Insights into the structure and function of the RNA ligase RtcB.

To be functional, some RNAs require a processing step involving splicing events. Each splicing event necessitates an RNA ligation step. RNA ligation is a process that can be achieved with various intermediaries such as self-catalysing RNAs, 5'-3' and 3'-5' RNA ligases. While several types of RNA ligation mechanisms occur in human, RtcB is the only 3'-5' RNA ligase identified in human cells to date. RtcB RNA ligation activity is well known to be essential for the splicing of XBP1, an essential transcription factor of the unfolded protein response; as well as for the maturation of specific intron-containing tRNAs. As such, RtcB is a core factor in protein synthesis and homeostasis. Taking advantage of the high homology between RtcB orthologues in archaea, bacteria and eukaryotes, this review will provide an introduction to the structure of RtcB and the mechanism of 3'-5' RNA ligation. This analysis is followed by a description of the mechanisms regulating RtcB activity and localisation, its known partners and its various functions from bacteria to human with a specific focus on human cancer.

Constitutive expression of spliced XBP1 causes perinatal lethality in mice.

Upon endoplasmic reticulum (ER) stress, inositol-requiring enzyme 1 (IRE1) is activated and catalyzes nonconventional splicing of an unspliced X-box binding protein 1 (XBP1U) mRNA to yield a spliced XBP1 (XBP1S) mRNA that encodes a potent XBP1S transcription factor. XBP1S is a key mediator of the IRE1 branch that is essential for alleviating ER stress. We generated a novel mouse strain (referred to as "Xbp1CS/+ " mice) that constitutively expressed XBP1S after Cre recombinase-mediated recombination. Further breeding of these mice with Twist2 Cre recombinase (Twist2-Cre) knock-in mice generated Twist2-Cre;Xbp1CS/+ mice. Most Twist2-Cre;Xbp1CS/+ mice died shortly after birth. Reverse-transcription polymerase chain reaction (RT-PCR) showed that constitutive expression of XBP1S occurred in various mouse tissues examined, but not in the brain. Immunohistochemistry confirmed that although the immunostaining signals for total XBP1 (XBP1U and XBP1S) were found in the calvarial bones in both Twist2-Cre;Xbp1CS/+ and control mice, the signals for XBP1S were only detected in the Twist2-Cre;Xbp1CS/+ mice, but not in the control mice. These results suggest that a precise control of XBP1S production is essential for normal mouse development.

Hypoxic ER stress suppresses ß-catenin expression and promotes cooperation between the transcription factors XBP1 and HIF1α for cell survival.

Hypoxia occurs in many human solid tumors and activates multiple cellular adaptive-response pathways, including the unfolded protein response (UPR) in the endoplasmic reticulum (ER). Wnt/ß-catenin signaling plays a critical role in tumorigenesis, and ß-catenin has been shown to enhance hypoxia-inducible factor 1α (HIF1α)-activated gene expression, thereby supporting cell survival during hypoxia. However, the molecular interplay between hypoxic ER stress, Wnt/ß-catenin signaling, and HIF1α-mediated gene regulation during hypoxia remains incompletely understood. Here, we report that hypoxic ER stress reduces ß-catenin stability, which, in turn, enhances the activity of spliced X-box-binding protein 1 (XBP1s), a transcription factor and signal transducer of the UPR, in HIF1α-mediated hypoxic responses. We observed that in the RKO colon cancer cell line, which possesses a Wnt-stimulated ß-catenin signaling cascade, increased ER stress during hypoxia is accompanied by a reduction in low-density lipoprotein receptor-related protein 6 (LRP6), and this reduction in LRP6 decreased ß-catenin accumulation and impaired Wnt/ß-catenin signaling. Of note, ß-catenin interacted with both XBP1s and HIF1α, suppressing XBP1s-mediated augmentation of HIF1α target gene expression. Furthermore, Wnt stimulation or ß-catenin overexpression blunted XBP1s-mediated cell survival under hypoxia. Together, these results reveal an unanticipated role for the Wnt/ß-catenin pathway in hindering hypoxic UPR-mediated responses that increase cell survival. Our findings suggest that the molecular cross-talks between hypoxic ER stress, LRP6/ß-catenin signaling, and the HIF1α pathway may represent an unappreciated mechanism that enables some tumor subtypes to survive and grow in hypoxic conditions.

Emerging roles for the ER stress sensor IRE1α in metabolic regulation and disease.

Inositol-requiring enzyme 1 (IRE1) is an endoplasmic reticulum (ER)-resident transmembrane protein that senses ER stress and is evolutionarily conserved from yeast to humans. IRE1 possesses both Ser/Thr protein kinase and endoribonuclease (RNase) activities within its cytoplasmic domain and is activated through autophosphorylation and dimerization/oligomerization. It mediates a critical arm of the unfolded protein response to manage ER stress provoked by lumenal overload of unfolded/misfolded proteins. Emerging lines of evidence have revealed that in mammals, IRE1α functions as a multifunctional signal transducer that responds to metabolic cues and nutrient stress conditions, exerting profound and broad effects on metabolic homeostasis. In this review, we cover recent advances in our understanding of how IRE1α integrates a variety of metabolic and stress signals and highlight its tissue-specific or context-dependent metabolic activities. We also discuss how dysregulation of this metabolic stress sensor during handling of excessive nutrients in cells contributes to the progression of obesity and metabolic disorders.

The cyclic phosphodiesterase CNP and RNA cyclase RtcA fine-tune noncanonical XBP1 splicing during ER stress.

The activity of X box-binding protein 1 (XBP1), a master transcriptional regulator of endoplasmic reticulum (ER) homeostasis and the unfolded protein response (UPR), is controlled by a two-step noncanonical splicing reaction in the cytoplasm. The first step of nuclease cleavage by inositol-requiring enzyme 1 (IRE1), a protein kinase/endoribonuclease, is conserved in all eukaryotic cells. The second step of RNA ligation differs biochemically among species. In yeast, tRNA ligase 1 (Trl1) and tRNA 2'-phosphotransferase 1 (Tpt1) act through a 5'-PO4/3'-OH pathway. In metazoans, RNA 2',3'-cyclic phosphate and 5'-OH ligase (RtcB) ligate XBP1 exons via a 3'-PO4/5'-OH reaction. Although RtcB has been identified as the primary RNA ligase, evidence suggests that yeast-like ligase components may also operate in mammals. In this study, using mouse and human cell lines along with in vitro splicing assays, we investigated whether these components contribute to XBP1 splicing during ER stress. We found that the mammalian 2'-phosphotransferase Trpt1 does not contribute to XBP1 splicing even in the absence of RtcB. Instead, we found that 2',3'-cyclic nucleotide phosphodiesterase (CNP) suppresses RtcB-mediated XBP1 splicing by hydrolyzing 2',3'-cyclic phosphate into 2'-phosphate on the cleaved exon termini. By contrast, RNA 3'-terminal cyclase (RtcA), which converts 2'-phosphate back to 2',3'-cyclic phosphate, facilitated XBP1 splicing by increasing the number of compatible RNA termini for RtcB. Taken together, our results provide evidence that CNP and RtcA fine-tune XBP1 output during ER stress.

ER stress signaling has an activating transcription factor 6α (ATF6)-dependent "off-switch".

In response to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen, three ER transmembrane signaling proteins, inositol-requiring enzyme 1 (IRE1), PRKR-like ER kinase (PERK), and activating transcription factor 6α (ATF6α), are activated. These proteins initiate a signaling and transcriptional network termed the unfolded protein response (UPR), which re-establishes cellular proteostasis. When this restoration fails, however, cells undergo apoptosis. To investigate cross-talk between these different UPR enzymes, here we developed a high-content live cell screening platform to image fluorescent UPR-reporter cell lines derived from human SH-SY5Y neuroblastoma cells in which different ER stress signaling proteins were silenced through lentivirus-delivered shRNA constructs. We observed that loss of ATF6 expression results in uncontrolled IRE1-reporter activity and increases X box-binding protein 1 (XBP1) splicing. Transient increases in both IRE1 mRNA and IRE1 protein levels were observed in response to ER stress, suggesting that IRE1 up-regulation is a general feature of ER stress signaling and was further increased in cells lacking ATF6 expression. Moreover, overexpression of the transcriptionally active N-terminal domain of ATF6 reversed the increases in IRE1 levels. Furthermore, inhibition of IRE1 kinase activity or of downstream JNK activity prevented an increase in IRE1 levels during ER stress, suggesting that IRE1 transcription is regulated through a positive feed-forward loop. Collectively, our results indicate that from the moment of activation, IRE1 signaling during ER stress has an ATF6-dependent "off-switch."

MicroRNA-326 suppresses iNOS expression and promotes autophagy of dopaminergic neurons through the JNK signaling by targeting XBP1 in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is characterized by a progressive loss of dopaminergic neurons, and substantia nigra is primarily one of the damaged brain regions. Evidence indicates that microRNAs (miRNAs) is involved in the pathophysiology of this disease. The present study aimed to investigate the biological function of miR-326 in PD through the JNK signaling pathway by targeting X-box binding protein 1 (XBP1). After liposome complexes were prepared, healthy male C57BL/6 mice were selected to construct a mouse model of PD. The targeting relationship between miR-326 and XBP1 was confirmed. The expression of miR-326 and XBP1 was measured in PD mice, and gain- and loss-function assay was conducted to examine the regulatory effect of miR-326 and XBP1 on inducible nitric oxide synthase (iNOS) expression and autophagy of dopaminergic neurons of PD mice. Mice treated with miR-326 mimic and siRNA-XBP1 showed increased traction test scores, activation of autophagy, expression of LC3-II, c-Jun, and p-α-Syn, but diminished climbing time and expressions of iNOS, α-Syn, and p-c-Jun. The siRNA-XBP1 treatment could reverse the effect of miR-326 inhibitor on PD mice. Overexpression of miR-326 inhibits iNOS expression and promotes autophagy of dopaminergic neurons through JNK signaling by targeting XBP1.

XBP1-s promotes colorectal cancer cell proliferation by inhibiting TAp73 transcriptional activity.

Endoplasmic reticulum (ER) stress activation could be found in a wide range of human tumors. ER stress induces the splicing of X-box binding protein 1 (XBP1) to form its splicing variant XBP1-s, which in turn activates various ER stress-related genes. XBP1-s is highly expressed in various tumors; however, its role in tumorigenesis is still largely unknown. Herein we showed that XBP1-s suppresses the expression of tumor suppressor TAp73, a member of p53 family with high homology with p53, by directly binds to TAp73 promoter and suppresses its transcriptional activity. We also found that overexpression of TAp73 cancelled the effect of XPB1-s on enhancing colorectal cancer cells proliferation and colony formation potential, indicating that TAp73 is critical for XBP1-s-induced tumorigenesis. Together, our findings not only reveal a novel mechanism of TAp73 aberrant regulation in tumor cells, but also link up tumor cells ER stress with tumor suppressive activity of TAp73.

Overexpression of CiIKKß enhances CIK cell viability against ER stress.

Recently, studies have shown that IκB kinase ß (IKKß), a critical kinase in the nucleus factor kappa-B (NF-κB) pathway, participates in inflammatory responses associated with unfolded protein response (UPR) and plays an important role in ER stress-induced cell death. The unfolded protein response (UPR), which is a regulatory system to restore cellular homeostasis in the endoplasmic reticulum (ER), such as oxidative stress, bacterial infection, and virus invasion. The UPR pathways have been reported to be involved in immune responses in mammals, including the classical NF-κB pathway. However, the molecular mechanism of their crosstalk remains to be elucidated. Previously, we demonstrated that IKKß also has some conserved functions between fish and human, as grass carp (Ctenopharyngodon idella) IKKß (CiIKKß) can activate NF-κB pathway. In this study, we found that CiIKKß level in nucleus was elevated under ER stress and CiIKKß can interact with grass carp X-box-binding protein 1 (CiXBP1S), a key transcription factor in UPR. Consistently, fluorescent histochemical analysis of grass carp kidney (CIK) cells indicated that CiIKKß and CiXBP1S colocalized under ER stress. Furthermore, overexpression of CiIKKß in CIK cells enhanced ER stress tolerance by regulating UPR signaling and resulted in the significant increase of cell viability.

Transcriptional Regulation of X-Box-binding Protein One (XBP1) by Hepatocyte Nuclear Factor 4α (HNF4Α) Is Vital to Beta-cell Function.

The transcription factor, X-box-binding protein-1 (XBP1), controls the development and maintenance of the endoplasmic reticulum (ER) in multiple secretory cell lineages. We show here that Hepatocyte Nuclear Factor 4α (HNF4α) directly induces XBP1 expression. Mutations in HNF4α cause Mature-Onset Diabetes of the Young I (MODYI), a subset of diabetes characterized by diminished GSIS. In mouse models, cell lines, and ex vivo islets, using dominant negative and human- disease-allele point mutants or knock-out and knockdown models, we show that disruption of HNF4α caused decreased expression of XBP1 and reduced cellular ER networks. GSIS depends on ER Ca(2+) signaling; we show that diminished XBP1 and/or HNF4α in ß-cells led to impaired ER Ca(2+) homeostasis. Restoring XBP1 expression was sufficient to completely rescue GSIS in HNF4α-deficient ß-cells. Our findings uncover a transcriptional relationship between HNF4α and Xbp1 with potentially broader implications about MODYI and the importance of transcription factor signaling in the regulation of secretion.

Hepatic Xbp1 Gene Deletion Promotes Endoplasmic Reticulum Stress-induced Liver Injury and Apoptosis.

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), a highly conserved signaling cascade that functions to alleviate stress and promote cell survival. If, however, the cell is unable to adapt and restore homeostasis, then the UPR activates pathways that promote apoptotic cell death. The molecular mechanisms governing the critical transition from adaptation and survival to initiation of apoptosis remain poorly understood. We aim to determine the role of hepatic Xbp1, a key mediator of the UPR, in controlling the adaptive response to ER stress in the liver. Liver-specific Xbp1 knockout mice (Xbp1(LKO)) and Xbp1(fl/fl) control mice were subjected to varying levels and durations of pharmacologic ER stress. Xbp1(LKO) and Xbp1(fl/fl) mice showed robust and equal activation of the UPR acutely after induction of ER stress. By 24 h, Xbp1(fl/fl) controls showed complete resolution of UPR activation and no liver injury, indicating successful adaptation to the stress. Conversely, Xbp1(LKO) mice showed ongoing UPR activation associated with progressive liver injury, apoptosis, and, ultimately, fibrosis by day 7 after induction of ER stress. These data indicate that hepatic XBP1 controls the adaptive response of the UPR and is critical to restoring homeostasis in the liver in response to ER stress.

Constitutive expression of spliced X-box binding protein 1 inhibits dentin formation in mice.

Upon endoplasmic reticulum (ER) stress, inositol-requiring enzyme 1 (IRE1) is activated, which subsequently converts an unspliced X-box binding protein 1 (XBP1U) mRNA to a spliced mRNA that encodes a potent XBP1S transcription factor. XBP1S is essential for relieving ER stress and secretory cell differentiation. We previously established Twist2-Cre;Xbp1 CS/+ mice that constitutively expressed XBP1S in the Twist2-expressing cells as well as in the cells derived from the Twist2-expressing cells. In this study, we analyzed the dental phenotype of Twist2-Cre;Xbp1 CS/+ mice. We first generated a mutant Xbp1s minigene that corresponds to the recombinant Xbp1 Δ26 allele (the Xbp1 CS allele that has undergone Cre-mediated recombination) and confirmed that the Xbp1s minigene expressed XBP1S that does not require IRE1α activation in vitro. Consistently, immunohistochemistry showed that XBP1S was constitutively expressed in the odontoblasts and other dental pulp cells in Twist2-Cre;Xbp1 CS/+ mice. Plain X-ray radiography and µCT analysis revealed that constitutive expression of XBP1S altered the dental pulp chamber roof- and floor-dentin formation, resulting in a significant reduction in dentin/cementum formation in Twist2-Cre;Xbp1 CS/+ mice, compared to age-matched Xbp1 CS/+ control mice. However, there is no significant difference in the density of dentin/cementum between these two groups of mice. Histologically, persistent expression of XBP1S caused a morphological change in odontoblasts in Twist2-Cre;Xbp1 CS/+ mice. Nevertheless, in situ hybridization and immunohistochemistry analyses showed that continuous expression of XBP1S had no apparent effects on the expression of the Dspp and Dmp1 genes. In conclusion, these results support that sustained production of XBP1S adversely affected odontoblast function and dentin formation.

Protein disulfide-isomerase A4 confers glioblastoma angiogenesis promotion capacity and resistance to anti-angiogenic therapy.

INTRODUCTION: Increasing evidence has revealed the key activity of protein disulfide isomerase A4 (PDIA4) in the endoplasmic reticulum stress (ERS) response. However, the role of PDIA4 in regulating glioblastoma (GBM)-specific pro-angiogenesis is still unknown. METHODS: The expression and prognostic role of PDIA4 were analyzed using a bioinformatics approach and were validated in 32 clinical samples and follow-up data. RNA-sequencing was used to search for PDIA4-associated biological processes in GBM cells, and proteomic mass spectrum (MS) analysis was used to screen for potential PDIA4 substrates. Western blotting, real-time quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assays (ELISA) were used to measure the levels of the involved factors. Cell migration and tube formation assays determined the pro-angiogenesis activity of PDIA4 in vitro. An intracranial U87 xenograft GBM animal model was constructed to evaluate the pro-angiogenesis role of PDIA4 in vivo. RESULTS: Aberrant overexpression of PDIA4 was associated with a poor prognosis in patients with GBM, although PDIA4 could also functionally regulate intrinsic GBM secretion of vascular endothelial growth factor-A (VEGF-A) through its active domains of Cys-X-X-Cys (CXXC) oxidoreductase. Functionally, PDIA4 exhibits pro-angiogenesis activity both in vitro and in vivo, and can be upregulated by ERS through transcriptional regulation of X-box binding protein 1 (XBP1). The XBP1/PDIA4/VEGFA axis partially supports the mechanism underlying GBM cell survival under ER stress. Further, GBM cells with higher expression of PDIA4 showed resistance to antiangiogenic therapy in vivo. CONCLUSIONS: Our findings revealed the pro-angiogenesis role of PDIA4 in GBM progression and its potential impact on GBM survival under a harsh microenvironment. Targeting PDIA4 might help to improve the efficacy of antiangiogenic therapy in patients with GBM.

Preservation of ∼12-h ultradian rhythms of gene expression of mRNA and protein metabolism in the absence of canonical circadian clock.

Introduction: Besides the ∼24-h circadian rhythms, ∼12-h ultradian rhythms of gene expression, metabolism and behaviors exist in animals ranging from crustaceans to mammals. Three major hypotheses were proposed on the origin and mechanisms of regulation of ∼12-h rhythms, namely, that they are not cell-autonomous and controlled by a combination of the circadian clock and environmental cues, that they are regulated by two anti-phase circadian transcription factors in a cell autonomous manner, or that they are established by a cell-autonomous ∼12-h oscillator. Methods: To distinguish among these possibilities, we performed a post hoc analysis of two high temporal resolution transcriptome dataset in animals and cells lacking the canonical circadian clock. Results: In both the liver of BMAL1 knockout mice and Drosophila S2 cells, we observed robust and prevalent ∼12-h rhythms of gene expression enriched in fundamental processes of mRNA and protein metabolism that show large convergence with those identified in wild-type mice liver. Bioinformatics analysis further predicted ELF1 and ATF6B as putative transcription factors regulating the ∼12-h rhythms of gene expression independently of the circadian clock in both fly and mice. Discussion: These findings provide additional evidence to support the existence of an evolutionarily conserved 12-h oscillator that controls ∼12-h rhythms of gene expression of protein and mRNA metabolism in multiple species.

RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response.

Introduction: The unfolded protein response (UPR) has emerged as an important signaling pathway mediating anti-viral defenses to Respiratory Syncytial Virus (RSV) infection. Earlier we found that RSV replication predominantly activates the evolutionarily conserved Inositol Requiring Enzyme 1α (IRE1α)-X-Box Binding Protein 1 spliced (XBP1s) arm of the Unfolded Protein Response (UPR) producing inflammation, metabolic adaptation and cellular plasticity, yet the mechanisms how the UPR potentiates inflammation are not well understood. Methods: To understand this process better, we examined the genomic response integrating RNA-seq and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) analyses. These data were integrated with an RNA-seq analysis conducted on RSV-infected small airway cells ± an IRE1α RNAse inhibitor. Results: We identified RSV induced expression changes in ~3.2K genes; of these, 279 required IRE1α and were enriched in IL-10/cytokine signaling pathways. From this data set, we identify those genes directly under XBP1s control by CUT&RUN. Although XBP1s binds to ~4.2 K high-confidence genomic binding sites, surprisingly only a small subset of IL10/cytokine signaling genes are directly bound. We further apply CUT&RUN to find that RSV infection enhances XBP1s loading on 786 genomic sites enriched in AP1/Fra-1, RELA and SP1 binding sites. These control a subset of cytokine regulatory factor genes including IFN response factor 1 (IRF1), CSF2, NFKB1A and DUSP10. Focusing on the downstream role of IRF1, selective knockdown (KD) and overexpression experiments demonstrate IRF1 induction controls type I and -III interferon (IFN) and IFN-stimulated gene (ISG) expression, demonstrating that ISG are indirectly regulated by XBP1 through IRF1 transactivation. Examining the mechanism of IRF1 activation, we observe that XBP1s directly binds a 5' enhancer sequence whose XBP1s loading is increased by RSV. The functional requirement for the enhancer is demonstrated by targeting a dCas9-KRAB silencer, reducing IRF1 activation. Chromatin immunoprecipitation shows that XBP1 is required, but not sufficient, for RSV-induced recruitment of activated phospho-Ser2 Pol II to the enhancer. Discussion: We conclude that XBP1s is a direct activator of a core subset of IFN and cytokine regulatory genes in response to RSV. Of these IRF1 is upstream of the type III IFN and ISG response. We find that RSV modulates the XBP1s binding complex on the IRF1 5' enhancer whose activation is required for IRF1 expression. These findings provide novel insight into how the IRE1α-XBP1s pathway potentiates airway mucosal anti-viral responses.

Prevention of alloimmune rejection using XBP1-deleted bone marrow-derived dendritic cells in heart transplantation.

BACKGROUND: Genetically modified dendritic cells (DCs) modulate the alloimmunity of T lymphocytes by regulating antigen presentation. METHODS: We generated mice with specific deletion of the X-box-binding protein 1 (XBP1) allele in bone marrow cells and cultured bone marrow-derived DCs (Xbp1-/- BMDCs) from these animals. We then tested the phenotype of Xbp1-/- BMDCs, evaluated their capability to activate allogeneic T cells and investigated their mechanistic actions. We developed a mouse model of allogeneic heart transplantation in which recipients received PBS, Xbp1-/- BMDCs, a suboptimal dose of cyclosporine A (CsA), or Xbp1-/- BMDCs combined with a suboptimal dose of CsA to evaluate the effects of Xbp1-/- BMDC transfusion on alloimmunity and on the survival of heart allografts. RESULTS: The deletion of XBP1 in BMDCs exploited the IRE1-dependent decay of TAPBP mRNA to reduce the expression of MHC-I on the cell surface, altered the capability of BMDCs to activate CD8+ T cells, and ultimately suppressed CD8+ T-cell-mediated allogeneic rejection. The adoptive transfer of Xbp1-/- BMDCs inhibited CD8+ T-cell-mediated rejection. In addition, XBP1-deficient BMDCs were weak stimulators of allogeneic CD4+ T cells despite expressing high levels of MHC-II and costimulatory molecules on their cell surface. Moreover, the adoptive transfer of Xbp1-/- BMDCs inhibited the production of circulating donor-specific IgG. The combination of Xbp1-/- BMDCs and CsA treatment significantly prolonged the survival of allografts compared to CsA alone. CONCLUSIONS: The deletion of XBP1 induces immunosuppressive BMDCs, and treatment with these immunosuppressive BMDCs prevents alloimmune rejection and improves the outcomes of heart transplantation. This finding provides a promising therapeutic target in combating transplant rejection and expands knowledge of inducing therapeutic DCs.

Analysis of XBP1 Contribution to Hyperosmolarity-Induced Lipid Synthesis.

The unfolded protein response (UPR) is a complex network of intracellular pathways that transmits signals from ER lumen and/or ER bilayer to the nuclear compartment in order to activate gene transcription. UPR is activated by the loss of ER capacities, known as ER stress, and occurs to restore ER properties. In this regard, glycerolipid (GL) synthesis activation contributes to ER membrane homeostasis and IRE1α-XBP1, one UPR pathway, has a main role in lipogenic genes transcription. Herein, we describe the strategy and methodology used to evaluate whether IRE1α-XBP1 pathway regulates lipid metabolism in renal epithelial cells subjected to hyperosmolar environment. XBP1s activity was hindered by blocking IRE1α RNAse activity and by impeding its expression; under these conditions, we determined GL synthesis and lipogenic enzymes expression.

IRE1α Inhibitors as a Promising Therapeutic Strategy in Blood Malignancies.

Synthesis, folding, and structural maturation of proteins occur in the endoplasmic reticulum (ER). Accumulation of misfolded or unfolded proteins in the ER lumen contributes to the induction of ER stress and activation of the unfolded protein response (UPR) signaling pathway. Under ER stress, the UPR tries to maintain cellular homeostasis through different pathways, including the inositol-requiring enzyme 1 alpha (IRE1α)-dependent ones. IRE1α is located in an ER membrane, and it is evolutionarily the oldest UPR sensor. Activation of IRE1α via ER stress triggers the formation of the spliced form of XBP1 (XBP1s), which has been linked to a pro-survival effect in cancer cells. The role of IRE1α is critical for blood cancer cells, and it was found that the levels of IRE1α and XBP1s are elevated in various hematological malignancies. This review paper is focused on summarizing the latest knowledge about the role of IRE1α and on the assessment of the potential utility of IRE1α inhibitors in blood cancers.

Inhibition of XBP1 Alleviates LPS-Induced Cardiomyocytes Injury by Upregulating XIAP through Suppressing the NF-κB Signaling Pathway.

Cardiomyocytes injury caused by sepsis is a complication of common clinical critical illness and an important cause of high mortality in intensive care unit (ICU) patients. Therefore, lipopolysaccharide (LPS)-induced H9c2 cells were used to simulate the cardiomyocytes injury in vitro. The aim of this study was to investigate whether X-box binding protein 1 (XBP1) exacerbated LPS-induced cardiomyocytes injury by downregulating Xlinked inhibitor of apoptosis protein (XIAP) through activating the NF-κB signaling pathway. After transfection or LPS induction, XBP1 expression was detected by RT-qPCR analysis and Western blot analysis. The viability and apoptosis of H9c2 cells was detected by MTT assay and TUNEL assay. The protein expression related to apoptosis and NF-κB signaling pathway was detected by Western blot analysis. The inflammation and oxidative stress in H9c2 cells was evaluated by their commercial kits. Dual-luciferase reporter assay and chromatin immunoprecipitation (CHIP) assay were used to determine the combination of XBP1 and XIAP. As a result, LPS promoted the XBP1 expression in H9c2 cells. XBP1 was combined with XIAP. Inhibition of XBP1 increased viability, and inhibited apoptosis, inflammation, and oxidative stress of LPS-induced H9c2 cells by suppressing the NF-κB signaling pathway, which was partially reversed by the inhibition of XIAP. In conclusion, inhibition of XBP1 alleviates LPS-induced cardiomyocytes injury by upregulating XIAP through suppressing the NF-κB signaling pathway.

The Structure, Activation and Signaling of IRE1 and Its Role in Determining Cell Fate.

Inositol-requiring enzyme type 1 (IRE1) is a serine/threonine kinase acting as one of three branches of the Unfolded Protein Response (UPR) signaling pathway, which is activated upon endoplasmic reticulum (ER) stress conditions. It is known to be capable of inducing both pro-survival and pro-apoptotic cellular responses, which are strictly related to numerous human pathologies. Among others, IRE1 activity has been confirmed to be increased in cancer, neurodegeneration, inflammatory and metabolic disorders, which are associated with an accumulation of misfolded proteins within ER lumen and the resulting ER stress conditions. Emerging evidence suggests that genetic or pharmacological modulation of IRE1 may have a significant impact on cell viability, and thus may be a promising step forward towards development of novel therapeutic strategies. In this review, we extensively describe the structural analysis of IRE1 molecule, the molecular dynamics associated with IRE1 activation, and interconnection between it and the other branches of the UPR with regard to its potential use as a therapeutic target. Detailed knowledge of the molecular characteristics of the IRE1 protein and its activation may allow the design of specific kinase or RNase modulators that may act as drug candidates.