Studies on gap-junctional intercellular communication as a mechanism regulating cell death in the cell society

缝隙连接被认为在调节细胞死亡（包括凋亡）中起重要作用，但缝隙连接在凋亡过程中何时何地发生改变的基本现象还没有很好的研究。本研究旨在探讨Cx43在细胞凋亡过程中的命运，以回答以下问题：（1）Cx43在细胞凋亡过程中的定位是否发生变化？(2)If什么时候（3）Cx43的改变是否依赖于caspase的激活？为了回答这些问题，我们分析了连接蛋白（Cx）的时空变化在紫外线诱导的细胞凋亡使用Cx43增强的绿色荧光蛋白（EGFP）表达的HeLa细胞，并比较它们与线粒体膜电位（MMP）使用四甲基罗丹明乙酯（TMRE）和核形态学观察使用Hoechst 33342。在UV照射后2小时，三分之一的细胞变成TMRE阴性，即，它们显示出MMP的丢失，但具有轻微的核碎裂，并且在TMRE阳性和TMRE阴性细胞中均发现高百分比的线性Cx43-EGFP斑块。在4小时后，紫外线照射，这些线性斑块的百分比减少，和Cx43-EGFP的点状和弥漫性定位在TMRE阴性细胞的细胞质中注意到没有核碎片。照射后8小时，Cx43-EGFP在TMRE阴性的细胞中出现点状细胞质定位，细胞核碎裂，caspase抑制剂Z-VAD-FMK处理阻断了细胞核碎裂，部分保留了缝隙连接斑块和MMP。这些结果表明，在细胞凋亡过程中，Cx动员到细胞质中发生MMP去极化后，但在核碎片，这种改变部分依赖于caspase。

Gap junctions are considered to play an important role in moderating cell death including apoptosis.However, the basic phenomena underlying when and where the alterations of gap junctions occur during apoptosis have not been well documented. In this study, we examined the fate of Cx43 during apoptosis in order to answer the following questions : (1)Does Cx43 localization change during apoptosis? (2)If so, when? (3)Does alteration of Cx43 depend on caspase activation? To answer these questions, we analyzed the spatiotemporal changes of connexin(Cx) during UV light-induced apoptosis using Cx43-enhanced green fluorescent protein(EGFP)-expressing HeLa cells, and compared them with those of mitochondrial membrane potential(MMP) using tetramethylrhodamine ethyl ester(TMRE) and nuclear morphological observation using Hoechst 33342. At 2 hr post-UV-irradiation, a third of the cells became TMRE-negative, i.e., they showed the loss of MMP, but with slight nuclear fragmentation, and high percentages of linear Cx43-EGFP plaques were found among both TMRE-positive and TMRE-negative cells. At 4 hr post-UV-irradiation, the percentage of these linear plaques was decreased, and both punctate and diffuse localization of Cx43-EGFP were noted in the cytoplasm of TMRE-negative cells without nuclear fragmentation. At 8 hr post-irradiation, punctate cytoplasmic localization of Cx43-EGFP was noted in TMRE-negative cells with nuclear fragmentation.Treatment with the caspase inhibitor Z-VAD-FMK blocked nuclear fragmentation and partially preserved both gap junctional plaques and MMP. These results indicate that, during apoptosis, Cx mobilization into the cytoplasm occurs after MMP depolarization but before nuclear fragmentation and that this alteration partly depends on caspase.