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.

Gene Expression of C2C12 Cells on Calcium Phosphate Ceramic In Vitro

2005 ◽  
Vol 288-289 ◽  
pp. 265-268 ◽  
Author(s):  
Yan Fei Tan ◽  
Ling Li Zhang ◽  
Xin Lai He ◽  
Wei Qiang Xiao ◽  
Hong Song Fan ◽  
...  
Keyword(s):  
Calcium Phosphate ◽  
Type I Collagen ◽  
C2c12 Cell ◽  
C2c12 Cells ◽  
Type I ◽  
Calcium Phosphate Ceramic ◽  
Mtt Method ◽  
Difference Expression

The osteoinduction of Calcium Phosphate (CaP) had been proved and generally been investigated by in vivo implantation. However, the mechanism of the osteoinductivity was not clear and it was difficult to judge the osteoinductivity in vitro. In this study, Mouse C2C12 cell line, a kind of myoblast precursor cell, was employed to co-culture with CaP. The induction of cell differentiation by materials was tested by MTT method, fluorescence observation, especially the mRNA expression of Osteocalcin, Type I collagen and Fibronectin by RT-PCR. It was founded that C2C12 cells could be induced to expression osteocalcin when growth on the surface of the HA/TCP ceramics. At the same time, the ceramics with different composition and sintering temperature seemed to induce difference expression level of the related genes. The results proved that phase composition was one of the most important factors in the regulation of bone-related genes. This study provided a potential model to evaluate the osteoinductivity of CaP ceramics in vitro.

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.

Phosphorylation of ERK and dystrophin S3059 protect against inflammation-associated C2C12 myotube atrophy

2021 ◽  
Author(s):  
Kristy Swiderski ◽  
Christopher J. Brock ◽  
Jennifer Trieu ◽  
Annabel Chee ◽  
Savant S. Thakur ◽  
...  
Keyword(s):  
Muscle Fiber ◽  
Protein Function ◽  
Muscle Wasting ◽  
Therapeutic Potential ◽  
Membrane Integrity ◽  
C2c12 Cells ◽  
Erk Activation ◽  
Over Expression ◽  
Fiber Size

The dystrophin-glycoprotein complex (DGC) is a multi-protein structure required to maintain muscle fiber membrane integrity, transmit force by linking the actin cytoskeleton with the extracellular matrix, and maintain muscle homeostasis. Membrane localization of dystrophin is perturbed in muscles wasting as a consequence of cancer cachexia, tenotomy and advanced ageing, which are all associated with low level, chronic inflammation. Strategies to preserve dystrophin expression at the sarcolemma might therefore combat muscle wasting. Phosphorylation of dystrophin serine 3059 (S3059) enhances the interaction between dystrophin and β-dystroglycan. To test the contribution of amino acid phosphorylation to muscle fiber size changes, dystrophin constructs with phospho-null and phosphomimetic mutations were transfected into C2C12 muscle cells or AAV-293 cells in the presence or absence of kinase inhibitors/activators to assess effects on myotube diameter and protein function. Over-expression of a dystrophin construct with a phospho-null mutation at S3059 in vitro reduced myotube size in healthy C2C12 cells. Conversely over-expression of a phosphomimetic mutation at S3059 attenuated inflammation-induced myotube atrophy. Increased ERK activation by addition of phorbol myristate acetate (PMA) also reduced inflammation-associated myotube atrophy, and increased the interaction between dystrophin and β-dystroglycan, which was partially attenuated in the presence of a phosphomimetic mutation at dystrophin S3059. These findings demonstrate a link between increased ERK activation, dystrophin S3059 phosphorylation, stabilization of the DGC, and the regulation of muscle fiber size. Interventions that increase dystrophin S3059 phosphorylation to promote stronger binding of dystrophin to β-dystroglycan may have therapeutic potential for attenuation of inflammation-associated muscle wasting.

Metalloprotease-disintegrin ADAM 12 interacts with α-actinin-1

2001 ◽  
Vol 357 (2) ◽  
pp. 353-361 ◽  
Author(s):  
Yi CAO ◽  
Qing KANG ◽  
Anna ZOLKIEWSKA
Keyword(s):  
Cell Adhesion ◽  
Fluorescent Protein ◽  
Critical Role ◽  
Cytoplasmic Domain ◽  
C2c12 Cells ◽  
Actin Binding ◽  
Myoblast Differentiation ◽  
Adam 12

ADAM 12, a member of the ADAM family of proteins (containing ADisintegrin And Metalloprotease domain), has been implicated in differentiation and fusion of myoblasts. While the extracellular domain of ADAM 12 contains an active metalloprotease and a region involved in cell adhesion, the function of the cytoplasmic tail of ADAM 12 has been less clear. Here we show that the cytoplasmic domain of ADAM 12 interacts in vitro and in vivo with α-actinin-1, an actin-binding and cross-linking protein. Green fluorescent protein fused to ADAM 12 cytoplasmic domain co-localizes with α-actinin-1-containing actin stress fibres in C2C12 cells. The interaction between ADAM 12 and α-actinin-1 is direct and involves the 58-amino acid C-terminal fragment of ADAM 12 and the 27kDa N-terminal domain of α-actinin-1. Consistently, expression of the 27kDa fragment of α-actinin-1 in C2C12 cells using a mitochondrial targeting system results in recruitment of the co-expressed ADAM 12 cytoplasmic domain to the mitochondrial surface. Moreover, α-actinin-1 co-purifies with a transmembrane, His6-tagged form of ADAM 12 expressed in C2C12 myoblasts, indicating that the transmembrane ADAM 12 forms a complex with α-actinin-1 in vivo. These results indicate that the actin cytoskeleton may play a critical role in ADAM 12-mediated cell–cell adhesion or cell signalling during myoblast differentiation and fusion.

Peculiarities of the course of type I allergic reactions in patients with blood diseases

2020 ◽  
pp. 40-50
Author(s):  
A. Nikitina
Keyword(s):  
Search Engines ◽  
Anaphylactic Shock ◽  
Type I ◽  
Allergic Reactions ◽  
Common Cause ◽  
Blood Diseases ◽  
Treatment Regimens ◽  
Modern Methods

Analysis of literature data presented in search engines — Elibrary, PubMed, Cochrane — concerning the risk of developing type I allergic reactions in patients with blood diseases is presented. It is shown that the most common cause of type I allergic reactions is drugs included in the treatment regimens of this category of patients. The article presents statistics on the increase in the number of drug allergies leading to cases of anaphylactic shock in patients with blood diseases. Modern methods for the diagnosis of type I allergic reactions in vivo and in vitro are considered.

scholarly journals Mechanism of CK2.3, a Novel Mimetic Peptide of Bone Morphogenetic Protein Receptor Type IA, Mediated Osteogenesis

2019 ◽  
Vol 20 (10) ◽  
pp. 2500 ◽  
Author(s):  
Vrathasha Vrathasha ◽  
Hilary Weidner ◽  
Anja Nohe
Keyword(s):  
Signaling Pathways ◽  
Treatment Options ◽  
Time Frame ◽  
Bmp Signaling ◽  
C2c12 Cells ◽  
Molecular Sequence ◽  
Sequence Of Events ◽  
Protein Receptor

Background: Osteoporosis is a degenerative skeletal disease with a limited number of treatment options. CK2.3, a novel peptide, may be a potential therapeutic. It induces osteogenesis and bone formation in vitro and in vivo by acting downstream of BMPRIA through releasing CK2 from the receptor. However, the detailed signaling pathways, the time frame of signaling, and genes activated remain largely unknown. Methods: Using a newly developed fluorescent CK2.3 analog, specific inhibitors for the BMP signaling pathways, Western blot, and RT-qPCR, we determined the mechanism of CK2.3 in C2C12 cells. We then confirmed the results in primary BMSCs. Results: Using these methods, we showed that CK2.3 stimulation activated OSX, ALP, and OCN. CK2.3 stimulation induced time dependent release of CK2β from BMPRIA and concurrently CK2.3 colocalized with CK2α. Furthermore, CK2.3 induced BMP signaling depends on ERK1/2 and Smad1/5/8 signaling pathways. Conclusion: CK2.3 is a novel peptide that drives osteogenesis, and we detailed the molecular sequence of events that are triggered from the stimulation of CK2.3 until the induction of mineralization. This knowledge can be applied in the development of future therapeutics for osteoporosis.

scholarly journals Collagen-Based Electrospun Materials for Tissue Engineering: A Systematic Review

2021 ◽  
Vol 8 (3) ◽  
pp. 39
Author(s):  
Britani N. Blackstone ◽  
Summer C. Gallentine ◽  
Heather M. Powell
Keyword(s):  
Systematic Review ◽  
Tissue Engineering ◽  
Collagen Type I ◽  
The Body ◽  
Pool Data ◽  
Type I ◽  
High Concentrations ◽  
Increased Strength

Collagen is a key component of the extracellular matrix (ECM) in organs and tissues throughout the body and is used for many tissue engineering applications. Electrospinning of collagen can produce scaffolds in a wide variety of shapes, fiber diameters and porosities to match that of the native ECM. This systematic review aims to pool data from available manuscripts on electrospun collagen and tissue engineering to provide insight into the connection between source material, solvent, crosslinking method and functional outcomes. D-banding was most often observed in electrospun collagen formed using collagen type I isolated from calfskin, often isolated within the laboratory, with short solution solubilization times. All physical and chemical methods of crosslinking utilized imparted resistance to degradation and increased strength. Cytotoxicity was observed at high concentrations of crosslinking agents and when abbreviated rinsing protocols were utilized. Collagen and collagen-based scaffolds were capable of forming engineered tissues in vitro and in vivo with high similarity to the native structures.

scholarly journals E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Peng Gao ◽  
Xianwei Ma ◽  
Ming Yuan ◽  
Yulan Yi ◽  
Guoke Liu ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Type I Interferon ◽  
Zinc Fingers ◽  
E3 Ligase ◽  
Type I ◽  
E3 Ligases ◽  
Protein Posttranslational Modifications ◽  
Antiviral Innate Immunity

AbstractUbiquitination is one of the most prevalent protein posttranslational modifications. Here, we show that E3 ligase Nedd4l positively regulates antiviral immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3. Deficiency of Nedd4l significantly impairs type I interferon and proinflammatory cytokine production induced by virus infection both in vitro and in vivo. Nedd4l deficiency inhibits virus-induced ubiquitination of TRAF3, the binding between TRAF3 and TBK1, and subsequent phosphorylation of TBK1 and IRF3. Nedd4l directly interacts with TRAF3 and catalyzes K29-linked ubiquitination of Cys56 and Cys124, two cysteines that constitute zinc fingers, resulting in enhanced association between TRAF3 and E3 ligases, cIAP1/2 and HECTD3, and also increased K48/K63-linked ubiquitination of TRAF3. Mutation of Cys56 and Cys124 diminishes Nedd4l-catalyzed K29-linked ubiquitination, but enhances association between TRAF3 and the E3 ligases, supporting Nedd4l promotes type I interferon production in response to virus by catalyzing ubiquitination of the cysteines in TRAF3.

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