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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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Effect of small-molecule inhibitors on C2C12 cell proliferation and differentiation. C2C12 cells were cultured without inhibitors (A-D) and with 1 μM FH535 (E-H), 1 μM GW9662 (I-L) and 3.6 nM K-252a (M-P), then immunostained with anti-troponin T (A, B, E, F, I, J, M, N) and anti-β-catenin (C, D, G, H, K, L, O, P) antibodies. FH535 interferes with β-catenin/Tcf complex formation, whereas GW9662 is structurally similar to FH535, but does not interfere with complex formation. K252a is a Trk kinase inhibitor, which phosphorylates β-catenin at tyrosine 654.
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Figure 7: Effect of small-molecule inhibitors on C2C12 cell proliferation and differentiation. C2C12 cells were cultured without inhibitors (A-D) and with 1 μM FH535 (E-H), 1 μM GW9662 (I-L) and 3.6 nM K-252a (M-P), then immunostained with anti-troponin T (A, B, E, F, I, J, M, N) and anti-β-catenin (C, D, G, H, K, L, O, P) antibodies. FH535 interferes with β-catenin/Tcf complex formation, whereas GW9662 is structurally similar to FH535, but does not interfere with complex formation. K252a is a Trk kinase inhibitor, which phosphorylates β-catenin at tyrosine 654.

Mentions: Small-molecule inhibitors of Wnt signaling have been developed for human cancer therapy and allow manipulation of intracellular signaling to decrease cell proliferation and induce differentiation or apoptosis [20]. To investigate Wnt signaling and β-catenin during C2C12 myoblast differentiation, we used chemical inhibitors FH535, GW9662 and K252a to observe troponin T expression and subcellular β-catenin localization by immunohistochemistry (Figure 7) and western blotting (Figure 8). FH535 interferes with β-catenin/Tcf complex formation, whereas GW9662 is structurally similar to FH535 but does not interfere with complex formation. K252a is a Trk kinase inhibitor, which phosphorylates β-catenin at tyrosine 654 [21,22]. FH535 and GW9662 did not affect myoblast differentiation at 1 μM, although FH535 exhibited cytotoxicity at concentrations > 10 μM (Figure 9). K252a dose-dependently increased troponin T-positive cells and promoted myoblast fusion, as reported elsewhere [21]. Subcellular β-catenin localization was determined by western blot analysis of cytoplasmic and membrane extracts from proliferative and differentiating C2C12 cells (Figure 9). FH535 increased the cytosolic level of β-catenin in proliferation medium, but decreased basal β-catenin in differentiation medium (Figure 8A, B). In contrast, K252a increased cytosolic and membrane-bound β-catenin during myogenic differentiation (Figure 9B).


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 small-molecule inhibitors on C2C12 cell proliferation and differentiation. C2C12 cells were cultured without inhibitors (A-D) and with 1 μM FH535 (E-H), 1 μM GW9662 (I-L) and 3.6 nM K-252a (M-P), then immunostained with anti-troponin T (A, B, E, F, I, J, M, N) and anti-β-catenin (C, D, G, H, K, L, O, P) antibodies. FH535 interferes with β-catenin/Tcf complex formation, whereas GW9662 is structurally similar to FH535, but does not interfere with complex formation. K252a is a Trk kinase inhibitor, which phosphorylates β-catenin at tyrosine 654.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Effect of small-molecule inhibitors on C2C12 cell proliferation and differentiation. C2C12 cells were cultured without inhibitors (A-D) and with 1 μM FH535 (E-H), 1 μM GW9662 (I-L) and 3.6 nM K-252a (M-P), then immunostained with anti-troponin T (A, B, E, F, I, J, M, N) and anti-β-catenin (C, D, G, H, K, L, O, P) antibodies. FH535 interferes with β-catenin/Tcf complex formation, whereas GW9662 is structurally similar to FH535, but does not interfere with complex formation. K252a is a Trk kinase inhibitor, which phosphorylates β-catenin at tyrosine 654.
Mentions: Small-molecule inhibitors of Wnt signaling have been developed for human cancer therapy and allow manipulation of intracellular signaling to decrease cell proliferation and induce differentiation or apoptosis [20]. To investigate Wnt signaling and β-catenin during C2C12 myoblast differentiation, we used chemical inhibitors FH535, GW9662 and K252a to observe troponin T expression and subcellular β-catenin localization by immunohistochemistry (Figure 7) and western blotting (Figure 8). FH535 interferes with β-catenin/Tcf complex formation, whereas GW9662 is structurally similar to FH535 but does not interfere with complex formation. K252a is a Trk kinase inhibitor, which phosphorylates β-catenin at tyrosine 654 [21,22]. FH535 and GW9662 did not affect myoblast differentiation at 1 μM, although FH535 exhibited cytotoxicity at concentrations > 10 μM (Figure 9). K252a dose-dependently increased troponin T-positive cells and promoted myoblast fusion, as reported elsewhere [21]. Subcellular β-catenin localization was determined by western blot analysis of cytoplasmic and membrane extracts from proliferative and differentiating C2C12 cells (Figure 9). FH535 increased the cytosolic level of β-catenin in proliferation medium, but decreased basal β-catenin in differentiation medium (Figure 8A, B). In contrast, K252a increased cytosolic and membrane-bound β-catenin during myogenic differentiation (Figure 9B).

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