IGF-II is regulated by microRNA-125b in skeletal myogenesis

MicroRNAs (miRNAs) have emerged as key regulators of skeletal myogenesis, but our knowledge of the identity of the myogenic miRNAs and their targets remains limited. In this study, we report the identification and characterization of a novel myogenic miRNA, miR-125b. We find that the levels of miR-125b decline during myogenesis and that miR-125b negatively modulates myoblast differentiation in culture and muscle regeneration in mice. Our results identify IGF-II (insulin-like growth factor 2), a critical regulator of skeletal myogenesis, as a direct and major target of miR-125b in both myocytes and regenerating muscles, revealing for the first time an miRNA mechanism controlling IGF-II expression. In addition, we provide evidence suggesting that miR-125b biogenesis is negatively controlled by kinase-independent mammalian target of rapamycin (mTOR) signaling both in vitro and in vivo as a part of a dual mechanism by which mTOR regulates the production of IGF-II, a master switch governing the initiation of skeletal myogenesis.     

Androgen receptor enhances myogenin expression and accelerates differentiation

Animal and clinical studies indicated that the androgen-AR signaling pathway is required for appropriate development of sexually dimorphic skeletal muscles and increases lean muscle mass, muscle strength, and muscle protein synthesis. However, the detailed mechanisms by which the androgen-AR signaling pathway regulates skeletal muscle development need further study at the molecular level. C2C12 myoblast cells stably transfected with the Flag-tagged AR were used to analyze the role of androgen-AR signaling pathway in skeletal muscle development. The results indicate that the androgen-AR signaling pathway may suppress skeletal myoblast cell growth and accelerate myoblast cell differentiation via enhanced myogenin expression. This is a first report showing the role of androgen-AR signaling pathway in regulation of myoblast cell growth and myogenic regulatory factors.     

Insulin-like growth factors (IGF) in muscle development. Expression of IGF-I, the IGF-I receptor, and an IGF binding protein during myoblast differentiation

The insulin-like growth factors (IGFs) I and II exert pleiotropic effects on diverse cell types through interaction with specific high affinity cell surface receptors and with locally produced binding proteins. In skeletal muscle and in myoblast cell lines, the functions of IGF-I and -II are complex. Both growth factors appear capable of stimulating cellular proliferation and differentiation, as well as exerting insulin-like effects on intermediary metabolism. We have demonstrated recently that the expression of IGF-II and its receptor is induced during the terminal differentiation of the myoblast cell line, C2, and have suggested that IGF-II may be an autocrine growth factor in these cells (Tollefsen, S.E., Sadow, J.L., and Rotwein, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1543-1547). We now have examined this cell line for expression of other components involved in IGF signaling. The synthesis of IGF-I is low during myoblast proliferation; IGF-I mRNA can be detected only through use of a sensitive solution hybridization assay. Typical IGF-I receptors can be measured in myoblasts, whereas IGF binding proteins cannot be detected in proliferating cells or in conditioned culture medium. During myogenic differentiation, IGF-I mRNA levels increase transiently by 6-10-fold within 48-72 h. The expression of IGF-I mRNA is accompanied by a 2.5-fold accumulation of IGF-I in the culture medium. IGF-I receptors also increase transiently, doubling by 48 h after the onset of differentiation. By contrast, secretion of a Mr 29,000 IGF binding protein is induced 30-fold to 100 ng/ml within 16 h and continues to increase throughout differentiation. These studies demonstrate that several components critical to IGF action are produced in a fusing skeletal muscle cell line in a differentiation-dependent manner and suggest that both IGF-I and IGF-II may be autocrine factors for muscle.     

TGM2 positively regulates myoblast differentiation via enhancing the mTOR signaling

Myoblast differentiation is an essential process for the control of muscle regeneration. However, the intrinsic mechanisms underlying this dynamic process are still not well clarified. Herein, we identified transglutaminase type 2 (TGM2) as a novel regulator of muscle differentiation and regeneration in vitro and in vivo. Specifically, knockdown of TGM2 suppresses whereas overexpression of TGM2 promotes myoblast differentiation in differentiating C2C12 cells. Mechanistic studies revealed that TGM2 promotes C2C12 myoblast differentiation via enhancing GPR56 mediated activation of the mTOR signaling. Additionally, lentivirus mediated knockdown of TGM2 hinders the regeneration of muscles in a BaCl₂ induced skeletal muscle injury model of mice. Finally, we found that both TGM2 and activation of the mTOR signaling are up-regulated in muscles of patients with immune-mediated necrotizing myopathy (IMNM), especially in the regenerating myofibers. Collectively, our research demonstrates that TGM2 positively regulates muscle differentiation and regeneration through facilitating the myogenic mTOR signaling, which might be a potential target of therapy for skeletal muscle injury.     

IGFBP-5 regulates muscle cell differentiation by binding to IGF-II and switching on the IGF-II auto-regulation loop

IGF-II stimulates both mitogenesis and myogenesis through its binding and activation of the IGF-I receptor (IGF-IR). How this growth factor pathway promotes these two opposite cellular responses is not well understood. We investigate whether local IGF binding protein-5 (IGFBP-5) promotes the myogenic action of IGF-II. IGFBP-5 is induced before the elevation of IGF-II expression during myogenesis. Knockdown of IGFBP-5 impairs myogenesis and suppresses IGF-II gene expression. IGF-II up-regulates its own gene expression via the PI3K-Akt signaling pathway. Adding IGF-II or constitutively activating Akt rescues the IGFBP-5 knockdown-caused defects. However, an IGF analogue that binds to the IGF-IR but not IGFBP has only a limited effect. When added with low concentrations of IGF-II, IGFBP-5 restores IGF-II expression and myogenic differentiation, whereas an IGF binding–deficient IGFBP-5 mutant has no effect. These findings suggest that IGFBP-5 promotes muscle cell differentiation by binding to and switching on the IGF-II auto-regulation loop.     

Muscle-specific overexpression of the type 1 IGF receptor results in myoblast-independent muscle hypertrophy via PI3K, and not calcineurin, signaling

The insulin-like growth factors (IGF-I and IGF-II), working through the type 1 IGF receptor (IGF-1R), are key mediators of skeletal muscle fiber growth and hypertrophy. These processes are largely dependent on stimulation of proliferation and differentiation of muscle precursor cells, termed myoblasts. It has not been rigorously determined whether the IGFs can also mediate skeletal muscle hypertrophy in a myoblast-independent fashion. Similarly, although the phosphatidylinositol 3-kinase (PI3K) and calcineurin signaling pathways have been implicated in skeletal muscle hypertrophy, these pathways are also involved in skeletal myoblast differentiation. To determine whether the IGFs can stimulate skeletal muscle hypertrophy in a myoblast-independent fashion, we developed and validated a retroviral expression vector that mediated overexpression of the human IGF-1R in rat L6 skeletal myotubes (immature muscle fibers), but not in myoblasts. L6 myotubes transduced with this vector accumulated significantly higher amounts of myofibrillar proteins, in a ligand- and receptor-dependent manner, than controls and demonstrated significantly increased rates of protein synthesis. Stimulation of myotube hypertrophy was independent of myoblast contributions, inasmuch as these cultures did not exhibit increased levels of myoblast proliferation or differentiation. Experiments with PI3K and calcineurin inhibitors indicated that myoblast-independent myotube hypertrophy was mediated by PI3K, but not calcineurin, signaling. This study demonstrates that IGF can mediate skeletal muscle hypertrophy in a myoblast-independent fashion and suggests that muscle-specific overexpression of the IGF-1R or stimulation of its signaling pathways could be used to develop strategies to ameliorate muscle wasting without stimulating proliferative pathways leading to carcinogenesis or other pathological sequelae.     

JAK2/STAT2/STAT3 are required for myogenic differentiation

Skeletal muscle satellite cell-derived myoblasts are mainly responsible for postnatal muscle growth and injury-induced regeneration. However, the cellular signaling pathways that control proliferation and differentiation of myoblasts remain poorly defined. Recently, we found that JAK1/STAT1/STAT3 not only participate in myoblast proliferation but also actively prevent them from premature differentiation. Unexpectedly, we found that a related pathway consisting of JAK2, STAT2, and STAT3 is required for early myogenic differentiation. Interference of this pathway by either a small molecule inhibitor or small interfering RNA inhibits myogenic differentiation. Consistently, all three molecules are activated upon differentiation. The pro-differentiation effect of JAK2/STAT2/STAT3 is partially mediated by MyoD and MEF2. Interestingly, the expression of the IGF2 gene and the HGF gene is also regulated by JAK2/STAT2/STAT3, suggesting that this pathway could also promote differentiation by regulating signaling molecules known to be involved in myogenic differentiation. In summary, our current study reveals a novel role for the JAK2/STAT2/STAT3 pathway in myogenic differentiation.     

Stac3 Inhibits Myoblast Differentiation into Myotubes

The functionally undefined Stac3 gene, predicted to encode a SH3 domain- and C1 domain-containing protein, was recently found to be specifically expressed in skeletal muscle and essential to normal skeletal muscle development and contraction. In this study we determined the potential role of Stac3 in myoblast proliferation and differentiation, two important steps of muscle development. Neither siRNA-mediated Stac3 knockdown nor plasmid-mediated Stac3 overexpression affected the proliferation of C2C12 myoblasts. Stac3 knockdown promoted the differentiation of C2C12 myoblasts into myotubes as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA and protein expression of myogenic markers including myogenin and myosin heavy chain. In contrast, Stac3 overexpression inhibited the differentiation of C2C12 myoblasts into myotubes as evidenced by decreased fusion index, decreased number of nuclei per myotube, and decreased mRNA and protein expression of myogenic markers. Compared to wild-type myoblasts, myoblasts from Stac3 knockout mouse embryos showed accelerated differentiation into myotubes in culture as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA expression of myogenic markers. Collectively, these data suggest an inhibitory role of endogenous Stac3 in myoblast differentiation. Myogenesis is a tightly controlled program; myofibers formed from prematurely differentiated myoblasts are dysfunctional. Thus, Stac3 may play a role in preventing precocious myoblast differentiation during skeletal muscle development.     

Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells

Differentiation of embryonic and adult myogenic progenitors undergoes a complex series of cell rearrangements and specification events which are controlled by distinct gene regulatory networks. Delineation of the molecular mechanisms that regulate skeletal muscle specification and formation should be important for understanding congenital myopathies and muscular degenerative diseases. Retinoic acid (RA) signaling plays an important role in development. However, the role of RA signaling in adult myogenic progenitors is poorly understood. Here, we investigate the role of RA signaling in regulating myogenic differentiation of myoblastic progenitor cells. Using the mouse myoblast progenitor C2C12 line as a model, we have found that the endogenous expression of most RAR and RXR isotypes is readily detected. While the nuclear receptor co-repressors are highly expressed, two of the three nuclear receptor co-activators and the enzymes involved in RA synthesis are expressed at low level or undetectable, suggesting that the RA signaling pathway may be repressed in myogenic progenitors. Using the α-myosin heavy chain promoter-driven reporter (MyHC-GLuc), we have demonstrated that either ATRA or 9CRA is able to effectively induce myogenic differentiation, which can be synergistically enhanced when both ATRA and 9CRA are used. Upon ATRA and 9CRA treatment of C2C12 cells the expression of late myogenic markers significantly increases. We have further shown that adenovirus-mediated exogenous expression of RARα and/or RXRα is able to effectively induce myogenic differentiation in a ligand-independent fashion. Morphologically, ATRA- and 9CRA-treated C2C12 cells exhibit elongated cell body and become multi-nucleated myoblasts, and even form myoblast fusion. Ultrastructural analysis under transmission electron microscope reveals that RA-treated myogenic progenitor cells exhibit an abundant presence of muscle fibers. Therefore, our results strongly suggest that RA signaling may play an important role in regulating myogenic differentiation.     

PDGF-PDGFR network differentially regulates the fate,migration, proliferation,and cell cycle progression of myogenic cells

Platelet-derived growth factors (PDGFs) regulate embryonic development, tissue regeneration, and wound healing through their binding to PDGF receptors, PDGFRα and PDGFRβ. However, the role of PDGF signaling in regulating muscle development and regeneration remains elusive, and the cellular and molecular responses of myogenic cells are understudied. Here, we explore the PDGF-PDGFR gene expression changes and their involvement in skeletal muscle myogenesis and myogenic fate. By surveying bulk RNA sequencing and single-cell profiling data of skeletal muscle stem cells, we show that myogenic progenitors and muscle stem cells differentially express PDGF ligands and PDGF receptors during myogenesis. Quiescent adult muscle stem cells and myoblasts preferentially express PDGFRβ over PDGFRα. Remarkably, cell culture- and injury-induced muscle stem cell activation altered PDGF family gene expression. In myoblasts, PDGF-AB and PDGF-BB treatments activate two pro-chemotactic and pro-mitogenic downstream transducers, RAS-ERK1/2 and PI3K-AKT. PDGFRs inhibitor AG1296 inhibited ERK1/2 and AKT activation, myoblast migration, proliferation, and cell cycle progression induced by PDGF-AB and PDGF-BB. We also found that AG1296 causes myoblast G0/G1 cell cycle arrest. Remarkably, PDGF-AA did not promote a noticeable ERK1/2 or AKT activation, myoblast migration, or expansion. Also, myogenic differentiation reduced the expression of both PDGFRα and PDGFRβ, whereas forced PDGFRα expression impaired myogenesis. Thus, our data highlight PDGF signaling pathway to stimulate satellite cell proliferation aiming to enhance skeletal muscle regeneration and provide a deeper understanding of the role of PDGF signaling in non-fibroblastic cells.     

Cortisone and dexamethasone inhibit myogenesis by modulating the AKT/mTOR signaling pathway in C2C12

Myogenesis occurs in both the prenatal and postnatal periods and the prenatal myogenesis is related to the postnatal myogenesis and the incidence of disease later in life. Glucocorticoids used as therapeutic agents for many diseases, but cause adverse effects on muscle homeostasis, including defects in fetal muscle development. The action of glucocorticoids on differentiated skeletal muscle was well studied, but their effects on myotube formation have not been well investigated. Dexamethasone (DEX) and cortisone (COR), two synthetic therapeutic glucocorticoids, suppress myotube formation in C2C12 cells. Both COR and DEX attenuated myotube formation through modulation of myogenic regulatory factors. In addition, they affected the IGF/PI3K/AKT/mTOR signaling pathway, resulting in increased proteolytic protein (atrogin-1 and MURF1) for muscle degradation and decreased ribosomal S6 phosphorylation. The current results conclude that COR and DEX inhibit myotube formation in C2C12 cells by modulating both the myogenic program via MRFs and protein metabolism via IGF/PI3K/AKT/mTOR signaling pathway.     

Raptor and Rheb negatively regulate skeletal myogenesis through suppression of insulin receptor substrate 1 (IRS1)

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.