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，我们和其他人发现了E3泛素连接酶Hect家族的两个新成员SMurf1和SMurf2，它们是Smads的相互作用伙伴，但SMurf在体内的功能以及在特定信号通路中对Smad的选择性仍有待确定。我们继续对这一领域的研究感兴趣，并开始解决这些重要的生物学问题。我们利用小鼠C2C12成肌细胞的体外分化过程，研究了SMurf1在成肌和成骨分化中的作用，C2C12成肌细胞受转化生长因子-β和骨形态发生蛋白(BMP)的控制。我们发现，SMurf1的表达增加促进了C2C12细胞的肌源性分化，并阻断了BMP诱导的成骨转化，但对转化生长因子-β诱导的分化停滞没有影响。与BMP信号通路中的抑制作用一致，升高的SMurf1显著降低内源性Smad5的水平，而不改变转化生长因子-β途径的组成部分Smad2、Smad3和Smad7的水平。将来自不同来源的Smad5重新加入到S-1过度表达的细胞中，可以恢复BMP介导的成骨细胞转换。最后，通过小干扰RNA介导的RNA干扰来耗尽内源性SMurf1，我们证明了SMurf1是C2C12细胞成肌分化所必需的，并在BMP-2介导的成骨细胞转化过程中发挥着重要的调节作用。 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。 此外，我们还感兴趣的是，转化生长因子-β信号如何与其他途径汇聚，以响应生长因子，从而激活丝裂原活化蛋白激酶(MAPK)途径。我们想要了解这种相互作用在控制转化生长因子-β调控的基因转录、细胞增殖、细胞外基质产生、细胞凋亡和肿瘤进展中的作用。与此同时，一项利用小鼠遗传学来解决转化生长因子-β/Smad信号在肿瘤发生中的生理和病理作用的长期研究计划也已经完成。

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 and others identified Smurf1 and Smurf2, two new members of the HECT family of E3 ubiquitin ligases, as interacting partners for Smads, but the in vivo function of Smurfs and the selectivity of Smurfs towards Smad in specific signaling pathways remain to be determined. We continued our research interest in this area and began to address these important biological issues. We examined the role of Smurf1 in myogenic and osteogenic differentiation by taking advantage of the in vitro differentiation process of mouse C2C12 myoblast cells, which is subject to control by both TGF-beta and bone morphogenetic protein (BMP). We found 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 while it leaves unaltered the levels 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 endogenous Smurf1 through small interfering RNA-mediated RNA interference, we demonstrated that Smurf1 is required for the myogenic differentiation of C2C12 cells and plays an important regulatory role in the BMP-2-mediated osteoblast conversion. 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. In addition, we are also interested in how TGF-beta signaling converges with other pathways in response to growth factors and consequent activation of mitogen-activated protein kinase(MAPK) pathways. We would like to understand the role of this cross-talk in controlling TGF-beta-regulated gene transcription, cell proliferation, extracellular matrix production,apoptosis and tumor progression. In the meantime, a long term research program using mouse genetics to address the physiological and pathological roles of TGF-beta/Smad signaling in tumorigenesis has also been