BAMBI Promotes C2C12 Myogenic Differentiation by Enhancing Wnt/β-Catenin Signaling

Abstract

Bone morphogenic protein and activin membrane-bound inhibitor (BAMBI) is regarded as an essential regulator of cell proliferation and differentiation that represses transforming growth factor-β and enhances Wnt/β-catenin signaling in various cell types. However, its role in skeletal muscle remains largely unknown. In the current study, we found that the expression level of BAMBI peaked in the early differentiation phase of the C2C12 rodent myoblast cell line. Knockdown of BAMBI via siRNA inhibited C2C12 differentiation, indicated by repressed MyoD, MyoG, and MyHC expression as well as reductions in the differentiation and fusion indices. BAMBI knockdown reduced the activity of Wnt/β-catenin signaling, as characterized by the decreased nuclear translocation of β-catenin and the lowered transcription of Axin2, which is a well-documented target gene of the Wnt/β-catenin signaling pathway. Furthermore, treatment with LiCl, an activator of Wnt/β-catenin signaling, rescued the reduction in C2C12 differentiation caused by BAMBI siRNA. Taken together, our data suggest that BAMBI is required for normal C2C12 differentiation, and that its role in myogenesis is mediated by the Wnt/β-catenin pathway.

Keywords: BAMBI; C2C12; LiCl; Wnt/β-catenin; myogenic differentiation.

Figure 1

The temporal mRNA expression profiles of BAMBI (a), MyoD (b) and MyHC (c) during C2C12 myogenic differentiation. The results were represented as mean ± SD.

Figure 2

Knockdown of BAMBI inhibited myogenic differentiation. All the cell samples were harvested after transfection and myogenic induction for 48 and 96 h. (a) The western blot images of BAMBI and GAPDH; (b) the efficiency of siRNA interference on the mRNA and protein expression of BAMBI; (c) the mRNA expression of MyoD at 48 h and that of MyoG and MyHC at 96 h; (d) the western blot images of MyoD at 48 h, MyHC at 96 h, and their corresponding GAPDH; (e) the protein expression of MyoD at 48 h and MyHC at 96 h; (f) immunofluorescence of MyHC in C2C12 myotubes at 96 h post differentiation, images captured at 100× magnification; (g) the populations of myotubes; (h) the differentiation index; and (i) the myotube fusion index. The results were represented as mean ± SD; n = 3; * p < 0.05; ** p < 0.01.

Figure 3

Knockdown of BAMBI suppressed Wnt/β-catenin signaling. All the cell samples were harvested after transfection and myogenic induction for 48 h. (a) The western blot images of nuclear β-catenin and laminB1; (b) the nuclear β-catenin protein levels; (c) the mRNA expression of Axin2; (d) the western blot images of nuclear β-catenin and laminB1; (e) the nuclear β-catenin protein levels; (f) the mRNA expression of Axin2; (g) the western blot images of BAMBI and GAPDH; and (h) the mRNA and protein expression of BAMBI. The results were represented as mean ± SD; n = 3; * p < 0.05; ** p < 0.01.

Figure 4

LiCl rescued the inhibitory effect of BAMBI siRNA on C2C12 myogenic differentiation. All the cell samples were harvested after transfection and myogenic induction for 48 and 96 h. (a) The mRNA expression of MyoD at 48 h and that of MyoG and MyHC at 96 h; (b) the western blot images of MyoD, MyHC, and GAPDH; (c) the protein expression of MyoD at 48 h and MyHC at 96 h; (d) immunofluorescence images of MyHC in C2C12 myotubes at 96 h post differentiation, images captured at 100× magnification; (e) the populations of myotubes; (f) the differentiation index and (g) the myotube fusion index. The results were represented as mean ± SD; n = 3; * p < 0.05; ** p < 0.01.