Hedgehog (Hh) signaling in vertebrates depends on primary cilia. Upon stimulation, Hh pathway components, including Gli transcription factors, accumulate at primary cilia to transduce the Hh signal, but the mechanisms underlying their ciliary targeting remains largely unknown. Here, we show that the PY-type nuclear localization signal (PY-NLS)/karyopherinβ2 (Kapβ2) nuclear import system regulates Gli ciliary localization and Hh pathway activation. Mutating the PY-NLS in Gli or knockdown of Kapβ2 diminished Gli ciliary localization. Kapβ2 is required for the formation of Gli activator (Gli^A) in wild-type but not in Sufu mutant cells. Knockdown of Kapβ2 affected Hh signaling in zebrafish embryos, as well as in vitro cultured cerebellum granule neuron progenitors (CGNPs) and SmoM2-driven medulloblastoma cells. Furthermore, Kapβ2 depletion impaired the growth of cultured medulloblastoma cells, which was rescued by Gli overexpression. Interestingly, Kapβ2 is a transcriptional target of the Hh pathway, thus forming a positive feedback loop for Gli activation. Our study unravels the molecular mechanism and cellular machinery regulating Gli ciliary localization and identifies Kapβ2 as a critical regulator of the Hh pathway and a potential drug target for Hh-driven cancers.

脊椎动物中的刺猬（Hh）信号依赖于初级纤毛。刺激后，包括Gli转录因子在内的Hh通路成分在初级纤毛上积累，以传导Hh信号，但其纤毛靶向作用的基本机制仍然未知。在这里，我们表明，PY型核定位信号（PY-NLS）/karyopherinβ2（Kapβ2）核输入系统调节Gli纤毛定位和Hh途径激活。Gli中的PY-NLS突变或Kapβ2的敲低减少了Gli睫状体的定位。Kapβ2是野生型中形成Gli激活剂（Gli ^A）所必需的，而在苏福州则不需要突变细胞。敲除Kapβ2会影响斑马鱼胚胎以及体外培养的小脑颗粒神经元祖细胞（CGNPs）和SmoM2驱动的髓母细胞瘤细胞中的Hh信号传导。此外，Kapβ2耗竭会损害培养的髓母细胞瘤细胞的生长，这可以通过Gli过表达来挽救。有趣的是，Kapβ2是Hh途径的转录靶标，因此形成了一个Gli激活的正反馈环。我们的研究揭示了调节Gli纤毛定位的分子机制和细胞机制，并确定Kapβ2是Hh途径的关键调节剂和Hh驱动的癌症的潜在药物靶标。