Secreted Frizzled-Related Proteins and Wnt Signaling

我的实验室的重点已经扩大到包括对分泌型FrizzledRelated Proteins(SFRP)和细胞对Wnt刺激的反应的研究。我们已经开始了对Wnt蛋白的纯化，不仅是为了提供试剂来促进SFRP功能的分析，而且也是为了促进对Wnt活性和相关信号的研究。确定WNT/FrizzledFZ/SFRP相互作用的特异性是我们的主要目标之一。目前，我们已经纯化了4个重组的sFRPs，并在分离3个Wnt蛋白方面取得了进展。我们还筛选了一个低背景Wnt/FZ表达的细胞系，并分别向这些细胞中导入了三种FZs，以帮助我们研究Wnt/FZ/SFRP的结合和信号转导。最近，我们发起了一项合作，以探索Cripto作为一种新的Wnt共同受体在典范/-连环蛋白途径中的所谓作用。我们开发了一个方便的模型系统来检测Wnt依赖的细胞运动和相关的细胞形状变化。我们证明了RhoA激活对细胞迁移是重要的，而β-catenin信号不是重要的，而Disheveled-2(DVL-2)在Transwell实验中作为Wnt活性的中介起着主要作用。这导致了对DVL调节的各个方面的重视，特别是被认为对非规范信号和细胞运动至关重要的磷酸化。我们还观察到WNT-3a刺激纤维连接蛋白(FN)纤维的组装，这一现象可能有助于细胞的扩散和运动。我们对SFRP-1的长期研究在不同的背景下继续进行。我们已经确定，SFRP1启动子中特定序列的甲基化破坏了核因子的结合，这可能有助于在几种癌症中记录的该基因的表观遗传学沉默。我们已经建立了SFRP1缺失的小鼠模型，我们正在与多个实验室共享该模型，以更好地了解SFRP-1的生理活性。在不同的实验系统中，我们一直在比较和对比SFRP-1和Dickkopf-1(DKK-1)的效果。虽然两者都抑制β-连环蛋白途径，但我们的结果表明，在被SFRP-1阻断的情况下，DKK-1可能会增强非典型信号转导。除了SFRP-1作为Wnt信号调节因子的公认作用外，我们还探索了它具有新的结合伙伴和额外的作用机制的可能性。利用噬菌体展示多肽文库，我们确定了一个在Wnt蛋白中不存在的SFRP-1结合基序，并可能为其他蛋白质-蛋白质相互作用提供线索。我们一度认为，这种相互作用可能是SFRP-1抑制破骨细胞形成的基础，我们与马修·吉莱斯皮博士合作展示了这一效果。RANKL是破骨细胞发生的关键调节因子，它含有一个类似的基序，并在ELISA实验中与SFRP-1结合。然而，随后的工作表明，SFRP-1的抑制活性不需要与RANKL直接相互作用。综上所述，我们的SFRP和WNT研究将有助于更好地了解它们在癌症、发育和组织动态平衡中的作用。

The focus of my lab has broadened to include both the study of secreted Frizzled-related proteins (sFRPs) and cellular responses to Wnt stimulation. We have embarked on the purification of Wnt proteins, not only to provide reagents to facilitate the analysis of sFRP function, but to foster investigation of Wnt activity and associated signaling. Defining the specificity of Wnt/Frizzled(Fz)/sFRP interactions is one of our primary goals. Currently, we have purified four recombinant sFRPs and have made progress in the isolation of three Wnt proteins. We also identified a cell line with a low background of Wnt/Fz expression, and separately introduced three Fzs into these cells to assist our study of Wnt/Fz/sFRP binding and signaling. Recently, we initiated a collaboration to explore the alleged role of Cripto as a novel Wnt co-receptor functioning in the canonical/?-catenin pathway. We developed a convenient model system to examine Wnt-dependent cell motility and associated changes in cell shape. We demonstrated that RhoA activation is important for cell migration, while ?-catenin signaling is not, and Dishevelled-2 (Dvl-2) had a predominant role as a mediator of Wnt activity in a transwell assay. This has led to an emphasis on aspects of Dvl regulation, particularly phosphorylation, thought to be important for non-canonical signaling and cell motility. We also have observed that Wnt-3a stimulates the assembly of fibronectin (FN) fibrils, a phenomenon that likely contributes to cell spreading and movement. Our longstanding study of sFRP-1 has continued in a variety of contexts. We have determined that methylation of specific sequences in the SFRP1 promoter disrupts binding of nuclear factors, which may contribute to epigenetic silencing of the gene that has been documented in several cancers. An Sfrp1 null mouse model has been developed, and we are sharing it with multiple labs to better understand the physiological activities of sFRP-1. In various experimental systems, we have been comparing and contrasting the effects of sFRP-1 and Dickkopf-1 (Dkk-1). While both inhibit the ?-catenin pathway, our results suggest that Dkk-1 might enhance non-canonical signaling in situations where it is blocked by sFRP-1. Besides the well-established role of sFRP-1 as a regulator of Wnt signaling, we have explored the possibility that it has novel binding partners and additional mechanisms of action. Using a phage-display peptide library, we identified a binding motif for sFRP-1 that is not present in Wnt proteins and might provide a clue to other protein-protein interactions. At one point, we thought that such interactions could be the basis for sFRP-1 inhibition of osteoclast formation, an effect which we demonstrated in collaboration with Dr. Matthew Gillespie. RANKL, a key mediator of osteoclastogenesis, contains a similar motif and bound to sFRP-1 in ELISA experiments. However, subsequent work suggests that sFRP-1 inhibitory activity does not require a direct interaction with RANKL. Taken together, our sFRP and Wnt studies will contribute to a better understanding of their roles in cancer, development and tissue homeostasis.