Wnt-Dependent Neurite Outgrowth in Ewing Tumor Cells

我们确定了尤文瘤细胞对Wnt-3a的反应是形成神经突，并开始确定这种作用的机制。Frizzled 3（Fzd 3）被鉴定为介导该过程的主要Wnt受体，该过程还需要Dishevelled-2（Dvl-2）、Dishevelled-3（Dvl-3）和氨基末端c-Jun激酶（JNK）。我们发现Dickkopf-1也促进这些细胞中的神经突生长，显然是通过促进内源性Wnt激活Fzd 3/JNK。Wnt-3a诱导的神经突生长与Dvl-2/3磷酸化相关;神经突形成和Dvl磷酸化均被酪蛋白激酶1 δ/β（CK 1d/e）抑制剂IC 261阻断。用小干扰RNA敲低CK 1d抑制Wnt-3a依赖的神经突形成，而敲低CK 1 e在没有外源性Wnt-3a的情况下刺激神经突形成。CK 1d和CK 1 e对神经突生长的对比效应可能是由于CK 1d优先定位于中心体，这被认为在神经突形成中具有关键作用。敲除非典型PKCiota也阻断Wnt-3a依赖性神经突生长。初步实验表明PKC 1 ota可能受CK 1d和/或Dvl调节。 这项工作意义重大，不仅因为它提供了有关神经突形成机制的见解。参与神经突生长的许多因子在细胞迁移的关键细胞延伸的形成中也具有重要作用。在尤因肿瘤细胞模型中获得的信息可能会增强我们对生理背景下细胞运动和转移的理解。此外，我们有初步证据表明，CK 1d也参与初级纤毛的形成。有缺陷的初级纤毛是负责几种疾病，包括神经管缺陷，多囊肾病和内脏逆位。异常的Wnt信号传导也可以引起这些异常。因此，我们对CK 1d和Dvl的研究可能为Wnt信号控制胚胎发育的方式提供新的见解。

We established that cells from Ewing tumors form neurites in response to Wnt-3a and have begun to define the mechanisms that account for this effect. Frizzled3 (Fzd3) was identified as the primary Wnt receptor that mediates the process, which also requires Dishevelled-2 (Dvl-2), Dishevelled-3 (Dvl-3), and amino-terminal c-Jun kinase (JNK). We showed that Dickkopf-1 also promotes neurite outgrowth in these cells, apparently by facilitating Fzd3/JNK activation by endogenous Wnts. Neurite outgrowth induced by Wnt-3a was associated with Dvl-2/3 phosphorylation; both neurite formation and Dvl phosphorylation were blocked by the casein kinase 1 delta/epsilon (CK1d/e) inhibitor, IC261. Knockdown of CK1d with small interfering RNA suppressed Wnt-3a-dependent neuritogenesis, whereas knockdown of CK1e stimulated neurite formation in the absence of exogenous Wnt-3a. The contrasting effects of CK1d and CK1e on neurite outgrowth might be due to the preferential localization of CK1d at the centrosome, which is thought to have a critical role in neurite formation. Knockdown of the atypical PKCiota also blocked Wnt-3a-dependent neurite outgrowth. Preliminary experiments suggest that PKCiota may be regulated by CK1d and/or Dvl. This work is significant not only because it provides insights about mechanisms involved in the formation of neurites. Many of the factors that participate in neurite outgrowth also have important roles in the formation of cellular extensions critical for cell migration. The information obtained in the Ewing tumor cell model is likely to enhance our understanding of cell movement in physiological contexts and metastasis. Moreover, we have preliminary evidence that CK1d also participates in the formation of primary cilia. Defective primary cilia are responsible for several disorders including neural tube defects, polycystic kidney disease and situs inversus. Aberrant Wnt signaling also can elicit these abnormalities. Thus, our studies of CK1d and Dvl may provide new insight about the ways in which Wnt signaling controls embryonic development.