SIGNAL TRANSDUCTION 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，这些受体的短路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。我们已经确定了共同介体Smad4中间连接区的一个45个氨基酸的区域，称为Smad激活结构域(SAD)。我们已经证明SAD是一个富含Pro的转录激活结构域，它与共激活因子p300/CBP相互作用，是Smad转录复合体活性的必要条件和充分条件。其他研究集中在我们从以Smad1为诱饵的酵母双杂交系统中分离到的两个新克隆的特征。其中一种蛋白质是核转录抑制因子SNIP1，它主要与Smad4和p300/CBP相互作用，从而阻断Smad4与这些共激活因子的激活作用。另一种蛋白最近被发现与其他分类连接素具有同源性，似乎既与转化生长因子-b家族的受体结合，也与其他受体结合，包括表皮生长因子和胰岛素受体。第三种蛋白，TRap1，通过使用激活的转化生长因子-b受体进行相互克隆而鉴定，似乎在Smad2和Smad4之间扮演着穿梭的角色，可能促进了这两个Smad在细胞质中的联系。对这些蛋白质及其功能活性的表征表明，Smad信号转导途径具有额外的复杂性。为了阐明Smad介导的信号转导在体内的作用，我们同时产生了转基因和基因敲除小鼠。为了研究转化生长因子-β激活的Smad3途径，我们与初夏邓楚霞(NIDDK)合作研究了Smad3基因敲除小鼠的特征。对这些小鼠伤口愈合的研究表明，这种信号中间产物的缺失对依赖于转化生长因子-b的基因靶点具有选择性作用，导致创面变窄，上皮化速度更快，并减少炎细胞渗透和肉芽组织。这可能会对人类伤口的愈合和纤维化产生影响。为了研究BMP激活的Smad1途径，我们还培育了Smad1基因缺失的小鼠，但这些小鼠在胚胎第9.5天因无法形成胚外循环而死亡。为了避免这种早期胚胎死亡，并研究Smad1基因缺失对成人的影响，我们正在产生Smad1条件基因敲除小鼠，它将以组织特异性的方式与各种表达Cre重组酶的小鼠杂交。我们还产生了表达Smad1显性阴性的小鼠。这些小鼠与Smad1杂合子的小鼠和过度表达Smad6的小鼠进行回交，Smad1杂合子的目的是减少活性Smad的数量，Smad6是该信号通路的抑制因子。这些小鼠的表型仍在调查中。-生长因子、受体、信号、转化生长因子β、肿瘤抑制因子、

The TGF-beta family of ligands signal through a unique heteromeric receptor complex distinguished by its serine-threonine kinase activity. Recently, a short-circuit signal transduction pathway from these receptors 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. We have identified a 45 amino acid region of the middle linker region of the common mediator, Smad4, called the Smad activation domain (SAD). We have shown the SAD to be a proline-rich transcriptional activation domain which interacts with the co-activator p300/CBP and is both necessary and sufficient for activity of the Smad transcriptional complex. Other studies are focused on characterization of two novel clones we have isolated from a yeast two-hybrid system using Smad1 as bait. One of these proteins, a nuclear transcriptional repressor which we have called SNIP1, interacts principally with Smad4 and with p300/CBP in such a manner as to block the activating interaction of Smad4 with these coactivators. Another protein has recently been found to have homology to other sorting nexins and appears to bind both to receptors of the TGF-b family and to other receptors including the EGF and insulin receptors. A third protein, Trap1, identified by interaction cloning using an activated TGF-b receptor, appears to act as a shuttle between Smad2 and Smad4, possibly facilitating the association of these two Smads in the cytoplasm. The characterization of these proteins and their functional activity suggests additional complexity to the Smad signal transduction pathways. To delineate the role of Smad-mediated signal transduction in vivo, we have generated both transgenic and knockout mice. To study the TGF-b-activated Smad3 pathway, we have collaborated with Chuxia Deng (NIDDK) on characterization of a Smad3 knockout mouse. Study of wound healing in these mice has shown that deletion of this signaling intermediate has selective effects on TGF-b-dependent gene targets, resulting in narrower wounds that epithelialization more rapidly and have reduced inflammatory cell infiltratrates and reduced granulation tissue. This has possible implications for healing of human wounds and for fibrosis. To study the BMP-activated Smad1 pathway, we also generated mice null for Smad1, but these mice die at embryonic day 9.5 from an inability to form extraembryonic circulation. To circumvent this early embryonic lethality and to study the effect of deletion of Smad1 in the adult, we are in the process of generating a Smad1 conditional knockout mouse, which will be crossed with various mice expressing Cre recombinase in a tissue-specific manner. We have also generated mice expressing a dominant negative from of Smad1. These mice are being backcrossed both with mice heterozygous for Smad1, to reduce the quantity of the active Smad, and with mice overexpressing Smad6, an inhibitor of this signaling pathway. The phenotypes of these mice are still under investigation. - growth factors, receptors, signaling, TGF beta, Tumor Suppressor,