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1.  Activation of Unfolded Protein Response in Transgenic Mouse Lenses 
Transgene overexpression in mouse lens can activate unfolded protein response (UPR) in the lens fiber cells. Activation of UPR may contribute to defective and degenerative changes in the fiber cells. This study implies the levels of UPR activation should be assessed when using transgenic techniques to study gene function in vivo.
Overloading of unfolded or misfolded proteins in the endoplasmic reticulum (ER) can cause ER stress and activate the unfolded protein response (UPR) in the cell. The authors tested whether transgene overexpression in the mouse lens would activate the UPR.
Transgenic mice expressing proteins that either enter the ER secretory pathway or are synthesized in cytosol were selected. Activation of the UPR was assessed by determining the expression levels of the ER chaperone protein BiP, the spliced form of X-box binding protein-1 (Xbp-1) mRNA, and the transcription factor CHOP. Changes in the ubiquitin-proteasome system in the mouse lens were detected by ubiquitin immunofluorescence.
BiP expression was upregulated in the fiber cells of transgenic mouse lenses expressing platelet-derived growth factor-A (PDGF-A), dominant-negative fibroblast growth factor receptor (DN-FGFR), or DN-Sprouty2 (DN-Spy2). BiP upregulation occurred around embryonic day 16.5, primarily in the fiber cells adjacent to the organelle free zone. Fiber cell differentiation was disrupted in the PDGF-A and DN-Spry2 lenses, whereas the fiber cells were degenerating in the DN-FGFR lens. High levels of UPR activation and ubiquitin-labeled protein aggregates were found in the DN-FGFR lens, indicating inefficient disposal of unfolded/misfolded proteins in the fiber cells.
This study implies that overexpression of some transgenes in the lens can induce ER or overall cell stress in fiber cells, resulting in the activation of UPR signaling pathways. Therefore, investigators should assess the levels of UPR activation when they analyze the downstream effects of transgene expression in the lens.
PMCID: PMC3080171  PMID: 21310900
2.  XBP-1 Regulates a Subset of Endoplasmic Reticulum Resident Chaperone Genes in the Unfolded Protein Response 
Molecular and Cellular Biology  2003;23(21):7448-7459.
The mammalian unfolded protein response (UPR) protects the cell against the stress of misfolded proteins in the endoplasmic reticulum (ER). We have investigated here the contribution of the UPR transcription factors XBP-1, ATF6α, and ATF6β to UPR target gene expression. Gene profiling of cell lines lacking these factors yielded several XBP-1-dependent UPR target genes, all of which appear to act in the ER. These included the DnaJ/Hsp40-like genes, p58IPK, ERdj4, and HEDJ, as well as EDEM, protein disulfide isomerase-P5, and ribosome-associated membrane protein 4 (RAMP4), whereas expression of BiP was only modestly dependent on XBP-1. Surprisingly, given previous reports that enforced expression of ATF6α induced a subset of UPR target genes, cells deficient in ATF6α, ATF6β, or both had minimal defects in upregulating UPR target genes by gene profiling analysis, suggesting the presence of compensatory mechanism(s) for ATF6 in the UPR. Since cells lacking both XBP-1 and ATF6α had significantly impaired induction of select UPR target genes and ERSE reporter activation, XBP-1 and ATF6α may serve partially redundant functions. No UPR target genes that required ATF6β were identified, nor, in contrast to XBP-1 and ATF6α, did the activity of the UPRE or ERSE promoters require ATF6β, suggesting a minor role for it during the UPR. Collectively, these results suggest that the IRE1/XBP-1 pathway is required for efficient protein folding, maturation, and degradation in the ER and imply the existence of subsets of UPR target genes as defined by their dependence on XBP-1. Further, our observations suggest the existence of additional, as-yet-unknown, key regulators of the UPR.
PMCID: PMC207643  PMID: 14559994
3.  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
4.  Activation of the unfolded protein response by a cataract-associated αA-crystallin mutation 
αA-crystallin is a lens chaperone that plays an essential role in the transparency and refractive properties of the lens. Mutations in αA-crystallin have been associated with the development of hereditary cataracts. The R49C mutation of αA-crystallin (αA-R49C) was identified in a four-generation Caucasian family with hereditary cataracts. The αA-R49C protein forms larger-than-normal oligomers in the lens and has decreased solubility. This aberrant αA-R49C oligomerization suggests that protein folding is altered. However, whether activation of the unfolded protein response (UPR) occurs during crystallin mutation-induced cataract formation and whether the UPR causes cell death under these conditions is unclear. We investigated UPR activation in an in vivo mouse model of αA-R49C using immunoblot analysis of lens extracts. We found that expression of the endoplasmic reticulum (ER) chaperone, BiP, was 5-fold higher in homozygous αA-R49C lenses than in wild type lenses. Analysis of proteins typically expressed during the UPR revealed that ATF-4 and CHOP levels were also higher in homozygous lenses than in wild type lenses, while the opposite was true of ATF-6 and XBP-1. Taken together, these findings show that mutation of αA-crystallin induces activation of the UPR during cataract formation. They also suggest that the UPR is an important mediator of cell death observed in homozygous αA-R49C lenses.
PMCID: PMC2956780  PMID: 20833134
Crystallin; cataract; mutation; lens; unfolded protein response
5.  The Unfolded Protein Response is activated in Connexin 50 mutant mouse lenses 
Experimental eye research  2012;102:28-37.
The unfolded protein response is a set of cell signaling pathways recently recognized to be activated in the lens during both normal development and endoplasmic reticulum stress induced by either unfolded proteins or oxidative damage. While mutations in the gene for connexin 50 are known to cause autosomal dominant cataracts, it has not been previously reported whether mutant connexins can activate the unfolded protein response in the lens. Mice homozygous for the S50P or G22R mutation of connexin 50 have reduced amounts of connexin 50 protein at the cell membrane, with some intracellular staining consistent with retention in the endoplasmic reticulum. Connexin 50 mutants have elevated levels of BiP expression in both lens epithelial and fiber cells from E15.5 with the most robust elevation detected in newborns. While this elevation decreases in magnitude postnatally, BiP expression is still abnormally high in adults, particularly in the perinuclear endoplasmic reticulum of cell nuclei that are inappropriately retained in adult homozygous mutant lenses. Xbp1 splicing was elevated in lenses from both connexin mutants studied, while Atf4 and Atf6 levels were not majorly affected. Overall, these data suggest that UPR may be a contributing factor to the phenotype of connexin 50 mutant lenses even though the relatively modest extent of the response suggests that it is unlikely to be a major driver of the pathology.
PMCID: PMC3461258  PMID: 22713599
UPR; BiP; Cataract; Connexin 50
6.  Induction of unfolded protein response during neuronal induction of rat bone marrow stromal cells and mouse embryonic stem cells 
Experimental & Molecular Medicine  2009;41(6):440-452.
When we treated rat bone marrow stromal cells (rBMSCs) with neuronal differentiation induction media, typical unfolded protein response (UPR) was observed. BIP/GRP78 protein expression was time-dependently increased, and three branches of UPR were all activated. ATF6 increased the transcription of XBP1 which was successfully spliced by IRE1. PERK was phosphorylated and it was followed by eIF2α phosphorylation. Transcription of two downstream targets of eIF2α, ATF4 and CHOP/GADD153, were transiently up-regulated with the peak level at 24 h. Immunocytochemical study showed clear coexpression of BIP and ATF4 with NeuN and Map2, respectively. UPR was also observed during the neuronal differentiation of mouse embryonic stem (mES) cells. Finally, chemical endoplasmic reticulum (ER) stress inducers, thapsigargin, tunicamycin, and brefeldin A, dose-dependently increased both mRNA and protein expressions of NF-L, and, its expression was specific to BIP-positive rBMSCs. Our results showing the induction of UPR during neuronal differentiations of rBMSCs and mES cells as well as NF-L expression by ER stress inducers strongly suggest the potential role of UPR in neuronal differentiation.
PMCID: PMC2705864  PMID: 19322020
bone marrow; cell differentiation; embryonic stem cells; endoplasmic reticulum; neuron; stem cells; stress, physiological; stromal cells
7.  Gene regulatory network of unfolded protein response genes in endoplasmic reticulum stress 
Cell Stress & Chaperones  2012;18(1):11-23.
In the endoplasmic reticulum (ER), secretory and membrane proteins are properly folded and modified, and the failure of these processes leads to ER stress. At the same time, unfolded protein response (UPR) genes are activated to maintain homeostasis. Despite the thorough characterization of the individual gene regulation of UPR genes to date, further investigation of the mutual regulation among UPR genes is required to understand the complex mechanism underlying the ER stress response. In this study, we aimed to reveal a gene regulatory network formed by UPR genes, including immunoglobulin heavy chain-binding protein (BiP), X-box binding protein 1 (XBP1), C/EBP [CCAAT/enhancer-binding protein]-homologous protein (CHOP), PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring 1 (IRE1), activating transcription factor 6 (ATF6), and ATF4. For this purpose, we focused on promoter-luciferase reporters for BiP, XBP1, and CHOP genes, which bear an ER stress response element (ERSE), and p5 × ATF6-GL3, which bears an unfolded protein response element (UPRE). We demonstrated that the luciferase activities of the BiP and CHOP promoters were upregulated by all the UPR genes, whereas those of the XBP1 promoter and p5 × ATF6-GL3 were upregulated by all the UPR genes except for BiP, CHOP, and ATF4 in HeLa cells. Therefore, an ERSE- and UPRE-centered gene regulatory network of UPR genes could be responsible for the robustness of the ER stress response. Finally, we revealed that BiP protein was degraded when cells were treated with DNA-damaging reagents, such as etoposide and doxorubicin; this finding suggests that the expression level of BiP is tightly regulated at the post-translational level, rather than at the transcriptional level, in the presence of DNA damage.
PMCID: PMC3508129  PMID: 22802018
ER stress; Unfolded protein response; ERSE; UPRE; BiP; Gene regulation
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.  Plasma cell differentiation initiates a limited ER stress response by specifically suppressing the PERK-dependent branch of the unfolded protein response 
Cell Stress & Chaperones  2009;15(3):281-293.
In response to terminal differentiation signals that enable B cells to produce vast quantities of antibodies, a dramatic expansion of the secretory pathway and a corresponding increase in the molecular chaperones and folding enzymes that aid and monitor immunoglobulin synthesis occurs. Recent studies reveal that the unfolded protein response (UPR), which is normally activated by endoplasmic reticulum (ER) stress, plays a critical role in this process. Although B cells activate all three branches of the UPR in response to pharmacological inducers of the pathway, plasma cell differentiation elicits only a partial UPR in which components of the PKR-like ER kinase (PERK) branch are not expressed. This prompted us to further characterize UPR activation during plasma cell differentiation. We found that in response to lipopolysaccharides (LPS)-induced differentiation of the I.29 μ+ B cell line, Ire1 was activated early, which led to splicing of XBP-1. PERK was partially phosphorylated with similar kinetics, but this was not sufficient to activate its downstream target eIF-2α, which initiates translation arrest, or to induce other targets like CHOP or GADD34. Both of these events preceded increased Ig synthesis, arguing this is not the signal for activating these two transducers. Targets of activating transcription factor 6 (ATF6) were up-regulated considerably later, arguing that the ATF6 branch is activated by a distinct signal. Pretreatment with LPS inhibited activation of the PERK branch by pharmacological inducers of the UPR, suggesting that differentiation-induced signals specifically silence this branch. This unique ability to differentially regulate various branches of the UPR allows B cells to accomplish distinct outcomes via the same UPR machinery.
PMCID: PMC2866998  PMID: 19898960
Plasma cell; Differentiation; B cell; UPR; ER stress
10.  Transcriptional induction of the human asparagine synthetase gene during the unfolded protein response does not require the ATF6 and IRE1/XBP1 arms of the pathway 
The Biochemical journal  2009;417(3):695-703.
The UPR (unfolded protein response) pathway is comprised of three signalling cascades mediated by the ER (endoplasmic reticulum) stress sensor proteins PERK [PKR (double-stranded RNA-activated protein kinase)-like ER kinase], IRE1 (inositol-requiring kinase 1) and ATF6 (activating transcription factor 6). The present study shows that ASNS (asparagine synthetase) transcription activity was up-regulated in HepG2 cells treated with the UPR activators thapsigargin and tunicamycin. ChIP (chromatin immunoprecipitation) analysis demonstrated that during ER stress, ATF4, ATF3 and C/EBPβ (CCAAT/enhancer-binding protein β) bind to the ASNS proximal promoter region that includes the genomic sequences NSRE (nutrient-sensing response element)-1 and NSRE-2, previously implicated by mutagenesis in UPR activation. Consistent with increased ASNS transcription, ChIP analysis also demonstrated that UPR signalling resulted in enhanced recruitment of general transcription factors, including RNA Pol II (polymerase II), to the ASNS promoter. The ASNS gene is also activated by the AAR (amino acid response) pathway following amino acid deprivation of tissue or cells. Immunoblot analysis of HepG2 cells demonstrated that simultaneous activation of the AAR and UPR pathways did not further increase the ASNS or ATF4 protein abundance when compared to triggering either pathway alone. In addition, siRNA (small interfering RNA)-mediated knockdown of XBP1 (X-box binding protein 1), ATF6α or ATF6β expression did not affect ASNS transcription, whereas siRNA against ATF4 suppressed ASNS transcription during UPR activation. Collectively, these results indicate that the PERK/p-eIF2α (phosphorylated eukaryotic initiation factor 2α)/ATF4 signalling cascade is the only arm of the UPR that is responsible for ASNS transcriptional induction during ER stress. Consequently, the ASNS NSRE-1 and NSRE-2 elements, in addition to ERSE (ER stress response element)-I, ERSE-II and the mUPRE (mammalian UPR element), function as mammalian UPR responsive sequences.
PMCID: PMC3595601  PMID: 18840095
activating transcription factor 3 (ATF3); activating transcription factor 4 (ATF4); asparagine synthetase (ASNS); CCAAT/enhancer-binding protein (C/EBP); endoplasmic reticulum stress; nutrient sensing; unfolded protein response (UPR)
11.  The Unfolded Protein Response of B-Lymphocytes: PERK-independent Development of Antibody-Secreting Cells 
Molecular immunology  2007;45(4):1035-1043.
When B-lymphocytes differentiate into plasma cells, immunoglobulin (Ig) heavy and light chain synthesis escalates and the entire secretory apparatus expands to support high-rate antibody secretion. These same events occur when murine B-cells are stimulated with lipopolysaccharide (LPS), providing an in vitro model in which to investigate the differentiation process. The unfolded protein response (UPR), a multi-pathway signaling response emanating from the endoplasmic reticulum (ER) membrane, allows cells to adapt to increasing demands on the protein folding capacity of the ER. As such, the UPR plays a pivotal role in the differentiation of antibody-secreting cells. Three specific stress sensors, IRE1, PERK/PEK and ATF6, are central to the recognition of ER stress and induction of the UPR. IRE1 triggers splicing of Xbp-1 mRNA, yielding a transcriptional activator of the UPR termed XBP-1(S), and activation of the IRE1/XBP-1 pathway has been reported to be required for expansion of the ER and antibody secretion. Here, we provide evidence that PERK is not activated in LPS-stimulated splenic B-cells, whereas XBP-1(S) and the UPR transcriptional activator ATF6 are both induced. We further demonstrate that Perk-/- B-cells develop and are fully competent for induction of Ig synthesis and antibody secretion when stimulated with LPS. These data provide clear evidence for differential activation and utilization of distinct UPR components as activated B-lymphocytes increase Ig synthesis and differentiate into specialized secretory cells.
PMCID: PMC2677759  PMID: 17822768
B-lymphocytes; plasma cells; antibody secretion; unfolded protein response; PERK
12.  Murine Cytomegalovirus Targets Transcription Factor ATF4 To Exploit the Unfolded-Protein Response 
Journal of Virology  2012;86(12):6712-6723.
The unfolded-protein response (UPR), activated by sensor molecules PERK, ATF6, and IRE1 to resolve endoplasmic reticulum (ER) stress, has emerged as a key target for host cells and viruses to control the infection outcomes. The UPR regulates ER protein folding, controls cell fate upon ER stress, and plays an important role in innate immunity. We and others have shown that human cytomegalovirus (HCMV) modulates the UPR. We show here that murine CMV (MCMV), the widely used CMV model for small animal infection, regulated the UPR in a manner similar to that of HCMV. This modulatory ability was triggered by virion entry and enhanced by viral immediate-early and early gene expression. Thus, while vulnerable at early times, MCMV became resistant to exogenous ER stress at late times of infection. MCMV activated the PERK-ATF4 pathway but only induced a subset of representative ATF4 targets at levels somewhat lower than those by the ER stress inducer tunicamycin. Moreover, MCMV induced ER chaperone Bip but actively blocked IRE1-mediated Xbp1(s) protein accumulation. ATF4 depletion severely attenuated viral growth at a low multiplicity of infection by modestly reducing viral DNA synthesis and more pronouncedly inhibiting late gene transcription. Collectively, we show that the UPR is a conserved target of CMVs and identify ATF4, a key UPR component, as a factor critical for MCMV infection. This work sets the stage for using the MCMV model to explore the role of this stress response in CMV biology, particularly during infection of the host, which is difficult to study in HCMV.
PMCID: PMC3393534  PMID: 22496230
13.  Comparative transcriptome analysis of epithelial and fiber cells in newborn mouse lenses with RNA sequencing 
Molecular Vision  2014;20:1491-1517.
The ocular lens contains only two cell types: epithelial cells and fiber cells. The epithelial cells lining the anterior hemisphere have the capacity to continuously proliferate and differentiate into lens fiber cells that make up the large proportion of the lens mass. To understand the transcriptional changes that take place during the differentiation process, high-throughput RNA-Seq of newborn mouse lens epithelial cells and lens fiber cells was conducted to comprehensively compare the transcriptomes of these two cell types.
RNA from three biologic replicate samples of epithelial and fiber cells from newborn FVB/N mouse lenses was isolated and sequenced to yield more than 24 million reads per sample. Sequence reads that passed quality filtering were mapped to the reference genome using Genomic Short-read Nucleotide Alignment Program (GSNAP). Transcript abundance and differential gene expression were estimated using the Cufflinks and DESeq packages, respectively. Gene Ontology enrichment was analyzed using GOseq. RNA-Seq results were compared with previously published microarray data. The differential expression of several biologically important genes was confirmed using reverse transcription (RT)-quantitative PCR (qPCR).
Here, we present the first application of RNA-Seq to understand the transcriptional changes underlying the differentiation of epithelial cells into fiber cells in the newborn mouse lens. In total, 6,022 protein-coding genes exhibited differential expression between lens epithelial cells and lens fiber cells. To our knowledge, this is the first study identifying the expression of 254 long intergenic non-coding RNAs (lincRNAs) in the lens, of which 86 lincRNAs displayed differential expression between the two cell types. We found that RNA-Seq identified more differentially expressed genes and correlated with RT-qPCR quantification better than previously published microarray data. Gene Ontology analysis showed that genes upregulated in the epithelial cells were enriched for extracellular matrix production, cell division, migration, protein kinase activity, growth factor binding, and calcium ion binding. Genes upregulated in the fiber cells were enriched for proteosome complexes, unfolded protein responses, phosphatase activity, and ubiquitin binding. Differentially expressed genes involved in several important signaling pathways, lens structural components, organelle loss, and denucleation were also highlighted to provide insights into lens development and lens fiber differentiation.
RNA-Seq analysis provided a comprehensive view of the relative abundance and differential expression of protein-coding and non-coding transcripts from lens epithelial cells and lens fiber cells. This information provides a valuable resource for studying lens development, nuclear degradation, and organelle loss during fiber differentiation, and associated diseases.
PMCID: PMC4225139  PMID: 25489224
14.  Endoplasmic Reticulum Stress, Unfolded Protein Response and Altered T Cell Differentiation in Necrotizing Enterocolitis  
PLoS ONE  2013;8(10):e78491.
Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) play important roles in chronic intestinal inflammation. Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants and is characterized by acute intestinal inflammation and necrosis. The objective of the study is to investigate the role of ER stress and the UPR in NEC patients.
Ileal tissues from NEC and control patients were obtained during surgical resection and/or at stoma closure. Splicing of XBP1 was detected using PCR, and gene expression was quantified using qPCR and Western blot.
Splicing of XBP1 was only detected in a subset of acute NEC (A-NEC) patients, and not in NEC patients who had undergone reanastomosis (R-NEC). The other ER stress and the UPR pathways, PERK and ATF6, were not activated in NEC patients. A-NEC patients showing XBP1 splicing (A-NEC-XBP1s) had increased mucosal expression of GRP78, CHOP, IL6 and IL8. Similar results were obtained by inducing ER stress and the UPR in vitro. A-NEC-XBP1s patients showed altered T cell differentiation indicated by decreased mucosal expression of RORC, IL17A and FOXP3. A-NEC-XBP1s patients additionally showed more severe morphological damage and a worse surgical outcome. Compared with A-NEC patients, R-NEC patients showed lower mucosal IL6 and IL8 expression and higher mucosal FOXP3 expression.
XBP1 splicing, ER stress and the UPR in NEC are associated with increased IL6 and IL8 expression levels, altered T cell differentiation and severe epithelial injury.
PMCID: PMC3806824  PMID: 24194940
15.  Integration of the Unfolded Protein and Oxidative Stress Responses through SKN-1/Nrf 
PLoS Genetics  2013;9(9):e1003701.
The Unfolded Protein Response (UPR) maintains homeostasis in the endoplasmic reticulum (ER) and defends against ER stress, an underlying factor in various human diseases. During the UPR, numerous genes are activated that sustain and protect the ER. These responses are known to involve the canonical UPR transcription factors XBP1, ATF4, and ATF6. Here, we show in C. elegans that the conserved stress defense factor SKN-1/Nrf plays a central and essential role in the transcriptional UPR. While SKN-1/Nrf has a well-established function in protection against oxidative and xenobiotic stress, we find that it also mobilizes an overlapping but distinct response to ER stress. SKN-1/Nrf is regulated by the UPR, directly controls UPR signaling and transcription factor genes, binds to common downstream targets with XBP-1 and ATF-6, and is present at the ER. SKN-1/Nrf is also essential for resistance to ER stress, including reductive stress. Remarkably, SKN-1/Nrf-mediated responses to oxidative stress depend upon signaling from the ER. We conclude that SKN-1/Nrf plays a critical role in the UPR, but orchestrates a distinct oxidative stress response that is licensed by ER signaling. Regulatory integration through SKN-1/Nrf may coordinate ER and cytoplasmic homeostasis.
Author Summary
Proteins that are placed in membranes or secreted are produced in a cellular structure called the endoplasmic reticulum (ER). An accumulation of misfolded proteins in the ER contributes to many disease states, including diabetes and neurodegeneration. The ER protects against a toxic buildup of misfolded proteins by activating the unfolded protein response (UPR), which maintains ER homeostasis by slowing protein synthesis and enhancing ER functions such as protein folding and degradation. Many of these processes are controlled by three canonical ER/UPR gene regulatory factors. Here we identify the gene regulator SKN-1/Nrf as also playing a critical role in the UPR. SKN-1/Nrf is well known for its functions in oxidative stress defense and longevity. We now report that SKN-1/Nrf mobilizes an ER stress gene network that is distinct from its oxidative stress response, and includes regulation of other central UPR factors. Surprisingly, we also find that ER- and UPR-associated mechanisms are needed to “license” SKN-1/Nrf to defend against oxidative stresses. Our findings show that UPR and oxidative stress defense mechanisms are integrated through SKN-1/Nrf, and suggest that this integration may help maintain a healthy balance between ER and cytoplasmic functions, and stress defenses.
PMCID: PMC3772064  PMID: 24068940
16.  HLA-B35 and dsRNA Induce Endothelin-1 via Activation of ATF4 in Human Microvascular Endothelial Cells 
PLoS ONE  2013;8(2):e56123.
Endothelin 1 (ET-1) is a key regulator of vascular homeostasis. We have recently reported that the presence of Human antigen class I, HLA-B35, contributes to human dermal microvascular endothelial cell (HDMEC) dysfunction by upregulating ET-1 and proinflammatory genes. Likewise, a Toll-like receptor 3 (TLR3) ligand, Poly(I:C), was shown to induce ET-1 expression in HDMECs. The goal of this study was to determine the molecular mechanism of ET-1 induction by these two agonists. Because HLA-B35 expression correlated with induction of Binding Immunoglobulin Protein (BiP/GRP78) and several heat shock proteins, we first focused on ER stress and unfolded protein response (UPR) as possible mediators of this response. ER stress inducer, Thapsigargin (TG), HLA-B35, and Poly(I:C) induced ET-1 expression with similar potency in HDMECs. TG and HLA-B35 activated the PERK/eIF2α/ATF4 branch of the UPR and modestly increased the spliced variant of XBP1, but did not affect the ATF6 pathway. Poly(I:C) also activated eIF2α/ATF4 in a protein kinase R (PKR)-dependent manner. Depletion of ATF4 decreased basal expression levels of ET-1 mRNA and protein, and completely prevented upregulation of ET-1 by all three agonists. Additional experiments have demonstrated that the JNK and NF-κB pathways are also required for ET-1 upregulation by these agonists. Formation of the ATF4/c-JUN complex, but not the ATF4/NF-κB complex was increased in the agonist treated cells. The functional role of c-JUN in responses to HLA-B35 and Poly(I:C) was further confirmed in ET-1 promoter assays. This study identified ATF4 as a novel activator of the ET-1 gene. The ER stress/UPR and TLR3 pathways converge on eIF2α/ATF4 during activation of endothelial cells.
PMCID: PMC3575387  PMID: 23441162
17.  Involvement of Endoplasmic Reticulum Stress in Inflammatory Bowel Disease: A Different Implication for Colonic and Ileal Disease? 
PLoS ONE  2011;6(10):e25589.
Endoplasmic reticulum (ER) stress has been suggested to play a role in inflammatory bowel disease (IBD). The three branches (ATF6, IRE1 and PERK) of the unfolded protein response (UPR) have different roles and are not necessarily activated simultaneously.
Methodology/Principal Findings
Expression of UPR-related genes was investigated in colonic and ileal biopsies of 23 controls, 15 ulcerative colitis (UC) and 54 Crohn's disease (CD) patients. This expression was confirmed at protein level in colonic and ileal samples of five controls, UC and CD patients. HSPA5, PDIA4 and XBP1s were significantly increased in colonic IBD at mRNA and/or protein levels, indicating activation of the ATF6 and IRE1 branch. Colonic IBD was associated with increased phosphorylation of EIF2A suggesting the activation of the PERK branch, but subsequent induction of GADD34 was not observed. In ileal CD, no differential expression of the UPR-related genes was observed, but our data suggested a higher basal activation of the UPR in the ileal mucosa of controls. This was confirmed by the increased expression of 16 UPR-related genes as 12 of them were significantly more expressed in ileal controls compared to colonic controls. Tunicamycin stimulation of colonic and ileal samples of healthy individuals revealed that although the ileal mucosa is exhibiting this higher basal UPR activation, it is still responsive to ER stress, even more than colonic mucosa.
Activation of the three UPR-related arms is seen in colonic IBD-associated inflammation. However, despite EIF2A activation, inflamed colonic tissue did not increase GADD34 expression, which is usually involved in re-establishment of ER homeostasis. This study also implies the presence of a constitutive UPR activation in healthy ileal mucosa, with no further activation during inflammation. Therefore, engagement of the UPR differs between colon and ileum and this could be a factor in the development of ileal or colonic disease.
PMCID: PMC3196494  PMID: 22028783
18.  Transcriptional Regulation of VEGF-A by the Unfolded Protein Response Pathway 
PLoS ONE  2010;5(3):e9575.
Angiogenesis is crucial to many physiological and pathological processes including development and cancer cell survival. Vascular endothelial growth factor-A (VEGFA) is the predominant mediator of angiogenesis in the VEGF family. During development, adverse environmental conditions like nutrient deprivation, hypoxia and increased protein secretion occur. IRE1α, PERK, and ATF6α, master regulators of the unfolded protein response (UPR), are activated under these conditions and are proposed to have a role in mediating angiogenesis.
Principal Findings
Here we show that IRE1α, PERK, and ATF6α powerfully regulate VEGFA mRNA expression under various stress conditions. In Ire1α−/− and Perk−/− mouse embryonic fibroblasts and ATF6α-knockdown HepG2 cells, induction of VEGFA mRNA by endoplasmic reticulum stress is attenuated as compared to control cells. Embryonic lethality of Ire1α−/− mice is due to the lack of VEGFA induction in labyrinthine trophoblast cells of the developing placenta. Rescue of IRE1α and PERK in Ire1α−/− and Perk−/− cells respectively, prevents VEGFA mRNA attenuation. We further report that the induction of VEGFA by IRE1α, PERK and ATF6 involves activation of transcription factors, spliced-XBP-1, ATF4 and cleaved ATF6 respectively.
Our results reveal that the IRE1α-XBP-1, PERK-ATF4, and ATF6α pathways constitute novel upstream regulatory pathways of angiogenesis by modulating VEGF transcription. Activation of these pathways helps the rapidly growing cells to obtain sufficient nutrients and growth factors for their survival under the prevailing hostile environmental conditions. These results establish an important role of the UPR in angiogenesis.
PMCID: PMC2833197  PMID: 20221394
19.  hCG-induced endoplasmic reticulum stress triggers apoptosis and reduces steroidogenic enzyme expression through activating transcription factor 6 in Leydig cells of the testis 
Endoplasmic reticulum (ER) stress generally occurs in secretory cell types. It has been reported that Leydig cells, which produce testosterone in response to human chorionic gonadotropin (hCG), express key steroidogenic enzymes for the regulation of testosterone synthesis. In this study, we analyzed whether hCG induces ER stress via three unfolded protein response (UPR) pathways in mouse Leydig tumor (mLTC-1) cells and the testis. Treatment with hCG induced ER stress in mLTC-1 cells via the ATF6, IRE1a/XBP1, and eIF2α/GADD34/ATF4 UPR pathways, and transient expression of 50 kDa protein activating transcription factor 6 (p50ATF6) reduced the expression level of steroidogenic 3β-hydroxy-steroid dehydrogenase Δ5-Δ4-isomerase (3β-HSD) enzyme. In an in vivo model, high-level hCG treatment induced expression of p50ATF6 while that of steroidogenic enzymes, especially 3β-HSD, 17α-hydroxylase/C17–20 lyase (CYP17), and 17β-hydrozysteroid dehydrogenase (17β-HSD), was reduced. Expression levels of steroidogenic enzymes were restored by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Furthermore, lentivirus-mediated transient expression of p50ATF6 reduced the expression level of 3β-HSD in the testis. Protein expression levels of phospho-JNK, CHOP, and cleaved caspases-12 and -3 as markers of ER stress-mediated apoptosis markedly increased in response to high-level hCG treatment in mLTC-1 cells and the testis. Based on transmission electron microscopy and H&E staining of the testis, it was shown that abnormal ER morphology and destruction of testicular histology induced by high-level hCG treatment were reversed by the addition of TUDCA. These findings suggest that hCG-induced ER stress plays important roles in steroidogenic enzyme expression via modulation of the ATF6 pathway as well as ER stress-mediated apoptosis in Leydig cells.
PMCID: PMC4111658  PMID: 23256993
Leydig cells; Steroidogenic enzyme expression; Activating transaction factor 6; ER stress; Testosterone
20.  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)
21.  Coronavirus Infection Modulates the Unfolded Protein Response and Mediates Sustained Translational Repression▿  
Journal of Virology  2008;82(9):4492-4501.
During coronavirus replication, viral proteins induce the formation of endoplasmic reticulum (ER)-derived double-membrane vesicles for RNA synthesis, and viral structural proteins assemble virions at the ER-Golgi intermediate compartment. We hypothesized that the association and intense utilization of the ER during viral replication would induce the cellular unfolded protein response (UPR), a signal transduction cascade that acts to modulate translation, membrane biosynthesis, and the levels of ER chaperones. Here, we report that infection by the murine coronavirus mouse hepatitis virus (MHV) triggers the proximal UPR transducers, as revealed by monitoring the IRE1-mediated splicing of XBP-1 mRNA and the cleavage of ATF6α. However, we detected minimal downstream induction of UPR target genes, including ERdj4, ER degradation-enhancing α-mannosidase-like protein, and p58IPK, or expression of UPR reporter constructs. Translation initiation factor eIF2α is highly phosphorylated during MHV infection, and translation of cellular mRNAs is attenuated. Furthermore, we found that the critical homeostasis regulator GADD34, which recruits protein phosphatase 1 to dephosphorylate eIF2α during the recovery phase of the UPR, is not expressed during MHV infection. These results suggest that MHV modifies the UPR by impeding the induction of UPR-responsive genes, thereby favoring a sustained shutdown of the synthesis of host cell proteins while the translation of viral proteins escalates. The role of this modified response and its potential relevance to viral mechanisms for the evasion of innate defense signaling pathways during coronavirus replication are discussed.
PMCID: PMC2293058  PMID: 18305036
22.  Endoplasmic Reticulum Stress in Liver Disease 
Journal of Hepatology  2010;54(4):795-809.
The unfolded protein response (UPR) is activated upon the accumulation of misfolded proteins in the endoplasmic reticulum (ER), that are sensed by the binding immunoglobulin protein (BiP)/glucose-regulated protein 78 (GRP78). The accumulation of unfolded proteins sequesters BiP so it dissociates from three ER-transmembrane transducers leading to their activation. These transducers are inositol requiring (IRE) 1α, PKR-like ER kinase (PERK) and activating transcription factor (ATF) 6α. PERK phosphorylates eukaryotic initiation factor 2 alpha (eIF2α) resulting in global mRNA translation attenuation, and concurrently selectively increases the translation of several mRNAs, including the transcription factor ATF4, and its downstream target CHOP. IRE1α has kinase and endoribonuclease (RNase) activities. IRE1α autophosphorylation activates the RNase activity to cleave XBP1 mRNA, to produce the active transcription factor sXBP1. IRE1α activation also recruits and activates the stress kinase JNK. ATF6α transits to the Golgi compartment where it is cleaved by intramembrane proteolysis to generate a soluble active transcription factor. These UPR pathways act in concert to increase ER content, expand the ER protein folding capacity, degrade misfolded proteins, and reduce the load of new proteins entering the ER. All of these are geared toward adaptation to resolve the protein folding defect. Faced with persistent ER stress, adaptation starts to fail and apoptosis occurs, possibly mediated through calcium perturbations, reactive oxygen species, and the proapoptotic transcription factor CHOP. The UPR is activated in several liver diseases; including obesity associated fatty liver disease, viral hepatitis and alcohol-induced liver injury, all of which are associated with steatosis, raising the possibility that ER stress-dependent alteration in lipid homeostasis is the mechanism that underlies the steatosis. Hepatocyte apoptosis is a pathogenic event in several liver diseases, and may be linked to unresolved ER stress. If this is true, restoration of ER homeostasis prior to ER stress-induced cell death may provide a therapeutic rationale in these diseases. Here we discuss each branch of the UPR and how they may impact hepatocyte function in different pathologic states.
PMCID: PMC3375108  PMID: 21145844
23.  The eIF2 kinase PERK and the integrated stress response facilitate activation of ATF6 during endoplasmic reticulum stress 
Molecular Biology of the Cell  2011;22(22):4390-4405.
This study shows that the eIF2 kinase PERK is required not only for translational control but also for activation of ATF6 and its target genes in the unfolded protein response. The PERK pathway facilitates both the synthesis of ATF6 and trafficking of ATF6 from the endoplasmic reticulum to the Golgi for intramembrane proteolysis and activation of ATF6.
Disruptions of the endoplasmic reticulum (ER) that perturb protein folding cause ER stress and elicit an unfolded protein response (UPR) that involves translational and transcriptional changes in gene expression aimed at expanding the ER processing capacity and alleviating cellular injury. Three ER stress sensors (PERK, ATF6, and IRE1) implement the UPR. PERK phosphorylation of the α subunit of eIF2 during ER stress represses protein synthesis, which prevents further influx of ER client proteins. Phosphorylation of eIF2α (eIF2α∼P) also induces preferential translation of ATF4, a transcription activator of the integrated stress response. In this study we show that the PERK/eIF2α∼P/ATF4 pathway is required not only for translational control, but also for activation of ATF6 and its target genes. The PERK pathway facilitates both the synthesis of ATF6 and trafficking of ATF6 from the ER to the Golgi for intramembrane proteolysis and activation of ATF6. As a consequence, liver-specific depletion of PERK significantly reduces both the translational and transcriptional phases of the UPR, leading to reduced protein chaperone expression, disruptions of lipid metabolism, and enhanced apoptosis. These findings show that the regulatory networks of the UPR are fully integrated and help explain the diverse biological defects associated with loss of PERK.
PMCID: PMC3216664  PMID: 21917591
24.  Activation of the Unfolded Protein Response Is Required for Defenses against Bacterial Pore-Forming Toxin In Vivo 
PLoS Pathogens  2008;4(10):e1000176.
Pore-forming toxins (PFTs) constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR) is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of the UPR is protective in vivo against PFTs since animals that lack either the ire-1-xbp-1 or the atf-6 arms of the UPR are more sensitive to PFT than wild-type animals. The UPR acts directly in the cells targeted by the PFT. Loss of the UPR leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its PFT-protective role is specific. The p38 mitogen-activated protein (MAPK) kinase pathway has been previously shown to be important for cellular defenses against PFTs. We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR. The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.
Author Summary
Pore-forming toxins (PFTs) are bacterial toxins that form holes at the plasma membrane of cells and play an important role in the pathogenesis of many important human pathogens. Although PFTs comprise an important and the single largest class of bacterial protein virulence factors, how cells respond to these toxins has been understudied. We describe here the surprising discovery that a fundamental pathway of eukaryotic cell biology, the endoplasmic reticulum unfolded protein response (UPR), is activated by pore-forming toxins in Caenorhabditis elegans and mammalian cells. We find that this activation is functionally important since loss of either of two of the three arms of UPR leads to hypersensitivity of the nematode to attack by PFTs. The response of the UPR to PFTs can be separated from its response to unfolded proteins both at the level of activation and functional relevance. The response of the UPR to PFTs is dependent on a central pathway of cellular immunity, the p38 MAPK pathway. Our data show that the response of cells to bacterial attack can reveal unanticipated uses and connections between fundamental cell biological pathways.
PMCID: PMC2553261  PMID: 18846208
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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