STUDY ON FUNCTION OF PHOSPHOLIPASE D2 IN NEURITE REMODELING

哺乳动物磷脂酶D（PLD）是一种新型的信号转导酶，被认为在多种细胞功能中发挥作用。已鉴定出两种哺乳动物PLD同工酶，PLD 1和PLD 2。尽管PLD 1的激活机制和生理功能已被充分证明，但PLD 2的激活机制和生理功能仍有待阐明。在本研究中，我们使用PC 12细胞和原代培养的小脑颗粒神经元来研究PLD 2是否参与轴突生长。（1）磷脂酶D2作为MAP激酶通路的下游信号分子在L1刺激的小脑颗粒神经突起生长中的作用：在出生后8天的小鼠小脑中，PLD 2蛋白大量表达，而PLD 1未检测到。L1刺激的神经突生长被过表达的脂肪酶缺陷型（LD）PLD 2抑制。此外，发现CGN中的L1刺激伴随着细胞外信号调节激酶（ERK）的磷酸化而增加PLD活性 关于我们 MAP激酶-ERK激酶（MEK）抑制剂可抑制这两种作用。这些结果提供的证据表明，PLD 2作为ERK的下游信号分子介导的L1依赖性的CGN的神经突生长，这可能是与酒精相关的神经发育障碍的机制。（2）ERK MAP激酶下游磷脂酶D2活化在PC 12细胞中NGF刺激的神经突生长的信号通路中的重要作用：在PC 12克隆细胞系中，野生型PLD 2（WT-PLD 2）而非WT-PLD 1的表达增加显著延长了由NGF刺激或MAP激酶-ERK激酶活性形式（MEK-CA）的瞬时表达诱导的神经突。与此相反，LD-PLD 2的表达抑制了神经突的伸长。此外，MEK抑制剂抑制PLD 2激活和NGF诱导的PC 12细胞诱导表达WT-PLD 2的肥大神经突延伸。MEK-CA刺激共表达的PLD 2的活性。这些结果提供了证据，PLD 2作为ERK的下游信号分子在NGF诱导的PC 12细胞的神经突生长的信号通路中起作用。少

Mammalian phospholipase D (PLD) is a novel signal transducing enzyme, which is believed to play roles in a wide variety of cell functions. Two mammalian PLD isozymes, PLD1 and PLD2,have been identified. Although activation mechanisms and physiological functions of PLD1 have been well documented, those of PLD2 still remain to be clarified. In the present study, we investigated whether PLD2 is involved in axonal outgrowth using PC12 cells and primary cultured cerebellar granule neurons.(1)Phospholipase D2 Functions as a Downstream Signaling Molecule of MAP kinase Pathway in L1-Stimulated Neurite Outgrowth of Cerebellar Granule:In the cerebellum of postnatal day 8 mice, PLD2 protein was abundantly expressed, while PLD1 was not detected. The L1-stimulated neurite outgrowth was inhibited by overexpression of lipase-deficient (LD) PLD2. Furthermore, it was found that L1 stimulation in CGNs increased PLD activity concomitantly with phosphorylation of extracellular signal-regulated kinase (ERK … More ), both of which were inhibited by the MAP kinase-ERK kinase (MEK) inhibitor. These results provide evidence that PLD2 functions as a downstream signaling molecule of ERK to mediate the L1-dependent neurite outgrowth of CGNs, a mechanism that may be related to alcohol-related neurodevelopmental disorders.(2)Essential Role of Phospholipase D2 Activation Downstream of ERK MAP Kinase in the Signaling Pathway of NGF-Stimulated Neurite Outgrowth in PC12 Cells:Increased expression of wild type PLD2 (WT-PLD2), but not WT-PLD1,in a PC12 clonal cell line dramatically elongated neurites induced by NGF stimulation or transient expression of the active form of MAP kinase-ERK kinase (MEK-CA). In contrast, neurite elongation was inhibited by expression of LD-PLD2. Furthermore, the MEK inhibitor suppressed PLD2 activation and the hypertrophic neurite extension induced by NGF in PC12 cells inducibly expressing WT-PLD2. MEK-CA stimulated the activity of co-expressed PLD2. These results provide evidence that PLD2 functions as a downstream signaling molecule of ERK in the signaling pathway of the NGF-induced neurite outgrowth of PC 12 cells. Less