Signaling Mechanisms of EphrinB1 in Cell Adhesion, Migration and Invasion

我们目前的研究兴趣是旨在研究Eph受体酪氨酸激酶及其ephrin配体信号事件影响细胞-细胞粘附和形态发生运动的机制。从这些信号转导途径的阐明，我们可以提高我们对肿瘤发生的认识。细胞-细胞粘附系统在细胞的正常发育和形态发生中起着重要作用。这种粘附系统的失活被认为在癌症侵袭和转移中起关键作用。非洲爪蟾胚胎非常适合研究这些过程，因为青蛙具有很好的特征和不变的细胞命运图，并且在实验中可以很容易地追踪细胞谱系。突变受体、配体和其他蛋白质可以在胚胎中异位表达。因此，它们对信号转导、运动和分化的影响可以在发育中的脊椎动物中进行形态学、组织学和生化评估。我们的实验室目前正在利用非洲爪蟾卵母细胞和胚胎系统以及人类培养的细胞系研究非洲爪蟾Eph受体酪氨酸激酶和ephrinB跨膜配体在细胞信号传导和功能中的作用。目前，我们的重点放在这些Eph家族成员发送影响形态发生运动的信号的机制上。Eph家族成员参与调节许多发育过程，并被发现在转移性癌症中不受调节，例如前列腺癌、卵巢癌、乳腺癌、结肠癌、神经母细胞瘤、肺癌和黑色素瘤。我们的实验室继续进行这些研究，检查控制细胞粘附和细胞运动的ephrinB1的近端和远端信号。我们最近发现的证据表明，ephrinB1通过其胞内结构域与Dishevelled （dsh）相互作用，并选择平面细胞极性（PCP）途径，控制视网膜祖细胞进入视野的运动。通过生化分析和功能增益或损失实验，我们的数据表明，在眼视野形成过程中，dsh与ephrinB1相关，并通过PCP通路下游成员介导ephrinB1信号传导。因此，我们使用视野作为模型系统来理解ephrinB1如何控制细胞运动。最近，我们研究了ephrinB1影响细胞-细胞连接的机制。越来越多的证据表明，在细胞的适当迁移以及细胞和组织边界的形成中需要ephrin配体。这些过程都依赖于细胞黏附系统，而细胞黏附系统在细胞发育过程中的正常形态发生过程以及侵袭和转移过程中起着至关重要的作用。尽管ephrinB配体是双向信号分子，但在上皮细胞中，ephrinB1通过其胞内结构域信号调控细胞-细胞粘附的确切机制尚不清楚。在这里，我们证明了蓝精灵通过与ephrinB1的拮抗相互作用来调节中胚层/外胚层的组织分离，ephrinB1是一种Eph受体配体，在调节胚胎胚层的分离中起关键作用。EphrinB1被Smurf2靶向降解；然而，Smurf1与ephrinB1的相互作用阻止了与Smurf2的结合，并阻止了ephrinB1的泛素化和降解，因为它是Smurf1的弱底物。抑制胚胎中胚层中Smurf1的表达会导致ephrinb1介导的该组织与外胚层的分离丧失，这可以通过同时抑制Smurf2的表达来挽救。蓝精灵和ephrinB1之间的差异相互作用系统通过控制ephrinB蛋白水平来调节组织边界的维持。我们相信这些发现大大拓宽了我们对胚胎发生过程中影响组织排斥和边界维持的Smurf泛素连接酶和ephrinB调控的概念和知识。此外，由于ephrinb和Smurfs都与癌症进展密切相关，因此在考虑对转移性疾病中的这些分子进行干预时，这项工作可能具有启示意义。

Our current research interests are aimed toward examining the mechanism by which Eph receptor tyrosine kinases and their ephrin ligands signal events affecting cell-cell adhesion and morphogenetic movements. From the elucidation of these signal transduction pathways we may improve our understanding of oncogenesis. The cell-cell adhesion system plays a major role in normal development and morphogenesis. Inactivation of this adhesion system is thought to play a critical role in cancer invasion and metastasis. The Xenopus embryo is well suited for investigations of these processes because the frog has a well characterized and invariant cell fate map and cell lineage can be easily traced during experiments. Mutant receptors, ligands, and other proteins can be ectopically expressed in embryos. Thus, their effects on signal transduction, motility, and differentiation can be assessed morphologically and histologically as well as biochemically in a developing vertebrate. Our laboratory is currently investigating the role of the Xenopus Eph receptor tyrosine kinases and ephrinB transmembrane ligands in cell signaling and function using the Xenopus oocyte and embryo systems, as well as human cultured cell lines. At present, our emphasis is placed upon the mechanism by which these Eph family members send signals affecting morphogenetic movements. Members of the Eph family have been implicated in regulating numerous developmental processes and have been found to be de-regulated in metastatic cancers, for example, prostate, ovarian, breast, colon, neuroblastoma, lung, and melanoma. Our laboratory has continued these studies examining proximal and distal signaling from ephrinB1 that controls cell adhesion and cell movement. We recently found evidence that ephrinB1 signals via its intracellular domain to control retinal progenitor movement into the eye field by interacting with Dishevelled (dsh), and co-opting the planar cell polarity (PCP) pathway. Using biochemical analysis and gain or loss of function experiments, our data suggest that dsh associates with ephrinB1 and mediates ephrinB1 signaling via downstream members of the PCP pathway during eye field formation. thus, we have used the eye field as a model system for understanding how ephrinB1 controls cell movement. Most recently we have examined the mechanisms by which ephrinB1 affects cell-cell junctions. A body of evidence is emerging that shows a requirement for ephrin ligands in the proper migration of cells, and the formation of cell and tissue boundaries. These processes are dependent on the cell cell adhesion system, which plays a crucial role in normal morphogenetic processes during development, as well as in invasion and metastasis19. Although ephrinB ligands are bi-directional signalling molecules, the precise mechanism by which ephrinB1 signals through its intracellular domain to regulate cell-cell adhesion in epithelial cells remains unclear. Here, we demonstrate that the Smurfs regulate tissue separation at mesoderm/ectoderm boundaries through antagonistic interactions with ephrinB1, an Eph receptor ligand that has a key role in regulating the separation of embryonic germ layers. EphrinB1 is targeted by Smurf2 for degradation; however, a Smurf1 interaction with ephrinB1 prevents the association with Smurf2 and precludes ephrinB1 from ubiquitination and degradation, since it is a substantially weaker substrate for Smurf1. Inhibition of Smurf1 expression in embryonic mesoderm results in loss of ephrinB1-mediated separation of this tissue from the ectoderm, which can be rescued by the coincident inhibition of Smurf2 expression. This system of differential interactions between Smurfs and ephrinB1 regulates the maintenance of tissue boundaries through the control of ephrinB protein levels.We believe that these findings significantly broaden our concept and knowledge of the Smurf ubiquitin ligases and ephrinB regulation that affects tissue repulsion and border maintenance during embryogenesis. Furthermore, since both ephrinBs and Smurfs have been strongly implicated in cancer progression, this work may have implications when considering interventions regarding these molecules in metastatic disease.