Extracellular Reactive Oxygen Species Production Research Articles

Therapeutic Efficacy of Topically Applied Antioxidant Medicinal Plant Extracts in a Mouse Model of Experimental Dry Eye.

Purpose. To investigate the therapeutic effects of topical administration of antioxidant medicinal plant extracts in a mouse model of experimental dry eye (EDE). Methods. Eye drops containing balanced salt solution (BSS) or 0.001%, 0.01%, and 0.1% extracts were applied for the treatment of EDE. Tear volume, tear film break-up time (BUT), and corneal fluorescein staining scores were measured 10 days after desiccating stress. In addition, we evaluated the levels of interleukin- (IL-) 1β, tumor necrosis factor- (TNF-) α, IL-6, interferon- (IFN-) γ, and IFN-γ associated chemokines, percentage of CD4+C-X-C chemokine receptor type 3 positive (CXCR3+) T cells, goblet cell density, number of 4-hydroxy-2-nonenal (4-HNE) positive cells, and extracellular reactive oxygen species (ROS) production. Results. Compared to the EDE and BSS control groups, the mice treated with topical application of the 0.1% extract showed significant improvements in all clinical parameters, IL-1β, IL-6, TNF-α, and IFN-γ levels, percentage of CD4+CXCR3+ T cells, goblet cell density, number of 4-HNE-positive cells, and extracellular ROS production (P < 0.05). Conclusions. Topical application of 0.1% medicinal plant extracts improved clinical signs, decreased inflammation, and ameliorated oxidative stress marker and ROS production on the ocular surface of the EDE model mice.

Fibulin-5 Blocks Microenvironmental ROS in Pancreatic Cancer.

Elevated oxidative stress is an aberration seen in many solid tumors, and exploiting this biochemical difference has the potential to enhance the efficacy of anticancer agents. Homeostasis of reactive oxygen species (ROS) is important for normal cell function, but excessive production of ROS can result in cellular toxicity, and therefore ROS levels must be balanced finely. Here, we highlight the relationship between the extracellular matrix and ROS production by reporting a novel function of the matricellular protein Fibulin-5 (Fbln5). We used genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDAC) and found that mutation of the integrin-binding domain of Fbln5 led to decreased tumor growth, increased survival, and enhanced chemoresponse to standard PDAC therapies. Through mechanistic investigations, we found that improved survival was due to increased levels of oxidative stress in Fbln5-mutant tumors. Furthermore, loss of the Fbln5-integrin interaction augmented fibronectin signaling, driving integrin-induced ROS production in a 5-lipooxygenase-dependent manner. These data indicate that Fbln5 promotes PDAC progression by functioning as a molecular rheostat that modulates cell-ECM interactions to reduce ROS production, and thus tip the balance in favor of tumor cell survival and treatment-refractory disease.

Discrete roles of intracellular phospholipases A2 in human neutrophil cytotoxicity.

The role of calcium-independent phospholipase A2 (iPLA2), a component of the three major PLA2 families, in acute/chronic inflammatory processes remains elusive. Previous investigations have documented iPLA2-mediated respiratory burst of neutrophils (PMNs); however, the causative isoform of iPLA2 is unidentified. We also demonstrated that the iPLA2γ-specific inhibitor attenuates trauma/hemorrhagic shock-induced lung injury. Therefore, iPLA2γ may be implicated in acute inflammation. In addition, arachidonic acid (AA), which is primarily produced by cytosolic PLA2 (cPLA2), is known to display PMN cytotoxicity, although the relationship between AA and the cytotoxic function is still being debated on. We therefore hypothesized that iPLA2γ regulates PMN cytotoxicity via AA-independent signaling pathways. The study aim was to distinguish the role of intracellular phospholipases A2, iPLA2, and cPLA2, in human PMN cytotoxicity and explore the possibility of the presence of signaling molecule(s) other than AA. Isolated human PMNs were incubated with the PLA2 inhibitor selective for iPLA2β, iPLA2γ, or cPLA2 and then activated with formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA). Superoxide production was assayed according to the superoxide dismutase-inhibitable cytochrome c reduction method, and the degree of elastase release was measured using a p-nitroanilide-conjugated elastase-specific substrate. In addition, chemotaxis toward platelet activating factor/fMLP was determined with a modified Boyden chamber system. The iPLA2γ-specific inhibitor reduced the fMLP/PMA-stimulated superoxide generation by 90% and 30%, respectively; in addition, the inhibitor completely blocked the fMLP/PMA-activated elastase release. However, the cPLA2-specific inhibitor did not abrogate these effects to any degree at all concentrations. Likewise, the inhibitor for iPLA2γ, but not iPLA2β or cPLA2, completely inhibited the platelet activating factor/fMLP-induced chemotaxis. iPLA2 is involved in extracellular reactive oxygen species production, elastase release, and chemotaxis in response to well-defined stimuli. In addition, the ineffectiveness of the cPLA2 inhibitor suggests that AA may not be relevant to these cytotoxic functions.

Glycated human serum albumin induces NF-κB activation and endothelial nitric oxide synthase uncoupling in human umbilical vein endothelial cells

AimsNon-enzymatic glycated proteins could mediate diabetes vascular complications, but the molecular mechanisms are unknown. Our objective was to find new targets involved in the glycated human serum albumin (gHSA)-enhanced extracellular reactive oxygen species (ROS) production in human endothelial cells. Methods & resultsSome nuclear factors and phosphorylation cascades were analysed. gHSA activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which up-regulated NOX4 and P22PHOX and enhanced ROS production. Pharmacological inhibition of NF-κB reversed gHSA-enhanced NOX4 expression and decreased gHSA-induced ROS production in extra- and intracellular spaces. The inhibition of activator protein-1 (AP-1) induced a rise in NOX4 and P22PHOX subunit expression and a down-regulation of endothelial nitric oxide synthase (eNOS). AP-1 inhibition also enhanced extracellular ROS production in the presence of serum albumin, but not with gHSA. These results were explained by the eNOS uncoupling induced by gHSA, also demonstrated in this study. Phosphatidylinositol 3-kinase or mitogen-activated protein kinase kinase 1/2 did not show to be involved in gHSA-induced ROS production. ConclusionsAll together, the results suggested that gHSA-enhanced ROS production in endothelium is mediated by: 1) NF-κB activation and subsequence up-regulation of NADPH oxidase, 2) eNOS uncoupling. AP-1, although is not directly affected by gHSA, is another target for regulating NADPH oxidase and eNOS expression in endothelial cells.

Chronic high fat diet decreases CD34/CD133 cell population in bone marrow and peripheral circulation in association with decreased level of serum MG53

Bone marrow (BM)-derived endothelial progenitor cells (EPCs) play an important role in maintaining the function and integrity of vasculature. The number of EPCs is significantly decreased in hyperlipidemic animals and patients. The present study was to determine if chronic high fat diet (HFD) treatment could modify the the EPC population in BM and blood and the relationship to the membrane protective protein MG53 and reactive oxygen species (ROS). EPC numbers and ROS formation in BM and blood were determined with flow cytometry in male C57BL/6 mice treated with HFD for 6 months. Serum MG53 level was determined using Western blot. Mice on normal diet were used as control. Chronic HFD treatment significantly increased the blood cholesterol and glucose levels. Surprisingly, HFD treatment did not significantly change the intracellular and extracellular ROS production in BM and blood. In addition, HFD treatment significantly suppressed the serum MG53 level. HFD also significantly decreased the number of CD34/CD133 double positive EPCs in both BM and blood. Taken together, chronic HFD treatment significantly decreased the population of CD34/CD133 double positive EPCs in both BM and blood in association with decreased serum MG53 but had no effect on intracellular and extracellular ROS production in BM and blood. These results suggest a potential role of circulating MG53 for the maintenance of EPCs population and integrity of vasculature.

Nontypeable Haemophilus influenzae induces sustained lung oxidative stress and protease expression.

Nontypeable Haemophilus influenzae (NTHi) is a prevalent bacterium found in a variety of chronic respiratory diseases. The role of this bacterium in the pathogenesis of lung inflammation is not well defined. In this study we examined the effect of NTHi on two important lung inflammatory processes 1), oxidative stress and 2), protease expression. Bronchoalveolar macrophages were obtained from 121 human subjects, blood neutrophils from 15 subjects, and human-lung fibroblast and epithelial cell lines from 16 subjects. Cells were stimulated with NTHi to measure the effect on reactive oxygen species (ROS) production and extracellular trap formation. We also measured the production of the oxidant, 3-nitrotyrosine (3-NT) in the lungs of mice infected with this bacterium. NTHi induced widespread production of 3-NT in mouse lungs. This bacterium induced significantly increased ROS production in human fibroblasts, epithelial cells, macrophages and neutrophils; with the highest levels in the phagocytic cells. In human macrophages NTHi caused a sustained, extracellular production of ROS that increased over time. The production of ROS was associated with the formation of macrophage extracellular trap-like structures which co-expressed the protease metalloproteinase-12. The formation of the macrophage extracellular trap-like structures was markedly inhibited by the addition of DNase. In this study we have demonstrated that NTHi induces lung oxidative stress with macrophage extracellular trap formation and associated protease expression. DNase inhibited the formation of extracellular traps.

Analysis of ROS-Responsive Genes in Mutant RAS Expressing Hematopoietic Progenitors Identifies the Glycolytic Pathway As a Major Target Promoting Both Proliferation and Survival

Acute myeloid leukemia (AML) is a heterogeneous clonal disorder with a generally poor clinical outcome. Previously we have shown that over-production of reactive oxygen species (ROS) occurs in >60% of AML patients due to NOX oxidase activation and that this promotes growth factor independent proliferation of AML blasts (Hole et al., 2013). Normal CD34+ cells overexpressing mutant NRAS serve as a model for this since these cells also overproduce ROS through NOX activation, which in turn promotes their proliferation (Hole et al., 2010). We used this model to investigate the mechanism by which ROS promote proliferation by examining the effect of ROS on gene expression. CD34+ expressing NRASG12D showed an 8-fold (p<0.05) increase in ROS production compared to empty vector controls. DPI, a NOX inhibitor, virtually ablated ROS production (>90%; p<0.05) and also selectively inhibited proliferation of CD34+-NRAS cells (>60%; p<0.05). We next compared the gene expression profile (GEP) of NRAS and control cells ±DPI to determine the ROS-specific gene expression profile (Affymetrix Human Exon 1.0ST). NRAS changed the expression of 342 genes (>1.2 fold; p<0.05) of which 24 were specifically attributed to ROS production (Table 1). Most of these were associated with metabolic change; particularly glycolysis (p<0.0001 n=4). Consistent with this we found a doubling in the level of extracellular lactate production (indicating increased glycolysis) from NRAS cells compared to controls (n=4). Extracellular ROS (generated from GOX) also directly promoted a 1.3 fold increase in lactate production (n=4). These data suggest that ROS directly promotes glycolysis in hematopoietic cells. To examine this in AML we analysed a GEP database of 139 AML patients; this showed that those with high ROS (defined by high NOX2 oxidase expression; Hole et al., 2013) had a distinct profile of glycolytic enzyme overexpression, particularly ALDOC (r=0.4; p=2x10-25), GPI (r=0.4; p=2x10-8) and FBP1(r=0.7; p=5x10-8). These are amongst the most significant ROS-responsive genes in Table 1 and suggest that promotion of glycolysis through extracellular ROS production is also seen in AML blasts.To establish the functional significance of upregulated expression of glycolytic enzymes, we focused on the aldolase enzyme, ALDOC, since it showed the biggest induction with ROS and because overexpression of this enzyme has been recently associated with elevated glycolysis and poor prognosis in primary AML (Chen et al., 2014). We found that ALDOC was directly induced (2 fold) by physiological levels (150 nM/hour) of ROS in both normal CD34+ cells and in AML cell lines. We next examined the effect of stable ALDOC knockdown in 3 myeloid leukemia cell lines: Mv4;11 (1.5 fold reduction at the protein level), K562 (3.5 fold) and THP-1 (1.5 fold). Knockdown was associated with a reduction in proliferation in Mv4;11 and THP-1 cells (2- and 5-fold respectively; p<0.05 n=3) and also reduction in survival of THP-1 (1.7 fold; p<0.05, n=3).These data have identified ROS-responsive genes in CD34+ hematopoietic cells and show for the first time that a major target of ROS are enzymes of the glycolytic pathway. We also show evidence that ROS promotes glycolysis in both cell lines and in AML patients and that myeloid leukemia cells show dependency on ALDOC, for their growth and survival. Given the frequent overexpression of ROS in primary AML, these data provide a plausible mechanism for the enhanced glycolysis seen in AML (Chen et al., 2014) and suggest that agents restoring the redox environment could be used to correct metabolic imbalances which contribute to treatment resistance in this disease.Table 1Effect of ROS on gene expressionGeneGene expression (fold and direction of change)p-valueProtein validation (fold and direction of change where available)ProcessALDOC+4.31×10-6+2GlycolysisENO2+2.61×10-4+1.5FBP1+1.82×10-7GPI+1.52×10-6PFK-1+1.49×10-6GATM+2.83×10-5+2Metabolism SULF2+2.12×10-5CKB+2.17×10-5ASPH+1.41×10-4PTPRD+2.22×10-5+2Signal transductionKIT-2.15×10-50CD32+1.57×10-50TNS1+1.78×10-6REC8+1.46×10-5STARD8+1.43×10-5CMTM8-1.21×10-4CNR2-3.62×10-60CD34+1.74×10-50OtherCITED1+1.76×10-5+1.5CYTL1-1.96×10-6CACNB1+1.31×10-4SLC6A8+1.71×10-5JAKMIP2+1.23×10-5WDR54+1.85×10-6 DisclosuresNo relevant conflicts of interest to declare.

Reactive Oxygen Species Produced By Myeloid Cells Block CD20-Antibody Dependent NK Cell-Mediated Cytotoxicity Against Chronic Lymphocytic Leukemia Cells

Introduction:The addition of the anti-CD20 antibodies rituximab or ofatumumab to chemotherapy has improved the outcome for patients with chronic lymphocytic leukemia (CLL), but the efficacy of anti-CD20 monoclonal antibodies (mAbs) as single agents is limited. Immunosuppressive reactive oxygen species (ROS), produced by myeloid cells, including monocytes, may be a limiting factor for the efficacy of ADCC-dependent therapy. With the aim of reducing a potentially inhibitory role of ROS on mAb-dependent NK cell cytotoxicity against CLL cells, we performed in vitro cytotoxicity experiments in the presence of monocytes and the ROS formation inhibitor histamine dihydrochloride (HDC).Methods:After informed consent, blood samples were collected from Binet stage A CLL patients seen at Sahlgrenska University Hospital, Gothenburg, Sweden. NK cells, monocytes and CLL cells were isolated from PBMCs by immunomagnetic negative selection or flow cytometry. ROS production by monocytes was assessed by isoluminol-enhanced chemiluminescense upon stimulation with the chemotactic peptide fMLF in the presence or absence of CLL cells, rituximab (Roche), ofatumumab (GSK), HDC or the NADPH oxidase inhibitor DPI. NK cell death was assessed by flow cytometry after over-night culture of monocytes and lymphocytes. Autologous ADCC assays were performed with NK cells, monocytes and CFSE-labeled CLL cells in the presence or absence of rituximab, ofatumumab, HDC, catalase and interleukin-2 (IL-2). CLL cell death was determined by flow cytometry after live/dead staining. The study was approved by the local ethical review board of Gothenburg, and conducted in accordance with the declaration of Helsinki.Results:Rituximab and ofatumumab both triggered extracellular ROS production by monocytes. ROS production was blocked by the NADPH oxidase inhibitor DPI. Furthermore, monocytes induced ROS-dependent apoptosis in adjacent NK cells. In the cytotoxicity experiments, rituximab-mediated NK cell dependent cytotoxicity against CLL cells was significantly inhibited by the presence of autologous monocytes. This suppression, as well as the induction of NK cell apoptosis, were dependent on ROS, since both HDC and catalase significantly increased cytotoxicity and rescued NK cells from monocyte-induced apoptosis. Expectedly, IL-2 augmented NK cell-mediated ADCC.Conclusion:The results imply that CLL patients harbor immunosuppressive myeloid cells that reduce CD20-antibody dependent NK cell-mediated cytotoxicity against leukemic cells by producing ROS. We propose that treatment with HDC, preferably in conjunction with NK cell stimulatory cytokines, may improve anti-CD20-based immunotherapy in CLL. DisclosuresNo relevant conflicts of interest to declare.

Hydrogen peroxide inhibits proliferation and endothelial differentiation of bone marrow stem cells partially via reactive oxygen species generation

AimsThe present study was to investigate the effect of hydrogen peroxide (H2O2) on bone marrow stem cells and their endothelial differentiation and the underlying mechanisms in vitro. Main methodsRat bone marrow multipotent adult progenitor cells (MAPCs) were used as the source of bone marrow stem cells, and treated with H2O2 (with the final concentration from 0 to 50μM) with or without N-acetylcysteine (NAC, 0.1mM). Reactive oxygen species (ROS) was measured by electron paramagnetic resonance (EPR) and fluorescent microscope. Flow cytometry and immunoblotting were used to determine apoptosis and differentiation of MAPCs. Key findingsH2O2 generated a significant amount of intracellular and extracellular ROS in the culture system, substantially inhibited the proliferation of MAPCs and Oct-4 expression, and induced their apoptosis in a dose-dependent manner. Exposure to H2O2 also significantly attenuated the endothelial differentiation of MAPCs with reduced expression of endothelial markers CD31 and FLK-1 as well as impaired in vitro vascular structure formation. Both intracellular and extracellular ROS production from H2O2 were blocked by NAC. NAC treatment effectively prevented H2O2-induced reduction of Oct-4 expression in the cells. However, NAC treatment only partially prevented H2O2-induced apoptosis, and inhibition of cell proliferation and endothelial differentiation of MAPCs. SignificanceH2O2 exposure suppressed Oct-4 expression in MAPCs through ROS-dependent mechanism, while increasing the apoptosis of MAPCs and inhibiting their proliferation and endothelial differentiation with a mechanism partially due to ROS generation in vitro.

Reprogramming to a pluripotent state modifies mesenchymal stem cell resistance to oxidative stress

Properties of induced pluripotent stem cells (iPSC) have been extensively studied since their first derivation in 2006. However, the modification in reactive oxygen species (ROS) production and detoxification caused by reprogramming still needs to be further elucidated. The objective of this study was to compare the response of iPSC generated from menstrual blood–derived mesenchymal stem cells (mb-iPSC), embryonic stem cells (H9) and adult menstrual blood–derived mesenchymal stem cells (mbMSC) to ROS exposure and investigate the effects of reprogramming on cellular oxidative stress (OS). mbMSC were extremely resistant to ROS exposure, however, mb-iPSC were 10-fold less resistant to H2O2, which was very similar to embryonic stem cell sensitivity. Extracellular production of ROS was also similar in mb-iPSC and H9 and almost threefold lower than in mbMSC. Furthermore, intracellular amounts of ROS were higher in mb-iPSC and H9 when compared with mbMSC. As the ability to metabolize ROS is related to antioxidant enzymes, we analysed enzyme activities in these cell types. Catalase and superoxide dismutase activities were reduced in mb-iPSC and H9 when compared with mbMSC. Finally, cell adhesion under OS conditions was impaired in mb-iPSC when compared with mbMSC, albeit similar to H9. Thus, reprogramming leads to profound modifications in extracellular ROS production accompanied by loss of the ability to handle OS.

A re-elicitation assay to correlate flg22-signaling competency with ligand-induced endocytic degradation of the FLS2 receptor.

In the model plant Arabidopsis, the best studied Pattern-triggered immunity (PTI) system is perception of the bacterial pathogen-associated molecular pattern (PAMP) flagellin, or its active peptide-derivative flg22, by the plasma membrane-localized receptor FLAGELLIN SENSING 2 (FLS2). Flg22 perception initiates an array of immune responses including the fast and transient production of reactive oxygen species (ROS). In addition, FLS2 undergoes ligand-induced endocytosis and subsequent degradation within 60 min of flg22-treatment. Luminol-based assays are routinely used to measure extracellular ROS production within minutes after flg22 treatment. Many mutants in flg22-response pathways display defects in flg22-induced ROS production. Here, we describe a luminol-based ROS Re-elicitation Assay that can be utilized to quantitatively assess flg22-signaling competency of FLS2 at times during which FLS2 is internalized, trafficked through endosomal compartments, and degraded in response to flg22. This assay may also be employed to correlate FLS2 signaling competency with receptor accumulation in vesicular trafficking mutants that either affect FLS2 endocytosis or replenishment of FLS2 through the secretory pathway. In addition, this assay can be extended to studies of other PAMP (ligand)-receptor pairs.

Overproduction of NOX-derived ROS in AML promotes proliferation and is associated with defective oxidative stress signaling

AbstractExcessive production of reactive oxygen species (ROS) is frequently observed in cancer and is known to strongly influence hematopoietic cell function. Here we report that extracellular ROS production is strongly elevated (mean >10-fold) in >60% of acute myeloid leukemia (AML) patients and that this increase is attributable to constitutive activation of nicotinamide adenine dinucleotide phosphate oxidases (NOX). In contrast, overproduction of mitochondrial ROS was rarely observed. Elevated ROS was found to be associated with lowered glutathione levels and depletion of antioxidant defense proteins. We also show for the first time that the levels of ROS generated were able to strongly promote the proliferation of AML cell lines, primary AML blasts, and, to a lesser extent, normal CD34+ cells, and that the response to ROS is limited by the activation of the oxidative stress pathway mediated though p38MAPK. Consistent with this, we observed that p38MAPK responses were attenuated in patients expressing high levels of ROS. These data show that overproduction of NOX-derived ROS can promote the proliferation of AML blasts and that they also develop mechanisms to suppress the stress signaling that would normally limit this response. Together these adaptations would be predicted to confer a competitive advantage to the leukemic clone.

Glucosinolate Degradation Products, Isothiocyanates, Nitriles, and Thiocyanates, Induce Stomatal Closure Accompanied by Peroxidase-Mediated Reactive Oxygen Species Production inArabidopsis thaliana

Isothiocyanates, nitriles, and thiocyanates are degradation products of glucosinolates in crucifer plants. In this study, we investigated the stomatal response to allyl isothiocyanate (AITC), 3-butenenitrile (3BN), and ethyl thiocyanate (ESCN) in Arabidopsis. AITC, 3BN, and ESCN induced stomatal closure in the wild type and the atrbohD atrbohF mutant. Stomatal closure was inhibited by catalase and salicylhydroxamic acid (SHAM). The degradation products induced extracellular reactive oxygen species (ROS) production in the rosette leaves, and intracellular ROS accumulation, NO production, and cytosolic free calcium concentration ([Ca(2+)]cyt) oscillations in guard cells, which were inhibited by SHAM. These results suggest that glucosinolate degradation products induce stomatal closure accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca(2+)]cyt oscillation in Arabidopsis.

Antioxidant activity of Bios-p peptide analogue in HEK293T cells and three-dimensional structure prediction

Studies had indicate that excessive production of reactive oxygen species (ROS) affect cellular signaling pathways, which is associated with pathological and physiological conditions such as cancer, diabetes and neurodegenerative diseases In this context, our laboratory has obtained the Bios-p, a ROS modulator, peptide analogue by sequencing from the seed of Bauhinia bauhinoides, which represents the active 12-amino acid, obtained from the inhibitor BbKI protease and we predicted the three-dimensional structure of Bios-p analogue peptide using homology modeling, being patented by the working group of Dr. Maria Luiza Vilela Oliva of UNIFESP, Brazil (a member of our cluster). The protective effect on the viability and antioxidant capacity of Bios-p was studied in HEK 293T cells under oxidative stress induced by hydrogen peroxide (H2O2) using SYTOXGREEN/DHE and luminescence assay. The three-dimensional structure of Bios-p peptide analogue was predicted by homology-based modeling using Modeller9v8. The pretreatment with different concentrations of Bios-p (1 μM - 10 μM) showed an increase of 53.83% ± 3.86% the cellular viability in under oxidative stress compared to control. Furthermore, the results to indicate that HEK293T cells by incubating for 24 h with Bios-p shown a significant decreased of basal extracellular ROS on total cell population in 89.67% ± 0.76%, compared to control in the absence of the analogue. Similarly it is observed that Bios-p has a significant antioxidant effect on extracellular ROS production when cells are subjected to oxidative stress induced by 200 μM H2O2 in 64.37% ± 4.63%, compared to control in absence of H2O2 and Bios-p. These results suggest that Bios-p has potential as antioxidant agent in cells HEK293T under H2O2-induced oxidative stress and that can protect the cells viability as concentration-dependent, and we propose a new biotechnological tool for modulate the ROS production.

MUNC13-4 Protein Regulates the Oxidative Response and Is Essential for Phagosomal Maturation and Bacterial Killing in Neutrophils

Neutrophils use diverse mechanisms to kill pathogens including phagocytosis, exocytosis, generation of reactive oxygen species (ROS), and neutrophil extracellular traps. These mechanisms rely on their ability to mobilize intracellular organelles and to deliver granular cargoes to specific cellular compartments or into the extracellular milieu, but the molecular mechanisms regulating vesicular trafficking in neutrophils are not well understood. MUNC13-4 is a RAB27A effector that coordinates exocytosis in hematopoietic cells, and its deficiency is associated with the human immunodeficiency familial hemophagocytic lymphohistiocytosis type 3. In this work, we have established an essential role for MUNC13-4 in selective vesicular trafficking, phagosomal maturation, and intracellular bacterial killing in neutrophils. Using neutrophils from munc13-4 knock-out (KO) mice, we show that MUNC13-4 is necessary for the regulation of p22(phox)-expressing granule trafficking to the plasma membrane and regulates extracellular ROS production. MUNC13-4 was also essential for the regulation of intracellular ROS production induced by Pseudomonas aeruginosa despite normal trafficking of p22(phox)-expressing vesicles toward the phagosome. Importantly, in the absence of MUNC13-4, phagosomal maturation was impaired as observed by the defective delivery of azurophilic granules and multivesicular bodies to the phagosome. Significantly, this mechanism was intact in RAB27A KO neutrophils. Intracellular bacterial killing was markedly impaired in MUNC13-4 KO neutrophils. MUNC13-4-deficient cells showed a significant increase in neutrophil extracellular trap formation but were unable to compensate for the impaired bacterial killing. Altogether, these findings characterize novel functions of MUNC13-4 in the innate immune response of the neutrophil and have direct implications for the understanding of immunodeficiencies in patients with MUNC13-4 deficiency.

Two guard cell‐preferential MAPKs, MPK9 and MPK12, regulate YEL signalling in Arabidopsis guard cells

We report that two mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate abscisic acid (ABA)-induced stomatal closure in Arabidopsis thaliana. Yeast elicitor (YEL) induced stomatal closure accompanied by intracellular reactive oxygen species (ROS) accumulation and cytosolic free calcium concentration ([Ca(2+) ]cyt ) oscillation. In this study, we examined whether these two MAP kinases are involved in YEL-induced stomatal closure using MAPKK inhibitors, PD98059 and U0126, and MAPK mutants, mpk9, mpk12 and mpk9 mpk12. Both PD98059 and U0126 inhibited YEL-induced stomatal closure. YEL induced stomatal closure in the mpk9 and mpk12 mutants but not in the mpk9 mpk12 mutant, suggesting that a MAPK cascade involving MPK9 and MPK12 functions in guard cell YEL signalling. However, YEL induced extracellular ROS production, intracellular ROS accumulation and cytosolic alkalisation in the mpk9, mpk12 and mpk9 mpk12 mutants. YEL induced [Ca(2+) ]cyt oscillations in both wild type and mpk9 mpk12 mutant. These results suggest that MPK9 and MPK12 function redundantly downstream of extracellular ROS production, intracellular ROS accumulation, cytosolic alkalisation and [Ca(2+) ]cyt oscillation in YEL-induced stomatal closure in Arabidopsis guard cells and are shared with ABA signalling.

Brassica carinata CIL1 mediates extracellular ROS production during auxin- and ABA-regulated lateral root development

In the plant cytoplasm signals from multiple sources interact to control development. Lateral root proliferation is controlled by the antagonistic action of the hormones auxin and abscisic acid (ABA). Auxin stimulates the separation of pericycle initials and cell expansion. ABA is required for lateral root initiation and elongation and acts as an antagonist through auxin-dependent pathways. These hormones also mediate reactive oxygen species (ROS) accumulation in growing roots and promote cell expansion. We describe the isolation and characterization of a copper- and auxin-induced gene, COPPER INDUCED in LEAVES (CIL1), from Brassica carinata. Transgenic B. carinata seedlings expressing antisense CIL1 were used to determine the biological function of this gene. Lines with reduced CIL1 expression showed a decrease in lateral root development, as well as reduced sensitivity to auxin and ABA. Steady-state analysis of redox components showed a decrease in NADPH oxidase, superoxide dismutase, and catalase activity, accompanied by an increase in hydrogen peroxide concentration. GFP-tagged CIL1 accumulated at the plasma membrane and in the apoplast indicating CIL1 is likely extracellular. From these data we propose that CIL1 is an extracellular protein involved in ROS cycling mediating auxin and ABA signals.

Abstract 2060: NADPH oxidase NOX5 regulates the expression of the cell cycle inhibitor p27kip1 in the human melanoma UACC-257 cancer cells

Abstract NADPH oxidase (NOX) family enzymes (NOX1-5 and DUOX1-2) generate reactive oxygen species (ROS) and modulate cellular pathways that regulate proliferation. While high doses of ROS are clearly toxic, the effects of low doses of NOX-mediated ROS as signaling molecules are less clear. Recent work has proposed that NOX5 plays a role in cell proliferation in Hairy cell leukemia, Barrett's esophageal adenocarcinoma and in prostate cancer cells, yet relatively little is known of the signaling pathways mediated by NOX5. In this study we explored the functional role of NOX5 in cancer cells obtained from the NCI-60 cell line panel. Initial screening of the NCI-60 cancer cell line panel for expression of the Nox family members by real time PCR revealed several melanoma cell lines that express Nox5. Of the NOX5 expressing NCI-60 human cancer cell lines, subsequent studies were carried out in human UACC-257 melanoma cancer cells. Two molecular approaches were undertaken to determine the functional role of NOX5 in UACC-257 cells: 1) generating NOX5 over-expressing stable clones and 2) transiently silencing endogenous NOX5 and the stably over-expressed NOX5. Redox-sensitive assays for both intra- and extracellular ROS production confirmed that NOX5 is functional and generates superoxide in both UACC-257 cells and stable Nox5-over-expressing clones. NOX5 can be activated both by the protein kinase C activating phorbol ester PMA and by the Ca2+ ionophore ionomycin. Furthermore, this enzymatic activity is calcium- and flavin dehydrogenase-dependent; both the calcium chelator BAPTA-AM and the flavoprotein inhibitor DPI (200nM) significantly decreased NOX5-mediated ROS production. Silencing NOX5 over-expression significantly inhibited both endogenous and PMA-stimulated ROS production, suggesting that the ROS production is NOX5-mediated. Furthermore, knockdown of both endogenous and over-expressed NOX5 in UACC-257 cells resulted in increased p27Kip1 expression. Conversely, over-expression of NOX5 in UACC-257 cells resulted in decreased p27Kip1 expression both at the mRNA and protein levels. As the AKT signaling pathways are known to modulate p27Kip1 levels, we observed that knockdown of both endogenous and over-expressed NOX5 in UACC-257 cells resulted in decreased Akt and GSK-3β phosphorylation. Studies to characterize additional mechanisms of translational and transcriptional regulation of p27Kip1 by NOX5 are ongoing. In summary, our findings suggest that NOX5 expression could contribute to cell proliferation in human cancers in part due to the generation of high local concentrations of extracellular ROS that regulate the Akt/GSK-3β/p27Kip1 signaling network. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2060. doi:1538-7445.AM2012-2060

Methylglyoxal-induced stomatal closure accompanied by peroxidase-mediated ROS production in Arabidopsis

Methylglyoxal (MG) is an oxygenated short aldehyde and a glycolytic intermediate that accumulates in plants under environmental stresses. Being a reactive α-oxoaldehyde, MG may act as a signaling molecule in plants during stresses. We investigated whether MG induces stomatal closure, reactive oxygen species (ROS) production, and cytosolic free calcium concentration ([Ca2+]cyt) to clarify roles of MG in Arabidopsis guard cells. MG induced production of ROS and [Ca2+]cyt oscillations, leading to stomatal closure. The MG-induced stomatal closure and ROS production were completely inhibited by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), but were not affected by an NAD(P)H oxidase mutation, atrbohD atrbohF. Furthermore, the MG-elicited [Ca2+]cyt oscillations were significantly suppressed by SHAM but not by the atrbohD atrbohF mutation. Neither endogenous abscisic acid nor endogenous methyl jasmonate was involved in MG-induced stomatal closure. These results suggest that intrinsic metabolite MG can induce stomatal closure in Arabidopsis accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca2+]cyt oscillations.

ROS-talk – how the apoplast, the chloroplast, and the nucleus get the message through

The production of reactive oxygen species (ROS) in different plant subcellular compartments is the hallmark of the response to many stress stimuli and developmental cues. The past two decades have seen a transition from regarding ROS as exclusively cytotoxic agents to being considered as reactive compounds which participate in elaborate signaling networks connecting various aspects of plant life. We have now arrived at a stage where it has become increasingly difficult to disregard the communication between different types and pools of ROS. Production of ROS in the extracellular space, the apoplast, can influence their generation in the chloroplast and both can regulate nuclear gene expression. In spite of existing information on these signaling events, we can still barely grasp the mechanisms of ROS signaling and communication between the organelles. In this review, we summarize evidence that supports the mutual influence of extracellular and chloroplastic ROS production on nuclear gene regulation and how this interaction might occur. We also reflect on how, and via which routes signals might reach the nucleus where they are ultimately integrated for transcriptional reprogramming. New ideas and approaches will be needed in the future to address the pressing questions of how ROS as signaling molecules can participate in the coordination of stress adaptation and development and how they are involved in the chatter of the organelles.