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。到目前为止，转化生长因子-β信号通路不同组成部分的遗传或表观遗传改变已在许多人类发育或过度增殖疾病以及各种形式的癌症中被报道。我们的研究集中在转化生长因子-β信号转导的三个方面，以期进一步了解其在发育和肿瘤发生中的调控、作用机制和功能。 首先是了解泛素-蛋白酶体系统在调节转化生长因子-β信号中的作用。我们和其他人已经鉴定了Hect结构域泛素连接酶家族的两个Smad泛素调节因子(SMurf)，并表明SMurf1和SMurf2能够直接与BMP途径的Smad1和Smad5相互作用并介导它们的降解。为了探讨蓝精灵在转化生长因子-β信号转导中的生理意义，我们培育了缺乏SMurf1或SMurf2的小鼠，并报告了SMurf1缺陷小鼠出生时正常，但由于成骨细胞活性增强和对BMP的反应性增强，表现出随年龄增加的骨量增加。令人惊讶的是，这种骨骼异常并不是由Smad介导的转化生长因子-β或骨形态发生蛋白信号的改变引起的。相反，SMurf1的缺失会导致成骨细胞中磷酸化的MEKK2的积累，并激活其下游的JNK信号级联。我们证明MEKK2是S-1泛素连接酶的直接生理靶点。我们的结果揭示了SMurf1在成骨细胞生理和骨稳态调节中的新功能，并为丝裂原活化蛋白激酶(MAPK)信号通路在形成对转化生长因子-β家族的特异性生物反应中的重要性提供了一个有趣的例子。目前，我们正在研究SMurf1和SMurf2双缺陷小鼠的表型，以研究SMurf介导的泛素化如何影响细胞生长、组织分化和受转化生长因子-β家族调控的其他生物学过程。 尽管Smads参与了转化生长因子-β超家族的大部分活动，但激活的转化生长因子-β受体也通过其他细胞内信号转导途径传递信号，特别是由MAP激酶介导的信号转导。我的团队的第二个研究领域集中在转化生长因子-β受体激活不依赖于Smads的MAP激酶的具体机制，以及这一非Smad依赖的途径在转化生长因子-β信号转导中的生物学意义。我们观察到，在小鼠乳腺上皮细胞(NMuMG)中，转化生长因子-β激活p38MAPK是转化生长因子-β诱导的细胞凋亡、上皮向间充质转化(EMT)所必需的，但不是生长停滞所必需的。这种激活是不依赖于Smad的，因为突变的I型受体缺乏与R-SMADs结合的能力，但仍然完全有能力激活p38MAPK。这些结果构成了Smad非依赖的转化生长因子-β受体信号传递的第一个直接生化证据，并表明转化生长因子-β受体通过多个细胞内途径传递信号。目前，我们试图通过鉴定与转化生长因子-βI型受体特异性相关的蛋白并鉴定它们的功能来确定Smad非依赖性激活映射蛋白激酶的分子机制。

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. To date, genetic or epigenetic alterations of different components of the TGF-beta signaling pathway have been reported in a number of human developmental or hyper-proliferative disorders and in various forms of cancers. Our research has focused on three aspects of TGF-beta signaling in an attempt to gain further appreciation of its regulation, mechanisms of action and function in development and tumorigenesis. The first of these is to understand the role of the ubiquitin-proteasome system in modulating TGF-beta signaling. We, and others, have previously identified two Smad ubiquitin regulatory factors (Smurfs) of the HECT domain-containing ubiquitin ligase family and shown that Smurf1 and Smurf2 have the ability to interact directly with Smad1 and Smad5 of the BMP pathway and mediate their degradation. To address the physiological significance of Smurfs in TGF-beta signaling, we have generated mice lacking either Smurf1 or Smurf2, and reported that Smurf1-deficient mice are perinatally normal but exhibit an age-dependent increase of bone mass due to enhanced osteoblast activity and increased responsiveness to BMP. Surprisingly, this skeletal abnormality is not caused by alteration in Smad-mediated TGF-beta or BMP signaling. Instead, loss of Smurf1 results in accumulation of phosphorylated MEKK2 in osteoblasts and activation of its downstream JNK signaling cascade. We demonstrate that MEKK2 is a direct physiological target of the Smurf1 ubiquitin ligase. Our results reveal a novel function of Smurf1 in the regulation of osteoblast physiology and bone homeostasis, and provide an interesting example for the importance of the mitogen-activated protein kinase (MAPK) signaling pathway in shaping specific biological response to the TGF-beta family of cytokines. Currently, we are characterizing the phenotypes of Smurf1 and Smurf2 double deficient mice to investigate how Smurf-mediated ubiquitination affects cell growth, tissue differentiation and other biological processes regulated by the TGF-beta family of ligands. Although Smads are involved in most actions of the TGF-beta superfamily, activated TGF-beta receptors also transduce signals through other intracellular signaling pathways, especially those mediated by MAP kinases. The second area of research of my group focuses on the specific mechanism by which TGF-beta receptors activate MAP kinases independent of Smads, and the biological significance of this non-Smad dependent pathway in TGF-beta signaling. We have observed that in mouse mammary epithelial (NMuMG) cells, activation of p38 MAPK by TGF-beta is required for TGF-beta-induced apoptosis, epithelial to mesenchymal transition (EMT), but not growth arrest. This activation is Smad independent since a mutant type I receptor, which lacks the ability to bind R-Smads, is nevertheless fully competent to activate p38 MAPK. These results constitute the first direct biochemical evidence for Smad-independent TGF-beta receptor signaling and indicate that TGF-beta receptor signals through multiple intracellular pathways. Currently, we seek to determine the molecular mechanism of the Smad-independent activation MAP kinases by identifying proteins that are specifically associated with TGF-beta type I receptor and characterizing their functions.