scholarly journals IGF-I increases the expression of fibronectin by Nox4-dependent Akt phosphorylation in renal tubular epithelial cells

2012 ◽  
Vol 302 (1) ◽  
pp. C122-C130 ◽  
Author(s):  
David D. New ◽  
Karen Block ◽  
Basant Bhandhari ◽  
Yves Gorin ◽  
Hanna E. Abboud
Keyword(s):  
Epithelial Cells ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Dominant Negative ◽  
Tubular Epithelial Cells ◽  
Nadph Oxidase Activity ◽  
Akt Phosphorylation ◽  
Fibronectin Expression ◽  
Igf I

Extracellular matrix accumulation contributes to the progression of chronic kidney disease. Many growth factors including insulin-like growth factor-I (IGF-I) enhance matrix protein accumulation. Proximal tubular epithelial cells (PTCs) synthesize matrix proteins. NADPH oxidases are major sources of reactive oxygen species (ROS), important signaling molecules that mediate biological responses in a variety of cells and tissue. We investigated the mechanism by which IGF-I regulates fibronectin accumulation in PTCs and the role of a potential redox-dependent signaling pathway. IGF-I induces an increase in NADPH-dependent superoxide generation, enhances the release of hydrogen peroxide, and increases the expression of NADPH oxidase 4 (Nox4) in PTCs. IGF-I also stimulates phosphorylation of Akt, and inhibition of Akt or its upstream activator phosphatidylinositol 3-kinase attenuates IGF-I-induced fibronectin accumulation. Expression of dominant negative Akt also inhibits IGF-I-induced expression of fibronectin, indicating a role for this kinase in fibronectin accumulation. Expression of dominant negative adenovirus Nox4 inhibits IGF-I-induced NADPH oxidase activity, Akt phosphorylation, and fibronectin protein expression. Moreover, transfection of small interfering RNA targeting Nox4 decreases Nox4 protein expression and blocks IGF-I-induced Akt phosphorylation and the increase in fibronectin, placing Nox4 and ROS upstream of Akt signaling pathway. To confirm the role of Nox4, PTCs were infected with adenovirus construct expressing wild-type Nox4. Ad-Nox4, but not control Ad-green fluorescent protein, upregulated Nox4 expression and increased NADPH oxidase activity as well as fibronectin expression. Taken together, these results provide the first evidence for a role of Nox4 in IGF-I-induced Akt phosphorylation and fibronectin expression in tubular epithelial cells.

scholarly journals PKC-α mediates flow-stimulated superoxide production in thick ascending limbs

2010 ◽  
Vol 298 (4) ◽  
pp. F885-F891 ◽  
Author(s):  
Nancy J. Hong ◽  
Guillermo B. Silva ◽  
Jeffrey L. Garvin
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Mesangial Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Oxidase Inhibitor ◽  
Dominant Negative ◽  
Aortic Endothelial Cells ◽  
Nadph Oxidase Activity ◽  
Measured Flow

We showed that luminal flow increases net superoxide (O2−) production via NADPH oxidase in thick ascending limbs. Protein kinase C (PKC) activates NADPH oxidase activity in phagocytes, cardiomyocytes, aortic endothelial cells, vascular smooth muscle cells, and renal mesangial cells. However, the flow-activated pathway that induces NADPH oxidase activity in thick ascending limbs is unclear. We hypothesized that PKC mediates flow-stimulated net O2− production by thick ascending limbs. Initiation of flow (20 nl/min) increased net O2− production from 4 ± 1 to 61 ± 12 AU/s ( P < 0.007; n = 5). The NADPH oxidase inhibitor apocynin completely blocked the flow-induced increase in net O2− production (2 ± 1 vs. 1 ± 1 AU/s; P > 0.05; n = 5). Flow-stimulated O2− was also blocked in p47phox-deficient mice. We measured flow-stimulated PKC activity with a fluorescence resonance energy transfer (FRET)-based membrane-targeted PKC activity reporter and found that the FRET ratio increased from 0.87 ± 0.02 to 0.96 ± 0.04 AU ( P < 0.05; n = 6). In the absence of flow, the PKC activator phorbol 12-myristate 13-acetate (200 nM) enhanced net O2− production from 5 ± 2 to 92 ± 6 AU/s ( P < 0.001; n = 6). The PKC-α- and βI-selective inhibitor Gö 6976 (100 nM) decreased flow-stimulated net O2− production from 54 ± 15 to 2 ± 1 AU/s ( P < 0.04; n = 5). Flow-induced net O2− production was inhibited in thick ascending limbs transduced with dominant-negative (dn)PKC-α but not dnPKCβI or LacZ (Δ = 11 ± 3 AU/s for dnPKCα, 55 ± 7 AU/s for dnPKCβI, and 63 ± 7 AU/s for LacZ; P < 0.001; n = 6). We concluded that flow stimulates net O2− production in thick ascending limbs via PKC-α-mediated activation of NADPH oxidase.

scholarly journals FcγR-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome

Blood ◽  
2008 ◽  
Vol 112 (9) ◽  
pp. 3867-3877 ◽  
Author(s):  
Wei Tian ◽  
Xing Jun Li ◽  
Natalie D. Stull ◽  
Wenyu Ming ◽  
Chang-Il Suh ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Superoxide Production ◽  
Resting Cells ◽  
Cellular Activation ◽  
Nadph Oxidase Activity ◽  
Phagocyte Nadph Oxidase ◽  
Before And After ◽  
Membrane Bound

AbstractThe phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b558 and cytosolic p67phox, p47phox, and p40phox subunits that undergo membrane translocation upon cellular activation. The function of p40phox, which binds p67phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40phox in FcγR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcγR-induced phagocytosis. YFP-tagged p67phox and p40phox translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67phox and p47phox accumulation on nascent and internalized phagosomes did not require p40phox or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40phox PI3P-binding domain or wortmannin. Translocation of p40phox to nascent phagosomes required binding to p67phox but not PI3P, although the loss of PI3P binding reduced p40phox retention after phagosome internalization. We conclude that p40phox functions primarily to regulate FcγR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization.

Novel Role of Protein Disulfide Isomerase in the Regulation of NADPH Oxidase Activity: Pathophysiological Implications in Vascular Diseases

2008 ◽  
Vol 10 (6) ◽  
pp. 1101-1114 ◽  
Author(s):  
Francisco R.M. Laurindo ◽  
Denise C. Fernandes ◽  
Angélica M. Amanso ◽  
Lucia R. Lopes ◽  
Célio X.C. Santos
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Protein Disulfide Isomerase ◽  
Vascular Diseases ◽  
Disulfide Isomerase ◽  
Nadph Oxidase Activity ◽  
Protein Disulfide

scholarly journals Inhibitory Effect of Korean Red Ginseng Extract on Interleukin-8 Expression in Helicobacter pylori-Infected Gastric Epithelial Cells

2021 ◽  
Vol 5 (Supplement_2) ◽  
pp. 1130-1130
Author(s):  
Haesou Kim ◽  
Soon Ok Cho ◽  
Joo Weon Lim ◽  
Hyeyoung Kim
Keyword(s):  
Epithelial Cells ◽  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Gastric Epithelial Cells ◽  
Red Ginseng ◽  
Korean Red Ginseng ◽  
Ags Cells ◽  
Ginseng Extract ◽  
Nadph Oxidase Activity ◽  
H Pylori

Abstract Objectives Infection of Helicobacter pylori (H. pylori), a gram-negative bacterium, leads to various gastric diseases, such as gastritis, peptic ulcer and gastric cancer. H. pylori increases cytokine release and activates inflammatory mediators in gastric mucosa. Particularly, H. pylori upregulates the inflammatory chemokine interleukin-8 (IL-8), which are activated by oxidative stress. IL-8 can cause severe inflammation of the stomach and gastric cancer. Korean red ginseng is the steamed root of 6-year-old Korean ginseng (Panax ginseng Meyer). Ginsenosides, triterpene glycosides, are the active components of Korean red ginseng. Ginsenosides have antioxidant, anti-inflammatory, and antitumor activities. The present study is aimed at determining whether Korean red ginseng extract inhibits H. pylori-induced IL-8 expression in gastric epithelial cells. Methods The human gastric epithelial cell line AGS was used. Gastric epithelial AGS cells were treated with Korean red ginseng extract, and infected with H. pylori (NCTC 11,637). Reactive oxygen species (ROS) levels were determined using dichlorofluorescein fluorescence. NADPH oxidase activity was measured using lucigenin chemiluminescence. IL-8 mRNA expression was measured by using real-time PCR. NADPH oxidase subunits were determined in cytosolic extract and membrane extract by using Western blotting. Results H. pylori increased NADPH oxidase activity, ROS levels, and upregulated IL-8 expression in gastric epithelial cells. Korean red ginseng extract inhibited IL-8 expression by suppressing NADPH oxidase activity and reducing ROS levels in gastric epithelial cells. H. pylori induced translocation of NADPH oxidase cytosolic subunits to membrane, which is a marker of NADPH oxidase activation, in AGS cells. Korean red ginseng extract inhibited translocation of cytosolic subunits of NADPH oxidase to membrane in AGS cells. Conclusions Korean red ginseng may be beneficial for preventing H. pylori-associated gastric inflammation by inhibiting oxidative stress and IL-8 expression. Funding Sources This study was supported by a Brain Korea 21 FOUR Project, Yonsei University, Seoul, Republic of Korea.

scholarly journals Composition of partially purified NADPH oxidase from pig neutrophils

1984 ◽  
Vol 223 (3) ◽  
pp. 639-648 ◽  
Author(s):  
P Bellavite ◽  
O T G Jones ◽  
A R Cross ◽  
E Papini ◽  
F Rossi
Keyword(s):  
Enzyme Activity ◽  
Cytochrome C ◽  
Nadph Oxidase ◽  
Cytochrome B ◽  
Oxidase Activity ◽  
Gel Filtration ◽  
Purification Procedure ◽  
Nadph Oxidase Activity ◽  
Cytochrome C Reductase

The superoxide (O2.-)-forming enzyme NADPH oxidase from pig neutrophils was solubilized and partially purified by gel-filtration chromatography. The purification procedure allowed the separation of NADPH oxidase activity from NADH-dependent cytochrome c reductase and 2,6-dichlorophenol-indophenol reductase activities. O2.-forming activity was co-purified with cytochrome b-245 and was associated with phospholipids. However, active fractions endowed with cytochrome b were devoid of ubiquinone and contained only little FAD. The cytochrome b/FAD ratio was 1.13:1 in the crude solubilized extract and increased to 18.95:1 in the partially purified preparations. Most of FAD was associated with fractions containing NADH-dependent oxidoreductases. These results are consistent with the postulated role of cytochrome b in O2.-formation by neutrophil NADPH oxidase, but raise doubts about the participation of flavoproteins in this enzyme activity.

Cigarette smoke-induced kinin B1 receptor promotes NADPH oxidase activity in cultured human alveolar epithelial cells

Peptides ◽  
2011 ◽  
Vol 32 (7) ◽  
pp. 1447-1456 ◽  
Author(s):  
Sébastien Talbot ◽  
James Chi-Jen Lin ◽  
Karim Lahjouji ◽  
Jean-Philippe Roy ◽  
Jacques Sénécal ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Nadph Oxidase ◽  
Cigarette Smoke ◽  
Oxidase Activity ◽  
Alveolar Epithelial Cells ◽  
Nadph Oxidase Activity ◽  
Alveolar Epithelial ◽  
B1 Receptor ◽  
Kinin B1 Receptor ◽  
Human Alveolar Epithelial Cells

NADPH oxidase activity is higher in cerebral versus systemic arteries of four animal species: role of Nox2

2009 ◽  
Vol 296 (1) ◽  
pp. H220-H225 ◽  
Author(s):  
Alyson A. Miller ◽  
Grant R. Drummond ◽  
T. Michael De Silva ◽  
Anja E. Mast ◽  
Haruyo Hickey ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Animal Species ◽  
Cerebral Arteries ◽  
Nadph Oxidase Activity ◽  
Amplex Red ◽  
Enhanced Chemiluminescence ◽  
Middle Cerebral Arteries ◽  
Systemic Arteries

We previously reported that NADPH oxidase activity is greater in intracranial cerebral versus systemic arteries of the rat. Here, we first tested whether NADPH oxidase activity is also greater in intracranial cerebral than systemic arteries of three other animal species, i.e., mouse, rabbit, and pig. Second, using Nox2-deficient mice, we evaluated the involvement of Nox2-containing NADPH oxidases in any such regional differences. NADPH-stimulated superoxide (O2−) production by basilar, middle cerebral arteries (MCA), and common carotid arteries (CA) and thoracic aorta (AO) from rat, mouse, rabbit, and pig was measured using lucigenin-enhanced chemiluminescence. Basal production of O2− and hydrogen peroxide (H2O2) by cerebral arteries, AO, and CA from wild-type (WT) and Nox2−/− mice was measured using L-012-enhanced chemiluminescence and Amplex Red fluorescence, respectively. Western blotting was used to measure Nox2 and SOD1–3 protein expression, and immunofluorescence was used to localize Nox2, in mouse arteries. In rats, WT mice, rabbits, and pigs, NADPH-stimulated O2− production by cerebral arteries was up to 40-fold greater than that in AO and CA. In WT mice, basal O2− and H2O2 production by cerebral arteries was ninefold and ∼2.5-fold higher, respectively, than that in AO and CA and was associated with ∼40% greater expression of Nox2 protein. Nox2 immunofluorescence was localized to the endothelium, and to a lesser extent the adventitia, in all mouse arteries and appeared to be more intense in endothelium of MCA than AO or CA. In Nox2−/− mice, NADPH-stimulated O2− production by cerebral arteries was ∼35% lower than that in WT mice, whereas Nox2 deletion had no significant effect on O2− production by AO or CA. Thus NADPH oxidase activity is greater in intracranial cerebral versus systemic arteries of several animal species and is associated with higher cerebrovascular expression and activity of Nox2.

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