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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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Wnt family members differentially regulate β-catenin phosphorylation and subcellular localization in C2C12 cells. (A-D) C2C12 cells were cultured with or without K252a, and then phospho-β-catenin (Y654) was immunostained. K252a caused an accumulation of vesicles containing phospho-β-catenin (Y654) in C2C12 cells. (E-L) Wnt3a, Wnt4 and Wnt5a are overexpressed in C2C12 cells by adenovirus-mediated gene transfer. After 2 day culture in differentiation medium commencing 24 h post-transfection, subcellular phospho-β-catenin (Y654) localization was determined by immunostaining. (M-T) C2C12 cells expressing eGFP, Wnt3a, Wnt4 and Wnt5a were immunostained with anti-pan-β-catenin antibodies. Cytoplasmic β-catenin is elevated with Wnt3a expression in proliferation medium and maintained at a higher level in differentiation medium, whereas nuclear and membrane-bound β-catenin is increased with Wnt4 expression.
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Figure 10: Wnt family members differentially regulate β-catenin phosphorylation and subcellular localization in C2C12 cells. (A-D) C2C12 cells were cultured with or without K252a, and then phospho-β-catenin (Y654) was immunostained. K252a caused an accumulation of vesicles containing phospho-β-catenin (Y654) in C2C12 cells. (E-L) Wnt3a, Wnt4 and Wnt5a are overexpressed in C2C12 cells by adenovirus-mediated gene transfer. After 2 day culture in differentiation medium commencing 24 h post-transfection, subcellular phospho-β-catenin (Y654) localization was determined by immunostaining. (M-T) C2C12 cells expressing eGFP, Wnt3a, Wnt4 and Wnt5a were immunostained with anti-pan-β-catenin antibodies. Cytoplasmic β-catenin is elevated with Wnt3a expression in proliferation medium and maintained at a higher level in differentiation medium, whereas nuclear and membrane-bound β-catenin is increased with Wnt4 expression.

Mentions: Next, we analyzed β-catenin phosphorylation at residue Y654 during myoblast proliferation and differentiation. Proliferation medium supplemented with FH535 increased phospho-β-catenin (Y654) by 2-fold, but decreased phospho-β-catenin to less than half in differentiation medium (Figure 8C). In contrast, K252a-supplemented proliferation medium decreased phospho-β-catenin (Y654) as anticipated, but increased phosphorylated β-catenin and total β-catenin in differentiation medium. This observation was consistent with the immunohistochemical analysis. K252a treatment in differentiation medium caused cytosolic accumulation of small vesicles containing phospho-β-catenin (Y654) (Figure 10A,B). Together with western blot data, these results suggested that K252a increases total β-catenin in C2C12 cells. Simultaneously, β-catenin may be phosphorylated at tyrosine 654 by a kinase that was not inhibited by K252a and induced in differentiation medium via serum starvation.


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)

Wnt family members differentially regulate β-catenin phosphorylation and subcellular localization in C2C12 cells. (A-D) C2C12 cells were cultured with or without K252a, and then phospho-β-catenin (Y654) was immunostained. K252a caused an accumulation of vesicles containing phospho-β-catenin (Y654) in C2C12 cells. (E-L) Wnt3a, Wnt4 and Wnt5a are overexpressed in C2C12 cells by adenovirus-mediated gene transfer. After 2 day culture in differentiation medium commencing 24 h post-transfection, subcellular phospho-β-catenin (Y654) localization was determined by immunostaining. (M-T) C2C12 cells expressing eGFP, Wnt3a, Wnt4 and Wnt5a were immunostained with anti-pan-β-catenin antibodies. Cytoplasmic β-catenin is elevated with Wnt3a expression in proliferation medium and maintained at a higher level in differentiation medium, whereas nuclear and membrane-bound β-catenin is increased with Wnt4 expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 10: Wnt family members differentially regulate β-catenin phosphorylation and subcellular localization in C2C12 cells. (A-D) C2C12 cells were cultured with or without K252a, and then phospho-β-catenin (Y654) was immunostained. K252a caused an accumulation of vesicles containing phospho-β-catenin (Y654) in C2C12 cells. (E-L) Wnt3a, Wnt4 and Wnt5a are overexpressed in C2C12 cells by adenovirus-mediated gene transfer. After 2 day culture in differentiation medium commencing 24 h post-transfection, subcellular phospho-β-catenin (Y654) localization was determined by immunostaining. (M-T) C2C12 cells expressing eGFP, Wnt3a, Wnt4 and Wnt5a were immunostained with anti-pan-β-catenin antibodies. Cytoplasmic β-catenin is elevated with Wnt3a expression in proliferation medium and maintained at a higher level in differentiation medium, whereas nuclear and membrane-bound β-catenin is increased with Wnt4 expression.
Mentions: Next, we analyzed β-catenin phosphorylation at residue Y654 during myoblast proliferation and differentiation. Proliferation medium supplemented with FH535 increased phospho-β-catenin (Y654) by 2-fold, but decreased phospho-β-catenin to less than half in differentiation medium (Figure 8C). In contrast, K252a-supplemented proliferation medium decreased phospho-β-catenin (Y654) as anticipated, but increased phosphorylated β-catenin and total β-catenin in differentiation medium. This observation was consistent with the immunohistochemical analysis. K252a treatment in differentiation medium caused cytosolic accumulation of small vesicles containing phospho-β-catenin (Y654) (Figure 10A,B). Together with western blot data, these results suggested that K252a increases total β-catenin in C2C12 cells. Simultaneously, β-catenin may be phosphorylated at tyrosine 654 by a kinase that was not inhibited by K252a and induced in differentiation medium via serum starvation.

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