Modulation Of Endoplasmic Reticulum Stress Research Articles

MiR-185-5p ameliorates endoplasmic reticulum stress and renal fibrosis by downregulation of ATF6

AbstractEndoplasmic reticulum (ER) stress is considered an important factor in the formation of fibrosis. Therefore, modulation of ER stress may represent a promising therapeutic strategy in renal fibrosis. MiR-185-5p has been identified to be implicated in TGF-β1-induced renal fibrosis; however, it is largely unknown whether and how miR-185-5p regulates ER stress in renal fibrosis. In this study, we demonstrated that miR-185-5p directly bound to ATF6, an ER stress-related protein, and downregulated the expression thereof. We subsequently constructed an in vitro model of renal fibrosis using HK2 cells treated with TGF-β1, and found that miR-185-5p attenuated ER stress and dedifferentiation of tubular epithelia by suppression of ATF6. In addition, we constructed an in vivo mouse model using unilateral urethral obstruction (UUO). Our in vivo findings showed that miR-185-5p reduced the expression of ER stress-related proteins and inhibited epithelial dedifferentiation via downregulation of ATF6, thereby improving UUO-induced renal fibrosis. Overall, our findings revealed that miR-185-5p exerts beneficial effects in renal fibrosis. Thus, the miR-185-5p/ATF6 regulatory pathway may be a potential target for therapeutic intervention in renal fibrosis.This study reveals that miR-185-5p alleviates endoplasmic reticulum (ER) stress, prevents epithelial dedifferentiation and subsequently improves renal fibrosis by downregulating activating transcription factor 6 (ATF6). The miR-185-5p/ATF6 pathway is proposed as a novel regulatory mechanism for renal fibrosis and may provide a therapeutic strategy for renal fibrosis associated with ER stress.

Role of binding immunoglobulin protein (BiP) in Graves' orbitopathy pathogenesis.

Inflammation and adipogenesis represent the main pathogenic mechanisms of Graves' orbitopathy (GO), and oxidative stress is a well-known inducer of GO pathology. Endoplasmic reticulum (ER) stress has been suggested as a major contributor to inflammation and reactive oxygen species (ROS) generation. In this study, we investigated the role of the ER-stress chaperone protein, binding immunoglobulin protein (BiP), in GO pathogenesis. Using primary cultures of orbital fibroblasts from patients with GO, we examined the role of BiP in GO pathogenesis by silencing its expression with small-interfering RNA (siRNA). Inflammatory cytokine expression was analysed by Western blotting and ELISA. Intracellular ROS levels induced by hydrogen peroxide or cigarette smoke extract were measured by 5-(and 6)-carboxy-20,70-dichlorodihydrofluorescein diacetate staining and flow cytometry. After adipogenic differentiation in BiP siRNA-transfected cells, the cells were stained with Oil Red O, and the levels of adipogenic transcription factors were determined by Western blot analysis. BiP mRNA expression levels were significantly higher in GO orbital tissues than in non-GO orbital tissues. Silencing BiP attenuated the expression of pro-inflammatory cytokines (interleukin-6, intercellular adhesion molecule-1, and monocyte chemotactic protein-1) in primary cultured GO orbital fibroblasts. Silencing BiP also reduced ROS generation, hyaluronan production, and adipocyte differentiation. These findings suggest that ER stress is involved in the aetiology of GO and that modulation of ER stress has therapeutic potential for GO.

Endoplasmic reticulum stress remodels alveolar bone formation after tooth extraction.

Bone healing in tooth extraction sockets occurs in a complex environment containing saliva and many microorganisms and is affected by many factors. Endoplasmic reticulum (ER) stress affects bone metabolism, but the role of ER stress in bone healing after tooth extraction remains unclear. We utilized a rat tooth extraction model, in which we promoted wound healing by using salubrinal to regulate the ER stress response. Western blot analysis showed increased expression of p‐eIF2α/eIF2α, Runx2 and alkaline phosphatase (ALP) in bone tissue, and histological assays showed irregularly arranged and new bone with more collagen fibres 14 days after tooth extraction and after modulating the degree of ER stress. Micro‐CT showed that modulating ER stress to an appropriate degree increases bone filling in regards to the density in the bottom and the surrounding bone wall of the tooth extraction wounds. Transmission electron microscopy showed rough ER expansion and newly formed collagen fibrils in osteoblasts after modulating ER stress to an appropriate degree. We also used different concentrations of salubrinal to evaluate the resistance to tunicamycin‐induced ER stress in an osteogenic induction environment. Salubrinal restored the tunicamycin‐induced decrease in the viability of primary calvarial osteoblasts and increased the expression of Runx2 and ALP, and decreased p‐eIF2α/eIF2α in a dose‐dependent manner. Taken together, the results demonstrate that ER stress occurred after tooth extraction, and regulating the degree of ER stress can promote bone healing in tooth extraction sockets, providing clinical evidence for bone healing.

Natural activators of SIRT1 protect cardiac cells from severe ER stress

Perturbations of endoplasmic reticulum (ER) functions result in the accumulation of misfolded proteins inside the ER lumen, a condition known as ER stress. As a compensatory pro-survival response, the unfolded protein response (UPR) is activated to restore normal ER function. However, in the case of severe or chronic ER stress, apoptosis is triggered to eliminate damaged cells. Recently, ER stress was implicated in the pathogenesis of various heart diseases including heart failure (HF). Previously, we have shown that SIRT1 is cardioprotective against severe ER stress by regulating the PERK pathway of the UPR, suggesting that activation of SIRT1 could be effective to limit cardiac ER stress. Different natural polyphenols have been shown to activate SIRT1 in a variety of models. In this study, we thus evaluated the protective effect of 11 polyphenols (butein, fisetin, catechin…) that have been reported to activate SIRT1, against severe ER stress-induced cardiac cell death. Tunicamycin (TN), an inhibitor of N-glycosylation of proteins, was used to induce a severe ER stress in H9c2 cardiomyoblasts and adult rat ventricle myocytes (ARVM). Cell death was determined by fluorescence microscopy and flow cytometry and ER stress was analysed by western blot. As expected, TN induced an increase of the ER stress markers GRP78, GRP94 and the pro-apoptotic transcription factor CHOP associated with cardiac cell death. Three of the tested polyphenols were shown to protect H9c2 cells and ARVM against cell death induced by TN. Protection was abrogated when SIRT1 was inhibited by EX527. In addition, the level of the pro-apoptotic factor CHOP was reduced with polyphenols. These results indicated that among the 11 polyphenols tested, 3 were able to protect cardiac cells from ER stress-induced cell death in a SIRT1-dependent manner. Activation of SIRT1 by selected polyphenols thus appears as a promising strategy to modulate ER stress and protect cardiac cells.

Tissue plasminogen activator attenuates outflow facility reduction in mouse model of juvenile open angle glaucoma

Tissue plasminogen activator (tPA) has been shown to prevent steroid-induced reduction in aqueous humor outflow facility via an upregulation in matrix metalloproteinase (Mmp) expression. The purpose of this study was to determine whether tPA can rescue outflow facility reduction in the Tg-MYOCY437H mouse model, which replicates human juvenile open angle glaucoma. Outflow facility was measured in Tg-MYOCY437H mice following: periocular steroid exposure and intraocular protein treatment with enzymatically active or enzymatically inactive tPA. Effects of tPA on outflow facility were compared to those of animals treated with topical sodium phenylbutarate (PBA), a modulator of endoplasmic reticulum stress. Gene expression of fibrinolytic pathway components (Plat, Plau, and Pai-1) and matrix metalloproteinases (Mmp-2, -9, and -13) was determined in angle ring tissues containing the trabecular meshwork. Tg-MYOCY437H mice did not display further outflow facility reduction following steroid exposure. Enzymatically active and enzymatically inactive tPA were equally effective in attenuating outflow facility reduction in Tg-MYOCY437H mice and caused enhanced expression of matrix metalloproteinases (Mmp-9 and Mmp-13). tPA was equally effective to topical PBA treatment in ameliorating outflow facility reduction in Tg-MYOCY437H mice. Both treatments were associated with an upregulation in Mmp-9 expression while tPA also upregulated Mmp-13 expression. tPA increases the expression of matrix metalloproteinases and may cause extracellular matrix remodeling at the trabecular meshwork, which results in reversal of outflow facility reduction in Tg-MYOCY437H mice.

A comparative study on normal and obese mice indicates that the secretome of mesenchymal stromal cells is influenced by tissue environment and physiopathological conditions

BackgroundThe term mesenchymal stromal cells (MSCs) designates an assorted cell population comprised of stem cells, progenitor cells, fibroblasts, and stromal cells. MSCs contribute to the homeostatic maintenance of many organs through paracrine and long-distance signaling.Tissue environment, in both physiological and pathological conditions, may affect the intercellular communication of MSCs.MethodsWe performed a secretome analysis of MSCs isolated from subcutaneous adipose tissue (sWAT) and visceral adipose tissue (vWAT), and from bone marrow (BM), of normal and obese mice.ResultsThe MSCs isolated from tissues of healthy mice share a common core of released factors: components of cytoskeletal and extracellular structures; regulators of basic cellular functions, such as protein synthesis and degradation; modulators of endoplasmic reticulum stress; and counteracting oxidative stress. It can be hypothesized that MSC secretome beneficially affects target cells by the horizontal transfer of many released factors. Each type of MSC may exert specific signaling functions, which could be determined by looking at the many factors that are exclusively released from every MSC type.The vWAT-MSCs release factors that play a role in detoxification activity in response to toxic substances and drugs. The sWAT-MSC secretome contains proteins involved in in chondrogenesis, osteogenesis, and angiogenesis. Analysis of BM-MSC secretome revealed that these cells exert a signaling function by remodeling extracellular matrix structures, such as those containing glycosaminoglycans. Obesity status profoundly modified the secretome content of MSCs, impairing the above-described activity and promoting the release of inflammatory factors.ConclusionWe demonstrated that the content of MSC secretomes depends on tissue microenvironment and that pathological condition may profoundly alter its composition.8V16rGN-xAmXY2STsyvvq8Video abstract

Endoplasmic reticulum stress in the dorsal root ganglia regulates large-conductance potassium channels and contributes to pain in a model of multiple sclerosis.

Neuropathic pain is a common symptom of multiple sclerosis (MS) and current treatment options are ineffective. In this study, we investigated whether endoplasmic reticulum (ER) stress in dorsal root ganglia (DRG) contributes to pain hypersensitivity in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Inflammatory cells and increased levels of ER stress markers are evident in post-mortem DRGs from MS patients. Similarly, we observed ER stress in the DRG of mice with EAE and relieving ER stress with a chemical chaperone, 4-phenylbutyric acid (4-PBA), reduced pain hypersensitivity. In vitro, 4-PBA and the selective PERK inhibitor, AMG44, normalize cytosolic Ca2+ transients in putative DRG nociceptors. We went on to assess disease-mediated changes in the functional properties of Ca2+ -sensitive BK-type K+ channels in DRG neurons. We found that the conductance-voltage (GV) relationship of BK channels was shifted to a more positive voltage, together with a more depolarized resting membrane potential in EAE cells. Our results suggest that ER stress in sensory neurons of MS patients and mice with EAE is a source of pain and that ER stress modulators can effectively counteract this phenotype.

Cognitive Improvement by Vorinostat through Modulation of Endoplasmic Reticulum Stress in a Corticosterone-Induced Chronic Stress Model in Mice.

Chronic stress is the leading cause of memory impairment today. Various stress-based models are being developed for studying cognitive impairment. Repurposing of existing drugs in a new pharmacology class is the safest and cheapest option for treatment instead of new drug discovery. Vorinostat (VOR) is the first histone deacetylase (HDAC) inhibitor approved for the treatment of cutaneous T-cell lymphoma by the U.S. FDA. VOR follows the rule of five and is reported to cross the blood-brain barrier. Therefore, we aimed to evaluate the procognitive potential of VOR (25 mg/kg) administered by intraperitoneal (ip) route in a stress-based model of chronic corticosterone (CORT) injections (20 mg/kg, subcutaneously (sc)). The study comprised six groups. Normal mice were administered vehicle (VEH) (days 1-21, sc) in the first group, VOR (days 8-21, 25 mg/kg, ip) in the second group, and fluoxetine (FLX) (days 8-21, 15 mg/kg, oral) in the third group. Mice in the remaining three groups were given 20 mg/kg (sc) CORT for 21 days, and VOR (days 8-21, 25 mg/kg, ip) or FLX (days 8-21, 15 mg/kg, oral) was additionally administered to the treatment groups. Behavioral tests such as Morris water maze test, novel object recognition test, and object in place test were performed at the end of the dosing schedule to assess cognition. After behavior tests, mice were sacrificed, and hippocampus was separated from brain tissue for reverse transcriptase polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry studies. VOR treatment attenuated endoplasmic reticulum (ER) stress in CORT mice as evident from the reduction in DNA damage-inducible transcript 3 (Ddit3) (gene encoding CHOP), caspase 12 (Casp12), and calpain-2 (Capn2) mRNA levels, and cleaved caspase 3 (CASP3) protein expression. Bax inhibitor-1 (BI-1) was significantly increased in VOR-treated CORT mice. VOR also reversed CORT induced increase in HDAC2 level in the CA3 region. The protective effects of VOR were comparable to that of FLX in CORT mice. Thus, VOR has the potential to reverse cognitive dysfunction via modulation of ER stress markers and HDAC2.

PpAOX regulates ER stress tolerance in Physcomitrella patens

Severe environments disturb the folding or assembly of newly synthesized proteins, resulting in accumulation of misfolded or unfolded proteins in the endoplasmic reticulum (ER) as well as cytotoxic aggregation of abnormal proteins. Therefore, ER stress is evoked due to disturbed ER homeostasis. Alternative oxidase (AOX) plays an important role in coping with various abiotic stresses and plant growth. Our previous study has reported that PpAOX is involved in the regulation of salt tolerance in moss Physcomitrella patens (P. patens), but its biological functions in modulating ER stress remain unknown. Here we report that the gametophyte of P. patens displays severe growth inhibition and developmental deficiency under tunicamycin (Tm, an elicitor of ER stress)-induced ER stress conditions. PpAOX and selected ER stress response-like genes in P. patens were induced under Tm treatment. PpAOX knockout (PpAOX KO) plants exhibited decreased resistance to Tm-induced ER stress, whereas PpAOX-overexpressing lines (PpAOX OX) plants were more tolerant to Tm-induced ER stress. Data showed that PpAOX contributes to redox homeostasis under Tm treatment. In addition, we observed that PpAOX completely restores the Tm-sensitive phenotype of Arabidopsis AOX1a mutant (Ataox1a). Taken together, our work reveals a functional link between PpAOX and ER stress tolerance regulation in P. patens.

The ER stress-autophagy axis: implications for cognitive dysfunction in diabetes mellitus.

Unfolded protein response (UPR) often coordinates with autophagy to maintain cellular proteostasis. Disturbance of proteostasis correlates with diseases including diabetes and neurological complications. In a recent article in Clinical Science, Kong et al. highlighted the critical role of endoplasmic reticulum (ER) stress-autophagy axis in maintaining cognitive functions and provided pharmacological evidence with respect to cognitive improvements in a diabetic mouse model. These novel findings present new insights into the pathological mechanisms and therapeutic implications with the ER stress modulators in diabetes-related cognitive dysfunction.

Loss of tumor susceptibility gene 101 (TSG101) perturbs endoplasmic reticulum structure and function

Tumor susceptibility gene 101 (TSG101), an ESCRT-I protein, is implicated in multiple cellular processes and its functional depletion can lead to blocked lysosomal degradation, cell cycle arrest, demyelination and neurodegeneration. Here, we show that loss of TSG101 results in endoplasmic reticulum (ER) stress and this causes ER membrane remodelling (EMR). This correlates with an expansion of ER, increased vacuolation, altered relative distribution of the rough and smooth ER and disruption of three-way junctions. Blocked lysosomal degradation due to TSG101 depletion leads to ER stress and Ca2+ leakage from ER stores, causing destabilization of actin cytoskeleton. Inhibiting Ca2+ release from the ER by blocking ryanodine receptors (RYRs) with Dantrolene partially rescues the ER stress phenotypes. Hence, in this study we have identified the involvement of TSG101 in modulating ER stress mediated remodelling by engaging the actin cytoskeleton. This is significant because functional depletion of TSG101 effectuates ER-stress, perturbs the structure, mobility and function of the ER, all aspects closely associated with neurodegenerative diseases. Summary statementWe show that tumor susceptibility gene (TSG) 101 regulates endoplasmic reticulum (ER) stress and its membrane remodelling. Loss of TSG101 perturbs structure, mobility and function of the ER as a consequence of actin destabilization.

Expression of cancer cell-associated Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) regulates protective anti-tumor immunity in melanoma

Abstract Activation of moderated endoplasmic reticulum (ER) stress in cancer cells promotes cellular adaptation and survival in response to the adverse conditions of the tumor microenvironment (TME). However, how modulation of ER stress in cancer cells culminates in the development of an immunosuppressive TME remains poorly understood and therapeutically unexploited. Upon disruption of ER homeostasis, mediator proteins IRE1a, ATF6 and PERK induce activation of the unfolded protein response (UPR). Although maladaptive priming of UPR mediators in immune cells deactivates their capacity for anti-tumor immunity, the immunosuppressive potential of UPR mediators in cancer cells, in particular PERK, remains unknown. Thus, in this study, we hypothesized that activation of PERK in tumor cells shapes the formation of the immunosuppressive TME and impairs the development of anti-tumor immunity. Elimination of PERK in cancer cells impaired survival upon chemically-induced ER stress and promoted caspase-independent cell death. Interestingly, in vivo ablation of PERK in melanoma impaired tumor growth, which surprisingly occurred in a T cell-dependent manner. Additionally we observed that ablation of PERK shifted the TME from an immunosuppressive to immunostimulatory environment, enhanced accumulation of effector T cells and directed reprogramming of myeloid derived suppressor cells (MDSCs) into immunostimulatory cells. Additional experiments indicated that ablation of PERK induced expression of mediators associated with immunogenic cell death. Collectively, our findings demonstrate the immunoregulatory role of PERK in tumor cells and highlight the therapeutic potential of targeting UPR mediators and tumor adaptive processes.

Extensive Unfolded Protein Response Stimulation in Colon Cancer Cells Enhances VEGF Expression and Secretion

Endoplasmic Reticulum (ER) is an important site for protein folding and maturation. Accumulation of unfolded or misfolded proteins in the ER leads to “ER stress”. The unfolded protein response (UPR) is a finely regulated cell-signaling program to re-establish ER folding capacity for building up cellular homeostasis. Although ER stress modulators have emerged as promising treatment options with their potential to trigger ER stress-mediated apoptosis in cancer cells, here we show that ER stress upregulates VEGF (Vascular Endothelial Growth Factor) expression and secretion in colon cancer cells, which may result in resistance against the treatment through enhanced autocrine/intracrine VEGF signaling and through supporting further angiogenesis.

Protection of retinal function and morphology in MNU-induced retinitis pigmentosa rats by ALDH2: an in-vivo study

BackgroundRetinitis pigmentosa (RP) is a kind of inherited retinal degenerative diseases characterized by the progressive loss of photoreceptors. RP has been a conundrum without satisfactory countermeasures in clinic until now. Acetaldehyde dehydrogenase 2 (ALDH2), a major enzyme involved in aldehyde detoxification, has been demonstrated to be beneficial for a growing number of human diseases, such as cardiovascular dysfunction, diabetes mellitus and neurodegeneration. However, its protective effect against RP remains unknown. Our study explored the impact of ALDH2 on retinal function and structure in N-methyl-N-nitrosourea (MNU)-induced RP rats.MethodsRats were gavaged with 5 mg/kg Alda-1, an ALDH2 agonist, 5 days before and 3 days after MNU administration. Assessments of retinal function and morphology as well as measurement of specific proteins expression level were conducted.ResultsElectroretinogram recordings showed that Alda-1 administration alleviated the decrease in amplitude caused by MNU, rendering protection of retinal function. Mitigation of photoreceptor degeneration in MNU-treated retinas was observed by optical coherence tomography and retinal histological examination. In addition, Western blotting results revealed that ALDH2 protein expression level was upregulatedwith increased expression of SIRT1 protein after the Alda-1 intervention. Besides, endoplasmic reticulum stress (ERS) was reduced according to the significant downregulation of GRP78 protein, while apoptosis was ameliorated as shown by the decreased expression of PARP1 protein.ConclusionsTogether, our data demonstrated that ALDH2 could provide preservation of retinal function and morphology against MNU-induced RP, with the underlying mechanism at least partly related to the modulation of SIRT1, ERS and apoptosis.

Grp78 Loss in Epithelial Progenitors Reveals an Age-linked Role for Endoplasmic Reticulum Stress in Pulmonary Fibrosis.

Rationale: Alveolar epithelial cell (AEC) injury and dysregulated repair are implicated in the pathogenesis of pulmonary fibrosis. Endoplasmic reticulum (ER) stress in AEC has been observed in idiopathic pulmonary fibrosis (IPF), a disease of aging.Objectives: To investigate a causal role for ER stress in the pathogenesis of pulmonary fibrosis (PF) and therapeutic potential of ER stress inhibition in PF.Methods: The role of ER stress in AEC dysfunction and fibrosis was studied in mice with tamoxifen (Tmx)-inducible deletion of ER chaperone Grp78, a key regulator of ER homeostasis, in alveolar type II (AT2) cells, progenitors of distal lung epithelium, and in IPF lung slice cultures.Measurements and Main Results:Grp78 deletion caused weight loss, mortality, lung inflammation, and spatially heterogeneous fibrosis characterized by fibroblastic foci, hyperplastic AT2 cells, and increased susceptibility of old and male mice, all features of IPF. Fibrosis was more persistent in more severely injured Grp78 knockout (KO) mice. Grp78 KO AT2 cells showed evidence of ER stress, apoptosis, senescence, impaired progenitor capacity, and activation of TGF-β (transforming growth factor-β)/SMAD signaling. Glucose-regulated protein 78 is reduced in AT2 cells from old mice and patients with IPF, and ER stress inhibitor tauroursodeoxycholic acid ameliorates ER stress and fibrosis in Grp78 KO mouse and IPF lung slice cultures.Conclusions: These results support a causal role for ER stress and resulting epithelial dysfunction in PF and suggest ER stress as a potential mechanism linking aging to IPF. Modulation of ER stress and chaperone function may offer a promising therapeutic approach for pulmonary fibrosis.

Effects of Arginine and Its Deprivation on Human Glioblastoma Physiology and Signaling.

The observations that numerous cancers are characterized by impairment in arginine synthesis and that deficit of exogenous arginine specifically affects their growth and viability are the ground for arginine deprivation-based anticancer treatment strategy. This review addresses molecular mechanisms of the human glioblastoma cell response to arginine deprivation. Our earlier studies have shown that arginine deprivation specifically impairs glioblastoma cell motility, adhesion and invasiveness. These changes were evoked by alterations in the actin cytoskeleton organization resulting from a decreased arginylation of β-actin isoform. Moreover, deficit of arginine induces prolonged endoplasmic reticulum (ER) stress and activation of the unfolded protein response, not leading, however, to a massive apoptosis in glioblastoma cells. Our current research indicates that cell death could be augmented by other compounds such as modulators of ER stress, for example arginine analogue of plant origin, canavanine. Implication of these studies on the development of new anti-glioma metabolic therapeutic modalities are discussed.

A Review of Mouse Models of Monogenic Diabetes and ER Stress Signaling.

Diabetes is a major public health problem: it is estimated that 420million people are affected globally. Monogenic forms of diabetes are less common, but variants in monogenic diabetes genes have been shown to contribute to type 2 diabetes risk. In vitro and in vivo models of monogenic forms of diabetes related to the endoplasmic reticulum (ER) stress response provided compelling evidence on the role of ER stress and dysregulated ER stress signaling on β cell demise in type 1 and type 2 diabetes. In this chapter, we describe the genetics, background, and phenotype of ER stress-related monogenic diabetes mouse models, and we comment on their advantages and disadvantages. We conclude that these mouse models are very useful tools for monogenic diabetes molecular pathogenesis studies, although there is a variability on the methodology that is used. Regarding the use of these models for therapeutic testing of ER stress modulators, a specific consideration should be given to the fact that they recapitulate some, but not all, the phenotypic characteristics of the human disease.

Effect of Berberine from Coptis chinensis on Apoptosis of Intestinal Epithelial Cells in a Mouse Model of Ulcerative Colitis: Role of Endoplasmic Reticulum Stress.

The purpose of this study was to verify the effect of berberine (BBR) on endoplasmic reticulum stress (ERS) and apoptosis of intestinal epithelial cells (IECs) in mice with ulcerative colitis (UC). BALB/c mice were randomly divided into five groups as follows: blank control, model, and low-, medium-, and high-dose BBR. A dextran sodium sulfate- (DSS-) induced model of UC was prepared, and the low-, medium-, and high-dose BBR groups were simultaneously gavaged with a BBR suspension for 7 d. Disease activity index (DAI) was assessed, and tissue damage index (TDI) was assessed from colon samples after the last administration. TUNEL assays were used to detect apoptosis of IECs. Immunohistochemistry and/or real-time PCR were applied to determine the expression of GRP78, caspase-12, and caspase-3. In all BBR treatment groups, clinical symptoms of colitis and histopathological damage were significantly reduced. The high-dose BBR group exhibited particularly pronounced decrease (p < 0.01) in both DAI (0.48 ± 0.36) and TDI (1.62 ± 0.64) relative to the model group (1.50 ± 0.65 and 3.88 ± 0.04, respectively). In colon tissues of the model group, the number of apoptotic IECs was significantly increased; the expression of GRP78, caspase-12, and caspase-3 proteins was significantly increased; and the expression of the GRP78 mRNA was upregulated. In low-, medium-, and high-dose BBR groups, the number of apoptotic IECs was significantly reduced. Moreover, GRP78 and caspase-3 expression levels were significantly decreased in the medium- and high-dose BBR groups, caspase-12 expression was significantly decreased in the high-dose BBR group, and the GRP78 mRNA expression level was significantly decreased in the high-dose BBR group. BBR can effectively reduce the rate of IEC apoptosis in UC mice and alleviate the inflammatory response in the colon. The underlying mechanism seems to involve ERS modulation and inhibition of ERS-mediated activation of the caspase-12/caspase-3 apoptosis signaling pathway.

BLISTER-regulated vegetative growth is dependent on the protein kinase domain of ER stress modulator IRE1A in Arabidopsis thaliana.

The unfolded protein response (UPR) is required for protein homeostasis in the endoplasmic reticulum (ER) when plants are challenged by adverse environmental conditions. Inositol-requiring enzyme 1 (IRE1), the bifunctional protein kinase / ribonuclease, is an important UPR regulator in plants mediating cytoplasmic splicing of the mRNA encoding the transcription factor bZIP60. This activates the UPR signaling pathway and regulates canonical UPR genes. However, how the protein activity of IRE1 is controlled during plant growth and development is largely unknown. In the present study, we demonstrate that the nuclear and Golgi-localized protein BLISTER (BLI) negatively controls the activity of IRE1A/IRE1B under normal growth condition in Arabidopsis. Loss-of-function mutation of BLI results in chronic up-regulation of a set of both canonical UPR genes and non-canonical UPR downstream genes, leading to cell death and growth retardation. Genetic analysis indicates that BLI-regulated vegetative growth phenotype is dependent on IRE1A/IRE1B but not their canonical splicing target bZIP60. Genetic complementation with mutation analysis suggests that the D570/K572 residues in the ATP-binding pocket and N780 residue in the RNase domain of IRE1A are required for the activation of canonical UPR gene expression, in contrast, the D570/K572 residues and D590 residue in the protein kinase domain of IRE1A are important for the induction of non-canonical UPR downstream genes in the BLI mutant background, which correlates with the shoot growth phenotype. Hence, our results reveal the important role of IRE1A in plant growth and development, and BLI negatively controls IRE1A’s function under normal growth condition in plants.

Loss of Ca2+ entry via Orai-TRPC1 induces ER stress, initiating immune activation in macrophages.

Activation of cellular stresses is associated with inflammation; however, the mechanisms are not well identified. Here, we provide evidence that loss of Ca2+ influx induces endoplasmic reticulum (ER) stress in primary macrophages and in murine macrophage cell line Raw 264.7, in which the unfolded protein response is initiated to modulate cytokine production, thereby activating the immune response. Stressors that initiate the ER stress response block store-dependent Ca2+ entry in macrophages prior to the activation of the unfolded protein response. The endogenous Ca2+ entry channel is dependent on the Orai1-TRPC1-STIM1 complex, and the presence of ER stressors decreased expression of TRPC1, Orai1 and STIM1. Additionally, blocking Ca2+ entry with SKF96365 also induced ER stress, promoted cytokine production, activation of autophagy, increased caspase activation and induced apoptosis. Furthermore, ER stress inducers inhibited cell cycle progression, promoted the inflammatory M1 phenotype, and increased phagocytosis. Mechanistically, restoration of Orai1-STIM1 expression inhibited the ER stress-mediated loss of Ca2+ entry that prevents ER stress and inhibits cytokine production, and thus induced cell survival. These results suggest an unequivocal role of Ca2+ entry in modulating ER stress and in the induction of inflammation.