Signal Pathways of Transforming Growth Factor Beta

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。最近，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被I型受体激酶直接磷酸化，并与共同的介体Smad 4/DPC 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描绘这一途径的生物化学在体外，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。我们已经利用酵母双杂交系统使用Smad 1作为诱饵，以确定两个新的克隆。一种蛋白质SNX 6是受体相互作用蛋白的分选连接蛋白家族的成员，可能在丝氨酸-苏氨酸激酶受体的运输中起作用。另一个，SNIP 1，是一种新的Smad信号的核阻遏物，并可能整合不同的信号通路之间的串扰，如NF-kappaB会聚在转录复合物依赖于转录辅激活因子p300和CBP的C/H1结构域。我们还阐明了一个新的作用，以前确定的TGF-β受体相互作用蛋白，TRAP 1，它作为一个伴侣的专性合作伙伴，Smad 4，提出Smad 4受体激活Smads。这些研究将扩大到进一步定义这些分子的作用，并包括额外的酵母双杂交筛选，以确定这些蛋白质是否反过来与信号复合物的其他组分相互作用。我们还使用微阵列分析来识别小鼠胚胎成纤维细胞中由Smad 3或Smad 2依赖性途径独特诱导的cDNA。这些研究已经导致这些信号通路的靶点的鉴定，其表征肯定会为TGF-β的即刻早期和二级基因靶点提供新的视角。 为了补充上述基础科学方法，我们还开发了一个强大的研究计划，该计划基于以下假设：在体内删除特定的信号传导组分，在概念上，与涉及配体或受体的靶向删除或过表达的更广泛的方法相比，具有更低的严重性和更高的选择性。我们有幸与NIDDK的Chuxia Deng合作，对Smad 3敲除小鼠进行表征，该小鼠现在为TGF-β在造血，伤口愈合和纤维化中的作用提供了新的见解。在与Angelo Russo和James Mitchell博士的合作中，我们专注于Smad 3在电离辐射中的保护作用，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。TGF-β依赖于这一特定途径的发病机制的阐明表明，Smad 3抑制剂的开发将具有广泛的临床应用。为了研究BMP依赖的信号通路，我们已经创建了Smad 1基因的条件性敲除，以克服Smad 1敲除小鼠的早期胚胎致死性，也是在我们组中产生的。

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/ DPC4, translocate to the nucleus where they participate in transcriptional complexes. We have taken a multi-faceted approach to delineate 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. We have utilized a yeast two-hybrid system using Smad1 as bait to identify two novel clones. One protein, SNX6, a member of the sorting nexin family of receptor interacting proteins, is likely to play a role in trafficking of serine-threonine kinase receptors. The other, SNIP1, is a novel nuclear repressor of Smad signaling, and may integrate cross-talk between different signaling pathways such as NF-kappaB converging on transcriptional complexes dependent on the C/H1 domain of the transcriptional coactivators p300 and CBP. We have also elucidated a novel role for the previously identified TGF-beta-receptor-interacting protein, TRAP1, which acts as a chaperone for the obligate partner, Smad4, presenting Smad4 to receptor-activated Smads. These studies will be expanded to define the roles of these molecules further and to include additional yeast two-hybrid screens to determine whether these proteins, in turn, interact with yet other components of the signaling complex. We are also using microarray analysis to identify cDNAs uniquely induced by Smad3- or Smad2-dependent pathways in mouse embryo fibroblasts. These studies have resulted in identification of targets of these signaling pathways, the characterization of which is certain to provide a new perspective on immediate-early and second-order gene targets of TGF-beta. To complement the above basic science approaches, we have also developed a strong program of research based on the hypothesis that deletion of specific signaling components 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. We have been fortunate to collaborate with Chuxia Deng, NIDDK, on the characterization of a Smad3 knockout mouse, which is now providing new insights into the roles of TGF-beta in hematopoiesis, in wound healing, and in fibrosis. In collaboration with Drs. Angelo Russo and James Mitchell, we are focusing on protective effects of loss of Smad3 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. Elucidation of pathogenetic mechanisms of TGF-beta dependent on this specific pathway now suggest that development of a Smad3 inhibitor will have wide-ranging clinical applications. To investigate BMP-dependent signaling pathways, we have created a conditional knockout of the Smad1 gene to overcome the early embryonic lethality of the Smad1 knockout mouse, also generated in our group.