Secreted Frizzled-Related Proteins and Wnt Signaling

我的实验室的重点已经扩大到包括分泌的卷曲相关蛋白（sFRPs）和细胞对Wnt刺激的反应的研究。确定Wnt/Frizzled（Fzd）/sFRP相互作用的特异性仍然是我们的长期目标之一。在过去，我们纯化了四种重组sFRPs，并在分离三种Wnt蛋白方面取得了进展。我们还鉴定了具有低背景Wnt/Fz表达的细胞系，并分别将三个表位标记的Fzds引入这些细胞中以帮助我们研究Wnt/Fz/sFRP结合和信号传导。在过去的一年中，我们设计了剩余Fzds家族成员的表位标记版本，这样我们现在就有了一整套10个具有相同标签的Fzds，以便于我们在各种模型系统中研究蛋白质-蛋白质相互作用和信号传导。特别是，我们一直专注于所谓的sFRPs与Fzds的关联，以及这些相互作用的潜在功能后果。到目前为止，我们的研究结果表明，这种sFRP-1/Fzd结合发生，并在某些情况下可能激活非经典Wnt信号转导。我们首次证明了Wnt刺激诱导尤文肉瘤细胞形成神经突，并已开始定义解释这种细胞反应的机制。Fzd 3是介导该事件的主要Wnt受体，其还涉及先前鉴定的Wnt效应分子Dishevelled-2和Dishevelled-3以及氨基末端c-Jun激酶（JNK）。与我们的总体目标之一（见上文）一致，我们观察到Dickkopf-1也促进尤文氏细胞中的神经突生长，显然是通过激活类似的非经典Wnt信号传导。这个实验模型应该有助于理解Wnt如何促进神经系统中细胞间连接的形成，并可能增强神经再生。这项研究也将提供有关尤文肉瘤细胞的特性的见解。描述这项工作的手稿已提交出版。去年，我们发起了一项合作，研究R-spondins在刺激Wnt/β-catenin信号传导和肿瘤发生中的作用。本财政年度的结果表明，R-spondins可能通过其他途径来调节基因表达和其他细胞反应。已经产生了各种R-spondin 2衍生物以确定R-spondin生物活性的结构要求，特别是基因表达的调节。这项研究应该提供关于Wnt，Rspondins和肿瘤发生之间假设联系的新信息。

The focus of my lab has broadened to include both the study of secreted Frizzled-related proteins (sFRPs) and cellular responses to Wnt stimulation. Defining the specificity of Wnt/Frizzled(Fzd)/sFRP interactions continues to be one of our long-term goals. In the past, we purified four recombinant sFRPs and 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 epitope-tagged Fzds into these cells to assist our study of Wnt/Fz/sFRP binding and signaling. During the past year, we engineered epitope-tagged versions of the remaining Fzds family members, such that we now have a full set of ten Fzds with the same tag to facilitate our investigation of protein-protein interactions and signaling in various model systems. In particular, we have been focusing on an alleged association of sFRPs with Fzds, and the potential functional consequences of these interactions. Thus far, our results suggest that such sFRP-1/Fzd binding occurs and in some instances may activate non-canonical Wnt signaling. We have demonstrated for the first time that Wnt stimulation induces Ewing's sarcoma cells to form neurites, and have begun to define the mechanisms that account for this cellular response. Fzd3 is the primary Wnt receptor that mediates this event, which also involves the previously identified Wnt effector molecules, Dishevelled-2 and Dishevelled-3, and amino-terminal c-Jun kinase (JNK). Consistent with one of our general objectives (see above), we observed that Dickkopf-1 also promoted neurite outgrowth in Ewing's cells, apparently by activating similar non-canonical Wnt signaling. This experimental model should prove useful in understanding how Wnts facilitate the formation of cell-cell connections in the nervous system, and perhaps enhance nerve regeneration. This investigation also will provide insight about the properties of Ewing's sarcoma cells. A manuscript describing this work has been submitted for publication. Last year we initiated a collaboration to investigate the role of R-spondins in the stimulation of Wnt/beta-catenin signaling and tumorigenesis. Results during the current fiscal year suggested that R-spondins may signal through additional pathways to regulate gene expression and presumably other cellular responses. Various R-spondin2 derivatives have been generated to determine the structural requirements for R-spondin biological activities, in particular, regulation of gene expression. This research should provide new information about a hypothesized connection between Wnts, Rspondins and tumorigenesis.