Skeletal muscle myogenesis is regulated by G protein-coupled receptor kinase 2

G protein-coupled receptor kinase 2 （GRK2） is an important serine/threonine-kinase regulating different membrane receptors and intraceUular proteins. Attenuation of Drosophila Gprk2 in embryos or adult flies induced a defective differentiation of somatic muscles, loss of fibers, and a flightless phenotype. In vertebrates, GRK2 hemizygous mice contained less but more hypertrophied skeletal muscle fibers than wild-type littermates. In C2C12 myoblasts, overexpression of a GRK2 kinase-deficient mutant （K220R） caused precocious differentiation of ceUs into immature myotubes, which were wider in size and contained more fused nuclei, while GRK2 overexpression blunted differentiation. Moreover, p38MAPK and Akt pathways were activated at an earlier stage and to a greater extent in K220R-expressing cells or upon kinase downregulation, while the activation of both kinases was impaired in GRK2-overexpressing cells. The impaired differentiation and fewer fusion events promoted by enhanced GRK2 levels were recapitulated by a p38MAPK mutant, which was able to mimic the inhibitory phosphorylation of p38MAPK by GRK2, whereas the blunted differentiation observed in GRK2-expressing clones was rescued in the presence of a constitutively active upstream stimulator of the p38MAPK pathway. These results suggest that balanced GRK2 function is necessary for a timely and complete myogenic process.     

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.     

PI3K/Akt/mTOR信号通路及临床相关肿瘤抑制剂

哺乳动物雷帕霉素靶（mTOR）和蛋白激酶B（Akt/PKB）与肿瘤发生的密切关系已被广泛地认可.mTOR是一种丝/苏氨酸激酶,可以通过影响mRNA转录、代谢、自噬等方式调控细胞的生长.它既是PI3K的效应分子,也可以是PI3K的反馈调控因子.mTORC1 和mTORC2是mTOR的两种不同复合物. 对雷帕霉素敏感的mTORC1受到营养、生长因子、能量和应激4种因素的影响.生长因子通过PI3K/Akt信号通路调控mTORC1是最具特征性调节路径.而mTORC2最为人熟知的是作为Akt473磷酸化位点的上游激酶. 同样,Akt/PKB在细胞增殖分化、迁移生长过程中发挥着重要作用. 随着Thr308和Ser473两个位点激活,Akt/PKB也得以全面活化.因此,mTORC2-Akt-mTORC1的信号通路在肿瘤形成和生长中是可以存在的.目前临床肿瘤治疗中,PI3K/Akt/mTOR是重要的靶向治疗信号通路.然而,仅抑制mTORC1活性,不是所有的肿瘤都能得到预期控制.雷帕霉素虽然能抑制mTORC1,但也能反馈性地增加PI3K信号活跃度,从而影响治疗预后.近来发现的第二代抑制剂可以同时抑制mTORC1/2和PI3K活性,这种抑制剂被认为在肿瘤治疗上颇具前景.本综述着重阐述了PI3K/Akt/mTOR信号通路的传导、各因子之间的相互调控以及相关抑制剂的发展.     

Effects of PI3K catalytic subunit and Akt isoform deficiency on mTOR and p70S6K activation in myoblasts

The PI3K/Akt/mTOR signaling pathway is critical for cellular growth and survival in skeletal muscle, and is activated in response to growth factors such as insulin-like growth factor-I (IGF-I). We found that in C2C12 myoblasts, deficiency of PI3K p110 catalytic subunits or Akt isoforms had distinct effects on phosphorylation of mTOR and p70S6K. siRNA-mediated knockdown of PI3K p110α, p110β, and simultaneous knockdown of p110α and p110β resulted in increased basal and IGF-I-stimulated phosphorylation of mTOR S2448 and p70S6K T389; however, phosphorylation of S6 was reduced in p110β-deficient cells, possibly due to reductions in total S6 protein. We found that IGF-I-stimulated Akt1 activity was enhanced in Akt2- or Akt3-deficient cells, and that knockdown of individual Akt isoforms increased mTOR/p70S6K activation in an isoform-specific fashion. Conversely, levels of IGF-I-stimulated p70S6K phosphorylation in cells simultaneously deficient in both Akt1 and Akt3 were increased beyond those seen with loss of any single Akt isoform, suggesting an alternate, Akt-independent mechanism that activates mTOR/p70S6K. Our results collectively suggest that mTOR/p70S6K is activated in a PI3K/Akt-dependent manner, but that in the absence of p110α or Akt, alternate pathway(s) may mediate activation of mTOR/p70S6K in C2C12 myoblasts.     

PI3K和Akt蛋白在异丙肾上腺素所致大鼠心肌肥厚中的表达

目的研究异丙肾上腺素(ISO)致大鼠心肌肥厚中PI3K和Akt在心肌组织中的表达,为探讨心肌肥厚的信号转导机制和逆转心肌肥厚提供形态学资料.方法健康成年SD大鼠20只,随机分为实验组、对照组,每组10只.实验组给予异丙肾上腺素处理.1周后处死大鼠,取心肌组织,常规石蜡切片,HE染色,观察心肌组织的病理变化,测量心肌肥厚指标;免疫组织化学染色和免疫荧光染色,检测p-PI3K和p-Akt的表达及分布.结果实验组大鼠心肌肥厚指标与对照组相比均明显升高;免疫组织化学检测显示,实验组心肌组织p-PI3K和p-Akt蛋白表达面积和平均光密度较对照组高.免疫荧光检测实验组心肌组织p-PI3K和p-Akt蛋白表达较对照组高.结论小剂量持续给予 ISO 能建立大鼠心肌肥厚模型;p-PI3K和p-Akt蛋白表达均与心肌肥厚的发生和发展过程相关,PI3K/Akt信号通路激活,可能是导致心肌肥厚的机制之一.     

Discovery and SAR exploration of a novel series of imidazo[4,5-b]pyrazin-2-ones as potent and selective mTOR kinase inhibitors

We report here the discovery of a novel series of selective mTOR kinase inhibitors. A series of imidazo[4,5-b]pyrazin-2-ones, represented by screening hit 1, was developed into lead compounds with excellent mTOR potency and exquisite kinase selectivity. Potent compounds from this series show >1000-fold selectivity over the related PI3Kα lipid kinase. Further, compounds such as 2 achieve mTOR pathway inhibition, blocking both mTORC1 and mTORC2 signaling, in PC3 cancer cells as measured by inhibition of pS6 and pAkt (S473).     

Apelin inhibits the proliferation and migration of rat PASMCs via the activation of PI3K/Akt/mTOR signal and the inhibition of autophagy under hypoxia

Apelin is highly expressed in the lungs, especially in the pulmonary vasculature, but the functional role of apelin under pathological conditions is still undefined. Hypoxic pulmonary hypertension is the most common cause of acute right heart failure, which may involve the remodeling of artery and regulation of autophagy. In this study, we determined whether treatment with apelin regulated the proliferation and migration of rat pulmonary arterial smooth muscle cells (SMCs) under hypoxia, and investigated the underlying mechanism and the relationship with autophagy. Our data showed that hypoxia activated autophagy significantly at 24 hrs. The addition of exogenous apelin decreased the level of autophagy and further inhibited pulmonary arterial SMC (PASMC) proliferation via activating downstream phosphatidylinositol‐3‐kinase (PI3K)/protein kinase B (Akt)/the mammalian target of Rapamycin (mTOR) signal pathways. The inhibition of the apelin receptor (APJ) system by siRNA abolished the inhibitory effect of apelin in PASMCs under hypoxia. This study provides the evidence that exogenous apelin treatment contributes to inhibit the proliferation and migration of PASMCs by regulating the level of autophagy.     

A hit to lead discovery of novel N-methylated imidazolo-, pyrrolo-, and pyrazolo-pyrimidines as potent and selective mTOR inhibitors

A series of N-7-methyl-imidazolopyrimidine inhibitors of the mTOR kinase have been designed and prepared, based on the hypothesis that the N-7-methyl substituent on imidazolopyrimidine would impart selectivity for mTOR over the related PI3Kα and δ kinases. The corresponding N-Me substituted pyrrolo[3,2-d]pyrimidines and pyrazolo[4,3-d]pyrimidines also show potent mTOR inhibition with selectivity toward both PI3α and δ kinases. The most potent compound synthesized is pyrazolo[4,3-d]pyrimidine 21c. Compound 21c shows a K_i of 2 nM against mTOR inhibition, remarkable selectivity (>2900×) over PI3 kinases, and excellent potency in cell-based assays.     

The Therapeutic Potential of PI3K/Akt/mTOR Inhibitors in Breast Cancer: Rational and Progress

Breast cancer (BC) is the most commonly diagnosed cancer in women. The PI3K/AKT/mTOR pathway is among the most frequently dysregulated pathways in patients with BC. The activation of this pathway is associated with increased cell growth and clinical outcome, and its overexpression is associated with a poor prognosis. It has been proposed that it may be of importance as a potential therapeutic target in the treatment of BC. The aim of current review is to provide an overview of the potential utility of PI3K/Akt/mTOR inhibitors in patients with BC, with particular emphasis on recent preclinical and clinical studies. J. Cell. Biochem. 119: 213–222, 2018. © 2017 Wiley Periodicals, Inc.     

Attenuation of cardiac hypertrophy by inhibiting both mTOR and NFkappaB activation in vivo

A role for the PI3K/Akt/mTOR pathway in cardiac hypertrophy has been well documented. We reported that NFκB activation is needed for cardiac hypertrophy in vivo. To investigate whether both NFκB activation and PI3K/Akt/mTOR signaling participate in the development of cardiac hypertrophy, two models of cardiac hypertrophy, namely, induction in caAkt-transgenic mice and by aortic banding in mice, were employed. Rapamycin (2 mg/kg/daily), an inhibitor of the mammalian target of rapamycin, and the antioxidant pyrrolidine dithiocarbamate (PDTC; 120 mg/kg/daily), which can inhibit NFκB activation, were administered to caAkt mice at 8 weeks of age for 2 weeks. Both rapamycin and PDTC were also administered to the mice immediately after aortic banding for 2 weeks. Administration of either rapamycin or PDTC separately or together to caAkt mice reduced the ratio of heart weight/body weight by 21.54, 32.68, and 42.07% compared with untreated caAkt mice. PDTC administration significantly reduced cardiac NFκB activation by 46.67% and rapamycin significantly decreased the levels of p70S6K by 34.20% compared with untreated caAkt mice. Similar results were observed in aortic-banding-induced cardiac hypertrophy in mice. Our results suggest that both NFκB activation and the PI3K/Akt signaling pathway participate in the development of cardiac hypertrophy in vivo.     

Melatonin protects against methotrexate hepatotoxicity in young rats: Impact of PI3K/Akt/mTOR signaling

With the improvement in children's acute lymphoblastic leukemia (ALL) care, the survival rate in children ALL has improved much. Methotrexate (MTX) plays an essential role in the success of children's ALL treatment. Since hepatotoxicity is commonly reported in individuals treated with intravenous or oral MTX, our study further examined the hepatic effect following intrathecal MTX treatment, which is an essential treatment for leukemia patients. Specifically, we examined the pathogenesis of MTX hepatotoxicity in young rats and explored the impact of melatonin treatment in protection against MTX hepatotoxicity. Successfully, we found that melatonin was able to protect against MTX hepatotoxicity.     

Exosome biogenesis,secretion and function of exosomal miRNAs in skeletal muscle myogenesis

Exosomes are membrane‐bound extracellular vesicles that are produced in the endosomal compartment of most mammalian cell types and then released. Exosomes are effective carriers for the intercellular material transfer of material that can influence a series of physiological and pathological processes in recipient cells. Among loaded cargoes, non‐coding RNAs (ncRNAs) vary for the exosome‐producing cell and its homeostatic state, and characterization of the biogenesis and secretion of exosomal ncRNAs and the functions of these ncRNAs in skeletal muscle myogenesis remain preliminary. In this review, we will describe what is currently known of exosome biogenesis, release and uptake of exosomal ncRNAs, as well as the varied functions of exosomal miRNAs in skeletal muscle myogenesis.     

GRP94 promotes muscle differentiation by inhibiting the PI3K/AKT/mTOR signaling pathway

The glucose-regulated endoplasmic reticulum chaperone protein 94 (GRP94) is required for many biological processes, such as secretion of immune factors and mesoderm induction. Here, we demonstrated that GRP94 promotes muscle differentiation in vitro and in vivo. Moreover, GRP94 inhibited the PI3K/AKT/mTOR signaling pathway. Using both in vitro and in vivo approaches, in myoblasts, we found that this inhibition resulted in reduced proliferation and increased differentiation. To further investigate the mechanism of GRP94-induced muscle differentiation, we used co-immunoprecipitation and proximity ligation assays and found that GRP94 interacted with PI3K-interacting protein 1 (Pik3ip1). The latter protein promoted muscle differentiation by inhibiting the PI3K/AKT/mTOR pathway. Furthermore, GRP94 was found to regulate Pik3ip1 expression. Finally, when Pik3ip1 expression was inhibited, GRP94-induced promotion of muscle differentiation was diminished. Taken together, our data demonstrated that GRP94 promoted muscle differentiation, mediated by Pik3ip1-dependent inhibition of the PI3K/AKT/mTOR signaling pathway.     

Follistatin could promote the proliferation of duck primary myoblasts by activating PI3K/Akt/mTOR signalling

FST (follistatin) is essential for skeletal muscle development, but the intracellular signalling networks that regulate FST-induced effects are not well defined. We sought to investigate whether FST promotes the proliferation of myoblasts through the PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) signalling. In the present study, we transfected the pEGFP-duFST plasmid and added PI3K and mTOR inhibitors to the medium of duck primary myoblasts. Then, we analysed the cellular phenotypic changes that occurred and analysed the expression of target genes. The results showed that FST promoted myoblast proliferation, induced the mRNA expression of PI3K, Akt, mTOR, 70-kDa ribosomal protein S6K (S6 kinase) and the protein expression of phospho-Akt (Thr³⁰⁸), mTOR, phospho-mTOR (serine 2448), phospho-S6K (Ser⁴¹⁷), inhibited the mRNA expression of FoxO1, MuRF1 (muscle RING finger-1) and the protein expression of phospho-FoxO1 (Ser²⁵⁶). Moreover, we found that the overexpression of FST could alleviate the inhibitory effect of myoblast proliferation caused by the addition of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor. Additionally, the overexpression of duck FST also relieved the inhibition of myoblast proliferation caused by the addition of rapamycin (an mTOR inhibitor) through PI3K/Akt/mTOR signalling. In light of the present results, we hypothesize that duck FST could promote myoblast proliferation, which is dependent on PI3K/Akt/mTOR signalling.     

Extracellular matrix is required for skeletal muscle differentiation but not myogenin expression

Skeletal muscle cells are a useful model for studying cell differentiation. Muscle cell differentiation is marked by myoblast proliferation followed by progressive fusion to form large multinucleated myotubes that synthesize muscle-specific proteins and contract spontaneously. The molecular analysis of myogenesis has advanced with the identification of several myogenic regulatory factors, including myod1, myd, and myogenin. These factors regulate each other's expression and that of muscle-specific proteins such as the acetylcholine receptor and acetylcholinesterase (AChE). In order to investigate the role of extracellular matrix (ECM) in myogenesis we have cultured myoblasts (C₂C₁₂) in the presence or absence of an exogenous ECM (Matrigel). In addition, we have induced differentiation of myoblasts in the presence or absence of Matrigel and/or chlorate, a specific inhibitor of proteoglycan sulfation. Our results indicated that the formation of fused myotubes and expression of AChE was stimulated by Matrigel. Treatment of myoblasts induced to differentiate with chlorate resulted in an inhibition of cell fusion and AChE activity. Chlorate treatment was also found to inhibit the deposition and assembly of ECM components such fibronectin and laminin. The expression of myogenin mRNA was observed when myoblasts were induced to differentiate, but was unaffected by the presence of Matrigel or by culture of the cells in the presence of chlorate. These results suggest that the expression of myogenin is independent of the presence of ECM, but that the presence of ECM is essential for the formation of myotubes and the expression of later muscle-specific gene products. © 1996 Wiley-Liss, Inc.