Faculty Opinions recommendation of Rb is required for progression through myogenic differentiation but not maintenance of terminal differentiation.

When a proliferating myoblast culture is induced to differentiate by deprivation of serum in the medium, a significant proportion of cells escape from terminal differentiation, while the rest of the cells differentiate. Using C2C12 mouse myoblast cells, this heterogeneity observed upon differentiation was investigated with an emphasis on the myogenic regulatory factors. The differentiating part of the cell population followed a series of well-described events, including expression of myogenin, p21(WAF1), and contractile proteins, permanent withdrawal from the cell cycle and cell fusion, whereas the rest of the cells did not initiate any of these events. Interestingly, the latter cells showed an undetectable or greatly reduced level of MyoD and Myf-5 expression, which had been originally expressed in the undifferentiated proliferating myoblasts. When these undifferentiated cells were isolated and returned to the growth conditions, they progressed through the cell cycle and regained MyoD expression. These cells demonstrated identical features with the original culture on the deprivation of serum. They produced both MyoD-positive differentiating and MyoD-negative undifferentiated populations once again. Thus the undifferentiated cells in the serum-deprived culture were designated ‘reserve cells’. Upon serum deprivation, MyoD expression rapidly decreased as a result of down-regulation in approximately 50% of the cells. After this heterogenization, MyoD positive cells expressed myogenin, which is the earliest known event of terminal differentiation and marks irreversible commitment to this, while MyoD-negative cells did not differentiate and became the reserve cells. We also demonstrated that ectopic expression of MyoD converted the reserve cells to differentiating cells, indicating that down-regulation of MyoD is a causal event in the formation of reserve cells.

Download Full-text

Rb is required for progression through myogenic differentiation but not maintenance of terminal differentiation

The Journal of Cell Biology ◽

10.1083/jcb.200403004 ◽

2004 ◽

Vol 166 (6) ◽

pp. 865-876 ◽

Cited By ~ 103

Author(s):

Michael S. Huh ◽

Maura H. Parker ◽

Anthony Scimè ◽

Robin Parks ◽

Michael A. Rudnicki

Keyword(s):

Myogenic Differentiation ◽

Terminal Differentiation ◽

S Phase ◽

Normal Muscle ◽

Serum Stimulation ◽

Primary Myoblasts ◽

Ability To Regenerate ◽

Induction Of Differentiation ◽

Multinucleated Myotubes ◽

Low Serum

To investigate the requirement for pRb in myogenic differentiation, a floxed Rb allele was deleted either in proliferating myoblasts or after differentiation. Myf5-Cre mice, lacking pRb in myoblasts, died immediately at birth and exhibited high numbers of apoptotic nuclei and an almost complete absence of myofibers. In contrast, MCK-Cre mice, lacking pRb in differentiated fibers, were viable and exhibited a normal muscle phenotype and ability to regenerate. Induction of differentiation of Rb-deficient primary myoblasts resulted in high rates of apoptosis and a total inability to form multinucleated myotubes. Upon induction of differentiation, Rb-deficient myoblasts up-regulated myogenin, an immediate early marker of differentiation, but failed to down-regulate Pax7 and exhibited growth in low serum conditions. Primary myoblasts in which Rb was deleted after expression of differentiated MCK-Cre formed normal multinucleated myotubes that did not enter S-phase in response to serum stimulation. Therefore, Rb plays a crucial role in the switch from proliferation to differentiation rather than maintenance of the terminally differentiated state.

Download Full-text

Simultaneous overexpression of Oct4 and Nanog abrogates terminal myogenesis

AJP Cell Physiology ◽

10.1152/ajpcell.00468.2008 ◽

2009 ◽

Vol 297 (1) ◽

pp. C43-C54 ◽

Cited By ~ 30

Author(s):

Kuan Chih Lang ◽

I. Hsuan Lin ◽

Han Fang Teng ◽

Yi Cheng Huang ◽

Chung Leung Li ◽

...

Keyword(s):

Myogenic Differentiation ◽

Severe Combined Immunodeficiency ◽

Embryonic Stem ◽

Terminal Differentiation ◽

C2c12 Cells ◽

Es Cell ◽

Nonobese Diabetic ◽

Nanog Expression ◽

Myogenic Markers ◽

Lineage Specific Genes

Oct4 and Nanog are two embryonic stem (ES) cell-specific transcription factors that play critical roles in the maintenance of ES cell pluripotency. In this study, we investigated the effects of Oct4 and Nanog expression on the differentiation state of myogenic cells, which is sustained by a strong positive feedback loop. Oct4 and Nanog, either independently or simultaneously, were overexpressed in C2C12 myoblasts, and the expression of myogenic lineage-specific genes and terminal differentiation was observed by RT-PCR. Overexpression of Oct4 in C2C12 cultures repressed, while exogenous Nanog did not significantly alter C2C12 terminal differentiation. The expression of Pax7 was reduced in all Oct4-overexpressing myoblasts, and we identified a major Oct4-binding site in the Pax7 promoter. Simultaneous expression of Oct4 and Nanog in myoblasts inhibited the formation of myotubes, concomitant with a reduction in the endogenous levels of hallmark myogenic markers. Furthermore, overexpression of Oct4 and Nanog induced the expression of their endogenous counterparts along with the expression of Sox2. Using mammalian two-hybrid assays, we confirmed that Oct4 functions as a transcriptional repressor whereas Nanog functions as a transcriptional activator during muscle terminal differentiation. Importantly, in nonobese diabetic (NOD) severe combined immunodeficiency (SCID) mice, the pluripotency of C2C12 cells was conferred by overexpression of Oct4 and Nanog. These results suggest that Oct4 in cooperation with Nanog strongly suppresses the myogenic differentiation program and promotes pluripotency in myoblasts.

Download Full-text

Coupled Transcriptional and Translational Control of Cyclin-Dependent Kinase Inhibitor p18INK4cExpression during Myogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.18.4.2334 ◽

1998 ◽

Vol 18 (4) ◽

pp. 2334-2343 ◽

Cited By ~ 56

Author(s):

Dawn E. Phelps ◽

Kuang-Ming Hsiao ◽

Yan Li ◽

Nanpin Hu ◽

David S. Franklin ◽

...

Keyword(s):

Translational Control ◽

Kinase Inhibitor ◽

Myogenic Differentiation ◽

Protein A ◽

Terminal Differentiation ◽

C2c12 Cells ◽

Cdk Inhibitor ◽

Cyclin Dependent Kinase ◽

C2c12 Myoblasts ◽

Exon 1

ABSTRACT Terminal differentiation of many cell types involves permanent withdrawal from the cell division cycle. The p18 INK4c protein, a member of the p16/INK4 cyclin-dependent kinase (CDK) inhibitor family, is induced more than 50-fold during myogenic differentiation of mouse C2C12 myoblasts to become the predominant CDK inhibitor complexed with CDK4 and CDK6 in terminally differentiated myotubes. We have found that the p18 INK4c gene expresses two mRNA transcripts—a 2.4-kb transcript, p18(L), and a 1.2-kb transcript, p18(S). In proliferating C2C12 myoblasts, only the larger p18(L) transcript is expressed from an upstream promoter. As C2C12 cells are induced to differentiate into permanently arrested myotubes, the abundance of the p18(L) transcript decreases. The smaller p18(S) transcript expressed from a downstream promoter becomes detectable by 12 h postinduction and is the predominant transcript expressed in terminally differentiated myotubes. Both transcripts contain coding exons 2 and 3, but p18(L) uniquely contains an additional noncoding 1.2-kb exon, exon 1, corresponding exclusively to the 5′ untranslated region (5′ UTR). The expression pattern of the shorter p18(S) transcript, but not that of the longer p18(L) transcript, correlates with terminal differentiation of muscle, lung, liver, thymus, and eye lens cells during mouse embryo development. The presence of the long 5′ UTR in exon 1 attenuated the translation of p18(L) transcript, while its absence from the shorter p18(S) transcript resulted in significantly more efficient translation of the p18 protein. Our results demonstrate that during terminal muscle cell differentiation, induction of the p18 protein is regulated by promoter switching coupled with translational control.

Download Full-text

p27Kip1 Acts Downstream of N-Cadherin-mediated Cell Adhesion to Promote Myogenesis beyond Cell Cycle Regulation

Molecular Biology of the Cell ◽

10.1091/mbc.e04-07-0612 ◽

2005 ◽

Vol 16 (3) ◽

pp. 1469-1480 ◽

Cited By ~ 37

Author(s):

Graziella Messina ◽

Cristiana Blasi ◽

Severina Anna La Rocca ◽

Monica Pompili ◽

Attilio Calconi ◽

...

Keyword(s):

Cell Cycle ◽

Myogenic Differentiation ◽

Cell Cycle Control ◽

C2c12 Cell ◽

Terminal Differentiation ◽

Active Role ◽

C2c12 Cells ◽

Low Density

It is widely acknowledged that cultured myoblasts can not differentiate at very low density. Here we analyzed the mechanism through which cell density influences myogenic differentiation in vitro. By comparing the behavior of C2C12 myoblasts at opposite cell densities, we found that, when cells are sparse, failure to undergo terminal differentiation is independent from cell cycle control and reflects the lack of p27Kip1 and MyoD in proliferating myoblasts. We show that inhibition of p27Kip1 expression impairs C2C12 cell differentiation at high density, while exogenous p27Kip1 allows low-density cultured C2C12 cells to enter the differentiative program by regulating MyoD levels in undifferentiated myoblasts. We also demonstrate that the early induction of p27Kip1 is a critical step of the N-cadherin-dependent signaling involved in myogenesis. Overall, our data support an active role of p27Kip1 in the decision of myoblasts to commit to terminal differentiation, distinct from the regulation of cell proliferation, and identify a pathway that, reasonably, operates in vivo during myogenesis and might be part of the phenomenon known as “community effect”.

Download Full-text

Distinct Effects of Rac1 on Differentiation of Primary Avian Myoblasts

Molecular Biology of the Cell ◽

10.1091/mbc.10.10.3137 ◽

1999 ◽

Vol 10 (10) ◽

pp. 3137-3150 ◽

Cited By ~ 45

Author(s):

Rita Gallo ◽

Marco Serafini ◽

Loriana Castellani ◽

Germana Falcone ◽

Stefano Alemà

Keyword(s):

Myogenic Differentiation ◽

Terminal Differentiation ◽

Dominant Negative ◽

Skeletal Muscle Differentiation ◽

Rho Family Gtpases ◽

Specific Proteins ◽

Rho Family ◽

Selective Disassembly ◽

Jnk Activation ◽

Constitutively Active

Rho family GTPases have been implicated in the regulation of the actin cytoskeleton in response to extracellular cues and in the transduction of signals from the membrane to the nucleus. Their role in development and cell differentiation, however, is little understood. Here we show that the transient expression of constitutively active Rac1 and Cdc42 in unestablished avian myoblasts is sufficient to cause inhibition of myogenin expression and block of the transition to the myocyte compartment, whereas activated RhoA affects myogenic differentiation only marginally. Activation of c-Jun N-terminal kinase (JNK) appears not to be essential for block of differentiation because, although Rac1 and Cdc42 GTPases modestly activate JNK in quail myoblasts, a Rac1 mutant defective for JNK activation can still inhibit myogenic differentiation. Stable expression of active Rac1, attained by infection with a recombinant retrovirus, is permissive for terminal differentiation, but the resulting myotubes accumulate severely reduced levels of muscle-specific proteins. This inhibition is the consequence of posttranscriptional events and suggests the presence of a novel level of regulation of myogenesis. We also show that myotubes expressing constitutively active Rac1 fail to assemble ordered sarcomeres. Conversely, a dominant-negative Rac1 variant accelerates sarcomere maturation and inhibits v-Src–induced selective disassembly of I-Z-I complexes. Collectively, our findings provide a role for Rac1 during skeletal muscle differentiation and strongly suggest that Rac1 is required downstream of v-Src in the signaling pathways responsible for the dismantling of tissue-specific supramolecular structures.

Download Full-text

Interaction between SNAI2 and MYOD enhances oncogenesis and suppresses differentiation in Fusion Negative Rhabdomyosarcoma

Nature Communications ◽

10.1038/s41467-020-20386-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Silvia Pomella ◽

Prethish Sreenivas ◽

Berkley E. Gryder ◽

Long Wang ◽

David Milewski ◽

...

Keyword(s):

Transcription Factors ◽

Myogenic Differentiation ◽

Terminal Differentiation ◽

Muscle Differentiation ◽

Chromatin Binding ◽

Pediatric Malignancy ◽

Ras Pathway ◽

Oncogenic Ras ◽

Binding Analysis ◽

Genome Wide

AbstractRhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis.

Download Full-text

Regulation of Myogenesis by Fibroblast Growth Factors Requires Beta-Gamma Subunits of Pertussis Toxin-Sensitive G Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.18.10.5780 ◽

1998 ◽

Vol 18 (10) ◽

pp. 5780-5787 ◽

Cited By ~ 47

Author(s):

Yuri V. Fedorov ◽

Nathan C. Jones ◽

Bradley B. Olwin

Keyword(s):

Skeletal Muscle ◽

Growth Factors ◽

Growth Factor ◽

G Proteins ◽

Pertussis Toxin ◽

Intracellular Signaling ◽

Mapk Pathway ◽

Myogenic Differentiation ◽

Terminal Differentiation ◽

Fibroblast Growth

ABSTRACT Terminal differentiation of skeletal muscle cells in culture is inhibited by a number of different growth factors whose subsequent intracellular signaling events are poorly understood. In this study, we have investigated the role of heterotrimeric G proteins in mediating fibroblast growth factor (FGF)-dependent signals that regulate myogenic differentiation. Pertussis toxin, which ADP-ribosylates and inactivates susceptible G proteins, promotes terminal differentiation in the presence of FGF-2, suggesting that Gα or Gβγ subunits or both are involved in transducing the FGF-dependent signal(s) that inhibits myogenesis. We found that Gβγ subunits are likely to be involved since the expression of the C terminus of β-adrenergic receptor kinase 1, a Gβγ subunit-sequestering agent, promotes differentiation in the presence of FGF-2, and expression of the free Gβγ dimer can replace FGF-2, rescuing cells from pertussis toxin-induced differentiation. Addition of pertussis toxin also blocked FGF-2-mediated activation of mitogen-activated protein kinases (MAPKs). Ectopic expression of dominant active mutants in the Ras/MAPK pathway rescued cells from pertussis toxin-induced terminal differentiation, suggesting that the Gβγ subunits act upstream of the Ras/MAPK pathway. It is unlikely that the pertussis toxin-sensitive pathway is activated by other, as yet unidentified FGF receptors since PDGF (platelet-derived growth factor)-stimulated MM14 cells expressing a chimeric receptor containing the FGF receptor-1 intracellular domain and the PDGF receptor extracellular domain were sensitive to pertussis toxin. Our data suggest that FGF-mediated signals involved in repression of myogenic differentiation are transduced by a pertussis toxin-sensitive G-protein-coupled mechanism. This signaling pathway requires the action of Gβγ subunits and activation of MAPKs to repress skeletal muscle differentiation.

Download Full-text

MyoD-induced expression of p21 inhibits cyclin-dependent kinase activity upon myocyte terminal differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.15.7.3823 ◽

1995 ◽

Vol 15 (7) ◽

pp. 3823-3829 ◽

Cited By ~ 272

Author(s):

K Guo ◽

J Wang ◽

V Andrés ◽

R C Smith ◽

K Walsh

Keyword(s):

Cell Cycle ◽

Growth Medium ◽

Myogenic Differentiation ◽

Terminal Differentiation ◽

Cyclin Dependent Kinase ◽

Heat Stable ◽

Cell Extracts ◽

Cdk2 Activity ◽

Functional Consequences ◽

C2c12 Skeletal Muscle Cells

The terminal differentiation of C2C12 skeletal muscle cells involves the activation of unique sets of genes and an irreversible withdrawal from the cell cycle. This process is associated with a decrease in cdk2 activity in cell extracts. The decrease in cdk2 activity correlates with diminished levels of cdk2 and cyclin A and with a marked induction of the p21 cyclin-dependent kinase (cdk) inhibitor. The upregulation of p21 occurred at the levels of mRNA and protein, and p21 formed a complex with the cyclin kinases in myotubes. Further, the immunodepletion of p21 from myotube extracts neutralized the heat-stable cdk2 inhibitory activity that was induced upon myogenic differentiation. The levels of p21 mRNA, protein, and activity remained constant in myotubes when they were reexposed to mitogen-rich growth medium, indicating that permanent changes in the cell's genetic program contribute to its sustained expression following terminal differentiation. Indeed, 10T1/2 fibroblasts transformed with the myogenic factor MyoD, but not the parental multipotent cells, upregulated p21 transcript levels when induced to differentiate by serum withdrawal, demonstrating that the upregulation is an integral feature of myogenic commitment and differentiation. The functional consequences of this upregulation were indicated by ectopically expressing p21 in myoblasts; this was sufficient for cell cycle arrest in mitogen-rich growth medium. The induction and sustained expression of p21 appears to be a contributory mechanism by which myocytes irreversibly exit the cell cycle upon terminal differentiation.

Download Full-text

The mTORC2 Complex Regulates Terminal Differentiation of C2C12 Myoblasts

Molecular and Cellular Biology ◽

10.1128/mcb.00764-09 ◽

2009 ◽

Vol 29 (17) ◽

pp. 4691-4700 ◽

Cited By ~ 37

Author(s):

Lili Shu ◽

Peter J. Houghton

Keyword(s):

Myogenic Differentiation ◽

Terminal Differentiation ◽

Myoblast Fusion ◽

Selective Inhibitor ◽

Myoblast Differentiation ◽

Rock Inhibitor ◽

C2c12 Myoblasts ◽

Rapamycin Treatment ◽

Mtorc1 Signaling ◽

Mtorc2 Complex

ABSTRACT Rapamycin, a selective inhibitor of mTORC1 signaling, blocks terminal myoblast differentiation. We found that downregulation of rictor, a component of the mTORC2 complex, but not downregulation of raptor, a component of the mTORC1 complex, prevented terminal differentiation (fusion) of C2C12 myoblasts. Both rapamycin and rictor downregulation suppressed the phosphorylation of AKT(S473), and rapamycin treatment of C2C12 myoblasts disrupted the mTORC2 complex. Importantly, downregulation of rictor inhibited TORC2 signaling without inhibiting mTORC1 signaling, suggesting that inhibition of mTORC1 by rapamycin may not be the cause of arrested differentiation. In support of this, expression of a phosphomimetic mutant AKT(S473D) in rictor-deficient cells rescued myoblast fusion even in the presence of rapamycin. mTORC2 signaling to AKT appears necessary for downregulation of the Rho-associated kinase (ROCK1) that occurs during myogenic differentiation. Rapamycin treatment prevented ROCK1 inactivation during differentiation, while suppression of ROCK1 activity during differentiation and myoblast fusion was restored through expression of AKT(S473D), even in the presence of rapamycin. Further, the ROCK inhibitor Y-27632 restored terminal differentiation in rapamycin-treated myoblasts. These results provide the first evidence of a specific role for mTORC2 signaling in terminal myogenic differentiation.

Download Full-text