Mediator Of Endoplasmic Reticulum Stress Research Articles

136 - Leukotriene C4 Is the Major Mediator of Endoplasmic Reticulum Stress-Triggered Oxidative Stress

Endoplasmic reticulum (ER) stress and major chemotherapeutic agents invariably trigger oxidative stress, but the exact mechanism was not known. We found that ER stress and major chemotherapeutic agents induce leukotriene C4 (LTC4) biosynthesis in cells of non-hematopoietic lineage by assembly on the nuclear envelope of an LTC4 biosynthetic machinery based on microsomal glutathione-S-transferase 2 (MGST2). ER stress and chemotherapy also triggered nuclear translocation of the two LTC4 receptors. Acting in an intracrine manner, LTC4 then elicits nuclear translocation of NADPH oxidase 4 (NOX4), resulting in accumulation of reactive oxygen species (ROS) and subsequent DNA damage. Mgst2 deficiency, RNAi and LTC4 receptor antagonists abolished ER stress- and chemotherapy-induced oxidative nuclear DNA damage and dsDNA breaks in vitro and in mouse kidneys. Cell death and mouse morbidity were also significantly attenuated. Hence, MGST2-generated LTC4 is a major mediator of ER stress- and chemotherapy-triggered oxidative stress, leading to oxidative DNA damage and dsDNA breaks. Previous studies demonstrated that LTC4 inhibitors, commonly used as approved asthma drugs, attenuated neurological damage, cardiovascular damage and drug toxicity. Our findings provide the mechanism by which these pathophysiological conditions lead to cellular damage, suggesting a broad use of LTC4 inhibitors in major human morbidities.

346 - Hypercholesterolemia Mediated Endoplasmic Reticulum Stress Response Might Be Related to Autophagic Death of Cardiomyocytes in Heart Failure

Proper synthesis and correct folding of proteins in endoplasmic reticulum (ER) is important for the proper function of many organs including heart. Cardiovascular diseases (CVDs) are the leading cause of death, with highest mortality and morbidity rates worldwide. High blood cholesterol levels are well recognized as a risk factor for atherosclerosis and the development of CVDs such as heart failure. Globally, high blood cholesterol levels are estimated to cause 56% of ischemic heart disease and 18% of strokes, which equals to 4.4 million deaths per year. Elevated oxLDL levels have significant roles in the induction of reactive oxygen species, unfolded/misfolded proteins and oxidative/ER stress related changes. Although autophagy plays a beneficial role in cellular homeostasis, excessive autophagy might be expected to lead to cellular destruction by high degrading rate of proteins and organelles such as mitochondria and ER. Heart failure, as a result of the disturbed unfolded protein response mediated cardiomyocyte death leads to many cardiovascular disorders. The role of apoptosis and necrosis in development of CVDs, especially heart failure, has been well documented. Recent evidence suggests the potential role of another cell death mechanism, autophagy, in loss of cardiomyocytes. In our study, protein levels of well-known ER stress markers (Grp78, Grp94, pIRE1, pPERK), autophagic activity markers (beclin-1, LC3-II), and apoptotic activity markers (bax, caspase 3), are investigated in the heart of hypercholesterolemic rabbits. Furthermore, autophagic activity in heart is also observed in hypercholesterolemic rabbits by electron microscopy along with apoptotic cell death by TUNEL. Additionally, colocalization studies of mitochondria and p62 in cryosections of heart tissue were applied by confocal microscopy. Our animal model showed that hypercholesterolemia mediated ER stress activation triggers autophagy/mitophagy, which might cause cell death of cardiomyocytes by organel depletion in juxtanuclear area, pathophysiological setting of autophagic cardiomyocyte death in heart failure needs to be further studied. Supported by Marmara University Research Funds SAG-A-130612-0202 and SAG-C-DRP-130515-0164.

OS 02-02 microRNA-204 AND SIRTUIN1 REGULATE VASCULAR ENDOPLASMIC RETICULUM STRESS AND ENDOTHELIUM-DEPENDENT VASORELAXATION.

Objective: Endoplasmic reticulum (ER) stress leads to vascular endothelial dysfunction in hypertension. The role of microRNAs in vascular ER stress is not fully understood. Here we examined the role of vascular microRNA-204 (miR-204) in inducing endothelial ER stress and dysfunction by governing the expression of the SIRTUIN1 (SIRT1) lysine deacetylase. Design and Method: Human Umbilical Vein cells (HUVECs) were treated with tunicamycin or transfected with miR-204 Mimic or siRNA SIRT1. In other set of experiments, HUVEC cells were treated with tunicamycin and either transfected with miR-204 inhibitor or transfected with adenovirus of SIRT1. Expression of ER stress markers were determined by Western blots, RTPCR and Immunostaining in all these conditions. Additionally, we treated C57/b6 mice and endothelial specific knockout mice for SIRT1 with tunicamycin and we infused them with miR-204 inhibitor and we studied the vessels reactivity and the expression of ER stress markers. Results: miR-204 mimic decreased expression of SIRT1 and promoted ER stress in HUVECs. Tunicamycin induced endothelial ER stress was also mediated by miR-204. Moreover, knockdown of SIRT1 induced ER stress in HUVECs, which were rescued by a miR-204 antagomir. Tunicamycin-induced ER stress upregulated miR-204, and downregulated SIRT1 in HUVECs. Similarly and in endothelium of aortas and mesenteric arteries of mice, antagonism of miR-204, or SIRT1 overexpression, rescued endothelial cells from tunicamycin-induced ER stress. Similarly, antagonism of miR-204 in vivo with anti-miR-204 mitigated tunicamycin-induced vascular ER stress, decline in vascular SIRT1 and impairment of endothelium-dependent vasorelaxation. Moreover, tunicamycin-induced vascular ER stress and endothelial dysfunction were exacerbated in mice with conditional deletion of endothelial SIRT1. Finally, Vascular miR-204 was upregulated with high-fat diet feeding, anti-miR-204 prevented vascular ER stress stimulated by high-fat diet feeding. Conclusions: These findings show an important role for miR-204 as a mediator of vascular ER stress, and ER stress-induced endothelial dysfunction, via downregulation of endothelial SIRT1.

KRE5 Suppression Induces Cell Wall Stress and Alternative ER Stress Response Required for Maintaining Cell Wall Integrity in Candida glabrata.

The maintenance of cell wall integrity in fungi is required for normal cell growth, division, hyphae formation, and antifungal tolerance. We observed that endoplasmic reticulum stress regulated cell wall integrity in Candida glabrata, which possesses uniquely evolved mechanisms for unfolded protein response mechanisms. Tetracycline-mediated suppression of KRE5, which encodes a predicted UDP-glucose:glycoprotein glucosyltransferase localized in the endoplasmic reticulum, significantly increased cell wall chitin content and decreased cell wall β-1,6-glucan content. KRE5 repression induced endoplasmic reticulum stress-related gene expression and MAP kinase pathway activation, including Slt2p and Hog1p phosphorylation, through the cell wall integrity signaling pathway. Moreover, the calcineurin pathway negatively regulated cell wall integrity, but not the reduction of β-1,6-glucan content. These results indicate that KRE5 is required for maintaining both endoplasmic reticulum homeostasis and cell wall integrity, and that the calcineurin pathway acts as a regulator of chitin-glucan balance in the cell wall and as an alternative mediator of endoplasmic reticulum stress in C. glabrata.

Expression of IGFBP6, IGFBP7, NOV, CYR61, WISP1 and WISP2 genes in U87 glioma cells in glutamine deprivation condition.

We have studied gene expression of insulin-like growth factor binding proteins in U87 glioma cells upon glutamine deprivation depending on the inhibition of IRE1 (inositol requiring enzyme-1), a central mediator of endoplasmic reticulum stress. We have shown that exposure of control glioma cells upon glutamine deprivation leads to down-regulation of NOV/IGFBP9, WISP1 and WISP2 gene expressions and up-regulation of CYR61/IGFBP10 gene expression at the mRNA level. At the same time, the expression of IGFBP6 and IGFBP7 genes in control glioma cells was resistant to glutamine deprivation. It was also shown that the inhibition of IRE1 modifies the effect of glutamine deprivation on the expression of all studied genes. Thus, the inhibition of IRE1 signaling enzyme enhances the effect of glutamine deprivation on the expression of CYR61 and WISP1 genes and suppresses effect of the deprivation on WISP2 gene expression in glioma cells. Moreover, the inhibition of IRE1 introduces sensitivity of the expression of IGFBP6 and IGFBP7 genes to glutamine deprivation and removes this sensitivity to NOV gene. We have also demonstrated that the expression of all studied genes in glioma cells growing with glutamine is regulated by IRE1 signaling enzyme, because the inhibition of IRE1 significantly down-regulates IGFBP6 and NOV genes and up-regulates IGFBP7, CYR61, WISP1, and WISP2 genes as compared to control glioma cells. The present study demonstrates that glutamine deprivation condition affects most studied IGFBP and WISP gene expressions in relation to IRE1 signaling enzyme function and possibly contributes to slower glioma cell proliferation upon inhibition of IRE1.

Effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells.

We have studied the effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells under the inhibition of IRE1 (inositol requiring enzyme-1), which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress. It was shown that hypoxia down-regulated gene expression of malate dehydrogenase 2 (MDH2), malic enzyme 2 (ME2), mitochondrial aspartate aminotransferase (GOT2), and subunit B of succinate dehydrogenase (SDHB) in control (transfected by empty vector) glioma cells in a gene specific manner. At the same time, the expression level of mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) and subunit D of succinate dehydrogenase (SDHD) genes in these cells does not significantly change in hypoxic conditions. It was also shown that the inhibition of ІRE1 signaling enzyme function in U87 glioma cells decreases the effect of hypoxia on the expression of ME2, GOT2, and SDHB genes and introduces the sensitivity of IDH2 gene to hypoxia. Furthermore, the expression of all studied genes depends on IRE1-mediated endoplasmic reticulum stress signaling in gene specific manner, because ІRE1 knockdown significantly decreases their expression in normoxic conditions, except for IDH2 gene, which expression level is strongly up-regulated. Therefore, changes in the expression level of nuclear genes encoding ME2, MDH2, IDH2, SDHB, SDHD, and GOT2 proteins possibly reflect metabolic reprogramming of mitochondria by hypoxia and IRE1-mediated endoplasmic reticulum stress signaling and correlate with suppression of glioma cell proliferation under inhibition of the IRE1 enzyme function.

Anticancer metal drugs and immunogenic cell death

Conventional chemotherapeutics, but also innovative precision anticancer compounds, are commonly perceived to target primarily the cancer cell compartment. However, recently it was discovered that some of these compounds can also exert immunomodulatory activities which might be exploited to synergistically enhance their anticancer effects. One specific phenomenon of the interplay between chemotherapy and the anticancer immune response is the so-called “immunogenic cell death” (ICD). ICD was discovered based on a vaccination effect exerted by cancer cells dying from pretreatment with certain chemotherapeutics, termed ICD inducers, in syngeneic transplantation mouse models. Interestingly, only a minority of drugs is able to trigger ICD without a clear-cut relation to chemical structures or their primary modes-of-action. Nevertheless, generation of reactive oxygen species (ROS) and induction of endoplasmic reticulum (ER) stress are clearly linked to ICD. With regard to metal drugs, oxaliplatin but not cisplatin is considered a bona fide ICD inducer. Taken into account that several experimental metal compounds are efficient ROS and ER stress mediators, presence of potent ICD inducers within the plethora of novel metal complexes seems feasible and has occasionally been reported. In the light of recent successes in cancer immunotherapy, here we review existing literature regarding anticancer metal drugs and ICD induction. We recommend a more profound investigation of the immunogenic features of experimental anticancer metal drugs.

Endoplasmic reticulum stress in bone marrow-derived cells prevents acute cardiac inflammation and injury in response to angiotensin II.

Inflammation plays an important role in hypertensive cardiac injury. The endoplasmic reticulum (ER) stress pathway is involved in the inflammatory response. However, the role of ER stress in elevated angiotensin II (Ang II)-induced cardiac injury remains unclear. In this study, we investigated the role of ER stress in Ang II-induced hypertensive cardiac injury. Transcriptome analysis and quantitative real-time PCR showed that Ang II infusion in mice increased ER stress-related genes expression in the heart. C/EBP homologous protein (CHOP) deficiency, a key mediator of ER stress, increased infiltration of inflammatory cells, especially neutrophils, the production of inflammatory cytokines, chemokines in Ang II-infused mouse hearts. CHOP deficiency increased Ang II-induced cardiac fibrotic injury: (1) Masson trichrome staining showed increased fibrotic areas, (2) immunohistochemistry staining showed increased expression of α-smooth muscle actin, transforming growth factor β1 and (3) quantitative real-time PCR showed increased expression of collagen in CHOP-deficient mouse heart. Bone marrow transplantation experiments indicated that CHOP deficiency in bone marrow cells was responsible for Ang II-induced cardiac fibrotic injury. Moreover, TUNEL staining and flow cytometry revealed that CHOP deficiency decreased neutrophil apoptosis in response to Ang II. Taken together, our study demonstrated that hypertension induced ER stress after Ang II infusion. ER stress in bone marrow-derived cells protected acute cardiac inflammation and injury in response to Ang II.

Acrolein Is a Pathogenic Mediator of Alcoholic Liver Disease andthe Scavenger Hydralazine Is Protective in Mice.

Background & AimsAlcoholic liver disease (ALD) remains a major cause of morbidity and mortality, with no Food and Drug Administration–approved therapy. Chronic alcohol consumption causes a pro-oxidant environment and increases hepatic lipid peroxidation, with acrolein being the most reactive/toxic by-product. This study investigated the pathogenic role of acrolein in hepatic endoplasmic reticulum (ER) stress, steatosis, and injury in experimental ALD, and tested acrolein elimination/scavenging (using hydralazine) as a potential therapy in ALD.MethodsIn vitro (rat hepatoma H4IIEC cells) and in vivo (chronic+binge alcohol feeding in C57Bl/6 mice) models were used to examine alcohol-induced acrolein accumulation and consequent hepatic ER stress, apoptosis, and injury. In addition, the potential protective effects of the acrolein scavenger, hydralazine, were examined both in vitro and in vivo.ResultsAlcohol consumption/metabolism resulted in hepatic accumulation of acrolein-protein adducts, by up-regulation of cytochrome P4502E1 and alcohol dehydrogenase, and down-regulation of glutathione-s-transferase-P, which metabolizes/detoxifies acrolein. Alcohol-induced acrolein adduct accumulation led to hepatic ER stress, proapoptotic signaling, steatosis, apoptosis, and liver injury; however, ER-protective/adaptive responses were not induced. Notably, direct exposure to acrolein in vitro mimicked the in vivo effects of alcohol, indicating that acrolein mediates the adverse effects of alcohol. Importantly, hydralazine, a known acrolein scavenger, protected against alcohol-induced ER stress and liver injury, both in vitro and in mice.ConclusionsOur study shows the following: (1) alcohol consumption triggers pathologic ER stress without ER adaptation/protection; (2) alcohol-induced acrolein is a potential therapeutic target and pathogenic mediator of hepatic ER stress, cell death, and injury; and (3) removal/clearance of acrolein by scavengers may have therapeutic potential in ALD.

Exosomal formulation enhances therapeutic response of celastrol against lung cancer

Celastrol (CEL), a plant-derived triterpenoid, is a known inhibitor of Hsp90 and NF-κB activation pathways and has recently been suggested to be of therapeutic importance in various cancers. However, the molecular mechanisms of celastrol-mediated effects in lung cancer are not systematically studied. Moreover, it suffers from poor bioavailability and off-site toxicity issues. This study aims to study the effect of celastrol loaded into exosomes against two non-small cell-lung carcinoma (NSCLC) cell lines and explore the molecular mechanisms to determine the proteins governing the cellular responses. We observed that celastrol inhibited the proliferation of A549 and H1299 NSCLC cells in a time- and concentration-dependent manner as indexed by MTT assay. Mechanistically, CEL pre-treatment of H1299 cells completely abrogated TNFα-induced NF-κB activation and upregulated the expression of ER-stress chaperones Grp 94, Grp78, and pPERK. These changes in ER-stress mediators were paralleled by an increase in apoptotic response as evidenced by higher annexin-V/PI positive cells evaluated by FACS and immunoblotting which showed upregulation of the ER stress specific pro-apoptotic transcription factor, GADD153/CHOP and alteration of Bax/Bcl2 levels. Exosomes loaded with CEL exhibited enhanced anti-tumor efficacy as compared to free CEL against lung cancer cell xenograft. CEL did not exhibit any gross or systemic toxicity in wild-type C57BL6 mice as determined by hematological and liver and kidney function test. Together, our data demonstrate the chemotherapeutic potential of CEL in lung cancer and that exosomal formulation enhances its efficacy and reduces dose related toxicity.

188. AAV Mediated Gene Therapy to Modulate Neurotropic Factors in the Retina and in Neuronal Cells in Culture

Neurotrophic factors, particularly Brain Derived Neurotrophic Factor (BDNF) and its high affinity receptor TrkB, play an important role in protecting the retinal ganglion cells (RGCs) in glaucoma. Translation of the immense neuro-protective potential of BDNF/TrkB activation has been largely unsuccessful and gene therapy to modulate BDNF and TrkB have also shown only short-lived protective effects on the RGCs. PTPN11 phosphatase plays a key role in the regulation of BDNF/TrkB signaling in the retina. Here, we investigated the effects of PTPN11 modulation involving both over-expression and knock-down paradigms on the TrkB activation. PTPN11 and PTPN11-shRNA sequences were separately incorporated into the AAV2 vector under cytomegalovirus chicken β actin hybrid promoter linked with green fluorescent protein (GFP). SH-SY5Y cells were transfected with each of the PTPN11 modulating and control GFP constructs. PTPN11 over-expression resulted in significant reduced TrkB phosphorylation and accordingly reduced viability of the SH-SY5Y cells was observed while PTPN11 knockdown did not exhibit any significant effects on either TrkB phosphorylation or cellular survival. PTPN11 over-expression in neuronal cells was also accompanied by elevation of GADD, PERK and XBP-1 endoplasmic reticulum (ER) stress marker proteins as detected using western blotting. The roles of PTPN11 and TrkB in mediating ER stress were evaluated by treating cells with PTPN11 inhibitor (PHPS1) or TrkB agonist (7,8-dihydroxyflavone) independently, both of which ablated the up-regulation of ER stress proteins upon PTPN11 over-expression. The effects of PTPN11 over-expression on the rat retina were evaluated by administering PTPN11 AAV2 construct along with corresponding GFP controls individually into the rat eyes. Briefly, 5µl of AAV2 constructs were injected intravitreally into SD rats (2×1010 vg). Anti-GFP staining in retinal sections demonstrated AAV2 transduction in the RGC layer. Anti-NeuN/ anti-PTPN11 staining confirmed the ganglion cell specific over-expression of PTPN11. Rat retinas were assessed for inner retinal functional changes using scotopic threshold response (STR) measurements. Retinal structural changes were assessed using histological analysis. Results indicate that pSTR amplitude which primarily reflects the function of the RGCs was significantly diminished in the PTPN11 over-expression model. Hematoxylin and eosin staining also revealed degenerative changes primarily associated with the inner retina. Assessment of optic nerve sections using Bielschowsky's staining further identified reduced axonal density. Concluding, our findings strongly indicate that PTPN11 genetic modulation holds the promise to regulate neuronal cell survival through its effects on TrkB activation. PTPN11 over-expression exerts detrimental impact on the inner retinal health which has significant implications in glaucoma and other retinal disorders. Further studies involving rescue of the disease phenotype using PTPN11 knockdown in animal models will substantiate these observations and provide mechanistic insights into the roles of neurotrophic factor regulation in retina.

Inhibition of IRE1 modifies the hypoxic regulation of GADD family gene expressions in U87 glioma cells.

We have studied hypoxic regulation of the expression of genes encoded GADD (growth arrest and DNA damage) family proteins in U87 glioma cells in relation to inhibition of IRE1 (inositol requiring enzyme-1), which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress. We have shown that hypoxia up-regulates the expression of GADD34, GADD45A, GADD45B, and GADD153 genes, which are related to cell proliferation and apoptosis, in control (transfected by empty vector) glioma cells in gene specific manner. At the same time, the expression level of EIF2AK 1 (eukaryotic translation initiation factor 2-alpha kinase 1) and AI FM1 (apoptosis inducing factor, mitochondria associated 1) genes in these cells is down-regulated upon hypoxic condition. It was also shown that inhibition of ІRE1 signaling enzyme function in U87 glioma cells enhances the effect of hypoxia on these genes expression, except EIF2AK 1 and AI FM1 genes. Furthermore, the expression of all studied genes in ІRE1 knockdown cells is significantly decreased upon normoxic condition, except GADD45B gene, which expression level is strongly up-regulated. Therefore, the expression level of genes encoding GADD34, GADD45A, GADD45B, GADD153, EIF2AK 1, and AI FM1 is affected by hypoxia and by inhibition of IRE1-mediated endoplasmic reticulum stress signaling in gene specific manner and correlates with suppression of glioma cell proliferation upon inhibition of the IRE1 enzyme function.

The p-eIF2α/ATF4 pathway links endoplasmic reticulum stress to autophagy following the production of reactive oxygen species in mouse spermatocyte-derived cells exposed to dibutyl phthalate

Dibutyl phthalate (DBP) is a widely used plasticizer that has been shown to induce germ cell apoptosis-related testicular atrophy and cause reproductive toxicity. Our previous results indicated that endoplasmic reticulum (ER) stress-activated autophagy served as a self-defense mechanism against DBP-induced germ cell apoptosis. However, the specific pathways that link ER stress and autophagy remain unclear. Here, we showed that exposure to DBP enhanced autophagic flux in mouse spermatocyte-derived GC-2 cells and that the eukaryotic translation initiation factor 2/activating transcription factor 4 pathway mediated ER stress-related autophagy independent of the mTOR and Beclin-1 pathways. Moreover, we demonstrated that DBP treatment led to the generation of reactive oxygen species (ROS) and that the inhibition of ROS by melatonin abrogated both ER stress and autophagy. The results indicated that excessive ROS production might be involved in DBP-induced ER stress and autophagy in GC-2 cells. Thus, ROS may serve as upstream mediators of ER stress and autophagy in DBP-treated GC-2 cells.

Hydrogen Sulfide Suppresses Diabetic Myocardial ER Stress: An Uncanonical Way

Diabetes critically inhibits cardiac endogenous production of hydrogen sulfide (H2S), a cardioprotective gaseous signaling molecule, by suppressing expression of CBS, CSE and 3MST (known H2S producers endogenously) in the mouse models of type 1 diabetes (Akita or C57BL/6‐Ins2Akita/J). Diabetes induced ER stress has been previously identified as an initiator of cardiomyocyte apoptosis and cardiomyopathy. However, the mechanisms of ER stress induction are not clearly illustrated. Furthermore, H2S has been implicated as an inducer or suppressor of ER stress in a tissue type specific manner and molecular mechanisms of H2S mediated ER stress regulation are not entirely known. Here, we show that chronic H2S supplementation resulted in enhanced cardiac function concurrent with suppression of ER stress with significant reversal of elevated CHOP levels in the diabetic hearts. Interestingly, there were no significant changes in the sXBP1 levels in the diabetic hearts with or without H2S treatment suggesting differential regulation of different arms (PERK, ATF6 and IRE1) of ER stress pathway. Importantly, the ER stress suppression is accompanied by suppression of GRP78 (AKA Bip) mRNA levels with a dramatic and paradoxical elevation in its protein levels. Differential translational regulation and/or protein degradation are implicated in regulation of GRP78 protein levels which further highlighted that H2S supplementation not only suppresses ER stress, but also raises the threshold for ER stress induction through elevation of GRP78 protein levels. Remarkably, the H2S mediated diabetic ER suppression was also accompanied with reversal of elevated TLR4 receptors, a well‐known inducer of ER stress upon stimulation. TLR4 mutant diabetic mice (Akita background) are currently studied to unravel relative contribution of TLR4 dependent and independent mechanisms for H2S mediated reversal of ER stress during diabetic cardiomyopathy. In conclusion, H2S mediated raise in threshold for ER stress induction involve GRP78 protein preservation and TLR4 level suppression in the diabetic hearts.Support or Funding InformationNIH grants : HL108621, HL074185 and DK104653.

Hypoxic regulation of the expression of cell proliferation related genes in U87 glioma cells upon inhibition of IRE1 signaling enzyme

We have studied the effect of inhibition of IRE1 (inositol requiring enzyme 1), which is a central mediator of endoplasmic reticulum stress and a controller of cell proliferation and tumor growth, on hypoxic regulation of the expression of different proliferation related genes in U87 glioma cells. It was shown that hypoxia leads to up-regulation of the expression of IL13RA2, CD24, ING1, ING2, ENDOG, and POLG genes and to down-regulation – of KRT18, TRAPPC3, TSFM, and MTIF2 genes at the mRNA level in control glioma cells. Changes for ING1 and CD24 genes were more significant. At the same time, inhibition of IRE1 modifies the effect of hypoxia on the expression of all studied genes. In particular, it increases sensitivity to hypoxia of the expression of IL13RA2, TRAPPC3, ENDOG, and PLOG genes and suppresses the effect of hypoxia on the expression of ING1 gene. Additionally, it eliminates hypoxic regulation of KRT18, CD24, ING2, TSFM, and MTIF2 genes expressions and introduces sensitivity to hypoxia of the expression of BET1 gene in glioma cells. The present study demonstrates that hypoxia, which often contributes to tumor growth, affects the expression of almost all studied genes. Additionally, inhibition of IRE1 can both enhance and suppress the hypoxic regulation of these gene expressions in a gene specific manner and thus possibly contributes to slower glioma growth, but several aspects of this regulation must be further clarified.

CHOP Contributes to, But Is Not the Only Mediator of, IAPP Induced β-Cell Apoptosis.

The islet in type 2 diabetes is characterized by β-cell loss, increased β-cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP). When protein misfolding protective mechanisms are overcome, human IAPP (h-IAPP) forms membrane permeant toxic oligomers that induce β-cell dysfunction and apoptosis. In humans with type 2 diabetes (T2D) and mice transgenic for h-IAPP, endoplasmic reticulum (ER) stress has been inferred from nuclear translocation of CCAAT/enhancer-binding protein homologous protein (CHOP), an established mediator of ER stress. To establish whether h-IAPP toxicity is mediated by ER stress, we evaluated diabetes onset and β-cell mass in h-IAPP transgenic (h-TG) mice with and without deletion of CHOP in comparison with wild-type controls. Diabetes was delayed in h-TG CHOP(-/-) mice, with relatively preserved β-cell mass and decreased β-cell apoptosis. Deletion of CHOP attenuates dysfunction of the autophagy/lysosomal pathway in β-cells of h-TG mice, uncovering a role for CHOP in mediating h-IAPP-induced dysfunction of autophagy. As deletion of CHOP delayed but did not prevent h-IAPP-induced β-cell loss and diabetes, we examined CHOP-independent stress pathways. JNK, a target of the IRE-1pTRAF2 complex, and the Bcl-2 family proapoptotic mediator BIM, a target of ATF4, were comparably activated by h-IAPP expression in the presence and absence of CHOP. Therefore, although these studies affirm that CHOP is a mediator of h-IAPP-induced ER stress, it is not the only one. Therefore, suppression of CHOP alone is unlikely to be a durable therapeutic strategy to protect against h-IAPP toxicity because multiple stress pathways are activated.

Downregulation of miR-384-5p attenuates rotenone-induced neurotoxicity in dopaminergic SH-SY5Y cells through inhibiting endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress has been linked to the pathogenesis of Parkinson's disease (PD). However, the role of microRNAs (miRNAs) in this process involved in PD remains poorly understood. Recent studies indicate that miR-384-5p plays an important role for cell survival in response to different insults, but the role of miR-384-5p in PD-associated neurotoxicity remains unknown. In this study, we investigated the role of miR-384-5p in an in vitro model of PD using dopaminergic SH-SY5Y cells treated with rotenone. We found that miR-384-5p was persistently induced by rotenone in neurons. Also, the inhibition of miR-384-5p significantly suppressed rotenone-induced neurotoxicity, while overexpression of miR-384-5p aggravated rotenone-induced neurotoxicity. Through bioinformatics and dual-luciferase reporter assay, miR-384-5p was found to directly target the 3'-untranslated region of glucose-regulated protein 78 (GRP78), the master regulator of ER stress sensors. Quantitative polymerase chain reaction and Western blotting analysis showed that miR-384-5p negatively regulated the expression of GRP78. Inhibition of miR-384-5p remarkably suppressed rotenone-evoked ER stress, which was evident by a reduction in the phosphorylation of activating transcription factor 4 (ATF4) and inositol-requiring enzyme 1 (IRE1α). The downstream target genes of ER stress including CCAAT/enhancer-binding protein-homologous protein (CHOP) and X box-binding protein-1 (XBP-1) were also decreased by the miR-384-5p inhibitor. In contrast, overexpression of miR-384-5p enhanced ER stress signaling. In addition, knockdown of GRP78 significantly abrogated the inhibitory effect of miR-384-5p inhibitors on cell apoptosis and ER stress signaling. Moreover, we observed a significant increase of miR-384-5p expression in primary neurons induced by rotenone. Taken together, our results suggest that miR-384-5p mediated ER stress by negatively regulating GRP78 and that miR-384-5p inhibition might be a novel and promising approach for the treatment of PD.

C-Jun N-terminal Kinase-Dependent Endoplasmic Reticulum Stress Pathway is Critically Involved in Arjunic Acid Induced Apoptosis in Non-Small Cell Lung Cancer Cells.

Though arjunic acid, a triterpene isolated from Terminalia arjuna, was known to have antioxidant, antiinflammatory, and cytotoxic effects, its underlying antitumor mechanism still remains unclear so far. Thus, in the present study, the molecular antitumor mechanism of arjunic acid was examined in A549 and H460 non-small cell lung cancer (NSCLC) cells. Arjunic acid exerted cytotoxicity by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay and significantly increased sub-G1 population in A549 and H460 cells by cell cycle analysis. Consistently, arjunic acid cleaved poly (ADP-ribose) polymerase (PARP), activated Bax, and phosphorylation of c-Jun N-terminal kinases (JNK), and also attenuated the expression of pro-caspase-3 and Bcl-2 in A549 and H460 cells. Furthermore, arjunic acid upregulated the expression of endoplasmic reticulum (ER) stress proteins such as IRE1 α, ATF4, p-eIF2α, and C/EBP homologous protein (CHOP) in A549 and H460 cells. Conversely, CHOP depletion attenuated the increase of sub-G1 population by arjunic acid, and also JNK inhibitor SP600125 blocked the cytotoxicity and upregulation of IRE1 α and CHOP induced by arjunic acid in A549 and H460 cells. Overall, our findings suggest that arjunic acid induces apoptosis in NSCLC cells via JNK mediated ER stress pathway as a potent chemotherapeutic agent for NSCLC.

EFFECT OF HYPOXIA ON THE EXPRESSION OF GENES THAT ENCODE SOME IGFBP AND CCN PROTEINS IN U87 GLIOMA CELLS DEPENDS ON IRE1 SIGNALING.

We have studied hypoxic regulation of the expression of different insulin-like growth factor binding protein genes in U87 glioma cells in relation to inhibition of IRE1 (inositol requiring enzyme-1), a central mediator of endoplasmic reticulum stress, which controls cell proliferation and tumor growth. We have demonstrated that hypoxia leads to up-regulation of the expression of IGFBP6, IGFBP7, IGFBP10/CYR61, WISP1, and WISP2 genes and down-regulation--of IGFBP9/NOV gene at the mRNA level in control glioma cells, being more signifcant changes for IGFBP10/CYR61 and WISP2 genes. At the same time, inhibition of IRE1 modifies the effect of hypoxia on the expression of all studied genes: eliminates sensitivity to hypoxia the expression of IGFBP7 and IGFBP9/NOV genes, suppresses effect of hypoxia on IGFBP6, IGFBP10/CYR61, and WISP2 genes, and slightly enhances hypoxic regulation of WISP1 gene expression in glioma cells. We have also demonstrated that the expression of all studied genes in glioma cells is regulated by IRE1 signaling enzyme upon normoxic condition, because inhibition of IRE1 significantly up-regulates IGFBP7, IGFBP10/CYR61, WISP1, and WISP2 genes and down-regulates IGFBP6 and IGFBP9/NOV genes as compared to control glioma cells. The present study demonstrates that hypoxia, which contributes to tumor growth, affects all studied IGFBP and WISP gene expressions and that inhibition of IRE1 preferentially abolishes or suppresses the hypoxic regulation of these gene expressions and thus possibly contributes to slower glioma growth. Moreover, inhibition of IRE1, which correlates with suppression of cell proliferation and glioma growth, is down-regulated expression of pro-proliferative IGFBP genes, attesting to the fact that endoplasmic reticulum stress is a necessary component of malignant tumor growth.

Intrarenal renin-angiotensin system mediates fatty acid-induced ER stress in the kidney

Obesity-related kidney disease is related to caloric excess promoting deleterious cellular responses. Accumulation of saturated free fatty acids in tubular cells produces lipotoxicity involving significant cellular dysfunction and injury. The objectives of this study were to elucidate the role of renin-angiotensin system (RAS) activation in saturated fatty acid-induced endoplasmic reticulum (ER) stress in cultured human proximal tubule epithelial cells (HK2) and in mice fed with a high-fat diet. Treatment with saturated fatty acid palmitic acid (PA; 0.8 mM) for 24 h induced ER stress in HK2, leading to an unfolded protein response as reflected by increased expressions of the ER chaperone binding immunoglobulin protein (BiP) and proapoptotic transcription factor C/EBP homologous protein (CHOP) protein as evaluated by immunoblotting. PA treatment also induced increased protein expression of inositol requiring protein 1α (IRE1α), phosphorylated eukaryotic initiation factor-α (eIF2α), and activating transcription factor 4 (ATF4) as well as activation of caspase-3. PA treatment was associated with increased angiotensin II levels in cultured medium. The angiotensin II type 1 receptor (AT1R) blocker valsartan or renin inhibitor aliskiren dramatically suppressed PA-induced upregulation of BiP, CHOP, IRE1α, p-eIF2α, and ATF4 in HK2 cells. In contrast, valsartan or aliskiren did not prevent ER stress induced by tunicamycin. C57BL/6 mice fed with a high-fat diet for 14 wk exhibited increased protein expressions of BiP and CHOP compared with control mice, which were significantly attenuated by the valsartan treatment. Increased angiotensin II levels in serum and urine were observed in mice fed with a high-fat diet when compared with controls. It is suggested that the intrarenal RAS activation may play an important role in diabetic kidney injury via mediating ER stress induced by saturated fatty acid.