Wnt-Dependent Neurite Outgrowth in Ewing Tumor Cells

尤因肿瘤细胞中 Wnt 依赖性神经突生长

• 批准号: 8349411
• 负责人: Jeffrey Rubin
• 金额: $ 34.88万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349411
• 关键词: Accounting; Cell Polarity; Cells; Centrosome; Chimera organism; Chimeric Proteins; Cilia; Disease; Embryonic Development; Ewings sarcoma; JUN gene; Length; Maps; Mediating; Neural Tube Defects; Neurites; Organelles; Pathogenesis; Phosphorylation; Phosphorylation Site; Phosphotransferases; Polycystic Kidney Diseases; Process; Regulation; Role; Signal Transduction; Site; Situs Inversus; Small Interfering RNA; Testing; Work; base; beta-arrestin; casein kinase I; cell motility; cilium biogenesis; deletion analysis; inhibitor/antagonist; insight; neoplastic cell; receptor; research study; response

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.

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，但没有CK 1 e检测中心体，与神经突起形成的细胞器。缺失分析将CK 1d的中心体定位信号（CLS）定位到其羧基端结构域。含有CLS和EGFP的融合蛋白从中心体置换全长CK 1d并抑制Wnt-3a依赖的神经突生长。与野生型CK 1 e相反，由CK 1 e的激酶结构域和CK 1d的CLS组成的嵌合体定位于中心体，并挽救了因CK 1d敲低而受到抑制的Wnt-3a依赖性神经突生长。这些结果提供了强有力的证据，CK 1d的中心体定位是Wnt-3a依赖的轴突发生所必需的。 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素在其他情况下有助于细胞极性，例如形成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，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信号控制胚胎发育及其失调导致发病机制的方式提供新的见解。