A WNT/beta-catenin signaling activator, R-spondin, plays positive regulatory roles during skeletal myogenesis.
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.
Center of Biomedical Research Excellence in Stem Cell Biology and Regenerative Medicine, Maine Medical Center Research Institute, Scarborough, Maine 04074, USA.
Xiang Hua
XH
Han
Yong-Ri
YR
Jin
Marianne
M
Seto
Jeong Kyo
JK
Yoon
D000818:Animals; D004622:Embryo, Mammalian / Q000196:embryology*; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008817:Mice, Mutant Strains; D024510:Muscle Development / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism*; D051787:Myogenic Regulatory Factor 5 / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D019699:Thrombospondins / Q000096:biosynthesis* / Q000235:genetics; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism*; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*; D000098907:R-Spondins
D036341:Intercellular Signaling Peptides and Proteins; D051787:Myogenic Regulatory Factor 5; D012333:RNA, Messenger; D019699:Thrombospondins; D051153:Wnt Proteins; D051176:beta Catenin; C497417:MYF5 protein, human; C497418:Myf5 protein, mouse; C503243:RSPO1 protein, human; D000098907:R-Spondins; C518903:RSPO1 protein, mouse; C518904:RSPO2 protein, mouse; C557408:Rspo2 protein, human
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