miR-22 regulates C2C12 myoblast proliferation and differentiation by targeting TGFBR1

Recently, miR-22 was found to be differentially expressed in different skeletal muscle growth period, indicated that it might have function in skeletal muscle myogenesis. In this study, we found that the expression of miR-22 was the most in skeletal muscle and was gradually up-regulated during mouse myoblast cell (C2C12 myoblast cell line) differentiation. Overexpression of miR-22 repressed C2C12 myoblast proliferation and promoted myoblast differentiation into myotubes, whereas inhibition of miR-22 showed the opposite results. During myogenesis, we predicted and verified transforming growth factor beta receptor 1 (TGFBR1), a key receptor of the TGF-β/Smad signaling pathway, was a target gene of miR-22. Then, we found miR-22 could regulate the expression of TGFBR1 and down-regulate the Smad3 signaling pathway. Knockdown of TGFBR1 by siRNA suppressed the proliferation of C2C12 cells but induced its differentiation. Conversely, overexpression of TGFBR1 significantly promoted proliferation but inhibited differentiation of the myoblast. Additionally, when C2C12 cells were treated with different concentrations of transforming growth factor beta 1 (TGF-β1), the level of miR-22 in C2C12 cells was reduced. The TGFBR1 protein level was significantly elevated in C2C12 cells treated with TGF-β1. Moreover, miR-22 was able to inhibit TGF-β1-induced TGFBR1 expression in C2C12 cells. Altogether, we demonstrated that TGF-β1 inhibited miR-22 expression in C2C12 cells and miR-22 regulated C2C12 cell myogenesis by targeting TGFBR1.     

c-Myb Inhibits Myoblast Fusion

Satellite cells represent a heterogeneous population of stem and progenitor cells responsible for muscle growth, repair and regeneration. We investigated whether c-Myb could play a role in satellite cell biology because our previous results using satellite cell-derived mouse myoblast cell line C2C12 showed that c-Myb was expressed in growing cells and downregulated during differentiation. We detected c-Myb expression in activated satellite cells of regenerating muscle. c-Myb was also discovered in activated satellite cells associated with isolated viable myofiber and in descendants of activated satellite cells, proliferating myoblasts. However, no c-Myb expression was detected in multinucleated myotubes originated from fusing myoblasts. The constitutive expression of c-Myb lacking the 3′ untranslated region (3′ UTR) strongly inhibited the ability of myoblasts to fuse. The inhibition was dependent on intact c-Myb transactivation domain as myoblasts expressing mutated c-Myb in transactivation domain were able to fuse. The absence of 3′ UTR of c-Myb was also important because the expression of c-Myb coding region with its 3′ UTR did not inhibit myoblast fusion. The same results were repeated in C2C12 cells as well. Moreover, it was documented that 3′ UTR of c-Myb was responsible for downregulation of c-Myb protein levels in differentiating C2C12 cells. DNA microarray analysis of C2C12 cells revealed that the expression of several muscle-specific genes was downregulated during differentiation of c-Myb-expressing cells, namely: ACTN2, MYH8, TNNC2, MYOG, CKM and LRRN1. A detailed qRT-PCR analysis of MYOG, TNNC2 and LRRN1 is presented. Our findings thus indicate that c-Myb is involved in regulating the differentiation program of myogenic progenitor cells as its expression blocks myoblast fusion.     

Polyomavirus genome and polyomavirus enhancer-driven gene expression during myogenesis.

The mRNAs for myogenic functions are coordinately transcribed with polyomavirus (Py) early mRNA during in vitro differentiation of mouse C2 myoblast cells. Sequence analysis shows that the A domain of the Py enhancer includes an E1A-like consensus sequence that is also found in the 5' upstream region of two genes expressed during myoblast differentiation: alpha-actin and myosin light chain. Therefore, the coordinate expression of such genes with Py early mRNA may be activated by a common cellular regulatory factor. In the present work, we report that C2 cells surviving Py infection are unable to differentiate and do not express alpha-actin and myosin light-chain mRNAs. Hybrids between such Py-resistant myoblast cells and the parental cells exhibited dominance of the permissibility to Py growth and of the expression of myogenic mRNAs. In C2 cells transiently transfected with a chimeric plasmid (pSVPy12CAT) harboring the bacterial chloramphenicol acetyltransferase (CAT) gene driven by the Py enhancer-promoter region, the CAT gene was expressed irrespective of their stage of differentiation. Moreover, undifferentiated stably transfected cells expressing the CAT gene restricted viral growth. Py-resistant C2 myoblasts transiently transfected with pSVPy12CAT also expressed the CAT gene driven by the Py enhancer. This contradictory finding is similar to results previously obtained by other investigators with cloned genes specific for myogenic functions, and it may be explained by a structural difference between the pSVPy12CAT and the Py genomic organizations in which the viral enhancer operates.     

RIP2 regulates growth and differentiation of normal myoblasts and of rhabdomyosarcoma cells

RIP2 is an important regulator of myoblast proliferation and differentiation. We have previously demonstrated that in the myoblast cell line C2C12 and in primary myoblasts, downregulation of rip2 gene expression is a prerequisite for differentiation. To further study the role of rip genes in myogenesis, we compared expression patterns of rip1–4 in two myoblast cell lines, C2C12 and C2F3, after the induction of differentiation. These two cell lines are derived from the same clonal origin, but differ with respect to their differentiation behaviour: specifically, the differentiation process is slower and more incomplete in C2F3 cells. When analyzing cells up to 4 days after the induction of differentiation, we found no downregulation of rip2 gene expression in C2F3 cells, which might be linked to the low differentiation potential of these cells. In addition, in contrast to C2C12 cells, the rip3 gene was not expressed in C2F3 cells. To further study the role of rip genes in the regulation of myoblast growth and differentiation, we analyzed expression patterns of rip1–4 in rhabdomyosarcoma cell lines. We found that in these cells, rip2 expression was not downregulated after the induction of differentiation. Furthermore, in contrast to normal myoblasts, they did not express the rip3 and rip4 genes. Thus, we focused on the functional role of RIP2 in rhabdomyosarcoma cells. Inhibition of rip2 gene expression in C2C12 and in rhabdomyosarcoma cells using specific siRNAs led to decreased proliferation and promoted the differentiation process of these cells. These data indicate that differential expression of rip genes can be associated with abnormal growth and differentiation behaviour of skeletal myoblasts.     

Stevioside counteracts the alpha-cell hypersecretion caused by long-term palmitate exposure

Long-term exposure to fatty acids impairs beta-cell function in type 2 diabetes, but little is known about the chronic effects of fatty acids on alpha-cells. We therefore studied the prolonged impact of palmitate on alpha-cell function and on the expression of genes related to fuel metabolism. We also investigated whether the antihyperglycemic agent stevioside was able to counteract these effects of palmitate. Clonal alpha-TC1-6 cells were cultured with palmitate in the presence or absence of stevioside. After 72 h, we evaluated glucagon secretion, glucagon content, triglyceride (TG) content, and changes in gene expression. Glucagon secretion was dose-dependently increased after 72-h culture, with palmitate at concentrations >or=0.25 mM (P< 0.05). Palmitate (0.5 mM) enhanced TG content of alpha-cells by 73% (P< 0.01). Interestingly, stevioside (10(-8) and 10(-6) M) reduced palmitate-stimulated glucagon release by 22 and 45%, respectively (P< 0.01). There was no significant change in glucagon content after 72-h culture with palmitate and/or stevioside. Palmitate increased carnitine palmitoyltransferase I (CPT I) mRNA level, whereas stevioside enhanced CPT I, peroxisome proliferator-activated receptor-gamma, and stearoyl-CoA desaturase gene expressions in the presence of palmitate (P<0.05). In conclusion, long-term exposure to elevated fatty acids leads to a hypersecretion of glucagon and an accumulation of TG content in clonal alpha-TC1-6 cells. Stevioside was able to counteract the alpha-cell hypersecretion caused by palmitate and enhanced the expression of genes involved in fatty acid metabolism. This indicates that stevioside may be a promising antidiabetic agent in treatment of type 2 diabetes.     

FHL1 Reduces Dystrophy in Transgenic Mice Overexpressing FSHD Muscular Dystrophy Region Gene 1 (FRG1)

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1.     

Transforming growth factor-beta isoform expression in insulin-like growth factor stimulated myogenesis

Transforming growth factor betas (TGF-b?s) are the defining members of a superfamily of small proteins that are involved in the regulation of development and morphogenesis in a wide array of systems. Previous studies have demonstrated that TGF-b?s both inhibit and, under specialized conditions, induce the differentiation of myoblasts. TGF-b?s have been shown to be secreted by mouse C2C12 myoblast cultures undergoing differentiation. Insulin-like growth factors (IGFs) have also been shown to be secreted by myoblasts and to induce myogenesis. This study characterizes the effects of IGF treatment on the expression and secretion of TGF-b?s in the IGF-sensitive L6A1 myoblast line. IGF downregulated the expression of TGF-b?3 in a concentration-dependent manner at 24 and 48 hours; TGF-b?1 was not sensitive to IGF treatment at 24 hours but was downregulated by IGFs at 48 hours. This downregulation was mediated by the type I IGF receptor and modulated by IGF binding proteins secreted by the myoblasts. Some reexpression of TGF-b?1 and TGF-b?3 mRNAs was observed after extensive morphological differentiation had occurred. These results support the hypothesis that IGFs act through the IGF type I receptor as part of a concerted mechanism to modulate expression of the TGF-b? genes, as part of a coordinated set of changes associated with terminal myogenic differentiation. © 1995 Wiley-Liss, Inc.     

人骨保护素(OPG)重组腺病毒的制备及其生物活性研究

采用RT-PCR法得到人OPG的编码区cDNA,克隆至穿梭质粒pShuttle,构建重组有OPG编码区cDNA的腺病毒DNA,经Pac I 酶切线性化,在脂质体介导下转染HEK293细胞,制备重组腺病毒并测定病毒滴度约为5×10⁶～1.5×10⁷ pfu/mL。体外感染小鼠成肌细胞C2C12,Western blot及ELISA检测证实有OPG蛋白的表达,并可在细胞培养上清中持续表达6周。感染OPG重组腺病毒的C2C12细胞生长状态良好、细胞周期无明显变化。将重组腺病毒加入体外培养的小鼠骨髓细胞的培养基中,诱导形成的破骨细胞数量及在象牙片上形成的吸收陷窝的数量显著减少（P<0.01）。      

Partial characterization of skeletal myoblast mitogens in mouse crushed muscle extract.

We have utilized a model system to investigate myotrophic factors released by normal adult mouse muscles following a crush injury. We found that saline extracts from gently crushed mouse muscles (CME) contain potent mitogenic activities which act on primary newborn mouse myoblast cultures, as well as on mouse C2 cells, a mouse myoblast cell line. We compared the activity of CME on mouse myoblasts with that of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I), two growth factors known to be mitogenic for primary myoblasts (Allen, Dodson, and Lutein: Exp. Cell. Res., 152:154-160, 1984; DiMario and Strohman: Differentiation, 39:42-49, 1988; Allen and Boxhorn: J. Cell. Physiol., 138:311-315, 1989; Dodson, Allen, and Hossner: Endocrinology, 117:2357-2363, 1985; Florini and Magri: Am. J. Physiol., 256:C701-C711, 1989). We found that CME could act in an additive fashion to saturating doses of bFGF to increase proliferation in myoblast cultures. Additionally, CME acted additively to the combination of saturating amounts of bFGF and IGF-I on both C2 and primary myoblast cultures. We also examined additivity of CME with the combination of saturating doses of bFGF, IGF-I, transferrin (Tf), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), adrenocorticotrophin (ACTH), and macrophage colony-stimulating factor (M-CSF). Our data indicate that CME contains Tf, as well as one or more uncharacterized mitogens for myoblasts which are distinct from Tf, the IGFs, bFGF, EGF, PDGF, M-CSF, and ACTH. These uncharacterized mitogens may act independently of known growth factors to stimulate myoblast proliferation, or may act through modulation of known growth factor activities.     

Transfection of a DNA locus that mediates the conversion of 10T1/2 fibroblasts to myoblasts

Stable myoblast cell lines were isolated after a brief exposure of mouse fibroblasts (10T1/2 cells) to 5-azacytidine. We show that transfection of 10T1/2 cells with DNA from these azacytidine-induced myoblasts (or from mouse C2C12 myoblasts) results in myogenic conversion of approximately 1 in 15,000 transfected colonies. In contrast, transfection of 10T1/2 cells with DNA from nonmyogenic cells (parental 10T1/2 cell DNA) does not give rise to myoblast colonies. These results indicate that an azacytidine-induced structural modification (presumably demethylation) in the DNA of a single locus is sufficient to convert 10T1/2 cells into determined myoblasts.     

Retrovirally mediated overexpression of insulin-like growth factor binding protein 4: Evidence that insulin-like growth factor is required for skeletal muscle differentiation

Recent studies have indicated that the insulin-like growth factors (IGFs) stimulate skeletal myoblast proliferation and differentiation. However, the question of whether IGFs are required for myoblast differentiation has not been resolved. To address this issue directly, we used a retroviral vector (LBP4SN) to develop a subline of mouse C2 myoblasts (C2-BP4) that constitutively overexpress IGF binding protein-4 (IGFBP-4). A control C2 myoblast subline (C2-LNL6) was also developed by using the LNL6 control retroviral vector. C2-BP4 myoblasts expressed sixfold higher levels of IGFBP-4 protein than C2-LNL6 myoblasts. ¹²⁵I-IGF-I cross linking indicated that IGFBP-4 overexpression reduced IGF access to the type-1 IGF receptor tenfold. At low plating densities, myoblast proliferation was inhibited, and myoblast differentiation was abolished in C2-BP4 cultures compared with C2-LNL6 cultures. At high plating densities in which nuclear numbers were equal in the two sets of cultures, C2-BP4 myoblast differentiation was inhibited completely. Differentiation was restored in C2-BP4 cells by treatment with high levels of exogenous IGF-I or with des(1–3)IGF-I, an analog of IGF-I with reduced affinity for IGFBPs. These findings confirm the hypothesis that positive differentiation signals from the IGFs are necessary for C2 myoblast differentiation, and they suggest that the present model of myogenic differentiation, which involves only negative external control of differentiation by mitogens, may be incomplete. J. Cell. Physiol. 175:109–120, 1998. © 1998 Wiley-Liss, Inc.     

DN-AMPK腺病毒载体的构建及其在小鼠成肌细胞系C2C12肌管分化中的影响

在研究AMPK的调控网络时,通常利用过表达显性失活突变型AMPK(dominant negative AMPK,DN-AMPK)作为研究手段来验证AMPK在某些重要生理病理调节通路中的关键作用。旨在利用Ad5腺病毒载体体系构建Ad-DN-AMPK表达载体,并在成肌细胞系C2C12中检测无活性AMPK高表达后对C2C12细胞分化为肌管细胞的影响。通过构建AMPKα1(D159A)和AMPKα2(K45R)的腺病毒表达载体,在HEK293细胞中成功包装并扩增出完整的腺病毒,待其感染能力基本稳定后,将腺病毒感染C2C12,利用激光定量成像仪检测其感染滴度,感染效率能高达100%,并且能够持续表达6 d。DN-AMPK高表达后,AMPK常用激活剂A769662(SN-5)不能激活AMPK,表现为AMPK下游蛋白活性丧失,如ACC磷酸化无变化。通过实时定量PCR的方法,检测DN-AMPK对C2C12分化为肌管细胞的影响,结果表明过表达DN-AMPK能够促进C2C12细胞分化为肌管细胞的标记蛋白(Myod和Myogenin)的表达,即促进C2C12分化为肌管细胞。     

Diminished G1 checkpoint after gamma-irradiation and altered cell cycle regulation by insulin-like growth factor II overexpression

High levels of insulin-like growth factor II (IGFII) mRNA expression are detected in many human tumors of different origins including rhabdomyosarcoma, a tumor of skeletal muscle origin. To investigate the role of IGFII in tumorigenesis, we have compared the mouse myoblast cell line C2C12-2.7, which was stably transfected with human IGFII cDNA and expressed high and constant amounts of IGFII, to a control cell line C2C12-1.1. A rhabdomyosarcoma cell line, RH30, which expresses high levels of IGFII and contains mutated p53, was also used in these studies. IGFII overexpression in mouse myoblast C2C12 cells causes a reduced cycling time and higher growth rate. After gamma-irradiation treatment, C2C12-1.1 cells were arrested mainly in G0/G1 phase. However, C2C12-2.7 and RH30 cells went through a very short G1 phase and then were arrested in an extended G2/M phase. To verify further the effect of IGFII on the cell cycle, we developed a Chinese hamster ovary (CHO) cell line with tetracycline-controlled IGFII expression. We found that CHO cells with high expression of IGFII have a shortened cycling time and a diminished G1 checkpoint after treatment with methylmethane sulfonate (MMS), a DNA base-damaging agent, when compared with CHO cells with very low IGFII expression. It was also found that IGFII overexpression in C2C12 cells was associated with increases in cyclin D1, p21, and p53 protein levels, as well as mitogen-activated protein kinase activity. These studies suggest that IGFII overexpression shortens cell cycling time and diminishes the G1 checkpoint after DNA damage despite an intact p53/p21 induction. In addition, IGFII overexpression is also associated with multiple changes in the levels and activities of cell cycle regulatory components following gamma-irradiation. Taken together, these changes may contribute to the high growth rate and genetic alterations that occur during tumorigenesis.