Wnt Signaling and Secreted Frizzled-Related Proteins

该项目的主要目的是更好地了解sFRP 1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sFRP 1来控制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。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。信号传导的增强与HEKSTF细胞和L/Fzd 5转染子中可溶性β-连环蛋白的增加相关。sFRP 1在亲本L细胞中的抑制作用与β-连环蛋白的减少有关。然而，在HEKSTF细胞中观察到不同的模式：用100和300 nM sFRP 1观察到的报告活性的抑制并没有导致可溶性β-连环蛋白水平的降低。CRDsFRP 1增强HEKSTF和C57 MG细胞中的Wnt 3a活性，但具有很少或没有抑制活性。如先前对于Wg所报道的，与全长sFRPl相反，CRDsFRPl在ELISA、Biacore或免疫共沉淀测定中对Wnt 3a结合表现出很小的亲合力。我们假设CRDsFRP 1通过与Fzd相互作用促进Wnt信号传导，最有可能是通过CRD-CRD结合。当在HEK 293细胞中共表达时，CRDsFRP 1与全长Fzds共免疫沉淀，尽管其与Fzd 2或Fzd 5的关联没有显著差异。然而，在Wnt 3a与Fzds或CRDFzd-Fc融合蛋白结合的基于细胞和无细胞测定中，我们观察到sFRP 1在低浓度或高浓度下几乎没有影响。总之，我们确定sFRP 1对Wnt 3a/β-连环蛋白信号传导具有多种剂量依赖性作用，这取决于细胞模型。增强和抑制之间的平衡可能取决于单个Fzd的相对表达，Fzd 5能够在低sFRP 1浓度下支持增强。虽然β-连环蛋白信号的增强与可溶性β-连环蛋白蛋白的增加有关，但抑制似乎取决于不同的机制：如在L细胞中所见的β-连环蛋白稳定的抑制或在HEKSTF细胞中观察到的尚未阐明的另一个过程。由于CRDsFRP 1模拟sFRP 1的增强活性，但不与Wnt 3a显著结合，因此我们怀疑其作用是通过CRDFzd或其他尚未表征的机制与Fzds相互作用介导的。32 D细胞是一个有吸引力的模型，用于研究信号，从特定的Wnt/Fzd相互作用的结果，因为在这些细胞中的内源性Wnt配体/受体的表达量有限。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。到目前为止，引入Fzd 3的尝试一直存在问题：仅检测到低水平的表达，甚至该信号也是瞬时的，表明负选择。使用Wnt 3a和32 D/Fzd转染子的子集进行β-连环蛋白稳定化测定已获得阳性结果。

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 sFRP1. Such knowledge would be beneficial in clinical applications that involve the use of sFRP1 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. 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. The potentiation of signaling correlated with an increase in soluble beta-catenin protein in HEKSTF cells and L/Fzd5 transfectants. The inhibitory effect of sFRP1 in parental L cells was associated with a decrease in beta-catenin protein. However, a different pattern was seen in HEKSTF cells: the suppression of reporter activity observed with 100 and 300 nM sFRP1 did not result in diminished levels of soluble beta-catenin. CRDsFRP1 potentiated Wnt3a activity in HEKSTF and C57MG cells, but had little or no inhibitory activity. As previously reported for Wg, CRDsFRP1 exhibited little avidity for Wnt3a binding in ELISA, Biacore or co-immunoprecipitation assays in contrast to full-length sFRP1. We hypothesized that CRDsFRP1 promoted Wnt signaling by interacting with Fzd, most likely via CRD-CRD binding. CRDsFRP1 co-immunoprecipitated with full-length Fzds when co-expressed in HEK293 cells, although there was not a significant difference in its association with Fzd2 or Fzd5. However, in cell-based and cell-free assays of Wnt3a binding to Fzds or CRDFzd-Fc fusion proteins, we observed little effect of sFRP1 either at low or high concentrations. In summary, we determined that sFRP1 had a variety of dose-dependent effects on Wnt3a/beta-catenin signaling depending on the cell model. The balance between potentiation and inhibition presumably depends on the relative expression of individual Fzds, with Fzd5 being able to support potentiation at low sFRP1 concentrations. While the enhancement of beta-catenin signaling was associated with increases in soluble beta-catenin protein, inhibition appears to depend on different mechanisms: either suppression of beta-catenin stabilization as seen in L cells or another process observed in HEKSTF cells that has not yet been elucidated. Because CRDsFRP1 mimicked the potentiating activity of sFRP1 but did not bind significantly to Wnt3a, we suspect that its effect is mediated by interaction with Fzds via the CRDFzd or by other, as yet uncharacterized mechanisms. The 32D cell is an attractive model for the study of signaling that results from specific Wnt/Fzd interactions because of the limited amount of endogenous Wnt ligand/receptor expression in these cells. We have stably transfected 32D cells with nine of the ten mammalian Fzds, all with N-terminal HA epitope tags. Thus far, attempts to introduce Fzd3 have been problematic: only low levels of expression have been detected and even this signal is transient, suggesting negative selection. Positive results have been obtained with a beta-catenin protein stabilization assay using Wnt3a and a subset of the 32D/Fzd transfectants.