Signal Transduction Pathways of Transforming Growth Fact

转化生长因子-β配体家族通过一种独特的异构体受体复合体发出信号，该复合体以丝氨酸-苏氨酸激酶的活性为特征。最近，从这些受体到核靶基因的直接信号转导途径已经被阐明，其中涉及一个新的蛋白质家族，称为Smads。在这个途径中，受体激活的Smad直接被I型受体激酶磷酸化，并与共同的介导物Smad4结合，转移到细胞核，在那里它们参与转录复合体。我们采取了多方面的方法，在体外深入了解这一途径的生物化学，并了解其在体内的意义。一种方法是确定调节受体或Smad活性的独特因素。在之前确定了转化生长因子-β受体相互作用蛋白TRAP1作为专有信号伙伴Smad4的伴侣的作用后，我们现在正在鉴定一种相关的蛋白质，我们称之为TRAP1样蛋白，或TLP。TLP似乎具有选择性地阻止来自Smad3而不是Smad2的信令的独特功能。因此，它可能在特定细胞类型或疾病中调节转化生长因子-β的信号转导途径和基因靶点方面发挥重要作用。这些研究将进一步扩大，以确定这些分子的作用，检查它们特定的细胞表达模式，并进行酵母双杂交筛选，以确定这些蛋白质是否反过来与信号复合体的其他成分相互作用。这种方法已经潜在地具有SNIP1的重要相互作用因子，SNIP1是另一种新的Smad相互作用蛋白，起到转录抑制因子的作用。其他研究的重点是确定Smad2和Smad3通路在细胞和组织中的独特功能。我们正在解决这个问题，通过使用微阵列分析来确定Smad3或Smad2依赖的通路在小鼠胚胎成纤维细胞中唯一诱导的cDNA为Smad2或Smad3缺失。这些研究已经确定了每一条信号通路的靶点，其特征为研究转化生长因子-β的即刻-早期和二级基因靶点提供了新的视角。我们目前正在研究两个依赖Smad的即刻早期基因的调控，这两个基因分别参与了细胞凋亡和上皮向间充质转化，分别是Gadd45beta和Snail。为了检测Smad2和Smad3在癌细胞中的特定靶点，我们正在通过检测Smad2或Smad3过表达在肿瘤发生和转移中的结果，通过检测在体外和体内改变这两个途径的平衡的影响来表征从亲本MCF10A系衍生的人乳腺癌细胞中这两个通路的平衡。 为了补充上述基本的生化方法，我们还开发了一个强大的研究计划，基于以下假设：从概念上讲，体内特定下游信号通路的删除应该比涉及靶向删除或过度表达配体或受体的更广泛的方法具有更少的严重和更多的选择性影响。由NIDDK楚霞·邓开发的SMAD3基因敲除小鼠现在为转化生长因子-β在造血、损伤修复和纤维化中的作用提供了新的见解。在CCR的Angelo Russo博士和James Mitchell博士的合作下，我们专注于Smad3在皮肤中丢失对电离辐射的保护作用，并试图将这些发现与体外辐射对原代角质形成细胞和真皮成纤维细胞影响的研究相关联。在与和歌山大学Shizuya Saika博士的合作中，我们已经证明，在缺乏SMAD3的小鼠中，眼睛损伤后晶状体上皮细胞向间充质表型的病理转分化被完全阻断。目前对依赖于Smad3途径的转化生长因子-β的致病机制的阐明表明，Smad3抑制剂的开发将在加速皮肤伤口上皮化、减少晶状体植入物混浊和减少纤维化方面具有广泛的临床应用。

The TGF-beta family of ligands signal through a unique heteromeric receptor complex distinguished by its serine-threonine kinase activity. Recently, a direct signal transduction pathway from these receptors to nuclear target genes has been elucidated which involves a novel family of proteins termed Smads. In this pathway, receptor-activated Smads are phosphorylated directly by the type I receptor kinase and, in association with a common mediator Smad4, translocate to the nucleus where they participate in transcriptional complexes. We have taken a multi-faceted approach to gain insight into the biochemistry of this pathway in vitro and to understand its significance in vivo. One approach has been to identify unique factors which modulate receptor or Smad activity. Having previously identified a role for the TGF-beta-receptor-interacting protein, TRAP1, as a chaperone for the obligate signaling partner, Smad4, we are now characterizing a related protein which we call TRAP1-like protein, or TLP. TLP appears to have the unique feature of selectively blocking signaling from Smad3, but not Smad2. As such, it could play an important role in modulating the signal transduction pathways and gene targets of TGF-beta in specific cell types or in disease. These studies will be expanded to define the roles of these molecules further, to examine their specific patterns of cellular expression, and to conduct yeast two-hybrid screens to determine whether these proteins, in turn, interact with yet other components of the signaling complex. This approach has already potentially important interactors of SNIP1, another novel Smad-interacting protein which acts as a transcriptional repressor. Other studies are focused on identifying the unique functions of the Smad2 and Smad3 pathways in cells and tissues. We are addressing this by using microarray analysis to identify cDNAs uniquely induced by Smad3- or Smad2-dependent pathways in mouse embryo fibroblasts null for either Smad2 or Smad3. These studies have resulted in identification of targets of each of these signaling pathways, the characterization of which is providing a new perspective on immediate-early and second-order gene targets of TGF-beta. We are presently investigating the regulation of two of the Smad-dependent immediate-early genes which have been implicated in apoptosis and in epithelial-to-mesenchymal transition, respectively, Gadd45beta and Snail. To examine specific targets of Smad2 and Smad3 in cancer cells, we are characterizing the outcome of overexpression of Smad2 or Smad3 in tumorigenesis and metastasis by examining both in vitro and in vivo effects of altering the balance of these two pathways in human breast cancer cells derived from the parental MCF10A line. To complement the above basic biochemical approaches, we have also developed a strong program of research based on the hypothesis that deletion of specific downstream signaling pathways in vivo should, conceptually, have a less severe and more selective effect than broader-based approaches involving targeted deletion or overexpression of ligand or receptors. The Smad3 knockout mouse, developed by Chuxia Deng, NIDDK, is now providing new insights into the roles of TGF-beta in hematopoiesis, in repair of injury, and in fibrosis. In collaboration with Drs. Angelo Russo and James Mitchell, CCR, we are focusing on protective effects of loss of Smad3 in the skin in response to ionizing radiation, and attempting to correlate these findings with studies of effects of irradiation on primary keratinocytes and dermal fibroblasts in vitro. In a collaboration with Dr. Shizuya Saika, Wakayama U., we have shown that the pathologic transdifferentiation of lens epithelial cells to a mesenchymal phenotype post injury to the eye is completely blocked in mice lacking Smad3. Elucidation of pathogenetic mechanisms of TGF-beta dependent on the Smad3 pathway now suggest that development of a Smad3 inhibitor will have wide-ranging clinical applications in acceleration of epithelization of cutaneous wounds, in reducing opacification of lens implants, and in reducing fibrosis.