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I δ /epsilon （CKId/e）抑制剂IC261阻断了神经突形成和Dvl磷酸化。用小干扰RNA敲低CKId可抑制Wnt-3a依赖性神经突的形成，而敲低CKIe可在缺乏外源Wnt-3a的情况下刺激神经突的形成。CKId和CKIe对神经突生长的不同影响可能是由于CKId优先定位于中心体，这被认为在神经突形成中起关键作用。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CKId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。许多参与神经突生长的因素也在细胞迁移的关键细胞延伸的形成中发挥重要作用。在Ewing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，我们已经确定CKId和Dvl也是初级纤毛形成所必需的。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CKId和Dvl的研究应该扩展我们对Wnt信号控制胚胎发育方式的认识。

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.