The MyoD-Inducible p204 Protein Overcomes the Inhibition of Myoblast Differentiation by Id Proteins

ABSTRACT The murine p204 protein level is highest in heart and skeletal muscle. During the fusion of cultured myoblasts to myotubes, the p204 level increases due to transcription dependent on the muscle-specific MyoD protein, and p204 is phosphorylated and translocated from the nucleus to the cytoplasm. p204 overexpression accelerates myoblast fusion in differentiation medium and triggers this process even in growth medium. Here we report that p204 is required for the differentiation of C2C12 myoblasts. We propose that it enables the differentiation, at least in part, by overcoming the inhibition of the activities of the MyoD and E47 proteins by the Id proteins: Id1, Id2, and Id3. These are known to inhibit skeletal muscle differentiation by binding and blocking the activity of MyoD, E12/E47, and other myogenic basic helix-loop-helix (bHLH) proteins. Our hypothesis is based on the following findings. (i) A decrease in the p204 level in C2C12 myoblasts by antisense RNA (a) increased the level of the Id2; (b) inhibited the MyoD-, E12/E47-, and other bHLH protein-dependent accumulation of the muscle-specific myosin heavy-chain protein; and (c) inhibited the fusion of myoblasts to myotubes in differentiation medium. (ii) p204 bound to the Id proteins in vitro and in vivo. (iii) In the binding of p204 to Id2, the b segment of p204 and the HLH segment of Id2 were involved. (iv) Addition of p204 overcame the inhibition by the Id proteins of the binding of MyoD and E47 to DNA in vitro. (v) Overexpression of p204 in myoblasts (a) decreased the level of the Id proteins, even in a culture in growth medium, and (b) overcame the inhibition by the Id proteins of MyoD- and E47 dependent transcription and also overcame the inhibition by Id2 of the fusion of myoblasts to myotubes.

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Tristetraprolin and LPS-inducible CXC chemokine are rapidly induced in presumptive satellite cells in response to skeletal muscle injury

Journal of Cell Science ◽

10.1242/jcs.115.13.2701 ◽

2002 ◽

Vol 115 (13) ◽

pp. 2701-2712 ◽

Cited By ~ 4

Author(s):

Chetana Sachidanandan ◽

Ramkumar Sambasivan ◽

Jyotsna Dhawan

Keyword(s):

Skeletal Muscle ◽

Satellite Cells ◽

Cell Biology ◽

Muscle Injury ◽

Rna Binding ◽

C2c12 Myoblasts ◽

Adult Skeletal Muscle ◽

Cxc Chemokine

Myogenic precursor cells known as satellite cells persist in adult skeletal muscle and are responsible for its ability to regenerate after injury. Quiescent satellite cells are activated by signals emanating from damaged muscle. Here we describe the rapid activation of two genes in response to muscle injury; these transcripts encode LPS-inducible CXC chemokine (LIX), a neutrophil chemoattractant, and Tristetraprolin (TTP), an RNA-binding protein implicated in the regulation of cytokine expression. Using a synchronized cell culture model we show that C2C12 myoblasts arrested in G0 exhibit some molecular attributes of satellite cells in vivo: suppression of MyoD and Myf5 expression during G0 and their reactivation in G1. Synchronization also revealed cell cycle dependent expression of CD34, M-cadherin, HGF and PEA3, genes implicated in satellite cell biology. To identify other genes induced in synchronized C2C12 myoblasts we used differential display PCR and isolated LIX and TTP cDNAs. Both LIX and TTP mRNAs are short-lived, encode molecules implicated in inflammation and are transiently induced during growth activation in vitro. Further, LIX and TTP are rapidly induced in response to muscle damage in vivo. TTP expression precedes that of MyoD and is detected 30 minutes after injury. The spatial distribution of LIX and TTP transcripts in injured muscle suggests expression by satellite cells. Our studies suggest that in addition to generating new cells for repair, activated satellite cells may be a source of signaling molecules involved in tissue remodeling during regeneration.

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Creating Interactions between Tissue-Engineered Skeletal Muscle and the Peripheral Nervous System

Cells Tissues Organs ◽

10.1159/000443634 ◽

2016 ◽

Vol 202 (3-4) ◽

pp. 143-158 ◽

Cited By ~ 21

Author(s):

Alec S.T. Smith ◽

Samantha L. Passey ◽

Neil R.W. Martin ◽

Darren J. Player ◽

Vivek Mudera ◽

...

Keyword(s):

Skeletal Muscle ◽

Motor Neurons ◽

Muscle Development ◽

Functional Performance ◽

Cell Types ◽

Original Data ◽

Synaptic Proteins ◽

Myoblast Differentiation

Effective models of mammalian tissues must allow and encourage physiologically (mimetic) correct interactions between co-cultured cell types in order to produce culture microenvironments as similar as possible to those that would normally occur in vivo. In the case of skeletal muscle, the development of such a culture model, integrating multiple relevant cell types within a biomimetic scaffold, would be of significant benefit for investigations into the development, functional performance, and pathophysiology of skeletal muscle tissue. Although some work has been published regarding the behaviour of in vitro muscle models co-cultured with organotypic slices of CNS tissue or with stem cell-derived neurospheres, little investigation has so far been made regarding the potential to maintain isolated motor neurons within a 3D biomimetic skeletal muscle culture platform. Here, we review the current state of the art for engineering neuromuscular contacts in vitro and provide original data detailing the development of a 3D collagen-based model for the co-culture of primary muscle cells and motor neurons. The devised culture system promotes increased myoblast differentiation, forming arrays of parallel, aligned myotubes on which areas of nerve-muscle contact can be detected by immunostaining for pre- and post-synaptic proteins. Quantitative RT-PCR results indicate that motor neuron presence has a positive effect on myotube maturation, suggesting neural incorporation influences muscle development and maturation in vitro. The importance of this work is discussed in relation to other published neuromuscular co-culture platforms along with possible future directions for the field.

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Divergent Regulation of Myotube Formation and Gene Expression by E2 and EPA during In-Vitro Differentiation of C2C12 Myoblasts

International Journal of Molecular Sciences ◽

10.3390/ijms21030745 ◽

2020 ◽

Vol 21 (3) ◽

pp. 745

Author(s):

Orly Lacham-Kaplan ◽

Donny M. Camera ◽

John A. Hawley

Keyword(s):

Synergistic Effect ◽

Striated Muscle ◽

Morphological Changes ◽

Estrogen Receptor Α ◽

Mitogen Activated Protein Kinase ◽

Myoblast Differentiation ◽

C2c12 Myoblasts ◽

Myotube Formation

Estrogen (E2) and polyunsaturated fatty acids (n-3PUFA) supplements independently support general wellbeing and enhance muscle regeneration in-vivo and myotube formation in-vitro. However, the combined effect of E2 and n-3PUFA on myoblast differentiation is not known. The purpose of the study was to identify whether E2 and n-3PUFA possess a synergistic effect on in-vitro myogenesis. Mouse C2C12 myoblasts, a reliable model to reiterate myogenic events in-vitro, were treated with 10nM E2 and 50μM eicosapentaenoic acid (EPA) independently or combined, for 0–24 h or 0–120 h during differentiation. Immunofluorescence, targeted qPCR and next generation sequencing (NGS) were used to characterize morphological changes and differential expression of key genes involved in the regulation of myogenesis and muscle function pathways. E2 increased estrogen receptor α (Erα) and the expression of the mitogen-activated protein kinase 11 (Mapk11) within 1 h of treatment and improved myoblast differentiation and myotube formation. A significant reduction (p < 0.001) in myotube formation and in the expression of myogenic regulatory factors Mrfs (MyoD, Myog and Myh1) and the myoblast fusion related gene, Tmem8c, was observed in the presence of EPA and the combined E2/EPA treatment. Additionally, EPA treatment at 48 h of differentiation inhibited the majority of genes associated with the myogenic and striated muscle contraction pathways. In conclusion, EPA and E2 had no synergistic effect on myotube formation in-vitro. Independently, EPA inhibited myoblast differentiation and overrides the stimulatory effect of E2 when used in combination with E2.

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Id Helix-Loop-Helix Proteins Antagonize Pax Transcription Factor Activity by Inhibiting DNA Binding

Molecular and Cellular Biology ◽

10.1128/mcb.21.2.524-533.2001 ◽

2001 ◽

Vol 21 (2) ◽

pp. 524-533 ◽

Cited By ~ 92

Author(s):

E. Claire Roberts ◽

Richard W. Deed ◽

Toshiaki Inoue ◽

John D. Norton ◽

Andrew D. Sharrocks

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Cellular Proliferation ◽

Regulatory Function ◽

Transcription Factor Family ◽

Helix Loop Helix ◽

Id Proteins ◽

Proliferation And Differentiation

ABSTRACT The Id subfamily of helix-loop-helix (HLH) proteins plays a fundamental role in the regulation of cellular proliferation and differentiation. The major mechanism by which Id proteins are thought to inhibit differentiation is through interaction with other HLH proteins and inhibition of their DNA-binding activity. However, Id proteins have also been shown to interact with other proteins involved in regulating cellular proliferation and differentiation, suggesting a more widespread regulatory function. In this study we demonstrate functional interactions between Id proteins and members of the Pax-2/-5/-8 subfamily of paired-domain transcription factors. Members of the Pax transcription factor family have key functions in regulating several developmental processes exemplified by B lymphopoiesis, in which Pax-5 plays an essential role. Id proteins bind to Pax proteins in vitro and in vivo. Binding occurs through the paired DNA-binding domain of the Pax proteins and results in the disruption of DNA-bound complexes containing Pax-2, Pax-5, and Pax-8. In vivo, Id proteins modulate the transcriptional activity mediated by Pax-5 complexes on the B-cell-specific mb-1 promoter. Our results therefore demonstrate a novel facet of Id function in regulating cellular differentiation by functionally antagonizing the action of members of the Pax transcription factor family.

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Basic Helix-Loop-Helix Transcription Factor Heterocomplex of Yas1p and Yas2p Regulates Cytochrome P450 Expression in Response to Alkanes in the Yeast Yarrowia lipolytica

Eukaryotic Cell ◽

10.1128/ec.00412-06 ◽

2007 ◽

Vol 6 (4) ◽

pp. 734-743 ◽

Cited By ~ 39

Author(s):

Setsu Endoh-Yamagami ◽

Kiyoshi Hirakawa ◽

Daisuke Morioka ◽

Ryouichi Fukuda ◽

Akinori Ohta

Keyword(s):

Cytochrome P450 ◽

Transcription Factors ◽

Yarrowia Lipolytica ◽

Bhlh Protein ◽

Genome Database ◽

Basic Helix Loop Helix ◽

Helix Loop Helix ◽

Bhlh Transcription Factors

ABSTRACT The expression of the ALK1 gene, which encodes cytochrome P450, catalyzing the first step of alkane oxidation in the alkane-assimilating yeast Yarrowia lipolytica, is highly regulated and can be induced by alkanes. Previously, we identified a cis-acting element (alkane-responsive element 1 [ARE1]) in the ALK1 promoter. We showed that a basic helix-loop-helix (bHLH) protein, Yas1p, binds to ARE1 in vivo and mediates alkane-dependent transcription induction. Yas1p, however, does not bind to ARE1 by itself in vitro, suggesting that Yas1p requires another bHLH protein partner for its DNA binding, as many bHLH transcription factors function by forming heterodimers. To identify such a binding partner of Yas1p, here we screened open reading frames encoding proteins with the bHLH motif from the Y. lipolytica genome database and identified the YAS2 gene. The deletion of the YAS2 gene abolished the alkane-responsive induction of ALK1 transcription and the growth of the yeast on alkanes. We revealed that Yas2p has transactivation activity. Furthermore, Yas1p and Yas2p formed a protein complex that was required for the binding of these proteins to ARE1. These findings allow us to postulate a model in which bHLH transcription factors Yas1p and Yas2p form a heterocomplex and mediate the transcription induction in response to alkanes.

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ROCK2 and Its Alternatively Spliced Isoform ROCK2m Positively Control the Maturation of the Myogenic Program

Molecular and Cellular Biology ◽

10.1128/mcb.01735-06 ◽

2007 ◽

Vol 27 (17) ◽

pp. 6163-6176 ◽

Cited By ~ 31

Author(s):

Michele Pelosi ◽

Francesco Marampon ◽

Bianca M. Zani ◽

Sabrina Prudente ◽

Emerald Perlas ◽

...

Keyword(s):

Skeletal Muscle ◽

Intracellular Signaling ◽

Elongation Factor ◽

Size Control ◽

Mitogen Activated Protein Kinase ◽

Myoblast Differentiation ◽

Myogenic Cells ◽

Ribosomal S6 Kinase

ABSTRACT Signal transduction cascades involving Rho-associated kinases (ROCK), the serine/threonine kinases downstream effectors of Rho, have been implicated in the regulation of diverse cellular functions including cytoskeletal organization, cell size control, modulation of gene expression, differentiation, and transformation. Here we show that ROCK2, the predominant ROCK isoform in skeletal muscle, is progressively up-regulated during mouse myoblast differentiation and is highly expressed in the dermomyotome and muscle precursor cells of mouse embryos. We identify a novel and evolutionarily conserved ROCK2 splicing variant, ROCK2m, that is preferentially expressed in skeletal muscle and strongly up-regulated during in vivo and in vitro differentiation processes. The specific knockdown of ROCK2 or ROCK2m expression in C2C12 myogenic cells caused a significant and selective impairment of the expression of desmin and of the myogenic regulatory factors Mrf4 and MyoD. We demonstrate that in myogenic cells, ROCK2 and ROCK2m are positive regulators of the p42 and p44 mitogen-activated protein kinase-p90 ribosomal S6 kinase-eucaryotic elongation factor 2 intracellular signaling pathways and, thereby, positively regulate the hypertrophic effect elicited by insulin-like growth factor 1 and insulin, linking the multifactorial functions of ROCK to an important control of the myogenic maturation.

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Mammalian target of rapamycin regulates miRNA-1 and follistatin in skeletal myogenesis

The Journal of Cell Biology ◽

10.1083/jcb.200912093 ◽

2010 ◽

Vol 189 (7) ◽

pp. 1157-1169 ◽

Cited By ~ 116

Author(s):

Yuting Sun ◽

Yejing Ge ◽

Jenny Drnevich ◽

Yong Zhao ◽

Mark Band ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Development ◽

Mammalian Target Of Rapamycin ◽

Micro Rna ◽

Skeletal Muscle Regeneration ◽

Target Of Rapamycin ◽

Myoblast Differentiation ◽

Mirna Biogenesis

Mammalian target of rapamycin (mTOR) has emerged as a key regulator of skeletal muscle development by governing distinct stages of myogenesis, but the molecular pathways downstream of mTOR are not fully understood. In this study, we report that expression of the muscle-specific micro-RNA (miRNA) miR-1 is regulated by mTOR both in differentiating myoblasts and in mouse regenerating skeletal muscle. We have found that mTOR controls MyoD-dependent transcription of miR-1 through its upstream enhancer, most likely by regulating MyoD protein stability. Moreover, a functional pathway downstream of mTOR and miR-1 is delineated, in which miR-1 suppression of histone deacetylase 4 (HDAC4) results in production of follistatin and subsequent myocyte fusion. Collective evidence strongly suggests that follistatin is the long-sought mTOR-regulated fusion factor. In summary, our findings unravel for the first time a link between mTOR and miRNA biogenesis and identify an mTOR–miR-1–HDAC4–follistatin pathway that regulates myocyte fusion during myoblast differentiation in vitro and skeletal muscle regeneration in vivo.

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Prion Infection of Muscle Cells In Vitro

Journal of Virology ◽

10.1128/jvi.02628-06 ◽

2007 ◽

Vol 81 (9) ◽

pp. 4615-4624 ◽

Cited By ~ 21

Author(s):

Wendy M. Dlakic ◽

Eric Grigg ◽

Richard A. Bessen

Keyword(s):

Skeletal Muscle ◽

Cell Line ◽

Muscle Cells ◽

C2c12 Cells ◽

C2c12 Myotubes ◽

C2c12 Myoblasts ◽

Peripheral Cell ◽

Prion Infection

ABSTRACT The prion agent has been detected in skeletal muscle of humans and animals with prion diseases. Here we report scrapie infection of murine C2C12 myoblasts and myotubes in vitro following coculture with a scrapie-infected murine neuroblastoma (N2A) cell line but not following incubation with a scrapie-infected nonneuronal cell line or a scrapie brain homogenate. Terminal differentiation of scrapie-infected C2C12 myoblasts into myotubes resulted in an increase in the expression of the disease-specific prion protein, PrPSc. The amount of scrapie infectivity or PrPSc in C2C12 myotubes was comparable to the levels found in scrapie-infected N2A cells, indicating that a high level of infection was established in muscle cells. Subclones of scrapie-infected C2C12 cells produced high levels of PrPSc in myotubes, and the C-terminal C2 polypeptide fragment of PrPSc was found based on deglycosylation and PrPSc-specific immunoprecipitation of cell lysates. This is the first report of a stable prion infection in muscle cells in vitro and of a long-term prion infection in a nondividing, differentiated peripheral cell type in culture. These in vitro studies also suggest that in vivo prion infection of skeletal muscle requires contact with prion-infected neurons or, possibly, nerve terminals.

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NLK is required for Ras/ERK/SRF/ELK signaling to tune skeletal muscle development by phosphorylating SRF and antagonizing the SRF/MKL pathway

Cell Death Discovery ◽

10.1038/s41420-021-00774-9 ◽

2022 ◽

Vol 8 (1) ◽

Author(s):

Shang-Ze Li ◽

Ze-Yan Zhang ◽

Jie Chen ◽

Ming-You Dong ◽

Xue-Hua Du ◽

...

Keyword(s):

Skeletal Muscle ◽

Molecular Mechanisms ◽

Muscle Development ◽

Serum Response Factor ◽

Conditional Knockout ◽

Myoblast Differentiation ◽

Knockout Mouse Model ◽

Hypertrophic Growth

AbstractSerum response factor (SRF) regulates differentiation and proliferation by binding to RhoA-actin-activated MKL or Ras-MAPK-activated ELK transcriptional coactivators, but the molecular mechanisms responsible for SRF regulation remain unclear. Here, we show that Nemo-like kinase (NLK) is required for the promotion of SRF/ELK signaling in human and mouse cells. NLK was found to interact with and phosphorylate SRF at serine residues 101/103, which in turn enhanced the association between SRF and ELK. The enhanced affinity of SRF/ELK antagonized the SRF/MKL pathway and inhibited mouse myoblast differentiation in vitro. In a skeletal muscle-specific Nlk conditional knockout mouse model, forming muscle myofibers underwent hypertrophic growth, resulting in an increased muscle and body mass phenotype. We propose that both phosphorylation of SRF by NLK and phosphorylation of ELKs by MAPK are required for RAS/ELK signaling, confirming the importance of this ancient pathway and identifying an important role for NLK in modulating muscle development in vivo.

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A defined N6-methyladenosine (m6A) profile conferred by METTL3 regulates muscle stem cell/myoblast state transitions

Cell Death Discovery ◽

10.1038/s41420-020-00328-5 ◽

2020 ◽

Vol 6 (1) ◽

Cited By ~ 1

Author(s):

Brandon J. Gheller ◽

Jamie E. Blum ◽

Ern Hwei Hannah Fong ◽

Olga V. Malysheva ◽

Benjamin D. Cosgrove ◽

...

Keyword(s):

Skeletal Muscle ◽

Stem Cell ◽

Muscle Regeneration ◽

Transcriptional Profile ◽

Skeletal Muscle Regeneration ◽

State Transitions ◽

C2c12 Myoblasts ◽

Primary Mouse

Abstract Muscle-specific adult stem cells (MuSCs) are required for skeletal muscle regeneration. To ensure efficient skeletal muscle regeneration after injury, MuSCs must undergo state transitions as they are activated from quiescence, give rise to a population of proliferating myoblasts, and continue either to terminal differentiation, to repair or replace damaged myofibers, or self-renewal to repopulate the quiescent population. Changes in MuSC/myoblast state are accompanied by dramatic shifts in their transcriptional profile. Previous reports in other adult stem cell systems have identified alterations in the most abundant internal mRNA modification, N6-methyladenosine (m6A), conferred by its active writer, METTL3, to regulate cell state transitions through alterations in the transcriptional profile of these cells. Our objective was to determine if m6A-modification deposition via METTL3 is a regulator of MuSC/myoblast state transitions in vitro and in vivo. Using liquid chromatography/mass spectrometry we identified that global m6A levels increase during the early stages of skeletal muscle regeneration, in vivo, and decline when C2C12 myoblasts transition from proliferation to differentiation, in vitro. Using m6A-specific RNA-sequencing (MeRIP-seq), a distinct profile of m6A-modification was identified, distinguishing proliferating from differentiating C2C12 myoblasts. RNAi studies show that reducing levels of METTL3, the active m6A methyltransferase, reduced global m6A levels and forced C2C12 myoblasts to prematurely differentiate. Reducing levels of METTL3 in primary mouse MuSCs prior to transplantation enhanced their engraftment capacity upon primary transplantation, however their capacity for serial transplantation was lost. In conclusion, METTL3 regulates m6A levels in MuSCs/myoblasts and controls the transition of MuSCs/myoblasts to different cell states. Furthermore, the first transcriptome wide map of m6A-modifications in proliferating and differentiating C2C12 myoblasts is provided and reveals a number of genes that may regulate MuSC/myoblast state transitions which had not been previously identified.

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