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Research on the mechanism of EBVLMP1-indused release of fibroblast growth factor2

EBVLMP1诱导成纤维细胞生长因子2释放的机制研究

基本信息

批准号：
17591780
负责人：
WAKISAKA Naohiro
金额：
$ 2.39万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17591780/
关键词：
EBV LMP1 FGF-2 exosome multivesicular body endosome lvsosome colocalization LMPI

项目摘要

Fibroblast growth factor (FGF)-2, a potent angiogenic factor that is involved in tumor invasion and angiogenesis, is known to be released extracellularly by a non-classical secretory pathway. Recently it has become clear that Epstein-Barrvirus, specifically its oncoprotein LMP1, can induce expression of angiogenic factors. Among these factors is FGF-2. LMP1 not only promotes expression of FGF-2, but also the release extracellularly of its 18-kDa isoform. We analyzed the mechanism of FGF-2 release induced by LMP1. Confocal immunofluorescence microscopy revealed colocalization of FGF-2 with LMP1 in small dots also stained positively for CD63 and cathepsin.D, markers of late endosomes or multivesicular bodies. Biochemical analysis and immunoelectron microscopy of purified exosomal fractions from cotransfected cells demonstrated increased release of exosomes and the concentration of LMP I and FGF-2 in these structures. Moreover, cotransfection appeared to induce partial redistribution of the Na/K-ATPase, which participates in FGF-2 release, from the plasma membrane to the intracellular LMPI/FGF-2 positive dots. Treatment with ouabain, which inhibits Na/K-ATPage activity, partially suppressed FGF-2 secretion via exosomes in a dose-dependent manner. The results suggest that exosomes may represent a previously unrecognized mechanism for FGF-2 release mediated by LMP1, and that this pathway, at least in part, involves the activity of Na/K-ATPase.

成纤维细胞生长因子（FGF）-2是一种参与肿瘤侵袭和血管生成的强有力的血管生成因子，已知其通过非经典分泌途径在细胞外释放。最近已经清楚，EB病毒，特别是其癌蛋白LMP 1，可以诱导血管生成因子的表达。这些因素中有FGF-2。LMP 1不仅促进FGF-2的表达，而且还促进其18-kDa同种型的细胞外释放。我们分析了LMP 1诱导FGF-2释放的机制。共聚焦免疫荧光显微镜检查显示FGF-2与LMP 1在小圆点中共定位，也对CD 63和组织蛋白酶D染色阳性，后者是晚期内体或多泡体的标志物。来自共转染细胞的纯化的外泌体组分的生化分析和免疫电子显微镜显示，这些结构中的外泌体的释放和LMP I和FGF-2的浓度增加。此外，共转染似乎诱导参与FGF-2释放的Na/K-ATP酶从质膜到细胞内LMPI/FGF-2阳性点的部分重新分布。用抑制Na/K-ATPage活性的哇巴因处理，以剂量依赖性方式部分抑制FGF-2通过外泌体的分泌。结果表明，外泌体可能代表了一种以前未被认识到的由LMP 1介导的FGF-2释放机制，并且该途径至少部分涉及Na/K-ATP酶的活性。