ◽  
Mobility Shift ◽  
Autoimmune Myocarditis ◽  
Iκb Kinase ◽  
Subsequent Degradation ◽  
Mobility Shift Assay

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a naturally occurring tetrapeptide that prevents inflammation and fibrosis in hypertension and other cardiovascular diseases. We previously showed that Ac-SDKP decreased transcription factor NF-κB activation in angiotensin II-induced hypertension and also reduced intercellular adhesion molecule-1 (ICAM-1) expression in an experimental autoimmune myocarditis and hypertension animal model. However, the mechanisms by which Ac-SDKP down-regulated ICAM-1 expression are unclear. TNFα is a proinflammatory cytokine that induces ICAM-1 expression on different cell types. We hypothesized that Ac-SDKP suppresses TNFα-induced ICAM-1 via inhibition of IκB kinase (IKK) and subsequently by blockade of NF-κB activation. Human coronary artery endothelial cells were treated with Ac-SDKP or vehicle and then stimulated with TNFα (0.5 ng/ml). ICAM-1 protein expression and phosphorylation of IKK (p-IKK), inhibitory κB (p-IκB), p38 (p-p38) and ERK (p-ERK) were measured by Western Blot. Activation of NF-κB was determined by electrophoretic mobility shift assay (EMSA). ICAM-1 expression was virtually undetectable under basal conditions, but greatly increased by TNFα. Ac-SDKP dose-dependently suppressed TNFα-induced ICAM-1 expression (set at a value of 1.0 arbitrary units, AU) to 0.67±0.13 (p<0.05), 0.51±0.12 (p<0.01) and 0.39±0.09 AU (p<0.01) at 0.1 nM, 1 nM and 10 nM, respectively. In addition, Ac-SDKP (10 nM) inhibited TNFα-induced p-IKK from 1.0 to 0.71±0.02 AU (p<0.01). Ac-SDKP also inhibited TNFα-induced IKKβ expression from 1.0 to 0.64±0.12 AU (p<0.05), without affecting IKKα expression. Furthermore, Ac-SDKP inhibited TNFα-induced p-IκB from 1.0 to 0.54±0.03 AU (p<0.01). EMSA results showed that Ac-SDKP inhibited TNFα-mediated activation of NF-κB, which was 0.63±0.04-fold of TNFα-treated cells. However, Ac-SDKP had no effect on TNFα-induced p-p38 and p-ERK. Thus, we conclude that Ac-SDKP inhibits TNFα-induced IKK and subsequent degradation of IκB, thereby preventing NF-κB activation and ICAM-1 expression. These inhibitory effects are independent of p38 and ERK.

scholarly journals Regulation of differential proton-coupled folate transporter gene expression in human tumors: transactivation by KLF15 with NRF-1 and the role of Sp1

2019 ◽  
Vol 476 (8) ◽  
pp. 1247-1266
Author(s):  
Zhanjun Hou ◽  
Carrie O'Connor ◽  
Josephine Frühauf ◽  
Steve Orr ◽  
Seongho Kim ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Transcriptional Control ◽  
Consensus Sequence ◽  
Minimal Promoter ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Ht1080 Cells ◽  
Promoter Construct ◽  
Mobility Shift Assay ◽  
Reporter Gene Assays

Abstract Tumors can be therapeutically targeted with novel antifolates (e.g. AGF94) that are selectively transported by the human proton-coupled folate transporter (hPCFT). Studies were performed to determine the transcription regulation of hPCFT in tumors and identify possible mechanisms that contribute to the highly disparate levels of hPCFT in HepG2 versus HT1080 tumor cells. Transfection of hPCFT-null HT1080 cells with hPCFT restored transport and sensitivity to AGF94. Progressive deletions of the hPCFT promoter construct (−2005 to +96) and reporter gene assays in HepG2 and HT1080 cells confirmed differences in hPCFT transactivation and localized a minimal promoter to between positions −50 and +96. The minimal promoter included KLF15, GC-Box and NRF-1 cis-binding elements whose functional importance was confirmed by promoter deletions and mutations of core consensus sequences and reporter gene assays. In HepG2 cells, NRF-1, KLF15 and Sp1 transcripts were increased over HT1080 cells by ∼5.1-, ∼44-, and ∼2.4-fold, respectively. In Drosophila SL2 cells, transfection with KLF15 and NRF-1 synergistically activated the hPCFT promoter; Sp1 was modestly activating or inhibitory. Chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA) and supershifts confirmed differential binding of KLF15, Sp1, and NRF-1 to the hPCFT promoter in HepG2 and HT1080 cells that paralleled hPCFT levels. Treatment of HT1080 nuclear extracts (NE) with protein kinase A increased Sp1 binding to its consensus sequence by EMSA, suggesting a role for Sp1 phosphorylation in regulating hPCFT transcription. A better understanding of determinants of hPCFT transcriptional control may identify new therapeutic strategies for cancer by modulating hPCFT levels in combination with hPCFT-targeted antifolates.

PDGF Up-regulates CSF-1 Gene Transcription in Ameloblast-like Cells

2008 ◽  
Vol 87 (1) ◽  
pp. 33-38 ◽  
Author(s):  
Y. Wittrant ◽  
B. Sriniketan Bhandari ◽  
H. Abboud ◽  
N. Benson ◽  
K. Woodruff ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Binding Motif ◽  
Rt Pcr ◽  
Mobility Shift ◽  
Protein Levels ◽  
Macrophage Colony Stimulating Factor ◽  
Cellular Pathways ◽  
Mobility Shift Assay ◽  
Macrophage Colony ◽  
Stimulating Factor

Macrophage colony-stimulating factor (CSF-1) is a key regulatory cytokine for amelogenesis, and ameloblasts synthesize CSF-1. We hypothesized that PDGF stimulates DNA synthesis and regulates CSF-1 in these cells. We determined the effect of PDGF on CSF-1 expression using MEOE-3M ameloblasts as a model. By RT-PCR, MEOE-3M expressed PDGFRs and PDGF A- and B-chain mRNAs. PDGF-BB increased DNA synthesis and up-regulated CSF-1 mRNA and protein in MEOE-3M. Cells transfected with CSF-1 promoter deletion constructs were analyzed. A PDGF-responsive region between −1.7 and −0.795 kb, containing a consensus Pea3 binding motif, was identified. Electrophoretic mobility shift assay (EMSA) showed that PDGF-BB stimulated protein binding to this motif that was inhibited in the presence of anti-Pea3 antibody. Analysis of these data provides the first evidence that PDGF-BB is a mitogen for MEOE-3M and increases CSF-1 protein levels, predominantly by transcription. Elucidation of the cellular pathways that control CSF-1 expression may provide novel strategies for the regulation of enamel matrix formation.

scholarly journals A Sequence of the CIS Gene Promoter Interacts Preferentially with Two Associated STAT5A Dimers: a Distinct Biochemical Difference between STAT5A and STAT5B

1998 ◽  
Vol 18 (10) ◽  
pp. 5852-5860 ◽  
Author(s):  
Frédérique Verdier ◽  
Raquel Rabionet ◽  
Fabrice Gouilleux ◽  
Christian Beisenherz-Huss ◽  
Paule Varlet ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Prolactin Receptor ◽  
Gene Promoter ◽  
Expression Vectors ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Mobility Shift Assay ◽  
Binding Sequence ◽  
Biochemical Difference

ABSTRACT Two distinct genes encode the closely related signal transducer and activator of transcription proteins STAT5A and STAT5B. The molecular mechanisms of gene regulation by STAT5 and, particularly, the requirement for both STAT5 isoforms are still undetermined. Only a few STAT5 target genes, among them the CIS (cytokine-inducible SH2-containing protein) gene, have been identified. We cloned the human CIS gene and studied the human CIS gene promoter. This promoter contains four STAT binding elements organized in two pairs. By electrophoretic mobility shift assay studies using nuclear extracts of UT7 cells stimulated with erythropoietin, we showed that these four sequences bound to STAT5-containing complexes that exhibited different patterns and affinities: the three upstream STAT binding sequences bound to two distinct STAT5-containing complexes (C0 and C1) and the downstream STAT box bound only to the slower-migrating C1 band. Using nuclear extracts from COS-7 cells transfected with expression vectors for the prolactin receptor, STAT5A, and/or STAT5B, we showed that the C1 complex was composed of a STAT5 tetramer and was dependent on the presence of STAT5A. STAT5B lacked this property and bound with a stronger affinity than did STAT5A to the four STAT sequences as a homodimer (C0 complex). This distinct biochemical difference between STAT5A and STAT5B was confirmed with purified activated STAT5 recombinant proteins. Moreover, we showed that the presence on the same side of the DNA helix of a second STAT sequence increased STAT5 binding and that only half of the palindromic STAT binding sequence was sufficient for the formation of a STAT5 tetramer. Again, STAT5A was essential for this cooperative tetrameric association. This property distinguishes STAT5A from STAT5B and could be essential to explain the transcriptional regulation diversity of STAT5.

Urea-associated oxidative stress and Gadd153/CHOP induction

1999 ◽  
Vol 276 (5) ◽  
pp. F786-F793 ◽  
Author(s):  
Zheng Zhang ◽  
Xiao-Yan Yang ◽  
David M. Cohen
Keyword(s):  
Oxidative Stress ◽  
Reporter Gene ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Kidney Medulla ◽  
Urea Treatment ◽  
Protein Levels ◽  
Enhancer Binding Protein ◽  
Mrna And Protein Expression ◽  
Reporter Gene Analysis

Urea treatment (100–300 mM) increased expression of the oxidative stress-responsive transcription factor, Gadd153/CHOP, at the mRNA and protein levels (at ≥4 h) in renal medullary mIMCD3 cells in culture, whereas other solutes did not. Expression of the related protein, CCAAT/enhancer-binding protein (C/EBP-β), was not affected, nor was expression of the sensor of endoplasmic reticulum stress, grp78. Urea modestly increased Gadd153 transcription by reporter gene analysis but failed to influence Gadd153 mRNA stability. Importantly, upregulation of Gadd153 mRNA and protein expression by urea was antioxidant sensitive. Accordingly, urea treatment was associated with oxidative stress, as quantitated by intracellular reduced glutathione content in mIMCD3 cells. In addition, antioxidant treatment partially inhibited the ability of urea to activate transcription of an Egr-1 luciferase reporter gene. Therefore oxidative stress represents a novel solute-signaling pathway in the kidney medulla and, potentially, in other tissues.

scholarly journals The NF-κB p50 Subunit Is Protective during Intestinal Entamoeba histolytica Infection of 129 and C57BL/6 Mice

2010 ◽  
Vol 78 (4) ◽  
pp. 1475-1481 ◽  
Author(s):  
Kyou-Nam Cho ◽  
Stephen M. Becker ◽  
Eric R. Houpt
Keyword(s):  
Entamoeba Histolytica ◽  
Electrical Resistance ◽  
Transepithelial Electrical Resistance ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mobility Shift ◽  
Protective Function ◽  
Targeted Deletion ◽  
Nuclear Extracts ◽  
Histolytica Infection ◽  
Mobility Shift Assay

ABSTRACT Entamoeba histolytica is the agent of amebic colitis. In this work we examined the intestinal NF-κB response to this parasite. Using an enzyme-linked immunosorbent assay (ELISA) and an electrophoretic mobility shift assay, we found that the NF-κB subunit p50 predominated in nuclear extracts of whole cecal tissue and of isolated crypts from mice inoculated with E. histolytica. p50 was protective, since C57BL/6 and 129 mice in which there was targeted deletion of this subunit were more susceptible to E. histolytica infection as measured by culture results, cecal parasite ELISA results, and/or histologic scores. The transepithelial electrical resistance of cecal explants from C57BL/6 and 129 p50 knockout mice decreased markedly in response to the parasite compared with the transepithelial electrical resistance of their wild-type counterparts, suggesting that a protective function of p50 was present in the epithelium itself. This work shows that NF-κB activity, particularly activity of the p50 subunit, is one factor that contributes to resistance of the gut to E. histolytica infection.

Signal Transducer and Activator of Transcription (Stat)-3 Activates the Receptor Tyrosine Kinase-Like Orphan (ROR)-1 Gene in Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 57-57
Author(s):  
Li Ping ◽  
David Harris ◽  
Zhiming Liu ◽  
Michael Keating ◽  
Zeev Estrov
Keyword(s):  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Luciferase Assay ◽  
Mobility Shift ◽  
Protein Levels ◽  
Cell Chip ◽  
Stat3 Phosphorylation ◽  
Mobility Shift Assay ◽  
Activation Sequence ◽  
Gene Mrna

Abstract Abstract 57 ROR1, an embryonic protein involved in organogenesis and Wnt signaling, is expressed in B-cell CLL. Because Stat3 is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors ψ-interferon activation sequence (GAS)-like elements typically activated by Stat3, we sought to determine whether Stat3 activates ROR1. In MM1 cells interleukin (IL)-6 induced Stat3 phosphorylation and upregulated ROR1 whereas STAT3-siRNA downregulated both Stat3 and ROR1 protein levels, suggesting that Stat3 transcribes ROR1. Therefore, we cloned the human ROR1 promoter, generated a series of truncated promoter constructs and assessed their activity by using the luciferase assay. We found that IL-6 augmented the luciferase activity of ROR1 -195, ROR1 -666, ROR1 -834, and co-transfection with Stat3-siRNA significantly attenuated it, suggesting that IL-6 enhanced ROR1 expression by activating Stat3. Furthermore, we established that a region, located between bp -122 and -134, harbors a GAS-like element and activates the ROR1 promoter upon exposure to IL-6. Binding of Stat3 to that region in IL-6-stimulated MM1 cells was confirmed by the electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). To test whether Stat3 transcribes ROR1 in CLL, we obtained fresh CLL cells and by using the same GAS-like element-containing probe we performed EMSA. CLL cell nuclear protein bound this probe and anti-Stat3 and -phsophoserine Stat3 antibodies induced a super-shift. CLL cell ChIP confirmed that Stat3 binds to the promoter of ROR1 as well as the promoters of the Stat3-regulated genes STAT3, c-Myc and P21, but not that of the control gene RPL30. Finally, using qRT-PCR and western immunoblotting we determined that STAT3-shRNA downregulated ROR1, STAT3 and STAT3-regulated gene mRNA by 4-6 fold, and Stat3 and ROR1 protein levels by 50%. Taken together, these data suggest that constitutively activated Stat3 binds to the ROR1 promoter, activates transcription, and induces production of ROR1 protein in CLL cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Molecular regulation of the human IL-3 gene: inducible T cell-restricted expression requires intact AP-1 and Elf-1 nuclear protein binding sites.

1993 ◽  
Vol 178 (5) ◽  
pp. 1681-1692 ◽  
Author(s):  
L R Gottschalk ◽  
D M Giannola ◽  
S G Emerson
Keyword(s):  
T Cells ◽  
Transcription Factors ◽  
T Cell ◽  
Protein Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Nuclear Protein ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Mobility Shift Assay

Interleukin 3 (IL-3) is a hematopoietic stem-cell growth and differentiation factor that is expressed solely in activated T and NK cells. Studies to date have identified elements 5' to the IL-3 coding sequences that regulate its transcription, but the sequences that confer T cell-specific expression remain to be clearly defined. We have now identified DNA sequences that are required for T cell-restricted IL-3 gene transcription. A series of transient transfections performed with human IL-3-chloramphenicol acetyltransferase (CAT) reporter plasmids in T and non-T cells revealed that a plasmid containing 319 bp of 5' flanking sequences was active exclusively in T cells. Deletion analysis revealed that T cell specificity was conferred by a 49-bp fragment (bp -319 to -270) that included a potential binding site for AP-1 transcription factors 6 bp upstream of a binding site for Elf-1, a member of the Ets family of transcription factors. DNaseI footprint and electrophoretic mobility shift assay analyses performed with MLA-144 T cell nuclear extracts demonstrated that this 49-bp region contains a nuclear protein binding region that includes consensus AP-1 and Elf-1 binding sites. In addition, extracts prepared from purified human T cells contained proteins that bound to synthetic oligonucleotides corresponding to the AP-1 and Elf-1 binding sites. In vitro-transcribed and -translated Elf-1 protein bound specifically to the Elf-1 site, and Elf-1 antisera competed and super shifted nuclear protein complexes present in MLA-144 nuclear extracts. Moreover, addition of anti-Jun family antiserum in electrophoretic mobility shift assay reactions completely blocked formation of the AP-1-related complexes. Transient transfection studies in MLA-144 T cells revealed that constructs containing mutations in the AP-1 site almost completely abolished CAT activity while mutation of the Elf-1 site or the NF-IL-3 site, a previously described nuclear protein binding site (bp. -155 to -148) in the IL-3 promoter, reduced CAT activity to &lt; 25% of the activity given by wild-type constructs. We conclude that expression of the human IL-3 gene requires the AP-1 and Elf-1 binding sites; however, unlike other previously characterized cytokine genes such as IL-2, the AP-1 and Elf-1 factors can bind independently in the IL-3 gene.(ABSTRACT TRUNCATED AT 400 WORDS)

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