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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

Effect of Wnt expression on cell migration. A scratch test was used to determine C2C12 cell proliferation and migration. Cell numbers were counted over 9 h, and mobility was monitored by time-lapse microscopy. Phase contrast images at 0 h (purple) and 9 h (green) were merged to show the migration of C2C12 cells expressing eGFP (A), Wnt3a (B), Wnt4(C) and Wnt5a (D). (E) Summary of cell numbers counted over 9 h (cells/field) with n = 3 for control, Wnt3a and Wnt4, and n = 4 for Wnt5a. *P < 0.01; † P < 0.02 vs. eGFP.
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Figure 4: Effect of Wnt expression on cell migration. A scratch test was used to determine C2C12 cell proliferation and migration. Cell numbers were counted over 9 h, and mobility was monitored by time-lapse microscopy. Phase contrast images at 0 h (purple) and 9 h (green) were merged to show the migration of C2C12 cells expressing eGFP (A), Wnt3a (B), Wnt4(C) and Wnt5a (D). (E) Summary of cell numbers counted over 9 h (cells/field) with n = 3 for control, Wnt3a and Wnt4, and n = 4 for Wnt5a. *P < 0.01; † P < 0.02 vs. eGFP.

Mentions: A scratch wound-healing assay was performed to examine proliferation and migration of cells overexpressing Wnt3a, Wnt4 and Wnt5a. After 48 h post-transfection, C2C12 cell monolayers were scratched to remove cells along the middle of the culture plate surface. Then, cell migration was monitored by time-lapse video microscopy (Additional files 5, 6, 7, 8). Cellular migration of Wnt5a-expressing cells was nearly identical to that of control cells expressing enhanced green florescent protein (eGFP), while Wnt4- and Wnt3a-expressing cells showed decreased migration to 69% and 77%, respectively (Figure 4). Although we observed cell migration in the scratch assay, we could not detect any differences between eGFP and Wnt5a transgenes during regeneration of the cells at the wound edges.


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)

Effect of Wnt expression on cell migration. A scratch test was used to determine C2C12 cell proliferation and migration. Cell numbers were counted over 9 h, and mobility was monitored by time-lapse microscopy. Phase contrast images at 0 h (purple) and 9 h (green) were merged to show the migration of C2C12 cells expressing eGFP (A), Wnt3a (B), Wnt4(C) and Wnt5a (D). (E) Summary of cell numbers counted over 9 h (cells/field) with n = 3 for control, Wnt3a and Wnt4, and n = 4 for Wnt5a. *P < 0.01; † P < 0.02 vs. eGFP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Effect of Wnt expression on cell migration. A scratch test was used to determine C2C12 cell proliferation and migration. Cell numbers were counted over 9 h, and mobility was monitored by time-lapse microscopy. Phase contrast images at 0 h (purple) and 9 h (green) were merged to show the migration of C2C12 cells expressing eGFP (A), Wnt3a (B), Wnt4(C) and Wnt5a (D). (E) Summary of cell numbers counted over 9 h (cells/field) with n = 3 for control, Wnt3a and Wnt4, and n = 4 for Wnt5a. *P < 0.01; † P < 0.02 vs. eGFP.
Mentions: A scratch wound-healing assay was performed to examine proliferation and migration of cells overexpressing Wnt3a, Wnt4 and Wnt5a. After 48 h post-transfection, C2C12 cell monolayers were scratched to remove cells along the middle of the culture plate surface. Then, cell migration was monitored by time-lapse video microscopy (Additional files 5, 6, 7, 8). Cellular migration of Wnt5a-expressing cells was nearly identical to that of control cells expressing enhanced green florescent protein (eGFP), while Wnt4- and Wnt3a-expressing cells showed decreased migration to 69% and 77%, respectively (Figure 4). Although we observed cell migration in the scratch assay, we could not detect any differences between eGFP and Wnt5a transgenes during regeneration of the cells at the wound edges.

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