Signal Transduction Pathways of Transforming Growth Fact

tgf - β家族的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-苏氨酸激酶活性和一种称为Smads的新型细胞质蛋白家族。在这一途径中，受体激活的Smads2和smad3直接被I型受体激酶磷酸化，并与共同的介质Smad4一起转运到细胞核，在那里它们参与转录复合物。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。一种方法是确定直接或间接调节受体或Smad活性的独特因素。该实验室首次发现的一种新型核蛋白SNIP1已被证明与Smad4相互作用并抑制tgf - β信号传导，现在，基于酵母双杂交筛选，与重要的tgf - β靶点c-Myc相互作用以增强其转录激活活性。SNIP1稳定c-Myc并增强其与辅激活子p300的相互作用。正在进行的研究检测了多种人类癌症中SNIP1的表达模式，表明它将在阻断tgf - β的肿瘤抑制功能和增强c-Myc的致癌性方面发挥复杂的作用。为了补充上述基本生化方法，我们还开发了一个强有力的研究项目，该研究基于以下假设：在体内删除特定的下游信号通路，从概念上讲，应该比涉及靶向删除或过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作用。目标过程包括伤口愈合，纤维化，尤其是癌变。在一种方法中，我们试图通过表征Smad2或Smad3在肿瘤发生和转移过程中选择性过表达或抑制的结果，来确定癌细胞中Smad2和Smad3的特定靶点。目前，这方面的工作主要集中在检查来自亲本MCF10A系的人乳腺癌细胞中改变这两种途径平衡的体外和体内影响。初步结果表明，Smad通路介导肿瘤细胞上tgf - β的肿瘤抑制和促转移活性，但Smad2和Smad3的具体作用仍有待确定。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型可以被操纵。初步结果表明，MMP-9是由成纤维细胞响应肿瘤细胞的信号而产生的，并取决于上皮成分的致瘤程度和成纤维细胞的信号通路。我们已经证明肿瘤细胞的MMP-9分泌和迁移部分依赖于tgf - β通过Smad3和MAPK途径发出的信号。我们正在研究的基质反应的另一个方面包括理解tgf -抑制免疫监视的机制，特别是那些涉及树突状细胞的机制。由NIDDK的Chuxia Deng开发的Smad3敲除小鼠继续为tgf - 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朵伤口模型来揭示Smad3在依赖于细胞外基质（特别是弹性蛋白、胶原蛋白、糖胺聚糖和整合素）变化的伤口愈合机械转导调节中的新作用。与CCR James Mitchell博士合作，我们继续关注电离辐射下皮肤中Smad3缺失的保护作用机制，使用皮肤移植物和微阵列分析来识别参与介导保护的特定细胞类型和途径。

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. In collaboration with Dr. James Mitchell, CCR, we continue to focus on mechanisms of protective effects of loss of Smad3 in the skin in response to ionizing radiation, using skin grafts and microarray analysis to identify specific cell types and pathways involved in mediating protection.