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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 reduces Wnt-3a-induced transcriptional activity in myoblasts. Reporter assays were performed using TOP/FLASH and FOP/FLASH (luciferase assay) in C2C12 cells. Wnt3a significantly increases luciferase activity in TOP over FOP. (A) Wnt4 has no effect on luciferase activity, whereas Wnt4 inhibits Wnt3a activity. (B) Wnt5a poorly inhibits Wnt3a activity. Bars are the means of four independent experiments. *P < 0.01 between TOP and FOP; P < 0.01 between Wnt3a and Wnt3a + Wnt4 in TOP; P < 0.02 between Wnt3a and Wnt3a + Wnt5a in TOP.
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Figure 5: Wnt4 reduces Wnt-3a-induced transcriptional activity in myoblasts. Reporter assays were performed using TOP/FLASH and FOP/FLASH (luciferase assay) in C2C12 cells. Wnt3a significantly increases luciferase activity in TOP over FOP. (A) Wnt4 has no effect on luciferase activity, whereas Wnt4 inhibits Wnt3a activity. (B) Wnt5a poorly inhibits Wnt3a activity. Bars are the means of four independent experiments. *P < 0.01 between TOP and FOP; P < 0.01 between Wnt3a and Wnt3a + Wnt4 in TOP; P < 0.02 between Wnt3a and Wnt3a + Wnt5a in TOP.

Mentions: We then examined whether Wnt4 mediated myogenesis induction by canonical or non-canonical signaling using a TOP/FOP reporter assay in C2C12 cells under a proliferative condition. Wnt4 and Wnt3a reporter constructs were transfected into C2C12 cells and luciferase activity was determined at 24 h post-transfection. Although Wnt3a significantly elevated reporter activity, Wnt4 did not show increased activity above FOP (Figure 5A). However, Wnt4 coexpressed with Wnt3a showed 47% inhibition. Weak inhibition was observed when Wnt5a was coexpressed with Wnt3a (Figure 5B). These results indicated that Wnt4 suppressed canonical Wnt signaling mediated by the β-catenin/TCF complex and promoted myogenic differentiation. This observation was consistent with down-regulated cyclin D1 and c-myc in differentiating myoblasts, both of which are direct targets of the β-catenin/TCF complex.


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 reduces Wnt-3a-induced transcriptional activity in myoblasts. Reporter assays were performed using TOP/FLASH and FOP/FLASH (luciferase assay) in C2C12 cells. Wnt3a significantly increases luciferase activity in TOP over FOP. (A) Wnt4 has no effect on luciferase activity, whereas Wnt4 inhibits Wnt3a activity. (B) Wnt5a poorly inhibits Wnt3a activity. Bars are the means of four independent experiments. *P < 0.01 between TOP and FOP; P < 0.01 between Wnt3a and Wnt3a + Wnt4 in TOP; P < 0.02 between Wnt3a and Wnt3a + Wnt5a in TOP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Wnt4 reduces Wnt-3a-induced transcriptional activity in myoblasts. Reporter assays were performed using TOP/FLASH and FOP/FLASH (luciferase assay) in C2C12 cells. Wnt3a significantly increases luciferase activity in TOP over FOP. (A) Wnt4 has no effect on luciferase activity, whereas Wnt4 inhibits Wnt3a activity. (B) Wnt5a poorly inhibits Wnt3a activity. Bars are the means of four independent experiments. *P < 0.01 between TOP and FOP; P < 0.01 between Wnt3a and Wnt3a + Wnt4 in TOP; P < 0.02 between Wnt3a and Wnt3a + Wnt5a in TOP.
Mentions: We then examined whether Wnt4 mediated myogenesis induction by canonical or non-canonical signaling using a TOP/FOP reporter assay in C2C12 cells under a proliferative condition. Wnt4 and Wnt3a reporter constructs were transfected into C2C12 cells and luciferase activity was determined at 24 h post-transfection. Although Wnt3a significantly elevated reporter activity, Wnt4 did not show increased activity above FOP (Figure 5A). However, Wnt4 coexpressed with Wnt3a showed 47% inhibition. Weak inhibition was observed when Wnt5a was coexpressed with Wnt3a (Figure 5B). These results indicated that Wnt4 suppressed canonical Wnt signaling mediated by the β-catenin/TCF complex and promoted myogenic differentiation. This observation was consistent with down-regulated cyclin D1 and c-myc in differentiating myoblasts, both of which are direct targets of the β-catenin/TCF complex.

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