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和一种称为Smads的新型细胞质蛋白来传递信号。在这个途径中，受体激活的Smads2和Smads3直接被I型受体激酶磷酸化，并与共同的介导物Smad4一起移位到细胞核，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。一种方法是确定直接或间接调节受体或Smad活性的独特因素。本实验室首次发现的一种新的核蛋白SNIP1，已被证明与Smad4相互作用并抑制转化生长因子-β信号，现在，基于酵母双杂交筛选，它与重要的转化生长因子-β靶标c-Myc相互作用，以增强其转录激活活性。SNIP1稳定c-Myc并增强其与辅活化子p300的相互作用。正在进行的研究表明，SNIP1在多种人类癌症中的表达模式表明，它将在阻断转化生长因子-β的肿瘤抑制功能和增强c-Myc的致瘤性方面发挥复杂的作用。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。目标过程包括伤口愈合、纤维化，特别是癌变。在一种方法中，我们试图通过表征选择性过表达或抑制Smad2或Smad3在肿瘤发生和转移中的结果，来确定Smad2和Smad3在癌细胞中的特定靶点。目前，这方面的大部分工作都集中在检测从亲本MCF10A系衍生的人乳腺癌细胞中改变这两个途径的平衡的体外和体内效应。初步结果表明，Smad2和Smad3在肿瘤细胞中的具体作用尚不清楚，但Smad2和Smad3在肿瘤细胞中的抑制和促进转移活性均可通过Smad途径实现。另一种方法是基于肿瘤细胞和基质细胞的体外共培养，其中每个组成细胞的基因可以被操纵。初步结果表明，基质金属蛋白酶-9是由成纤维细胞对肿瘤细胞信号的反应而产生的，并且取决于成纤维细胞中上皮成分的致瘤性程度和信号转导途径。我们已经证明，肿瘤细胞的基质金属蛋白酶-9的分泌和迁移在一定程度上依赖于通过SMAD3和MAPK通路的转化生长因子-β信号。我们正在研究的基质反应的另一个方面涉及了解转化生长因子-β抑制免疫监视的机制，特别是涉及树突状细胞的机制。由NIDDK的楚霞·邓开发的Smad3基因敲除小鼠继续为转化生长因子-β在损伤修复和纤维化中的作用提供新的见解。我们正在使用一种新的耳部创伤模型来揭示Smad3在依赖于细胞外基质变化的伤口闭合的机械转导中的新作用，特别是弹性蛋白、胶原、糖胺聚糖和整合素的变化。

The TGF-beta family of ligands signal through a unique heteromeric receptor complex distinguished by its serine-threonine kinase activity and a novel family cytoplasmic proteins termed Smads. In this pathway, receptor-activated Smads2 and 3 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 that modulate receptor or Smad activity either directly or indirectly. A novel nuclear protein first identified in this laboratory, SNIP1, has been shown to interact with Smad4 and to suppress TGF-beta signaling, and now, based on a yeast two-hybrid screen, to interact with an important TGF-beta target, c-Myc to enhance its transcriptional activating activity. SNIP1 stabilizes c-Myc and enhances its interaction with the coactivator, p300. Ongoing studies examining the expression patterns of SNIP1 in a variety of human cancers suggest that it will play a complex role both in blocking the tumor suppressor functions of TGF-beta and enhancing the oncogenicity of c-Myc. 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. Processes targeted include wound healing, fibrosis, and especially carcinogenesis. In one approach we are attempting to identify specific targets of Smad2 and Smad3 in cancer cells, by characterizing the outcome of selective overexpression or suppression of Smad2 or Smad3 in tumorigenesis and metastasis. Much of this effort is presently focused on 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. Initial results show that the Smad pathway mediates both tumor suppressor and pro-metastatic activities of TGF-beta on tumor cells, though specific roles of Smad2 and Smad3 remain to be identified. Another approach is based on in vitro co-culture of tumor cells and stromal cells, where the genotype of each component cell can be manipulated. Initial results show that MMP-9 is produced by fibroblasts in response to signals from tumor cells and depends both on the degree of tumorigenicity of the epithelial component and the signaling pathways in the fibroblasts. We have shown that both MMP-9 secretion and migration of the tumor cells is dependent, in part, on TGF-beta signaling through Smad3 as well as through MAPK pathways. Another aspect of the stromal response that we are investigating involves understanding mechanisms of suppression of immune surveillance by TGF-beta, especially those involving dendritic cells.The Smad3 knockout mouse, developed by Chuxia Deng, NIDDK, continues to provide new insights into the roles of TGF-beta in repair of injury and in fibrosis. We are using a novel wound model in the ear to unravel novel roles of Smad3 in modulation of mechanotransduction of wound closure dependent on changes in the extracellular matrix, including especially elastin, collagen, glycosaminoglycans and the integrins.