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Prorenin receptor (PRR)-mediated NADPH oxidase (Nox) signaling regulates VEGF synthesis under hyperglycemic condition in ARPE-19 cells.

ABSTRACT: The stimulation of angiotensin II (Ang II), the effector peptide of renin-angiotensin system, has been reported to increase the expression of vascular endothelial growth factor (VEGF) through the activation of the Ang II type 1 receptor (AT1R). In this study, we investigated whether hyperglycemia (HG, 33?mM glucose) in ARPE-19 cells could promote the expression of VEGF independently of Ang II through prorenin receptor (PRR), via an NADPH oxidase (Nox)-dependent mechanism. ARPE-19 cells were treated with the angiotensin converting enzyme (ACE) inhibitor perindopril to block the synthesis of Ang II. Treatment with HG induced VEGF expression in ARPE-19 cells, which was attenuated by pretreatment with the inhibitors of Nox, but not those of nitric oxide synthase, xanthine oxidase and mitochondrial O2 synthesis. In addition, Nox-derived [Formula: see text] and H2O2 signaling in the regulation of VEGF was determined by using both polyethylene glycol (PEG)-catalase (CAT) and PEG-superoxide dismutase (SOD). We demonstrated that small interfering RNA (siRNA)-mediated knockdown of PRR, Nox2 and Nox4 significantly reduced the HG-induced stimulation of VEGF. On the other hand, Nox4 overexpression significantly potentiated PRR-induced stimulation of VEGF under hyperglycemia in ARPE-19 cells. Furthermore, Nox4 was shown to be associated with enhanced activities of ERK1/2 and NF-?B (p65), indicating their involvement in PRR-induced activation of VEGF under HG in ARPE-19 cells. Our results support the hypothesis that Nox4-derived reactive oxygen species (ROS) signaling is implicated in the hyperglycemia-induced increase of VEGF expression through PRR in ARPE-19 cells. However, further work is needed to evaluate the role of PRR and Nox-s in HG-induced stimulation of VEGF in vivo.

SUBMITTER: Haque R

PROVIDER: S-EPMC5994760 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Prorenin receptor (PRR)-mediated NADPH oxidase (Nox) signaling regulates VEGF synthesis under hyperglycemic condition in ARPE-19 cells.

Haque Rashidul R Iuvone P Michael PM He Li L Hur Elizabeth H EH Chung Choi Kimberly Su KS Park Daniel D Farrell Annie N AN Ngo Ashley A Gokhale Samantha S Aseem Madiha M Kumar Bhavna B

Journal of receptor and signal transduction research 20170825 6

The stimulation of angiotensin II (Ang II), the effector peptide of renin-angiotensin system, has been reported to increase the expression of vascular endothelial growth factor (VEGF) through the activation of the Ang II type 1 receptor (AT1R). In this study, we investigated whether hyperglycemia (HG, 33 mM glucose) in ARPE-19 cells could promote the expression of VEGF independently of Ang II through prorenin receptor (PRR), via an NADPH oxidase (Nox)-dependent mechanism. ARPE-19 cells were trea ...[more]

PMID: 28840773

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Project description:PurposeMicroRNAs (miRNAs/miRs) are involved in a large number of biological functions and diseases, such as cancer, cardiovascular diseases, and diabetes. MiR-21 has been reported to target Sprouty homolog 1 (SPRY1), SMAD7, and PTEN. In this study, we examined the underlying role of miR-21 in the regulation of prorenin receptor (PRR)-mediated induction of vascular endothelial growth factor (VEGF) expression via targeting SMAD7, SPRY1, and PTEN in a hyperglycemic condition.MethodsPRR-mediated induction of VEGF under a hyperglycemic condition (high glucose, 33mM) was studied by treating ARPE-19 cells with perindopril (10 µmol/l), which inhibits angiotensin II-mediated signaling. ARPE-19 cells exposed to normal glucose (NG, 5.5 mM) were considered as the control. To examine the role of miR-21 in the regulation of SPRY1, SMAD7, PTEN, and VEGF, ARPE-19 cells cultured in NG or high glucose were transfected with scramble negative control (Scr), a miR-21 mimic, or a miR-21 antagomir. To investigate the role of PRR and the small GTP-binding protein RAC1 in the regulation of miR-21, the expression of PRR and RAC1 was silenced by transfecting ARPE-19 cells with their corresponding siRNAs.ResultsCompared with the NG control, high glucose significantly induced the expression of PRR, VEGF, VEGFR2, and miR-21 but significantly suppressed the expression of SPRY1, SMAD7, and PTEN at the transcript and protein levels. In contrast, silencing the expression of PRR significantly abolished the high glucose-induced expression of VEGF, VEGFR2, and miR-21. Knockdown of RAC1 significantly attenuated the high glucose-induced expression of LOX, CTGF, and miR-21, suggesting that PRR and RAC1 are involved in the CTGF/LOX-mediated regulation of miR-21. Furthermore, high glucose dramatically increased the levels of pERK (p44), hypoxia-inducible factor (HIF-1α), and VEGF. However, this effect was antagonized by the miR-21 antagomir, indicative of the involvement of high glucose-induced miR-21 in the regulation of VEGF through ERK signaling.ConclusionsOur findings, for the first time, showed that the pleiotropic action of miR-21 induced the expression of pERK, HIF-1α, and VEGF in the high glucose condition by simultaneously targeting SPRY1, SMAD7, and PTEN in ARPE-19 cells. Therefore, miR-21 may serve as a potential therapeutic target for diabetes-induced retinal pathology.

Project description:Vascular Nox (NADPH oxidase)-derived reactive oxygen species and endoplasmic reticulum (ER) stress have been implicated in hypertension. However, relationships between these processes are unclear. We hypothesized that Nox isoforms localize in a subcellular compartment-specific manner, contributing to oxidative and ER stress, which influence the oxidative proteome and vascular function in hypertension. Nox compartmentalization (cell fractionation), O2- (lucigenin), H2O2 (amplex red), reversible protein oxidation (sulfenylation), irreversible protein oxidation (protein tyrosine phosphatase, peroxiredoxin oxidation), and ER stress (PERK [protein kinase RNA-like endoplasmic reticulum kinase], IRE1? [inositol-requiring enzyme 1], and phosphorylation/oxidation) were studied in spontaneously hypertensive rat (SHR) vascular smooth muscle cells (VSMCs). VSMC proliferation was measured by fluorescence-activated cell sorting, and vascular reactivity assessed in stroke-prone SHR arteries by myography. Noxs were downregulated by short interfering RNA and pharmacologically. In SHR, Noxs were localized in specific subcellular regions: Nox1 in plasma membrane and Nox4 in ER. In SHR, oxidative stress was associated with increased protein sulfenylation and hyperoxidation of protein tyrosine phosphatases and peroxiredoxins. Inhibition of Nox1 (NoxA1ds), Nox1/4 (GKT137831), and ER stress (4-phenylbutyric acid/tauroursodeoxycholic acid) normalized SHR vascular reactive oxygen species generation. GKT137831 reduced IRE1? sulfenylation and XBP1 (X-box binding protein 1) splicing in SHR. Increased VSMC proliferation in SHR was normalized by GKT137831, 4-phenylbutyric acid, and STF083010 (IRE1-XBP1 disruptor). Hypercontractility in the stroke-prone SHR was attenuated by 4-phenylbutyric acid. We demonstrate that protein hyperoxidation in hypertension is associated with oxidative and ER stress through upregulation of plasmalemmal-Nox1 and ER-Nox4. The IRE1-XBP1 pathway of the ER stress response is regulated by Nox4/reactive oxygen species and plays a role in the hyperproliferative VSMC phenotype in SHR. Our study highlights the importance of Nox subcellular compartmentalization and interplay between cytoplasmic reactive oxygen species and ER stress response, which contribute to the VSMC oxidative proteome and vascular dysfunction in hypertension.

Project description:BackgroundNADPH-oxidases (Nox) and the related Dual oxidases (Duox) play varied biological and pathological roles via regulated generation of reactive oxygen species (ROS). Members of the Nox/Duox family have been identified in a wide variety of organisms, including mammals, nematodes, fruit fly, green plants, fungi, and slime molds; however, little is known about the molecular evolutionary history of these enzymes.ResultsWe assembled and analyzed the deduced amino acid sequences of 101 Nox/Duox orthologs from 25 species, including vertebrates, urochordates, echinoderms, insects, nematodes, fungi, slime mold amoeba, alga and plants. In contrast to ROS defense enzymes, such as superoxide dismutase and catalase that are present in prokaryotes, ROS-generating Nox/Duox orthologs only appeared later in evolution. Molecular taxonomy revealed seven distinct subfamilies of Noxes and Duoxes. The calcium-regulated orthologs representing 4 subfamilies diverged early and are the most widely distributed in biology. Subunit-regulated Noxes represent a second major subdivision, and appeared first in fungi and amoeba. Nox5 was lost in rodents, and Nox3, which functions in the inner ear in gravity perception, emerged the most recently, corresponding to full-time adaptation of vertebrates to land. The sea urchin Strongylocentrotus purpuratus possesses the earliest Nox2 co-ortholog of vertebrate Nox1, 2, and 3, while Nox4 first appeared somewhat later in urochordates. Comparison of evolutionary substitution rates demonstrates that Nox2, the regulatory subunits p47phox and p67phox, and Duox are more stringently conserved in vertebrates than other Noxes and Nox regulatory subunits. Amino acid sequence comparisons identified key catalytic or regulatory regions, as 68 residues were highly conserved among all Nox/Duox orthologs, and 14 of these were identical with those mutated in Nox2 in variants of X-linked chronic granulomatous disease. In addition to canonical motifs, the B-loop, TM6-FAD, VXGPFG-motif, and extreme C-terminal regions were identified as important for Nox activity, as verified by mutational analysis. The presence of these non-canonical, but highly conserved regions suggests that all Nox/Duox may possess a common biological function remained in a long history of Nox/Duox evolution.ConclusionThis report provides the first comprehensive analysis of the evolution and conserved functions of Nox and Duox family members, including identification of conserved amino acid residues. These results provide a guide for future structure-function studies and for understanding the evolution of biological functions of these enzymes.

Project description:The NADPH oxidases (NOX) catalyze the production of superoxide by transferring electrons from NADPH to O2, in a regulated manner. In Neurospora crassa NOX-1 is required for normal growth of hyphae, development of aerial mycelium and asexual spores, and it is essential for sexual differentiation and cell-cell fusion. Determining the subcellular localization of NOX-1 is a critical step in understanding the mechanisms by which this enzyme can regulate all these different processes. Using fully functional versions of NOX-1 tagged with mCherry, we show that in growing hyphae NOX-1 shows only a minor association with the endoplasmic reticulum (ER) markers Ca2+-ATPase NCA-1 and an ER lumen-targeted GFP. Likewise, NOX-1 shows minor co-localization with early endosomes labeled with YPT-52, a GTPase of the Rab5 family. In contrast, NOX-1 shows extensive co-localization with two independent markers of the entire vacuolar system; the vacuolar ATPase subunit VMA-1 and the fluorescent molecule carboxy-DFFDA. In addition, part of NOX-1 was detected at the plasma membrane. The NOX-1 regulatory subunit NOR-1 displays a very different pattern of localization, showing a fine granular distribution along the entire hypha and some accumulation at the hyphal tip. In older hyphal regions, germinating conidia, and conidiophores it forms larger and discrete puncta some of which appear localized at the plasma membrane and septa. Notably, co-localization of NOX-1 and NOR-1 was mainly observed under conidial cell-cell fusion conditions in discrete vesicular structures. NOX functions in fungi have been evaluated mainly in mutants that completely lacked this protein, also eliminating interactions between hyphal growth regulatory proteins NOR-1, the GTPase RAC-1 and the scaffold protein BEM-1. To dissect NOX-1 roles as scaffold and as ROS-producing enzyme, we analyzed the function of NOX-1::mCherry proteins carrying proline 382 by histidine (P382H) or cysteine 524 by arginine (C524R) substitutions, predicted to only affect NADPH-binding. Without notably affecting NOX-1 localization or protein levels, each of these substitutions resulted in lack of function phenotypes, indicating that NOX-1 multiple functions are all dependent on its oxidase activity. Our results open new interpretations to possible NOX functions, as components of the fungal vacuolar system and the plasma membrane, as well as to new vacuolar functions.

Dataset Information

Prorenin receptor (PRR)-mediated NADPH oxidase (Nox) signaling regulates VEGF synthesis under hyperglycemic condition in ARPE-19 cells.

Publications

Prorenin receptor (PRR)-mediated NADPH oxidase (Nox) signaling regulates VEGF synthesis under hyperglycemic condition in ARPE-19 cells.

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