Regulation of plant NADPH oxidase.
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ABSTRACT:
SUBMITTER: Elmayan T
PROVIDER: S-EPMC2634351 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:NADPH oxidase5 (Nox5) is a novel Nox isoform which has recently been recognized as having important roles in the pathogenesis of coronary artery disease, acute myocardial infarction, fetal ventricular septal defect and cancer. The activity of Nox5 and production of reactive oxygen species is regulated by intracellular calcium levels and phosphorylation. However, the kinases that phosphorylate Nox5 remain poorly understood. Previous studies have shown that the phosphorylation of Nox5 is PKC dependent, but this contention was based on the use of pharmacological inhibitors and the isoforms of PKC involved remain unknown. Thus, the major goals of this study were to determine whether PKC can directly regulate Nox5 phosphorylation and activity, to identify which isoforms are involved in the process, and to understand the functional significance of this pathway in disease. We found that a relatively specific PKCα inhibitor, Ro-32-0432, dose-dependently inhibited PMA-induced superoxide production from Nox5. PMA-stimulated Nox5 activity was significantly reduced in cells with genetic silencing of PKCα and PKCε, enhanced by loss of PKCδ and the silencing of PKCθ expression was without effect. A constitutively active form of PKCα robustly increased basal and PMA-stimulated Nox5 activity and promoted the phosphorylation of Nox5 on Ser490, Thr494, and Ser498. In contrast, constitutively active PKCε potently inhibited both basal and PMA-dependent Nox5 activity. Co-IP and in vitro kinase assay experiments demonstrated that PKCα directly binds to Nox5 and modifies Nox5 phosphorylation and activity. Exposure of endothelial cells to high glucose significantly increased PKCα activation, and enhanced Nox5 derived superoxide in a manner that was in prevented by a PKCα inhibitor, Go 6976. In summary, our study reveals that PKCα is the primary isoform mediating the activation of Nox5 and this maybe of significance in our understanding of the vascular complications of diabetes and other diseases with increased ROS production.
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Project description:Respiratory burst oxidase homologue (Rboh), which is found in the plasma membrane, is a generator of reactive oxygen species (ROS) in plants. Many studies have indicated that the ROS produced by Rboh play critical roles in various cellular activities, including plant defence against pathogens. Crystals of the N-terminal domain of Oryza sativa RbohB (OsRbohB) have been obtained. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 60.4, b = 72.2, c = 118.9 A. An intensity data set was collected to 2.4 A resolution.
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Project description:Exposure to microgravity results in post-flight cardiovascular deconditioning and orthostatic intolerance in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been indicated in this process. To elucidate the mechanism for this condition, we investigated whether mitochondria regulated NADPH oxidase in hindlimb unweighting (HU) rat cerebral and mesenteric arteries. Four-week HU was used to simulate microgravity in rats. Vascular superoxide generation, protein and mRNA levels of Nox2/Nox4, and the activity of NADPH oxidase were examined in the present study. Compared with control rats, the levels of superoxide increased in cerebral (P<0.001) but not in mesenteric vascular smooth muscle cells. The protein and mRNA levels of Nox2 and Nox4 were upregulated significantly (P<0.001 and P<0.001 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) in HU rat cerebral arteries but not in mesenteric arteries. NADPH oxidases were activated significantly by HU (P<0.001) in cerebral arteries but not in mesenteric arteries. Chronic treatment with mitochondria-targeted antioxidant mitoTEMPO attenuated superoxide levels (P<0.001), decreased the protein and mRNA expression levels of Nox2/Nox4 (P<0.01 and P<0.05 for Nox2, respectively; P<0.001 and P<0.001 for Nox4, respectively) and the activity of NADPH oxidase (P<0.001) in HU rat cerebral arteries, but exerted no effects on HU rat mesenteric arteries. Therefore, mitochondria regulated the expression and activity of NADPH oxidases during simulated microgravity. Both mitochondria and NADPH oxidase participated in vascular redox status regulation.
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Project description:Oral treatment of apolipoprotein E-knockout (ApoE-KO) mice with the putative sirtuin 1 (SIRT1) activator resveratrol led to a reduction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the heart. In contrast, the SIRT1 inhibitor EX527 enhanced the superoxide production in isolated human polymorphonuclear granulocytes. In human monocytic THP-1 cells, phorbol ester-stimulated superoxide production was enhanced by inhibitors of histone deacetylases (HDACs; including quisinostat, trichostatin A (TSA), PCI34051, and tubastatin A) and decreased by inhibitors of histone acetyltransferases [such as garcinol, curcumin, and histone acetyltransferase (HAT) Inhibitor II]. These results indicate that protein acetylation and deacetylation may represent crucial mechanisms regulating NADPH oxidase-mediated superoxide production. In cell-free systems, incubation of recombinant Rac1 with SIRT1 resulted in decreased Rac1 acetylation. Mass spectrometry analyses identified lysine 166 (K166) in Rac1 as a residue targeted by SIRT1. Deacetylation of Rac1 by SIRT1 markedly reduced the interaction of Rac1 with p67phox in in vitro assays. Computational modeling analyses revealed that K166 deacetylation of Rac1 led to a 5-fold reduction in its binding affinity to guanosine-5'-triphosphate, and a 21-fold decrease in its binding potential to p67phox. The latter is crucial for Rac1-mediated recruitment of p67phox to the membrane and for p67phox activation. In conclusion, both SIRT1 and non-sirtuin deacetylases play a role in regulating NADPH oxidase activity. Rac1 can be directly deacetylated by SIRT1 in a cell-free system, leading to an inhibition of Rac1-p67phox interaction. The downstream targets of non-sirtuin deacetylases are still unknown. The in vivo significance of these findings needs to be investigated in future studies.
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Project description:Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase catalyzes the transfer of electrons from NADPH to O2, which is the main source of reactive oxygen species (ROS) in nonphagocytic cells. Excess ROS are toxic; therefore, keeping ROS in homeostasis in cells can protect cells from oxidative damage. It is meaningful to further understand the molecular mechanism by which ROS homeostasis is mediated. Human protein HSCARG is a newly identified oxidative sensor and a negative regulator of NF-?B. Here, we find that HSCARG represses the cellular ROS generation through inhibiting mRNA and protein expression of p47phox, a subunit of NADPH oxidase. In contrast, shRNA-mediated HSCARG knockdown increases endogenous p47phox expression level. And HSCARG has no obvious effect on ROS production in p47phox-depleted cells. Furthermore, HSCARG regulates p47phox through inhibition of NF-?B activity. Our findings identify HSCARG as a novel regulator in regulation of the activity of NADPH oxidase and ROS homeostasis.
| S-EPMC3602244 | biostudies-literature
Project description:NADPH oxidase 5 (NOX5) is a transmembrane signaling enzyme that produces superoxide in response to elevated cytosolic calcium. In addition to its association with numerous human diseases, NOX5 has recently been discovered to play crucial roles in the immune response and cardiovascular system. Details of NOX5 maturation, and specifically its response to changes in intracellular heme levels have remained unclear. Here we establish an experimental system in mammalian cells that allows us to probe the influence of heme availability on ROS production by NOX5. We identified a mode of dynamic regulatory control over NOX5 activity through modulation of its heme saturation and oligomeric state by intracellular heme levels and Hsp90 binding. This regulatory mechanism allows for fine-tuning and reversible modulation of NOX5 activity in response to stimuli.
| S-EPMC7394750 | biostudies-literature
Project description:BackgroundNormal cells are sensitive to anoikis, which is a cell detachment-induced apoptosis. However, cancer cells acquire anoikis resistance that is essential for successful metastasis. This study aimed to demonstrate the function and potential mechanism of NADPH oxidase 4 (NOX4) and EGFR activation in regulating anoikis resistance in lung cancer.MethodsCells were cultured either in the attached or suspended condition. Cell viability was measured by cell counting and live and dead cell staining. Expression levels of NOX4 and EGFR were measured by PCR and immunoblotting. Reactive oxygen species (ROS) levels were measured by flow cytometry. Effects of NOX4 overexpression or NOX4 knockdown by si-NOX4 on anoikis sensitivity were explored. Levels of NOX4 and EGFR in lung cancer tissues were evaluated by IHC staining.ResultsNOX4 was upregulated but EGFR decreased in suspended cells compared with attached cells. Accordingly, ROS levels were increased in suspended cells, resulting in the activation of Src and EGFR. NOX4 knockdown decreased activation of Src and EGFR, and thus sensitised cells to anoikis. NOX4 overexpression increased EGFR levels and attenuated anoikis. NOX4 expression is upregulated and is positively correlated with EGFR levels in the lung cancer patient tissues.ConclusionsNOX4 upregulation confers anoikis resistance by ROS-mediated activation of EGFR and Src, and by maintaining EGFR levels, which is critical for cell survival.
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Project description:RNA-seq analysis was performed in samples from WT and NOX4 Knock-out mice after two-thirds Partial Hepatectomy (PH). Transcriptomic analysis through RNA-seq revealed significant changes mainly 24 hours after PH in NOX4-/- mice and support a relevant role for Myc in a node of regulation of proliferation-related genes.
2021-08-05 | GSE181476 | GEO
Project description:The vascular endothelium plays an important role in the regulation of inflammatory responses after trauma and hemorrhage. Interactions of neutrophils with endothelial cells (ECs) contribute to the activation of specific EC responses involved in innate immunity. We have previously reported that oxidants derived from the neutrophil reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a critical regulator to EC activation. Our objective was to test the role of neutrophil NADPH oxidase-derived oxidants in mediating and enhancing hemorrhagic shock (HS)-induced activation of lung endothelial NADPH oxidase. Mice were subjected to HS and neutrophil depletion. The mice were also replenished with the neutrophil from NADPH oxidase-deficient mice. The resultant activation of lung NADPH oxidase was analyzed. The in vivo studies were also recapitulated with in vitro neutrophil-EC coculture system. HS induces NADPH oxidase activation in neutrophils and lung through high-mobility group box 1/Toll-like receptor 4-dependent signaling. In neutropenic mice, shock-induced NADPH oxidase activation in the lung was reduced significantly, but was restored upon repletion with neutrophils obtained from wild-type mice subjected to shock, but not with neutrophils from shock mice lacking the gp91(phox) subunit of NADPH oxidase. The findings were recapitulated in mouse lung vascular ECs cocultured with neutrophils. The data further demonstrate that neutrophil-derived oxidants are key factors mediating augmented High mobility group box 1 (HMGB1)-induced endothelial NADPH oxidase activation through a Rac1-dependent, but p38 mitogen-activated protein kinase-independent, pathway. Oxidant signaling by neutrophil NADPH oxidase is an important determinant of activation of endothelial NADPH oxidase after HS.
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Project description:Revascularization of ischemic tissue is a highly desirable outcome in multiple diseases, including cardiovascular diseases and ischemic retinopathies. Oxidative stress and inflammation are both known to play a role in suppressing reparative angiogenesis in ischemic disease models including oxygen-induced retinopathy (OIR), but the regulatory molecules governing these pathophysiologic processes in retinal ischemia are largely unknown. Nrf2 is a major stress-response transcription factor that has been implicated in regulating ischemic angiogenesis in the retina and other tissue beds. Using Nrf2-deficient mice, we investigated the effects of Nrf2 in regulating revascularization and modulating the retinal tissue milieu during ischemia. Strikingly, Nrf2's beneficial effect on reparative angiogenesis only became manifested in the later phase of ischemia in OIR, from postnatal day 14 (P14) to P17. This was temporally associated with a reduction in both oxidative stress and inflammatory mediators in wild-type compared to Nrf2-/- mice. Nrf2-/- retinas exhibited an increase in VEGF but also induction of anti-angiogenic Dll4/Notch signaling. NADPH oxidase (NOX), and especially NOX2, is a major pathogenic molecule and a particularly important contributor to oxidative stress in multiple retinal disease processes. Nrf2-/- mice exhibited a significant exacerbation of NOX2 induction in OIR that manifested in the later phases of ischemia. Pharmacologic inhibition of NADPH oxidase abrogated the adverse effect of Nrf2 deficiency on reparative angiogenesis. Taken together, this suggests that Nrf2 is an important regulator of the retinal milieu during tissue ischemia, and that the Nrf2/NOX2 balance may play a critical role in determining the fate of ischemic revascularization.
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