Open Access Research article

Retinoic acid enhances skeletal muscle progenitor formation and bypasses inhibition by bone morphogenetic protein 4 but not dominant negative β-catenin

Karen AM Kennedy3†, Tammy Porter1†, Virja Mehta1, Scott D Ryan1,2, Feodor Price4, Vian Peshdary1,2, Christina Karamboulas1,3, Josée Savage1, Thomas A Drysdale5, Shun-Cheng Li3, Steffany AL Bennett1,2 and Ilona S Skerjanc1,3*

Author Affiliations

1 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada

2 Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, Canada

3 Department of Biochemistry, Medical Sciences Building, The University of Western Ontario, London, Ontario, Canada

4 Ottawa Health Research Institute, Molecular Medicine Program, Ottawa, Ontario, Canada

5 Department of Pediatrics and Physiology and Pharmacology, The University of Western Ontario, Children's Health Research Institute, London, Ontario, Canada

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BMC Biology 2009, 7:67 doi:10.1186/1741-7007-7-67

Published: 8 October 2009

Abstract

Background

Understanding stem cell differentiation is essential for the future design of cell therapies. While retinoic acid (RA) is the most potent small molecule enhancer of skeletal myogenesis in stem cells, the stage and mechanism of its function has not yet been elucidated. Further, the intersection of RA with other signalling pathways that stimulate or inhibit myogenesis (such as Wnt and BMP4, respectively) is unknown. Thus, the purpose of this study is to examine the molecular mechanisms by which RA enhances skeletal myogenesis and interacts with Wnt and BMP4 signalling during P19 or mouse embryonic stem (ES) cell differentiation.

Results

Treatment of P19 or mouse ES cells with low levels of RA led to an enhancement of skeletal myogenesis by upregulating the expression of the mesodermal marker, Wnt3a, the skeletal muscle progenitor factors Pax3 and Meox1, and the myogenic regulatory factors (MRFs) MyoD and myogenin. By chromatin immunoprecipitation, RA receptors (RARs) bound directly to regulatory regions in the Wnt3a, Pax3, and Meox1 genes and RA activated a β-catenin-responsive promoter in aggregated P19 cells. In the presence of a dominant negative β-catenin/engrailed repressor fusion protein, RA could not bypass the inhibition of skeletal myogenesis nor upregulate Meox1 or MyoD. Thus, RA functions both upstream and downstream of Wnt signalling. In contrast, it functions downstream of BMP4, as it abrogates BMP4 inhibition of myogenesis and Meox1, Pax3, and MyoD expression. Furthermore, RA downregulated BMP4 expression and upregulated the BMP4 inhibitor, Tob1. Finally, RA inhibited cardiomyogenesis but not in the presence of BMP4.

Conclusion

RA can enhance skeletal myogenesis in stem cells at the muscle specification/progenitor stage by activating RARs bound directly to mesoderm and skeletal muscle progenitor genes, activating β-catenin function and inhibiting bone morphogenetic protein (BMP) signalling. Thus, a signalling pathway can function at multiple levels to positively regulate a developmental program and can function by abrogating inhibitory pathways. Finally, since RA enhances skeletal muscle progenitor formation, it will be a valuable tool for designing future stem cell therapies.