Signal Transduction Pathways of Transforming Growth Fact

TGF-β配体家族通过独特的异聚受体复合物发出信号，该异聚受体复合物以其丝氨酸-苏氨酸激酶活性为特征。从这些受体到核靶基因的直接信号转导途径已被阐明，其中涉及一个新的蛋白质家族，称为Smads。在该途径中，受体激活的Smads直接被I型受体激酶磷酸化，并与共同的介体Smad 4结合，易位到细胞核，在那里它们参与转录复合物。我们已经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，并了解其在体内的意义。一种方法是鉴定调节受体或Smad活性的独特因子。之前已经确定了TGF-β受体相互作用蛋白TRAP 1作为专性信号伴侣Smad 4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TRAP 1样蛋白或TLP。TLP似乎具有来自Smad 3而不是Smad 2的信号传导所需的独特特征。我们正在研究改变细胞中TLP的表达是否可能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。其中的目标是两个Smad 3依赖的立即早期基因，这两个基因分别与细胞凋亡和上皮细胞向间质细胞转化有关，Gadd 45 β和Snail。我们还试图确定癌细胞中Smad 2和Smad 3的特异性靶点，通过检测在来自亲本MCF 10A系的人乳腺癌细胞中改变这两种途径的平衡的体外和体内效应，来表征Smad 2或Smad 3在肿瘤发生和转移中的过表达或抑制的结果。初步结果令人惊讶地证明，Smad途径介导TGF-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，SNIP 1，我们首先确定为TGF-β信号转导抑制分子，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研究SNIP 1在各种人类癌症中的表达模式将揭示这种分子在致癌作用中的复杂作用。 为了补充上述基本的生物化学方法，我们还开发了一个强有力的研究计划，该计划基于以下假设：在概念上，体内特定下游信号传导途径的缺失应该比涉及配体或受体的靶向缺失或过表达的更广泛的方法具有更少的严重性和更高的选择性。由NIDDK的Chuxia Deng开发的Smad 3基因敲除小鼠现在为TGF-β在造血、损伤修复和纤维化中的作用提供了新的见解。与Angelo Russo和James Mitchell博士合作，CCR，我们专注于电离辐射引起皮肤Smad 3损失的保护作用机制，并试图将我们在小鼠中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。与和歌山大学Shizuya Saika博士和Akira Ooshima合作，我们已经表明，在缺乏Smad 3的小鼠中，损伤诱导的上皮细胞向损伤后间充质表型的转分化（如在眼睛损伤或肾脏阻塞性疾病中）被完全阻断。TGF-β依赖于Smad 3途径的发病机制的阐明现在表明，Smad 3抑制剂的开发将在加速皮肤伤口的上皮形成、减少透镜植入物的混浊和减少由肺损伤、视网膜脱离或输尿管阻塞引起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.