The Regulation of Reactive Oxygen Species Production during Programmed Cell Death

Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. The role of ROS in cell death caused by oxidative glutamate toxicity was studied in an immortalized mouse hippocampal cell line (HT22). The causal relationship between ROS production and glutathione (GSH) levels, gene expression, caspase activity, and cytosolic Ca²⁺ concentration was examined. An initial 5–10-fold increase in ROS after glutamate addition is temporally correlated with GSH depletion. This early increase is followed by an explosive burst of ROS production to 200–400-fold above control values. The source of this burst is the mitochondrial electron transport chain, while only 5–10% of the maximum ROS production is caused by GSH depletion. Macromolecular synthesis inhibitors as well as Ac-YVAD-cmk, an interleukin 1β–converting enzyme protease inhibitor, block the late burst of ROS production and protect HT22 cells from glutamate toxicity when added early in the death program. Inhibition of intracellular Ca²⁺ cycling and the influx of extracellular Ca²⁺ also blocks maximum ROS production and protects the cells. The conclusion is that GSH depletion is not sufficient to cause the maximal mitochondrial ROS production, and that there is an early requirement for protease activation, changes in gene expression, and a late requirement for Ca²⁺ mobilization.     

Neuronal Cell Death and Reactive Oxygen Species

1. We have investigated the role of reactive oxygen species (ROS) in cell death induced by ischemia or application of the excitatory amino acid agonist, N-methyl-D-aspartate (NMDA) or kainate (KA), in acutely isolated rat cerebellar granule cell neurons, studied by flow cytometry. Various fluorescent dyes were used to monitor intracellular calcium concentration, ROS concentration, membrane potential, and viability in acutely dissociated neurons subjected to ischemia and reoxygenation alone, NMDA or kainate alone, and ischemia and reoxygenation plus NMDA or kainate.2. With ischemia followed by reoxygenation, ROS concentrations rose slightly and there was only a modest increase in cell death after 60 min.3. When NMDA or kainate alone was applied to the cells there was a large increase in ROS and in intracellular calcium concentration but only a small loss of cellular viability. However, when NMDA or kainate was applied during the reoxygenation period there was a large loss of viability, accompanied by membrane depolarization, but the elevations of ROS and intracellular calcium concentration were not greater than seen with the excitatory amino acids alone.4. These observations indicate that other factors beyond ROS and intracellular calcium concentration contribute to cell death in cerebellar granule cell neurons.     

Reactive Oxygen Species Generated by NADPH Oxidase 2 and 4 Are Required for Chondrogenic Differentiation

Although generation of reactive oxygen species (ROS) by NADPH oxidases (Nox) is thought to be important for signal transduction in nonphagocytic cells, little is known of the role ROS plays in chondrogenesis. We therefore examined the possible contribution of ROS generation to chondrogenesis using both ATDC5 cells and primary chondrocytes derived from mouse embryos. The intracellular level of ROS was increased during the differentiation process, which was then blocked by treatment with the ROS scavenger N-acetylcysteine. Expression of Nox1 and Nox2 was increased upon differentiation of ATDC5 cells and primary mouse chondrocytes, whereas that of Nox4, which was relatively high initially, was decreased gradually during chondrogenesis. In developing limb, Nox1 and Nox2 were highly expressed in prehypertrophic and hypertrophic chondrocytes. However, Nox4 was highly expressed in proliferating chondrocytes and prehypertrophic chondrocytes. Depletion of Nox2 or Nox4 expression by RNA interference blocked both ROS generation and differentiation of ATDC5 cells, whereas depletion of Nox1 had no such effect. We also found that ATDC5 cells depleted of Nox2 or Nox4 underwent apoptosis. Further, inhibition of Akt phosphorylation along with subsequent activation of ERK was observed in the cells. Finally, depletion of Nox2 or Nox4 inhibited the accumulation of proteoglycan in primary chondrocytes. Taken together, our data suggest that ROS generated by Nox2 or Nox4 are essential for survival and differentiation in the early stage of chondrogenesis.     

Urotensin-II-Mediated Reactive Oxygen Species Generation via NADPH Oxidase Pathway Contributes to Hepatic Oval Cell Proliferation

Urotensin II (UII), a somatostatin-like cyclic peptide, is involved in tumor progression due to its mitogenic effect. Our previous study demonstrated that UII and its receptor UT were up-regulated in human hepatocellular carcinoma (HCC), and exogenous UII promoted proliferation of human hepatoma cell line BEL-7402. Hepatic progenitor cell (HPCs) are considered to be one of the origins of liver cancer cells, but their relationship with UII remains unclear. In this work, we aimed to investigate the effect of UII on ROS generation in HPCs and the mechanisms of UII-induced ROS in promoting cell proliferation. Human HCC samples were used to examine ROS level and expression of NADPH oxidase. Hepatic oval cell line WB-F344 was utilized to investigate the underlying mechanisms. ROS level was detected by dihydroethidium (DHE) or 2’, 7’-dichlorofluorescein diacetate (DCF-DA) fluorescent probe. For HCC samples, ROS level and expression of NADPH oxidase were significantly up-regulated. In vitro, UII also increased ROS generation and expression of NADPH oxidase in WB-F344 cells. NADPH oxidase inhibitor apocynin pretreatment partially abolished UII-increased phosphorylation of PI3K/Akt and ERK, expression of cyclin E/cyclin-dependent kinase 2. Cell cycle was then analyzed by flow cytometry and UII-elevated S phase proportion was inhibited by apocynin pretreatment. Finally, bromodeoxyuridine (Brdu) incorporation assay showed that apocynin partially abolished UII induced cell proliferation. In conclusion, this study indicates that UII-increased ROS production via the NADPH oxidase pathway is partially associated with activation of the PI3K/Akt and ERK cascades, accelerates G1/S transition, and contributes to cell proliferation. These results showed that UII plays an important role in growth of HPCs, which provides novel evidence for the involvement of HPCs in the formation and pathogenesis of HCC.     

Chitosan-Induced Stomatal Closure Accompanied by Peroxidase-Mediated Reactive Oxygen Species Production in Arabidopsis

Chitosan induced stomatal closure in wild type-plants and NADPH oxidase knock-out mutants (atrbohD atrbohF), and reactive oxygen species (ROS) production in wild-type guard cells. Closure and production were completely abolished by catalase and a peroxidase inhibitor. These results indicate that chitosan induces ROS production mediated by peroxidase, resulting in stomatal closure.     

Reactive Oxygen Generated by NADPH Oxidase 1 (Nox1) Contributes to Cell Invasion by Regulating Matrix Metalloprotease-9 Production and Cell Migration

A mediating role of the reactive oxygen species-generating enzyme Nox1 has been suggested for Ras oncogene transformation phenotypes including anchorage-independent cell growth, augmented angiogenesis, and tumorigenesis. However, little is known about whether Nox1 signaling regulates cell invasiveness. Here, we report that the cell invasion activity was augmented in K-Ras-transformed normal rat kidney cells and attenuated by transfection of Nox1 small interference RNAs (siRNAs) into the cells. Diphenyleneiodonium (DPI) or Nox1 siRNAs blocked up-regulation of matrix metalloprotease-9 at both protein and mRNA levels in K-Ras-transformed normal rat kidney cells. Furthermore, DPI and Nox1 siRNAs inhibited the activation of IKKα kinase and the degradation of IκBα, suppressing the NFκB-dependent matrix metalloprotease-9 promoter activity. Additionally, epidermal growth factor-stimulated migration of CaCO-2 cells was abolished by DPI and Nox1 siRNAs, indicating the requirement of Nox1 activity for the motogenic effect of epidermal growth factor. This Nox1 action was mediated by down-regulation of the Rho activity through the low molecular weight protein-tyrosine phosphatase-p190RhoGAP-dependent mechanism. Taken together, our findings define a mediating role of Nox1-generated reactive oxygen species in cell invasion processes, most notably metalloprotease production and cell motile activity.     

活性氧与植物细胞编程性死亡

在各种条件诱导的植物细胞编程性死亡(PCD)过程中都有活性氧的参与,H2O2和O2可能是参与PCD调节的最重要的活性氧.文中概述了活性氧与植物细胞编程性死亡的关系以及活性氧的生成调节和与其它一些信号物质之间可能的相互作用.     

Ca2+-Activated Reactive Oxygen Species Production by Arabidopsis RbohH and RbohJ Is Essential for Proper Pollen Tube Tip Growth

In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca²⁺ binding EF-hand motifs, possessed Ca²⁺-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca²⁺-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca²⁺-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca²⁺. Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca²⁺-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.     

Dynamin 2 and c-Abl Are Novel Regulators of Hyperoxia-mediated NADPH Oxidase Activation and Reactive Oxygen Species Production in Caveolin-enriched Microdomains of the Endothelium

Reactive oxygen species (ROS) generation, particularly by the endothelial NADPH oxidase family of proteins, plays a major role in the pathophysiology associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. We examined potential regulators of ROS production and discovered that hyperoxia treatment of human pulmonary artery endothelial cells induced recruitment of the vesicular regulator, dynamin 2, the non-receptor tyrosine kinase, c-Abl, and the NADPH oxidase subunit, p47^phox, to caveolin-enriched microdomains (CEMs). Silencing caveolin-1 (which blocks CEM formation) and/or c-Abl expression with small interference RNA inhibited hyperoxia-mediated tyrosine phosphorylation and association of dynamin 2 with p47^phox and ROS production. In addition, treatment of human pulmonary artery endothelial cells with dynamin 2 small interfering RNA or the dynamin GTPase inhibitor, Dynasore, attenuated hyperoxia-mediated ROS production and p47^phox recruitment to CEMs. Using purified recombinant proteins, we observed that c-Abl tyrosine-phosphorylated dynamin 2, and this phosphorylation increased p47^phox/dynamin 2 association (change in the dissociation constant (K_d) from 85.8 to 6.9 nm). Furthermore, exposure of mice to hyperoxia increased ROS production, c-Abl activation, dynamin 2 association with p47^phox, and pulmonary leak, events that were attenuated in the caveolin-1 knock-out mouse confirming a role for CEMs in ROS generation. These results suggest that hyperoxia induces c-Abl-mediated dynamin 2 phosphorylation required for recruitment of p47^phox to CEMs and subsequent ROS production in lung endothelium.     

Production of Reactive Oxygen Species and Release of l-Glutamate During Superoxide Anion-Induced Cell Death of Cerebellar Granule Neurons

Abstract: Enhanced production of superoxide anion (O₂⁻) is considered to play a pivotal role in the pathogenesis of CNS neurons. Here, we report that O₂⁻ generated by xanthine (XA) + xanthine oxidase (XO) triggered cell death associated with nuclear condensation and DNA fragmentation in cerebellar granule neuron. XA + XO induced significant increases in amounts of intracellular reactive oxygen species (ROS) before initiating loss of cell viability, as determined by measurement of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA) for O₂⁻ and other ROS and hydroethidine (HEt) specifically for O₂⁻ by using fluorescence microscopy and flow cytometry. Catalase, but not superoxide dismutase (SOD), significantly protected granule neurons from the XA + XO-induced cell death. Catalase effectively reduced C-DCDHF-DA but not HEt fluorescence, whereas SOD reduced HEt but not C-DCDHF-DA fluorescence, indicating that HEt and C-DCDHF-DA fluorescence correlated with O₂⁻ and hydrogen peroxide, respectively. The NMDA antagonist MK-801 prevented the death. XA + XO induced an increase in l -glutamate release from cerebellar granule neurons. These results indicate that elevation of O₂⁻ induces cell death associated with increasing ROS production in cerebellar granule neurons and that XA + XO enhanced release of l -glutamate.     

NADPH Oxidase NOX4 Mediates Stellate Cell Activation and Hepatocyte Cell Death during Liver Fibrosis Development

A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2^−/−/p19^ARF−/−, Stat3^Δhc/Mdr2^−/−) and a model of experimental induced fibrosis (CCl₄) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-β pathway activation. In vitro TGF-β-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-β is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-β-induced epithelial-mesenchymal transition (EMT), but was required for TGF-β-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-β-induced death of hepatocytes.     

Reactive Oxygen Species Production by Potato Tuber Mitochondria Is Modulated by Mitochondrially Bound Hexokinase Activity

Potato tuber (Solanum tuberosum) mitochondria (PTM) have a mitochondrially bound hexokinase (HK) activity that exhibits a pronounced sensitivity to ADP inhibition. Here we investigated the role of mitochondrial HK activity in PTM reactive oxygen species generation. Mitochondrial HK has a 10-fold higher affinity for glucose (Glc) than for fructose (K_MGlc = 140 μm versus K_MFrc = 1,375 μm). Activation of PTM respiration by succinate led to an increase in hydrogen peroxide (H₂O₂) release that was abrogated by mitochondrial HK activation. Mitochondrial HK activity caused a decrease in the mitochondrial membrane potential and an increase in oxygen consumption by PTM. Inhibition of Glc phosphorylation by mannoheptulose or GlcNAc induced a rapid increase in H₂O₂ release. The blockage of H₂O₂ release sustained by Glc was reverted by oligomycin and atractyloside, indicating that ADP recycles through the adenine nucleotide translocator and F₀F₁ATP synthase is operative during the mitochondrial HK reaction. Inhibition of mitochondrial HK activity by 60% to 70% caused an increase of 50% in the maximal rate of H₂O₂ release. Inhibition in H₂O₂ release by mitochondrial HK activity was comparable to, or even more potent, than that observed for StUCP (S. tuberosum uncoupling protein) activity. The inhibition of H₂O₂ release in PTM was two orders of magnitude more selective for the ADP produced from the mitochondrial HK reaction than for that derived from soluble yeast (Saccharomyces cerevisiae) HK. Modulation of H₂O₂ release and oxygen consumption by Glc and mitochondrial HK inhibitors in potato tuber slices shows that hexoses and mitochondrial HK may act as a potent preventive antioxidant mechanism in potato tubers.Production of reactive oxygen species (ROS) is an unavoidable consequence of aerobic respiration (Chance et al., 1979). The mitochondrial electron transport system (ETS) is the major site of ROS production in mammalian and nonphotosynthesizing plant cells (Puntarulo et al., 1991; Halliwell and Gutteridge, 2007). Depending on the mitochondrial respiratory states, a small portion of the consumable oxygen is partially reduced to generate ROS (Skulachev, 1996; Liu, 1997; Turrens, 1997; Møller, 2001; Considine et al., 2003; Smith et al., 2004). In plants, the monoelectronic reduction of oxygen by ETS leads to the production of superoxide radicals (O₂^·−) that can be dismutated by SOD, producing hydrogen peroxide (H₂O₂), and further decomposed by catalase and/or ascorbate-glutathione peroxidase cycles (Møller, 2001). An imbalance between the ROS production and antioxidant defenses can lead to an oxidative stress condition. Increased levels of ROS may be a consequence of the action of plant hormones, environmental stress, pathogens, or high levels of sugars and fatty acids (Bolwell et al., 2002; Couée et al., 2006; Gechev et al., 2006; Liu et al., 2007; Rhoads and Subbaiah, 2007). These conditions may lead to storage deterioration or impairment of seedling growth decreasing on crop yield. To avoid the harmful accumulation of ROS or to fine tune the steady-state levels of ROS, various enzymatic systems control the rate of ROS production in mitochondria (Schreck and Baeuerle, 1991; Møller, 2001).Mitochondrial ROS production is highly dependent on the membrane potential (ΔΨ_m) generated by the proton gradient formed across the inner mitochondrial membrane. High ΔΨ_m was shown to stimulate ROS production when the ETS is predominantly in a reduced state (i.e. when NADH, FADH₂, and O₂ are present in abundance but ADP or Pi levels are low). This condition is reached in resting metabolic states after a full oxidation of Glc or fatty acids. Stimulating electron flow by decreasing ΔΨ_m, either by the use of uncouplers or by coupling respiration to ATP synthesis, slows the ROS generation rate (Boveris and Chance, 1973; Korshunov et al., 1997). It has been observed that in isolated potato tuber (Solanum tuberosum) mitochondria (PTM) the uncoupling protein (referred to as PUMP in plants, or UCP in animals) causes a small decrease in ΔΨ_m when this proton carrier protein is activated by the presence of anionic fatty acids, a condition that blocks ROS generation (Vercesi et al., 1995, 2006). Nucleotides, such as ATP, antagonize this effect (Considine et al., 2003; Vercesi et al., 2006). On the other hand, fluctuations in free hexose levels due to environmental or developmental conditions (Morrell and ap Rees, 1986; Geigenberger and Stitt, 1993; Renz and Stitt, 1993) lead to variations in the oxygen consumption rate in heterotrophic tissues of plant (Brouquisse et al., 1991; Dieuaide et al., 1992). As a result, ROS-producing pathways may be either stimulated or repressed (Couée et al., 2006). Unlike PUMP activity, which is activated by an excess of free fatty acids, a specific mechanism for mitochondrial ROS production caused by an excess of hexose remains elusive.The metabolism of free hexoses begins by their phosphorylation in a reaction catalyzed by the hexokinase (HK):HK is a ubiquitous enzyme found in many organisms. In plants, the binding mechanism of HK to the outer mitochondrial membrane is not fully established, but some reports indicate that it may differ considerably from those properties described for mammal cells (Dry et al., 1983; Miernyk and Dennis, 1983; Rezende et al., 2006). It has been shown that in several mature and developing plant tissues, multiple HK isoforms are expressed with different kinetic properties and subcellular localizations. The HKs are found in cytosol, bound to the mitochondrial membrane, or in stroma of plastids in plant cells (Miernyk and Dennis, 1983; Galina et al., 1995; Damari-Weissler et al., 2007). Beyond its obvious role in glycolysis regulation, HK activity may also function as a sugar sensor, triggering a signal transduction pathway in plants (Rolland et al., 2006).In mammals, HK types I and II are associated with the mitochondrial outer membrane through the voltage-dependent anion channel (VDAC) and adenine nucleotide transporter (ANT). These associations were found in tissues with a high energy demand, such as heart, brain, and tumor cells (Arora and Pedersen, 1988; BeltrandelRio and Wilson, 1992; Wilson, 2003). In addition, recent evidence in mammalian cells has shown that binding of HK to VDAC located at the outer mitochondrial membrane is somehow involved in the protection against proapoptotic stimuli (Nakashima et al., 1986; Gottlob et al., 2001; Vander Heiden et al., 2001; Pastorino et al., 2002; Cesar and Wilson, 2004). Similar observations were reported for tobacco (Nicotiana tabacum) plant mitochondrial HK (mt-HK; Kim et al., 2006). However, it has been shown that drugs such as the fungicide clotrimazole and the anesthetic thiopental, which promptly disrupt the association between mt-HK and VDAC in mammalian mitochondria, are unable to promote this effect in maize (Zea mays) root mitochondria (Rezende et al., 2006). These observations suggest a different type of association of mt-HK with plant mitochondria. The binding of mt-HK with mitochondria in many plants involves a common N-terminal hydrophobic membrane anchor domain of about 24 amino acids that is related to the membrane targeting, but the exact mechanism of association is unknown (Damari-Weissler et al., 2007).Recently, our group demonstrated that mt-HK activity plays a key preventive antioxidant role by reducing mitochondrial ROS generation through a steady-state ADP recycling mechanism in rat brain neurons. The mitochondrial ADP recycling leads to a decrease in the ΔΨ_m coupled to the synthesis of ATP by oxidative phosphorylation (da-Silva et al., 2004; Meyer et al., 2006).Although plant HK is recognized to fulfill a catalytic function, the role of mt-HK activity in the regulation of both mitochondrial respiration and ROS production in plants is unknown. Recently, an authentic HK activity was detected in PTM (Graham et al., 2007) and its involvement in potato tuber glycolysis suggested, but its involvement in PTM ROS generation was not explored. We then raise the hypothesis that HK bound to PTM would contribute to produce a steady-state ADP recycling that regulates ROS formation. However, whether this association is capable of controlling the rate of ROS generation in plant mitochondria is unknown. Here, we aim to investigate the role of mt-HK activity in PTM physiology. The data indicate that mt-HK activity plays a key role as a regulator of ROS levels in respiring plant tissues exposed to high hexose levels.     

Reactive Oxygen Species Formation and Cell Death in Catalase-Deficient Tobacco Leaf Discs Exposed to Paraquat

In the present work, the response of tobacco (Nicotiana tabaccum L.) wild-type SR1 and transgenic CAT1AS plants (with a basal reduced CAT activity) was evaluated after exposure to the herbicide paraquat (PQ). Superoxide anion (O₂^.−) formation was inhibited at 3 or 21 h of exposure, but H₂O₂ production and ion leakage increased significantly, both in SR1 or CAT1AS leaf discs. NADPH oxidase activity was constitutively 57% lower in non-treated transgenic leaves than in SR1 leaves and was greatly reduced both at 3 or 21 h of PQ treatment. Superoxide dismutase (SOD) activity was significantly reduced by PQ after 21 h, showing a decrease from 70% to 55%, whereas catalase (CAT) activity decreased an average of 50% after 3 h of treatment, and of 90% after 21 h, in SR1 and CAT1AS, respectively. Concomitantly, total CAT protein content was shown to be reduced in non-treated CAT1AS plants compared to control SR1 leaf discs at both exposure times. PQ decreased CAT expression in SR1 or CAT1AS plants at 3 and 21 h of treatment. The mechanisms underlying PQ-induced cell death were possibly not related exclusively to ROS formation and oxidative stress in tobacco wild-type or transgenic plants.     

Thioredoxin-interacting Protein Mediates High Glucose-induced Reactive Oxygen Species Generation by Mitochondria and the NADPH Oxidase,Nox4, in Mesangial Cells

Thioredoxin-interacting protein (TxNIP) is up-regulated by high glucose and is associated with oxidative stress. It has been implicated in hyperglycemia-induced β-cell dysfunction and apoptosis. As high glucose and oxidative stress mediate diabetic nephropathy (DN), the contribution of TxNIP was investigated in renal mesangial cell reactive oxygen species (ROS) generation and collagen synthesis. To determine the role of TxNIP, mouse mesangial cells (MC) cultured from wild-type C3H and TxNIP-deficient Hcb-19 mice were incubated in HG. Confocal microscopy was used to measure total and mitochondrial ROS production (DCF and MitoSOX) and collagen IV. Trx and NADPH oxidase activities were assayed and NADPH oxidase isoforms, Nox2 and Nox4, and antioxidant enzymes were determined by immunoblotting. C3H MC exposed to HG elicited a significant increase in cellular and mitochondrial ROS as well as Nox4 protein expression and NADPH oxidase activation, whereas Hcb-19 MC showed no response. Trx activity was attenuated by HG only in C3H MC. These defects in Hcb-19 MC were not due to increased antioxidant enzymes or scavenging of ROS, but associated with decreased ROS generation. Adenovirus-mediated overexpression of TxNIP in Hcb-19 MC and TxNIP knockdown with siRNA in C3H confirmed the specific role of TxNIP. Collagen IV accumulation in HG was markedly reduced in Hcb-19 cells. TxNIP is a critical component of the HG-ROS signaling pathway, required for the induction of mitochondrial and total cell ROS and the NADPH oxidase isoform, Nox4. TxNIP is a potential target to prevent DN.     

NADPH Oxidase-Dependent Production of Reactive Oxygen Species Induces Endoplasmatic Reticulum Stress in Neutrophil-Like HL60 Cells

Reactive oxygen species (ROS) primarily produced via NADPH oxidase play an important role for killing microorganisms in neutrophils. In this study we examined if ROS production in Human promyelocytic leukemia cells (HL60) differentiated into neutrophil-like cells (dHL60) induces ER stress and activates the unfolded protein response (UPR). To cause ROS production cells were treated with PMA or by chronic hyperglycemia. Chronic hyperglycemia failed to induce ROS production and did not cause activation of the UPR in dHL60 cells. PMA, a pharmacologic NADPH oxidase activator, induced ER stress in dHL60 cells as monitored by IRE-1 and PERK pathway activation, and this was independent of calcium signaling. The NADPH oxidase inhibitor, DPI, abolished both ROS production and UPR activation. These results show that ROS produced by NADPH oxidase induces ER stress and suggests a close association between the redox state of the cell and the activation of the UPR in neutrophil-like HL60 cells.     

Ca2+ Regulates Reactive Oxygen Species Production and pH during Mechanosensing in Arabidopsis Roots

Mechanical stimulation of plants triggers a cytoplasmic Ca²⁺ increase that is thought to link the touch stimulus to appropriate growth responses. We found that in roots of Arabidopsis thaliana, external and endogenously generated mechanical forces consistently trigger rapid and transient increases in cytosolic Ca²⁺ and that the signatures of these Ca²⁺ transients are stimulus specific. Mechanical stimulation likewise elicited an apoplastic alkalinization and cytoplasmic acidification as well as apoplastic reactive oxygen species (ROS) production. These responses showed the same kinetics as mechanically induced Ca²⁺ transients and could be elicited in the absence of a mechanical stimulus by artificially increasing Ca²⁺ concentrations. Both pH changes and ROS production were inhibited by pretreatment with a Ca²⁺ channel blocker, which also inhibited mechanically induced elevations in cytosolic Ca²⁺. In trichoblasts of the Arabidopsis root hair defective2 mutant, which lacks a functional NADPH oxidase RBOH C, touch stimulation still triggered pH changes but not the local increase in ROS production seen in wild-type plants. Thus, mechanical stimulation likely elicits Ca²⁺-dependent activation of RBOH C, resulting in ROS production to the cell wall. This ROS production appears to be coordinated with intra- and extracellular pH changes through the same mechanically induced cytosolic Ca²⁺ transient.