Wnt Signaling and Secreted Frizzled-Related Proteins

(A)该项目的主要目的是更好地了解通过分泌的卷曲相关蛋白（sFRPs）增强或抑制Wnt3a/ β -catenin信号传导的因素。这些知识将有利于临床应用，包括使用sFRPs来控制Wnt信号。细胞环境是sFRP1对Wnt3a/ β -连环蛋白信号传导作用的决定因素。sFRP1在亲代HEK293细胞和HEKSTF细胞（稳定表达SuperTopFlash报告基因构建的克隆系）中具有双相活性，在1-10 nM增强β -catenin信号传导，在100-300 nM抑制β -catenin信号传导。相反，在该浓度范围内，sFRP1主要刺激小鼠乳腺上皮细胞系C57MG中的Wnt3a活性，但即使在低浓度范围内，sFRP1也严格抑制L929成纤维细胞（L细胞）。在sFRP2中观察到类似的细胞特异性活性模式。受体表达是对sFRP1反应的关键因素。在L细胞中，Fzd5而非Fzd2的异位过表达使Wnt3a活性增强。我们假设Fzd5在低sFRP1浓度下有助于增强作用，因为它与DFz2同源，果蝇Fzd在S2细胞中表达，之前在Wingless存在时表现出对sFRP1的双相反应。CRDsFRP1在多种情况下模仿了sFRP1的增强作用，这与最初认为该结构域会抑制Wnt信号传导的预期相矛盾。此外，与sFRP1相比，CRDsFRP1对Wnt3a的亲和性较低，这意味着CRDsFRP1增强Wnt3a的机制可能不需要与Wnt蛋白相互作用。总之，这些发现表明，sFRPs可以促进或抑制Wnt/ β -连环蛋白信号传导，这取决于细胞环境、浓度和最有可能的Fzd受体的表达模式。(B)该项目的第二个目标是开发32D髓系祖细胞系，作为研究Wnt/Fzd相互作用特异性的模型系统。32D细胞很少或不表达内源性Fzd转录本。我们用10个哺乳动物Fzds中的9个稳定地转染了32D细胞，所有Fzds都带有n端HA表位标签。用特定的Wnt处理表达单个Fzds的32D细胞，会引起不同Wnt信号通路的反应，这表明该模型将有助于将特定的Wnt/Fzd组合与不同的下游信号事件联系起来。

(A) The primary objective of this project has been to obtain a better understanding of the factors that account for the potentiation or inhibition of Wnt3a/beta-catenin signaling by secreted Frizzled-related proteins (sFRPs). Such knowledge would be beneficial in clinical applications that involve the use of sFRPs to control Wnt signaling. Cell context is a determinant of the effect sFRP1 has on Wnt3a/beta-catenin signaling. sFRP1 has biphasic activity in parental HEK293 cells and HEKSTF cells (a clonal line stably expressing a SuperTopFlash reporter construct), enhancing beta-catenin signaling at 1-10 nM and inhibiting it at 100-300 nM. In contrast, sFRP1 primarily stimulated Wnt3a activity in the mouse mammary epithelial line C57MG in this concentration range, but was a strict inhibitor in L929 fibroblasts (L cells) even at low concentrations. A similar pattern of cell-specific activity was observed with sFRP2. Receptor expression is a key factor in the response to sFRP1. Ectopic over-expression of Fzd5, but not Fzd2, in L cells enabled potentiation of Wnt3a activity. We had hypothesized that Fzd5 would contribute to a potentiating effect at low sFRP1 concentrations, because of its homology to DFz2, the Drosophila Fzd expressed in S2 cells that previously had shown a biphasic response to sFRP1 in the presence of Wingless. CRDsFRP1 mimicked the potentiating effect of sFRP1 in multiple settings, contradicting initial expectations that this domain would inhibit Wnt signaling. Moreover, CRDsFRP1 showed little avidity for Wnt3a compared with sFRP1, implying that the mechanism for potentiation by CRDsFRP1 probably does not require an interaction with Wnt protein. Together, these findings demonstrate that sFRPs can either promote or suppress Wnt/beta-catenin signaling, depending on cellular context, concentration and most likely the expression pattern of Fzd receptors. (B) A secondary objective of this project has been to develop the 32D myeloid progenitor cell line as a model system for the study of specificity in Wnt/Fzd interactions. 32D cells express little or no endogenous Fzd transcripts. We have stably transfected 32D cells with nine of the ten mammalian Fzds, all with N-terminal HA epitope tags. Treatment of 32D cells expressing individual Fzds with particular Wnts elicits responses in different Wnt signaling pathways, suggesting that this model would be useful in associating specific Wnt/Fzd combinations with distinct downstream signaling events.