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。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似乎有一个独特的功能，即Smad3而不是Smad2的信令需要TLP。我们正在研究改变TLP在细胞中的表达是否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。在这些靶点中，有两个依赖于SMAD3的即刻早期基因，分别与细胞凋亡和上皮向间充质转化有关，分别是Gadd45beta和Snail。我们还试图通过检测Smad2或Smad3过表达或抑制在肿瘤发生和转移中的结果来鉴定Smad2和Smad3在癌细胞中的特定靶点，通过检测在体外和体内改变这两条途径的平衡的效果来描述来自亲本MCF10A系的人乳腺癌细胞这两条通路的平衡。初步结果令人惊讶地表明，Smad通路同时介导了转化生长因子-β对肿瘤细胞的肿瘤抑制和促转移活性。其他方法是基于新的发现，SNIP1，一个我们首先确定为转化生长因子-β信号抑制因子的分子，与c-Myc相互作用，并增强其转录激活活性。人们希望，正在进行的检测SNIP1在各种人类癌症中的表达模式的研究将有助于阐明该分子在癌症发生中的复杂作用。 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。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。与CCR的Angelo Russo博士和James Mitchell博士合作，我们专注于Smad3在皮肤中丢失对电离辐射的保护作用机制，并试图将我们在小鼠身上的发现与体外辐射对原代角质形成细胞和成纤维细胞的影响研究相关联。在Wakayama大学的Shizuya Saika博士和Akira Ooshima博士的合作中，我们已经证明，在缺乏Smad3的小鼠中，损伤诱导的上皮细胞转分化为间充质表型，就像在眼睛损伤或肾脏阻塞性疾病中一样，完全被阻断。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以及减少肺损伤、视网膜脱离或输尿管梗阻所致的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. 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 being required for signaling from Smad3, but not Smad2. We are investigating whether altering the expression of TLP in cells might modulating the signal transduction pathways and gene targets of TGF-beta in specific cell types or in disease. Among the targets are two Smad3-dependent immediate-early genes which have been implicated in apoptosis and in epithelial-to-mesenchymal transition, respectively, Gadd45beta and Snail. We are also attempting to identify specific targets of Smad2 and Smad3 in cancer cells, by characterizing the outcome of overexpression or suppression 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. Initial results surprisingly demonstrate that the Smad pathway mediates both tumor suppressor and pro-metastatic activities of TGF-beta on tumor cells. Other approaches are based on the novel finding that SNIP1, a molecule which we first identified as a suppressor of TGF-beta signaling, interacts with c-Myc and enhances its transcriptional activating activity. It is hoped that ongoing studies examining the expression patterns of SNIP1 in a variety of human cancers will shed light onto the complex role of this molecule in carcinogenesis. 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 mechanisms of protective effects of loss of Smad3 in the skin in response to ionizing radiation, and attempting to correlate our findings in mice with studies of effects of irradiation on primary keratinocytes and fibroblasts in vitro. In collaborations with Dr. Shizuya Saika and Akira Ooshima, Wakayama U., we have shown that injury-induced transdifferentiation of epithelial cells to a mesenchymal phenotype post injury, as in injury to the eye or obstructive disease in the kidney, 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 resulting from lung injury, retinal detachment, or ureteral obstruction.