Phospholipase Cδ3 Regulates RhoA/Rho Kinase Signaling and Neurite Outgrowth
Phospholipase Cδ3 Regulates RhoA/Rho Kinase Signaling and Neurite Outgrowth
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DOI：
10.1074/jbc.m110.171223
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发表时间：
2011-03-11
影响因子：
4.8
通讯作者：
Fukami, Kiyoko
中科院分区：
文献类型：
--
作者：
Kouchi, Zen;Igarashi, Takahiro;Fukami, Kiyoko
磷脂酶C δ 3（PLC δ 3）是调节磷酸肌醇代谢的关键酶，但其生理功能尚不清楚。由于PLC δ 3在小脑和大脑皮层中高度富集，我们研究了PLC δ 3在神经元迁移和生长中的作用。PLC delta 3敲低（KD）抑制小脑颗粒细胞的神经突形成，并且在发育中的脑中使用子宫内电穿孔的PLC delta 3 KD的应用导致大脑皮质中神经元的径向迁移的延迟。此外，PLC delta 3 KD抑制原代皮层神经元中的轴突和树突生长。PLC delta 3 KD还抑制由血清撤除或用视黄酸处理诱导的Neuro 2a神经母细胞瘤细胞的神经突形成。这种抑制通过重新引入野生型PLC δ 3而释放。有趣的是，缺乏磷脂酰肌醇4，5-二磷酸水解活性的H393 A突变体产生额外的突起，组成型活性突变体促进广泛的神经突生长，表明PLC活性对正常神经突生长很重要。引入显性负性RhoA（RhoA-DN）或用Y-27632（Rho激酶特异性抑制剂）处理可挽救PLC delta 3 KD Neuro 2a细胞中的神经突延伸。在初级皮质神经元中也检测到类似的效应。此外，RhoA的表达水平显着降低血清撤出或维甲酸在对照细胞中，虽然这种减少在PLC δ 3 KD细胞中没有观察到。我们还发现PLC delta 3的外源表达下调RhoA蛋白，并且组成型活性PLC delta 3在分化时比PLC delta 3更显著地促进RhoA下调。这些结果表明PLC delta 3负调节RhoA表达，抑制RhoA/Rho激酶信号传导，从而促进神经突延伸。
Phospholipase C delta 3 (PLC delta 3) is a key enzyme regulating phosphoinositide metabolism; however, its physiological function remains unknown. Because PLC delta 3 is highly enriched in the cerebellum and cerebral cortex, we examined the role of PLC delta 3 in neuronal migration and outgrowth. PLC delta 3 knockdown (KD) inhibits neurite formation of cerebellar granule cells, and application of PLC delta 3KD using in utero electroporation in the developing brain results in the retardation of the radial migration of neurons in the cerebral cortex. In addition, PLC delta 3KD inhibits axon and dendrite outgrowth in primary cortical neurons. PLC delta 3KD also suppresses neurite formation of Neuro2a neuroblastoma cells induced by serum withdrawal or treatment with retinoic acid. This inhibition is released by the reintroduction of wild-type PLC delta 3. Interestingly, the H393A mutant lacking phosphatidylinositol 4,5-bisphosphate hydrolyzing activity generates supernumerary protrusions, and a constitutively active mutant promotes extensive neurite outgrowth, indicating that PLC activity is important for normal neurite outgrowth. The introduction of dominant negative RhoA (RhoA-DN) or treatment with Y-27632, a Rho kinase-specific inhibitor, rescues the neurite extension in PLC delta 3KD Neuro2a cells. Similar effects were also detected in primary cortical neurons. Furthermore, the RhoA expression level was significantly decreased by serum withdrawal or retinoic acid in control cells, although this decrease was not observed in PLC delta 3KD cells. We also found that exogenous expression of PLC delta 3 down-regulated RhoA protein, and constitutively active PLC delta 3 promotes the RhoA down-regulation more significantly than PLC delta 3 upon differentiation. These results indicate that PLC delta 3 negatively regulates RhoA expression, inhibits RhoA/Rho kinase signaling, and thereby promotes neurite extension.