Canonical Wnt signaling is involved in switching from cell proliferation to myogenic differentiation of mouse myoblast cells

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.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.Wnt signaling plays key roles in stem cell maintenance and adult tissue homeostasis [1,2]. In addition, Wnt signaling controls cell proliferation and differentiation, as well as organized cell movements and tissue polarity establishment. Wnt signaling dysregulation can induce degenerative and cancerous disorders. 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.Members of the Wnt family are involved in various stages of skeletal muscle development and regeneration [3]. Wnt1 and Wnt3a expression in the developing neural tube initiate myogenic differentiation in dorsal and medial somites [4,5]. Wnt3a overexp