Endoplasmic Reticulum Stress-related Molecules Research Articles

Cell division cyclin 25C knockdown inhibits hepatocellular carcinoma development by inducing endoplasmic reticulum stress

BACKGROUND Cell division cyclin 25C (CDC25C ) is a protein that plays a critical role in the cell cycle, specifically in the transition from the G2 phase to the M phase. Recent research has shown that CDC25C could be a potential therapeutic target for cancers, particularly for hepatocellular carcinoma (HCC). However, the specific regulatory mechanisms underlying the role of CDC25C in HCC tumorigenesis and development remain incompletely understood. AIM To explore the impact of CDC25C on cell proliferation and apoptosis, as well as its regulatory mechanisms in HCC development. METHODS Hepa1-6 and B16 cells were transduced with a lentiviral vector containing shRNA interference sequences (LV-CDC25C shRNA) to knock down CDC25C . Subsequently, a xenograft mouse model was established by subcutaneously injecting transduced Hepa1-6 cells into C57BL/6 mice to assess the effects of CDC25C knockdown on HCC development in vivo . Cell proliferation and migration were evaluated using a Cell Counting Kit-8 cell proliferation assays and wound healing assays, respectively. The expression of endoplasmic reticulum (ER) stress-related molecules (glucose-regulated protein 78, X-box binding protein-1, and C/EBP homologous protein) was measured in both cells and subcutaneous xenografts using quantitative real-time PCR (qRT-PCR) and western blotting. Additionally, apoptosis was investigated using flow cytometry, qRT-PCR, and western blotting. RESULTS CDC25C was stably suppressed in Hepa1-6 and B16 cells through LV-CDC25C shRNA transduction. A xenograft model with CDC25C knockdown was successfully established and that downregulation of CDC25C expression significantly inhibited HCC growth in mice. CDC25C knockdown not only inhibited cell proliferation and migration but also significantly increased the ER stress response, ultimately promoting ER stress-induced apoptosis in HCC cells. CONCLUSION The regulatory mechanism of CDC25C in HCC development may involve the activation of ER stress and the ER stress-induced apoptosis signaling pathway.

Endoplasmic reticulum stress drives macrophages to produce IL-33 to favor Th2 polarization in the airways.

Interleukin (IL)-33 is a key driver of T helper 2 (Th2) cell polarization. Endoplasmic reticulum (ER) stress plays a role in the skewed T cell activation. The objective of this project is to elucidate the role of IL-33 derived from macrophages in inducing Th2 polarization in the airways. In this study, bronchoalveolar lavage fluids (BALF) were collected from patients with asthma and healthy control subjects. Macrophages were isolated from the BALF by flow cytometry cell sorting. An asthmatic mouse model was established using the ovalbumin/alum protocol. The results showed that increased IL33 gene activity and ER stress-related molecules in BALF-derived M2a macrophages was observed in asthmatic patients. Levels of IL33 gene activity in M2a cells were positively correlated with levels of asthma response in asthma patients. Sensitization exacerbated the ER stress in the airway macrophages, which increased the expression of IL-33 in macrophages of airway in sensitized mice. Conditional ablation of Il33 or Perk or Atf4 genes in macrophages prevented induction of airway allergy in mice. In conclusion, asthma airway macrophages express high levels of IL-33 and at high ER stress status. Inhibition of IL-33 or ER stress in macrophages can effectively alleviate experimental asthma.

Decreased Proteasomal Function Exacerbates Endoplasmic Reticulum Stress-Induced Chronic Inflammation in Obese Adipose Tissue

Low-grade chronic inflammation contributes to both aging and the pathogenesis of age-related diseases. White adipose tissue (WAT) in obese individuals exhibits chronic inflammation, which is associated with obesity-related disorders. Aging exacerbates obesity-related inflammation in WAT; however, the molecular mechanisms underlying chronic inflammation and its exacerbation by aging remain unclear. Age-related decline in activity of the proteasome, a multi-subunit proteolytic complex, has been implicated in age-related diseases. This study employed a mouse model with decreased proteasomal function that exhibits age-related phenotypes to investigate the impact of adipocyte senescence on WAT inflammation. Transgenic mice expressing proteasomal subunit β5t with weak chymotrypsin-like activity suffer from reduced lifespan and develop age-related phenotypes. Mice fed with a high-fat diet (HFD) and suffering proteasomal dysfunction exhibited increased WAT inflammation, increased infiltration of pro-inflammatory M1-like macrophages, and increased pro-inflammatory adipocytokines like monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, and tumor necrosis factor αwhich are all associated with activation of endoplasmic reticulum (ER) stress-related pathways. Impaired proteasomal activity also activated ER stress-related molecules and induced expression of pro-inflammatory adipocytokines in adipocyte-like cells differentiated from 3T3-L1 cells. Collective evidence suggests that impaired proteasomal activity increases ER stress and that subsequent inflammatory pathways play pivotal roles in WAT inflammation. Since proteasomal function declines with age, age-related proteasome impairment may be involved in obesity-related inflammation among the elderly.

Roles of Neuronal Protein Kinase Cε on Endoplasmic Reticulum Stress and Autophagic Formation in Diabetic Neuropathy.

In chronic diabetic neuropathy (DN), the cellular mechanisms of neuropathic pain remain unclear. Protein kinase C epsilon (PKCε) is an intracellular signaling molecule that mediates chronic pain. This paper addresses the long-term upregulated PKCε in DN associated with endoplasmic reticulum (ER) stress and autophagic formation and correlates to chronic neuropathic pain. We found that thermal hyperalgesia and mechanical allodynia course development were associated with PKCε upregulation after DN but not skin denervation. Pathologically, PKCε upregulation was associated with the expression of inositol-requiring enzyme 1α (IRE1α; ER stress-related molecule) and ubiquitin D (UBD), which are involved in the ubiquitin-proteasome system (UPS)-mediated degradation of misfolded proteins under ER stress. Manders coefficient analyses revealed an approximately 50% colocalized ratio for IRE1α(+):PKCε(+) neurons (0.34-0.48 for M1 and 0.40-0.58 for M2 Manders coefficients). The colocalized coefficients of UBD/PKCε increased (M1: 0.33 ± 0.03 vs. 0.77 ± 0.04, p < 0.001; M2: 0.29 ± 0.05 vs. 0.78 ± 0.04; p < 0.001) in the acute DN stage. In addition, the regulatory subunit p85 of phosphoinositide 3-kinase, which is involved in regulating insulin signaling, exhibited similar expression patterns to those of IRE1α and UBD; for example, it had highly colocalized ratios to PKCε. The ultrastructural examination further confirmed that autophagic formation was associated with PKCε upregulation. Furthermore, PKCεv1-2, a PKCε specific inhibitor, reverses neuropathic pain, ER stress, and autophagic formation in DN. This finding suggests PKCε plays an upstream molecule in DN-associated neuropathic pain and neuropathology and could provide a potential therapeutic target.

LncRNA SNHG14/miR-497a-5p/BACE1 axis modulates obesity-induced adipocyte inflammation and endoplasmic reticulum stress.

Obesity is a metabolic disease with excess weight. LncRNA SNHG14 is abnormally expressed in numerous diseases. This research aimed to enucleate the lncRNA SNHG14 role in obesity. Adipocytes were treated with free fatty acid (FFA) to establish an in vitro model for obesity. Mice were fed a high-fat diet to construct an in vivo model. Gene levels were determined using quantitative real-time PCR (RT-PCR).The protein level was checked by western blot. The lncRNA SNHG14 role in obesity was assessed using western blot and enzyme-linked immunosorbent assay. The mechanism was estimated by Starbase, dual-luciferase reporter gene assay, and RNA pull-down. LncRNA SNHG14 function in obesity was estimated using mouse xenograft models, RT-PCR, western blot, and enzyme-linked immunosorbent assay. LncRNA SNHG14 and BACE1 levels were increased, but the miR-497a-5p level was decreased in FFA-induced adipocytes. Interference with lncRNA SNHG14 reduced endoplasmic reticulum (ER) stress-related molecules GRP78 and CHOP expressions in FFA-induced adipocytes, and decreased IL-1β, IL-6, and TNF-α expressions, indicating that lncRNA SNHG14 knockdown mitigated FFA-induced ER stress and inflammation in adipocytes. Mechanistically, lncRNA SNHG14 combined with miR-497a-5p, and miR-497a-5p targeted BACE1. Meanwhile, lncRNA SNHG14 knockdown reduced levels of GRP78, CHOP, IL-1β, IL-6, and TNF-α, while cotransfection with anti-miR-497a-5p or pcDNA-BACE1 abolished these trends. Rescue assays illustrated that lncRNA SNHG14 knockdown relieved FFA-induced adipocyte ER stress and inflammation through miR-497a-5p/BACE1. Meanwhile, lncRNA SNHG14 knockdown restrained adipose inflammation and ER stress caused by obesity in vivo. LncRNA SNHG14 mediated obesity-induced adipose inflammation and ER stress through miR-497a-5p/BACE1.

Early-life cadmium exposure elevates susceptibility to allergic asthma in ovalbumin-sensitized and challenged mice

Increasing evidence have demonstrated that early-life exposure to environmental toxicants elevates risk of allergic asthma. Cadmium (Cd) is widely present in the environment. The purposes of this study were to evaluate the impact of early-life Cd exposure on susceptibility to ovalbumin (OVA)-evoked allergic asthma. Newly weaned mice were subjected to a low concentration of CdCl2 (1 mg/L) by drinking water for 5 consecutive weeks. Penh value, an index of airway obstruction, was increased in OVA-stimulated and challenged pups. Abundant inflammatory cells were observed in the lung of OVA-exposed pups. Goblet cell hyperplasia and mucus secretion were shown in the airway of OVA-stimulated and challenged pups. Early-life Cd exposure exacerbated OVA-evoked airway hyperreactivity, Goblet cell hyperplasia and mucus secretion. The in vitro experiments showed that mucoprotein gene MUC5AC mRNA was upregulated in Cd-exposed bronchial epithelial cells. Mechanistically, endoplasmic reticulum (ER) stress-related molecules GRP78, p-eIF2α, CHOP, p-IRE1α and spliced XBP-1 (sXBP-1) were elevated in Cd-subjected bronchial epithelial cells. The blockade of ER stress, using chemical inhibitor 4-PBA or sXBP-1 siRNA interference, attenuated Cd-induced MUC5AC upregulation in bronchial epithelial cells. These results indicate that early-life Cd exposure aggravates OVA-induced allergic asthma partially through inducing ER stress in bronchial epithelial cells.

Role of IRE1 in autophagy induced by vitamin E succinate in human gastric cancer cell

To investigate the role of inositol-requiring enzyme 1(IRE1) in autophagy of human gastric cancer cells induced by vitamin E succinate(VES). Human gastric cancer SGC-7901 cells were cultured in vitro and divided into solvent control group(0.1% ethanol absolute), different doses(5, 10, 15 and 20 μg/mL) VES group, 4μ8C group, and VES + 4μ8C group. The endoplasmic reticulum stress-related molecules glucose regulated protein 78(GRP78) and C/EBP homologous protein(CHOP), autophagy marker microtubule associated Protein1 light chain 3(LC3), Beclin-1, unfolded protein response branching pathway Inositol-requiring enzyme 1(IRE1), X box-binding protein 1(XBP1), c-Jun n-terminal kinase(JNK) and p-JNK were detected by Western blot in the solvent control group and different doses of VES group. IRE1 was inhibited by 4μ8C. The expressions of IRE1, XBP1, JNK, p-JNK, GRP78 and CHOP were detected by Western blot, and the expressions of LC3 and Beclin-1 were detected. The expression of GRP78(1.16±0.06) and CHOP(1.36±0.11) in 20 μg/mL VES group were significantly higher than those in solvent control group GRP78(0.36±0.10) and CHOP(0.48±0.05)(P&lt;0.001). The expression of Beclin-1(1.09±0.20) and LC3-Ⅱ/LC3-Ⅰ(1.29±0.03) in 20 μg/mL VES group were significantly higher than those in solvent control group(0.27±0.07) and LC3-Ⅱ/LC3-Ⅰ(0.43±0.06)(P&lt;0.001). The expression levels of IRE1(1.07±0.20), XBP1(1.33±0.07) and p-JNK/JNK(1.19±0.31) in 20 μg/mL VES group were significantly higher than those in the solvent control group(P&lt;0.01). After IRE1 is inhibited: The expression level of IRE1(0.63±0.27), XBP1(0.74±0.09), p-JNK/JNK(0.35±0.04), GRP78(0.66±0.02), CHOP(0.51±0.02), LC3-Ⅱ/LC3-Ⅰ(0.72±0.01), Beclin-1(0.70±0.15) was significantly lower than that of VES group(P&lt;0.05). VES may participate in the regulation of autophagy in gastric cancer cells by upregulating IRE1 pathway.

Effect of acupuncture on endoplasmic reticulum stress-related factors in hippocampus of post-traumatic stress disorder rats

To observe the effect of acupuncture on endoplasmic reticulum stress-related molecules glucose regulated protein 78 kD (GRP78), C/EBP homologous protein (CHOP), cysteinyl aspartate specific proteinase-12 (Caspase-12) and cysteinyl aspartate specific proteinase-3 (Caspase-3)in the hippocampus of rats with post-traumatic stress disorder, so as to explore the possible mechanism of acupuncture in treating post-traumatic stress disorder (PTSD). Twenty-eight SD rats were randomly divided into normal control, model, acupuncture and sertraline groups, with 7 rats in each group. The PTSD rat model was established by single prolonged stress. After modeling, acupuncture was applied to "Baihui" (GV20) and "Dazhui" (GV14) for rats of the acupuncture group for 10 min, once a day for 7 days. Sertraline (10 mg/kg) was given by gavage to rats of the sertraline group daily for 7 days. Rats' behavior was assessed by open field test and novelty-suppressed test. The mRNA expression levels of GRP78 and CHOP in the hippocampus were detected by real-time PCR. The expression le-vels of Caspase-12 and Caspase-3 in the hippocampus were detected by Western blot. Compared with the normal control group, the rearing and crossing times were decreased (P<0.05), the time remaining in the central zone and the total distance of movement were significantly reduced (P<0.01, P<0.05), the time of entering the central area for the first time was significantly increased (P<0.01), the latency of the novelty-suppressed feeding was significantly increased (P<0.05) in the model group, meanwhile the expression level of GRP78 and CHOP mRNAs, Caspase-12 and Caspase-3 proteins in the hippocampus were increased (P<0.05, P<0.01). In comparison with the model group, the crossing times, the time remaining in the central zone and total distance of movement were increased significantly (P<0.05, P<0.01), while the time of entering the central area for the first time, the expression levels of GRP78 and CHOP mRNAs, and Caspase-12 protein in the hippocampus were obviously decreased (P<0.05, P<0.01) in the acupuncture and sertraline groups. In addition, the rearing times were increased significantly (P<0.05), the latency of the novelty-suppressed feeding and the expression of Caspase-3 were decreased significantly (P<0.05) in the sertraline group than in the model group. Acupuncture can significantly down-regulate the expression of endoplasmic reticulum stress-related molecules GRP78, CHOP and Caspase-12 in PTSD rats, which may be one of the mechanisms of acupuncture in treating PTSD.

Methylmercury Induces Mitochondria- and Endoplasmic Reticulum Stress-Dependent Pancreatic β-Cell Apoptosis via an Oxidative Stress-Mediated JNK Signaling Pathway.

Methylmercury (MeHg), a long-lasting organic pollutant, is known to induce cytotoxic effects in mammalian cells. Epidemiological studies have suggested that environmental exposure to MeHg is linked to the development of diabetes mellitus (DM). The exact molecular mechanism of MeHg-induced pancreatic β-cell cytotoxicity is still unclear. Here, we found that MeHg (1-4 μM) significantly decreased insulin secretion and cell viability in pancreatic β-cell-derived RIN-m5F cells. A concomitant elevation of mitochondrial-dependent apoptotic events was observed, including decreased mitochondrial membrane potential and increased proapoptotic (Bax, Bak, p53)/antiapoptotic (Bcl-2) mRNA ratio, cytochrome c release, annexin V-Cy3 binding, caspase-3 activity, and caspase-3/-7/-9 activation. Exposure of RIN-m5F cells to MeHg (2 μM) also induced protein expression of endoplasmic reticulum (ER) stress-related signaling molecules, including C/EBP homologous protein (CHOP), X-box binding protein (XBP-1), and caspase-12. Pretreatment with 4-phenylbutyric acid (4-PBA; an ER stress inhibitor) and specific siRNAs for CHOP and XBP-1 significantly inhibited their expression and caspase-3/-12 activation in MeHg-exposed RIN-mF cells. MeHg could also evoke c-Jun N-terminal kinase (JNK) activation and reactive oxygen species (ROS) generation. Antioxidant N-acetylcysteine (NAC; 1mM) or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox; 100 μM) markedly prevented MeH-induced ROS generation and decreased cell viability in RIN-m5F cells. Furthermore, pretreatment of cells with SP600125 (JNK inhibitor; 10 μM) or NAC (1 mM) or transfection with JNK-specific siRNA obviously attenuated the MeHg-induced JNK phosphorylation, CHOP and XBP-1 protein expression, apoptotic events, and insulin secretion dysfunction. NAC significantly inhibited MeHg-activated JNK signaling, but SP600125 could not effectively reduce MeHg-induced ROS generation. Collectively, these findings demonstrate that the induction of ROS-activated JNK signaling is a crucial mechanism underlying MeHg-induced mitochondria- and ER stress-dependent apoptosis, ultimately leading to β-cell death.

Nelfinavir Induces Cytotoxicity towards High-Grade Serous Ovarian Cancer Cells, Involving Induction of the Unfolded Protein Response, Modulation of Protein Synthesis, DNA Damage, Lysosomal Impairment, and Potentiation of Toxicity Caused by Proteasome Inhibition.

Simple SummaryHigh-grade serous ovarian cancer (HGSOC) accounts for 70% of all ovarian-cancer-related deaths. Mainstay treatment with platinum-based drugs following surgery results in favorable outcomes in the majority of patients; however, in >80% of cases, the disease relapses with eventual drug resistance. As such, urgent development of improved alternative therapies is necessary for HGSOC patients with lower life expectancy. Rapid repurposing of market available drugs for cancer therapy is a cost-effective alternative to bypass the decade-long traditional drug development pipeline. Among potential drug-repurposing candidates, nelfinavir (NFV)—an anti-infective agent to treat acquired immunodeficiency syndrome (AIDS)—has shown anti-cancer effects against diverse cancers; however, its remedial benefits against HGSOC are unknown. In this study, we explored how NFV targets HGSOC cells obtained from patients at platinum-sensitive and -resistant stages. We observed beneficial efficacy elicited by NFV against HGSOC in both disease conditions through multiple mechanistic avenues, suggesting positive drug-repurposing prospects.High-grade serous ovarian cancer (HGSOC) is a significant cause of mortality among women worldwide. Traditional treatment consists of platinum-based therapy; however, rapid development of platinum resistance contributes to lower life expectancy, warranting newer therapies to supplement the current platinum-based protocol. Repurposing market-available drugs as cancer therapeutics is a cost- and time-effective way to avail new therapies to drug-resistant patients. The anti-HIV agent nelfinavir (NFV) has shown promising toxicity against various cancers; however, its role against HGSOC is unknown. Here, we studied the effect of NFV against HGSOC cells obtained from patients along disease progression and carrying different sensitivities to platinum. NFV triggered, independently of platinum sensitivity, a dose-dependent reduction in the HGSOC cell number and viability, and a parallel increase in hypo-diploid DNA content. Moreover, a dose-dependent reduction in clonogenic survival of cells escaping the acute toxicity was indicative of long-term residual damage. In addition, dose- and time-dependent phosphorylation of H2AX indicated NFV-mediated DNA damage, which was associated with decreased survival and proliferation signals driven by the AKT and ERK pathways. NFV also mediated a dose-dependent increase in endoplasmic reticulum stress-related molecules associated with long-term inhibition of protein synthesis and concurrent cell death; such events were accompanied by a proapoptotic environment, signaled by increased phospho-eIF2α, ATF4, and CHOP, increased Bax/Bcl-2 ratio, and cleaved executer caspase-7. Finally, we show that NFV potentiates the short-term cell cycle arrest and long-term toxicity caused by the proteasome inhibitor bortezomib. Overall, our in vitro study demonstrates that NFV can therapeutically target HGSOC cells of differential platinum sensitivities via several mechanisms, suggesting its prospective repurposing benefit considering its good safety profile.

Genetic loss-of-function of activating transcription factor 3 but not C-type lectin member 5A prevents diabetic peripheral neuropathy

We investigated the mediating roles of activating transcription factor 3 (ATF3), an injury marker, or C-type lectin member 5A (CLEC5A), an inflammatory response molecule, in the induction of endoplasmic reticulum (ER) stress and neuroinflammation in diabetic peripheral neuropathy in ATF3 and CLEC5A genetic knockout (aft3−/− and clec5a−/−, respectively) mice. ATF3 was expressed intranuclearly and was upregulated in mice with diabetic peripheral neuropathy (DN) and clec5a−/− mice. The DN and clec5a−/− groups also exhibited neuropathic behavior, but not in the aft3−/− group. The upregulation profiles of cytoplasmic polyadenylation element-binding protein, a protein translation–regulating molecule, and the ER stress-related molecules of inositol-requiring enzyme 1α and phosphorylated eukaryotic initiation factor 2α in the DN and clec5a−/− groups were correlated with neuropathic behavior. Ultrastructural evidence confirmed ER stress induction and neuroinflammation, including microglial enlargement and proinflammatory cytokine release, in the DN and clec5a−/− mice. By contrast, the induction of ER stress and neuroinflammation did not occur in the aft3−/− mice. Furthermore, the mRNA of reactive oxygen species–removing enzymes such as superoxide dismutase, heme oxygenase-1, and catalase were downregulated in the DN and clec5a−/− groups but were not changed in the aft3−/− group. Taken together, the results indicate that intraneuronal ATF3, but not CLEC5A, mediates the induction of ER stress and neuroinflammation associated with diabetic neuropathy.Neuroinflammation and endoplasmic reticulum stress is a critical process that leads to diabetic peripheral neuropathy (DPN). Activating transcription factor 3 (ATF3) is a susceptible molecule, and loss-of-function of ATF3, but not C-type lectin member 5A (CLEC5A), prevents the development of neuroinflammation and ER stress. Functional blockade of ATF3 may be a potential treatment for diabetic peripheral neuropathy by inhibiting neuroinflammation and cellular stress-induced ER stress.

Protective effect of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress

To explore the protective effect and mechanism of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress. Tunicamycin was used to establish the damage model in L02 cells. Methyl thiazolyl tetrazolium(MTT) colorimetric assay was used to investigate the survival rate of ethyl acetate extract from B. bipinnata in L02 cells injury induced by endoplasmic reticulum stress; the protein expressions of endoplasmic reticulum stress-related molecule glucose regulated protein 78(GRP78), PKR-like ER kinase(PERK), eukaryotic initiation factor-2(eIF2α), activating transcription factor 4(ATF4), C/EBP homologous protein(CHOP), B-cell CLL/lymphoma 2(Bcl-2), Bal-2 associated X apoptosis regulator(Bax) were examined by Wes-tern blot. The expressions of the above proteins were also detected after endoplasmic reticulum stress inhibitor(4-phenyl butyric acid) and CHOP shRNA-mediated knockdowns were added. The expressions of GRP78, PERK, CHOP in L02 cells were observed by immunofluorescence method. The results showed that ethyl acetate extract from B. bipinnata could significantly increase the survival rate of L02 cell injury caused by endoplasmic reticulum stress in a dose and time-dependent manner(P&lt;0.05 or P&lt;0.01). The expression levels of GRP78, PERK, eIF2α, ATF4, CHOP and Bax in the drug treatment groups were significantly down-regulated(P&lt;0.05 or P&lt;0.01), while Bcl-2 was significantly up-regulated(P&lt;0.01). After endoplasmic reticulum stress inhibitor and CHOP shRNA-mediated knockdowns were added, the expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax in the drug treatment groups were significantly down-regulated(P&lt;0.01), whereas Bcl-2 was significantly up-regulated(P&lt;0.01). Immunofluorescence results showed that the expressions of GRP78, PERK, CHOP were consistent with the Western blot method. In conclusion, ethyl acetate extract from B. bipinnata has a significant protective effect on the damage of L02 cells caused by endoplasmic reticulum stress. The mechanism may be related to the inhibition of endoplasmic reticulum stress and the down-regulation of apoptosis in cells through the PERK/eIF2α/ATF4/CHOP signaling pathway.

1169-P: PNPLA3 148M Is More Susceptible to PA-Induced Endoplasmic Reticulum Stress-Associated Apoptosis in HepG2 Cells

PNPLA3 is a major susceptibility gene for MAFLD and its polymorphic locus of rs738409 (I148M) is associated with MAFLD occurrence and progression. Endoplasmic reticulum (ER) stress and hepatocyte apoptosis induced by free fatty acids lipotoxicity in fatty liver cells play an important role in the progression of NAFLD [9]. PNPLA3 is a member of the non-calcium-dependent phospholipase A2 (iPLA2) family, iPLA2ε, and whether PNPLA3 148M has stronger phospholipase activity than 148I to promote apoptosis in hepatocyte is not clear. In this study, we constructed 148I/I knock-in HepG2 cell model by Cas9/sgRNA system on the basis of HepG2 carrying PNPLA3 148M/M. The PNPLA3 148MM and I/I cells were intervened with palmitic acid (PA) for 24 h, and cell lipid deposition was detected using oil red staining, apoptosis was observed by TUNEL, and western blot to detect PNPLA3 expression and ER stress-related CHOP apoptotic signaling pathway molecule expression. The results showed no difference in PNPLA3 expression and hepatocyte lipid deposition between PNPLA3 148M/M and 148I/I cells at baseline. However, after PA intervention, PNPLA3 148M/M cells exhibited more significant lipid deposition and apoptosis than 148I/I, as well as ER-related apoptotic molecules Bip, PERK, p-eIF2α, CHOP PUMA, BAX and cleaved caspase3 expression were more significantly increased. In conclusion, PNPLA3 148M/M cells were more significantly susceptible to PA-induced lipid deposition and endoplasmic reticulum stress-related apoptosis than 148I/I cells. Disclosure H. Liang: None. Funding Guangdong Natural Science Foundation (2020A1515010226)

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.

MicroRNA-211-5p attenuates spinal cord injury via targeting of activating transcription factor 6

The recovery of spinal cord injury (SCI) involves multiple factors, of which miRNAs take an important part. In this study, we evaluated the function of microRNA-211−5p (miR-211−5p) on SCI in a rat model. SCI model was established using modified Allen's weight-drop method and Basso-Bcattie-Bresnahan score was applied to assess the locomotor function. MiR-211−5p agomir was utilized to increase miR-211−5p expression and endoplasmic reticulum (ER) stress inhibitor, 4-PBA (4-phenylbutyric acid), was utilized to suppress ER stress. Neuron apoptosis and the expressions of miR-211−5p, activating transcription factor 6 (ATF6), apoptosis-related proteins, pro-inflammatory cytokines and endoplasmic reticulum stress-related proteins were detected. Dual luciferase reporter gene assay was performed to verify the binding between miR-211−5p and ATF6. The results showed that miR-211−5p directly targeted ATF6. MiR-211−5p was down-regulated and ATF6 was up-regulated in SCI rats. Both interferences with miR-211−5p agomir and 4-PBA effectively attenuated neuron apoptosis and reversed the expressions of apoptosis, inflammation and endoplasmic reticulum stress-related molecules post SCI in rats. These findings demonstrated that miR-211−5p could effectively alleviate SCI-induced neuron apoptosis and inflammation via directly targeting ATF-6 and regulating ER stress.

Stage-specific expression patterns of ER stress-related molecules in mice molars: Implications for tooth development

The endoplasmic reticulum (ER) is a site where protein folding and posttranslational modifications occur, but when unfolded or misfolded proteins accumulate in the ER lumen, an unfolded protein response (UPR) occurs. A UPR activates ER-stress signalling genes, including inositol-requiring enzyme-1 (Ire1), activating transcription factor 6 (Atf6), and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (Perk), to maintain homeostasis. The involvement of ER stress molecules in metabolic disease and hard tissue matrix formation has been established; however, an understanding of the role of ER-stress signalling molecules in tooth development is lacking. The aims of this study are to define the stage-specific expression patterns of ER stress-related molecules and to elucidate their putative functions in the organogenesis of teeth. This study leverages knowledge of the tissue morphology and expression patterns of a range of signalling molecules during tooth development. RT-qPCR, in situ hybridization, and immunohistochemistry analyses were performed to determine the stage-specific expression patterns of ER-stress-related signalling molecules at important stages of tooth development. RT-qPCR analyses showed that Atf6 and Perk have similar expression levels during all stages of tooth development; however, the expression levels of Ire1 and its downstream target X-box binding protein (Xbp1) increased significantly from the cap to the secretory stage of tooth development. In situ hybridization results revealed that Atf6 and Xbp1 were expressed in cells that form the enamel knot at cap stage and ameloblasts and odontoblasts at secretory stage in stage-specific patterns. In addition, Atf6, Ire1, and Xbp1 expression exhibited distinct localization patterns in secretory odontoblasts and ameloblasts of PN0 molars. Overall, our results strongly suggest that ER-stress molecules are involved in tooth development in response to protein overload that occurs during signaling modulations from enamel knots at cap stage and extracellular matrix secretion at secretory stage.

Excretory/secretory products of Angiostrongylus cantonensis fifth-stage larvae induce endoplasmic reticulum stress via the Sonic hedgehog pathway in mouse astrocytes

BackgroundAngiostrongylus cantonensis is an important food-borne zoonotic parasite. Humans are non-permissive hosts, and this parasite develops into fifth-stage larvae (L5) in the brain and subarachnoid cavity and then induces eosinophilic meningitis and eosinophilic meningoencephalitis. Excretory/secretory products (ESPs) are valuable targets for the investigation of host-parasite interactions. These products contain a wide range of molecules for penetrating defensive barriers and avoiding the immune response of the host. Endoplasmic reticulum (ER) stress has been found to be associated with a wide range of parasitic infections and inflammation. ER stress can increase cell survival via the activation of downstream signalling. However, the mechanisms of ER stress in A. cantonensis infection have not yet been clarified. This study was designed to investigate the molecular mechanisms of ER stress in astrocytes after treatment with the ESPs of A. cantonensis L5.ResultsThe results demonstrated that A. cantonensis infection activated astrocytes in the mouse hippocampus and induced the expression of ER stress-related molecules. Next, the data showed that the expression of ER stress-related molecules and the Ca2+ concentration were significantly increased in activated astrocytes after treatment with the ESPs of L5 of A. cantonensis. Ultimately, we found that ESPs induced GRP78 expression via the Sonic hedgehog (Shh) signalling pathway.ConclusionsThese findings suggest that in astrocytes, the ESPs of A. cantonensis L5 induce ER stress and that the Shh signalling pathway plays an important role in this process.

Newer perspective on the coupling between glucose-mediated signaling and β-cell functionality.

Insulin secretion by the pancreatic β-cells is elicited in response to elevated extracellular glucose concentration. In addition to triggering insulin secretion, glucose-induced signal regulates β-cell proliferation and survival. However, the molecular mechanism underlying the effects of glucose on the β-cell functionality still remains unclear. Glucokinase, a hexokinase isozyme that catalyzes the phosphorylation of glucose, acts as the glucose sensor in the β-cells. To investigate the mechanisms of glucose signaling in the regulation of β-cell functions, we analyzed the role of glucokinase in insulin secretion, β-cell proliferation and β-cell apoptosis, using β-cell-specific glucokinase-haploinsufficient (Gck+/-) mice and allosteric glucokinase activators (GKAs). Glucokinase-mediated glucose metabolism (1) suppresses endoplasmic reticulum (ER) stress-induced β-cell apoptosis via inducing insulin receptor substrate-2 (IRS-2) expression and expression of ER stress-related molecules, (2) promotes adaptive β-cell proliferation through activation of the Forkhead Box M1 (FoxM1)/polo-like kinase-1 (PLK1)/centromere protein-A (CENP-A) pathway, (3) induces islet inflammation by promoting interaction of islet-derived S100 calcium-binding protein A8 (S100A8) with macrophages, (4) induces the expression of Fibulin-5 (Fbln5), an extracellular matrix protein to regulate β-cell functions, and (5) activates other unknown pathways. Glucagon-like peptide-1 (GLP-1) receptor agonists and dipeptidyl peptidase 4 (DPP-4) inhibitors have been found to possibly compensate for dysregulation of glucose metabolism in the β-cells. This review provides an update and overview of the recent advances in the study of β-cell pathophysiology and some therapeutic possibilities focusing on glucose-/glucokinase-mediated signaling.

Autophagy activated via GRP78 to alleviate endoplasmic reticulum stress for cell survival in blue light-mediated damage of A2E-laden RPEs

BackgroundRetinal pigment epithelium cells (RPEs) are critical for maintaining retinal homeostasis. Accumulation of age-related lipofuscin, N-retinylidene-N-retinylethanolamine (A2E), makes RPEs vulnerable to blue light-mediated damage, which represents an initial cause of some retinal degenerative diseases. This study investigated the activation of autophagy and the signaling pathway involved in glucose-related protein 78 (GRP78) induced autophagy in blue light-mediated damage of A2E-laden RPEs. In addition, we explored whether autophagy could play a protective role by alleviating endoplasmic reticulum (ER) stress to promote RPEs survival.MethodsRPEs were incubated with 25 μM A2E for 2 h and exposed to blue light for 20 min. The expression of ER stress-related apoptotic proteins, CHOP and caspase-12, as well as autophagy marker LC3 were measured by western blot analysis. Autophagosomes were observed by both transmission electron microscopy and immunofluorescence assays. GRP78 interference performed by short hairpin RNA (shRNA) was used to identify the signaling pathway involved in GRP78 induced autophagy. Cell death was assessed using TUNEL analysis.ResultsTreatment with A2E and blue light markedly increased the expression of ER stress-related apoptotic molecules CHOP and caspase-12. The activation of autophagy was recognized by observing autophagosomes at ultrastructural level. Additionally, punctate distributions of LC3 immunofluorescence and enhanced conversions of LC3-I to LC3-II were found in A2E and blue light-treated RPEs. Moreover, GRP78 interference reduced AMPK phosphorylation and promoted mTOR activity, thereby downregulating autophagy. In addition, the inhibition of autophagy made RPEs vulnerable to A2E and blue light damage. In contrast, the autophagy inducer rapamycin alleviated ER stress to promote RPEs survival.ConclusionsGRP78 activates autophagy via AMPK/mTOR in blue light-mediated damage of A2E-laden RPEs in vitro. Autophagy may be a vital endogenous cytoprotective process to alleviate stress for RPEs survival in retinal degenerative diseases.

Medroxyprogesterone acetate causes the alterations of endoplasmic reticulum related mRNAs and lncRNAs in endometrial cancer cells

BackgroundProgestin is effective to promote endometrial cancer (EC) cells apoptosis, however, continuous progestin administration causes low level of progestin receptor B (PRB), further resulting in progestin resistance. Here, we performed microarray analysis on Ishikawa cells (PRB+) treated with medroxyprogesterone acetate (MPA) to explore the molecular mechanism underlying the inhibitory influence of MPA on PRB+ EC cells.MethodsMicroarray analysis was performed by using Ishikawa cells (PRB+) treated with MPA. Differentially expressed mRNA and long noncoding RNAs (lncRNAs) were identified. Furthermore, the functions of these mRNAs and lncRNAs were predicted by functional enrichment analysis. QRT-PCR was further performed to verify the microarray data.ResultsA total of 358 differentially expressed genes and 292 lncRNAs were identified in Ishikawa cells (PRB+) treated with MPA. QRT-PCR verified these data. Functional enrichment analysis identified endoplasmic reticulum (ER) stress as the key pathway involved in the inhibitory effect of MPA on EC cells. And the ER stress apoptotic molecule CHOP and ER stress related molecule HERPUD1 were both highly expressed in Ishikawa cells (PRB+) treated with MPA. Co-expression analysis showed lnc-CETP-3 was highly correlated with CHOP and HERPUD1, suggesting it might participate in ER stress pathway-related EC cell apoptosis caused by MPA. In addition, compared with untreated cells, lnc-CETP-3, CHOP and HERPUD1 were significantly up-regulated in Ishikawa cells (PRB+) treated with MPA, whereas they have no statistical significance in KLE cells (PRB-).ConclusionsMPA may activate ER stress by progesterone-PRB pathway to up-regulate CHOP expression, which may be one of the molecular mechanisms underlying the inhibitory effect of MPA on EC cells with PRB+. Lnc-CETP-3 might be involved in this process. These findings may provide therapeutic targets for EC patients with PRB-, and resistance-related targets to increase the sensitivity of MPA on EC cells.