Endoplasmic Reticulum Stress Response Genes Research Articles

A Role for SIR-2.1 Regulation of ER Stress Response Genes in Determining C. elegans Life Span

C. elegans SIR-2.1, a member of the Sir-2 family of NAD(+)-dependent protein deacetylases, has been shown to regulate nematode aging via the insulin/IGF pathway transcription factor daf-16. Treatment of C. elegans with the small molecule resveratrol, however, extends life span in a manner fully dependent upon sir-2.1, but independent of daf-16. Microarray analysis of worms treated with resveratrol demonstrates the transcriptional induction of a family of genes encoding prion-like glutamine/asparagine-rich proteins involved in endoplasmic reticulum (ER) stress response to unfolded proteins. RNA interference of abu-11, a member of this ER stress gene family, abolishes resveratrol-mediated life span extension, and overexpression of abu-11 extends the life span of transgenic animals. Furthermore, SIR-2.1 normally represses transcription of abu-11 and other ER stress gene family members, indicating that resveratrol extends life span by inhibiting sir-2.1-mediated repression of ER stress genes. Our findings demonstrate that abu-11 and other members of its ER stress gene family are positive determinants of C. elegans life span.

Modulating the Endoplasmic Reticulum Stress Response with trans-4,5-Dihydroxy-1,2-Dithiane Prevents Chemically Induced Renal Injury In Vivo

Agents that disrupt functions of the endoplasmic reticulum (ER) induce expression of ER stress-response genes including ER chaperones. Increased expression of the major ER chaperone, Grp78, protects cells, including renal epithelial cells, from chemically induced injury and death in vitro. In this study, we determined if pharmacological manipulation of the ER stress-response gene is an effective strategy to protect the kidney from chemical stress in vivo. Treatment with trans-4,5-dihydroxy-1,2-dithiane (DTTox), a novel inducer of ER stress proteins, stimulated a time-and dose-dependent increase in Grp78 expression in the kidney, but it did not cause detectable injury. Furthermore, prior treatment with DTTox protected the proximal tubular epithelium against a subsequent challenge with the nephrotoxicant S-(1,1,2,2,-tetrafluoroethyl)-L-cysteine (TFEC). In contrast, activating a heat shock response did not have a protective effect. Prior treatment with DTTox did not reduce covalent binding of radiolabeled reactive metabolites of (35)S-TFEC to renal proteins, indicating that protection was not due to an effect on the metabolic activation of TFEC to the reactive metabolite(s) responsible for renal injury. Antisense grp78 expression in the renal epithelial cell line LLC-PK1 blocked the DTTox-induced Grp78 increase and ablated the protective effect against TFEC damage, indicating that the induction of grp78 expression and the ER stress response were critical for the protective effect of DTTox. These findings suggest that increased expression of Grp78 plays a major role in the protection of renal epithelial cells from reactive intermediate-induced chemical injury in vivo and that pharmacological manipulation is an effective strategy to prevent damage by some classes of nephrotoxicants.

Role of CHOP in Hepatic Apoptosis in the Murine Model of Intragastric Ethanol Feeding

CHOP is a transcriptional regulator involved in apoptosis caused by endoplasmic reticulum (ER) stress. We previously reported that CHOP as well as other ER stress response genes is induced in the liver of a murine model of intragastric ethanol feeding. This study was undertaken to determine the role of CHOP in hepatocellular apoptosis and liver injury in this model. CHOP wild-type (+/+) mice and CHOP null (-/-) mice were fed alcohol for four weeks with glucose as control. Hematoxylin-eosin staining, TUNEL, and caspase 3 staining of liver tissues were performed for assessment of fatty liver, necroinflammation, and apoptosis. Total RNA was extracted for microarray and reverse transcription-PCR analyses, and proteins were used for western blotting. Significant increased liver/body ratio, steatosis, liver triglyceride levels, and plasma homocysteine concentrations were observed in alcohol-fed mice as compared with controls in both genotypes. There was no significant difference between wild-type and CHOP null (-/-) mice in the parameters related to fatty liver. Alcohol-induced increased serum alanine aminotransferase levels and necroinflammatory foci were not significantly reduced in CHOP null (-/-) mice. However, apoptosis was present in alcohol-fed wild-type mice but virtually absent in alcohol-fed CHOP null (-/-) mice. The ER stress response indicated by increased Grp78 mRNA was observed in both types of mice fed alcohol. Of 12,423 transcripts analyzed for >or= two-fold changes, several related to apoptosis were influenced by CHOP: Gadd45 and cathepsin B were up-regulated in ethanol-fed wild-type mice but not in CHOP null (-/-) mice, whereas Jun D and Bcl-xL were down-regulated in ethanol-fed wild-type mice but not in ethanol-fed CHOP null (-/-) mice. CHOP null (-/-) mice have remarkable absence of hepatocellular apoptosis in response to alcohol feeding but no protection against hyperhomocysteinemia, ER stress, and fatty liver. Thus, CHOP up-regulation occurs downstream of and contributes to one manifestation of ER stress, namely, apoptosis. Microarray studies confirmed by PCR analysis and western blotting indicate that genes affected by CHOP are both proapoptotic and antiapoptotic and CHOP induction by ethanol may tip the balance of cell survival and death toward apoptosis.

Fluoride Induces Endoplasmic Reticulum Stress in Ameloblasts Responsible for Dental Enamel Formation

The mechanism of how fluoride causes fluorosis remains unknown. Exposure to fluoride can inhibit protein synthesis, and this may also occur by agents that cause endoplasmic reticulum (ER) stress. When translated proteins fail to fold properly or become misfolded, ER stress response genes are induced that together comprise the unfolded protein response. Because ameloblasts are responsible for dental enamel formation, we used an ameloblast-derived cell line (LS8) to characterize specific responses to fluoride treatment. LS8 cells were growth-inhibited by as little as 1.9-3.8 ppm fluoride, whereas higher doses induced ER stress and caspase-mediated DNA fragmentation. Growth arrest and DNA damage-inducible proteins (GADD153/CHOP, GADD45alpha), binding protein (BiP/glucose-responsive protein 78 (GRP78), the non-secreted form of carbonic anhydrase VI (CA-VI), and active X-box-binding protein-1 (Xbp-1) were all induced significantly after exposure to 38 ppm fluoride. Unexpectedly, DNA fragmentation increased when GADD153 expression was inhibited by short interfering RNA treatment but remained unaffected by transient GADD153 overexpression. Analysis of control and GADD153(-/-) embryonic fibroblasts demonstrated that caspase-3 mediated the increased DNA fragmentation observed in the GADD153 null cells. We also demonstrate that mouse incisor ameloblasts are sensitive to the toxic effects of high dose fluoride in drinking water. Activated Ire1 initiates an ER stress response pathway, and mouse ameloblasts were shown to express activated Ire1. Ire1 levels appeared induced by fluoride treatment, indicating that ER stress may play a role in dental fluorosis. Low dose fluoride, such as that present in fluoridated drinking water, did not induce ER stress.

PPARgamma ligands induce ER stress in pancreatic beta-cells: ER stress activation results in attenuation of cytokine signaling.

Peroxisome proliferator-activated receptor (PPAR)gamma ligands are known to have anti-inflammatory properties that include the inhibition of cytokine signaling, transcription factor activation, and inflammatory gene expression. We have recently observed that increased expression of heat shock protein (HSP)70 correlates with, but is not required for, the anti-inflammatory actions of PPARgamma ligands on cytokine signaling. In this study, we provide evidence that the inhibitory actions of PPARgamma ligands on cytokine signaling are associated with endoplasmic reticulum (ER) stress or unfolded protein response (UPR) activation in pancreatic beta-cells. 15-Deoxy-Delta(12,14)-prostaglandin J(2), at concentrations that inhibit cytokine signaling, stimulates phosphorylation of eukaryotic initiation factor-2alpha, and this event is followed by a rapid inhibition of protein translation. Under conditions of impaired translation, PPARgamma ligands stimulate the expression of a number of ER stress-responsive genes, such as GADD 153, BiP, and HSP70. Importantly, ER stress activation in response to PPARgamma ligands or known UPR activators results in the attenuation of IL-1 and IFN-gamma signaling. These findings indicate that PPARgamma ligands induce ER stress, that ER stress activation is associated with an attenuation of cytokine signaling in beta-cells, and that the attenuation of responsiveness to extracellular stimuli appears to be a novel protective action of the UPR in cells undergoing ER stress.

Activation of mesangial cell MAPK in responseto homocysteine

Alteration in mesangial cell function is central to the progression of glomerular disease in numerous models of chronic renal failure (CRF). Animal models of chronic glomerular disease are characterized by mesangial cell proliferation and elaboration of extracellular matrix protein (ECM), resulting in glomerulosclerosis. Elevated plasma levels of homocysteine (Hcy) are seen in both animal models and humans with CRF, and have been proposed to contribute to the high prevalence of vascular disease in this group. Some of the pathogenetic effects of Hcy are thought to be mediated via the induction of endoplasmic reticulum stress. Thus, Hcy effects on mesangial cells could contribute to the progression of CRF. Previous work has shown Hcy- mediated induction of Erk mitogen-activated protein kinase (MAPK) in vascular smooth muscle cells (VSMCs). Erk induces increases in activator protein-1 (AP-1) transcription factor activity which may augment mesangial cell proliferation and ECM protein production. Consequently, we studied the effect of Hcy on mesangial cell Erk signaling. Mesangial cells were exposed to Hcy after 24 hours of serum starvation and Erk activity assessed. Nuclear translocation of phospho-Erk was visualized by confocal microscopy. AP-1 nuclear protein binding was measured in response to Hcy by mobility shift assay. Hcy-induced mesangial cell calcium flux was measured in Fura-2 loaded cells. Mesangial cell DNA synthesis in response to Hcy was assessed by [3H]-thymidine incorporation and proliferation by Western blotting for proliferating cell nuclear antigen (PCNA). Expression of endoplasmic reticulum stress response genes were determined by Northern and Western analysis. Hcy led to an increase in Erk activity that was maximal at 50 micromol/L and 20 minutes of treatment. Subsequent experiments used this concentration and time point. Erk activity in response to Hcy was insensitive to n-acetylcysteine and catalase, indicating oxidative stress did not play a role. However, Hcy50 micromol/L induced a brief increase in intracellular mesangial cell calcium within 5 minutes, and the calcium ionophores A23187 and ionomycin increased Erk activity while chelation of intracellular calcium with BAPTA-AM abrogated the Erk response to Hcy. Confocal microscopy of activated Erk nuclear translocation mirrored these results as did mesangial cell nuclear protein binding to AP-1 consensus sequences. Hcy- induced increases in thymidine incorporation and PCNA expression at 24 hours were Erk dependent. The expression of endoplasmic reticulum stress response genes was significantly elevated by Hcy in an Erk-dependent manner. Hcy increases Erk activity in mesangial cells via a calcium-dependent mechanism, resulting in increased AP-1 nuclear protein binding, cell DNA synthesis and proliferation and induction of endoplasmic reticulum stress. These observations suggest potential mechanisms by which Hcy may contribute to progressive glomerular injury.

Opposing Roles for ATF6α and ATF6β in Endoplasmic Reticulum Stress Response Gene Induction

The endoplasmic reticulum (ER) transmembrane proteins, ATF6alpha and ATF6beta, are cleaved in response to ER stress, which can be induced by tunicamycin. The resulting N-terminal fragments of both ATF6 isoforms, which have conserved basic leucine-zipper and DNA binding domains but divergent transcriptional activation domains, translocate to the nucleus where they bind to ER stress-response elements (ERSE) in ER stress-response genes (ERSRG), such as GRP78. Although it is known that ATF6alpha is a potent activator of ERSRGs, the transcriptional potency and functions of ATF6beta remain to be explored. Accordingly, N-terminal fragments of each ATF6 isoform (N-ATF6alpha and N-ATF6beta) were overexpressed in HeLa cells and the effects on GRP78 induction were assessed. When expressed at similar levels, N-ATF6alpha conferred approximately 200-fold greater GRP78 promoter activation than N-ATF6beta. Because ER stress activates nuclear translocation of both ATF6alpha and beta and because both bind to ERSEs, the effect of co-expressing them on GRP78 induction was assessed. Surprisingly, N-ATF6beta inhibited N-ATF6alpha-mediated GRP78 promoter activation in a dominant-negative manner. Moreover, N-ATF6beta inhibited TN-mediated GRP78 promoter activation, which requires endogenous ATF6alpha. ATF6 isoform-specific small inhibitory RNAs were used to show that, as expected, endogenous ATF6alpha was required for maximal ERSRG induction; however, endogenous ATF6beta moderated ERSRG induction. These results indicate that compared with ATF6alpha, ATF6beta is a very poor activator of ERSRG induction and it represses ATF6alpha-mediated ERSRG induction. Thus, ATF6beta may serve as a transcriptional repressor functioning in part to regulate the strength and duration of ATF6alpha-mediated ERSRG activation during the ER stress response.

Manganese superoxide dismutase is induced by endoplasmic reticulum stress through IRE1-mediated nuclear factor (NF)-kappaB and AP-1 activation.

Manganese superoxide dismutase (MnSOD) is an antioxidative enzyme that scavenges superoxide radicals and is localized in the mitochondrial matrix. MnSOD is induced by a variety of stimuli through nuclear factor (NF)-kappaB and AP-1 activation. We investigated the expression of MnSOD in HeLa cells exposed to various agents interfering with endoplasmic reticulum (ER) functions. All agents caused an increase in the mRNA and protein levels of MnSOD. Although ER stress-responsive genes often are up-regulated by ATF6, IRE1 and XBP1, which are ER stress-related transcription factors/transducers, the overexpression of neither molecule affected the levels of MnSOD mRNA and protein. Furthermore, we showed that ER stress reagents induced NF-kappaB and AP-1 activation that were inhibited by a dominant-negative IRE1 mutant. We finally demonstrated that ER stress-induced MnSOD expression was reduced by the IRE1 mutant. These results suggest that the MnSOD expression is controlled by ER stress through IRE1-mediated NF-kappaB and AP-1 activation.

Expression of the pro-angiogenic factors vascular endothelial growth factor and interleukin-8/CXCL8 by human breast carcinomas is responsive to nutrient deprivation and endoplasmic reticulum stress

BackgroundThe expression of pro-angiogenic cytokines, such as vascular endothelial growth factor (VEGF) and interleukin-8/CXCL8 (IL-8), plays an important role in tumor growth and metastasis. Low oxygen tension within poorly-vascularized tumors is thought to be the prime stimulus causing the secretion of VEGF. The expression of IL-8 by solid tumors is thought to be primarily due to intrinsic influences, such as constitutive activation of nuclear factor kappa B (NF-κB). However, VEGF expression is responsive to glucose deprivation, suggesting that low concentrations of nutrients other than oxygen may play a role in triggering the pro-angiogenic phenotype. Glucose deprivation causes endoplasmic reticulum (ER) stress and alters gene expression through the unfolded protein response (UPR) signaling pathway. A branch of the UPR, known as the ER overload response (EOR), can cause NF-κB activation. Thus, we hypothesized that treatments that cause ER stress and deprivation of other nutrients, such as amino acids, would trigger the expression of angiogenic cytokines by breast cancer cell lines.ResultsWe found that glutamine deprivation and treatment with a chemical inducer of ER stress (tunicamycin) caused a marked induction of the secretion of both VEGF and IL-8 protein by a human breast adenocarcinoma cell line (TSE cells). Glutamine deprivation, glucose deprivation and several chemical inducers of ER stress increased VEGF and IL-8 mRNA expression in TSE and other breast cancer cell lines cultured under both normoxic and hypoxic conditions, though hypoxia generally diminished the effects of glucose deprivation. Of all amino acids tested, ambient glutamine availability had the largest effect on VEGF and IL-8 mRNA expression. The induction of VEGF mRNA expression, but not IL-8, was sustained and closely corresponded with the upregulated expression of the ER stress-responsive genes glucose-regulated protein 78 (GRP78) and growth arrest and DNA damage inducible gene 153 (GADD153).ConclusionThese results suggest that nutrient deprivation within the solid tumor microenvironment might contribute to the activation of a pro-angiogenic phenotype. The angiogenic switch may act to increase blood supply in response to nutrient deprivation as well as hypoxia.

The acute neurotoxicity of mefloquine may be mediated through a disruption of calcium homeostasis and ER function in vitro

BackgroundThere is no established biochemical basis for the neurotoxicity of mefloquine. We investigated the possibility that the acute in vitro neurotoxicity of mefloquine might be mediated through a disruptive effect of the drug on endoplasmic reticulum (ER) calcium homeostasis.MethodsLaser scanning confocal microscopy was employed to monitor real-time changes in basal intracellular calcium concentrations in embryonic rat neurons in response to mefloquine and thapsigargin (a known inhibitor of the ER calcium pump) in the presence and absence of external calcium. Changes in the transcriptional regulation of known ER stress response genes in neurons by mefloquine were investigated using Affymetrix arrays. The MTT assay was employed to measure the acute neurotoxicity of mefloquine and its antagonisation by thapsigargin.ResultsAt physiologically relevant concentrations mefloquine was found to mobilize neuronal ER calcium stores and antagonize the pharmacological action of thapsigargin, a specific inhibitor of the ER calcium pump. Mefloquine also induced a sustained influx of extra-neuronal calcium via an unknown mechanism. The transcription of key ER proteins including GADD153, PERK, GRP78, PDI, GRP94 and calreticulin were up-regulated by mefloquine, suggesting that the drug induced an ER stress response. These effects appear to be related, in terms of dose effect and kinetics of action, to the acute neurotoxicity of the drug in vitro.ConclusionsMefloquine was found to disrupt neuronal calcium homeostasis and induce an ER stress response at physiologically relevant concentrations, effects that may contribute, at least in part, to the neurotoxicity of the drug in vitro.

Androgen-induced expression of endoplasmic reticulum (ER) stress response genes in prostate cancer cells.

Evaluations of androgen regulated gene (ARG) repertoire provide new insights into the androgen receptor (AR) mediated signaling at the transcriptional level. Definition of ARGs having critical functions in the biology of normal and malignant prostate should aid in identifying new bio-markers and therapeutic targets for prostate cancer (CaP). Using Affymetrix HuGene FL oligonucleotide arrays, temporal expression profiles of ARGs in widely used hormone responsive LNCaP cells, were analysed by hierarchical clustering methods and functional classification. ARGs in response to different androgen concentrations showed temporal co-regulation of genes involved in specific biochemical pathways. This study focuses on our new observations of the coordinated androgen induction of genes (NDRG1, PDIR, HERPUD1, ORP150) involved in the endoplasmic reticulum (ER) stress response pathway. Expression analysis of the two selected ER stress responsive genes, NDRG1 and HERPUD1 in primary CaPs revealed a significantly reduced tumor associated expression. Intriguing linkage of the androgen signaling to ER stress responsive genes, a protective response to protein unfolding or protein damage resulting from cellular stress signals, suggests that androgens may induce such stress signals in CaP cells. Decreased CaP associated expression of two ER stress responsive genes also suggests that possible abrogation of this pathway in prostate tumorigenesis.

New Player in the UPR Game

The unfolded protein response (UPR) is a signaling cascade triggered by conditions that cause the accumulation of improperly folded proteins in the endoplasmic reticulum (ER). The ER stress response results in increased expression of genes containing the ER stress response element in their promoters and include the genes for ER chaperones, such as grp78 and ERp72. Parker et al. identified the molecular identity of the ER stress-response-element binding factor (ERSF) involved in regulating ER stress-responsive gene activity. ERSF is the ubiquitous transcriptional regulator TFII-I (best known for its role in activating TATA-less promoters), and its role in UPR is dependent on a consensus tyrosine phosphorylation site, and its activity is enhanced by interaction with the transcriptional regulator ATF6. UPR triggers an increase in TFII-I itself, suggesting a positive feedback system for up-regulation of ERSF genes. R. Parker, T. Phan, P. Baumeister, B. Roy, V. Cheriyath, A. L. Roy, A. S. Lee, Identification of TFII-I as the endoplasmic reticulum stress response binding element factor ERSF: Its autoregulation by stress and interaction with ATF6. Mol. Cell. Biol. 21 , 3220-3233 (2001). [Abstract] [Full Text]

The Mammalian Odz Gene Family: Homologs of a Drosophila Pair-Rule Gene with Expression Implying Distinct yet Overlapping Developmental Roles

The Drosophila pair-rule gene odz (Tenm) has many patterning roles throughout development. We have identified four mammalian homologs of this gene, including one previously described as a mouse ER stress response gene, Doc4 (Wang et al., 1998). The Odz genes encode large polypeptides displaying the hallmarks of Drosophila Odz: a putative signal peptide; eight EGF-like repeats; and a putative transmembrane domain followed by a 1800-amino-acid stretch without homology to any proteins outside of this family. The mouse genes Odz3 and Doc4/Odz4 exhibit partially overlapping, but clearly distinct, embryonic expression patterns. The major embryonic sites of expression are in the nervous system, including the tectum, optic recess, optic stalk, and developing eye. Additional sites of expression include trachea and mesodermally derived tissues, such as mesentery, and forming limb and bone. Expression of the Odz2 gene is restricted to the nervous system. The expression patterns suggest that each of the genes has its own distinct developmental role. Comparisons of Drosophila and vertebrate Odz expression patterns suggest evolutionarily conserved functions.

Plasmapheresis in the initial treatment of insulin-dependent diabetes mellitus in children.

Several factors indicate that autoimmune mechanisms may play a part in the aetiology of insulin-dependent diabetes mellitus. At the onset of the disease in 10 children (aged 11-16 years) plasmapheresis was performed four times over one to two weeks. Seventeen age-matched children with the same clinical features served as controls. The C-peptide concentrations at onset were the same in the two groups, but after one month the children treated with plasmapheresis had significantly higher values. This difference became even more pronounced after three, nine, and 18 months, both during fasting and at the maximum response to a standardised meal. The study group also had a significantly more stable metabolism, longer partial remission, and no higher insulin requirement. Of the 10 treated children islet-cell cytoplasmic antibodies were present in seven before plasmapheresis and in nine during treatment. The antibodies remained detectable in five and six out of nine patients at one and six months respectively after plasmapheresis. Although the mechanisms are obscure, plasmapheresis performed at the onset of insulin-dependent diabetes mellitus may help to preserve beta-cell function.