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Acute kidney injury (AKI) is closely related to lysosomal dysfunction and ferroptosis in renal tubular epithelial cells (TECs), for which effective treatments are urgently needed. Although selenium nanoparticles (SeNPs) have emerged as promising candidates for AKI therapy, their underlying mechanisms have not been fully elucidated. Here, we investigated the effect of SeNPs on hypoxia/reoxygenation (H/R)-induced ferroptosis and lysosomal dysfunction in TECs in vitro and evaluated their efficacy in a murine model of ischemia/reperfusion (I/R)-AKI. We observed that H/R-induced ferroptosis was accompanied by lysosomal Fe2+ accumulation and dysfunction in TECs, which was ameliorated by SeNPs administration. Furthermore, SeNPs protected C57BL/6 mice against I/R-induced inflammation and ferroptosis. Mechanistically, we found that lysosomal Fe2+ accumulation and ferroptosis were associated with the excessive activation of NCOA4-mediated ferritinophagy, a process mitigated by SeNPs through the upregulation of X-box binding protein 1 (XBP1). Downregulation of XBP1 promoted ferritinophagy and partially counteracted the protective effects of SeNPs on ferroptosis inhibition in TECs. Overall, our findings revealed a novel role for SeNPs in modulating ferritinophagy, thereby improving lysosomal function and attenuating ferroptosis of TECs in I/R-AKI. These results provide evidence for the potential application of SeNPs as therapeutic agents for the prevention and treatment of AKI. [ABSTRACT FROM AUTHOR]

Abstract Acute kidney injury (AKI) is closely related to lysosomal dysfunction and ferroptosis in renal tubular epithelial cells (TECs), for which effective treatments are urgently needed. Although selenium nanoparticles (SeNPs) have emerged as promising candidates for AKI therapy, their underlying mechanisms have not been fully elucidated. Here, we investigated the effect of SeNPs on hypoxia/reoxygenation (H/R)-induced ferroptosis and lysosomal dysfunction in TECs in vitro and evaluated their efficacy in a murine model of ischemia/reperfusion (I/R)-AKI. We observed that H/R-induced ferroptosis was accompanied by lysosomal Fe2+ accumulation and dysfunction in TECs, which was ameliorated by SeNPs administration. Furthermore, SeNPs protected C57BL/6 mice against I/R-induced inflammation and ferroptosis. Mechanistically, we found that lysosomal Fe2+ accumulation and ferroptosis were associated with the excessive activation of NCOA4-mediated ferritinophagy, a process mitigated by SeNPs through the upregulation of X-box binding protein 1 (XBP1). Downregulation of XBP1 promoted ferritinophagy and partially counteracted the protective effects of SeNPs on ferroptosis inhibition in TECs. Overall, our findings revealed a novel role for SeNPs in modulating ferritinophagy, thereby improving lysosomal function and attenuating ferroptosis of TECs in I/R-AKI. These results provide evidence for the potential application of SeNPs as therapeutic agents for the prevention and treatment of AKI.

Inositol requiring enzyme 1 (IRE1) is generally thought to control the most conserved pathway in the unfolded protein response (UPR). Two isoforms of IRE1, IRE1α and IRE1β, have been reported in mammals. IRE1α is a ubiquitously expressed protein whose knockout shows marked lethality. In contrast, the expression of IRE1β is exclusively restricted in the epithelial cells of the respiratory and gastrointestinal tracts, and IRE1β-knockout mice are phenotypically normal. As research continues to deepen, IRE1α was showed to be tightly linked to inflammation, lipid metabolism regulation, cell death and so on. Growing evidence also suggests an important role for IRE1α in promoting atherosclerosis (AS) progression and acute cardiovascular events through disrupting lipid metabolism balance, facilitating cells apoptosis, accelerating inflammatory responses and promoting foam cell formation. In addition, IRE1α was recognized as novel potential therapeutic target in AS prevention. This review provides some clues about the relationship between IRE1α and AS, hoping to contribute to further understanding roles of IRE1α in atherogenesis and to be helpful for the design of novel efficacious therapeutics agents targeting IRE1α-related pathways. [ABSTRACT FROM AUTHOR]

Immune cells must be able to adjust their metabolic programs to effectively carry out their effector functions. Here, we show that the endoplasmic reticulum (ER) stress sensor Inositol-requiring enzyme 1 alpha (IRE1α) and its downstream transcription factor X box binding protein 1 (XBP1) enhance the upregulation of glycolysis in classically activated macrophages (CAMs). The IRE1α-XBP1 signaling axis supports this glycolytic switch in macrophages when activated by lipopolysaccharide (LPS) stimulation or infection with the intracellular bacterial pathogen Brucella abortus. Importantly, these different inflammatory stimuli have distinct mechanisms of IRE1α activation; while Toll-like receptor 4 (TLR4) supports glycolysis under both conditions, TLR4 is required for activation of IRE1α in response to LPS treatment but not B. abortus infection. Though IRE1α and XBP1 are necessary for maximal induction of glycolysis in CAMs, activation of this pathway is not sufficient to increase the glycolytic rate of macrophages, indicating that the cellular context in which this pathway is activated ultimately dictates the cell's metabolic response and that IRE1α activation may be a way to fine-tune metabolic reprogramming. IMPORTANCE The immune system must be able to tailor its response to different types of pathogens in order to eliminate them and protect the host. When confronted with bacterial pathogens, macrophages, frontline defenders in the immune system, switch to a glycolysis-driven metabolism to carry out their antibacterial functions. Here, we show that IRE1α, a sensor of ER stress, and its downstream transcription factor XBP1 support glycolysis in macrophages during infection with Brucella abortus or challenge with Salmonella LPS. Interestingly, these stimuli activate IRE1α by independent mechanisms. While the IRE1α-XBP1 signaling axis promotes the glycolytic switch, activation of this pathway is not sufficient to increase glycolysis in macrophages. This study furthers our understanding of the pathways that drive macrophage immunometabolism and highlights a new role for IRE1α and XBP1 in innate immunity.

Objective To detect the expressions of X-box binding protein 1 unspliced （xbp1u） and X-box binding protein 1 spliced （xbp1s） mRNA in peripheral blood CD19+B cells of patients with lupus nephritis （LN） and healthy subjects and explore the clinical significance of XBP1 in LN patients. Methods The expression levels of xbp1u and xbp1s in peripheral blood CD19+B cells of 65 LN patients and 30 healthy controls were detected by real-time polymerase chain reaction （RT-PCR）. And the ratio of CD19−CD138+plasma cells/CD19+B cells in peripheral blood was detected by flow cytometry. Results The expression level of xbp1u mRNA in CD19+B cells of active LN group （0.078±0.034） was higher than that in control group（0.035±0.011）（P＜0.05） and in stable group（0.049±0.016）（P＜0.05）. The expression level of xbp1s mRNA in CD19+B cells of active LN group（0.076±0.037）was higher than that in control group（0.022±0.010）（P＜0.05） and stable group（0.043±0.017）（P＜0.05）. The ratio of CD19−CD138+ plasma cells /CD19+B cells in LN group（0.059±0.024）was significantly higher than that in control group （P＜0.01） and stable group （0.073±0.036）（P＜0.05）; XBP1 expression in LN patients was correlated positively with the ratio of CD19−CD138+ plasma cells/CD19+B cells （r value = 0.138 & 0.036）, positively with anti-ds-DNA antibody and systemic lupus erythematosus disease activity index（SLEDAI）score （r value = 0.417 & 0.058） and negatively with hemoglobin and complement C3 （r value = −0.559 & −0.219）. Conclusion The expressions of xbp1u and xbp1s mRNA in peripheral blood mononuclear cells of LN patients in active group are higher than those in healthy group. Both are correlated positively with SLEDAI score and anti-DS DNA antibody. The expression of XBP1 is up-regulated in B cells of LN patients and it is significantly correlated with plasma cells. It implies that XBP1 may participate in the pathogenesis of LN through promoting the differentiation of B cells.

Aging is a major risk factor to develop neurodegenerative diseases and is associated with decreased buffering capacity of the proteostasis network. We investigated the significance of the unfolded protein response (UPR), a major signaling pathway activated to cope with endoplasmic reticulum (ER) stress, in the functional deterioration of the mammalian brain during aging. We report that genetic disruption of the ER stress sensor IRE1 accelerated age-related cognitive decline. In mouse models, overexpressing an active form of the UPR transcription factor XBP1 restored synaptic and cognitive function, in addition to reducing cell senescence. Proteomic profiling of hippocampal tissue showed that XBP1 expression significantly restore changes associated with aging, including factors involved in synaptic function and pathways linked to neurodegenerative diseases. The genes modified by XBP1 in the aged hippocampus where also altered. Collectively, our results demonstrate that strategies to manipulate the UPR in mammals may help sustain healthy brain aging.

Introduction: The gut microbiota plays an important role in the development of non-alcoholic steatohepatitis (NASH), but the underlying mechanism is unclear. It has been found that the transcription factor XBP1s plays an important role in regulating inflammation and lipid metabolism and maintaining the integrity of intestinal barrier. However, whether XBP1s modulates the development of NASH by regulating the integrity of the intestinal barrier and altering the composition of the gut microbiota remains unknown. Methods: Mice fed with a fat-, fructose-, cholesterol-rich (FFC) diet for 24 weeks successfully established the NASH model, as demonstrated by significant hepatic steatosis, inflammation, hepatocyte injury and fibrosis. The profile of gut microbiota dynamically changed with the dierent stages of NAFLD via 16S rDNA sequencing the feces from mice fed with FFC diet for 0, 12, or 24 weeks or NASH mice treated with siRNA-loaded folic acid-modified TPGS (hereafter named FT@XBP1). Results: NASH mice had significantly higher abundance of Firmicutes, Blautia and Bacteroides, and lower abundance of Bifidobacterium and GCA-900066575. FT@XBP1 supplementation had a significantly attenuated eect on FFC dietinduced weight gain, hepatic fat accumulation, dyslipidemia, inflammatory cytokines, ER stress and fibrosis. In particularly, FT@XBP1 modulates the composition of the intestinal flora; for example, NASH mice demonstrated higher abundance of Blautia and Bacteroides, and lower abundance of Actinobacteriota, Muribaculaceae and Bifidobacterium, which were partially restored by FT@XBP1 treatment. Mechanistically, FT@XBP1 increased the expression of ZO-1 in the intestine and had the potential to restore intestinal barrier integrity and improve antimicrobial defense to alleviate enterogenic endotoxemia and activation of inflammatory signaling pathways. Discussion: Regulation of the key transcription factor XBP1s can partially restore the intestinal microbiota structure, maintain the integrity of intestinal mucosal barrier, and prevent the progression of NASH, providing new evidence for treating NASH [ABSTRACT FROM AUTHOR]

Estrogen receptor α (ERα) is a hormone receptor and key driver for over 70% of breast cancers that has been studied for decades as a transcription factor. Unexpectedly, we discover that ERα is a potent non-canonical RNA-binding protein. We show that ERα RNA binding function is uncoupled from its activity to bind DNA and critical for breast cancer progression. Employing genome-wide cross-linking immunoprecipitation (CLIP) sequencing and a functional CRISPRi screen, we find that ERα-associated mRNAs sustain cancer cell fitness and elicit cellular responses to stress. Mechanistically, ERα controls different steps of RNA metabolism. In particular, we demonstrate that ERα RNA binding mediates alternative splicing of XBP1 and translation of the eIF4G2 and MCL1 mRNAs, which facilitates survival upon stress conditions and sustains tamoxifen resistance of cancer cells. ERα is therefore a multifaceted RNA-binding protein, and this activity transforms our knowledge of post-transcriptional regulation underlying cancer development and drug response.

X-box binding protein-1 (XBP1) is a transcription factor that plays a central role in controlling cellular responses to endoplasmic reticulum (ER) stress. Under stress conditions, the transcriptionally active form of XBP1 is generated via splicing of Xbp1 mRNA by the ER-resident protein inositol-requiring enzyme-1 (IRE1α). Genetic deletion of XBP1 has multiple consequences: some resulting from the loss of the transcription factor per se, and others related to compensatory activation of IRE1α. The objective of the current study was to investigate the effects of XBP1 deletion in adult mouse liver and determine to what extent they are direct or indirect. XBP1 was deleted from hepatocytes in adult Xbp1fl/fl mice using AAV8-Transthyretin-Cre (Xbp1Δhep). Xbp1Δhep mice exhibited no liver disease at baseline, but developed acute biochemical and histologic liver injury in response to a dietary challenge with fructose for 4 weeks. Fructose-mediated liver injury in Xbp1Δhep mice coincided with heightened IRE1α activity, as demonstrated by Xbp1 mRNA splicing, JNK activation, and regulated IRE1α-dependent RNA decay (RIDD). Activation of eIF2α was also evident, with associated up-regulation of the pro-apoptotic molecules CHOP, BIM, and PUMA. To determine whether the adverse consequences of liver-specific XBP1 deletion were due to XBP1 loss or heightened IRE1α activity, we repeated a fructose challenge in mice with liver-specific deletion of both XBP1 and IRE1α (Xbp1Δhep;Ire1aΔhep). Xbp1Δhep;Ire1aΔhep mice were protected from fructose-mediated liver injury and failed to exhibit any of the signs of ER stress seen in mice lacking XBP1 alone. The protective effect of IRE1α deletion persisted even with long-term exposure to fructose. Xbp1Δhep mice developed liver fibrosis at 16 weeks, but Xbp1Δhep;Ire1aΔhep mice did not. Overall, the results indicate that the deleterious effects of hepatocyte-specific XBP1 deletion are due primarily to hyperactivation of IRE1α. They support further exploration of IRE1α as a contributor to acute and chronic liver diseases.

The α6β4 nicotinic acetylcholine receptor (nAChR) is enriched in dorsal root ganglia neurons and is an attractive non-opioid therapeutic target for pain. However, difficulty expressing human α6β4 receptors in recombinant systems has precluded drug discovery. Here, genome-wide screening identified accessory proteins that enable reconstitution of human α6β4 nAChRs. BARP, an auxiliary subunit of voltage-dependent calcium channels, promoted α6β4 surface expression while IRE1α, an unfolded protein response sensor, enhanced α6β4 receptor assembly. Effects on α6β4 involve BARPs N-terminal region and IRE1αs splicing of XBP1 mRNA. Furthermore, clinical efficacy of nicotinic agents in relieving neuropathic pain best correlated with their activity on α6β4. Finally, BARP-knockout, but not NACHO-knockout mice lacked nicotine-induced antiallodynia, highlighting the functional importance of α6β4 in pain. These results identify roles for IRE1α and BARP in neurotransmitter receptor assembly and unlock drug discovery for the previously elusive α6β4 receptor.

Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) stress caused by cellular oncogene activation and a hostile tumor microenvironment (TME). The key UPR sensor IRE1α resides in the ER and deploys a cytoplasmic kinase-endoribonuclease module to activate the transcription factor XBP1s, which facilitates ER-mediated protein folding. Studies of triple-negative breast cancer (TNBC)-a highly aggressive malignancy with a dismal posttreatment prognosis-implicate XBP1s in promoting tumor vascularization and progression. However, it remains unknown whether IRE1α adapts the ER in TNBC cells and modulates their TME, and whether IRE1α inhibition can enhance antiangiogenic therapy-previously found to be ineffective in patients with TNBC. To gauge IRE1α function, we defined an XBP1s-dependent gene signature, which revealed significant IRE1α pathway activation in multiple solid cancers, including TNBC. IRE1α knockout in TNBC cells markedly reversed substantial ultrastructural expansion of their ER upon growth in vivo. IRE1α disruption also led to significant remodeling of the cellular TME, increasing pericyte numbers while decreasing cancer-associated fibroblasts and myeloid-derived suppressor cells. Pharmacologic IRE1α kinase inhibition strongly attenuated growth of cell line-based and patient-derived TNBC xenografts in mice and synergized with anti-VEGFA treatment to cause tumor stasis or regression. Thus, TNBC cells critically rely on IRE1α to adapt their ER to in vivo stress and to adjust the TME to facilitate malignant growth. TNBC reliance on IRE1α is an important vulnerability that can be uniquely exploited in combination with antiangiogenic therapy as a promising new biologic approach to combat this lethal disease. SIGNIFICANCE: Pharmacologic IRE1α kinase inhibition reverses ultrastructural distension of the ER, normalizes the tumor vasculature, and remodels the cellular TME, attenuating TNBC growth in mice.

Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) stress caused by cellular oncogene activation and a hostile tumor microenvironment (TME). The key UPR sensor IRE1α resides in the ER and deploys a cytoplasmic kinase-endoribonuclease module to activate the transcription factor XBP1s, which facilitates ER-mediated protein folding. Studies of triple-negative breast cancer (TNBC)-a highly aggressive malignancy with a dismal posttreatment prognosis-implicate XBP1s in promoting tumor vascularization and progression. However, it remains unknown whether IRE1α adapts the ER in TNBC cells and modulates their TME, and whether IRE1α inhibition can enhance antiangiogenic therapy-previously found to be ineffective in patients with TNBC. To gauge IRE1α function, we defined an XBP1s-dependent gene signature, which revealed significant IRE1α pathway activation in multiple solid cancers, including TNBC. IRE1α knockout in TNBC cells markedly reversed substantial ultrastructural expansion of their ER upon growth in vivo. IRE1α disruption also led to significant remodeling of the cellular TME, increasing pericyte numbers while decreasing cancer-associated fibroblasts and myeloid-derived suppressor cells. Pharmacologic IRE1α kinase inhibition strongly attenuated growth of cell line-based and patient-derived TNBC xenografts in mice and synergized with anti-VEGFA treatment to cause tumor stasis or regression. Thus, TNBC cells critically rely on IRE1α to adapt their ER to in vivo stress and to adjust the TME to facilitate malignant growth. TNBC reliance on IRE1α is an important vulnerability that can be uniquely exploited in combination with antiangiogenic therapy as a promising new biologic approach to combat this lethal disease. SIGNIFICANCE: Pharmacologic IRE1α kinase inhibition reverses ultrastructural distension of the ER, normalizes the tumor vasculature, and remodels the cellular TME, attenuating TNBC growth in mice.

Abstract Background Endothelial cells (ECs) play an important role in angiogenesis and vascular reconstruction in the pathophysiology of ischemic stroke. Previous investigations have provided a profound cerebral vascular atlas under physiological conditions, but have failed to identify new disease-related cell subtypes. We aimed to identify new EC subtypes and determine the key modulator genes. Methods Two datasets GSE174574 and GSE137482 were included in the study. Seurat was utilized as the standard quality-control pipeline. UCell was used to calculate single-cell scores to validate cellular identity. Monocle3 and CytoTRACE were utilized in aid of pseudo-time differentiation analysis. CellChat was utilized to infer the intercellular communication pathways. The angiogenesis ability of ECs was validated by MTS, Transwell, tube formation, flow cytometry, and immunofluorescence assays in vitro and in vivo. A synchrotron radiation-based propagation contrast imaging was introduced to comprehensively portray cerebral vasculature. Results We successfully identified a novel subtype of EC named “healing EC” that highly expressed pan-EC marker and pro-angiogenic genes but lowly expressed all the arteriovenous markers identified in the vascular single-cell atlas. Further analyses showed its high stemness to differentiate into other EC subtypes and potential to modulate inflammation and angiogenesis via excretion of signal molecules. We therefore identified X-box binding protein 1 (Xbp1) as a key modulator in the healing EC phenotype. In vitro and in vivo experiments confirmed its pro-angiogenic roles under both physiological and pathological conditions. Synchrotron radiation-based propagation contrast imaging further proved that Xbp1 could promote angiogenesis and recover normal vasculature conformation, especially in the corpus striatum and prefrontal cortex under middle cerebral artery occlusion (MCAO) condition. Conclusions Our study identified a novel disease-related EC subtype that showed high stemness to differentiate into other EC subtypes. The predicted molecule Xbp1 was thus confirmed as a key modulator that can promote angiogenesis and recover normal vasculature conformation.

Multiple myeloma (MM) arises from malignant immunoglobulin (Ig)-secreting plasma cells and remains an incurable, often lethal disease despite therapeutic advances. The unfolded-protein response sensor IRE1α supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may co-opt the IRE1α-XBP1s pathway; however, the validity of IRE1α as a potential MM therapeutic target is controversial. Genetic disruption of IRE1α or XBP1s, or pharmacologic IRE1α kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice and augmented efficacy of two established frontline antimyeloma agents, bortezomib and lenalidomide. Mechanistically, IRE1α perturbation inhibited expression of key components of the endoplasmic reticulum-associated degradation machinery, as well as secretion of Ig light chains and of cytokines and chemokines known to promote MM growth. Selective IRE1α kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells derived from bone marrows of newly diagnosed or posttreatment-relapsed MM patients, in both US- and European Union-based cohorts. Effective IRE1α inhibition preserved glucose-induced insulin secretion by pancreatic microislets and viability of primary hepatocytes in vitro, as well as normal tissue homeostasis in mice. These results establish a strong rationale for developing kinase-directed inhibitors of IRE1α for MM therapy.

Neointimal hyperplasia, stimulated by injury and certain vascular diseases, promotes artery obstruction and tissue ischemia. In vascular smooth muscle cell (VSMCs), multiple modulators of protein handling machinery regulate intimal hyperplasia. These include elements of the VSMC unfolded protein response to endoplasmic reticulum stress (UPRER), and transglutaminase 2 (TG2), which catalyzes post-translational protein modification. Previous results for deficiency of UPRER-specific mediator XBP1, and of TG2, have been significant, but in multiple instances contradictory, for effects on cultured VSMC function, and, using multiple models, for neointimal hyperplasia in vivo. Here, we engineered VSMC-specific deficiency of XBP1, and studied cultured VSMCs, and neointimal hyperplasia in response to carotid artery ligation in vivo. Intimal area almost doubled in Xbp1fl/fl SM22α-CRE+ mice 21 days post-ligation. Cultured murine Xbp1 deficient VSMCs migrated more in response to platelet derived growth factor (PDGF) than control VSMCs, and had an increased level of inositol-requiring enzyme 1α (Ire1α), a PDGF receptor-binding UPRER transmembrane endonuclease whose substrates include XBP1. Cultured XBP1-deficient VSMCs demonstrated decreased levels of TG2 protein, in association with increased TG2 polyubiquitination, but with increased TG transamidation catalytic activity. Moreover, IRE1α, and TG2-specific transamidation cross-links were increased in carotid artery neointima in Xbp1fl/fl SM22α-CRE+ mice. Cultured TG2-deficient VSMCs had decreased XBP1 associated with increased IRE1α, and increased migration in response to PDGF. Neointimal hyperplasia also was significantly increased in Tgm2fl/fl SM22α-CRE+ mice at 21 days after carotid ligation. In conclusion, a VSMC regulatory circuit between XBP1 and TG2 limits neointimal hyperplasia in response to carotid ligation.

Classically, the unfolded protein response (UPR) safeguards secretory pathway proteostasis. The most ancient arm of the UPR, the IRE1-activated spliced X-box binding protein 1 (XBP1s)-mediated response, has roles in secretory pathway maturation beyond resolving proteostatic stress. Understanding the consequences of XBP1s activation for cellular processes is critical for elucidating mechanistic connections between XBP1s and development, immunity, and disease. Here, we show that a key functional output of XBP1s activation is a cell type-dependent shift in the distribution of N-glycan structures on endogenous membrane and secreted proteomes. For example, XBP1s activity decreased levels of sialylation and bisecting GlcNAc in the HEK293 membrane proteome and secretome, while substantially increasing the population of oligomannose N-glycans only in the secretome. In HeLa cell membranes, stress-independent XBP1s activation increased the population of high-mannose and tetraantennary N-glycans, and also enhanced core fucosylation. mRNA profiling experiments suggest that XBP1s-mediated remodeling of the N-glycome is, at least in part, a consequence of coordinated transcriptional resculpting of N-glycan maturation pathways by XBP1s. The discovery of XBP1s-induced N-glycan structural remodeling on a glycome-wide scale suggests that XBP1s can act as a master regulator of N-glycan maturation. Moreover, because the sugars on cell-surface proteins or on proteins secreted from an XBP1s-activated cell can be molecularly distinct from those of an unactivated cell, these findings reveal a potential new mechanism for translating intracellular stress signaling into altered interactions with the extracellular environment.

Accumulating evidence has demonstrated that metformin improved hypertriglyceridemia. The present study aim to investigate the molecular mechanism by which metformin improves hypertriglyceridemia via regulation of diacylglycerol O-acyltransferase 2 (DGAT2) and X-box binding protein 1 (XBP1) in the liver and whether AMP-activated protein kinase (AMPK) is involved. Mice were fed a high-fat diet (HFD) or high-fat diet with metformin for 5 weeks to evaluate the effect of metformin on triglyceride (TG) levels and expression of DGAT2 and XBP1 in the liver. In vitro HepG2 cells or XBP1 knockout AML12 hepatocytes were stimulated with metformin, palmitic acid or small interfering RNA inducing XBP1 knockdown, or dominant-negative mutant AMPK plasmid. Metformin treatment reduced hepatic TG levels in the liver of HFD-fed mice. Expression of nuclear and cytoplasmic XBP1 protein and its downstream target gene DGAT2 decreased in the liver of HFD-fed mice and HepG2 cells after metformin treatment. AMPK inactivation or overexpression of XBP1 attenuates this effect. Our preliminary results demonstrate that metformin activates AMPK to reduce TG synthesis by inhibiting the XBP1-mediated DGAT2 pathway, at least in part, suggesting that XBP1 is a new metabolic mediator for metformin treatment of hypertriglyceridemia and associated metabolic disease. [ABSTRACT FROM AUTHOR]

Background: Endothelial cells (ECs) play an important role in angiogenesis and vascular reconstruction in the pathophysiology of ischemic stroke. Previous investigations have provided a profound cerebral vascular atlas under physiological conditions, but have failed to identify new disease-related cell subtypes. We aimed to identify new EC subtypes and determine the key modulator genes. Methods: Two datasets GSE174574 and GSE137482 were included in the study. Seurat was utilized as the standard quality-control pipeline. UCell was used to calculate single-cell scores to validate cellular identity. Monocle3 and CytoTRACE were utilized in aid of pseudo-time differentiation analysis. CellChat was utilized to infer the intercellular communication pathways. The angiogenesis ability of ECs was validated by MTS, Transwell, tube formation, flow cytometry, and immunofluorescence assays in vitro and in vivo. A synchrotron radiation-based propagation contrast imaging was introduced to comprehensively portray cerebral vasculature. Results: We successfully identified a novel subtype of EC named "healing EC" that highly expressed pan-EC marker and pro-angiogenic genes but lowly expressed all the arteriovenous markers identified in the vascular single-cell atlas. Further analyses showed its high stemness to differentiate into other EC subtypes and potential to modulate inflammation and angiogenesis via excretion of signal molecules. We therefore identified X-box binding protein 1 (Xbp1) as a key modulator in the healing EC phenotype. In vitro and in vivo experiments confirmed its pro-angiogenic roles under both physiological and pathological conditions. Synchrotron radiation-based propagation contrast imaging further proved that Xbp1 could promote angiogenesis and recover normal vasculature conformation, especially in the corpus striatum and prefrontal cortex under middle cerebral artery occlusion (MCAO) condition. Conclusions: Our study identified a novel disease-related EC subtype that showed high stemness to differentiate into other EC subtypes. The predicted molecule Xbp1 was thus confirmed as a key modulator that can promote angiogenesis and recover normal vasculature conformation. [ABSTRACT FROM AUTHOR]

Background: The activation of X-box binding protein 1 (XBP1) plays an essential role in the unfolded protein response (UPR) of the endoplasmic reticulum (ER). XBP1 is commonly expressed in various tumors and is closely related to tumorigenesis and progression. However, the role of XBP1 in lung adenocarcinoma (LUAD), especially the prognostic value of its alternative splicing isoforms, remains largely unknown. Methods: The LUAD datasets were retrieved from the The Cancer Genome Atlas, ArrayExpress and Gene Expression Omnibus. GEPIA2 and metaanalysis were employed to explore the prognostic value, and bioinformatics analysis with the TIMER2.0 database was used to investigate immune cell infiltration. We performed single-cell analyses to identify cell types with high XBP1 expression. In addition, polymerase chain reaction (PCR) and DNA sequencing were performed to verify the authenticity of the new spliceosome. Results: In this study, we found that high expression of XBP1 was significantly associated with a good prognosis, and XBP1 expression was significantly positively correlated with B cell infiltration in LUAD. In addition, we found that high-level expression of a novel splicing isoform, XBP1 (XBP1-003), improved the prognosis of LUAD. Protein structural analysis demonstrated that XBP1-003 has several specific protein domains that are different from those of other XBP1 isoforms, indicating a unique function of this isoform in LUAD. Conclusion: All these results suggest that XBP1 plays an antitumorigenic role in LUAD through alternative splicing, which may be related to the adaptation of plasma cells. This sheds new light on the potential strategy for LUAD prognosis evaluation and immunotherapy. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress is commonly observed in intestinal epithelial cells (IECs) and can, if excessive, cause spontaneous intestinal inflammation as shown by mice with IEC-specific deletion of X-box-binding protein 1 (Xbp1), an unfolded protein response-related transcription factor. In this study, Xbp1 deletion in the epithelium (Xbp1ΔIEC ) is shown to cause increased expression of natural killer group 2 member D (NKG2D) ligand (NKG2DL) mouse UL16-binding protein (ULBP)-like transcript 1 and its human orthologue cytomegalovirus ULBP via ER stress-related transcription factor C/EBP homology protein. Increased NKG2DL expression on mouse IECs is associated with increased numbers of intraepithelial NKG2D-expressing group 1 innate lymphoid cells (ILCs; NK cells or ILC1). Blockade of NKG2D suppresses cytolysis against ER-stressed epithelial cells in vitro and spontaneous enteritis in vivo. Pharmacological depletion of NK1.1+ cells also significantly improved enteritis, whereas enteritis was not ameliorated in Recombinase activating gene 1-/-;Xbp1ΔIEC mice. These experiments reveal innate immune sensing of ER stress in IECs as an important mechanism of intestinal inflammation.

Background: Chronic obstructive pulmonary disease (COPD) is caused by exposure to noxious external particles, air pollution, and the inhalation of cigarette smoke. Airway mucus hypersecretion particularly mucin5AC (MUC5AC), is a crucial pathological feature of COPD and is associated with its initiation and progression. In this study, we aimed to investigate the effects of cigarette smoke extract (CSE) on MUC5AC expression, particularly the mechanisms by which reactive oxygen species (ROS) induce MUC5AC expression., Methods: The effects of CSE on the expression of MUC5AC and mucin5B (MUC5B) were investigated in vitro in Calu-3 cells. MUC5AC and MUC5B expression levels were measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), immunofluorescence staining, and enzyme-linked immunosorbent assay (ELISA). Total cellular levels of ROS and Ca 2+ were determined using DCFH-DA and Fluo-4 AM. Subsequently, the expression levels of IP3R, IRE1α, p-IRE1α and XBP1s were measured by Western blotting. Gene silencing was achieved by using small-interfering RNAs., Results: Our findings revealed that exposure to CSE increased MUC5AC levels and upregulated ROS, IP3R/Ca 2+ and unfolded protein response (UPR)-associated factors. In addition, knockdown of IP3R using siRNA decreased CSE-induced Ca 2+ production, UPR-associated factors, and MUC5AC expression. Furthermore, 10 mM N-acetyl-l-cysteine (NAC) treatment suppressed the effects of CSE, including ROS generation, IP3R/ Ca 2+ , UPR activation, and MUC5AC overexpression., Conclusion: Our results suggest that ROS regulates CSE-induced UPR and MUC5AC overexpression through IP3R/ Ca 2+ signaling. Additionally, we identified NAC as a promising therapeutic agent for mitigating CSE-induced MUC5AC overexpression., Competing Interests: The authors declare that they have no competing interests in this work., (© 2024 Wu et al.)

Macrophages show high plasticity and play a vital role in the progression of metabolic dysfunction-associated steatohepatitis (MASH). X-box binding protein 1 (XBP1), a key sensor of the unfolded protein response, can modulate macrophage-mediated pro-inflammatory responses in the pathogenesis of MASH. However, how XBP1 influences macrophage plasticity and promotes MASH progression remains unclear. Herein, we formulated an Xbp1 siRNA delivery system based on folic acid modified D-α-tocopheryl polyethylene glycol 1000 succinate nanoparticles (FT@XBP1) to explore the precise role of macrophage-specific Xbp1 deficiency in the progression of MASH. FT@XBP1 was specifically internalized into hepatic macrophages and subsequently inhibited the expression of spliced XBP1 both in vitro and in vivo . It promoted M1-phenotype macrophage repolarization to M2 macrophages, reduced the release of pro-inflammatory factors, and alleviated hepatic steatosis, liver injury, and fibrosis in mice with fat-, fructose- and cholesterol-rich diet-induced MASH. Mechanistically, FT@XBP1 promoted macrophage polarization toward the M2 phenotype and enhanced the release of exosomes that could inhibit the activation of hepatic stellate cells. A promising macrophage-targeted siRNA delivery system was revealed to pave a promising strategy in the treatment of MASH., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Objective: The need to perform genetic sequencing to diagnose the polymerase epsilon exonuclease (POLE) subtype of endometrial cancer (EC) hinders the adoption of molecular classification. We investigated clinicopathologic and protein markers that distinguish the POLE from the copy number (CN)-low subtype in EC. Methods: Ninety-one samples (15 POLE, 76 CN-low) were selected from The Cancer Genome Atlas EC dataset. Clinicopathologic and normalized reverse phase protein array expression data were analyzed for associations with the subtypes. A logistic model including selected markers was constructed by stepwise selection using area under the curve (AUC) from 5-fold cross-validation (CV). The selected markers were validated using immunohistochemistry (IHC) in a separate cohort. Results: Body mass index (BMI) and tumor grade were significantly associated with the POLE subtype. With BMI and tumor grade as covariates, 5 proteins were associated with the EC subtypes. The stepwise selection method identified BMI, cyclin B1, caspase 8, and X-box binding protein 1 (XBP1) as markers distinguishing the POLE from the CN-low subtype. The mean of CV AUC, sensitivity, specificity, and balanced accuracy of the selected model were 0.97, 0.91, 0.87, and 0.89, respectively. IHC validation showed that cyclin B1 expression was significantly higher in the POLE than in the CN-low subtype and receiver operating characteristic curve of cyclin B1 expression in IHC revealed AUC of0.683. Conclusion: BMI and expression of cyclin B1, caspase 8, and XBP1 are candidate markers distinguishing the POLE from the CN-low subtype. Cyclin B1 IHC may replace POLE sequencing in molecular classification of EC. [ABSTRACT FROM AUTHOR]

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It is widely believed that inflammation associated with obesity has an important role in the development of type 2 diabetes. IκB kinase beta (IKKβ) is a crucial kinase that responds to inflammatory stimuli such as tumor necrosis factor α (TNF-α) by initiating a variety of intracellular signaling cascades and is considered to be a key element in the inflammation-mediated development of insulin resistance. We show here, contrary to expectation, that IKKβ-mediated inflammation is a positive regulator of hepatic glucose homeostasis. IKKβ phosphorylates the spliced form of X-Box Binding Protein 1 (XBP1s) and increases the activity of XBP1s. We have used three experimental approaches to enhance the IKKβ activity in the liver of obese mice and observed increased XBP1s activity, reduced ER stress, and a significant improvement in insulin sensitivity and consequently in glucose homeostasis. Our results reveal a beneficial role of IKKβ-mediated hepatic inflammation in glucose homeostasis.

Diets rich in saturated fatty acids (SFAs) produce a form of tissue inflammation driven by "metabolically activated" macrophages. We show that SFAs, when in excess, induce a unique transcriptional signature in both mouse and human macrophages that is enriched by a subset of ER stress markers, particularly IRE1α and many adaptive downstream target genes. SFAs also activate the NLRP3 inflammasome in macrophages, resulting in IL-1β secretion. We found that IRE1α mediates SFA-induced IL-1β secretion by macrophages and that its activation by SFAs does not rely on unfolded protein sensing. We show instead that the ability of SFAs to stimulate either IRE1α activation or IL-1β secretion can be specifically reduced by preventing their flux into phosphatidylcholine (PC) or by increasing unsaturated PC levels. Thus, IRE1α is an unrecognized intracellular PC sensor critical to the process by which SFAs stimulate macrophages to secrete IL-1β, a driver of diet-induced tissue inflammation.

The kinase/endonuclease IRE1 is the most conserved signal transducer of the unfolded protein response (UPR), an intracellular signaling network that monitors and regulates the protein folding capacity of the endoplasmic reticulum (ER). Upon sensing protein folding perturbations in the ER, IRE1 initiates the unconventional splicing of XBP1 mRNA culminating in the production of the transcription factor XBP1s, which expands the ER's protein folding capacity. We show that an RNA-intrinsic conformational change causes the intron of XBP1 mRNA to be ejected and the exons to zipper up into an extended stem, juxtaposing the RNA ends for ligation. These conformational rearrangements are important for XBP1 mRNA splicing in vivo. The features that point to such active participation of XBP1 mRNA in the splicing reaction are highly conserved throughout metazoan evolution, supporting their importance in orchestrating XBP1 mRNA processing with efficiency and fidelity.

Glioblastoma multiforme (GBM) almost invariably acquires an invasive phenotype, resulting in limited therapeutic options. Protein palmitoylation markedly affects tumorigenesis and malignant progression in GBM. The role of protein palmitoylation in GBM, however, has not been systematically reported. This study aimed to investigate the effect of protein palmitoylation on GBM cell survival and the cell cycle. In this study, most palmitoyltransferases were upregulated in GBM and its cell lines, and protein palmitoylation participated in signaling pathways controlling cell survival and the GBM cell cycle. Inhibition of protein palmitoylation with substrate-analog inhibitors, that is, 2-bromopalmitate, cerulenin, and tunicamycin, induced G2 cell cycle arrest and cell death in GBM cells through enhanced endoplasmic reticulum (ER) stress. These effects are primarily attributed to the palmitoylation inhibitors activating pro-apoptotic pathways and ER stress signals. Further analysis revealed was the accumulation of SUMOylated XBP1 (X-box binding protein 1) and its transcriptional repression, along with a reduction in XBP1 palmitoylation. Taken together, the present results indicate that protein palmitoylation plays an important role in the survival of GBM cells, further providing a potential therapeutic strategy for GBM.

Long-term dietary supplementation with resveratrol protects against cardiovascular disease, osteoporesis, and metabolic decline. This study determined how long-term dietary resveratrol treatment protects against retinal ganglion cell (RGC) dendrite loss after optic nerve injury and alters the resolution of the unfolded protein response. Associated changes in markers of endoplasmic reticulum stress in RGCs also were investigated. Young-adult Thy1-yellow fluorescent protein (YFP) and C57BL/6 mice received either control diet or diet containing resveratrol for approximately 1 year. Both groups then received optic nerve crush (ONC). Fluorescent RGC dendrites in the Thy1-YFP mice were imaged weekly for 4 weeks after ONC. There was progressive loss of dendrite length in all RGC types within the mice that received control diet. Resveratrol delayed loss of dendrite complexity and complete dendrite loss for most RGC types. However, there were variations in the rate of retraction among different RGC types. Three weeks after ONC, cytoplasmic binding immunoglobulin protein (BiP) suppression observed in control diet ganglion cell layer neurons was reversed in mice that received resveratrol, nuclear C/EBP homologous protein (CHOP) was near baseline in control diet eyes but was moderately increased by resveratrol; and increased nuclear X-box-binding protein-1 (XBP-1) observed in control diet eyes was reduced in eyes that received resveratrol to the same level as in control diet uncrushed eyes. These results indicate that protection of dendrites by resveratrol after ONC differs among RGC types and suggest that alterations in long-term expression of binding immunoglobulin protein, CHOP, and XBP-1 may contribute to the resveratrol-mediated protection of RGC dendrites after ONC.

The disruption of the energy or nutrient balance triggers endoplasmic reticulum (ER) stress, a process that mobilizes various strategies, collectively called the unfolded protein response (UPR), which reestablish homeostasis of the ER and cell. Activation of the UPR stress sensor IRE1α (inositol-requiring enzyme 1α) stimulates its endoribonuclease activity, leading to the generation of the mRNA encoding the transcription factor XBP1 (X-box binding protein 1), which regulates the transcription of genes encoding factors involved in controlling the quality and folding of proteins. We found that the activity of IRE1α was regulated by the ER oxidoreductase PDIA6 (protein disulfide isomerase A6) and the microRNA miR-322 in response to disruption of ER Ca2+ homeostasis. PDIA6 interacted with IRE1α and enhanced IRE1α activity as monitored by phosphorylation of IRE1α and XBP1 mRNA splicing, but PDIA6 did not substantially affect the activity of other pathways that mediate responses to ER stress. ER Ca2+ depletion and activation of store-operated Ca2+ entry reduced the abundance of the microRNA miR-322, which increased PDIA6 mRNA stability and, consequently, IRE1α activity during the ER stress response. In vivo experiments with mice and worms showed that the induction of ER stress correlated with decreased miR-322 abundance, increased PDIA6 mRNA abundance, or both. Together, these findings demonstrated that ER Ca2+, PDIA6, IRE1α, and miR-322 function in a dynamic feedback loop modulating the UPR under conditions of disrupted ER Ca2+ homeostasis.

The unfolded protein response (UPR) pathway, a stress-induced signaling cascade emanating from the endoplasmic reticulum (ER), regulates the expression and activity of molecules including BiP (HSPA5), IRE1 (ERN1), Blimp-1 (PRDM1), and X-box binding protein 1 (XBP1). These molecules are required for terminal differentiation of B cells into plasma cells and expressed at high levels in plasma cell-derived multiple myeloma. Although these molecules have no known role at early stages of B-cell development, here we show that their expression transiently peaks at the pre-B-cell receptor checkpoint. Inducible, Cre-mediated deletion of Hspa5, Prdm1, and Xbp1 consistently induces cellular stress and cell death in normal pre-B cells and in pre-B-cell acute lymphoblastic leukemia (ALL) driven by BCR-ABL1- and NRAS(G12D) oncogenes. Mechanistically, expression and activity of the UPR downstream effector XBP1 is regulated positively by STAT5 and negatively by the B-cell-specific transcriptional repressors BACH2 and BCL6. In two clinical trials for children and adults with ALL, high XBP1 mRNA levels at the time of diagnosis predicted poor outcome. A small molecule inhibitor of ERN1-mediated XBP1 activation induced selective cell death of patient-derived pre-B ALL cells in vitro and significantly prolonged survival of transplant recipient mice in vivo. Collectively, these studies reveal that pre-B ALL cells are uniquely vulnerable to ER stress and identify the UPR pathway and its downstream effector XBP1 as novel therapeutic targets to overcome drug resistance in pre-B ALL.

Objective Among stress responses, the unfolded protein response (UPR) is a well-known mechanism related to endoplasmic reticulum (ER) stress. ER stress is induced by a variety of external and environmental factors such as starvation, ischemia, hypoxia, oxidative stress, and heat stress. Inositol requiring enzyme 1α (IRE1α)-X-box protein 1 (XBP1) is the most conserved pathway involved in the UPR and is the main component that mediates IRE1α signalling to downstream ER-associated degradation (ERAD)- or UPR-related genes. XBP1 is a transcription factor synthesised via a novel mechanism called ‘frame switch splicing’, and this process has not yet been studied in the horse XBP1 gene. Therefore, the aim of this study was to confirm the frame switch splicing of horse XBP1 and characterise its dynamics using Thoroughbred muscle cells exposed to heat stress. Methods Primary horse muscle cells were used to investigate heat stress-induced frame switch splicing of horse XBP1. Frame switch splicing was confirmed by sequencing analysis. XBP1 amino acid sequences and promoter sequences of various species were aligned to confirm the sequence homology and to find conserved cis-acting elements, respectively. The expression of the potential XBP1 downstream genes were analysed by quantitative real-time polymerase chain reaction. Results We confirmed that splicing of horse XBP1 mRNA was affected by the duration of thermal stress. Twenty-six nucleotides in the mRNA of XBP1 were deleted after heat stress. The protein sequence and the cis-regulatory elements on the promoter of horse XBP1 are highly conserved among the mammals. Induction of putative downstream genes of horse XBP1 was dependent on the duration of heat stress. We confirmed that both the mechanisms of XBP1 frame switch splicing and various binding elements found in downstream gene promoters are highly evolutionarily conserved. Conclusion The frame switch splicing of horse XBP1 and its dynamics were highly conserved among species. These results facilitate studies of ER-stress in horse.

Objectives Endoplasmic reticulum (ER) stress is known to be associated with inflammatory airway diseases, and three major transmembrane receptors: double-stranded RNA-activated protein kinase-like ER kinase, inositol requiring enzyme 1, and activating transcription factor 6 (ATF6) play important roles in ER stress-related proinflammatory signaling. However, the effects of ER stress and these three major signaling pathways on the regulation of the production of airway mucins in human nasal airway epithelial cells have not been elucidated. Methods In primary human nasal epithelial cells, the effect of tunicamycin (an ER stress inducer) and 4-phenylbutyric acid (4-PBA, ER stress inhibitor) on the expression of MUC5AC and MUC5B was investigated by reverse transcriptasepolymerase chain reaction, real-time polymerase chain reaction, enzyme immunoassay, and immunoblot analysis. Small interfering RNA (siRNA) transfection was used to identify the mechanisms involved. Results Tunicamycin increased the expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules, including spliced X-box binding protein 1 (XBP-1), transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP), and ATF6. In addition, 4-PBA attenuated the tunicamycin-induced expressions of MUC5AC and MUC5B and the mRNA expressions of ER stress-related signaling molecules. Furthermore, siRNA knockdowns of XBP-1, CHOP, and ATF6 blocked the tunicamycin-induced mRNA expressions and glycoprotein productions of MUC5AC and MUC5B. Conclusion. These results demonstrate that ER stress plays an important role in the regulation of MUC5AC and MUC5B via the activations of XBP-1, CHOP, and ATF6 in human nasal airway epithelial cells.

Pseudomonas aeruginosa infections are associated with high mortality rates and occur in diverse conditions including pneumonias, cystic fibrosis and neutropenia. Quorum sensing, mediated by small molecules including N-(3-oxo-dodecanoyl) homoserine lactone (C12), regulates P. aeruginosa growth and virulence. In addition, host cell recognition of C12 initiates multiple signalling responses including cell death. To gain insight into mechanisms of C12-mediated cytotoxicity, we studied the role of endoplasmic reticulum stress in host cell responses to C12. Dramatic protection against C12-mediated cell death was observed in cells that do not produce the X-box binding protein 1 transcription factor (XBP1s). The leucine zipper and transcriptional activation motifs of XBP1s were sufficient to restore C12-induced caspase activation in XBP1s-deficient cells, although this polypeptide was not transcriptionally active. The XBP1s polypeptide also regulated caspase activation in cells stimulated with N-(3-oxo-tetradecanoyl) homoserine lactone (C14), produced by Yersinia enterolitica and Burkholderia pseudomallei, and enhanced homoserine lactone-mediated caspase activation in the presence of endogenous XBP1s. In C12-tolerant cells, responses to C12 including phosphorylation of p38 MAPK and eukaryotic initiation factor 2α were conserved, suggesting that C12 cytotoxicity is not heavily dependent on these pathways. In summary, this study reveals a novel and unconventional role for XBP1s in regulating host cell cytotoxic responses to bacterial acyl homoserine lactones.

Introduction: The gut microbiota plays an important role in the development of non-alcoholic steatohepatitis (NASH), but the underlying mechanism is unclear. It has been found that the transcription factor XBP1s plays an important role in regulating inflammation and lipid metabolism and maintaining the integrity of intestinal barrier. However, whether XBP1s modulates the development of NASH by regulating the integrity of the intestinal barrier and altering the composition of the gut microbiota remains unknown., Methods: Mice fed with a fat-, fructose-, cholesterol-rich (FFC) diet for 24 weeks successfully established the NASH model, as demonstrated by significant hepatic steatosis, inflammation, hepatocyte injury and fibrosis. The profile of gut microbiota dynamically changed with the different stages of NAFLD via 16S rDNA sequencing the feces from mice fed with FFC diet for 0, 12, or 24 weeks or NASH mice treated with siRNA-loaded folic acid-modified TPGS (hereafter named FT@XBP1)., Results: NASH mice had significantly higher abundance of Firmicutes, Blautia and Bacteroides, and lower abundance of Bifidobacterium and GCA-900066575. FT@XBP1 supplementation had a significantly attenuated effect on FFC diet-induced weight gain, hepatic fat accumulation, dyslipidemia, inflammatory cytokines, ER stress and fibrosis. In particularly, FT@XBP1 modulates the composition of the intestinal flora; for example, NASH mice demonstrated higher abundance of Blautia and Bacteroides, and lower abundance of Actinobacteriota, Muribaculaceae and Bifidobacterium, which were partially restored by FT@XBP1 treatment. Mechanistically, FT@XBP1 increased the expression of ZO-1 in the intestine and had the potential to restore intestinal barrier integrity and improve antimicrobial defense to alleviate enterogenic endotoxemia and activation of inflammatory signaling pathways., Discussion: Regulation of the key transcription factor XBP1s can partially restore the intestinal microbiota structure, maintain the integrity of intestinal mucosal barrier, and prevent the progression of NASH, providing new evidence for treating NASH., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhu, Cheng, Tang, Li, Rao, Zhang, Xiao and Liu.)

Objective: To investigate the mechanism and clinical value of nicotinamide phosphoribosyltransferase (NAMPT) in multiple myeloma (MM)., Methods: RT-qPCR and Western blot were used to detect the expression of NAMPT in MM cells and normal bone marrow mononuclear cells. The biological function of NAMPT was analyzed by cell proliferation and apoptosis assay, small interfering RNA silencing, overexpression assay and chromatin immunoprecipitation assay., Results: The mRNA and protein expression levels of NAMPT in MM cell lines (MM1R, MM1S, U266 and RPMI-8226) were significantly higher than those in normal bone marrow mononuclear cells ( P < 0.001), and were most obvious in U266 cells. Compared with Si-NC group, the proliferation of U266 cells in Si- NAMPT group was significantly inhibited at 24, 48 and 72 h after transfection ( P =0.006, P < 0.001, P =0.001), and the apoptosis rate of U266 cells was significantly increased at 48 h after transfection ( P < 0.001). Compared with Flag-NC group, U266 cell proliferation in Flag- NAMPT group was significantly increased ( P =0.003, P =0.002, P < 0.001), while the apoptosis rate decreased significantly at 48 h after transfection. The expression of NAMPT in U266 cells was regulated by XBP1 at transcriptional level. The proliferation rate of U266 cells with XBP1 or NAMPT stable knockout or MKC3946 pretreated with bortezomib was significantly decreased, the levels of BCL-2 mRNA and protein were also significantly decreased, while the levels of BAX mRNA and protein were significantly increased, moreover, the cleavage degree of caspase-3 significantly decreased, while caspase-3/7 activity increased dramatically ( P < 0.05)., Conclusions: The high expression of NAMPT in MM cell line can promote MM cell proliferation and inhibit apoptosis. NAMPT is regulated by IRE1α-XBP1 signaling pathway in U266 cells. Stable knockdown of NAMPT or blocking of IRE1α-XBP1 pathway can significantly increase the sensitivity of U266 cells to bortezomib.

Atrazine (ATR) is a widely used herbicide and due to its persistence in environment and bioaccumulation, it can cause harmful impacts on human health. ATR exposure can lead to disorders of lipid metabolism in the liver, but its underlying mechanism is still unclear. 40 eight-week-old rats were given different doses of ATR (0, 0.5, 5 and 50 mg/kg/d) for 90 days. The liver tissue and serum were collected for histological observation and biochemical analysis. The levels of lipid and oxidative stress were assessed using colorimetry. Changes in MMP and ROS of liver cells were observed through flow cytometry. The expression of mRNA and protein was detected using Real-Time PCR and western blot. The results showed that TC and HDL-C levels in both the liver and serum were increased in the ATR-treated groups. The levels of MDA were accumulated, while the levels of SOD and GSH were depleted in the liver with ATR exposure. The expression of liver lipid metabolism related genes (SCD1, DGAT2, ACC1, PPARγ) was elevated. The liver ERS was activated and the gene expression of IRE1α/XBP1 signal pathway and GRP78, GRP94 in the liver was increased. There was a correlation between the levels of ERS and the levels of lipid metabolism. These results suggested that ATR can activate ERS and promote the expression of IRE1α/XBP1 signaling pathway, and further lead to lipid metabolism disorders in rat liver. This study can provide valuable insights as a reference for the prevention and control of hazards associated with agricultural residues., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. I declare on behalf of my co-authors that the research content described is unpublished original research and is not considered for publication elsewhere. All listed authors acknowledge the attached manuscript., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Plasminogen activator inhibitor 1 (PAI‐1) is a stress‐responsive gene that is highly induced in nonalcoholic steatohepatitis (NASH). Endoplasmic reticulum (ER) stress is a salient feature of NASH, yet it is unknown whether ER stress contributes to hepatic PAI‐1 induction in this disorder. Therefore, we aimed to (a) establish the role of ER stress in the regulation of hepatic Pai‐1 expression, and (b) determine whether induction of Pai‐1 in murine NASH is driven by ER stress. Hepatic Pai‐1 expression was measured in C57BL/6 J mice and human HepG2 cells subjected to acute or prolonged pharmacologic ER stress. We found that hepatic Pai‐1 expression was acutely suppressed in murine liver in response to severe ER stress followed by marked induction during the recovery phase of the ER stress response. Hepatic Pai‐1 expression was induced in response to prolonged low‐grade ER stress in mice. Induction of PAI‐1 by ER stress in HepG2 cells was prevented by pharmacologic inhibition of MEK1/ERK signaling or by siRNA‐mediated knockdown of XBP1, mediators of the recovery response to ER stress. Inhibiting ER stress with 4‐phenylbutyric acid prevented hepatic Pai‐1 induction in mice with diet‐induced steatohepatitis. We conclude that hepatic Pai‐1 is induced by ER stress via a pathway involving XBP1 and MEK1/ERK signaling, and induction of hepatic Pai‐1 in murine NASH is mediated by ER stress. These data implicate ER stress as a novel mechanistic link between Pai‐1 induction and NASH. [ABSTRACT FROM AUTHOR]

Under endoplasmic reticulum stress, unfolded protein accumulation leads to activation of the endoplasmic reticulum transmembrane kinase/endoRNase (RNase) IRE1α. IRE1α oligomerizes, autophosphorylates and initiates splicing of XBP1 mRNA, thus triggering the unfolded protein response (UPR). Here we show that IRE1αs kinase-controlled RNase can be regulated in two distinct modes with kinase inhibitors: one class of ligands occupies IRE1αs kinase ATP-binding site to activate RNase-mediated XBP1 mRNA splicing even without upstream endoplasmic reticulum stress, whereas a second class can inhibit the RNase through the same ATP-binding site, even under endoplasmic reticulum stress. Thus, alternative kinase conformations stabilized by distinct classes of ATP-competitive inhibitors can cause allosteric switching of IRE1αs RNase--either on or off. As dysregulation of the UPR has been implicated in a variety of cell degenerative and neoplastic disorders, small-molecule control over IRE1α should advance efforts to understand the UPRs role in pathophysiology and to develop drugs for endoplasmic reticulum stress-related diseases.

X-box binding protein 1 (XBP1) is a unique basic-region leucine zipper (bZIP) transcription factor whose dynamic form is controlled by an alternative splicing response upon disturbance of homeostasis in the endoplasmic reticulum (ER) and activation of the unfolded protein response (UPR). XBP1 was first distinguished as a key regulator of major histocompatibility complex (MHC) class II gene expression in B cells. XBP1 communicates with the foremost conserved signalling component of the UPR and is essential for cell fate determination in response to ER stress (ERS). Here, we review recent advances in our understanding of this multifaceted translation component in cancer. In this review, we briefly discuss the role of XBP1 mediators in the UPR and the transcriptional function of XBP1. In addition, we describe how XBP1 operates as a key factor in tumour progression and metastasis. We mainly review XBP1′s expression, function and prognostic value in research on solid tumours. Finally, we discuss multiple approaches, especially those involving XBP1, that overcome the immunosuppressive effect of the UPR in cancer that could potentially be useful as antitumour therapies.

Abstract Background Aging is the strongest risk factor for neurodegenerative diseases and extended age results in neuronal degeneration and functional decline in the visual system. Among many contributing factors to age-related deterioration of neurons is an insufficient activation of the Unfolded Protein Response (UPR) in the endoplasmic reticulum (ER) in response to cellular stress. X-box binding protein 1 (XBP1) is a major component of the UPR and is essential for maintaining protein homeostasis and reducing cellular stresses. Herein, we investigate the role of XBP1 in maintaining morphological and functional integrity in retinal neurons during adulthood and the early stages of aging. Methods The basal and induced levels of XBP1 activation in the retina were measured in young adult and aged mice. Conditional knockout (cKO) of XBP1 in retinal neurons was achieved by crossing XBP1 floxed mice with a retina specific Cre-recombinase line (Chx10-Cre). Retinal morphology, neuronal populations including photoreceptors, bipolar cells, and retinal ganglion cells (RGCs), synaptic structure, and microglial activation were examined with immunohistochemistry and staining of retinal sections. Retinal function was evaluated with light-adapted (photopic) and dark adapted (scotopic) electroretinograms. Retinal mitochondrial function and metabolism was assessed by Seahorse XFe24 Extracellular Flux Analyzer. Results The retinas of aged wild type (WT) mice display a significantly reduced basal level of Xbp1s and compromised activation of ER stress response. In XBP1 cKO mice, significant structural degeneration of the retina, evidenced by thinning of retinal layers and a loss of RGCs, and functional defects indicated by diminished photopic and scotopic ERG b-waves are observed at the age of 12–14 months. Furthermore, discontinuous and disorganized synaptic laminae, colocalized with activated microglia, in the inner plexiform layer is found in the XBP1 cKO retinas. In addition, cKO mice demonstrate a significant increase in ectopic synapses between bipolar cells and photoreceptors, which is strikingly similar to WT mice at 20–24 months of age. These changes are associated with defective retinal glycolysis while mitochondrial respiratory function appears normal in the cKO retina. Conclusions XBP1 cKO mice at 12–14 months of age show significant structural, functional, and metabolic deficits that closely resemble WT mice twice that age. Our findings suggest that the absence of XBP1, a critical component of the UPR, accelerates age-related retinal neurodegeneration.