scholarly journals Endothelin-1 expression is strongly repressed by AU-rich elements in the 3′-untranslated region of the gene

2005 ◽  
Vol 387 (3) ◽  
pp. 763-772 ◽  
Author(s):  
Francisco M. REIMUNDE ◽  
Cristina CASTAÑARES ◽  
Mariano REDONDO-HORCAJO ◽  
Santiago LAMAS ◽  
Fernando RODRÍGUEZ-PASCUAL
Keyword(s):  
Untranslated Region ◽  
Transcriptional Control ◽  
Transforming Growth Factor ◽  
Mrna Level ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Regulation Of Expression ◽  
Control Of Gene Expression ◽  
Cytosolic Proteins ◽  
Endothelin 1

The regulation of the synthesis of the endothelial-derived vasoconstrictor ET-1 (endothelin-1) is a complex process that occurs mainly at the mRNA level. Transcription of the gene accounts for an important part of the regulation of expression, as already described for different modulators such as the cytokine TGF-β (transforming growth factor-β). However, very little is known about mechanisms governing ET-1 expression at the post-transcriptional level. The aim of the present study was to investigate the regulation of the ET-1 expression at this level. Since the 3′-UTR (3′-untranslated region) of mRNAs commonly contains genetic determinants for the post-transcriptional control of gene expression, we focused on the potential role of the 3′-UTR of ET-1 mRNA. Experiments performed with luciferase reporter constructs containing the 3′-UTR showed that this region exerts a potent destabilizing effect. Deletional analyses allowed us to locate this activity within a region at positions 924–1127. Some (but not all) of the AREs (AU-rich elements) present in this region were found to be essential for this mRNA-destabilizing activity. We also present evidence that cytosolic proteins from endothelial cells interact specifically with these RNA elements, and that a close correlation exists between the ability of the AREs to destabilize ET-1 mRNA and the binding of proteins to these elements. Our results are compatible with the existence of a strong repressional control of ET-1 expression mediated by destabilization of the mRNA exerted through the interaction of specific cytosolic proteins with AREs present in the 3′-UTR of the gene.

Down-regulation of hepatic nuclear factor 4α on expression of human hepatic stimulator substance via its action on the proximal promoter in HepG2 cells

2008 ◽  
Vol 415 (1) ◽  
pp. 111-121 ◽  
Author(s):  
Duo Guo ◽  
Ling-yue Dong ◽  
Yuan Wu ◽  
Lin Yang ◽  
Wei An
Keyword(s):  
Hepg2 Cells ◽  
Promoter Activity ◽  
Transcriptional Control ◽  
Mrna Level ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Hepatic Stimulator Substance

hHSS (human hepatic stimulator substance) stimulates hepatocyte growth. To understand the mechanism controlling hHSS expression, we analysed the proximal promoter activity and identified two regulatory regions (−212/−192 and −152/−132) that were important for transcription in HepG2 cells. Using the luciferase reporter assay, gel-shift experiments and ChIP (chromatin immunoprecipitation), we found that the transcription factors HNF4α (hepatocyte nuclear factor 4α) and Sp1 (stimulating protein-1) were essential for hHSS promoter activity and could directly bind to regions −209/−204 and −152/−145 respectively. We also confirmed that activation and repression of hHSS transcription induced by Sp1 and HNF4α resulted from binding of these factors to these two cis-elements respectively. Overexpression of HNF4α led to a dramatic repression of the promoter activity and, in contrast, the activity was markedly elevated by overexpression of Sp1. Furthermore, overexpression of HNF4α1, one of the HNF4α isoforms, resulted in a dramatic suppression of the promoter activity. Moreover, repression of HNF4α expression by siRNA (small interfering RNA) remarkably enhanced the hHSS mRNA level. It has been reported previously that expression of HNF4α is functionally regulated by dexamethasone. To further confirm the transcriptional control of HNF4α on hHSS, we tested the effect of dexamethasone on hHSS transcription in HepG2 cells. In the present study we have demonstrated that the expression of the hHSS gene was down-regulated at the transcriptional level by dexamethasone in HepG2 cells. A deletion and decoy assay revealed that binding of HNF4α to nucleotides −209/−204 was responsible for the suppression of hHSS promoter activity by dexamethasone. Increases in the HNF4α-binding activity and expression were simultaneously observed in an electrophoretic mobility-shift assay and Western blot analysis. These results suggested that Sp1 activates hHSS basal expression, but HNF4α inhibits hHSS gene expression.

scholarly journals Glyceraldehyde-3-Phosphate Dehydrogenase Regulates Endothelin-1 Expression by a Novel, Redox-Sensitive Mechanism Involving mRNA Stability

2008 ◽  
Vol 28 (23) ◽  
pp. 7139-7155 ◽  
Author(s):  
Fernando Rodríguez-Pascual ◽  
Mariano Redondo-Horcajo ◽  
Noemi Magán-Marchal ◽  
David Lagares ◽  
Antonio Martínez-Ruiz ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Untranslated Region ◽  
Oxidant Stress ◽  
Cytosolic Proteins ◽  
Endothelin 1 ◽  
Complex Process ◽  
Catalytically Active ◽  
Active Residue ◽  
Enhanced Stability

ABSTRACT The regulation of the synthesis of the endothelial-derived vasoconstrictor endothelin-1 (ET-1) is a complex process encompassing transcriptional as well as mRNA stability mechanisms. We have described recently the existence of a mechanism for the control of ET-1 expression based on the mRNA-destabilizing capacity of specific cytosolic proteins through interaction with AU-rich elements (AREs) present in the 3′ untranslated region of the gene. We now identify glyceraldehyde-3′-phosphate dehydrogenase (GAPDH) as a protein which binds to the AREs and is responsible for the destabilization of the mRNA. Oxidant stress alters the binding of GAPDH to the mRNA and its capacity to modulate ET-1 expression, a phenomenon occurring through specific S glutathionylation of the catalytically active residue Cys 152. Finally, we provide data consistent with a role for GAPDH in mRNA unwinding, yielding this molecule more prone to degradation. In contrast, S-thiolated GAPDH appears unable to modify mRNA unwinding, thus facilitating enhanced stability. Taken together, these results describe a novel, redox-based mechanism regulating mRNA stability and add a new facet to the panoply of GAPDH cellular homeostatic actions.

Hypoxia induces differentiation of pulmonary artery adventitial fibroblasts into myofibroblasts

2004 ◽  
Vol 286 (2) ◽  
pp. C416-C425 ◽  
Author(s):  
Megan Short ◽  
Raphel A. Nemenoff ◽  
W. Michael Zawada ◽  
Kurt R. Stenmark ◽  
Mita Das
Keyword(s):  
Pulmonary Artery ◽  
Signaling Pathways ◽  
Promoter Activity ◽  
Transforming Growth Factor ◽  
Essential Feature ◽  
Smooth Muscle Actin ◽  
Neutralizing Antibody ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Adventitial Fibroblasts

Activation of the α-smooth muscle actin (α-SMA) gene during the conversion of fibroblasts into myofibroblasts is an essential feature of various fibrotic conditions. Microvascular compromise and thus local environmental hypoxia are important components of the fibrotic response. The present study was thus undertaken to test the hypothesis that hypoxia can induce transdifferentiation of vascular fibroblasts into myofibroblasts and also to evaluate potential signaling mechanisms governing this process. We found that hypoxia significantly upregulates α-SMA protein levels in bovine pulmonary artery adventitial fibroblasts. Increased α-SMA expression is controlled at the transcriptional level because the α-SMA gene promoter activity, assayed via a luciferase reporter, was markedly increased in transfected fibroblasts exposed to hypoxia. Hypoxic induction of the α-SMA gene was mimicked by overexpression of constitutively active Gαi2 (αi2Q205L) but not Gα16 (α-16Q212L). Blockade of hypoxia-induced α-SMA expression with pertussis toxin, a Gαi antagonist, confirmed a role for Gαi in the hypoxia-induced transdifferentiation process. c-Jun NH2-terminal kinase (JNK) inhibitor II and SB202190, but not U0126, also attenuated α-SMA expression in hypoxic fibroblasts, suggesting the importance of JNK in the differentiation process. Hypoxia-induced increase in bromodeoxyuridine incorporation, which occurred concomitantly with hypoxia-induced differentiation, was blocked by U0126, suggesting that DNA synthesis and α-SMA expression take place through simultaneously activated parallel signaling pathways. Neutralizing antibody against transforming growth factor-β1 blocked only 30% of the hypoxia-induced α-SMA promoter activity. Taken together, our results suggest that hypoxia induces differentiation of vascular fibroblasts into myofibroblasts by upregulating the expression of α-SMA, and this increase in α-SMA level occurs through Gαi- and JNK-dependent signaling pathways.

scholarly journals Transcriptional regulation of expression of the rainbow trout albumin gene by estrogen

1998 ◽  
Vol 20 (3) ◽  
pp. 355-362 ◽  
Author(s):  
G Flouriot ◽  
B Ducouret ◽  
L Byrnes ◽  
Y Valotaire
Keyword(s):  
Rainbow Trout ◽  
Mrna Level ◽  
Primary Cultures ◽  
Transcriptional Level ◽  
Down Regulation ◽  
Dramatic Decrease ◽  
Regulation Of Expression ◽  
Albumin Gene ◽  
Run Off ◽  
Vitellogenin Genes

Estrogens modulate the expression of many liver-specific genes in oviparous species. For instance, expression of the estrogen receptor and vitellogenin genes is strongly up-regulated by estradiol in rainbow trout liver. Using hepatocyte primary cultures, we demonstrate that trout albumin (Alb) gene is also regulated by this hormone. Indeed, treatment of hepatocytes with 1 microM estradiol led, after 24 h, to a dramatic decrease in Alb mRNA level. To investigate the mechanism of this down-regulation, run-off experiments were performed and mRNA half-lives were determined in the presence and absence of estradiol. The results show that the down-regulation of Alb mRNA expression by estrogens occurs only at the transcriptional level.

scholarly journals Low Affinity Binding Sites in an Activating CRM Mediate Negative Autoregulation of the Drosophila Hox Gene Ultrabithorax

2019 ◽  
Author(s):  
Rebecca K Delker ◽  
Vikram Ranade ◽  
Ryan Loker ◽  
Roumen Voutev ◽  
Richard S Mann
Keyword(s):  
Gene Expression ◽  
Binding Sites ◽  
Transcriptional Control ◽  
Regulation Of Gene Expression ◽  
Cell Types ◽  
Transcriptional Level ◽  
Regulation Of Transcription ◽  
Control Of Gene Expression ◽  
Hox Gene ◽  
Endogenous Locus

AbstractSpecification of cell identity and the proper functioning of a mature cell depend on precise regulation of gene expression. Both binary ON/OFF regulation of transcription, as well as more fine-tuned control of transcription levels in the ON state, are required to define cell types. The Drosophila melanogaster Hox gene, Ultrabithorax (Ubx), exhibits both of these modes of control during development. While ON/OFF regulation is needed to specify the fate of the developing wing (Ubx OFF) and haltere (Ubx ON), the levels of Ubx within the haltere differ between compartments along the proximal-distal axis. Here, we identify and molecularly dissect the novel contribution of a previously identified Ubx cis-regulatory module (CRM), anterobithorax (abx), to a negative auto-regulatory loop that maintains decreased Ubx expression in the proximal compartment of the haltere as compared to the distal compartment. We find that Ubx, in complex with the known Hox cofactors, Homothorax (Hth) and Extradenticle (Exd), acts through low-affinity Ubx-Exd binding sites to reduce the levels of Ubx transcription in the proximal compartment. Importantly, we also reveal that Ubx-Exd-binding site mutations sufficient to result in de-repression of abx activity in the proximal haltere in a transgenic context are not sufficient to de-repress Ubx expression when mutated at the endogenous locus, suggesting the presence of multiple mechanisms through which Ubx-mediated repression occurs. Our results underscore the complementary nature of CRM analysis through transgenic reporter assays and genome modification of the endogenous locus; but, they also highlight the increasing need to understand gene regulation within the native context to capture the potential input of multiple genomic elements on gene control.Author SummaryOne of the most fundamental questions in biology is how information encoded in the DNA is translated into the diversity of cell-types that exist within a multicellular organism, each with the same genome. Regulation at the transcriptional level, mediated through the activity of transcription factors bound to cis-regulatory modules (CRMs), plays a key role in this process. While we typically distinguish cell-type by the specific subset of genes that are transcriptionally ON or OFF, it is also important to consider the more fine-tuned transcriptional control of gene expression level. We focus on the regulatory logic of the Hox developmental regulator, Ultrabithorax (Ubx), in fruit flies, which exhibits both forms of transcriptional control. While ON/OFF control of Ubx is required to define differential appendage fate in the T2 and T3 thoracic segments, respectively, more fine-tuned control of transcription levels is observed in distinct compartments within the T3 appendage, itself, in which all cells exhibit a Ubx ON state. Through genetic analysis of regulatory inputs, and dissection of a Ubx CRM in a transgenic context and at the endogenous locus, we reveal a compartment-specific negative autoregulatory loop that dampens Ubx transcription to maintain distinct transcriptional levels within a single developing tissue.

scholarly journals Heparin suppresses endothelin-1 peptide and mRNA expression in cultured endothelial cells of spontaneously hypertensive rats.

1994 ◽  
Vol 4 (9) ◽  
pp. 1683-1689
Author(s):  
K Yokokawa ◽  
M Kohno ◽  
A K Mandal ◽  
H Tahara ◽  
M Yanagisawa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Mrna Expression ◽  
Spontaneously Hypertensive Rats ◽  
Actinomycin D ◽  
Mrna Level ◽  
Transcriptional Level ◽  
Hypertensive Rats ◽  
Spontaneously Hypertensive ◽  
Endothelin 1 ◽  
Wistar Kyoto

Heparin given sc consistently lowers blood pressure in spontaneously hypertensive rats (SHR). This study was designed to examine heparin's effect on vasoconstrictor endothelin-1 production in cultured aortic endothelial cells (EC). Aortic EC from SHR and normotensive Wistar Kyoto rats (WKY) were cultured and incubated with or without different concentrations of heparin. Heparin suppressed endothelin-1 release and endothelin-1 mRNA expression in a dose- and a time-dependent fashion in both WKY and SHR. The suppressive effects were more augmented in SHR than in WKY: SHR versus WKY--endothelin-1 level at 6 h = 8 +/- 1.8 versus 14 +/- 2.2 pg/10(6) cells (P < 0.01) and mRNA expression--85 versus 52% maximal inhibition by heparin, 10 U/mL (=70 micrograms/mL) (P < 0.01). When heparin was added with transcriptional inhibitor actinomycin D and incubated for 30 min, no further inhibition of endothelin-1 mRNA level measured after another 30 minutes was observed compared with the endothelin-1 mRNA level in cultured EC of SHR treated with just actinomycin D at 30 min. These results suggest that heparin regulates endogenous endothelin-1 production by cultured EC, probably at the transcriptional level, and that this effect is more marked in SHR than in WKY.

scholarly journals Identification and characterization of a novel GGA/C-binding protein, GBP-i, that is rapidly inducible by cytokines.

1994 ◽  
Vol 14 (12) ◽  
pp. 7770-7781 ◽  
Author(s):  
G V Raj ◽  
K Khalili
Keyword(s):  
Phorbol Myristate Acetate ◽  
Transforming Growth Factor Beta ◽  
Transcriptional Control ◽  
Transforming Growth Factor ◽  
Jc Virus ◽  
Interleukin 1 ◽  
Transcriptional Level ◽  
Sequence Specificity ◽  
Myristate Acetate ◽  
Necrosis Factor Alpha

Immunosuppressive states with accompanying alterations in cytokine profiles have been postulated to play a vital role in the reactivation of viruses from latency. Cytokines regulate gene expression by activating transcription factors via well-characterized signal transduction pathways. In this study, we report the identification of a novel inducible protein, GBP-i, that binds to a double-stranded GGA/C-rich region of the transcriptional control region of the human papovavirus JC virus (JCV), specifically within the origin of viral DNA replication. GBP-i is distinct from previously characterized GC-box-binding proteins with respect to both its sequence specificity and its electrophoretic mobility on native and denaturing gels. GBP-i responds within 90 min to phorbol myristate acetate stimulation; however, unlike typical phorbol myristate acetate-inducible factors, this rapid induction is regulated primarily at the transcriptional level. Further, the induction of GBP-i appears to be widespread and mediated by many inflammatory cytokines, including interleukin-1 beta, tumor necrosis factor alpha, gamma interferon, and transforming growth factor beta. Interestingly, the induced protein acts as a transcriptional repressor in its native context in the JCVL promoter. However, when its binding sequence is transposed to a heterologous promoter, GBP-i appears to function as a transcriptional activator. The data presented here suggest a role for GBP-i in cytokine-mediated induction of viral and cellular genes.

scholarly journals IL-1β-Mediated Up-Regulation of WT1D via miR-144-3p and Their Synergistic Effect with NF-κB/COX-2/HIF-1α Pathway on Cell Proliferation in LUAD

2018 ◽  
Vol 48 (6) ◽  
pp. 2493-2502 ◽  
Author(s):  
Chen Wu ◽  
Xiaodong Li ◽  
Dachuan Zhang ◽  
Bin Xu ◽  
Wenwei Hu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
De Novo ◽  
Lentiviral Vector ◽  
Mrna Level ◽  
A549 Cells ◽  
Xenograft Model ◽  
Suppressive Effect ◽  
Luciferase Reporter ◽  
Transcriptional Level ◽  
Cox 2

Background/Aims: IL-1β is an important mediator of “inflammation-cancer" transformation through IL-1β/NF-κB/COX-2/HIF-1α signaling pathway, whereas certain portion of patients with lung adenocarcinoma (LUAD) still suffer from rapid tumor progression in clinical practice, indicating the occurrence of potential bypass. Methods: Real-time polymerase chain reaction was applied to examine the expressions of mir-144-3p, WT1, NF-κB, COX2 and HIF-1α at the mRNA level in 127 LUAD samples and corresponding adjacent tissues. miR-144-3p mimic and antagormiR were used to trigger activation and suppression of miR-144-3p in A549 cells, respectively. MTT assay and Western blotting analysis were carried out to evaluate the cell proliferation. Stable clones with over-expression or knockdown of WT1 were generated with plasmid or shRNA by lentiviral vector technology in H1568 and H1650 NSCLC cell lines, respectively. Dual luciferase reporter assay was performed to validate the effect of miR-144-3p on WT1D. Xenograft model was established for in vivo experiment, and TCGA data were extracted for validation. Results: miR-144-3p could suppress the WT1D expression at the post-transcriptional level, hence regulating cell proliferation in LUAD. WT1 and COX-2 were independent prognostic factors of LUAD patients. In addition, inhibition of IL-1β/miR-144-3p/WT1D and IL-1β/NF-κB/COX-2/HIF-1α pathways using miR-144-3p mimic and Celecoxib, respectively, displayed synergistic suppressive effect on cell proliferation in LUAD. Conclusion: A de novo IL-1β/miR-144-3p/WT1D axis was involved in proliferative regulation of LUAD. Moreover, simultaneous blockade of both IL-1β/miR-144-3p/WT1D and IL-1β/NF-κB/COX-2/ HIF-1α pathways might have synergistic suppressive effect on cell proliferation in LUAD.

scholarly journals Regulation of TG-interacting factor by transforming growth factor-beta

2003 ◽  
Vol 371 (2) ◽  
pp. 257-263 ◽  
Author(s):  
Feifei CHEN ◽  
Kenji OGAWA ◽  
Raman P. NAGARAJAN ◽  
Meiyu ZHANG ◽  
Chenzhong KUANG ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Transforming Growth Factor ◽  
Mrna Level ◽  
Transcriptional Level ◽  
Dependent Manner ◽  
Human T Cell ◽  
Tgf Β1

TG-interacting factor (TGIF) is a transcriptional co-repressor that directly associates with Smad (Sma- and Mad-related protein) proteins and inhibits Smad-mediated transcriptional activation. By using Affymetrix (Santa Clara, CA, U.S.A.) oligonucleotide microarray analysis, we found that TGIF mRNA level was elevated by transforming-growth-factor-β (TGF-β) treatment in a human T-cell line, HuT78. Subsequent reverse-transcription PCR assays indicated that TGF-β1 and activin were able to induce a rapid and transient increase in the level of TGIF in both HuT78 and HepG2 hepatoma cells. To analyse whether or not the regulation of TGIF mRNA occurs at the transcriptional level, a 2.4kb human TGIF promoter was isolated. A primer extension assay was performed to localize the putative transcription initiation site of the promoter. When transiently expressed in HepG2 cells, this promoter was stimulated by TGF-β1 and activin treatment in a time-dependent manner. A series of deletion mutants of the TGIF promoter were also generated to further characterize the TGF-β responsive region of the promoter. In addition, expression of TGIF was able to cause a dose-dependent inhibition of TGF-β and activin signalling. Taken together, these experiments indicated that TGIF is a novel transcriptional target of TGF-β and activin signalling and is likely involved in a negative feedback loop to desensitize TGF-β/activin action.

Identification and characterization of a novel GGA/C-binding protein, GBP-i, that is rapidly inducible by cytokines

1994 ◽  
Vol 14 (12) ◽  
pp. 7770-7781
Author(s):  
G V Raj ◽  
K Khalili
Keyword(s):  
Phorbol Myristate Acetate ◽  
Transforming Growth Factor Beta ◽  
Transcriptional Control ◽  
Transforming Growth Factor ◽  
Jc Virus ◽  
Interleukin 1 ◽  
Transcriptional Level ◽  
Sequence Specificity ◽  
Myristate Acetate ◽  
Necrosis Factor Alpha

Immunosuppressive states with accompanying alterations in cytokine profiles have been postulated to play a vital role in the reactivation of viruses from latency. Cytokines regulate gene expression by activating transcription factors via well-characterized signal transduction pathways. In this study, we report the identification of a novel inducible protein, GBP-i, that binds to a double-stranded GGA/C-rich region of the transcriptional control region of the human papovavirus JC virus (JCV), specifically within the origin of viral DNA replication. GBP-i is distinct from previously characterized GC-box-binding proteins with respect to both its sequence specificity and its electrophoretic mobility on native and denaturing gels. GBP-i responds within 90 min to phorbol myristate acetate stimulation; however, unlike typical phorbol myristate acetate-inducible factors, this rapid induction is regulated primarily at the transcriptional level. Further, the induction of GBP-i appears to be widespread and mediated by many inflammatory cytokines, including interleukin-1 beta, tumor necrosis factor alpha, gamma interferon, and transforming growth factor beta. Interestingly, the induced protein acts as a transcriptional repressor in its native context in the JCVL promoter. However, when its binding sequence is transposed to a heterologous promoter, GBP-i appears to function as a transcriptional activator. The data presented here suggest a role for GBP-i in cytokine-mediated induction of viral and cellular genes.

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