Molecular mechanisms of TGF-beta signaling pathway

转化生长因子-β是多肽生长因子家族的成员，包括转化生长因子-β、骨形态发生蛋白(BMPs)和激活素，调节从细胞生长、分化到凋亡的广泛的细胞过程。对转化生长因子-β和其他家族成员的信号反应是由细胞表面两种跨膜型丝氨酸/苏氨酸激酶受体及其胞内底物Smad蛋白组成的异构体复合体介导的。 正确的转化生长因子-β超家族信号传递需要对Smad功能的精确控制。泛素-蛋白酶体介导的降解是控制Smad活性的重要机制之一。此前，我们已经确定SMurf2是E3泛素连接酶Hect家族的一个新成员，作为Smads的相互作用伙伴。我们已经发现，SMurf2及其相关的SMurf1与受体调节的Smads相互作用，并优先靶向Smad1或Smad5，以实现泛素化和蛋白酶体介导的降解。然而，蓝精灵活动的重要性和整体生物影响仍不清楚。为了了解Smads降解的功能，我们利用C2C12细胞分化系统研究了SmRf在转化生长因子-β和骨形态发生蛋白对成肌和成骨细胞分化的调控中的作用。转化生长因子-β抑制C2C12细胞向多核肌管分化。BMP-2不仅抑制C2C12细胞的肌源性分化，而且诱导成骨细胞表型。因此，C2C12模型对于分析转化生长因子-β和骨形态发生蛋白的共同和特异性信号机制是有用的。我们发现SMurf1在C2C12细胞中的稳定表达促进了成肌分化。在这些细胞中加入转化生长因子-β可以有效地抑制C2C12和C2C12-SMurf1细胞中肌球蛋白重链的表达和肌管的形成。因此，在C2C12-SMurf1细胞中，转化生长因子-β信号通路是完整的，并且SMurf1的表达不会导致转化生长因子-β受体或转化生长因子-β途径特异的Smads的降解。然后，我们评估了C2C12-S-1细胞在BMP2存在的情况下进行成骨分化的能力。与转化生长因子-β处理相比，在C2C12-SMurf1细胞中加入BMP2未能抑制肌管的形成。此外，SMurf1的稳定表达也抑制了BMP-2诱导的成骨细胞分化。这些结果表明，SMurf1抑制了这些细胞中的BMP信号通路。相应地，C2C12-SMurf1细胞中BMP途径特异的Smad5水平显著降低。因此，我们的结果表明，SMurf1通过降低Smad5蛋白水平促进肌源性分化，抑制BMP诱导的成骨细胞分化，从而减少BMP而不是转化生长因子-β信号转导。 尽管Smads参与了转化生长因子-β超家族的大部分活动，但许多报告表明，转化生长因子-β可能通过其他途径发出信号。为了研究Smad非依赖性途径中转化生长因子-β信号转导的机制，了解Smad非依赖性转化生长因子-β受体信号的功能，我们产生了一种突变型转化生长因子-βI型受体，该受体不能激活Smads，但仍能保持激酶活性。我们发现这种突变的转化生长因子-βI型受体能够激活p38激酶，并且p38的激活是转化生长因子-β诱导的细胞凋亡和上皮向间质转化所必需的。这些结果表明，转化生长因子-β受体通过多种细胞内途径传递信号，为Smad非依赖性转化生长因子-β受体信号的存在提供了第一手的生化证据。目前，我们正在努力寻找负责Smad非依赖性转化生长因子-β受体信号传递的下游介体。这些研究可能揭示新的分子机制，解释许多Smad非依赖的转化生长因子-β信号反应。

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.