Regulation of Gli ciliary localization and Hedgehog signaling by the PY-NLS/karyopherin-β2 nuclear import system.
Regulation of Gli ciliary localization and Hedgehog signaling by the PY-NLS/karyopherin-β2 nuclear import system.
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PY-NLS/karyopherin-beta 2 核输入系统对 Gli 纤毛定位和 Hedgehog 信号传导的调节
DOI：
10.1371/journal.pbio.2002063
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发表时间：
2017-08
期刊：
影响因子：
9.8
通讯作者：
Jiang J
中科院分区：
文献类型：
--
作者：
Han Y;Xiong Y;Shi X;Wu J;Zhao Y;Jiang J
脊椎动物中的Hedgehog（Hh）信号传导依赖于初级纤毛。在刺激后，Hh通路组分，包括Gli转录因子，在初级纤毛处积累以抑制Hh信号，但其纤毛靶向的机制在很大程度上仍然未知。在这里，我们表明，PY-type核定位信号（PY-NLS）/karyopherinβ2（Kapβ2）核输入系统调节Gli纤毛定位和Hh通路激活。突变Gli中的PY-NLS或敲低Kapβ2可减少Gli纤毛定位。Kapβ2是野生型细胞中Gli激活因子（GliA）形成所必需的，但在Sufu突变体细胞中则不然。Kapβ2的敲低影响斑马鱼胚胎中的Hh信号，以及体外培养的小脑颗粒神经元祖细胞（CGNP）和SmoM 2驱动的髓母细胞瘤细胞。此外，Kapβ2耗竭损害了培养的髓母细胞瘤细胞的生长，Gli过表达挽救了这种生长。有趣的是，Kapβ2是Hh通路的转录靶点，从而形成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，并将Kapβ2鉴定为Hh通路的关键调节剂和Hh驱动的癌症的潜在药物靶点。分泌的Hedgehog（Hh）蛋白在胚胎发育和成体组织稳态中起着进化上保守的作用。Hh信号传导活性的功能障碍导致广泛的人类疾病，包括出生缺陷和癌症。脊椎动物中的Hh信号传导主要依赖于初级纤毛，初级纤毛是存在于大多数哺乳动物细胞表面的基于微管的质膜突起。在配体刺激后，Hh通路组分，包括七跨膜蛋白Smoothened（Smo）和Gli转录因子，在初级纤毛处积累以抑制Hh信号，但其纤毛靶向的机制仍然知之甚少。本研究发现，PY-type nuclear localization signal（PY-NLS）和核内输入因子karyopherinβ2（Kapβ2）参与调控Gli纤毛定位和Hh通路活性。突变Gli中的PY-NLS或敲低Kapβ2可减少Gli纤毛定位，而不影响Smo纤毛对Hh的反应。Kapβ2调节Gli活性形式的形成，这是斑马鱼胚胎和培养的小脑颗粒神经元祖细胞（CGNP）中适当的Hh信号传导所必需的。Kapβ2耗竭损害了由Smo致癌形式驱动的髓母细胞瘤细胞的生长。最后，Kapβ2是Hh通路的转录靶点，形成促进Gli激活的正反馈环。我们的研究揭示了调控Gli纤毛靶向的分子机制，并确定Kapβ2为潜在的癌症药物靶点。
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 (GliA) 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. The secreted Hedgehog (Hh) protein plays an evolutionarily conserved role in both embryonic development and adult tissue homeostasis. Malfunction of Hh signaling activity contributes to a wide range of human diseases, including birth defects and cancer. Hh signaling in vertebrates critically depends on the primary cilium, a microtubule-based plasma membrane protrusion present on the surface of most mammalian cells. Upon ligand stimulation, Hh pathway components, including the seven-transmembrane protein Smoothened (Smo) and Gli transcription factors, accumulate at primary cilia to transduce the Hh signal, but the mechanisms underlying their ciliary targeting are still poorly understood. Here, we discover that the PY-type nuclear localization signal (PY-NLS) and the nuclear import factor karyopherinβ2 (Kapβ2) regulate Gli ciliary localization and Hh pathway activity. Mutating the PY-NLS in Gli or knockdown of Kapβ2 diminished Gli ciliary localization without affecting Smo ciliary accumulation in response to Hh. Kapβ2 regulates the formation of the active form of Gli, which is required for proper Hh signaling in zebrafish embryos and cultured cerebellum granule neuron progenitors (CGNPs). Kapβ2 depletion impaired the growth of medulloblastoma cells driven by an oncogenic form of Smo. Finally, Kapβ2 is a transcriptional target of the Hh pathway, forming a positive feedback loop to promote Gli activation. Our study reveals the molecular mechanism underlying the regulation of Gli ciliary targeting and identifies Kapβ2 as a potential cancer drug target.
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