Disease relevance of NOX4

• Elevated NOX4 mRNA levels were detectable as early as 24 h after the onset of ischemia and persisted throughout the 30 days of follow-up period, reaching a maximum between days 7 and 15 [1].
• Melanoma proliferation was reduced by NAD(P)H oxidase inhibitors and by transfection of antisense but not sense oligonucleotides for p22(phox) and NOX4 [2].
• The present study was carried out to examine the expression of NOX4, one of the new NADPH oxidase isoforms in a mouse model of focal permanent brain ischemia [1].
• In HEK293 cells expressing NOX4 endogenously, the activity of expressed TASK-1 was moderately inhibited by hypoxia, and this oxygen response was significantly augmented by NOX4 [3].
• Here, we show that both a flavoprotein inhibitor, diphenylene iodonium (DPI), and small interfering RNAs designed to target Nox4 mRNA (siNox4RNAs) inhibited superoxide production in PANC-1 pancreatic cancer cells, and depletion of ROS by DPI or siNox4RNAs induced apoptosis [4].

High impact information on NOX4

• Identification of renox, an NAD(P)H oxidase in kidney [5].
• Our data suggest that Renox, as a renal source of reactive oxygen species, is a likely candidate for the oxygen sensor function regulating oxygen-dependent gene expression and may also have a role in the development of inflammatory processes in the kidney [5].
• NIH 3T3 fibroblasts overexpressing transfected Renox show increased production of superoxide and develop signs of cellular senescence [5].
• In situ RNA hybridization revealed that renox is highly expressed at the site of erythropoietin production in the renal cortex, showing the greatest accumulation of renox mRNA in proximal convoluted tubule epithelial cells [5].
• Transfection of specific small interfering RNA oligonucleotides reduced Nox4 protein abundance and also inhibited the insulin signaling cascade [6].

Chemical compound and disease context of NOX4

• The oxidases are also upregulated in vascular disease and are involved in the development of atherosclerosis and a significant part of angiotensin II-induced hypertension, possibly via nox1 and nox4 [7].
• Inhibiting ROS with the antioxidants, Nox4 antisense, or MnSOD overexpression all stimulated apoptosis in pancreatic cancer cells as measured by internucleosomal DNA fragmentation, phosphatidylserine externalization, cytochrome c release, and effector caspase activation [8].
• The crucial flavin-containing catalytic subunits Nox1 and Nox4 are not present in leucocytes, but are highly expressed in vascular cells and upregulated in vascular remodeling, such as that found in hypertension and atherosclerosis [9].

Biological context of NOX4

• The human NOX4 gene, comprising 18 exons, is located on chromosome 11q14.2-q21, and its expression is almost exclusively restricted to adult and fetal kidneys [10].
• The NOX4 protein of 578 amino acids exhibits 39% identity to gp91(phox) with special conservation in membrane-spanning regions and binding sites for heme, FAD, and NAD(P)H, indicative of its function as a superoxide-producing NAD(P)H oxidase [10].
• The present findings thus suggest that the novel NAD(P)H oxidase NOX4 may serve as an oxygen sensor and/or a regulator of cell growth in kidney [10].
• NOX4, widely expressed in kidney, vascular cells, osteoclasts etc.; it might be a constitutively active enzyme, regulated on the level of gene expression but its precise physiological function remains unknown [11].
• Furthermore, transient transfection of endothelial cells with Nox4 siRNA led to a decrease in migration and adhesion of monocytes in response to LPS by 36% and 52%, respectively [12].

Anatomical context of NOX4

• In addition, overexpression of NOX4 in cultured cells leads to increased superoxide production and decreased rate of growth [10].
• Expression and localization of NOX2 and NOX4 in primary human endothelial cells [13].
• NOX4-green fluorescent protein largely localizes to an intracellular compartment that costained with a marker for the endoplasmic reticulum, and its distribution did not overlap with lysosomes, Weibel-Palade bodies, or mitochondria [13].
• NOX2 and NOX4 co-localized with the endoplasmic reticulum (ER) marker calreticulin [14].
• By spectral analysis, flow cytometry, reverse transcriptase-PCR, immunocytochemistry, and immunoprecipitation it was shown that the extra-mitochondrial oxygen consumption was contributed by the NOX2 and NOX4 isoforms of the O2-*. producer plasma membrane NAD(P)H oxidase with low constitutive activity [15].

Associations of NOX4 with chemical compounds

• Here we describe the cloning of human cDNA that encodes a novel NAD(P)H oxidase, designated NOX4 [10].
• The membrane fraction of kidney-derived human embryonic kidney (HEK) 293 cells, expressing NOX4, exhibits NADH- and NADPH-dependent superoxide-producing activities, both of which are inhibited by diphenylene iodonium, an agent known to block oxygen sensing, and decreased in cells expressing antisense NOX4 mRNA [10].
• Role of NADPH oxidase 4 in lipopolysaccharide-induced proinflammatory responses by human aortic endothelial cells [12].
• Furthermore, the flavoprotein inhibitor diphenyleniodonium chloride significantly attenuated LPS-mediated AP-1-dependent CXCR6 expression, as did inhibition of NOX4 NADPH oxidase by siRNA [16].
• We further showed that the lower-level tyrosine phosphorylation in response to growth factors results from the downregulation of an NADPH oxidase, Nox4, which in turn results in the reduction of ROS generation [17].

Physical interactions of NOX4

• Yeast two hybrid and GST pull-down assays indicated that the COOH-terminal region of Nox4 interacted with the cytoplasmic tail of TLR4 [18].

Regulatory relationships of NOX4

• Interestingly, NOX4 was expressed at 100-fold higher levels compared with NOX2 [13].
• Inhibition of NADPH oxidase 4 activates apoptosis via the AKT/apoptosis signal-regulating kinase 1 pathway in pancreatic cancer PANC-1 cells [4].
• Finally, inhibition of ROS with ROS scavengers or dominant-negative Nox4 blocked the TGF-beta effect on cytoskeleton changes in endothelial cells [19].
• EGF blocks up-regulation of nox4 by TGF-beta [20].
• Transfection with Nox4 antisense oligonucleotide inhibited NAD(P)H oxidase activity and ROS production in MIA PaCa-2 and PANC-1 cells [8].

Other interactions of NOX4

• METHODS AND RESULTS: Yeast two-hybrid and glutathione-S-transferase pull-down assays indicated that the cytosolic Toll/IL-1R region of Toll-like receptor 4 (TLR4) (amino acids 739-769) is the responsible domain for interaction with the COOH terminal of Nox4 (amino acids 451-530) [12].
• NOX2 and NOX4 were abundantly expressed, whereas NOX1 expression was less prominent [14].
• In human renal cortex, high amounts of the NOX4 protein are present in distal tubular cells, which reside near erythropoietin-producing cells [10].
• Nox4 associates with the protein p22phox on internal membranes, where ROS generation occurs [21].
• Aortic lesions also expressed Thox1 and Nox4, and although their expression also increases with lesion severity, their expression is less frequent than that of gp91(phox) [22].

Analytical, diagnostic and therapeutic context of NOX4

• We demonstrate that NOX4 is expressed in neurons using in situ hybridization and immunohistochemistry [1].
• The p22(phox), gp91(phox), and p67(phox) components of the human phagocyte NAD(P)H oxidase and the gp91(phox) homolog NOX4 were demonstrated in melanomas by RT-PCR and sequencing, and protein product for both p22(phox) and gp91(phox) was detected in cell membranes by immunoassay [2].
• We and others have shown that although gp91phox mRNA expression is upregulated during atherogenesis in human and animal models, expression of the Nox4 subunit remains unchanged [23].
• 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 [24].
• Using reverse transcription-PCR and Northern blot analyses, we found that Nox4, a homolog of gp91phox, the phagocytic NAD(P)H oxidase catalytic subunit, is prominently expressed in insulin-sensitive adipose cells [6].

References