The superoxide-producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells
The superoxide-producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells
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DOI：
10.1111/j.1365-2443.2005.00907.x
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发表时间：
2005-12-01
期刊：
影响因子：
2.1
通讯作者：
Sumimoto, H
中科院分区：
文献类型：
--
作者：
Kuroda, J;Nakagawa, K;Sumimoto, H
产生超氧化物的 NAD(P)H 氧化酶 Nox4 最初被鉴定为一种在肾脏中高表达的酶，可能参与氧传感和细胞衰老。尽管氧化酶在血管内皮细胞中也丰富，但其作用仍有待阐明。在这里，我们使用三种针对人 Nox4 不同免疫原的亲和纯化抗体，通过免疫细胞化学和免疫电子显微镜显示，Nox4 优先定位于人脐静脉内皮细胞 (HUVEC) 的细胞核。通过 RNA 干扰 (RNAi) 沉默 Nox4 会消除抗体发出的核信号，从而证实了 Nox4 的核定位。 HUVEC 的核部分以依赖于 Nox4 的方式表现出 NAD(P)H 依赖性超氧化物生成活性，该活性可以在用佛波醇 12-肉豆蔻酸酯 13-乙酸酯刺激细胞后增强。这种刺激剂还促进基因表达，正如在目前的 HUVEC 转染测定中所估计的，使用由 Maf 识别元件 MARE（构成氧化应激反应一部分的 DNA 序列）调节的报告基因。 RNAi 介导的 Nox4 沉默会损害基础转录活性和刺激转录活性。因此，Nox4 似乎在 HUVEC 细胞核中产生超氧化物，从而通过氧化应激反应机制调节基因表达。
The superoxide-producing NAD(P)H oxidase Nox4 was initially identified as an enzyme that is highly expressed in the kidney and is possibly involved in oxygen sensing and cellular senescence. Although the oxidase is also abundant in vascular endothelial cells, its role remains to be elucidated. Here we show that Nox4 preferentially localizes to the nucleus of human umbilical vein endothelial cells (HUVECs), by immunocytochemistry and immunoelectron microscopy using three kinds of affinity-purified antibodies raised against distinct immunogens from human Nox4. Silencing of Nox4 by RNA interference (RNAi) abrogates nuclear signals given with the antibodies, confirming the nuclear localization of Nox4. The nuclear fraction of HUVECs exhibits an NAD(P)H-dependent superoxide-producing activity in a manner dependent on Nox4, which activity can be enhanced upon cell stimulation with phorbol 12-myristate 13-acetate. This stimulant also facilitates gene expression as estimated in the present transfection assay of HUVECs using a reporter regulated by the Maf-recognition element MARE, a DNA sequence that constitutes a part of oxidative stress response. Both basal and stimulated transcriptional activities are impaired by RNAi-mediated Nox4 silencing. Thus Nox4 appears to produce superoxide in the nucleus of HUVECs, thereby regulating gene expression via a mechanism for oxidative stress response.