Phosphorylation Of PERK Research Articles

Nicotine Directly Induces Endoplasmic Reticulum Stress Response in Rat Placental Trophoblast Giant Cells.

Nicotine exposure during pregnancy leads to placental insufficiency impairing both fetal and neonatal development. Previous studies from our laboratory have demonstrated that in rats, nicotine augmented endoplasmic reticulum (ER) stress in association with placental insufficiency; however, the underlying mechanisms remain elusive. Therefore, we sought to investigate the possible direct effect of nicotine on ER stress in Rcho-1 rat placental trophoblast giant (TG) cells during differentiation. Protein and/or mRNA expression of markers involved in ER stress (eg, phosphorylated PERK, eIF2α, CHOP, and BiP/GRP78) and TG cell differentiation and function (eg, Pl-1, placental growth factor [Pgf], Hsd11b1, and Hsd11b2) were quantified via Western blot or real-time polymerase chain reaction. Nicotine treatment led to dose-dependent increases in the phosphorylation of PERK[Thr981] and eIF2α[Ser51], whereas pretreatment with a nicotinic acetylcholine receptor (nAChR) antagonist (mecamylamine hydrochloride) blocked the induction of PERK phosphorylation, verifying the direct involvement of nicotine and nAChR binding. We next investigated select target genes known to play essential roles in placental TG cell differentiation and function (Pl-1, Pgf, Hsd11b1, and Hsd11b2), and found that nicotine significantly augmented the mRNA levels of Hsd11b1 in a dose-dependent manner. Furthermore, using tauroursodeoxycholic acid, a safe bile acid known to improve protein chaperoning and folding, we were able to prevent nicotine-induced increases in both PERK phosphorylation and Hsd11b1 mRNA levels, revealing a potential novel therapeutic approach to reverse the deleterious effects of nicotine exposure in pregnancy. Collectively, these results implicate that nicotine, acting through its receptor, can directly augment ER stress and impair placental function.

Abstract A039: Caspase-2 and oxidative-ER stress crosstalk regulates the exposure of “eat me” signal calreticulin by high hydrostatic pressure treated cancer cells

Abstract Immunogenic cell death (ICD) is a type of cell death induced by various chemotherapeutics, physical modalities such as photodynamic therapy, radiotherapy and high hydrostatic pressure (HHP). HHP induces immunogenic cell death of cancer cell is currently tested in running II. and III. phase of clinical trial for preparation of active cellular cancer immunotherapy (DCVAC). Previous studies have shown that the immunogenicity of ICD mainly relies on surface exposed calreticulin (ecto-CRT); CRT is normally located in the lumen of the endoplasmic reticulum (ER) and may translocate to the cell surface within hours after treatment. Although ICD can be triggers by HHP, underlying danger signaling pathways remain unclear. Therefore ovarian (OV90) cancer cell line was treated by HHP, UV-B and anthracycline (Idarubicin). The kinetics of ICD markers and key components of ER stress-mediated apoptotic pathway were analyzed by flow cytometry, western blotting and confocal microscopy. In this work we described the ability of HHP triggers oxidative ER-stress (the production of reactive oxygen species (ROS) together with ER stress response) which results in phosphorylation of PERK and eIF2α. We also observed a rapid caspase-8 activation subsequently followed by BAP31 cleavage, Bid activation and caspase-3 activation. While caspase-8 activation was necessary for cell surface exposure of calreticulin (ecto-CRT) after HHP treatment, proapoptotic proteins Bax and Bak were dispensable for this process. Moreover, we identified that HHP treatment leads to caspase-2 activation. Whereas Idarubicin treatment has no effect on ecto-CRT exposure in Caspase-2 knockdown tumor cells, ecto-CRT was significantly decreased in these cells after HHP treatment. In addition, phagocytosis of HHP-treated Caspase-2 knockdown tumor cells was compromised in contrast to Idarubicin-treated knockout tumor cells. In conclusion, we characterized the molecular mechanism of ecto-CRT exposure pathway induced by HHP which is analogous to that induced by anthracycline treatment. For the first time we provide evidence that caspase-2 might play an important role in the HHP-mediated ecto-CRT exposure. Citation Format: Irena Kusova Moserova, Iva Truxova, Abhishek G, Garg, Patrizia Agostinis, Piere Francois Cartron, Sarka Vosahlikova, Radek Spisek, Jitka Fucikova. Caspase-2 and oxidative-ER stress crosstalk regulates the exposure of “eat me” signal calreticulin by high hydrostatic pressure treated cancer cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A039.

Spermine inhibits Endoplasmic Reticulum Stress - induced Apoptosis: a New Strategy to Prevent Cardiomyocyte Apoptosis

Background/Aims: Endoplasmic reticulum stress (ERS) plays an important role in the progression of acute myocardial infarction (AMI), in part by mediating apoptosis. Polyamines, including putrescine, spermidine, and spermine, are polycations with anti-oxidative, anti-aging, and cell growth-promoting activities. This study aimed to determine the mechanisms by which spermine protects against ERS-induced apoptosis in rats following AMI. Methods and Results: AMI was established by ligation of the left anterior descending coronary artery (LAD) in rats, and exogenous spermine was administered by intraperitoneal injection (2.5 mg/ml daily for 7 days pre-AMI). Spermine treatment limited infarct size, attenuated cardiac troponin I and creatinine kinase-MB release, improved cardiac function, and decreased ERS and apoptosis related protein expression. Isolated cardiomyocytes subjected to hypoxia showed significant increase in reactive oxygen species (ROS) and the expression of apoptosis and ERS related proteins; these effects occurred through PERK and eIF2α phosphorylation. The addition of spermine attenuated cardiomyocyte apoptosis, suppressed the production of ROS, and inhibited ERS related pathways. Conclusions: Spermine was an effective pre-treatment strategy to attenuate cardiac ERS injury in rats, and the cardioprotective mechanism occurring through inhibition of ROS production and down regulation of the PERK-eIF2α pathway. These findings provide a novel target for the prevention of apoptosis in the setting of AMI.

Mitigating ER stress and oxidative stress is beneficial after traumatic brain injury

Event Abstract Back to Event Mitigating ER stress and oxidative stress is beneficial after traumatic brain injury Raghu Vemuganti1*, S L. Mehta1, L Bodden1, V Nakka1, T H. Kim1, R Chandran1, E Udho2, P Cengiz2 and R J. Dempsey1 1 University of Wisconsin, Department of Neurological Surgery, United States 2 University of Wisconsin, Department of Pediatrics, United States Endoplasmic reticulum (ER) stress and oxidative stress are known proponents of secondary neuronal damage and neurological dysfunction following traumatic brain injury (TBI). A moderate controlled cortical impact-induced TBI in adult mice significantly induced the phosphorylation of PERK and eIF2α and also increased the expression of the down-stream CHOP and ATF4 indicating activation of PERK signaling pathway that kills neurons if uncontrolled. We further show that treating adult mice with salubrinal (an inhibitor of PERK pathway) leads to a significant decrease in the secondary cortical contusion with a good window of efficacy (5 min to 4h after injury). In the cohort of mice treated with salubrinal at 2h after TBI, there was a significant improvement in the motor function recovery measured by rotarod test and beam walk test. TBI also increased the expression NADPH oxidase subunit NOX2 (gp91phox; promotes formation of reactive oxygen species) and the transcription factor Nrf2 (induces down-stream anti-oxidant enzymes). We tested apocynin (an inhibitor of NOX2; 10 mg/Kg) and TBHQ (an activator of Nrf2; 25 mg/Kg) alone or in combination in cohorts of mice subjected to CCI injury. Either apocynin or TBHQ treatment alone significantly improved the motor function after TBI compared to vehicle control. The drug treated mice stayed longer on rotarod and made fewer foot faults in beam walk test between day 1 to 7 after injury. The combination therapy (apocynin + TBHQ) also significantly improved the post-TBI motor function compared to vehicle control, but there was no additive effect of the 2 drugs. Following the combination therapy, mice spent significantly greater time with higher frequency in the platform quadrant as compared to vehicle control when tested for cognition by Morris Water maze test at 3 weeks after injury. The cortical contusion volume was also significantly lower in the combo therapy group compared to vehicle control. In conclusion, these studies show that controlling either ER stress or oxidative stress significantly improves the functional outcome and tissue sparing after TBI. Keywords: Endoplasmic Reticulum Stress, Oxidative Stress, Motor function, in vivo, Brain Injury Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Free Paper Session 2: Molecular Mechanisms of the Nervous System Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Vemuganti R, Mehta SL, Bodden L, Nakka V, Kim TH, Chandran R, Udho E, Cengiz P and Dempsey RJ (2016). Mitigating ER stress and oxidative stress is beneficial after traumatic brain injury. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00105 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Prof. Raghu Vemuganti, University of Wisconsin, Department of Neurological Surgery, Madison, WI, United States, vemuganti@neurosurgery.wisc.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Raghu Vemuganti S L Mehta L Bodden V Nakka T H Kim R Chandran E Udho P Cengiz R J Dempsey Google Raghu Vemuganti S L Mehta L Bodden V Nakka T H Kim R Chandran E Udho P Cengiz R J Dempsey Google Scholar Raghu Vemuganti S L Mehta L Bodden V Nakka T H Kim R Chandran E Udho P Cengiz R J Dempsey PubMed Raghu Vemuganti S L Mehta L Bodden V Nakka T H Kim R Chandran E Udho P Cengiz R J Dempsey Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

The role of heme oxygenase-1 in drug metabolizing dysfunction in the alcoholic fatty liver exposed to ischemic injury

This study was designed to investigate the role of heme oxygenase-1 (HO-1) in hepatic drug metabolizing dysfunction after ischemia/reperfusion (IR) in alcoholic fatty liver (AFL). Rats were fed a Lieber–DeCarli diet for five weeks to allow for development of AFL and were then subjected to 90min of hepatic ischemia and 5h of reperfusion. Rats were pretreated with hemin (HO-1 inducer) or ZnPP (HO-1 inhibitor) for 16h and 3h before hepatic ischemia. After hepatic IR, ethanol diet (ED)-fed rats had higher serum aminotransferase activities and more severe hepatic necrosis compared to the control diet (CD)-fed rats. These changes were attenuated by hemin and exacerbated by ZnPP. The activity and gene expression of HO-1 and its transcription factor (Nrf2) level increased significantly after 5h of reperfusion in CD-fed rats but not in ED-fed rats. After reperfusion, cytochrome P450 (CYP) 1A1, 1A2, and 2B1 activities were reduced to levels lower than those observed in sham group, whereas CYP2E1 activity increased. The decrease in CYP2B1 activity and the increase in CYP2E1 activity were augmented after hepatic IR in ED-fed animals. These changes were significantly attenuated by hemin but aggravated by ZnPP. Finally, CHOP expression and PERK phosphorylation, microsomal lipid peroxidation, and levels of proinflammatory mediators increased in ED-fed rats compared to CD-fed rats after reperfusion. These increases were attenuated by hemin. Our results suggest that AFL exacerbates hepatic drug metabolizing dysfunction during hepatic IR via endoplasmic reticulum stress and lipid peroxidation and this is associated with impaired HO-1 induction.

ONC201 Induces p53-Independent Apoptosis and Abrogates Stem Cell Function in Hematological Malignancies By Induction of ATF4 through Integrated Stress Response

The clinical challenge posed by p53-deficiency in hematological malignancies needs novel therapeutic strategies. ONC201, a first-in-class small molecule, was discovered as a p53-independent activator of apoptosis with a benign preclinical safety profile (Allen et al, Sci Transl Med 2013). Here we report that ONC201 exerts anti-tumor effects in hematological malignancies and leukemia stem cells (LSC) via the induction of ATF4 in the integrated stress response (ISR).ONC201 induced p53-independent apoptosis in cell lines and primary patient samples from mantle cell lymphomas (MCL) and acute myeloid leukemias (AML), independent of genetic alterations that correlate with poor prognosis (e.g., TP53 mutation, FLT3-ITD, or complex karyotype). ONC201 also induced apoptosis in LSC, while sparing normal bone marrow progenitors, as measured in vitro and after transplantation. Specifically, we recovered unfractionated LSC-containing populations of AML cells (t(9;11)(p22; q23), CEBPA and ATM mutant) from secondarily-engrafted mice and cultured them in vitro for 48 hr in two groups, ONC201-treated (5 uM) or Control. For both groups, the same number of Trypan Blue-negative cells was then re-transplanted. The frequency of human CD45+ cells 4 weeks after transplantation was 38.09 ± 2.59 % of tibial BM cells in untreated mice and 0.10 ± 0.05% in treated mice (n = 3 for each, p < 0.01). The survival of the treated group was dramatically prolonged (Figure 1).Gene Set Enrichment Analysis (GSEA) of gene expression profiling (GEP) data of Jeko-1 and Z-138 cells treated with ONC201 implicated up-regulated Endoplasmic Reticulum (ER) stress-related genes, such as targets of the ER stress-induced transcription factor CHOP (DDIT3; FDR q = 0.016), and ER component proteins (FDR q = 0.039). We confirmed the ONC201-induced increased mRNA levels of DDIT3, GADD34, DR5 and TRIB3, and increased protein levels of CHOP, ATF4 and IRE1-a in Jeko-1 cells. Knockdown of ATF4 and IRE1-a revealed that ATF4 is an essential protein for ONC201-induced apoptosis in most AML cells and MCL cells, while IRE1-a is only necessary for apoptosis in MCL cells. However, unlike other ER stress inducers, ONC201 did not cause phosphorylation of an eIF2a kinase PERK, a hallmark of classical ER stress. Importantly, ONC201 was also effective in lymphoma cells resistant to bortezomib, an ER stress inducer, supporting the notion that ONC201 uses a unique mechanism to trigger the ISR. In addition, ONC201 inhibited mTORC1 signaling, likely secondary to ATF4 activation via the induction of DDIT4, a negative regulator of mTORC1, which presumably increases the cytotoxicity of ONC201 by global translation inhibition.Investigating the type of apoptosis induced by ONC201, we examined protein levels of anti-apoptotic BCL-2 family members (BCL-2, BCL-XL and MCL-1) after ONC201 treatment. MCL-1 was reduced most notably after 12 hr. We then tested the effect of ONC201 on cells with overexpression or knockdown of BCL-2 family proteins. MCL-1 knockdown in OCI-AML3 cells increased their sensitivity to ONC201 only slightly, but ONC201 efficacy was dramatically reduced in BCL-2-overexpressing HL-60 cells, even more so than in BCL-XL-overexpressing HL-60 cells. Therefore, we investigated whether ONC201 sensitivity could be increased by ABT-199, a small-molecule BH3 mimetic that specifically inhibits BCL-2, and is known to be ineffective in cell lines with MCL-1 overexpression; accordingly, the combination of ONC201 and BCL-2 antagonist ABT-199 was highly synergistic (Figure 2).In conclusion, ONC201 induces p53-independent apoptosis and abrogates LSC function by ATF4 induction, via the ISR. By suppressing MCL-1, ONC201 can also increase the effectiveness of the Bcl-2 inhibitor ABT-199. These findings suggest that ONC201 may provide promising novel therapeutic strategies for TP53 -wild type and TP53 -mutant hematological malignancies. Phase I/II clinical trials have been initiated at the MD Anderson Cancer Center in leukemias and lymphomas to determine safety, efficacy and further characterize mechanism of action. [Display omitted] [Display omitted] DisclosuresIshizawa:Karyopharm: Research Funding. Allen:Oncoceutics, Inc: Employment, Equity Ownership. Orlowski:Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Array BioPharma: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; Forma Therapeutics: Consultancy; Onyx Pharmaceuticals: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Spectrum Pharmaceuticals: Research Funding. Wang:Celgene: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Andreeff:Oncoceutics, Inc.: Membership on an entity's Board of Directors or advisory committees.

Abstract C142: Functional characterization of P58IPK-PERK pathway in prostate cancer cells

Abstract Androgen signaling is important for the normal development and function of the prostate as well as for prostate cancer (PCa). We recently found that androgens activated the IRE1α-XBP1 arm of the canonical unfolded protein response (UPR) pathways and simultaneously inhibited PERK-eIF2α signaling. Activation of the IRE1α-XBP1 arm was mediated by direct binding of the androgen receptor (AR) in the vicinity of IRE1, as well as XBP1s target genes, and increase in their expression. Consistently, AR and IRE1α pathway gene expression are correlated in human PCa samples and XBP1s protein expression is significantly increased in cancer compared to normal prostate. However, the mechanisms behind androgen mediated inhibition of PERK-eIF2α signaling are not clear at present. One possible mediator in this regard is P58IPK, an XBP1s target gene that can interact with and inhibit PERK and eIF2α phosphorylation. Here, we show that P58IPK expression is increased by androgens in a time-dependent manner and that it plays a pro-survival role in PCa cells. P58IPK knockdown activated PERK expression as well as subsequent eIF2α phosphorylation when induced with the UPR activator thapsigargin. These findings suggest that P58IPK mediates, at least in part, the differential androgen effects between the IRE1α and PERK signaling in PCa and may be a potential therapeutic target. Citation Format: Xia Sheng, Margrethe Storm, Yang Jin, Fahri Saatcioglu. Functional characterization of P58IPK-PERK pathway in prostate cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C142.

Endoplasmic reticulum pathology and stress response in neurons precede programmed necrosis after neonatal hypoxia-ischemia

The endoplasmic reticulum (ER) is tasked, among many other functions, with preventing excitotoxicity from killing neurons following neonatal hypoxia-ischemia (HI). With the search for delayed therapies to treat neonatal HI, the study of delayed ER responses becomes relevant. We hypothesized that ER stress is a prominent feature of delayed neuronal death via programmed necrosis after neonatal HI. Since necrostatin-1 (Nec-1), an inhibitor of programmed necrosis, provides delayed neuroprotection against neonatal HI in male mice, Nec-1 is an ideal tool to study delayed ER responses. C57B6 male mice were exposed to right carotid ligation followed by exposure to FiO2=0.08 for 45min at p7. Mice were treated with vehicle or Nec-1 (0.1μl of 8μmol) intracerebroventricularly with age-matched littermates as controls. Biochemistry assays at 3 and 24h and electron microscopy (EM) and immunohistochemistry at 96h after HI were performed. EM showed ER dilation and mitochondrial swelling as apparent early changes in neurons. With advanced neurodegeneration, large cytoplasmic fragments containing dilated ER “shed” into the surrounding neuropil and calreticulin immunoreactivity was lost concurrent with nuclear features suggestive of programmed necrosis. Nec-1 attenuated biochemical markers of ER stress after neonatal HI, including PERK and eIF2α phosphorylation, and unconventional XBP-1 splicing, consistent with the mitigation of later ER pathology. ER pathology may be an indicator of severity of neuronal injury and potential for recovery characterized by cytoplasmic shedding, distinct from apoptotic blebbing, that we term neuronal macrozeiosis. Therapies to attenuate ER stress applied at delayed stages may rescue stressed neurons after neonatal HI.

High-intensity training reduces intermittent hypoxia-induced ER stress and myocardial infarct size.

Chronic intermittent hypoxia (IH) is described as the major detrimental factor leading to cardiovascular morbimortality in obstructive sleep apnea (OSA) patients. OSA patients exhibit increased infarct size after a myocardial event, and previous animal studies have shown that chronic IH could be the main mechanism. Endoplasmic reticulum (ER) stress plays a major role in the pathophysiology of cardiovascular disease. High-intensity training (HIT) exerts beneficial effects on the cardiovascular system. Thus, we hypothesized that HIT could prevent IH-induced ER stress and the increase in infarct size. Male Wistar rats were exposed to 21 days of IH (21-5% fraction of inspired O2, 60-s cycle, 8 h/day) or normoxia. After 1 wk of IH alone, rats were submitted daily to both IH and HIT (2 × 24 min, 15-30m/min). Rat hearts were either rapidly frozen to evaluate ER stress by Western blot analysis or submitted to an ischemia-reperfusion protocol ex vivo (30 min of global ischemia/120 min of reperfusion). IH induced cardiac proapoptotic ER stress, characterized by increased expression of glucose-regulated protein kinase 78, phosphorylated protein kinase-like ER kinase, activating transcription factor 4, and C/EBP homologous protein. IH-induced myocardial apoptosis was confirmed by increased expression of cleaved caspase-3. These IH-associated proapoptotic alterations were associated with a significant increase in infarct size (35.4 ± 3.2% vs. 22.7 ± 1.7% of ventricles in IH + sedenary and normoxia + sedentary groups, respectively, P < 0.05). HIT prevented both the IH-induced proapoptotic ER stress and increased myocardial infarct size (28.8 ± 3.9% and 21.0 ± 5.1% in IH + HIT and normoxia + HIT groups, respectively, P = 0.28). In conclusion, these findings suggest that HIT could represent a preventive strategy to limit IH-induced myocardial ischemia-reperfusion damages in OSA patients.

Endoplasmic Reticulum Stress in Hepatic Stellate Cells Promotes Liver Fibrosis via PERK-Mediated Degradation of HNRNPA1 and Up-regulation of SMAD2

Endoplasmic reticulum (ER) stress has been implicated in a variety of diseases. Hepatic stellate cells (HSCs) contribute to the development of liver fibrosis. Information on the link between ER stress and HSC activation is scarce. We investigated the effects of ER stress in HSCs on the progression of liver fibrosis and the regulation of this process in cells and mice. Proteins and messenger RNAs were measured in 2 sets of liver samples (n= 25 and n= 44) collected from patients with chronic hepatitis C virus infection and/or fibrosis. ER stress was induced in cells and mice using chemical agents. Lentiviral vectors were constructed to express glucose-regulated protein 78 (GRP78; also known as HSPA5) or heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) from the α-smooth muscle actin promoter and injected into C57BL/6 mice for HSC-specific gene expression. Liver tissues and HSCs were collected from mice or rats and analyzed using immunoblottings and quantitative reverse-transcription polymerase chain reaction. LX-2 cells were transfected with small interfering RNAs, microRNA mimics, or overexpression vectors. Hepatic ER stress was much higher in liver tissues from patients with severe vs mild fibrosis. ER stress induced fibrogenic genes in HSCs. Targeted lentiviral delivery of glucose-regulated protein 78 to HSCs in mice reduced fiber accumulation in liver. Levels of SMAD2, but not SMAD3, were increased in fibrotic liver tissues from patients or mice exposed to ER stress; small interfering RNA-mediated knockdown of SMAD2 reduced ER stress-mediated activation of HSCs. In rat HSCs, ER stress increased levels of SMAD2 messenger RNA by decreasing levels of microRNA 18a (MIR18A), an inhibitor of SMAD2 expression, rather than transactivating the SMAD2 gene. ER stress-activated PKR-like endoplasmic reticulum kinase, also known as EIF2AK3 (PERK) phosphorylated HNRNPA1, a protein required for the maturational processing of primary MIR18A, at Thr51, accelerating its degradation. Overexpression of HNRNPA1 (or its T51A mutant) in HSCs of mice inhibited liver fibrosis. Severe fibrotic liver tissues from patients had increased levels of phosphorylated PERK and reduced levels of HNRNPA1 in HSCs, compared with mild fibrotic liver tissues. ER stress in HSCs promotes liver fibrosis by inducing overexpression of SMAD2, via dysregulation of MIR18A; this dysregulation is mediated by PERK phosphorylation and destabilization of HNRNPA1.

Endoplasmic reticulum stress-mediated autophagy contributes to bluetongue virus infection via the PERK-eIF2α pathway

Bluetongue virus (BTV) is an important pathogen of wild and domestic ruminants. We have previously reported that BTV1 infection induced autophagy for its own benefit, but how this occurs remains unclear. Here, the classical autophagy features including autophagsomes formation, GFP-LC3 dots and LC3-II conversation were shown in BTV1-infected cells, we also found the endoplasmic reticulum (ER) stress was triggered by BTV1 infection, which was demonstrated by the increased transcription level of the ER stress marker GRP78 and the expanded morphology of ER. During ER stress, PERK and eIF2α phosphorylation increased along with BTV1 infection, consistent with the elevated LC3 level, indicating that the PERK pathway of the unfolded protein response (UPR) was activated. In addition, both the blockage of PERK by GSK2656157 or knockdown of eIF2α by siRNA reduced the level of LC3, which suggested that the PERK-eIF2α pathway contributed to autophagy induced by BTV1. Furthermore, inactivation of PERK or silencing of eIF2α both significantly reduced the expression of VP2 protein and the viral yields in the supernatants. In sum, these data suggest that ER stress mediates autophagy via the PERK-eIF2α pathway and contributes to BTV1 replication, thus offering new insight into the molecular mechanisms of the BTV-host interaction.

Dietary Fish Oil Inhibits Pro-Inflammatory and ER Stress Signalling Pathways in the Liver of Sows during Lactation.

Lactating sows have been shown to develop typical signs of an inflammatory condition in the liver during the transition from pregnancy to lactation. Hepatic inflammation is considered critical due to the induction of an acute phase response and the activation of stress signaling pathways like the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR), both of which impair animal´s health and performance. Whether ER stress-induced UPR is also activated in the liver of lactating sows and whether dietary fish oil as a source of anti-inflammatory effects n-3 PUFA is able to attenuate hepatic inflammation and ER stress-induced UPR in the liver of sows is currently unknown. Based on this, two experiments with lactating sows were performed. The first experiment revealed that ER stress-induced UPR occurs also in the liver of sows during lactation. This was evident from the up-regulation of a set of genes regulated by the UPR and numerically increased phosphorylation of the ER stress-transducer PERK and PERK-mediated phosphorylation of eIF2α and IκB. The second experiment showed that fish oil inhibits ER stress-induced UPR in the liver of lactating sows. This was demonstrated by decreased mRNA levels of a number of UPR-regulated genes and reduced phosphorylation of PERK and PERK-mediated phosphorylation of eIF2α and IκB in the liver of the fish oil group. The mRNA levels of various nuclear factor-κB-regulated genes encoding inflammatory mediators and acute phase proteins in the liver of lactating sows were also reduced in the fish oil group. In line with this, the plasma levels of acute phase proteins were reduced in the fish oil group, although differences to the control group were not significant. In conclusion, ER stress-induced UPR is present in the liver of lactating sows and fish oil is able to inhibit inflammatory signaling pathways and ER stress-induced UPR in the liver.

Panax quinquefolium saponin attenuates cardiomyocyte apoptosis induced by thapsigargin through inhibition of endoplasmic reticulum stress.

BackgroundEndoplasmic reticulum (ER) stress-related apoptosis is involved in the pathophysiology of many cardiovascular diseases, and Panax quinquefolium saponin (PQS) is able to inhibit excessive ER stress-related apoptosis of cardiomyocytes following hypoxia/reoxygenation and myocardial infarction. However, the pathway by which PQS inhibits the ER stress-related apoptosis is not well understood. To further investigate the protective effect of PQS against ER stress-related apoptosis, primary cultured cardiomyocytes were stimulated with thapsigargin (TG), which is widely used to model cellular ER stress, and it could induce apoptotic cell death in sufficient concentration.MethodsPrimary cultured cardiomyocytes from neonatal rats were exposed to TG (1 µmol/L) treatment for 24 h, following PQS pre-treatment (160 µg/mL) for 24 h or pre-treatment with small interfering RNA directed against protein kinase-like endoplasmic reticulum kinase (Si-PERK) for 6 h. The viability and apoptosis rate of cardiomyocytes were detected by cell counting kit-8 and flow cytometry respectively. ER stress-related protein expression, such as glucose-regulated protein 78 (GRP78), calreticulin, PERK, eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) were assayed by western blotting.ResultsBoth PQS pre-treatment and PERK knockdown remarkably inhibited the cardiomyocyte apoptosis induced by TG, increased cell viability, decreased phosphorylation of both PERK and eIF2α, and decreased protein levels of both ATF4 and CHOP. There was no statistically significant difference between PQS pre-treatment and PERK knockdown in the cardioprotective effect.ConclusionsOur data indicate that the PERK-eIF2α-ATF4-CHOP pathway of ER stress is involved in the apoptosis induced by TG, and PQS might prevent TG-induced cardiomyocyte apoptosis through a mechanism involving the suppression of this pathway. These findings provide novel data regarding the molecular mechanisms by which PQS inhibits cardiomyocyte apoptosis.

Abstract 1382: 2-Deoxy-glucose downregulates endothelial Akt and Erk via interference with N-linked glycosylation, induction of endoplasmic reticulum stress, and GSK-3β activation

Abstract BACKGROUND: Interference with endothelial cell metabolism is a promising, yet unexploited strategy for angiogenesis inhibition. We reported that the glucose analog, 2-deoxy-D-Glucose (2-DG) inhibits angiogenesis at significantly lower concentrations than those required for tumor cytotoxicity (PloS One2010;5(10):e13699), and down regulates endothelial Akt and Erk pathways (Kovacs et al. 2012 AACR annual meeting; abstract no 2232). The aims of this study are to characterize the mechanisms of Akt and Erk down regulation by 2-DG. METHODS: Differences in 2-DG uptake between endothelial and tumor cells were assessed by determination of radioactive labeled 2-DG uptake at different time points. The effects of 2-DG on VEGFR2 status were determined by immunocytochemistry (ICC), N-glycan digestions and cell surface biotinylation experiments. 2-DG's effects on ER stress pathways, apoptosis, and differential effects of other ER stress inducers were assessed by Western blot (WB). The in vivo effects of 2-DG were further evaluated in the LHBETATAG retinoblastoma model by immunofluorescence staining (IF) for microvessel density and markers of unfolded protein response (UPR). In vivo UPR induction was further determined in Laser capture microdissection (LCM) isolated tumor cells using immunoblots for GRP 78. RESULTS/CONCLUSIONS: No significant differences in 2-DG uptake were found between endothelial cells (HUVECs/HMVECs) or tumor cells (MDA-MB231, HT-29). 2-DG mediated ERK and AKT down regulation were associated with changes in VEGFR2 migration pattern and downstream signaling, as evidenced by decreased phosphorylation of the downstream effector PLC-γ1 pTyr783. Changes in VEGFR2 migration pattern were due to receptor hypoglycosylation, as supported by mannose reversal of these effects, and N-glycan digestion showing that Endo H and PNGase F digestion of 2-DG treated samples resulted in less number of cleaved bands than ctrl. Biotinylation experiments showed that in 2-DG treated cells, VEGFR2 was not present on the cell surface. 2-DG induced endoplasmic reticulum (ER) stress (increased PERK and eIF2-α phosphorylation), leading to GSK-3β activation and apoptosis (increased cleaved caspase-3 and PARP) in a mannose reversible manner. Chemical inhibition of GSK-3β prevented 2-DG induced apoptosis. In vivo, periocular administration of 2-DG in LHBETATAG mice was associated with significant reduction of newly formed (CD 105 +) tumor capillaries and induction of ER stress (GRP 78 expression). These findings uniquely link N-linked glycosylation inhibition, ER stress and endothelial Erk/Akt down regulation, and provide a novel strategy for angiogenesis inhibition, which may have the potential to overcome resistance to standard antiangiogenic agents. Citation Format: Krisztina Kovacs, Christina Decatur, Dien G. Pham, Huaping Liu, Yuqi Jing, Timothy G. Murray, Theodore J. Lampidis, Jaime R. Merchan. 2-Deoxy-glucose downregulates endothelial Akt and Erk via interference with N-linked glycosylation, induction of endoplasmic reticulum stress, and GSK-3β activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1382. doi:10.1158/1538-7445.AM2015-1382

Pharmacological activation of AMPK prevents Drp1-mediated mitochondrial fission and alleviates endoplasmic reticulum stress-associated endothelial dysfunction

Background and purposeThis study aims to investigate whether and how pharmacological activation of AMP-activated protein kinase (AMPK) improves endothelial function by suppressing mitochondrial ROS-associated endoplasmic reticulum stress (ER stress) in the endothelium.Experimental approachPalmitate stimulation induced mitochondrial fission and ER stress-associated endothelial dysfunction. The effects of AMPK activators salicylate and AICA riboside (AICAR) on mitochondrial ROS production, Drp1 phosphorylation, mitochondrial fission, ER stress, thioredoxin-interacting protein (TXNIP)/NLRP3 inflammasome activation, inflammation, cell apoptosis and endothelium-dependent vasodilation were observed.Key results“Silencing” of TXNIP by RNA interference inhibited NLRP3 inflammasome activation in response to ER stress, indicating that TXNIP was a key link between ER stress and NLRP3 inflammasome activation. AMPK activators salicylate and AICAR prevented ROS-induced mitochondrial fission by enhancing dynamin-related protein 1 (Drp1) phosphorylation (Ser 637) and thereby attenuated IRE-1α and PERK phosphorylation, but their actions were blocked by knockdown of AMPK. Salicylate and AICAR reduced TXNIP induction and inhibited NLRP3 inflammasome activation by reducing NLRP3 and caspase-1 expression, leading to a reduction in IL-1β secretion. As a result, salicylate and AICAR inhibited inflammation and reduced cell apoptosis. Meanwhile, salicylate and AICAR enhanced eNOS phosphorylation and restored the loss of endothelium-dependent vasodilation in the rat aorta. Immunohistochemistry staining showed that AMPK activation inhibited ER stress and NLRP3 inflammasome activation in the vascular endothelium. Conclusion and implicationsPharmacological activation of AMPK regulated mitochondrial morphology and ameliorated endothelial dysfunction by suppression of mitochondrial ROS-associated ER stress and subsequent TXNIP/NLRP3 inflammasome activation. These findings suggested that regulation of Drp1 phosphorylation by AMPK activation contributed to suppression of ER stress and thus presented a potential therapeutic strategy for AMPK activation in the regulation of endothelium homeostasis.

Palmitate induces cisternal ER expansion via the activation of XBP-1/CCTα-mediated phospholipid accumulation in RAW 264.7 cells

BackgroundEndoplasmic reticulum (ER) stress induces ER expansion. The expansion of the intracisternal space of the ER was found in macrophages associated with human atherosclerotic lesions. We also previously reported that palmitate induces cisternal ER expansion and necrosis in RAW 264.7 cells. In this study, we report on an investigation of the likely mechanism responsible for this palmitate-induced cisternal ER expansion in a mouse macrophage cell line, RAW 264.7 cells.MethodsRAW 264.7 cells were pre-treated with the designated inhibitor or siRNA, followed by treatment with palmitate. Changes in the ER structure were examined by transmission electron microscopy. The induction of ER stress was confirmed by an increase in the extent of phosphorylation of PERK, the expression of BiP and CHOP, and the splicing of XBP-1 mRNA. Phospholipid staining was performed with the LipidTOX Red phospholipidosis detection reagent. Related gene expressions were detected by quantitative real time-RT-PCR or RT-PCR.ResultsPalmitate was found to induce ER stress and cisternal ER expansion. In addition, palmitate-induced cisternal ER expansion was attenuated by ER stress inhibitors, such as 4-phenylbutyric acid (4-PBA) and tauroursodeoxycholic acid (TUDCA). The findings also show that palmitate induced-mRNA expression of CCTα, which increases phospholipid synthesis, was attenuated by the down-regulation of XBP-1, a part of ER stress. Furthermore, palmitate-induced phospholipid accumulation and cisternal ER expansion were attenuated by the down-regulation of XBP-1 or CCTα.ConclusionsThe findings reported herein indicate that palmitate-induced cisternal ER expansion is dependent on the activation of XBP-1/CCTα-mediated phospholipid accumulation in RAW 264.7 cells.

PPARγ Agonists Attenuate Palmitate-Induced ER Stress through Up-Regulation of SCD-1 in Macrophages.

BackgroundClinical trials have shown that treatment of patients with type 2 diabetes with pioglitazone, a peroxisome proliferator-activated receptor (PPAR)γ agonist, reduces cardiovascular events. However, the effect of PPARγ agonists on endoplasmic reticulum (ER) stress that plays an important role in the progression of atherosclerosis has not been determined. We sought to determine the effect of PPARγ agonists on ER stress induced by palmitate, the most abundant saturated fatty acid in the serum.Methods and ResultsProtein expression of ER stress marker was evaluated by Western blot analysis and stearoyl-CoA desaturase1 (SCD-1) mRNA expression was evaluated by qRT-PCR. Macrophage apoptosis was detected by flowcytometry. Pioglitazone and rosiglitazone reduced palmitate-induced phosphorylation of PERK, a marker of ER stress, in RAW264.7, a murine macrophage cell line. Pioglitazone also suppressed palmitate-induced apoptosis in association with inhibition of CHOP expression, JNK phosphorylation and cleavage of caspase-3. These effects of pioglitazone were reversed by GW9662, a PPARγ antagonist, indicating that PPARγ is involved in this process. PPARγ agonists increased expression of SCD-1 that introduces a double bond on the acyl chain of long-chain fatty acid. 4-(2-Chlorophenoxy)-N-(3-(3-methylcarbamoyl)phenyl)piperidine-1-carboxamide, an inhibitor of SCD-1, abolished the anti-ER stress and anti-apoptotic effects of pioglitazone. These results suggest that PPARγ agonists attenuate palmitate-induced ER stress and apoptosis through SCD-1 induction. Up-regulation of SCD-1 may contribute to the reduction of cardiovascular events by treatment with PPARγ agonists.

The Impact of Small RNA Interference Against Homer1 on Rats with Type 2 Diabetes and ERK Phosphorylation.

The objective of the study is to evaluate Homer1 expression in rats with Type 2 diabetes mellitus (T2DM) and investigate the mechanism by which Homer1 influences the pathogenesis of diabetes through study on rat model with decreased Homer1 expression. Rat model of T2DM was constructed and blood insulin concentration was measured. Homer1 mRNA and protein expressions in rat pancreatic tissue were determined using RT-PCR as well as Western blotting. Homer1 expression in human monocytic THP-1 cells was interfered using short hairpin RNA, and its effect on phosphorylation of extracellular signal-regulated kinase (ERK) was assessed. Fasting glucose concentration in rat model of T2DM was significantly higher than that of normal rats (13.1±2.4 vs 5.1±1.1mmol/L), and fasting blood insulin concentration of diabetic group was significantly lower than that of normal group (13.6±1.9 18.3±2.2 mIU/L) (P<0.05). Homer1 mRNA and protein expressions in pancreatic tissue of rats with T2DM were significantly higher than those of normal rats (P<0.05). Level of ERK phosphorylation in pancreatic tissue of rats with T2DM was significantly higher than that of normal rats. Homer1 mRNA level in rat pancreatic tissue of T2DM was positively correlated with the area of pancreatic islets (r=0.526, P=0.014). Homer1 mRNA level was significantly inhibited in high-glucose and high-fat stimulated human monotypic THP-1 cells with interfered Homer1. Compared with controls, P-ERK phosphorylation was significantly decreased in THP-1 cells with interfered Homer1 (P<0.05). Homer1 can promote the progression of T2DM, which may be achieved through affecting ERK phosphorylation.

Fucosylated chondroitin sulphate from sea cucumber reduces hepatic endoplasmic reticulum stress-associated inflammation in obesity mice

Endoplasmic reticulum (ER) stress-associated inflammation positively induces many diseases, including insulin resistance. Fucosylated chondroitin sulphate from Acaudina molpadioides (Am-CHS) can mitigate insulin resistance. However, its effects on ER stress and inflammation are unclear. We investigated whether Am-CHS influenced ER stress, inflammatory cytokines, and JNK and IKKβ/NFκB pathways in insulin resistant mice. Am-CHS lowered serum and hepatic ROS, NO, and FFA levels. Meanwhile, Am-CHS decreased serum proinflammatory cytokines TNF-α, CRP, MIP-1, IL-1β and IL-6 concentrations and their hepatic mRNA expression, and increased anti-inflammatory cytokine IL-10 levels. Am-CHS reduced ER stress markers Bip, ATF6, PERK, and XBP1 mRNA or protein expression, and PERK, eIF2α, and IRE1α phosphorylation. These reductions were accompanied by reduced JNK1 and IKKβ activation, NFκB nuclear translocation, and IR/IRS-2 serine phosphorylation in Am-CHS-treated mice. These findings suggest that Am-CHS could be an alternative against inflammation and ER stress as a functional food ingredient and natural health product.

Endoplasmic reticulium protein profiling of heat-stressed Jurkat cells by one dimensional electrophoresis and liquid chromatography tandem mass spectrometry.

Proteomic study on membrane-integrated proteins in endoplasmic reticulum (ER) fractions was performed. In this study, we examined the effects of heat stress on Jurkat cells. The ER fractions were highly purified by differential centrifugation with sodium carbonate washing and acetone methanol precipitations. The ER membrane proteins were separated by one dimensional electrophoresis (1-DE), and some of the protein bands changed their abundance by heat stress, 12 of the 14 bands containing 40 and 60 ribosomal proteins whose expression level were decreased, on the contrary, 2 of the 14 bands containing ubiquitin and eukaryotic translation initiation factor 3 were increased. Heat treatment of human Jurkat cells led to an increase in the phosphorylation of PERK and eIF2α within 30min of exposure. This was followed by an increase in the expression of the GRP78. Protein ubiquitination and subsequent degradation by the proteasome are important mechanisms regulating cell cycle, growth and differentiation, the result showed that heat stress enhanced ubiquitination modification of the microsomal proteins. The data of this study strongly suggest that heat treatment led to a significant reduction in protein expression and activated UPR, concomitant with protein hyperubiqutination in ER.