Abstract
BackgroundWnt/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.ResultsUsing 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.ConclusionsThese 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.
Highlights
Wnt/b-catenin signaling is involved in various aspects of skeletal muscle development and regeneration
Expression of Wnt signaling molecules during myogenic differentiation of C2C12 cells Mouse mesenchymal C2C12 cells can differentiate into muscle cells under a low-serum condition [16]
Using realtime PCR, we investigated the expression of endogenous Wnt signaling components before and after C2C12 cell differentiation
Summary
Wnt/b-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. Wnt3a and b-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. Both target genes are suppressed during differentiation of mouse myoblast cells, C2C12. Wnt signaling plays key roles in stem cell maintenance and adult tissue homeostasis [1,2]. Wnt signaling controls cell proliferation and differentiation, as well as organized cell movements and tissue polarity establishment. The Wnt signaling pathway has gained attention as a potential therapeutic target for cancer treatment, as well as research interest in regenerative medicine and stem cell biology. Wnt and Wnt3a expression in the developing neural muscle development and homeostasis is not fully understood
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