Raptor and Rheb negatively regulate skeletal myogenesis through suppression of insulin receptor substrate 1 (IRS1).
ABSTRACT The mammalian target of rapamycin (mTOR) is essential for skeletal myogenesis through controlling distinct cellular pathways. The importance of the canonical mTOR complex 1 signaling components, including raptor, S6K1, and Rheb, had been suggested in muscle maintenance, growth, and metabolism. However, the role of those components in myogenic differentiation is not entirely clear. In this study we have investigated the functions of raptor, S6K1, and Rheb in the differentiation of C2C12 mouse myoblasts. We find that although mTOR knockdown severely impairs myogenic differentiation as expected, the knockdown of raptor, as well as Rheb, enhances differentiation. Consistent with a negative role for these proteins in myogenesis, overexpression of raptor or Rheb inhibits C2C12 differentiation. On the other hand, neither knockdown nor overexpression of S6K1 has any effect. Moreover, the enhanced differentiation elicited by raptor or Rheb knockdown is accompanied by increased Akt activation, elevated IRS1 protein levels, and decreased Ser-307 (human Ser-312) phosphorylation on IRS1. Finally, IRS1 knockdown eliminated the enhancement in differentiation elicited by raptor or Rheb knockdown, suggesting that IRS1 is a critical mediator of the myogenic functions of raptor and Rheb. In conclusion, the Rheb-mTOR/raptor pathway negatively regulates myogenic differentiation by suppressing IRS1-PI3K-Akt signaling. These findings underscore the versatility of mTOR signaling in biological regulations and implicate the existence of novel mTOR complexes and/or signaling mechanism in skeletal myogenesis.
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ABSTRACT: Hair follicles (HFs) undergo cycles of degeneration (catagen), rest (telogen), and regeneration (anagen) phases. Anagen begins when the hair follicle stem cells (HFSCs) obtain sufficient activation cues to overcome suppressive signals mainly through the BMP pathway from their niche cells. Here, we unveil that mTOR complex 1 (mTORC1) signaling is activated in HFSCs, which coincides with the HFSC activation at the telogen-to-anagen transition. By using both an inducible conditional gene targeting strategy and a pharmacological inhibition method to ablate or inhibit mTOR signaling in adult skin epithelium before anagen initiation, we demonstrate that HFs that cannot respond to mTOR signaling display significantly delayed HFSC activation and extended telogen. Unexpectedly, BMP signaling activity is dramatically prolonged in mTOR signaling-deficient HFs. Through both gain- and loss-of-function studies in vitro, we show that mTORC1 signaling negatively affects BMP signaling, which serves as a main mechanism whereby mTORC1 signaling facilitates HFSC activation. Indeed, in vivo suppression of BMP by its antagonist Noggin rescues the HFSC activation defect in mTORC1-null skin. Our findings reveal a critical role for mTOR signaling in regulating stem cell activation through counterbalancing BMP-mediated repression during hair regeneration. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.Journal of Molecular Cell Biology 01/2015; · 8.43 Impact Factor
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ABSTRACT: Background Domestication of the wild pig has led to obese and lean phenotype breeds, and evolutionary genome research has sought to identify the regulatory mechanisms underlying this phenotypic diversity. However, revealing the molecular mechanisms underlying muscle phenotype variation based on differentially expressed genes has proved to be difficult. To characterize the mechanisms regulating muscle phenotype variation under artificial selection, we aimed to provide an integrated view of genome organization by weighted gene coexpression network analysis.ResultsOur analysis was based on 20 publicly available next-generation sequencing datasets of lean and obese pig muscle generated from 10 developmental stages. The evolution of the constructed coexpression modules was examined using the genome resequencing data of 37 domestic pigs and 11 wild boars. Our results showed the regulation of muscle development might be more complex than had been previously acknowledged, and is regulated by the coordinated action of muscle, nerve and immunity related genes. Breed-specific modules that regulated muscle phenotype divergence were identified, and hundreds of hub genes with major roles in muscle development were determined to be responsible for key functional distinctions between breeds. Our evolutionary analysis showed that the role of changes in the coding sequence under positive selection in muscle phenotype divergence was minor.Conclusions Muscle phenotype divergence was found to be regulated by the divergence of coexpression network modules under artificial selection, and not by changes in the coding sequence of genes. Our results present multiple lines of evidence suggesting links between modules and muscle phenotypes, and provide insights into the molecular bases of genome organization in muscle development and phenotype variation.BMC Genomics 02/2015; 16(1):50. · 4.04 Impact Factor
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ABSTRACT: During myogenesis, satellite stem cells (SCs) are induced to proliferate and differentiate to myogenic precursors. The role of energy sensors such as the AMP activated protein kinase (AMPK) and the mammalian Target Of Rapamycin (mTOR) in SC activation is unclear. We previously observed that upregulation of ATP through RNA-mediated mitochondrial restoration (MR) accelerates SC activation following skeletal muscle injury. We show here that during regeneration, the AMPK-CRTC2-CREB and Raptor-mTORC-4EBP1 pathways were rapidly activated. The phosho-CRTC2-CREB complex was essential for myogenesis and activated transcription of the critical cell cycle regulator cyclin D1 (Ccnd1). Knockdown (KD) of either mTORC or its subunit Raptor delayed SC activation without influencing the differentiation program. KD of 4EBP1 had no effect on SC activation but enhanced myofiber size. mTORC1 positively regulated Ccnd1 translation but destabilized Ccnd1 mRNA. These antithetical effects of mTORC1 were mediated by two microRNAs (miRs) targeted to the 3'-UTR of Ccnd1 mRNA: miR-1 was downregulated in mTORC-KD muscle, and depletion of miR-1 resulted in increased levels of mRNA without any effect on Ccnd1 protein. In contrast, miR-26a was upregulated upon mTORC depletion, while anti-miR-26a oligonucleotide specifically stimulated Ccnd1 protein expression. Thus, mTORC may act as a timer of satellite cell proliferation during myogenesis.Molecular and Cellular Biology 07/2014; · 5.04 Impact Factor