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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.


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Localization of β-catenin and troponin T in proliferative and differentiating C2C12 cells. C2C12 cells cultured in proliferation and differentiation medium were double immunostained for β-catenin (A, B; red) and troponin T (C, D; green), and counterstained with DAPI (blue). Immunofluorescent images with various filters are merged (E, F).
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Figure 6: Localization of β-catenin and troponin T in proliferative and differentiating C2C12 cells. C2C12 cells cultured in proliferation and differentiation medium were double immunostained for β-catenin (A, B; red) and troponin T (C, D; green), and counterstained with DAPI (blue). Immunofluorescent images with various filters are merged (E, F).

Mentions: Wnt4, represented by luciferase activity, inhibited transcriptional activation via β-catenin/TCF. In HEK-293T cells, Wnt4 relocates β-catenin to the cell membrane [19]. We observed subcellular β-catenin localization by immunohistochemistry in C2C12 cells cultured in proliferation and differentiation medium (Figure 6). Under a proliferative condition, β-catenin was predominantly localized in the cytoplasm. β-Catenin was also localized within the nucleus of round proliferating cells and weak β-catenin was coexpressed with differentiation marker troponin T (Figure 6E). This observation coincided with changing morphology to flattened multinucleated cells. In differentiation medium, cellular β-catenin accumulated in the membrane and nuclei as differentiation proceeded into myotubes (Figure 6B,F).


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)

Localization of β-catenin and troponin T in proliferative and differentiating C2C12 cells. C2C12 cells cultured in proliferation and differentiation medium were double immunostained for β-catenin (A, B; red) and troponin T (C, D; green), and counterstained with DAPI (blue). Immunofluorescent images with various filters are merged (E, F).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Localization of β-catenin and troponin T in proliferative and differentiating C2C12 cells. C2C12 cells cultured in proliferation and differentiation medium were double immunostained for β-catenin (A, B; red) and troponin T (C, D; green), and counterstained with DAPI (blue). Immunofluorescent images with various filters are merged (E, F).
Mentions: Wnt4, represented by luciferase activity, inhibited transcriptional activation via β-catenin/TCF. In HEK-293T cells, Wnt4 relocates β-catenin to the cell membrane [19]. We observed subcellular β-catenin localization by immunohistochemistry in C2C12 cells cultured in proliferation and differentiation medium (Figure 6). Under a proliferative condition, β-catenin was predominantly localized in the cytoplasm. β-Catenin was also localized within the nucleus of round proliferating cells and weak β-catenin was coexpressed with differentiation marker troponin T (Figure 6E). This observation coincided with changing morphology to flattened multinucleated cells. In differentiation medium, cellular β-catenin accumulated in the membrane and nuclei as differentiation proceeded into myotubes (Figure 6B,F).

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