Wnt-Dependent Neurite Outgrowth in Ewing Tumor Cells

我们建立了尤文肿瘤细胞对WNT-3a的反应形成神经突起，并开始定义解释这一效应的机制。Frizzled3(Fzd3)是介导这一过程的主要Wnt受体，它还需要脱发-2(DVL-2)、脱发-3(DVL-3)和氨基末端c-Jun激酶(JNK)。我们发现Dickkopf-1还促进了这些细胞中的轴突生长，显然是通过促进内源性Wnts激活Fzd3/JNK来实现的。Wnt-3a诱导的轴突生长与DVL-2/3的磷酸化有关；酪蛋白激酶1β/epsilon(CK1d/e)抑制剂IC261可阻断轴突的形成和DVL的磷酸化。用小干扰RNA敲除CK1d可抑制Wnt-3a依赖的神经发生，而CK1e在缺乏外源Wnt-3a的情况下可促进神经突起的形成。中心体是与轴突形成有关的细胞器，CK1d未检测到CK1e。缺失分析将CK1d的中心体定位信号(CLS)定位到其羧基末端结构域。含有CLS和EGFP的融合蛋白取代了中心体上的全长CK1d，并抑制了依赖Wnt-3a的轴突生长。与野生型CK1e不同，由CK1e的激酶域和CK1d的CLS组成的嵌合体定位于中心体，并挽救了被CK1d基因敲除抑制的Wnt-3a依赖的轴突生长。这些结果提供了强有力的证据，证明CK1d的中心体定位是Wnt-3a依赖的神经发生所必需的。敲除非典型的PKCiota也阻止了Wnt-3a依赖的神经突起的生长。初步实验表明，PKCiota可能受CK1d和/或DVL的调控。这项工作意义重大，不仅是因为它提供了对神经突起形成机制的洞察。许多参与轴突生长的因素在其他环境中对细胞极性有贡献，例如对细胞迁移至关重要的细胞延伸的形成。此外，我们有初步证据表明，CK1d也参与了初生纤毛的形成。初级纤毛缺陷是几种疾病的原因，包括神经管缺陷、多囊肾病和内翻。Wnt信号的异常也可以引起这些异常。因此，我们对CK1d和DVL的研究可能为Wnt信号控制胚胎发育及其失调在发病机制中的作用提供新的见解。

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. CK1d but not CK1e was detected at the centrosome, an organelle associated with neurite formation. Deletion analysis mapped the centrosomal localization signal (CLS) of CK1d to its carboxyl-terminal domain. A fusion protein containing the CLS and EGFP displaced full-length CK1d from the centrosome and inhibited Wnt-3a-dependent neurite outgrowth. In contrast to wild-type CK1e, a chimera comprised of the kinase domain of CK1e and the CLS of CK1d localized to the centrosome and rescued Wnt-3a-dependent neurite outgrowth suppressed by CK1d knockdown. These results provide strong evidence that the centrosomal localization of CK1d is required for Wnt-3a-dependent neuritogenesis. 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 contribute to cell polarity in other contexts such as the formation of cellular extensions critical for cell migration. 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 and its dysregulation contributes to pathogenesis.