Wnt Signaling and Secreted Frizzled-Related Proteins

(A)该项目的主要目的是更好地了解分泌型卷曲相关蛋白（sFRPs）增强或抑制Wnt 3a/β-连环蛋白信号传导的因素。这样的知识在涉及使用sFRPs控制Wnt信号传导的临床应用中将是有益的。细胞环境是sFRP 1对Wnt 3a/β-连环蛋白信号传导的影响的决定因素。sFRP 1在亲本HEK 293细胞和HEKSTF细胞（稳定表达SuperTopFlash报告基因构建体的克隆系）中具有双相活性，在1-10 nM时增强β-连环蛋白信号传导，在100-300 nM时抑制β-连环蛋白信号传导。相反，sFRP 1在此浓度范围内主要刺激小鼠乳腺上皮细胞系C57 MG中的Wnt 3a活性，但即使在低浓度下也是L929成纤维细胞（L细胞）中的严格抑制剂。在sFRP 2中观察到类似的细胞特异性活性模式。受体表达是对sFRP 1应答的关键因素。在L细胞中Fzd 5而非Fzd 2的异位过表达能够增强Wnt 3a活性。我们曾假设Fzd 5在低sFRP 1浓度下有助于增强效应，因为它与DFz 2同源，DFz 2是在S2细胞中表达的果蝇Fzd，先前在Wingless存在下对sFRP 1表现出双相反应。CRDsFRP 1在多种环境中模拟sFRP 1的增强作用，与最初的预期相矛盾，即该结构域将抑制Wnt信号传导。此外，与sFRP 1相比，CRDsFRP 1对Wnt 3a的亲合力很小，这意味着CRDsFRP 1的增强机制可能不需要与Wnt蛋白的相互作用。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、浓度和最可能的Fzd受体的表达模式。(B)该项目的第二个目的是开发32 D骨髓祖细胞系作为研究Wnt/Fzd相互作用特异性的模型系统。32 D细胞表达很少或不表达内源性Fzd转录物。我们已经用十种哺乳动物Fzd中的九种稳定转染了32 D细胞，所有Fzd都具有N-末端HA表位标签。用特定的Wnt处理表达单个Fzd的32 D细胞在不同的Wnt信号传导途径中激发应答，表明该模型可用于将特定的Wnt/Fzd组合与不同的下游信号传导事件相关联。

(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.