SIGNAL TRANSDUCTION 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，从这些受体的短路信号转导途径已被阐明，其中涉及一个新的家庭的蛋白质称为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。我们已经确定了一个45个氨基酸的共同介质，Smad 4，称为Smad激活结构域（SAD）的中间连接区。我们已经证明SAD是一个富含脯氨酸的转录激活结构域，它与共激活因子p300/CBP相互作用，并且对于Smad转录复合物的活性是必要的和足够的。其他研究的重点是表征两个新的克隆，我们已经从酵母双杂交系统中分离出Smad 1作为诱饵。这些蛋白质之一，我们称之为SNIP 1的核转录抑制因子，主要与Smad 4和p300/CBP相互作用，以这种方式阻断Smad 4与这些辅激活因子的激活相互作用。最近发现另一种蛋白质与其他分选连接蛋白具有同源性，并且似乎与TGF-β家族的受体和其他受体（包括EGF和胰岛素受体）结合。第三个蛋白质，Trap 1，确定使用激活的TGF-β受体的相互作用克隆，似乎作为Smad 2和Smad 4之间的穿梭，可能促进这两个Smads在细胞质中的协会。这些蛋白质及其功能活性的表征表明Smad信号转导途径的额外复杂性。为了描述Smad介导的体内信号转导的作用，我们产生了转基因小鼠和基因敲除小鼠。为了研究TGF-β激活的Smad 3通路，我们与Chuxia Deng（NIDDK）合作对Smad 3敲除小鼠进行了表征。对这些小鼠伤口愈合的研究表明，这种信号传导中间体的缺失对TGF-β依赖性基因靶点具有选择性作用，导致更窄的伤口，其上皮化更快，并具有减少的炎性细胞浸润和减少的肉芽组织。这可能对人类伤口愈合和纤维化有影响。为了研究BMP激活的Smad 1通路，我们还产生了Smad 1无效的小鼠，但这些小鼠在胚胎第9.5天死于不能形成胚外循环。为了避免这种早期胚胎致死性和研究Smad 1在成年人中缺失的影响，我们正在产生Smad 1条件性敲除小鼠的过程中，该小鼠将与以组织特异性方式表达Cre重组酶的各种小鼠杂交。我们还产生了表达Smad 1的显性阴性的小鼠。这些小鼠与Smad 1杂合子小鼠回交，以减少活性Smad的数量，并与过表达Smad 6的小鼠回交，Smad 6是该信号通路的抑制剂。这些小鼠的表型仍在研究中。 - 生长因子，受体，信号传导，TGF 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,