Endoplasmic reticulum stress contributes to the cell death induced by UCH-L1 inhibitor

At the neuropathological level, Parkinson's disease (PD) is characterized by the accumulation of misfolded proteins, which can trigger the unfolded protein response (UPR). UCH-L1 is a component of ubiquitin proteasome system (UPS). It is reported that the loss of its function will impair ubiquitin proteasome system and cause toxicity to cells. But its mechanism has not been illustrated. In this study, we detected the protein expression of Bip/Grp78 and the spliced form of XBP-1 to examine the activation of unfolded protein response after SK-N-SH cells being treated with LDN-57444, a UCH-L1 inhibitor which could inhibit UCH-L1 hydrolase activity. Our data showed that UCH-L1 inhibitor was able to cause cell death through the apoptosis pathway by decreasing the activity of ubiquitin proteasome system and increasing the levels of highly ubiquitinated proteins, both of which can activate unfolded protein response. There is a lot of evidence that unfolded protein response is activated as a protective response at the early stage of the stress; this protective response can switch to a pro-apoptotic response when the stress persists. In this study, we demonstrated this switch by detecting the upregulation of CHOP/Gadd153. Taken together, our data indicated that the apoptosis induced by UCH-L1 inhibitor may be triggered by the activation of endoplasmic reticulum stress (ERS). Moreover, we provide a new cell model for studying the roles of UCH-L1 in Parkinson's disease.     

Expression of mutant or cytosolic PrP in transgenic mice and cells is not associated with endoplasmic reticulum stress or proteasome dysfunction

The cellular pathways activated by mutant prion protein (PrP) in genetic prion diseases, ultimately leading to neuronal dysfunction and degeneration, are not known. Several mutant PrPs misfold in the early secretory pathway and reside longer in the endoplasmic reticulum (ER) possibly stimulating ER stress-related pathogenic mechanisms. To investigate whether mutant PrP induced maladaptive responses, we checked key elements of the unfolded protein response (UPR) in transgenic mice, primary neurons and transfected cells expressing two different mutant PrPs. Because ER stress favors the formation of untranslocated PrP that might aggregate in the cytosol and impair proteasome function, we also measured the activity of the ubiquitin proteasome system (UPS). Molecular, biochemical and immunohistochemical analyses found no increase in the expression of UPR-regulated genes, such as Grp78/Bip, CHOP/GADD153, or ER stress-dependent splicing of the mRNA encoding the X-box-binding protein 1. No alterations in UPS activity were detected in mutant mouse brains and primary neurons using the Ub(G76V)-GFP reporter and a new fluorogenic peptide for monitoring proteasomal proteolytic activity in vivo. Finally, there was no loss of proteasome function in neurons in which endogenous PrP was forced to accumulate in the cytosol by inhibiting cotranslational translocation. These results indicate that neither ER stress, nor perturbation of proteasome activity plays a major pathogenic role in prion diseases.     

Involvement of dopamine receptors in binge methamphetamine-induced activation of endoplasmic reticulum and mitochondrial stress pathways

Single large doses of methamphetamine (METH) cause endoplasmic reticulum (ER) stress and mitochondrial dysfunctions in rodent striata. The dopamine D(1) receptor appears to be involved in these METH-mediated stresses. The purpose of this study was to investigate if dopamine D(1) and D(2) receptors are involved in ER and mitochondrial stresses caused by single-day METH binges in the rat striatum. Male Sprague-Dawley rats received 4 injections of 10 mg/kg of METH alone or in combination with a putative D(1) or D(2) receptor antagonist, SCH23390 or raclopride, respectively, given 30 min prior to each METH injection. Rats were euthanized at various timepoints afterwards. Striatal tissues were used in quantitative RT-PCR and western blot analyses. We found that binge METH injections caused increased expression of the pro-survival genes, BiP/GRP-78 and P58(IPK), in a SCH23390-sensitive manner. METH also caused up-regulation of ER-stress genes, Atf2, Atf3, Atf4, CHOP/Gadd153 and Gadd34. The expression of heat shock proteins (HSPs) was increased after METH injections. SCH23390 completely blocked induction in all analyzed ER stress-related proteins that included ATF3, ATF4, CHOP/Gadd153, HSPs and caspase-12. The dopamine D(2)-like antagonist, raclopride, exerted small to moderate inhibitory influence on some METH-induced changes in ER stress proteins. Importantly, METH caused decreases in the mitochondrial anti-apoptotic protein, Bcl-2, but increases in the pro-apoptotic proteins, Bax, Bad and cytochrome c, in a SCH23390-sensitive fashion. In contrast, raclopride provided only small inhibition of METH-induced changes in mitochondrial proteins. These findings indicate that METH-induced activation of striatal ER and mitochondrial stress pathways might be more related to activation of SCH23390-sensitive receptors.     

Progression of apoptic signaling from mesenteric ischemia–reperfusion injury to lungs: correlation in the level of ER chaperones expression

Multiple organ dysfunction syndrome (MODS) is characterized by the development of probably reversible, progressive dysfunction of vital systems in two or more organs, directly undamaged by surgery or other trauma. The organs which have the most common potential dysfunction are lungs, liver, kidneys, heart and gastrointestinal tract. The small intestine is the source of production of proinflammatory mediators leading and contributing to multiorgan failure. The endoplasmic reticulum (ER), after ischemia and post-ischemic reperfusion, is significantly involved in the activation of enterocyte apoptosis. The purpose of this study was to determine the stage of apoptosis in the lungs, initiated through inflammatory response from the small intestine. We analyzed changes in mRNA levels of pro-apoptotic genes Gadd153 (Chop) and anti-apoptotic genes Grp78 (Bip) in the small intestine wall and lung parenchyma. During experimental procedure the rats underwent 60 min of ischemia, caused by complete occlusion of the mesenteric arteria cranialis, with subsequent reperfusion and evaluation after 1 h, 24 h and 30 days (from R1, R24 to R30, respectively, each group n = 8). The gene expression levels were measured using RT-PCR followed by electrophoresis and visualization under UV. In the lungs we detected significantly lower level of expression Grp78 by 45 ± 6.9%. This suggests that ischemic attack and subsequent reperfusion did not promote ER stress in the lungs through induction of Gadd153 expression in the small intestine. There is still no effective approach to the treatment of affected ischemic intestine tissue, to stop the processes with could eventually lead to MODS. Therefore it is necessary to study changes in the damaged tissue at the molecular level and try to suggest possible therapeutic defined routes to the protection of tissue.     

CHOP双重调控衣霉素诱导的DU-145细胞凋亡及自噬的研究

目的研究内质网应激分子CHOP调控细胞凋亡与自噬的作用和机制。 方法利用衣霉素诱导DU-145细胞产生内质网应激,Western Blot法检测内质网应激相关分子Grp78、Grp94、p-eIF2α和CHOP及自噬蛋白LC3Ⅱ、Atg5和Beclin1的表达;用流式细胞术检测细胞凋亡水平;沉默CHOP基因,用Western Blot法检测凋亡蛋白PARP、Caspase3的表达,流式细胞术检测细胞凋亡;并利用免疫荧光检测自噬标志性蛋白LC3B的表达。 结果衣霉素诱导DU-145细胞内质网应激能诱导一定程度的细胞凋亡,衣霉素处理8、12、24?h的细胞凋亡率分别为3.27﹪±1.02﹪,8.97﹪±0.71﹪和11.67﹪±1.41﹪,处理12?h及24?h的细胞凋亡率与对照组相比差异具有统计学意义（P < 0.01）。同时也能通过抑制PI3K/AKt/mTOR信号通路激活DU-145细胞自噬。CHOP基因沉默抑制细胞凋亡,shCtrl组细胞凋亡率为32.17﹪±3.93﹪,shCHOP-1组细胞凋亡率为23.53﹪±3.41﹪,两组相比差异具有统计学意义（P < 0.05）。且CHOP基因沉默能促进细胞自噬分子LC3B的表达。 结论衣霉素诱导DU-145细胞内质网应激状态下,CHOP在细胞凋亡与自噬之间发挥双重调控作用。     

Unfolding protein response signaling is involved in development,maintenance, and regression of the corpus luteum during the bovine estrous cycle

The corpus luteum (CL) is a transient endocrine organ. Development, maintenance, and regression of CL are effectively controlled by dynamic changes in gene expression. However, it is unknown what types of gene are affected during the CL life span of the estrous cycle in bovine. Here, we determined whether unfolded protein response (UPR) signaling via eIF2α/ATF4/GADD34, p90ATF6/p50ATF6, and IRE1/XBP1, which is a cellular stress response associated with the endoplasmic reticulum (ER), is involved in the bovine CL life span. Our results indicated that expression of Grp78/Bip, the master UPR regulator, was increased during the maintenance stage and rapidly decreased at the regression stage. Additionally, UPR signaling pathways genes were found to be involved in luteal phase progression during the estrous cycle. Our findings suggested that Grp78/Bip, ATF6, and XBP1 act as ER chaperones for initiating CL development and maintaining the CL. In addition, we investigated whether ER stress-mediated apoptosis is occurred through three UPR signaling pathways in CL regression stage. Interestingly, pIRE1 and CHOP were found to be involved in both the adaptive response and ER stress-mediated apoptosis. During the CL regression stage, increased expression of pJNK and CHOP, two components of ER stress-mediated apoptotic cascades, occurred before increased level of cleaved caspase 3 were observed. The present investigation was performed to identify a functional link between UPR signaling and CL life span during the bovine estrous cycle. Taken together, results from this study demonstrated that UPR protein/gene expression levels were different at various stages of the bovine CL life span. Variations in the expression of these protein/genes may play important roles in luteal stage progression during the estrous cycle.     

Proteomic characterization of the striatum and midbrain treated with 6-hydroxydopamine: Alteration of 58-kDa glucose-regulated protein and C/EBP homologous protein

Abstract

The present study performed proteomic analysis of the midbrain and striatum of 6-hydroxydopamine (6-OHDA)-treated neonatal rats—a model of attention-deficit hyperactivity disorder (ADHD). Proteomic analysis revealed that a 58-kDa glucose-regulated protein (Grp58) was temporarily phosphorylated and its level was elevated by 6-OHDA. Furthermore, 6-OHDA increased the expression level of C/EBP homologous protein (CHOP), a mediator of endoplasmic reticulum (ER) stress response, in the midbrain and striatum. In vitro experiments using PC12 cells revealed that 6-OHDA or hydrogen peroxide could induce the elevation of Grp58 and CHOP. 6-OHDA could induce the elevation of Grp58 and CHOP in the presence of catalase, a hydrogen peroxide-removing enzyme, suggesting that the elevation of Grp58 and CHOP are induced by both hydrogen peroxide and p-quinone generated by 6-OHDA. Collectively, these findings suggest that ER stress involving the alteration of Grp58 and CHOP play a significant role in the induction of insults by 6-OHDA in vivo.     

Apoptosis induced by all-trans retinoic acid in N-acetylglucosaminyltransferase V repressed human hepatocarcinoma cells is mediated through endoplasmic reticulum stress

We previously demonstrated that endoplasmic reticulum (ER) stress was triggered in human hepatocarcinoma 7721 cells transfected with antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V-AS/7721) which were more susceptible to apoptosis induced by all-trans retinoic acid (ATRA). In the present study, we report that ATRA-induced apoptosis in GnT-V-AS/7721 cells is mediated through ER stress. We show here that ER stress is enhanced in GnT-V-AS/7721 cells with 80 microM ATRA treatment for 24 h, which is evidenced by the increase of GRP78/Bip, C/EBP-homologous protein-10 (CHOP, also known as GADD153) and spliced XBP1. Additionally, activation of caspase-12, caspase-9, and -3 was detected, and apoptosis morphology was observed in GnT-V-AS/7721 cells with ATRA treatment. These results suggest that ATRA enhances the ER stress triggered in GnT-V-AS/7721 cells, which represents a novel mechanism of ATRA to induce apoptosis. We further observed that GnT-V was significantly repressed and the structure of N-glycans was changed in GnT-V-AS/7721 cells with 80 microM ATRA treatment for 24 h, suggesting that repression of GnT-V by ATRA causes the enhanced ER stress and ER stress-mediated apoptosis in GnT-V-AS/7721 cells.     

The presence of extracellular matrix alters the chondrocyte response to endoplasmic reticulum stress

The objective of this study was to test the hypothesis that extracellular matrix (ECM) would alter the endoplasmic reticulum (ER) stress response of chondrocytes. Chondrocytes were isolated from calf knees and maintained in monolayer culture or suspended in collagen I to form spot cultures (SCs). Our laboratory has shown that bovine chondrocytes form cartilage with properties similar to native cartilage after 2-4 weeks in SCs. Monolayer cultures treated with ER stressors glucose withdrawal (-Glu), tunicamycin (TN), or thapsigargin (TG) up-regulated Grp78 and Gadd153, demonstrating a complete ER stress response. SCs were grown at specific times from 1 day to 6 weeks before treatment with ER stressors. Additionally, SCs grown for 1, 2, or 6 weeks were treated with increasing concentrations of TN or TG. Western blotting of SCs for Grp78 indicated that increased ECM accumulation results in delayed expression; however, Grp78 mRNA is up-regulated in response to ER stressors even after 6 weeks in culture. SCs treated with ER stressors did not up-regulate Gadd153, suggesting that the cells experienced ER stress but would not undergo apoptosis. In fact, SCs undergo apoptosis upon ER stress treatment after 0-1 day of growth; however, after 4 days and to 6 weeks, apoptosis in treated samples was not different than controls. Pro-survival molecules Bcl-2 and Bag-1 were up-regulated upon ER stress in SCs. These results suggest that presence of ECM confers protection from ER stressors. Future studies involving chondrocyte physiology should focus on responses in conditions more closely mimicking the in vivo cartilage environment.     

小鼠脑组织及培养神经元Neuro-2a 在内质网应激反应时的基因表达谱分析

内质网是真核细胞的重要细胞器。某些细胞内外因素如病原体感染等能引起从内质网到胞浆和胞核的信号传导途径活化,即内质网应激反应。但是,目前国内外尚无针对内质网应激反应的基因表达谱分析报道。本研究中,用3种已报道的内质网应激反应诱导剂,包括蛋白质糖基化抑制剂衣霉素(tunicamycin)、内质网Ca ²⁺-ATPases抑制剂毒胡萝卜素（thapsigargin）和乙脑病毒(Japanese encephalitis virus, JEV),分别处理小鼠颅腔和小鼠脑神经瘤细胞（Neuro-2a）,试剂处理组与未处理组的第二代RNA测序分析发现,衣霉素、毒胡萝卜素和乙脑病毒在体外和体内均引起分子伴侣基因Hsp70表达上调,诱导内质网应激反应。衣霉素、毒胡萝卜素和乙脑病毒体外处理诱导的内质网应激反应信号通路中,基因差异表达相似性高于体内处理组。乙脑病毒和糖基化抑制剂衣霉素体内外处理,主要诱导内质网应激反应的非折叠蛋白质反应信号通路,引起相关基因Atf4、Bip、Edem和Perk等表达上调。内质网Ca ²⁺-ATPases抑制剂毒胡萝卜素主要诱导内质网超负荷反应,激活NF-κB信号通路。乙脑病毒诱导的内质网应激反应相关差异表达基因数量最多,体外与体内合计有40种。乙脑病毒体内外处理上调的基因包括Bax、Casp12、Atf4、Bip、Edem和Perk等,下调的基因包括Sec23/24、Nef、Svip和Jnk等。糖基化抑制剂衣霉素体内外处理上调基因包括Gadd34、Atf4、Ermani和Bip等,下调基因包括Grp94、Atf6、Sec23/24和Nef等。内质网Ca ^-2+-ATPases抑制剂毒胡萝卜素体内外处理上调的基因包括Sec61、Trap和Ask1等。衣霉素、毒胡萝卜素和乙脑病毒体内外处理也通过内质网应激反应,调控与炎症或凋亡相关的MAPK信号通路和P53信号通路。本研究首次通过使用3种内质网应激反应诱导剂分别处理小鼠和细胞,揭示了体内外内质网应激反应引起的基因表达谱变化,为内质网应激反应相关疾病的治疗提供了新思路。     

Salsolinol causing parkinsonism activates endoplasmic reticulum-stress signaling pathways in human dopaminergic SK-N-SH cells

The endoplasmic reticulum (ER) is a small intracellular organelle to which one-third of cellular proteins are translocated after translation and post-translational modification, folding and the formation of a three- or four-dimensional structure. ER also has a role in the transportation of proteins to other intracellular organelles, the cell surface or the outer space of the cell membrane. Thus, ER is an important intermediate which maintains intracellular homeostasis through complex control systems. Once these control systems are disrupted, serious disturbances occur. Many neurodegenerative diseases including Parkinson's disease involve aggregation and deposition of misfolded proteins such as alpha-synuclein. Endogenously occurring neurotoxins such as Salsolinol and 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ) causing Parkinsonism may foster misfolded proteins and bring forth ER stress in dopaminergic neurons. In the present study we examined translational changes fostered by ER stress and mediated by the Parkinsonian endogenous neurotoxins, salsolinol and 1BnTIQ, in dopaminergic cell line. Treatment with salsolinol and 1BnTIQ induced several genes involved in ER stress and unfolded protein response (UPR), such as ER chaperones and GADD153 (CHOP). Immunoblotting confirmed phosphorylation of the key endoplasmic reticulum stress kinase PERK (PKR-like-ER kinase) and eIF2alpha and induction of their downstream targets such as Bip and GADD153. These findings suggest a widespread involvement of ER stress and unfolded protein response in the pathophysiology of Parkinson's disease.