Myostatin is an inhibitor of myogenic differentiation

2002 ◽  
Vol 282 (5) ◽  
pp. C993-C999 ◽  
Author(s):  
Ramón Rı́os ◽  
Isabel Carneiro ◽  
Víctor M. Arce ◽  
Jesús Devesa
Keyword(s):  
Transforming Growth Factor ◽  
Myogenic Differentiation ◽  
Muscle Growth ◽  
Precursor Cell ◽  
Myoblast Differentiation ◽  
Mrna Levels ◽  
Downstream Target ◽  
Muscle Regulatory Factors ◽  
Muscle Precursor Cell ◽  
Autocrine Mechanism

Myostatin (MSTN), a transforming growth factor (TGF)-β superfamily member, has been shown to negatively regulate muscle growth by inhibiting muscle precursor cell proliferation. Here, we stably transfected C2C12 cells with mouse MSTN cDNA to investigate its possible role in myoblast differentiation. We found that MSTN cDNA overexpression reversibly inhibits the myogenic process by downregulating mRNA levels of the muscle regulatory factors myoD and myogenin, as well as the activity of their downstream target creatine kinase. Taking into consideration that MSTN expression during development is restricted to muscle, our results suggest that MSTN probably regulates myogenic differentiation by an autocrine mechanism.

scholarly journals Human myostatin negatively regulates human myoblast growth and differentiation

2011 ◽  
Vol 301 (1) ◽  
pp. C195-C203 ◽  
Author(s):  
Craig McFarlane ◽  
Gu Zi Hui ◽  
Wong Zhi Wei Amanda ◽  
Hiu Yeung Lau ◽  
Sudarsanareddy Lokireddy ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Target Genes ◽  
Transforming Growth Factor ◽  
Myogenic Differentiation ◽  
Muscle Growth ◽  
Notch Pathway ◽  
Transforming Growth Factor Β ◽  
Myoblast Differentiation ◽  
Myogenic Regulatory Factor ◽  
Human Myoblast

Myostatin, a member of the transforming growth factor-β superfamily, has been implicated in the potent negative regulation of myogenesis in murine models. However, little is known about the mechanism(s) through which human myostatin negatively regulates human skeletal muscle growth. Using human primary myoblasts and recombinant human myostatin protein, we show here that myostatin blocks human myoblast proliferation by regulating cell cycle progression through targeted upregulation of p21. We further show that myostatin regulates myogenic differentiation through the inhibition of key myogenic regulatory factors including MyoD, via canonical Smad signaling. In addition, we have for the first time demonstrated the capability of myostatin to regulate the Notch signaling pathway during inhibition of human myoblast differentiation. Treatment with myostatin results in the upregulation of Hes1, Hes5, and Hey1 expression during differentiation; moreover, when we interfere with Notch signaling, through treatment with the γ-secretase inhibitor L-685,458, we find enhanced myotube formation despite the presence of excess myostatin. Therefore, blockade of the Notch pathway relieves myostatin repression of differentiation, and myostatin upregulates Notch downstream target genes. Immunoprecipitation studies demonstrate that myostatin treatment of myoblasts results in enhanced association of Notch1-intracellular domain with Smad3, providing an additional mechanism through which myostatin targets and represses the activity of the myogenic regulatory factor MyoD. On the basis of these results, we suggest that myostatin function and mechanism of action are very well conserved between species, and that myostatin regulation of postnatal myogenesis involves interactions with numerous downstream signaling mediators, including the Notch pathway.

scholarly journals JAK1–STAT1–STAT3, a key pathway promoting proliferation and preventing premature differentiation of myoblasts

2007 ◽  
Vol 179 (1) ◽  
pp. 129-138 ◽  
Author(s):  
Luguo Sun ◽  
Kewei Ma ◽  
Haixia Wang ◽  
Fang Xiao ◽  
Yan Gao ◽  
...  
Keyword(s):  
Myogenic Differentiation ◽  
Muscle Growth ◽  
Janus Kinase ◽  
Myoblast Differentiation ◽  
Muscle Stem Cell ◽  
Janus Kinase 1 ◽  
Binding Factor ◽  
Proliferation And Differentiation ◽  
Myoblast Proliferation ◽  
Concomitant Reduction

Skeletal muscle stem cell–derived myoblasts are mainly responsible for postnatal muscle growth and injury-induced muscle regeneration. However, the cellular signaling pathways controlling the proliferation and differentiation of myoblasts are not fully understood. We demonstrate that Janus kinase 1 (JAK1) is required for myoblast proliferation and that it also functions as a checkpoint to prevent myoblasts from premature differentiation. Deliberate knockdown of JAK1 in both primary and immortalized myoblasts induces precocious myogenic differentiation with a concomitant reduction in cell proliferation. This is caused, in part, by an accelerated induction of MyoD, myocyte enhancer–binding factor 2 (MEF2), p21Cip1, and p27Kip1, a faster down-regulation of Id1, and an increase in MEF2-dependent gene transcription. Downstream of JAK1, of all the signal transducer and activator of transcriptions (STATs) present in myoblasts, we find that only STAT1 knockdown promotes myogenic differentiation in both primary and immortalized myoblasts. Leukemia inhibitory factor stimulates myoblast proliferation and represses differentiation via JAK1–STAT1–STAT3. Thus, JAK1–STAT1–STAT3 constitutes a signaling pathway that promotes myoblast proliferation and prevents premature myoblast differentiation.

scholarly journals The oncogenic forms of N-ras or H-ras prevent skeletal myoblast differentiation.

1987 ◽  
Vol 7 (6) ◽  
pp. 2104-2111 ◽  
Author(s):  
E N Olson ◽  
G Spizz ◽  
M A Tainsky
Keyword(s):  
Skeletal Muscle ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Transforming Growth Factor ◽  
Myogenic Differentiation ◽  
Myoblast Differentiation ◽  
Specific Gene ◽  
Gene Products ◽  
Fibroblast Growth ◽  
Ras Genes

Differentiation of skeletal muscle involves withdrawal of myoblasts from the cell cycle, fusion to form myotubes, and the coordinate expression of a variety of muscle-specific gene products. Fibroblast growth factor and type beta transforming growth factor specifically inhibit myogenesis; however, the transmembrane signaling pathways responsible for suppression of differentiation by these growth factors remain elusive. Because ras proteins have been implicated in the transduction of growth factor signals across the plasma membrane, we used DNA-mediated gene transfer to investigate the potential involvement of this family of regulatory proteins in the control of myogenesis. Transfection of the mouse skeletal muscle cell line C2 with the oncogenic forms of H-ras or N-ras completely suppressed both myoblast fusion and induction of the muscle-specific gene products nicotinic acetylcholine receptor and creatine kinase. Inhibition of differentiation by activated ras genes occurred at the level of muscle-specific mRNA accumulation. In contrast, proto-oncogenic forms of N-ras or H-ras had no apparent effects on the ability of C2 cells to differentiate. Myoblasts transfected with activated ras genes exhibited normal growth properties and ceased proliferating in the absence of mitogens, indicating that ras inhibited differentiation through a mechanism independent of cell proliferation. These results demonstrate that activated ras gene products mimic the inhibitory effects of fibroblast growth factor and type beta transforming growth factor on myogenic differentiation and suggest that each of these regulators of myogenesis may operate through a common intracellular pathway.

Regulation of myostatin expression and myoblast differentiation by FoxO and SMAD transcription factors

2007 ◽  
Vol 292 (1) ◽  
pp. C188-C199 ◽  
Author(s):  
David L. Allen ◽  
Terry G. Unterman
Keyword(s):  
Transcription Factors ◽  
Promoter Activity ◽  
Transforming Growth Factor ◽  
Active Form ◽  
Muscle Growth ◽  
Myoblast Differentiation ◽  
Binding Assays ◽  
Myostatin Gene

Myostatin, a member of the transforming growth factor (TGF)-β family, plays an important role in regulating skeletal muscle growth and differentiation. Here we examined the role of FoxO1 and SMAD transcription factors in regulating myostatin gene expression and myoblast differentiation in C2C12 myotubes in vitro. Both myostatin and FoxO1 mRNA expression were greater in fast- vs. slow-twitch skeletal muscles in vivo. Moreover, expression of a constitutively active form of FoxO1 increased myostatin mRNA and increased activity of a myostatin promoter reporter construct in differentiated C2C12 myotubes. Mutagenesis of highly conserved FoxO or SMAD binding sites significantly decreased myostatin promoter activity, and binding assays showed that both FoxO1 and SMADs bind to their respective sites in the myostatin promoter. Treatment with TGF-β and/or overexpression of SMAD2, -3, or -4 also resulted in a significant increase in myostatin promoter activity. Treatment with TGF-β along with overexpression of SMAD2 and FoxO1 resulted in the largest increase in myostatin promoter activity. Finally, TGF-β treatment and SMAD2 overexpression greatly potentiated FoxO1-mediated suppression of myoblast differentiation. Together these data demonstrate that FoxO1 and SMAD transcription factors regulate the expression of myostatin and contribute to the control of muscle cell growth and differentiation.

scholarly journals Regulation of calpain and calpastatin in differentiating myoblasts: mRNA levels, protein synthesis and stability

2000 ◽  
Vol 351 (2) ◽  
pp. 413-420 ◽  
Author(s):  
Sivia BARNOY ◽  
Lia SUPINO-ROSIN ◽  
Nechama S. KOSOWER
Keyword(s):  
Transforming Growth Factor ◽  
Protein Translation ◽  
Transforming Growth Factor Β ◽  
Myoblast Fusion ◽  
Myoblast Differentiation ◽  
Mrna Levels ◽  
Endogenous Inhibitor ◽  
Drug Induced ◽  
Dividing Cells ◽  
The Stability

Calpain (Ca2+-dependent intracellular protease)-induced proteolysis has been considered to play a role in myoblast fusion to myotubes. We found previously that calpastatin (the endogenous inhibitor of calpain) diminishes transiently during myoblast differentiation. To gain information about the regulation of calpain and calpastatin in differentiating myoblasts, we evaluated the stability and synthesis of calpain and calpastatin, and measured their mRNA levels in L8 myoblasts. We show here that µ-calpain and m-calpain are stable, long-lived proteins in both dividing and differentiating L8 myoblasts. Calpain is synthesized in differentiating myoblasts, and calpain mRNA levels do not change during differentiation. In contrast, calpastatin (though also a long-lived protein in myoblasts), is less stable in differentiating myoblasts than in the dividing cells, and its synthesis is inhibited upon initiation of differentiation. Inhibition of calpastatin synthesis is followed by a diminution in calpastatin mRNA levels. A similar calpastatin mRNA diminution is observed upon drug-induced inhibition of protein translation. On the other hand, transforming growth factor β (which inhibits differentiation) allows calpastatin synthesis and prevents the diminution in calpastatin mRNA. The overall results suggest that at the onset of myoblast differentiation, calpastatin is regulated mainly at the level of translation and that an inhibition of calpastatin synthesis leads to the decrease in its mRNA stability. The existing calpastatin then diminishes, resulting in decreased calpastatin activity in the fusing myoblasts, allowing calpain activation and protein degradation required for fusion.

The oncogenic forms of N-ras or H-ras prevent skeletal myoblast differentiation

1987 ◽  
Vol 7 (6) ◽  
pp. 2104-2111
Author(s):  
E N Olson ◽  
G Spizz ◽  
M A Tainsky
Keyword(s):  
Skeletal Muscle ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Transforming Growth Factor ◽  
Myogenic Differentiation ◽  
Myoblast Differentiation ◽  
Specific Gene ◽  
Gene Products ◽  
Fibroblast Growth ◽  
Ras Genes

Differentiation of skeletal muscle involves withdrawal of myoblasts from the cell cycle, fusion to form myotubes, and the coordinate expression of a variety of muscle-specific gene products. Fibroblast growth factor and type beta transforming growth factor specifically inhibit myogenesis; however, the transmembrane signaling pathways responsible for suppression of differentiation by these growth factors remain elusive. Because ras proteins have been implicated in the transduction of growth factor signals across the plasma membrane, we used DNA-mediated gene transfer to investigate the potential involvement of this family of regulatory proteins in the control of myogenesis. Transfection of the mouse skeletal muscle cell line C2 with the oncogenic forms of H-ras or N-ras completely suppressed both myoblast fusion and induction of the muscle-specific gene products nicotinic acetylcholine receptor and creatine kinase. Inhibition of differentiation by activated ras genes occurred at the level of muscle-specific mRNA accumulation. In contrast, proto-oncogenic forms of N-ras or H-ras had no apparent effects on the ability of C2 cells to differentiate. Myoblasts transfected with activated ras genes exhibited normal growth properties and ceased proliferating in the absence of mitogens, indicating that ras inhibited differentiation through a mechanism independent of cell proliferation. These results demonstrate that activated ras gene products mimic the inhibitory effects of fibroblast growth factor and type beta transforming growth factor on myogenic differentiation and suggest that each of these regulators of myogenesis may operate through a common intracellular pathway.

A muscle precursor cell-dependent pathway contributes to muscle growth after atrophy

2001 ◽  
Vol 281 (5) ◽  
pp. C1706-C1715 ◽  
Author(s):  
Patrick O. Mitchell ◽  
Grace K. Pavlath
Keyword(s):  
Muscle Mass ◽  
Recovery Period ◽  
Muscle Growth ◽  
Precursor Cells ◽  
Precursor Cell ◽  
Hindlimb Suspension ◽  
Γ Irradiation ◽  
Muscle Precursor ◽  
Muscle Precursor Cells ◽  
Muscle Precursor Cell

Slow-twitch skeletal muscle atrophies greatly in response to unloading conditions. The cellular mechanisms that contribute to the restoration of muscle mass after atrophy are largely unknown. Here, we show that atrophy of the mouse soleus is associated with a 36% decrease in myonuclear number after 2 wk of hindlimb suspension. Myonuclear number is restored to control values during the 2-wk recovery period in which muscle mass returns to normal, suggesting that muscle precursor cells proliferate and fuse with myofibers. Inhibition of muscle precursor cell proliferation by local γ-irradiation of the hindlimb completely prevents this increase in myonuclear number. Muscle growth occurs normally during the first week in irradiated muscles, but growth during the second week is inhibited, leading to a 50% attenuation in the restoration of muscle mass. Thus early muscle growth occurs independently of an increase in myonuclear number, whereas later growth requires proliferating muscle precursor cells leading to myonuclear accretion. These results suggest that increasing the proliferative capacity of muscle precursor cells may enhance restoration of muscle mass after atrophy.

Differentiation in C2C12 myoblasts depends on the expression of endogenous IGFs and not serum depletion

2002 ◽  
Vol 283 (4) ◽  
pp. C1278-C1286 ◽  
Author(s):  
Yuji Yoshiko ◽  
Keita Hirao ◽  
Norihiko Maeda
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Myogenic Differentiation ◽  
Negative Control ◽  
Mrna Levels ◽  
Rich Medium ◽  
Medium Conditioning ◽  
Serum Components

Myogenic differentiation in vitro has been usually viewed as being negatively controlled by serum mitogens. A depletion of critical serum components from medium has been considered to be essential for permanent withdrawal from the cell cycle and terminal differentiation of myoblasts. Removal of serum mitogens induces the expression of insulin-like growth factors (IGFs), whereas it inhibits that of basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-β in myoblasts. These responses of growth factors to medium conditioning seem to be well matched to their functions in proliferation/differentiation. In the present study, we showed that C2C12 myoblasts differentiated actively, even in mitogen-rich medium, and that this medium offered an advantage over mitogen-poor medium in terms of increasing differentiation. Our attention focused on endogenous growth factors, as described above, especially IGFs in mitogen-rich medium. During differentiation, IGF-I and IGF-II mRNA levels increased, but bFGF and TGF-β1 mRNAs decreased. Differentiation was commensurable with IGF mRNA levels and suppressed by antisense oligodeoxynucleotides and neutralizing monoclonal antibodies against IGFs. These results suggest that an autocrine/paracrine loop of IGFs, bFGF, and TGF-β1 is active in proliferating and differentiating C2C12 cells without a depletion of serum and that endogenous IGFs actively override the negative control of differentiation by serum mitogens.

scholarly journals Overlapping Functions of Nuclear Envelope Proteins NET25 (Lem2) and Emerin in Regulation of Extracellular Signal-Regulated Kinase Signaling in Myoblast Differentiation

2009 ◽  
Vol 29 (21) ◽  
pp. 5718-5728 ◽  
Author(s):  
Michael D. Huber ◽  
Tinglu Guan ◽  
Larry Gerace
Keyword(s):  
Nuclear Envelope ◽  
Transforming Growth Factor ◽  
Myogenic Differentiation ◽  
Ectopic Expression ◽  
Transforming Growth Factor Β ◽  
Mitogen Activated Protein Kinase ◽  
Myoblast Differentiation ◽  
Extracellular Signal Regulated Kinase ◽  
Pharmacological Inhibition ◽  
Extracellular Signal

ABSTRACT Mutations in certain nuclear envelope (NE) proteins cause muscular dystrophies and other disorders, but the disease mechanisms remain unclear. The nuclear envelope transmembrane protein NET25 (Lem2) is a truncated paralog of MAN1, an NE component linked to bone disorders. NET25 and MAN1 share an ∼40-residue LEM homology domain with emerin, the protein mutated in X-linked Emery-Dreifuss muscular dystrophy. However, roles for NET25 and MAN1 in myogenesis have not yet been described. Using RNA interference in C2C12 myoblasts, we show for the first time that both NET25 and MAN1 are required for myogenic differentiation. NET25 depletion causes hyperactivation of extracellular signal-regulated kinase 1/2 at the onset of differentiation, and pharmacological inhibition of this transient overactivation rescues myogenesis. In contrast, pharmacological inhibition of both mitogen-activated protein kinase and transforming growth factor β signaling is required to rescue differentiation after MAN1 depletion. Ectopic expression of silencing-resistant NET25 rescues myogenesis after depletion of emerin but not after MAN1 silencing. Thus, NET25 and emerin have at least partially overlapping functions during myogenic differentiation, which are distinct from those of MAN1. Our work supports the hypothesis that deregulation of cell signaling contributes to NE-linked disorders and suggests that mutations in NET25 and MAN1 may cause muscle diseases.

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