Molecular Mechanisms of TGF-beta Signaling Pathway

转化生长因子-β的信号在多个水平上受到调节，以产生特异性并微调这些信号。这些调控机制之一是泛素-蛋白酶体途径，它通过降解控制Smad蛋白的水平和/或活性，从而调节细胞对转化生长因子-β配体的反应。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，我们正在对SMurf2基因缺陷小鼠和SMurf1/SMurf2双基因缺陷小鼠的表型进行研究，以研究SMurf介导的泛素化如何影响细胞生长、组织分化和其他受配体家族调控的生物学过程。尽管Smads参与了TGF-β超家族的大部分活动，但激活的TGF-β受体也通过其他细胞内信号通路传递信号，特别是由MAP激酶介导的那些信号通路。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。为此，我们发现TRAF6是JNK和p38 Smad非依赖性激活所必需的，其羧基TRAF同源结构域与转化生长因子-β受体发生物理作用。转化生长因子-β诱导K63连接的TRAF6泛素化，并促进TRAF6和TAK1之间的关联。我们的结果表明，转化生长因子-β激活JNK和p38的机制与IL-1β/Toll样受体途径类似。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质并表征它们的功能来扩展这一发现，以表征Smad非依赖性通路的分子机制。我的小组的第三个方向集中在异常的Smad信号在肿瘤发生中的作用。在四环素抑制启动子(tet-off)的控制下，我们已经产生了不同的转基因小鼠系，携带野生型、显性阴性或Smad3。我们将这些小鼠与LAP-TTA小鼠杂交，LAP-TTA小鼠允许四环素调控的四环素反式激活蛋白(TTA)在肝细胞中特异性表达Smad3及其变异体。我们发现，Smad3的高表达保护肝脏免受化学诱导的癌变，这是由于肝脏对凋亡刺激的反应增强所致。我们计划继续使用这个模型来进一步探索Smad3在肝脏肿瘤进展和转移的晚期阶段中的作用。此外，我们还对Smad信号如何与其他途径融合以及这些信号通路在控制转化生长因子-β调控的基因转录、细胞增殖、分化、凋亡和肿瘤进展中发挥的作用感兴趣。

Signaling by TGF-beta is regulated at multiple levels in order to generate specificity and to finely tune these signals. One of these regulatory mechanisms is the ubiquitin-proteasome pathway, which controls the level and/or activity of Smad proteins through degradation, thereby modulating cellular responsiveness to TGF-beta ligands. 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. 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 Smurf2 deficient mice and Smurf1/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. Toward this goal, we found that TRAF6 is specifically required for the Smad-independent activation of JNK and p38 and its carboxyl TRAF homology domain physically interacts with TGF-beta receptors. TGF-beta induces K63-linked ubiquitination of TRAF6, and promotes association between TRAF6 and TAK1. Our results indicate that TGF-beta activates JNK and p38 through a mechanism similar to that operating in the interleukin-1beta/Toll-like receptor pathway. Currently, we seek to expand this findings to characterize molecular mechanisms of the Smad-independent pathways by identifying proteins that are specifically associated with TGF-beta type I receptor and characterizing their functions. The third direction of my group focuses on the effect of aberrant Smad signaling in tumorigenesis. We have generated different lines of transgenic mice carrying either wild type, or dominant negative or Smad3 under the control of a tetracycline-repressible promoter (tet-off). We crossed these mice to LAP-tTA mice, which allow tetracycline-regulated expression of tetracycline-transactivating protein (tTA) specifically in hepatocytes, to express Smad3 and its variants in liver. We find that elevated Smad3 expression protects liver from chemically induced carcinogenesis due to a heightened hepatic response to apoptotic stimuli. We plan to continue using this model to further explore the role of Smad3 in late stages of liver tumor progression and metastasis. In addition, we are also interested in how Smad signaling converges with other pathways and what kind of roles these cross-talks play in controlling TGF-beta-regulated gene transcription, cell proliferation, differenciation, apoptosis and tumor progression.