Center of Biomedical Research Excellence in Stem Cell Biology and Regenerative Medicine, Maine Medical Center Research Institute, Scarborough, Maine 04074, USA.
J Biol Chem. 2011 Mar 25;286(12):10649-59. doi: 10.1074/jbc.M110.169391. Epub 2011 Jan 20.
R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/β-catenin signaling. In this study, we investigated the potential roles of the RSPO proteins during myogenic differentiation. Overexpression of the Rspo1 gene or administration of recombinant RSPO2 protein enhanced mRNA and protein expression of a basic helix-loop-helix (bHLH) class myogenic determination factor, MYF5, in both C2C12 myoblasts and primary satellite cells, whereas MYOD or PAX7 expression was not affected. RSPOs also promoted myogenic differentiation and induced hypertrophic myotube formation in C2C12 cells. In addition, Rspo2 and Rspo3 gene knockdown by RNA interference significantly compromised MYF5 expression, myogenic differentiation, and myotube formation. Furthermore, Myf5 expression was reduced in the developing limbs of mouse embryos lacking the Rspo2 gene. Finally, we demonstrated that blocking of WNT/β-catenin signaling by DKK1 or a dominant-negative form of TCF4 reversed MYF5 expression, myogenic differentiation, and hypertrophic myotube formation induced by RSPO2, indicating that RSPO2 exerts its activity through the WNT/β-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive regulators of skeletal myogenesis acting through the WNT/β-catenin signaling pathway.
RSPO（R-spondin）蛋白是最近被鉴定的一类分泌蛋白家族，可激活 WNT/β-连环蛋白信号通路。在这项研究中，我们研究了 RSPO 蛋白在成肌分化过程中的潜在作用。Rspo1 基因的过表达或重组 RSPO2 蛋白的给药增强了 C2C12 成肌细胞和原代卫星细胞中碱性螺旋-环-螺旋（bHLH）类成肌决定因子 MYF5 的 mRNA 和蛋白表达，而 MYOD 或 PAX7 的表达不受影响。RSPO 还促进了 C2C12 细胞的成肌分化，并诱导了肥大肌管的形成。此外，通过 RNA 干扰敲低 Rspo2 和 Rspo3 基因显著削弱了 MYF5 的表达、成肌分化和肌管形成。此外，缺乏 Rspo2 基因的小鼠胚胎发育肢体中的 Myf5 表达减少。最后，我们证明通过 DKK1 或 TCF4 的显性失活形式阻断 WNT/β-连环蛋白信号通路可逆转 RSPO2 诱导的 MYF5 表达、成肌分化和肥大肌管形成，表明 RSPO2 通过 WNT/β-连环蛋白信号通路发挥其活性。我们的研究结果提供了有力的证据表明，RSPO 是通过 WNT/β-连环蛋白信号通路发挥作用的骨骼成肌的关键正调控因子。
