Autophagy inhibition induces podocyte apoptosis by activating the pro-apoptotic pathway of endoplasmic reticulum stress

Podocyte apoptosis is a major factor inducing podocyte depletion that predicts the progressive course of glomerulosclerosis. However, the molecular mechanisms underlying podocyte apoptosis are still not well understood. Autophagy is a lysosomal degradation system involving the degradation and recycling of obsolete, damaged, or harmful cytoplasmic materials and organelles. Recent advances in the understanding of the molecular processes contributing to autophagy have provided insight into the relationship between autophagy and apoptosis. However, their cross-talk remains largely obscure until now. Here, we found that podocytes both in vivo and in vitro always exhibited high basal levels of autophagy, whereas autophagy inhibition could induce podocyte apoptosis, suggesting the pro-survival role of autophagy in podocytes. Besides, we found that autophagy inhibition by 3-methyladenine (3-MA) could induce the activation of endoplasmic reticulum stress even without any external stimulations, whereas knockdown of CHOP could effectively improve podocyte apoptosis and down-regulated expression of slit-diaphragm proteins induced by autophagy inhibition. Collectively, this study demonstrated that autophagy might act as a crucial regulatory mechanism of apoptotic cell death by modulating the balance between the pro-survival pathway and the pro-apoptotic pathway of endoplasmic reticulum stress, which might provide a novel mechanistic insight into the interface between autophagy and apoptosis in the progression of podocyte injury.     

Hypoxia induces the activation of the phosphatidylinositol 3-kinase/Akt cell survival pathway in PC12 cells: protective role in apoptosis

Hypoxia is a common environmental stress that influences signaling pathways and cell function. Several cell types, including neuroendocrine chromaffin cells, have evolved to sense oxygen levels and initiate specific adaptive responses to hypoxia. Here we report that under hypoxic conditions, rat pheochromocytoma PC12 cells are resistant to apoptosis induced by serum withdrawal and chemotherapy treatment. This effect is also observed after treatment with deferoxamine, a compound that mimics many of the effects of hypoxia. The hypoxia-dependent protection from apoptosis correlates with activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is detected after 3-4 h of hypoxic or deferoxamine treatment and is sustained while hypoxic conditions are maintained. Hypoxia-induced Akt activation can be prevented by treatment with cycloheximide or actinomycin D, suggesting that de novo protein synthesis is required. Finally, inhibition of PI3K impairs both the protection against apoptosis and the activation of Akt in response to hypoxia, suggesting a functional link between these two phenomena. Thus, reduced oxygen tension regulates apoptosis in PC12 cells through activation of the PI3K/Akt survival pathway.     

Neuroprotective effect of the stearic acid against oxidative stress via phosphatidylinositol 3-kinase pathway

Stearic acid is a long-chain saturated fatty acid consisting of 18 carbon atoms without double bonds. In the present study, we reported the neuroprotective effects and mechanism of stearic acid on cortical or hippocampal slices insulted by oxygen-glucose deprivation, NMDA or hydrogen peroxide (H(2)O(2)) in vitro. Different types of models of brain slice injury in vitro were developed by 10 min of oxygen/glucose deprivation, 0.5 mM NMDA or 2 mM H(2)O(2), respectively. After 30 min of preincubation with stearic acid (3-30 microM), cortical or hippocampal slices were subjected to oxygen-glucose deprivation, NMDA or H(2)O(2). Then the tissue activities were evaluated by using the 2,3,5-triphenyltetrazolium chloride (TTC) method. Population spikes were recorded in randomly selected hippocampal slices. Stearic acid (3-30 microM) dose-dependently protected brain slices from oxygen-glucose deprivation, NMDA and H(2)O(2) insults. Its neuroprotective effect against H(2)O(2) insults can be completely blocked by wortmannin (inhibitor of PI3K) and partially blocked by H7 (inhibitor of PKC) or genistein (inhibitor of TPK). Treatment of cortical or hippocampal slices with 30 microM stearic acid resulted in a significant increase in PI3K activity at 5, 10, 30 and 60 min. These observations reveal that stearic acid can protect cortical or hippocampal slices against injury induced by oxygen-glucose deprivation, NMDA or H(2)O(2), and its neuroprotective effects are via phosphatidylinositol 3-kinase dependent mechanism.     

Taurine protection of PC12 cells against endoplasmic reticulum stress induced by oxidative stress

Background

Taurine is a free amino acid present in high concentrations in a variety of organs of mammalians. As an antioxidant, taurine has been found to protect cells against oxidative stress, but the underlying mechanism is still unclear.

Methods

In this report, we present evidence to support the conclusion that taurine exerts a protective function against endoplasmic reticulum (ER) stress induced by H₂O₂ in PC 12 cells. Oxidative stress was introduced by exposure of PC 12 cells to 250 uM H₂O₂ for 4 hours.

Results

It was found that the cell viability of PC 12 cells decreased with an increase of H₂O₂ concentration ranging from approximately 76% cell viability at 100 uM H₂O₂ down to 18% at 500 uM H₂O₂. At 250 uM H₂O₂, cell viability was restored to 80% by taurine at 25 mM. Furthermore, H₂O₂ treatment also caused a marked reduction in the expression of Bcl-2 while no significant change of Bax was observed. Treatment with taurine restored the reduced expression of Bcl-2 close to the control level without any obvious effect on Bax. Furthermore, taurine was also found to suppress up-regulation of GRP78, GADD153/CHOP and Bim induced by H₂O₂, suggesting that taurine may also exert a protective function against oxidative stress by reducing the ER stress.

Conclusion

In summary, taurine was shown to protect PC12 cells against oxidative stress induced by H₂O₂. ER stress was induced by oxidative stress and can be suppressed by taurine.

     

Changes in mitochondrial function induced by dequalinium precede oxidative stress and apoptosis in the human prostate-cancer cell line PC-3

Mitochondria play central roles in diverse physiological and pathological conditions associated with cell survival and death. Delocalized lipophilic cations, such as dequalinium (DQA), are accumulated in cancer cells attracted by the highly negative mitochondrial transmembrane potential of these cells. DQA showed a potent anticancer activity in cells from different malignancies. Here, we report the effect of DQA on PC-3 prostate cancer cells. Incubation with DQA at concentrations between 1.5 and 100 μM from 24 to 48 h decreases cell viability. The decrease in cell viability together with a loss of mitochondrial transmembrane potential induced an increase in reactive oxygen species production and cell death via caspase-3 dependent apoptotic pathway. DQA was shown to cause moderate to strong cell death in a time and concentration dependent manner, causing a most advantageous effect at a concentration of 10 μM applied for a long 48 h time period, which might be a consequence of the kinetics of intracellular DQA accumulation in mitochondria, but also of the mechanisms of DQA-induced cell death. This data shows DQA as a promising agent against the human prostate cancer PC-3 cell line, activating the caspase-3 dependent apoptotic pathway. This fact might be beneficial for possible future applications in cancer therapy.     

Mitochondrial reactive oxygen species signal hepatocyte steatosis by regulating the phosphatidylinositol 3-kinase cell survival pathway

Abnormal dietary intake of macronutrients is implicated in the development of obesity and fatty liver disease. Steatosis develops in cultured hepatocytes exposed to medium containing either a high concentration of long chain free fatty acids (HFFA) or medium deficient in methionine and choline (MCD). This study examined the mitochondrial reactive oxygen species (ROS)-dependent regulation of the phosphoinositol (PI) 3-kinase pathway in steatosis induced by exposure of AML-12 mouse hepatocytes to MCD or HFFA medium. Exposure to either MCD or HFFA medium resulted in increased production of superoxide anions and H(2)O(2), transduction of the PI 3-kinase pathway and steatosis. Inhibition of PI 3-kinase with LY294002 prevented steatosis. Pharmacologically inhibiting electron transport chain complex III production of ROS prevented activation of PI 3-kinase during macronutrient perturbation, whereas pharmacologically promoting electron transport chain complex III ROS production activated PI 3-kinase independent of nutrient input. The data suggest that H(2)O(2) is the ROS species involved in signal transduction; promoting the rapid conversion of superoxide to H(2)O(2) does not inhibit PI 3-kinase pathway activation during nutrient perturbation, and exogenous H(2)O(2) activates it independent of nutrient input. In addition to transducing PI 3-kinase, the ROS-dependent signal cascade amplifies the PI 3-kinase signal by maintaining phosphatase and tensin homolog in its inactive phosphorylated state. Knockdown of phosphatase and tensin homolog by small interfering RNA independently activated the PI 3-kinase pathway. Our findings suggest a common path for response to altered nutrition involving mitochondrial ROS-dependent PI 3-kinase pathway regulation, leading to steatosis.     

A dibenzoylmethane derivative protects dopaminergic neurons against both oxidative stress and endoplasmic reticulum stress

The enhancement of intracellular stresses such as oxidative stress and endoplasmic reticulum (ER) stress has been implicated in several neurodegenerative disorders including Parkinson's disease (PD). During a search for compounds that regulate ER stress, a dibenzoylmethane (DBM) derivative 14-26 (2,2'-dimethoxydibenzoylmethane) was identified as a novel neuroprotective agent. Analysis in SH-SY5Y cells and in PC12 cells revealed that the regulation of ER stress by 14-26 was associated with its anti-oxidative property. 14-26 prevented the production of reactive oxygen species (ROS) when the cells were exposed to oxidants such as hydrogen peroxide and 6-hydroxydopamine (6-OHDA) or an ER stressor brefeldin A (BFA). 14-26 also prevented ROS-induced damage in both the ER and the mitochondria, including the protein carbonylation in the microsome and the reduction of the mitochondrial membrane potential. Further examination disclosed the presence of the iron-chelating activity in 14-26. In vivo, 14-26 suppressed both oxidative stress and ER stress and prevented neuronal death in the substantia nigra pars compacta (SNpc) after injection of 6-OHDA in mice. These results suggest that 14-26 is an antioxidant that protects dopaminergic neurons against both oxidative stress and ER stress and could be a therapeutic candidate for the treatment of PD.     

Coupling endoplasmic reticulum stress to the cell death program. An Apaf-1-independent intrinsic pathway

Accumulation of misfolded proteins and alterations in Ca2+ homeostasis in the endoplasmic reticulum (ER) causes ER stress and leads to cell death. However, the signal-transducing events that connect ER stress to cell death pathways are incompletely understood. To discern the pathway by which ER stress-induced cell death proceeds, we performed studies on Apaf-1(-/-) (null) fibroblasts that are known to be relatively resistant to apoptotic insults that induce the intrinsic apoptotic pathway. While these cells were resistant to cell death initiated by proapoptotic stimuli such as tamoxifen, they were susceptible to apoptosis induced by thapsigargin and brefeldin-A, both of which induce ER stress. This pathway was inhibited by catalytic mutants of caspase-12 and caspase-9 and by a peptide inhibitor of caspase-9 but not by caspase-8 inhibitors. Cleavage of caspases and poly(ADP-ribose) polymerase was observed in cell-free extracts lacking cytochrome c that were isolated from thapsigargin or brefeldin-treated cells. To define the molecular requirements for this Apaf-1 and cytochrome c-independent apoptosis pathway further, we developed a cell-free system of ER stress-induced apoptosis; the addition of microsomes prepared from ER stress-induced cells to a normal cell extract lacking mitochondria or cytochrome c resulted in processing of caspases. Immunodepletion experiments suggested that caspase-12 was one of the microsomal components required to activate downstream caspases. Thus, ER stress-induced programmed cell death defines a novel, mitochondrial and Apaf-1-independent, intrinsic apoptotic pathway.     

Molecular components of a cell death pathway activated by endoplasmic reticulum stress

Alterations in Ca2+ homeostasis and accumulation of misfolded proteins in the endoplasmic reticulum (ER) cause ER stress that ultimately leads to programmed cell death. Recent studies have shown that ER stress triggers programmed cell death via an alternative intrinsic pathway of apoptosis that, unlike the intrinsic pathway described previously, is independent of Apaf-1 and cytochrome c. In the present work, we have used a set of complementary approaches, including two-dimensional gel electrophoresis coupled with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and nano-liquid chromatography-electrospray ionization mass spectrometry with tandem mass spectrometry, RNA interference, co-immunoprecipitation, immunodepletion of candidate proteins, and reconstitution studies, to identify mediators of the ER stress-induced cell death pathway. Our data identify two molecules, valosin-containing protein and apoptosis-linked gene-2 (ALG-2), that appear to play a role in mediating ER stress-induced cell death.     

Shikonin selectively induces apoptosis in human prostate cancer cells through the endoplasmic reticulum stress and mitochondrial apoptotic pathway

Background

Despite the recent progress in screening and therapy, a majority of prostate cancer cases eventually attain hormone refractory and chemo-resistant attributes. Conventional chemotherapeutic strategies are effective at very high doses for only palliative management of these prostate cancers. Therefore chemo-sensitization of prostate cancer cells could be a promising strategy for increasing efficacy of the conventional chemotherapeutic agents in prostate cancer patients. Recent studies have indicated that the chemo-preventive natural agents restore the pro-apoptotic protein expression and induce endoplasmic reticulum stress (ER stress) leading to the inhibition of cellular proliferation and activation of the mitochondrial apoptosis in prostate cancer cells. Therefore reprogramming ER stress-mitochondrial dependent apoptosis could be a potential approach for management of hormone refractory chemoresistant prostate cancers. We aimed to study the effects of the natural naphthoquinone Shikonin in human prostate cancer cells.

Results

The results indicated that Shikonin induces apoptosis in prostate cancer cells through the dual induction of the endoplasmic reticulum stress and mitochondrial dysfunction. Shikonin induced ROS generation and activated ER stress and calpain activity. Moreover, addition of antioxidants attenuated these effects. Shikonin also induced the mitochondrial apoptotic pathway mediated through the enhanced expression of the pro-apoptotic Bax and inhibition of Bcl-2, disruption of the mitochondrial membrane potential (MMP) followed by the activation of caspase-9, caspase-3, and PARP cleavage.

Conclusion

The results suggest that shikonin could be useful in the therapeutic management of hormone refractory prostate cancers due to its modulation of the pro-apoptotic ER stress and mitochondrial apoptotic pathways.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0127-1) contains supplementary material, which is available to authorized users.     

Polystyrene nanoplastics aggravates lipopolysaccharide-induced apoptosis in mouse kidney cells by regulating IRE1/XBP1 endoplasmic reticulum stress pathway via oxidative stress

Nanoplastics (NPs) pollution poses a huge threat to the ecosystem and has become one of the environmental pollutants that have attracted much attention. There is increasing evidence that both oxidative stress and endoplasmic reticulum stress (ERS) are associated with polystyrene nanoplastics (PS-NPs) exposure. Lipopolysaccharide (LPS) has been shown to induce apoptotic damage in various tissues, but whether PS-NPs can aggravate LPS-induced apoptosis in mouse kidneys through oxidative stress-regulated inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) ERS pathway remains unclear. In this study, based on the establishment of in vitro and in vivo PS-NPs and LPS exposure models alone and in combination in mice and HEK293 cells, the effects and mechanisms of PS-NPs on LPS-induced renal cell apoptosis were investigated. The results showed that PS-NPs could aggravate LPS-induced apoptosis. PS-NPs/LPS can induce ERS through oxidative stress, activate the IRE1/XBP1 pathway, and promote the expression of apoptosis markers (Caspase-3 and Caspase-12). Kidney oxidative stress, ERS, and apoptosis in PS-NPs + LPS combined exposure group were more severe than those in the single exposure group. Interestingly, 4-phenylbutyric acid-treated HEK293 cells inhibited the expression of the IRE1/XBP1 ERS pathway and apoptotic factors in the PS-NPs + LPS combined exposure group. N-acetyl-L-cysteine effectively blocked the activation of the IRE1/XBP1 ERS pathway, suggesting that PS-NPs-induced oxidative stress is an early event that triggers ERS. Collectively, these results confirmed that PS-NPs aggravated LPS-induced apoptosis through the oxidative stress-induced IRE1/XBP1 ERS pathway. Our study provides new insights into the health threats of PS-NPs exposed to mammals and humans.     

氧化应激对原代培养乳鼠心房肌细胞中内质网应激及凋亡因子的影响

目的：研究氧化应激对原代培养乳鼠心房肌细胞凋亡、内质网应激及凋亡因子的影响。方法：实验分2组：对照组、氧化应激组。原代培养乳鼠心房肌细胞,氧化应激组在培养的原代心房肌细胞中加入终浓度为100μmol/L的H₂O₂培养2 h,检测氧化和抗氧化指标超氧化物歧化酶（SOD）活力、丙二醛（MDA）及还原型谷胱甘肽（GSH）含量;检测细胞凋亡、细胞GRP78、GRP94及chop、bax、bcl-2 mRNA表达。结果：与对照组相比较,氧化应激组心房肌细胞SOD活力和GSH含量下降、MDA含量增加（P < 0.01）,细胞凋亡增加（P < 0.01）,细胞GRP78、GRP94、chop、bax mRNA表达增加、bcl-2 mRNA表达减少（P < 0.01）。结论：氧化应激反应可能介导内质网应激反应并激活促凋亡因子表达,抑制抗凋亡因子表达,引起心房肌细胞凋亡增加。这可能与心房纤颤的发生有一定关联性。     

NADPH oxidase links endoplasmic reticulum stress, oxidative stress, and PKR activation to induce apoptosis

Endoplasmic reticulum (ER)-induced apoptosis and oxidative stress contribute to several chronic disease processes, yet molecular and cellular mechanisms linking ER stress and oxidative stress in the setting of apoptosis are poorly understood and infrequently explored in vivo. In this paper, we focus on a previously elucidated ER stress-apoptosis pathway whose molecular components have been identified and documented to cause apoptosis in vivo. We now show that nicotinamide adenine dinucleotide phosphate reduced oxidase (NOX) and NOX-mediated oxidative stress are induced by this pathway and that apoptosis is blocked by both genetic deletion of the NOX subunit NOX2 and by the antioxidant N-acetylcysteine. Unexpectedly, NOX and oxidative stress further amplify CCAAT/enhancer binding protein homologous protein (CHOP) induction through activation of the double-stranded RNA-dependent protein kinase (PKR). In vivo, NOX2 deficiency protects ER-stressed mice from renal cell CHOP induction and apoptosis and prevents renal dysfunction. These data provide new insight into how ER stress, oxidative stress, and PKR activation can be integrated to induce apoptosis in a pathophysiologically relevant manner.     

Activation of phosphatidylinositol 3-kinase by cellular prion protein and its role in cell survival

The cellular prion protein (PrP(C)) is thought to be involved in protection against cell death, however the exact cellular mechanisms involved are still controversial. Herein we present data that strongly indicate a functional link between PrP(C) expression and phosphatidylinositol 3-kinase (PI 3-kinase) activation, a protein kinase that plays a pivotal role in cell survival. Both mouse neuroblastoma N2a cells and immortalized murine hippocampal neuronal cell lines expressing wild-type PrP(C) had significantly higher PI 3-kinase activity levels than their respective controls. Moreover, PI 3-kinase activity was found to be elevated in brain lysates from wild-type mice, as compared to prion protein-knockout mice. Recruitment of PI 3-kinase by PrP(C) was shown to contribute to cellular survival toward oxidative stress by using 3-morpholinosydnonimine (SIN-1) and serum deprivation. Moreover, both PI 3-kinase activation and cytoprotection by PrP(C) appeared to rely on copper binding to the N-terminal octapeptide of PrP(C). Thus, we propose a model in which the interaction of copper(II) with the N-terminal domain of PrP(C) enables transduction of a signal to PI 3-kinase; the latter, in turn, mediates downstream regulation of cell survival.     

Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress

The ability to respond to perturbations in endoplasmic reticulum (ER) function is a fundamentally important property of all cells, but ER stress can also lead to apoptosis. In settings of chronic ER stress, the associated apoptosis may contribute to pathophysiological processes involved in a number of prevalent diseases, including neurodegenerative diseases, diabetes, atherosclerosis and renal disease. The molecular mechanisms linking ER stress to apoptosis are the topic of this review, with emphases on relevance to pathophysiology and integration and complementation among the various apoptotic pathways induced by ER stress.     

The inhibition of phosphatidylinositol 3-kinase by quercetin and analogs.

Phosphatidylinositol (PtdIns) 3-kinase is an enzyme involved in cellular responses to growth factors. Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyrano-4-one), a naturally occuring bioflavinoid, was found to inhibit PtdIns 3-kinase with an IC50 of 1.3 micrograms/ml (3.8 microM); inhibition appears to be directed towards the ATP binding site of the kinase. Analogs of quercetin were also investigated as PtdIns 3-kinase inhibitors, with the most potent compounds exhibiting IC50's in the range of 1.7-8.4 micrograms/ml (5-19 microM). In contrast, genistein, a potent tyrosine kinase inhibitor of the isoflavone class, did not inhibit PtdIns 3-kinase significantly (IC50 greater than 30 micrograms/ml). These findings suggest that flavinoids may serve as potent inhibitors of PtdIns 3-kinase. Furthermore, the enzyme is much more sensitive to substituents at the 3-position of the flavinoid ring than are other protein and PtdIns kinases, suggesting that specific inhibitors of PtdIns 3-kinase can be developed to explore the biological role of the enzyme in cellular proliferation and growth factor response.     

Exercise protects against diabetic cardiomyopathy by the inhibition of the endoplasmic reticulum stress pathway in rats

To explore the protective effect of exercise training on the injury of myocardium tissues induced by streptozotocin (STZ) in diabetic rats and the relationship with endoplasmic reticulum stress (ERS), the male sprague-dawley (SD) rats were fed with high-fat and high-sugar diet for 4 weeks, followed by intraperitoneal injection of STZ, 40 mg/kg, to establish a diabetes model, and then 10 rats were randomly selected as diabetes mellitus (DM) controls and 20 eligible diabetic rats were randomized into two groups: low-intensity exercise training (n = 10) and high-intensity exercise training (n = 10). After 12 weeks of exercise training, rats were killed and serum samples were used to determine cardiac troponin-I (cTn-I). Myocardial tissues were sampled for morphological analysis to detect myocardial cell apoptosis, and to analyze protein expression of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and caspase-12. Different intensities (low and high) significantly reduced serum cTn-I levels compared with the DCM group (p < 0.01), and significantly reduced the percentage of apoptotic myocardial cells and improved the parameters of cardiac function. Hematoxylin and eosin and Masson staining indicated that exercise training could attenuate myocardial apoptosis. Additionally, exercise training significantly reduced GRP78, CHOP, and cleaved caspase-12 protein expression in an intensity-dependent manner. These findings suggest that exercise appeared to ameliorate diabetic cardiomyopathy by inhibiting endoplasmic reticulum stress-induced apoptosis in diabetic rats.     

Ischemia-induced neuronal cell death is mediated by the endoplasmic reticulum stress pathway involving CHOP

Brain ischemia induces apoptosis in neuronal cells, but the mechanism is not well understood. When wild-type mice were subjected to bilateral common carotid arteries occlusion (BCCAO) for 15 min, apoptosis-associated morphological changes and appearance of TUNEL-positive cells were observed in the striatum and in the hippocampus at 48 h after occlusion. RT-PCR analysis revealed that mRNAs for ER stress-associated proapoptotic factor CHOP and an ER chaperone BiP are markedly induced at 12 h after BCCAO. Immunohistochemical analysis showed that CHOP protein is induced in nuclei of damaged neurons at 24 h after occlusion. In contrast, ischemia-associated apoptotic loss of neurons was decreased in CHOP(-/-) mice. Primary hippocampal neurons from CHOP(-/-) mice were more resistant to hypoxia-reoxygenation-induced apoptosis than those from wild-type animals. These results indicate that ischemia-induced neuronal cell death is mediated by the ER stress pathway involving CHOP induction.