Retinoic acid enhances skeletal muscle progenitor formation and bypasses inhibition by bone morphogenetic protein 4 but not dominant negative beta-catenin.
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 beta-catenin-responsive promoter in aggregated P19 cells. In the presence of a dominant negative beta-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.


CONCLUSIONS
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 beta-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.
Kennedy
Karen A M
KA
Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada. kark61@hotmail.com
Porter
Tammy
T
Mehta
Virja
V
Ryan
Scott D
SD
Price
Feodor
F
Peshdary
Vian
V
Karamboulas
Christina
C
Savage
Josée
J
Drysdale
Thomas A
TA
Li
Shun-Cheng
SC
Bennett
Steffany A L
SA
Skerjanc
Ilona S
IS
D000818:Animals; D019521:Body Patterning / Q000187:drug effects; D055415:Bone Morphogenetic Protein 4 / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D019070:Cell Lineage / Q000187:drug effects; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D005799:Genes, Dominant; D006801:Humans; D051379:Mice; D008954:Models, Biological; D024510:Muscle Development / Q000187:drug effects; D018482:Muscle, Skeletal / Q000166:cytology*; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D038081:Organogenesis / Q000187:drug effects; D051419:Paxillin / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects*; D014212:Tretinoin / Q000494:pharmacology*; D051176:beta Catenin / Q000378:metabolism*
D055415:Bone Morphogenetic Protein 4; D017570:MyoD Protein; D051419:Paxillin; D051176:beta Catenin; D014212:Tretinoin; D004121:Dimethyl Sulfoxide