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Cells Apr 2024SARS-Co-V2 infection can induce ER stress-associated activation of unfolded protein response (UPR) in host cells, which may contribute to the pathogenesis of COVID-19....
BACKGROUND
SARS-Co-V2 infection can induce ER stress-associated activation of unfolded protein response (UPR) in host cells, which may contribute to the pathogenesis of COVID-19. To understand the complex interplay between SARS-Co-V2 infection and UPR signaling, we examined the effects of acute pre-existing ER stress on SARS-Co-V2 infectivity.
METHODS
Huh-7 cells were treated with Tunicamycin (TUN) and Thapsigargin (THA) prior to SARS-CoV-2pp transduction (48 h p.i.) to induce ER stress. Pseudo-typed particles (SARS-CoV-2pp) entry into host cells was measured by Bright Glo luciferase assay. Cell viability was assessed by cell titer Glo luminescent assay. The mRNA and protein expression was evaluated by RT-qPCR and Western Blot.
RESULTS
TUN (5 µg/mL) and THA (1 µM) efficiently inhibited the entry of SARS-CoV-2pp into host cells without any cytotoxic effect. TUN and THA's attenuation of virus entry was associated with differential modulation of ACE2 expression. Both TUN and THA significantly reduced the expression of stress-inducible ER chaperone GRP78/BiP in transduced cells. In contrast, the IRE1-XBP1s and PERK-eIF2α-ATF4-CHOP signaling pathways were downregulated with THA treatment, but not TUN in transduced cells. Insulin-mediated glucose uptake and phosphorylation of Ser IRS-1 and downstream p-AKT were enhanced with THA in transduced cells. Furthermore, TUN and THA differentially affected lipid metabolism and apoptotic signaling pathways.
CONCLUSIONS
These findings suggest that short-term pre-existing ER stress prior to virus infection induces a specific UPR response in host cells capable of counteracting stress-inducible elements signaling, thereby depriving SARS-Co-V2 of essential components for entry and replication. Pharmacological manipulation of ER stress in host cells might provide new therapeutic strategies to alleviate SARS-CoV-2 infection.
Topics: Humans; Thapsigargin; Unfolded Protein Response; Tunicamycin; Apoptosis; SARS-CoV-2; Endoplasmic Reticulum Chaperone BiP; Signal Transduction; Proto-Oncogene Proteins c-akt; Endoplasmic Reticulum Stress; COVID-19; Virus Internalization
PubMed: 38727305
DOI: 10.3390/cells13090769 -
Human & Experimental Toxicology 2024To explore the effect of acacetin on subarachnoid hemorrhage (SAH) and its possible mechanism.
PURPOSE
To explore the effect of acacetin on subarachnoid hemorrhage (SAH) and its possible mechanism.
METHODS
SAH model of rat was established, and intraperitoneally injected with three doses of acacetin. To verify the role of PERK pathway, we used the CCT020312 (PERK inhibitor) and Tunicamycin (activators of endoplasmic reticulum stress). The SAH score, neurological function score, brain edema content, and Evans blue (EB) exudate were evaluated. Western blot was used to determine the expression of inflammation-associated proteins and PERK pathway. The activation of microglia was also determined through Iba-1 detection. TEM and immunofluorescence staining of LC3B were performed to observe the autophagy degree of SAH rats after acacetin. Tunel/NeuN staining, HE and Nissl' staining were performed for neuronal damage.
RESULTS
Acacetin increased the neurological function score, reduce brain water content, Evans blue exudation and SAH scores. The microglia in cerebral cortex were activated after SAH, while acacetin could inhibit its activation, and decreased the expression of TNF-α and IL-6 proteins. The pathological staining showed the severe neuronal damage and increased neuronal apoptosis after SAH, while acacetin could improve these pathological changes. We also visualized the alleviated autophagy after acacetin. The expression of Beclin1 and ATF4 proteins were increased, but acacetin could inhibit them. Acacetin also inactivated PERK pathway, which could improve the neuronal injury and neuroinflammation after SAH, inhibit the microglia activation and the overactivated autophagy through PERK pathway.
CONCLUSION
Acacetin may alleviate neuroinflammation and neuronal damage through PERK pathway, thus having the protective effect on EBI after SAH.
Topics: Animals; Subarachnoid Hemorrhage; Microglia; Autophagy; eIF-2 Kinase; Male; Neuroinflammatory Diseases; Rats, Sprague-Dawley; Rats; Signal Transduction; Flavones
PubMed: 38720657
DOI: 10.1177/09603271241251447 -
The Journal of Biological Chemistry May 2024Endoplasmic reticulum (ER) stress, a common cellular stress response induced by various factors that interfere with cellular homeostasis, may trigger cell apoptosis....
Endoplasmic reticulum (ER) stress, a common cellular stress response induced by various factors that interfere with cellular homeostasis, may trigger cell apoptosis. Autophagy is an important and conserved mechanism for eliminating aggregated proteins and maintaining protein stability of cells, which is closely associated with ER stress and ER stress-induced apoptosis. In this paper, we report for the first time that Hhatl, an ER-resident protein, is downregulated in response to ER stress. Hhatl overexpression alleviated ER stress and ER stress induced apoptosis in cells treated with tunicamycin or thapsigargin, whereas Hhatl knockdown exacerbated ER stress and apoptosis. Further study showed that Hhatl attenuates ER stress by promoting autophagic flux. Mechanistically, we found that Hhatl promotes autophagy by associating with autophagic protein LC3 (microtubule-associated protein 1A/1B-light chain 3) via the conserved LC3-interacting region (LIR) motif. Noticeably, the LIR motif was essential for Hhatl-regulated promotion of autophagy and reduction of ER stress. These findings demonstrate that Hhatl ameliorates ER stress via autophagy activation by interacting with LC3, thereby alleviating cellular pressure. The study indicates that pharmacological or genetic regulation of Hhatl-autophagy signaling might be potential for mediating ER stress and related diseases.
PubMed: 38705394
DOI: 10.1016/j.jbc.2024.107335 -
Current Issues in Molecular Biology Apr 2024Corneal ulcers, characterized by severe inflammation of the cornea, can lead to serious, debilitating complications and may be vision-threatening for horses. In this...
Corneal ulcers, characterized by severe inflammation of the cornea, can lead to serious, debilitating complications and may be vision-threatening for horses. In this study, we aimed to investigate the role of endoplasmic reticulum (ER) stress in corneal stem progenitor cell (CSSC) dysfunction and explore the potential of equine adipose-derived stromal stem cell (ASC)-derived extracellular vesicles (EVs) to improve corneal wound healing. We showed that CSSCs expressed high levels of CD44, CD45, and CD90 surface markers, indicating their stemness. Supplementation of the ER-stress-inducer tunicamycin to CSSCs resulted in reduced proliferative and migratory potential, accumulation of endoplasmic reticulum (ER)-stressed cells in the G0/G1 phase of the cell cycle, increased expression of proinflammatory genes, induced oxidative stress and sustained ER stress, and unfolded protein response (UPR). Importantly, treatment with EVs increased the proliferative activity and number of cells in the G2/Mitosis phase, enhanced migratory ability, suppressed the overexpression of proinflammatory cytokines, and upregulated the anti-inflammatory , compared to control and/or ER-stressed cells. Additionally, EVs lowered the expression of ER-stress master regulators and effectors (, , , and ), increased the number of mitochondria, and reduced the expression of and , thereby promoting metabolic homeostasis and protecting against apoptosis in equine CSSCs. Our findings demonstrate that MSCs-derived EVs represent an innovative and promising therapeutic strategy for the transfer of bioactive mediators which regulate various cellular and molecular signaling pathways.
PubMed: 38666934
DOI: 10.3390/cimb46040204 -
Cartilage Apr 2024Oxidative stress and endoplasmic reticulum (ER) stress play pivotal roles in disrupting the homeostasis of chondrocytes by producing catalytic proteases and enhancing...
OBJECTIVE
Oxidative stress and endoplasmic reticulum (ER) stress play pivotal roles in disrupting the homeostasis of chondrocytes by producing catalytic proteases and enhancing chondrocyte senescence, consequently contributing to the progression of osteoarthritis (OA). Despite their close interaction, the underlying molecular mechanisms remain poorly understood. Here, we show that ER stress and oxidative stress reciprocally modulate each other to promote cartilage degradation.
METHODS
Primary chondrocytes were obtained from the articular cartilage of 5-day-old C57BL/6J mice by excising distal femur and proximal tibia. Tunicamycin was applied to induce ER stress in primary chondrocytes. Surgical OA was induced in 12-week-old male C57BL/6J mice by destabilizing the medial meniscus (DMM).
RESULTS
Tunicamycin-induced ER stress led to an increase in the production of reactive oxygen species (ROS) and catalytic proteases, including MMP13 and Adamts5, in primary chondrocytes, and it was primarily dependent on the NADPH oxidase (NOX) system. ER stress directly increased the expression of NOX2, NOX3, NOX4, and p22phox. Specifically, the protein kinase RNA-like ER kinase (PERK) pathway is involved in the expression of NOX4 and p22phox, the inositol-requiring enzyme 1 alpha (IRE1α) pathway in NOX2 and NOX3 expression, and the activating transcription factor 6 (ATF6) pathway influences NOX3 expression in chondrocytes. Conversely, inhibiting NOX function significantly reduced both ER stress sensor-related signaling and chondrocyte catabolism, thereby decelerating the progression of surgically induced OA .
CONCLUSIONS
Our findings highlight the positive feedback loop between ER stress and oxidative stress in OA pathogenesis, suggesting that targeting NOX isoforms is a promising therapeutic strategy for OA.
PubMed: 38641979
DOI: 10.1177/19476035241245803 -
Gene Jul 2024Cells sense, respond, and adapt to environmental conditions that cause stress. In a previous study using HeLa cells, we isolated reporter cells responding to the...
Cells sense, respond, and adapt to environmental conditions that cause stress. In a previous study using HeLa cells, we isolated reporter cells responding to the endoplasmic reticulum (ER) stress inducers, thapsigargin and tunicamycin, using a highly sensitive promoter trap vector system. Splinkerette PCR and 5' rapid amplification of cDNA ends (5' RACE) identified a novel transcript that is upregulated by ER stress. Its endogenous expression increased approximately 10-fold in response to thapsigargin and tunicamycin within 1 h, but was down-regulated after 4 h. Because the transcript starts from an intron of a long noncoding RNA known as LINC-PINT, we designated the newly identified transcript TISPL (transcript induced by stressors from LINC-PINTlocus). TISPL was also expressed under several other stress conditions. It was particularly increased > 10-fold upon glucose starvation and 7-fold by arsenite exposure. Furthermore, in silico analyses, including a ChIP-atlas search, revealed that there is an ATF4-binding region with a c/ebp-Atf response element (CARE) downstream of the transcription start site of TISPL. Based on these results, we hypothesized that TISPL may be induced by the phospho-eIF2α and ATF4- axis of the integrated stress response pathway, which is known to be activated by the stress conditions listed above. As expected, knockout of ATF4 abolished the stress-induced upregulation of TISPL. Our results indicate that TISPL may be a useful biomarker for detecting stress conditions that activate ATF4. Our highly sensitive trap vector system proved beneficial in discovering new biomarkers.
Topics: Activating Transcription Factor 4; Humans; HeLa Cells; Up-Regulation; Endoplasmic Reticulum Stress; RNA, Long Noncoding; Thapsigargin; Tunicamycin; Arsenites
PubMed: 38615981
DOI: 10.1016/j.gene.2024.148464 -
International Journal of Molecular... Mar 2024There is a growing body of evidence that ER stress and the unfolded protein response (UPR) play a key role in numerous diseases. Impaired liver perfusion and ER stress...
There is a growing body of evidence that ER stress and the unfolded protein response (UPR) play a key role in numerous diseases. Impaired liver perfusion and ER stress often accompany each other in liver diseases. However, the exact impact of ER stress and UPR on the hepatic perfusion is not fully understood. The aim of this study was to disclose the effect of ER stress and UPR on the size of liver vessels and on the levels of Ca and nitric oxide (NO), critical regulators of vascular tonus. This study was carried out in precisely cut liver tissue slices. Confocal microscopy was used to create 3D images of vessels. NO levels were determined either using either laser scan microscopy (LSM) in cells or by NO-analyser in medium. Ca levels were analysed by LSM. We show that tunicamycin, an inducer of ER stress, acts similarly with vasodilator acetylcholine. Both exert a similar effect on the NO and Ca levels; both induce significant vasodilation. Notably, this vasodilative effect persisted despite individual inhibition of UPR pathways-ATF-6, PERK, and IRE1-despite confirming the activation of UPR. Experiments with HUVEC cells showed that elevated NO levels did not result from endothelial NO synthase (eNOS) activation. Our study suggests that tunicamycin-mediated ER stress induces liver vessel vasodilation in an NO-dependent manner, which is mediated by intracellular nitrodilator-activatable NO store (NANOS) in smooth muscle cells rather than by eNOS.
Topics: Vasodilation; Tunicamycin; Endoplasmic Reticulum Stress; Unfolded Protein Response; Liver
PubMed: 38612675
DOI: 10.3390/ijms25073865 -
Life Science Alliance Jul 2024Elevated ER stress has been linked to the pathogenesis of several disease conditions including neurodegeneration. In this study, we have holistically determined the...
Elevated ER stress has been linked to the pathogenesis of several disease conditions including neurodegeneration. In this study, we have holistically determined the differential expression of all the nuclear receptors (NRs) in the presence of classical ER stress inducers. Activation of Nr1h4 and Thrb by their cognate ligands (GW4064 and T3) ameliorates the tunicamycin (TM)-induced expression of ER stress genes. A combination of both ligands is effective in mitigating cell death induced by TM. Further exploration of their protective effects in the Parkinson's disease (PD) model shows that they reduce MPP-induced dissipation of mitochondrial membrane potential and ROS generation in an in vitro PD model in neuronal cells. Furthermore, the generation of an experimental murine PD model reveals that simultaneous treatment of GW4064 and T3 protects mice from ER stress, dopaminergic cell death, and functional deficits in the MPTP mouse model of PD. Thus, activation of Nr1h4 and Thrb by their respective ligands plays an indispensable role in ER stress amelioration and mounts protective effects in the MPTP mouse model of PD.
Topics: Animals; Mice; Cell Death; Disease Models, Animal; Dopamine; Dopaminergic Neurons; Parkinson Disease; Thyroid Hormone Receptors beta
PubMed: 38609183
DOI: 10.26508/lsa.202302416 -
Journal of Hepatology Apr 2024Endoplasmic reticulum (ER) membrane protein complex subunit 10 (EMC10) has been implicated in obesity. Here we investigated the roles of the two isoforms of EMC10,...
BACKGROUND & AIMS
Endoplasmic reticulum (ER) membrane protein complex subunit 10 (EMC10) has been implicated in obesity. Here we investigated the roles of the two isoforms of EMC10, including a secreted isoform (scEMC10) and an ER membrane-bound isoform (mEMC10), in MASLD.
METHODS
Manifold steatotic mouse models and HepG2 cells were employed to investigate the role of EMC10 in the regulation of hepatic PERK-eIF2α-ATF4 signaling and hepatosteatosis. The therapeutic effect of scEMC10-neutralizing antibody on mouse hepatosteatosis was explored. Associations of MASLD with serum scEMC10 and hepatic mEMC10 were determined in two cohorts of participants with MASLD.
RESULTS
scEMC10 promoted, while mEMC10 suppressed the activation of hepatocytic PERK-eIF2α-ATF4 signaling. Emc10 gene knockout exacerbated, while hepatic overexpression of mEMC10 ameliorated hepatic ER stress and steatosis in mice challenged with either a MCD diet or tunicamycin, highlighting a direct, suppressive role of mEMC10 in MASLD via modulation of hepatic ER stress. Overexpression of scEMC10 promoted, whereas neutralization of circulating scEMC10 prevented hepatosteatosis in mice with fatty liver, suggesting a progressive role of scEMC10 in MASLD. Clinically, serum scEMC10 increased, while hepatic mEMC10 decreased in participants with MASLD. Correlative analysis indicated serum scEMC10 positively, whereas hepatic mEMC10 negatively correlated with liver fat content and serum ALT, AST, and GGT.
CONCLUSIONS
These findings demonstrate a novel, isoform specific role for EMC10 in the pathogenesis of MASLD and identify the secreted isoform as a tractable therapeutic target for MASLD via antibody-based neutralization.
IMPACT AND IMPLICATIONS
We have shown the role of EMC10 in the regulation of energy homeostasis and obesity. In this study, we determine the distinct roles of the two isoforms of EMC10 in the regulation of hepatic ER stress and steatosis in mice, and associations of MASLD with different EMC10 isoforms in humans. Our findings delineate a novel regulatory axis for hepatosteatosis and identify EMC10 as a modulator of the PERK-eIF2α-ATF4 signaling cascade that may be of broad physiological significance. Moreover, our pre-clinical and clinical studies clearly provide the foundations for translation of scEMC10 modulation for the treatment of MASLD.
PubMed: 38599383
DOI: 10.1016/j.jhep.2024.03.047 -
Experimental and Therapeutic Medicine May 2024The complex manifestation of diabetic hearing loss and the relative inaccessibility of the inner ear contribute to the lack of research. The present study aimed to...
The complex manifestation of diabetic hearing loss and the relative inaccessibility of the inner ear contribute to the lack of research. The present study aimed to reveal the role of Apelin-13, a critical regulator of lipid metabolism, in diabetes-induced hearing loss. Cochlear hair cells treated with high glucose (HG) were adopted as an research model, and the impacts of Apelin-13 on cellular oxidative stress, apoptosis, mitochondrial dysfunction and endoplasmic reticulum (ER) stress were determined. In addition, cells were treated with the ER stress agonist tunicamycin to further explore its potential role in the regulatory effects of Apelin-13. Apelin-13 inhibited oxidative stress and apoptosis in the HG-induced cells. Additionally, Apelin-13 elevated mitochondrial membrane potential and ATP production, whereas it reduced mitochondrial reactive oxygen species levels. The levels of ER stress-related proteins exhibited a downward trend in response to Apelin-13. By contrast, tunicamycin reversed the effects of Apelin-13 on the aforementioned aspects, suggesting the role of ER stress in the regulatory effects of Apelin-13. In conclusion, the present study elucidated the protective role of Apelin-13 in ameliorating HG-induced mitochondrial functional impairment in cochlear hair cells by inhibiting ER stress.
PubMed: 38596659
DOI: 10.3892/etm.2024.12515