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Canonical Wnt Signaling Induces BMP-4 to Specify Slow Myofibrogenesis of Fetal Myoblasts

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Canonical Wnt Signaling Induces BMP-4 to Specify Slow Myofibrogenesis of Fetal Myoblasts

Kazuki Kuroda et al. Skelet Muscle.

Abstract

Background: The Wnts are secreted proteins that play important roles in skeletal myogenesis, muscle fiber type diversification, neuromuscular junction formation and muscle stem cell function. How Wnt proteins orchestrate such diverse activities remains poorly understood. Canonical Wnt signaling stabilizes β-catenin, which subsequently translocate to the nucleus to activate the transcription of TCF/LEF family genes.

Methods: We employed TCF-reporter mice and performed analysis of embryos and of muscle groups. We further isolated fetal myoblasts and performed cell and molecular analyses.

Results: We found that canonical Wnt signaling is strongly activated during fetal myogenesis and weakly activated in adult muscles limited to the slow myofibers. Muscle-specific transgenic expression of a stabilized β-catenin protein led to increased oxidative myofibers and reduced muscle mass, suggesting that canonical Wnt signaling promotes slow fiber types and inhibits myogenesis. By TCF-luciferase reporter assay, we identified Wnt-1 and Wnt-3a as potent activators of canonical Wnt signaling in myogenic progenitors. Consistent with in vivo data, constitutive overexpression of Wnt-1 or Wnt-3a inhibited the proliferation of both C2C12 and primary myoblasts. Surprisingly, Wnt-1 and Wnt-3a overexpression up-regulated BMP-4, and inhibition of BMP-4 by shRNA or recombinant Noggin protein rescued the myogenic inhibitory effect of Wnt-1 and Wnt-3a. Importantly, Wnt-3a or BMP-4 recombinant proteins promoted slow myosin heavy chain expression during myogenic differentiation of fetal myoblasts.

Conclusions: These results demonstrate a novel interaction between canonical Wnt and BMP signaling that induces myogenic differentiation towards slow muscle phenotype.

Figures

Figure 1
Figure 1
Activation of canonical Wnt signaling in embryonic, neonatal and adult skeletal muscles. Tcf-lacZ reporter mice were used to report activation of TCF promoter, the nuclear target of canonical Wnt signaling. X-gal staining (in blue) was used to reveal LacZ (b-gal) activity. (A) Whole mount staining of an E14.5 embryo. (B) Forelimbs and hind limbs at E14.5. (C) Ventral and (D) dorsal view of an E14.5 embryo revealing intensive staining in some muscles. (E) Hind limb and (F) forelimb at P0 (postnatal Day 0). (G) Diaphragm and (H) soleus muscles of adult mouse showing staining in a subset of myofibers with intensive signals at the neuromuscular junction area.
Figure 2
Figure 2
Activation of canonical Wnt signaling in adult slow myofibers. (A-D) Soleus myofibers from adult Tcf-lacZ mice were stained with antibodies for pan-myosin heavy chain (White) or slow myosin heavy chain (White), and β-gal (Red) and FITC conjugated BTX (Green). (E-F) Whole mount muscles co-stained with antibodies for slow (Blue) and fast (Red) myosin heavy together with beta-gal (Green) and BTX (Red). (G) Percentage of β-gal positive (n = 69) and negative (n = 60) myofibers that co-express slow myosin heavy chain.
Figure 3
Figure 3
Muscle-specific constitutive activation of canonical Wnt signaling promotes oxidative myofiber phenotype. The MCK-Cre/Ctnnblox(ex3) mice were used to express constitutively active β-cateninΔex3, which mimics canonical Wnt signaling. The MCK promoter-drive Cre expression limits Wnt activation only in mature skeletal muscles. (A-B) X-Gal staining (blue signal) of whole mount TA muscles of Ctnnblox(ex3)/Tcf-LacZ (WT) and MCK-Cre/Ctnnblox(ex3)/Tcf-LacZ (Mut) mice. Blue signal, indicative of canonical Wnt signaling, is only detectable in the Mut TA muscles (A and B represent the dorsal and ventral view of the same muscles). (C-D) NADH-TR staining of TA muscle sections from the WT (C) and Mut (D) mice. (E) Percentage of high oxidative (black), middle oxidative (gray) and low oxidative (white) myofibers of WT and Mut mice (n = 3, each). Scale bar: 1 mm.
Figure 4
Figure 4
Canonical Wnt signaling induces BMP-4 in C2C12 myoblasts. (A) qPCR analysis of the relative expression of various genes in C2C12 myoblasts overexpressing Wnt-1 and Wnt-3a under growth condition. (B) BMP-4 knockdown reduced the expression of alkaline phosphatase (Akp2) but not the Axin2 gene. (C-F) BMP-4 shRNA rescued the myogenic differentiation of Wnt-1 and Wnt-3a infected C2C12 cells. (C-D) Wnt-1 and Wnt-3a overexpressing myoblasts treated with Scramble shRNA; (E-F) Wnt-1 and Wnt-3a overexpressing myoblasts treated with BMP-4 shRNA. Black signaling is myosin heavy chain antibody staining reacted with 3, 3'-diaminobenzidine (DAB) substrate. (G) Western blotting showing myosin heavy chain protein expression after BMP-4 shRNA treatment. (H) qPCR analysis of myosin heavy chain gene expression. (I-L) BMP-4 antagonist Noggin-Fc dose-dependently rescued the myogenic differentiation of Wnt-3a infected C2C12 cells. The myosin heavy chain was labeled in black and nuclei were labeled in white.
Figure 5
Figure 5
Wnt-3a induces slow myosin heavy chain expression in fetal myoblasts via BMP-4. Fetal myoblasts were isolated by fluorescence-activated cell sorting (FACS) from E14.5-15.5 embryos of Myf5-Cre/Rosa26-YFP mice. FACS isolated fetal myoblasts were cultured and induced to differentiate for three days. (A-D) Immunofluorescence showing slow myosin heavy chain (green) expression in myotubes treated with vehicle control medium or Wnt-3a recombinant protein (50 ng/ml). (E) Relative expression of myosin heavy chain and BMP-4 genes based on qPCR analysis. (F) Western blotting showing slow myosin heavy chain protein expression. (G-J) Immunofluorescence showing slow- (green) and pan- (red) myosin heavy chain expression in myotubes treated with vehicle control medium, Wnt-3a, and BMP-4 recombinant protein at concentration shown. (K) qPCR analysis showing relative expression of myosin heavy chain isoform genes. Scale bars: 40 μm.

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