Activated NADPH Oxidase Research Articles

Abstract 876: CD40-induced growth inhibition of Burkitt lymphoma: A possible role for NADPH oxidase by upregulation of p67phox

Abstract CD40 stimulation together with IL4 has been shown to promote growth of normal B cells in vitro. In contrast to this, CD40 stimulation in several B cell lymphoma cell lines has been demonstrated to be growth inhibitory. Though the mechanism is not completely known, CD40-stimulated cells exhibit increased levels of reactive oxygen species (ROS). In B lymphocytes, a major source of ROS production is the NADPH oxidase (NOX) that is comprised of a membrane-bound Nox2 catalytic subunit and four cytosolic regulatory components, with the p67phox cytosolic protein being the activator factor. Upon stimulation, by either cross linking of the B-cell receptor or following treatment with PMA, the active NADPH oxidase generates superoxide. We have investigated the role of NADPH oxidase in CD40-mediated growth inhibition in Burkitt lymphoma (BL) cells. For this study, we first evaluated the effect of CD40 stimulation on the expression of Nox2 and p67phox in BL cells that demonstrate different levels of endogenous Nox2 and p67phox. Accordingly, Daudi and Raji cell lines, with endogenously high Nox2 and low p67phox, NAMALWA cells which have high Nox2 and no p67phox expression, and Ramos cells that contain no Nox2 or p67phox were analyzed. Treatment with anti-CD40 or CD40 ligand (CD40L) for 24 h induced up-regulation of p67phox in Daudi, Ramos, and NAMALWA cells, but not in the Raji line. No significant change in the expression of Nox2 was observed. Additionally, Daudi, Ramos and NAMALWA cells showed higher production of ROS after CD40 stimulation and activation with anti-IgM or PMA, in a chemiluminescence assay. To determine whether the change in ROS level was related to the upregulation of p67phox, Daudi cells that express either stable p67phox shRNA or control shRNA were generated. Treatment with CD40L for 24 h demonstrated significant ROS production only in control shRNA and not in the p67shRNA cells, confirming that the ROS production is NOX2-p67phox mediated. This enhanced ROS production was observed following treatment with CD40L alone and was enhanced further in conjunction with anti-IgM or PMA. However, cells with the p67phox shRNA showed detectable level of ROS only after activation with anti IgM, albeit to much lower levels than the control. Co-treatment of Daudi cells with anti-CD40 or CD40L and IL4 further increased the expression of p67phox. Although the basal level of ROS in the cells was unchanged upon co-treatment, the ROS generated following stimulation with anti-IgM or PMA was significantly enhanced. Preliminary MTT studies confirmed the growth inhibition of CD40-stimulated Daudi cells but not of Raji cells, suggesting a role for p67phox-dependent, ROS-related inhibition of lymphoma cell proliferation. Experiments are currently ongoing to delineate further the role of CD40 stimulation and Nox2-p67phox mediated ROS in affecting the growth of Burkitt lymphomas and signaling pathways downstream of CD40. Citation Format: Mazal Iris Dahan, Smitha Antony, Agnes Juhasz, Guojian Jiang, Mariam Konate, Jiamo Lu, Jennifer Meitzler, YongZhong Wu, Krishnendu Roy, James H. Doroshow. CD40-induced growth inhibition of Burkitt lymphoma: A possible role for NADPH oxidase by upregulation of p67phox [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 876.

Superoxide generation via the NR2B-NMDAR/RasGRF1/NOX2 pathway promotes dendritogenesis.

N-methyl-D-aspartate receptors (NMDARs) that contain the NR2A and NR2B subunits play a critical role in neuronal plasticity and dendritogenesis. Gain-and-loss-of function studies indicate that NR2B, but not NR2A, promotes dendritic branching. Accumulating evidence indicates that stimulation of NMDARs activates NADPH oxidase (NOX2), thereby generating superoxide. However, the molecular underpinnings of this process are not understood. RasGRF1, a guanine nucleotide exchange factor, is key for several forms of neuronal plasticity and interacts directly with the tail of NR2B. We investigated whether the NR2B-NMDAR/RasGRF1 pathway regulates the activity of NOX2 and whether superoxide production is required for dendritogenesis. We measured superoxide production in developing primary cultures of hippocampal neurons from 3 to 25 days in vitro (DIV) with the probe dihydroethidium (dHE). We found the highest dHE levels at early and intermediate developmental stages (3-15 DIV), when the NR2B-NMDAR expression is abundant. During these early/intermediate developmental stages, but not in mature neurons (>15 DIV), NMDAR activity is required for superoxide production. We also found that disrupting the NR2B-RasGRF1 interaction led to reduced dHE fluorescence intensity and moreover inhibited dendritic branching in hippocampal neurons. Together, our data indicate that superoxide production is induced by the NR2B-NMDARs/RasGRF1/NOX2 pathway and promotes dendritogenesis.

Sleep deprivation alters neutrophil functions and levels of Th1-related chemokines and CD4+ T cells in the blood.

The state of knowledge about the effect of sleep deprivation on the immune system is scarce and conflicting. It would therefore be useful to investigate the consequences of sleep deprivation on the immune system. We have studied the effect of sleep deprivation on the changes in neutrophil functions, and the ex vivo proliferative pattern of CD4+ T lymphocytes in relationship with blood cytokine and chemokine levels due to the crucial role of these cells in mounting potent immune responses. Healthy volunteers were followed for 3weeks. They had normal sleep in weeks 1 and 3 and they were sleep-deprived on week 2, sleeping < 6h per 24h, a pattern similar to sleep behaviors of many chronically sleep-deprived individuals. We assessed the levels of Th1/Th2 and inflammatory cytokines and chemokines, CD4+ T cells, and the NADPH oxidase activation and phagocytic functions in neutrophils. Our results suggest that sleep deprivation leads to a decreased neutrophil capacity to phagocytose bacteria and activate NADPH oxidase (p < 0.05). Sleep deprivation was associated with a potential increase in CXCL9 levels and decrease in CXCL10/CXCL9 and CCL5/CXCL9 ratios (p < 0.05). Furthermore, our results suggest that the decrease in CD4+ T cell due to sleep deprivation was not associated with changes in their proliferation as observed by Ki67 levels, but rather, it correlated with changes in CXCL10/CXCL9 ratio (p < 0.05). Sleep deprivation may lead to a decreased phagocytosis and NADPH oxidase activity in neutrophils and a decrease in the levels of CD4+ T cells which is related to changes in the Th1-related chemokine balance.

Hydrogen peroxide accelerated the lignification process of bamboo shoots by activating the phenylpropanoid pathway and programmed cell death in postharvest storage

Lignification is a typical characteristic of senescence of bamboo shoots during postharvest storage. In this study, exogenous hydrogen peroxide (H2O2) and diphenyliodonium iodide (DPI) were applied to postharvest bamboo shoots to explore the regulation of H2O2 in the lignification process. Bamboo shoots, soaked in 10 mM H2O2 or 5 mM DPI for 10 min, were stored at 20 ± 1 °C for 12 d. H2O2 treatment accelerated the accumulation of endogenous H2O2 by activating NADPH oxidase, while DPI treatment inhibited NADPH oxidase activity, leading to the decrease of endogenous H2O2. After 12 d of storage, the firmness and lignin content in H2O2-treated shoots was 4.79% and 5.14% higher, respectively, than in the control shoots. However, DPI treatment showed the opposite effects, with 10.88% less firmness and 19.23% less lignin content observed as compared with the control group. High level of endogenous H2O2 activated the activities of PAL, C4H and 4CL, thus promoting lignin biosynthesis. Also, H2O2 upregulated DNase, RNase and caspase 3-like activities, leading to the acceleration of the programmed cell death process, which finally contributed to the resulting lignification. Transmission electron microscopy further revealed thickened cell walls and the decompartmentalization of cellular structure in H2O2-treated shoots. Our results indicated that endogenous H2O2 may play a vital role in the lignification process of bamboo shoots.

Apoptosis signal-regulating kinase 1 activation by Nox1-derived oxidants is required for TNFα receptor endocytosis.

Tumor necrosis factor-α (TNFα) is a proinflammatory cytokine that is closely linked to the development of cardiovascular disease. TNFα activates NADPH oxidase 1 (Nox1) and reactive oxygen species (ROS), including superoxide (O2·-), production extracellularly is required for subsequent signaling in vascular smooth muscle cells (VSMCs). Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that is activated by oxidation of associated thioredoxin. The role of ASK1 in Nox1-mediated signaling by TNFα is poorly defined. We hypothesized that ASK1 is required for TNFα receptor endocytosis and subsequent inflammatory TNFα signaling. We employed a knockdown strategy to explore the role of ASK1 in TNFα signaling in VSMCs. siRNA targeting ASK1 had no effect on TNFα-induced extracellular O2·- production. However, siASK1 inhibited receptor endocytosis as well as phosphorylation of two endocytosis-related proteins, dynamin1 and caveolin1. Intracellular O2·- production was subsequently reduced, as were other inflammatory signaling steps including NF-κB activation, IL-6 production, inducible nitric oxide synthase and VCAM expression, and VSMC proliferation. Prolonged exposure to TNFα (24 h) increased tumor necrosis factor receptor (TNFR) subtype 1 and 2 expression, and these effects were also attenuated by siASK1. ASK1 coimmunoprecipitated with both Nox1 and the leucine rich repeat containing 8A anion channel, two essential components of the TNFR1 signaling complex. Activation of ASK1 by autophosphorylation at Thr845 occurs following thioredoxin dissociation, and this requires the presence of Nox1. Thus, Nox1 is part of the multiprotein ASK1 signaling complex. In response to TNFα, ASK1 is activated by Nox1-derived oxidants, and this plays a critical role in translating these ROS into a physiologic response in VSMCs. NEW & NOTEWORTHY Apoptosis signal-regulating kinase 1 (ASK1) drives dynamin1 and caveolin1 phosphorylation and TNFα receptor endocytosis. ASK1 modulates TNFα-induced NF-κB activation, survival, and proliferation. ASK1 and NADPH oxidase 1 (Nox1) physically associate in a multiprotein signaling complex. Nox1 is required for TNFα-induced ASK1 activation.

Stimulation of the class-A scavenger receptor induces neutrophil extracellular traps (NETs) by ERK dependent NOX2 and ROMO1 activation

Neutrophil extracellular traps (NETs) play a critical role in host antimicrobial response whereas they are also implicated in the pathogenesis of inflammatory and autoimmunediseases. Generation of reactiveoxygen species (ROS) is key to NETs formation. A variety of stimulatory ligands have been found to enhance ROS production and thus trigger NETs. However, the mechanisms that connect receptor stimuli with ROS production and NETs formation remain unclear. In this study, we described a new mechanism of NETs generation in neutrophils triggered by stimulation of the class A scavenger receptor (SRA), a major subtype of scavenger receptors in response to various stimuli during infection and inflammatory disorders. By using polyinosinic acid (Poly I), a ribonucleotide ligand of SRA, we demonstrated that SRA stimulation lead to selective ERK phosphorylation, which upregulated cytosol ROS levels and induced canonical NETs formation by activating NADPH oxidase 2 (NOX2). Interestingly, our results showed that mitochondrial ROS (mtROS) production was also enhanced by the SRA dependent ERK activation through upregulation and activation of reactive oxygen species modulator 1(ROMO1), a mitochondrial membrane protein and a key mediator of mtROS. Moreover, inhibition of the SRA elicited ROMO1 activation dampened NETs release upon SRA stimulation. Overall, our study describes a new insight into the NETs release triggered by membrane SRA stimulation and mediated by ERK dependent NOX2 and ROMO1 activation.

BIAM switch assay coupled to mass spectrometry identifies novel redox targets of NADPH oxidase 4

AimNADPH oxidase (Nox) -derived reactive oxygen species have been implicated in redox signaling via cysteine oxidation in target proteins. Although the importance of oxidation of target proteins is well known, the specificity of such events is often debated. Only a limited number of Nox-oxidized proteins have been identified thus far; especially little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4.In this study, HEK293 cells with tetracycline-inducible Nox4 overexpression (HEK-tet-Nox4), as well as podocytes of WT and Nox4-/- mice, were utilized to identify Nox4-dependent redox-modified proteins. ResultsTGFβ1 induced an elevation in Nox4 expression in podocytes from WT but not Nox4-/- mice. Using BIAM based redox switch assay in combination with mass spectrometry and western blot analysis, 142 proteins were identified as differentially oxidized in podocytes from wild type vs. Nox4-/- mice and 131 proteins were differentially oxidized in HEK-tet-Nox4 cells upon Nox4 overexpression. A predominant overlap was found for peroxiredoxins and thioredoxins, as expected. More interestingly, the GRB2-associated-binding protein 1 (Gab1) was identified as being differentially oxidized in both approaches. Further analysis using mass spectrometry-coupled BIAM switch assay and site directed mutagenesis, revealed Cys374 and Cys405 as the major Nox4 targeted oxidation sites in Gab1. Innovation & conclusionBIAM switch assay coupled to mass spectrometry is a powerful and versatile tool to identify differentially oxidized proteins in a global untargeted way. Nox4, as a source of hydrogen peroxide, changes the redox-state of numerous proteins. Of those, we identified Gab1 as a novel redox target of Nox4.

Sulfate is Incorporated into Cysteine to Trigger ABA Production and Stomatal Closure.

Plants close stomata when root water availability becomes limiting. Recent studies have demonstrated that soil-drying induces root-to-shoot sulfate transport via the xylem and that sulfate closes stomata. Here we provide evidence for a physiologically relevant signaling pathway that underlies sulfate-induced stomatal closure in Arabidopsis (Arabidopsis thaliana). We uncovered that, in the guard cells, sulfate activates NADPH oxidases to produce reactive oxygen species (ROS) and that this ROS induction is essential for sulfate-induced stomata closure. In line with the function of ROS as the second-messenger of abscisic acid (ABA) signaling, sulfate does not induce ROS in the ABA-synthesis mutant, aba3-1, and sulfate-induced ROS were ineffective at closing stomata in the ABA-insensitive mutant abi2-1 and a SLOW ANION CHANNEL1 loss-of-function mutant. We provided direct evidence for sulfate-induced accumulation of ABA in the cytosol of guard cells by application of the ABAleon2.1 ABA sensor, the ABA signaling reporter ProRAB18:GFP, and quantification of endogenous ABA marker genes. In concordance with previous studies, showing that ABA DEFICIENT3 uses Cys as the substrate for activation of the ABSCISIC ALDEHYDE OXIDASE3 (AAO3) enzyme catalyzing the last step of ABA production, we demonstrated that assimilation of sulfate into Cys is necessary for sulfate-induced stomatal closure and that sulfate-feeding or Cys-feeding induces transcription of NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3, limiting the synthesis of the AAO3 substrate. Consequently, Cys synthesis-depleted mutants are sensitive to soil-drying due to enhanced water loss. Our data demonstrate that sulfate is incorporated into Cys and tunes ABA biosynthesis in leaves, promoting stomatal closure, and that this mechanism contributes to the physiological water limitation response.

Evidence for a direct link between PAD4-mediated citrullination and the oxidative burst in human neutrophils

Neutrophils are critical for the defense against pathogens, in part through the extrusion of extracellular DNA traps, phagocytosis, and the production of reactive oxygen species. Neutrophils may also play an important role in the pathogenesis of rheumatoid arthritis (RA) through the activation of protein arginine deiminases (PADs) that citrullinate proteins that subsequently act as autoantigens. We report that PAD4 is physically associated with the cytosolic subunits of the oxidative burst machinery, p47phox (also known as neutrophil cytosol factor 1, NCF1) and p67phox (NCF2). Activation of PAD4 by membranolytic insults that result in high levels of intracellular calcium (higher than physiological neutrophil activation) leads to rapid citrullination of p47phox/NCF1 and p67phox/NCF2, as well as their dissociation from PAD4. This dissociation prevents the assembly of an active NADPH oxidase complex and an oxidative burst in neutrophils stimulated by phorbol-ester or immune complexes. In further support of a substrate-to-inactive enzyme interaction, small-molecule PAD inhibitors also disrupt the PAD4-NCF complex and reduce oxidase activation and phagocytic killing of Staphylococcus aureus. This novel role of PAD4 in the regulation of neutrophil physiology suggests that targeting PAD4 with active site inhibitors for the treatment of RA may have a broader impact on neutrophil biology than just inhibition of citrullination.

Astaxanthin Inhibits Mitochondrial Dysfunction and Interleukin-8 Expression in Helicobacter pylori-Infected Gastric Epithelial Cells.

Helicobacter pylori (H. pylori) infection leads to gastric inflammation, peptic ulcer and gastric carcinoma. H. pylori activates NADPH oxidase and increases reactive oxygen species (ROS), which induce NF-κB activation and IL-8 expression in gastric epithelial cells. Dysfunctional mitochondria trigger inflammatory cytokine production. Peroxisome proliferator-activated receptors-γ (PPAR-γ) regulate inflammatory response. Astaxanthin is a powerful antioxidant that protects cells against oxidative stress. The present study was aimed at determining whether astaxanthin inhibits H. pylori-induced mitochondrial dysfunction, NF-κB activation, and IL-8 expression via PPAR-γ activation in gastric epithelial cells. Gastric epithelial AGS cells were treated with astaxanthin, NADPH oxidase inhibitor apocynin and PPAR-γ antagonist GW9662, and infected with H. pylori. As a result, H. pylori caused an increase in intracellular and mitochondrial ROS, NF-κB activation and IL-8 expression, but decreased mitochondrial membrane potential and ATP level. Astaxanthin inhibited H. pylori-induced alterations (increased ROS, mitochondrial dysfunction, NF-κB activation, and IL-8 expression). Astaxanthin activated PPAR-γ and its target gene catalase in H. pylori-infected cells. Apocynin reduced ROS and inhibited IL-8 expression while astaxanthin did not affect NADPH oxidase activity. Inhibitory effects of astaxanthin on ROS levels and IL-8 expression were suppressed by addition of GW9662. In conclusion, astaxanthin inhibits H. pylori-induced mitochondrial dysfunction and ROS-mediated IL-8 expression by activating PPAR-γ and catalase in gastric epithelial cells. Astaxanthin may be beneficial for preventing oxidative stress-mediated gastric inflammation-associated H. pylori infection.

Mechanisms underlying modulation of podocyte TRPC6 channels by suPAR: Role of NADPH oxidases and Src family tyrosine kinases

The soluble urokinase receptor (suPAR) has been implicated in the pathogenesis of chronic kidney diseases (CKD) and may function as a circulating “permeability factor” driving primary focal and segmental glomerulosclerosis (FSGS). Here we examined the mechanisms whereby suPAR causes mobilization and increased activation of Ca2+-permeable TRPC6 channels, which are also implicated in FSGS. Treatment of immortalized mouse podocytes with recombinant suPAR for 24 h caused a marked increase in cytosolic reactive oxygen species (ROS) that required signaling through integrins. This effect was associated with increased assembly of active cell surface NADPH oxidase 2 (Nox2) complexes and was blocked by the Nox2 inhibitor apoycynin. Treatment with suPAR also evoked a functionally measurable increase in TRPC6 channels that was blocked by concurrent treatment with the ROS-quencher TEMPOL as well as by inhibition of Rac1, an essential component of active Nox2 complexes. Elevated ROS evoked by exposing cells to suPAR or H2O2 caused a marked increase in the abundance of tyrosine-phosphorylated proteins including Src, and suPAR-evoked Src activation was blocked by TEMPOL. Moreover, mobilization and increased activation of TRPC6 by suPAR or H2O2 was blocked by concurrent exposure to PP2, an inhibitor of Src family tyrosine kinases. These data suggest that suPAR induces oxidative stress in podocytes that in turn drives signaling through Src family kinases to upregulate TRPC6 channels. The combination of oxidative stress and altered Ca2+ signaling may contribute to loss of podocytes and progression of various forms of CKD.

NAD(P)H Oxidase Activity in the Small Intestine Is Predominantly Found in Enterocytes, Not Professional Phagocytes.

The balance between various cellular subsets of the innate and adaptive immune system and microbiota in the gastrointestinal tract is carefully regulated to maintain tolerance to the normal flora and dietary antigens, while protecting against pathogens. The intestinal epithelial cells and the network of dendritic cells and macrophages in the lamina propria are crucial lines of defense that regulate this balance. The complex relationship between the myeloid compartment (dendritic cells and macrophages) and lymphocyte compartment (T cells and innate lymphoid cells), as well as the impact of the epithelial cell layer have been studied in depth in recent years, revealing that the regulatory and effector functions of both innate and adaptive immune compartments exhibit more plasticity than had been previously appreciated. However, little is known about the metabolic activity of these cellular compartments, which is the basic function underlying all other additional tasks the cells perform. Here we perform intravital NAD(P)H fluorescence lifetime imaging in the small intestine of fluorescent reporter mice to monitor the NAD(P)H-dependent metabolism of epithelial and myeloid cells. The majority of myeloid cells which comprise the surveilling network in the lamina propria have a low metabolic activity and remain resting even upon stimulation. Only a few myeloid cells, typically localized at the tip of the villi, are metabolically active and are able to activate NADPH oxidases upon stimulation, leading to an oxidative burst. In contrast, the epithelial cells are metabolically highly active and, although not considered professional phagocytes, are also able to activate NADPH oxidases, leading to massive production of reactive oxygen species. Whereas the oxidative burst in myeloid cells is mainly catalyzed by the NOX2 isotype, in epithelial cells other isotypes of the NADPH oxidases family are involved, especially NOX4. They are constitutively expressed by the epithelial cells, but activated only on demand to ensure rapid defense against pathogens. This minimizes the potential for inadvertent damage from resting NOX activation, while maintaining the capacity to respond quickly if needed.

Novel redox-targets of NADPH oxidase 4

Nox-derived ROS have been implicated in redox-signaling as they can elicit cysteine oxidation in target proteins. Only a limited number of Nox-differentially oxidized proteins have been identified so far and particularly little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4. We set out to identify redox-targets of Nox4 by redox-proteomics, therefore podocytes of WT and Nox4-/- were used. Redox-modified proteins were identified by the BIAM switch assay in combination with mass spectrometry. Increased Nox4 expression in response to TGFβ-1 was detected in podocytes of WT mice. In response to TGFβ-1, oxidation of 138 proteins increased in podocytes of wildtype but not in podocytes of Nox4-/- mice. Identified proteins clustered in different groups of cellular processes like “cellular oxidant detoxification” and “activation of protein kinase activity”. The difference in oxidation of identified proteins could be validated for selected proteins by the BIAM-switch assay and western blot analysis and show up some interesting novel redox-targets of Nox4. The BIAM-Switch assay coupled to Mass spec is a powerful and versatile tool to identify differentially oxidized proteins. Nox4 is a source of ROS which changes the redox-state of numerous proteins.

ß-Cyanoalanine Synthase Action in Root Hair Elongation is Exerted at Early Steps of the Root Hair Elongation Pathway and is Independent of Direct Cyanide Inactivation of NADPH Oxidase.

In Arabidopsis thaliana, cyanide is produced concomitantly with ethylene biosynthesis and is mainly detoxified by the ß-cyanoalanine synthase CAS-C1. In roots, CAS-C1 activity is essential to maintain a low level of cyanide for proper root hair development. Root hair elongation relies on polarized cell expansion at the growing tip, and we have observed that CAS-C1 locates in mitochondria and accumulates in root hair tips during root hair elongation, as shown by observing the fluorescence in plants transformed with the translational construct ProC1:CASC1-GFP, containing the complete CAS-C1 gene fused to green fluorescent protein (GFP). Mutants in the SUPERCENTIPEDE (SCN1) gene, that regulate the NADPH oxidase gene ROOT HAIR DEFECTIVE 2 (RHD2)/AtrbohC, are affected at the very early steps of the development of root hair that do not elongate and do not show a preferential localization of the GFP accumulation in the tips of the root hair primordia. Root hairs of mutants in CAS-C1 or RHD2/AtrbohC, whose protein product catalyzes the generation of ROS and the Ca2+ gradient, start to grow out correctly, but they do not elongate. Genetic crosses between the cas-c1 mutant and scn1 or rhd2 mutants were performed, and the detailed phenotypic and molecular characterization of the double mutants demonstrates that scn1 mutation is epistatic to cas-c1 and cas-c1 is epistatic to rhd2 mutation, indicating that CAS-C1 acts in early steps of the root hair development process. Moreover, our results show that the role of CAS-C1 in root hair elongation is independent of H2O2 production and of a direct NADPH oxidase inhibition by cyanide.

FcγR/ROS/CK2α Is the Key Inducer of NF-κB Activation in a Murine Model of Asthma

Background: The transcription factor nuclear factor (NF)-κB plays a pivotal role in the development of allergic airway inflammation. However, the mechanism of NF-κB activation in asthma remains to be elucidated. Methods: CK2α activation was assessed by CK2α phosphorylation and protein expression. Airway levels of histamine and cytokines were determined by ELISA. We used 2 (active and passive) forms of allergic pulmonary inflammation models. In the active form, the animals were immunized with ovalbumin (OVA) intraperitoneally, followed by an airway challenge with OVA. In the passive form, the animals were passively sensitized by intratracheal instillation with either anti-OVA IgE or anti-OVA IgG, followed by an airway challenge with OVA. The role of NADPH oxidase (NOX) in CK2α activation was assessed using NOX2^-/- and NOX4^-/- mice because NOX2 and NOX4 contribute to many inflammatory diseases. Results: The second airway challenge increased CK2α phosphorylation and protein expression in airway epithelial cells as well as nuclear translocation of the p50 and p65 subunits of NF-κB, all of which were inhibited by the CK2α inhibitor 4,5,6,7-tetrabromobenzotriazole and the antioxidant N-acetyl-<smlcap>L</smlcap>-cysteine. CK2α phosphorylation and protein expression were significantly impaired in NOX2^-/-, but not in NOX4^-/-, mice. Induction of passive sensitization using anti-OVA IgE activated neither CK2α nor NF-κB. In contrast, induction of passive sensitization using anti-OVA IgG activated both CK2α and NF-κB. Conclusions: These data suggest that Fcγ receptor/reactive oxygen species/CK2α is a key inducer of NF-κB activation in airway epithelial cells in a murine model of asthma.

Arginase Inhibition Suppresses Native Low-Density Lipoprotein-Stimulated Vascular Smooth Muscle Cell Proliferation by NADPH Oxidase Inactivation.

PurposeVascular smooth muscle cell (VSMC) proliferation induced by native low-density lipoprotein (nLDL) stimulation is dependent on superoxide production from activated NADPH oxidase. The present study aimed to investigate whether the novel arginase inhibitor limonin could suppress nLDL-induced VSMC proliferation and to examine related mechanisms.Materials and MethodsIsolated VSMCs from rat aortas were treated with nLDL, and cell proliferation was measured by WST-1 and BrdU assays. NADPH oxidase activation was evaluated by lucigenin-induced chemiluminescence, and phosphorylation of protein kinase C (PKC) βII and extracellular signal-regulated kinase (ERK) 1/2 was determined by western blot analysis. Mitochondrial reactive oxygen species (ROS) generation was assessed using MitoSOX-red, and intracellular L-arginine concentrations were determined by high-performance liquid chromatography (HPLC) in the presence or absence of limonin.ResultsLimonin inhibited arginase I and II activity in the uncompetitive mode, and prevented nLDL-induced VSMC proliferation in a p21Waf1/Cip1-dependent manner without affecting arginase protein levels. Limonin blocked PKCβII phosphorylation, but not ERK1/2 phosphorylation, and translocation of p47phox to the membrane was decreased, as was superoxide production in nLDL-stimulated VSMCs. Moreover, mitochondrial ROS generation was increased by nLDL stimulation and blocked by preincubation with limonin. Mitochondrial ROS production was responsible for the phosphorylation of PKCβII. HPLC analysis showed that arginase inhibition with limonin increases intracellular L-arginine concentrations, but decreases polyamine concentrations. L-Arginine treatment prevented PKCβII phosphorylation without affecting ERK1/2 phosphorylation.ConclusionIncreased L-arginine levels following limonin-dependent arginase inhibition prohibited NADPH oxidase activation in a PKCβII-dependent manner, and blocked nLDL-stimulated VSMC proliferation.

PKCδ-dependent p47phox activation mediates methamphetamine-induced dopaminergic neurotoxicity.

Protein kinase C (PKC) has been recognized to activate NADPH oxidase (PHOX). However, the interaction between PKC and PHOX in vivo remains elusive. Treatment with methamphetamine (MA) resulted in a selective increase in PKCδ expression out of PKC isoforms. PKCδ co-immunoprecipitated with p47phox, and facilitated phosphorylation and membrane translocation of p47phox. MA-induced increases in PHOX activity and reactive oxygen species were attenuated by knockout of p47phox or PKCδ. In addition, MA-induced impairments in the Nrf-2-related glutathione synthetic system were also mitigated by knockout of p47phox or PKCδ. Glutathione-immunoreactivity was co-localized in Iba-1-labeled microglial cells and in NeuN-labeled neurons, but not in GFAP-labeled astrocytes, reflecting the necessity for self-protection against oxidative stress by mainly microglia. Buthionine-sulfoximine, an inhibitor of glutathione biosynthesis, potentiated microglial activation and pro-apoptotic changes, leading to dopaminergic losses. These neurotoxic processes were attenuated by rottlerin, a pharmacological inhibitor of PKCδ, genetic inhibitions of PKCδ [i.e., PKCδ knockout mice (KO) and PKCδ antisense oligonucleotide (ASO)], or genetic inhibition of p47phox (i.e., p47phox KO or p47phox ASO). Rottlerin did not exhibit any additive effects against the protective activity offered by genetic inhibition of p47phox. Therefore, we suggest that PKCδ is a critical regulator for p47phox activation induced by MA, and that Nrf-2-dependent GSH induction via inhibition of PKCδ or p47phox, is important for dopaminergic protection against MA insult.

Microtubules tune mechanotransduction through NOX2 and TRPV4 to decrease sclerostin abundance in osteocytes.

The adaptation of the skeleton to its mechanical environment is orchestrated by mechanosensitive osteocytes, largely by regulating the abundance of sclerostin, a secreted inhibitor of bone formation. We defined a microtubule-dependent mechanotransduction pathway that linked fluid shear stress to reactive oxygen species (ROS) and calcium (Ca2+) signals that led to a reduction in sclerostin abundance in cultured osteocytes. We demonstrated that microtubules stabilized by detyrosination, a reversible posttranslational modification of polymerized α-tubulin, determined the stiffness of the cytoskeleton, which set the mechanoresponsive range of cultured osteocytes to fluid shear stress. We showed that fluid shear stress through the microtubule network activated NADPH oxidase 2 (NOX2)-generated ROS that target the Ca2+ channel TRPV4 to elicit Ca2+ influx. Furthermore, tuning the abundance of detyrosinated tubulin affected cytoskeletal stiffness to define the mechanoresponsive range of cultured osteocytes to fluid shear stress. Finally, we demonstrated that NOX2-ROS elicited Ca2+ signals that activated the kinase CaMKII to decrease the abundance of sclerostin protein. Together, these discoveries may identify potentially druggable targets for regulating osteocyte mechanotransduction to affect bone quality.

The Regulation Of Nox In Vascular Disease

Despite significant advances, cardiovascular disease remains the leading global cause of death. Studies over the past twenty years have shown that the NADPH oxidases regulate diverse signaling pathways in the development of cardiovascular disease, thus identifying this enzyme complex as a potential therapeutic target. Our laboratory has examined the regulation and function of the Nox1 and Nox4 NADPH oxidases in vascular smooth muscle cell (SMC) function. Activation of Nox1 contributes to matrix degradation, migration and proliferation of SMCs, central common processes to progression of multiple vascular diseases. Protein kinase C-beta1-mediated phosphorylation of Nox1 is necessary for its interaction with the NoxA1 subunit and generation of superoxide. A peptide inhibitor of this process prevents SMC migration. Meanwhile, the constitutively active Nox4 NADPH oxidase maintains SMCs in a quiescent contractile phenotype, in part through regulation of myocardin, a master regulator of SMC gene expression. In response to vascular injury, an increase in specific microRNAs cause a decrease in Nox4 levels through both translational suppression and proteasomal degradation. These changes in miRNA and Nox4 levels decrease myocardin expression allowing for SMC phenotypic switching. The coordination of Nox1 and Nox4-dependent signaling coordinates the de-differentiation and subsequent migration and proliferation of SMCs. Strategies to inhibit Nox1 phosphorylation or Nox4 silencing may mitigate the development of cardiovascular disease.

198 - Novel Redox-Targets of NADPH Oxidase 4 Identified by Untargeted BIAM Switch Assay

NADPH oxidases of the Nox family are important sources of reactive oxygen species. Nox-derived ROS have been implicated in redox-signaling as they can elicit cysteine oxidation in target proteins. Only a limited number of Nox-differentially oxidized proteins have been identified so far and particularly little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4. We set out to identify redox-targets of Nox4 by redox-proteomics. Tetracycline-inducible HEK293 cells overexpressing Nox4 (HEK-TET-Nox4) and podocytes of WT and Nox4-/- mice were used. Redox-modified proteins were identified by the BIAM switch assay in combination with mass spectrometry and western blot analysis. Increased Nox4 expression in response to TGFβ-1 was detected by western blot analysis in podocytes of WT mice. In response to TGFβ-1, oxidation of 138 proteins increased in podocytes of wildtype but not in podocytes of Nox4-/- mice. Identified proteins clustered in different groups of cellular processes like “cellular oxidant detoxification” and “activation of protein kinase activity”. The difference in oxidation of identified proteins could be validated for selected proteins by the BIAM-switch assay and western blot analysis. As a second approach, HEK-TET-Nox4 cells were used in which Nox4 overexpression and ROS production can be stimulated by tetracycline. In this protocol, an overlap in the oxidized proteins to the WT/KO system was found for proteins with antioxidant capacity as well for some interesting novel redox-targets of Nox4. The BIAM-Switch assay coupled to mass spectrometry is a powerful and versatile tool to identify differentially oxidized proteins. Nox4 is a source of ROS which changes the redox-state of numerous proteins.