Cardiomyocyte-specific loss of RNA polymerase II subunit 5-mediating protein causes myocardial dysfunction and heart failure

2018 ◽  
Vol 115 (11) ◽  
pp. 1617-1628
Author(s):  
Jian Zhang ◽  
Jingyi Sheng ◽  
Liwei Dong ◽  
Yinli Xu ◽  
Liming Yu ◽  
...  
Keyword(s):  
Heart Failure ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genome Stability ◽  
Transforming Growth Factor ◽  
Myocardial Dysfunction ◽  
Transforming Growth Factor Β ◽  
And Function ◽  
Deficient Mice

AbstractAimsMyocardial dysfunction is an important cause of heart failure (HF). RNA polymerase II subunit 5 (RPB5)-mediating protein (RMP) is a transcriptional mediating protein which co-ordinates cellular processes including gene expression, metabolism, proliferation, and genome stability. However, its role in cardiac disease remains unknown. We aimed to determine the role and regulatory mechanisms of RMP in cardiomyocyte function and the development of HF.Methods and resultsMyocardial RMP expression was examined in human heart tissues from healthy controls and patients with advanced HF. Compared to normal cardiac tissues, RMP levels were significantly decreased in the myocardium of patients with advanced HF. To investigate the role of RMP in cardiac function, Cre-loxP recombinase technology was used to generate tamoxifen-inducible cardiomyocyte-specific Rmp knockout mice. Unexpectedly, cardiomyocyte-specific deletion of Rmp in mice resulted in contractile dysfunction, cardiac dilatation, and fibrosis. Furthermore, the lifespan of cardiac-specific Rmp-deficient mice was significantly shortened when compared with littermates. Mechanistically, we found that chronic HF in Rmp-deficient mice was associated with impaired mitochondrial structure and function, which may be mediated via a transforming growth factor-β/Smad3-proliferator-activated receptor coactivator1α (PGC1α)-dependent mechanism. PGC1α overexpression partially rescued chronic HF in cardiomyocyte-specific Rmp-deficient mice, and Smad3 blockade protected against the loss of PGC1α and adenosine triphosphate content that was induced by silencing RMP in vitro.ConclusionsRMP plays a protective role in chronic HF. RMP may protect cardiomyocytes from injury by maintaining PGC1α-dependent mitochondrial biogenesis and function. The results from this study suggest that RMP may be a potential therapeutic agent for treating HF.

Expression and function of CD105 during the onset of hematopoiesis from Flk1+ precursors

Blood ◽  
2001 ◽  
Vol 98 (13) ◽  
pp. 3635-3642 ◽  
Author(s):  
Sarah K. Cho ◽  
Annie Bourdeau ◽  
Michelle Letarte ◽  
Juan Carlos Zúñiga-Pflücker
Keyword(s):  
Transforming Growth Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Transforming Growth Factor Β ◽  
Hematopoietic Development ◽  
Mesoderm Specification ◽  
Molecular Events ◽  
Regulated Expression ◽  
And Function

Abstract During ontogeny, the hematopoietic system is established from mesoderm-derived precursors; however, molecular events regulating the onset of hematopoiesis are not well characterized. Several members of the transforming growth factor β (TGF-β) superfamily have been implicated as playing a role during mesoderm specification and hematopoiesis. CD105 (endoglin) is an accessory receptor for members of the TGF-β superfamily. Here it is reported that during the differentiation of murine embryonic stem (ES) cells in vitro, hematopoietic commitment within Flk1+ mesodermal precursor populations is characterized by CD105 expression. In particular, CD105 is expressed during the progression from the Flk1+CD45− to Flk1−CD45+ stage. The developmentally regulated expression of CD105 suggests that it may play a role during early hematopoiesis from Flk1+ precursors. To determine whether CD105 plays a functional role during early hematopoietic development, the potential of CD105-deficient ES cells to differentiate into various hematopoietic lineages in vitro was assessed. In the absence of CD105, myelopoiesis and definitive erythropoiesis were severely impaired. In contrast, lymphopoiesis appeared to be only mildly affected. Thus, these findings suggest that the regulated expression of CD105 functions to support lineage-specific hematopoietic development from Flk1+ precursors.

scholarly journals Domains in the SPT5 Protein That Modulate Its Transcriptional Regulatory Properties

2000 ◽  
Vol 20 (9) ◽  
pp. 2970-2983 ◽  
Author(s):  
Dmitri Ivanov ◽  
Youn Tae Kwak ◽  
Jun Guo ◽  
Richard B. Gaynor
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Repression ◽  
Transcriptional Elongation ◽  
Tat Protein ◽  
C Terminus ◽  
Heptad Repeats ◽  
And Function ◽  
Hiv 1

ABSTRACT SPT5 and its binding partner SPT4 regulate transcriptional elongation by RNA polymerase II. SPT4 and SPT5 are involved in both 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB)-mediated transcriptional inhibition and the activation of transcriptional elongation by the human immunodeficiency virus type 1 (HIV-1) Tat protein. Recent data suggest that P-TEFb, which is composed of CDK9 and cyclin T1, is also critical in regulating transcriptional elongation by SPT4 and SPT5. In this study, we analyze the domains of SPT5 that regulate transcriptional elongation in the presence of either DRB or the HIV-1 Tat protein. We demonstrate that SPT5 domains that bind SPT4 and RNA polymerase II, in addition to a region in the C terminus of SPT5 that contains multiple heptad repeats and is designated CTR1, are critical for in vitro transcriptional repression by DRB and activation by the Tat protein. Furthermore, the SPT5 CTR1 domain is a substrate for P-TEFb phosphorylation. These results suggest that C-terminal repeats in SPT5, like those in the RNA polymerase II C-terminal domain, are sites for P-TEFb phosphorylation and function in modulating its transcriptional elongation properties.

scholarly journals Subnuclear Localization of Ku Protein: Functional Association with RNA Polymerase II Elongation Sites

2002 ◽  
Vol 22 (22) ◽  
pp. 8088-8099 ◽  
Author(s):  
Xianming Mo ◽  
William S. Dynan
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Genome Stability ◽  
Strand Break ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Rnap Ii

ABSTRACT Ku is an abundant nuclear protein with an essential function in the repair of DNA double-strand breaks. Various observations suggest that Ku also interacts with the cellular transcription machinery, although the mechanism and significance of this interaction are not well understood. In the present study, we investigated the subnuclear distribution of Ku in normally growing human cells by using confocal microscopy, chromatin immunoprecipitation, and protein immunoprecipitation. All three approaches indicated association of Ku with RNA polymerase II (RNAP II) elongation sites. This association occurred independently of the DNA-dependent protein kinase catalytic subunit and was highly selective. There was no detectable association with the initiating isoform of RNAP II or with the general transcription initiation factors. In vitro protein-protein interaction assays demonstrated that the association of Ku with elongation proteins is mediated, in part, by a discrete C-terminal domain in the Ku80 subunit. Functional disruption of this interaction with a dominant-negative mutant inhibited transcription in vitro and in vivo and suppressed cell growth. These results suggest that association of Ku with transcription sites is important for maintenance of global transcription levels. Tethering of double-strand break repair proteins to defined subnuclear structures may also be advantageous in maintenance of genome stability.

scholarly journals Ameliorative role of SIRT1 in peritoneal fibrosis: an in vivo and in vitro study

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yanhong Guo ◽  
Liuwei Wang ◽  
Rong Gou ◽  
Yulin Wang ◽  
Xiujie Shi ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
In Vitro Study ◽  
Transforming Growth Factor Β ◽  
Peritoneal Fibrosis ◽  
Mesenchymal Transition ◽  
Protein Levels ◽  
And Function

Abstract Background Peritoneal fibrosis is one of the major complications induced by peritoneal dialysis (PD). Damaged integrity and function of peritoneum caused by peritoneal fibrosis not only limits the curative efficacy of PD and but affects the prognosis of patients. However, the detailed mechanisms underlying the process remain unclear and therapeutic strategy targeting TGF‐β is deficient. Transforming growth factor‐β (TGF‐β) signaling participates in the progression of peritoneal fibrosis through enhancing mesothelial-mesenchymal transition of mesothelial cells. Methods The study aims to demonstrate the regulatory role of Sirtuin1 (SIRT1) to the TGF‐β signaling mediated peritoneal fibrosis. SIRT1−/− mice were used to establish animal model. Masson’s staining and peritoneal equilibration assay were performed to evaluate the degree of peritoneal fibrosis. QRT-PCR assays were used to estimate the RNA levels of Sirt1 and matrix genes related to peritoneal fibrosis, and their protein levels were examined by Western blot assays. Results SIRT1 significantly decreased in vivo post PD treatment. SIRT1 knockout exacerbated peritoneal fibrosis both in vivo and vitro. Overexpression of SIRT1 efficiently inhibited peritoneal fibrosis by inhibiting the peritoneal inflammation and the activation of TGF‐β signaling. Conclusion SIRT1 ameliorated peritoneal fibrosis both in vivo and in vitro through inhibiting the expression of protein matrix induced by TGF‐β signaling.

scholarly journals Human cardiac fibrosis-on-a-chip model recapitulates disease hallmarks and can serve as a platform for drug screening

2019 ◽  
Author(s):  
Olya Mastikhina ◽  
Byeong-Ui Moon ◽  
Kenneth Williams ◽  
Rupal Hatkar ◽  
Dakota Gustafson ◽  
...  
Keyword(s):  
Heart Failure ◽  
Cardiac Fibrosis ◽  
Transforming Growth Factor ◽  
Force Measurement ◽  
Interstitial Fibrosis ◽  
Transforming Growth Factor Β ◽  
Tissue Stiffness ◽  
Human Heart Failure ◽  
Transcriptomic Signature

AbstractWhile interstitial fibrosis plays a significant role in heart failure, our understanding of disease progression in humans is limited. To address this limitation, we have engineered a cardiac-fibrosis-on-a-chip model consisting of a microfabricated device with live force measurement capabilities using co-cultured human cardiac fibroblasts and pluripotent stem cell-derived cardiomyocytes. Transforming growth factor-β was used as a trigger for fibrosis. Here, we have reproduced the classic hallmarks of fibrosis-induced heart failure including high collagen deposition, increased tissue stiffness, BNP secretion, and passive tension. Force of contraction was significantly decreased in fibrotic tissues that displayed a transcriptomic signature consistent with human cardiac fibrosis/heart failure. Treatment with an anti-fibrotic drug decreased tissue stiffness and BNP secretion, with corresponding changes in the transcriptomic signature. This model represents an accessible approach to study human heart failure in vitro, and allows for testing anti-fibrotic drugs while facilitating the real-time assessment of cardiomyocyte function.

scholarly journals Perivascular Cells in Diffuse Cutaneous Systemic Sclerosis Overexpress Activated ADAM12 and Are Involved in Myofibroblast Transdifferentiation and Development of Fibrosis

2016 ◽  
Vol 43 (7) ◽  
pp. 1340-1349 ◽  
Author(s):  
Paola Cipriani ◽  
Paola Di Benedetto ◽  
Piero Ruscitti ◽  
Vasiliki Liakouli ◽  
Onorina Berardicurti ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Transforming Growth Factor Β ◽  
Potential Contribution ◽  
Microvascular Damage ◽  
Fibrotic Process ◽  
Ethical Approval ◽  
And Function

Objective.Microvascular damage is pivotal in the pathogenesis of systemic sclerosis (SSc), preceding fibrosis, and whose trigger is not still fully understood. Perivascular progenitor cells, with profibrotic activity and function, are identified by the expression of the isoform 12 of ADAM (ADAM12) and this molecule may be upregulated by transforming growth factor-β (TGF-β). The goal of this work was to evaluate whether pericytes in the skin of patients with diffuse cutaneous SSc (dcSSc) expressed ADAM12, suggesting their potential contribution to the fibrotic process, and whether TGF-β might modulate this molecule.Methods.After ethical approval, mesenchymal stem cells (MSC) and fibroblasts (FB) were isolated from bone marrow and skin samples collected from 20 patients with dcSSc. ADAM12 expression was investigated in the skin and in isolated MSC and FB treated with TGF-β by immunofluorescence, quantitative real-time PCR, and western blot. Further, we silenced ADAM12 expression in both dcSSc-MSC and -FB to confirm the TGF-β modulation.Results.Pericytes and FB of dcSSc skin showed an increased expression of ADAM12 when compared with healthy control skin. TGF-β in vitro treatment induced a significant increase of ADAM12 in both SSc-MSC and -FB, with the higher levels observed in dcSSc cells. After ADAM12 silencing, the TGF-β ability to upregulate α-smooth muscle actin in both SSc-MSC and SSc-FB was inhibited.Conclusion.Our results suggest that in SSc, pericytes that transdifferentiate toward activated FB are present in the vascular tree, and TGF-β, while increasing ADAM12 expression, may modulate this transdifferentiation.

Severely Impaired Erythropoiesis in Mice Lacking Mediator Subunit Med1/TRAP220.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1611-1611
Author(s):  
Tilman Borggrefe ◽  
Claudia Waskow ◽  
Robert G. Roeder ◽  
Melanie Stumpf
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Regulatory Proteins ◽  
Double Positive ◽  
Gata Factors ◽  
Colony Forming Units ◽  
Gata Transcription Factors ◽  
Hepatic Development ◽  
Deficient Mice

Abstract GATA transcription factors are essential for diverse developmental processes including erythropoiesis. Med1/TRAP220 was shown to interact with GATA factors in vitro (Crawford et al. (2002): J Biol Chem. 277(5):3585-92.). Med1/TRAP220 is part of the multiprotein Mediator complex that forms a bridge between gene-specific regulatory proteins and the RNA polymerase II (Borggrefe (2004): Research Adv. in Biol. Chem. (2: 9-20), Bourbon et al. (2004) Mol. Cell 14(5):553-7). Mediator subunit Med1/TRAP220 deficient mice die during mid-embryonic development (day 11.5dpc) due to defects in placental, cardiac and hepatic development (Ito et al. (2000): Mol Cell. 5 (4):683–93., Landles et al. (2004): Mol Endocrinol. 17(12):2418–35). In order to analyze hematopoiesis in TRAP220-deficient embryos, AGMs (aorta-gonad-mesonephros) and Yolk-sacs were isolated from day 10.5 dpc embryos and analyzed by FACS and methylcellulose assays. TRAP220-deficient embryos contain c-Kit positive cells and BFU-E (Burst forming units erythrocytes) activity. However, CFU-E (Colony-forming units erythrocytes) activity was 10-fold reduced. This correlated with severe decrease of the CD71/Ter119 double-positive cells, the CFU-E containing fraction, in FACS-analysis. Our data shows that Mediator subunit Med1/TRAP220 is required for erythropoiesis just before the CFU-E stage. Because GATA factors affect early erythropoiesis and because MED1/TRAP220 binds to GATA factors in vitro, our data suggest that the Mediator of transcriptional regulation forms the bridge between GATA factors and the RNA polymerase II machinery.

scholarly journals RNA polymerase II progression through H3K27me3-enriched gene bodies requires JMJD3 histone demethylase

2013 ◽  
Vol 24 (3) ◽  
pp. 351-360 ◽  
Author(s):  
Conchi Estarás ◽  
Raquel Fueyo ◽  
Naiara Akizu ◽  
Sergi Beltrán ◽  
Marian A. Martínez-Balbás
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Target Genes ◽  
Transforming Growth Factor ◽  
Transcriptional Response ◽  
Transforming Growth Factor Β ◽  
Genome Wide ◽  
Pathway Activation

JMJD3 H3K27me3 demethylase plays an important role in the transcriptional response to different signaling pathways; however, the mechanism by which it facilitates transcription has been unclear. Here we show that JMJD3 regulates transcription of transforming growth factor β (TGFβ)–responsive genes by promoting RNA polymerase II (RNAPII) progression along the gene bodies. Using chromatin immunoprecipitation followed by sequencing experiments, we show that, upon TGFβ treatment, JMJD3 and elongating RNAPII colocalize extensively along the intragenic regions of TGFβ target genes. According to these data, genome-wide analysis shows that JMJD3-dependent TGFβ target genes are enriched in H3K27me3 before TGFβ signaling pathway activation. Further molecular analyses demonstrate that JMJD3 demethylates H3K27me3 along the gene bodies, paving the way for the RNAPII progression. Overall these findings uncover the mechanism by which JMJD3 facilitates transcriptional activation.

Apigenin Alleviates Renal Fibroblast Activation through AMPK and ERK Signaling Pathways In Vitro

2020 ◽  
Vol 21 (11) ◽  
pp. 1107-1118
Author(s):  
Ningning Li ◽  
Zhan Wang ◽  
Tao Sun ◽  
Yanfei Lei ◽  
Xianghua Liu ◽  
...  
Keyword(s):  
Renal Fibrosis ◽  
Transforming Growth Factor ◽  
Therapeutic Potential ◽  
Protective Role ◽  
Fibroblast Proliferation ◽  
Fibroblast Activation ◽  
And Function ◽  
Activated Fibroblasts ◽  
Tgf Β1

Objective: Renal fibrosis is a common pathway leading to the progression of chronic kidney disease. Activated fibroblasts contribute remarkably to the development of renal fibrosis. Although apigenin has been demonstrated to play a protective role from fibrotic diseases, its pharmacological effect on renal fibroblast activation remains largely unknown. Materials and Methods: Here, we examined the functional role of apigenin in the activation of renal fibroblasts response to transforming growth factor (TGF)-β1 and its potential mechanisms. Cultured renal fibroblasts (NRK-49F) were exposed to apigenin (1, 5, 10 and 20 μM), followed by the stimulation of TGF-β1 (2 ng/mL) for 24 h. The markers of fibroblast activation were determined. In order to confirm the anti-fibrosis effect of apigenin, the expression of fibrosis-associated genes in renal fibroblasts was assessed. As a consequence, apigenin alleviated fibroblast proliferation and fibroblastmyofibroblast differentiation induced by TGF-β1. Result: Notably, apigenin significantly inhibited the fibrosis-associated genes expression in renal fibroblasts. Moreover, apigenin treatment significantly increased the phosphorylation of AMP-activated protein kinase (AMPK). Apigenin treatment also obviously reduced TGF-β1 induced phosphorylation of ERK1/2 but not Smad2/3, p38 and JNK MAPK in renal fibroblasts. Conclusion: In a summary, these results indicate that apigenin inhibits renal fibroblast proliferation, differentiation and function by AMPK activation and reduced ERK1/2 phosphorylation, suggesting it could be an attractive therapeutic potential for the treatment of renal fibrosis.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

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