Down regulation of DJ-1 enhances cell death by oxidative stress, ER stress, and proteasome inhibition

Mutations in DJ-1 gene have been linked to autosomal recessive early onset parkinsonism (AR-EOP). Although the mechanism of neuronal cell death due to DJ-1 mutation has not been fully elucidated, loss of DJ-1 function was considered to cause the phenotype. Here, we demonstrated that the down regulation of endogenous DJ-1 of the neuronal cell line by siRNA enhanced the cell death which was induced by oxidative stress, ER stress, and proteasome inhibition, but not by pro-apoptotic stimulus. The cell death with hydrogen peroxide was dramatically rescued by over-expression of wild-type DJ-1, but not by that of L166P mutant DJ-1. Furthermore, DJ-1 rescued the cell death caused by over-expression of Pael receptor, which was a substrate of Parkin, another gene product for autosomal recessive juvenile parkinsonism. These results suggest that loss of protective activity of DJ-1 from neuro-toxicity induced by these stresses contributes to neuronal cell death in AR-EOP with mutant DJ-1.     

DJ-1 regulation of mitochondrial function and autophagy through oxidative stress

The dysregulation of mitochondrial function has been implicated in the pathogenesis of Parkinson disease.

Mutations in the parkin, PINK1 and DJ-1 genes all result in recessive parkinsonism. Although the protein products of these genes have not been fully characterized, it has been established that all three contribute to the maintenance of mitochondrial function. PINK1 and parkin act in a common pathway to regulate the selective autophagic removal of depolarized mitochondria, but the relationship between DJ-1 and PINK1- and/or parkin-mediated effects on mitochondria and autophagy is less clear. We have shown that loss of DJ-1 leads to mitochondrial phenotypes including reduced membrane potential, increased fragmentation and accumulation of autophagic markers. Supplementing DJ-1-deficient cells with glutathione reverses both mitochondrial and autophagic changes suggesting that DJ-1 may act to maintain mitochondrial function during oxidative stress and thereby alter mitochondrial dynamics and autophagy indirectly.     

DJ-1 regulation of mitochondrial function and autophagy through oxidative stress

The dysregulation of mitochondrial function has been implicated in the pathogenesis of Parkinson disease. Mutations in the parkin, PINK1 and DJ-1 genes all result in recessive parkinsonism. Although the protein products of these genes have not been fully characterized, it has been established that all three contribute to the maintenance of mitochondrial function. PINK1 and parkin act in a common pathway to regulate the selective autophagic removal of depolarized mitochondria, but the relationship between DJ-1 and PINK1- and/or parkin-mediated effects on mitochondria and autophagy is less clear. We have shown that loss of DJ-1 leads to mitochondrial phenotypes including reduced membrane potential, increased fragmentation and accumulation of autophagic markers. Supplementing DJ-1-deficient cells with glutathione reverses both mitochondrial and autophagic changes suggesting that DJ-1 may act to maintain mitochondrial function during oxidative stress and thereby alter mitochondrial dynamics and autophagy indirectly.     

Nuclear ULK1 promotes cell death in response to oxidative stress through PARP1

Reactive oxygen species (ROS) may cause cellular damage and oxidative stress-induced cell death. Autophagy, an evolutionarily conserved intracellular catabolic process, is executed by autophagy (ATG) proteins, including the autophagy initiation kinase Unc-51-like kinase (ULK1)/ATG1. Although autophagy has been implicated to have both cytoprotective and cytotoxic roles in the response to ROS, the role of individual ATG proteins, including ULK1, remains poorly characterized. In this study, we demonstrate that ULK1 sensitizes cells to necrotic cell death induced by hydrogen peroxide (H₂O₂). Moreover, we demonstrate that ULK1 localizes to the nucleus and regulates the activity of the DNA damage repair protein poly (ADP-ribose) polymerase 1 (PARP1) in a kinase-dependent manner. By enhancing PARP1 activity, ULK1 contributes to ATP depletion and death of H₂O₂-treated cells. Our study provides the first evidence of an autophagy-independent prodeath role for nuclear ULK1 in response to ROS-induced damage. On the basis of our data, we propose that the subcellular distribution of ULK1 has an important role in deciding whether a cell lives or dies on exposure to adverse environmental or intracellular conditions.Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide (H₂O₂), are formed by the incomplete reduction of oxygen during oxidative phosphorylation and other enzymatic processes. ROS are signaling molecules that regulate cell proliferation, differentiation, and survival.^{1, 2, 3} Accumulation of ROS (i.e., oxidative stress) on exposure to xenobiotic agents or environmental toxins can cause cellular damage and death via apoptotic or nonapoptotic pathways.^{4, 5, 6} Oxidative stress-induced cellular damage and death have been implicated in aging, ischemia-reperfusion injury, inflammation, and the pathogenesis of diseases (e.g., neurodegeneration and cancer).⁷ Oxidative stress also contributes to the antitumor effects of many chemotherapeutic drugs, including camptothecin^{8, 9} and selenite.^{10, 11}Autophagy, an evolutionarily conserved intracellular catabolic process, involves lysosome-dependent degradation of superfluous and damaged cytosolic organelles and proteins.¹² It is typically upregulated under conditions of perceived stress and in response to cellular damage. The consequence of autophagy activation – whether cytoprotective or cytotoxic – appears to depend on the cell type and the nature and extent of stress. Although most studies indicate a cytoprotective role for autophagy, some evidence suggests that it contributes to cell death in response to oxidative stress.^{13, 14, 15, 16, 17} Studies have also indicated that autophagy may be suppressed in response to oxidative stress, thereby sensitizing certain cells to apoptosis.^{18, 19} Unc-51-like kinase/autophagy 1 (ULK1/ATG1) is a mammalian serine–threonine kinase that regulates flux through the autophagy pathway by activating the VPS34 PI(3) kinase complex and facilitating ATG9-dependent membrane recycling.²⁰ Results from two studies suggest that ULK1 expression is altered in response to oxidative stress, and that the corresponding effects on autophagy contribute to cell death.^{18, 21}For example, p53-mediated upregulation of ULK1 and increase in autophagy promote cell death in osteosarcoma cells exposed to sublethal doses of camptothecin,²¹ yet mutant p53-mediated suppression of ULK1 impairs autophagic flux and promotes apoptosis in selenite-treated NB4 cells.¹⁸ Here we investigated the role of ULK1 in cells exposed to H₂O₂.     

Involvement of ERK1/2 signaling pathway in DJ-1-induced neuroprotection against oxidative stress

Parkinson’s disease (PD) is a progressive neurodegenerative disorder. Although the precise mechanism remains unclear, mounting evidence suggests that oxidative stress plays an important role in the pathogenesis of PD. DJ-1 gene is associated with oxidative stress and mutations in DJ-1 are involved in an autosomal recessive, early onset familial form of PD. The ERK1/2 signaling pathway contributes to neuroprotection during oxidative stress. However, the correlation between DJ-1 and the ERK1/2 signaling pathway remains unknown. To test for an association of DJ-1 with the ERK1/2 signaling pathway, we transfected wild-type and L166P mutated DJ-1 into COS-7 and MN9D cells. The results showed that over-expression of WT-DJ-1 dramatically enhanced the phosphorylation of ERK1/2 and its upstream kinase MEK1/2. Meanwhile, WT-DJ-1, but not L166P-DJ-1 inhibited the expression of protein phosphatase 2A (PP2A), an inhibitor of the ERK1/2 signaling pathway. Moreover, over-expression of WT-DJ-1 increased cell viability and decreased cell sensitivity to H₂O₂-induced neurotoxicity. Inhibition of the ERK1/2 signaling pathway with a MEK1/2 inhibitor reversed these changes. We conclude that DJ-1 does affect the ERK1/2 signaling pathway and change the susceptibility of cells to oxidative stress.     

DJ-1 has a role in antioxidative stress to prevent cell death

Deletion and point (L166P) mutations of DJ-1 have recently been shown to be responsible for the onset of familial Parkinson's disease (PD, PARK7). The aim of this study was to determine the role of DJ-1 in PD. We first found that DJ-1 eliminated hydrogen peroxide in vitro by oxidizing itself. We then found that DJ-1 knockdown by short interfering RNA rendered SH-SY5Y neuroblastoma cells susceptible to hydrogen peroxide-, MPP+- or 6-hydroxydopamine-induced cell death and that cells harbouring mutant forms of DJ-1, including L166P, became susceptible to death in parallel with the loss of oxidized forms of DJ-1. These results clearly showed that DJ-1 has a role in the antioxidative stress reaction and that mutations of DJ-1 lead to cell death, which is observed in PD.     

Deoxycholic acid modulates cell death signaling through changes in mitochondrial membrane properties

Cytotoxic bile acids, such as deoxycholic acid (DCA), are responsible for hepatocyte cell death during intrahepatic cholestasis. The mechanisms responsible for this effect are unclear, and recent studies conflict, pointing to either a modulation of plasma membrane structure or mitochondrial-mediated toxicity through perturbation of mitochondrial outer membrane (MOM) properties. We conducted a comprehensive comparative study of the impact of cytotoxic and cytoprotective bile acids on the membrane structure of different cellular compartments. We show that DCA increases the plasma membrane fluidity of hepatocytes to a minor extent, and that this effect is not correlated with the incidence of apoptosis. Additionally, plasma membrane fluidity recovers to normal values over time suggesting the presence of cellular compensatory mechanisms for this perturbation. Colocalization experiments in living cells confirmed the presence of bile acids within mitochondrial membranes. Experiments with active isolated mitochondria revealed that physiologically active concentrations of DCA change MOM order in a concentration- and time-dependent manner, and that these changes preceded the mitochondrial permeability transition. Importantly, these effects are not observed on liposomes mimicking MOM lipid composition, suggesting that DCA apoptotic activity depends on features of mitochondrial membranes that are absent in protein-free mimetic liposomes, such as the double-membrane structure, lipid asymmetry, or mitochondrial protein environment. In contrast, the mechanism of action of cytoprotective bile acids is likely not associated with changes in cellular membrane structure.     

Curcumin attenuates endothelial cell oxidative stress injury through Notch signaling inhibition

Previous studies have demonstrated that Notch signaling pathway plays a regulatory role in cellular oxidative stress injury (OSI). In this study, our aim was to explore the role of the Notch signaling pathway in hydrogen peroxide (H₂O₂)-induced OSI and the protective effect of curcumin during (H₂O₂)-induced injury in human umbilical vein endothelial cells (HUVECs). DAPT, a specific inhibitor of the Notch signaling pathway, and Notch1 siRNA were used to study Notch activity. Further, HUVECs were exposed to H₂O₂ in the absence or presence of curcumin. DAPT and Notch1 siRNA significantly inhibited OSI and the expression of Notch1 and Hes1. Curcumin conferred a protective effect on the HUVECs against H₂O₂, which was evidenced by improved cell viability, adhesive ability and migratory ability and a decreased apoptotic index, decreased production of reactive oxygen species (ROS) and a reduction in several biochemical parameters. Immunofluorescence and Western blotting analyses demonstrated that H₂O₂ treatment upregulated the expression of Notch1, Hes1, Caspase3, Bax and cytochrome c downregulated the expression of Bcl2, and treatment with curcumin reversed these effects. We demonstrated for the first time that the inhibition of Notch signaling pathway imparts a protective effect against endothelial OSI. The protective effects of curcumin against OSI are at least in part dependent on Notch1 inhibition.     

Avian influenza A virus H5N1 causes autophagy-mediated cell death through suppression of mTOR signaling

Of the few avian influenza viruses that have crossed the species barrier to infect humans,the highly pathogenic influenza A (H5N1) strain has claimed the lives of more than half of the infected patients.With largely unknown mechanism of lung injury by H5N1 infection,acute respiratory distress syndrome (ARDS) is the major cause of death among the victims.Here we present the fact that H5N1 caused autophagic cell death through suppression of mTOR signaling.Inhibition of autophagy,either by depletion of autophagy gene Beclinl or by autophagy inhibitor 3-methyladenine (3-MA),significantly reduced H5N1 mediated cell death.We suggest that autophagic cell death may contribute to the development of ARDS in H5N1 influenza patients and inhibition of autophagy could therefore become a novel strategy for the treatment of H5N1 infection.     

Jasmonic acid signaling modulates ozone-induced hypersensitive cell death

Recent studies suggest that cross-talk between salicylic acid (SA)-, jasmonic acid (JA)-, and ethylene-dependent signaling pathways regulates plant responses to both abiotic and biotic stress factors. Earlier studies demonstrated that ozone (O(3)) exposure activates a hypersensitive response (HR)-like cell death pathway in the Arabidopsis ecotype Cvi-0. We now have confirmed the role of SA and JA signaling in influencing O(3)-induced cell death. Expression of salicylate hydroxylase (NahG) in Cvi-0 reduced O(3)-induced cell death. Methyl jasmonate (Me-JA) pretreatment of Cvi-0 decreased O(3)-induced H(2)O(2) content and SA concentrations and completely abolished O(3)-induced cell death. Cvi-0 synthesized as much JA as did Col-0 in response to O(3) exposure but exhibited much less sensitivity to exogenous Me-JA. Analyses of the responses to O(3) of the JA-signaling mutants jar1 and fad3/7/8 also demonstrated an antagonistic relationship between JA- and SA-signaling pathways in controlling the magnitude of O(3)-induced HR-like cell death.     

Accelerated cell death 2 suppresses mitochondrial oxidative bursts and modulates cell death in Arabidopsis

The Arabidopsis ACCELERATED CELL DEATH 2 (ACD2) protein protects cells from programmed cell death (PCD) caused by endogenous porphyrin‐related molecules like red chlorophyll catabolite or exogenous protoporphyrin IX. We previously found that during bacterial infection, ACD2, a chlorophyll breakdown enzyme, localizes to both chloroplasts and mitochondria in leaves. Additionally, acd2 cells show mitochondrial dysfunction. In plants with acd2 and ACD2 ⁺ sectors, ACD2 functions cell autonomously, implicating a pro‐death ACD2 substrate as being cell non‐autonomous in promoting the spread of PCD. ACD2 targeted solely to mitochondria can reduce the accumulation of an ACD2 substrate that originates in chloroplasts, indicating that ACD2 substrate molecules are likely to be mobile within cells. Two different light‐dependent reactive oxygen bursts in mitochondria play prominent and causal roles in the acd2 PCD phenotype. Finally, ACD2 can complement acd2 when targeted to mitochondria or chloroplasts, respectively, as long as it is catalytically active: the ability to bind substrate is not sufficient for ACD2 to function in vitro or in vivo. Together, the data suggest that ACD2 localizes dynamically during infection to protect cells from pro‐death mobile substrate molecules, some of which may originate in chloroplasts, but have major effects on mitochondria.     

Mitochondria,oxidative stress and cell death

In addition to the well-established role of the mitochondria in energy metabolism, regulation of cell death has recently emerged as a second major function of these organelles. This, in turn, seems to be intimately linked to their role as the major intracellular source of reactive oxygen species (ROS), which are mainly generated at Complex I and III of the respiratory chain. Excessive ROS production can lead to oxidation of macromolecules and has been implicated in mtDNA mutations, ageing, and cell death. Mitochondria-generated ROS play an important role in the release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation and apoptosis. Cytochrome c release occurs by a two-step process that is initiated by the dissociation of the hemoprotein from its binding to cardiolipin, which anchors it to the inner mitochondrial membrane. Oxidation of cardiolipin reduces cytochrome c binding and results in an increased level of “free” cytochrome c in the intermembrane space. Conversely, mitochondrial antioxidant enzymes protect from apoptosis. Hence, there is accumulating evidence supporting a direct link between mitochondria, oxidative stress and cell death.     

肾素-血管紧张素系统通过氧化应激机制参与血管反应性的调节

肾素-血管紧张素系统过度激活导致血管氧化应激损伤,进而影响血管功能.xanthine氧化酶、NAD(P)H氧化酶和脱耦联的NO合酶是血管组织中活性氧的主要来源.超氧化物阴离子和活性氧簇中的其他成分通过多种机制失活NO在心血管疾病的发生和发展中具有重要作用.随着对氧化应激损伤参与血管反应性调节机制的认识逐渐加深,有希望通过抑制氧化应激损伤改善血管内皮功能.     

Zinc chelator TPEN induces pancreatic cancer cell death through causing oxidative stress and inhibiting cell autophagy

The essential trace element zinc (Zn) is widely required in cellular functions, and abnormal Zn homeostasis causes a variety of health problems including immunodeficiency and sensory dysfunctions. Previous studies had shown that Zn availability was also important for tumor growth and progression. The aim of the present study was to investigate the potential mechanisms of N,N,N,N-Tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) (a membrane permeable zinc chelator) induced pancreatic cancer cell death. The text of inductively coupled plasma-mass spectrometry (ICP-MS) showed in human pancreatic cancer samples that the zinc content in cancer was higher than that in adjacent tissues. The pancreatic cancer cell lines Panc-1, 8988T, BxPc-3, and L3.6 were used in this study. Our results indicated that TPEN markedly induced cell death, via increasing reactive oxygen species (ROS) and restraining autophagy. Our data also indicated that TPEN-stimulated mitochondrial metabolism produced much ROS. Meanwhile, TPEN reduced the levels of glutathione (GSH) and triggered ROS outbreak, which were the main causes of cell death. In addition, cell autophagy was significantly depressed in Panc-1 cells treated by TPEN, which was due to the ability of disrupting lysosomal by TPEN. Thus, we thought zinc depletion by TPEN was a potential therapeutic strategy for pancreatic cancer.     

Roles of Drosophila DJ-1 in survival of dopaminergic neurons and oxidative stress

The loss of dopaminergic neurons in the substantia nigra is the pathological hallmark of Parkinson's disease (PD). While the etiology of sporadic PD remains elusive, an inherited form of early-onset familial PD is linked to mutations of DJ-1. To understand the biological function of DJ-1 and its relevance to the pathogenesis of PD, we investigated the function of DJ-1 using Drosophila. Drosophila possesses two homologs of human DJ-1: DJ-1alpha and DJ-1beta. We found that DJ-1alpha is expressed predominantly in the testis, while DJ-1beta is ubiquitously present in most tissues, resembling the expression pattern of human DJ-1. Loss-of-function DJ-1beta mutants demonstrated an extended survival of dopaminergic neurons and resistance to paraquat stress, but showed acute sensitivity to hydrogen peroxide treatment. We showed a compensatory upregulation of DJ-1alpha expression in the brain of the DJ-1beta mutant and demonstrated that overexpression of DJ-1alpha in dopaminergic neurons is sufficient to confer protection against paraquat insult. These results suggest that Drosophila homologs of DJ-1 play critical roles in the survival of dopaminergic neurons and response to oxidative stress.     

Melatonin inhibits breast cancer cell invasion through modulating DJ-1/KLF17/ID-1 signaling pathway

Breast cancer is the most common neoplastic disorder diagnosed in women. The main goal of this study was to explore the effect of melatonin against breast cancer metastasis and compared this with the actions of taxol (a well-known chemotherapeutic drug), and the impact of their combination against breast cancer metastasis. Melatonin showed no cytotoxic effect while taxol showed antiproliferative and cytotoxic effects on MCF-7 and MDA-MB-231 cells. Furthermore, melatonin inhibited the generation of reactive oxygen species. Melatonin and taxol clearly decreased cell migration and invasion at low doses, especially those matching the normal physiological concentration at night. Melatonin and taxol markedly reduced DJ-1 and ID-1 and increased KLF17 messenger RNA and protein expression levels. The present results also showed that melatonin and taxol induced GSK3-β nuclear and Snail cytosolic localization. These changes were accompanied by a concurrent rise in E-cadherin expression. The above data show that normal levels of melatonin may help in preventing breast cancer metastasis through inhibiting DJ-1/KLF17/ID-1 signaling pathway. The combination of melatonin and taxol is a potent candidate against breast cancer metastasis, better than using melatonin or taxol as a single drug.