X-box Binding Protein 1 mRNA Splicing Research Articles

Mammalian IRE1α dynamically and functionally coalesces with stress granules.

Upon endoplasmic reticulum (ER) stress, activation of the ER-resident transmembrane protein kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1) initiates a key branch of the unfolded protein response (UPR) through unconventional splicing generation of the transcription factor X-box-binding protein 1 (XBP1s). Activated IRE1 can form large clusters/foci, whose exact dynamic architectures and functional properties remain largely elusive. Here we report that, in mammalian cells, formation of IRE1α clusters is an ER membrane-bound phase separation event that is coupled to the assembly of stress granules (SGs). In response to different stressors, IRE1α clusters are dynamically tethered to SGs at the ER. The cytosolic linker portion of IRE1α possesses intrinsically disordered regions and is essential for its condensation with SGs. Furthermore, disruption of SG assembly abolishes IRE1α clustering and compromises XBP1 mRNA splicing, and such IRE1α-SG coalescence engenders enrichment of the biochemical components of the pro-survival IRE1α-XBP1 pathway during ER stress. Our findings unravel a phase transition mechanism for the spatiotemporal assembly of IRE1α-SG condensates to establish a more efficient IRE1α machinery, thus enabling higher stress-handling capacity.

Spliced X-box binding protein 1 (XBP1s) protects spermatogonial stem cells (SSCs) from lipopolysaccharide (LPS)-induced damage by regulating the testicular microenvironment

XBP1 is a transcription factor that plays a central role in controlling cellular responses to endoplasmic reticulum stress (ERS). Under stress conditions, the transcriptionally active form of XBP1 is generated by splicing of XBP1 mRNA by the ER-resident protein inositol-requiring enzyme-1α (IRE1α). This study aimed to investigate the role of XBP1 in male reproductive disorders. XBP1s-overexpressing goat spermatogonial stem cells (gSSCs) showed higher proliferative ability in vitro and in vivo. These cells also showed higher antioxidant capacity. In comparison, XBP1 knockdown significantly suppressed proliferation. Further analysis showed that XBP1 could stimulate the secretion of IL-6 from macrophages. Overall, the results indicate that XBP1s functions to enhance the proliferation ability and antioxidant capacity of gSSCs, potentially through a mechanism involving the regulation of gSSCs by macrophages.

IL‐10 Attenuates ER‐Stress Induced Apoptosis in Doxorubicin Treated Cardiomyocytes

Despite its antitumor efficacy, use of Doxorubicin (Dox) is limited by its cardiotoxic side effects. In Dox‐induced cardiomyopathy (DIC), a causal role of oxidative stress (OS) has gained a lot of traction. Among other subcellular changes, Dox causes vacuolization of the cytoplasm and dilation of endoplasmic reticulum (ER). Dox also causes an excessive production of reactive oxygen species as well as imbalance in redox state both of which can affect ER homeostasis and protein folding. An accumulation of unfolded/misfolded proteins triggers the unfolded protein response (UPR) in the ER, which activates transcriptional and translation pathways involving: a) Protein‐kinase like endoplasmic reticulum kinase (PERK); b) activating transcription factor 6α (ATF6α); and c) inositol requiring kinase1α (IRE1α) (Fig.1). Thus leading to the activation of ER chaperons, protein foldases and induce the expression of various genes, including X‐box binding protein 1 (XBP1). UPR plays an important role in the attenuation of protein translation, upregulation of chaperones and antioxidant expression, and thus maintains ER homeostasis. However, if UPR fails to restore ER homeostasis, ER stress‐initiated apoptotic signaling pathways are activated via CCAAT/enhancer homologous protein (CHOP), caspase‐12 activation, phosphorylation of c‐JUN NH2‐terminal kinase (JNK) and pro‐apoptotic gene expression. Understanding the role of these pathways of the ER under Dox treatment will highlight potential targets for new interventions as an adjunct therapy to restore ER functioning.In this study on isolated rat cardiomyocytes, we analyzed ER‐stress markers, spliced XBP1 mRNA, and ER‐apoptotic proteins. We also assessed protective effects of Interleukin‐10 (IL‐10) against Dox initiated ER‐induced apoptosis and overall ER‐stress. Dox significantly decreased the viability of cardiomyocytes and upregulated ER stress markers (GRP78, GRP94, and PDI). Interestingly, the exogenous administration of IL‐10, one hour prior to the exposure of cardiomyocytes to Dox for 24hrs, was found to be effective in suppressing the expressions of ER‐stress markers as well as ER‐related apoptotic proteins and preserved cell viability. Additionally, significant changes in downstream targets of IRE1α, XBP1 in IL‐10 treatment were recorded. IL‐10 significantly increased the expression of unspliced XBP1 (U), an inhibitor of spliced XBP1(S) particularly in Dox‐treated cells. This was followed by a reduction in the UPR mediated cell death in cardiomyocytes confirmed by a decrease in expression of GRP78, GRP94, and spliced XBP1 (S). IRE1 α arm of UPR initiates phosphorylation of JNK, which was reduced by IL‐10 in Dox treated cells. Dox also initiated activation of procaspase‐12, which is known to activate caspase‐3 and apoptosis. IL‐10 significantly reduced the activation of procaspase‐12 and cell death. These data suggest that IL‐10 provides protection against Dox‐induced cardiotoxicity by restoring ER homeostasis.

Nrf2-mediated induction of Cyp2a5 partially protects against reductive endoplasmic reticulum stress in mouse hepatocytes

Cytochrome P450 2a5 (Cyp2a5) is distinct from other P450 enzymes in that it is induced in the endoplasmic reticulum (ER) of mouse hepatocytes in conditions that are injurious to the liver. These conditions cause ER stress eventually resulting in apoptosis if not rectified. We previously showed that mouse hepatic Cyp2a5 is induced during reductive ER stress caused by the intramolecular disulfide form of dithiothreitol, trans-4,5-dihydroxy-1,2-dithiane (DTTox), and that overexpression of Cyp2a5 provides partial protection against apoptosis due to bilirubin (BR), a compound known to cause ER stress. The purpose of this study was to investigate the mechanism of Cyp2a5 gene regulation by DTTox and to determine if Cyp2a5 plays a cytoprotective role during reductive ER stress. Exposure to DTTox (10 mM) and another reductive ER stressor, 2-mercaptoethanol (1 mM), for 48 h markedly increased Cyp2a5 protein levels in primary mouse hepatocytes. In addition, DTTox transactivated Cyp2a5 via a mechanism involving the transcription factor nuclear factor-(erythroid-derived 2)-like 2 (Nrf2). Expression of the BR-conjugating enzyme, UDP glucuronosyl transferase 1A1 (UGT1A1) was also increased after DTTox treatment, however, this was reduced by Cyp2a5 overexpression. Hemin, a porphyrin inducer of Cyp2a5, induced mRNA splicing of X-box binding protein 1 (XBP-1), a transcription factor involved in the ER stress response, however, this was also reduced by Cyp2a5 overexpression. Finally, overexpression of Cyp2a5 partially blocked DTTox-mediated caspase-3 cleavage in Hepa 1–6 cells suggesting a cytoprotective role during ER stress. These findings demonstrate that Nrf2-mediated induction of Cyp2a5 in a reducing ER environment provides partial protection against ER stress-induced apoptosis by decreasing XBP-1 mRNA splicing and caspase-3 cleavage.

Post-treatment with apocynin at a lower dose regulates the UPR branch of eIF2α and XBP-1 pathways after stroke

Stroke leads to disturbance in the physiology of the ER (Endoplasmic Reticulum) that triggers UPR (Unfolded Protein Response) pathways aimed to compensate neuronal cell damage. However, sustained UPR causes stressful conditions in the ER lumen forming abnormal protein aggregates. Stroke-induced oxidative stress also amalgamates with UPR to safeguard and ensure the proper functioning of brain cells. Thus we tested the effect of apocynin (a potent antioxidant) post-treatment in experimental stroke on the outcome of ER stress and UPR branch pathways. We administered a low dose of apocynin at 1 mg/kg (intraperitoneal) to adult Sprague-Dawley rats subjected to Middle Cerebral Artery Occlusion (MCAO) for two-time points. The first dose immediately after re-establishing the blood flow and another at 6 h of reperfusion. Apocynin post-treatment significantly reduced ROS (Reactive Oxygen Species) generation at an early reperfusion time point of 4 h. It preserved neuronal morphology, dendritic spine density, reduced protein aggregation, and brain damage after 24 h of reperfusion. Apocynin post-treatment regulates the two UPR branch pathways in our experimental paradigm. 1) Down-regulation of eIF2α (Eukaryotic Initiation Factor 2α) phosphorylation, and CHOP (C/EBP homologous protein) 2) by reducing the XBP-1 (X-Box binding Protein-1) mRNA splicing downstream to PERK (Protein Kinase RNA-Like ER Kinase) and IRE1α (Inositol Requiring Enzyme 1alpha) UPR pathways, respectively. Bioinformatics prediction showed that apocynin has binding sites for PERK (Protein Kinase RNA-Like ER Kinase) and IRE1α proteins. The amino acid residues interacting with apocynin were Cys891 and Gln889 (for PERK), and the amino acids Ser726, Arg722, and Ala719 (for IRE1α) lying within their activation loop. Overall, these studies indicate that apocynin post-treatment might regulate ER stress/UPR pathways and minimize stroke brain damage, thus having implications for developing newer strategies for stroke treatment.

Exposure to Stearate Activates the IRE1α/XBP-1 Pathway in 3T3-L1 Adipocytes.

In the endoplasmic reticulum (ER), accumulation of abnormal proteins with malformed higher-order structures activates signaling pathways (inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 (XBP-1) pathway, protein kinase RNA-activated-like endoplasmic reticulum kinase (PERK)/CCAAT/enhancer binding protein-homologous protein (CHOP) pathway and activating transcription factor 6α (ATF6α) pathway) that result in a cellular response suppressing the production of abnormal proteins or inducing apoptosis. These responses are collectively known as the unfolded protein response (UPR). Recently, it has been suggested that the UPR induced by saturated fatty acids in hepatocytes and pancreatic β cells is involved in the development of metabolic diseases such as diabetes. The effect of palmitate, a saturated fatty acid, on the UPR has also been investigated in adipocytes, which are associated with the development of metabolic disorders, but the results were inconclusive. Therefore, as the major saturated fatty acids present in the daily diet are palmitate and stearate, we examined the effects of these saturated fatty acids on UPR in adipocytes. Here, we show that saturated fatty acids caused limited activation of the UPR in adipocytes. Exposure to stearate for several hours elevated the ratio of spliced XBP-1 mRNA, and this effect was stronger than that of palmitate. Moreover, the phosphorylation level of IRE1α, upstream of XBP-1 and expression levels of its downstream targets such as DNAJB9 and Pdia6 were elevated in 3T3-L1 adipocytes exposed to stearate. On the other hand, stearate did not affect the phosphorylation of PERK, its activation of CHOP, or the cleavage of ATF6α. Thus, in adipocytes, exposure to stearate activates the UPR via the IRE1α/XBP-1 pathway, but not the PERK/CHOP and ATF6α pathway.

Genome-wide mRNA profiling identifies X-box-binding protein 1 (XBP1) as an IRE1 and PUMA repressor

Accumulation of misfolded proteins in ER activates the unfolded protein response (UPR), a multifunctional signaling pathway that is important for cell survival. The UPR is regulated by three ER transmembrane sensors, one of which is inositol-requiring protein 1 (IRE1). IRE1 activates a transcription factor, X-box-binding protein 1 (XBP1), by removing a 26-base intron from XBP1 mRNA that generates spliced XBP1 mRNA (XBP1s). To search for XBP1 transcriptional targets, we utilized an XBP1s-inducible human cell line to limit XBP1 expression in a controlled manner. We also verified the identified XBP1-dependent genes with specific silencing of this transcription factor during pharmacological ER stress induction with both an N-linked glycosylation inhibitor (tunicamycin) and a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) (thapsigargin). We then compared those results to the XBP1s-induced cell line without pharmacological ER stress induction. Using next‐generation sequencing followed by bioinformatic analysis of XBP1-binding motifs, we defined an XBP1 regulatory network and identified XBP1 as a repressor of PUMA (a proapoptotic gene) and IRE1 mRNA expression during the UPR. Our results indicate impairing IRE1 activity during ER stress conditions accelerates cell death in ER-stressed cells, whereas elevating XBP1 expression during ER stress using an inducible cell line correlated with a clear prosurvival effect and reduced PUMA protein expression. Although further studies will be required to test the underlying molecular mechanisms involved in the relationship between these genes with XBP1, these studies identify a novel repressive role of XBP1 during the UPR.

The enigmatic role of HLA-B*27 in spondyloarthritis pathogenesis.

Establishing a clear role for HLA-B*27 in the pathogenesis of spondyloarthritis continues to be challenging. Aberrant properties of the heavy chain as well as a potential role presenting arthritogenic peptides continue to be pursued as plausible mechanisms. Recent studies implicate HLA-B*27 in aberrant bone formation. An unanticipated cell surface interaction between HLA-B*27 and the bone morphogenetic protein pathway receptor subunit ALK2 may augment TGFβ superfamily signaling pathways, increasing responsiveness to Activin A and TGFβ. This has the potential to increase bone formation as well as Th17 T cell development, presenting an attractive model to explain several aspects of axial and peripheral spondyloarthritis. In a separate study, intracellular effects of misfolded HLA-B*27 implicate this mechanism in increased osteoblast mineralization and bone formation. HLA-B*27 expression in early osteoblasts activates unfolded protein response-mediated X-box binding protein-1 mRNA splicing and induction of the retinoic acid receptor-β gene, with downstream increases in expression of tissue non-specific alkaline phosphatase. Increased TNAP expression in osteoblasts was linked to increased mineralization in vitro and bone formation in vivo. In the ongoing search for evidence of arthritogenic peptides, high-throughput TCR (T cell receptor) sequencing has provided evidence for reduced clonal expansion and increased TCR diversity in ankylosing spondylitis. In addition to two common CD8+ TCR sequences identified in one study, similar CD8 and CD4 TCR motifs were found in another study. Further work will be needed to shed light on the nature of the peptide-HLA class I complex recognized by these T cells and its role in disease.

IBD: Role of intestinal compartments in the mucosal immune response.

Laser capture microdissection (LCM) is a powerful tool for the isolation of specific tissue compartments. We aimed to investigate the mucosal immune response that takes place in different intestinal compartments of IBD patients, dissected by LCM, analyzing cytokines expression profile and endoplasmic reticulum (ER) stress markers. Frozen sections of gut were obtained from patients with Crohn's disease (CD), ulcerative colitis (UC) and from controls. Using LCM, surface epithelium (SE) and lamina propria (LP) compartments were isolated and total RNA extracted. The relative expression of Th1, Th17 and Treg cytokines was evaluated by quantitative reverse transcriptase real-time PCR (qRT-PCR), in addition to the assessment of mRNA splicing of the transcription factor X-box binding protein-1 (XBP1). Human neutrophil elastase (HNE) and the transcription factor forkhead box P3 (Foxp3) were also analyzed by immunohistochemistry. The increased expression of IL-17 was observed in both intestinal compartments of IBD patients when compared to controls. IFN- γ, TNF-α , IL-10, HNE and Foxp3 were overexpressed in the LP compartment of both IBD patients as compared to controls. An upregulation of IFN-γ and an infiltration of HNE+ cells was found in the SE of patients with UC. Splicing of XBP1 mRNA was recognized in both intestinal compartments of IBD patients when compared to controls. In IBD patients, both intestinal compartments are involved in Th17 response, whereas, LP compartment plays a prominent role in Th1 and Treg immune responses. Nevertheless, high level of IFN- γ was found in the SE of UC patients, suggesting that this compartment is involved in the Th1 immune response. Our data also suggested that ER stress signalling is active in both LP and SE compartment of IBD patients, thus advocating that ER stress and immunity are intertwined.

Real-time PCR quantification of spliced X-box binding protein 1 (XBP1) using a universal primer method.

X-box binding protein 1 (XBP1) mRNA processing plays a crucial role in the unfolded protein response (UPR), which is activated in response to endoplasmic reticulum (ER) stress. Upon accumulation of the UPR-converted XBP1 mRNA splicing from an unspliced (u) XBP1 (inactive) isoform to the spliced (s) XBP1 (active) isoform, inositol-requiring enzyme 1 α (IRE1α) removes a 26-nucleotide intron from uXBP1 mRNA. Recent studies have reported the assessment of ER stress by examining the ratio of sXBP1 to uXBP1 mRNA (s/uXBP1 ratio) via densitometric analysis of PCR bands relative to increased levels of sXBP1 to uXBP1 using a housekeeping gene for normalization. However, this measurement is visualized by gel electrophoresis, making it very difficult to quantify differences between the two XBP1 bands and complicating data interpretation. Moreover, most commonly used housekeeping genes display an unacceptably high variable expression pattern of the s/uXBP1 ratio under different experimental conditions, such as various phases of development and different cell types, limiting their use as internal controls. For a more quantitative determination of XBP1 splicing activity, we measured the expression levels of total XBP1 (tXBP1: common region of s/uXBP1) and sXBP1 via real-time PCR using specific primer sets. We also designed universal real-time PCR primer sets capable of amplifying a portion of each u/s/tXBP1 mRNA that is highly conserved in eukaryotes, including humans, monkeys, cows, pigs, and mice. Therefore, we provide a more convenient and easily approachable quantitative real-time PCR method that can be used in various research fields to assess ER stress.

Pre-pregnancy maternal obesity associates with endoplasmic reticulum stress in human umbilical vein endothelium

Obesity associates with the endoplasmic reticulum (ER) stress-induced endothelial dysfunction. Pregnant women with pre-pregnancy maternal obesity (PGMO) may transfer this potential risk to their offspring; however, whether ER stress occurs and associates with foetoplacental endothelial dysfunction in PGMO is unknown. We studied the l-arginine transport and nitric oxide (NO) synthesis in human umbilical vein endothelial cells (HUVECs) from women with PGMO or with a normal pre-pregnancy weight. We analysed the expression and activation of the ER stress sensors protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6). PGMO associated with lower endothelial NO synthase activity due to increased Thr495-inhibitor and decreased Ser1177-stimulator phosphorylation. However, higher expression and activity of the human cationic amino acid transporter 1 was found. PGMO caused activation of PERK and its downstream targets eukaryotic initiation factor 2 (eIF2α), C/EBP homologous protein 10 (CHOP), and tribbles-like protein 3 (TRB3). Increased IRE1α protein abundance (but not its phosphorylation or X-box binding protein 1-mRNA splicing) and increased c-Jun N-terminal kinase 1 phosphorylation was seen in PGMO. A preferential nuclear location of the activating transcription factor 6 (ATF6) was found in HUVECs from PGMO. All the changes seen in PGMO were blocked by TUDCA but unaltered by tunicamycin. Thus, PGMO may determine a state of ER stress via upregulation of the PERK–eIF2α–CHOP–TRB3 axis signalling in HUVECs. This phenomenon results in foetoplacental vascular endothelial dysfunction at birth.

Variation in x-box binding protein 1 (XBP1) expression and its dependent endoplasmic reticulum chaperones does not regulate adiponectin secretion in dairy cows

Adiponectin is an insulin-sensitizing hormone produced predominantly by adipose tissue; it circulates as oligomers of 3, 6, 18, or more units. Plasma adiponectin might be involved in the development of insulin resistance in transition dairy cows because it falls to a nadir around parturition. The possibility that this regulation occurs through a post-transcriptional mechanism was suggested in a previous study that showed unchanged adiponectin mRNA abundance combined with reduced expression of endoplasmic reticulum (ER) chaperones implicated in assembly of adiponectin oligomers. Expression of ER chaperones is controlled by x-box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6), suggesting a model whereby transcriptional regulation of ER chaperones during the transition period contributes to the regulation of adiponectin production. In support of this model, XBP1 expression in adipose tissue, measured either as the active spliced XBP1 mRNA or as the total of all XBP1 mRNA isoforms, was 45% lower on d 8 of lactation than 4 wk before parturition; ATF6 mRNA abundance remained unchanged over the same period. To assess the functional importance of XBP1, preadipocytes isolated from pregnant cows were differentiated into adipocytes that secrete adiponectin. Infection of differentiating cells with an adenovirus expressing the active spliced version of bovine XBP1 did not alter adiponectin mRNA but increased the expression of ER chaperones 1.5- to 5-fold. Despite the latter, XBP1 overexpression did not affect the total amount of adiponectin secreted in medium. In additional experiments, adiponectin production was dependent on exogenous lipid in the medium and was reduced during incubation with tumor necrosis factor-α (TNFα). Accordingly, we asked whether the repressive effects of these factors on adiponectin production were related to a reduction in the expression of adiponectin or determinants of ER function (XBP1, ATF6, and ER chaperones). Exogenous lipid had no effect on the expression of any of these genes, whereas TNFα repressed adiponectin mRNA abundance by 61% but had little effect on determinants of ER function. Overall, this work shows that XBP1 is a positive regulator of ER chaperone expression in adipose tissue but provides no support for XBP1 and its dependent ER chaperones in the regulation of adiponectin production in bovine adipocytes. Mechanisms accounting for reduced plasma adiponectin in transition cows remain poorly understood.

Inhibition of IRE1α-mediated XBP1 mRNA cleavage by XBP1 reveals a novel regulatory process during the unfolded protein response.

Background: The mammalian endoplasmic reticulum (ER) continuously adapts to the cellular secretory load by the activation of an unfolded protein response (UPR). This stress response results in expansion of the ER, upregulation of proteins involved in protein folding and degradation, and attenuation of protein synthesis. The response is orchestrated by three signalling pathways each activated by a specific signal transducer, either inositol requiring enzyme α (IRE1α), double-stranded RNA-activated protein kinase-like ER kinase (PERK) or activating transcription factor 6 (ATF6). Activation of IRE1α results in its oligomerisation, autophosphorylation and stimulation of its ribonuclease activity. The ribonuclease initiates the splicing of an intron from mRNA encoding the transcription factor, X-box binding protein 1 (XBP1), as well as degradation of specific mRNAs and microRNAs. Methods: To investigate the consequence of expression of exogenous XBP1, we generated a stable cell-line expressing spliced XBP1 mRNA under the control of an inducible promotor. Results: Following induction of expression, high levels of XBP1 protein were detected, which allowed upregulation of target genes in the absence of induction of the UPR. Remarkably under stress conditions, the expression of exogenous XBP1 repressed splicing of endogenous XBP1 mRNA without repressing the activation of PERK. Conclusions: These results illustrate that a feedback mechanism exists to attenuate Ire1α ribonuclease activity in the presence of XBP1.

Inhibition of IRE1α-mediated XBP1 mRNA cleavage by XBP1 reveals a novel regulatory process during the unfolded protein response

Background: The mammalian endoplasmic reticulum (ER) continuously adapts to the cellular secretory load by the activation of an unfolded protein response (UPR). This stress response results in expansion of the ER, upregulation of proteins involved in protein folding and degradation, and attenuation of protein synthesis. The response is orchestrated by three signalling pathways each activated by a specific signal transducer, either inositol requiring enzyme α (IRE1α), double-stranded RNA-activated protein kinase-like ER kinase (PERK) or activating transcription factor 6 (ATF6). Activation of IRE1α results in its oligomerisation, autophosphorylation and stimulation of its ribonuclease activity. The ribonuclease initiates the splicing of an intron from mRNA encoding the transcription factor, X-box binding protein 1 (XBP1), as well as degradation of specific mRNAs and microRNAs. Methods: To investigate the consequence of expression of exogenous XBP1, we generated a stable cell-line expressing spliced XBP1 mRNA under the control of an inducible promotor. Results: Following induction of expression, high levels of XBP1 protein were detected, which allowed upregulation of target genes in the absence of induction of the UPR. Remarkably under stress conditions, the expression of exogenous XBP1 repressed splicing of endogenous XBP1 mRNA without repressing the activation of PERK. Conclusions: These results illustrate that a feedback mechanism exists to attenuate Ire1α ribonuclease activity in the presence of XBP1.

Inhibition of IRE1α-mediated XBP1 mRNA cleavage by XBP1 reveals a novel regulatory process during the unfolded protein response

Background: The mammalian endoplasmic reticulum (ER) continuously adapts to the cellular secretory load by the activation of an unfolded protein response (UPR). This stress response results in expansion of the ER, upregulation of proteins involved in protein folding and degradation, and attenuation of protein synthesis. The response is orchestrated by three signalling pathways each activated by a specific signal transducer, either inositol requiring enzyme α (IRE1α), double-stranded RNA-activated protein kinase-like ER kinase (PERK) or activating transcription factor 6 (ATF6). Activation of IRE1α results in its oligomerisation, autophosphorylation and stimulation of its ribonuclease activity. The ribonuclease initiates the splicing of an intron from mRNA encoding the transcription factor, X-box binding protein 1 (XBP1), as well as degradation of specific mRNAs and microRNAs. Methods: To investigate the consequence of expression of exogenous XBP1, we generated a stable cell-line expressing spliced XBP1 mRNA under the control of an inducible promotor. Results: Following induction of expression, high levels of XBP1 protein were detected, which allowed upregulation of target genes in the absence of induction of the UPR. Remarkably under stress conditions, the expression of exogenous XBP1 repressed splicing of endogenous XBP1 mRNA without repressing the activation of PERK. Conclusions: These results illustrate that a feedback mechanism exists to attenuate activation of the Ire1α ribonuclease activity in the presence of XBP1.

TLR7-mediated activation of XBP1 correlates with the IFNα production in humans

The transcription factor X-box binding protein 1 (XBP1) represents a key component of the endoplasmatic reticulum (ER) stress response and is required for the production of several pro-inflammatory cytokines. XBP1 is furthermore essential for the development and survival of plasmacytoid dendritic cells (pDCs), and has recently been suggested to be involved in toll-like receptor (TLR) 2/4 signaling. Activation of TLR7 on pDCs results in an upregulation of pro-inflammatory cytokines, such as type I interferons (IFN-I), and has been implicated in several autoimmune and inflammatory diseases, but the role of XBP1 in this process remains unknown. Here, we show that signaling via TLR7 leads to an upregulation of XBP1 and IFNα-production. XBP1 mRNA expression levels positively correlated with the production of IFNα, while blocking of XBP1 mRNA splicing significantly reduced mRNA transcripts of IFNα. In conclusion, these data suggest a central role of XBP1 in TLR7-induced IFNα production and identify XBP1 as a potential novel therapeutic target in IFNα-driven autoimmune and inflammatory diseases.

Withaferin A Induces ROS-Mediated Paraptosis in Human Breast Cancer Cell-Lines MCF-7 and MDA-MB-231

Advancement in cancer therapy requires a better understanding of the detailed mechanisms that induce death in cancer cells. Besides apoptosis, themode of other types of cell death has been increasingly recognized in response to therapy. Paraptosis is a non-apoptotic alternative form of programmed cell death, morphologically) distinct from apoptosis and autophagy. In the present study, Withaferin-A (WA) induced hyperpolarization of mitochondrial membrane potential and formation of many cytoplasmic vesicles. This was due to progressive swelling and fusion of mitochondria and dilation of endoplasmic reticulum (ER), forming large vacuolar structures that eventually filled the cytoplasm in human breast cancer cell-lines MCF-7 and MDA-MB-231. The level of indigenous paraptosis inhibitor, Alix/AIP-1 (Actin Interacting Protein-1) was down-regulated by WA treatment. Additionally, prevention of WA-induced cell death and vacuolation on co-treatment with protein-synthesis inhibitor indicated requirement of de-novo protein synthesis. Co-treatment with apoptosis inhibitor resulted in significant augmentation of WA-induced death in MCF-7 cells, while partial inhibition in MDA-MB-231 cells; implyingthat apoptosis was not solely responsible for the process.WA-mediated cytoplasmic vacuolationcould not be prevented by autophagy inhibitor wortmanninas well, claiming this process to be a non-autophagic one. Early induction of ROS (Reactive Oxygen Species)by WA in both the cell-lines was observed. ROS inhibitorabrogated the effect of WA on: cell-death, expression of proliferation-associated factor andER-stress related proteins,splicing of XBP-1 (X Box Binding Protein-1) mRNA and formation of paraptotic vacuoles.All these results conclusively indicate thatWA induces deathin bothMCF-7 and MDA-MB-231 cell lines byROS-mediated paraptosis.

The Role of IRE-XBP1 Pathway in Regulation of Retinal Pigment Epithelium Tight Junctions.

PurposeThe retinal pigment epithelium (RPE) tight junctions play a pivotal role in maintaining the homeostatic environment of the neural retina. Herein, we investigated the role of X-box binding protein 1 (XBP1), an endoplasmic reticulum (ER) stress-responsive transcription factor, in regulation of RPE tight junctions.MethodsHuman RPE cell line (ARPE-19) and primary primate RPE cells were used for in vitro experiments and RPE-specific XBP1 knockout (KO) mice were used for in vivo study. Endoplasmic reticulum stress was induced by a sublethal dose of thapsigargin or tunicamycin. XBP1 activation was manipulated by IRE inhibitor 4μ8C, which suppresses XBP1 mRNA splicing. The integrity of tight junctions and the involvement of calcium-dependent RhoA/Rho kinase pathway were examined.ResultsInduction of ER stress by thapsigargin, but not tunicamycin, disrupted RPE tight junctions in ARPE-19 cells. Inhibition of XBP1 activation by 4μ8C resulted in a remarkable downregulation of tight junction proteins (ZO-1 and occludin) and defects in tight junction formation in the presence or absence of ER stress inducers. Overexpression of active XBP1 partially reversed 4μ8C-induced anomalies in tight junctions. Mechanistically, XBP1 inhibition resulted in increased intracellular Ca2+ concentration, upregulation of RhoA expression, redistribution of F-actin, and tight junction damage, which was attenuated by Rho kinase inhibitor Y27632. In vivo, deletion of XBP1 in the RPE resulted in defective RPE tight junctions accompanied by increased VEGF expression.ConclusionsTaken together, these results suggest a protective role of XBP1 in maintaining RPE tight junctions possibly through regulation of calcium-dependent RhoA/Rho kinase signaling and actin cytoskeletal reorganization.

Lipopolysaccharide Activates the Unfolded Protein Response in Human Periodontal Ligament Fibroblasts.

The activation of the unfolded protein response (UPR) has been demonstrated in periodontal diseases. However, the cellular and molecular mechanisms by which the UPR is induced in periodontitis remain unclear. In this study, the effects of lipopolysaccharide (LPS) on the induction of the UPR in human periodontal ligament fibroblasts (HPDLFs) in vitro are investigated. HPDLFs isolated from human PDLs were stimulated with various concentrations of Escherichia coli LPS (0.1, 1, and 10 μg/mL) for the indicated time points (0, 3, 6, 9, 12, and 18 hours). The messenger RNA (mRNA) and protein levels of molecular markers associated with UPR activation, such as glucose-regulated protein 78 (GRP78), X-box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP), were measured at different time points of LPS treatment. Apoptosis of HPDLFs was assessed by Annexin V-FITC and propidium iodide staining, followed by flow cytometry. LPS treatment of HPDLFs increased GRP78 mRNA and protein levels in a concentration-dependent manner. Additionally, LPS also induced the expression, splicing, and activation of XBP1 mRNA. Moreover, LPS-induced CHOP expression was concentration dependent: a lower concentration of LPS (0.1 μg/mL) had no effect on CHOP mRNA levels, but higher concentrations of LPS (1 and 10 μg/mL) markedly increased CHOP mRNA and protein expression without inducing apoptosis. The findings demonstrate that activating the Toll-like receptor-4 signaling pathway in HPDLFs using LPS triggers the UPR in vitro, warranting additional investigation into the precise mechanisms by which pathways promote this response under inflammatory conditions.

Novel mechanism of enhancing IRE1α-XBP1 signalling via the PERK-ATF4 pathway.

Mammalian inositol-requiring enzyme 1α (IRE1α) is the most conserved of all endoplasmic reticulum (ER) stress sensors, which includes activating transcription factor (ATF) 6 and double-stranded RNA-dependent protein kinase (PKR)-like ER kinase (PERK). IRE1α has been known to splice X-box binding protein 1 (XBP1) mRNA, which is induced by ATF6 under ER stress. This spliced XBP1 mRNA is translated into the active transcription factor that promotes the expression of specific genes to alleviate ER stress. Herein, we report that in addition to the induction of XBP1 expression by ATF6, IRE1α expression is induced by ATF4, which is downstream of PERK, under ER stress. Increased IRE1α expression results in a higher splicing ratio of XBP1 mRNA. This effect was not transient and affected not only the intensity but also the duration of the activated state of this pathway. These multiple regulatory mechanisms may modulate the response to various levels or types of ER stress.