The superoxide‐producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells

Abstract

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)Hdependent 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.

Subcellular localization and function of alternatively spliced Noxo1 isoforms

“…A similar dual interaction with the membrane is well described in the case of p47 phox , although both interactions are prevented in the absence of cell stimulation [34,36,42]. Nuclear localization of Nox1 and of p22 phox has been reported in transformed human gingival keratinocytes [43] and in human umbilical vein endothelial cells (HUVEC) [44], respectively. Although we could not detect p22 phox in the nucleus in HEK293 cells (Fig.…”

NADPH Oxidase-generated Hydrogen Peroxide Induces DNA Damage in Mutant FLT3-expressing Leukemia Cells

“…Up to now, NOX4 have been localized to mitochondria (41,42), nucleus (31)(32)(33), ER (43)(44)(45)(46), and plasma membrane (45). Here for the first time, we localize NOX4 expression in FLT3-ITD expressing cells.…”

“…NOX4 and p22 phox Colocalized to the Nuclear Membrane in MV4-11-Given that NOX4 knockdown caused a decrease in both H 2 O 2 and DNA damage, we investigated if NOX4 localizes to the nucleus as previously shown in the literature in other cell types (31)(32)(33). We observed no clear NOX4 foci in the nucleus (Fig.…”

Atorvastatin inhibits homocysteine-induced dysfunction and apoptosis in endothelial progenitor cells

“…It has been reported that ROS increase the phosphorylation of a tyrosine or/and serine of MAPK [20] , while siRNAs against NADPH oxidases [36] suppress the phosphorylation of p38MAPK. Endogenous Nox4 [37] has been located to the nucleus of endothelial cells, which pointed to the nucleus as an intracellular site of ROS production. The nuclear localization of Nox4 suggests that it might regulate gene expression and activate a serine/ threonine protein kinase through the production of ROS.…”