Wnt-Dependent Neurite Outgrowth in Ewing Tumor Cells

我们确定来自Ewing肿瘤的细胞在Wnt-3a的作用下形成神经突，并开始确定这种作用的机制。Frizzled3 （Fzd3）被确定为介导该过程的主要Wnt受体，该过程还需要disheveled -2 （Dvl-2）、disheveled -3 （Dvl-3）和氨基末端c-Jun激酶（JNK）。我们发现Dickkopf-1也促进这些细胞的神经突生长，显然是通过促进内源性wnt激活Fzd3/JNK。Wnt-3a诱导的神经突生长与Dvl-2/3磷酸化有关；酪蛋白激酶1 δ /epsilon （CK1d/e）抑制剂IC261阻断了神经突的形成和Dvl的磷酸化。用小干扰RNA敲低CK1d抑制Wnt-3a依赖性神经突的发生，而敲低CK1e则在缺乏外源Wnt-3a的情况下刺激神经突的形成。在中心体中检测到CK1d，但未检测到CK1e，中心体是与神经突形成相关的细胞器。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的融合蛋白将全长CK1d从中心体中移出，并抑制wnt -3a依赖性神经突的生长。与野生型CK1e相反，由CK1e的激酶结构域和CK1d的CLS组成的嵌合体定位于中心体并拯救wnt -3a依赖性神经突生长，CK1d敲低抑制。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依赖性神经细胞发生所必需的。非典型PKCiota的敲除也阻断了wnt -3a依赖性神经突的生长。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，不仅因为它提供了有关神经突形成机制的见解。在其他情况下，许多参与神经突生长的因素有助于细胞极性的形成，例如对细胞迁移至关重要的细胞延伸。此外，我们有初步证据表明CK1d也参与初级纤毛的形成。原发纤毛缺陷是导致神经管缺陷、多囊肾病和倒立位等多种疾病的原因。异常的Wnt信号也可引起这些异常。因此，我们对CK1d和Dvl的研究可能为Wnt信号控制胚胎发育的途径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.