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Segregation of myoblast fusion and muscle-specific gene expression by distinct ligand-dependent inactivation of GSK-3β.

Pansters NA, van der Velden JL, Kelders MC, Laeremans H, Schols AM, Langen RC - Cell. Mol. Life Sci. (2010)

Bottom Line: Furthermore, LiCl and IGF-I, but not Wnt-3a, increased NFATc3 transcriptional activity.In contrast, β-catenin-dependent transcriptional activity was increased by Wnt-3a and LiCl, but not IGF-I.These results for the first time reveal a segregated regulation of myoblast fusion and muscle-specific gene expression following stimulation of myogenic differentiation in response to distinct ligand-specific signaling routes of GSK-3β inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 5800, 6202 AZ Maastricht, The Netherlands.

ABSTRACT
Myogenic differentiation involves myoblast fusion and induction of muscle-specific gene expression, which are both stimulated by pharmacological (LiCl), genetic, or IGF-I-mediated GSK-3β inactivation. To assess whether stimulation of myogenic differentiation is common to ligand-mediated GSK-3β inactivation, myoblast fusion and muscle-specific gene expression were investigated in response to Wnt-3a. Moreover, crosstalk between IGF-I/GSK-3β/NFATc3 and Wnt/GSK-3β/β-catenin signaling was assessed. While both Wnt-3a and LiCl promoted myoblast fusion, muscle-specific gene expression was increased by LiCl, but not by Wnt-3a or β-catenin over-expression. Furthermore, LiCl and IGF-I, but not Wnt-3a, increased NFATc3 transcriptional activity. In contrast, β-catenin-dependent transcriptional activity was increased by Wnt-3a and LiCl, but not IGF-I. These results for the first time reveal a segregated regulation of myoblast fusion and muscle-specific gene expression following stimulation of myogenic differentiation in response to distinct ligand-specific signaling routes of GSK-3β inactivation.

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Muscle-specific gene expression during differentiation is stimulated by LiCl but not by Wnt-3a. C2C12 myoblast cells were cultured in DM with or without LiCl (10 mM), control, or Wnt-3a CM (diluted 1/10, or as indicated, in DM). a After 72 h, lysates were prepared for determination of muscle creatine kinase activity and total protein. Results are expressed as specific enzyme activity (units/mg protein). b After 72 h, lysates were prepared to determine mRNA expression levels of MCK, MyHC IIB, and perinatal. c Myoblasts containing a stable genomically integrated troponin I (TnI) luciferase reporter construct were cultured for 48 h in DM, ±LiCl (10 mM), or Wnt-3a or control-CM (1/10 diluted in DM). Alternatively, (d) C2C12 myoblasts were transfected with a 4RTK luciferase-reporter construct and plasmid encoding β-gal (0.25μg each) and cultured in DM with or without LiCl (10 mM), control, or Wnt-3a CM (diluted 1/10 in DM). Lysates were prepared for luciferase and β-galactosidase enzyme activity (RLU/mg protein) or (RLU luciferase/β-gal activity). Shown are representative graphs of three independent experiments (n = 3 ± SEM), *p < 0.05, #p < 0.01, $p < 0.001, and NS non-significant
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Fig2: Muscle-specific gene expression during differentiation is stimulated by LiCl but not by Wnt-3a. C2C12 myoblast cells were cultured in DM with or without LiCl (10 mM), control, or Wnt-3a CM (diluted 1/10, or as indicated, in DM). a After 72 h, lysates were prepared for determination of muscle creatine kinase activity and total protein. Results are expressed as specific enzyme activity (units/mg protein). b After 72 h, lysates were prepared to determine mRNA expression levels of MCK, MyHC IIB, and perinatal. c Myoblasts containing a stable genomically integrated troponin I (TnI) luciferase reporter construct were cultured for 48 h in DM, ±LiCl (10 mM), or Wnt-3a or control-CM (1/10 diluted in DM). Alternatively, (d) C2C12 myoblasts were transfected with a 4RTK luciferase-reporter construct and plasmid encoding β-gal (0.25μg each) and cultured in DM with or without LiCl (10 mM), control, or Wnt-3a CM (diluted 1/10 in DM). Lysates were prepared for luciferase and β-galactosidase enzyme activity (RLU/mg protein) or (RLU luciferase/β-gal activity). Shown are representative graphs of three independent experiments (n = 3 ± SEM), *p < 0.05, #p < 0.01, $p < 0.001, and NS non-significant

Mentions: Next we investigated whether enhanced myotube formation by Wnt-3a was associated with increased muscle-specific gene expression. Muscle creatine kinase (MCK) activity was increased by LiCl compared with control after 72 h (Fig. 2a), which is in line with previous results [8]. In contrast, MCK activity was not increased by Wnt-3a when compared to controls (Fig. 2a). Similarly, mRNA expression levels of MCK, but also myosin heavy chain (MyHC)-IIB and MyHC-perinatal at 72 h were only increased by LiCl, but not by Wnt-3a treatment (Fig. 2b). Differentiation-induced transcriptional activation, of the troponin I (TnI) promoter, evaluated in a stable reporter cell line, was increased by LiCl but not Wnt-3a (Fig. 2c) as increasing concentration of Wnt-3a even slightly decreased TnI-promoter transactivation. In line with this, over-expression of β-catenin, did not promote TnI-promoter transactivation (Supplemental Fig. 4A). Next, MRF activity was assessed with a transiently transfected, MyoD-sensitive (supplemental Fig. 4B), 4RTK luciferase reporter. Only LiCl induced an increase in MRF transcriptional activity compared to control, whereas Wnt-3a did not affect MRF activity (Fig. 2d), in line with the absence of a stimulatory effect on muscle-specific gene expression.Fig. 2


Segregation of myoblast fusion and muscle-specific gene expression by distinct ligand-dependent inactivation of GSK-3β.

Pansters NA, van der Velden JL, Kelders MC, Laeremans H, Schols AM, Langen RC - Cell. Mol. Life Sci. (2010)

Muscle-specific gene expression during differentiation is stimulated by LiCl but not by Wnt-3a. C2C12 myoblast cells were cultured in DM with or without LiCl (10 mM), control, or Wnt-3a CM (diluted 1/10, or as indicated, in DM). a After 72 h, lysates were prepared for determination of muscle creatine kinase activity and total protein. Results are expressed as specific enzyme activity (units/mg protein). b After 72 h, lysates were prepared to determine mRNA expression levels of MCK, MyHC IIB, and perinatal. c Myoblasts containing a stable genomically integrated troponin I (TnI) luciferase reporter construct were cultured for 48 h in DM, ±LiCl (10 mM), or Wnt-3a or control-CM (1/10 diluted in DM). Alternatively, (d) C2C12 myoblasts were transfected with a 4RTK luciferase-reporter construct and plasmid encoding β-gal (0.25μg each) and cultured in DM with or without LiCl (10 mM), control, or Wnt-3a CM (diluted 1/10 in DM). Lysates were prepared for luciferase and β-galactosidase enzyme activity (RLU/mg protein) or (RLU luciferase/β-gal activity). Shown are representative graphs of three independent experiments (n = 3 ± SEM), *p < 0.05, #p < 0.01, $p < 0.001, and NS non-significant
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Fig2: Muscle-specific gene expression during differentiation is stimulated by LiCl but not by Wnt-3a. C2C12 myoblast cells were cultured in DM with or without LiCl (10 mM), control, or Wnt-3a CM (diluted 1/10, or as indicated, in DM). a After 72 h, lysates were prepared for determination of muscle creatine kinase activity and total protein. Results are expressed as specific enzyme activity (units/mg protein). b After 72 h, lysates were prepared to determine mRNA expression levels of MCK, MyHC IIB, and perinatal. c Myoblasts containing a stable genomically integrated troponin I (TnI) luciferase reporter construct were cultured for 48 h in DM, ±LiCl (10 mM), or Wnt-3a or control-CM (1/10 diluted in DM). Alternatively, (d) C2C12 myoblasts were transfected with a 4RTK luciferase-reporter construct and plasmid encoding β-gal (0.25μg each) and cultured in DM with or without LiCl (10 mM), control, or Wnt-3a CM (diluted 1/10 in DM). Lysates were prepared for luciferase and β-galactosidase enzyme activity (RLU/mg protein) or (RLU luciferase/β-gal activity). Shown are representative graphs of three independent experiments (n = 3 ± SEM), *p < 0.05, #p < 0.01, $p < 0.001, and NS non-significant
Mentions: Next we investigated whether enhanced myotube formation by Wnt-3a was associated with increased muscle-specific gene expression. Muscle creatine kinase (MCK) activity was increased by LiCl compared with control after 72 h (Fig. 2a), which is in line with previous results [8]. In contrast, MCK activity was not increased by Wnt-3a when compared to controls (Fig. 2a). Similarly, mRNA expression levels of MCK, but also myosin heavy chain (MyHC)-IIB and MyHC-perinatal at 72 h were only increased by LiCl, but not by Wnt-3a treatment (Fig. 2b). Differentiation-induced transcriptional activation, of the troponin I (TnI) promoter, evaluated in a stable reporter cell line, was increased by LiCl but not Wnt-3a (Fig. 2c) as increasing concentration of Wnt-3a even slightly decreased TnI-promoter transactivation. In line with this, over-expression of β-catenin, did not promote TnI-promoter transactivation (Supplemental Fig. 4A). Next, MRF activity was assessed with a transiently transfected, MyoD-sensitive (supplemental Fig. 4B), 4RTK luciferase reporter. Only LiCl induced an increase in MRF transcriptional activity compared to control, whereas Wnt-3a did not affect MRF activity (Fig. 2d), in line with the absence of a stimulatory effect on muscle-specific gene expression.Fig. 2

Bottom Line: Furthermore, LiCl and IGF-I, but not Wnt-3a, increased NFATc3 transcriptional activity.In contrast, β-catenin-dependent transcriptional activity was increased by Wnt-3a and LiCl, but not IGF-I.These results for the first time reveal a segregated regulation of myoblast fusion and muscle-specific gene expression following stimulation of myogenic differentiation in response to distinct ligand-specific signaling routes of GSK-3β inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, PO Box 5800, 6202 AZ Maastricht, The Netherlands.

ABSTRACT
Myogenic differentiation involves myoblast fusion and induction of muscle-specific gene expression, which are both stimulated by pharmacological (LiCl), genetic, or IGF-I-mediated GSK-3β inactivation. To assess whether stimulation of myogenic differentiation is common to ligand-mediated GSK-3β inactivation, myoblast fusion and muscle-specific gene expression were investigated in response to Wnt-3a. Moreover, crosstalk between IGF-I/GSK-3β/NFATc3 and Wnt/GSK-3β/β-catenin signaling was assessed. While both Wnt-3a and LiCl promoted myoblast fusion, muscle-specific gene expression was increased by LiCl, but not by Wnt-3a or β-catenin over-expression. Furthermore, LiCl and IGF-I, but not Wnt-3a, increased NFATc3 transcriptional activity. In contrast, β-catenin-dependent transcriptional activity was increased by Wnt-3a and LiCl, but not IGF-I. These results for the first time reveal a segregated regulation of myoblast fusion and muscle-specific gene expression following stimulation of myogenic differentiation in response to distinct ligand-specific signaling routes of GSK-3β inactivation.

Show MeSH
Related in: MedlinePlus