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Canonical Wnt signaling is involved in switching from cell proliferation to myogenic differentiation of mouse myoblast cells.

Tanaka S, Terada K, Nohno T - J Mol Signal (2011)

Bottom Line: Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4.TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts.Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated β-catenin (Tyr654) during myogenic differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Developmental Biology, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan. nohno@bcc.kawasaki-m.ac.jp.

ABSTRACT

Background: Wnt/β-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. In addition, Wnt3a and β-catenin are required for muscle-specific gene transcription in embryonic carcinoma cells and satellite-cell proliferation during adult skeletal muscle regeneration. Downstream targets of canonical Wnt signaling are cyclin D1 and c-myc. However both target genes are suppressed during differentiation of mouse myoblast cells, C2C12. Underlying molecular mechanisms of β-catenin signaling during myogenic differentiation remain unknown.

Results: Using C2C12 cells, we examined intracellular signaling and gene transcription during myoblast proliferation and differentiation. We confirmed that several Wnt signaling components, including Wnt9a, Sfrp2 and porcupine, were consistently upregulated in differentiating C2C12 cells. Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4. TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts. FH535, a small-molecule inhibitor of β-catenin/Tcf complex formation, reduced basal β-catenin in the cytoplasm and decreased myoblast proliferation. K252a, a protein kinase inhibitor, increased both cytosolic and membrane-bound β-catenin and enhanced myoblast fusion. Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated β-catenin (Tyr654) during myogenic differentiation.

Conclusions: These results suggest that various Wnt ligands control subcellular β-catenin localization, which regulate myoblast proliferation and myotube formation. Wnt signaling via β-catenin likely acts as a molecular switch that regulates the transition from cell proliferation to myogenic differentiation.

No MeSH data available.


Related in: MedlinePlus

Wnt4 promotes myogenic differentiation and induces fusion of C2C12 cells. (A) Troponin T expression after 3 day culture in proliferation or differentiation medium with recombinant adenoviruses expressing Wnt3a, Wnt4, Wnt5a or eGFP (control). Cell numbers are the means and standard deviations of three independent experiments performed in triplicate, *P < 0.01 vs. control. (B) Differentiated myotubes were evaluated by counting nuclei within fused multinucleated cells expressing troponin T. Four fields in triplicate cultures were used for counting fused cells cultured under a differentiation condition, *P < 0.04 vs. control.
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Figure 3: Wnt4 promotes myogenic differentiation and induces fusion of C2C12 cells. (A) Troponin T expression after 3 day culture in proliferation or differentiation medium with recombinant adenoviruses expressing Wnt3a, Wnt4, Wnt5a or eGFP (control). Cell numbers are the means and standard deviations of three independent experiments performed in triplicate, *P < 0.01 vs. control. (B) Differentiated myotubes were evaluated by counting nuclei within fused multinucleated cells expressing troponin T. Four fields in triplicate cultures were used for counting fused cells cultured under a differentiation condition, *P < 0.04 vs. control.

Mentions: DNA transfection by cationic liposomes is generally less effective compared with that of recombinant virus infection concerning proportion of the cells expressing HA-tagged Wnt proteins. Therefore, we evaluated myogenic activity using a recombinant adenovirus encoding Wnt3a, Wnt4 and Wnt5a. Within the Wnt family, we selected Wnt3a, Wnt4 and Wnt5a, because these Wnts exhibit significantly varying biological activities in chick embryos [6-9], although endogenous expression levels of Wnt3a and Wnt5a in C2C12 cells was less than one-tenth of Wnt4 (Additional files 2, 3). C2C12 cells cultured in 24 well plates were infected by the recombinant adenovirus with a multiplicity of infection of 400, because C2C12 cells lack coxsackie and adenovirus receptor expression. Under these conditions, uniform weak expression of the transgene was usually detected at 48 h post-infection. Troponin T immunostaining showed that Wnt4-overexpressing cells expressed troponin T and differentiated into myofibers (Figure 2). However, cells that overexpressed Wnt3a showed decreased troponin T staining and poor differentiation compared with that of the control expressing eGFP. Wnt5a overexpression did not significantly affect myoblast differentiation and myofiber formation. Wnt3a overexpression significantly reduced troponin T-positive cells in proliferation and differentiation medium (Figure 3A). However, Wnt4 overexpression significantly increased troponin T-positive cells in proliferation medium. In differentiation medium, Wnt4 overexpression enhanced myotube formation as indicated by fused multinuclear troponin T-positive cells (Figure 3B). Wnt5a overexpression demonstrated no effect on C2C12 differentiation under identical conditions.


Canonical Wnt signaling is involved in switching from cell proliferation to myogenic differentiation of mouse myoblast cells.

Tanaka S, Terada K, Nohno T - J Mol Signal (2011)

Wnt4 promotes myogenic differentiation and induces fusion of C2C12 cells. (A) Troponin T expression after 3 day culture in proliferation or differentiation medium with recombinant adenoviruses expressing Wnt3a, Wnt4, Wnt5a or eGFP (control). Cell numbers are the means and standard deviations of three independent experiments performed in triplicate, *P < 0.01 vs. control. (B) Differentiated myotubes were evaluated by counting nuclei within fused multinucleated cells expressing troponin T. Four fields in triplicate cultures were used for counting fused cells cultured under a differentiation condition, *P < 0.04 vs. control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3198762&req=5

Figure 3: Wnt4 promotes myogenic differentiation and induces fusion of C2C12 cells. (A) Troponin T expression after 3 day culture in proliferation or differentiation medium with recombinant adenoviruses expressing Wnt3a, Wnt4, Wnt5a or eGFP (control). Cell numbers are the means and standard deviations of three independent experiments performed in triplicate, *P < 0.01 vs. control. (B) Differentiated myotubes were evaluated by counting nuclei within fused multinucleated cells expressing troponin T. Four fields in triplicate cultures were used for counting fused cells cultured under a differentiation condition, *P < 0.04 vs. control.
Mentions: DNA transfection by cationic liposomes is generally less effective compared with that of recombinant virus infection concerning proportion of the cells expressing HA-tagged Wnt proteins. Therefore, we evaluated myogenic activity using a recombinant adenovirus encoding Wnt3a, Wnt4 and Wnt5a. Within the Wnt family, we selected Wnt3a, Wnt4 and Wnt5a, because these Wnts exhibit significantly varying biological activities in chick embryos [6-9], although endogenous expression levels of Wnt3a and Wnt5a in C2C12 cells was less than one-tenth of Wnt4 (Additional files 2, 3). C2C12 cells cultured in 24 well plates were infected by the recombinant adenovirus with a multiplicity of infection of 400, because C2C12 cells lack coxsackie and adenovirus receptor expression. Under these conditions, uniform weak expression of the transgene was usually detected at 48 h post-infection. Troponin T immunostaining showed that Wnt4-overexpressing cells expressed troponin T and differentiated into myofibers (Figure 2). However, cells that overexpressed Wnt3a showed decreased troponin T staining and poor differentiation compared with that of the control expressing eGFP. Wnt5a overexpression did not significantly affect myoblast differentiation and myofiber formation. Wnt3a overexpression significantly reduced troponin T-positive cells in proliferation and differentiation medium (Figure 3A). However, Wnt4 overexpression significantly increased troponin T-positive cells in proliferation medium. In differentiation medium, Wnt4 overexpression enhanced myotube formation as indicated by fused multinuclear troponin T-positive cells (Figure 3B). Wnt5a overexpression demonstrated no effect on C2C12 differentiation under identical conditions.

Bottom Line: Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4.TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts.Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated β-catenin (Tyr654) during myogenic differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Developmental Biology, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan. nohno@bcc.kawasaki-m.ac.jp.

ABSTRACT

Background: Wnt/β-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. In addition, Wnt3a and β-catenin are required for muscle-specific gene transcription in embryonic carcinoma cells and satellite-cell proliferation during adult skeletal muscle regeneration. Downstream targets of canonical Wnt signaling are cyclin D1 and c-myc. However both target genes are suppressed during differentiation of mouse myoblast cells, C2C12. Underlying molecular mechanisms of β-catenin signaling during myogenic differentiation remain unknown.

Results: Using C2C12 cells, we examined intracellular signaling and gene transcription during myoblast proliferation and differentiation. We confirmed that several Wnt signaling components, including Wnt9a, Sfrp2 and porcupine, were consistently upregulated in differentiating C2C12 cells. Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4. TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts. FH535, a small-molecule inhibitor of β-catenin/Tcf complex formation, reduced basal β-catenin in the cytoplasm and decreased myoblast proliferation. K252a, a protein kinase inhibitor, increased both cytosolic and membrane-bound β-catenin and enhanced myoblast fusion. Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated β-catenin (Tyr654) during myogenic differentiation.

Conclusions: These results suggest that various Wnt ligands control subcellular β-catenin localization, which regulate myoblast proliferation and myotube formation. Wnt signaling via β-catenin likely acts as a molecular switch that regulates the transition from cell proliferation to myogenic differentiation.

No MeSH data available.


Related in: MedlinePlus