Smurf1 facilitates myogenic differentiation and antagonizes the bone morphogenetic protein-2-induced osteoblast conversion by targeting Smad5 for degradation.
Controlled proteolysis mediated by Smad ubiquitination regulatory factors (Smurfs) plays a crucial role in modulating cellular responses to signaling of the transforming growth factor-beta (TGF-beta) superfamily. However, it is not clear what influences the selectivity of Smurfs in the individual signaling pathway, nor is it clear the biological function of Smurfs in vivo. Using a mouse C2C12 myoblast cell differentiation system, which is subject to control by both TGF-beta and bone morphogenetic protein (BMP), here we examine the role of Smurf1 in myogenic differentiation. We show that increased expression of Smurf1 promotes myogenic differentiation of C2C12 cells and blocks the BMP-induced osteogenic conversion but has no effect on the TGF-beta-induced differentiation arrest. Consistent with an inhibitory role in the BMP signaling pathway, the elevated Smurf1 markedly reduces the level of endogenous Smad5, whereas it leaves unaltered that of Smad2, Smad3, and Smad7, which are components of the TGF-beta pathway. Adding back Smad5 from a different source to the Smurf1-overexpressing cells restores the BMP-mediated osteoblast conversion. Finally, by depletion of the endogenous Smurf1 through small interfering RNA-mediated RNA interference, we demonstrate that Smurf1 is required for the myogenic differentiation of C2C12 cells and plays an important regulatory role in the BMP-2-mediated osteoblast conversion.
Ying
Sai-Xia
SX
Laboratory of Cellular and Molecular Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Hussain
Zareena J
ZJ
Zhang
Ying E
YE
D000199:Actins / Q000235:genetics; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D003402:Creatine Kinase / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D008025:Ligases / Q000378:metabolism / Q000502:physiology*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D010006:Osteoblasts / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction; D051899:Smad2 Protein; D051900:Smad3 Protein; D051902:Smad5 Protein; D051906:Smad7 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
D000199:Actins; C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497797:SMAD5 protein, human; C497806:SMAD7 protein, human; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D051902:Smad5 Protein; C497798:Smad5 protein, mouse; D051906:Smad7 Protein; C497807:Smad7 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D012313:RNA; D008156:Luciferases; C471641:SMURF2 protein, human; C492381:Smurf1 protein, mouse; D044767:Ubiquitin-Protein Ligases; D003402:Creatine Kinase; D000469:Alkaline Phosphatase; D008025:Ligases