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Distinct amino acid-sensing mTOR pathways regulate skeletal myogenesis.

Yoon MS, Chen J - Mol. Biol. Cell (2013)

Bottom Line: On the other hand, Vps34 is required for myogenic differentiation.The product of PLD, phosphatidic acid, activates the enhancer in a rapamycin-sensitive but mTOR kinase-independent manner.Our results uncover amino acid-sensing mechanisms controlling the homeostasis of myogenesis and underline the versatility and context dependence of mTOR signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801.

ABSTRACT
Signaling through the mammalian target of rapamycin (mTOR) in response to amino acid availability controls many cellular and developmental processes. mTOR is a master regulator of myogenic differentiation, but the pathways mediating amino acid signals in this process are not known. Here we examine the Rag GTPases and the class III phosphoinositide 3-kinase (PI3K) Vps34, two mediators of amino acid signals upstream of mTOR complex 1 (mTORC1) in cell growth regulation, for their potential involvement in myogenesis. We find that, although both Rag and Vps34 mediate amino acid activation of mTORC1 in C2C12 myoblasts, they have opposing functions in myogenic differentiation. Knockdown of RagA/B enhances, whereas overexpression of active RagB/C mutants impairs, differentiation, and this inhibitory function of Rag is mediated by mTORC1 suppression of the IRS1-PI3K-Akt pathway. On the other hand, Vps34 is required for myogenic differentiation. Amino acids activate a Vps34-phospholipase D1 (PLD1) pathway that controls the production of insulin-like growth factor II, an autocrine inducer of differentiation, through the Igf2 muscle enhancer. The product of PLD, phosphatidic acid, activates the enhancer in a rapamycin-sensitive but mTOR kinase-independent manner. Our results uncover amino acid-sensing mechanisms controlling the homeostasis of myogenesis and underline the versatility and context dependence of mTOR signaling.

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Rag inhibits differentiation through the IRS1/Akt pathway. (A) C2C12 cells were infected with lentiviruses expressing shRNAs for RagA and RagB. Cell lysates were analyzed by Western blotting. (B) Cells stably expressing RagB-GTP and RagC-GDP mutants were induced to differentiate for 3 d, followed by Western analysis. (C) Cells were infected with lentiviruses expressing various shRNAs as indicated, followed by differentiation for 3 d and Western analysis of cell lysates. (D) Cells treated as in C were stained for MHC (green) and DAPI (magenta) and quantified for fusion index. Data shown are mean ± SD or representative blots from three independent experiments. A paired t test was performed to compare each sample to the control. *p < 0.05. Scale bar, 100 μm.
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Figure 2: Rag inhibits differentiation through the IRS1/Akt pathway. (A) C2C12 cells were infected with lentiviruses expressing shRNAs for RagA and RagB. Cell lysates were analyzed by Western blotting. (B) Cells stably expressing RagB-GTP and RagC-GDP mutants were induced to differentiate for 3 d, followed by Western analysis. (C) Cells were infected with lentiviruses expressing various shRNAs as indicated, followed by differentiation for 3 d and Western analysis of cell lysates. (D) Cells treated as in C were stained for MHC (green) and DAPI (magenta) and quantified for fusion index. Data shown are mean ± SD or representative blots from three independent experiments. A paired t test was performed to compare each sample to the control. *p < 0.05. Scale bar, 100 μm.

Mentions: Because Rag depletion suppressed mTORC1 activation in C2C12 cells (Supplemental Figure S1) and we previously reported that mTORC1 plays a negative role in myogenic differentiation through a feedback inhibition of IRS1-Akt signaling (Ge et al., 2011b), we considered the possibility of Rag acting via this feedback pathway. Indeed, IRS1 phosphorylation on Ser-307 was decreased by the knockdown of RagA/B in myoblasts, which was accompanied by increased Akt phosphorylation (Figure 2A), mirroring the results of Rheb and raptor knockdown (Ge et al., 2011b). Conversely, overexpression of constitutively active RagB/C decreased IRS1 levels and pAkt (Figure 2B). More important, knockdown of IRS1 eliminated the enhancement of differentiation resulted from Rag depletion, as assessed by both MHC expression (Figure 2C) and myotube formation (Figure 2D). Therefore, by activating mTORC1, Rag induces IRS1 serine phosphorylation and suppresses IRS1 signaling to Akt, subsequently inhibiting myogenic differentiation.


Distinct amino acid-sensing mTOR pathways regulate skeletal myogenesis.

Yoon MS, Chen J - Mol. Biol. Cell (2013)

Rag inhibits differentiation through the IRS1/Akt pathway. (A) C2C12 cells were infected with lentiviruses expressing shRNAs for RagA and RagB. Cell lysates were analyzed by Western blotting. (B) Cells stably expressing RagB-GTP and RagC-GDP mutants were induced to differentiate for 3 d, followed by Western analysis. (C) Cells were infected with lentiviruses expressing various shRNAs as indicated, followed by differentiation for 3 d and Western analysis of cell lysates. (D) Cells treated as in C were stained for MHC (green) and DAPI (magenta) and quantified for fusion index. Data shown are mean ± SD or representative blots from three independent experiments. A paired t test was performed to compare each sample to the control. *p < 0.05. Scale bar, 100 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Rag inhibits differentiation through the IRS1/Akt pathway. (A) C2C12 cells were infected with lentiviruses expressing shRNAs for RagA and RagB. Cell lysates were analyzed by Western blotting. (B) Cells stably expressing RagB-GTP and RagC-GDP mutants were induced to differentiate for 3 d, followed by Western analysis. (C) Cells were infected with lentiviruses expressing various shRNAs as indicated, followed by differentiation for 3 d and Western analysis of cell lysates. (D) Cells treated as in C were stained for MHC (green) and DAPI (magenta) and quantified for fusion index. Data shown are mean ± SD or representative blots from three independent experiments. A paired t test was performed to compare each sample to the control. *p < 0.05. Scale bar, 100 μm.
Mentions: Because Rag depletion suppressed mTORC1 activation in C2C12 cells (Supplemental Figure S1) and we previously reported that mTORC1 plays a negative role in myogenic differentiation through a feedback inhibition of IRS1-Akt signaling (Ge et al., 2011b), we considered the possibility of Rag acting via this feedback pathway. Indeed, IRS1 phosphorylation on Ser-307 was decreased by the knockdown of RagA/B in myoblasts, which was accompanied by increased Akt phosphorylation (Figure 2A), mirroring the results of Rheb and raptor knockdown (Ge et al., 2011b). Conversely, overexpression of constitutively active RagB/C decreased IRS1 levels and pAkt (Figure 2B). More important, knockdown of IRS1 eliminated the enhancement of differentiation resulted from Rag depletion, as assessed by both MHC expression (Figure 2C) and myotube formation (Figure 2D). Therefore, by activating mTORC1, Rag induces IRS1 serine phosphorylation and suppresses IRS1 signaling to Akt, subsequently inhibiting myogenic differentiation.

Bottom Line: On the other hand, Vps34 is required for myogenic differentiation.The product of PLD, phosphatidic acid, activates the enhancer in a rapamycin-sensitive but mTOR kinase-independent manner.Our results uncover amino acid-sensing mechanisms controlling the homeostasis of myogenesis and underline the versatility and context dependence of mTOR signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801.

ABSTRACT
Signaling through the mammalian target of rapamycin (mTOR) in response to amino acid availability controls many cellular and developmental processes. mTOR is a master regulator of myogenic differentiation, but the pathways mediating amino acid signals in this process are not known. Here we examine the Rag GTPases and the class III phosphoinositide 3-kinase (PI3K) Vps34, two mediators of amino acid signals upstream of mTOR complex 1 (mTORC1) in cell growth regulation, for their potential involvement in myogenesis. We find that, although both Rag and Vps34 mediate amino acid activation of mTORC1 in C2C12 myoblasts, they have opposing functions in myogenic differentiation. Knockdown of RagA/B enhances, whereas overexpression of active RagB/C mutants impairs, differentiation, and this inhibitory function of Rag is mediated by mTORC1 suppression of the IRS1-PI3K-Akt pathway. On the other hand, Vps34 is required for myogenic differentiation. Amino acids activate a Vps34-phospholipase D1 (PLD1) pathway that controls the production of insulin-like growth factor II, an autocrine inducer of differentiation, through the Igf2 muscle enhancer. The product of PLD, phosphatidic acid, activates the enhancer in a rapamycin-sensitive but mTOR kinase-independent manner. Our results uncover amino acid-sensing mechanisms controlling the homeostasis of myogenesis and underline the versatility and context dependence of mTOR signaling.

Show MeSH
Related in: MedlinePlus