Endoplasmic Reticulum Stress-mediated Pathway Research Articles

P149 Bacteroides uniformis ameliorates pro-inflammatory diet-exacerbated colitis by targeting endoplasmic reticulum stress-mediated ferroptosis

Abstract Background Pro-inflammatory diet is positively associated with the risk and progression of inflammatory bowel diseases. Recently, ferroptosis was observed in patients with dietary pattern-associated colitis. However, the mechanisms underlying the effects of a pro-inflammatory diet and whether it mediates ferroptosis are unknown. This study aimed to clarify the mechanisms of pro-inflammatory diet in colitis. Methods Mice were fed a dietary inflammatory index-based pro-inflammatory diet for 12 weeks. Subsequently, colitis was chemically induced using 2.5% dextran sulfate sodium with or without a ferroptosis inhibitor (ferrostatin-1). Body weight, histological scores, immune response, and mucosal barrier function were evaluated to assess intestinal inflammation.Colon tissue transcriptomics, fecal microbiome, and serum metabolomics were applied to identify diet-microbe-host interactions. Dietary inflammatory index of 36 patients with Crohn’s disease were evaluated. Their intestinal specimens were also collected. The biological functions of Bacteroides uniformis were observed in vitro and in vivo. Results Pro-inflammatory diet induced low-grade intestinal inflammation and exacerbated colitis by activating glutathione peroxidase 4-associated ferroptosis in the endoplasmic reticulum stress-mediated pathway; ferrostatin-1 treatment reversed this pro-inflammatory potential. Additionally, the pro-inflammatory diet triggered colitis by modulating the gut microbiota and metabolites. Notably, supplementation with B. uniformis improved the pro-inflammatory diet-aggravated colitis by inhibiting endoplasmic reticulum stress-mediated ferroptosis. Co-culturing B. uniformis with intestinal epithelial cells revealed it is non-enterotoxigenic and non-enteroinvasive. Conclusion Pro-inflammatory diet drives colitis by targeting endoplasmic reticulum stress-mediated ferroptosis, possibly in a gut microbiota-dependent manner. Pro-inflammatory diet restriction and microbial-based therapies may be effective strategies for preventing and treating inflammatory bowel disease.

Triggering of Endoplasmic Reticulum Stress by Tannic Acid Inhibits the Proliferation and Migration of Colorectal Cancer Cells.

Due to the pivotal role of endoplasmic reticulum (ER) stress in cancers, interfering with its function can cause the accumulation of unfolded proteins, which ultimately leads to the activation of the unfolded protein response (UPR) signaling pathway and apoptosis. Therefore, the use of plant compounds such as tannic acid with UPR-inducing properties can be proposed as a possible treatment method for cancer. In this study, we investigated the effect of tannic acid on cell migration, colony formation, growth, and UPR-induced apoptosis in the SW48 colorectal cancer cell line. The MTT assay was performed to investigate the cytotoxic effect of tannic acid. We performed the qPCR method to elucidate the effect of tannic acid on the expression of Bim, MMP-9, Bcl-xL, cyclin D1, CHOP, and ATF4 genes. We also used the colony formation and migration experiments to investigate the effect of this compound on the colony formation and migration ability of tumor cells. Finally, we used Hoechst staining to measure cell apoptosis. Tannic acid inhibited the cell survival, clonogenic, and migration of colon cancer cells. This compound increased the expression of ER stress-mediated UPR genes, ATF4 and CHOP. Moreover; tannic acid increased the expression of pro-apoptotic proteins like Bim, while at the same time causing a sharp decline in the expression of anti-apoptotic protein Bcl-xL. A decline in MMP-9 expression confirmed the anti-metastatic role of this compound. Taken together, tannic acid can induce apoptosis via ER stress-mediated UPR pathway, and has a suppressive effect on cell viability, growth, migration, colony formation, and metastasis, suggesting it may be a potential drug in colorectal cancer treatment.

Houttuynia cordata Thunb. Hexane fraction induces MDA-MB-231 cell apoptosis via caspases, ER stress, cell cycle arrest and attenuated Akt/ERK signaling

Houttuynia cordata Thunb. (HCT) is a perennial plant used in traditional Thai medicine for many centuries. This study aimed to investigate the antiproliferative effect of the hexane fraction, which has not been explored before. HCT ethanol extract (crude extract) was sequentially fractionated to obtain a hexane (H) fraction. GC-MS was used to determine the phytochemicals. The H fraction consisted of lipids, mainly α-linolenic acid and some terpenoids. MTT assay was used to determine the cytotoxic effects of H fraction in MCF-7, MDA-MB-231, NIH3T3 and PBMCs. The mode of cell death and cell cycle analysis were determined by flow cytometry. The mechanisms of cell death were defined by mitochondrial transmembrane potential (MTP) reduction and activation of caspase-3, -8 and -9. The expression levels of the Bcl-2 family, cell cycle-related, endoplasmic reticulum (ER) stress-associated proteins; and Akt/ERK signaling molecules were investigated by immunoblotting. The H fraction was toxic to MDA-MB-231 more than MCF-7 cells but not to NIH3T3 and PBMCs. The growth of MDA-MB-231 cells was inhibited through apoptosis. MTP was disrupted whereas caspase-3, -8 and -9 were activated. The expression of pro-apoptotic Bax and Bak was upregulated, while Bid and anti-apoptotic Bcl-xL proteins were downregulated. Cyclin D1 and CDK4 levels were downregulated. The cell cycle was arrested at G1. Moreover, GRP78 and CHOP elevation indicated ER stress-mediated pathway. The expression ratio of pAkt/Akt and pERK/ERK were reduced. Taken together, the molecular mechanisms of MDA-MB-231 cell apoptosis were via intrinsic/extrinsic pathways, cell cycle arrest, ER stress and abrogation of Akt/ERK survival pathways. According to the most current research, the H fraction may be used as an adjuvant in the BC treatment; however, before the anticancer strategy can be applied to patients, it is important to determine each active compound's effects in cell lines and in vivo when compared with a combined mixture.

The Proliferative and Functional Roles of CEBP/A in Ins-1 832/13 Cells and Primary Rat Islets

The beta cell transcription factor Nkx6.1, when overexpressed in primary rodent and human islets, is sufficient to enhance beta cell proliferation, increase insulin secretion and enhance cell survival. We have sought to define the transcriptional targets of Nkx6.1 that allow for its ability to increase functional beta cell mass. We have shown that Nkx6.1 induces expression of the transcription factor CEBP/A. CEBP/A overexpression is sufficient to induce proliferation of Ins-1 832/13 cells and primary rat islets. CEBP/A overexpression enhances glucose stimulated insulin secretion from primary rat islets, while decreasing total insulin content. Finally, CEBPa overexpression protects Ins-1 832/13 cells from thapsigargin and glucolipotoxicity induced cell death but fails to protect against etoposide and camptothecin induced cell death. These data suggest that CEBP/A may play a critical role in Nkx6.1 mediated expansion of functional beta cell mass through a endoplasmic reticulum stress-mediated pathway. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MSX1 Regulates Goat Endometrial Function by Altering the Plasma Membrane Transformation of Endometrial Epithelium Cells during Early Pregnancy

MSX1 is an important member of the muscle segment homeobox gene (Msh) family and acts as a transcription factor to regulate tissue plasticity, yet its role in goat endometrium remodeling remains elusive. In this study, an immunohistochemical analysis showed that MSX1 was mainly expressed in the luminal and glandular epithelium of goat uterus, and the MSX1 expression was upregulated in pregnancy at days 15 and 18 compared with pregnancy at day 5. In order to explore its function, goat endometrial epithelial cells (gEECs) were treated with 17 β-estrogen (E2), progesterone (P4), and/or interferon-tau (IFNτ), which were used to mimic the physiological environment of early pregnancy. The results showed that MSX1 was significantly upregulated with E2- and P4-alone treatment, or their combined treatment, and IFNτ further enhanced its expression. The spheroid attachment and PGE2/PGF2α ratio were downregulated by the suppression of MSX1. The combination of E2, P4, and IFNτ treatment induced the plasma membrane transformation (PMT) of gEECs, which mainly showed the upregulation of N-cadherin (CDH2) and concomitant downregulation of the polarity-related genes (ZO-1, α-PKC, Par3, Lgl2, and SCRIB). The knockdown of MSX1 partly hindered the PMT induced by E2, P4, and IFNτ treatment, while the upregulation of CDH2 and the downregulation of the partly polarity-related genes were significantly enhanced when MSX1 was overexpressed. Moreover, MSX1 regulated the CDH2 expression by activating the endoplasmic reticulum (ER) stress-mediated unfolded protein response (UPR) pathway. Collectively, these results suggest that MSX1 was involved in the PMT of the gEECs through the ER stress-mediated UPR pathway, which affects endometrial adhesion and secretion function.

Endoplasmic reticulum stress-controlled autophagic pathway promotes polystyrene microplastics-induced myocardial dysplasia in birds

In complex ecosystems, birds are generally long-lived and occupy high trophic positions, making them good bioindicators for monitoring environmental contaminants. The effects of microplastics (MPs) on myocardial development in bird is currently unknown. Chicks, as a high trophic level terrestrial bird, may be more affected by MPs exposure and. Therefore, we established an in vivo model of chicks exposed to different concentrations of polystyrene microplastics (PS-MPs) and selected 12-day-old chicken embryos in vitro to extract primary cardiomyocytes to further investigate the potential molecular mechanisms of the effect of PS-MPs on myocardial development in birds. Histopathological observations revealed that the PS-MPs treated exhibited loose and irregular myocardial arrangement, large cell gaps and broken myocardial fiber bundles. More mechanistically, TnnT2, Nkx2-5, Gata4, TBX5 and ACTN2 were down-regulated, endoplasmic reticulum (ER) stress markers GRP78, PERK, eIF2α, IRE1, ATF4, ATF6 and CHOP were overexpressed, autophagy-related genes LC3, ATG5, Beclin1 and P62 were down-expressed after PS-MPs exposure, and the addition of 4PBA effectively deregulated the above aberrant expression. Hence, our report indicated that PS-MPs induced myocardial dysplasia in birds is mainly attributed to the ER stress-mediated autophagic pathway. This provided data supporting the protection of birds from the health risks of MPs pollution. More critically, the study of cardiac developmental toxicity in birds may help to better explain or solve the problem of MPs pollution in complex ecosystems.

Ginsenoside Rb1 protects from Staphylococcus aureus-induced oxidative damage and apoptosis through endoplasmic reticulum-stress and death receptor-mediated pathways

Acute lung injury (ALI) is acute uncontrolled inflammation of lung tissue that leads to high fatality both in human and animals. Staphylococcus aureus (S. aureus) could be an opportunistic, versatile bacterial etiology of ALI. Ginsenoside Rb1 (Rb1) is extracted from the Panax ginseng, which displays a wide range of biological and pharmacological effects. However, protective effects of Rb1 in S. aureus-induced ALI though endoplasmic reticulum (ER) stress and death receptor-mediated pathways have not yet been reported. Therefore, present study was planned with the aims to investigate the antioxidant and anti-apoptotic properties of Rb1 through regulation of ER stress as well as death receptor-mediated pathways in ALI induced by S. aureus in mice. In this study, four groups of healthy Kunming mice (n = 48) were used. The S. aureus (80 µl; 1 ×107 CFU/10 µl) was administered intranasally to establish mice model of ALI. After 24 h of onset of S. aureus-induced ALI, the mice were injected thrice with Rb1 (40 mg/kg) intraperitoneally six hours apart. Histopathology, enzyme linked immunosorbent assay (ELISA), real time quantitative polymerase chain reaction (RT-qPCR), Immunohistochemistry and western blotting assay were employed in the current study. Our results suggested that Rb1 administration save lungs from pulmonary injury by reducing wet to dry (W/D) ratio, protein levels, total cells, neutrophilic count, reactive oxygen species (ROS), myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx)1 depletion. Meanwhile, Rb1 therapy ameliorated histopathology alteration of lung tissue and pro-inflammatory cytokines secretion. The gene expression of ER stress marker (PERK, AFT-6, IRE1 and CHOP) were upregulated markedly (P < .05) in S. aureus-instilled groups, which was reduced by Rb1 administration that is reveled from the result findings of the RT-qPCR and immunoblot assay. The results of immunohistochemistry for CHOP indicated the increased expression in S. aureus groups which in turn ameliorated by Rb1 treatment. The mRNA expression demonstrated that death receptor-associated genes (FasL, Fas, FADD and caspase-8) showed up-regulation in S. aureus group. The similar findings were observed for the protein expression of caspase-8, FADD and Fas. Rb1 treatment markedly (P < .05) reversed protein and mRNA expression levels of these death receptor-associated genes when compared to the S. aureus group. Taken together, Rb1 attenuated S. aureus-induced oxidative damage via the ER stress-mediated pathway and apoptosis through death receptor-mediated pathway. Conclusively, our findings provide an insight into preventive mechanism of Rb1 in ALI caused by S. aureus and hence proven a scientific baseline for the therapeutic application of Rb1.

Decreased Proteasomal Function Induces Neuronal Loss and Memory Impairment

Alzheimer disease (AD) is a progressive neurodegenerative disorder and the most common type of dementia worldwide. There is considerable evidence of age-related disruption of proteostasis being responsible for the development of AD. The proteasome is a multicatalytic enzyme complex that degrades both normal and damaged proteins, and an age-related decline in its activity has been implicated in age-related pathologies. Although proteasomal dysfunction is assumed to be a key AD hallmark, it remains unclear whether its role in disease onset is causative or secondary. In this study, we demonstrate that mice with proteasomal dysfunction exhibited memory impairment with associated neuronal loss, accumulation of phosphorylated tau, and activation of endoplasmic reticulum (ER) stress-related apoptosis pathways. Impaired proteasomal activity also activated ER stress-related apoptosis pathways in HT-22, a murine hippocampal neuronal cell line. HT-22 cell death, caused by proteasomal inhibition, was prevented by an inhibitor of c-Jun N-terminal kinase, an ER stress-related molecule. Collective evidence suggests that impaired proteasomal activity alters proteostasis, and subsequent ER stress-mediated pathways play pivotal roles in neuronal loss. Because aging decreases proteasomal function, age-related impairment of proteasomes may be involved in the development and progression of AD in elderly patients.

Teglicar Induces Apoptosis in HeLa Cervical Cancer Cells via Endoplasmic Reticulum Stress-Mediated Pathway

Carnitine palmitoyltransferase 1A (CPT1A) is a potential therapeutic target for several cancers. The inhibition of CPT1A has anticancer effects. Teglicar is a new reversible CPT1A inhibitor. The aim of this research was to expound the effect of teglicar in cervical cancer cells. Cell proliferation was conducted using the CCK8 assay. The apoptotic rate was examined by Annexin V-APC/PI staining and flow cytometry. The expression levels of apoptosis-related proteins and endoplasmic reticulum (ER) stress-related proteins were determined by western blot assay. Teglicar was found to inhibit the proliferation of HeLa cells in a dose-dependent manner, with an IC50 value of 18.38 µM at 24 h. The apoptotic rate of HeLa cells was significantly increased following treatment with teglicar. Western blot analysis indicated that the treatment of teglicar enhanced ER stress and apoptosis in HeLa cells. However, inhibition of proliferation and enhancement of apoptosis in teglicar-treated HeLa cells was reversed by 4-PBA. These results revealed that teglicar promoted apoptosis of HeLa cells via ER stress.

Ambient particulate matter triggers dysfunction of subcellular structures and endothelial cell apoptosis through disruption of redox equilibrium and calcium homeostasis

Ambient particulate matter (APM) is becoming a global environmental problem that seriously jeopardizes public health. Previous evidence hinted that APM correlates to cardiovascular diseases. As a potential target, equilibrium of endothelial cell is a prerequisite for vascular health which could be vulnerably attacked by particles, but the specific mechanisms whereby APM damages endothelial cells have not been fully elucidated. In the current study, based on two classical mechanisms of oxidative stress and intracellular calcium overload, we aimed to explore their roles in APM-induced endothelial cell apoptosis from the perspective of subcellular levels, including endoplasmic reticulum (ER) stress and mitochondrial dysfunction. As a result, PM SRM1648a results in oxidative stress and calcium overload in EA.hy926 cells. Additionally, ERs and mitochondria could be severely disturbed by particles in morphology and function, characterized by swelling ERs, mitochondrial fission and disappearance of cristae, coupled with ER damage, mtROS overproduction and significant reduction in mitochondrial membrane potential (MMP). Adverse effects on these organelles are the prime culprits of following apoptosis in endothelial cells. Fortunately, additional antioxidants and calcium inhibitors could mitigate cellular lesion through improvement of subcellular function. Intriguingly, antioxidants relieve cell stress via both mitochondrial and ER stress-mediated pathways, whereas the role of calcium modulators in cell apoptosis is independent of the mitochondrial pathway but could be explained by amelioration of ER stress. In conclusion, our data basically revealed that internalized PM SRM1648a triggers oxidative stress and calcium influx in EA.hy926 endothelial cells, followed by multiple subcellular damage and eventually contributes to cell death, during which antioxidants and calcium inhibitors confer protective effects.

GPR177 promotes gastric cancer proliferation by suppressing endoplasmic reticulum stress-induced cell death.

Gastric cancer is the fourth most common cancer worldwide. Despite the high incidence of gastric cancer, efficient chemotherapy treatments still need to be developed. In this study, we examined the anticancer effects of endoplasmic reticulum (ER) stress inducer tunicamycin in gastric cancer. Previously, we found that overexpression of WLS1/GPR177 correlated with poor prognosis in patients with gastric cancer. Furthermore, tunicamycin treatment downregulated GPR177 expression in a dose-dependent manner. GPR177 transports WNT ligand from ER to the plasma membrane, mediating its secretion to the extracellular matrix. In gastric cancer cells, GPR177 preferentially localizes to the ER. Small interfering RNA-mediated knockdown of GPR177 leads to sensitization to ER stress and induces apoptosis of cancer cells along with tunicamycin treatment. GPR177 suppression promoted the ER stress-mediated proapoptotic pathway, such as PERK-CHOP cascade. Furthermore, fluorouracil treatment combined with tunicamycin dramatically reduced cancer cell proliferation. Efficacy of tunicamycin chemotherapy treatments depended on GPR177 expression in gastric cancer cell lines. Together, our results indicate that ER stress can potentiate anticancer effects and suggest GPR177 as a potential gastric cancer therapeutic target.

Endoplasmic reticulum stress/autophagy pathway is involved in diabetes-induced neuronal apoptosis and cognitive decline in mice.

Diabetes mellitus is a significant global public health problem depicting a rising prevalence worldwide. As a serious complication of diabetes, diabetes-associated cognitive decline is attracting increasing attention. However, the underlying mechanisms are yet to be fully determined. Both endoplasmic reticulum (ER) stress and autophagy have been reported to modulate neuronal survival and death and be associated with several neurodegenerative diseases. Here, a streptozotocin-induced diabetic mouse model and primary cultured mouse hippocampal neurons were employed to investigate the possible role of ER stress and autophagy in diabetes-induced neuronal apoptosis and cognitive impairments, and further explore the potential molecular mechanisms. ER stress markers GRP78 and CHOP were both enhanced in diabetic mice, as was phosphorylation of PERK, IRE1α, and JNK. In addition, the results indicated an elevated level of autophagy in diabetic mice, as demonstrated by up-regulated expressions of autophagy markers LC3-II, beclin 1 and down-regulated level of p62, and increased formation of autophagic vacuoles and LC3-II aggregates. Meanwhile, we found that these effects could be abolished by ER stress inhibitor 4-phenylbutyrate or JNK inhibitor SP600125 in vitro. Furthermore, neuronal apoptosis of diabetic mice was attenuated by pretreatment with 4-phenylbutyrate, while aggravated by application of inhibitor of autophagy bafilomycin A1 in vitro. These results suggest that ER stress pathway may be involved in diabetes-mediated neurotoxicity and promote the following cognitive impairments. More important, autophagy was induced by diabetes possibly through ER stress-mediated JNK pathway, which may protect neurons against ER stress-associated cell damages.

Phytochemical-induced reactive oxygen species and endoplasmic reticulum stress-mediated apoptosis and differentiation in malignant melanoma cells

BackgroundPhytochemicals are derived from plants, vegetables and daily products and exert chemopreventive effects. Malignant melanoma is highly metastatic, and melanoma patients can develop chemotherapeutic resistance against conventional melanoma therapies. MethodsIn the present study, we investigated the anti-cancer effect of the phytochemicals kaempferol (Kaem), genistein (Gen), and 3′3-diindolylmethane (DIM) on melanoma cell viability. We also evaluated the altered expression of cell cycle-related genes. We verified the production of intracellular reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress at both the protein and cellular level using a western blot, TUNEL assay, and Dihydrodichlorofluorescein diacetate (DCF-DA) assay. ResultsTreatment of A375SM melanoma cells with phytochemicals resulted in inhibition of cell growth. Treatment with phytochemicals increased the gene expression of p21 and decreased the gene expression of cyclin E and/or cyclin B. The three phytochemicals activated the ROS-p38-p53 apoptotic pathway by increasing the level of phosphorylated p38 MAPK and p53, and they activated the ER stress-mediated apoptotic pathway by increasing the level of phosphorylated eIF2α and C/EBP homologous protein (CHOP). Both the ROS-p38-p53 and ER stress-mediated pathway induced the mitochondrial apoptotic pathway by attenuating Bcl-2 expression and upregulating BAX. Detection of morphological changes demonstrated that Kaem and Gen can induce differentiation in A375SM cell line. ConclusionThese results indicate that phytochemicals are potentially useful in treatments for melanoma due to their ability to inhibit melanoma cell growth and division via the ROS and ER stress pathway.

Dihydroartemisinin inhibits ER stress-mediated mitochondrial pathway to attenuate hepatocyte lipoapoptosis via blocking the activation of the PI3K/Akt pathway

Alcoholic liver disease (ALD), characterized by accumulation of fatty acids in liver cells, is usually caused by Chronic alcohol consumption. Our previous study has identified that DA protects against alcoholic liver injury in alcohol-fed rats through alleviating hepatocyte steatosis. It has emerged that saturated fatty acids could provoke endoplasmic reticulum (ER) stress and apoptosis in hepatocytes. This study was aimed to explore the impact of DA on ALD and further elaborate the underlying mechanisms. Results demonstrated that DA attenuates alcoholic liver injury in mice. Our results also indicated that DA attenuated lipid accumulation in hepatocytes exposed to ethanol. DA attenuates ethanol-induced hepatocyte apoptosis. Results demonstrated that DA dose-dependently ameliorated activation of mitochondrial pathway activation, which plays a critical role in apoptosis attributed to lipotoxicity. Further, DA suppressed the activation of JNK and the expression of CHOP, attributed to the inhibition of ER stress. It has emerged that activation of ER stress-JNK/CHOP-mitochondria cascade is considered as the key mechanisms underlying hepatocyte lipoapoptosis. In addition, DA attenuates PI3K/Akt Pathway in hepatocytes, consistent with our previous finding in HSCs. DA effects were reinforced by PI3K specific inhibitor LY294002. In summary, DA significantly protected hepatocytes against lipoapoptosis via a PI3K/Akt Pathway inhibition-dependent mechanism.

Cooperation between ER stress and calcineurin signaling contributes to the maintenance of cell wall integrity in Candida glabrata

Candida glabrata is the second most common source of Candida infections in humans. In this pathogen, the maintenance of cell wall integrity (CWI) frequently precludes effective pharmacological treatment by antifungal agents. In numerous fungi, cell wall modulation is reported to be controlled by endoplasmic reticulum (ER) stress, but how the latter affects CWI maintenance in C. glabrata is not clearly understood. Here, we characterized a C. glabrata strain harboring a mutation in the CNE1 gene, which encodes a molecular chaperone associated with nascent glycoprotein maturation in the ER. Disruption of cne1 induced ER stress and caused changes in the normal cell wall structure, specifically a reduction in the β-1,6-glucan content and accumulation of chitin. Conversely, a treatment with the typical ER stress inducer tunicamycin up-regulated the production of cell wall chitin but did not affect β-1,6-glucan content. Our results also indicated that C. glabrata features a uniquely evolved ER stress-mediated CWI pathway, which differs from that in the closely related species Saccharomyces cerevisiae. Furthermore, we demonstrated that ER stress-mediated CWI pathway in C. glabrata is also induced by the disruption of other genes encoding proteins that function in a correlated manner in the quality control of N-linked glycoproteins in the ER. These results suggest that calcineurin and ER quality control system act as a platform for maintaining CWI in C. glabrata.

Sustained Activation of the Unfolded Protein Response Induces Cell Death in Fuchs' Endothelial Corneal Dystrophy.

The unfolded protein response (UPR) is believed to play a role in the pathogenesis of Fuchs' endothelial corneal dystrophy (FECD). The purpose of this study was to investigate whether unfolded proteins accumulate in the corneal endothelium in FECD and if they are involved in triggering cell death. Descemet's membranes with corneal endothelial cells (CECs) were obtained during keratoplasty, and expression of aggresomes, type 1 collagen, fibronectin, and agrin was evaluated. Endoplasmic reticulum (ER) stress of immortalized human CECs from non-FECD subjects and from FECD patients (iHCEC and iFECD, respectively) were evaluated. The effect of MG132-mediated aggresome formation on the UPR and intrinsic pathway and the effect of mitochondrial damage on UPR were also examined. The effect of CHOP knockdown on the ER stress-mediated intrinsic pathway was also evaluated. Aggresome formation was higher in iFECD than in iHCEC and was colocalized with type 1 collagen, fibronectin, and agrin. GRP78, phosphorylated IRE1, PERK, and CHOP showed higher activation in iFECD than in iHCEC. MG132-mediated aggresome formation upregulated ER stress sensors, the mitochondrial membrane potential drop, cytochrome c release to the cytoplasm, and activation of caspase-9 and -3. By contrast, staurosporine-mediated mitochondrial damage did not induce ER stress. Knockdown of CHOP attenuated the ER stress-induced cleavage of caspase-9, which is caused by intrinsic pathway activation. Excessive synthesis of extracellular matrix proteins induced unfolded protein accumulation in FECD. Prolonged ER stress-mediated cell death, occurring via the intrinsic apoptotic signaling pathway, therefore might be associated with the pathogenesis of FECD.

Molecular chaperones and hypoxic-ischemic encephalopathy.

Hypoxic-ischemic encephalopathy (HIE) is a disease that occurs when the brain is subjected to hypoxia, resulting in neuronal death and neurological deficits, with a poor prognosis. The mechanisms underlying hypoxic-ischemic brain injury include excitatory amino acid release, cellular proteolysis, reactive oxygen species generation, nitric oxide synthesis, and inflammation. The molecular and cellular changes in HIE include protein misfolding, aggregation, and destruction of organelles. The apoptotic pathways activated by ischemia and hypoxia include the mitochondrial pathway, the extrinsic Fas receptor pathway, and the endoplasmic reticulum stress-induced pathway. Numerous treatments for hypoxic-ischemic brain injury caused by HIE have been developed over the last half century. Hypothermia, xenon gas treatment, the use of melatonin and erythropoietin, and hypoxic-ischemic preconditioning have proven effective in HIE patients. Molecular chaperones are proteins ubiquitously present in both prokaryotes and eukaryotes. A large number of molecular chaperones are induced after brain ischemia and hypoxia, among which the heat shock proteins are the most important. Heat shock proteins not only maintain protein homeostasis; they also exert anti-apoptotic effects. Heat shock proteins maintain protein homeostasis by helping to transport proteins to their target destinations, assisting in the proper folding of newly synthesized polypeptides, regulating the degradation of misfolded proteins, inhibiting the aggregation of proteins, and by controlling the refolding of misfolded proteins. In addition, heat shock proteins exert anti-apoptotic effects by interacting with various signaling pathways to block the activation of downstream effectors in numerous apoptotic pathways, including the intrinsic pathway, the endoplasmic reticulum-stress mediated pathway and the extrinsic Fas receptor pathway. Molecular chaperones play a key role in neuroprotection in HIE. In this review, we provide an overview of the mechanisms of HIE and discuss the various treatment strategies. Given their critical role in the disease, molecular chaperones are promising therapeutic targets for HIE.

Cytotoxicity of CdTe quantum dots in human umbilical vein endothelial cells: the involvement of cellular uptake and induction of pro-apoptotic endoplasmic reticulum stress

Cadmium telluride quantum dots (CdTe QDs) have been proposed to induce oxidative stress, which plays a crucial role in CdTe QDs-mediated mitochondrial-dependent apoptosis in human umbilical vein endothelial cells (HUVECs). However, the direct interactions of CdTe QDs with HUVECs and their potential impairment of other organelles like endoplasmic reticulum (ER) in HUVECs are poorly understood. In this study, we reported that the negatively charged CdTe QDs (−21.63±0.91 mV), with good dispersity and fluorescence stability, were rapidly internalized via endocytosis by HUVECs, as the notable internalization could be inhibited up to 95.52% by energy depletion (NaN3/deoxyglucose or low temperature). The endocytosis inhibitors (methyl-β-cyclodextrin, genistein, sucrose, chlorpromazine, and colchicine) dramatically decreased the uptake of CdTe QDs by HUVECs, suggesting that both caveolae/raft- and clathrin-mediated endocytosis were involved in the endothelial uptake of CdTe QDs. Using immunocytochemistry, a striking overlap of the internalized CdTe QDs and ER marker was observed, which indicates that QDs may be transported to ER. The CdTe QDs also caused remarkable ER stress responses in HUVECs, confirmed by significant dilatation of ER cisternae, upregulation of ER stress markers GRP78/GRP94, and activation of protein kinase RNA-like ER kinase-eIF2α-activating transcription factor 4 pathway (including phosphorylation of both protein kinase RNA-like ER kinase and eIF2α and elevated level of activating transcription factor 4). CdTe QDs further promoted an increased C/EBP homologous protein expression, phosphorylation of c-JUN NH2-terminal kinase, and cleavage of ER-resident caspase-4, while the specific inhibitor (SP600125, Z-LEVD-fmk, or salubrinal) significantly attenuated QDs-triggered apoptosis, indicating that all three ER stress-mediated apoptosis pathways were activated and the direct participation of ER in the CdTe QDs-caused apoptotic cell death in HUVECs. Our findings provide important new insights into QDs toxicity and reveal potential cardiovascular risks for the future applications of QDs.

Targeting Glutamine Metabolism in Breast Cancer with Aminooxyacetate.

Glutamine addiction in c-MYC-overexpressing breast cancer is targeted by the aminotransferase inhibitor, aminooxyacetate (AOA). However, the mechanism of ensuing cell death remains unresolved. A correlation between glutamine dependence for growth and c-MYC expression was studied in breast cancer cell lines. The cytotoxic effects of AOA, its correlation with high c-MYC expression, and effects on enzymes in the glutaminolytic pathway were investigated. AOA-induced cell death was assessed by measuring changes in metabolite levels by magnetic resonance spectroscopy (MRS), the effects of amino acid depletion on nucleotide synthesis by cell-cycle and bromodeoxyuridine (BrdUrd) uptake analysis, and activation of the endoplasmic reticulum (ER) stress-mediated pathway. Antitumor effects of AOA with or without common chemotherapies were determined in breast cancer xenografts in immunodeficient mice and in a transgenic MMTV-rTtA-TetO-myc mouse mammary tumor model. We established a direct correlation between c-MYC overexpression, suppression of glutaminolysis, and AOA sensitivity in most breast cancer cells. MRS, cell-cycle analysis, and BrdUrd uptake measurements indicated depletion of aspartic acid and alanine leading to cell-cycle arrest at S-phase by AOA. Activation of components of the ER stress-mediated pathway, initiated through GRP78, led to apoptotic cell death. AOA inhibited growth of SUM159, SUM149, and MCF-7 xenografts and c-myc-overexpressing transgenic mouse mammary tumors. In MDA-MB-231, AOA was effective only in combination with chemotherapy. AOA mediates its cytotoxic effects largely through the stress response pathway. The preclinical data of AOA's effectiveness provide a strong rationale for further clinical development, particularly for c-MYC-overexpressing breast cancers.

Activation of sphingosine kinase 2 by endoplasmic reticulum stress ameliorates hepatic steatosis and insulin resistance in mice.

The endoplasmic reticulum (ER) is the principal organelle in the cell for protein folding and trafficking, lipid synthesis, and cellular calcium homeostasis. Perturbation of ER function results in activation of the unfolded protein response (UPR) and is implicated in abnormal lipid biosynthesis and development of insulin resistance. In this study, we investigated whether transcription of sphingosine kinase (Sphk)2 is regulated by ER stress-mediated UPR pathways. Sphk2, a major isotype of sphingosine kinase in the liver, was transcriptionally up-regulated by tunicamycin and lipopolysaccharides. Transcriptional regulation of Sphk2 was mediated by activation of activating transcription factor (ATF)4 as demonstrated by promoter assays, immunoblotting, and small interfering RNA analyses. In primary hepatocytes, adenoviral Sphk2 expression elevated cellular sphingosine 1 phosphate (S1P) and activated protein kinase B phosphorylation, with no alteration of insulin receptor substrate phosphorylation. Hepatic overexpression of Sphk2 in mice fed a high-fat diet (HFD) led to elevated S1P and reduced ceramide, sphingomyelin, and glucosylceramide in plasma and liver. Hepatic accumulation of lipid droplets by HFD feeding was reduced by Sphk2-mediated up-regulation of fatty acid (FA) oxidizing genes and increased FA oxidation in liver. In addition, glucose intolerance and insulin resistance were ameliorated by improved hepatic insulin signaling through Sphk2 up-regulation. Sphk2 is transcriptionally up-regulated by acute ER stress through activation of ATF4 and improves perturbed hepatic glucose and FA metabolism.