Signal Transduction Pathways of Transforming Growth Fact

tgf - β家族配体通过一种独特的异聚体受体复合物进行信号传递，其特征是丝氨酸-苏氨酸激酶活性。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。在这一途径中，受体激活的smad直接被I型受体激酶磷酸化，并与共同的介质Smad4结合，转运到细胞核，参与转录复合物。我们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作用，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。之前已经确定了tgf - β受体相互作用蛋白TRAP1作为专性信号伴侣Smad4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TRAP1样蛋白，或TLP。TLP似乎具有来自Smad3而不是Smad2的信令所必需的独特特性。我们正在研究是否改变细胞中TLP的表达可能会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。其中的靶标是两个依赖smad3的直接早期基因，它们分别与细胞凋亡和上皮-间质转化有关，分别是Gadd45beta和Snail。我们还试图通过在体外和体内检测源自亲本MCF10A系的人乳腺癌细胞中改变Smad2和Smad3通路平衡的影响，来表征Smad2或Smad3在肿瘤发生和转移中的过表达或抑制的结果，从而确定Smad2和Smad3在癌细胞中的特定靶点。最初的结果令人惊讶地表明，Smad途径介导肿瘤细胞上tgf - β的肿瘤抑制和促转移活性。其他方法是基于我们首次发现的tgf - β信号抑制分子SNIP1与c-Myc相互作用并增强其转录激活活性的新发现。希望正在进行的研究在多种人类癌症中检测SNIP1的表达模式，将揭示该分子在致癌作用中的复杂作用。

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.