Endoplasmic reticulum stress activates telomerase

Telomerase contributes to cell proliferation and survival through both telomere‐dependent and telomere‐independent mechanisms. In this report, we discovered that endoplasmic reticulum (ER) stress transiently activates the catalytic components of telomerase (TERT) expression in human cancer cell lines and murine primary neural cells. Importantly, we show that depletion of hTERT sensitizes cells to undergo apoptosis under ER stress, whereas increased hTERT expression reduces ER stress‐induced cell death independent of catalytically active enzyme or DNA damage signaling. Our findings establish a functional link between ER stress and telomerase, both of which have important implications in the pathologies associated with aging and cancer.     

Endoplasmic reticulum stress is implicated in retinal inflammation and diabetic retinopathy

Diabetic retinopathy is a chronic low-grade inflammatory disease; however, the mechanisms remain elusive. In the present study, we demonstrated that endoplasmic reticulum (ER) stress was activated in the retina in animal models of diabetes and oxygen-induced retinopathy (OIR). Induction of ER stress by tunicamycin resulted in significantly increased expression of inflammatory molecules in the retina. Inhibition of ER stress by chemical chaperone 4-phenyl butyric acid ameliorated inflammation in cultured human retinal endothelial cells exposed to hypoxia, and in the retinas of diabetic and OIR mice. These findings indicate that ER stress is a potential mediator of retinal inflammation in diabetic retinopathy.     

Bufotalin-induced apoptosis in osteoblastoma cells is associated with endoplasmic reticulum stress activation

The search for novel and more efficient chemo-agents against malignant osteoblastoma is important. In this study, we examined the potential anti-osteoblastoma function of bufotalin, and studied the underlying mechanisms. Our results showed that bufotalin induced osteoblastoma cell death and apoptosis in dose- and time-dependent manners. Further, bufotalin induced endoplasmic reticulum (ER) stress activation in osteoblastoma cells, the latter was detected by the induction of C/EBP homologous protein (CHOP), phosphorylation of inositol-requiring enzyme 1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), as well as caspase-12 activation. Conversely, the ER stress inhibitor salubrinal, the caspase-12 inhibitor z-ATAD-fmk as well as CHOP depletion by shRNA significantly inhibited bufotalin-induced osteoblastoma cell death and apoptosis. Finally, by using a mice xenograft model, we demonstrated that bufotalin inhibited U2OS osteoblastoma cell growth in vivo. In summary, our results suggest that ER stress contributes to bufotalin-induced apoptosis in osteoblastoma cells. Bufotalin might be investigated as a novel anti-osteoblastoma agent.     

Unexpected blockade of adipocyte differentiation by K-7174: implication for endoplasmic reticulum stress

Preadipocytes constitutively express GATA-2 and GATA-3 that are required to halt the cells at the undifferentiated stage. However, we unexpectedly found that K-7174, a GATA-specific inhibitor, did not induce but rather inhibited differentiation of 3T3-L1 preadipocytes. It was associated with lack of lipid accumulation, blunted expression of adipocyte markers including adiponectin and peroxisome proliferator-activated receptor gamma (PPARgamma), and sustained expression of a preadipocyte marker monocyte chemoattractant protein 1 (MCP-1). Subsequent experiments revealed that K-7174 had the potential to induce endoplasmic reticulum (ER) stress evidenced by induction of GRP78 and CHOP. Other inducers of ER stress completely reproduced the effects of K-7174 including suppression of lipid accumulation, blockade of induction of adiponection and PPARgamma and maintenance of MCP-1 expression. These results indicated a possibility that ER stress suppresses adipocyte differentiation and that GATA inhibitor K-7174 has the potential for interfering with adipogenesis through induction of ER stress.     

Endoplasmic reticulum: ER stress regulates mitochondrial bioenergetics

Endoplasmic reticulum (ER) stress activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. The molecular mechanisms responsible for execution of the cell death program are relatively well characterized, but the metabolic events taking place during the adaptive phase of ER stress remain largely undefined. Here we discuss emerging evidence regarding the metabolic changes that occur during the onset of ER stress and how ER influences mitochondrial function through mechanisms involving calcium transfer, thereby facilitating cellular adaptation. Finally, we highlight how dysregulation of ER-mitochondrial calcium homeostasis during prolonged ER stress is emerging as a novel mechanism implicated in the onset of metabolic disorders.     

ER stress impairs MHC Class I surface expression and increases susceptibility of thyroid cells to NK-mediated cytotoxicity

We recently reported that, in thyroid cells, ER stress triggered by thapsigargin or tunicamycin, two well known ER stressing agents, induced dedifferentiation and loss of the epithelial phenotype in rat thyroid cells. In this study, we sought to evaluate if, in thyroid cells, ER stress could affect MHC class I expression and the possible implications of this effect in the alteration of function of natural killer cells, suggesting a role in thyroid pathology. In both, a human line of fetal thyroid cells (TAD-2 cells) and primary cultures of human thyroid cells, thapsigargin and tunicamicin triggered ER stress evaluated by BiP mRNA levels and XBP-1 splicing. In both cell types, TAD-2 cell line and primary cultures, major histocompatibility complex class I (MHC-I) plasmamembrane expression was significantly reduced by ER stress. This effect was accompanied by signs of natural killer activation. Thus, natural killer cells dramatically increased IFN-γ production and markedly increased their cytotoxicity against thyroid cells. Together, these data indicate that ER stress induces a decrease of MHC class I surface expression in thyroid cells, resulting in reduced natural killer-cell self-tolerance.     

Active mixture of serum-circulating small molecules selectively inhibits proliferation and triggers apoptosis in cancer cells via induction of ER stress

Genetic and epigenetic regulation as well as immune surveillance are known defense mechanisms to protect organisms from developing cancer. Based on experimental evidence, we proposed that small metabolically active molecules accumulating in cancer cells may play a role in an alternative antitumor surveillance system. Previously, we reported that treatment with a mixture of experimentally selected small molecules, usually found in the serum (defined ‘active mixture’, AM), selectively induces apoptosis in cancer cells and significantly inhibits tumor formation in vivo. In this study, we show that the AM elicits gene expression changes characteristic of endoplasmic reticulum (ER) stress in HeLa, MCF-7, PC-3 and Caco-2 cancer cells, but not in primary human renal epithelial cells. The activation of the ER stress pathway was confirmed by the upregulation of ATF3, ATF4, CHAC1, DDIT3 and GDF15 proteins. Mechanistically, our investigation revealed that eIF2α, PERK and IRE1α are phosphorylated upon treatment with the AM, linking the induction of ER stress to the antiproliferative and proapoptotic effects of the AM previously demonstrated. Inhibition of ER stress in combination with BBC3 and PMAIP1 knockdown completely abrogated the effect of the AM. Moreover, we also demonstrated that the AM induces mIR-3189-3p, which in turn enhances the expression of ATF3 and DDIT3, thus representing a possible new feedback mechanism in the regulation of ATF3 and DDIT3 during ER stress. Our results highlight small molecules as attractive anticancer agents and warrant further evaluation of the AM in cancer therapy, either alone or in combination with other ER stress inducing agents.     

Lead-induced endoplasmic reticulum (ER) stress responses in the nervous system

Lead (Pb) poisoning continues to be a significant health risk because of its pervasiveness in the environment, its known neurotoxic effects in children, and potential endogenous exposure from Pb deposited in bone. New information about mechanisms by which Pb enters cells and its organelle targets within cells are briefly reviewed. Toxic effects of Pb on the endoplasmic reticulum (ER) are considered in detail, based on recent evidence that Pb induces the expression of the gene for 78-kD glucose-regulated protein (GRP78) and other ER stress genes. GRP78 is a molecular chaperone that binds transiently to proteins traversing through the ER and facilitates their folding, assembly, and transport. Models are presented for the induction of ER stress by Pb in astrocytes, the major cell type of the central nervous system, in which Pb accumulates. A key feature of the models is disruption of GRP78 function by direct Pb binding. Possible pathways by which Pb-bound GRP78 stimulates the unfolded protein response (UPR) in the ER are discussed, specifically transduction by IRE1/ATF6 and/or IRE1/JNK. The effect of Pb binding to GRP78 in the ER is expected to be a key component for understanding mechanisms of Pb-induced ER stress gene expression.     

Autophagy-dependent EIF2AK3 activation compromises ursolic acid-induced apoptosis through upregulation of MCL1 in MCF-7 human breast cancer cells

Ursolic acid (UA) is a pentacyclic triterpenoid with promising cancer chemopreventive properties. A better understanding of the mechanisms underlying anticancer activity of UA is needed for further development as a clinically useful chemopreventive agent. Here, we found that both endoplasmic reticulum (ER) stress and autophagy were induced by UA in MCF-7 human breast cancer cells. Surprisingly, ER stress was identified as an effect rather than a cause of UA-induced autophagy. Autophagy-dependent ER stress protected the cells from UA-induced apoptosis through EIF2AK3-mediated upregulation of MCL1. Activation of MAPK1/3 but not inhibition of MTOR pathway contributed to UA-induced cytoprotective autophagy in MCF-7 cells. Our findings uncovered a novel cellular mechanism involved in the anticancer activity of UA, and also provided a useful model to study biological significance and mechanisms of autophagy-mediated ER stress.     

Downregulation of gap junction expression and function by endoplasmic reticulum stress

Gap junctional intercellular communication (GJIC) plays a critical role in the control of multiple cell behavior as well as in the maintenance of tissue and organ homeostasis. However, mechanisms involved in the regulation of gap junctions (GJs) have not been fully understood. Given endoplasmic reticulum (ER) stress and dysfunction of GJs coexist in several pathological situations, we asked whether GJs could be regulated by ER stress. Incubation of mesangial cells with ER stress‐inducing agents (thapsigargin, tunicamycin, and AB₅ subtilase cytotoxin) resulted in a decrease in connexin 43 (Cx43) expression at both protein and mRNA levels. This was accompanied by a loss of GJIC, as evidenced by the reduced numbers of dye‐coupled cells after single cell microinjection or scrape loading dye transfer. Further studies demonstrated that ER stress significantly inhibited the promoter activity of the Cx43 gene, reduced [³⁵S]‐methionine incorporation into Cx43 protein and accelerated degradation of Cx43. ER stress also decreased the Cx43 protein levels in several different cell types, including human umbilical vein endothelial cells, mouse‐derived renin‐secreting cells and human hepatoma cells. Furthermore, induction of ER stress by hypoxic chemicals thenoyltrifluoroacetone and cobalt chloride was found to be associated with a reduction in Cx43. Our findings thus reveal a close link between ER stress and GJs. ER stress may represent a novel mechanism underlying the altered GJs in a variety of pathological situations. J. Cell. Biochem. 107: 973–983, 2009. © 2009 Wiley‐Liss, Inc.     

A rare variant in human fibroblast activation protein associated with ER stress,loss of enzymatic function and loss of cell surface localisation

Fibroblast activation protein (FAP) is a focus of interest as a potential cancer therapy target. This membrane bound protease possesses the unique catalytic activity of hydrolysis of the post-proline bond two or more residues from the N-terminus of substrates. FAP is highly expressed in activated fibroblastic cells in tumours, arthritis and fibrosis. A rare, novel, human polymorphism, C1088T, encoding Ser363 to Leu, occurring in the sixth blade of the β propeller domain, was identified in a family. Both in primary human fibroblasts and in Ser363LeuFAP transfected cells, we showed that this single substitution ablates FAP dimerisation and causes loss of enzyme activity. Ser363LeuFAP was detectable only in endoplasmic reticulum (ER), in contrast to the distribution of wild-type FAP on the cell surface. The variant FAP showed decreased conformational antibody binding, consistent with an altered tertiary structure. Ser363LeuFAP expression was associated with upregulation of the ER chaperone BiP/GRP78, ER stress sensor ATF6, and the ER stress response target phospho-eIF2α, all indicators of ER stress. Proteasomal inhibition resulted in accumulation of Ser363LeuFAP, indicating the involvement of ER associated degradation (ERAD). Neither CHOP expression nor apoptosis was elevated, so ERAD is probably important for protecting Ser363LeuFAP expressing cells. These data on the first loss of function human FAP gene variant indicates that although the protein is vulnerable to an amino acid substitution in the β-propeller domain, inactive, unfolded FAP can be tolerated by cells.     

High levels of the molecular chaperone Mdg1/ERdj4 reflect the activation state of endothelial cells

Mdg1/ERdj4, a mammalian chaperone that belongs to the HSP40 protein family, has been reported to be located in the endoplasmic reticulum (ER), is induced by ER stress, and protects ER stressed cells from apoptosis. Here we show that under normal physiological conditions, Mdg1/ERdj4 is expressed at various levels in the vasculature due to different activation states of the endothelium. To elucidate the stimuli that induce ER stress and thus upregulate Mdg1/ERdj4, we investigated the effect of several endothelium specific stressors on its expression. Mdg1/ERdj4 mRNA is induced by activated macrophages, by nitric oxide (NO) and heat shock, and during terminal cell differentiation, whereas shear stress does not affect Mdg1/ERdj4 expression levels. While the mRNA stability of BiP/GRP78 is unaffected in ER stressed cells, the stability of Mdg1/ERdj4 mRNA is prolonged during ER stress resulting in rapid increases and high levels of Mdg1/ERdj4 mRNA. Mdg1/ERdj4 protein is localized in the ER under control conditions. While heat shock induces a rapid translocation of Mdg1/ERdj4 to the nucleoli, no translocation could be observed during ER stress. This indicates that Mdg1/ERdj4 protein has diverse mechanisms to protect stressed cells from apoptosis.     

A novel role for Bcl-2 associated-athanogene-1 (Bag-1) in regulation of the endoplasmic reticulum stress response in mammalian chondrocytes

BAG-1 (Bcl-2 associated athanogene-1) is a multifunctional protein, linking cell proliferation, cell death, protein folding, and cell stress. In vivo, BAG-1 is expressed in growth plate and articular cartilage, and the expression of BAG-1 is decreased with aging. Chondrocytes respond to endoplasmic reticulum (ER) stress with decreased expression of extracellular matrix proteins, and prolonged ER stress leads to chondrocyte apoptosis. Here we demonstrate for the first time that BAG-1 is involved in ER stress-induced apoptosis in chondrocytes. Induction of ER stress through multiple mechanisms all resulted in downregulation of BAG-1 expression. In addition, direct suppression of BAG-1 expression resulted in chondrocyte growth arrest and apoptosis, while stable overexpression of BAG-1 delayed the onset of ER stress-mediated apoptosis. In addition to regulating apoptosis, we also observed decreased expression of collagen type II in BAG-1 deficient chondrocytes. In contrast, overexpression of BAG-1 resulted in increased expression of collagen type II. Moreover, under ER stress conditions, the reduced expression of collagen type II was delayed in chondrocytes overexpressing BAG-1. These results suggest a novel role for BAG-1 in supporting viability and matrix expression of chondrocytes.     

Attenuation of unfolded protein response and apoptosis by mReg2 induced GRP78 in mouse insulinoma cells

Murine regenerating (mReg) genes have been implicated in preserving islet cell biology. Expanding on our previous work showing that overexpression of mReg2 protects MIN6 insulinoma cells against streptozotocin-induced apoptosis, we now demonstrate that mReg2 induces glucose-regulated peptide 78 (GRP78) expression via the Akt–mTORC1 axis and protects MIN6 cells against ER stress induced by thapsigargin and glucolipotoxicity. Activation of mTORC1 activity results from both mReg2-induced increased mTOR phosphorylation as well as increased expression of Raptor and GβL. Inhibition of Akt and mTORC1 blunted the ability of mReg2 to induce GRP78 and attenuate unfolded protein response (UPR). Knockdown of GRP78 sensitized the cells overexpressing mReg2 to UPR without affecting its ability to activate Akt–mTORC1 signaling. Induced expression of mReg2 may protect insulin producing cells from ER stress in diabetes.     

Glucosamine-induced endoplasmic reticulum stress attenuates apolipoprotein B100 synthesis via PERK signaling

Glucosamine impairs hepatic apolipoprotein B100 (apoB100) production by inducing endoplasmic reticulum (ER) stress and enhancing cotranslational and posttranslational apoB100 degradation (Qiu, W., R. K. Avramoglu, A. C. Rutledge, J. Tsai, and K. Adeli. Mechanisms of glucosamine-induced suppression of the hepatic assembly and secretion of apolipoprotein B-100-containing lipoproteins. J. Lipid Res. 2006. 47: 1749–1761). Here, we report that glucosamine also regulates apoB100 protein synthesis via ER-stress-induced PERK activation. Short-term (4 h) glucosamine treatment of HepG2 cells reduced both cellular (by 62%) and secreted apoB100 (by 43%) without altering apoB100 mRNA. Treatment with proteasomal inhibitors only partially prevented the suppressive effects of glucosamine, suggesting that mechanisms other than proteasomal degradation may also be involved. Glucosamine-induced ER stress was associated with a significantly reduced apoB100 synthesis with no significant change in posttranslational decay rates, suggesting that glucosamine exerted its effect early during apoB biosynthesis. The role of PERK and its substrate, α-subunit of eukaryotic initiation factor 2 (eIF2α), in the suppressive effects of glucosamine on apoB synthesis was then investigated. Coexpression of apoB15 (normally resistant to intracellular degradation) with wild-type double stranded (ds) RNA activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) in COS-7 cells resulted in a dramatic reduction in the levels of newly synthesized apoB15. Interestingly, cotransfection with apoB15 and a kinase inactive PERK mutant (K618A) increased apoB15 expression. In addition, short-term glucosamine treatment stimulated an increase in phosphorylation of PERK and eIF2α. Taken together, these data suggest that in addition to the induction of ER-associated degradation and other degradative pathways, ER stress is associated with suppression of apoB synthesis via a PERK-dependent mechanism.     

Decreased vitamin B12 availability induces ER stress through impaired SIRT1-deacetylation of HSF1

Vitamin B12 (cobalamin) is a key determinant of S-adenosyl methionine (SAM)-dependent epigenomic cellular regulations related to methylation/acetylation and its deficiency produces neurodegenerative disorders by elusive mechanisms. Sirtuin 1 deacetylase (SIRT1) triggers cell response to nutritional stress through endoplasmic reticulum (ER) stress. Recently, we have established a N1E115 dopaminergic cell model by stable expression of a transcobalamin–oleosin chimera (TO), which impairs cellular availability of vitamin B12, decreases methionine synthase activity and SAM level, and reduces cell proliferation. In contrast, oleosin-transcobalamin chimera (OT) does not modify the phenotype of transfected cells. Presently, the impaired cellular availability of vitamin B12 in TO cells activated irreversible ER stress pathways, with increased P-eIF-2α, P-PERK, P-IRE1α, ATF6, ATF4, decreased chaperon proteins and increased pro-apoptotic markers, CHOP and cleaved caspase 3, through reduced SIRT1 expression and consequently greater acetylation of heat-shock factor protein 1 (HSF1). Adding either B12, SIRT1, or HSF1 activators as well as overexpressing SIRT1 or HSF1 dramatically reduced the activation of ER stress pathways in TO cells. Conversely, impairing SIRT1 and HSF1 by siRNA, expressing a dominant negative form of HSF1, or adding a SIRT1 inhibitor led to B12-dependent ER stress in OT cells. Addition of B12 abolished the activation of stress transducers and apoptosis, and increased the expression of protein chaperons in OT cells subjected to thapsigargin, a strong ER stress stimulator. AdoX, an inhibitor of methyltransferase activities, produced similar effects than decreased B12 availability on SIRT1 and ER stress by a mechanism related to increased expression of hypermethylated in cancer 1 (HIC1). Taken together, these data show that cellular vitamin B12 has a strong modulating influence on ER stress in N1E115 dopaminergic cells. The impaired cellular availability in vitamin B12 induces irreversible ER stress by greater acetylation of HSF1 through decreased SIRT1 expression, whereas adding vitamin B12 produces protective effects in cells subjected to ER stress stimulation.     

Decreased protein and mRNA expression of ER stress proteins GRP78 and GRP94 in HepG2 cells over-expressing CYP2E1

CYP2E1 causes oxidative stress mediated cell death; the latter is one mechanism for endoplasmic reticulum (ER) stress in the cell. Unfolded proteins accumulate during ER stress and ER resident proteins GRP78 and GRP94 protect cells against ER dysfunction. We examined the possible role of GRP78 and GRP94 as protective factors against CYP2E1-mediated toxicity in HepG2 cells expressing CYP2E1 (E47 cells). E47 cells expressed high levels of CYP2E1 protein and catalytic activity which is associated with increased ROS generation, lipid peroxidation and the elevated presence of ubiquinated and aggregated proteins as compared to control HepG2 C34 cells which do not express CYP2E1. The mRNA and protein expression of GRP78 and GRP94 were decreased in E47 cells compared to the C34 cells, which may explain the accumulation of ubiquinated and aggregated proteins. Expression of these GRP proteins was induced with the ER stress agent thapsigargin in E47 cells, and E47 cells were more resistant to the toxicity caused by thapsigargin and calcimycin, possibly due to this upregulation and also because of the high expression of GSH and antioxidant enzymes in E47 cells. Antioxidants such as trolox and N-acetylcysteine increased GRP78 and GRP94 levels in the E47 cells, suggesting that CYP2E1- derived oxidant stress was responsible for down regulation of these GRPs in the E47 cells. Thapsigargin mediated toxicity was decreased in cells treated with the antioxidant trolox indicating a role for oxidative stress in this toxicity. These results suggest that CYP2E1 mediated oxidative stress downregulates the expression of GRP proteins in HepG2 cells and oxidative stress is an important mechanism in causing ER dysfunction in these cells.