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Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism

View Article: PubMed Central - PubMed

ABSTRACT

Background:: The transforming growth factor β (TGF-β) signaling pathways modulate skeletal muscle growth, regeneration, and cellular metabolism. Several recent gene expression studies have shown that inhibition of myostatin and TGF-β1 signaling consistently leads to a significant reduction in expression of Mss51, also named Zmynd17. The function of mammalian Mss51 is unknown although a putative homolog in yeast is a mitochondrial translational activator.

Objective:: The objective of this work was to characterize mammalian MSS51.

Methods:: Quantitative RT-PCR and immunoblot of subcellular fractionation were used to determine expression patterns and localization of Mss51. The CRISPR/Cas9 system was used to reduce expression of Mss51 in C2C12 myoblasts and the function of Mss51 was evaluated in assays of proliferation, differentiation and cellular metabolism.

Results:: Mss51 was predominantly expressed in skeletal muscle and in those muscles dominated by fast-twitch fibers. In vitro, its expression was upregulated upon differentiation of C2C12 myoblasts into myotubes. Expression of Mss51 was modulated in response to altered TGF-β family signaling. In human muscle, MSS51 localized to the mitochondria. Its genetic disruption resulted in increased levels of cellular ATP, β-oxidation, glycolysis, and oxidative phosphorylation.

Conclusions:: Mss51 is a novel, skeletal muscle-specific gene and a key target of myostatin and TGF-β1 signaling. Unlike myostatin, TGF-β1 and IGF-1, Mss51 does not regulate myoblast proliferation or differentiation. Rather, Mss51 appears to be one of the effectors of these growth factors on metabolic processes including fatty acid oxidation, glycolysis and oxidative phosphorylation.

No MeSH data available.


Mss51 expression in vitro. (A) Mss51 mRNA expression measured by qRT-PCR in proliferating and differentiating C2C12 cells. Expression level is normalized to proliferating (Day 0) cells using the reference gene TBP. (B) Mss51 expression in differentiated C2C12 myotubes treated with various growth factors and inhibitors: 300 ng/mL myostatin, 5μg/mL ActRIIB-Fc, 50 ng/mL TGF-β1, 10μg/mL TGF-β neutralizing antibody 1D11, 20 ng/mL Activin A, and 100 ng/mL IGF-1. Expression level is normalized to control (untreated) cells using reference genes β2 m and TBP. For (A) and (B), significant differences between groups were determined by a one-way ANOVA (n = 3, p <  0.01) with Bonferroni post hoc comparisons. The annotation above each bar indicates statistically significant differences between the means in each group at p <  0.05: Groups sharing a letter designation were not significantly different from each other, while groups not sharing a letter were significantly different.
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jnd-2-4-jnd150119-g001: Mss51 expression in vitro. (A) Mss51 mRNA expression measured by qRT-PCR in proliferating and differentiating C2C12 cells. Expression level is normalized to proliferating (Day 0) cells using the reference gene TBP. (B) Mss51 expression in differentiated C2C12 myotubes treated with various growth factors and inhibitors: 300 ng/mL myostatin, 5μg/mL ActRIIB-Fc, 50 ng/mL TGF-β1, 10μg/mL TGF-β neutralizing antibody 1D11, 20 ng/mL Activin A, and 100 ng/mL IGF-1. Expression level is normalized to control (untreated) cells using reference genes β2 m and TBP. For (A) and (B), significant differences between groups were determined by a one-way ANOVA (n = 3, p <  0.01) with Bonferroni post hoc comparisons. The annotation above each bar indicates statistically significant differences between the means in each group at p <  0.05: Groups sharing a letter designation were not significantly different from each other, while groups not sharing a letter were significantly different.

Mentions: To examine modulation of Mss51 expression, we first determined the time course of its expression in vitro. Mss51 was expressed at low levels in proliferating C2C12 myoblasts, and significantly higher levels as cells differentiated into myotubes (Fig. 1A). Myotubes were then differentiated for three days and treated with various TGF-β superfamily members as well as various inhibitors (Fig. 1B). TGF-β superfamily members (myostatin, TGF-β1 and activin A) all increased Mss51 expression in myotubes. The TGF-β1/2/3 neutralizing antibody 1D11 decreased Mss51 expression and there was a trend toward decreased expression with ActRIIB-Fc treatment. IGF-1, which has several similar effects on skeletal muscle as myostatin inhibition including increasing proliferation and differentiation in vitro and muscle growth in vivo [34], also decreased Mss51 expression. We examined other time points and saw the greatest effects on Mss51 expression levels when the myotubes were differentiated for three days and no significant change seen when proliferating myoblasts were treated (data not shown). This may be because during differentiation, the cells are actively increasing Mss51 expression and are therefore more responsive to exogenous signaling cues.


Mammalian Mss51 is a Skeletal Muscle-Specific Gene Modulating Cellular Metabolism
Mss51 expression in vitro. (A) Mss51 mRNA expression measured by qRT-PCR in proliferating and differentiating C2C12 cells. Expression level is normalized to proliferating (Day 0) cells using the reference gene TBP. (B) Mss51 expression in differentiated C2C12 myotubes treated with various growth factors and inhibitors: 300 ng/mL myostatin, 5μg/mL ActRIIB-Fc, 50 ng/mL TGF-β1, 10μg/mL TGF-β neutralizing antibody 1D11, 20 ng/mL Activin A, and 100 ng/mL IGF-1. Expression level is normalized to control (untreated) cells using reference genes β2 m and TBP. For (A) and (B), significant differences between groups were determined by a one-way ANOVA (n = 3, p <  0.01) with Bonferroni post hoc comparisons. The annotation above each bar indicates statistically significant differences between the means in each group at p <  0.05: Groups sharing a letter designation were not significantly different from each other, while groups not sharing a letter were significantly different.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664537&req=5

jnd-2-4-jnd150119-g001: Mss51 expression in vitro. (A) Mss51 mRNA expression measured by qRT-PCR in proliferating and differentiating C2C12 cells. Expression level is normalized to proliferating (Day 0) cells using the reference gene TBP. (B) Mss51 expression in differentiated C2C12 myotubes treated with various growth factors and inhibitors: 300 ng/mL myostatin, 5μg/mL ActRIIB-Fc, 50 ng/mL TGF-β1, 10μg/mL TGF-β neutralizing antibody 1D11, 20 ng/mL Activin A, and 100 ng/mL IGF-1. Expression level is normalized to control (untreated) cells using reference genes β2 m and TBP. For (A) and (B), significant differences between groups were determined by a one-way ANOVA (n = 3, p <  0.01) with Bonferroni post hoc comparisons. The annotation above each bar indicates statistically significant differences between the means in each group at p <  0.05: Groups sharing a letter designation were not significantly different from each other, while groups not sharing a letter were significantly different.
Mentions: To examine modulation of Mss51 expression, we first determined the time course of its expression in vitro. Mss51 was expressed at low levels in proliferating C2C12 myoblasts, and significantly higher levels as cells differentiated into myotubes (Fig. 1A). Myotubes were then differentiated for three days and treated with various TGF-β superfamily members as well as various inhibitors (Fig. 1B). TGF-β superfamily members (myostatin, TGF-β1 and activin A) all increased Mss51 expression in myotubes. The TGF-β1/2/3 neutralizing antibody 1D11 decreased Mss51 expression and there was a trend toward decreased expression with ActRIIB-Fc treatment. IGF-1, which has several similar effects on skeletal muscle as myostatin inhibition including increasing proliferation and differentiation in vitro and muscle growth in vivo [34], also decreased Mss51 expression. We examined other time points and saw the greatest effects on Mss51 expression levels when the myotubes were differentiated for three days and no significant change seen when proliferating myoblasts were treated (data not shown). This may be because during differentiation, the cells are actively increasing Mss51 expression and are therefore more responsive to exogenous signaling cues.

View Article: PubMed Central - PubMed

ABSTRACT

Background:: The transforming growth factor &beta; (TGF-&beta;) signaling pathways modulate skeletal muscle growth, regeneration, and cellular metabolism. Several recent gene expression studies have shown that inhibition of myostatin and TGF-&beta;1 signaling consistently leads to a significant reduction in expression of Mss51, also named Zmynd17. The function of mammalian Mss51 is unknown although a putative homolog in yeast is a mitochondrial translational activator.

Objective:: The objective of this work was to characterize mammalian MSS51.

Methods:: Quantitative RT-PCR and immunoblot of subcellular fractionation were used to determine expression patterns and localization of Mss51. The CRISPR/Cas9 system was used to reduce expression of Mss51 in C2C12 myoblasts and the function of Mss51 was evaluated in assays of proliferation, differentiation and cellular metabolism.

Results:: Mss51 was predominantly expressed in skeletal muscle and in those muscles dominated by fast-twitch fibers. In vitro, its expression was upregulated upon differentiation of C2C12 myoblasts into myotubes. Expression of Mss51 was modulated in response to altered TGF-&beta; family signaling. In human muscle, MSS51 localized to the mitochondria. Its genetic disruption resulted in increased levels of cellular ATP, &beta;-oxidation, glycolysis, and oxidative phosphorylation.

Conclusions:: Mss51 is a novel, skeletal muscle-specific gene and a key target of myostatin and TGF-&beta;1 signaling. Unlike myostatin, TGF-&beta;1 and IGF-1, Mss51 does not regulate myoblast proliferation or differentiation. Rather, Mss51 appears to be one of the effectors of these growth factors on metabolic processes including fatty acid oxidation, glycolysis and oxidative phosphorylation.

No MeSH data available.