Neddylated Cullin 3 is required for vascular endothelial-cadherin-mediated endothelial barrier function.
Neddylated Cullin 3 is required for vascular endothelial-cadherin-mediated endothelial barrier function.
复制标题
Neddylated Cullin 3 是血管内皮钙粘蛋白介导的内皮屏障功能所必需的。
DOI：
10.1111/cas.13133
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发表时间：
2017-02
期刊：
影响因子：
5.7
通讯作者：
Higashiyama S
中科院分区：
文献类型：
--
作者：
Sakaue T;Fujisaki A;Nakayama H;Maekawa M;Hiyoshi H;Kubota E;Joh T;Izutani H;Higashiyama S
血管内皮细胞(VE)-钙粘附素是一种主要的内皮细胞黏附分子，调节血管通透性，在一些癌症中观察到血管通透性增加。本研究的目的是阐明NEDD8-cullin E3连接酶在维持屏障通透性中的作用。为此，我们通过使用抑制剂和基因敲除技术研究了cullin E3连接酶在人脐静脉内皮细胞中的抑制作用。此外，我们还分别用定量RT-PCR和Western blotting分析了连接酶的mRNA和蛋白水平。结果表明，VE-钙粘附素介导的内皮屏障功能需要NEDD8偶联的cullin 3。用NEDD8激活酶的化学抑制剂MLN4924处理HUVEC时，由于细胞与细胞之间的接触受损，导致血管通透性增加。当针对NEDD8的靶底物之一cullin 3的siRNA处理HUVEC时，也得到了类似的结果。免疫细胞化学染色显示，两种处理均使定位于细胞-细胞边界的VE-钙粘附素蛋白消耗殆尽。而定量RT-PCR结果显示，治疗组和对照组之间VE-cadherin基因表达水平差异无统计学意义。此外，放线菌亚胺追赶实验表明，缺失cullin 3后，VE-钙粘蛋白的半衰期显著缩短。综上所述，这些发现表明neddylated cullin 3通过调节VE-cadherin在内皮细胞屏障功能中发挥关键作用。
Vascular endothelial (VE)‐cadherin, a major endothelial adhesion molecule, regulates vascular permeability, and increased vascular permeability has been observed in several cancers. The aim of this study was to elucidate the role of the NEDD8‐Cullin E3 ligase, in maintaining barrier permeability. To this end, we investigated the effects of the inhibition of Cullin E3 ligases, by using inhibitors and knockdown techniques in HUVECs. Furthermore, we analyzed the mRNA and protein levels of the ligases by quantitative RT‐PCR and Western blotting, respectively. The results revealed that NEDD8‐conjugated Cullin 3 is required for VE‐cadherin‐mediated endothelial barrier functions. Treatment of HUVECs with MLN4924, a chemical inhibitor of the NEDD8‐activating enzyme, led to high vascular permeability due to impaired cell–cell contact. Similar results were obtained when HUVECs were treated with siRNA directed against Cullin 3, one of the target substrates of NEDD8. Immunocytochemical staining showed that both treatments equally depleted VE‐cadherin protein localized at the cell–cell borders. However, quantitative RT‐PCR showed that there was no significant difference in the VE‐cadherin mRNA levels between the treatment and control groups. In addition, cycloheximide chase assay revealed that the half‐life of VE‐cadherin protein was dramatically reduced by Cullin 3 depletion. Together, these findings suggest that neddylated Cullin 3 plays a crucial role in endothelial cell barrier function by regulating VE‐cadherin.