Signal Transduction Pathways of Transforming Growth Fact

tgf - β家族配体通过一种独特的异聚体受体复合物进行信号传递，其特征是丝氨酸-苏氨酸激酶活性。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。在这一途径中，受体激活的smad直接被I型受体激酶磷酸化，并与共同的介质Smad4结合，转运到细胞核，参与转录复合物。我们采取了多方面的方法来深入了解这一途径在体外的生物化学作用，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。之前已经确定了tgf - β受体相互作用蛋白TRAP1作为专性信号伴侣Smad4的伴侣的作用，我们现在正在表征一种相关蛋白，我们称之为TRAP1样蛋白，或TLP。TLP似乎具有选择性阻断Smad3而不是Smad2信号的独特特征。因此，它可能在调节特定细胞类型或疾病中tgf - β的信号转导途径和基因靶点方面发挥重要作用。这些研究将扩展到进一步定义这些分子的作用，检查它们的细胞表达的特定模式，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。这种方法已经发现了潜在的重要的SNIP1相互作用因子，SNIP1是另一种新的smad相互作用蛋白，作为转录抑制因子。其他研究的重点是确定细胞和组织中Smad2和Smad3通路的独特功能。我们正在解决这个问题，通过使用微阵列分析来鉴定Smad3或Smad2依赖性途径在小鼠胚胎成纤维细胞中诱导的dna， Smad2或Smad3均为零。这些研究已经确定了这些信号通路的靶点，这些靶点的表征为tgf - β的即时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于smad的即时早期基因的调控，它们分别与细胞凋亡和上皮-间质转化有关，分别是Gadd45beta和Snail。为了研究Smad2和Smad3在癌细胞中的特异性靶点，我们在体外和体内检测了源自母体MCF10A系的人乳腺癌细胞中Smad2或Smad3过表达在肿瘤发生和转移中的结果，改变了这两种途径的平衡。

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.