Genome-Wide Analysis of Smad7-Mediated Transcription in Mouse Embryonic Stem Cells

Smad7 has been identified as a negative regulator of the transforming growth factor TGF-β pathway by direct interaction with the TGF-β type I receptor (TβR-I). Although Smad7 has also been shown to play TGF-β unrelated functions in the cytoplasm and in the nucleus, a comprehensive analysis of its nuclear function has not yet been performed. Here, we show that in ESCs Smad7 is mainly nuclear and acts as a general transcription factor regulating several genes unrelated to the TGF-β pathway. Loss of Smad7 results in the downregulation of several key stemness master regulators, including Pou5f1 and Zfp42, and in the upregulation of developmental genes, with consequent loss of the stem phenotype. Integrative analysis of genome-wide mapping data for Smad7 and ESC self-renewal and pluripotency transcriptional regulators revealed that Smad7 co-occupies promoters of highly expressed key stemness regulators genes, by binding to a specific consensus response element NCGGAAMM. Altogether, our data establishes Smad7 as a new, integral component of the regulatory circuitry that controls ESC identity.

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Genome-wide CRISPR-Cas9 screen identified KLF11 as a druggable suppressor for sarcoma cancer stem cells

Science Advances ◽

10.1126/sciadv.abe3445 ◽

2021 ◽

Vol 7 (5) ◽

pp. eabe3445

Author(s):

Yicun Wang ◽

Jinhui Wu ◽

Hui Chen ◽

Yang Yang ◽

Chengwu Xiao ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

Negative Feedback ◽

Direct Interaction ◽

Therapy Resistance ◽

Negative Regulator ◽

Chemotherapy Response ◽

Genome Wide

Cancer stem cells (CSCs) are involved in tumorigenesis, recurrence, and therapy resistance. To identify critical regulators of sarcoma CSCs, we performed a reporter-based genome-wide CRISPR-Cas9 screen and uncovered Kruppel-like factor 11 (KLF11) as top candidate. In vitro and in vivo functional annotation defined a negative role of KLF11 in CSCs. Mechanistically, KLF11 and YAP/TEAD bound to adjacent DNA sites along with direct interaction. KLF11 recruited SIN3A/HDAC to suppress the transcriptional output of YAP/TEAD, which, in turn, promoted KLF11 transcription, forming a negative feedback loop. However, in CSCs, this negative feedback was lost because of epigenetic silence of KLF11, causing sustained YAP activation. Low KLF11 was associated with poor prognosis and chemotherapy response in patients with sarcoma. Pharmacological activation of KLF11 by thiazolidinedione effectively restored chemotherapy response. Collectively, our study identifies KLF11 as a negative regulator in sarcoma CSCs and potential therapeutic target.

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Collagen Type I Containing Hybrid Hydrogel Enhances Cardiomyocyte Maturation in a 3D Cardiac Model

Polymers ◽

10.3390/polym11040687 ◽

2019 ◽

Vol 11 (4) ◽

pp. 687 ◽

Cited By ~ 4

Author(s):

Sam G. Edalat ◽

Yongjun Jang ◽

Jongseong Kim ◽

Yongdoo Park

Keyword(s):

Growth Factor ◽

Collagen Type ◽

Transforming Growth Factor ◽

Embryonic Stem ◽

Collagen Type I ◽

Transforming Growth Factor Β1 ◽

Type I ◽

Hybrid Hydrogel ◽

Cardiac Model

In vitro maturation of cardiomyocytes in 3D is essential for the development of viable cardiac models for therapeutic and developmental studies. The method by which cardiomyocytes undergoes maturation has significant implications for understanding cardiomyocytes biology. The regulation of the extracellular matrix (ECM) by changing the composition and stiffness is quintessential for engineering a suitable environment for cardiomyocytes maturation. In this paper, we demonstrate that collagen type I, a component of the ECM, plays a crucial role in the maturation of cardiomyocytes. To this end, embryonic stem-cell derived cardiomyocytes were incorporated into Matrigel-based hydrogels with varying collagen type I concentrations of 0 mg, 3 mg, and 6 mg. Each hydrogel was analyzed by measuring the degree of stiffness, the expression levels of MLC2v, TBX18, and pre-miR-21, and the size of the hydrogels. It was shown that among the hydrogel variants, the Matrigel-based hydrogel with 3 mg of collagen type I facilitates cardiomyocyte maturation by increasing MLC2v expression. The treatment of transforming growth factor β1 (TGF-β1) or fibroblast growth factor 4 (FGF-4) on the hydrogels further enhanced the MLC2v expression and thereby cardiomyocyte maturation.

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High doses of TGF-β potently suppress type I collagen via the transcription factor CUX1

Molecular Biology of the Cell ◽

10.1091/mbc.e10-08-0669 ◽

2011 ◽

Vol 22 (11) ◽

pp. 1836-1844 ◽

Cited By ~ 26

Author(s):

Maria Fragiadaki ◽

Tetsurou Ikeda ◽

Abigail Witherden ◽

Roger M Mason ◽

David Abraham ◽

...

Keyword(s):

Type I Collagen ◽

Transforming Growth Factor ◽

Interstitial Fibrosis ◽

Transforming Growth Factor Β ◽

Negative Regulator ◽

Mitogen Activated Protein Kinase ◽

Type I ◽

Aberrant Expression

Transforming growth factor-β (TGF-β) is an inducer of type I collagen, and uncontrolled collagen production leads to tissue scarring and organ failure. Here we hypothesize that uncovering a molecular mechanism that enables us to switch off type I collagen may prove beneficial in treating fibrosis. For the first time, to our knowledge, we provide evidence that CUX1 acts as a negative regulator of TGF-β and potent inhibitor of type I collagen transcription. We show that CUX1, a CCAAT displacement protein, is associated with reduced expression of type I collagen both in vivo and in vitro. We show that enhancing the expression of CUX1 results in effective suppression of type I collagen. We demonstrate that the mechanism by which CUX1 suppresses type I collagen is through interfering with gene transcription. In addition, using an in vivo murine model of aristolochic acid (AA)-induced interstitial fibrosis and human AA nephropathy, we observe that CUX1 expression was significantly reduced in fibrotic tissue when compared to control samples. Moreover, silencing of CUX1 in fibroblasts from kidneys of patients with renal fibrosis resulted in increased type I collagen expression. Furthermore, the abnormal CUX1 expression was restored by addition of TGF-β via the p38 mitogen-activated protein kinase pathway. Collectively, our study demonstrates that modifications of CUX1 expression lead to aberrant expression of type I collagen, which may provide a molecular basis for fibrogenesis.

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Immunoproteasome Subunit β5i Promotes Ang II (Angiotensin II)–Induced Atrial Fibrillation by Targeting ATRAP (Ang II Type I Receptor–Associated Protein) Degradation in Mice

Hypertension ◽

10.1161/hypertensionaha.118.11813 ◽

2019 ◽

Vol 73 (1) ◽

pp. 92-101 ◽

Cited By ~ 16

Author(s):

Jing Li ◽

Shuai Wang ◽

Yun-Long Zhang ◽

Jie Bai ◽

Qiu-Yue Lin ◽

...

Keyword(s):

Atrial Fibrillation ◽

Angiotensin Ii ◽

Connexin 43 ◽

Transforming Growth Factor ◽

Negative Regulator ◽

Atrial Remodeling ◽

Type I ◽

Ang Ii ◽

Adeno Associated Virus ◽

Virus Serotype

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and increases the risk of stroke, heart failure, and death. Ang II (angiotensin II) triggers AF, mainly through stimulation of the AT1R (Ang II type I receptor). The immunoproteasome is a highly efficient proteolytic machine derived from the constitutive proteasome, but the role it plays in regulating AT1R activation and triggering AF remains unknown. Here, we show that among the catalytic subunits, β5i (PSMB8) expression, and chymotrypsin-like activity were the most significantly upregulated in atrial tissue of Ang II–infused mice or serum from patients with AF. β5i KO (β5i knockout) in mice markedly attenuated Ang II-induced AF incidence, atrial fibrosis, inflammatory response, and oxidative stress compared with WT (wild type) animals, but injection with recombinant adeno-associated virus serotype 9–β5i increased these effects. Moreover, we found that ATRAP (AT1R-associated protein) was a target of β5i. Overexpression of ATRAP significantly attenuated Ang II-induced atrial remodeling and AF in recombinant adeno-associated virus serotype 9–β5i-injected mice. Mechanistically, Ang II upregulated β5i expression to promote ATRAP degradation, which resulted in activation of AT1R-mediated NF-κB signaling, increased NADPH oxidase activity, increased TGF (transforming growth factor)-β1/Smad signaling, and altered the expression of Kir2.1 and CX43 (connexin 43) in the atria, thereby affecting atrial remodeling and AF. In summary, this study identifies β5i as a negative regulator of ATRAP stability that contributes to AT1R activation and to AF, highlighting that targeting β5i activity may represent a potential therapeutic approach for the treatment of hypertensive AF.

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VprBP mitigates TGF-β and Activin signaling by promoting Smurf1-mediated type I receptor degradation

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjz057 ◽

2019 ◽

Vol 12 (2) ◽

pp. 138-151 ◽

Cited By ~ 2

Author(s):

Yihao Li ◽

Chao Cui ◽

Feng Xie ◽

Szymon Kiełbasa ◽

Hailiang Mei ◽

...

Keyword(s):

Transforming Growth Factor ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Transforming Growth Factor Β ◽

Proteasomal Degradation ◽

Fine Tuning ◽

Mesoderm Induction ◽

Type I ◽

Critical Determinant ◽

Β Receptor

Abstract The transforming growth factor-β (TGF-β) family controls embryogenesis, stem cell differentiation, and tissue homeostasis. However, how post-translation modifications contribute to fine-tuning of TGF-β family signaling responses is not well understood. Inhibitory (I)-Smads can antagonize TGF-β/Smad signaling by recruiting Smurf E3 ubiquitin ligases to target the active TGF-β receptor for proteasomal degradation. A proteomic interaction screen identified Vpr binding protein (VprBP) as novel binding partner of Smad7. Mis-expression studies revealed that VprBP negatively controls Smad2 phosphorylation, Smad2–Smad4 interaction, as well as TGF-β target gene expression. VprBP was found to promote Smad7–Smurf1–TβRI complex formation and induce proteasomal degradation of TGF-β type I receptor (TβRI). Moreover, VprBP appears to stabilize Smurf1 by suppressing Smurf1 poly-ubiquitination. In multiple adult and mouse embryonic stem cells, depletion of VprBP promotes TGF-β or Activin-induced responses. In the mouse embryo VprBP expression negatively correlates with mesoderm marker expression, and VprBP attenuated mesoderm induction during zebrafish embryogenesis. Our findings thereby uncover a novel regulatory mechanism by which Smurf1 controls the TGF-β and Activin cascade and identify VprBP as a critical determinant of embryonic mesoderm induction.

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FRI0022 THE ANALYSIS OF THE INVOLVEMENT OF TRANSFORMING-GROWTH INTERACTING FACTOR POLYMORPHISMS IN THE BONE METABOLISM OF RHEUMATOID ARTHRITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.3126 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 583.1-583

Author(s):

H. Tan ◽

L. Zhang ◽

B. Yang ◽

L. Zhou

Keyword(s):

Rheumatoid Arthritis ◽

Bone Metabolism ◽

Type I Collagen ◽

Transforming Growth Factor ◽

Bone Erosion ◽

Serum Levels ◽

Negative Regulator ◽

Type I ◽

Nucleotide Polymorphisms ◽

The Relationship

Background:Rheumatoid arthritis (RA) is an autoimmune disease that mainly invades synovial membranes and further damage to articular cartilage and bone. The abnormal activation of transforming-growth factor β (TGF-β) in the subchondral bone is related to the onset of RA joint cartilage degeneration and transforming-growth interacting factor (TGIF) is a negative regulator of TGF-β signaling, while there is no literature addressing the relationship between TGIF polymorphisms and the bone metabolism of RA.Objectives:The aim of the study was to comprehensively explore the possible association for single nucleotide polymorphisms (SNPs) in the TGIF gene with serum bone metabolism markers and RA susceptibility.Methods:Three SNPs within the TGIF gene were genotyped in 155 RA patients and 168 healthy controls by high resolution melting (HRM) analysis in a case-control study. The serum levels of osteocalcin, bone alkaline phosphatase (BALP) and β type I collagen-crosslinked C telopeptide (β-CTX) were detected by electrochemical luminescence in 108 RA patients randomly selected from RA patients group.Results:Genotypes and alleles frequency analysis showed rs7362020 was associated with bone erosion in RA (P=0.012, P=0.003, respectively) and individuals carrying T allele for rs7362020 showed a decreased RA risk (OR=0.59, 95% CI = 0.42-0.84; P= 0.003). In the gender-specific analysis, rs73620203 polymorphism was associated with bone erosion of female RA patients (P values of the distribution of genotypes and alleles were 0.022 and 0.006, respectively). In addition, RA patients with CC, CT and TT genotypes at rs73620203 locus had statistically significant differences in serum osteocalcin and BALP (P=0.006, P=0.037, respectively) and the serum levels in TT genotype RA patients were significantly lower than CC and CT genotype RA patients. The serum levels of β-CTX in rs85440 AA genotype male RA patients were significantly higher than female RA patients (P=0.001), while the serum levels of osteocalcin and BALP in genotype AA, AG and GG female and male RA patients were not significantly different (P all>0.05).Conclusion:Our study provided the first evidence that rs73620203 is associated with bone erosion of RA and provided new insight into the relationship between three SNPs within TGIF gene and regulation of bone metabolism in RA patients of different genders.Acknowledgments:This work was supported by the National Natural Science Foundation of China (Nos. 81772258) and Science and Technology Agency of Sichuan Province (Nos. 2019YFS0310, 2018FZ0106).Disclosure of Interests:None declared

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SnoN facilitates ALK1–Smad1/5 signaling during embryonic angiogenesis

The Journal of Cell Biology ◽

10.1083/jcb.201208113 ◽

2013 ◽

Vol 202 (6) ◽

pp. 937-950 ◽

Cited By ~ 10

Author(s):

Qingwei Zhu ◽

Yong Hwan Kim ◽

Douglas Wang ◽

S. Paul Oh ◽

Kunxin Luo

Keyword(s):

Endothelial Cells ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Negative Regulator ◽

Endothelial Cell Proliferation ◽

Type I ◽

Positive Role ◽

And Migration ◽

Proliferation And Migration

In endothelial cells, two type I receptors of the transforming growth factor β (TGF-β) family, ALK1 and ALK5, coordinate to regulate embryonic angiogenesis in response to BMP9/10 and TGF-β. Whereas TGF-β binds to and activates ALK5, leading to Smad2/3 phosphorylation and inhibition of endothelial cell proliferation and migration, BMP9/10 and TGF-β also bind to ALK1, resulting in the activation of Smad1/5. SnoN is a negative regulator of ALK5 signaling through the binding and repression of Smad2/3. Here we uncover a positive role of SnoN in enhancing Smad1/5 activation in endothelial cells to promote angiogenesis. Upon ligand binding, SnoN directly bound to ALK1 on the plasma membrane and facilitated the interaction between ALK1 and Smad1/5, enhancing Smad1/5 phosphorylation. Disruption of this SnoN–Smad interaction impaired Smad1/5 activation and up-regulated Smad2/3 activity. This resulted in defective angiogenesis and arteriovenous malformations, leading to embryonic lethality at E12.5. Thus, SnoN is essential for TGF-β/BMP9-dependent biological processes by its ability to both positively and negatively modulate the activities of Smad-dependent pathways.

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Hepatic stellate cell–specific knockout of transcriptional intermediary factor 1γ aggravates liver fibrosis

Journal of Experimental Medicine ◽

10.1084/jem.20190402 ◽

2020 ◽

Vol 217 (6) ◽

Cited By ~ 2

Author(s):

Eun Ju Lee ◽

Injoo Hwang ◽

Ji Yeon Lee ◽

Jong Nam Park ◽

Keun Cheon Kim ◽

...

Keyword(s):

Stem Cells ◽

Growth Factor ◽

Liver Fibrosis ◽

Hepatic Stellate Cell ◽

Transforming Growth Factor ◽

Stellate Cell ◽

Embryonic Stem ◽

Transforming Growth Factor Β ◽

Negative Regulator ◽

Promising Therapeutic Approach

Transforming growth factor β (TGFβ) is a crucial factor in fibrosis, and transcriptional intermediary factor 1γ (TIF1γ) is a negative regulator of the TGFβ pathway; however, its role in liver fibrosis is unknown. In this study, mesenchymal stem cells derived from human embryonic stem cells (hE-MSCs) that secrete hepatocyte growth factor (HGF) were used to observe the repair of thioacetamide (TAA)-induced liver fibrosis. Our results showed that TIF1γ was significantly decreased in LX2 cells when exposed to TGFβ1. Such decrease of TIF1γ was significantly prevented by co-culture with hE-MSCs. Interaction of TIF1γ with SMAD2/3 and binding to the promoter of the α-smooth muscle gene (αSMA) suppressed αSMA expression. Phosphorylation of cAMP response element–binding protein (CREB) and binding on the TIF1γ promoter region induced TIF1γ expression. Furthermore, hepatic stellate cell–specific TIF1γ-knockout mice showed aggravation of liver fibrosis. In conclusion, loss of TIF1γ aggravates fibrosis, suggesting that a strategy to maintain TIF1γ during liver injury would be a promising therapeutic approach to prevent or reverse liver fibrosis.

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Myostatin Signals through a Transforming Growth Factor β-Like Signaling Pathway To Block Adipogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.23.20.7230-7242.2003 ◽

2003 ◽

Vol 23 (20) ◽

pp. 7230-7242 ◽

Cited By ~ 386

Author(s):

A. Rebbapragada ◽

H. Benchabane ◽

J. L. Wrana ◽

A. J. Celeste ◽

L. Attisano

Keyword(s):

Signal Transduction ◽

Growth Factor ◽

Signaling Pathway ◽

Transforming Growth Factor ◽

Signal Transduction Pathway ◽

Transforming Growth Factor Β ◽

Negative Regulator ◽

Type I ◽

Type Ii ◽

Transduction Pathway

ABSTRACT Myostatin, a transforming growth factor β (TGF-β) family member, is a potent negative regulator of skeletal muscle growth. In this study we characterized the myostatin signal transduction pathway and examined its effect on bone morphogenetic protein (BMP)-induced adipogenesis. While both BMP7 and BMP2 activated transcription from the BMP-responsive I-BRE-Lux reporter and induced adipogenic differentiation, myostatin inhibited BMP7- but not BMP2-mediated responses. To dissect the molecular mechanism of this antagonism, we characterized the myostatin signal transduction pathway. We showed that myostatin binds the type II Ser/Thr kinase receptor. ActRIIB, and then partners with a type I receptor, either activin receptor-like kinase 4 (ALK4 or ActRIB) or ALK5 (TβRI), to induce phosphorylation of Smad2/Smad3 and activate a TGF-β-like signaling pathway. We demonstrated that myostatin prevents BMP7 but not BMP2 binding to its receptors and that BMP7-induced heteromeric receptor complex formation is blocked by competition for the common type II receptor, ActRIIB. Thus, our results reveal a strikingly specific antagonism of BMP7-mediated processes by myostatin and suggest that myostatin is an important regulator of adipogenesis.

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TGFβ induces SIK to negatively regulate type I receptor kinase signaling

The Journal of Cell Biology ◽

10.1083/jcb.200804107 ◽

2008 ◽

Vol 182 (4) ◽

pp. 655-662 ◽

Cited By ~ 55

Author(s):

Marcin Kowanetz ◽

Peter Lönn ◽

Michael Vanlandewijck ◽

Katarzyna Kowanetz ◽

Carl-Henrik Heldin ◽

...

Keyword(s):

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Cell Types ◽

Negative Regulator ◽

Type I ◽

Gene Target ◽

Smad Proteins ◽

Tgfβ Receptor ◽

Receptor Kinase Signaling ◽

Feedback Program

Signal transduction by transforming growth factor β (TGFβ) coordinates physiological responses in diverse cell types. TGFβ signals via type I and type II receptor serine/threonine kinases and intracellular Smad proteins that regulate transcription. Strength and duration of TGFβ signaling is largely dependent on a negative-feedback program initiated during signal progression. We have identified an inducible gene target of TGFβ/Smad signaling, the salt-inducible kinase (SIK), which negatively regulates signaling together with Smad7. SIK and Smad7 form a complex and cooperate to down-regulate the activated type I receptor ALK5. We further show that both the kinase and ubiquitin-associated domain of SIK are required for proper ALK5 degradation, with ubiquitin functioning to enhance SIK-mediated receptor degradation. Loss of endogenous SIK results in enhanced gene responses of the fibrotic and cytostatic programs of TGFβ. We thus identify in SIK a negative regulator that controls TGFβ receptor turnover and physiological signaling.

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