Abstract
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.
Highlights
Using small interfering RNA to knock down endogenous Smurf1 in C2C12 cells, we demonstrate that, at the physiological level, Smurf1 is required for myogenic differentiation and does operate in C2C12 cells to modulate BMPmediated osteogenic differentiation
Having established the stable clones of C2C12 cells that overexpress Smurf1, we examined the impact of elevated Smurf1 on myogenic differentiation
When grown to confluence and deprived of growth factors, C2C12 cells spontaneously commit to a myogenic differentiation process that can be blocked by the action of both TGF- and bone morphogenetic protein (BMP) (14 –17, 27, 30)
Summary
Vol 278, No 40, Issue of October 3, pp. 39029 –39036, 2003 Printed in U.S.A. Smurf Facilitates Myogenic Differentiation and Antagonizes the Bone Morphogenetic Protein-2-induced Osteoblast Conversion by Targeting Smad 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- (TGF-) superfamily. 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. To address the biological significance of Smurf-mediated Smad degradation and the specificity of Smurfs toward TGF- or BMP signaling, we took advantage of the in vitro differentiation of C2C12 myoblast cells. C2C12 cells exit cell cycle, up-regulate muscle-specific genes, and fuse into multinucleated myotubes Both TGF- and BMP inhibit the myogenic differentiation of C2C12 myoblasts, but the outcomes of inhibition by these two factors are very different. Using small interfering RNA (siRNA) to knock down endogenous Smurf in C2C12 cells, we demonstrate that, at the physiological level, Smurf is required for myogenic differentiation and does operate in C2C12 cells to modulate BMPmediated osteogenic differentiation
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