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

成纤维细胞生长因子-2是一种强大的血管生成因子，参与肿瘤的侵袭和血管生成，已知通过非经典的分泌途径在细胞外释放。最近发现EB病毒，特别是其癌蛋白LMP1，可以诱导血管生成因子的表达。这些因素中包括成纤维细胞生长因子-2。LMP1不仅能促进成纤维细胞生长因子-2的表达，还能促进其18-kDa亚型的细胞外释放。我们分析了LMP1诱导成纤维细胞生长因子-2释放的机制。免疫荧光共聚焦显微镜下可见成纤维细胞生长因子-2和LMP1在小结节内共存，CD63和组织蛋白D染色阳性。生化分析和免疫电子显微镜显示，共转染细胞纯化的胞外体组分增加了胞外体的释放，并在这些结构中增加了LMP I和FGF2的浓度。此外，共转染可诱导参与成纤维细胞生长因子-2释放的Na/K-ATPase从细胞膜部分重分布到胞内LMPI/成纤维细胞生长因子-2阳性斑点。哇巴因抑制Na/K-ATPage活性，以剂量依赖的方式部分抑制通过外切体分泌成纤维细胞生长因子-2。这些结果表明，Exosome可能代表了LMP1介导的成纤维细胞生长因子-2释放的一种以前未知的机制，并且这一途径至少部分地涉及Na/K-ATPase的活性。

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.