Members of the transforming growth factor-beta (TGF-beta) family of peptide growth factors, which include TGF-beta, bone morphogenetic proteins (BMPs) and activins, regulate a broad range of cellular processes from cell growth and differentiation to apoptosis. The signaling responses to TGF-beta and other family members are mediated by a heteromeric complex of two types of transmembrane serine/threonine kinase receptors at the cell surface, and their intracellular substrates, the Smad proteins. Proper TGF-beta superfamily signaling requires precise control of Smad functions. One of the important mechanisms that control Smad activity is ubiquitin-proteasome-mediated degradation. Previously, we have identified Smurf2, a new member of the HECT family of E3 ubiquitin ligases, as an interacting partner for Smads. We have found that Smurf2 and the related Smurf1 interact with receptor-regulated Smads and preferentially target Smad1 or Smad5 for ubiquitination and proteasome-mediated degradation. However, the importance and overall biological impact of Smurf activity has remained unclear. In order to understand the function of the Smurf-mediated degradation of Smads, we characterized the role of Smurfs in the regulation of myogenic and osteoblastic differentiation in response to TGF-beta and BMP using the C2C12 cell differentiation system. TGF-beta inhibits the differentiation of C2C12 cells into multinucleated myotubes. BMP-2 not only inhibits myogenic differentiation of C2C12 cells but also induces an osteoblast phenotype. Therefore, the C2C12 model is useful for analyzing both the common and specific signaling mechanisms of TGF-beta and BMPs. We found that stable expression of Smurf1 in C2C12 cells promoted myogenic differentiation. Addition of TGF-beta to these cells effectively blocked myotube formation and expression of the muscle specific marker, myosin heavy chain, in both control C2C12 and C2C12-Smurf1 cells. Therefore, TGF-beta signaling pathway was intact in the C2C12-Smurf1 cells, and expression of Smurf1 did not result in degradation of TGF-beta receptors or Smads specific for TGF-beta pathway. We then assessed the ability of C2C12-Smurf1 cells to undergo osteoblastic differentiation in the presence of BMP2. In contrast to TGF-beta treatment, addition of BMP2 to C2C12-Smurf1 cells failed to inhibit myotube formation. Moreover, stable expression of Smurf1 also inhibited BMP-2-induced osteoblastic differentiation. These results suggest that Smurf1 inhibited BMP signaling pathway in these cells. Accordingly, BMP pathway- specific Smad5 levels were dramatically reduced in C2C12-Smurf1 cells. Therefore, our results indicate that Smurf1 promotes myogenic differentiation and inhibits BMP-induced osteoblastic differentiation by reducing Smad5 protein level, thereby decreasing BMP but not TGF-beta signaling. Although Smads are involved in most actions of the TGF-beta superfamily, many reports have suggested that TGF-beta may signal through alternative pathways. In order to characterize the mechanism of TGF-beta signaling through Smad-independent pathways and to understand the function of Smad-independent TGF-beta receptor signaling, we have generated a mutant TGF-beta type I receptor that is unable to activate Smads but retains kinase activity. We found that this mutant TGF-beta type I receptor is able to activate p38 kinase, and the p38 activation is required for TGF-beta induced apoptosis and epithelial to mesenchymal transition. These results indicate that the TGF-beta receptor exerts its signals through multiple intracellular pathways and provide first hand biochemical evidence to support the existence of Smad-independent TGF-beta receptor signaling. Currently we are working to identify downstream mediators that are responsible for Smad-independent TGF-beta receptor signaling. These studies could uncover novel molecular mechanisms that account for a number of Smad-independent TGF-beta signaling responses.
