The superoxide-producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells.
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.
Kuroda
Junya
J
Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Japan.
Nakagawa
Kazunori
K
Yamasaki
Tomoko
T
Nakamura
Kei-ichiro
K
Takeya
Ryu
R
Kuribayashi
Futoshi
F
Imajoh-Ohmi
Shinobu
S
Igarashi
Kazuhiko
K
Shibata
Yosaburo
Y
Sueishi
Katsuo
K
Sumimoto
Hideki
H
D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism*; D005786:Gene Expression Regulation; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D050977:Maf Transcription Factors / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism* / Q000494:pharmacology; D018384:Oxidative Stress / Q000235:genetics; D010750:Phosphoproteins / Q000378:metabolism; D034622:RNA Interference; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects; D014471:Umbilical Veins / Q000201:enzymology / Q000378:metabolism; D015854:Up-Regulation
D050977:Maf Transcription Factors; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C086986:CYBA protein, human; D013755:Tetradecanoylphorbol Acetate