Spliced X-box Binding Protein Research Articles

The modulation of autophagy and unfolded protein response by ent-kaurenoid derivative CPUK02 in human colorectal cancer cells.

CPUK02 (15-Oxosteviol benzyl ester) is a semi-synthetic derivative of stevioside known for its anticancer effects. It has been reported that the natural compound of stevioside and its associated derivatives enhances the sensitivity of cancer cells to conventional anti-cancer agents by inducing endoplasmic reticulum (ER) stress. In response to ER stress, autophagy and unfolded protein responses (UPR) are activated to restore cellular homeostasis. Consequently, the primary aim of this study is to investigate the impact of CPUK02 treatment on UPR and autophagy markers in two colorectal cancer cell lines. HCT116 and SW480 cell lines were treated with various concentrations of CPUK02 for 72h. The expression levels of several proteins and enzymes were evaluated to investigate the influence of CPUK02 on autophagy and UPR pathways. These include glucose-regulated protein 78 (GRP78), Inositol-requiring enzyme 1-α (IRE1-α),spliced X-box binding protein 1 (XBP-1s), protein kinase R-like ER kinase (PERK), C/EBP homologous protein (CHOP), Beclin-1, P62 and Microtubule-associated protein1 light chain 3 alpha (LC3βII). The evaluation was conducted using western blotting and quantitative real-time PCR techniques. The results obtained indicate that the treatment with CPUK02 reduced the expression of UPR markers, including GRP78 and IRE1-α at protein levels and XBP-1s, PERK, and CHOP at mRNA levels in both HCT116 and SW480 cell lines. Furthermore, CPUK02 also influenced autophagy by decreasing Beclin-1 and increasing P62 and LC3βII at mRNA levels in both HCT116 and SW480 treated cells. The study findings suggest CPUK02 may exert its cytotoxic effects by inhibiting UPR and autophagy flux in colorectal cancer cells.

Synergistic promotion of transient transgene expression in CHO cells by PDI/XBP-1s co-transfection and mild hypothermia.

Transient gene expression system is an important tool for rapid production of recombinant proteins in Chinese hamster ovary (CHO) cells. However, their low productivity is the main hurdle to overcome. An effective approach through which to obtain high protein yield involves targeting transcriptional, post-transcriptional events (PTEs), and culture conditions. Here, we investigated the effects of protein disulfide isomerase (PDI) and spliced X-box binding protein 1 (XBP-1s) co-overexpression combined with mild hypothermia on the transient yields of recombinant proteins in CHO cells. The results showed that the gene of interest (GOI) and the PDI/XBP-1s helper vector at a co-transfection ratio of 10:1 could obviously increase transient expression level of recombinant protein in CHO cells. However, PDI/XBP-1s overexpression had no significance effect on the mRNA levels of the recombinant protein, suggesting that it targeted PTEs. Moreover, the increased production was due to the enhancing of cell specific productivity, not related to cell growth, viability, and cell cycle. In addition, combined PDI/XBP-1s co-overexpression and mild hypothermia could further improve Adalimumab expression, compared to the control/37°C and PDI/XBP-1s/37°C, the Adalimumab volume yield of PDI/XBP-1s/33°C increased by 203% and 142%, respectively. Mild hypothermia resulted in 3.52- and 2.33-fold increase in the relative mRNA levels of PDI and XBP-1s, respectively. In conclusion, the combination of PDI/XBP-1s overexpression and culture temperature optimization can achieve higher transient expression of recombinant protein, which provides a synergetic strategy to improve transient production of recombinant protein in CHO cells.

Molecular mechanisms underlying TNFα-induced mitochondrial fragmentation in human airway smooth muscle cells.

Tumor necrosis factor α (TNFα), a proinflammatory cytokine, plays a significant role in mediating the effects of acute inflammation in response to allergens, pollutants, and respiratory infections. Previously, we showed that acute exposure to TNFα induces mitochondrial fragmentation in human airway smooth muscle (hASM) cells, which is associated with increased expression of dynamin-related protein 1 (DRP1). Phosphorylation of DRP1 at serine 616 (pDRP1S616) promotes its translocation and binding to the outer mitochondrial membrane (OMM) and mediates mitochondrial fragmentation. Previously, we reported that TNFα exposure triggers protein unfolding and triggers an endoplasmic reticulum (ER) stress response involving phosphorylation of inositol-requiring enzyme 1α (pIRE1α) at serine 724 (pIRE1αS724) and subsequent splicing of X-box binding protein 1 (XBP1s) in hASM cells. We hypothesize that TNFα-mediated activation of the pIRE1αS724/XBP1s ER stress pathway in hASM cells transcriptionally activates genes that encode kinases responsible for pDRP1S616 phosphorylation. Using 3-D confocal imaging of MitoTracker green-labeled mitochondria, we found that TNFα treatment for 6 h induces mitochondrial fragmentation in hASM cells. We also confirmed that 6 h TNFα treatment activates the pIRE1α/XBP1s ER stress pathway. Using in silico analysis and ChIP assay, we showed that CDK1 and CDK5, kinases involved in the phosphorylation of pDRP1S616, are transcriptionally targeted by XBP1s. TNFα treatment increased the binding affinity of XBP1s on the promoter regions of CDK1 and CDK5, and this was associated with an increase in pDRP1S616 and mitochondria fragmentation. This study reveals a new underlying molecular mechanism for TNFα-induced mitochondrial fragmentation in hASM cells.NEW & NOTEWORTHY Airway inflammation is increasing worldwide. Proinflammatory cytokines mediate an adaptive mechanism to overcome inflammation-induced cellular stress. Previously, we reported that TNFα mediates hASM cellular responses, leading to increased force and ATP consumption associated with increased O2 consumption, and oxidative stress. This study indicates that TNFα induces ER stress, which induces mitochondrial fragmentation via pIRE1αS724/XBP1s mediated CDK1/5 upregulation and pDRP1S616 phosphorylation. Mitochondrial fragmentation may promote hASM mitochondrial biogenesis to maintain healthy mitochondrial pool.

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.

Loss of function of XBP1 splicing activity of IRE1α favors B cell tolerance breakdown

Anti-nuclear antibodies are the hallmark of autoimmune diseases such as systemic lupus erythematosus (SLE) and scleroderma. However, the molecular mechanisms of B cell tolerance breakdown in these pathological contexts are poorly known. The study of rare familial forms of autoimmune diseases could therefore help to better describe common biological mechanisms leading to B cell tolerance breakdown. By Whole-Exome Sequencing, we identified a new heterozygous mutation (p.R594C) in ERN1 gene, encoding IRE1α (Inositol-Requiring Enzyme 1α), in a multiplex family with several members presenting autoantibody-mediated autoimmunity. Using human cell lines and a knock-in (KI) transgenic mouse model, we showed that this mutation led to a profound defect of IRE1α ribonuclease activity on X-Box Binding Protein 1 (XBP1) splicing. The KI mice developed a broad panel of autoantibodies, however in a subclinical manner. These results suggest that a decrease of spliced form of XBP1 (XBP1s) production could contribute to B cell tolerance breakdown and give new insights into the function of IRE1α which are important to consider for the development of IRE1α targeting strategies.

CDN1163 alleviates SERCA2 dysfunction-induced pulmonary vascular remodeling by inhibiting the phenotypic transition of pulmonary artery smooth muscle cells

ABSTRACT Background and Purpose Substitution of Cys674 (C674) in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) causes SERCA2 dysfunction which leads to activated inositol requiring enzyme 1 alpha (IRE1α) and spliced X-box binding protein 1 (XBP1s) pathway accelerating cell proliferation of pulmonary artery smooth muscle cells (PASMCs) followed by significant pulmonary vascular remodeling resembling human pulmonary hypertension. Based on this knowledge, we intend to investigate other potential mechanisms involved in SERCA2 dysfunction-induced pulmonary vascular remodeling. Experimental Approach Heterozygous SERCA2 C674S knock-in (SKI) mice of which half of cysteine in 674 was substituted by serine to mimic the partial irreversible oxidation of C674 were used. The lungs of SKI mice and their littermate wild-type mice were collected for PASMC culture, protein expression, and pulmonary vascular remodeling analysis. Results SERCA2 dysfunction increased intracellular Ca2+ levels, which activated Ca2+-dependent calcineurin (CaN) and promoted the nuclear translocation and protein expression of the nuclear factor of activated T-lymphocytes 4 (NFAT4) in an IRE1α/XBP1s pathway-independent manner. In SKI PASMCs, the scavenge of intracellular Ca2+ by BAPTA-AM or inhibition of CaN by cyclosporin A can prevent PASMC phenotypic transition. CDN1163, a SERCA2 agonist, suppressed the activation of CaN/NFAT4 and IRE1α/XBP1s pathways, reversed the protein expression of PASMC phenotypic transition markers and cell cycle-related proteins, and inhibited cell proliferation and migration when given to SKI PASMCs. Furthermore, CDN1163 ameliorated pulmonary vascular remodeling in SKI mice. Conclusions and Implications SERCA2 dysfunction promotes PASMC phenotypic transition and pulmonary vascular remodeling by multiple mechanisms, which could be improved by SERCA2 agonist CDN1163.

XBP1 splicing contributes to endoplasmic reticulum stress-induced human islet amyloid polypeptide up-regulation

As a pathological hallmark of type 2 diabetes, islet amyloid is formed by the aggregation of islet amyloid polypeptide (IAPP). Endoplasmic reticulum (ER) stress interacts with IAPP aggregates and has been implicated in the pathogenesis of type 2 diabetes. To examine the role of ER stress in type 2 diabetes, we cloned the hIAPP promoter and analyzed its promoter activity in human β-cells. We found that ER stress significantly enhanced hIAPP promoter activity and expression in human β-cells via triggering X-box binding protein 1 (XBP1) splicing. We identified a binding site of XBP1 in the hIAPP promoter. Disruption of this binding site by substitution or deletion mutagenesis significantly diminished the effects of ER stress on hIAPP promoter activity. Blockade of XBP splicing by MKC3946 treatment inhibited ER stress-induced hIAPP up-regulation and improved human β-cell survival and function. Our study uncovers a link between ER stress and IAPP at the transcriptional level and may provide novel insights into the role of ER stress in IAPP cytotoxicity and the pathogenesis of type 2 diabetes.

SIRT1-dependent deacetylation of Txnip H3K9ac is critical for exenatide-improved diabetic kidney disease

Glucagon-like peptide 1 receptor agonist exenatide (exendin-4) has potential protective capabilities against diabetic kidney disease (DKD). However, the underlying mechanism has not been fully elucidated. The expression of thioredoxin-interacting protein (Txnip) is upregulated during DKD progression by histone acetylation. Sirtuin 1 (SIRT1) is a deacetylase and is decreased in DKD, which indicates that it may regulate Txnip in this disease. Here, we used whole-body heterozygous Sirt1 knockout (Sirt1+/-) and kidney-specific Sirt1 knockout (KSK) mice to investigate whether SIRT1 regulates Txnip via histone deacetylation in DKD and exenatide-alleviated DKD. Exenatide substantially improved renal pathological damage, decreased the albumin-to-creatinine ratio (ACR), upregulated SIRT1 expression, and downregulated Txnip expression in kidneys of high-fat diet-treated C57BL/6J mice. However, these effects diminished in Sirt1+/- and KSK mice under exenatide treatment. The downregulation of Txnip expression by exendin-4 in high-glucose-treated SV40 MES13 cells was hampered during Sirt1 knockdown. These results demonstrate that kidney SIRT1 is indispensable in exenatide-improved DKD and downregulation of Txnip expression. Exendin-4 mechanistically downregulated Txnip histone 3 lysine 9 acetylation (H3K9ac) in a SIRT1-dependent manner and decreased spliced X-box binding protein 1 (XBP1s) recruitment to the Txnip promoter. These findings provide epigenetic evidence elucidating the specific mechanism for exenatide-mediated DKD alleviation and highlight the importance of Txnip as a promising therapeutic target for DKD.

The expression of lncRNAs CASC2, NEAT1, LINC00299 in breast cancer tissues and their relationship with the XBP1 splicing rate in Iranian patients during 2014-2019: A cross-sectional study.

Breast cancer is a leading cause of incidence and mortality in women globally. Identifying new molecular markers can aid in cancer diagnosis, targeted therapy, and treatment monitoring. This study aimed to measure the expression of the X-box binding protein 1 (XBP1) gene, an index of the unfolded protein response (UPR), and long noncoding RNAs (lncRNAs), including Nuclear Enriched Abundant Transcript 1 (NEAT1), Cancer Susceptibility Candidate 2 (CASC2), and Long Intergenic Nonprotein Coding RNA 299 (LINC00299), as possible regulators of the UPR pathway. Total RNA was extracted from 40 samples of breast tumor tissues and their respective controls. The expression level of lncRNAs CASC2, NEAT1, and LINC00299 was quantified using reverse transcription-polymerase chain reaction (RT-PCR). The ratio of the spliced form of XBP1 to its unspliced form (XBP1u) was determined by PCR and electrophoresis. The results showed a 2.8-fold increase in the ratio of XBP1s/u in breast cancer tissues compared to adjacent nonmalignant samples (p < 0.05). Additionally, the level of lncRNAs NEAT1, CASC2, and LINC00299 in breast tumor tissues increased significantly by twofold, 1.5-fold, and 2.3-fold, respectively, compared to adjacent nonmalignant samples (p < 0.05). Based on the association between the expression of lncRNAs CASC2, LINC00299, and NEAT1 and the XBP1s/u ratio, these lncRNAs could be potential regulators of the UPR pathway. Also, CASC2 and NEAT1 genes could be suggested as suitable biomarkers to distinguish cancerous tissue from noncancerous breast tissue due to their significant increase in expression in cancerous samples compared to adjacent noncancerous.

Downregulation of XBP1s aggravates lipopolysaccharide-induced inflammation by promoting NF-κB and NLRP3 pathways’ activation in goat endometrial epithelial cells

After delivery, bacterial contamination and uterine tissue degeneration in animals can lead to the development of uterine diseases, such as endometritis, accompanied by endoplasmic reticulum stress (ERS). Increasing evidence suggests that spliced X-box binding protein 1 (XBP1s), a critical component of ERS, is involved in several pathological processes in various organisms. However, the specific molecular mechanisms by which XBP1s mediates the inflammatory response in goat endometrial epithelial cells (gEECs) remain largely unknown. In the present study, XBP1s protein was induced into the nucleus in the lipopolysaccharide (LPS, 5 μg/mL)-induced inflammatory response of gEECs. Lipopolysaccharide-induced expression and nucleation of XBP1s were reduced by the inhibition of Toll-like receptor 4 (TLR4) using TAK-242 (1 μM; a TLR4 inhibitor). Expression and nucleation of XBP1s were similarly reduced when the activity of inositol-requiring enzyme 1α (IRE1α) was inhibited using 4μ8C (10 μM; an IRE1α inhibitor). In addition, inhibition of IRE1a increased IL-1β, TNF-α, and IL-8 levels and secretion of IL-6 induced by LPS. Notably, phosphorylation of nuclear factor kappa-B (NF-κB) P65 protein and expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) were similarly increased. Furthermore, knockdown of XBP1s in gEECs consistently promoted NF-κB P65 protein phosphorylation, NLRP3 protein expression, and inflammatory cytokine secretion. In summary, the current results suggest that in the LPS-induced inflammatory response in gEECs, LPS generates intracellular signaling cascades in gEECs via TLR4, which may promote XBP1s protein expression and nucleation by activating IRE1a. However, downregulation of XBP1s expression exacerbates inflammation by promoting activation of the NF-κB and NLRP3 inflammatory vesicle pathways. These results will potentially contribute to the treatment and prevention of endometritis in ruminants.

The UFMylated ribosome-recognition protein SAYSD1 is predominantly expressed in spermatids but is dispensable for fertility in mice

SAYSVFN domain-containing protein 1 (SAYSD1) is an evolutionarily conserved membrane protein that has recently been identified as a ubiquitin-fold modifier 1 (UFM1)-conjugated ribosome-recognition protein that plays a critical role in translocation-associated quality control (TAQC). However, its expression and roles in mammals in vivo remain largely unknown. We found that SAYSD1 is predominantly expressed in round and elongating spermatids and localizes in the endoplasmic reticulum (ER) of mouse testes, but not in differentiated spermatozoa. Mice deficient in Saysd1 developed normally post-partum. Furthermore, Saysd1-deficient mice were fertile, with no apparent differences in sperm morphology or motility compared with wild-type sperm, although the cauda epididymis contained slightly less sperm. Expression of the ER stress markers spliced X-box binding protein 1s (XBP1s) and CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP) in the testes was comparable between Saysd1-deficient and wild-type mice. These results suggested that SAYSD1 is involved in sperm production in mice but is dispensable for their development and fertility.

A decrease of mitochondrial ubiquitin ligase increases the secretion of matrix metalloproteinase-1 by dermal fibroblasts through the induction of ER stress.

We previously reported that the level of mitochondrial ubiquitin ligase (MITOL) protein in fibroblasts was decreased by UVA and that the knock-down (KD) of MITOL increased the secretion of matrix metalloprotease-1 (MMP-1) by fibroblasts. A recent study reported that MITOL suppresses endoplasmic reticulum (ER) stress by stabilizing the interaction between ER and mitochondria (MT) through the ubiquitination of mitofusin 2. These facts suggest that a decrease of MITOL would increase the secretion of MMP-1 through ER stress, but the detailed mechanism of that process in dermal fibroblasts remains unclear. Thus, this study was conducted to clarify the involvement of ER stress in the oversecretion of MMP-1 induced by the decreased MT quality caused by MITOL-KD. MITOL-KD normal human dermal fibroblast (NHDFs) were prepared by treating them with MITOL-small interfering RNA, after which their MMP-1 protein levels were measured. ER stress in NHDFs was evaluated by measuring the mRNA levels of spliced X-box binding protein 1 (sXBP1) and the protein levels of inositol-requiring enzyme 1α (IRE1α). MITOL-KD NHDFs enhanced the secretion of MMP-1 via interleukin-6 (IL-6) elicited by the activation of nuclear factor-kappa B (NF-κB). The secretion of MMP-1 could be abrogated by a neutralizing IL-6 antibody and by JSH23, which is an inhibitor of NF-κB activation. Furthermore, MITOL-KD NHDFs as well as UVA-irradiated NHDFs showed increased ER stress levels. In addition, tunicamycin, which is an inducer of ER stress, also increased MMP-1 secretion. These results suggested that the decrease of MITOL caused the oversecretion of MMP-1 via NF-κB-IL-6 signaling through the activation of ER stress in fibroblasts.

ROS scavenger TEMPOL mitigates TNF alpha induced ER stress in hASM cells

The impact of airway inflammation is mediated by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα), which underlies several respiratory diseases, including COVID-19, asthma, chronic bronchitis, and chronic obstructive pulmonary disease (COPD). In human airway smooth muscle (hASM) cells, exposure to TNFα leads to an accumulation of unfolded proteins in the ER, which triggers a homeostatic ER stress response activating a signaling cascade to restore normal function. Previously, we showed that TNFα selectively activates the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 (XBP1) ER stress pathway in hASM cells. Most likely, the protein unfolding in hASM cells caused by TNFα results from an increase in reactive oxygen species (ROS) formation. We hypothesize that ER stress induced by TNFα is mitigated by the ROS scavenger TEMPOL. Primary hASM cells were isolated from bronchiolar tissue samples obtained from 5 patients (both sexes included) who had no history of respiratory diseases. hASM cells were serum deprived for 48 h before treatment with 1) TNFα (20 ng/ml) for 12 h; 2) TEMPOL (500 μM) for 30 min followed by TNFα (20 ng/ml) for 12 h; and 3) untreated control for 12 h. At the end of the treatment period, hASM cells were processed for determination of protein expression through Western blotting. Activation of the ER stress response was assessed by phosphorylation of IRE1α and splicing of XBP1. Confirming our previous results, we found that 12 h TNFα exposure induced an ER stress response in hASM cells, evidence by phosphorylation of IRE1α and XBP1 splicing. Treating hASM cells with TEMPOL before exposure to TNFα mitigated the ER stress response. These results support our hypothesis and indicate that ROS scavenging prevents TNFα induced protein unfolding and triggering of the IRE1α/XBP1 ER stress pathway. R01HL 157984 (GCS) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

β-Cell glucokinase expression was increased in type 2 diabetes subjects with better glycemic control.

Type 2 diabetes (T2D) is characterized by a progressive deterioration of β-cell function with a continuous decline in insulin secretion. Glucokinase (GCK) facilitates the rate-limiting step of glycolysis in pancreatic β-cells, to acquire the proper glucose-stimulated insulin secretion. Multiple glucokinase activators (GKAs) have been developed and clinically tested. However, the dynamic change of human pancreatic GCK expression during T2D progression has not been investigated. We evaluated GCK expression by measuring the average immunoreactivity of GCK in insulin+ or glucagon+ cells from pancreatic sections of 11 nondiabetic subjects (ND), 10 subjects with impaired fasting glucose (IFG), 9 with well-controlled T2D (wT2D), and 5 individuals with poorly controlled T2D (uT2D). We also assessed the relationship between GCK expression and adaptive unfolded protein response (UPR) in human diabetic β-cells. We did not detect changes of GCK expression in IFG islets. However, we found β-cell GCK levels were significantly increased in T2D with adequate glucose control (wT2D) but not in T2D with poor glucose control (uT2D). Furthermore, there was a strong positive correlation between GCK expression and adaptive UPR (spliced X-box binding protein 1 [XBP1s] and activating transcription factor 4 [ATF4]), as well as functional maturity marker (urocortin-3 [UCN3]) in human diabetic β-cells. Our study demonstrates that inductions of GCK enhanced adaptive UPR and UCN3 in human β-cells, which might be an adaptive mechanism during T2D progression. This finding provides a rationale for exploring novel molecules that activate β-cell GCK and thereby improve pharmacological treatment of T2D.

Targeting XBP1 mRNA splicing sensitizes glioblastoma to chemotherapy.

Glioblastoma (GBM) is the most frequent and deadly primary brain tumor in adults. Temozolomide (TMZ) is the standard systemic therapy in GBM but has limited and restricted efficacy. Better treatments are urgently needed. The role of endoplasmic reticulum stress (ER stress) is increasingly described in GBM pathophysiology. A key molecular mediator of ER stress, the spliced form of the transcription factor x-box binding protein 1 (XBP1s) may constitute a novel therapeutic target; here we report XBP1s expression and biological activity in GBM. Tumor samples from patients with GBM (n= 85) and low-grade glioma (n= 20) were analyzed by immunohistochemistry for XBP1s with digital quantification. XBP1s expression was significantly increased in GBM compared to low-grade gliomas. XBP1s mRNA showed upregulation by qPCR analysis in a panel of patient-derived GBM cell lines. Inhibition of XBP1 splicing using the small molecular inhibitor MKC-3946 significantly reduced GBM cell viability and potentiated the effect of TMZ in GBM cells, particularly in those with methylated O6-methylguanine-DNA methyl transferase gene promoter. GBM cells resistant to TMZ were also responsive to MKC-3946 and the long-term inhibitory effect of MKC-3946 was confirmed by colony formation assay. In conclusion, this data reveals that XBP1s is overexpressed in GBM and contributes to cancer cell growth. XBP1s warrants further investigation as a clinical biomarker and therapeutic target in GBM.

Sustained overexpression of spliced X-box-binding protein-1 in neurons leads to spontaneous seizures and sudden death in mice

The underlying etiologies of seizures are highly heterogeneous and remain incompletely understood. While studying the unfolded protein response (UPR) pathways in the brain, we unexpectedly discovered that transgenic mice (XBP1s-TG) expressing spliced X-box–binding protein-1 (Xbp1s), a key effector of UPR signaling, in forebrain excitatory neurons, rapidly develop neurologic deficits, most notably recurrent spontaneous seizures. This seizure phenotype begins around 8 days after Xbp1s transgene expression is induced in XBP1s-TG mice, and by approximately 14 days post induction, the seizures evolve into status epilepticus with nearly continuous seizure activity followed by sudden death. Animal death is likely due to severe seizures because the anticonvulsant valproic acid could significantly prolong the lives of XBP1s-TG mice. Mechanistically, our gene profiling analysis indicates that compared to control mice, XBP1s-TG mice exhibit 591 differentially regulated genes (mostly upregulated) in the brain, including several GABAA receptor genes that are notably downregulated. Finally, whole-cell patch clamp analysis reveals a significant reduction in both spontaneous and tonic GABAergic inhibitory responses in Xbp1s-expressing neurons. Taken together, our findings unravel a link between XBP1s signaling and seizure occurrence.

The activation of spliced X-box binding protein 1 by isorhynchophylline therapy improves diabetic encephalopathy.

The primary symptom of diabetic encephalopathy (DE), a kind of central diabetic neuropathy caused by diabetes mellitus (DM), is cognitive impairment. In addition, the tetracyclic oxindole alkaloid isorhynchophylline (IRN) helps lessen cognitive impairment. However, it is still unclear how IRN affects DM and DE and what mechanisms are involved. The effectiveness of IRN on brain insulin resistance was carefully examined in this work, both in vitro and in vivo. We found that IRN accelerates spliced form of X-box binding protein 1 (sXBP1) translocation into the nucleus under high glucose conditions in vitro. IRN also facilitates the nuclear association of pCREB with sXBP1 and the binding of regulatory subunits of phosphatidylinositol 3-kinase (PI3K) p85α or p85β with XBP1 to restore high glucose impairment. Also, IRN treatment improves high glucose-mediated impairment of insulin signaling, endoplasmic reticulum stress, and pyroptosis/apoptosis by depending on sXBP1 in vitro. In vivo studies suggested that IRN attenuates cognitive impairment, ameliorating peripheral insulin resistance, activating insulin signaling, inactivating activating transcription factor 6 (ATF6) and C/EBP homology protein (CHOP), and mitigating pyroptosis/apoptosis by stimulation of sXBP1 nuclear translocation in the brain. In summary, these data indicate that IRN contributes to maintaining insulin homeostasis by activating sXBP1 in the brain. Thus, IRN is a potent antidiabetic agent as well as an sXBP1 activator that has promising potential for the prevention or treatment of DE.

Molecular Mechanism Underlying Role of the XBP1s in Cardiovascular Diseases.

Spliced X-box binding protein-1 (XBP1s) is a protein that belongs to the cAMP-response element-binding (CREB)/activating transcription factor (ATF) b-ZIP family with a basic-region leucine zipper (bZIP). There is mounting evidence to suggest that XBP1s performs a critical function in a range of different cardiovascular diseases (CVDs), indicating that it is necessary to gain a comprehensive knowledge of the processes involved in XBP1s in various disorders to make progress in research and clinical therapy. In this research, we provide a summary of the functions that XBP1s performs in the onset and advancement of CVDs such as atherosclerosis, hypertension, cardiac hypertrophy, and heart failure. Furthermore, we discuss XBP1s as a novel therapeutic target for CVDs.

Spliced X-Box binding protein 1 predicts satisfying responsiveness and survival benefit toward bortezomib-based therapy in multiple myeloma patients

ABSTRACT Objective Spliced X-Box binding protein 1 (sXBP1) modulates malignant cell activities and enhances the bortezomib sensitivity in multiple myeloma (MM) cells, while its clinical value in MM patients remains elusive. Hence, the current study aimed to explore this issue, particularly the correlation of sXBP1 with treatment outcomes of bortezomib-based therapy in MM patients. Methods Totally, 97 newly-diagnosed MM patients undergoing bortezomib-based therapy, 20 disease controls (DCs), and 20 health controls (HCs) were enrolled. Bone marrow plasma cell samples were acquired to determine sXBP1 by RT-qPCR. Results sXBP1 was lowest in MM patients, followed by DCs, and highest in HCs (P < 0.001). Beyond that, sXBP1 discriminated MM patients from DCs with area under curve (AUC) of 0.728 (95% confidence interval (CI): 0.610–0.847) and HCs with AUC of 0.855 (95% CI: 0.771–0.939). sXBP1 was negatively associated with t (4; 14) (P = 0.047), Revised International Staging System stage (P = 0.008). There was a trend that sXBP1 was negatively correlated with β2-MG, LDH, and t (14; 16) (without statistical significance). sXBP1 was higher in patients with complete response (CR) compared to those with non-CR (P = 0.017) and higher in patients with objective response rate (ORR) compared to those with non-ORR (P = 0.006). sXBP1 (high vs. low) was linked with longer progression-free survival (PFS) (P = 0.011) and overall survival (P = 0.045) in MM patients. Additionally, sXBP1 (high vs. low) (P = 0.025) independently estimated a longer PFS. Conclusion sXBP1 forecasts a favorable treatment response and survival benefit toward bortezomib-based therapy in multiple myeloma patients.

Aging Impairs Adaptive Unfolded Protein Response and Drives Beta Cell Dedifferentiation in Humans.

Diabetes is an age-related disease; however, the mechanism underlying senescent beta cell failure is still unknown. The present study was designed to investigate whether and how the differentiated state was altered in senescent human beta cells by excluding the effects of impaired glucose tolerance. We calculated the percentage of hormone-negative/chromogranin A-positive endocrine cells and evaluated the expressions of forkhead box O1 (FoxO1) and Urocortin 3 (UCN3) in islets from 31 nondiabetic individuals, divided into young (<40 years), middle-aged (40-60 years) and elderly (>60 years) groups. We also assessed adaptive unfolded protein response markers glucose-regulated protein 94 (GRP94), and spliced X-box binding protein 1 (XBP1s) in senescent beta cells and their possible contributions to maintaining beta cell identity and differentiation state. We found an almost 2-fold increase in the proportion of dedifferentiated cells in elderly and middle-aged groups compared with the young group (3.1 ± 1.0% and 3.0 ± 0.9% vs 1.7 ± 0.5%, P < .001). This was accompanied by inactivation of FoxO1 and loss of UCN3 expression in senescent human beta cells. In addition, we demonstrated that the expression levels of adaptive unfolded protein response (UPR) components GRP94 and XBP1s declined with age. In vitro data showed knockdown GRP94 in Min6-triggered cells to dedifferentiate and acquire progenitor features, while restored GRP94 levels in H2O2-induced senescent Min6 cells rescued beta cell identity. Our finding highlights that the failure to establish proper adaptive UPR in senescent human beta cells shifts their differentiated states, possibly representing a crucial step in the pathogenesis of age-related beta cell failure.