scholarly journals C/EBPα activates the transcription of triacylglycerol hydrolase in 3T3-L1 adipocytes

2005 ◽  
Vol 388 (3) ◽  
pp. 959-966 ◽  
Author(s):  
Enhui WEI ◽  
Richard LEHNER ◽  
Dennis E. VANCE
Keyword(s):  
Transcriptional Activation ◽  
Promoter Region ◽  
Ectopic Expression ◽  
3T3 Cells ◽  
Mobility Shift ◽  
Protein Levels ◽  
Triacylglycerol Hydrolase ◽  
Enhancer Binding Protein ◽  
Nih 3T3 ◽  
Electrophoretic Mobility Shift Assays

TGH (triacylglycerol hydrolase) catalyses the lipolysis of intracellular stored triacylglycerol. To explore the mechanisms that regulate TGH expression in adipose tissue, we studied the expression of TGH during the differentiation of 3T3-L1 adipocytes. TGH mRNA and protein levels increased dramatically in 3T3-L1 adipocytes compared with pre-adipocytes. Electrophoretic mobility shift assays demonstrated enhanced binding of nuclear proteins of adipocytes to the distal murine TGH promoter region (−542/−371 bp), yielding one adipocyte-specific migrating complex. Competitive and supershift assays demonstrated that the distal TGH promoter fragment bound C/EBPα (CCAAT/enhancer-binding protein α). Transient transfections of different mutant TGH promoter–luciferase constructs into 3T3-L1 adipocytes and competitive electromobility shift assays showed that the C/EBP-binding elements at positions −470/−459 bp and −404/−390 bp are important for transcriptional activation. Co-transfection with C/EBPα cDNA and TGH promoter constructs in 3T3-L1 pre-adipocytes demonstrated that C/EBPα increased TGH promoter activity. Ectopic expression of C/EBPα in NIH 3T3 cells activated TGH mRNA expression without causing differentiation into adipocytes. These experiments directly link increased TGH expression in adipocytes to transcriptional regulation by C/EBPα. This is the first evidence that C/EBPα participates directly in the regulation of an enzyme associated with lipolysis.

scholarly journals Induction of the RelB NF-κB Subunit by the Cytomegalovirus IE1 Protein Is Mediated via Jun Kinase and c-Jun/Fra-2 AP-1 Complexes

2005 ◽  
Vol 79 (1) ◽  
pp. 95-105 ◽  
Author(s):  
Xiaobo Wang ◽  
Gail E. Sonenshein
Keyword(s):  
Promoter Activity ◽  
Ectopic Expression ◽  
Distal Region ◽  
3T3 Cells ◽  
Mobility Shift ◽  
Gene Encoding ◽  
3T3 Fibroblasts ◽  
Mobility Shift Assay ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT We recently demonstrated that the cytomegalovirus (CMV) immediate-early 1 (IE1) protein induces transcription of the gene encoding the RelB NF-κB subunit. The mechanism of this activation has been explored here. We report that the induction of the relB promoter by IE1 protein is mediated via activation of JNK and AP-1. The region controlling relB promoter induction was mapped to the upstream ∼600-bp region between −1694 and −1096 bp. IE1 stimulated AP-1 activity in NIH 3T3 cells. Competition electrophoretic mobility shift assay (EMSA) confirmed the presence of one bona fide AP-1 element centered at −1503 bp. Introduction of a G-to-C mutation in the AP-1 binding site within the distal region of the relB promoter eliminated its activation by IE1 in both NIH 3T3 fibroblasts and vascular smooth muscle cells (SMCs). Supershift EMSA identified c-Jun, Fra-2, and c-Fos in AP-1 binding complexes in IE1 transfected NIH 3T3 cells. IE1 induced c-Jun phosphorylation, and treatment with SP600125, a selective JNK inhibitor, as well as overexpression of JNK-binding domain of JIP1, blocked IE1-mediated induction of AP-1 and relB promoter activity in NIH 3T3 cells and SMCs. Ectopic expression of c-Jun plus Fra-2, but not c-Fos, induced relB promoter activity. The relB promoter has two proximal NF-κB elements, and c-Jun/Fra-2 worked in synergy with p50/p65 NF-κB complexes. Overall, these findings demonstrate for the first time the role of AP-1 in transcriptional regulation of a gene encoding an NF-κB subunit, and its involvement in induction of RelB activity by the CMV IE1 protein.

scholarly journals GATA-6 mediates transcriptional activation of aquaporin-5 through interactions with Sp1

2008 ◽  
Vol 295 (5) ◽  
pp. C1141-C1150 ◽  
Author(s):  
Beiyun Zhou ◽  
Tricia A. Francis ◽  
Hui Yang ◽  
Wanru Tseng ◽  
Qian Zhong ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Alveolar Epithelial Cells ◽  
Proximal Promoter ◽  
Type I ◽  
3T3 Cells ◽  
Aquaporin 5 ◽  
Mobility Shift ◽  
Alveolar Epithelial ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We investigated mechanisms underlying GATA-6-mediated transcriptional activation of the alveolar epithelial type I cell-enriched gene aquaporin-5 (AQP5). GATA-6 expression increases in alveolar epithelial cells in primary culture, concurrent with upregulation of AQP5 and transition to a type I cell-like phenotype. Cotransfections in MLE-15 and NIH 3T3 cells demonstrated trans-activation by GATA-6 of a rat 1,716-bp-AQP5-luciferase (−1716-AQP5-Luc) reporter. Electrophoretic mobility shift assay and chromatin immunoprecipitation identified an interaction between GATA-6 and putative binding sites in the AQP5 promoter. However, mutation of these sites did not reduce GATA-6-mediated activation, implicating mechanisms in addition to direct binding of GATA-6 to DNA. A 5′-deletion construct, −358-AQP5-Luc, that does not encompass GATA motifs was still activated by GATA-6 by as much as 50% relative to −1716-AQP5-Luc. Internal deletion of the −358/−173 GC-rich domain, which includes several putative Sp1 consensus sites, reduced trans-activation by ∼60%, suggesting importance of this region for GATA-mediated activity. −358-AQP5-Luc was similarly activated by both GATA-6 and a GATA DNA-binding defective mutant, whereas cotransfections in Schneider S2 cells demonstrated dose-dependent trans-activation of −358-AQP5-Luc by Sp1. Activation of −358-AQP5-Luc by GATA-6 was dramatically reduced by Sp1 small-interfering RNA, and −358-AQP5-Luc was activated synergistically by GATA-6 and Sp1 in NIH 3T3 cells. Furthermore, association between endogenous GATA-6 and Sp1 was demonstrated by coimmunoprecipitation. These results suggest that transcriptional activation of AQP5 by GATA-6 is mediated at least in part through cooperative interactions with Sp1 occurring at the proximal promoter.

scholarly journals Identification of a core sequence for the binding of BosR to the rpoS promoter region in Borrelia burgdorferi

Microbiology ◽  
2014 ◽  
Vol 160 (5) ◽  
pp. 851-862 ◽  
Author(s):  
Zhiming Ouyang ◽  
Jianli Zhou ◽  
Chad A. Brautigam ◽  
Ranjit Deka ◽  
Michael V. Norgard
Keyword(s):  
Borrelia Burgdorferi ◽  
Transcriptional Activation ◽  
Promoter Region ◽  
Regulatory Function ◽  
Adaptive Responses ◽  
Mobility Shift ◽  
Core Sequence ◽  
Dyad Symmetry ◽  
Electrophoretic Mobility Shift Assays ◽  
Binding Sequence

The alternative sigma factor RpoS in Borrelia burgdorferi plays a central role in modulating host adaptive responses when spirochaetes cycle between ticks and mammals. The transcriptional activation of σ54-dependent rpoS requires a Fur homologue designated BosR. Previously, BosR was shown to directly activate rpoS transcription by binding to the rpoS promoter. However, many other DNA binding features of BosR have remained obscure. In particular, the precise DNA sequence targeted by BosR has not yet been completely elucidated. The prediction of a putative Per box within the rpoS promoter region has further confounded the identification of the BosR binding sequence. Herein, by using electrophoretic mobility shift assays, we demonstrate that the putative Per box predicted in the rpoS promoter region is not involved in the binding of BosR. Rather, a 13 bp palindromic sequence (ATTTAANTTAAAT) with dyad symmetry, which we denote as the ‘BosR box’, functions as the core sequence recognized by BosR in the rpoS promoter region of Borrelia burgdorferi. Similar to a Fur box and a Per box, the BosR box probably comprises a 6–1–6 inverted repeat composed of two hexamers (ATTTAA) in a head-to-tail orientation. Selected mutations in the BosR box prevented recombinant BosR from binding to rpoS. In addition, we found that sequences neighbouring the BosR box also are required for the formation of BosR–DNA complexes. Identification of the BosR box advances our understanding of how BosR recognizes its DNA target(s), and provides new insight into the mechanistic details behind the unique regulatory function of BosR.

scholarly journals The histone acetyltransferases CBP/p300 are degraded in NIH 3T3 cells by activation of Ras signalling pathway

2006 ◽  
Vol 398 (2) ◽  
pp. 215-224 ◽  
Author(s):  
Sara Sánchez-Molina ◽  
José Luis Oliva ◽  
Susana García-Vargas ◽  
Ester Valls ◽  
José M. Rojas ◽  
...  
Keyword(s):  
Binding Protein ◽  
Camp Response Element Binding ◽  
Signalling Pathway ◽  
3T3 Cells ◽  
Protein Levels ◽  
3T3 Fibroblasts ◽  
Differentiation And Proliferation ◽  
Nih 3T3 ◽  
Ras Signalling ◽  
Nih 3T3 Cells

The CBP [CREB (cAMP-response-element-binding protein)-binding protein]/p300 acetyltransferases function as transcriptional co-activators and play critical roles in cell differentiation and proliferation. Accumulating evidence shows that alterations of the CBP/p300 protein levels are linked to human tumours. In the present study, we show that the levels of the CBP/p300 co-activators are decreased dramatically by continuous PDGF (platelet-derived growth factor) and Ras signalling pathway activation in NIH 3T3 fibroblasts. This effect occurs by reducing the expression levels of the CBP/p300 genes. In addition, CBP and p300 are degraded by the 26 S proteasome pathway leading to an overall decrease in the levels of the CBP/p300 proteins. Furthermore, we provide evidence that Mdm2 (murine double minute 2), in the presence of active H-Ras or N-Ras, induces CBP/p300 degradation in NIH 3T3 cells. These findings support a novel mechanism for modulating other signalling transduction pathways that require these common co-activators.

scholarly journals RbsR Activates Capsule but Represses therbsUDKOperon in Staphylococcus aureus

2015 ◽  
Vol 197 (23) ◽  
pp. 3666-3675 ◽  
Author(s):  
Mei G. Lei ◽  
Chia Y. Lee
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factor ◽  
Transcriptional Activation ◽  
Promoter Region ◽  
Mobility Shift ◽  
Content Type ◽  
Virulence Regulation ◽  
Important Virulence Factor ◽  
Dna Fragment ◽  
Mobility Shift Assay

ABSTRACTStaphylococcus aureuscapsule is an important virulence factor that is regulated by a large number of regulators. Capsule genes are expressed from a major promoter upstream of thecapoperon. A 10-bp inverted repeat (IR) located 13 bp upstream of the −35 region of the promoter was previously shown to affect capsule gene transcription. However, little is known about transcriptional activation of thecappromoter. To search for potential proteins which directly interact with thecappromoter region (Pcap), we directly analyzed the proteins interacting with the PcapDNA fragment from shifted gel bands identified by electrophoretic mobility shift assay. One of these regulators, RbsR, was further characterized and found to positively regulatecapgene expression by specifically binding to thecappromoter region. Footprinting analyses showed that RbsR protected a DNA region encompassing the 10-bp IR. Our results further showed thatrbsRwas directly controlled by SigB and that RbsR was a repressor of therbsUDKoperon, involved in ribose uptake and phosphorylation. The repression ofrbsUDKby RbsR could be derepressed byd-ribose. However,d-ribose did not affect RbsR activation of capsule.IMPORTANCEStaphylococcus aureusis an important human pathogen which produces a large number of virulence factors. We have been using capsule as a model virulence factor to study virulence regulation. Although many capsule regulators have been identified, the mechanism of regulation of most of these regulators is unknown. We show here that RbsR activates capsule by direct promoter binding and that SigB is required for the expression ofrbsR. These results define a new pathway wherein SigB activates capsule through RbsR. Our results further demonstrate that RbsR inhibits therbsoperon involved in ribose utilization, thereby providing an example of coregulation of metabolism and virulence inS. aureus. Thus, this study further advances our understanding of staphylococcal virulence regulation.

Ectopic Expression of Protein Kinase CβII, -δ, and -ϵ, but Not -βI or -ζ, Provide for Insulin Stimulation of Glucose Uptake in NIH-3T3 Cells

1999 ◽  
Vol 372 (1) ◽  
pp. 69-79 ◽  
Author(s):  
Denise R. Cooper ◽  
James E. Watson ◽  
Niketa Patel ◽  
Philip Illingworth ◽  
Mildred Acevedo-Duncan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
Ectopic Expression ◽  
3T3 Cells ◽  
Insulin Stimulation ◽  
Nih 3T3 ◽  
Stimulation Of ◽  
Nih 3T3 Cells

scholarly journals Tumour necrosis factor-α regulates expression of the CCAAT-enhancer-binding proteins (C/EBPs) α and β and determines the occupation of the C/EBP site in the promoter of the insulin-responsive glucose-transporter gene in 3T3-L1 adipocytes

1999 ◽  
Vol 338 (3) ◽  
pp. 737-743 ◽  
Author(s):  
Renu JAIN ◽  
Shailaja POLICE ◽  
Kelle PHELPS ◽  
Phillip H. PEKALA
Keyword(s):  
Tumour Necrosis Factor ◽  
Tumour Necrosis ◽  
Protein C ◽  
Glucose Transporter ◽  
Tumour Necrosis Factor Α ◽  
Mobility Shift ◽  
Enhancer Binding Protein ◽  
Factor Α ◽  
Electrophoretic Mobility Shift Assays ◽  
Necrosis Factor

We have demonstrated previously that treatment of 3T3-L1 adipocytes with tumour necrosis factor-α (TNF) results in a rapid (4 h) and significant (75–80%) reduction in the rate of transcription of the GLUT4 gene. Control of GLUT4 gene transcription has been suggested at least in part to reside with the CCAAT-enhancer-binding protein (C/EBP) family (α, β and δ isoforms) of transcription factors. Using electrophoretic mobility shift assays, we have examined the ability of TNF to alter the occupation of the C/EBP site in the GLUT4 promoter. The data suggest that in fully differentiated adipocytes the C/EBP site is a ligand for predominantly α/α homodimers; however, after exposure to TNF, a shift in occupancy of the site occurs and the ligands become α/β heterodimers and β/β homodimers. Partner selection in dimer formation appears to be controlled by selective translocation of the β-isoform from the cytosol to the nucleus after exposure of the cells to TNF.

scholarly journals ATF4-Dependent Oxidative Induction of the DNA Repair Enzyme Ape1 Counteracts Arsenite Cytotoxicity and Suppresses Arsenite-Mediated Mutagenesis

2007 ◽  
Vol 27 (24) ◽  
pp. 8834-8847 ◽  
Author(s):  
Hua Fung ◽  
Pingfang Liu ◽  
Bruce Demple
Keyword(s):  
Dna Repair ◽  
Transcriptional Activation ◽  
Activity Levels ◽  
Cellular Mechanisms ◽  
Mobility Shift ◽  
Repair Enzyme ◽  
Base Excision ◽  
Transcription Suppression ◽  
Tk6 Cells ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT Arsenite is a human carcinogen causing skin, bladder, and lung tumors, but the cellular mechanisms underlying these effects remain unclear. We investigated expression of the essential base excision DNA repair enzyme apurinic endonuclease 1 (Ape1) in response to sodium arsenite. In mouse 10T½ fibroblasts, Ape1 induction in response to arsenite occurred about equally at the mRNA, protein, and enzyme activity levels. Analysis of the APE1 promoter region revealed an AP-1/CREB binding site essential for arsenite-induced transcriptional activation in both mouse and human cells. Electrophoretic mobility shift assays indicated that an ATF4/c-Jun heterodimer was the responsible transcription factor. RNA interference targeting c-Jun or ATF4 eliminated arsenite-induced APE1 transcription. Suppression of Ape1 or ATF4 sensitized both mouse fibroblasts (10T½) and human lymphoblastoid cells (TK6) to arsenite cytotoxicity. Expression of Ape1 from a transgene did not efficiently restore arsenite resistance in ATF4-depleted cells but did offset initial accumulation of abasic DNA damage following arsenite treatment. Mutagenesis by arsenite (at the TK and HPRT loci in TK6 cells) was observed only for ATF4-depleted cells, which was strongly offset by Ape1 expression from a transgene. Therefore, the ATF4-mediated up-regulation of Ape1 and other genes plays a key role against arsenite-mediated toxicity and mutagenesis.

scholarly journals Transformation properties of the E2a-Pbx1 chimeric oncoprotein: fusion with E2a is essential, but the Pbx1 homeodomain is dispensable.

1994 ◽  
Vol 14 (12) ◽  
pp. 8304-8314 ◽  
Author(s):  
K Monica ◽  
D P LeBrun ◽  
D A Dedera ◽  
R Brown ◽  
M L Cleary
Keyword(s):  
Transcriptional Activation ◽  
Binding Sites ◽  
Chromosomal Translocation ◽  
Wild Type ◽  
3T3 Cells ◽  
Homeodomain Proteins ◽  
Malignant Lymphomas ◽  
Oncogenic Potential ◽  
Related Proteins ◽  
Nih 3T3

The t(1;19) chromosomal translocation in acute lymphoblastic leukemias creates chimeric E2a-Pbx1 oncoproteins that can act as DNA-binding activators of transcription. A structural analysis of the functional domains of E2a-Pbx1 showed that portions of both E2a and Pbx1 were essential for transformation of NIH 3T3 cells and transcriptional activation of synthetic reporter genes containing PBX1 consensus binding sites. Hyperexpression of wild-type or experimentally truncated Pbx1 proteins was insufficient for transformation, consistent with their inability to activate transcription. When fused with E2a, the Pbx-related proteins Pbx2 and Pbx3 were also transformation competent, demonstrating that all known members of this highly similar subfamily of homeodomain proteins have latent oncogenic potential. The oncogenic contributions of E2a to the chimeras were localized to transactivation motifs AD1 and AD2, as their mutation significantly impaired transformation. Either the homeodomain or Pbx1 amino acids flanking this region could mediate transformation when fused to E2a. However, the homeodomain was not essential for transformation, since a mutant E2a-Pbx1 protein (E2a-Pbx delta HD) lacking the homeodomain efficiently transformed fibroblasts and induced malignant lymphomas in transgenic mice. Thus, transformation mediated by the chimeric oncoprotein E2a-Pbx1 is absolutely dependent on motifs acquired from E2a but the Pbx1 homeodomain is optional. The latter finding suggests that E2a-Pbx1 may interact with cellular proteins that assist or mediate alterations in gene expression responsible for oncogenesis even in the absence of homeodomain-DNA interactions.

scholarly journals Alpha B-crystallin expression in mouse NIH 3T3 fibroblasts: glucocorticoid responsiveness and involvement in thermal protection.

1993 ◽  
Vol 13 (3) ◽  
pp. 1824-1835 ◽  
Author(s):  
A Aoyama ◽  
E Fröhli ◽  
R Schäfer ◽  
R Klemenz
Keyword(s):  
Heat Shock ◽  
Thermal Protection ◽  
Cell Clone ◽  
Ectopic Expression ◽  
Small Heat Shock Protein ◽  
Ras Oncogene ◽  
3T3 Cells ◽  
3T3 Fibroblasts ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

alpha B-crystallin, a major soluble protein of vertebrate eye lenses, is a small heat shock protein which transiently accumulates in response to heat shock and other kinds of stress in mouse NIH 3T3 fibroblasts. Ectopic expression of an alpha B-crystallin cDNA clone renders NIH 3T3 cells thermoresistant. alpha B-crystallin accumulates in response to the synthetic glucocorticoid hormone dexamethasone. Dexamethasone-treated NIH 3T3 cells become thermoresistant to the same extent as they accumulate alpha B-crystallin. A cell clone in which alpha B-crystallin is superinduced upon heat shock acquires augmented thermotolerance. Expression of the ras oncogene causes a rapid but transient accumulation of alpha B-crystallin within 1 day. Later, sustained ras oncogene expression suppresses the dexamethasone-mediated alpha B-crystallin accumulation. Thus, oncogenic transformation triggered by the ras oncogene interferes with hormone-mediated accumulation of alpha B-crystallin and concomitant acquisition of thermoresistance. Other known heat shock proteins do not accumulate in response to ectopic alpha B-crystallin expression or to dexamethasone treatment. These results indicate that alpha B-crystallin can protect NIH 3T3 fibroblasts from thermal shock.

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