Secreted Frizzled-Related Proteins and Wnt Signaling

分泌的卷曲相关蛋白和 Wnt 信号传导

• 批准号: 7592634
• 负责人: JEFFREY RUBIN
• 金额: $ 85.8万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592634
• 关键词: Accounting; Binding; Biological; Biological Models; Cell Line; Cell Shape; Cells; Collaborations; Engineering; Epitopes; Event; Ewings sarcoma; Experimental Models; Family member; Gene Expression; Gene Expression Regulation; Goals; Investigation; Manuscripts; Mediating; Modeling; Nerve Regeneration; Nervous system structure; Neurites; Oncogenes; Pathway interactions; Property; Proteins; Publications; Recombinants; Research; Role; Signal Pathway; Signal Transduction; Specificity; Structure-Activity Relationship; Testing; Time; Wnt proteins; Work; beta catenin; cell motility; human SFRP4 protein; insight; novel; protein protein interaction; receptor; response; stress-activated protein kinase 1; tumorigenesis

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.

我的实验室的重点已经扩大到包括分泌卷曲相关蛋白（sFRPs）和细胞对Wnt刺激的反应的研究。确定Wnt/ frizzed (Fzd)/sFRP相互作用的特异性仍然是我们的长期目标之一。过去，我们纯化了4个重组sFRPs，并在3个Wnt蛋白的分离方面取得了进展。我们还鉴定了一个低背景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，以协助我们研究Wnt/Fz/sFRP结合和信号传导。在过去的一年里，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完整的10个具有相同标签的Fzds，以促进我们在各种模型系统中蛋白质-蛋白质相互作用和信号传导的研究。特别是，我们一直在关注所谓的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，在某些情况下可能激活非规范的Wnt信号。我们首次证明了Wnt刺激诱导尤文氏肉瘤细胞形成神经突，并开始确定这种细胞反应的机制。Fzd3是介导这一事件的主要Wnt受体，它还涉及到先前鉴定的Wnt效应分子，disheveled -2和disheveled -3，以及氨基末端c-Jun激酶（JNK）。与我们的总体目标一致（见上文），我们观察到Dickkopf-1也促进了尤因氏细胞的神经突生长，显然是通过激活类似的非规范Wnt信号。该实验模型将有助于理解wnt如何促进神经系统中细胞-细胞连接的形成，并可能增强神经再生。这项研究也将提供关于尤文氏肉瘤细胞特性的见解。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 -连环蛋白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可能通过其他途径调节基因表达和可能的其他细胞反应。各种R-spondin2衍生物已经产生，以确定R-spondin2生物活性的结构要求，特别是基因表达的调节。这项研究应该为wnt、应答和肿瘤发生之间的假设联系提供新的信息。