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1.  Selective inhibition of unfolded protein response induces apoptosis in pancreatic cancer cells 
Oncotarget  2014;5(13):4881-4894.
Endoplasmic reticulum stress from unfolded proteins is associated with the proliferation of pancreatic tumor cells, making the many regulatory molecules of this pathway appealing targets for therapy. The objective of our study was to assess potential therapeutic efficacy of inhibitors of unfolded protein response (UPR) in pancreatic cancers focusing on IRE1α inhibitors. IRE1α-mediated XBP-1 mRNA splicing encodes a transcription factor that enhances transcription of chaperone proteins in order to reverse UPR. Proliferation assays using a panel of 14 pancreatic cancer cell lines showed a dose- and time-dependent growth inhibition by IRE1α-specific inhibitors (STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, toyocamycin). Growth inhibition was also noted using a clonogenic growth assay in soft agar, as well as a xenograft in vivo model of pancreatic cancer. Cell cycle analysis showed that these IRE1α inhibitors caused growth arrest at either the G1 or G2/M phases (SU8686, MiaPaCa2) and induced apoptosis (Panc0327, Panc0403). Western blot analysis showed cleavage of caspase 3 and PARP, and prominent induction of the apoptotic molecule BIM. In addition, synergistic effects were found between either STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, or toyocamycin and either gemcitabine or bortezomib. Our data suggest that use of an IRE1α inhibitor is a novel therapeutic approach for treatment of pancreatic cancers.
PMCID: PMC4148107  PMID: 24952679
UPR; pancreatic cancer; IRE1α
2.  Inhibition of Endoplasmic Reticulum Stress Improves Mouse Embryo Development 
PLoS ONE  2012;7(7):e40433.
X-box binding protein-1 (XBP-1) is an important regulator of a subset of genes during endoplasmic reticulum (ER) stress. In the current study, we analyzed endogenous XBP-1 expression and localization, with a view to determining the effects of ER stress on the developmental competency of preimplantation embryos in mice. Fluorescence staining revealed that functional XBP-1 is localized on mature oocyte spindles and abundant in the nucleus at the germinal vesicle (GV) stage. However, in preimplantation embryos, XBP-1 was solely detected in the cytoplasm at the one-cell stage. The density of XBP-1 was higher in the nucleus than the cytoplasm at the two-cell, four-cell, eight-cell, morula, and blastocyst stages. Furthermore, RT-PCR analysis confirmed active XBP-1 mRNA splicing at all preimplantation embryo stages, except the one-cell stage. Tunicamycin (TM), an ER stress inducer used as a positive control, promoted an increase in the density of nuclear XBP-1 at the one-cell and two-cell stages. Similarly, culture medium supplemented with 25 mM sorbitol displayed a remarkable increase active XBP-1 expression in the nuclei of 1-cell and 2-cell embryos. Conversely, high concentrations of TM or sorbitol led to reduced nuclear XBP-1 density and significant ER stress-induced apoptosis. Tauroursodeoxycholic acid (TUDCA), a known inhibitor of ER stress, improved the rate of two-cell embryo development to blastocysts by attenuating the expression of active XBP-1 protein in the nucleus at the two-cell stage. Our data collectively suggest that endogenous XBP-1 plays a role in normal preimplantation embryonic development. Moreover, XBP-1 splicing is activated to generate a functional form in mouse preimplantation embryos during culture stress. TUDCA inhibits hyperosmolar-induced ER stress as well as ER stress-induced apoptosis during mouse preimplantation embryo development.
PMCID: PMC3396646  PMID: 22808162
3.  Resveratrol triggers the pro-apoptotic endoplasmic reticulum stress response and represses the pro-survival XBP1 signaling in human multiple myeloma cells 
Experimental Hematology  2011;39(10):999-1006.
Resveratrol, trans-3, 4’, 5,-trihydroxystilbene, suppresses multiple myeloma (MM). The endoplasmic reticulum (ER) stress response component IRE1α/XBP1 axis is essential for MM pathogenesis. We investigated the molecular action of resveratrol on IRE1α/XBP1 axis in human MM cells.
Human MM cell lines ANBL-6, OPM2, and MM.1S were utilized to determine the molecular signaling events following the treatment with resveratrol. The stimulation of IRE1α/XBP1 axis was analyzed by Western blot and reverse transcription polymerase chain reaction. The effect of resveratrol on the transcriptional activity of spliced XBP1 was assessed by luciferase assays. Chromatin immunoprecipitation (ChIP) was performed to determine the effects of resveratrol on the DNA binding activity of XBP1 in MM cells.
Resveratrol activated IRE1α as evidenced by XBP1 mRNA splicing and the phosphorylation of both IRE1α and its downstream kinase JNK in MM cells. These responses were associated with resveratrol-induced cytotoxicity of MM cells. Resveratrol selectively suppressed the transcriptional activity of XBP1s while it stimulated gene expression of the molecules that are regulated by non-IRE1/XBP1 axis of the ER stress response. Luciferase assays indicated that resveratrol suppressed the transcriptional activity of XBP1s through sirtuin 1 (SIRT1), a downstream molecular target of resveratrol. ChIP studies revealed that resveratrol decreased the DNA binding capacity of XBP1 and increased the enrichment of SIRT1 at the XBP1 binding region in the XBP1 promoter.
Resveratrol exerts its chemotherapeutic effect on human MM cells through mechanisms involving the impairment of the pro-survival XBP1 signaling and the activation of pro-apoptotic ER stress response.
PMCID: PMC3261654  PMID: 21723843
Endoplasmic Reticulum Stress Response; IRE1α; Multiple Myeloma; Resveratrol; XBP1
4.  Genetic Interactions Due to Constitutive and Inducible Gene Regulation Mediated by the Unfolded Protein Response in C. elegans 
PLoS Genetics  2005;1(3):e37.
The unfolded protein response (UPR) is an adaptive signaling pathway utilized to sense and alleviate the stress of protein folding in the endoplasmic reticulum (ER). In mammals, the UPR is mediated through three proximal sensors PERK/PEK, IRE1, and ATF6. PERK/PEK is a protein kinase that phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 to inhibit protein synthesis. Activation of IRE1 induces splicing of XBP1 mRNA to produce a potent transcription factor. ATF6 is a transmembrane transcription factor that is activated by cleavage upon ER stress. We show that in Caenorhabditis elegans, deletion of either ire-1 or xbp-1 is synthetically lethal with deletion of either atf-6 or pek-1, both producing a developmental arrest at larval stage 2. Therefore, in C. elegans, atf-6 acts synergistically with pek-1 to complement the developmental requirement for ire-1 and xbp-1. Microarray analysis identified inducible UPR (i-UPR) genes, as well as numerous constitutive UPR (c-UPR) genes that require the ER stress transducers during normal development. Although ire-1 and xbp-1 together regulate transcription of most i-UPR genes, they are each required for expression of nonoverlapping sets of c-UPR genes, suggesting that they have distinct functions. Intriguingly, C. elegans atf-6 regulates few i-UPR genes following ER stress, but is required for the expression of many c-UPR genes, indicating its importance during development and homeostasis. In contrast, pek-1 is required for induction of approximately 23% of i-UPR genes but is dispensable for the c-UPR. As pek-1 and atf-6 mainly act through sets of nonoverlapping targets that are different from ire-1 and xbp-1 targets, at least two coordinated responses are required to alleviate ER stress by distinct mechanisms. Finally, our array study identified the liver-specific transcription factor CREBh as a novel UPR gene conserved during metazoan evolution.
The endoplasmic reticulum (ER) is an intracellular organelle where proteins fold and assemble prior to transport to the cell surface. The ER contains a finely tuned quality control apparatus to ensure that improperly folded proteins are retained in the ER lumen. A variety of physiological demands, environmental perturbations, and pathological conditions compromise protein folding in the ER and lead to the accumulation of unfolded proteins. The unfolded protein response (UPR) is an evolutionarily conserved intracellular adaptive signaling pathway that alleviates protein-folding defects in the ER. The unfolded protein signal is transmitted from the ER to the nucleus by three pathways involving the proteins ATF-6, PEK-1, and IRE-1/XBP-1. However, it is not known how these three pathways coordinate downstream transcriptional activation to mediate either cell adaptation or cell death. The authors have studied the nematode Caenorhabditis elegans to present a comprehensive genetic and gene expression analysis of the three UPR pathways. The findings demonstrate that the UPR regulates the expression of hundreds of genes in the presence, as well as the absence, of ER stress in a manner that is more complex and diverse than previously known.
PMCID: PMC1231716  PMID: 16184190
5.  Suppression of XBP1S Mediates High Glucose-Induced Oxidative Stress and Extracellular Matrix Synthesis in Renal Mesangial Cell and Kidney of Diabetic Rats 
PLoS ONE  2013;8(2):e56124.
Recent evidences suggest that endoplasmic reticulum (ER) stress was involved in multi pathological conditions, including diabetic nephropathy (DN). X-box binding protein 1(XBP1), as a key mediator of ER stress, has been proved having the capability of preventing oxidative stress. In this study, we investigated the effects of spliced XBP1 (XBP1S), the dominant active form of XBP1, on high glucose (HG)-induced reactive oxygen species (ROS) production and extracellular matrix (ECM) synthesis in cultured renal mesangial cells (MCs) and renal cortex of STZ-induced diabetic rats. Real time PCR and Western blot were used to evaluate the mRNA and protein levels respectively. Transfection of recombinant adenovirus vector carrying XBP1S gene (Ad-XBP1S) was used to upregulate XBP1S expression. XBP1S siRNA was used to knockdown XBP1S expression. ROS level was detected by dihydroethidium (DHE) fluorescent probe assay. The results showed that HG treatment significantly reduced XBP1S protein and mRNA level in the cultured MCs while no obvious change was observed in unspliced XBP1 (XBP1U). In the mean time, the ROS production, collagen IV and fibronectin expressions were increased. Diphenylene-chloride iodonium (DPI), a NADPH oxidase inhibtor, prevented HG-induced increases in ROS as well as collagen IV and fibronectin expressions. Transfection of Ad-XBP1S reversed HG-induced ROS production and ECM expressions. Knockdown intrinsic XBP1S expression induced increases in ROS production and ECM expressions. Supplementation of supreoxide reversed the inhibitory effect of Ad-XBP1S transfection on ECM synthesis. P47phox was increased in HG-treated MCs. Ad-XBP1S transfection reversed HG-induced p47phox increase while XBP1S knockdown upregulated p47phox expression. In the renal cortex of diabetic rats, the expression of XBP1S was reduced while p47phox, collagen IV and fibronectin expression were elevated. These results suggested that XBP1S pathway of ER stress was involved in HG-induced oxidative stress and ECM synthesis. A downstream target of XBP1S in regulating ROS formation might be NADPH oxidase.
PMCID: PMC3573021  PMID: 23457509
6.  Reconstitution and characterization of the unconventional splicing of XBP1u mRNA in vitro 
Nucleic Acids Research  2011;39(12):5245-5254.
Upon endoplasmic reticulum (ER) stress, mammalian cells induce the synthesis of a transcriptional activator XBP1s to alleviate the stress. Under unstressed conditions, the messenger RNA (mRNA) for XBP1s exists in the cytosol as an unspliced precursor form, XBP1u mRNA. Thus, its intron must be removed for the synthesis of XBP1s. Upon ER stress, a stress sensor IRE1α cleaves XBP1u mRNA to initiate the unconventional splicing of XBP1u mRNA on the ER membrane. The liberated two exons are ligated to form the mature XBP1s mRNA. However, the mechanism of this splicing is still obscure mainly because the enzyme that joins XBP1s mRNA halves is unknown. Here, we reconstituted the whole splicing reaction of XBP1u mRNA in vitro. Using this assay, we showed that, consistent with the in vivo studies, mammalian cytosol indeed had RNA ligase that could join XBP1s mRNA halves. Further, the cleavage of XBP1u mRNA with IRE1α generated 2′, 3′-cyclic phosphate structure at the cleavage site. Interestingly, this phosphate was incorporated into XBP1s mRNA by the enzyme in the cytosol to ligate the two exons. These studies reveal the utility of the assay system and the unique properties of the mammalian cytosolic enzyme that can promote the splicing of XBP1u mRNA.
PMCID: PMC3130286  PMID: 21398633
7.  The Role of XBP-1 in Tumorigenicity 
Drug news & perspectives  2009;22(5):241-246.
Rapid growth of a tumor can overwhelm the vasculature that supplies it with nutrients and oxygen. Inside such tumors, cells undergo endoplasmic reticulum (EnR) stress but can survive such adverse microenvironments by an adaptive mechanism called the unfolded protein response (UPR). X-box binding protein-1 (XBP-1) is a critical transcriptional activator of the UPR and is responsible for regulating the function of genes in cell survival. An unconventional splicing of the XBP-1(U) mRNA results in two proteins: XBP-1(S) that is often increased in a variety of human cancers, and any translated proteins from the unspliced XBP-1(U) mRNA that acts as a dominant negative of endogenous XBP-1(S) action. In cancer cells, over-expression of XBP-1 can confer drug resistance by preventing drug-induced cell cycle arrest and mitochondrial permeability and apoptosis while down-regulation of XBP-1 increases the sensitivity to killing by hypoxia. XBP-1 is also implicated in cellular de-differentiation, oncovirus infection and the epithelial-to-mesenchymal transition (EMT). Given that XBP-1 mediates a wide range of responses in tumorigenesis, it is logical to focus on XBP-1 as an anti-cancer therapeutic target. Furthermore, combining inhibitors of XBP-1 with other anti-UPR drugs may enhance the activity of some anti-neoplastic therapies.
PMCID: PMC3510657  PMID: 19609461
8.  Coxsackievirus B3 Infection Activates the Unfolded Protein Response and Induces Apoptosis through Downregulation of p58IPK and Activation of CHOP and SREBP1▿  
Journal of Virology  2010;84(17):8446-8459.
Cardiomyocyte apoptosis is a hallmark of coxsackievirus B3 (CVB3)-induced myocarditis. We used cardiomyocytes and HeLa cells to explore the cellular response to CVB3 infection, with a focus on pathways leading to apoptosis. CVB3 infection triggered endoplasmic reticulum (ER) stress and differentially regulated the three arms of the unfolded protein response (UPR) initiated by the proximal ER stress sensors ATF6a (activating transcription factor 6a), IRE1-XBP1 (X box binding protein 1), and PERK (PKR-like ER protein kinase). Upon CVB3 infection, glucose-regulated protein 78 expression was upregulated, and in turn ATF6a and XBP1 were activated via protein cleavage and mRNA splicing, respectively. UPR activity was further confirmed by the enhanced expression of UPR target genes ERdj4 and EDEM1. Surprisingly, another UPR-associated gene, p58IPK, which often is upregulated during infections with other types of viruses, was downregulated at both mRNA and protein levels after CVB3 infection. These findings were observed similarly for uninfected Tet-On HeLa cells induced to overexpress ATF6a or XBP1. In exploring potential connections between the three UPR pathways, we found that the ATF6a-induced downregulation of p58IPK was associated with the activation of PKR (PERK) and the phosphorylation of eIF2α, suggesting that p58IPK, a negative regulator of PERK and PKR, mediates cross-talk between the ATF6a/IRE1-XBP1 and PERK arms. Finally, we found that CVB3 infection eventually produced the induction of the proapoptoic transcription factor CHOP and the activation of SREBP1 and caspase-12. Taken together, these data suggest that CVB3 infection activates UPR pathways and induces ER stress-mediated apoptosis through the suppression of P58IPK and induction/activation of CHOP, SREBP1, and caspase-12.
PMCID: PMC2918999  PMID: 20554776
9.  A Novel Feedback Loop Regulates the Response to Endoplasmic Reticulum Stress via the Cooperation of Cytoplasmic Splicing and mRNA Translation 
Molecular and Cellular Biology  2012;32(5):992-1003.
The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers transcriptional and translational reprogramming. This unfolded protein response (UPR) protects cells during transient stress and can lead to apoptosis during prolonged stress. Two key mediators of the UPR are PKR-like ER kinase (PERK), which phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in decreased protein synthesis, and the α subunit of inositol-requiring enzyme 1 (IRE1α), which initiates cytoplasmic splicing of the mRNA encoding the transcription factor X-box binding protein 1 (XBP1). XBP1 induces transcription of genes involved in protein quality control. This report describes cross talk between these two pathways: phosphorylation of eIF2α was required for maximal induction of spliced XBP1 (XBP1s) protein levels via a mechanism that involved stabilization of XBP1s mRNA. By using mouse embryo fibroblasts deficient in UPR signaling pathways, we demonstrate that stress-induced stabilization of XBP1s mRNA requires cytoplasmic splicing of the mRNA and inhibition of its translation. Because the XBP1s protein promotes transcription of its own gene, the UPR-induced mRNA stabilization is part of a positive feedback loop that induces XBP1s protein accumulation and transcription of target genes during stress. We propose a model in which eIF2α phosphorylation-mediated control of mRNA turnover is a molecular switch that regulates the stress response transcription program and the ER's capacity for protein folding during stress.
PMCID: PMC3295193  PMID: 22215619
10.  Potent and Selective Inhibitors of the Inositol-requiring Enzyme 1 Endoribonuclease* 
The Journal of Biological Chemistry  2011;286(14):12743-12755.
Inositol-requiring enzyme 1 (IRE1) is the most highly conserved signaling node of the unfolded protein response (UPR) and represents a potential therapeutic target for a number of diseases associated with endoplasmic reticulum stress. IRE1 activates the XBP-1 transcription factor by site-specific cleavage of two hairpin loops within its mRNA to facilitate its nonconventional splicing and alternative translation. We screened for inhibitors using a construct containing the unique cytosolic kinase and endoribonuclease domains of human IRE1α (hIRE1α-cyto) and a mini-XBP-1 stem-loop RNA as the substrate. One class compounds was salicylaldehyde analogs from the hydrolyzed product of salicylaldimines in the library. Salicylaldehyde analogs were active in inhibiting the site-specific cleavage of several mini-XBP-1 stem-loop RNAs in a dose-dependent manner. Salicyaldehyde analogs were also active in inhibiting yeast Ire1 but had little activity inhibiting RNase L or the unrelated RNases A and T1. Kinetic analysis revealed that one potent salicylaldehyde analog, 3-ethoxy-5,6-dibromosalicylaldehyde, is a non-competitive inhibitor with respect to the XBP-1 RNA substrate. Surface plasmon resonance studies confirmed this compound bound to IRE1 in a specific, reversible and dose-dependent manner. Salicylaldehydes inhibited XBP-1 splicing induced pharmacologically in human cells. These compounds also blocked transcriptional up-regulation of known XBP-1 targets as well as mRNAs targeted for degradation by IRE1. Finally, the salicylaldehyde analog 3-methoxy-6-bromosalicylaldehyde strongly inhibited XBP-1 splicing in an in vivo model of acute endoplasmic reticulum stress. To our knowledge, salicylaldehyde analogs are the first reported specific IRE1 endoribonuclease inhibitors.
PMCID: PMC3069474  PMID: 21303903
Enzyme Inhibitors; ER Stress; Protein Folding; RNA Processing; Transcription Factors; IRE1; RNase; XBP-1
11.  Functional characterization of the ER stress induced X-box-binding protein-1 (Xbp-1) in the porcine system 
BMC Molecular Biology  2011;12:25.
The unfolded protein response (UPR) is an evolutionary conserved adaptive reaction for increasing cell survival under endoplasmic reticulum (ER) stress conditions. X-box-binding protein-1 (Xbp1) is a key transcription factor of UPR that activates genes involved in protein folding, secretion, and degradation to restore ER function. The UPR induced by ER stress was extensively studied in diseases linked to protein misfolding and aggregations. However, in the porcine system, genes in the UPR pathway were not investigated. In this study, we isolated and characterized the porcine Xbp1 (pXbp1) gene in ER stress using porcine embryonic fibroblast (PEF) cells and porcine organs. ER stress was induced by the treatment of tunicamycin and cell viability was investigated by the MTT assay. For cloning and analyzing the expression pattern of pXbp1, RT-PCR analysis and Western blot were used. Knock-down of pXbp1 was performed by the siRNA-mediated gene silencing.
We found that the pXbp1 mRNA was the subject of the IRE1α-mediated unconventional splicing by ER stress. Knock-down of pXbp1 enhanced ER stress-mediated cell death in PEF cells. In adult organs, pXbp1 mRNA and protein were expressed and the spliced forms were detected.
It was first found that the UPR mechanisms and the function of pXbp1 in the porcine system. These results indicate that pXbp1 plays an important role during the ER stress response like other animal systems and open a new opportunity for examining the UPR pathway in the porcine model system.
PMCID: PMC3112107  PMID: 21605464
12.  Interaction of Rio1 Kinase with Toyocamycin Reveals a Conformational Switch That Controls Oligomeric State and Catalytic Activity 
PLoS ONE  2012;7(5):e37371.
Rio1 kinase is an essential ribosome-processing factor required for proper maturation of 40 S ribosomal subunit. Although its structure is known, several questions regarding its functional remain to be addressed. We report that both Archaeoglobus fulgidus and human Rio1 bind more tightly to an adenosine analog, toyocamycin, than to ATP. Toyocamycin has antibiotic, antiviral and cytotoxic properties, and is known to inhibit ribosome biogenesis, specifically the maturation of 40 S. We determined the X-ray crystal structure of toyocamycin bound to Rio1 at 2.0 Å and demonstrated that toyocamycin binds in the ATP binding pocket of the protein. Despite this, measured steady state kinetics were inconsistent with strict competitive inhibition by toyocamycin. In analyzing this interaction, we discovered that Rio1 is capable of accessing multiple distinct oligomeric states and that toyocamycin may inhibit Rio1 by stabilizing a less catalytically active oligomer. We also present evidence of substrate inhibition by high concentrations of ATP for both archaeal and human Rio1. Oligomeric state studies show both proteins access a higher order oligomeric state in the presence of ATP. The study revealed that autophosphorylation by Rio1 reduces oligomer formation and promotes monomerization, resulting in the most active species. Taken together, these results suggest the activity of Rio1 may be modulated by regulating its oligomerization properties in a conserved mechanism, identifies the first ribosome processing target of toyocamycin and presents the first small molecule inhibitor of Rio1 kinase activity.
PMCID: PMC3358306  PMID: 22629386
13.  Activation of Hepatitis B Virus S Promoter by a Cell Type-Restricted IRE1-Dependent Pathway Induced by Endoplasmic Reticulum Stress 
Molecular and Cellular Biology  2005;25(17):7522-7533.
IRE1-alpha is an integral membrane protein of the endoplasmic reticulum (ER) that is a key sensor in the cellular transcriptional response to stress in the ER. Upon induction of ER stress, IRE1-alpha is activated, resulting in the synthesis of the active form of the transcription factor XBP1 via IRE1-mediated splicing of its mRNA. In this report, we have examined the role of IRE1-alpha and XBP1 in activation of the hepatitis B virus S promoter by ER stress. Cotransfection experiments revealed that overexpression of either IRE1-alpha or XBP1 activated this promoter. Conversely, cotransfected dominant-negative IRE1-alpha or small interfering RNA directed against XBP1 decreased the activation of the S promoter by ER stress, confirming an important role for the IRE1-alpha/XBP1 signaling pathway in activation of the S promoter. However, XBP1 does not bind directly to the S promoter; rather, a novel S promoter-binding complex that does not contain XBP1 is induced in cells undergoing ER stress in an XBP1-dependent manner. This complex, as well as transcriptional activation of the S promoter, is induced by ER stress in hepatocytes but not in fibroblasts, despite the presence of active XBP1 in the latter. Thus, the hepatitis B virus S promoter responds to a novel, cell type-restricted transcriptional pathway downstream of IRE1-alpha and XBP1.
PMCID: PMC1190304  PMID: 16107700
14.  HSP72 Protects Cells from ER Stress-induced Apoptosis via Enhancement of IRE1α-XBP1 Signaling through a Physical Interaction 
PLoS Biology  2010;8(7):e1000410.
The cytosolic chaperone Hsp72 directly modulates stress sensing in response to the accumulation of unfolded proteins in the endoplasmic reticulum and promotes cell survival.
Endoplasmic reticulum (ER) stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR). Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1α, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1α or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1α. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1α in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1α enhances IRE1α/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.
Author Summary
The endoplasmic reticulum (ER) is responsible for production and folding of secreted proteins. When the protein folding machinery cannot keep up with demand, misfolded proteins accumulate, leading to a state of ER stress that contributes to diseases such as cancer, neurodegeneration, diabetes, and myocardial infarct. The unfolded protein response (UPR) is an intracellular signaling network activated in response to ER stress. It initially tries to restore normal ER homeostasis, but if the damage is too severe cell death pathways mediated by cytosolic and mitochondrial proteins are activated. The molecular mechanisms involved in the transition of the UPR from a protective to an apoptotic phase are unclear. IRE1α is an ER membrane protein that acts as a sensor of ER stress. A number of proteins can interact with IRE1α to regulate its function, which includes an RNase activity responsible for inducing the unconventional splicing of the transcript for a downstream signaling protein called XBP-1. Here, we report that Hsp72, a stress-inducible cytosolic molecular chaperone, can bind to and enhance the RNase activity of IRE1α, providing an important molecular link between the heat shock response and the ER stress response. Importantly, increased production of active XBP-1 was necessary for Hsp72 to exert its prosurvival effect under conditions of ER stress. Our results suggest a mechanism whereby Hsp72 overexpression helps cells adapt to long-term ER stress in vivo by enhancing the pro-survival effects of the IRE1α/XBP1 branch of the UPR.
PMCID: PMC2897763  PMID: 20625543
15.  A modified UPR stress sensing system reveals a novel tissue distribution of IRE1/XBP1 activity during normal Drosophila development 
Cell Stress & Chaperones  2012;18(3):307-319.
Eukaryotic cells respond to stress caused by the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum by activating the intracellular signaling pathways referred to as the unfolded protein response (UPR). In metazoans, UPR consists of three parallel branches, each characterized by its stress sensor protein, IRE1, ATF6, and PERK, respectively. In Drosophila, IRE1/XBP1 pathway is considered to function as a major branch of UPR; however, its physiological roles during the normal development and homeostasis remain poorly understood. To visualize IRE1/XBP1 activity in fly tissues under normal physiological conditions, we modified previously reported XBP1 stress sensing systems (Souid et al., Dev Genes Evol 217: 159–167, 2007; Ryoo et al., EMBO J 26: 242-252, 2007), based on the recent reports regarding the unconventional splicing of XBP1/HAC1 mRNA (Aragon et al., Nature 457: 736–740, 2009; Yanagitani et al., Mol Cell 34: 191–200, 2009; Science 331: 586–589, 2011). The improved XBP1 stress sensing system allowed us to detect new IRE1/XBP1 activities in the brain, gut, Malpighian tubules, and trachea of third instar larvae and in the adult male reproductive organ. Specifically, in the larval brain, IRE1/XBP1 activity was detected exclusively in glia, although previous reports have largely focused on IRE1/XBP1 activity in neurons. Unexpected glial IRE1/XBP1 activity may provide us with novel insights into the brain homeostasis regulated by the UPR.
PMCID: PMC3631089  PMID: 23160805
UPR; ER stress; XBP1; Drosophila; Glia
16.  Regulation of unfolded protein response modulator XBP1s by acetylation and deacetylation 
The Biochemical journal  2011;433(1):245-252.
X-box-binding protein 1 (XBP1) is a key modulator of unfolded protein response (UPR), which is involved in a wide range of pathological and physiological processes. The active/spliced form of XBP1 (XBP1s) messenger RNA is generated from unspliced form by IRE1 during UPR. However, post-translational modulation of XBP1s remains largely unknown. Here, we demonstrate that XBP1s is a target of acetylation and deacetylation mediated by p300 and SIRT1 respectively. p300 increases acetylation and protein stability of XBP1s, and enhances the transcriptional activity of XBP1s. SIRT1 deacetylates XBP1s and inhibits the transcriptional activity of XBP1s. Deficiency of SIRT1 enhances the XBP1s-mediated luciferase reporter activity in HEK293 cells and the upregulation of XBP1s target gene expression under ER stress in mouse embryonic fibroblasts (MEFs). Consistent with XBP1s favoring cell survival under ER stress, Sirt1−/− MEFs display a greater resistance to the ER stress-induced apoptotic cell death compared with Sirt1+/+ MEFs. Taken together, these results suggest that acetylation/deacetylation constitutes an important post-translational mechanism in controlling protein levels as well as transcriptional activity of XBP1s. This study provides a novel insight into molecular mechanisms by which SIRT1 regulates UPR signaling.
PMCID: PMC3477812  PMID: 20955178
acetylation; SIRT1; p300; unfolded protein response; X-box-binding protein 1; cell death
17.  Human Leptin Protein Induces Proliferation of A549 Cells via Inhibition of PKR-Like ER Kinase and Activating Transcription Factor-6 Mediated Apoptosis 
Yonsei Medical Journal  2013;54(6):1407-1415.
To investigate the anti-apoptotic mechanism of leptin in non-small cell lung cancer.
Materials and Methods
The influences of leptin on apoptosis were investigated, analyzing the mechanism that triggers growth of A549 cells. The effects of leptin on cell proliferation were examined by XTT analysis. Leptin, C/EBP homologous protein (CHOP), phosphorylated-PKR-like ER kinase (p-Perk), inositol requiring proteins-1, spliced X-box transcription factor-1 (XBP1), cleaved activating transcription factor-6 (ATF6), eukaryotic translation initiation factor-2α, caspase-12 and CHOP protein were detected in four groups by western blot, and endoplasmic reticulum (ER) stress related mRNA were detected by reverse transcription PCR.
The expression of leptin in A549 and leptin transfected cells inhibited cisplatin activated ER stress-associated mRNA transcription and protein activation. Two ER stress unfolded protein response pathways, PERK and ATF6, were involved, and XBP1 and tumor necrosis factor receptor-associated factor 2 (TRAF2) were increased significantly when treated with cisplatin in A549-siRNA against leptin cells. Furthermore, CHOP expression was inhibited upon leptin expression in A549, LPT-PeP and LPT-EX cells.
Leptin serves as an important factor that promotes the growth of A549 cells through blocking ER stress-mediated pathways. This blocking is triggered by p-Perk and ATF6 via inhibition of CHOP expression.
PMCID: PMC3809871  PMID: 24142645
Apoptosis; ER stress; cell growth; leptin; TRAF2; XPB1
18.  X Box-Binding Protein 1 Regulates Angiogenesis in Human Pancreatic Adenocarcinomas12 
Translational Oncology  2009;2(1):31-38.
PURPOSE: Tumors encounter endoplasmic reticulum stress during tumor growth and activate an adaptive pathway known as the unfolded protein response (UPR). Because this pathway is induced by the tumor microenvironment, it is a promising target for cancer therapy. We have previously demonstrated that X-box binding protein 1 (XBP-1), a key regulator of the UPR, was required for survival under hypoxia and critical for tumor growth in tumor xenografts. In this study, we investigated the role of XBP-1 in regulating tumor angiogenesis. METHODS: We used an intradermal angiogenesis model to quantify the effect of XBP-1 on angiogenesis. We also used a human tumor xenograft model to assay for tumor growth delay. We determined vascular endothelial growth factor (VEGF) expression by quantitative polymerase chain reaction and ELISA. Finally, we stained human pancreatic adenocarcinoma specimens for XBP-1 expression and correlated the expression pattern of XBP-1 with CD31 (endothelial cell marker) expression. RESULTS: We demonstrated that XBP-1 is essential for angiogenesis during early tumor growth. Inhibiting XBP-1 expression by short-hairpin RNA sequence specific for XBP-1 reduced blood vessel formation in tumors from mouse embryonic fibroblast cells and human fibrosarcoma tumor cells (HT1080). Expressing a dominant-negative form of IRE1α also reduced blood vessel formation in tumors. Moreover, expression of spliced XBP-1 (XBP-1s) restored angiogenesis in IRE1α dominant-negative expressing cells. We further demonstrated that XBP-1-mediated angiogenesis does not depend on VEGF. CONCLUSIONS: We propose that the IRE1α-XBP-1 branch of the UPR modulates a complex proangiogenic, VEGF-independent response that depends on signals received from the tumor microenvironment.
PMCID: PMC2647700  PMID: 19252749
19.  Sulforaphane synergistically enhances the cytotoxicity of arsenic trioxide in multiple myeloma cells via stress-mediated pathways 
Oncology Reports  2012;28(5):1851-1858.
Persistent paraprotein production in plasma cells necessitates a highly developed rough endoplasmic reticulum (ER) that is unusually susceptible to perturbations in protein synthesis. This biology is believed to account for the exquisite sensitivity of multiple myeloma (MM) to the proteasomal inhibitor bortezomib (BTZ). Despite remarkable response rates to BTZ in MM, BTZ carries the potential for serious side-effects and development of resistance. We, therefore, sought to identify therapeutic combinations that effectively disrupt proteostasis in order to provide new potential treatments for MM. We found that sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, inhibits TNFα-induced Iκβ proteasomal degradation in a manner similar to BTZ. Like BTZ, sulforaphane synergistically enhances the cytotoxicity of arsenic trioxide (ATO), an agent with clinical activity in MM. ATO and sulforaphane co-treatment augmented apoptotic induction as demonstrated by cleavage of caspase-3, -4 and PARP. The enhanced apoptotic response was dependent upon production of reactive oxygen species (ROS) as demonstrated by glutathione depletion and partial inhibition of the apoptotic cascade after pretreatment with the radical scavenger N-acetyl-cysteine (NAC). Combination treatment resulted in enhanced ER stress signaling and activation of the unfolded protein response (UPR), indicative of perturbation of proteostasis. Specifically, combination treatment caused elevated expression of the molecular chaperone HSP90 (heat shock protein 90) along with increased PERK (protein kinase RNA-like endoplasmic reticulum kinase) and eIF2α phosphorylation and XBP1 (X-box binding protein 1) splicing, key indicators of UPR activation. Moreover, increased splicing of XBP1 was apparent upon combination treatment compared to treatment with either agent alone. Sulforaphane in combination with ATO effectively disrupts protein homeostasis through ROS generation and induction of ER stress to culminate in inhibition of protein secretion and apoptotic induction in MM. Our results suggest that sulforaphane deserves further investigation in combination with ATO in the treatment of MM.
PMCID: PMC3981004  PMID: 22922937
sulforaphane; arsenic trioxide; unfolded protein response; Iκβ; endoplasmic reticulum stress; reactive oxygen species
20.  Endoplasmic Reticulum Stress Regulator XBP-1 Contributes to Effector CD8+ T Cell Differentiation during Acute Infection1 
The transcription factor X-box-binding protein-1 (XBP-1) plays an essential role in activating the unfolded protein response in the endoplasmic reticulum (ER). Transcribed XBP-1 mRNA is converted to its active form by unconventional cytoplasmic splicing mediated by inositol-requiring enzyme-1 (IRE-1) upon ER stress. We report activation of the IRE-1/XBP-1 pathway in effector CD8+ T cells during the response to acute infection. Transcription of unspliced XBP-1 mRNA is up-regulated by IL-2 signals, while its splicing is induced after TCR ligation. Splicing of XBP-1 mRNA was evident during the expansion of Ag-specific CD8+ T cells in response to viral or bacterial infection. An XBP-1 splicing reporter revealed that splicing activity was enriched in terminal effector cells expressing high levels of killer cell lectin-like receptor G1 (KLRG1). Overexpression of the spliced form of XBP-1 in CD8+ T cells enhanced KLRG1 expression during infection, whereas XBP-1−/− CD8+ T cells or cells expressing a dominant-negative form of XBP-1 showed a decreased proportion of KLRG1high effector cells. These results suggest that, in the response to pathogen, activation of ER stress sensors and XBP-1 splicing contribute to the differentiation of end-stage effector CD8+ T cells.
PMCID: PMC2776092  PMID: 18832700
21.  Role of GRP78/BiP Degradation and ER Stress in Deoxynivalenol-Induced Interleukin-6 Upregulation in the Macrophage 
Toxicological Sciences  2009;109(2):247-255.
The trichothecene mycotoxin deoxynivalenol (DON) induces systemic expression of the interleukin-6 (IL-6) and other proinflammatory cytokines in the mouse. The purpose of this study was to test the hypothesis that DON triggers an endoplasmic reticulum (ER) stress response in murine macrophages capable of driving IL-6 gene expression. DON at concentrations up 5000 ng/ml. was not cytotoxic to peritoneal cells. However, DON markedly decreased protein levels but not the mRNA levels of glucose-regulated protein (GRP) 78 (BiP), a chaperone known to mediate ER stress. Inhibitor studies suggested that DON-induced GRP78 degradation was cathepsin and calpain dependent but was proteosome-independent. RNAi-mediated knockdown of GRP78 resulted in increased IL-6 gene expression indicating a potential downregulatory role for this chaperone. GRP78 is critical to the regulation of the two transcription factors, X-box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6), which bind to cAMP-response element (CRE) and drive expression of CRE-dependent genes such as IL-6. DON exposure was found to increase IRE1α protein, its modified products spliced XBP1 mRNA and XBP1 protein as well as ATF6. Knockdown of ATF6 but not XBP1 partially inhibited DON-induced IL-6 expression in the macrophages. Three other trichothecenes (satratoxin G, roridin, T-2 toxin) and the ribosome inhibitory protein ricin were also found to induce GRP78 degradation suggesting that other translation inhibitors might evoke ER stress. Taken together, these data suggest that in the macrophage DON induces GRP78 degradation and evokes an ER stress response that could contribute, in part, to DON-induced IL-6 gene expression.
PMCID: PMC2721661  PMID: 19336499
deoxynivalenol (DON); interleukin-6; ER stress, translation inhibition
22.  XBP-1, a Novel Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Binding Protein, Activates HTLV-1 Basal and Tax-Activated Transcription▿  
Journal of Virology  2008;82(9):4343-4353.
X-box binding protein 1 (XBP-1), a basic leucine zipper transcription factor, plays a key role in the cellular unfolded protein response (UPR). There are two XBP-1 isoforms in cells, spliced XBP-1S and unspliced XBP-1U. XBP-1U has been shown to bind to the 21-bp Tax-responsive element of the human T-lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR) in vitro and transactivate HTLV-1 transcription. Here we identify XBP-1S as a transcription activator of HTLV-1. Compared to XBP-1U, XBP-1S demonstrates stronger activating effects on both basal and Tax-activated HTLV-1 transcription in cells. Our results show that both XBP-1S and XBP-1U interact with Tax and bind to the HTLV-1 LTR in vivo. In addition, elevated mRNA levels of the gene for XBP-1 and several UPR genes were detected in the HTLV-1-infected C10/MJ and MT2 T-cell lines, suggesting that HTLV-1 infection may trigger the UPR in host cells. We also identify Tax as a positive regulator of the expression of the gene for XBP-1. Activation of the UPR by tunicamycin showed no effect on the HTLV-1 LTR, suggesting that HTLV-1 transcription is specifically regulated by XBP-1. Collectively, our study demonstrates a novel host-virus interaction between a cellular factor XBP-1 and transcriptional regulation of HTLV-1.
PMCID: PMC2293026  PMID: 18287238
23.  Human Melanoma Cells under Endoplasmic Reticulum Stress Acquire Resistance to Microtubule-Targeting Drugs through XBP-1-Mediated Activation of Akt1 
Neoplasia (New York, N.Y.)  2009;11(5):436-447.
Past studies have shown that melanoma cells have largely adapted to endoplasmic reticulum (ER) stress. In this study, we report that melanoma cells under ER stress are more resistant to apoptosis induced by the microtubule-targeting chemotherapeutic drugs, docetaxel and vincristine, and this is, at least in part, due to activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway mediated by the X-box-binding protein 1 (XBP-1) axis of the unfolded protein response. Treatment with the ER stress-inducer tunicamycin (TM) or thapsigargin before the addition of docetaxel or vincristine reduced the levels of apoptosis induced by the drugs. This was associated with inhibition of mitochondrial release of apoptogenic proteins and activation of Bax and Bak. Induction of ER stress resulted in the rapid activation of the PI3K/Akt pathway that seemed to be important in antagonizing docetaxel and vincristine, in that inhibition of Akt blocked the effect of pretreatment with TM on apoptosis induced by the drugs. Neither docetaxel nor vincristine triggered ER stress in melanoma cells, but the basal activity of XBP-1 signaling seemed to play a role in the protection against the drugs because small interfering RNA knockdown of XBP-1 enhanced docetaxel- and vincristine-induced apoptosis. In addition, inhibition of XBP-1 decreased the constitutive levels of activation of Akt and blocked the activation of Akt induced by TM. Taken together, these results identify activation of the PI3K/Akt pathway by XBP-1-mediated signaling of the unfolded protein response as a resistance mechanism against docetaxel and vincristine in melanoma cells under ER stress.
PMCID: PMC2671856  PMID: 19412428
24.  Temporal regulation of Cat-1 (cationic amino acid transporter-1) gene transcription during endoplasmic reticulum stress 
The Biochemical journal  2010;429(1):215-224.
Expression of the Cat-1 gene (cationic amino acid transporter-1) is induced in proliferating cells and in response to a variety of stress conditions. The expression of the gene is mediated via a TATA-less promoter. In the present study we show that an Sp1 (specificity protein 1)-binding site within a GC-rich region of the Cat-1 gene controls its basal expression and is important for induction of the gene during the UPR (unfolded protein response). We have shown previously that induction of Cat-1 gene expression during the UPR requires phosphorylation of the translation initiation factor eIF2α (eukaryotic initiation factor 2α) by PERK (protein-kinase-receptor-like endoplasmic reticulum kinase), one of the signalling pathways activated during the UPR. This leads to increased translation of the transcription factor ATF4 (activating transcription factor 4). We also show that a second signalling pathway is required for sustained transcriptional induction of the Cat-1 gene during the UPR, namely activation of IRE1 (inositol-requiring enzyme 1) leading to alternative splicing of the mRNA for the transcription factor XBP1 (X-box-binding protein 1). The resulting XBP1s (spliced XBP1) can bind to an ERSE (endoplasmic-reticulum-stress-response-element), ERSE-II-like, that was identified within the Cat-1 promoter. Surprisingly, eIF2α phosphorylation is required for accumulation of XBP1s. We propose that the signalling via phosphorylated eIF2α is required for maximum induction of Cat-1 transcription during the UPR by inducing the accumulation of both ATF4 and XBP1s.
PMCID: PMC3684439  PMID: 20408811
activating transcription factor 4 (ATF4); cationic amino acid transporter-1 (Cat-1); endoplasmic reticulum stress; specificity protein 1 (Sp1); unfolded protein response; X-box-binding protein 1 (XBP1)
25.  Human Cytomegalovirus Infection Activates and Regulates the Unfolded Protein Response 
Journal of Virology  2005;79(11):6890-6899.
Viral infection causes stress to the endoplasmic reticulum. The response to endoplasmic reticulum stress, known as the unfolded protein response (UPR), is designed to eliminate misfolded proteins and allow the cell to recover by attenuating translation and upregulating the expression of chaperones, degradation factors, and factors that regulate the cell's metabolic and redox environment. Some consequences of the UPR (e.g., expression of chaperones and regulation of the metabolism and redox environment) may be advantageous to the viral infection; however, translational attenuation would not. Thus, viruses may induce mechanisms which modulate the UPR, maintaining beneficial aspects and suppressing deleterious aspects. We demonstrate that human cytomegalovirus (HCMV) infection induces the UPR but specifically regulates the three branches of UPR signaling, PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE-1), to favor viral replication. HCMV infection activated the eIF2α kinase PERK; however, the amount of phosphorylated eIF2α was limited and translation attenuation did not occur. Interestingly, translation of select mRNAs, which is dependent on eIF2α phosphorylation, did occur, including the transcription factor ATF4, which activates genes which may benefit the infection. The endoplasmic reticulum stress-induced activation of the transcription factor ATF6 was suppressed in HCMV-infected cells; however, specific chaperone genes, normally activated by ATF6, were activated by a virus-induced, ATF6-independent mechanism. Lastly, HCMV infection activated the IRE-1 pathway, as indicated by splicing of Xbp-1 mRNA. However, transcriptional activation of the XBP-1 target gene EDEM (ER degradation-enhancing α-mannosidase-like protein, a protein degradation factor) was inhibited. These results suggest that, although HCMV infection induces the unfolded protein response, it modifies the outcome to benefit viral replication.
PMCID: PMC1112127  PMID: 15890928

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