Subcellular localization of Nox4 and regulation in diabetes

Abstract

Oxidative stress is implicated in human diseases. Some of the oxidative pathways are harbored in the mitochondria. NAD(P)H oxidases have been identified not only in phagocytic but also in somatic cells. Nox4 is the most ubiquitous of these oxidases and is a major source of reactive oxygen species (ROS) in many cell types and in kidney tissue of diabetic animals. We generated specific Nox4 antibodies, and found that Nox4 localizes to mitochondria. (i) Immunoblot analysis in cultured mesangial cells and kidney cortex revealed that Nox4 is present in crude mitochondria, in mitochondria-enriched heavy fractions, and in purified mitochondria; (ii) immunofluorescence confocal microscopy also revealed that Nox4 localizes with the mitochondrial marker Mitotracker; and (iii) the mitochondrial localization prediction program MitoProt indicated that the probability score for Nox4 is identical to mitochondrial protein cytochrome c oxidase subunit IV. We also show that in purified mitochondria, siRNAmediated knockdown of Nox4 significantly reduces NADPH oxidase activity in pure mitochondria and blocks glucose-induced mitochondrial superoxide generation. In a rat model of diabetes, mitochondrial Nox4 expression is increased in kidney cortex. Our data provide evidence that a functional Nox4 is present and regulated in mitochondria, indicating the existence of a previously undescribed source of ROS in this organelle.kidney ͉ mitochondria ͉ oxidative stress

myo-Inositol Oxygenase Overexpression Accentuates Generation of Reactive Oxygen Species and Exacerbates Cellular Injury following High Glucose Ambience

“…From Amersham Biosciences, we purchased the Enhanced Chemiluminescence (ECL) kit. Polyclonal NOX4-antibody was a gift from Dr. Karen Block, and it was prepared by using recombinant mouse NOX4 as the immunogen (30). The mink lung epithelial cell line stably transfected with plasminogen activator-inhibitor-1 promoter linked to the luciferase reporter gene was a gift from Dr. Daniel Rifkin (31).…”

“…Total TGF-␤1 was measured using a sandwich ELISA kit according to the manufacturer's instructions (Assaypro), and its amount in the samples was expressed as pg/mg of total cell protein. In addition to the amount of TGF-␤1 in the culture medium, its bioactivity was determined using a mink lung epithelial cell line stably transfected with plasminogen activator-inhibitor-1 promoter linked to the luciferase reporter gene (30). Mink cells were seeded onto a 96-well tissue culture plate (1.6 ϫ 10 4 /well) and allowed to adhere for 3 h. A serial dilution of TGF-␤1 standard or conditioned medium from LLC-PK1 cells (1:1 dilution) was added to the medium of the mink cells and incubated for 15 h. Luciferase activity was determined using Promega Luciferase assay reagent, as described previously (31).…”

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.…”

Mitochondrial Reactive Oxygen Species Mediate Cardiomyocyte Formation from Embryonic Stem Cells in High Glucose

“…Interestingly, Nox4 has been recently reported to reside in the mitochondrion of the kidney cortex, and its expression is sensitive to glucose levels [46]. This mitochondrial location thus places Nox4 in an ideal position to potentially regulate ROS levels in the cell.…”