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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.

nur77 is an immediate-early gene inducible by nerve growth factor or membrane depolarization in the rat pheochromocytoma cell line PC12 and by serum growth factors in fibroblasts. The nur77-encoded protein is a member of the steroid/thyroid hormone receptor superfamily and can act as a potent transcription activator. The induction of nur77 in PC12 cells is rapid and transient, with kinetics similar to those of the c-fos protooncogene. Induction does not require de novo protein synthesis. Whereas transcriptional activation of c-fos by nerve growth factor in PC12 cells requires a 20-base pair serum response element in its promoter, there is no such sequence in the nur77 promoter. To understand the mechanism for the activation of nur77, we have analyzed the inducibility of a series of transfected nur77 minigenes using an S1 nuclease protection assay. We identified the sequence 22-86 nucleotides upstream of the transcription start site as necessary and sufficient for nur77 induction by nerve growth factor and membrane depolarization in PC12 cells. Sequences farther upstream enhance the induction. Analysis of base substitution mutations allowed us to identify three sequence elements within this region that are essential for induction. These sequence elements include two copies of an AP1-like element and a GC-rich sequence. Unlike transcriptional activation of c-fos, the sequence requirements for the activation of nur77 by nerve growth factor and membrane depolarization cannot be readily separated. Taken together, our data suggest that activation of nur77 and c-fos by nerve growth factor occurs through different mechanisms in PC12 cells.