Klotho Suppresses Cardiomyocyte Apoptosis in Mice with Stress-Induced Cardiac Injury via Downregulation of Endoplasmic Reticulum Stress

Cardiomyocyte apoptosis is a common pathological alteration in heart disease which results in systolic dysfunction or sudden death. Klotho is a novel anti-aging hormone. We tested the effects of klotho on cell apoptosis in isoproterenol-treated cardiomyocytes. In BALB/c mice, cardiac injury was induced by subcutaneous injection of isoproterenol (5mg/kg, for 9days, sc). Klotho (0.01 mg/kg, every other day for 4days, ip) was administered to determine the changes in isoproterenol-induced apoptosis. Mouse heart was harvested at day 2, day 5, and day 9 after isoproterenol injection. Isoproterenol induced cardiac apoptosis and endoplasmic reticulum (ER) stress in a time-dependent manner. However, klotho partly reversed isoproterenol-induced cardiac apoptosis and ER stress. These same effects were observed in cultured cardiomyocytes. Furthermore, the results also showed that SB203580, a p38 inhibitor, and SP600125, a c-Jun NH2-terminal kinase (JNK) inhibitor, reduced cardiomyocyte apoptosis and ER stress, however, klotho suppressed isoproterenol-induced activation of p38 and JNK. Taken together, these results indicated that cardioprotection by klotho was related to the attenuation of ER stress and ER stress-induced apoptosis, at least partly, through suppressing activation of the p38 and JNK pathway.     

Derlin-1 Regulates Mutant VCP-Linked Pathogenesis and Endoplasmic Reticulum Stress-Induced Apoptosis

Mutations in VCP (Valosin-containing protein), an AAA ATPase critical for ER-associated degradation, are linked to IBMPFD (Inclusion body myopathy with Paget disease and frontotemporal dementia). Using a Drosophila IBMPFD model, we have identified the ER protein Derlin-1 as a modifier of pathogenic TER94 (the fly VCP homolog) mutants. Derlin-1 binds to TER94 directly, and this interaction is essential for Derlin-1 overexpression to suppress the pathogenic TER94-induced neurodegeneration. Derlin-1 overexpression reduces the elevated ATPase activity of pathogenic TER94, implying that IBMPFD is caused by ATPase hyper-activation. Under physiological condition, Derlin-1 expression is increased upon ER stress to recruit TER94 to the ER. However, in response to severe ER stress, Derlin-1 is required for activating apoptosis to eliminate damaged cells. This pro-apoptotic response is mimicked by Derlin-1 overexpression, which elicits acute ER stress and triggers apoptosis via a novel C-terminal motif (α). As this Derlin-1-dependent cell death is negated by TER94 overexpression, we propose that while Derlin-1 and VCP work cooperatively in ER stress response, their imbalance has a role in removing cells suffering prolonged ER stress.     

Endoplasmic Reticulum Stress and Autophagy in Homocystinuria Patients with Remethylation Defects

Proper function of endoplasmic reticulum (ER) and mitochondria is crucial for cellular homeostasis, and dysfunction at either site as well as perturbation of mitochondria-associated ER membranes (MAMs) have been linked to neurodegenerative and metabolic diseases. Previously, we have observed an increase in ROS and apoptosis levels in patient-derived fibroblasts with remethylation disorders causing homocystinuria. Here we show increased mRNA and protein levels of Herp, Grp78, IP₃R1, pPERK, ATF4, CHOP, asparagine synthase and GADD45 in patient-derived fibroblasts suggesting ER stress and calcium perturbations in homocystinuria. In addition, overexpressed MAM-associated proteins (Grp75, σ-1R and Mfn2) were found in these cells that could result in mitochondrial calcium overload and oxidative stress increase. Our results also show an activation of autophagy process and a substantial degradation of altered mitochondria by mitophagy in patient-derived fibroblasts. Moreover, we have observed that autophagy was partially abolished by antioxidants suggesting that ROS participate in this process that may have a protective role. Our findings argue that alterations in Ca²⁺ homeostasis and autophagy may contribute to the development of this metabolic disorder and suggest a therapeutic potential in homocystinuria for agents that stabilize calcium homeostasis and/or restore the proper function of ER-mitochondria communications.     

内质网应激与自噬及其交互作用影响内皮细胞凋亡

内质网应激是普遍存在于真核细胞中的应激-防御机制。在内环境稳态遭到破坏的情况下,未折叠蛋白质反应的3条信号通路,分别通过增强蛋白质折叠能力、减少蛋白质生成和促进内质网相关蛋白质降解等途径缓解细胞内压力。同时,也通过多种分子信号机制调控细胞凋亡。自噬是一种生理性的降解机制。通过形成自噬泡并与溶酶体结合摄取并水解胞内受损细胞器和蛋白质等,清除代谢废物,维持细胞正常功能。自噬缺陷或过度激活均可导致细胞凋亡或非程序性死亡。自噬的程度和细胞内压力水平有关。内质网应激通过未折叠蛋白质反应和Ca²⁺浓度变化及其相关分子信号调控自噬。自噬又可反馈性调节内质网应激反应,二者相互作用,在内皮细胞凋亡过程中发挥重要作用。未来内质网应激和自噬可作为药物靶点为内皮相关性疾病提供诊疗策略。     

内质网应激诱导自噬的分子机制及意义

细胞对内质网应激(endoplasmic reticulum stress,ER stress)的早期反应是启动未折叠反应(unfolded protein response,UPR),ER stress及UPR与人类多种疾病的发病机制相关.近年来的研究表明ER stress可诱导细胞自噬(autophagy).内质网应激诱导的自噬反应既可以帮助细胞减轻ER stress反应,又可以引导细胞发生非凋亡性死亡.本文将对ER stress诱导自噬的分子机制及可能的临床意义进行阐述.     

PERK-Dependent Activation of JAK1 and STAT3 Contributes to Endoplasmic Reticulum Stress-Induced Inflammation

Neuroinflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. Here, we have examined the interaction between ER stress and JAK/STAT-dependent inflammation in glial cells. We show that ER stress is present in the central nervous system (CNS) concomitant with inflammation and astrogliosis in the multiple sclerosis (MS) mouse model of experimental autoimmune encephalomyelitis (EAE). Astrocytes do not easily succumb to ER stress but rather activate an inflammatory program involving activation of STAT3 in a JAK1-dependent fashion. ER stress-induced activation of the JAK1/STAT3 axis leads to expression of interleukin 6 (IL-6) and several chemokines. Moreover, the activation of STAT3 signaling is dependent on PERK, a central component of the ER stress response, which we show is phosphorylated by JAK1. Disruption of PERK abrogates ER stress-induced activation of STAT3 and subsequent gene expression. Additionally, ER-stressed astrocytes, via paracrine signaling, can stimulate activation of microglia, leading to production of IL-6 and oncostatin M (OSM). These IL-6 cytokines can then synergize with ER stress in astrocytes to drive inflammation. Together, this work describes a new PERK/JAK1/STAT3 signaling pathway that elicits a feed-forward inflammatory loop involving astrocytes and microglia to drive neuroinflammation, which may be relevant in diseases such as MS.     

The Roles of Synoviolin in Crosstalk Between Endoplasmic Reticulum Stress-Induced Apoptosis and p53 Pathway

Endopalsmic reticulum (ER) is specialized organelle to maintain the integrity of secreted and membranous proteins. ER also senses so-called “ER stress”, which is a resulted from a various internal and external stresses, and triggers apoptosis when the diverse attempts to accommodate with the stress are in fail. The impairment these ER functions has been implicated in several human diseases, in which aberrant ER stress induced apoptosis is observed. We discuss about another disease model related with ER mediated apoptosis based on the recent studies about Synoviolin, an E3 ubiquitin ligase inherently utilized for ER associated degradation (ERAD). In addition to its canonical role in ERAD, Synoviolin targets tumor suppressor gene p53 for proteasomal degradation, suggesting the crosstalk between ERAD and p53 mediated apoptotic pathway under ER stress. Together with the anti-apoptotic property of Synoviolin previously elucidated by both in vitro and in vivo analyses, its new function in p53 regulation may provide a new insight into the pathomechanism of proliferative diseases such as cancer or rheumatoid arthritis.     

Phenylbutyric Acid Rescues Endoplasmic Reticulum Stress-Induced Suppression of APP Proteolysis and Prevents Apoptosis in Neuronal Cells

Background

The familial and sporadic forms of Alzheimer''s disease (AD) have an identical pathology with a severe disparity in the time of onset [1]. The pathological similarity suggests that epigenetic processes may phenocopy the Familial Alzheimer''s disease (FAD) mutations within sporadic AD. Numerous groups have demonstrated that FAD mutations in presenilin result in ‘loss of function’ of γ-secretase mediated APP cleavage [2], [3], [4], [5]. Accordingly, ER stress is prominent within the pathologically impacted brain regions in AD patients [6] and is reported to inhibit APP trafficking through the secretory pathway [7], [8]. As the maturation of APP and the cleaving secretases requires trafficking through the secretory pathway [9], [10], [11], we hypothesized that ER stress may block trafficking requisite for normal levels of APP cleavage and that the small molecular chaperone 4-phenylbutyrate (PBA) may rescue the proteolytic deficit.

Methodology/Principal Findings

The APP-Gal4VP16/Gal4-reporter screen was stably incorporated into neuroblastoma cells in order to assay γ-secretase mediated APP proteolysis under normal and pharmacologically induced ER stress conditions. Three unrelated pharmacological agents (tunicamycin, thapsigargin and brefeldin A) all repressed APP proteolysis in parallel with activation of unfolded protein response (UPR) signaling—a biochemical marker of ER stress. Co-treatment of the γ-secretase reporter cells with PBA blocked the repressive effects of tunicamycin and thapsigargin upon APP proteolysis, UPR activation, and apoptosis. In unstressed cells, PBA stimulated γ-secretase mediated cleavage of APP by 8–10 fold, in the absence of any significant effects upon amyloid production, by promoting APP trafficking through the secretory pathway and the stimulation of the non-pathogenic α/γ-cleavage.

Conclusions/Significance

ER stress represses γ-secretase mediated APP proteolysis, which replicates some of the proteolytic deficits associated with the FAD mutations. The small molecular chaperone PBA can reverse ER stress induced effects upon APP proteolysis, trafficking and cellular viability. Pharmaceutical agents, such as PBA, that stimulate α/γ-cleavage of APP by modifying intracellular trafficking should be explored as AD therapeutics.     

Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis

Purpose

Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells.

Methods

Human colorectal cancer cell lines (HCT-116 and HT-29) were treated with sodium butyrate at concentrations ranging from 0.5–5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining), and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot.

Results

Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II), beclin-1, and autophagocytosis-associated protein (Atg)3. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin) and genetic (siRNA targeting BIP and CHOP) methods, the induction of BIP, PDI, IRE1a, and LC3-II was blocked, but PARP cleavage was markedly enhanced.

Discussion

Taken together, these results suggested that sodium butyrate-induced autophagy was mediated by endoplasmic reticulum stress, and that preventing autophagy by blocking the endoplasmic reticulum stress response enhanced sodium butyrate-induced apoptosis. These results provide novel insights into the anti-tumor mechanisms of butyric acid.     

Bufalin Induces the Interplay between Apoptosis and Autophagy in Glioma Cells through Endoplasmic Reticulum Stress

Malignant gliomas are common primary tumors of the central nervous system. The prognosis of patients with malignant glioma is poor in spite of current intensive therapy and thus novel therapeutic modalities are necessary. Bufalin is the major component of Chan-Su (a traditional Chinese medicine) extracts from the venom of Bufo gargarizan. In this study, we evaluated the growth inhibitory effect of bufalin on glioma cells and explored the underlying molecular mechanisms. Our results showed that bufalin inhibited the growth of glioma cells significantly. Mechanistic studies demonstrated that bufalin induced apoptosis through mitochondrial apoptotic pathway. In addition, bufalin was also found to induce ER stress-mediated apoptosis, which was supported by the up- regulation of ER stress markers, CHOP and GRP78, and augmented phosphorylation of PERK and eIF2α as well as cleavage of caspase-4. Downregulation of CHOP using siCHOP RNA attenuated bufalin-induced apoptosis, further confirming the role of ER stress response in mediating bufalin-induced apoptosis. Evidence of bufalin-induced autophagy included formation of the acidic vesicular organelles, increase of autophagolysosomes and LC3-II accumulation. Further experiments showed that the mechanism of bufalin-induced autophagy associated with ATP deleption involved an increase in the active form of AMPK, decreased phosphorylation levels of mTOR and its downstream targets 4EBP1 and p70S6K1. Furthermore, TUDC and silencing of eIF2α or CHOP partially blocked bufalin-induced accumulation of LC3-II, which indicated that ER stress preceded bufalin-induced autophagy and PERK/eIF2α/CHOP signaling pathway played a major part in the process. Blockage of autophagy increased expression of ER stress associated proteins and the ratio of apoptosis, indicating that autophagy played a cytoprotective role in bufalin induced ER stress and cell death. In conclusion, bufalin inhibits glioma cell growth and induces interplay between apoptosis and autophagy through endoplasmic reticulum stress. It will provide molecular bases for developing bufalin into a drug candidate for the treatment of maglinant glioma.     

Endoplasmic Reticulum Stress in Heat- and Shake-Induced Injury in the Rat Small Intestine

We investigated the mechanisms underlying damage to rat small intestine in heat- and shake-induced stress. Eighteen Sprague-Dawley rats were randomly divided into a control group and a 3-day stressed group treated 2 h daily for 3 days on a rotary platform at 35°C and 60 r/min. Hematoxylin and eosin-stained paraffin sections of the jejunum following stress revealed shedding of the villus tip epithelial cells and lamina propria exposure. Apoptosis increased at the villus tip and extended to the basement membrane. Photomicrographs revealed that the microvilli were shorter and sparser; the nuclear envelope invaginated and gaps in the karyolemma increased; and the endoplasmic reticulum (ER) swelled significantly. Gene microarray analysis assessed 93 differentially expressed genes associated with apoptosis, ER stress, and autophagy. Relevant genes were compiled from the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Forty-one genes were involved in the regulation of apoptosis, fifteen were related to autophagy, and eleven responded to ER stress. According to KEGG, the apoptosis pathways, mitogen-activated protein kinase(MAPK) signaling pathway, the mammalian target of rapamycin (mTOR) signaling pathway, and regulation of autophagy were involved. Caspase3 (Casp3), caspase12 (Casp12), and microtubule-associate proteins 1 light chain 3(LC3) increased significantly at the villus tip while mTOR decreased; phosphorylated-AKT (P-AKT) decreased. ER stress was involved and induced autophagy and apoptosis in rat intestinal damage following heat and shake stress. Bioinformatic analysis will help determine the underlying mechanisms in stress-induced damage in the small intestine.     

Hepatic Xbp1 Gene Deletion Promotes Endoplasmic Reticulum Stress-induced Liver Injury and Apoptosis

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), a highly conserved signaling cascade that functions to alleviate stress and promote cell survival. If, however, the cell is unable to adapt and restore homeostasis, then the UPR activates pathways that promote apoptotic cell death. The molecular mechanisms governing the critical transition from adaptation and survival to initiation of apoptosis remain poorly understood. We aim to determine the role of hepatic Xbp1, a key mediator of the UPR, in controlling the adaptive response to ER stress in the liver. Liver-specific Xbp1 knockout mice (Xbp1^LKO) and Xbp1^fl/fl control mice were subjected to varying levels and durations of pharmacologic ER stress. Xbp1^LKO and Xbp1^fl/fl mice showed robust and equal activation of the UPR acutely after induction of ER stress. By 24 h, Xbp1^fl/fl controls showed complete resolution of UPR activation and no liver injury, indicating successful adaptation to the stress. Conversely, Xbp1^LKO mice showed ongoing UPR activation associated with progressive liver injury, apoptosis, and, ultimately, fibrosis by day 7 after induction of ER stress. These data indicate that hepatic XBP1 controls the adaptive response of the UPR and is critical to restoring homeostasis in the liver in response to ER stress.     

Sec22 Regulates Endoplasmic Reticulum Morphology but Not Autophagy and Is Required for Eye Development in Drosophila

The endoplasmic reticulum (ER) is a highly dynamic organelle that plays a critical role in many cellular processes. Abnormal ER morphology is associated with some human diseases, although little is known regarding how ER morphology is regulated. Using a forward genetic screen to identify genes that regulated ER morphology in Drosophila, we identified a mutant of Sec22, the orthologs of which in yeast, plants, and humans are required for ER to Golgi trafficking. However, the physiological function of Sec22 has not been previously investigated in animal development. A loss of Sec22 resulted in ER proliferation and expansion, enlargement of late endosomes, and abnormal Golgi morphology in mutant larvae fat body cells. However, starvation-induced autophagy was not affected by a loss of Sec22. Mosaic analysis of the eye revealed that Sec22 was required for photoreceptor morphogenesis. In Sec22 mutant photoreceptor cells, the ER was highly expanded and gradually lost normal morphology with aging. The rhabdomeres in mutants were small and sometimes fused with each other. The morphology of Sec22 mutant eyes resembled the eye morphology of flies with overexpressed eyc (eyes closed). eyc encodes for a Drosophila p47 protein that is required for membrane fusion. A loss of Syntaxin5 (Syx5), encoding for a t-SNARE on Golgi, also phenocopied the Sec22 mutant. Sec22 formed complexes with Syx5 and Eyc. Thus, we propose that appropriate trafficking between the ER and Golgi is required for maintaining ER morphology and for Drosophila eye morphogenesis.     

自噬对慢性间歇性低氧大鼠颏舌肌损伤的作用及机制

目的:探讨自噬在慢性间歇性低氧状态下大鼠颏舌肌损伤中的作用及其机制。方法:将36只SD大鼠随机均分为对照组,慢性间歇性低氧组(chronic intermittent hypoxia,CIH组),慢性间歇性低氧+氯喹组(CIH+CQ组)。苏木素-伊红(HE)染色观察颏舌肌组织形态学变化;激光共聚焦显微镜下观察自噬标记物LC3在颏舌肌中的表达;免疫组织化学染色法观察组织中细胞色素c(cytochrome,Cyt c)的表达。结果:激光共聚焦结果显示,对照组颏舌肌中未见明显LC3表达,CIH组中LC3的平均荧光强度较对照组明显增强(P0.05),CIH+CQ组中LC3的平均荧光强度较CIH组显著增加(P0.05)。免疫组织化学结果显示:CIH组中Cyt c的阳性表达较对照组增加(P0.05),CIH+CQ组中Cyt c表达与CIH组相比显著增加(P0.05)。结论:慢性间歇性低氧引起颏舌肌线粒体损伤,触发细胞凋亡,同时诱发自噬。抑制自噬加重线粒体损伤,促进细胞凋亡。说明自噬可能通过抑制凋亡而在慢性间歇性低氧状态下的大鼠颏舌肌中起维护肌肉功能的作用。     

Induction of the Endoplasmic Reticulum Stress and Autophagy in Human Lung Carcinoma A549 Cells by Anacardic Acid

Anacardic acid (AA, 2-hydroxy-6-pentadecylbenzoic acid), a constituent of the cashew-nut shell, has a variety of beneficial effects on the treatment of cancer and tumors. However, the fact that AA induces ER stress and autophagy in cancer cell is not known. We investigated the effect of AA on ER-stress and autophagy-induced cell death in cancer cells. Because of our interest in lung cancer, we used the non-small cell lung adenocarcinoma A549 cells treated with 3.0 μg/ml of AA for this research. In this research we found that AA induces intracellular Ca²⁺ mobilization and ER stress. AA induced the ER stress-inducing factors, especially IRE1α, and the hallmarks of UPR, Grp78/Bip and GADD153/CHOP. AA inhibited the expression of p-PERK and its downstream substrate, p-elF2α. We also demonstrated that AA induces autophagy. Up-regulation of autophagy-related genes and the appearance of autophagosome in transfected cells with green fluorescent protein (GFP)-LC3 and GFP-Beclin1 plasmids showed the induction of autophagy in AA-treated A549 cells. The morphological analysis of intracellular organelles by TEM also showed the evidence that AA induces ER stress and autophagy. For the first time, our research showed that AA induces ER stress and autophagy in cancer cells.