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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

Endogenous Wnt signaling component expression during myogenic differentiation of C2C12 cells. Wnt signaling molecule expression was measured by quantitative RT-PCR. After 2 or 4 days culture in differentiation medium, cells were harvested, total RNA prepared and relative gene expression quantified by real-time PCR as follows: (A) wingless-related MMTV integration, Wnt; secreted frizzled-related protein, Sfrp; and porcupine, Porcn. (B) frizzled, Fzd; and low density lipoprotein receptor-related protein, Lrp. (C) cyclin D, Ccnd; myelocytomatosis oncogene, Myc; fos-like antigen 1, Fosl1; casein kinase 1, alpha 1, Csnk1a1; catenin, beta 1, Ctnnb1; dishevelled 1, Dvl1; glycogen synthase kinase 3 beta, Gsk3b; transcription factor 3, Tcf3; and transducin-like enhancer of split 2, Tle2. Day 2 and 4 time points were normalized to expression levels at day 0. Data are from three independent experiments, *P < 0.005 and † P < 0.05.
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Figure 1: Endogenous Wnt signaling component expression during myogenic differentiation of C2C12 cells. Wnt signaling molecule expression was measured by quantitative RT-PCR. After 2 or 4 days culture in differentiation medium, cells were harvested, total RNA prepared and relative gene expression quantified by real-time PCR as follows: (A) wingless-related MMTV integration, Wnt; secreted frizzled-related protein, Sfrp; and porcupine, Porcn. (B) frizzled, Fzd; and low density lipoprotein receptor-related protein, Lrp. (C) cyclin D, Ccnd; myelocytomatosis oncogene, Myc; fos-like antigen 1, Fosl1; casein kinase 1, alpha 1, Csnk1a1; catenin, beta 1, Ctnnb1; dishevelled 1, Dvl1; glycogen synthase kinase 3 beta, Gsk3b; transcription factor 3, Tcf3; and transducin-like enhancer of split 2, Tle2. Day 2 and 4 time points were normalized to expression levels at day 0. Data are from three independent experiments, *P < 0.005 and † P < 0.05.

Mentions: Wnt9a, Wnt10a and Wnt6 expression increased up to day 4, while Wnt2b, Wnt4 and Wnt5a expression reached a maximum at day 2 (Figure 1). Net expression levels of Wnt members were as follows in descending order: Wnt10a > Wnt9a > Wnt2b > Wnt4, Wnt6 > Wnt5a. This expression pattern was consistent with the expression of downstream signaling molecules involved in non-canonical Wnt signaling. Among transmembrane receptors, frizzled2 and frizzled8 were expressed during early differentiation stages compared with those of frizzled1, frizzled4 and frizzled5. For canonical Wnt signaling target genes, fosl1 and c-myc expression were decreased, while Tle2, a negative regulator of canonical signaling, was increased compared with those of β-catenin (Ctnnb1), TCF7 and Apc. Cyclin D1 and c-myc expression decreased, while cyclin D3 and Gsk3β increased during differentiation [17]. This was consistent with a report that describes Gsk3β regulation of cyclin D1 degradation and subcellular localization [18]. We also found that porcupine (Porcn) expression was 13-fold and 19-fold higher at days 2 and 4, respectively, compared with those of cells in a proliferative state (Additional files 1, 2, 3). Porcn encodes an O-acyltransferase involved in lipid modification of Wnt proteins in the endoplasmic reticulum. Thus, Porcn upregulation indicated that Wnt protein transport and secretion was stimulated during myoblast differentiation. Wnt signaling downstream also dynamically changed toward non-canonical pathways following myogenic differentiation.


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)

Endogenous Wnt signaling component expression during myogenic differentiation of C2C12 cells. Wnt signaling molecule expression was measured by quantitative RT-PCR. After 2 or 4 days culture in differentiation medium, cells were harvested, total RNA prepared and relative gene expression quantified by real-time PCR as follows: (A) wingless-related MMTV integration, Wnt; secreted frizzled-related protein, Sfrp; and porcupine, Porcn. (B) frizzled, Fzd; and low density lipoprotein receptor-related protein, Lrp. (C) cyclin D, Ccnd; myelocytomatosis oncogene, Myc; fos-like antigen 1, Fosl1; casein kinase 1, alpha 1, Csnk1a1; catenin, beta 1, Ctnnb1; dishevelled 1, Dvl1; glycogen synthase kinase 3 beta, Gsk3b; transcription factor 3, Tcf3; and transducin-like enhancer of split 2, Tle2. Day 2 and 4 time points were normalized to expression levels at day 0. Data are from three independent experiments, *P < 0.005 and † P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Endogenous Wnt signaling component expression during myogenic differentiation of C2C12 cells. Wnt signaling molecule expression was measured by quantitative RT-PCR. After 2 or 4 days culture in differentiation medium, cells were harvested, total RNA prepared and relative gene expression quantified by real-time PCR as follows: (A) wingless-related MMTV integration, Wnt; secreted frizzled-related protein, Sfrp; and porcupine, Porcn. (B) frizzled, Fzd; and low density lipoprotein receptor-related protein, Lrp. (C) cyclin D, Ccnd; myelocytomatosis oncogene, Myc; fos-like antigen 1, Fosl1; casein kinase 1, alpha 1, Csnk1a1; catenin, beta 1, Ctnnb1; dishevelled 1, Dvl1; glycogen synthase kinase 3 beta, Gsk3b; transcription factor 3, Tcf3; and transducin-like enhancer of split 2, Tle2. Day 2 and 4 time points were normalized to expression levels at day 0. Data are from three independent experiments, *P < 0.005 and † P < 0.05.
Mentions: Wnt9a, Wnt10a and Wnt6 expression increased up to day 4, while Wnt2b, Wnt4 and Wnt5a expression reached a maximum at day 2 (Figure 1). Net expression levels of Wnt members were as follows in descending order: Wnt10a > Wnt9a > Wnt2b > Wnt4, Wnt6 > Wnt5a. This expression pattern was consistent with the expression of downstream signaling molecules involved in non-canonical Wnt signaling. Among transmembrane receptors, frizzled2 and frizzled8 were expressed during early differentiation stages compared with those of frizzled1, frizzled4 and frizzled5. For canonical Wnt signaling target genes, fosl1 and c-myc expression were decreased, while Tle2, a negative regulator of canonical signaling, was increased compared with those of β-catenin (Ctnnb1), TCF7 and Apc. Cyclin D1 and c-myc expression decreased, while cyclin D3 and Gsk3β increased during differentiation [17]. This was consistent with a report that describes Gsk3β regulation of cyclin D1 degradation and subcellular localization [18]. We also found that porcupine (Porcn) expression was 13-fold and 19-fold higher at days 2 and 4, respectively, compared with those of cells in a proliferative state (Additional files 1, 2, 3). Porcn encodes an O-acyltransferase involved in lipid modification of Wnt proteins in the endoplasmic reticulum. Thus, Porcn upregulation indicated that Wnt protein transport and secretion was stimulated during myoblast differentiation. Wnt signaling downstream also dynamically changed toward non-canonical pathways following myogenic differentiation.

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