Sato Takuma, Takino Jun-Ichi, Nagamine Kentaro, Nishio Kazuto, Hori Takamitsu
Laboratory of Biochemistry, Hiroshima International University, Hiroshima, Japan.
Department of Genome Biology, Kindai University Faculty of Medicine, Osaka, Japan.
ScientificWorldJournal. 2019 Jan 1;2019:4639165. doi: 10.1155/2019/4639165. eCollection 2019.
We have identified () as a blood vessel related gene from Xenopus embryo. In addition, we reported that RASGRP2 is also expressed in human umbilical vein endothelial cells (HUVEC). It is known that RASGRP2 activates Ras-related protein 1 (Rap1). However, the function of RASGRP2 in human vascular endothelium remains unknown. Therefore, we performed functional analysis of RASGRP2 using immortalized HUVEC (TERT HUVEC). We established a stable RASGRP2 overexpressing cell line (TERT HUVEC R) and mock cell line (mock). Furthermore, we compared the activity of Rap1 and the generation of intracellular reactive oxygen species (ROS), which is related to cell death, in both cell lines. Significant increase in Rap1 activity was observed in the TERT HUVEC R compared to the mock. Furthermore, apoptosis by tumor necrosis factor- (TNF-) stimulation was significantly more reduced in the TERT HUVEC R than in the mock. In the mock, apoptosis induced by TNF- stimulation was decreased by pretreatment with diphenyleneiodonium (DPI), which is an inhibitor of NADPH oxidase (NOX). However, in the TERT HUVEC R, apoptosis induced by TNF- stimulation was not reduced after pretreatment of DPI. Furthermore, there was no reduction in ROS production in the TERT HUVEC R after DPI pretreatment. In addition, the difference in the degree of apoptosis induced by TNF- stimulation in both cell lines was consistent with the difference in ROS production in the cell lines. From these results, it was suggested that RASGRP2 activates Rap1 and the activated Rap1 suppresses apoptosis via NOX inhibition.
我们已从非洲爪蟾胚胎中鉴定出（）作为一种血管相关基因。此外，我们报道RASGRP2也在人脐静脉内皮细胞（HUVEC）中表达。已知RASGRP2可激活Ras相关蛋白1（Rap1）。然而，RASGRP2在人血管内皮中的功能仍不清楚。因此，我们使用永生化的HUVEC（TERT HUVEC）对RASGRP2进行了功能分析。我们建立了一个稳定的RASGRP2过表达细胞系（TERT HUVEC R）和对照细胞系（mock）。此外，我们比较了这两种细胞系中Rap1的活性以及与细胞死亡相关的细胞内活性氧（ROS）的产生。与对照相比，TERT HUVEC R中观察到Rap1活性显著增加。此外，与对照相比，TERT HUVEC R中肿瘤坏死因子-（TNF-）刺激诱导产生的凋亡显著减少。在对照中，用二苯基碘鎓（DPI）预处理可减少TNF-刺激诱导的凋亡，DPI是烟酰胺腺嘌呤二核苷酸磷酸氧化酶（NOX）的抑制剂。然而，在TERT HUVEC R中，DPI预处理后TNF-刺激诱导的凋亡并未减少。此外，DPI预处理后TERT HUVEC R中的ROS产生也没有减少。另外，两种细胞系中TNF-刺激诱导的凋亡程度差异与细胞系中ROS产生的差异一致。从这些结果表明，RASGRP2激活Rap1，且激活的Rap1通过抑制NOX来抑制凋亡。
