Molecular mechanisms for membrane lipid signaling in apoptosis

细胞凋亡中膜脂信号传导的分子机制

• 批准号: 07457036
• 负责人: NOZAWA Yoshinori
• 金额: $ 4.67万
• 依托单位: GIFU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457036/
• 关键词: Signal transduction; C2 ceramide; Phospholipase D; Apoptosis; C6 cells; CPP32; ホスホリパーゼ

There are several lines of evidence indicating that tumor-nectosis factor (TNF)-induced apoptosis involves various signaling phospholipases, e.g. phospholipase A_2, phospatidylinositol-specific phospholipase C (PI-PLC), PC-specific PLC (PC-PLC), phospholipase D (PLD), sphingomyelinase (SMase). However, the regulatory mechanisms of PC-PLC,PLD,SMase except PI-PLC and PLA_2 have not yet been disclosed. Especially, the activation mechanisms of SMase participated in apoptosis has been poorly understood. In this research project, we have first investigated the downstream signaling of ceramide, the immediate haydrolytic product of SM via SMase, on the expressed PLD's mRNA levels by Northen blotting and also the activity of interleukin 1beta-converting enzyme (ICE) and CPP32 during ceramide-induced apoptosis in rat basophilic leuchemia RBL cells, glioma C6 cells and pheochromcytoma PC12 cells.C6 cells were found to undergo the apoptotic process by exposure to membrane-permeable ceramide (C2-ce … More ramide) for 6-12 h, as inferred by nuclear DNA fragmentation and laddering. During the progress of apoptotic process, the PLD activity measured by the in vitro assay was considerably decreased to less than 10%. The levels of mRNA of PLD isozymes (rPLDa, b, c) were examined in the course of the apoptotic process induced by C2-ceramide. The mRNA levels of PLDa, b were greatly decreased whereas the level of PLDc was in contrast increased.In PC12 cells, C2-ceramide induced DNA fragmentation due to apoptosis. There was a trandient elevation of CPP32 activity after the 12h-treatment with C2-ceramide. However, no canges were observed in cell morphology and CPP32 activity in BCL-2 overexpressed PC12 cells.It has also demonstrated in basophilic leukemia RBL cells that the pretreatment of cells with C2-ceramide inhibited the IgE receptor-mediated PLD activation. This and those data by other groups have provided evidence that ceramide generated from SM via SMase may partake in induction of apoptosis. Less

肿瘤坏死因子（tumor-nectosis factor，TNF）诱导的细胞凋亡涉及多种信号磷脂酶，如磷脂酶A_2、磷脂酰肌醇特异性磷脂酶C（phosphatidylinositol-specific phospholipase C，PI-PLC）、磷脂酶D（phospholipase D，PLD）、鞘磷脂酶（sphingomyelinase，SMase）等。然而，除PI-PLC和PLA_2外，PC-PLC、PLD、SMase的调控机制尚不清楚。特别是SMase参与细胞凋亡的激活机制还不清楚。在本研究项目中，我们首先通过Northen印迹法研究了SM通过SMase的直接水解产物神经酰胺对PLD表达的mRNA水平的下游信号传导，以及神经酰胺诱导大鼠嗜碱性白血病RBL细胞凋亡过程中白细胞介素1 β转换酶（ICE）和CPP 32的活性，神经胶质瘤C6细胞和嗜铬细胞瘤PC 12细胞，发现C6细胞暴露于膜渗透性神经酰胺（C2-ce）后发生凋亡过程， ...更多信息 酰胺）6-12小时，如通过核DNA片段化和梯状化所推断的。在凋亡过程中，PLD活性在体外实验中被显著降低至低于10%。检测C2-神经酰胺诱导细胞凋亡过程中PLD同工酶（rPLDa，B，c）mRNA的表达水平。C2-神经酰胺可诱导PC 12细胞凋亡，导致DNA片段化，使PLDa、B的mRNA水平显著降低，而PLDc的mRNA水平显著升高。C2-神经酰胺处理12 h后，CPP 32活性呈一过性升高。而BCL-2过表达的PC 12细胞形态和CPP 32活性无明显改变，在嗜碱性白血病RBL细胞中，C2-神经酰胺预处理可抑制IgE受体介导的PLD激活。这些数据和其他研究组的数据提供了证据，表明SM通过SMase产生的神经酰胺可能参与诱导细胞凋亡。少

项目成果

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Nakamura, Y.：“C2-神经酰胺抑制大鼠嗜碱性白血病 (RBL-2H3) 细胞中 IgG 介导的磷脂酶 D，但不抑制 C 激活”J.Immunol.156。

Banno,Y.: "Differential translocation of phospholipase C isozymes to integrin-mediated cytoskeletal complexes in thrombin-stimulated human platelets" J.Biol.Chem.271. 14989-14994 (1996)

Banno,Y.：“在凝血酶刺激的人血小板中，磷脂酶 C 同工酶差异易位至整合素介导的细胞骨架复合物”J.Biol.Chem.271。

Ohguchi, K.: "Regulation of membrane-bound phospholipase D by protein kinase C in HL-60 cells: Synergistic action of small GTP-binding protein RhoA" J. Biol. Chem.271. 4366-4373 (1996)

Ohguchi, K.：“HL-60 细胞中蛋白激酶 C 对膜结合磷脂酶 D 的调节：小 GTP 结合蛋白 RhoA 的协同作用”J. Biol。

Higashida, H.: Progress in Brain Reserch. Elsevier Science Publisher B.V., 15 (1996)

Higashida, H.：脑研究进展。

Yoshimura,S.: "Differential mRNA expression of PLD isozymes during cAMP-induced differentiation in C6 glioma cells" Biochem.Biophys.Res.Commun.225. 494-499 (1996)

Yoshimura,S.：“cAMP 诱导的 C6 神经胶质瘤细胞分化过程中 PLD 同工酶的差异 mRNA 表达”Biochem.Biophys.Res.Commun.225。