Cell Biology and Stem Cells Unit, CIC bioGUNE, Bilbao, Spain.
Department of Physiology, Anatomy and Genetics, Oxford University, Oxford, UK.
Mol Neurobiol. 2017 Oct;54(8):6213-6224. doi: 10.1007/s12035-016-0151-5. Epub 2016 Oct 5.
Wnt proteins preferentially activate either β-catenin-dependent or β-catenin-independent signals, but the activity of a particular Wnt also depends on cellular context and receptor availability. We previously reported that Wnt-3a induces neural differentiation of human embryonic stem cell-derived neural stem cells (NSCs) in a β-catenin-independent manner by activating a signal involving JNK and the AP-1 family member ATF-2. Here, we report the results of a gene silencing approach to identify the Wnt receptors that mediate this response to Wnt-3a. Silencing of ROR2 increased neuronal differentiation, as measured by expression of the genes DCX, NEUROD1, and NGN1, suggesting ROR2 signals normally prevent differentiation. Silencing of the other Wnt receptors singly did not affect Wnt-3a-induced neuronal differentiation. However, pairwise silencing of ROR1 and FZD4 or FZD5 and of LRP6 and FZD4 or FZD5 inhibited neuronal differentiation, as detected by reductions in the expression of neuronal genes and immunocytochemical detection of DCX, NEUROD1 and DCX. Ectopic expression of these receptors in HEK 293 cells increased ATF2-dependent transcription. In addition, ROR1 coimmunoprecipitated with FZD4 and LRP6 in transfected HEK 293 cells and colocalized with FZD4 and with LRP6 at the cell surface of transfected L cells. Wnt-3a did not appear to affect these interactions but did alter the interactions between LRP6 and FZD4/5. Together, these observations highlight roles for ROR1, LRP6, FZD4, and FZD5 in neural stem cell differentiation and provide support for a model in which dynamic interactions among these receptors mediate Wnt-3a activation of ATF2 signaling.
Wnt 蛋白优先激活β-连环蛋白依赖性或β-连环蛋白非依赖性信号，但特定 Wnt 的活性也取决于细胞环境和受体可用性。我们之前报道过，Wnt-3a 通过激活涉及 JNK 和 AP-1 家族成员 ATF-2 的信号，以β-连环蛋白非依赖性方式诱导人胚胎干细胞衍生的神经干细胞（NSC）的神经分化。在这里，我们报告了一种基因沉默方法的结果，以鉴定介导对 Wnt-3a 反应的 Wnt 受体。沉默 ROR2 增加了神经元分化，如 DCX、NEUROD1 和 NGN1 基因的表达所测量的，表明 ROR2 信号通常阻止分化。单独沉默其他 Wnt 受体不会影响 Wnt-3a 诱导的神经元分化。然而，ROR1 和 FZD4 或 FZD5 以及 LRP6 和 FZD4 或 FZD5 的成对沉默抑制了神经元分化，如神经元基因表达的减少和 DCX、NEUROD1 和 DCX 的免疫细胞化学检测所检测到的。这些受体在 HEK 293 细胞中的异位表达增加了 ATF2 依赖性转录。此外，在转染的 HEK 293 细胞中，ROR1 与 FZD4 共免疫沉淀，并且在转染的 L 细胞的细胞表面与 FZD4 和 LRP6 共定位。Wnt-3a 似乎不会影响这些相互作用，但确实改变了 LRP6 和 FZD4/5 之间的相互作用。总之，这些观察结果突出了 ROR1、LRP6、FZD4 和 FZD5 在神经干细胞分化中的作用，并为一个模型提供了支持，即这些受体之间的动态相互作用介导了 Wnt-3a 对 ATF2 信号的激活。
