6-Hydroxydopamine induces cystatin C-mediated cysteine protease suppression and cathepsin D activation

Alteration in the lysosomal system (LS) may represent a central mechanism in neurodegeneration. 6-Hydroxydopamine (6-OHDA) induces oxidative stress and cell death in catecholaminergic cells. The LS and caspases participate in apoptosis, although the mechanism(s) that is involved is not completely understood. Here, we show that Pheochromocytoma (PC12) cells exposed to 6-OHDA results in lysosomal dysregulation, caspase activation and cell death. Cells exposed to 6-OHDA increased expression and release of cystatin C (CC) and suppressed cathepsin B (CB). CB activity significantly declined 24h following exposure to 6-OHDA, however neutralization of CC restored CB activity. Cathepsin D (CD) and caspase-3 activity also increased following exposure to 6-OHDA. Inhibition of CD and caspase-3 with pepstatin A (PA) and DEVD-Cho, respectively, attenuated the 6-OHDA induced cell death at 48 and 72 h. However, the CB inhibitor CA-074 Me failed to protect cells. Additionally, poly-ADP-ribose polymerase (PARP) cleavage was evaluated after exposure to 6-OHDA and PA, CA-074 Me, and DEVD-Cho. Only DEVD-Cho significantly decreased PARP cleavage following exposure to 6-OHDA. Hence, caspase-3 mediated PARP cleavage following exposure to 6-OHDA appears independent of CB and CD alterations. These studies suggest alternate pathways and potential therapeutic targets of cell death associated with oxidative stress, CC, and lysosomal dysregulation.     

Nicotine mediates oxidative stress and apoptosis through cross talk between NOX1 and Bcl-2 in lung epithelial cells

Nicotine contributes to the onset and progression of several pulmonary diseases. Among the various pathophysiological mechanisms triggered by nicotine, oxidative stress and cell death are reported in several cell types. We found that chronic exposure to nicotine (48 h) induced NOX1-dependent oxidative stress and apoptosis in primary pulmonary cells. In murine (MLE-12) and human (BEAS-2B) lung epithelial cell lines, nicotine acted as a sensitizer to cell death and synergistically enhanced apoptosis when cells were concomitantly exposed to hyperoxia. The precise signaling pathway was investigated in MLE-12 cells in which NOX1 was abrogated by a specific inhibitor or stably silenced by shRNA. In the early phase of exposure (1 h), nicotine mediated intracellular Ca²⁺ fluxes and activation of protein kinase C, which in its turn activated NOX1, leading to cellular and mitochondrial oxidative stress. The latter triggered the intrinsic apoptotic machinery by modulating the expression of Bcl-2 and Bax. Overexpression of Bcl-2 completely prevented nicotine’s detrimental effects, suggesting Bcl-2 as a downstream key regulator in nicotine/NOX1-induced cell damage. These results suggest that NOX1 is a major contributor to the generation of intracellular oxidative stress induced by nicotine and might be an important molecule to target in nicotine-related lung pathologies.     

Autophagic proteins regulate cigarette smoke-induced apoptosis: protective role of heme oxygenase-1

Cigarette smoke-induced cell death contributes to the pathogenesis of chronic obstructive pulmonary disease, though the relative roles of apoptosis and autophagy remain unclear. The inducible stress protein heme oxygenase-1 (HO-1) confers cytoprotection against oxidative stress. We examined the relationships between these processes in human bronchial epithelial cells (Beas-2b) exposed to cigarette smoke extract (CSE). CSE induced morphological and biochemical markers of autophagy in Beas-2b cells and induced autophagosome formation as evidenced by formation of GFP-LC3 puncta and electron microscopic analysis. Furthermore, CSE increased the processing of microtubule-associated protein-1 light chain-3 (LC3B-I) to LC3B-II, within 1 hr of exposure. Increased LC3B-II was associated with increased autophagy, since inhibitors of lysosomal proteases and of autophagosome-lysosome fusion further increased LC3B-II levels during CSE exposure. CSE concurrently induced extrinsic apoptosis in Beas-2b cells involving early activation of death-inducing-signaling-complex (DISC) formation and downstream activation of caspases (-8,-9,-3). The induction of extrinsic apoptosis by CSE was dependent in part on autophagic proteins. Reduction of Beclin 1 levels with beclin 1 siRNA inhibited DISC formation and caspase-3/8 activation in response to CSE. LC3B siRNA also inhibited caspase-3/8 activation. The stress protein HO-1 protected against CSE-induced cell death by concurrently downregulating apoptosis and autophagy-related signaling. Adenoviral mediated expression of HO-1 inhibited DISC formation and caspase-3/9 activation in CSE-treated epithelial cells, diminished the expression of Beclin 1, and partially inhibited the processing of LC3B-I to LC3B-II. Conversely, transfection of Beas-2b with ho-1 siRNA augmented CSE-induced DISC formation and increased intracellular reactive oxygen species formation. HO-1 expression augmented CSE-induced phosphorylation of NFkappaB p65 in Beas-2b cells. Consistently, expression of IkappaB, the inhibitor of NFkappaB, increased CSE-induced DISC formation. LC3B siRNA also enhanced p65 phosphorylation. In fibroblasts from beclin 1 heterozygous knockout mice, p65 phosphorylation was dramatically upregulated, while CSE-induced DISC formation was inhibited, consistent with an anti-apoptotic role for NFkappaB and a pro-apoptotic role for Beclin 1. These studies demonstrated an interdependence of autophagic and apoptogenic signaling in CSE-induced cell death, and their coordinated downregulation by HO-1. An understanding of the regulation of cell death pathways during smoke exposure may provide therapeutic strategies in smoke-related illness.     

Heme Oxygenase-1 Protects Retinal Endothelial Cells against High Glucose- and Oxidative/Nitrosative Stress-Induced Toxicity

Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2',7'-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H(2)O(2) and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H(2)O(2) and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.     

Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

BackgroundExposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associated with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood.PurposeIn this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells.MethodThe human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process.ResultsHyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally, phosphorylation of ERK1/2, p38, and Akt were affected by CHI3L1 knockdown.ConclusionThis study indicates that CHI3L1 is involved in hyperoxia-induced cell death, suggesting that CHI3L1 may be one of several cell death regulators influencing the MAPK and PI3K pathways during oxidative stress in human airway epithelial cells.     

Induction of heat shock proteins (HSPs) by sodium arsenite in cultured astrocytes and reduction of hydrogen peroxide-induced cell death

Induction of heat shock proteins (HSPs) protects cells from oxidative injury. Here Hsp72, Hsp27 and heme oxygenase-1 (HO-1) were induced in cultured rat astrocytes, and protection against oxidative stress was investigated. Astrocytes were treated with sodium arsenite (20-50 micro m) for 1 h, which was non-toxic to cells, 24 h later they were exposed to 400 micro m H2O2 for 1 h, and cell death was evaluated at different time points. Arsenite triggered strong induction of HSPs, which was prevented by 1 micro g/mL cycloheximide (CXH). H2O2 caused cell loss and increased cell death with features of apoptosis, i.e. TdT-mediated dUTP nick-end labelling (TUNEL) reaction and caspase-3 activation. These features were abrogated by pre-treatment with arsenite, which prevented cell loss and significantly reduced the number of dead cells. The protective effect of arsenite was not detected in the presence of CHX. Pre-treatment with arsenite increased protein kinase B (Akt) and extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation after H2O2. However, while Akt phosphorylation was prevented by CHX, Erk1/2 phosphorylation was further enhanced by CHX. The results show that transient arsenite pre-treatment induces Hsp72, HO-1 and, to a lesser extent, Hsp27; it reduces H2O2-induced astrocyte death; and it causes selective activation of Akt following H2O2. It is suggested that HSP expression at the time of H2O2 exposure protects astrocytes from oxidative injury and apoptotic cell death by means of pro-survival Akt.     

Proteome profiling reveals potential toxicity and detoxification pathways following exposure of BEAS-2B cells to engineered nanoparticle titanium dioxide

Oxidative stress is known to play important roles in engineered nanomaterial‐induced cellular toxicity. However, the proteins and signaling pathways associated with the engineered nanomaterial‐mediated oxidative stress and toxicity are largely unknown. To identify these toxicity pathways and networks that are associated with exposure to engineered nanomaterials, an integrated proteomic study was conducted using human bronchial epithelial cells, BEAS‐2B and nanoscale titanium dioxide. Utilizing 2‐DE and MS, we identified 46 proteins that were altered at protein expression levels. The protein changes detected by 2‐DE/MS were verified by functional protein assays. These identified proteins include some key proteins involved in cellular stress response, metabolism, adhesion, cytoskeletal dynamics, cell growth, cell death, and cell signaling. The differentially expressed proteins were mapped using Ingenuity Pathway Analyses^? canonical pathways and Ingenuity Pathway Analyses tox lists to create protein‐interacting networks and proteomic pathways. Twenty protein canonical pathways and tox lists were generated, and these pathways were compared to signaling pathways generated from genomic analyses of BEAS‐2B cells treated with titanium dioxide. There was a significant overlap in the specific pathways and lists generated from the proteomic and the genomic data. In addition, we also analyzed the phosphorylation profiles of protein kinases in titanium dioxide‐treated BEAS‐2B cells for a better understanding of upstream signaling pathways in response to the titanium dioxide treatment and the induced oxidative stress. In summary, the present study provides the first protein‐interacting network maps and novel insights into the biological responses and potential toxicity and detoxification pathways of titanium dioxide.     

Oxidative preconditioning and apoptosis in L-cells. Roles of protein kinase B and mitogen-activated protein kinases

Oxidative stress can cause significant cell death by apoptosis. We performed studies in L-cells to explore whether prior exposure to oxidative stress ("oxidative preconditioning") can protect the cell against the apoptotic consequences of subsequent oxidative insults and to establish the mediators in the preconditioning signaling cascade. Cells were preconditioned with three 5-min exposures to H(2)O(2), followed by 10-h recovery and subsequent exposure to 600 microm H(2)O(2) for 10 h. A single 10-h exposure to H(2)O(2) induced substantial apoptotic cell death (approximately 90%), as determined by enzyme-linked immunosorbent assay, TUNEL (terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling), and Annexin V methods, but apoptosis was largely prevented in preconditioned cells. The degree of cytoprotection depended on the strength of preconditioning or H(2)O(2) concentration (20 approximately 600 microm). Transient increases in mitogen-activated protein kinase (MAPK), p38, and JNK/SAPK activities and sustained protein kinase B (Akt) activation, accompanied by drastically reduced caspase 3 activity, were seen after preconditioning. The expression levels of these kinases were unaltered. Inhibitors of p38 (SB203580) and phosphoinositide 3-kinase (PI3K, LY294002) pathways abolished the protection provided by preconditioning. We conclude that oxidative preconditioning protects cells against apoptosis and that this effect involves MAPK and PI3K/Akt pathways. This system may be important in regulating apoptotic cell death in development and disease states.     

Sensory plasticity of carotid body is correlated with oxidative stress in paraventricular nucleus during chronic intermittent hypoxia

Chronic intermittent hypoxia (CIH) is known to induce hypertension, but the mechanism is not well understood. We hypothesized that sensory plasticity of the carotid body (CB) and oxidative stress in the paraventricular nucleus (PVN) are involved in CIH-induced hypertension. In this study, rats were exposed to CIH for 28 days (intermittent hypoxia of 21% O₂ for 60 s and 5% O₂ for 30 s, cyclically repeated for 8 hr/day) and then randomly grouped for intracerebroventricular injection of 5-HT2 receptor antagonist ritanserin, Rho-associated protein kinase (ROCK) inhibitor Y-27632, and NADPH oxidase (NOX) inhibitor diphenyleneiodonium (DPI), respectively. We found that CIH increased blood pressure (BP), elevated carotid sinus nerve (CSN) and renal sympathetic nerve (RSN) activities, oxidative stress, and cell apoptosis in PVN. NOX-derived reactive oxygen species (ROS) production and cell apoptosis decreased when CIH-induced activation of 5-HT/5-HT2AR/PKC signaling was inhibited by ritanserin. In addition, RhoA expression was downregulated when oxidative stress was attenuated by DPI, while Y-27632 decreased the expression of endothelin-1, which is overexpressed in the vascular wall during hypertension. Moreover, treatment with ritanserin, DPI or Y-27632 attenuated the sensory plasticity and sympathetic hyperactivity as well as CIH-induced elevation of BP. In conclusion, CIH-induced activation of 5-HT/5-HT2AR/PKC signaling contributes to NOX-derived oxidative stress in PVN, which may cause sensory plasticity of CB, RSN hyperactivity, and elevated BP.     

Effects of nitric oxide synthase-3 overexpression on post-translational modifications and cell survival in HepG2 cells

Hepatocarcinoma is the fifth most common neoplasm and the third cause of cancer-related death. The development of genetic- and/or molecular-based therapies is urgently required. The administration of high doses of nitric oxide (NO) promotes cell death in hepatocytes. NO contributes to cell signaling by inducing oxidative/nitrosative-dependent post-translational modifications. The aim of the present study was to investigate protein modifications and its relation with alteration of cell proliferation and death in hepatoma cells. Increased intracellular NO production was achieved by stable nitric oxide synthase-3 (NOS-3) overexpression in HepG2 cells. We assessed the pattern of nitration, nitrosylation and carbonylation of proteins by proteomic analysis. The results showed that NOS-3 cell overexpression increased oxidative stress, which affected proteins mainly involved in cell protein folding. Carbonylation also altered metabolism, as well as immune and antioxidant responses. The interaction of nitrosative and oxidative stress generated tyrosine nitration, which affected the tumor marker Serpin B3, ATP synthesis and cytoskeleton. All these effects were associated with a decrease in chaperone activity, a reduction in cell proliferation and an increased cell death. Our study showed that alteration of nitration, nitrosylation and carbonylation pattern of proteins by NO-dependent oxidative/nitrosative stress was related to a reduction of cell survival in a hepatoma cell line.     

Proteomic analysis of the response to high-salinity stress in <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Physcomitrella patens is well known because of its importance in the study of plant systematics and evolution. The tolerance of P. patens for high-salinity environments also makes it an ideal candidate for studying the molecular mechanisms by which plants respond to salinity stresses. We measured changes in the proteome of P. patens gametophores that were exposed to high-salinity (250, 300, and 350 mM NaCl) using two-dimensional gel electrophoresis (2-DE) via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sixty-five protein spots were significantly altered by exposure to the high-salinity environment. Among them, 16 protein spots were down-regulated and 49 protein spots were up-regulated. These proteins were associated with a variety of functions, including energy and material metabolism, protein synthesis and degradation, cell defense, cell growth/division, transport, signal transduction, and transposons. Specifically, the up-regulated proteins were primarily involved in defense, protein folding, and ionic homeostasis. In summary, we outline several novel insights into the response of P. patens to high-salinity; (1) HSP70 is likely to play a significant role in protecting proteins from denaturation and degradation during salinity stress, (2) signaling proteins, such as 14-3-3 and phototropin, may work cooperatively to regulate plasma membrane H(+)-ATPase and maintain ion homeostasis, (3) an increase in photosynthetic activity may contribute to salinity tolerance, and (4) ROS scavengers were up-regulated suggesting that the antioxidative system may play a crucial role in protecting cells from oxidative damage following exposure to salinity stress in P. patens.     

A novel function of poly(ADP-ribose) polymerase-1 in modulation of autophagy and necrosis under oxidative stress

Under oxidative stress, poly(ADP-ribose) polymerase-1 (PARP-1) is activated and contributes to necrotic cell death through ATP depletion. On the other hand, oxidative stress is known to stimulate autophagy, and autophagy may act as either a cell death or cell survival mechanism. This study aims to explore the regulatory role of PARP-1 in oxidative stress-mediated autophagy and necrotic cell death. Here, we first show that hydrogen peroxide (H(2)O(2)) induces necrotic cell death in Bax-/- Bak-/- mouse embryonic fibroblasts through a mechanism involving PARP-1 activation and ATP depletion. Next, we provide evidence that autophagy is activated in cells exposed to H(2)O(2). More importantly, we identify a novel autophagy signaling mechanism linking PARP-1 to the serine/threonine protein kinase LKB1-AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway, leading to stimulation of autophagy. Finally, we demonstrate that autophagy plays a cytoprotective role in H(2)O(2)-induced necrotic cell death, as suppression of autophagy by knockdown of autophagy-related gene ATG5 or ATG7 greatly sensitizes H(2)O(2)-induced cell death. Taken together, these findings demonstrate a novel function of PARP-1: promotion of autophagy through the LKB1-AMPK-mTOR pathway to enhance cell survival in cells under oxidative stress.     

Protective mechanism of FSH against oxidative damage in mouse ovarian granulosa cells by repressing autophagy

Oxidative stress-induced granulosa cell (GCs) death represents a common reason for follicular atresia. Follicle-stimulating hormone (FSH) has been shown to prevent GCs from oxidative injury, although the underlying mechanism remains to be elucidated. Here we first report that the suppression of autophagic cell death via some novel signaling effectors is engaged in FSH-mediated GCs protection against oxidative damage. The decline in GCs viability caused by oxidant injury was remarkably reduced following FSH treatment, along with impaired macroautophagic/autophagic flux under conditions of oxidative stress both in vivo and in vitro. Blocking of autophagy displayed similar levels of suppression in oxidant-induced cell death compared with FSH treatment, but FSH did not further improve survival of GCs pretreated with autophagy inhibitors. Further investigations revealed that activation of the phosphoinositide 3-kinase (PI3K)-AKT-MTOR (mechanistic target of rapamycin [serine/threonine kinase]) signaling pathway was required for FSH-mediated GCs survival from oxidative stress-induced autophagy. Additionally, the FSH-PI3K-AKT axis also downregulated the autophagic response by targeting FOXO1, whereas constitutive activation of FOXO1 in GCs not only abolished the protection from FSH, but also emancipated the autophagic process, from the protein level of MAP1LC3B-II to autophagic gene expression. Furthermore, FSH inhibited the production of acetylated FOXO1 and its interaction with Atg proteins, followed by a decreased level of autophagic cell death upon oxidative stress. Taken together, our findings suggest a new mechanism involving FSH-FOXO1 signaling in defense against oxidative damage to GCs by restraining autophagy, which may be a potential avenue for the clinical treatment of anovulatory disorders.     

β-cell death and proliferation after intermittent hypoxia: Role of oxidative stress

Intermittent hypoxia (IH), such as occurs in sleep apnea, induces increased oxidative stress and is associated with altered glucose homeostasis. Because pancreatic β cells are very sensitive to oxidative stress we tested whether they could be affected by IH. The effects of IH exposure (24 h/day, 5.7 and 21% O₂ alternation) in mice on β-cell proliferation and β-cell death were tested using Ki67 staining and TUNEL staining, respectively. To assess the role of oxidative stress in these processes, transgenic mice with β-cell-specific overexpression of the antioxidant protein MnSOD were exposed to IH. After 4 days of IH exposure, β-cell proliferation was increased almost fourfold. Coinciding with the increase in proliferation, the subcellular localization of the cell cycle regulator cyclin D2 was increased in the nucleus. In addition, β-cell death was increased approximately fourfold. MnSOD transgene did not alter the effects of IH on β-cell proliferation, but completely abrogated the IH effects on cell death. Thus, IH exposure that mimics sleep apnea can lead to increased β-cell proliferation and cell death. Furthermore, the cell death response seems to be due to oxidative stress.     

Modification of Proteins in Endothelial Cell Death during Oxidative Stress

Exposure of bovine aortic endothelial cells in vitro to oxidative stress causes a cascade of changes in cell function, culminating in cell death if the stress is sufficiently severe. Oxidative modification of proteins, as measured by the reaction of 2,4-dinitrophenylhydrazine with carbonyl groups of oxidized proteins, increased three- to fourfold in endothelial cells exposed to hydrogen peroxide or to a xanthine/xanthine oxidase system. The increase in oxidative modification of protein occurred rapidly, preceding loss of cellular ATP and eventual cell death. Oxidative modification of protein was paralleled by loss of activity of the key metabolic enzymes, glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. The finding that oxidative modification of protein is an early event following oxidative stress suggests that oxidative modification of protein is not only a marker for oxidative damage but also a causal factor in oxidative injury. Published by Elsevier Science Inc.