Local Reactive Oxygen Species Production Research Articles

The antagonistic roles of PDGF and integrin αvβ3 in regulating ROS production at focal adhesions

Reactive oxygen species (ROS) have been shown to play crucial roles in regulating various cellular functions, e.g. focal adhesion (FA) dynamics and cell migration upon growth factor stimulation. However, it is not clear how ROS are regulated at subcellular FA sites to impact cell migration. We have developed a biosensor capable of monitoring ROS production at FA sites in live cells with high sensitivity and specificity, utilizing fluorescence resonance energy transfer (FRET). The results revealed that platelet derived growth factor (PDGF) can induce ROS production at FA sites, which is mediated by Rac1 activation. In contrast, integrins, specifically integrin αvβ3, inhibits this local ROS production. The RhoA activity can mediate this inhibitory role of integrins in regulating ROS production. Therefore, PDGF and integrin αvβ3 coordinate to have an antagonistic effect in the ROS production at FA sites to regulate cell adhesion and migration.

Rate-Dependent X-Ros Signaling in Cardiac Ventricular Myocytes

X-ROS is the novel signaling pathway seen in ventricular myocytes1 and skeletal muscle2 that arises when cell stretch triggers local ROS (reactive oxygen species) production by NADPH oxidase 2. A sustained (10-20s) stretch produces a transient rise in ROS with a half-time of decay of ∼5 seconds. In cardiomyocytes, this ROS sensitizes local Ca2+ release channels (ryanodine receptors) in the sarcoplasmic reticulum. In normal heart cells, this produces a transient burst of Ca2+ sparks and ensures the fidelity of excitation-contraction coupling, but can trigger arrhythmogenic Ca2+ waves in diverse pathologies.In reports to date, X-ROS has been characterized in cells that were stretched and held at a constant length. However, this is not how a cardiomyocyte is stretched in situ - instead it is stretched (during diastole) and shortens (during systole) in rhythmic fashion. Additionally, this stretch is regulated by two factors that vary with changing physiologic demand: 1) preload (the amount of blood that fills the ventricles during diastole) grades the magnitude of cell stretch, and 2) heart rate varies the frequency of cell stretch. Thus we investigated how the magnitude and frequency of stretch affect X-ROS signaling. Briefly, we find a critical effect: rhythmic stretch elevates the steady state level of ROS production in the cell, and this level is graded by both the magnitude and frequency of stretch. In turn, the elevated ROS proportionately modulates Ca2+ spark rate. Thus our findings hold the critical implication that the redox state and Ca2+ signaling sensitivity of cardiomyocytes is coupled to mechanical changes and graded by both preload and heart rate.1. Prosser et al. “X-ROS signaling: Rapid mechano-chemo transduction in heart.” Science. 333, 1440 (2011 9 Sept).2. Khairallah et al. “Microtubules underlie dysfunction in Duchenne muscular dystrophy.” Sci. Signal. 5, ra56 (2012).

Neutrophil-derived reactive oxygen species in SSc

Reactive oxygen species (ROS) are implicated in the pathogenesis of SSc. Neutrophils constitute a major source of ROS during inflammation. Here, we examined endogenous and stimulated ex vivo ROS production of SSc neutrophils compared with control neutrophils with and without prior priming with TNF-α. ROS generation was measured using luminol-enhanced chemiluminescence. Neutrophils isolated from SSc patients and healthy controls were unprimed or were primed with TNF-α. ROS production was stimulated in vitro with phorbol 12-myristate 13-acetate (PMA) and formyl-met-leu-phe (fMLP). To examine the effects of serum mediators on ROS generation, control neutrophils were also stimulated with SSc or control serum. Neutrophil stimulation with PMA and fMLP resulted in a greater increase in ROS generation in SSc neutrophils compared with controls. However, unstimulated SSc neutrophils generated lower levels of ROS than controls. SSc neutrophils demonstrated an increased response to fMLP in the absence of in vitro TNF-α priming indicating priming of SSc neutrophils in vivo. SSc serum did not stimulate neutrophil ROS generation in vitro. SSc neutrophils are primed for ROS generation. Neutrophils binding to activated endothelium in SSc, may induce local production of ROS, perpetuating endothelial dysfunction and mediating fibrosis.

Enteric Commensal Bacteria Induce Extracellular Signal-regulated Kinase Pathway Signaling via Formyl Peptide Receptor-dependent Redox Modulation of Dual Specific Phosphatase 3

The normal microbial occupants of the mammalian intestine are crucial for maintaining gut homeostasis, yet the mechanisms by which intestinal cells perceive and respond to the microbiota are largely unknown. Intestinal epithelial contact with commensal bacteria and/or their products has been shown to activate noninflammatory signaling pathways, such as extracellular signal-related kinase (ERK), thus influencing homeostatic processes. We previously demonstrated that commensal bacteria stimulate ERK pathway activity via interaction with formyl peptide receptors (FPRs). In the current study, we expand on these findings and show that commensal bacteria initiate ERK signaling through rapid FPR-dependent reactive oxygen species (ROS) generation and subsequent modulation of MAP kinase phosphatase redox status. ROS generation induced by the commensal bacteria Lactobacillus rhamnosus GG and the FPR peptide ligand, N-formyl-Met-Leu-Phe, was abolished in the presence of selective inhibitors for G protein-coupled signaling and FPR ligand interaction. In addition, pretreatment of cells with inhibitors of ROS generation attenuated commensal bacteria-induced ERK signaling, indicating that ROS generation is required for ERK pathway activation. Bacterial colonization also led to oxidative inactivation of the redox-sensitive and ERK-specific phosphatase, DUSP3/VHR, and consequent stimulation of ERK pathway signaling. Together, these data demonstrate that commensal bacteria and their products activate ROS signaling in an FPR-dependent manner and define a mechanism by which cellular ROS influences the ERK pathway through a redox-sensitive regulatory circuit.

Homocysteine-Induced Apoptosis in Endothelial Cells Coincides With Nuclear NOX2 and Peri-nuclear NOX4 Activity

Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01–2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47phox expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47phox, and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47phox in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47phox was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.

Angiotensin II Inhibits Neuronal Nitric Oxide Synthase Activation Through the ERK1/2-RSK Signaling Pathway to Modulate Central Control of Blood Pressure

Angiotensin (Ang) II exerts diverse physiological actions in both the peripheral and central neural systems. It was reported that the activity of Ang II is higher in the nucleus tractus solitarii (NTS) of spontaneously hypertensive rats (SHRs) and that angiotensin type-1 receptors are colocalized with NAD(P)H oxidase in the neurons of the NTS, resulting in the induction of local reactive oxygen species production by Ang II. However, the signaling mechanisms of Ang II that induce hypertension remain unclear. The aim of this study was to investigate the possible signaling pathways involved in Ang II-mediated blood pressure regulation in the NTS. Male SHRs were treated with losartan or tempol for 2 weeks, after which systolic blood pressure was observed to decrease significantly. Dihydroethidium staining showed many cells with high reactive oxygen species in the NTS of SHRs. The addition of losartan or tempol decreased the numbers of reactive oxygen species-positive cells in the NTS. The systemic administration of losartan or tempol reduced the systolic blood pressure and increased NO production. Immunoblotting and immunohistochemical analysis further showed that inhibition of Ang II activity by losartan or tempol significantly increased the expression extracellular signal-regulated kinase (ERK)1/2, ribosomal protein S6 kinase (RSK), and also increased neuronal NO synthase (nNOS) phosphorylation. RSK was also found to bind directly to nNOS and induce phosphorylation at the Ser1416 position. Taken together, these results suggest that the ERK1/2-RSK-nNOS signaling pathway may play a significant role in Ang II-mediated central blood pressure regulation.

Compartmentalization of Redox Signaling Through NADPH Oxidase–Derived ROS

Reactive oxygen species (ROS) are generated in response to growth factors, cytokines, G protein-coupled receptor agonists, or shear stress, and function as signaling molecules in nonphagocytes. However, it is poorly understood how freely diffusible ROS can activate specific signaling, so-called "redox signaling." NADPH oxidases are a major source of ROS and now recognized to have specific subcellular localizations, and this targeting to specific compartments is required for localized ROS production. One important mechanism may involve the interaction of oxidase subunits with various targeting proteins localized in lamellipodial leading edge and focal adhesions/complexes. ROS are believed to inactivate protein tyrosine phosphatases, thereby establishing a positive-feedback system that promotes activation of specific redox signaling pathways involved in various functions. Additionally, ROS production may be localized through interactions of NADPH oxidase with signaling platforms associated with caveolae/lipid rafts, endosomes, and nucleus. These indicate that the specificity of ROS-mediated signal transduction may be modulated by the localization of Nox isoforms and their regulatory subunits within specific subcellular compartments. This review summarizes the recent progress on compartmentalization of redox signaling via activation of NADPH oxidase, which is implicated in cell biology and pathophysiologies.

Parallels of snipe hunting and ROS research: the challenges of studying ROS and redox signalling in response to exercise

Parallels of snipe hunting and ROS research: the challenges of studying ROS and redox signalling in response to exercise

Regulation of Apical Dominance inAspergillus nidulansHyphae by Reactive Oxygen Species

In fungal hyphae, apical dominance refers to the suppression of secondary polarity axes in the general vicinity of a growing hyphal tip. The mechanisms underlying apical dominance remain largely undefined, although calcium signaling may play a role. Here, we describe the localized accumulation of reactive oxygen species (ROS) in the apical region of Aspergillus nidulans hyphae. Our analysis of atmA (ATM) and prpA (PARP) mutants reveals a correlation between localized production of ROS and enforcement of apical dominance. We also provide evidence that NADPH oxidase (Nox) or related flavoproteins are responsible for the generation of ROS at hyphal tips and characterize the roles of the potential Nox regulators NoxR, Rac1, and Cdc42 in this process. Notably, our genetic analyses suggest that Rac1 activates Nox, whereas NoxR and Cdc42 may function together in a parallel pathway that regulates Nox localization. Moreover, the latter pathway may also include Bem1, which we propose represents a p40phox analog in fungi. Collectively, our results support a model whereby localized Nox activity generates a pool of ROS that defines a dominant polarity axis at hyphal tips.

A tip-high, Ca(2+) -interdependent, reactive oxygen species gradient is associated with polarized growth in Fucus serratus zygotes.

We report the existence of a tip-high reactive oxygen species (ROS) gradient in growing Fucus serratus zygotes, using both 5-(and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein and nitroblue tetrazolium staining to report ROS generation. Suppression of the ROS gradient inhibits polarized zygotic growth; conversely, exogenous ROS generation can redirect zygotic polarization following inhibition of endogenous ROS. Confocal imaging of fluo-4 dextran distributions suggests that the ROS gradient is interdependent on the tip-high [Ca(2+)](cyt) gradient which is known to be associated with polarized growth. Our data support a model in which localized production of ROS at the rhizoid tip stimulates formation of a localized tip-high [Ca(2+)](cyt) gradient. Such modulation of intracellular [Ca(2+)](cyt) signals by ROS is a common motif in many plant and algal systems and this study extends this mechanism to embryogenesis.

Localizing NADPH Oxidase–Derived ROS

Reactive oxygen species (ROS) function as signaling molecules to mediate various biological responses, including cell migration, growth, and gene expression. ROS are diffusible and short-lived molecules. Thus, localizing the ROS signal at the specific subcellular compartment is essential for activating redox signaling events after receptor activation. NADPH (nicotinamide adenine dinucleotide phosphate) oxidase is one of the major sources of ROS in vasculature; it consists of a catalytic subunit (Nox1, Nox2, Nox3, Nox4, or Nox5), p22phox, p47phox, p67phox, and the small guanosine triphosphatase Rac1. Targeting of NADPH oxidase to focal complexes in lamellipodia and membrane ruffles through the interaction of p47phox with the scaffold proteins TRAF4 and WAVE1 provides a mechanism for achieving localized ROS production, which is required for directed cell migration. ROS are believed to inactivate protein tyrosine phosphatases, which concentrate in specific subcellular compartments, thereby establishing a positive feedback system that activates redox signaling pathways to promote cell movement. Additionally, ROS production may be localized through interactions of NADPH oxidase with signaling platforms associated with lipid rafts and caveolae, as well as with endosomes. There is also evidence that NADPH oxidase is found in the nucleus, indicating its involvement in redox-responsive gene expression. This review focuses on targeting of NADPH oxidase to discrete subcellular compartments as a mechanism of localizing ROS and activation of downstream redox signaling events that mediate various cell functions.

Localizing the ROS Signal

Reactive oxygen species (ROS) are short-lived molecules. Thus, localizing the production of these molecules is essential if they are to participate in cell signaling events. Two groups report mechanisms for localized production of ROS by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in migrating endothelial cells (Wu et al. ) and in the growing tips of plant root hair cells (Carol et al. ). Wu et al. report that in endothelial cells, recruitment of the adaptor TRAF4, which binds the p47 phox subunit of NADPH oxidase, to focal adhesion sites through interaction with Hic-5, a focal adhesion site scaffold, allowed the local activation of NADPH oxidase, which biased the focal adhesion site environment toward tyrosine phosphorylation through the oxidative inactivation of the tyrosine phosphatase PTP-PEST, and the activation of redox-sensitive kinases such as Pyk2. Forcing TRAF4 to lipid rafts by addition of a myristoylation site also promoted activation of RhoA guanosine triphosphatase (GTPase), as well as the GTPases Cdc42 and Rac1, which increased the activity of the kinase PAK1, leading to phosphorylation and activation of p47 phox , which in turn inactivated PTP-PEST by oxidation. Cells expressing the myristoylated TRAF4 also exhibited extensive membrane ruffling, which was blocked in cells also expressing catalytically inactive versions of the tyrosine kinases Pyk2 or Src. Endothelial cell migration was blocked if the abundance of either TRAF4 or Hic-5 was decreased by RNAi or if ROS accumulation was prevented by the addition of a superoxide dismutase mimetic. Thus, TRAF4 provides a mechanism for localized ROS generation, which contributes to a permissive environment for formation of transient peripheral focal adhesion sites. Trichoblasts of Arabidopsis roots form a single root hair by a process involving polarized growth and localized ROS production. Carol et al. identified a Rho GTPase guanine nucleotide dissociation inhibitor (GDI) encoded by the supercentipede ( SCN1 ) gene that, when inactivated, caused multiple root hairs to form. In wild-type roots, ROS are locally produced by RHD2, the Arabidopsis NADPH oxidase. In the scn1 mutants, ROS was produced at the ectopic root hair cell sites and was more broadly distributed. The rhd2 scn1 double-mutant plants exhibited single root hair growth sites that failed to elongate properly. The Rho GTPase ROP2 was also mislocalized in the scn1 mutant root hair cells to the ectopic hair formation sites. These two articles show that localized Rho GTPase activity combined with localized ROS production contribute to polarized growth and movement in plants and animals. R. F. Wu, Y. C. Xu, Z. Ma, F. E. Nwariaku, G. A. Sarosi Jr., L. S. Terada, Subcellular targeting of oxidants during endothelial cell migration. J. Cell Biol. 171 , 893-904 (2005). [Abstract] [Full Text] R. J. Carol, S. Takeda, P. Linstead, M. C. Durrant, H. Kakesova, P. Derbyshire, S. Drea, V. Zarsky, L. Dolan, A RhoGDP dissociation inhibitor spatially regulates growth in root hair cells. Nature 438 , 1013-1016 (2005). [PubMed]

Signals from reactive oxygen species

Reactive oxygen species (ROS) can arise from normal metabolic activity such as organelle-based electron transport or be intermediates in signal transduction pathways activated by plant respiratory burst oxidase homologue (Rboh). UV-B exposure induces a pathogenesis-like response in leaves that can be abrogated by ROS scavengers [1]. A local signal is propagated by hydrogen peroxide and is sensitive to the application of catalase [2]. Similarly, local ROS production initiated by elicitors or pathogens can arise from stimulation of superoxide producing Rboh activity [3,4]. In this case as well, propagation of a ROS signal to adjoining cells is sensitive to catalase. Antisense of Rboh homologues in tomato lead to reduced ROS production [5]. The tomato plants show compromised wound-dependent responses. In addition, the antisense plants have a highly branched phenotype and fasciated-like reproductive organs. Transcriptome analysis of these plants revealed ectopic expression of homeotic MADS box genes that are normally expressed only in the reproductive organs. In addition, various applications of hormones were found to regulate Rboh levels. Thus, regulated ROS bursts and the general effect of Rboh activity on the steady state cellular redox milieu control short term physiological reactions and plant development. Divergent stress including temperature, drought and UV-B exposure yield overlapping transcriptome response profiles whose origin can be traced to the use of reactive oxygen signaling intermediates. Cellular scavenging systems and local production of NO are likely to temper these signalling properties by interacting with ROS and thus help to contribute to the specificity of particular responses.

Effects of thiol antioxidant on Reduced Nicotinamide Adenine Dinucleotide Phosphate oxidase in hypertensive Dahl salt-sensitive rats

Recent studies implicate of reactive oxygen species (ROS) in hypertension; however, whether reactive oxygen species promote hypertensive derangements is not fully clear. We thus investigated the effects of an antioxidant, n -acetyl- l-cysteine, on hypertensive Dahl salt-sensitive rats. High-salt intake for 4 weeks markedly elevated systolic arterial pressure, urinary excretion of protein, 8-isoprostane, and H 2O 2, and the enzyme activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase along with the elevated expression of its subunits gp91phox and p47phox at the levels of mRNA and protein. Supplement with n -acetyl- l-cysteine reduced the increase in systolic arterial pressure and counteracted the elevation of urinary excretion of protein, 8-isoprostane, and H 2O 2, and the increases in NADPH oxidase activity/expression in high-salt-loaded Dahl salt-sensitive rats. n -acetyl- l-cysteine supplement ameliorated plasma and urinary levels of thromboxane B 2 (an end metabolite of thromboxane A 2), associated with improvement of both the abnormal contraction and the impaired nitric oxide-dependent relaxation in renal arteries. These results revealed that oxidative stress mediates hypertensive changes in Dahl salt-sensitive rats, because thiol antioxidant n -acetyl- l-cysteine attenuated the augmentation of local ROS production by diminishing the elevation of NADPH oxidase expression and ameliorated renal/vascular hypertensive changes.

NADPH oxidase mediates oxidative stress in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder of uncertain pathogenesis characterized by a loss of substantia nigra pars compacta (SNpc) dopaminergic (DA) neurons, and can be modeled by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Both inflammatory processes and oxidative stress may contribute to MPTP- and PD-related neurodegeneration. However, whether inflammation may cause oxidative damage in MPTP and PD is unknown. Here we show that NADPH-oxidase, the main reactive oxygen species (ROS)-producing enzyme during inflammation, is up-regulated in SNpc of human PD and MPTP mice. These changes coincide with the local production of ROS, microglial activation, and DA neuronal loss seen after MPTP injections. Mutant mice defective in NADPH-oxidase exhibit less SNpc DA neuronal loss and protein oxidation than their WT littermates after MPTP injections. We show that extracellular ROS are a main determinant in inflammation-mediated DA neurotoxicity in the MPTP model of PD. This study supports a critical role for NADPH-oxidase in the pathogenesis of PD and suggests that targeting this enzyme or enhancing extracellular antioxidants may provide novel therapies for PD.

Oxidative stress caused by glycation of Cu,Zn-superoxide dismutase and its effects on intracellular components.

It is now evident that the redox state of the cell is a pivotal determinant of the fate of cells. Extensive production of reactive oxygen species (ROI) causes necrotic cell death. Even transient or localized production of ROI may mediate a signal for apoptotic cell death, whereas small amounts of ROI function as an intracellular messenger of some growth stimulants. Accumulating evidence supports the concept that decreases in Cu,Zn-superoxide dismutase (SOD) activity causes apoptotic cell death in neuronal cells. Our data using mutant Cu,Zn-SOD related to familial amyotrophic lateral sclerosis (FALS) suggest that glycation itself and ROI produced from the glycated proteins are involved in many diseases, including diabetic complications. Glycation of important cellular components, including lipid, DNA and proteins, induces dysfunction of these components. Mutant proteins in patients with various hereditary diseases would be destabilized by the glycation reaction, as shown in the case of mutant Cu,Zn-SODs, thereby hyperglycaemic conditions would trigger the onset of some hereditary diseases such as FALS and Alzheimer's disease. Glycation, particularly of antioxidative enzymes, would enhance production of ROI, resulting in oxidative damage to the cells.