Signal Pathways of Transforming Growth Factor Beta

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。最近，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/DPC4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描述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。我们利用以Smad1为诱饵的酵母双杂交系统鉴定了两个新的克隆。SNX6是受体相互作用蛋白家族中的一员，它可能在丝氨酸苏氨酸激酶受体的运输中发挥作用。另一个是SNIP1，它是一种新的Smad信号的核抑制因子，可能整合不同信号通路之间的串扰，如NF-kappaB聚集在依赖于转录共激活因子p300和CBP的C/H1结构域的转录复合体上。我们还阐明了先前发现的转化生长因子-β受体相互作用蛋白TRAP1的一个新角色，它作为专性伴侣Smad4的伴侣，将Smad4递送给受体激活的Smads。这些研究将进一步扩大，以确定这些分子的作用，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体的其他成分相互作用。我们还使用微阵列分析来鉴定由Smad3或Smad2依赖的通路在小鼠胚胎成纤维细胞中唯一诱导的cDNA。这些研究已经确定了这些信号通路的靶点，其特征肯定会为研究转化生长因子-β的即刻-早期和二级基因靶点提供一个新的视角。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，基于以下假设：从概念上讲，体内特定信号组件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。我们有幸与NIDDK的邓楚霞合作，确定了Smad3基因敲除小鼠的特征，这为转化生长因子-β在造血、伤口愈合和纤维化中的作用提供了新的见解。在Angelo Russo博士和James Mitchell博士的合作下，我们专注于Smad3在电离辐射中丢失的保护作用，并试图将我们在老鼠身上的发现与辐射对体外原代角质形成细胞和成纤维细胞的影响研究相关联。依赖于这一特定途径的转化生长因子-β致病机制的阐明表明，开发一种Smad3抑制剂将具有广泛的临床应用。为了研究BMP依赖的信号通路，我们创建了Smad1基因的条件性敲除，以克服Smad1基因敲除小鼠的早期胚胎死亡，该基因敲除小鼠也是在我们的团队中产生的。

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.