ER Stress Inducer Thapsigargin Research Articles

Thapsigargin-induced ER stress suppressive compounds from the mushroom Mycoleptodonoides aitchisonii

Two novel compounds 1 and 2, along with two known ones (3 and 4) were isolated from the edible mushroom Mycoleptodonoides aitchisonii. The structures of 1–4 were determined and identified by interpreting their spectroscopic data. All the compounds showed protective activity against endoplasmic reticulum stress-dependent cell death.

Protective activity of carnosine and anserine against zinc-induced neurotoxicity: a possible treatment for vascular dementia.

Carnosine (β-alanyl-L-histidine) is a small dipeptide with numerous beneficial effects, including the maintenance of the acid-base balance, antioxidant properties, chelating agent, anti-crosslinking, and anti-glycation activities. High levels of carnosine and its analogue anserine (1-methyl carnosine) are found in skeletal muscle and the brain. Zinc (Zn)-induced neurotoxicity plays a crucial role in the pathogenesis of vascular dementia (VD), and carnosine inhibits Zn-induced neuronal death. Here, the protective activity of carnosine against Zn-induced neurotoxicity and its molecular mechanisms such as cellular Zn influx and Zn-induced gene expression were investigated using immortalised hypothalamic neurons (GT1-7 cells). Carnosine and anserine protected against Zn-induced neurotoxicity not by preventing increases in intracellular Zn(2+) but by participating in the regulation of the endoplasmic reticulum (ER) stress pathway and the activity-regulated cytoskeletal protein (Arc). Accordingly, carnosine and anserine protected against neurotoxicity induced by ER-stress inducers thapsigargin and tunicamycin. Hence, carnosine and anserine are expected to have future therapeutic potential for VD and other neurodegenerative diseases.

Ki-1/57 and CGI-55 ectopic expression impact cellular pathways involved in proliferation and stress response regulation

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events.

Impact of 60‐GHz millimeter waves and corresponding heat effect on endoplasmic reticulum stress sensor gene expression

Emerging high data rate wireless communication systems, currently under development, will operate at millimeter waves (MMW) and specifically in the 60 GHz band for broadband short-range communications. The aim of this study was to investigate potential effects of MMW radiation on the cellular endoplasmic reticulum (ER) stress. Human skin cell lines were exposed at 60.4 GHz, with incident power densities (IPD) ranging between 1 and 20 mW/cm(2) . The upper IPD limits correspond to the ICNIRP local exposure limit for the general public. The expression of ER-stress sensors, namely BIP and ORP150, was then examined by real-time RT-PCR. Our experimental data demonstrated that MMW radiations do not change BIP or ORP150 mRNA basal levels, whatever the cell line, the exposure duration or the IPD level. Co-exposure to the well-known ER-stress inducer thapsigargin (TG) and MMW were then assessed. Our results show that MMW exposure at 20 mW/cm(2) inhibits TG-induced BIP and ORP150 over expression. Experimental controls showed that this inhibition is linked to the thermal effect resulting from the MMW exposure.

TRIB3 alters endoplasmic reticulum stress-induced β-cell apoptosis via the NF-κB pathway

ObjectiveTo examine the effect of TRIB3 on endoplasmic reticulum stress induced β-cell apoptosis and to investigate the mechanism with a specific emphasis on the role of NF-κB pathway. Materials/MethodsWe investigated the effect of TRIB3 on ER stress-induced β-cell apoptosis in INS-1 cells and primary rodent islets. The potential role of TRIB3 in ER stress inducer thapsigargin (Tg)-induced β-cell apoptosis was assessed using overexpression and siRNA knockdown approaches. Inducible TRIB3 β-cells, regulated by the tet-on system, were used for sub-renal capsule transplantation in streptozotocin (STZ)-diabetic mice, to study the effect of TRIB3 on ER stress-induced β-cell apoptosis in vivo. Apoptosis was determined by TUNEL staining both in vivo and in vitro, while the molecular mechanisms of NF-κB activation were investigated. ResultsTRIB3 was induced in ER-stressed INS-1 cells and rodent islets, and its overexpression was accompanied by increased β-cell apoptosis. Specifically, TRIB3 overexpression enhanced Tg-induced INS-1 derived β-cell apoptosis both in vitro and in sub-renal capsular transplantation animal model. Additionally, knockdown of Trib3 blocked Tg-induced apoptosis. Mechanistically, the induction of TRIB3 during ER stress resulted in the activation of NF-κB and aggravated INS-1 derived β-cell apoptosis, while inhibiting the NF-κB pathway significantly abrogated this response and prevented β-cell apoptosis, both in vitro and in sub-renal capsular transplantation animal model. ConclusionTRIB3 mediated ER stress-induced β-cell apoptosis via the NF-κB pathway.

DON shares a similar mode of action as the ribotoxic stress inducer anisomycin while TBTO shares ER stress patterns with the ER stress inducer thapsigargin based on comparative gene expression profiling in Jurkat T cells

Previously, we studied the effects of deoxynivalenol (DON) and tributyltin oxide (TBTO) on whole genome mRNA expression profiles of human T lymphocyte Jurkat cells. These studies indicated that DON induces ribotoxic stress and both DON and TBTO induced ER stress which resulted into T-cell activation and apoptosis. The first goal of the present study was to provide final proof for these mode of actions by comparing the effects of 6h exposure to DON and TBTO on mRNA expression to those of positive controls of ribotoxic stress (anisomycin), ER stress (thapsigargin) and T cell activation (ionomycin). Genes affected by anisomycin and the majority of genes affected by thapsigargin were affected in the same direction by DON and TBTO, respectively, confirming the expected modes of action. Pathway analysis further sustained that DON induces ribotoxic stress and both DON and TBTO induce unfolded protein response (UPR), ER stress, T cell activation and apoptosis. The second goal was to assess whether DON and/or TBTO affect other pathways above those detected before. TBTO induced groups of genes that are involved in DNA packaging and heat shock response that were not affected by thapsigargin. DON did not affect other genes than anisomycin indicating the effect of DON to be restricted to ribotoxic stress. This study also demonstrates that comparative gene expression analysis is a very promising tool for the identification of modes of action of immunotoxic compounds.

Selection of autophagy or apoptosis in cells exposed to ER-stress depends on ATF4 expression pattern with or without CHOP expression

SummaryCells exposed to ER-stress undergo the Unfolded Protein Response (UPR) to avoid apoptosis, but may also activate autophagy. However, the signal for selection of one of these two protective responses is unknown. To clarify the key switch between autophagy and apoptosis, we examined the correlation of UPR-related signals with autophagy and/or apoptosis inductions in HepG2 cells exposed to three ER-stress inducers (NaF, tunicamycin, and thapsigargin) with time, including the effect of small interfering RNA on the cell responses. Thapsigargin-induced ER-stress caused only apoptosis after ∼2 hr with Ire1 phosphorylation, and Grp78, ATF4, and CHOP expressions. On the other hand, NaF- and tunicamycin-induced ER-stress caused only autophagy in the early stage by ∼8 hr with ATF4 expression and without CHOP expression. ATF4-siRNA completely inhibited the autophagy induced by NaF or tunicamycin with suppressed ATF4 protein and mRNA expressions, and also inhibited apoptosis by thapsigargin with suppression of both ATF4 and CHOP. CHOP-siRNA had no effect on autophagy activation by NaF and tunicamycin. On the other hand, CHOP-siRNA activated autophagy in thapsigargin-induced ER-stress with significant ATF4 expression, and suppressed apoptosis with CHOP suppression. These results showed that ATF4 is the key signal for autophagy induced by ER-stress, and that autophagy is switched to apoptosis by subsequent CHOP upregulation, suggesting that the changeover switch between autophagy and apoptosis is located between ATF4 to CHOP in the PERK pathway.

Cardiomyocyte specific overexpression of C3orf58 activates ER stress leading to impaired cardiac function

Although accumulating evidence supports the crucial role of ER stress in cardiovascular diseases, essential information is still required for its role in the pathogenesis of these disorders. We previously reported the identification of C3orf58 (aka HASF) as a novel stem cell derived factor involved in cytoprotection. Transgenic mice with a high‐level of cardiac specific overexpression of HASF showed reduced fractional shortening as early as 4 weeks old. Neonatal cardiomyocytes isolated from these mice showed reduction in basal Ca2+ levels, abnormal calcium oscillation and a dramatic reduction in magnitude of store‐operated Ca2+ entry after stimulation. Adult transgenic cardiomyocytes exhibited reduced L type channel currents. Notable, all three pathways involved in ER stress, PERK, ATF6 and IRE1, were activated in the hearts of transgenic mice as early as 2 weeks old. Moreover, to demonstrate the specific effect of HASF on ER stress, fibroblasts isolated from HASF heterologous KO were treated with ER stress inducers thapsigargin and tunicamycin. The induction of ER stress sensor GPR78 was significantly lower in these treated fibroblasts. Based on our findings we propose that C3orf58 is a novel regulator of ER stress pathways whose overexpression is involved in cardiac pathology and disease.The study was supported by NHLBI/NIH grants RO1 HL35610, HL81744, HL72010, HL73219 and Foundation Leducq (to VJD).

MicroRNA-34b Inhibits Pancreatic Cancer Metastasis Through Repressing Smad3

Pancreatic cancer is characterized by extremely poor prognosis because of early recurrence and metastasis, and increasing evidence supports the critical role of microRNA in cancer progression. Here we identified that microRNA-34b functioned as a tumor-suppressing microRNA by targeting oncogenic Smad3 in pancreatic cancer. As a hypovascular tumor with a potential endoplasmic reticulum stress microenvironment, miR-34b was silenced after ER stress inducer thapsigargin (Tg) treatment and negatively regulated by ER stress chaperone glucose regulated protein 78 (GRP78) in pancreatic cancer cells. In human specimens, we found that miR-34b was down-regulated in pancreatic cancer tissues and low level of miR-34b expression was positively correlated with tumor-node-metastasis (TNM) stage, lymph-node metastasis and overall survival. Functional assays showed that over-expression of miR-34b inhibited pancreatic cancer progression in vitro and in vivo. In addition, Smad3 was demonstrated as a direct target of miR-34b and negatively regulated by miR- 34b at mRNA and protein levels. Luciferase assays confirmed that miR-34b could directly bind to the 3'untranslated region of Smad3. An inverse correlation between miR-34b and Smad3 was observed in 64 pancreatic cancer tissues. Our findings indicate that miR-34b acts as a tumor metastasis suppressor through negatively modulating Smad3, which may provide a potential therapeutic strategy for pancreatic cancer.

Mitochondrial aldehyde dehydrogenase obliterates endoplasmic reticulum stress-induced cardiac contractile dysfunction via correction of autophagy

ER stress triggers myocardial contractile dysfunction while effective therapeutic regimen is still lacking. Mitochondrial aldehyde dehydrogenase (ALDH2), an essential mitochondrial enzyme governing mitochondrial and cardiac function, displays distinct beneficial effect on the heart. This study was designed to evaluate the effect of ALDH2 on ER stress-induced cardiac anomalies and the underlying mechanism involved with a special focus on autophagy. WT and ALDH2 transgenic mice were subjected to the ER stress inducer thapsigargin (1mg/kg, i.p., 48h). Echocardiographic, cardiomyocyte contractile and intracellular Ca2+ properties as well as myocardial histology, autophagy and autophagy regulatory proteins were evaluated. ER stress led to compromised echocardiographic indices (elevated LVESD, reduced fractional shortening and cardiac output), cardiomyocyte contractile and intracellular Ca2+ properties and cell survival, associated with upregulated autophagy, dampened phosphorylation of Akt and its downstream signal molecules TSC2 and mTOR, the effects of which were alleviated or mitigated by ALDH2. Thapsigargin promoted ER stress proteins Gadd153 and GRP78 without altering cardiomyocyte size and interstitial fibrosis, the effects of which were unaffected by ALDH2. Treatment with thapsigargin in vitro mimicked in vivo ER stress-induced cardiomyocyte contractile anomalies including depressed peak shortening and maximal velocity of shortening/relengthening as well as prolonged relengthening duration, the effect of which was abrogated by the autophagy inhibitor 3-methyladenine and the ALDH2 activator Alda-1. Interestingly, Alda-1-induced beneficial effect against ER stress was obliterated by autophagy inducer rapamycin, Akt inhibitor AktI and mTOR inhibitor RAD001. These data suggest a beneficial role of ALDH2 against ER stress-induced cardiac anomalies possibly through autophagy reduction.

Endoplasmic reticulum stress in HepG2 cells inhibits apolipoprotein A-I secretion

AimsEndoplasmic reticulum (ER) stress modulates gene expression and has been implicated in causing dyslipidemias. To determine if ER stress may contribute to hypoalphalipoproteinemia through suppression of apo A-I gene expression, human hepatoma cell line Hep G2 was treated with ER stress inducers and the changes in apo A-I gene expression were compared to albumin gene expression. Main methodsHepG2 cells were treated with tunicamycin (TM) and thapsigargin (TG), two potent inducers of ER stress, and apo A-I and albumin protein levels, mRNA levels, and promoter activity were measured. ER stress was measured using the ER stress-responsive alkaline phosphatase assay and by Western blot quantitation of ER stress markers such as glucose-regulated protein-78 (GRP-78), phosphorylated Jun N-terminal kinase (phospho-JNK), total JNK, phosphorylated eukaryotic initiation factor 2 alpha (phospho eIF2α), and total eIF2α. Key findingsTM and TG induced ER stress in HepG2 cells and reduced apo A-I and albumin secretion in a dose-dependent manner. Intracellular albumin levels increased in cells treated with TM and TG while intracellular apo A-I levels decreased. Albumin mRNA and albumin gene promoter activity were reduced in proportion to the decrease in albumin secreted while changes in the apo A-I mRNA levels and promoter activity were modest and did not account for the reduction in apo A-I secretion. SignificanceThese results indicate that apo A-I secretion is inhibited by ER stress possibly by affecting cellular degradation pathways. However, ER stress does not affect apo A-I secretion by regulating gene expression.

Role of calcium-independent phospholipase A2β in human pancreatic islet β-cell apoptosis

Death of β-cells due to apoptosis is an important contributor to β-cell dysfunction in both type 1 and type 2 diabetes mellitus. Previously, we described participation of the Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)β) in apoptosis of insulinoma cells due to ER stress. To examine whether islet β-cells are similarly susceptible to ER stress and undergo iPLA(2)β-mediated apoptosis, we assessed the ER stress response in human pancreatic islets. Here, we report that the iPLA(2)β protein is expressed predominantly in the β-cells of human islets and that thapsigargin-induced ER stress promotes β-cell apoptosis, as reflected by increases in activated caspase-3 in the β-cells. Furthermore, we demonstrate that ER stress is associated with increases in islet iPLA(2)β message, protein, and activity, iPLA(2)β-dependent induction of neutral sphingomyelinase and ceramide accumulation, and subsequent loss of mitochondrial membrane potential. We also observe that basal activated caspase-3 increases with age, raising the possibility that β-cells in older human subjects have a greater susceptibility to undergo apoptotic cell death. These findings reveal for the first time expression of iPLA(2)β protein in human islet β-cells and that induction of iPLA(2)β during ER stress contributes to human islet β-cell apoptosis. We hypothesize that modulation of iPLA(2)β activity might reduce β-cell apoptosis and this would be beneficial in delaying or preventing β-cell dysfunction associated with diabetes.

Abstract 353: Sunitinib Induces Endoplasmic Reticulum Stress in Cardiomyocytes

Introduction: Chemotherapy-induced cardiac toxicities are the main obstacle for effective treatment of cancer, particularly in patients with pre-existing heart disease. This often results in premature termination of cancer treatment. Prominent among these agents is sunitinib (Sutent), which causes cardiac dysfunction in nearly 20% of cancer patients. It has recently been suggested that cardiac microvascular dysfunction, particularly loss of pericyte coverage, underlies sunitinib-induced cardiac dysfunction. However the exact mechanisms of sunitinib-induced cardiotoxicites are still unknown. Results: Neonatal rat ventricular myocytes were treated with increasing concentrations (100nM-5uM) of sunitinib. Irrespective of the dose, there was a significant increase in markers of ER stress (Grp78, Grp 94, Erp72 and phospo/total-eIF2a) after 24 hours of treatment. The ER stress inducer thapsigargin was used as a positive control. Concordant with an increase in ER stress was the activation of mTOR in sunitinib (but not thapsigargin) treated NRVMs, suggesting a possible role of mTOR-mediated proteotoxicity in sunitinib-induced cardiac dysfunction. Protein synthesis, evaluated by 14 C-phenylalanine incorporation into proteins, was moderately elevated in cells treated with sunitinib. Conclusions: In addition to impairment of angiogenesis and pericyte loss, sunitinib induces mTOR activation and ER stress. We propose that the cardiac myocyte secretes paracrine factors that stabilize the cardiac microcirculation. This process is disrupted by sunitinib and ultimately leads to heart failure.

Abstract 2793: Targeting the unfolded protein response as a therapeutic approach in hematological malignancies

Abstract The unfolded protein response (UPR) is a complex signaling pathway activated in response to endoplasmic reticulum stress. In recent years, the UPR has been implicated in cancer and chemosensitivity, particularly solid tumors. We have investigated the potential value of targeting the UPR as a novel therapeutic approach in hematological malignancies using a panel of cell lines representing AML, lymphoma and multiple myeloma. UPR activation was determined from expression of the key ER chaperones GRP78 and GRP94 and UPR mediators using Western blot analysis. The effect of cytotoxic agents and agents that induce (thapsigargin, tunicamycin), target (versipelostatin) or silence (siRNA) UPR proteins were studied, alone and in combination, using cytotoxicity, apoptosis and colony formation assays. The UPR was constitutively active in these hematological cancer cell lines, with differential activation of key UPR proteins across the tumor types studied. There were also differences in UPR activation between the hematological cell line panel, peripheral blood mononuclear cells and the colorectal cancer cell line HT-29. Further UPR activation was seen at the translational level following minimally toxic (EC5), and cytotoxic (EC25 and EC50), concentrations of ER stress inducers, proteasome and HSP90 inhibitors. A number of strategies were used to study the effect of UPR modulation on chemosensitivity. Minimally toxic concentrations of the ER stress inducer thapsigargin protected cells from cytotoxic agents, with a reduction in antiproliferative drug effect. The activity of the novel small molecule versipelostatin, reported to downregulate the ER molecular chaperones GRP78 and GRP94, was also investigated. The downregulation of these chaperone proteins previously reported in solid tumor cell lines (Park et al. 2004) was confirmed in HT-29 cells, but downregulation was not observed in the hematological cell lines studied, although versipelostatin was an effective cytotoxic agent at low micromolar concentration. Combination experiments with the chemical chaperone 4-phenylbutyric acid (PBA) resulted in a small increase in apoptosis when PBA was combined with ER stress inducing agents. However, PBA also showed HDAC inhibitory activity at the concentrations used, suggesting the interaction seen may be due to HDAC inhibition in addition to, or instead of, its chemical chaperone activity. Finally, siRNA mediated silencing of GRP78 and GRP94 in the THP1 (AML) and U266 (multiple myeloma) cells resulted in a decrease in the targeted protein, but showed only minimal effects on the activity of chemotherapy. In conclusion, the UPR is activated in these hematological cancer cell lines and plays a complex role in chemosensitivity. In contrast to previous reports in solid tumor cells, modulating the UPR in these hematological malignancies had only a modest effect on sensitivity to cytotoxic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2793. doi:1538-7445.AM2012-2793

α-Mangostin Reduced ER Stress-mediated Tumor Growth through Autophagy Activation

α-Mangostin is a xanthon derivative contained in the fruit hull of mangosteen (Garcinia mangostana L.), and the administration of α-Mangostin inhibited the growth of transplanted colon cancer, Her/CT26 cells which expressed Her-2/neu as tumor antigen. Although α-Mangostin was reported to have inhibitory activity against sarco/endoplasmic reticulum Ca2+ ATPase like thapsigargin, it showed different activity for autophagy regulation. In the current study, we found that α-Mangostin induced autophagy activation in mouse intestinal epithelial cells, as GFP-LC3 transgenic mice were orally administered with 20 mg/kg of α-Mangostin daily for three days. However, the activation of autophagy by α-Mangostin did not significantly increase OVA-specific T cell proliferation. As we assessed ER stress by using XBP-1 reporter system and phosphorylation of eIF2α, thapsigargin-induced ER stress was significantly reduced by α-Mangostin. However, coadministration of thapsigargin with α-Mangostin completely blocked the antitumor activity of α-Mangostin, suggesting ER stress with autophagy blockade accelerated tumor growth in mouse colon cancer model. Thus the antitumor activity of α-Mangostin can be ascribable to the autophagy activation rather than ER stress induction.

Endoplasmic reticulum stress causes EBV lytic replication

AbstractEndoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)–specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication. SAL showed ER-stress–dependent and –independent antiviral effects, preventing virus release in human LCLs and abrogating gp350 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)–treated B95-8 cells. TG resulted in sustained BCL6 but not BLIMP1 or CD138 expression, which is consistent with maintenance of a germinal center B-cell, rather than plasma-cell, phenotype. Microarray analysis identified candidate genes governing lytic replication in LCLs undergoing ER stress.

RETRACTED: Thapsigargin triggers cardiac contractile dysfunction via NADPH oxidase-mediated mitochondrial dysfunction: Role of Akt dephosphorylation

ER stress triggers myocardial contractile dysfunction although the underlying mechanism is still elusive. Given that NADPH oxidase was recently implicated in ER stress-induced tissue injury, this study was designed to examine the role of NADPH oxidase in ER stress-induced cardiac mechanical defects and the impact of Akt activation on ER stress-induced cardiac anomalies. Wild-type and transgenic mice with cardiac-specific overexpression of an active mutant of Akt (MyAkt) were subjected to the ER stress inducer thapsigargin (1 and 3mg/kg, ip, for 48h). Thapsigargin compromised echocardiographic parameters, including elevating LVESD and reducing fractional shortening; suppressed cardiomyocyte contractile function, intracellular Ca(2+) handling, and cell survival; and enhanced carbonyl formation, apoptosis, superoxide production, NADPH oxidase expression, and mitochondrial damage. Interestingly, these anomalies were attenuated or mitigated by chronic Akt activation. Treatment with thapsigargin also dephosphorylated Akt and its downstream signal GSK3β (leading to activation of GSK3β), the effect of which was abrogated in MyAkt hearts. Knockdown of the cytosolic subunit of NADPH oxidase, p47(phox), using siRNA abrogated thapsigargin-induced apoptosis and cell death in H9C2 myoblasts. In vitro exposure to thapsigargin induced murine cardiomyocyte dysfunction reminiscent of the in vivo setting, the effects of which were ablated by the NADPH oxidase inhibitor apocynin and the mitochondrial Ca(2+) uptake inhibitor Ru360. In addition, apocynin abrogated thapsigargin-induced loss of mitochondrial membrane potential and permeability transition pore opening, similar to chronic Akt activation. In summary, these data suggest that ER stress interrupts cardiac contractile and intracellular Ca(2+) homeostasis, cell survival, and mitochondrial integrity through an Akt dephosphorylation- and NADPH oxidase-dependent mechanism.

Caspase‐11 mediates ischemia‐induced astrocyte death: Involvement of endoplasmic reticulum stress and C/EBP homologous protein

Astrocytes are essential cells for maintaining brain integrity. We have recently shown that the transcription factor C/EBP homologous protein (CHOP), associated with endoplasmic reticulum (ER) stress, plays a key role in the astrocyte death induced by ischemia. Meanwhile, mediators of apoptosis downstream of CHOP in the ER stress-dependent pathway remain to be elucidated. Our aim in this work was to determine whether caspase-11, able to activate apoptotic and proinflammatory pathways, is implicated in ER stress-dependent astrocyte death in ischemic conditions. According to our results, caspase-11 is up-regulated in primary astrocyte cultures following either oxygen and glucose deprivation (OGD) or treatment with the ER-stress inducers thapsigargin and tunicamycin. Moreover, these same stimuli increased caspase-11 mRNA levels and luciferase activity driven by a caspase-11 promoter, indicating that caspase-11 is regulated at the transcriptional level. Our data also illustrate the involvement of ER stress-associated CHOP in caspase-11 regulation, insofar as CHOP overexpression by means of an adenoviral vector caused a significant raise in caspase-11. In turn, caspase-11 suppression with siRNA rescued astrocytes from OGD- and ER stress-induced death, supporting the idea that caspase-11 is responsible for the deleterious effects of ischemia on astrocytes. Finally, inhibition of caspase-1 and caspase-3 significantly reduced astrocyte death, which indicates that these proteases act as death effectors of caspase-11. In conclusion, our work contributes to clarifying the pathways leading to astrocyte death in response to ischemia by defining caspase-11 as a key mediator of the ER stress response acting downstream of CHOP.

Docosahexaenoic Acid Activates Some SREBP‐2 Targets Independent of Cholesterol and ER Stress in SW620 Colon Cancer Cells

The SREBP-2 transcription factor is mainly activated by low cellular cholesterol levels. However, other factors may also cause SREBP-2 activation. We have previously demonstrated activation of SREBP-2 by the polyunsaturated fatty acid docosahexaenoic acid (DHA) in SW620 colon cancer cells. Despite activation of SREBP-2, only a few target genes were induced and cholesterol biosynthesis was reduced. In the present study, gene expression analysis at early time points verified the previously observed SREBP-2 target gene expression pattern. Activation of SREBP-2 using siRNAs targeting Niemann Pick C1 protein (NPC1) led to increased expression of all SREBP target genes examined, indicating that activation of some SREBP-2 target genes is inhibited during DHA-treatment. Cholesterol supplementation during DHA treatment did not abolish SREBP-2 activation. We also demonstrate that activation of SREBP-2 is independent of ER stress and eIF2alpha phosphorylation, which we have previously observed in DHA-treated cells. Thapsigargin-induced ER stress repressed expression of SREBP-2 target genes, but with a different pattern than observed in DHA-treated cells. Moreover, oleic acid (OA) treatment, which does not induce ER stress in SW620 cells, led to activation of SREBP-2 and induced a target gene expression pattern similar to that of DHA-treated cells. These results indicate that DHA and OA may activate SREBP-2 and inhibit activation of SREBP-2 target genes through a mechanism independent of cholesterol level and ER stress.

Time Series Proteome Profiling To Study Endoplasmic Reticulum Stress Response

Time series profiling is a powerful approach for obtaining information on protein expression dynamics and prevailing biochemical pathways. To date, such information could only be obtained at the mRNA level using mature and highly parallel technologies such as microarray gene expression profiling. The generation of time series data at the protein level has lagged due to the lack of robust and highly reproducible methodologies. Using a combination of SILAC strategy, SDS-PAGE and LC-MS/MS, we demonstrate successful monitoring of expression levels of the same set of proteins across different time points within the ER compartment of human primary fibroblast cells when exposed to ER stress inducers tunicamycin and thapsigargin. Data visualization was facilitated using GeneSpring GX analysis platform that was designed to process Affymetrix microarray data. This software also facilitated the generation of important parameters such as data normalization, calculation of statistical values to extract significant changes in protein expression, and the cross comparison of data sets.