scholarly journals L6E9 Myoblasts Are Deficient of Myostatin and Additional TGF-βMembers Are Candidates to Developmentally Control Their Fiber Formation

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Stefania Rossi ◽  
Elena Stoppani ◽  
Massimiliano Gobbo ◽  
Anna Caroli ◽  
Alessandro Fanzani
Keyword(s):  
Developmental Regulation ◽  
Fiber Formation ◽  
Dominant Negative ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
Knock Out ◽  
Fiber Size ◽  
Vitro Model ◽  
Activin Receptors

This work provides evidence that the robust myoblast differentiation observed in L6E9 cells is causally linked to deficiency of myostatin, which, conversely, has been found to be expressed in C2C12 cells. However, despite the absence of endogenous myostatin, L6E9 myoblasts expressed functional Activin receptors type II (ActRIIs) and follistatin as well as the highly related TGF-βmembers Activins and GDF11, suggesting that in this cell line the regulation of fiber size might be under the control of multiple regulators regardless of myostatin. In line with this hypothesis, delivery of a dominant-negative ActRIIb form or the increase of follistatin, as obtained via Trichostatin treatment or stable transfection of a short human follistatin form, enhanced the L6E9 cell differentiation and further increased the size of myotubes, suggesting that L6E9 myoblasts provide a spontaneous myostatin knock-out in vitro model to study TGF-βligands involved in developmental regulation of fiber size.

scholarly journals The Leucine Catabolite and Dietary Supplement β-Hydroxy-β-Methyl Butyrate (HMB) as an Epigenetic Regulator in Muscle Progenitor Cells

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 512
Author(s):  
Virve Cavallucci ◽  
Giovambattista Pani
Keyword(s):  
Histone Acetylation ◽  
Dietary Supplement ◽  
Hdac Inhibitor ◽  
Muscle Wasting ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
Epigenetic Mark ◽  
Epigenetic Regulator ◽  
Methyl Butyrate

β-Hydroxy-β-Methyl Butyrate (HMB) is a natural catabolite of leucine deemed to play a role in amino acid signaling and the maintenance of lean muscle mass. Accordingly, HMB is used as a dietary supplement by sportsmen and has shown some clinical effectiveness in preventing muscle wasting in cancer and chronic lung disease, as well as in age-dependent sarcopenia. However, the molecular cascades underlying these beneficial effects are largely unknown. HMB bears a significant structural similarity with Butyrate and β-Hydroxybutyrate (βHB), two compounds recognized for important epigenetic and histone-marking activities in multiple cell types including muscle cells. We asked whether similar chromatin-modifying actions could be assigned to HMB as well. Exposure of murine C2C12 myoblasts to millimolar concentrations of HMB led to an increase in global histone acetylation, as monitored by anti-acetylated lysine immunoblotting, while preventing myotube differentiation. In these effects, HMB resembled, although with less potency, the histone deacetylase (HDAC) inhibitor Sodium Butyrate. However, initial studies did not confirm a direct inhibitory effect of HMB on HDACs in vitro. β-Hydroxybutyrate, a ketone body produced by the liver during starvation or intense exercise, has a modest effect on histone acetylation of C2C12 cells or in vitro HDAC inhibitor activities, and, unlike Butyrate and HMB, did not interfere with myotube formation in a myoblast differentiation assay. Instead, βHB dramatically increased lysine β-hydroxybutyrylation (Kbhb) of histone tails, an epigenetic mark associated with fasting responses and muscle catabolic states. However, when C2C12 cells were exposed to βHB in the presence of equimolar HMB this chromatin modification was drastically reduced, pointing to a role for HMB in attenuating ketosis-associated muscle wasting. In conclusion, while their mechanistic underpinnings remain to be clarified, these preliminary observations highlight novel and potentially important activities of HMB as an epigenetic regulator and βHB antagonist in muscle precursor cells, to be further explored in their biomedical implications.

scholarly journals Hypoxia induces stress fiber formation in adipocytes in the early stage of obesity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Golnaz Anvari ◽  
Evangelia Bellas
Keyword(s):  
In Vitro Model ◽  
Nuclear Translocation ◽  
Early Stage ◽  
Coiled Coil ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Actin Stress Fiber ◽  
Stress Fiber Formation ◽  
Vitro Model

AbstractIn obese adipose tissue (AT), hypertrophic expansion of adipocytes is not matched by new vessel formation, leading to AT hypoxia. As a result, hypoxia inducible factor-1⍺ (HIF-1⍺) accumulates in adipocytes inducing a transcriptional program that upregulates profibrotic genes and biosynthetic enzymes such as lysyl oxidase (LOX) synthesis. This excess synthesis and crosslinking of extracellular matrix (ECM) components cause AT fibrosis. Although fibrosis is a hallmark of obese AT, the role of fibroblasts, cells known to regulate fibrosis in other fibrosis-prone tissues, is not well studied. Here we have developed an in vitro model of AT to study adipocyte-fibroblast crosstalk in a hypoxic environment. Further, this in vitro model was used to investigate the effect of hypoxia on adipocyte mechanical properties via ras homolog gene family member A (RhoA)/Rho-associated coiled-coil kinases (ROCK) signaling pathways. We confirmed that hypoxia creates a diseased phenotype by inhibiting adipocyte maturation and inducing actin stress fiber formation facilitated by myocardin-related transcription factor A (MRTF-A/MKL1) nuclear translocation. This work presents new potential therapeutic targets for obesity by improving adipocyte maturation and limiting mechanical stress in obese AT.

scholarly journals Ganoderma microsporum immunomodulatory protein, GMI, promotes C2C12 myoblast differentiation in vitro via upregulation of Tid1 and STAT3 acetylation

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244791
Author(s):  
Wan-Huai Teo ◽  
Jeng-Fan Lo ◽  
Yu-Ning Fan ◽  
Chih-Yang Huang ◽  
Tung-Fu Huang
Keyword(s):  
Myogenic Differentiation ◽  
Atp Synthesis ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Muscle Loss ◽  
C2c12 Myoblasts ◽  
Immunomodulatory Protein ◽  
Pre Treatment

Ageing and chronic diseases lead to muscle loss and impair the regeneration of skeletal muscle. Thus, it’s crucial to seek for effective intervention to improve the muscle regeneration. Tid1, a mitochondrial co-chaperone, is important to maintain mitochondrial membrane potential and ATP synthesis. Previously, we demonstrated that mice with skeletal muscular specific Tid1 deficiency displayed muscular dystrophy and postnatal lethality. Tid1 can interact with STAT3 protein, which also plays an important role during myogenesis. In this study, we used GMI, immunomodulatory protein of Ganoderma microsporum, as an inducer in C2C12 myoblast differentiation. We observed that GMI pretreatment promoted the myogenic differentiation of C2C12 myoblasts. We also showed that the upregulation of mitochondria protein Tid1 with the GMI pre-treatment promoted myogenic differentiation ability of C2C12 cells. Strikingly, we observed the concomitant elevation of STAT3 acetylation (Ac-STAT3) during C2C12 myogenesis. Our study suggests that GMI promotes the myogenic differentiation through the activation of Tid1 and Ac-STAT3.

scholarly journals Ezrin regulated myoblast differentiation/fusion and muscle fiber specialization through PKA-NFAT-MyoD/MEF2csignalling pathway

2020 ◽  
Author(s):  
Ruo-nan Zhang ◽  
Yan Wang ◽  
Yun Liu ◽  
Xin Bao ◽  
Wei Xu ◽  
...  
Keyword(s):  
Muscle Fiber ◽  
Neuromuscular Diseases ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
Type I ◽  
Over Expression ◽  
Myoblast Cells ◽  
Myofiber Type

Abstract Backgorund:Neuromuscular diseases are a kind of nervous system diseases that have a high disability rate.Ezrin’ role in skeletal muscle has not been identified. This study aims to confirm the effect and mechanism of Ezrin on myoblast differentiation and fusion, myotube size, and myofiber type.Method:By using immunoassaying and western blot analyses, Ezrin, MyHC,MEF2c, MyoG, PKAα/β/γ, PKA reg Iα, PKA reg IIβand NFATc1-c4 were detected in myoblast cells treated with Ad-Ezrin or Ad-shEzrin. Real-time PCR were used to evaluate MyoD, Myf5, MyHC-I , MyHC-IIa/b and MyHC-IIx in myoblast cells. PKA inhibitor H-89 or PKAreg I activator N6-Bz-cAMP were added into medium to confirm their relationship between Ezrin and PKA during myoblast differentiation/fusion. In vitro, Ad-NFATc1/c2 or Ad-shNFATc3/c4 were respectively transfected into C2C12 cells, myoblast differentiation/fusion, myotube size and myofiber type were assessed by using immunostaining of MyHC, MEF2c and MyoG. In vivo, transfection of Ad-Ezrin into gastrocnemius and soleus muscles for 7 days, the numbers of MyHC-1 postivemyofibers were analyzed after immunostaining of MyHC-1.Results: Ezrin expression were time-dependently increased during myoblast differentiation/fusion. Knockdown of Ezrin by shRNA delayed myoblast differentiation and fusion in a time dose-dependent pattern, as shown by immunostaining of MyHC. Conversely, over-expression of Ezrin by adenovirus time- and dosage-dependently promoted myoblastdifferentiation/fusion, and muscle fiber specialization characterized by increased MyHC I and MyHCIIa/b. Forced expression of Ezrin did not alter PKA, and PKAreg II α levels, but altered the levels of PKAreg I α/β, Myf5 and MyoD, and leading to the accumulation of MyoG+/MEF2c+ nuclei. By contrast, Ezrin knockdown significantly decreased the PKA reg I/II ratio and MyoG+/MEF2c+ nuclei. The PKA inhibitor H-89 remarkably abolished the beneficial effect of over-expressingEzrin on the numbers of MyHC+ myotubes and MyoG+/MEF2c nuclei. These opposite changes mediated by knocking down Ezrin were almost eliminated by PKAreg I activator N6-Bz-cAMP. Furthermore, over-expression of NFATc2 or knockdown of NFATc4reversed the inhibitory effect of Ezrin knockdown on myoblast differentiation/fusion, resulting in the recovery of the numbers ofMyoG+/MEF2c+ nucleiin3-nuclei+myotubes. Meanwhile, overexpression of Ezrin specifically induced type I muscle fiber specialization, which was associated with increased levels of NFATc1/c2. Furthermore, in vivo transfection ofAd-Ezrin into gastrocnemius and soleus muscles increased the numbers of MyHC-1 postivemyofibers. By contrast, knockdown of NFATc4resulted in the recovery to normal levels of MyHC-2b in Ezrin-knockdown myoblast cells, attributingtoregainingMyoDand MEF2c expression. Conclusions: Ezrin trigger myoblast differentiation and fusion, myotube size, and alters muscle fiber specialization through PKA-NFAT-MyoD/MEF2C signalling pathway.

scholarly journals In vivo dissection of Rhoa function in vascular development using zebrafish

2021 ◽  
Author(s):  
Laura M. Pillay ◽  
Joseph J. Yano ◽  
Andrew E. Davis ◽  
Matthew G. Butler ◽  
Keith A. Barnes ◽  
...  
Keyword(s):  
Vascular Dysfunction ◽  
Vascular Development ◽  
Vascular Endothelial Cell ◽  
Fiber Formation ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Vascular Integrity ◽  
Rhoa Activity

Rationale: The small monomeric GTPase RHOA acts as a master regulator of signal transduction cascades by activating effectors of cellular signaling, including the Rho-associated protein kinases ROCK1/2. Previous in vitro cell culture studies suggest that RHOA can regulate many critical aspects of vascular endothelial cell (EC) biology, including focal adhesion, stress fiber formation, and angiogenesis. However, the specific in vivo roles of RHOA during vascular development and homeostasis are still not well understood. Objective: In this study we examine the in vivo functions of RHOA in regulating vascular development and integrity in zebrafish. Methods and Results: We use zebrafish RHOA-ortholog (rhoaa) mutants, transgenic embryos expressing wild type, dominant-negative, or constitutively active forms of rhoaa in ECs, and a pharmacologic inhibitor of ROCK1/2 to study the in vivo consequences of RHOA gain- and loss-of-function in the vascular endothelium. Our findings document roles for RHOA in vascular integrity, developmental angiogenesis, and vascular morphogenesis. Conclusions: Our results indicate that either too much or too little RHOA activity leads to vascular dysfunction in vivo.

scholarly journals The Transcriptional Repressor ZEB Regulates p73 Expression at the Crossroad between Proliferation and Differentiation

2001 ◽  
Vol 21 (24) ◽  
pp. 8461-8470 ◽  
Author(s):  
Giulia Fontemaggi ◽  
Aymone Gurtner ◽  
Sabrina Strano ◽  
Yujiro Higashi ◽  
Ada Sacchi ◽  
...  
Keyword(s):  
Biological Activities ◽  
Ectopic Expression ◽  
Transcriptional Repressor ◽  
Dominant Negative ◽  
C2c12 Cells ◽  
Monocytic Differentiation ◽  
Hl60 Cells ◽  
Proliferation And Differentiation

ABSTRACT The newly discovered p73 gene encodes a nuclear protein that has high homology with p53. Furthermore, ectopic expression of p73 in p53+/+ and p53−/− cancer cells recapitulates some of the biological activities of p53 such as growth arrest, apoptosis, and differentiation. p73−/−-deficient mice exhibit severe defects in proper development of the central nervous system and pheromone sensory pathway. They also suffer from inflammation and infections. Here we studied the transcriptional regulation of p73 at the crossroad between proliferation and differentiation. p73 mRNA is undetectable in proliferating C2C12 cells and is expressed at very low levels in undifferentiated P19 and HL60 cells. Conversely, it is upregulated during muscle and neuronal differentiation as well as in response to tetradecanoyl phorbol acetate-induced monocytic differentiation of HL60 cells. We identified a 1-kb regulatory fragment located within the first intron of p73, which is positioned immediately upstream to the ATG codon of the second exon. This fragment exerts silencer activity on p73 as well as on heterologous promoters. The p73 intronic fragment contains six consensus binding sites for transcriptional repressor ZEB, which binds these sites in vitro and in vivo. Ectopic expression of dominant-negative ZEB (ZEB-DB) restores p73 expression in proliferating C2C12 and P19 cells. Thus, transcriptional repression of p73 expression by ZEB binding may contribute to the modulation of p73 expression during differentiation.

scholarly journals Development of an In Vitro Model to Screen CYP1B1-Targeted Anticancer Prodrugs

2016 ◽  
Vol 21 (10) ◽  
pp. 1090-1099
Author(s):  
Zhiying Wang ◽  
Yao Chen ◽  
Laura M. Drbohlav ◽  
Judy Qiju Wu ◽  
Michael Zhuo Wang
Keyword(s):  
Appreciable Amount ◽  
G1 Arrest ◽  
Screening Assay ◽  
Knock Out ◽  
Chemical Libraries ◽  
Cytochrome P450 1B1 ◽  
Endometrial Carcinoma Cell Line ◽  
Vitro Model ◽  
Apoptotic Effects

Cytochrome P450 1B1 (CYP1B1) is an anticancer therapeutic target due to its overexpression in a number of steroid hormone–related cancers. One anticancer drug discovery strategy is to develop prodrugs specifically activated by CYP1B1 in malignant tissues to cytotoxic metabolites. Here, we aimed to develop an in vitro screening model for CYP1B1-targeted anticancer prodrugs using the KLE human endometrial carcinoma cell line. KLE cells demonstrated superior stability of CYP1B1 expression relative to transiently transfected cells and did not express any appreciable amount of cognate CYP1A1 or CYP1A2, which would have compromised the specificity of the screening assay. The effect of two CYP1B1-targeted probe prodrugs on KLE cells was evaluated in the absence and presence of a CYP1B1 inhibitor to chemically “knock out” CYP1B1 activity (CYP1B1 inhibited). Both probe prodrugs were more toxic to KLE cells than to CYP1B1-inhibited KLE cells and significantly induced G0/G1 arrest and decreased the S phase in KLE cells. They also exhibited pro-apoptotic effects in KLE cells, which were attenuated in CYP1B1-inhibited KLE cells. In summary, a KLE cell–based model has been characterized to be suitable for identifying CYP1B1-targeted anticancer prodrugs and should be further developed and employed for screening chemical libraries.

scholarly journals Hes6 regulates myogenic differentiation

Development ◽  
2002 ◽  
Vol 129 (9) ◽  
pp. 2195-2207
Author(s):  
Judy Cossins ◽  
Ann E. Vernon ◽  
Yun Zhang ◽  
Anna Philpott ◽  
Philip H. Jones
Keyword(s):  
Protein Interactions ◽  
Kinase Inhibitor ◽  
Myogenic Differentiation ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Xenopus Embryos ◽  
Enhancer Of Split

Hes6 is a basic helix-loop-helix transcription factor homologous to Drosophila Enhancer of Split (EoS) proteins. It is known to promote neural differentiation and to bind to Hes1, a related protein that is part of the Notch signalling pathway, affecting Hes1-regulated transcription. We show that Hes6 is expressed in the murine embryonic myotome and is induced on C2C12 myoblast differentiation in vitro. Hes6 binds DNA containing the Enhancer of Split E box (ESE) motif, the preferred binding site of Drosophila EoS proteins, and represses transcription of an ESE box reporter. When overexpressed in C2C12 cells, Hes6 impairs normal differentiation, causing a decrease in the induction of the cyclin-dependent kinase inhibitor, p21Cip1, and an increase in the number of cells that can be recruited back into the cell cycle after differentiation in culture. In Xenopus embryos, Hes6 is co-expressed with MyoD in early myogenic development. Microinjection of Hes6 RNA in vivo in Xenopus embryos results in an expansion of the myotome, but suppression of terminal muscle differentiation and disruption of somite formation at the tailbud stage. Analysis of Hes6 mutants indicates that the DNA-binding activity of Hes6 is not essential for its myogenic phenotype, but that protein-protein interactions are. Thus, we demonstrate a novel role for Hes6 in multiple stages of muscle formation.

Abstract 17273: Long Non-Coding RNA Ppp1r1b Regulates Myogenic Differentiation Through Modulating Histone 3 Methylation

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Xuedong Kang ◽  
Yan Zhao ◽  
Marlin Touma
Keyword(s):  
Histone Modification ◽  
Protein Complex ◽  
Myogenic Differentiation ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
Mouse Heart ◽  
Histone 3

Introduction: Long noncoding RNAs (lncRNAs), emerged as critical epigenetic regulators of transcriptome, play important roles in cardiac development and might be targeted to treat human cardiomyocyte dysfunction. In our work, we identified a novel lncRNA that regulates myogenesis. Hypothesis: LncRNA Ppp1r1b regulates myogenesis by modulating Histone 3 methylation Methods: After treated with antisense oligonucleotides (GapmeR) or siRNA against Ppp1r1b-LncRNA, real time PCR and Western blot analyses were performed to examine the expression of myogenic and sarcomere genes. Chromatin immunoprecipitation (CHIP) was used to comparatively analyze gene specific histone modification level. RNA pull-down was employed to identify the protein molecules that interact with Ppp1r1b-LncRNA. Results: By silencing Ppp1r1b-LncRNA with GapmeR, C2C12, a skeletal myoblast cell line, did not develop fully differentiated myotubes, but tend to remain in a quiescent mono-nucleated status. In vivo analysis of GapmeR injected neonatal mouse heart and in vitro siRNA silenced human skeletal myoblasts further confirmed the important role of Ppp1r1b-LncRNA on myogenesis. Members of the MyoD family of muscle-specific transcription factors (MyoD and myogenin) failed to be up-regulated during myogenic differentiation when treated with Ppp1r1b-LncRNA specific GapmeR or siRNA. Key proteins essential for establishing and maintaining normal skeletal muscle architecture, including Tcap and Dystropnin, are also suppressed in Ppp1r1b LncRNA- deficient heart. Analysis of histone modification levels at Myogenin, MyoD1 and Tcap in C2C12 cells revealed more histone tri-methylation at these myogenic and sarcomere structural genes compared to untreated cells. Additional lncRNA- protein complex isolation has further revealed insight into the biological roles of Ppp1r1b-LncRNA. Conclusions: Our results support the role of Ppp1r1b-LncRNA in promoting myogenic differentiation. Ppp1r1b-lncRNA function is mediated by inhibiting histone methylation on promoters of multiple myogenic and sarcomere genes. In particular, the identification of EZH2 in pulled Pp1r1b LncRNA: protein complex implies that Polycomb repressive complex 2 (PRC2) is involved in Ppp1r1b-LncRNA modulated myoblast differentiation.

scholarly journals Myostatin Inhibits Myoblast Differentiation by Down-regulating MyoD Expression

2002 ◽  
Vol 277 (51) ◽  
pp. 49831-49840 ◽  
Author(s):  
Brett Langley ◽  
Mark Thomas ◽  
Amy Bishop ◽  
Mridula Sharma ◽  
Stewart Gilmour ◽  
...  
Keyword(s):  
Culture Media ◽  
Myogenic Differentiation ◽  
Critical Role ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Myoblast Differentiation ◽  
Proliferation And Differentiation ◽  
Smad 3 ◽  
Low Serum

Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3.In vitro, increasing concentrations of recombinant mature myostatin reversibly blocked the myogenic differentiation of myoblasts, cultured in low serum media. Western and Northern blot analysis indicated that addition of myostatin to the low serum culture media repressed the levels of MyoD, Myf5, myogenin, and p21 leading to the inhibition of myogenic differentiation. The transient transfection of C2C12myoblasts with MyoD expressing constructs did not rescue myostatin-inhibited myogenic differentiation. Myostatin signaling specifically induced Smad 3 phosphorylation and increased Smad 3·MyoD association, suggesting that Smad 3 may mediate the myostatin signal by interfering with MyoD activity and expression. Consistent with this, the expression of dominant-negativeSmad3rescued the activity of a MyoD promoter-reporter in C2C12myoblasts treated with myostatin. Taken together, these results suggest that myostatin inhibits MyoD activity and expression via Smad 3 resulting in the failure of the myoblasts to differentiate into myotubes. Thus we propose that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that lack functional myostatin is because of deregulated proliferation and differentiation of myoblasts.

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