Activation Of Unfolded Protein Response Research Articles

Cannabidivarin and cannabigerol induce unfolded protein response and angiogenesis dysregulation in placental trophoblast HTR-8/SVneo cells.

Cannabidivarin (CBDV) and cannabigerol (CBG) are minor phytocannabinoids from Cannabis sativa, whose health benefits have been reported. However, studies about the impact of these cannabinoids on fundamental cellular processes in placentation are scarce. Placental development involves physiological endoplasmic reticulum (ER) stress, however when exacerbated it can lead to altered angiogenesis and pregnancy disorders, such as intrauterine growth restriction and preeclampsia. In this work, the effects of CBDV and CBG (1-10µM) on placental extravillous trophoblasts were studied, using the in vitro model HTR-8/SVneo cells. Both cannabinoids induced anti-proliferative effects and reactive oxygen/nitrogen species generation, which was dependent on transient receptor potential vanilloid 1 (TRPV1) activation. Moreover, CBDV and CBG significantly upregulated, in a TRPV-1 dependent manner, the gene expression of HSPA5/Glucose-regulated protein 78 (GRP78/BiP), a critical chaperone involved in ER stress and unfolded protein response (UPR) activation. Nevertheless, the UPR pathways were differentially activated. Both cannabinoids were able to recruit the IRE branch, while only CBDV enhanced the expression of downstream effectors of the PERK pathway, namely p-eIF2α, ATF4 and CHOP. It also augmented the activity of the apoptotic initiator caspases-8 and -9, though the effector caspases-3/-7 were not activated. TRB3 expression was increased by CBDV, which may hinder apoptosis termination. Moreover, both compounds upregulated the mRNA levels of the angiogenic factors VEGFA, PGF and sFLT1, and disrupted the endothelial-like behavior of HTR-8/SVneo cells, by reducing tube formation. Thus, CBDV and CBG treatment interferes with EVTs functions and may have a negative impact in placentation and in pregnancy outcome.

Bax Inhibitor-1 preserves pancreatic β-cell proteostasis by limiting proinsulin misfolding and programmed cell death

The prevalence of diabetes steadily increases worldwide mirroring the prevalence of obesity. Endoplasmic reticulum (ER) stress is activated in diabetes and contributes to β-cell dysfunction and apoptosis through the activation of a terminal unfolded protein response (UPR). Our results uncover a new role for Bax Inhibitor-One (BI-1), a negative regulator of inositol-requiring enzyme 1 (IRE1α) in preserving β-cell health against terminal UPR-induced apoptosis and pyroptosis in the context of supraphysiological loads of insulin production. BI-1-deficient mice experience a decline in endocrine pancreatic function in physiological and pathophysiological conditions, namely obesity induced by high-fat diet (HFD). We observed early-onset diabetes characterized by hyperglycemia, reduced serum insulin levels, β-cell loss, increased pancreatic lipases and pro-inflammatory cytokines, and the progression of metabolic dysfunction. Pancreatic section analysis revealed that BI-1 deletion overburdens unfolded proinsulin in the ER of β-cells, confirmed by ultrastructural signs of ER stress with overwhelmed IRE1α endoribonuclease (RNase) activity in freshly isolated islets. ER stress led to β-cell dysfunction and islet loss, due to an increase in immature proinsulin granules and defects in insulin crystallization with the presence of Rod-like granules. These results correlated with the induction of autophagy, ER phagy, and crinophagy quality control mechanisms, likely to alleviate the atypical accumulation of misfolded proinsulin in the ER. In fine, BI-1 in β-cells limited IRE1α RNase activity from triggering programmed β-cell death through apoptosis and pyroptosis (caspase-1, IL-1β) via NLRP3 inflammasome activation and metabolic dysfunction. Pharmaceutical IRE1α inhibition with STF-083010 reversed β-cell failure and normalized the metabolic phenotype. These results uncover a new protective role for BI-1 in pancreatic β-cell physiology as a stress integrator to modulate the UPR triggered by accumulating unfolded proinsulin in the ER, as well as autophagy and programmed cell death, with consequences on β-cell function and insulin secretion.In pancreatic β-cells, BI-1–/– deficiency perturbs proteostasis with proinsulin misfolding, ER stress, terminal UPR with overwhelmed IRE1α/XBP1s/CHOP activation, inflammation, β-cell programmed cell death, and diabetes.

Increased ER Stress and Unfolded Protein Response Activation in Epithelial and Inflammatory Cells in Hypersensitivity Pneumonitis.

Several types of cytotoxic insults disrupt endoplasmic reticulum (ER) homeostasis, cause ER stress, and activate the unfolded protein response (UPR). The role of ER stress and UPR activation in hypersensitivity pneumonitis (HP) has not been described. HP is an immune-mediated interstitial lung disease that develops following repeated inhalation of various antigens in susceptible and sensitized individuals. The aim of this study was to investigate the lung expression and localization of the key effectors of the UPR, BiP/GRP78, CHOP, and sXBP1 in HP patients compared with control subjects. Furthermore, we developed a mouse model of HP to determine whether ER stress and UPR pathway are induced during this pathogenesis. In human control lungs, we observed weak positive staining for BiP in some epithelial cells and macrophages, while sXBP1 and CHOP were negative. Conversely, strong BiP, sXBP1- and CHOP-positive alveolar and bronchial epithelial, and inflammatory cells were identified in HP lungs. We also found apoptosis and autophagy markers colocalization with UPR proteins in HP lungs. Similar results were obtained in lungs from an HP mouse model. Our findings suggest that the UPR pathway is associated with the pathogenesis of HP.

Thapsigargin and Tunicamycin Block SARS-CoV-2 Entry into Host Cells via Differential Modulation of Unfolded Protein Response (UPR), AKT Signaling, and Apoptosis.

SARS-Co-V2 infection can induce ER stress-associated activation of unfolded protein response (UPR) in host cells, which may contribute to the pathogenesis of COVID-19. To understand the complex interplay between SARS-Co-V2 infection and UPR signaling, we examined the effects of acute pre-existing ER stress on SARS-Co-V2 infectivity. Huh-7 cells were treated with Tunicamycin (TUN) and Thapsigargin (THA) prior to SARS-CoV-2pp transduction (48 h p.i.) to induce ER stress. Pseudo-typed particles (SARS-CoV-2pp) entry into host cells was measured by Bright GloTM luciferase assay. Cell viability was assessed by cell titer Glo® luminescent assay. The mRNA and protein expression was evaluated by RT-qPCR and Western Blot. TUN (5 µg/mL) and THA (1 µM) efficiently inhibited the entry of SARS-CoV-2pp into host cells without any cytotoxic effect. TUN and THA's attenuation of virus entry was associated with differential modulation of ACE2 expression. Both TUN and THA significantly reduced the expression of stress-inducible ER chaperone GRP78/BiP in transduced cells. In contrast, the IRE1-XBP1s and PERK-eIF2α-ATF4-CHOP signaling pathways were downregulated with THA treatment, but not TUN in transduced cells. Insulin-mediated glucose uptake and phosphorylation of Ser307 IRS-1 and downstream p-AKT were enhanced with THA in transduced cells. Furthermore, TUN and THA differentially affected lipid metabolism and apoptotic signaling pathways. These findings suggest that short-term pre-existing ER stress prior to virus infection induces a specific UPR response in host cells capable of counteracting stress-inducible elements signaling, thereby depriving SARS-Co-V2 of essential components for entry and replication. Pharmacological manipulation of ER stress in host cells might provide new therapeutic strategies to alleviate SARS-CoV-2 infection.

Mechanism of Decision Making between Autophagy and Apoptosis Induction upon Endoplasmic Reticulum Stress.

Dynamic regulation of the cellular proteome is mainly controlled in the endoplasmic reticulum (ER). Accumulation of misfolded proteins due to ER stress leads to the activation of unfolded protein response (UPR). The primary role of UPR is to reduce the bulk of damages and try to drive back the system to the former or a new homeostatic state by autophagy, while an excessive level of stress results in apoptosis. It has already been proven that the proper order and characteristic features of both surviving and self-killing mechanisms are controlled by negative and positive feedback loops, respectively. The new results suggest that these feedback loops are found not only within but also between branches of the UPR, fine-tuning the response to ER stress. In this review, we summarize the recent knowledge of the dynamical characteristic of endoplasmic reticulum stress response mechanism by using both theoretical and molecular biological techniques. In addition, this review pays special attention to describing the mechanism of action of the dynamical features of the feedback loops controlling cellular life-and-death decision upon ER stress. Since ER stress appears in diseases that are common worldwide, a more detailed understanding of the behaviour of the stress response is of medical importance.

Deficient glycan extension and endoplasmic reticulum stresses in ALG3-CDG.

ALG3-CDG is a rare congenital disorder of glycosylation (CDG) with a clinical phenotype that includes neurological manifestations, transaminitis, and frequent infections. The ALG3 enzyme catalyzes the first step of endoplasmic reticulum (ER) luminal glycan extension by adding mannose from Dol-P-Man to Dol-PP-Man5GlcNAc2 (Man5) forming Dol-PP-Man6. Such glycan extension is the first and fastest cellular response to ER stress, which is deficient in ALG3-CDG. In this study, we provide evidence that the unfolded protein response (UPR) and ER-associated degradation activities are increased in ALG3-CDG patient-derived cultured skin fibroblasts and there is constitutive activation of UPR mediated by the IRE1-α pathway. In addition, we show that N-linked Man3-4 glycans are increased in cellular glycoproteins and secreted plasma glycoproteins with hepatic or non-hepatic origin. We found that like other CDGs such as ALG1- or PMM2-CDG, in transferrin, the assembling intermediate Man5 in ALG3-CDG, are likely further processed into a distinct glycan, NeuAc1Gal1GlcNAc1Man3GlcNAc2, probably by Golgi mannosidases and glycosyltransferases. We predict it to be a mono-antennary glycan with the same molecular weight as the truncated glycan described in MGAT2-CDG. In summary, this study elucidates multiple previously unrecognized biochemical consequences of the glycan extension deficiency in ALG3-CDG which will have important implications in the pathogenesis of CDG.

Abstract LB192: AGR2 knockdown reduces cell proliferation and increases endoplasmic reticulum proteotoxic stress in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal solid malignancy with low survival rates due to a dense stroma, abnormal gene expression patterns, and limited treatment options. Anterior gradient 2 (AGR2) is often overexpressed in many cancers, including PDAC. AGR2 belongs to the protein disulfide isomerase family of proteins and is located in the endoplasmic reticulum, where it helps with protein-disulfide isomerase reactions to ensure proper protein folding. However, the molecular mechanisms underpinning AGR2 overexpression in PDAC pathophysiology are not well understood. Gene expression data obtained from the pancreatic adenocarcinoma (PAAD) dataset shows that AGR2 is overexpressed in PDAC tumors compared to normal tissues. We confirmed this gene over-expression in PDAC tumors and multiple pancreatic cancer cell lines using fluorescent microscopy, RT-qPCR, and western blotting. Using stable inducible short hairpin (shRNA) technology, we showed that AGR2 knockdown significantly decreases the viability and colony-forming ability of HPAF-II and PANC-1 cells. Gene Ontology (GO) enrichment analysis revealed that AGR2 knockdown suppresses a host of protein biosynthesis pathways, including protein targeting and localization to the endoplasmic reticulum (ER). Significantly, ER stress was induced in knockdown cells, shown by the activation of unfolded protein response, suggesting an accumulation of unfolded proteins. To determine the effects of unfolded protein accumulation on the ER, we performed transmission electron microscopy. Here, the results show an increase in the ER lumen size of knockdown cells compared to controls. Interestingly, we observed a decrease in mitochondria size and confirmed the increased expression of mitochondria fission proteins DRP1 and MFF. Finally, flow cytometry analysis revealed increased apoptosis in AGR2 knockdown cells compared to controls. Overall, our data provide insights into the role of AGR2 in PDAC tumorigenesis. However, more research is warranted to determine if AGR2 could be a potential therapeutic target. Citation Format: Philip Salu, Katie Reindl. AGR2 knockdown reduces cell proliferation and increases endoplasmic reticulum proteotoxic stress in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB192.

Endoplasmic Reticulum Stress Induces Vasodilation in Liver Vessels That Is Not Mediated by Unfolded Protein Response.

There is a growing body of evidence that ER stress and the unfolded protein response (UPR) play a key role in numerous diseases. Impaired liver perfusion and ER stress often accompany each other in liver diseases. However, the exact impact of ER stress and UPR on the hepatic perfusion is not fully understood. The aim of this study was to disclose the effect of ER stress and UPR on the size of liver vessels and on the levels of Ca2+ and nitric oxide (NO), critical regulators of vascular tonus. This study was carried out in precisely cut liver tissue slices. Confocal microscopy was used to create 3D images of vessels. NO levels were determined either using either laser scan microscopy (LSM) in cells or by NO-analyser in medium. Ca2+ levels were analysed by LSM. We show that tunicamycin, an inducer of ER stress, acts similarly with vasodilator acetylcholine. Both exert a similar effect on the NO and Ca2+ levels; both induce significant vasodilation. Notably, this vasodilative effect persisted despite individual inhibition of UPR pathways-ATF-6, PERK, and IRE1-despite confirming the activation of UPR. Experiments with HUVEC cells showed that elevated NO levels did not result from endothelial NO synthase (eNOS) activation. Our study suggests that tunicamycin-mediated ER stress induces liver vessel vasodilation in an NO-dependent manner, which is mediated by intracellular nitrodilator-activatable NO store (NANOS) in smooth muscle cells rather than by eNOS.

Salubrinal promotes phospho-eIF2α-dependent activation of UPR leading to autophagy-mediated attenuation of iron-induced insulin resistance

Identification of new mechanisms mediating insulin sensitivity is important to allow validation of corresponding therapeutic targets. In this study, we first used a cellular model of skeletal muscle cell iron overload and found that endoplasmic reticulum (ER) stress and insulin resistance occurred after iron treatment. Insulin sensitivity was assessed using cells engineered to express an Akt biosensor, based on nuclear FoxO localization, as well as western blotting for insulin signaling proteins. Use of salubrinal to elevate eIF2α phosphorylation and promote the unfolded protein response (UPR) attenuated iron-induced insulin resistance. Salubrinal induced autophagy flux and its beneficial effects on insulin sensitivity were not observed in autophagy-deficient cells generated by overexpressing a dominant-negative ATG5 mutant or via knockout of ATG7. This indicated the beneficial effect of salubrinal-induced UPR activation was autophagy-dependent. We translated these observations to an animal model of systemic iron overload-induced skeletal muscle insulin resistance where administration of salubrinal as pretreatment promoted eIF2α phosphorylation, enhanced autophagic flux in skeletal muscle and improved insulin responsiveness. Together, our results show that salubrinal elicited an eIF2α-autophagy axis leading to improved skeletal muscle insulin sensitivity both in vitro and in mice.

Abstract 6096: Targeting premalignant lung cancer to intercept progression to invasive disease

Abstract Increased implementation of low dose CT-guided screens in the clinic has led to the identification of premalignant lung nodules (ground glass opacity, GGO) which progress to invasive adenocarcinoma1,2. Using integrated genomic analysis of a cohort of >300 patients with premalignant lesions, we constructed the first high-resolution landscape of composition, lineage/functional states, developmental trajectories, and multicellular crosstalk networks, which revealed potential targets for therapeutic intervention. We uncovered that early lesions exhibit marked immune-suppressive phenotypes characterized by increased T lymphocyte exhaustion to cytotoxic scores, decrease in NKT cells, elevated Tregs, increased myeloid suppressor activity, accumulation of immunosuppressive myeloid cells activation of unfolded protein response (UPR) sensor IRE1α that activates the multitasking transcription factor XBP1 known to drive malignant progression. Targeting IRE1α endoribouclease with a small molecule drug limited disease progression with marked immunomodulation in our newly developed Kras and EGFR driven mouse models, which mirror progression of human precursor lesions to invasive disease. As an immunoprevention approach, we have generated LNP-mRNA vaccines targeting top two shared neoantigens in premalignant lesions, which have shown marked immunogenicity and efficacy in early disease. Our work provides the molecular foundation for precision oncology strategies to intercept transition of preinvasive to invasive adenocarcinoma. 1. Altorki, N.K., et al. Nat Rev Cancer, 2019. 2. Altorki, N.K., et al. Cell Rep, 2022. Citation Format: Mitchell Martin, Yongfeng He, Bhavneet Bhinder, Liron Yoffe, Sijin Luo Zhong, Arshdeep Singh, Olivier Elemento, Shaoyi Jiang, Nasser Altorki, Vivek Mittal. Targeting premalignant lung cancer to intercept progression to invasive disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6096.

Abstract 6952: The role of interleukin-11 (IL11) in cancer-associated cachexia (CAC)

Abstract Cancer-associated cachexia (CAC), characterized by loss of skeletal muscle and involuntary body weight loss, is responsible for increased chemotherapy toxicity, reduced quality of life, and impaired survival in cancer patients. While interleukin-11 (IL11), one of the IL6 family cytokines, was originally thought to be involved in hematopoiesis, studies increasingly suggest its involvement in tumorigenesis including chemoresistance, metastasis, and survival of cancer cells. However, the precise role of IL11 in CAC has not been determined. Our lab has previously shown that IL11 treatment suppressed the myogenic differentiation of C2C12 myoblasts, suggesting that IL11 may interfere with differentiation and subsequent regeneration of muscle. In this study, we investigated the role of IL11 in cancer-induced muscle wasting using cell culture and mouse models. The mRNA expression of IL11 was highly up-regulated in C26 and Lewis lung carcinoma (LLC) cells, two well-known cachectic mouse cancer cell lines, while its expression was low in a non-cachectic MC38 mouse colon cancer cell line. Knockdown of IL11 in C26 cells using two independent lentiviral shRNAs significantly reduced the mRNA levels of the cytokines involved in CAC including GDF15, LIF, and VEGFA, while the mRNA level of IL6 was not significantly altered. Additionally, IL11 knockdown significantly reduced the mRNA and protein levels of ATF4, CHOP, and TRIB3, suggesting that IL11 may be responsible for the activation of unfolded protein response, an adaptive mechanism for growth, survival, and angiogenesis of cancer. Knockdown of IL11 in C26 and LLC cell lines also significantly reduced the volume of tumors in syngeneic mouse models, while over-expression of IL11 in MC38 cells increased tumor size. Moreover, the knockdown of IL11 in cachectic cancer cells partially prevented the loss of muscle and body weight in tumor-bearing mice, although the amelioration of muscle loss by IL11 knockdown may be associated with the decreased tumor burden. Together, these results indicate that IL11 may contribute to muscle wasting and provide novel insight into the previously unestablished role of IL11 in CAC. Citation Format: Kimberly Drinkwater, Ashlyn Meyers, Samin Kargari, Natalie Crisan, Aaron Mody, Minsub Shim. The role of interleukin-11 (IL11) in cancer-associated cachexia (CAC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6952.

Abstract 584: Synergistic action of ATF6 and EZH2 in sensing metabolic stress to boost anti-tumor immunity

Abstract Although immune checkpoint blockade (ICB) marked a breakthrough in melanoma treatment, half of patients fail to respond, underscoring the crucial need for complementary adjuvant and neo-adjuvant therapies. Due to the low overall response rate and the stark contrast in outcomes between patients that respond and those that fail to respond, identifying the defining characteristics of immune checkpoint blockade (ICB) response is essential for enhancing the efficacy of melanoma treatments. To recapitulate and investigate the ICB-responsive phenotype in vitro, melanoma cells were metabolically adapted to an environment lacking glucose which successfully drove the dependence on oxidative metabolism and restored MHC-I expression, two established characteristics of ICB responsive tumors. Further, metabolic adaptations significantly enhance the tumor sensitivity to T-cell killing. Surprisingly even in JAK1 and IFNAR1 deficient cells, metabolic adaptation restored MHC I antigen presentation, indicating an interferon-independent mechanism of MHC-I regulation. Proteomic analysis of three metabolically conditioned melanoma cells lines (human and mouse) identified downregulation of the histone methyltransferase EZH2 and the activating transcription factor ATF6 as key corollaries of MHC-I expression. Interestingly, EZH2 and ATF6 activity were found to be inversely related in metabolically adapted cells and patient tumor samples. These studies mechanistically explore the epigenomic control of antigen presentation machinery and the transcriptional targets of ATF6 controlling sensitivity T cell killing. Here, through in vitro and in vivo studies, we demonstrate that constitutive activation of ATF6, and thereby activation of the adaptive unfolded protein response (UPR), sensitizes melanoma to immune control. These data suggest that EZH2 and ATF6 act synergistically to balance immune mediated killing during metabolic stress whereby loss of EZH2 increases MHC-I antigen presentation and activation of ATF6 sensitizes tumor cells to T cell-mediated control. Ongoing therapeutic studies combining EZH2 inhibition, ATF6 activation, and ICB in pre-clinical models further suggest targeting the synergy between EZH2 and ATF6 is a putative adjuvant or neo-adjuvant target to improve the clinical response of ICB. Citation Format: Jacob L. Edmondson, Megan R. Reed, Daniel Fil, Billie Heflin, Brian Koss, Alan J. Tackett. Synergistic action of ATF6 and EZH2 in sensing metabolic stress to boost anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 584.

Glitches in the brain: the dangerous relationship between radiotherapy and brain fog.

Up to approximately 70% of cancer survivors report persistent deficits in memory, attention, speed of information processing, multi-tasking, and mental health functioning, a series of symptoms known as "brain fog." The severity and duration of such effects can vary depending on age, cancer type, and treatment regimens. In particular, every year, hundreds of thousands of patients worldwide undergo radiotherapy (RT) for primary brain tumors and brain metastases originating from extracranial tumors. Besides its potential benefits in the control of tumor progression, recent studies indicate that RT reprograms the brain tumor microenvironment inducing increased activation of microglia and astrocytes and a consequent general condition of neuroinflammation that in case it becomes chronic could lead to a cognitive decline. Furthermore, radiation can induce endothelium reticulum (ER) stress directly or indirectly by generating reactive oxygen species (ROS) activating compensatory survival signaling pathways in the RT-surviving fraction of healthy neuronal and glial cells. In particular, the anomalous accumulation of misfolding proteins in neuronal cells exposed to radiation as a consequence of excessive activation of unfolded protein response (UPR) could pave the way to neurodegenerative disorders. Moreover, exposure of cells to ionizing radiation was also shown to affect the normal proteasome activity, slowing the degradation rate of misfolded proteins, and further exacerbating ER-stress conditions. This compromises several neuronal functions, with neuronal accumulation of ubiquitinated proteins with a consequent switch from proteasome to immunoproteasome that increases neuroinflammation, a crucial risk factor for neurodegeneration. The etiology of brain fog remains elusive and can arise not only during treatment but can also persist for an extended period after the end of RT. In this review, we will focus on the molecular pathways triggered by radiation therapy affecting cognitive functions and potentially at the origin of so-called "brain fog" symptomatology, with the aim to define novel therapeutic strategies to preserve healthy brain tissue from cognitive decline.

IRE1α inhibits osteogenic differentiation of mouse embryonic fibroblasts by limiting Shh signaling.

This study aimed to investigate the effect of endoplasmic reticulum (ER) stress sensor inositol-requiring enzyme 1α (IRE1α) on the sonic hedgehog N-terminus (N-Shh)-enhanced-osteogenic differentiation process in mouse embryonic fibroblasts (MEFs). Osteogenesis of MEFs was observed by alkaline phosphatase (ALP) staining, alizarin red staining, and Von Kossa staining assays. Activation of unfolded protein response and Shh signaling were examined using real-time quantitative PCR and western blot assays. IRE1α-deficient MEFs were used to explore the effect of IRE1α on N-Shh-driven osteogenesis. N-Shh increased ALP activity, matrix mineralization, and the expression of Alp and Col-I in MEFs under osteogenic conditions; notably, this was reversed when combined with the ER stress activator Tm treatment. Interestingly, the administration of N-Shh decreased the expression of IRE1α. Abrogation of IRE1α increased the expression of Shh pathway factors in osteogenesis-induced MEFs, contributing to the osteogenic effect of N-Shh. Moreover, IRE1α-deficient MEFs exhibited elevated levels of osteogenic markers. Our findings suggest that the IRE1α-mediated unfolded protein response may alleviate the ossification of MEFs by attenuating Shh signaling. Our research has identified a strategy to inhibit excessive ossification, which may have clinical significance in preventing temporomandibular joint bony ankylosis.

Protein Glycosylation Patterns Shaped By the IRE1‐XBP1s Arm of the Unfolded Protein Response

AbstractThe unfolded protein response (UPR) is a sensing and signaling pathway that surveys the endoplasmic reticulum (ER) for protein folding challenges and responds whenever issues are detected. UPR activation leads to upregulation of secretory pathway chaperones and quality control factors, as well as reduces the nascent protein load on the ER, thereby restoring and maintaining proteostasis. This paradigm‐defining view of the role of the UPR is accurate, but it elides additional key functions of the UPR in cell biology. In particular, recent work has revealed that the UPR can shape the structure and function of N‐ and O‐glycans installed on ER client proteins. This crosstalk between the UPR's reaction to protein misfolding and the regulation of glycosylation remains insufficiently understood. Still, emerging evidence makes it clear that the UPR, and particularly the IRE1‐XBP1s arm of the UPR, may be a central regulator of protein glycosylation, with important biological consequences. In this review, we discuss the crosstalk between proteostasis, the UPR, and glycosylation, present progress towards understanding biological functions of this crosstalk, and examine potential roles in diseases such as cancer.

Coated sodium butyrate ameliorates high-energy and low-protein diet induced hepatic dysfunction via modulating mitochondrial dynamics, autophagy and apoptosis in laying hens

BackgroundFatty liver hemorrhagic syndrome (FLHS), a fatty liver disease in laying hens, poses a grave threat to the layer industry, stemming from its ability to trigger an alarming plummet in egg production and usher in acute mortality among laying hens. Increasing evidence suggests that the onset and progression of fatty liver was closely related to mitochondria dysfunction. Sodium butyrate was demonstrated to modulate hepatic lipid metabolism, alleviate oxidative stress and improve mitochondrial dysfunction in vitro and mice models. Nevertheless, there is limited existing research on coated sodium butyrate (CSB) to prevent FLHS in laying hens, and whether and how CSB exerts the anti-FLHS effect still needs to be explored. In this experiment, the FLHS model was induced by administering a high-energy low-protein (HELP) diet in laying hens. The objective was to investigate the effects of CSB on alleviating FLHS with a focus on the role of CSB in modulating mitochondrial function.MethodsA total of 288 healthy 28-week-old Huafeng laying hens were arbitrarily allocated into 4 groups with 6 replicates each, namely, the CON group (normal diet), HELP group (HELP diet), CH500 group (500 mg/kg CSB added to HELP diet) and CH750 group (750 mg/kg CSB added to HELP diet). The duration of the trial encompassed a period of 10 weeks.ResultsThe result revealed that CSB ameliorated the HELP-induced FLHS by improving hepatic steatosis and pathological damage, reducing the gene levels of fatty acid synthesis, and promoting the mRNA levels of key enzymes of fatty acid catabolism. CSB reduced oxidative stress induced by the HELP diet, upregulated the activity of GSH-Px and SOD, and decreased the content of MDA and ROS. CSB also mitigated the HELP diet-induced inflammatory response by blocking TNF-α, IL-1β, and F4/80. In addition, dietary CSB supplementation attenuated HELP-induced activation of the mitochondrial unfolded protein response (UPRmt), mitochondrial damage, and decline of ATPase activity. HELP diet decreased the autophagosome formation, and downregulated LC3B but upregulated p62 protein expression, which CSB administration reversed. CSB reduced HELP-induced apoptosis, as indicated by decreases in the Bax/Bcl-2, Caspase-9, Caspase-3, and Cyt C expression levels.ConclusionsDietary CSB could ameliorate HELP diet-induced hepatic dysfunction via modulating mitochondrial dynamics, autophagy, and apoptosis in laying hens. Consequently, CSB, as a feed additive, exhibited the capacity to prevent FLHS by modulating autophagy and lipid metabolism.Graphical

Investigating the Role of 17-Beta Estradiol in the Regulation of the Unfolded Protein Response (UPR) in Pancreatic Beta Cells.

Diabetes mellitus is clinically defined by chronic hyperglycemia. Sex differences in the presentation and outcome of diabetes exist with premenopausal women having a reduced risk of developing diabetes, relative to men, or women after menopause. Accumulating evidence shows a protective role of estrogens, specifically 17-beta estradiol, in the maintenance of pancreatic beta cell health; however, the mechanisms underlying this protection are still unknown. To elucidate these potential mechanisms, we used a pancreatic beta cell line (BTC6) and a mouse model of hyperglycemia-induced atherosclerosis, the ApoE-/-:Ins2+/Akita mouse, exhibiting sexual dimorphism in glucose regulation. In this study we hypothesize that 17-beta estradiol protects pancreatic beta cells by modulating the unfolded protein response (UPR) in response to endoplasmic reticulum (ER) stress. We observed that ovariectomized female and male ApoE-/-:Ins2+/Akita mice show significantly increased expression of apoptotic UPR markers. Sham operated female and ovariectomized female ApoE-/-:Ins2+/Akita mice supplemented with exogenous 17-beta estradiol increased the expression of adaptive UPR markers compared to non-supplemented ovariectomized female ApoE-/-:Ins2+/Akita mice. These findings were consistent to what was observed in cultured BTC6 cells, suggesting that 17-beta estradiol may protect pancreatic beta cells by repressing the apoptotic UPR and enhancing the adaptive UPR activation in response to pancreatic ER stress.

Endoplasmic reticulum stress and autophagy in cerebral ischemia/reperfusion injury: PERK as a potential target for intervention

Abstract Several studies have shown that activation of unfolded protein response and endoplasmic reticulum (ER) stress plays a crucial role in severe cerebral ischemia/reperfusion injury. Autophagy occurs within hours after cerebral ischemia, but the relationship between ER stress and autophagy remains unclear. In this study, we established experimental models using oxygen-glucose deprivation/reoxygenation in PC12 cells and primary neurons to simulate cerebral ischemia/reperfusion injury. We found that prolongation of oxygen-glucose deprivation activated the ER stress pathway protein kinase-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2 subunit alpha (eIF2α)-activating transcription factor 4 (ATF4)-C/EBP homologous protein (CHOP), increased neuronal apoptosis, and induced autophagy. Furthermore, inhibition of ER stress using inhibitors or by siRNA knockdown of the PERK gene significantly attenuated excessive autophagy and neuronal apoptosis, indicating an interaction between autophagy and ER stress and suggesting PERK as an essential target for regulating autophagy. Blocking autophagy with chloroquine exacerbated ER stress-induced apoptosis, indicating that normal levels of autophagy play a protective role in neuronal injury following cerebral ischemia/reperfusion injury. Findings from this study indicate that cerebral ischemia/ reperfusion injury can trigger neuronal ER stress and promote autophagy, and suggest that PERK is a possible target for inhibiting excessive autophagy in cerebral ischemia/reperfusion injury.

Mitochondrial-to-nuclear communications through multiple routes regulate cardiomyocyte proliferation

The regenerative capacity of the adult mammalian heart remains a formidable challenge in biological research. Despite extensive investigations into the loss of regenerative potential during evolution and development, unlocking the mechanisms governing cardiomyocyte proliferation remains elusive. Two recent groundbreaking studies have provided fresh perspectives on mitochondrial-to-nuclear communication, shedding light on novel factors that regulate cardiomyocyte proliferation. The studies identified two mitochondrial processes, fatty acid oxidation and protein translation, as key players in restricting cardiomyocyte proliferation. Inhibition of these processes led to increased cell cycle activity in cardiomyocytes, mediated by reduction in H3k4me3 levels through accumulated α-ketoglutarate (αKG), and activation of the mitochondrial unfolded protein response (UPRmt), respectively. In this research highlight, we discuss the novel insights into mitochondrial-to-nuclear communication presented in these studies, the broad implications in cardiomyocyte biology and cardiovascular diseases, as well as the intriguing scientific questions inspired by the studies that may facilitate future investigations into the detailed molecular mechanisms of cardiomyocyte metabolism, proliferation, and mitochondrial-to-nuclear communications.

MUCOSAL SINGLE-CELL PROFILING OF CROHN’S-LIKE DISEASE OF THE POUCH REVEALS COMMON PATHOGENICS AND THERAPEUTIC TARGETS

Abstract BACKGROUND & AIMS After restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC), a subset of patients develop Crohn’s-like disease of the pouch (CDP), an inflammatory condition that affects both the ileoanal pouch and extra-pouch organs including pre-pouch ileum. The cellular and molecular identities of CDP are unknown, and its diagnosis and treatment remain challenging. To define the pathophysiology of CDP, we examined mucosal cells from patients with and without CDP using single cell analyses. METHODS Endoscopic samples from the pouch and pre-pouch ileum of 50 patients with an IPAA were collected for single-cell RNA sequencing (scRNA-seq) or mass cytometry (or CyTOF). We analyzed immune and non-immune cells from pouch and ileal tissues of patients with normal pouch/ileum and CDP (n = 5 or 6) using scRNA-seq. CyTOF was performed on mucosal immune cells from independent cohorts of patients with normal pouch/ileum, CDP, and pouchitis (n = 9 or 10). Mucosal samples from patients with familial adenomatous polyposis (FAP) after pouch formation were also analyzed by CyTOF. Some scRNA-seq and CyTOF findings were independently validated using immunohistochemistry (n = 6 or 8). RESULTS We revealed distinct immune and non-immune cell clusters in normal pouch and pre-pouch ileum. Colitogenic immune cells expanded in normal UC pouch compared to normal FAP pouch. Compared to normal pouch/pre-pouch ileum, CDP tissues exhibited expanded TCR clonotypes, elevated Th17 signaling, and diminished T cell exhaustion markers. Elevated frequencies of plasma cells, inflammatory fibroblasts and monocytes were noted in CDP pouch and ileum (Fig 1; only pouch data were shown). CDP also harbored elevated Th17-inducing cytokines such as IL23, IL1B, and IL6 produced by myeloid cells. ScRNA-seq and CyTOF identified increased CD14+TREM1+ pathogenic monocytes in both pouch and pre-pouch ileum of CDP. Ligand-receptor interactions further uncovered an essential role of proinflammatory myeloid cells in the pathogenesis of CDP. In addition, pouch and pre-pouch ileum of CDP showed prominent activation of the unfolded protein response (UPR) across all major cell compartments (Fig 2), which was not present in UC, CD or pouchitis based on integrated analysis of published databases. CONCLUSIONS CDP demonstrates altered immune and non-immune cell populations/states in the pouch and pre-pouch ileum. CDP represents a distinct entity of inflammatory bowel disease with extensive UPR activation, a unique feature that may serve as novel diagnostic markers and therapeutic targets using ER stress-alleviating agents including chemical chaperones. Fig 1 CDP reshapes immune and non-immune landscapes in pouch and pre-pouch ileum. (A) UMAP visualization of all cells from the pouch and ileum of CLDP and normal controls. (B) Diversity of epithelial cells, stromal cells, myeloid cells, T cells, innate lymphoid cells (ILCs), B cells and plasma cells in CLDP vs. normal controls in the pouch estimated by scRNA-seq (n = 5 or 6). *p<0.01. Fig 2 Extensive UPR activation in CDP tissues. (A) UPR gene expression in major cell compartments in CDP and normal controls, highlighted genes including CALR, XBP1, and SERP1 were upregulated in all 5 cell compartments. (B) ER stress/UPR pathways are enriched in CDP. (C) IHC of UPR proteins XBP1 and HSPA5/BiP.