Development of Oxytolerant Salmonella typhimurium Using Radiation Mutation Technology (RMT) for Cancer Therapy.

A critical limitation of Salmonella typhimurium (S. typhimurium) as an anti-cancer agent is the loss of their invasive or replicative activities, which results in no or less delivery of anti-cancer agents inside cancer cells in cancer therapy. Here we developed an oxytolerant attenuated Salmonella strain (KST0650) from the parental KST0649 (ΔptsIΔcrr) strain using radiation mutation technology (RMT). The oxytolerant KST0650 strain possessed 20-times higher replication activity in CT26 cancer cells and was less virulent than KST0649. Furthermore, KST0650 migrated effectively into tumor tissues in mice. KST0650 was further equipped with a plasmid harboring a spliced form of the intracellular pro-apoptotic protein sATF6, and the expression of sATF6 was controlled by the radiation-inducible recN promoter. The new strain was named as KST0652, in which sATF6 protein expression was induced in response to radiation in a dose-dependent manner. This strain was effectively delivered inside cancer cells and tumor tissues via the Salmonella type III secretion system (T3SS). In addition, combination treatment with KST0652 and radiation showed a synergistic anti-tumor effect in murine tumor model with complete inhibition of tumor growth and protection against death. In conclusion, we showed that RMT can be used to effectively develop an anti-tumor Salmonella strain for delivering anti-cancer agents inside tumors.

Expression of Endoplasmic Reticulum Stress-Related mRNAs in Chronic Otitis Media.

OBJECTIVES: Unfolded proteins in the endoplasmic reticulum (ER) cause an ER stress response and can result in various pathologic conditions, including inflammation. Otitis media is the most common disease in otolaryngology and is associated with inflammation. The pathophysiology of chronic otitis media is not well understood; we therefore investigated the expression pattern of ER stress-related mRNAs in chronic otitis media. METHODS: Specimens were obtained from 47 patients with chronic otitis media over a period of 2 years. Expression levels of 6 ER stress transcription factors were quantitatively assessed using real-time RT-PCR. RESULTS: The mRNA expression of sXBP1 was significantly higher in the otorrhea present group than in the otorrhea absent group (p < 0.05). ATF6 expression was significantly higher in the ossicle destruction group than in the ossicle intact group (p < 0.05). mRNA expression of the 6 ER stress-related genes did not differ significantly between those patients with positive microbial cultures versus those with negative cultures (p > 0.05) or those with facial nerve dehiscence versus those without facial nerve dehiscence (p > 0.05). CONCLUSIONS: sXBP1 appears to be involved in chronic otitis media-associated inflammation, including otorrhea. ATF6 is associated with the destruction of ossicles. Our results suggest that certain ER stress-related genes are expressed in chronic otitis media-associated inflammation.

4-Phenylbutyric Acid Protects Against Ethanol-Induced Damage in the Developing Mouse Brain.

BACKGROUND: Ethanol (EtOH) exposure during pregnancy may result in fetal alcohol spectrum disorders (FASD). One of the most deleterious consequences of EtOH exposure is neuronal loss in the developing brain. Previously, we showed that EtOH exposure induced neuroapoptosis in the brain of postnatal day 4 (PD4) mice but not PD12 mice. This differential susceptibility may result from an insufficient cellular stress response system such as unfolded protein response (also known as endoplasmic reticulum [ER] stress) in PD4 mice. In this study, we compared the effect of EtOH on ER stress in PD4 and PD12 mice and determined whether the inhibition of ER stress could protect the developing brain against EtOH damage. METHODS: We used a third-trimester equivalent mouse model of FASD. PD4 and PD12 C57BL/6 mice were subcutaneously injected with saline (control), EtOH, EtOH plus 4-phenylbutyric acid (4-PBA), a chemical chaperone known as ER stress inhibitor, and 4-PBA alone. The expression of apoptosis marker, ER stress markers, and markers for glial cell activation was examined in the cerebral cortex. RESULTS: EtOH induced neuroapoptosis and increased the expression of ER stress markers, such as activating transcription factor 6, 78-kDa glucose-regulated protein, inositol-requiring enzyme 1α, mesencephalic astrocyte-derived neurotrophic factor, and caspase-12 in PD4 but not PD12 mice. EtOH exposure also activated microglia and astrocytes. Interestingly, treatment with 4-PBA attenuated EtOH-induced neuroapoptosis. Moreover, 4-PBA inhibited the expression of the aforementioned ER stress markers and EtOH-induced glial activation in PD4 mice. CONCLUSIONS: ER stress plays an important role in EtOH-induced damage to the developing brain. Inhibition of ER stress is neuroprotective and may provide a new therapeutic strategy for treating FASD.

Pharmacologic inhibition of S1P attenuates ATF6 expression, causes ER stress and contributes to apoptotic cell death.

Mammalian cells express unique transcription factors embedded in the endoplasmic reticulum (ER) membrane, such as the sterol regulatory element-binding proteins (SREBPs), that promote de novo lipogenesis. Upon their release from the ER, the SREBPs require proteolytic activation in the Golgi by site-1-protease (S1P). As such, inhibition of S1P, using compounds such as PF-429242 (PF), reduces cholesterol synthesis and may represent a new strategy for the management of dyslipidemia. In addition to the SREBPs, the unfolded protein response (UPR) transducer, known as the activating transcription factor 6 (ATF6), is another ER membrane-bound transcription factor that requires S1P-mediated activation. ATF6 regulates ER protein folding capacity by promoting the expression of ER chaperones such as the 78-kDa glucose-regulated protein (GRP78). ER-resident chaperones like GRP78 prevent and/or resolve ER polypeptide accumulation and subsequent ER stress-induced UPR activation by folding nascent polypeptides. Here we report that pharmacological inhibition of S1P reduced the expression of ATF6 and GRP78 and induced the activation of UPR transducers inositol-requiring enzyme-1α (IRE1α) and protein kinase RNA-like ER kinase (PERK). As a consequence, S1P inhibition also increased the susceptibility of cells to ER stress-induced cell death. Our findings suggest that S1P plays a crucial role in the regulation of ER folding capacity and also identifies a compensatory cross-talk between UPR transducers in order to maintain adequate ER chaperone expression and activity.

Endoplasmic Reticulum Protein TXNDC5 Augments Myocardial Fibrosis by Facilitating Extracellular Matrix Protein Folding and Redox-Sensitive Cardiac Fibroblast Activation.

RATIONALE: Cardiac fibrosis plays a critical role in the pathogenesis of heart failure. Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited, and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting coexpression gene network analysis on RNA sequencing data from failing human heart, we identified TXNDC5 (thioredoxin domain containing 5), a cardiac fibroblast (CF)-enriched endoplasmic reticulum protein, as a potential novel mediator of cardiac fibrosis, and we completed experiments to test this hypothesis directly. OBJECTIVE: The objective of this study was to determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis. METHODS AND RESULTS: RNA sequencing and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles and in hypertrophied/failing mouse left ventricle. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor ß1 and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under transforming growth factor ß1 stimulation in vitro. Knockdown of TXNDC5 attenuated transforming growth factor ß1-induced hCF activation and ECM protein upregulation independent of SMAD3 (SMAD family member 3), whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing c-Jun N-terminal kinase activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4. Transforming growth factor ß1-induced TXNDC5 upregulation in hCF was dependent on endoplasmic reticulum stress and activating transcription factor 6-mediated transcriptional control. Targeted disruption of Txndc5 in mice (Txndc5-/-) revealed protective effects against isoproterenol-induced cardiac hypertrophy, reduced fibrosis (by ≈70%), and markedly improved left ventricle function; post-isoproterenol left ventricular ejection fraction was 59.1±1.5 versus 40.1±2.5 (P<0.001) in Txndc5-/- versus wild-type mice, respectively. CONCLUSIONS: The endoplasmic reticulum protein TXNDC5 promotes cardiac fibrosis by facilitating ECM protein folding and CF activation via redox-sensitive c-Jun N-terminal kinase signaling. Loss of TXNDC5 protects against ß agonist-induced cardiac fibrosis and contractile dysfunction. Targeting TXNDC5, therefore, could be a powerful new therapeutic approach to mitigate excessive cardiac fibrosis, thereby improving cardiac function and outcomes in patients with heart failure.

High expression of active ATF6 aggravates endoplasmic reticulum stress­induced vascular endothelial cell apoptosis through the mitochondrial apoptotic pathway.

Activating transcription factor 6 (ATF6), one of three sensor proteins in the endoplasmic reticulum (ER), is an important regulatory factor in the ER stress­induced apoptosis pathway. Although recent studies have made some progress in elucidating the regulation mechanism of ATF6, the specific regulatory mechanism of ER stress­induced vascular endothelial cell (VEC) apoptosis is still unclear. The present study was designed to investigate the role of ATF6 in VECs under thapsigargin (TG)­induced ER stress. ATF6 (1­366aa; ATF6 high­expressed plasmid) and ATF6 (151­366aa; plasmid without transcriptional activity) were transfected into VECs to yield an ATF6 high­expression model and a positive control model, respectively. High expression of ATF6 decreased viability and aggravated ER stress­induced apoptosis in VECs. Increased expression of apoptosis­related genes, including those encoding caspase­3, caspase­9, C/EBP homologous protein (CHOP), cytochrome c and B­cell lymphoma­associated protein X (Bax)/B­cell lymphoma (Bcl­)2, was detected by polymerase chain reaction and western blotting in the ATF6 (1­366aa) + TG group. No significant effect of TG treatment and high ATF6 expression was indicated on the expression of death receptor­related genes, including those encoding caspase­8 and Fas. The results demonstrated that high expression of activated ATF6 aggravates ER stress­induced VEC apoptosis through the mitochondrial apoptotic pathway. Furthermore, in response to ER stress, ATF6 upregulates the expression of caspase­3, caspase­9, CHOP, cytochrome c and Bax/Bcl­2.

ATF6 Decreases Myocardial Ischemia/Reperfusion Damage and Links ER Stress and Oxidative Stress Signaling Pathways in the Heart.

RATIONALE: Endoplasmic reticulum (ER) stress causes the accumulation of misfolded proteins in the ER, activating the transcription factor, ATF6 (activating transcription factor 6 alpha), which induces ER stress response genes. Myocardial ischemia induces the ER stress response; however, neither the function of this response nor whether it is mediated by ATF6 is known. OBJECTIVE: Here, we examined the effects of blocking the ATF6-mediated ER stress response on ischemia/reperfusion (I/R) in cardiac myocytes and mouse hearts. METHODS AND RESULTS: Knockdown of ATF6 in cardiac myocytes subjected to I/R increased reactive oxygen species and necrotic cell death, both of which were mitigated by ATF6 overexpression. Under nonstressed conditions, wild-type and ATF6 knockout mouse hearts were similar. However, compared with wild-type, ATF6 knockout hearts showed increased damage and decreased function after I/R. Mechanistically, gene array analysis showed that ATF6, which is known to induce genes encoding ER proteins that augment ER protein folding, induced numerous oxidative stress response genes not previously known to be ATF6-inducible. Many of the proteins encoded by the ATF6-induced oxidative stress genes identified here reside outside the ER, including catalase, which is known to decrease damaging reactive oxygen species in the heart. Catalase was induced by the canonical ER stressor, tunicamycin, and by I/R in cardiac myocytes from wild-type but not in cardiac myocytes from ATF6 knockout mice. ER stress response elements were identified in the catalase gene and were shown to bind ATF6 in cardiac myocytes, which increased catalase promoter activity. Overexpression of catalase, in vivo, restored ATF6 knockout mouse heart function to wild-type levels in a mouse model of I/R, as did adeno-associated virus 9-mediated ATF6 overexpression. CONCLUSIONS: ATF6 serves an important role as a previously unappreciated link between the ER stress and oxidative stress gene programs, supporting a novel mechanism by which ATF6 decreases myocardial I/R damage.

Expression and regulation of ATF6α in the mouse uterus during embryo implantation.

BACKGROUND: ATF6α, one of the sensor proteins in the stress signaling pathway of the endoplasmic reticulum, is located in the membrane of the endoplasmic reticulum. To date, the physiological function of ATF6α in the process of embryo implantation has not been reported. METHODS: In this study, the expression pattern of ATF6α in the mouse uterus during peri-implantation and the estrous cycle was detected by real-time PCR, western blot and immunohistochemistry. RESULTS: ATF6α mRNA and protein levels were higher in the uterus near the implantation site on day 5 and were intensely expressed in the secondary decidual zone (SDZ) on days 7-8. In the uteri of pseudopregnant mice, ATF6α mRNA and protein levels were lower on day 5 than on other days. The activating blastocyst and artificial decidualization had an obvious effect of increasing the expression of ATF6α. In addition, the expression of ATF6α was affected by progesterone (P4) and estrogen (E2) in ovariectomized mice. This finding is further supported by evidence from mice during the estrous cycle. CONCLUSIONS: Thus, we have concluded that ATF6α may play an important role during embryo implantation and decidualization.

Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation.

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases.

ELANE mutant-specific activation of different UPR pathways in congenital neutropenia.

A number of studies have demonstrated induction of the unfolded protein response (UPR) in patients with severe congenital neutropenia (CN) harbouring mutations of ELANE, encoding neutrophil elastase. Why UPR is not activated in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations is unclear. We evaluated the effects of ELANE mutants on UPR induction in myeloid cells from CN and CyN patients, and analysed whether additional CN-specific defects contribute to the differences in UPR induction between CN and CyN patients harbouring identical ELANE mutations. We investigated CN-specific p.C71R and p.V174_C181del (NP_001963.1) and CN/CyN-shared p.S126L (NP_001963.1) ELANE mutants. We found that transduction of haematopoietic cells with p.C71R, but not with p.V174_C181del or p.S126L ELANE mutants induced expression of ATF6, and the ATF6 target genes PPP1R15A, DDIT3 and HSPA5. Recently, we found that levels of secretory leucocyte protease inhibitor (SLPI), a natural ELANE inhibitor, are diminished in myeloid cells from CN patients, but not CyN patients. Combined knockdown of SLPI by shRNA and transduction of ELANE p.S126L in myeloid cells led to elevated levels of ATF6, PPP1R15A and HSPA5 RNA, suggesting that normal levels of SLPI in CyN patients might protect them from the UPR induced by mutant ELANE. In summary, different ELANE mutants have different effects on UPR activation, and SLPI regulates the extent of ELANE-triggered UPR.

The dysregulation of endoplasmic reticulum stress response in acute-on-chronic liver failure patients caused by acute exacerbation of chronic hepatitis B.

Although endoplasmic reticulum (ER) stress is critical in various liver diseases, its role in acute-on-chronic liver failure (AoCLF) caused by acute exacerbation of chronic hepatitis B (CHB) is still elusive. This study aimed to analyse ER stress responses in the progression of HBV-related AoCLF. Normal liver tissues (n = 10), liver tissues of CHB (n = 12) and HBV-related patients with AoCLF (n = 19) were used. Electron microscopy of the ultrastructure of the ER was carried out on liver specimens. The gene and protein expression levels of ER stress-related genes were measured. We further analysed the correlation between the expression levels of ER stress-related molecules and liver injury. Electron microscopy identified typical features of the ER microstructure in AoCLF subjects. Among the three pathways of unfolded protein responses, the PKR-like ER kinase and inositol-requiring enzyme 1 signalling pathway were activated in CHB subjects and inactivated in AoCLF subjects, while the activating transcription factor 6 signalling pathway was sustained in the activated form during the progression of AoCLF; the expression of glucose-regulated protein (Grp)78 and Grp94 was gradually decreased in AoCLF subjects compared to healthy individuals and CHB subjects, showing a negative correlation with serum ALT, AST and TBIL; moreover, the ER stress-related apoptosis molecules were activated in the progression of acute exacerbation of CHB. The dysregulated ER stress response may play a complicated role in the pathogenesis of AoCLF, and a severe ER stress response may predict the occurrence of AoCLF caused by acute exacerbation of CHB.

Transmission of ER stress response by ATF6 promotes endochondral bone growth.

BACKGROUND: We reported earlier that X-box binding protein1 spliced (XBP1S), a key regulator of the unfolded protein response (UPR), as a bone morphogenetic protein 2 (BMP2)-inducible transcription factor, positively regulates endochondral bone formation by activating granulin-epithelin precursor (GEP) chondrogenic growth factor. Under the stress of misfolded or unfolded proteins in the endoplasmic reticulum (ER), the cells can be protected by the mammalian UPR. However, the influence of activating transcription factor 6 (ATF6), another transcriptional arm of UPR, in BMP2-induced chondrocyte differentiation has not yet been elucidated. In the current study, we investigate and explore the role of ATF6 in endochondral bone formation, focus on associated molecules of hypertrophic chondrocyte differentiation, as well as the molecular events underlying this process. METHODS: High-cell-density micromass cultures were used to induce ATDC5 and C3H10T1/2 cell differentiation into chondrocytes. Quantitative real-time PCR, immunoblotting analysis, and immunohistochemistry were performed to examine (1) the expression of ATF6, ATF6α, collagen II, collagen X, and matrix metalloproteinase-13 (MMP13) and (2) whether ATF6 stimulates chondrogenesis and whether ATF6 enhances runt-related transcription factor 2 (Runx2)-mediated chondrocyte hypertrophy. Culture of fetal mouse bone explants was to detect whether ATF6 stimulates chondrocyte hypertrophy, mineralization, and endochondral bone growth. Coimmunoprecipitation was employed to determine whether ATF6 associates with Runx2 in chondrocyte differentiation. RESULTS: ATF6 is differentially expressed in the course of BMP2-triggered chondrocyte differentiation. Overexpression of ATF6 accelerates chondrocyte differentiation, and the ex vivo studies reveal that ATF6 is a potent stimulator of chondrocyte hypertrophy, mineralization, and endochondral bone growth. Knockdown of ATF6 via a siRNA approach inhibits chondrogenesis. Furthermore, ATF6 associates with Runx2 and enhances Runx2-induced chondrocyte hypertrophy. And, the stimulation effect of ATF6 is reduced during inhibition of Runx2 via a siRNA approach, suggesting that the promoting effect is required for Runx2. CONCLUSIONS: Our observations demonstrate that ATF6 positively regulates chondrocyte hypertrophy and endochondral bone formation through activating Runx2-mediated hypertrophic chondrocyte differentiation.

Insulin demand regulates ß cell number via the unfolded protein response.

Although stem cell populations mediate regeneration of rapid turnover tissues, such as skin, blood, and gut, a stem cell reservoir has not been identified for some slower turnover tissues, such as the pancreatic islet. Despite lacking identifiable stem cells, murine pancreatic ß cell number expands in response to an increase in insulin demand. Lineage tracing shows that new ß cells are generated from proliferation of mature, differentiated ß cells; however, the mechanism by which these mature cells sense systemic insulin demand and initiate a proliferative response remains unknown. Here, we identified the ß cell unfolded protein response (UPR), which senses insulin production, as a regulator of ß cell proliferation. Using genetic and physiologic models, we determined that among the population of ß cells, those with an active UPR are more likely to proliferate. Moreover, subthreshold endoplasmic reticulum stress (ER stress) drove insulin demand-induced ß cell proliferation, through activation of ATF6. We also confirmed that the UPR regulates proliferation of human ß cells, suggesting that therapeutic UPR modulation has potential to expand ß cell mass in people at risk for diabetes. Together, this work defines a stem cell-independent model of tissue homeostasis, in which differentiated secretory cells use the UPR sensor to adapt organ size to meet demand.

Expression of markers of endoplasmic reticulum stress-induced apoptosis in the placenta of women with early and late onset severe pre-eclampsia.

OBJECTIVES: Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in severe pre-eclampsia (SPE) and is characterized by the activation of three signaling pathways: PKR-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring 1 (Ire1). This study was designed to investigate the role of ER stress in the pathogenesis of SPE. MATERIALS AND METHODS: Placental tissues were collected from 32 women with normal pregnancies and two cohorts of women with early (n = 32) or late onset (n = 32) SPE. The expression of glucose-regulated protein 78 (GRP78), PERK, eukaryotic initiation factor 2 subunit a (eIF2α), activating transcription factor 6 (ATF4), ATF6, Ire1, CHOP (ClEBP homologus protein), and caspase 12 mRNA and protein in the placentas was analyzed using real-time reverse transcription-polymerase chain reaction and Western blotting, respectively. RESULTS: The levels of GRP78, PERK, eIF2α, CHOP, ATF6, and caspase 12 mRNA and protein expression were significantly higher in the placentas of women with early and late SPE than in the control women, whereas there were no differences in ATF6 and Ire1 mRNA and protein. CONCLUSION: ER stress-induced apoptosis was important in the development of SPE, especially in early onset SPE and was probably due to the activation of the PERK signaling pathway.

P58(IPK) inhibits coxsackievirus-induced apoptosis via the PI3K/Akt pathway requiring activation of ATF6a and subsequent upregulation of mitofusin 2.

Previously we found that prolonged endoplasmic reticulum (ER) stress caused by coxsackievirus B3 (CVB3) infection led to p58(IPK) downregulation and subsequent cell apoptosis. This finding implies that p58(IPK) expression benefits cell survival and counteracts CVB3-induced apoptosis. In testing this hypothesis, we first found that PI3K/Akt survival pathway is more sensitive than ERK1/2 in response to p58(IPK) expression. This finding was further verified by silencing p58(IPK) with specific siRNAs, which led to the significant suppression of phosphorylation of Akt (p-Akt) but not ERK1/2. Further, using CVB3-infected cell line expressing dominant negative ATF6a (DN-ATF6a), we found that expression of p58(IPK) and p-Akt was significantly reduced, which led to the decreased cell viability. However, when the DN-ATF6a cells were transiently transfected with p58(IPK) , an opposite result was obtained. Finally, by CVB3 infection of cells stably expressing p58(IPK) , we found that CVB3-induced mitochondria-mediated apoptosis was suppressed, which was evidenced by the reduced cytochrome c release and upregulation of the mitochondrial membrane protein mitofusin 2. However, silencing p58(IPK) with either specific siRNAs or DN-ATF6a sensitized cells to CVB3-induced apoptosis. These results suggest that p58(IPK) suppresses CVB3-induced apoptosis through selective activation of PI3K/Akt pathway that requires activation of ATF6a and subsequently upregulates mitofusin 2.

Effect of lithium chloride on endoplasmic reticulum stress-related PERK/ROCK signaling in a rat model of glaucoma.

Elevated intraocular pressure (IOP) is considered as the major risk factor for the loss of retinal ganglion cells (RGCs) and their axons in glaucoma. Lithium chloride (LiCl) inhibits glycogen synthase kinase-3 beta (GSK-3ß) and attends PERK-induced endoplasmic reticulum stress (ERs) transition. PERK is a type I transmembrane protein located in the endoplasmic reticulum. PERK pathway activation takes place in ERs early inhibiting protein synthnesis to protect cell and promote cell survival. Here, we firstly evaluate that LiCl reduced IOP when administered intraperitoneally. After 6 weeks, IOP dropped by around 21.9% in LiCl treated rats. Then we investigated the effects of LiCI on PERK-mediated signaling pathways. LiCl treatment activated PERK and inhibited the expression of ROCK-1 and ROCK-2 in a rat model of glaucoma. Collectively, these results suggest that LiCl reduced the IOP through the phosphorylation of PERK by the regulation of PERK/ROCK signaling in glaucoma rat model.

Transcriptional cross talk between orphan nuclear receptor ERRγ and transmembrane transcription factor ATF6α coordinates endoplasmic reticulum stress response.

Orphan nuclear receptor ERRγ is a member of nuclear receptor superfamily that regulates several important cellular processes including hepatic glucose and alcohol metabolism. However, mechanistic understanding of transcriptional regulation of the ERRγ gene remains to be elucidated. Here, we report that activating transcription factor 6α (ATF6α), an endoplasmic reticulum (ER)-membrane-bound basic leucine zipper (bZip) transcription factor, directly regulates ERRγ gene expression in response to ER stress. ATF6α binds to ATF6α responsive element in the ERRγ promoter. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) is required for this transactivation. Chromatin immunoprecipitation (ChIP) assay confirmed the binding of both ATF6α and PGC1α on the ERRγ promoter. ChIP assay demonstrated histone H3 and H4 acetylation occurs at the ATF6α and PGC1α binding site. Of interest, ERRγ along with PGC1α induce ATF6α gene transcription upon ER stress. ERRγ binds to an ERRγ responsive element in the ATF6α promoter. ChIP assay confirmed that both ERRγ and PGC1α bind to a site in the ATF6α promoter that exhibits histone H3 and H4 acetylation. Overall, for the first time our data show a novel pathway of cross talk between nuclear receptors and ER-membrane-bound transcription factors and suggest a positive feed-forward loop regulates ERRγ and ATF6α gene transcription.

Cage food location alters energy balance and endoplasmic reticulum stress in the brain of mice.

We have previously shown that voluntary wheel running activity in mice is associated with an increase in the Endoplasmic Reticulum (ER) Unfolded Protein stress response in multiple regions of the brain. Mice that are given access to running wheels show large variations in individual running activity. In contrast, when food is placed on the lid of their cages, rather than within the cage, all mice must undertake significant physical activity in order to gain access to their food. Hence we investigated the effects of food location on food intake and growth of C57BL/6 mice and on the activity of the ER stress system in the brain as reflected in the expression of two marker genes, Xbp1 and Atf6. Mice that had food in cups within their cages and allowed access to running wheels showed the anticipated changes in food intake, body weight and ER stress in the hippocampus compared to mice with no access to running wheels. Locating the food on the lid had no effect on food intake but reduced weight gain significantly. Likewise, locating food on the lid increased the expression of both Xbp1 and Atf6 in the hippocampi in the absence of any running wheel activity. Voluntary wheel running activity was reduced in mice whose food was located on the cage lid and this running actually reduced the expression of the two marker ER stress genes. We conclude that the usual practice of providing food for mice on their cage lids provides a significant level of physical activity that alters the metabolic status and increases ER stress. As such, this may not be the optimal model for the majority of mouse studies that are reported in the literature and it may significantly alter the interpretation of the effect of wheel running activity on ER stress. The differential effects of food location on hippocampal Bdnf gene expression also suggest that BDNF does not directly regulate UPR activity but may be coordinately regulated in response to running activity.

Endoplasmic reticulum stress signaling is involved in silver nanoparticles-induced apoptosis.

Although silver nanoparticles (AgNPs) have been reported to exert strong acute toxic effects on various cultured cells by inducing oxidative stress, the molecular mechanisms by which AgNPs-damaged cells are unknown. Because the endoplasmic reticulum (ER) may play an important role in the response to oxidative stress-induced damage and is quite sensitive to oxidative damage, we hypothesized that AgNPs may exert cytotoxic effects on cells by modulating ER stress. In our study, AgNPs resulted in cytotoxicity and apoptotic cell death when analyzing cell viability, DNA fragmentation and the apoptotic sub-G(1) population. Flow cytometry and confocal microscopy indicated that the cells were sensitive to AgNPs with respect to the induction of mitochondrial Ca(2+) overloading and enhancement of ER stress. AgNPs induced a number of signature ER stress markers, including phosphorylation of RNA-dependent protein kinase-like ER kinase (PERK) and its downstream eukaryotic initiation factor 2α, phosphorylation of inositol-requiring protein 1 (IRE1), splicing of ER stress-specific X-box transcription factor-1, cleavage of activating transcription factor 6 (ATF6) and up-regulation of glucose-regulated protein-78 and CCAAT/enhancer-binding protein-homologous protein (CHOP/GADD153). Down-regulation of PERK, IRE1 and ATF6 expression using siRNA significantly decreased AgNPs-induced the enhancement of ER stress. In addition, down-regulation of CHOP expression with siRNA CHOP attenuated AgNPs-induced apoptosis. Taken together, the present study supports an important role for the ER stress response in mediating AgNPs-induced apoptosis.

Endoplasmic reticulum stress is induced and modulated by enterovirus 71.

Picornavirus infection alters the endoplasmic reticulum (ER) membrane but it is unclear whether this induces ER stress. Infection of rhabdomyosarcoma cells with enterovirus 71 (EV71), a picornavirus, caused overexpression of the ER-resident chaperone proteins, BiP and calreticulin, and phosphorylation of eIF2alpha, but infection with UV-inactivated virus did not, indicating that ER stress was induced by viral replication and not by viral attachment or entry. Silencing (si)RNA knockdown demonstrated that phosphorylation of eIF2alpha was dependent on PKR: eIF2alpha phosphorylation was reduced by siPKR but not by siPERK. We provided evidence showing that PERK is upstream of PKR and is thus able to negatively regulate the PKR-eIF2alpha pathway. Pulse-chase experiments revealed that EV71 infection inhibited translation and activation of ATF6. Expression of BiP at the protein level was activated by a virus-dependent, ATF6-independent mechanism. EV71 upregulated XBP1 mRNA level, but neither IRE1-mediated XBP1 splicing nor its active spliced protein was detected, and its downstream gene, EDEM, was not activated. Epigenetic BiP overexpression alleviated EV71-induced ER stress and reduced viral protein expression and replication. Our results suggest that EV71 infection induces ER stress but modifies the outcome to assist viral replication.