Reactive oxygen species participate in the p38-mediated apoptosis induced by potassium deprivation and staurosporine in cerebellar granule neurons

Apoptosis induced by low potassium (K5) or staurosporine in cerebellar granule neurons triggers an increase in reactive oxygen species (ROS) levels. ROS inhibition by antioxidants or inhibitors of the NADPH oxidase (NOX) activity reduces the apoptosis induced by both stimuli. It has been reported that JNK mediates the apoptosis induced by K5 but not by staurosporine. No information is available about the role of other signaling pathways such as p38 in staurosporine-induced apoptosis, and whether p38 activation could be related to ROS levels induced by both K5 and staurosporine. Here, we explored this possibility and found that K5 activates p38 and ATF2 and that the inhibition of p38 activity prevents the apoptosis induced by this treatment. We also found that p38 is downstream of ROS generation induced by K5. On the other hand, staurosporine promotes a sustained activation of p38. We found that p38 inhibition markedly decreases ROS generation, NOX activity and apoptosis induced by staurosporine. Furthermore, antioxidants inhibit p38 activation induced by staurosporine. These data indicate that apoptosis induced by both K5 and staurosporine is dependent on p38 activation, which is mediated by ROS. In addition, p38 activation by staurosporine induces a further production of ROS through NOX activation.     

Role of reactive oxygen species and NADPH-oxidase in the development of rat cerebellum

Experimental evidence suggests that reactive oxygen species (ROS) could participate in the regulation of some physiological conditions. In the nervous system, ROS have been suggested to act as signaling molecules involved in several developmental processes including cell differentiation, proliferation and programmed of cell death. Although ROS can be generated by several sources, it has been suggested that NADPH oxidase (NOX) could be critical in the production of ROS acting as a signal in some of these events. It has been reported that ROS production by NOX enzymes participate in neuronal maturation and differentiation during brain development. In the present study, we found that during rat cerebellar development there was a differential ROS generation at different ages and areas of the cerebellum. We also found a differential expression of NOX homologues during rat cerebellar development. When we treated developing rats with an antioxidant or with apocynin, an inhibitor of NOX, we found a marked decrease of the ROS levels in all the cerebellar layers at all the ages tested. Both treatments also induced a significant change in the cerebellar foliation as well as an alteration in motor behavior. These results suggest that both ROS and NOX have a critical role during cerebellar development.     

Role of NADPH oxidase in the apoptotic death of cultured cerebellar granule neurons

Cerebellar granule neurons (CGN) cultured in a medium containing 25 mM KCl and treated with staurosporine (ST) or transferred to a medium with 5 mM KCl (K5) die apoptotically. CGN death is mediated by an increase in reactive oxygen species (ROS) production. When CGN are treated with antioxidants all apoptotic parameters and cell death are markedly diminished, showing a central role for ROS in this process. Recently, it has been suggested that a possible ROS source involved in cell death is a NADPH oxidase. In that regard, we found expression in CGN of the components of NADPH proteins, p40phox, p47phox and p67phox, and p22phox, as well as three homologues of the catalytic subunit of this complex, NOX1, 2, and 4. The inhibition of NADPH oxidase with diphenylene iodonium or 4-(2-aminoethyl)benzenesulfonyl fluoride significantly reduced ROS production, NADPH oxidase activity, all the apoptotic events, and cell death induced by both K5 and ST. We conclude that ROS could be an early signal of apoptotic neuronal death and that NADPH oxidase, including NOX1, 2, and/or 4, could have a central role in apoptotic death induced by different conditions in these neurons.     

Bioactive lipoxygenase metabolites stimulation of NADPH oxidases and reactive oxygen species

In mammalian cells, reactive oxygen species (ROS) are produced via a variety of cellular oxidative processes, including the activity of NADPH oxidases (NOX), the activity of xanthine oxidases, the metabolism of arachidonic acid (AA) by lipoxygenases (LOX) and cyclooxygenases (COX), and the mitochondrial respiratory chain. Although NOX-generated ROS are the best characterized examples of ROS in mammalian cells, ROS are also generated by the oxidative metabolism (e.g., via LOX and COX) of AA that is released from the membrane phospholipids via the activity of cytosolic phospholipase A₂ (cPLA₂). Recently, growing evidence suggests that LOX- and COX-generated AA metabolites can induce ROS generation by stimulating NOX and that a potential signaling connection exits between the LOX/COX metabolites and NOX. In this review, we discuss the results of recent studies that report the generation of ROS by LOX metabolites, especially 5-LOX metabolites, via NOX stimulation. In particular, we have focused on the contribution of leukotriene B₄ (LTB₄), a potent bioactive eicosanoid that is derived from 5-LOX, and its receptors, BLT1 and BLT2, to NOX stimulation through a signaling mechanism that leads to ROS generation.     

PTEN inhibits replicative senescence‐induced MMP‐1 expression by regulating NOX4‐mediated ROS in human dermal fibroblasts

The biological function of NADPH oxidase (NOX) is the generation of reactive oxygen species (ROS). ROS, primarily arising from oxidative cell metabolism, play a major role in both chronological ageing and photoageing. ROS in extrinsic and intrinsic skin ageing may be assumed to induce the expression of matrix metalloproteinases. NADPH oxidase is closely linked with phosphatidylinositol 3‐OH kinase (PI3K) signalling. Protein kinase C (PKC), a downstream molecule of PI3K, is essential for superoxide generation by NADPH oxidase. However, the effect of PTEN and NOX4 in replicative‐aged MMPs expression has not been determined. In this study, we confirmed that inhibition of the PI3K signalling pathway by PTEN gene transfer abolished the NOX‐4 and MMP‐1 expression. Also, NOX‐4 down‐expression of replicative‐aged skin cells abolished the MMP‐1 expression and ROS generation. These results suggest that increase of MMP‐1 expression by replicative‐induced ROS is related to the change in the PTEN and NOX expression.     

BJ-1108, a 6-Amino-2,4,5-Trimethylpyridin-3-ol Analog,Inhibits Serotonin-Induced Angiogenesis and Tumor Growth through PI3K/NOX Pathway

5-Hydroxytryptamine (5-HT) induces proliferation of cancer cells and vascular cells. In addition to 5-HT production by several cancer cells including gastrointestinal and breast cancer, a significant level of 5-HT is released from activated platelets in the thrombotic environment of tumors, suggesting that inhibition of 5-HT signaling may constitute a new target for antiangiogenic anticancer drug discovery. In the current study we clearly demonstrate that 5-HT-induced angiogenesis was mediated through the 5-HT₁ receptor-linked Gβγ/Src/PI3K pathway, but not through the MAPK/ERK/p38 pathway. In addition, 5-HT induced production of NADPH oxidase (NOX)-derived reactive oxygen species (ROS). In an effort to develop new molecularly targeted anticancer agents against 5-HT action in tumor growth, we demonstrate that BJ-1108, a derivative of 6-amino-2,4,5-trimethylpyridin-3-ol, significantly inhibited 5-HT-induced angiogenesis. In addition, BJ-1108 induced a significant reduction in the size and weight of excised tumors in breast cancer cell-inoculated CAM assay, showing proportionate suppression of tumor growth along with inhibition of angiogenesis. In human umbilical vein endothelial cells (HUVECs), BJ-1108 significantly suppressed 5-HT-induced ROS generation and phosphorylation of PI3K/Akt but not of Src. Unlike NOX inhibitors, BJ-1108, which showed better antioxidant activity than vitamin C, barely suppressed superoxide anion induced by mevalonate or geranylgeranyl pyrophosphate which directly activates NOX without help from other signaling molecules in HUVECs, implying that the anti-angiogenic action of BJ-1108 was not mediated through direct action on NOX activation, or free radical scavenging activity. In conclusion, BJ-1108 inhibited 5-HT-induced angiogenesis through PI3K/NOX signaling but not through Src, ERK, or p38.     

Role of TASK2 in the control of apoptotic volume decrease in proximal kidney cells

Apoptotic volume decrease (AVD) is prerequisite to apoptotic events that lead to cell death. In a previous study, we demonstrated in kidney proximal cells that the TASK2 channel was involved in the K+ efflux that occurred during regulatory volume decrease. The aim of the present study was to determine the role of the TASK2 channel in the regulation of AVD and apoptosis phenomenon. For this purpose renal cells were immortalized from primary cultures of proximal convoluted tubules (PCT) from wild type and TASK2 knock-out mice (task2-/-). Apoptosis was induced by staurosporine, cyclosporin A, or tumor necrosis factor alpha. Cell volume, K+ conductance, caspase-3, and intracellular reactive oxygen species (ROS) levels were monitored during AVD. In wild type PCT cells the K+ conductance activated during AVD exhibited characteristics of TASK2 currents. In task2-/- PCT cells, AVD and caspase activation were reduced by 59%. Whole cell recordings indicated that large conductance calcium-activated K+ currents inhibited by iberiotoxin (BK channels) partially compensated for the deletion of TASK2 K+ currents in the task2-/- PCT cells. This result explained the residual AVD measured in these cells. In both cell lines, apoptosis was mediated via intracellular ROS increase. Moreover AVD, K+ conductances, and caspase-3 were strongly impaired by ROS scavenger N-acetylcysteine. In conclusion, the main K+ channels involved in staurosporine, cyclosporin A, and tumor necrosis factor-alpha-induced AVD are TASK2 K+ channels in proximal wild type cells and iberiotoxin-sensitive BK channels in proximal task2-/- cells. Both K+ channels could be activated by ROS production.     

NADPH oxidase-derived reactive oxygen species increases expression of monocyte chemotactic factor genes in cultured adipocytes

Excess glucose and free fatty acids delivered to adipose tissue causes local inflammation, which contributes to insulin resistance. Glucose and palmitate generate reactive oxygen species (ROS) in adipocytes, leading to monocyte chemotactic factor gene expression. Docosahexaenoate (DHA) has the opposite effect. In this study, we evaluated the potential sources of ROS in the presence of excess nutrients. Differentiated 3T3-L1 adipocytes were exposed to palmitate and DHA (250 μM) in either 5 or 25 mM glucose to evaluate the relative roles of mitochondrial electron transport and NADPH oxidases (NOX) as sources of ROS. Excess glucose and palmitate did not increase mitochondrial oxidative phosphorylation. However, glucose exposure increased glycolysis. Of the NOX family members, only NOX4 was expressed in adipocytes. Moreover, its activity was increased by excess glucose and palmitate and decreased by DHA. Silencing NOX4 inhibited palmitate- and glucose-stimulated ROS generation and monocyte chemotactic factor gene expression. NADPH, a substrate for NOX, and pentose phosphate pathway activity increased with glucose but not palmitate and decreased with DHA exposure. Inhibition of the pentose phosphate pathway by glucose-6-phosphate dehydrogenase inhibitors and siRNA suppressed ROS generation and monocyte chemotactic factor gene expression induced by both glucose and palmitate. Finally, both high glucose and palmitate induced NOX4 translocation into lipid rafts, effects that were blocked by DHA. Excess glucose and palmitate generate ROS via NOX4 rather than by mitochondrial oxidation in cultured adipocytes. NOX4 is regulated by both NADPH generated in the PPP and translocation of NOX4 into lipid rafts, leading to expression of monocyte chemotactic factors.     

活性氧参与一氧化氮诱导的神经细胞凋亡

采用激光共聚焦成像技术，用氧化还原敏感的特异性荧光探针(DCFH-DA和DHR123)直接研究了一氧化氮供体S-亚硝基-N-乙酰基青霉胺(SNAP)诱导未成熟大鼠小脑颗粒神经元凋亡过程中的细胞胞浆、线粒体中活性氧水平的变化，发现神经细胞经0.5 mmol/L SNAP处理1 h后，细胞胞浆及线粒体中活性氧水平大大增加.一氧化氮清除剂血红蛋白能够有效抑制细胞胞浆、线粒体中活性氧的产生，防止细胞凋亡.外源性谷胱甘肽对细胞也具有良好的保护作用，而当细胞中谷胱甘肽的合成被抑制后，一氧化氮的神经毒性大大增强.实验结果表明一氧化氮通过促进神经细胞产生内源性活性氧而启动细胞凋亡程序，而谷胱甘肽可能是重要的防止一氧化氮引发神经损伤的内源性抗氧化剂.     

活性氧参与-氧化氮诱导的神经细胞凋亡

采用激光共聚焦成像技术,用氧化还原敏感的特异性荧光探针(DCFH-DA和DHR123)直接研究了一氧 化氮供体S-亚硝基-N-乙酰基青霉胺(SNAP)诱导未成熟大鼠小脑颗粒神经元凋亡过程中的细胞胞浆、线粒体 中活性氧水平的变化,发现神经细胞经0.5mmol/LSNAP处理1h后,细胞胞浆及线粒体中活性氧水平大大增 加.一氧化氮清除剂血红蛋白能够有效抑制细胞胞浆、线粒体中活性氧的产生,防止细胞凋亡.外源性谷胱甘 肽对细胞也具有良好的保护作用,而当细胞中谷胱甘肽的合成被抑制后,一氧化氮的神经毒性大大增强.实验 结果表明一氧化氮通过促进神经细胞产生内源性活性氧而启动细胞凋亡程序,而谷胱甘肽可能是重要的防止一 氧化氮引发神经损伤的内源性抗氧化剂     

Intracellular acidification by inhibition of the Na+/H+-exchanger leads to caspase-independent death of cerebellar granule neurons resembling paraptosis

Potassium withdrawal is commonly used to induce caspase-mediated apoptosis in cerebellar granule neurons in vitro. However, the underlying and cell death-initiating mechanisms are unknown. We firstly investigated potassium efflux through the outward delayed rectifier K+ current (Ik) as a potential mediator. However, tetraethylammoniumchloride, an inhibitor of Ik, was ineffective to block apoptosis after potassium withdrawal. Since potassium withdrawal reduced intracellular pH (pHi) from 7.4 to 7.2, we secondly investigated the effects of intracellular acidosis. To study intracellular acidosis in cerebellar granule neurons, we inhibited the Na+/H+ exchanger (NHE) with 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (HOE 642) and 5-(N-ethyl-N-isopropyl)-amiloride. Both inhibitors concentration-dependently induced cell death and potentiated cell death after potassium withdrawal. Although inhibition of the NHE induced cell death with morphological criteria of apoptosis in light and electron microscopy including chromatin condensation, positive TUNEL staining and cell shrinkage, no internucleosomal DNA cleavage or activation of caspases was detected. In contrast to potassium withdrawal-induced apoptosis, cell death induced by intracellular acidification was not prevented by insulin-like growth factor-1, cyclo-adenosine-monophosphate, caspase inhibitors and transfection with an adenovirus expressing Bcl-XL. However, cycloheximide protected cerebellar granule neurons from death induced by potassium withdrawal as well as from death after treatment with HOE 642. Therefore, the molecular mechanisms leading to cell death after acidification appear to be different from the mechanisms after potassium withdrawal and resemble the biochemical but not the morphological characteristics of paraptosis.     

ROS Produced by NOX2 Controls In Vitro Development of Cerebellar Granule Neurons Development

Reactive oxygen species (ROS) act as signaling molecules that regulate nervous system physiology. ROS have been related to neural differentiation, neuritogenesis, and programmed cell death. Nevertheless, little is known about the mechanisms involved in the regulation of ROS during neuronal development. In this study, we evaluated the mechanisms by which ROS are regulated during neuronal development and the implications of these molecules in this process. Primary cultures of cerebellar granule neurons (CGN) were used to address these issues. Our results show that during the first 3 days of CGN development in vitro (days in vitro; DIV), the levels of ROS increased, reaching a peak at 2 and 3 DIV under depolarizing (25 mM KCl) and nondepolarizing (5 mM KCl) conditions. Subsequently, under depolarizing conditions, the ROS levels markedly decreased, but in nondepolarizing conditions, the ROS levels increased gradually. This correlated with the extent of CGN maturation. Also, antioxidants and NADPH-oxidases (NOX) inhibitors reduced the expression of Tau and MAP2. On the other hand, the levels of glutathione markedly increased at 1 DIV. We inferred that the ROS increase at this time is critical for cell survival because glutathione depletion leads to axonal degeneration and CGN death only at 2 DIV. During the first 3 DIV, NOX2 was upregulated and expressed in filopodia and growth cones, which correlated with the hydrogen peroxide (H₂O₂) distribution in the cell. Finally, NOX2 KO CGN showed shorter neurites than wild-type CGN. Taken together, these results suggest that the regulation of ROS is critical during the early stages of CGN development.     

Hydrogen peroxide induces neurite degeneration: Prevention by tocotrienols

Reactive oxygen species (ROS) may attack several types of tissues and chronic exposure to ROS may attenuate various biological functions and increase the risk of several types of serious disorders. It is known that treatments with ROS attack neurons and induce cell death. However, the mechanisms of neuronal change by ROS prior to induction of cell death are not yet understood. Here, it was found that treatment of neurons with low concentrations of hydrogen peroxide induced neurite injury, but not cell death. Unusual bands located above the original collapsin response mediator protein (CRMP)-2 protein were detected by western blotting. Treatment with tocopherol or tocotrienols significantly inhibited these changes in neuro2a cells and cerebellar granule neurons (CGCs). Furthermore, prevention by tocotrienols of hydrogen peroxide-induced neurite degeneration was stronger than that by tocopherol. These findings indicate that neurite beading is one of the early events of neuronal degeneration prior to induction of death of hydrogen peroxide-treated neurons. Treatment with tocotrienols may protect neurite function through its neuroprotective function.     

p38 mitogen-activated protein kinase regulates low potassium-induced c-Jun phosphorylation and apoptosis in cultured cerebellar granule neurons

Cultured rat cerebellar granule neurons are widely used as a model system for studying neuronal apoptosis. After maturation by culturing in medium containing 26 mm potassium (high K(+)), changing to medium containing 5 mm potassium (low K(+); LK) rapidly induces neuronal apoptosis. Then over 50% of granule cells die within 24 h. However, the molecular mechanisms by which the LK-induced apoptosis occurs in cultured cerebellar granule cells remain unclear. In the present study, we found that p38 MAP kinase (p38) was an important factor for LK-induced apoptosis. Three hours after changing to LK medium, p38 was markedly activated. In addition, SB203580, a specific inhibitor of p38, strongly inhibited the phosphorylation and expression of c-Jun in LK-induced apoptosis of cultured cerebellar granule cells. In vitro kinase assay using glutathione S-transferase-c-Jun as a substrate showed that p38 directly phosphorylated c-Jun. Furthermore, in the presence of SB203580, about 80% of neurons survived. These results indicate that p38 regulates LK-induced apoptosis of cerebellar granule neurons.     

Critical Role of H2O2 Generated by NOX4 during Cellular Response under Glucose Deprivation

Glucose is the most efficient energy source, and various cancer cells depend on glycolysis for energy production. For maintenance of survival and proliferation, glucose sensing and adaptation to poor nutritional circumstances must be well organized in cancer cells. While the glucose sensing machinery has been well studied in yeasts, the molecular mechanism of glucose sensing in mammalian cells remains to be elucidated. We have reported glucose deprivation rapidly induces AKT phosphorylation through PI3K activation. We assumed that regulation of AKT is relevant to glucose sensing and further investigated the underlying mechanisms. In this study, AKT phosphorylation under glucose deprivation was inhibited by galactose and fructose, but induced by 2-deoxyglucose (2-DG). Both 2-DG treatment and glucose deprivation were found to induce AKT phosphorylation in HepG2 cells. These findings suggested that glucose transporter may not be involved in the sensing of glucose and induction of AKT phosphorylation, and that downstream metabolic events may have important roles. A variety of metabolic stresses reportedly induce the production of reactive oxygen species (ROS). In the present study, glucose deprivation was found to induce intracellular hydrogen peroxide (H₂O₂) production in HepG2 cells. N-acetylcysteine (NAC), an antioxidant reagent, reduced both the increase in cellular H₂O₂ levels and AKT phosphorylation induced by glucose deprivation. These results strongly suggest that the glucose deprivation-induced increase of H₂O₂ in the cells mediated the AKT phosphorylation. RNA interference of NOX4, but not of NOX5, completely suppressed the glucose deprivation-induced AKT phosphorylation as well as increase of the intracellular levels of ROS, whereas exogenous H₂O₂ could still induce AKT phosphorylation in the NOX4-knockdown cells. In this study, we demonstrated that the ROS generated by NOX4 are involved in the intracellular adaptive responses by recognizing metabolic flux.     

Entamoeba histolytica Induces Cell Death of HT29 Colonic Epithelial Cells via NOX1-Derived ROS

Entamoeba histolytica, which causes amoebic colitis and occasionally liver abscess in humans, is able to induce host cell death. However, signaling mechanisms of colon cell death induced by E. histolytica are not fully elucidated. In this study, we investigated the signaling role of NOX in cell death of HT29 colonic epithelial cells induced by E. histolytica. Incubation of HT29 cells with amoebic trophozoites resulted in DNA fragmentation that is a hallmark of apoptotic cell death. In addition, E. histolytica generate intracellular reactive oxygen species (ROS) in a contact-dependent manner. Inhibition of intracellular ROS level with treatment with DPI, an inhibitor of NADPH oxidases (NOXs), decreased Entamoeba-induced ROS generation and cell death in HT29 cells. However, pan-caspase inhibitor did not affect E. histolytica-induced HT29 cell death. In HT29 cells, catalytic subunit NOX1 and regulatory subunit Rac1 for NOX1 activation were highly expressed. We next investigated whether NADPH oxidase 1 (NOX1)-derived ROS is closely associated with HT29 cell death induced by E. histolytica. Suppression of Rac1 by siRNA significantly inhibited Entamoeba-induced cell death. Moreover, knockdown of NOX1 by siRNA, effectively inhibited E. histolytica-triggered DNA fragmentation in HT29 cells. These results suggest that NOX1-derived ROS is required for apoptotic cell death in HT29 colon epithelial cells induced by E. histolytica.     

Generation of cerebellar neuron precursors from embryonic stem cells

Here, we report in vitro generation of Math1+ cerebellar granule cell precursors and Purkinje cells from ES cells by using soluble patterning signals. When neural progenitors induced from ES cells in a serum-free suspension culture are subsequently treated with BMP4 and Wnt3a, a significant proportion of these neural cells become Math1+. The induced Math1+ cells are mitotically active and express markers characteristic of granule cell precursors (Pax6, Zic1, and Zipro1). After purification by FACS and coculture with postnatal cerebellar neurons, ES cell-derived Math1+ cells exhibit typical features of neurons of the external granule cell layer, including extensive motility and a T-shaped morphology. Interestingly, differentiation of L7+/Calbindin-D28K+ neurons (characteristic of Purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by Fgf8 rather than by Wnt3a. This is the first report of in vitro recapitulation of early differentiation of cerebellar neurons by using the ES cell system.     

NADPH oxidase 4 is required for interleukin-1β-mediated activation of protein kinase Cδ and downstream activation of c-jun N-terminal kinase signaling in smooth muscle

Reactive oxygen species (ROS) are generated in the vascular wall upon stimulation by proinflammatory cytokines and are important mediators of diverse cellular responses that occur as a result of vascular injury. Members of the NADPH oxidase (NOX) family of proteins have been identified in vascular smooth muscle (VSM) cells as important sources of ROS. In this study, we tested the hypothesis that NOX4 is a proximal mediator of IL-1β-dependent activation of PKCδ and increases IL-1β-stimulated c-Jun kinase (JNK) signaling in primary rat aortic VSM cells. We found that stimulation of VSM cells with IL-1β increased PKCδ activity and intracellular ROS generation. SiRNA silencing of NOX4 but not NOX1 ablated the IL-1β-dependent increase in ROS production. Pharmacological inhibition of PKCδ activity as well as siRNA depletion of PKCδ or NOX4 blocked the IL-1β-dependent activation of JNK. Further studies showed that the IL-1β-dependent upregulation of inducible NO synthase expression was inhibited through JNK inhibition and NOX4 silencing. Taken together, these results indicate that IL-1β-dependent activation of PKCδ is modulated by NOX4-derived ROS. Our study positions PKCδ as an important redox-sensitive mediator of IL-1β-dependent signaling and downstream activation of inflammatory mediators in VSM cells.