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诱导的突起生长与DVL-2/3的磷酸化有关；酪蛋白激酶Iβ/epsilon(CKiD/e)抑制剂IC261可阻断突起的形成和DVL的磷酸化。用小干扰RNA敲除CKiD抑制Wnt-3a依赖的神经发生，而敲除CKIE则在没有外源Wnt-3a的情况下刺激神经突起的形成。CKiD和CKIE对轴突生长的不同影响可能是由于CKiD优先定位于中心体，这被认为在轴突形成中起着关键作用。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CKiD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细胞延伸的形成中也起着重要作用，这些细胞延伸对细胞迁移至关重要。在尤文肿瘤细胞模型中获得的信息可能会增强我们对细胞在生理环境中的运动和转移的理解。此外，我们已经确定CkiD和DVL也是形成初生纤毛所必需的。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CKiD和DVL的研究应该扩大我们对Wnt信号控制胚胎发育的方式的了解。

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 I delta/epsilon (CKId/e) inhibitor, IC261. Knockdown of CKId with small interfering RNA suppressed Wnt-3a-dependent neuritogenesis, whereas knockdown of CKIe stimulated neurite formation in the absence of exogenous Wnt-3a. The contrasting effects of CKId and CKIe on neurite outgrowth might be due to the preferential localization of CKId 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 CKId 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 determined that CKId and Dvl also are required for 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 CKId and Dvl should extend our knowledge about the ways in which Wnt signaling controls embryonic development.