A WNT/β-Catenin Signaling Activator, R-spondin, Plays Positive Regulatory Roles during Skeletal Myogenesis

Abstract

R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/␤-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-loophelix (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/␤-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/␤-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive regulators of skeletal myogenesis acting through the WNT/␤-catenin signaling pathway.WNT signaling plays diverse roles in normal tissue development during embryogenesis and tissue function in adulthood. The importance of WNT signaling in skeletal myogenesis was initially demonstrated in embryonic skeletal myogenesis (1-4). The WNT1 and WNT3A ligands derived from the dorsal neural tube and the surface ectoderm positively regulate skeletal myogenesis within somites via the canonical WNT/␤-catenin signaling pathway (3, 4). Furthermore, noncanonical WNT signaling, which transmits signals through the RAC/RHO-dependent planar cell polarity and calcium-PKC pathways, regulates PAX3 and subsequently MYOD expression during myogenic differentiation (5) and a directional elongation of myofibers within the myotome (6).Recently, progress has been made in the understanding of the roles of WNT signaling in postnatal myogenesis. For instance, ␤-catenin was shown to promote self-renewal of satellite cells, the muscle stem/progenitor cells residing in adult skeletal muscle (7). In contrast, WNT/␤-catenin signaling was shown to initiate myogenic differentiation of satellite cells by replacing NOTCH signaling, which is critical for self-renewal of satellite cells, through the inhibition of GSK-3␤ (8). It was also demonstrated that the activation of the WNT/␤-catenin pathway occurs within the CD45-positive stem cell population present in regenerating skeletal muscles but not in satellite cells (9). In aged skeletal muscle, WNT10B regulates the balance between myogenic and adipogenic lineage determination (10). WNT signaling was also suggested to induce a fibroblastic phenotype in aged skeletal muscle (11). Furthermore, WNT7A regulates self-renewal of satellite cells via noncanonical WNT signaling (12). These ...

Wnt/β-catenin signaling via Axin2 is required for myogenesis and, together with YAP/Taz and Tead1, active in IIa/IIx muscle fibers

“…This condition is called hypertrophy and multiple studies identified different roles for canonical and non-canonical Wnt signaling in muscle fiber hypertrophy (Armstrong and Esser, 2005;Bernardi et al, 2011;Han et al, 2011;Rochat et al, 2004;von Maltzahn et al, 2011). Furthermore, expression of β-catenin is a prerequisite for the physiological growth of adult skeletal muscle, underscoring the importance of canonical Wnt signaling (Armstrong et al, 2006).…”

WNT/β-Catenin Signaling Regulates Multiple Steps of Myogenesis by Regulating Step-Specific Targets

“…For example, inhibition of GSK3␤ results in activated WNT/␤-catenin signaling and leads to enhanced differentiation of C2 myogenic cells (60). The RSPO family of proteins, WNT/␤-catenin signaling agonists, can promote myogenic differentiation in cell culture (55). Pharmacological activations of WNT/␤-catenin signaling can also facilitate the differentiation of satellite cells and myoblasts (60)(61)(62).…”

“…Members of the R-spondin family (RSPO) of secreted cysteine-rich proteins (RSPO1, -2, -3, and -4) activate the WNT/␤-catenin signaling pathway at the receptor level (53,54). RSPO2 promotes myogenic differentiation and myocyte fusion via activation of WNT/␤-catenin signaling in C2C12 cells (55). RSPOs can regulate WNT receptor expression and control myogenesis through gene expression of the transforming growth factor ␤ (TGF␤) antagonist Fst (56).…”

Role and Function of Wnts in the Regulation of Myogenesis: When Wnt Meets Myostatin

“…Likewise, Armstrong et al have reported that the expression of -catenin is necessary for physiological growth of skeletal muscle and that Wnt signaling pathway induces -catenin activation of growth-control genes during overload induced skeletal muscle hypertrophy [209,210]. By using C2C12 and satellite cells, Han et al reported recently that activation of canonical pathway by R-spondin2, promote myogenic differentiation and hypertrophic myofiber formation [211]. When LiCl was added to the culture medium, the area of myotubes and index fusion were increased in both C2C12 and satellite cells, reaching values similar to those observed after Wnt4-induced hypertrophy [37].…”