scholarly journals Smad3-mSin3A-HDAC1 Complex is Required for TGF-β1-Induced Transcriptional Inhibition of PPARγ in Mouse Cardiac Fibroblasts

2016 ◽  
Vol 40 (5) ◽  
pp. 908-920 ◽  
Author(s):  
Kaizheng Gong ◽  
Mingxing Chen ◽  
Rujun Li ◽  
Yanghong He ◽  
Huajiang Zhu ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Cardiac Fibroblasts ◽  
Luciferase Reporter ◽  
Transcriptional Inhibition ◽  
Transcriptional Regulatory ◽  
Promoter Deletion Analysis ◽  
Regulatory Complex ◽  
Pparγ Gene ◽  
Tgf Β1

Background: We have recently demonstrated that activated transforming growth factor-β (TGF-β) signaling suppresses myocardial peroxisome proliferator-activated receptor γ (PPARγ) expression in the pressure overloaded heart. In this study, we aim to further define the molecular mechanisms that underlie TGF-β-induced PPARγ transcriptional inhibition. Methods: Adult mouse cardiac fibroblasts were isolated and cultured. PPARγ promoter activity was measured by the dual-Luciferase reporter assay. Interactions between transcription factors and the target gene were identified. Results: In cultured cardiac fibroblasts transfected with a plasmid containing a human PPARγ promoter, co-transfection of Smad3 and Smad4, but not Smad2, plasmids significantly enhanced TGF-β1-induced inhibition of PPARγ promoter activity. Promoter deletion analysis and site-directed mutagenesis assays defined two Smad binding elements on the promoter of the PPARγ gene. Utilizing chromatin immunoprecipitation analysis and DNA-affinity precipitation methods, we demonstrated that the transcriptional regulatory complex consisting of Smad3, mSin3A and HDAC1 bound to the promoter of the PPARγ gene in cardiac fibroblasts in response to TGF-β1 stimulation. Either silencing endogenous mSin3A expression by Lentivirus-mediated transduction of mSin3A shRNA or pretreatment with the specific HDAC1 inhibitor MS-275 effectively attenuated TGF-β-induced transcriptional suppression of PPARγ. Conclusion: These results suggest that TGF-β1-induced inhibition of PPARγ transcription depends on formation of a functional transcriptional regulatory complex that includes Smad3, mSin3A and HDAC1 at the PPARγ promoter.

HDAC1-Smad3-mSin3Acomplex is required for Smad3-induced transcriptional inhibition of hepatocyte growth factor receptor in human lung cancers

2020 ◽  
Author(s):  
Hao-Xin Gui ◽  
Jun Peng ◽  
Ze-Ping Yang ◽  
Lu-Yao Chen ◽  
Hong Zeng ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Molecular Mechanisms ◽  
Growth Factor Receptor ◽  
Mrna Levels ◽  
Lung Cancers ◽  
Transcriptional Inhibition ◽  
Transcriptional Suppression ◽  
Transcriptional Regulatory ◽  
Regulatory Complex ◽  
Cancer Tissues

Abstract c-Met hyperactivity has been observed in numerous neoplasms. Several researchers have shown that the abnormal activation of c-Met is mainly caused by transcriptional activation. However, the molecular mechanism behind this transcriptional regulation is poorly understood. Here, we suggest that Smad3 negatively regulates the expression and activation of c-Met via a transcriptional mechanism. We explore the molecular mechanisms that underlie Smad3-induced c-Met transcription inhibition. We found in contrast to the high expression of c-Met, Smad3 showed low protein and mRNA levels. Smad3 and c-Met expression was inconsistent between lung cancer tissues and cell lines. We also found that Smad3 overexpression suppresses whereas Smad3 knockdown significantly promotes EMT and production of the angiogenic factors VEGF, CTGF and COX-2 through the ERK1/2 pathway. In addition, Smad3 overexpression decreases whereas Smad3 knockdown significantly increases protein and mRNA levels of invasion related β-catenin and FAK through the PI3K/Akt pathway. Furthermore, using the ChIP analysis method, we demonstrate that a transcriptional regulatory complex consisting of HDAC1, Smad3 and mSin3A binds to the promoter of the c-Met gene. By either silencing endogenous mSin3A expression with siRNA or by pretreating cells with a specific HDAC1 inhibitor (MS-275), Smad3-induced transcriptional suppression of c-Met could be effectively attenuated. These results demonstrate that Smad3-induced inhibition of c-Met transcription depends on of a functional transcriptional regulatory complex that includes Smad3, mSin3A and HDAC1 at the c-Met promoter. Collectively, our findings reveal a new regulatory mechanism of c-Met signaling, and suggest a potential molecular target for the development of anticancer drugs.

scholarly journals Circular RNA mmu_circ_0005019 inhibits fibrosis of cardiac fibroblasts and reverses electrical remodeling of cardiomyocytes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Na Wu ◽  
Chengying Li ◽  
Bin Xu ◽  
Ying Xiang ◽  
Xiaoyue Jia ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cardiac Fibroblasts ◽  
Circular Rna ◽  
Protective Role ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Candidate Target ◽  
Paired Control ◽  
Reporter Assays ◽  
And Migration

Abstract Background Circular RNA (circRNA) have been reported to play important roles in cardiovascular diseases including myocardial infarction and heart failure. However, the role of circRNA in atrial fibrillation (AF) has rarely been investigated. We recently found a circRNA hsa_circ_0099734 was significantly differentially expressed in the AF patients atrial tissues compared to paired control. We aim to investigate the functional role and molecular mechanisms of mmu_circ_0005019 which is the homologous circRNA in mice of hsa_circ_0099734 in AF. Methods In order to investigate the effect of mmu_circ_0005019 on the proliferation, migration, differentiation into myofibroblasts and expression of collagen of cardiac fibroblasts, and the effect of mmu_circ_0005019 on the apoptosis and expression of Ito, INA and SK3 of cardiomyocytes, gain- and loss-of-function of cell models were established in mice cardiac fibroblasts and HL-1 atrial myocytes. Dual-luciferase reporter assays and RIP were performed to verify the binding effects between mmu_circ_0005019 and its target microRNA (miRNA). Results In cardiac fibroblasts, mmu_circ_0005019 showed inhibitory effects on cell proliferation and migration. In cardiomyocytes, overexpression of mmu_circ_0005019 promoted Kcnd1, Scn5a and Kcnn3 expression. Knockdown of mmu_circ_0005019 inhibited the expression of Kcnd1, Kcnd3, Scn5a and Kcnn3. Mechanistically, mmu_circ_0005019 exerted biological functions by acting as a miR-499-5p sponge to regulate the expression of its target gene Kcnn3. Conclusions Our findings highlight mmu_circ_0005019 played a protective role in AF development and might serve as an attractive candidate target for AF treatment.

Bioactive factors secreted by Bifidobacterium breve B-3 enhance barrier function in human intestinal Caco-2 cells

2019 ◽  
Vol 10 (1) ◽  
pp. 89-100 ◽  
Author(s):  
Y. Kurose ◽  
J. Minami ◽  
A. Sen ◽  
N. Iwabuchi ◽  
F. Abe ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Barrier Function ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Intestinal Barrier ◽  
Intestinal Barrier Function ◽  
Luciferase Reporter ◽  
Barrier Integrity ◽  
Bifidobacterium Breve ◽  
Enhancing Effect

Intestinal barrier function is closely related to intestinal health and diseases. Recent studies demonstrate that some probiotic and commensal bacteria secrete metabolites that are capable of affecting the intestinal functions. The present study examined an enhancing effect of bioactive factors secreted by Bifidobacterium breve strain B-3 on the intestinal tight junction (TJ) barrier integrity in human intestinal Caco-2 cells. Administration of conditioned medium obtained from B. breve strain B-3 (B3CM) to Caco-2 cells for 24 h increased trans-epithelial electrical resistance (TER), a TJ barrier indicator, across their monolayers. Immunoblot, immunofluorescence, and qPCR analyses demonstrated that B3CM increased an integral TJ protein, claudin-4 expression. In luciferase reporter assay, the administration of B3CM enhanced the claudin-4 promoter activity, indicating the transcriptional upregulation of claudin-4. Site-directed mutation of specificity protein 1 (Sp1) binding sites in the claudin-4 promoter sequence and suppression of Sp1 expression by siRNA technology clearly reduced the enhancing effect of B3CM on claudin-4 promoter activity. Liquid chromatography/mass spectrometry detected a significant amount of acetic acid in B3CM (28.3 mM). The administration of acetic acid to Caco-2 cells partially mimicked a B3CM-mediated increase in TER, but failed to increase claudin-4 expression. Taken together, bioactive factors secreted by B. breve B-3 enhanced the TJ barrier integrity in intestinal Caco-2 cells. Transcriptional regulation of claudin-4 through Sp1 is at least in part one of the underlying molecular mechanisms. In addition, acetic acid contributes to the B3CM-mediated barrier effect independently of claudin-4 expression.

scholarly journals Effects of paeonol on proliferation and collagen synthesis of rat cardiac fibroblasts induced by aldosterone

2021 ◽  
Vol 16 (2) ◽  
pp. 42-48
Author(s):  
Qian Xu ◽  
Li Na Wang ◽  
Jing Yi Zhao ◽  
Yan Hong Xiao ◽  
Chao Du
Keyword(s):  
Cell Viability ◽  
Molecular Mechanisms ◽  
Type I Collagen ◽  
Ventricular Remodeling ◽  
Cardiac Fibroblasts ◽  
Neonatal Rat ◽  
Type Iii Collagen ◽  
Type I ◽  
Rt Pcr ◽  
Tgf Β1

The aim of this study was to explore the possible molecular mechanisms of paeonol in preventing ventricular remodeling. The cell viability of neonatal rat cardiac fibroblasts was detected by the method of MTT. RT-PCR and Western blot were used to measure the expression of TGF-β1, type I collagen and type III collagen. After treating the cardiac fibroblasts with paeonol, the cell viability decreased (p<0.01), and the expression of TGF-β1, type I collagen and types III collagen was significantly reduced (p<0.01). Thus, paeonol can inhibit the proliferation of fibroblast cells induced by aldosterone. The molecular mechanism is related to the down-regulation of TGF-β1 and type I and III collagen gene expression.

scholarly journals MiRNA-1202 promotes the TGF-β1-induced proliferation, differentiation and collagen production of cardiac fibroblasts by targeting nNOS

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0256066
Author(s):  
Jingwen Xiao ◽  
Yan Zhang ◽  
Yuan Tang ◽  
Hengfen Dai ◽  
Yu OuYang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Myocardial Fibrosis ◽  
Cardiac Fibroblasts ◽  
Collagen I ◽  
Luciferase Reporter ◽  
Control Group ◽  
Protein Levels ◽  
Collagen Iii ◽  
Human Cardiac Fibroblasts ◽  
Tgf Β1

Background Atrial fibrillation (AF) is a clinically common arrhythmia that affects human health. Myocardial fibrosis serves as an important contributor to AF. Recently, miRNA-1202 have been reported to be up-regulated in AF. However, the role of miRNA-1202 and its mechanism in myocardial fibrosis remain unclear. Methods Human cardiac fibroblasts (HCFs) were used to construct a fibrosis model by TGF-β1 induction. The expression of miR-1202 was measured by qRT-PCR. Cell proliferation was assessed by CCK-8 assays. Protein expression levels were measured by western blot. Collagen accumulation was measured by ELISA. The relationship between miR-1202 and nNOS was investigated by luciferase reporter assays. Results MiR-1202 expression was obviously increased in HCFs and was both time- and dose-independent. MiR-1202 could increase the proliferation and collagen I, collagen III, and α-SMA levels with or without TGF-β1. MiR-1202 could also increase TGF-β1 and p-Smad2/3 protein levels in comparison to the control group. However, they were obviously decreased after inhibitor transfection. MiR-1202 targets nNOS for negative regulation of HCFs fibrosis by decreasing cell differentiation, collagen deposition and the activity of the TGF-β1/Smad2/3 pathway. Co-transfection of miR-1202 inhibitor and siRNA of nNOS inhibited nNOS protein expression, thereby enhancing the HCFs proliferation. Furthermore, co-transfection of the miR-1202 inhibitor and siRNA of nNOS significantly promoted collagen I, collagen III, TGF-β1, Smad2/3 and α-SMA protein expression and Smad2/3 protein phosphorylation. These findings suggested that miR-1202 promotes HCFs transformation to a pro-fibrotic phenotype by targeting nNOS through activating the TGF-β1/Smad2/3 pathway.

scholarly journals Coordinate Transcription of the ADAMTS-1 Gene by Luteinizing Hormone and Progesterone Receptor

2004 ◽  
Vol 18 (10) ◽  
pp. 2463-2478 ◽  
Author(s):  
Kari M. H. Doyle ◽  
Darryl L. Russell ◽  
Venkataraman Sriraman ◽  
JoAnne S. Richards
Keyword(s):  
Progesterone Receptor ◽  
Granulosa Cells ◽  
Binding Sites ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Luciferase Reporter ◽  
Nuclear Factor ◽  
Nuclear Factor 1

Abstract ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin-like motifs) is a multifunctional protease that is expressed in periovulatory follicles. Herein we show that induction of ADAMTS-1 message in vivo and transcription of the ADAMTS-1 promoter in cultured granulosa cells are dependent on separable but coordinate actions of LH and the progesterone receptor (PR). To analyze the molecular mechanisms by which LH and PR regulate this gene, truncations and site-specific mutants of ADAMTS-1 promoter-luciferase reporter constructs (ADAMTS-1-Luc) were generated and transfected into rat granulosa cell cultures. Three regions of the promoter were found to be important for basal activity, two of which were guanine cytosine-rich binding sites for specificity proteins Sp1/Sp3 and the third bound a nuclear factor 1-like factor. Despite the absence of a consensus PR DNA response element in the proximal ADAMTS-1 promoter, cotransfection of a PRA (or PRB) expression vector stimulated ADAMTS-1 promoter activity, a response that was reduced by the PR antagonist ZK98299. Forskolin plus phorbol myristate acetate also increased promoter activity and, when added to cells cotransfected with PRA, ADAMTS-1 promoter activity increased further. Activation of the ADAMTS-1 promoter by PRA involves functional CAAT enhancer binding protein β, nuclear factor 1-like factor, and three Sp1/Sp3 binding sites as demonstrated by transfection of mutated promoter constructs. In summary, LH and PRA/B exert distinct but coordinate effects on transactivation of the ADAMTS-1 gene in granulosa cells in vivo and in vitro with PR acting as an inducible coregulator of the ADAMTS-1 gene.

Genomic structure and promoter characterization of the human Sprouty4 gene, a novel regulator of lung morphogenesis

2004 ◽  
Vol 287 (1) ◽  
pp. L52-L59 ◽  
Author(s):  
Wei Ding ◽  
Saverio Bellusci ◽  
Wei Shi ◽  
David Warburton
Keyword(s):  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Core Promoter ◽  
Genomic Structure ◽  
Inducible Promoter ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Significant Similarity ◽  
The Core ◽  
Flanking Region

The expression of Sprouty4 ( Spry4), an intracellular FGF receptor antagonist, shows a temporally and spatially restricted pattern in embryonic lung and is induced by ERK signaling. To clarify the molecular mechanisms regulating Spry4 transcription, the genomic structure of the human Sprouty4 ( hSpry4) gene was first determined by using the GenomeWalker kit. The hSpry4 gene spans > 14 kb and is organized in three exons and two introns. Multiple transcription start sites were subsequently mapped by 5′-rapid amplification of cDNA ends. Analysis of up to 4 kb of sequence in the 5′-flanking region of the gene showed the presence of multiple potential transcription factor binding sites but no TATA or CAAT boxes. Transient transfection using luciferase reporter gene constructs with progressive deletions of the hSpry4 5′-flanking region revealed that the core promoter activity is located within the proximal 0.4-kb region, whereas the minimal ERK-inducible promoter activity is between −69 and −31. Homology analysis further showed that the core promoter region of the hSpry4 gene exhibits significant similarity to the 5′-flanking region of the mouse gene.

scholarly journals AP-1 Proteins Mediate Hyperglycemia-Induced Activation of the Human TGF-β1 Promoter in Mesangial Cells

2000 ◽  
Vol 11 (11) ◽  
pp. 2007-2016
Author(s):  
CORA WEIGERT ◽  
ULRICH SAUER ◽  
KATRIN BRODBECK ◽  
ANDREAS PFEIFFER ◽  
HANS U. HÄRING ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
High Glucose ◽  
Binding Sites ◽  
Promoter Activity ◽  
Mesangial Cells ◽  
Luciferase Reporter ◽  
Kinase C ◽  
Tgf Β1

Abstract. Hyperglycemia-induced overproduction of the prosclerotic cytokine transforming growth factor-β1 (TGF-β1) has been implicated in the pathogenesis of diabetic nephropathy. Because high glucose and phorbol esters (PMA) increase TGF-β1 mRNA levels in mesangial cells, this study was designed to characterize these effects on the human TGF-β1 promoter activity. With the use of luciferase reporter gene constructs containing TGF-β1 5′-flanking sequence (from -453 to +11 bp) transfected into mesangial cells, it was found that 30 mM glucose induced a nearly twofold increase in TGF-β1 promoter activity after 24 h of incubation in human and porcine mesangial cells. Stimulation by PMA was more effective (2.3-fold). Mutagenesis in either one of the two or both activating protein-1 (AP-1) binding sites abolished the high glucose and the PMA effect. Furthermore, addition of the AP-1 inhibitor curcumin obliterated the glucose response. Corresponding experiments revealed that the transcription factor stimulating protein 1 was not involved in mediating the glucose effect. The high glucose-induced TGF-β1 promoter activation was also prevented by inhibitors of protein kinase C and p38 mitogen-activated proteinkinase. Electrophoretic mobility shift assays with oligonucleotides containing one of the two AP-1 binding sites showed that glucose treatment markedly enhanced the binding activity of nuclear proteins of mesangial cells, particularly to box B. Supershift assays demonstrated that JunD and c-Fos were present in the protein-DNA complexes under control and hyperglycemic conditions. The functional and structural results show that glucose regulates human TGF-β1 gene expression through two adjacent AP-1 binding sites and gives rise to the involvement of protein kinase C and p38 mitogen-activated proteinkinase in hyperglycemia-induced TGF-β1 gene expression.

scholarly journals Genetic exploration of a nuclear receptor transcriptional regulatory complex

2020 ◽  
Author(s):  
Masako Asahina ◽  
Deborah Thurtle-Schmidt ◽  
Keith R. Yamamoto
Keyword(s):  
Nuclear Receptor ◽  
Molecular Mechanisms ◽  
Genetic Screen ◽  
Fluorescent Reporter ◽  
Response Elements ◽  
C Elegans ◽  
Transcriptional Regulatory ◽  
Regulatory Complex ◽  
Composition Structure ◽  
Context Specific

ABSTRACTMetazoan transcriptional regulatory factors (TFs) bind to genomic response elements and assemble with co-regulators into transcriptional regulatory complexes (TRCs) whose composition, structure and activities are gene-, cell- and physiological-context specific. Each TRC is a “regulatory logic module,” integrating incoming signaling information, which defines context and thereby recruits a distinct combination of co-regulators that together specify outgoing regulatory activity. Analyzing TRCs unique to every context is daunting, yet justified by their properties as self-contained regulatory modules. As proof-of-concept, we performed a forward genetic screen in C. elegans carrying a synthetic simple response element for nuclear receptor NHR-25 upstream of a fluorescent reporter gene. We isolated independent mutations in uba-2, a component of the sumoylation signaling machinery, and in lir-2, which we demonstrated to be a novel co-regulator, interacting with NHR-25 through LxxLL motifs and modulating target gene expression. Our studies establish that an unbiased genetic screen readily identifies both afferent and efferent components that specify TRC function, and suggest that screening natural response elements of interest could illuminate molecular mechanisms of both context-specificity and transcriptional regulation.

scholarly journals LncRNA TUG1 Regulates Proliferation of Cardiac Fibroblast via the miR-29b-3p/TGF-β1 Axis

2021 ◽  
Vol 8 ◽  
Author(s):  
Yini Guo ◽  
Zongli Sun ◽  
Minghe Chen ◽  
Junjie Lun
Keyword(s):  
Target Gene ◽  
Cardiac Fibroblasts ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Healthy Controls ◽  
Non Coding Rna ◽  
Long Non Coding Rna ◽  
Taurine Upregulated Gene 1 ◽  
Tgf Β1

Background: Atrial fibrillation (AF) is a very common clinical arrhythmia, accompanied by the overproliferation of cardiac fibroblasts (CFs). This study aimed to investigate the role of the long non-coding RNA(lncRNA) taurine upregulated gene 1 (TUG1) in the proliferation of CFs and further investigated its underlying mechanism.Methods: One hundred four paroxysmal AF patients and 94 healthy controls were recruited. Human cardiac fibroblasts (HCFs) were applied to establish an AF cell model through treatment with angiotensin II (AngII). qRT-PCR was used for the measurement of gene levels. The cell proliferation was detected by cell counting kit-8 (CCK-8). Luciferase reporter assay was performed for target gene analysis.Results: Elevated levels of TUG1 and low expression of miR-29b-3p were detected in the serum of AF patients compared with the healthy controls. Pearson's correlation analysis exhibited an inverse relationship between TUG1 and miR-29b-3p expression in AF patients (r = −7.106, p &lt; 0.001). Knockdown of TUG1 inhibited AngII-induced CF proliferation. Taurine upregulated gene 1 (TUG1) functions as a competing endogenous RNA (ceRNA) for miR-29b-3p, and downregulation of miR-29b-3p reversed the role of TUG1 in CF proliferation. TGF-β1 is a direct target gene of miR-29b-3p.Conclusions: Long non-coding RNA taurine upregulated gene 1 is a key regulator in the occurrence of AF. Slicing TUG1 inhibits CF proliferation by regulating the miR-29b-3p/TGF-β1 axis.

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